Research ArticleOlive Ridley Sea Turtle Hatching Success as a Function ofMicrobial Abundance and the Microenvironment of In Situ NestSand at Ostional Costa Rica
Vanessa S Beacutezy1 Roldaacuten A Valverde2 and Craig J Plante1
1Department of Biology College of Charleston 66 George Street Charleston SC 29424 USA2Department of Biological Sciences Southeastern Louisiana University SLU Box 10736 Hammond LA 70402 USA
Correspondence should be addressed to Vanessa S Bezy vanessabezygmailcom
Received 11 August 2014 Revised 18 November 2014 Accepted 21 November 2014 Published 22 December 2014
Academic Editor Horst Felbeck
Copyright copy 2014 Vanessa S Bezy et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Sea turtle hatching success at mass nesting beaches is typically lower than at solitary nesting beaches presumably due in partto high rates of microbial metabolism resulting from the large input of organic matter from turtle eggs Therefore we tested thehypothesis that hatching success varies across areas of the beach in conjunction with differences in the physical nest environmentandmicrobial abundance of in situ olive ridley sea turtle nests atOstional Costa RicaWemarked natural nests in high-density low-density and tidal-wash nesting areas of the beach and monitored clutch pO
2and temperature throughout the incubation period
We quantified hatching success and collected samples of nest sand during nest excavations We quantified microbial abundance(bacteria and fungi) with a quantitative polymerase chain reaction (qPCR) analysis Hatching success was lower in nests with lowerpO2 higher temperatures higher organic matter content and higher microbial abundance Our results suggest that the lower
oxygen within the nest environment is likely a result of the high microbial abundance and rates of decomposition in the nest sandand that these factors along with increased temperature of clutches in the high-density nesting area are collectively responsible forthe low hatching success at Ostional
1 Introduction
The olive ridley sea turtle (Lepidochelys olivacea) is listedby the IUCN as a vulnerable species The major threats tothis species include fisheries bycatch habitat degradationand the unsustainable harvest of eggs [1] This species ischaracterized by a nesting behavioral polymorphism withsome females nesting solitarily and others nesting en masseMass nesting events at Ostional Costa Rica are some of thelargest in the world with events (or arribadas) estimatedat up to approximately 500000 nesting females [2] Yearlyarribada abundance at Ostional has exhibited a decreaseover the past 15 years which has been attributed in partto the particularly low hatching success at this beach (8[2 3]) While nest density can adversely affect hatchingsuccess (due to competition between developing embryosfor physiological requirements) the mean nest density at
Ostional is not high enough to reduce hatching success to thedrastically low levels observed [4 5]
Rather several studies have suggested that embryo mor-tality is associatedwith the highmicrobial load resulting fromthe decomposition of eggs broken by subsequent nestingturtles during these high-density arribada events [4 6 7]For example a previous study at an arribada beach observedhigher temperature and partial pressure of carbon dioxide(pCO
2) and lower partial pressure of oxygen (pO
2) in egg
clutches incubated in situ versus a hatchery [8] This samestudy also reported increased hatching success in the cleaner(tidal washed and sieved) hatchery sand in comparison tonatural beach sand Therefore microbial load and organicmatter could presumably be responsible for these differencesand may influence hatching success In fact nutrient loadsand bacterial abundance in the sand at mass nesting beaches
Hindawi Publishing CorporationJournal of Marine BiologyVolume 2014 Article ID 351921 10 pageshttpdxdoiorg1011552014351921
2 Journal of Marine Biology
aremuch higher than at neighboring beaches [9 10] Further-more Cornelius et al [6] suggested that observed increases inhatching success in nests below the high tide line may be dueto natural tidal washing which could remove microbes thatare intolerant to salt water Another study at arribada beachesfound that while bacterial phylotype richness and diversityincreased with increasing nest density in the high andmiddlezones of the beach this was not true in the low zone of thebeach perhaps due to the less stable osmotic environmentcaused by the tides [11]
Bacteria and fungi have been cultured and isolated fromnest sand and failed eggs as well as from the cloacal fluidof nesting females [10 12 13] However the literature onthe infection of sea turtle eggs by microbes is controversialMicrobial infection was traditionally thought to be oppor-tunistic and limited laboratory studies found no significanteffect of the presence of bacteria or fungi on the hatchlingproduction of olive ridley sea turtle eggs [10 12 14] Morerecent studies on the Fusarium solani species complex haveidentified several fungal pathogens to sea turtle eggs [15 16]However eggs infected with F solani can be asymptomatic[15] and other studies challenge this causality [17 18] Thesestudies reflect the difficulty in directly linking embryo mor-tality to the presence ofmicrobes in part due to limitations inconducting research on protected species as well as the highoccurrence of total nest failure in such studies [14 19]
While the negative effect of higher microbial densitieson hatching success at Ostional has long been presumedno previous research has ever directly quantified microbialabundance or the associated conditions of the nest envi-ronment with respect to hatching success in natural nestsBecause sea turtle embryo mortality is likely linked to manyfactors it is necessary to evaluate the entire nest environmentin order to fully understand the interactions between a clutchof eggs and the microecosystem that comprises a sea turtlenest [13 20]
The aim of this study was to describe the natural con-ditions of the sea turtle nest environment at Ostional inorder to identify factors that may be adversely impactingsea turtle embryonic development We tested the hypothesisthat hatching success varies across the beach in associationwith differences in the nest environment and microbialabundance We predicted that higher hatching success wouldoccur in beach areas exposed to tidal washing and that aninverse relationship exists between microbial abundance andhatching success To accomplish this we monitored nestconditions (ie temperature oxygen and organic mattercontent) and hatching success in natural nests located indifferent areas of the beach and quantified the microbialabundance in nest sand
2 Methodology
21 Study Site The Ostional National Wildlife Refuge(ONWR) is located on the Pacific coast of the NicoyaPeninsula in Costa Rica (9996471∘N 85697800∘W) WithinONWR the Nosara and Ostional beaches make up approx-imately 7 km of beach with variable width This study wasconducted at Ostional beach during the rainy season (May
through November) of 2012 when arribadas are typicallymore abundant All study nests were laid during the samenight of the arribada that occurred during the last quartermoon of August 2012 (hereinafter called the sample arrib-ada) This ensured that all nests incubated at the same timeand helped standardize any uncontrollable variables thatcould affect hatching success such as ambient temperatureand rainfall
We compared the microbial abundance in relation tohatching success in natural (in situ) nests located in a tidal-wash area a high-density nesting area where arribadas tendto concentrate and a low-density nesting areaThe tidal-washarea of the beachwas locatedwithin 25meters of a lagoon andin the tidal-wash zone where the tide is known to wash overthe berm and cause sand turnover The high-density nestingarea was located at the main nesting beach where the samplearribada was recorded to have the highest density of nestingfemales The low-density nesting area was located where thesample arribada was recorded to have the lowest densityof nesting females Nest densities for the sample arribadawere determined by the strip-transect-in-time method andconfirmed with quadrat sampling at the time of excavation[5 21]
We randomly selected five nests above the average hightide line in the tidal-wash high-nest-density and low-nest-density beach areas described above Eggs were countedduring oviposition to determine the total clutch count Adatalogger and oxygen tubing were placed within the centerof the nest chamber after approximately 50 eggs were laidA 5-sided 50 times 50 times 15 cm wire mesh cage with a woodenframe was placed over the nest and buried entirely below thesurface of the sand to prevent predation and excavation bysubsequently nesting females This cage was pulled above thesurface of the sand onday 40 of incubation to allowhatchlingsto emerge to the surface unobstructed Nests were thenmonitored three times daily (sunrise sunset and midnight)for signs of hatching in order to count and release hatchlingsas soon as possible
22 Nest pO2 Nest pO2 was monitored by placing an airstone fitted with the tip of 60 cm nylon tubing into the centerof the egg mass that ran from inside of the nest chamberto the top layer of sand where a shut-off valve impeded anyadditional gas exchange [4 8 22] The pO
2within the egg
clutch was measured using a flow-through oxygen sensor(S108 Oxygen Analyzer Qubit Systems) that was calibratedprior to the field season using nitrogen and prior to each set ofsamples using atmospheric air Dead air space (approximately10mL) was drawn from within the tubing and expelled priorto sampling to ensure the air sample was from within thenest cavity Air samples (approximately 60mL) were drawnusing an airtight syringe and analyzedwithin 1 h of collectionSamples were slowly injected through an air pump flowmeter and desiccant column and through the O
2sensor at
a flow rate of approximately 50mLminminus1 Air samples wereanalyzed every 5 days for the first 30 days of incubationand every 4 days through the end of the incubation periodGas percentages were converted to partial pressures usingambient barometric pressure
Journal of Marine Biology 3
23 Nest Temperature Nest temperature was monitoredusing HOBO pendant temperature dataloggers (Onset Com-puter Corporation) placed in the center of each clutch andprogrammed to record temperature at 3 h intervals startingat midnight on the night of oviposition through hatchlingemergence Mean daily nest temperatures were used tocompare nest temperatures across the different areas of thebeach
24 Nest Excavations Single-use sterile gloves were wornfor all excavations and changed before contact with differentnests Nests were exhumed three days after the last observedhatchling emergence to quantify hatching success [23]
25 Sand Collection and Characterization A sample of sandwas collected directly into sterile collection tubes from thecenter of the nest chamber during the excavation of the nest atthe end of the incubation period Samples were placed on iceimmediately after collection and then either frozen (minus20∘C)or preserved in formalin (2 formaldehyde) within 6 h ofcollection and until analysis The organic matter analysisconsisted of a loss-on-ignition method with the organicmatter content being the loss of mass after dry combustionThe sample was transferred to a porcelain container anddesiccated in a drying oven (24 h at 100∘C) before combustion(6 h at 500∘C) Prior to combustion samples of dried sandwere fractionated with a set of sieves (0063 0075 015 025045 and 085mm) to determine the particle-size distributionbymassMean grain size (120601) sorting (120590
120601) and skewness (Sk
120601)
were calculated using the logarithmic mathematical ldquomethodof momentsrdquo in GRADISTAT [24 25]
26 Microbial Abundance Samples preserved in formalinwere used for microscopy counts as a secondary methodof quantifying bacterial abundance These samples werecentrifuged for 10min at 16000 g in a microcentrifuge beforecarefully removing excess formalin The sand was thendiluted (approximately 1 2) with sterile water and sonicatedon ice for 20 s at 30W (Sonifier S-250A Branson) Theresulting supernatant fluid was stained for approximately 5minutes with a 1 10 dilution of 1x SybrGold and sterile waterMicroscopy counts were performed at 1000x magnificationon an epifluorescence microscope (Optiphot-2 Nikon) bycounting 10 fields per slide The number of cells gminus1 of sandwas then calculated based on the original weight of sandvolume of diluent and supernatant and the average numberof cells fieldminus1 using the number of fields per slide at 1000x(479 times 104) and correction factor for the addition of formalin(times116)
We used a quantitative real-time polymerase chain reac-tion (qPCR) analysis to determine the abundance of bacteriabased on the number of 16S rRNA gene copies gminus1 nestsand and the abundance of fungi based on the number of18S rRNA gene copies gminus1 nest sand While the resultingquantification of gene copies from a qPCR analysis cannot bedirectly transformed into the number of cells (given that copynumber can vary greatly between species) this can be used asa proxy for overall abundance ofmicrobial communities [26ndash28]
27 DNA Extraction Each sample of sand was thawed andhomogenized by vortexing before collecting a 1 g subsamplefor DNA extraction DNA was extracted from all samplesof sand using an EZNA Soil DNA kit (Omega Biotek) withthe following modifications to the protocol to increase DNAyields Samples were subjected to 5min at approximately2000 oscillations minminus1 in a bead beater (Mini Beadbeater-8Biospec Products) and three freeze-thaw cycles (minus20∘C and70∘C for 30min each) during the lysis step Additionally inthe final step of extraction only 50 120583L of elution buffer wasusedwhichwas reapplied to the column in the second elutionstep DNA samples were diluted in order to reduce inhibitionand optimize efficiency and Ct values to within the range ofthe standard curve
28 qPCR Analysis Absolute qPCR was run using an iCycleriQ Real-Time PCR Detection System (Bio-Rad LaboratoriesInc) on a 96-well plate Results were analyzed using iQ5software (Bio-Rad Laboratories Inc)The universal bacterialprimers 926F (51015840-AAA CTC AAA KGA ATT GAC GG-31015840)and 1062R (51015840-CTCACRRCACGAGCTGAC-31015840) that targetthe 16S rRNA gene were used based on a previous studyby de Gregoris et al [29] Each 10 120583L reaction containedthe following 5120583L of ABsolute qPCR Master Mix (ABgene)01 120583L bovine serum albumin (10 120583g 120583Lminus1Thermo Scientific)03 120583L of each primer (10 120583M 300 nM final concentrationIntegrated DNA Technologies) 39 120583L H
2O and 04 120583L tem-
plate DNA PCR conditions were 15min at 95∘C followed by40 cycles of 95∘C for 15 s 15 s at the annealing temperature of57∘C and 72∘C for 20 s
The universal fungal primers FR1 (51015840-AICCATTCAATC-GGTAIT-31015840) and FF390 (51015840-CGATAACGAACGAGACCT-31015840) that target the 18S rRNAgenewere used based on previousstudies [26 30] Each 10 120583L reaction contained the following5 120583L of ABsolute qPCR Master Mix (ABgene) 01 120583L bovineserum albumin (10 120583g 120583Lminus1Thermo Scientific) 01 120583L of eachprimer (10120583M 100 nM final concentration Integrated DNATechnologies) 43 120583L H
2O and 04 120583L template DNA PCR
conditions were 15min at 95∘C followed by 40 cycles of 95∘Cfor 15 s 30 s at the annealing temperature of 50∘C and 72∘Cfor 1min
29 qPCR Standards External fixed standards were cre-ated by amplifying and quantifying bacterial and fun-gal DNA using primer sets that targeted the full 16S18SrRNA sequence Template DNA extractions for bacterialand fungal standards were kindly provided from culturesof Bacillus pumilus (W Hook Grice Marine LaboratoryCollege of Charleston Charleston SC) and Phytophthoracapsici (J Ikerd US Vegetable Laboratory USDA ARSCharleston SC) respectively For bacteria the univer-sal primers 8F (51015840-AGAGTTTGATCCTGGCTCAG-31015840) and1492R (51015840-GGTTACCTTGTTACGACTT-31015840) were used withthe following PCR conditions 3min at 95∘C followed by30 cycles of 95∘C for 1min 1min at the annealing temper-ature of 45∘C and 72∘C for 15min with a final elongationstep of 4min at 72∘C [31 32] For fungi the universalprimers NS1 (51015840-GTAGTCATATGCTTGTCTC-31015840) and NS8(51015840-TCCGCAGGTTCACCTACGGA-31015840) were used with the
4 Journal of Marine Biology
following PCR conditions 4min at 95∘C followed by 30cycles of 95∘C for 1min 2min at the annealing temperatureof 55∘C and 72∘C for 15min with a final elongation step of10min at 72∘C [33 34] Each 25 120583L PCR reaction containedthe following 5 120583L of buffer (5x colorless GoTaq Flexi BufferPromega) 3 120583L of MgCl
2(25mM Promega) 025 120583L bovine
serum albumin (10 120583g 120583Lminus1 Promega) 05120583L dNTP (10mMThermo Scientific) 05120583L of each primer (5120583M IntegratedDNA Technologies) 01 120583L of DNA polymerase (5U120583Lminus1GoTaq Flexi DNA polymerase Promega) 1315 120583L H
2O and
2 120583L template DNA Bacterial and fungal PCR products werethen run on a 05 agarose gel purified with a QIAquickGel Extraction Kit (Qiagen) and eluted in 30 120583L at the finalstep Standards were quantified using the Qubit dsDNA BRAssay Kit (Invitrogen Life Technologies) and a Qubit 20Fluorometer (Qubit Systems) to determine the quantity ofDNA in the final sample The amplicon length was thenused to calculate the number of copies per 120583L under theassumption that the molecular weight of each bp is 650A standard curve was generated using 10-fold dilutions ofthese standards across 8 orders of magnitude thereforestandardizing qPCR results by copy number
Each plate included triplicate reactions per DNA sampleand for the standard curve as well as a no-template control tocheck for contamination Amelt curve (1min at 95∘C 1min at55∘C +05∘C 10 sminus1 to 95∘C) was used at the end of each qPCRrun to ensure the fluorescence signal resulted from specificityto the PCR product rather than from primer dimers orother nonspecific products The fluorescein dye included inthe master mix allowed for standard normalization of erroracross samples Threshold cycles (Ct) were automaticallycalculated by the software based on the average backgroundnoise Samples with less than one order of magnitude ofseparation (33 Ct value) from the no-template control wereexcluded given that this was outside of the detection limit ofthe present analysis [35 36] These samples were thereforereanalyzed using the same standard curve with a less dilutetemplate in order to ensure accurate quantification Samplesthat fell below the detection limit regardless of dilution wereexcluded from the analysis
210 Statistical Analysis Statistical analyses were conductedusing JMP 10 (SAS Institute httpwwwjmpcom) Hatch-ing success temperature pO
2 bacterial and fungal abun-
dance organic matter content and mean grain size data wereall analyzed using ANOVA When assumptions of normalityand homogeneity of variance were not met nonparametrictests were used (WilcoxonKruskal-Wallis) Statistically sig-nificant results were further testedwith Tukeyrsquos HSDpost hoctest to determine significant differences between beach areasRelationships between variables (hatching success pO
2 tem-
perature grain size organic matter content and bacterialand fungal abundance) were evaluated using Spearmanrsquos rankcorrelation coefficients
Because the metabolic demand of sea turtle embryos islow within the first half of incubation given their limiteddevelopment differences in oxygen content and temperaturein the egg clutch within this time period can presumablybe attributed to microbial activity [8 37] Mean nest pO
2
and temperature were therefore analyzed for the first andsecond half of incubation separately Repeatedmeasurementsof temperature and oxygen were also analyzed with repeatedmeasures MANOVA To allow for sample comparison acrossqPCR plates an ANCOVA was used to ensure there was nosignificant difference between standard curves from differentruns We also used a positive control on each plate tocalculate a coefficient of variation (CV) in order to ensurereproducibility within and between all plates All valuesare expressed as means plusmn standard error (SE) Percentagedata (hatching success) and microbial abundance data (copynumber) were arcsine- and log-transformed respectivelybefore analysis with parametric statistics Mean grain sizewas converted from phi units (120601) to mm for presentationThe number of 16S18S copies ulminus1 of template was convertedto a number of 16S18S copies gminus1 of nest sand to allowfor comparison across samples All analyses were tested forstatistical significance at 120572 lt 005
3 Results
The sample arribada occurring in August 2012 (estimated at72152 egg-laying females) and another arribada (September2012 estimated at 211954 egg-laying females) occurringbefore nest excavations were carried out in the middleof October An earthquake measuring 76 on the Richterscale occurred on September 5 2012 the epicenter locatedapproximately 50 km south of Ostional which caused visiblestructural changes to the beach topography Nest density forthe entire beach at the time of excavation was 36 plusmn 04 nestsmminus2 and overall hatching success for nests laid on the mainnesting beach during the August arribada was 34 plusmn 54 (RValverde unpublished data) At the time of excavation thenest densities in the high-nest-density tidal-wash and low-nest-density areas were 52 plusmn 04 20 plusmn 06 and 30 plusmn 05 nestsmminus2 respectively Two nests were excluded from the analysisdue to unforeseeable factors that likely caused a change inthe variables that we attempted to control in this study (ietemperature and pO
2of the nest) Specifically we excluded a
nest in the high-density nesting area that was partially shadedby a palm tree and another in the tidal-wash nesting area thatremained permanently inundated by the nearby estuary afterthe earthquake Hatching success in the high-nest-densityarea (06) was significantly lower than in the low-nest-density (682) and tidal-wash (547) areas of the beach(119875 lt 0001 Figure 1)
The pO2within egg clutches in the high-nest-density area
was significantly lower than that in other areas of the beachin both the first (154 plusmn 09 kPa 119875 lt 0001) and second halfof incubation (115 plusmn 05 kPa 119875 lt 0001 Figure 2) The meanminimumpO
2among nests in the high-nest-density area was
97plusmn 03 kPa with one clutch exhibiting pO2as low as 87 kPa
Nest pO2varied significantly with beach area and incubation
day with a significant interaction occurring between thesetwo factors (119865
was positively correlatedwith pO2in both the first and second
half of the incubation period (119903 = 0872 119875 lt 00001 and119903 = 0591 119875 = 0020 resp Table 1)
Journal of Marine Biology 5
100
80
60
40
20
0
Hat
chin
g su
cces
s (
)
Low-density Tidal-wash High-densityBeach area
A
B
Figure 1 Mean (plusmn SE) hatching success of nests located in differentnesting areas of the beach (119899 = 5) Different letters denote statisticaldifference Hatching success in the high-nest-density area (06)was significantly lower than in the low-nest-density (682) andtidal-wash (547) areas of the beach (119875 lt 0001)
20
18
16
14
12
10
pO2
(kPa
)
Low-densityTidal-wash
High-density
0 10 20 30 40 50
Incubation day
Figure 2 Mean (plusmn SE) partial pressure of oxygen (pO2 kPa) in
nests throughout the incubation period for nests located in thehigh-density tidal-wash and low-density nesting areas of the beach(119899 = 5) The pO
2within nests in the high-nest-density area was
significantly lower than that in other areas of the beach in both thefirst and the second half of incubation (119875 lt 0001 and 119875 lt 00001resp)
Nest temperatures were significantly lower in the tidal-wash area in the first and secondhalf of incubation (119875 = 0001and 119875 = 0001 resp Figure 3) Temperatures in the high-nest-density area of the beach were the highest in the secondhalf of incubation (357plusmn02∘C) and remained above the lethallimit (35∘C [38]) for a significantly longer duration of timein comparison to other areas of the beach (170 plusmn 07 d 119875 =0009) Nest temperature varied significantly with beach areaand incubation day with a significant interaction occurringbetween these two factors (119865
Table 1 Relationships between hatching success and partial pres-sure of oxygen (pO2) and temperature in the first (1st) and second(2nd) half of incubation bacterial and fungal abundance andorganic matter content for all study nests Copy number data fromseveral nests fell outside the range of detection for the presentanalysis and were therefore left out (see Section 3 for more details)Hatching success was positively correlated with nest pO2 in both thefirst and second half of incubation and negatively correlatedwith thebacterial abundance in nest sand
Variable Half of incubationperiod
Hatching success119903 119899 119875
pO21st 0872 13 lt00012nd 0591 13 0020
Temperature 1st minus0025 13 09292nd minus0207 13 0459
Figure 3 Mean nest temperature throughout the incubation periodin nests located in the high-density tidal-wash and low-densitynesting areas of the beach (119899 = 5) The field lethal incubationtemperature is indicated with a horizontal grey dashed line (35∘CValverde et al 2010) Nest temperatures were significantly lower inthe tidal-wash area in the first and the second half of incubation(119875 = 0001 and 119875 = 0001 resp)
However hatching success was not significantly correlatedwith nest temperatures in the first or second half of incuba-tion (Table 1)
The organic matter content of nest sand was significantlyhigher in the high-nest-density area (40) than in the low-nest-density (32) and tidal-wash (30) areas of the beach(119875 = 0015 and 119875 = 0003 resp) The sand from nestsin the low-nest-density area had a significantly larger meangrain size (0366mm) than in both the high-nest-density(0282mm) and tidal-wash (0283mm) areas of the beach
6 Journal of Marine Biology
(119875 = 0020 for both comparisons) Additionally the sandfrom all nests in the high-nest-density tidal-wash and low-nest-density areas of the beach wasmoderately sorted (120590
12060110
101 and 098 resp) and finely skewed (Sk120601035 036 and
103 resp)There was no significant difference in the slope of the
standard curve across assays for bacteria (119864 = 10223plusmn1411198772= 099 slope = minus327plusmn003 intercept = 3683plusmn056 CV =
341) or fungi (119864 = 8455 plusmn 225 1198772 = 100 plusmn 000 slope =minus376plusmn007 intercept = 4852plusmn080 CV = 168) indicatingthat copy numbers were comparable across assays (119875 = 0856and 119875 = 0088 resp) Microscopy counts (range 572 times 106ndash743 times 107 cells gminus1 of nest sand) were closely correlated withqPCR results for 16S rRNA gene copy number gminus1 of sand forall nests (119903 = 0404 119875 = 0013) Both the 16S and 18S copynumbers for one nest in the low-nest-density area as well asthe 18S copy number for two nests in the tidal-wash area werebelow the detection limit of the present analysis and weretherefore omitted
There was a significantly higher abundance of bacteriain nest sand from the high-nest-density area in comparisonto other areas of the beach (119875 = 0029) There was also ahigher abundance of fungi in nest sand from the high-nest-density area of the beach though this difference wasmarginal(119875 = 0063) While there was a negative correlation betweenhatching success and bacterial abundance for all nests thecorrelation between hatching success and fungal abundancewas marginal (119903 = minus0585 119875 = 0046 and 119903 = minus0607119875 = 0063 resp Table 1 and Figure 4) There was a negativecorrelation between the number of bacteria and egg clutchpO2in both the first and second half of incubation (119903 =
minus0741 119875 = 0006 and 119903 = minus0797 119875 = 0002 resp) aswell as a positive correlation between the number of bacteriaand nest temperatures in the second half of incubation (119903 =0818 119875 = 0001 Table 2) Furthermore there was a positivecorrelation between bacterial abundance and organic mattercontent in nest sand across all areas of the beach (119903 = 0636119875 = 0026)
4 Discussion
In this study we monitored nest conditions throughout theincubation period and quantified the microbial abundanceof nest sand in association with sea turtle hatching successWe found a negative relationship between the microbialabundance of nest sand and hatching success and observedsignificantly low pO
2in egg clutches located in the high-
nest-density area of the beach Egg clutches located in thetidal-wash area of the beach had higher hatching successand pO
2as well as lower organic matter content than those
located in the high-nest-density area of the beach Theseresults are consistent with previous reports that seawatermay have a positive effect on hatching success [39 40]presumably by decreasing microbial abundance incubationtemperature andor organic matter content Our results alsosupport our hypothesis that the low pO
2associated with
high microbial abundance in nest sand is responsible for thelower hatching rates observed at Ostional beach Collectively
105
106
107
108
109
1010
1011
BacteriaFungi
100
80
60
40
20
0
Hat
chin
g su
cces
s (
)
Log copy number gminus1 nest sand
Figure 4 Relationship between hatching success and microbialabundance (bacteria and fungi) for nests located in each nesting areaof the beach (119899 = 12 and 119899 = 10 resp) Regression lines for bacteria(solid) and fungi (dashed) are included for visualization purposesWhile therewas a negative correlation between hatching success andbacterial abundance for all nests the correlation between hatchingsuccess and fungal abundance was marginal (119875 = 0046 and119875 = 0063 resp) For Spearmanrsquos rank correlation coefficients seeTable 1
these data suggest a nonpathogenic mechanism for sea turtleembryonic mortality resulting from high rates of microbialdecomposition altering the nest environment beyond theoptimal range for embryonic development
Because all nests in this study incubated during the sametime period we are fairly confident that a majority of factorsaffecting embryonic development and hatching success werereasonably controlled However there may be factors that wedid notmonitor that caused incongruences in nest conditionsand could have influenced hatching success in this studyAdditionally an earthquake of large magnitude such as theone that occurred during this study could have feasiblycaused movement-induced mortality of sea turtle embryosHowever the earthquake occurred on day 25 of incubationafter the critical period (12 h to 14 d) during whichmovementsignificantly reduced hatching success [41] and thereforelikely had no adverse effects on the egg clutches in this study
41 Hatching Success Hatching success was highest in nestslocated in the low-nest-density area High-nest-densitieshave previously been observed to have direct physiologicaleffects (ie lower pO
2andhigher pCO
2within the egg clutch)
that negatively impacted hatching success [4] Nest densitiesof up to 5 nests mminus2 are unlikely to have a significant effect onhatching success (ge56) while densities of 9 nests mminus2 maysignificantly reduce hatching success to 30 [4]However thenests in this studywere subject to relatively low-nest-densities(2ndash5 nests mminus2) which likely were not high enough to causethe significant drop in hatching success observed
Journal of Marine Biology 7
Table 2 Relationships between bacterial and fungal abundance and the partial pressure of oxygen (pO2) and temperature in the first (1st)and second (2nd) half of incubation for all study nests There was a negative correlation between bacterial abundance and nest pO2 in boththe first and second half of incubation and a positive correlation with nest temperatures in the second half of incubation There was also anegative correlation between fungal abundance and nest pO2 in the first half of incubation
42 Nest Oxygen Content pO2for the low-nest-density and
tidal-wash areas of the beach was similar to that previouslyobserved in other studies on olive ridley sea turtles [4 8]Those studies also observed a change in the pO
2of nests
over the incubation period with pO2decreasing later in
the incubation period as a result of embryo respiration Thesignificant interaction effect of beach area and incubation dayon egg clutch pO
2indicates that the pO
2within egg clutches
changed differently across the areas of the beachOn the other hand the pO
2observed in egg clutches
located in the high-nest-density area in this study is lowerthan that previously observed in other studies [8 22 42]Specifically the drop in pO
2that we observed early in
the incubation period is uncharacteristic of sea turtle eggclutches given that the metabolic activity of the embryos istypically negligible at this time [42] Clutch pO
2typically
remains constant through the first half of incubation andthen decreases and tends to be negatively correlated with thenumber ofmetabolizing embryos per clutch [8 22] Howeverthis was not the case for clutches located in the high-nest-density area of the beach where pO
2began to drop from the
start of incubation and was lower in both the first and secondhalf of the incubation period in comparison to other areas ofthe beach
The negative correlation between microbial abundance(of both bacteria and fungi) and clutch pO
2at Ostional
suggests that microbial decomposition was responsible forthese decreases in pO
2 In particular our observations of high
abundance of bacteria and organic matter in nest sand fromthe high-nest-density area of the beach in association withlow pO
2further support this hypothesis Additionally if this
decrease in pO2resulted from the decomposition of embryos
killed by pathogens in these nests one would expect a lagtime (ie time for infection) and an abrupt change in oxy-gen and temperature following embryo death Furthermorewhile nest density alone affects the oxygen content of neststhe minimum pO
2observed in the high-nest-density area
(97 kPa) in this study was lower than previously observedat similar and even higher nest densities (approximately145ndash170 kPa) [4 8] Given that these previous studies inldquocleanrdquo sand (with presumably low organic matter content)did not find such discrepancies in the pO
2of egg clutches
at similar densities it is likely that microbial decompositionis responsible for the differences observed here Howeverfurther studies that include a controlled measure of pO
2in
beach sand outside of egg clutches and within egg clutches
at controlled densities are necessary to properly test thishypothesis
Because sea turtle embryos have a relatively low toleranceto hypoxia early in the incubation period [43] it is likelythat the drop in pO
2that we observed in the high-nest-
density area of the beach led to total nest failure While thecritical oxygen tension of olive ridley sea turtle eggs hasnot been studied this value was as high as 165 kPa on day22 of incubation for loggerhead sea turtle (Caretta caretta)eggs which are comparable in size [43] Assuming that thecritical oxygen tension of olive ridley sea turtle eggs is similarand given that egg clutch pO
2fell below 165 kPa on day 15
of incubation in the high-nest-density area it is likely thatany eggs located here were exposed to pO
2below a lethal
threshold
43 Nest Temperature The nest temperatures observed inthis study are similar to those observed in other studieson olive ridley sea turtles in this region [4 8 38] Ingeneral mean incubation temperatures were above the meanpivotal temperature of 305∘C for olive ridleys [44 45]indicating that the hatchling sex ratio could be female-biasedat Ostional as previously suggested [38] Previous studieshave also observed a change in nest temperatures throughoutthe incubation period with temperatures increasing laterin incubation in conjunction with increasing embryonicmetabolism [4 8] Nest temperatures were fairly consistentacross the different areas of the beach throughout the incu-bation period with the exception of nests located in thetidal-wash area where lower nest temperatures likely resultedfrom higher water content in the sandThe incubation periodwas 2 to 3 days longer in this group of nests and is likely aresult of slower embryonic development due to lower nesttemperatures [46]
While most nest temperatures fell within the wide rangeof tolerance for sea turtle embryological development (25ndash35∘C [39]) early in the incubation period the slightly highertemperatures observed in the high-nest-density area of thebeach later in incubation may have exceeded this optimalrange long enough to affect hatching success Though thefield lethal limit for olive ridley sea turtles at this beach isapproximately 35∘C embryos may be capable of recoveringfrom short-term exposure to high temperatures while longerdurations are lethal [38] Results from this study supportthis notion in that nests with lower hatching success (high-nest-density area) were also exposed to temperatures above
8 Journal of Marine Biology
the lethal limit for a longer duration of time The differentcombination of heat released from embryonic metabolismandor microbial decomposition is likely responsible for theobserved differences in temperature later in incubation
44 Characteristics of Nest Sand The mean grain size andsorting coefficient of sand from nests in this study werecomparable to those previously observed at a variety ofsea turtle nesting beaches [47] Given the relatively smallrange of mean grain size observed across study nests (0282ndash0366mm) granulometry likely did not drive the differencesin hatching success across the areas of the beach
The high amounts of organic matter in the sand fromthis study in comparison to other sea turtle nesting beaches[47] are not surprising as this is likely a result of the highrates of nest destruction due to the large number of nestingfemales and high-nest-density at this beach [9 19 48] Theassociation of high organic matter content with a higherabundance of bacteria in the sand from the high-nest-densityarea in this study supports the hypothesis that high organicmatter is associated with elevated microbial proliferation anddecomposition at Ostional and may also explain the low eggclutch pO
2in comparison to previous studies [7 8]
45 Microbial Abundance The number of bacterial 16S andfungal 18S rRNA gene copies in this study ranged from 105to 1010 copies gminus1 of sea turtle nest sand A previous studyfound that the sand at an arribada beach contained six ordersof magnitude more bacterial colonies gminus1 in comparison toa neighboring beach although no specific abundance datawere provided [10 14] Previous studies that have quantified16S or 18S rRNA gene copies in similar sediments have foundbetween 106 and 109 copies gminus1 [26 49]
The negative relationship between bacterial abundanceand hatching success that we observed supports the hypoth-esis that the microbial abundance in nest sand at Ostionalis high enough to adversely affect embryonic developmentSpecifically we suggest that the mechanism behind thisrelationship is not pathogenic but rather a result of high ratesof microbial decomposition of organic matter altering thenest environment beyond the optimal range for embryonicdevelopment Studies on underground nesting species ofbirds and reptiles suggest similar influences of microbialdecomposition on nest pO
2and temperature [50 51] It is
possible that the relationship between microbial activity andhatching success is nonlinear with a threshold beyond whichhigh rates of microbial respiration disrupt the diffusion ofoxygen to the nest to the point of impeding embryonic devel-opment For example the relationship between microbialabundance and hatching success appears to reach a thresholdat sim1 times 109ndash1010 16S18S copies gminus1 nest sand Furtherstudies that span a broader range in microbial abundanceare required to confirm the true nature of the relationshipbetween the microbial abundance of nest sand and sea turtlehatching success The coinciding occurrence of lower pO
2
higher temperatures higher organicmatter and low hatchingsuccess in the high-nest-density area of the beach serves asan extreme example of the adverse conditions imposed bymicrobial decomposition at Ostional
In addition to the indirect effect of microbial abundanceon the nest environment there are likely other effects ofmicrobial presence on sea turtle hatching success that wedid not consider because they were not within the scope ofthe present study For example we did not explore speciescomposition or diversity nor did we evaluate the presence orabsence of pathogens The potential presence of pathogenicmicrobes in the sand at Ostional cannot be ruled out Severalspecies of fungi in the Fusarium solani species complex havebeen identified as pathogens to sea turtle eggs and are knownto occur at nesting beaches around the world [15 16] It isalso possible that the effects of microbial abundance andthe nest environment on hatching success are synergisticwith the effects of lethal pathogens [15] For example onceembryos are killed due to the adverse effects of microbialoxygen depletion on the nest environment this may resultin the proliferation of fungi which could then spread toadjacent eggs Further studies should therefore focus onsampling from several components of the nest environment(ie cloacal fluid nest sand eggshell unhatched embryosand live hatchlings) as well as assessing the abundancediversity and activity of both fungi and bacteria to providea more complete understanding of the relationship betweenmicrobial activity and the reproductive success of sea turtles
Based on our comparative study it appears that tidalwashing decreases the organic matter and microbial abun-dance in the sand thus increasing hatching success Alter-natively the high humidity in the sand resulting fromtidal washing may also keep incubation temperatures fromreaching lethal temperaturesTherefore seawater inundationmay be critical in maintaining the population of sea turtlesat Ostional Using these natural mechanisms as a model forconservation management practices could be adopted topromote improved embryo survival and hatchling produc-tion For example seawater could be used to decrease organicmatter and microbial abundance in hatchery sand or byperiodically irrigating the beachwith seawaterThe treatmentof sand or relocation of hatcheries on a regular basis is a con-ventional management practice in conservation programs toavoid organic matter build-up as well as microbial and larvalinfestations In fact the leatherback sea turtle conservationprogram at Ostional successfully increased hatching successin the leatherback hatchery by using sand from below thehigh tide line [40]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank G Bran Y Arguello-GomezM Gomez S Steele and all the employees and volunteers atRNVS Ostional for their help in the field The authors wouldalso like to thank S Berthrong A Oporta McCarthy and RLeisen for their contributions to themolecular and laboratoryanalyses This material is based upon work supported by the
Journal of Marine Biology 9
National Science Foundation Graduate Research Fellowshipunder Grant no 511182 Additional funds to support the laband field research for this study were provided by the PADIFoundation National Geographic Young Explorers Grant(C220-12) USFWS Marine Turtle Conservation Act (96200-0-G037) and internal grants from the College of CharlestonThis research was approved by the College of CharlestonIACUC and permits were granted by MINAET (Resolucionno ACT-OR-DR-078) and CONAGEBio (R-020-2012-OT-CONAGEBIO) of Costa Rica
References
[1] A Abreu-Grobois and P T Plotkin ldquoIUCNSMTSG (2008) Lep-idochelys olivaceardquo The IUCN Red List of Threatened SpeciesVersion 20142 httpwwwiucnredlistorg
[2] R A Valverde C M Orrego M T Tordoir et al ldquoOlive ridleymass nesting ecology and egg harvest at ostional beach CostaRicardquoChelonian Conservation and Biology vol 11 no 1 pp 1ndash112012
[3] S E Cornelius R Arauz J Fretey et al ldquoEffect of land basedharvest of Lepidochelysrdquo in The Biology and Conservation ofRidley Sea Turtles P T Plotkin Ed pp 231ndash251 Johns HopkinsUniversity Press Baltimore Md USA 2007
[4] S Honarvar M P OrsquoConnor and J R Spotila ldquoDensity-dependent effects on hatching success of the olive ridley turtleLepidochelys olivaceardquo Oecologia vol 157 no 2 pp 221ndash2302008
[5] V S Beezy and R A Valverde ldquoA comparison ofmethodologiesfor estimating the nest density of olive ridley arribadas atOstional Costa Ricardquo in Proceedings of the 31st Annual Sympo-sium on Sea Turtle Biology andConservation T T Jones andB PWallace Eds NOAA Technical Memorandum NMFS-SEFSC-631 pp 194ndash195 2012
[6] S E Cornelius M A Ulloa J C Castro M Mata del Valleand D C Robinson ldquoManagement of olive ridley sea turtles(Lepidochelys olivacea) nesting at Playas Nancite and OstionalCosta Ricardquo in Neotropical Wildlife Use and Conservation JRobinson and K Redford Eds vol 1 pp 111ndash135 Universityof Chicago Press Chicago Ill USA 1991
[7] R A Valverde S E Cornelius and C L Mo ldquoDecline of theolive ridley sea turtle (Lepidochelys olivacea) nesting assemblageat Nancite Beach Santa Rosa National Park Costa RicardquoChelonian Conservation and Biology vol 3 pp 58ndash63 1998
[8] S Clusella Trullas and F V Paladino ldquoMicro-environment ofolive ridley turtle nests deposited during an aggregated nestingeventrdquo Journal of Zoology vol 272 no 4 pp 367ndash376 2007
[9] D McPherson and D Kibler ldquoEcological impact of olive ridleynesting at Ostional Costa Ricardquo in Proceedings of the 25thAnnual Symposium on Sea Turtle Biology and ConservationH Kalb A Rohde K Gayheart and K Shanker Eds NOAATechnical Memorandum NMFS-SEFSC-582 p 204 2008
[10] C L Mo I Salas and M Caballero ldquoAre fungi and bacteriaresponsible for olive ridleyrsquos egg lossrdquo in Proceedings of the 10thAnnual Workshop on Sea Turtle Biology and Conservation T HRichardson J I Richardson and M Donnelly Eds pp 249ndash252 NOAA Techn 1990
[11] S Honarvar J R Spotila andM P OrsquoConnor ldquoMicrobial com-munity structure in sand on two olive ridley arribada nestingbeaches Playa La Flor Nicaragua and Playa Nancite Costa
Ricardquo Journal of Experimental Marine Biology and Ecology vol409 no 1-2 pp 339ndash344 2011
[12] A D Phillott and C J Parmenter ldquoThe distribution of failedeggs and the appearance of fungi in artificial nests of green(Chelonia mydas) and loggerhead (Caretta caretta) sea turtlesrdquoAustralian Journal of Zoology vol 49 no 6 pp 713ndash718 2001
[13] J Wyneken T J Burke M Salmon and D K Pedersen ldquoEggfailure in natural and relocated sea turtle nestsrdquo Journal ofHerpetology vol 22 no 1 pp 88ndash96 1988
[14] C L Mo M Caballero and I Salas ldquoMicroorganism infectionof olive ridley eggsrdquo in Proceedings of the 12th Annual Workshopon Sea Turtle Biology and Conservation J I Richardson and TH Richardson Eds NOAA Technical Memorandum NMFS-SEFSC-361274 pp 81ndash84 1995
[15] J M Sarmiento-Ramırez E Abella M P Martın et alldquoFusarium solaniis responsible for mass mortalities in nests ofloggerhead sea turtle Caretta caretta in Boavista Cape VerderdquoFEMS Microbiology Letters vol 312 no 2 pp 192ndash200 2010
[16] J M Sarmiento-Ramırez E Abella-Perez A D Phillott etal ldquoGlobal distribution of two fungal pathogens threateningendangered sea turtlesrdquo PLoS ONE vol 9 Article ID e858532014
[17] J Patino-Martinez A Marco L Quinones E Abella R MAbad and J Dieguez-Uribeondo ldquoHow do hatcheries influenceembryonic development of sea turtle eggs Experimental anal-ysis and isolation of microorganisms in leatherback turtle eggsrdquoJournal of Experimental Zoology Part A Ecological Genetics andPhysiology vol 317 no 1 pp 47ndash54 2012
[18] A D Phillott and C J Parmenter ldquoFungal colonization of greensea turtle (Chelonia mydas) nests is unlikely to affect hatchlingconditionrdquo Herpetological Conservation amp Biology vol 9 pp297ndash301 2014
[19] MOcanaMHarfush-Melendez and SHeppell ldquoMass nestingof olive ridley sea turtles Lepidochelys olivacea at La EscobillaMexico linking nest density and rates of destructionrdquo Endan-gered Species Research vol 16 pp 45ndash54 2012
[20] DMadden J Ballestero C Calvo R Carlson E Christians andE Madden ldquoSea turtle nesting as a process influencing a sandybeach ecosystemrdquo Biotropica vol 40 no 6 pp 758ndash765 2008
[21] R Valverde and C Gates ldquoPopulation surveys on mass nestingbeachesrdquo in Research and Management Techniques for theConservation of Sea Turtles K L Eckert K A Bjorndal FA Abreu-Gobrois and M Donnelly Eds IUCNSSC MarineTurtle Specialist Group Publication No 4 1999
[22] B P Wallace P R Sotherland J R Spotila R D ReinaB F Franks and F V Paladino ldquoBiotic and abiotic factorsaffect the nest environment of embryonic leatherback turtlesDermochelys coriaceardquo Physiological and Biochemical Zoologyvol 77 no 3 pp 423ndash432 2004
[23] J D Miller ldquoDetermining clutch size and hatching successrdquo inResearch and Management Techniques for the Conservation ofSea Turtles K L Eckert K A Bjorndal F A Abreu-Groboisand M Donnelly Eds IUCNSSC Marine Turtle SpecialistGroup Publication No 4 1999
[24] S J Blott and K Pye ldquoGradistat a grain size distribution andstatistics package for the analysis of unconsolidated sedimentsrdquoEarth Surface Processes and Landforms vol 26 no 11 pp 1237ndash1248 2001
[25] W Krumbein and F Pettijohn Manual of Sedimentary Petrog-raphy Appleton-Century-Crofts New York NY USA 1938
10 Journal of Marine Biology
[26] N C Prevost-Boure R Christen S Dequiedt et al ldquoValidationand application of a PCR primer set to quantify fungal commu-nities in the soil environment by real-time quantitative PCRrdquoPLoS ONE vol 6 no 9 Article ID e24166 2011
[27] N Fierer J A Jackson R Vilgalys and R B Jackson ldquoAssess-ment of soil microbial community structure by use of taxon-specific quantitative PCR assaysrdquo Applied and EnvironmentalMicrobiology vol 71 no 7 pp 4117ndash4120 2005
[28] A Guidot J-C Debaud and R Marmeisse ldquoSpatial distribu-tion of the below-ground mycelia of an ectomycorrhizal fungusinferred from specific quantification of its DNA in soil samplesrdquoFEMS Microbiology Ecology vol 42 no 3 pp 477ndash486 2002
[29] T B de Gregoris N Aldred A S Clare and J G BurgessldquoImprovement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxardquo Journal of Microbio-logical Methods vol 86 no 3 pp 351ndash356 2011
[30] E J Vainio and J Hantula ldquoDirect analysis of wood-inhabitingfungi using denaturing gradient gel electrophoresis of amplifiedribosomal DNArdquoMycological Research vol 104 no 8 pp 927ndash936 2000
[31] S Turner K M Pryer V P W Miao and J D Palmer ldquoInves-tigating deep phylogenetic relationships among cyanobacteriaand plastids by small subunit rRNA sequence analysisrdquo TheJournal of Eukaryotic Microbiology vol 46 no 4 pp 327ndash3381999
[32] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash174 John Wiley amp Sons Chichester UK1991
[33] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications pp 315ndash322 1990
[34] N Uemura KMakimuraM Onozaki et al ldquoDevelopment of aloop-mediated isothermal amplificationmethod for diagnosingPneumocystis pneumoniardquo Journal of Medical Microbiology vol57 no 1 pp 50ndash57 2008
[35] C J Smith D B Nedwell L F Dong and A M OsbornldquoEvaluation of quantitative polymerase chain reaction-basedapproaches for determining gene copy and gene transcriptnumbers in environmental samplesrdquo Environmental Microbiol-ogy vol 8 no 5 pp 804ndash815 2006
[36] C J Smith and A M Osborn ldquoAdvantages and limitationsof quantitative PCR (Q-PCR)-based approaches in microbialecologyrdquo FEMS Microbiology Ecology vol 67 no 1 pp 6ndash202009
[37] R A Ackerman ldquoOxygen consumption by sea turtle (CheloniaCaretta) eggs during developmentrdquo Physiological Zoology vol54 pp 316ndash324 1981
[38] R A Valverde S Wingard F Gomez M T Tordoir and C MOrrego ldquoField lethal incubation temperature of olive ridley seaturtle Lepidochelys olivacea embryos at a mass nesting rookeryrdquoEndangered Species Research vol 12 no 1 pp 77ndash86 2010
[39] L G Fonseca W N Villachica R E Matarrita and R AValverde ldquoReporte final de la anidacioon de tortuga lora(Lepidochelys olivacea) Playa Nancite Parque Nacional SantaRosa Costa Rica (Temporada 2010ndash2011)rdquo Reporte Final deTemporada al USFWS 2011
[40] C Figgener A Castillo-MacCarthy J Mora-Sandoval YArguelo-Gomez and W Quiros-Pereira ldquoRaising the hatchingsuccess of leatherback nests at the olive ridley mass-nesting
beach inOstional Guanacaste Costa Ricardquo in Proceedings of the34thAnnual Symposiumon SeaTurtle Biology andConservation2014
[41] C Limpus V Baker and J Miller ldquoMovement induced mortal-ity of loggerhead eggsrdquoHerpetologica vol 35 pp 335ndash338 1979
[42] R A Ackerman ldquoThe respiratory gas exchange of sea turtlenests (Chelonia Caretta)rdquo Respiration Physiology vol 31 no 1pp 19ndash38 1977
[43] Y-C Kam ldquoPhysiological effects of hypoxia onmetabolism andgrowth of turtle embryosrdquo Respiration Physiology vol 92 no 2pp 127ndash138 1993
[44] TWibbels D Rostal andR Byles ldquoHigh pivotal temperature inthe sex determination of the olive ridley sea turtle Lepidochelysolivacea from Playa Nancite Costa Ricardquo Copeia no 4 pp1086ndash1088 1998
[45] C J McCoy R C Vogt and E J Censky ldquoTemperature-controlled sex determination in the sea turtle Lepidochelysolivaceardquo Journal of Herpetology vol 17 no 4 pp 404ndash4061983
[46] N A Miller ldquoPO2in loggerhead sea turtle (Caretta caretta)
[47] J A Mortimer ldquoThe influence of beach sand characteristicson the nesting behavior and clutch survival of green turtles(Chelonia mydas)rdquo Copeia vol 1990 no 3 pp 802ndash817 1990
[48] S E Cornelius The Sea Turtles of Santa Rosa National ParkFundacion de Parques Nacionales San Jose Costa Rica 1986
[49] X Chen E Peltier B S M Sturm and C B Young ldquoNitrogenremoval and nitrifying and denitrifying bacteria quantificationin a stormwater bioretention systemrdquo Water Research vol 47no 4 pp 1691ndash1700 2013
[50] R S Seymour and R A Ackerman ldquoAdaptations to under-ground nesting in birds and reptilesrdquo American Zoologist vol20 no 2 pp 437ndash447 1980
[51] R S Seymour D Vleck and C M Vleck ldquoGas exchange in theincubationmounds of megapode birdsrdquo Journal of ComparativePhysiology B vol 156 no 6 pp 773ndash782 1986
aremuch higher than at neighboring beaches [9 10] Further-more Cornelius et al [6] suggested that observed increases inhatching success in nests below the high tide line may be dueto natural tidal washing which could remove microbes thatare intolerant to salt water Another study at arribada beachesfound that while bacterial phylotype richness and diversityincreased with increasing nest density in the high andmiddlezones of the beach this was not true in the low zone of thebeach perhaps due to the less stable osmotic environmentcaused by the tides [11]
Bacteria and fungi have been cultured and isolated fromnest sand and failed eggs as well as from the cloacal fluidof nesting females [10 12 13] However the literature onthe infection of sea turtle eggs by microbes is controversialMicrobial infection was traditionally thought to be oppor-tunistic and limited laboratory studies found no significanteffect of the presence of bacteria or fungi on the hatchlingproduction of olive ridley sea turtle eggs [10 12 14] Morerecent studies on the Fusarium solani species complex haveidentified several fungal pathogens to sea turtle eggs [15 16]However eggs infected with F solani can be asymptomatic[15] and other studies challenge this causality [17 18] Thesestudies reflect the difficulty in directly linking embryo mor-tality to the presence ofmicrobes in part due to limitations inconducting research on protected species as well as the highoccurrence of total nest failure in such studies [14 19]
While the negative effect of higher microbial densitieson hatching success at Ostional has long been presumedno previous research has ever directly quantified microbialabundance or the associated conditions of the nest envi-ronment with respect to hatching success in natural nestsBecause sea turtle embryo mortality is likely linked to manyfactors it is necessary to evaluate the entire nest environmentin order to fully understand the interactions between a clutchof eggs and the microecosystem that comprises a sea turtlenest [13 20]
The aim of this study was to describe the natural con-ditions of the sea turtle nest environment at Ostional inorder to identify factors that may be adversely impactingsea turtle embryonic development We tested the hypothesisthat hatching success varies across the beach in associationwith differences in the nest environment and microbialabundance We predicted that higher hatching success wouldoccur in beach areas exposed to tidal washing and that aninverse relationship exists between microbial abundance andhatching success To accomplish this we monitored nestconditions (ie temperature oxygen and organic mattercontent) and hatching success in natural nests located indifferent areas of the beach and quantified the microbialabundance in nest sand
2 Methodology
21 Study Site The Ostional National Wildlife Refuge(ONWR) is located on the Pacific coast of the NicoyaPeninsula in Costa Rica (9996471∘N 85697800∘W) WithinONWR the Nosara and Ostional beaches make up approx-imately 7 km of beach with variable width This study wasconducted at Ostional beach during the rainy season (May
through November) of 2012 when arribadas are typicallymore abundant All study nests were laid during the samenight of the arribada that occurred during the last quartermoon of August 2012 (hereinafter called the sample arrib-ada) This ensured that all nests incubated at the same timeand helped standardize any uncontrollable variables thatcould affect hatching success such as ambient temperatureand rainfall
We compared the microbial abundance in relation tohatching success in natural (in situ) nests located in a tidal-wash area a high-density nesting area where arribadas tendto concentrate and a low-density nesting areaThe tidal-washarea of the beachwas locatedwithin 25meters of a lagoon andin the tidal-wash zone where the tide is known to wash overthe berm and cause sand turnover The high-density nestingarea was located at the main nesting beach where the samplearribada was recorded to have the highest density of nestingfemales The low-density nesting area was located where thesample arribada was recorded to have the lowest densityof nesting females Nest densities for the sample arribadawere determined by the strip-transect-in-time method andconfirmed with quadrat sampling at the time of excavation[5 21]
We randomly selected five nests above the average hightide line in the tidal-wash high-nest-density and low-nest-density beach areas described above Eggs were countedduring oviposition to determine the total clutch count Adatalogger and oxygen tubing were placed within the centerof the nest chamber after approximately 50 eggs were laidA 5-sided 50 times 50 times 15 cm wire mesh cage with a woodenframe was placed over the nest and buried entirely below thesurface of the sand to prevent predation and excavation bysubsequently nesting females This cage was pulled above thesurface of the sand onday 40 of incubation to allowhatchlingsto emerge to the surface unobstructed Nests were thenmonitored three times daily (sunrise sunset and midnight)for signs of hatching in order to count and release hatchlingsas soon as possible
22 Nest pO2 Nest pO2 was monitored by placing an airstone fitted with the tip of 60 cm nylon tubing into the centerof the egg mass that ran from inside of the nest chamberto the top layer of sand where a shut-off valve impeded anyadditional gas exchange [4 8 22] The pO
2within the egg
clutch was measured using a flow-through oxygen sensor(S108 Oxygen Analyzer Qubit Systems) that was calibratedprior to the field season using nitrogen and prior to each set ofsamples using atmospheric air Dead air space (approximately10mL) was drawn from within the tubing and expelled priorto sampling to ensure the air sample was from within thenest cavity Air samples (approximately 60mL) were drawnusing an airtight syringe and analyzedwithin 1 h of collectionSamples were slowly injected through an air pump flowmeter and desiccant column and through the O
2sensor at
a flow rate of approximately 50mLminminus1 Air samples wereanalyzed every 5 days for the first 30 days of incubationand every 4 days through the end of the incubation periodGas percentages were converted to partial pressures usingambient barometric pressure
Journal of Marine Biology 3
23 Nest Temperature Nest temperature was monitoredusing HOBO pendant temperature dataloggers (Onset Com-puter Corporation) placed in the center of each clutch andprogrammed to record temperature at 3 h intervals startingat midnight on the night of oviposition through hatchlingemergence Mean daily nest temperatures were used tocompare nest temperatures across the different areas of thebeach
24 Nest Excavations Single-use sterile gloves were wornfor all excavations and changed before contact with differentnests Nests were exhumed three days after the last observedhatchling emergence to quantify hatching success [23]
25 Sand Collection and Characterization A sample of sandwas collected directly into sterile collection tubes from thecenter of the nest chamber during the excavation of the nest atthe end of the incubation period Samples were placed on iceimmediately after collection and then either frozen (minus20∘C)or preserved in formalin (2 formaldehyde) within 6 h ofcollection and until analysis The organic matter analysisconsisted of a loss-on-ignition method with the organicmatter content being the loss of mass after dry combustionThe sample was transferred to a porcelain container anddesiccated in a drying oven (24 h at 100∘C) before combustion(6 h at 500∘C) Prior to combustion samples of dried sandwere fractionated with a set of sieves (0063 0075 015 025045 and 085mm) to determine the particle-size distributionbymassMean grain size (120601) sorting (120590
120601) and skewness (Sk
120601)
were calculated using the logarithmic mathematical ldquomethodof momentsrdquo in GRADISTAT [24 25]
26 Microbial Abundance Samples preserved in formalinwere used for microscopy counts as a secondary methodof quantifying bacterial abundance These samples werecentrifuged for 10min at 16000 g in a microcentrifuge beforecarefully removing excess formalin The sand was thendiluted (approximately 1 2) with sterile water and sonicatedon ice for 20 s at 30W (Sonifier S-250A Branson) Theresulting supernatant fluid was stained for approximately 5minutes with a 1 10 dilution of 1x SybrGold and sterile waterMicroscopy counts were performed at 1000x magnificationon an epifluorescence microscope (Optiphot-2 Nikon) bycounting 10 fields per slide The number of cells gminus1 of sandwas then calculated based on the original weight of sandvolume of diluent and supernatant and the average numberof cells fieldminus1 using the number of fields per slide at 1000x(479 times 104) and correction factor for the addition of formalin(times116)
We used a quantitative real-time polymerase chain reac-tion (qPCR) analysis to determine the abundance of bacteriabased on the number of 16S rRNA gene copies gminus1 nestsand and the abundance of fungi based on the number of18S rRNA gene copies gminus1 nest sand While the resultingquantification of gene copies from a qPCR analysis cannot bedirectly transformed into the number of cells (given that copynumber can vary greatly between species) this can be used asa proxy for overall abundance ofmicrobial communities [26ndash28]
27 DNA Extraction Each sample of sand was thawed andhomogenized by vortexing before collecting a 1 g subsamplefor DNA extraction DNA was extracted from all samplesof sand using an EZNA Soil DNA kit (Omega Biotek) withthe following modifications to the protocol to increase DNAyields Samples were subjected to 5min at approximately2000 oscillations minminus1 in a bead beater (Mini Beadbeater-8Biospec Products) and three freeze-thaw cycles (minus20∘C and70∘C for 30min each) during the lysis step Additionally inthe final step of extraction only 50 120583L of elution buffer wasusedwhichwas reapplied to the column in the second elutionstep DNA samples were diluted in order to reduce inhibitionand optimize efficiency and Ct values to within the range ofthe standard curve
28 qPCR Analysis Absolute qPCR was run using an iCycleriQ Real-Time PCR Detection System (Bio-Rad LaboratoriesInc) on a 96-well plate Results were analyzed using iQ5software (Bio-Rad Laboratories Inc)The universal bacterialprimers 926F (51015840-AAA CTC AAA KGA ATT GAC GG-31015840)and 1062R (51015840-CTCACRRCACGAGCTGAC-31015840) that targetthe 16S rRNA gene were used based on a previous studyby de Gregoris et al [29] Each 10 120583L reaction containedthe following 5120583L of ABsolute qPCR Master Mix (ABgene)01 120583L bovine serum albumin (10 120583g 120583Lminus1Thermo Scientific)03 120583L of each primer (10 120583M 300 nM final concentrationIntegrated DNA Technologies) 39 120583L H
2O and 04 120583L tem-
plate DNA PCR conditions were 15min at 95∘C followed by40 cycles of 95∘C for 15 s 15 s at the annealing temperature of57∘C and 72∘C for 20 s
The universal fungal primers FR1 (51015840-AICCATTCAATC-GGTAIT-31015840) and FF390 (51015840-CGATAACGAACGAGACCT-31015840) that target the 18S rRNAgenewere used based on previousstudies [26 30] Each 10 120583L reaction contained the following5 120583L of ABsolute qPCR Master Mix (ABgene) 01 120583L bovineserum albumin (10 120583g 120583Lminus1Thermo Scientific) 01 120583L of eachprimer (10120583M 100 nM final concentration Integrated DNATechnologies) 43 120583L H
2O and 04 120583L template DNA PCR
conditions were 15min at 95∘C followed by 40 cycles of 95∘Cfor 15 s 30 s at the annealing temperature of 50∘C and 72∘Cfor 1min
29 qPCR Standards External fixed standards were cre-ated by amplifying and quantifying bacterial and fun-gal DNA using primer sets that targeted the full 16S18SrRNA sequence Template DNA extractions for bacterialand fungal standards were kindly provided from culturesof Bacillus pumilus (W Hook Grice Marine LaboratoryCollege of Charleston Charleston SC) and Phytophthoracapsici (J Ikerd US Vegetable Laboratory USDA ARSCharleston SC) respectively For bacteria the univer-sal primers 8F (51015840-AGAGTTTGATCCTGGCTCAG-31015840) and1492R (51015840-GGTTACCTTGTTACGACTT-31015840) were used withthe following PCR conditions 3min at 95∘C followed by30 cycles of 95∘C for 1min 1min at the annealing temper-ature of 45∘C and 72∘C for 15min with a final elongationstep of 4min at 72∘C [31 32] For fungi the universalprimers NS1 (51015840-GTAGTCATATGCTTGTCTC-31015840) and NS8(51015840-TCCGCAGGTTCACCTACGGA-31015840) were used with the
4 Journal of Marine Biology
following PCR conditions 4min at 95∘C followed by 30cycles of 95∘C for 1min 2min at the annealing temperatureof 55∘C and 72∘C for 15min with a final elongation step of10min at 72∘C [33 34] Each 25 120583L PCR reaction containedthe following 5 120583L of buffer (5x colorless GoTaq Flexi BufferPromega) 3 120583L of MgCl
2(25mM Promega) 025 120583L bovine
serum albumin (10 120583g 120583Lminus1 Promega) 05120583L dNTP (10mMThermo Scientific) 05120583L of each primer (5120583M IntegratedDNA Technologies) 01 120583L of DNA polymerase (5U120583Lminus1GoTaq Flexi DNA polymerase Promega) 1315 120583L H
2O and
2 120583L template DNA Bacterial and fungal PCR products werethen run on a 05 agarose gel purified with a QIAquickGel Extraction Kit (Qiagen) and eluted in 30 120583L at the finalstep Standards were quantified using the Qubit dsDNA BRAssay Kit (Invitrogen Life Technologies) and a Qubit 20Fluorometer (Qubit Systems) to determine the quantity ofDNA in the final sample The amplicon length was thenused to calculate the number of copies per 120583L under theassumption that the molecular weight of each bp is 650A standard curve was generated using 10-fold dilutions ofthese standards across 8 orders of magnitude thereforestandardizing qPCR results by copy number
Each plate included triplicate reactions per DNA sampleand for the standard curve as well as a no-template control tocheck for contamination Amelt curve (1min at 95∘C 1min at55∘C +05∘C 10 sminus1 to 95∘C) was used at the end of each qPCRrun to ensure the fluorescence signal resulted from specificityto the PCR product rather than from primer dimers orother nonspecific products The fluorescein dye included inthe master mix allowed for standard normalization of erroracross samples Threshold cycles (Ct) were automaticallycalculated by the software based on the average backgroundnoise Samples with less than one order of magnitude ofseparation (33 Ct value) from the no-template control wereexcluded given that this was outside of the detection limit ofthe present analysis [35 36] These samples were thereforereanalyzed using the same standard curve with a less dilutetemplate in order to ensure accurate quantification Samplesthat fell below the detection limit regardless of dilution wereexcluded from the analysis
210 Statistical Analysis Statistical analyses were conductedusing JMP 10 (SAS Institute httpwwwjmpcom) Hatch-ing success temperature pO
2 bacterial and fungal abun-
dance organic matter content and mean grain size data wereall analyzed using ANOVA When assumptions of normalityand homogeneity of variance were not met nonparametrictests were used (WilcoxonKruskal-Wallis) Statistically sig-nificant results were further testedwith Tukeyrsquos HSDpost hoctest to determine significant differences between beach areasRelationships between variables (hatching success pO
2 tem-
perature grain size organic matter content and bacterialand fungal abundance) were evaluated using Spearmanrsquos rankcorrelation coefficients
Because the metabolic demand of sea turtle embryos islow within the first half of incubation given their limiteddevelopment differences in oxygen content and temperaturein the egg clutch within this time period can presumablybe attributed to microbial activity [8 37] Mean nest pO
2
and temperature were therefore analyzed for the first andsecond half of incubation separately Repeatedmeasurementsof temperature and oxygen were also analyzed with repeatedmeasures MANOVA To allow for sample comparison acrossqPCR plates an ANCOVA was used to ensure there was nosignificant difference between standard curves from differentruns We also used a positive control on each plate tocalculate a coefficient of variation (CV) in order to ensurereproducibility within and between all plates All valuesare expressed as means plusmn standard error (SE) Percentagedata (hatching success) and microbial abundance data (copynumber) were arcsine- and log-transformed respectivelybefore analysis with parametric statistics Mean grain sizewas converted from phi units (120601) to mm for presentationThe number of 16S18S copies ulminus1 of template was convertedto a number of 16S18S copies gminus1 of nest sand to allowfor comparison across samples All analyses were tested forstatistical significance at 120572 lt 005
3 Results
The sample arribada occurring in August 2012 (estimated at72152 egg-laying females) and another arribada (September2012 estimated at 211954 egg-laying females) occurringbefore nest excavations were carried out in the middleof October An earthquake measuring 76 on the Richterscale occurred on September 5 2012 the epicenter locatedapproximately 50 km south of Ostional which caused visiblestructural changes to the beach topography Nest density forthe entire beach at the time of excavation was 36 plusmn 04 nestsmminus2 and overall hatching success for nests laid on the mainnesting beach during the August arribada was 34 plusmn 54 (RValverde unpublished data) At the time of excavation thenest densities in the high-nest-density tidal-wash and low-nest-density areas were 52 plusmn 04 20 plusmn 06 and 30 plusmn 05 nestsmminus2 respectively Two nests were excluded from the analysisdue to unforeseeable factors that likely caused a change inthe variables that we attempted to control in this study (ietemperature and pO
2of the nest) Specifically we excluded a
nest in the high-density nesting area that was partially shadedby a palm tree and another in the tidal-wash nesting area thatremained permanently inundated by the nearby estuary afterthe earthquake Hatching success in the high-nest-densityarea (06) was significantly lower than in the low-nest-density (682) and tidal-wash (547) areas of the beach(119875 lt 0001 Figure 1)
The pO2within egg clutches in the high-nest-density area
was significantly lower than that in other areas of the beachin both the first (154 plusmn 09 kPa 119875 lt 0001) and second halfof incubation (115 plusmn 05 kPa 119875 lt 0001 Figure 2) The meanminimumpO
2among nests in the high-nest-density area was
97plusmn 03 kPa with one clutch exhibiting pO2as low as 87 kPa
Nest pO2varied significantly with beach area and incubation
day with a significant interaction occurring between thesetwo factors (119865
was positively correlatedwith pO2in both the first and second
half of the incubation period (119903 = 0872 119875 lt 00001 and119903 = 0591 119875 = 0020 resp Table 1)
Journal of Marine Biology 5
100
80
60
40
20
0
Hat
chin
g su
cces
s (
)
Low-density Tidal-wash High-densityBeach area
A
B
Figure 1 Mean (plusmn SE) hatching success of nests located in differentnesting areas of the beach (119899 = 5) Different letters denote statisticaldifference Hatching success in the high-nest-density area (06)was significantly lower than in the low-nest-density (682) andtidal-wash (547) areas of the beach (119875 lt 0001)
20
18
16
14
12
10
pO2
(kPa
)
Low-densityTidal-wash
High-density
0 10 20 30 40 50
Incubation day
Figure 2 Mean (plusmn SE) partial pressure of oxygen (pO2 kPa) in
nests throughout the incubation period for nests located in thehigh-density tidal-wash and low-density nesting areas of the beach(119899 = 5) The pO
2within nests in the high-nest-density area was
significantly lower than that in other areas of the beach in both thefirst and the second half of incubation (119875 lt 0001 and 119875 lt 00001resp)
Nest temperatures were significantly lower in the tidal-wash area in the first and secondhalf of incubation (119875 = 0001and 119875 = 0001 resp Figure 3) Temperatures in the high-nest-density area of the beach were the highest in the secondhalf of incubation (357plusmn02∘C) and remained above the lethallimit (35∘C [38]) for a significantly longer duration of timein comparison to other areas of the beach (170 plusmn 07 d 119875 =0009) Nest temperature varied significantly with beach areaand incubation day with a significant interaction occurringbetween these two factors (119865
Table 1 Relationships between hatching success and partial pres-sure of oxygen (pO2) and temperature in the first (1st) and second(2nd) half of incubation bacterial and fungal abundance andorganic matter content for all study nests Copy number data fromseveral nests fell outside the range of detection for the presentanalysis and were therefore left out (see Section 3 for more details)Hatching success was positively correlated with nest pO2 in both thefirst and second half of incubation and negatively correlatedwith thebacterial abundance in nest sand
Variable Half of incubationperiod
Hatching success119903 119899 119875
pO21st 0872 13 lt00012nd 0591 13 0020
Temperature 1st minus0025 13 09292nd minus0207 13 0459
Figure 3 Mean nest temperature throughout the incubation periodin nests located in the high-density tidal-wash and low-densitynesting areas of the beach (119899 = 5) The field lethal incubationtemperature is indicated with a horizontal grey dashed line (35∘CValverde et al 2010) Nest temperatures were significantly lower inthe tidal-wash area in the first and the second half of incubation(119875 = 0001 and 119875 = 0001 resp)
However hatching success was not significantly correlatedwith nest temperatures in the first or second half of incuba-tion (Table 1)
The organic matter content of nest sand was significantlyhigher in the high-nest-density area (40) than in the low-nest-density (32) and tidal-wash (30) areas of the beach(119875 = 0015 and 119875 = 0003 resp) The sand from nestsin the low-nest-density area had a significantly larger meangrain size (0366mm) than in both the high-nest-density(0282mm) and tidal-wash (0283mm) areas of the beach
6 Journal of Marine Biology
(119875 = 0020 for both comparisons) Additionally the sandfrom all nests in the high-nest-density tidal-wash and low-nest-density areas of the beach wasmoderately sorted (120590
12060110
101 and 098 resp) and finely skewed (Sk120601035 036 and
103 resp)There was no significant difference in the slope of the
standard curve across assays for bacteria (119864 = 10223plusmn1411198772= 099 slope = minus327plusmn003 intercept = 3683plusmn056 CV =
341) or fungi (119864 = 8455 plusmn 225 1198772 = 100 plusmn 000 slope =minus376plusmn007 intercept = 4852plusmn080 CV = 168) indicatingthat copy numbers were comparable across assays (119875 = 0856and 119875 = 0088 resp) Microscopy counts (range 572 times 106ndash743 times 107 cells gminus1 of nest sand) were closely correlated withqPCR results for 16S rRNA gene copy number gminus1 of sand forall nests (119903 = 0404 119875 = 0013) Both the 16S and 18S copynumbers for one nest in the low-nest-density area as well asthe 18S copy number for two nests in the tidal-wash area werebelow the detection limit of the present analysis and weretherefore omitted
There was a significantly higher abundance of bacteriain nest sand from the high-nest-density area in comparisonto other areas of the beach (119875 = 0029) There was also ahigher abundance of fungi in nest sand from the high-nest-density area of the beach though this difference wasmarginal(119875 = 0063) While there was a negative correlation betweenhatching success and bacterial abundance for all nests thecorrelation between hatching success and fungal abundancewas marginal (119903 = minus0585 119875 = 0046 and 119903 = minus0607119875 = 0063 resp Table 1 and Figure 4) There was a negativecorrelation between the number of bacteria and egg clutchpO2in both the first and second half of incubation (119903 =
minus0741 119875 = 0006 and 119903 = minus0797 119875 = 0002 resp) aswell as a positive correlation between the number of bacteriaand nest temperatures in the second half of incubation (119903 =0818 119875 = 0001 Table 2) Furthermore there was a positivecorrelation between bacterial abundance and organic mattercontent in nest sand across all areas of the beach (119903 = 0636119875 = 0026)
4 Discussion
In this study we monitored nest conditions throughout theincubation period and quantified the microbial abundanceof nest sand in association with sea turtle hatching successWe found a negative relationship between the microbialabundance of nest sand and hatching success and observedsignificantly low pO
2in egg clutches located in the high-
nest-density area of the beach Egg clutches located in thetidal-wash area of the beach had higher hatching successand pO
2as well as lower organic matter content than those
located in the high-nest-density area of the beach Theseresults are consistent with previous reports that seawatermay have a positive effect on hatching success [39 40]presumably by decreasing microbial abundance incubationtemperature andor organic matter content Our results alsosupport our hypothesis that the low pO
2associated with
high microbial abundance in nest sand is responsible for thelower hatching rates observed at Ostional beach Collectively
105
106
107
108
109
1010
1011
BacteriaFungi
100
80
60
40
20
0
Hat
chin
g su
cces
s (
)
Log copy number gminus1 nest sand
Figure 4 Relationship between hatching success and microbialabundance (bacteria and fungi) for nests located in each nesting areaof the beach (119899 = 12 and 119899 = 10 resp) Regression lines for bacteria(solid) and fungi (dashed) are included for visualization purposesWhile therewas a negative correlation between hatching success andbacterial abundance for all nests the correlation between hatchingsuccess and fungal abundance was marginal (119875 = 0046 and119875 = 0063 resp) For Spearmanrsquos rank correlation coefficients seeTable 1
these data suggest a nonpathogenic mechanism for sea turtleembryonic mortality resulting from high rates of microbialdecomposition altering the nest environment beyond theoptimal range for embryonic development
Because all nests in this study incubated during the sametime period we are fairly confident that a majority of factorsaffecting embryonic development and hatching success werereasonably controlled However there may be factors that wedid notmonitor that caused incongruences in nest conditionsand could have influenced hatching success in this studyAdditionally an earthquake of large magnitude such as theone that occurred during this study could have feasiblycaused movement-induced mortality of sea turtle embryosHowever the earthquake occurred on day 25 of incubationafter the critical period (12 h to 14 d) during whichmovementsignificantly reduced hatching success [41] and thereforelikely had no adverse effects on the egg clutches in this study
41 Hatching Success Hatching success was highest in nestslocated in the low-nest-density area High-nest-densitieshave previously been observed to have direct physiologicaleffects (ie lower pO
2andhigher pCO
2within the egg clutch)
that negatively impacted hatching success [4] Nest densitiesof up to 5 nests mminus2 are unlikely to have a significant effect onhatching success (ge56) while densities of 9 nests mminus2 maysignificantly reduce hatching success to 30 [4]However thenests in this studywere subject to relatively low-nest-densities(2ndash5 nests mminus2) which likely were not high enough to causethe significant drop in hatching success observed
Journal of Marine Biology 7
Table 2 Relationships between bacterial and fungal abundance and the partial pressure of oxygen (pO2) and temperature in the first (1st)and second (2nd) half of incubation for all study nests There was a negative correlation between bacterial abundance and nest pO2 in boththe first and second half of incubation and a positive correlation with nest temperatures in the second half of incubation There was also anegative correlation between fungal abundance and nest pO2 in the first half of incubation
42 Nest Oxygen Content pO2for the low-nest-density and
tidal-wash areas of the beach was similar to that previouslyobserved in other studies on olive ridley sea turtles [4 8]Those studies also observed a change in the pO
2of nests
over the incubation period with pO2decreasing later in
the incubation period as a result of embryo respiration Thesignificant interaction effect of beach area and incubation dayon egg clutch pO
2indicates that the pO
2within egg clutches
changed differently across the areas of the beachOn the other hand the pO
2observed in egg clutches
located in the high-nest-density area in this study is lowerthan that previously observed in other studies [8 22 42]Specifically the drop in pO
2that we observed early in
the incubation period is uncharacteristic of sea turtle eggclutches given that the metabolic activity of the embryos istypically negligible at this time [42] Clutch pO
2typically
remains constant through the first half of incubation andthen decreases and tends to be negatively correlated with thenumber ofmetabolizing embryos per clutch [8 22] Howeverthis was not the case for clutches located in the high-nest-density area of the beach where pO
2began to drop from the
start of incubation and was lower in both the first and secondhalf of the incubation period in comparison to other areas ofthe beach
The negative correlation between microbial abundance(of both bacteria and fungi) and clutch pO
2at Ostional
suggests that microbial decomposition was responsible forthese decreases in pO
2 In particular our observations of high
abundance of bacteria and organic matter in nest sand fromthe high-nest-density area of the beach in association withlow pO
2further support this hypothesis Additionally if this
decrease in pO2resulted from the decomposition of embryos
killed by pathogens in these nests one would expect a lagtime (ie time for infection) and an abrupt change in oxy-gen and temperature following embryo death Furthermorewhile nest density alone affects the oxygen content of neststhe minimum pO
2observed in the high-nest-density area
(97 kPa) in this study was lower than previously observedat similar and even higher nest densities (approximately145ndash170 kPa) [4 8] Given that these previous studies inldquocleanrdquo sand (with presumably low organic matter content)did not find such discrepancies in the pO
2of egg clutches
at similar densities it is likely that microbial decompositionis responsible for the differences observed here Howeverfurther studies that include a controlled measure of pO
2in
beach sand outside of egg clutches and within egg clutches
at controlled densities are necessary to properly test thishypothesis
Because sea turtle embryos have a relatively low toleranceto hypoxia early in the incubation period [43] it is likelythat the drop in pO
2that we observed in the high-nest-
density area of the beach led to total nest failure While thecritical oxygen tension of olive ridley sea turtle eggs hasnot been studied this value was as high as 165 kPa on day22 of incubation for loggerhead sea turtle (Caretta caretta)eggs which are comparable in size [43] Assuming that thecritical oxygen tension of olive ridley sea turtle eggs is similarand given that egg clutch pO
2fell below 165 kPa on day 15
of incubation in the high-nest-density area it is likely thatany eggs located here were exposed to pO
2below a lethal
threshold
43 Nest Temperature The nest temperatures observed inthis study are similar to those observed in other studieson olive ridley sea turtles in this region [4 8 38] Ingeneral mean incubation temperatures were above the meanpivotal temperature of 305∘C for olive ridleys [44 45]indicating that the hatchling sex ratio could be female-biasedat Ostional as previously suggested [38] Previous studieshave also observed a change in nest temperatures throughoutthe incubation period with temperatures increasing laterin incubation in conjunction with increasing embryonicmetabolism [4 8] Nest temperatures were fairly consistentacross the different areas of the beach throughout the incu-bation period with the exception of nests located in thetidal-wash area where lower nest temperatures likely resultedfrom higher water content in the sandThe incubation periodwas 2 to 3 days longer in this group of nests and is likely aresult of slower embryonic development due to lower nesttemperatures [46]
While most nest temperatures fell within the wide rangeof tolerance for sea turtle embryological development (25ndash35∘C [39]) early in the incubation period the slightly highertemperatures observed in the high-nest-density area of thebeach later in incubation may have exceeded this optimalrange long enough to affect hatching success Though thefield lethal limit for olive ridley sea turtles at this beach isapproximately 35∘C embryos may be capable of recoveringfrom short-term exposure to high temperatures while longerdurations are lethal [38] Results from this study supportthis notion in that nests with lower hatching success (high-nest-density area) were also exposed to temperatures above
8 Journal of Marine Biology
the lethal limit for a longer duration of time The differentcombination of heat released from embryonic metabolismandor microbial decomposition is likely responsible for theobserved differences in temperature later in incubation
44 Characteristics of Nest Sand The mean grain size andsorting coefficient of sand from nests in this study werecomparable to those previously observed at a variety ofsea turtle nesting beaches [47] Given the relatively smallrange of mean grain size observed across study nests (0282ndash0366mm) granulometry likely did not drive the differencesin hatching success across the areas of the beach
The high amounts of organic matter in the sand fromthis study in comparison to other sea turtle nesting beaches[47] are not surprising as this is likely a result of the highrates of nest destruction due to the large number of nestingfemales and high-nest-density at this beach [9 19 48] Theassociation of high organic matter content with a higherabundance of bacteria in the sand from the high-nest-densityarea in this study supports the hypothesis that high organicmatter is associated with elevated microbial proliferation anddecomposition at Ostional and may also explain the low eggclutch pO
2in comparison to previous studies [7 8]
45 Microbial Abundance The number of bacterial 16S andfungal 18S rRNA gene copies in this study ranged from 105to 1010 copies gminus1 of sea turtle nest sand A previous studyfound that the sand at an arribada beach contained six ordersof magnitude more bacterial colonies gminus1 in comparison toa neighboring beach although no specific abundance datawere provided [10 14] Previous studies that have quantified16S or 18S rRNA gene copies in similar sediments have foundbetween 106 and 109 copies gminus1 [26 49]
The negative relationship between bacterial abundanceand hatching success that we observed supports the hypoth-esis that the microbial abundance in nest sand at Ostionalis high enough to adversely affect embryonic developmentSpecifically we suggest that the mechanism behind thisrelationship is not pathogenic but rather a result of high ratesof microbial decomposition of organic matter altering thenest environment beyond the optimal range for embryonicdevelopment Studies on underground nesting species ofbirds and reptiles suggest similar influences of microbialdecomposition on nest pO
2and temperature [50 51] It is
possible that the relationship between microbial activity andhatching success is nonlinear with a threshold beyond whichhigh rates of microbial respiration disrupt the diffusion ofoxygen to the nest to the point of impeding embryonic devel-opment For example the relationship between microbialabundance and hatching success appears to reach a thresholdat sim1 times 109ndash1010 16S18S copies gminus1 nest sand Furtherstudies that span a broader range in microbial abundanceare required to confirm the true nature of the relationshipbetween the microbial abundance of nest sand and sea turtlehatching success The coinciding occurrence of lower pO
2
higher temperatures higher organicmatter and low hatchingsuccess in the high-nest-density area of the beach serves asan extreme example of the adverse conditions imposed bymicrobial decomposition at Ostional
In addition to the indirect effect of microbial abundanceon the nest environment there are likely other effects ofmicrobial presence on sea turtle hatching success that wedid not consider because they were not within the scope ofthe present study For example we did not explore speciescomposition or diversity nor did we evaluate the presence orabsence of pathogens The potential presence of pathogenicmicrobes in the sand at Ostional cannot be ruled out Severalspecies of fungi in the Fusarium solani species complex havebeen identified as pathogens to sea turtle eggs and are knownto occur at nesting beaches around the world [15 16] It isalso possible that the effects of microbial abundance andthe nest environment on hatching success are synergisticwith the effects of lethal pathogens [15] For example onceembryos are killed due to the adverse effects of microbialoxygen depletion on the nest environment this may resultin the proliferation of fungi which could then spread toadjacent eggs Further studies should therefore focus onsampling from several components of the nest environment(ie cloacal fluid nest sand eggshell unhatched embryosand live hatchlings) as well as assessing the abundancediversity and activity of both fungi and bacteria to providea more complete understanding of the relationship betweenmicrobial activity and the reproductive success of sea turtles
Based on our comparative study it appears that tidalwashing decreases the organic matter and microbial abun-dance in the sand thus increasing hatching success Alter-natively the high humidity in the sand resulting fromtidal washing may also keep incubation temperatures fromreaching lethal temperaturesTherefore seawater inundationmay be critical in maintaining the population of sea turtlesat Ostional Using these natural mechanisms as a model forconservation management practices could be adopted topromote improved embryo survival and hatchling produc-tion For example seawater could be used to decrease organicmatter and microbial abundance in hatchery sand or byperiodically irrigating the beachwith seawaterThe treatmentof sand or relocation of hatcheries on a regular basis is a con-ventional management practice in conservation programs toavoid organic matter build-up as well as microbial and larvalinfestations In fact the leatherback sea turtle conservationprogram at Ostional successfully increased hatching successin the leatherback hatchery by using sand from below thehigh tide line [40]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank G Bran Y Arguello-GomezM Gomez S Steele and all the employees and volunteers atRNVS Ostional for their help in the field The authors wouldalso like to thank S Berthrong A Oporta McCarthy and RLeisen for their contributions to themolecular and laboratoryanalyses This material is based upon work supported by the
Journal of Marine Biology 9
National Science Foundation Graduate Research Fellowshipunder Grant no 511182 Additional funds to support the laband field research for this study were provided by the PADIFoundation National Geographic Young Explorers Grant(C220-12) USFWS Marine Turtle Conservation Act (96200-0-G037) and internal grants from the College of CharlestonThis research was approved by the College of CharlestonIACUC and permits were granted by MINAET (Resolucionno ACT-OR-DR-078) and CONAGEBio (R-020-2012-OT-CONAGEBIO) of Costa Rica
References
[1] A Abreu-Grobois and P T Plotkin ldquoIUCNSMTSG (2008) Lep-idochelys olivaceardquo The IUCN Red List of Threatened SpeciesVersion 20142 httpwwwiucnredlistorg
[2] R A Valverde C M Orrego M T Tordoir et al ldquoOlive ridleymass nesting ecology and egg harvest at ostional beach CostaRicardquoChelonian Conservation and Biology vol 11 no 1 pp 1ndash112012
[3] S E Cornelius R Arauz J Fretey et al ldquoEffect of land basedharvest of Lepidochelysrdquo in The Biology and Conservation ofRidley Sea Turtles P T Plotkin Ed pp 231ndash251 Johns HopkinsUniversity Press Baltimore Md USA 2007
[4] S Honarvar M P OrsquoConnor and J R Spotila ldquoDensity-dependent effects on hatching success of the olive ridley turtleLepidochelys olivaceardquo Oecologia vol 157 no 2 pp 221ndash2302008
[5] V S Beezy and R A Valverde ldquoA comparison ofmethodologiesfor estimating the nest density of olive ridley arribadas atOstional Costa Ricardquo in Proceedings of the 31st Annual Sympo-sium on Sea Turtle Biology andConservation T T Jones andB PWallace Eds NOAA Technical Memorandum NMFS-SEFSC-631 pp 194ndash195 2012
[6] S E Cornelius M A Ulloa J C Castro M Mata del Valleand D C Robinson ldquoManagement of olive ridley sea turtles(Lepidochelys olivacea) nesting at Playas Nancite and OstionalCosta Ricardquo in Neotropical Wildlife Use and Conservation JRobinson and K Redford Eds vol 1 pp 111ndash135 Universityof Chicago Press Chicago Ill USA 1991
[7] R A Valverde S E Cornelius and C L Mo ldquoDecline of theolive ridley sea turtle (Lepidochelys olivacea) nesting assemblageat Nancite Beach Santa Rosa National Park Costa RicardquoChelonian Conservation and Biology vol 3 pp 58ndash63 1998
[8] S Clusella Trullas and F V Paladino ldquoMicro-environment ofolive ridley turtle nests deposited during an aggregated nestingeventrdquo Journal of Zoology vol 272 no 4 pp 367ndash376 2007
[9] D McPherson and D Kibler ldquoEcological impact of olive ridleynesting at Ostional Costa Ricardquo in Proceedings of the 25thAnnual Symposium on Sea Turtle Biology and ConservationH Kalb A Rohde K Gayheart and K Shanker Eds NOAATechnical Memorandum NMFS-SEFSC-582 p 204 2008
[10] C L Mo I Salas and M Caballero ldquoAre fungi and bacteriaresponsible for olive ridleyrsquos egg lossrdquo in Proceedings of the 10thAnnual Workshop on Sea Turtle Biology and Conservation T HRichardson J I Richardson and M Donnelly Eds pp 249ndash252 NOAA Techn 1990
[11] S Honarvar J R Spotila andM P OrsquoConnor ldquoMicrobial com-munity structure in sand on two olive ridley arribada nestingbeaches Playa La Flor Nicaragua and Playa Nancite Costa
Ricardquo Journal of Experimental Marine Biology and Ecology vol409 no 1-2 pp 339ndash344 2011
[12] A D Phillott and C J Parmenter ldquoThe distribution of failedeggs and the appearance of fungi in artificial nests of green(Chelonia mydas) and loggerhead (Caretta caretta) sea turtlesrdquoAustralian Journal of Zoology vol 49 no 6 pp 713ndash718 2001
[13] J Wyneken T J Burke M Salmon and D K Pedersen ldquoEggfailure in natural and relocated sea turtle nestsrdquo Journal ofHerpetology vol 22 no 1 pp 88ndash96 1988
[14] C L Mo M Caballero and I Salas ldquoMicroorganism infectionof olive ridley eggsrdquo in Proceedings of the 12th Annual Workshopon Sea Turtle Biology and Conservation J I Richardson and TH Richardson Eds NOAA Technical Memorandum NMFS-SEFSC-361274 pp 81ndash84 1995
[15] J M Sarmiento-Ramırez E Abella M P Martın et alldquoFusarium solaniis responsible for mass mortalities in nests ofloggerhead sea turtle Caretta caretta in Boavista Cape VerderdquoFEMS Microbiology Letters vol 312 no 2 pp 192ndash200 2010
[16] J M Sarmiento-Ramırez E Abella-Perez A D Phillott etal ldquoGlobal distribution of two fungal pathogens threateningendangered sea turtlesrdquo PLoS ONE vol 9 Article ID e858532014
[17] J Patino-Martinez A Marco L Quinones E Abella R MAbad and J Dieguez-Uribeondo ldquoHow do hatcheries influenceembryonic development of sea turtle eggs Experimental anal-ysis and isolation of microorganisms in leatherback turtle eggsrdquoJournal of Experimental Zoology Part A Ecological Genetics andPhysiology vol 317 no 1 pp 47ndash54 2012
[18] A D Phillott and C J Parmenter ldquoFungal colonization of greensea turtle (Chelonia mydas) nests is unlikely to affect hatchlingconditionrdquo Herpetological Conservation amp Biology vol 9 pp297ndash301 2014
[19] MOcanaMHarfush-Melendez and SHeppell ldquoMass nestingof olive ridley sea turtles Lepidochelys olivacea at La EscobillaMexico linking nest density and rates of destructionrdquo Endan-gered Species Research vol 16 pp 45ndash54 2012
[20] DMadden J Ballestero C Calvo R Carlson E Christians andE Madden ldquoSea turtle nesting as a process influencing a sandybeach ecosystemrdquo Biotropica vol 40 no 6 pp 758ndash765 2008
[21] R Valverde and C Gates ldquoPopulation surveys on mass nestingbeachesrdquo in Research and Management Techniques for theConservation of Sea Turtles K L Eckert K A Bjorndal FA Abreu-Gobrois and M Donnelly Eds IUCNSSC MarineTurtle Specialist Group Publication No 4 1999
[22] B P Wallace P R Sotherland J R Spotila R D ReinaB F Franks and F V Paladino ldquoBiotic and abiotic factorsaffect the nest environment of embryonic leatherback turtlesDermochelys coriaceardquo Physiological and Biochemical Zoologyvol 77 no 3 pp 423ndash432 2004
[23] J D Miller ldquoDetermining clutch size and hatching successrdquo inResearch and Management Techniques for the Conservation ofSea Turtles K L Eckert K A Bjorndal F A Abreu-Groboisand M Donnelly Eds IUCNSSC Marine Turtle SpecialistGroup Publication No 4 1999
[24] S J Blott and K Pye ldquoGradistat a grain size distribution andstatistics package for the analysis of unconsolidated sedimentsrdquoEarth Surface Processes and Landforms vol 26 no 11 pp 1237ndash1248 2001
[25] W Krumbein and F Pettijohn Manual of Sedimentary Petrog-raphy Appleton-Century-Crofts New York NY USA 1938
10 Journal of Marine Biology
[26] N C Prevost-Boure R Christen S Dequiedt et al ldquoValidationand application of a PCR primer set to quantify fungal commu-nities in the soil environment by real-time quantitative PCRrdquoPLoS ONE vol 6 no 9 Article ID e24166 2011
[27] N Fierer J A Jackson R Vilgalys and R B Jackson ldquoAssess-ment of soil microbial community structure by use of taxon-specific quantitative PCR assaysrdquo Applied and EnvironmentalMicrobiology vol 71 no 7 pp 4117ndash4120 2005
[28] A Guidot J-C Debaud and R Marmeisse ldquoSpatial distribu-tion of the below-ground mycelia of an ectomycorrhizal fungusinferred from specific quantification of its DNA in soil samplesrdquoFEMS Microbiology Ecology vol 42 no 3 pp 477ndash486 2002
[29] T B de Gregoris N Aldred A S Clare and J G BurgessldquoImprovement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxardquo Journal of Microbio-logical Methods vol 86 no 3 pp 351ndash356 2011
[30] E J Vainio and J Hantula ldquoDirect analysis of wood-inhabitingfungi using denaturing gradient gel electrophoresis of amplifiedribosomal DNArdquoMycological Research vol 104 no 8 pp 927ndash936 2000
[31] S Turner K M Pryer V P W Miao and J D Palmer ldquoInves-tigating deep phylogenetic relationships among cyanobacteriaand plastids by small subunit rRNA sequence analysisrdquo TheJournal of Eukaryotic Microbiology vol 46 no 4 pp 327ndash3381999
[32] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash174 John Wiley amp Sons Chichester UK1991
[33] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications pp 315ndash322 1990
[34] N Uemura KMakimuraM Onozaki et al ldquoDevelopment of aloop-mediated isothermal amplificationmethod for diagnosingPneumocystis pneumoniardquo Journal of Medical Microbiology vol57 no 1 pp 50ndash57 2008
[35] C J Smith D B Nedwell L F Dong and A M OsbornldquoEvaluation of quantitative polymerase chain reaction-basedapproaches for determining gene copy and gene transcriptnumbers in environmental samplesrdquo Environmental Microbiol-ogy vol 8 no 5 pp 804ndash815 2006
[36] C J Smith and A M Osborn ldquoAdvantages and limitationsof quantitative PCR (Q-PCR)-based approaches in microbialecologyrdquo FEMS Microbiology Ecology vol 67 no 1 pp 6ndash202009
[37] R A Ackerman ldquoOxygen consumption by sea turtle (CheloniaCaretta) eggs during developmentrdquo Physiological Zoology vol54 pp 316ndash324 1981
[38] R A Valverde S Wingard F Gomez M T Tordoir and C MOrrego ldquoField lethal incubation temperature of olive ridley seaturtle Lepidochelys olivacea embryos at a mass nesting rookeryrdquoEndangered Species Research vol 12 no 1 pp 77ndash86 2010
[39] L G Fonseca W N Villachica R E Matarrita and R AValverde ldquoReporte final de la anidacioon de tortuga lora(Lepidochelys olivacea) Playa Nancite Parque Nacional SantaRosa Costa Rica (Temporada 2010ndash2011)rdquo Reporte Final deTemporada al USFWS 2011
[40] C Figgener A Castillo-MacCarthy J Mora-Sandoval YArguelo-Gomez and W Quiros-Pereira ldquoRaising the hatchingsuccess of leatherback nests at the olive ridley mass-nesting
beach inOstional Guanacaste Costa Ricardquo in Proceedings of the34thAnnual Symposiumon SeaTurtle Biology andConservation2014
[41] C Limpus V Baker and J Miller ldquoMovement induced mortal-ity of loggerhead eggsrdquoHerpetologica vol 35 pp 335ndash338 1979
[42] R A Ackerman ldquoThe respiratory gas exchange of sea turtlenests (Chelonia Caretta)rdquo Respiration Physiology vol 31 no 1pp 19ndash38 1977
[43] Y-C Kam ldquoPhysiological effects of hypoxia onmetabolism andgrowth of turtle embryosrdquo Respiration Physiology vol 92 no 2pp 127ndash138 1993
[44] TWibbels D Rostal andR Byles ldquoHigh pivotal temperature inthe sex determination of the olive ridley sea turtle Lepidochelysolivacea from Playa Nancite Costa Ricardquo Copeia no 4 pp1086ndash1088 1998
[45] C J McCoy R C Vogt and E J Censky ldquoTemperature-controlled sex determination in the sea turtle Lepidochelysolivaceardquo Journal of Herpetology vol 17 no 4 pp 404ndash4061983
[46] N A Miller ldquoPO2in loggerhead sea turtle (Caretta caretta)
[47] J A Mortimer ldquoThe influence of beach sand characteristicson the nesting behavior and clutch survival of green turtles(Chelonia mydas)rdquo Copeia vol 1990 no 3 pp 802ndash817 1990
[48] S E Cornelius The Sea Turtles of Santa Rosa National ParkFundacion de Parques Nacionales San Jose Costa Rica 1986
[49] X Chen E Peltier B S M Sturm and C B Young ldquoNitrogenremoval and nitrifying and denitrifying bacteria quantificationin a stormwater bioretention systemrdquo Water Research vol 47no 4 pp 1691ndash1700 2013
[50] R S Seymour and R A Ackerman ldquoAdaptations to under-ground nesting in birds and reptilesrdquo American Zoologist vol20 no 2 pp 437ndash447 1980
[51] R S Seymour D Vleck and C M Vleck ldquoGas exchange in theincubationmounds of megapode birdsrdquo Journal of ComparativePhysiology B vol 156 no 6 pp 773ndash782 1986
23 Nest Temperature Nest temperature was monitoredusing HOBO pendant temperature dataloggers (Onset Com-puter Corporation) placed in the center of each clutch andprogrammed to record temperature at 3 h intervals startingat midnight on the night of oviposition through hatchlingemergence Mean daily nest temperatures were used tocompare nest temperatures across the different areas of thebeach
24 Nest Excavations Single-use sterile gloves were wornfor all excavations and changed before contact with differentnests Nests were exhumed three days after the last observedhatchling emergence to quantify hatching success [23]
25 Sand Collection and Characterization A sample of sandwas collected directly into sterile collection tubes from thecenter of the nest chamber during the excavation of the nest atthe end of the incubation period Samples were placed on iceimmediately after collection and then either frozen (minus20∘C)or preserved in formalin (2 formaldehyde) within 6 h ofcollection and until analysis The organic matter analysisconsisted of a loss-on-ignition method with the organicmatter content being the loss of mass after dry combustionThe sample was transferred to a porcelain container anddesiccated in a drying oven (24 h at 100∘C) before combustion(6 h at 500∘C) Prior to combustion samples of dried sandwere fractionated with a set of sieves (0063 0075 015 025045 and 085mm) to determine the particle-size distributionbymassMean grain size (120601) sorting (120590
120601) and skewness (Sk
120601)
were calculated using the logarithmic mathematical ldquomethodof momentsrdquo in GRADISTAT [24 25]
26 Microbial Abundance Samples preserved in formalinwere used for microscopy counts as a secondary methodof quantifying bacterial abundance These samples werecentrifuged for 10min at 16000 g in a microcentrifuge beforecarefully removing excess formalin The sand was thendiluted (approximately 1 2) with sterile water and sonicatedon ice for 20 s at 30W (Sonifier S-250A Branson) Theresulting supernatant fluid was stained for approximately 5minutes with a 1 10 dilution of 1x SybrGold and sterile waterMicroscopy counts were performed at 1000x magnificationon an epifluorescence microscope (Optiphot-2 Nikon) bycounting 10 fields per slide The number of cells gminus1 of sandwas then calculated based on the original weight of sandvolume of diluent and supernatant and the average numberof cells fieldminus1 using the number of fields per slide at 1000x(479 times 104) and correction factor for the addition of formalin(times116)
We used a quantitative real-time polymerase chain reac-tion (qPCR) analysis to determine the abundance of bacteriabased on the number of 16S rRNA gene copies gminus1 nestsand and the abundance of fungi based on the number of18S rRNA gene copies gminus1 nest sand While the resultingquantification of gene copies from a qPCR analysis cannot bedirectly transformed into the number of cells (given that copynumber can vary greatly between species) this can be used asa proxy for overall abundance ofmicrobial communities [26ndash28]
27 DNA Extraction Each sample of sand was thawed andhomogenized by vortexing before collecting a 1 g subsamplefor DNA extraction DNA was extracted from all samplesof sand using an EZNA Soil DNA kit (Omega Biotek) withthe following modifications to the protocol to increase DNAyields Samples were subjected to 5min at approximately2000 oscillations minminus1 in a bead beater (Mini Beadbeater-8Biospec Products) and three freeze-thaw cycles (minus20∘C and70∘C for 30min each) during the lysis step Additionally inthe final step of extraction only 50 120583L of elution buffer wasusedwhichwas reapplied to the column in the second elutionstep DNA samples were diluted in order to reduce inhibitionand optimize efficiency and Ct values to within the range ofthe standard curve
28 qPCR Analysis Absolute qPCR was run using an iCycleriQ Real-Time PCR Detection System (Bio-Rad LaboratoriesInc) on a 96-well plate Results were analyzed using iQ5software (Bio-Rad Laboratories Inc)The universal bacterialprimers 926F (51015840-AAA CTC AAA KGA ATT GAC GG-31015840)and 1062R (51015840-CTCACRRCACGAGCTGAC-31015840) that targetthe 16S rRNA gene were used based on a previous studyby de Gregoris et al [29] Each 10 120583L reaction containedthe following 5120583L of ABsolute qPCR Master Mix (ABgene)01 120583L bovine serum albumin (10 120583g 120583Lminus1Thermo Scientific)03 120583L of each primer (10 120583M 300 nM final concentrationIntegrated DNA Technologies) 39 120583L H
2O and 04 120583L tem-
plate DNA PCR conditions were 15min at 95∘C followed by40 cycles of 95∘C for 15 s 15 s at the annealing temperature of57∘C and 72∘C for 20 s
The universal fungal primers FR1 (51015840-AICCATTCAATC-GGTAIT-31015840) and FF390 (51015840-CGATAACGAACGAGACCT-31015840) that target the 18S rRNAgenewere used based on previousstudies [26 30] Each 10 120583L reaction contained the following5 120583L of ABsolute qPCR Master Mix (ABgene) 01 120583L bovineserum albumin (10 120583g 120583Lminus1Thermo Scientific) 01 120583L of eachprimer (10120583M 100 nM final concentration Integrated DNATechnologies) 43 120583L H
2O and 04 120583L template DNA PCR
conditions were 15min at 95∘C followed by 40 cycles of 95∘Cfor 15 s 30 s at the annealing temperature of 50∘C and 72∘Cfor 1min
29 qPCR Standards External fixed standards were cre-ated by amplifying and quantifying bacterial and fun-gal DNA using primer sets that targeted the full 16S18SrRNA sequence Template DNA extractions for bacterialand fungal standards were kindly provided from culturesof Bacillus pumilus (W Hook Grice Marine LaboratoryCollege of Charleston Charleston SC) and Phytophthoracapsici (J Ikerd US Vegetable Laboratory USDA ARSCharleston SC) respectively For bacteria the univer-sal primers 8F (51015840-AGAGTTTGATCCTGGCTCAG-31015840) and1492R (51015840-GGTTACCTTGTTACGACTT-31015840) were used withthe following PCR conditions 3min at 95∘C followed by30 cycles of 95∘C for 1min 1min at the annealing temper-ature of 45∘C and 72∘C for 15min with a final elongationstep of 4min at 72∘C [31 32] For fungi the universalprimers NS1 (51015840-GTAGTCATATGCTTGTCTC-31015840) and NS8(51015840-TCCGCAGGTTCACCTACGGA-31015840) were used with the
4 Journal of Marine Biology
following PCR conditions 4min at 95∘C followed by 30cycles of 95∘C for 1min 2min at the annealing temperatureof 55∘C and 72∘C for 15min with a final elongation step of10min at 72∘C [33 34] Each 25 120583L PCR reaction containedthe following 5 120583L of buffer (5x colorless GoTaq Flexi BufferPromega) 3 120583L of MgCl
2(25mM Promega) 025 120583L bovine
serum albumin (10 120583g 120583Lminus1 Promega) 05120583L dNTP (10mMThermo Scientific) 05120583L of each primer (5120583M IntegratedDNA Technologies) 01 120583L of DNA polymerase (5U120583Lminus1GoTaq Flexi DNA polymerase Promega) 1315 120583L H
2O and
2 120583L template DNA Bacterial and fungal PCR products werethen run on a 05 agarose gel purified with a QIAquickGel Extraction Kit (Qiagen) and eluted in 30 120583L at the finalstep Standards were quantified using the Qubit dsDNA BRAssay Kit (Invitrogen Life Technologies) and a Qubit 20Fluorometer (Qubit Systems) to determine the quantity ofDNA in the final sample The amplicon length was thenused to calculate the number of copies per 120583L under theassumption that the molecular weight of each bp is 650A standard curve was generated using 10-fold dilutions ofthese standards across 8 orders of magnitude thereforestandardizing qPCR results by copy number
Each plate included triplicate reactions per DNA sampleand for the standard curve as well as a no-template control tocheck for contamination Amelt curve (1min at 95∘C 1min at55∘C +05∘C 10 sminus1 to 95∘C) was used at the end of each qPCRrun to ensure the fluorescence signal resulted from specificityto the PCR product rather than from primer dimers orother nonspecific products The fluorescein dye included inthe master mix allowed for standard normalization of erroracross samples Threshold cycles (Ct) were automaticallycalculated by the software based on the average backgroundnoise Samples with less than one order of magnitude ofseparation (33 Ct value) from the no-template control wereexcluded given that this was outside of the detection limit ofthe present analysis [35 36] These samples were thereforereanalyzed using the same standard curve with a less dilutetemplate in order to ensure accurate quantification Samplesthat fell below the detection limit regardless of dilution wereexcluded from the analysis
210 Statistical Analysis Statistical analyses were conductedusing JMP 10 (SAS Institute httpwwwjmpcom) Hatch-ing success temperature pO
2 bacterial and fungal abun-
dance organic matter content and mean grain size data wereall analyzed using ANOVA When assumptions of normalityand homogeneity of variance were not met nonparametrictests were used (WilcoxonKruskal-Wallis) Statistically sig-nificant results were further testedwith Tukeyrsquos HSDpost hoctest to determine significant differences between beach areasRelationships between variables (hatching success pO
2 tem-
perature grain size organic matter content and bacterialand fungal abundance) were evaluated using Spearmanrsquos rankcorrelation coefficients
Because the metabolic demand of sea turtle embryos islow within the first half of incubation given their limiteddevelopment differences in oxygen content and temperaturein the egg clutch within this time period can presumablybe attributed to microbial activity [8 37] Mean nest pO
2
and temperature were therefore analyzed for the first andsecond half of incubation separately Repeatedmeasurementsof temperature and oxygen were also analyzed with repeatedmeasures MANOVA To allow for sample comparison acrossqPCR plates an ANCOVA was used to ensure there was nosignificant difference between standard curves from differentruns We also used a positive control on each plate tocalculate a coefficient of variation (CV) in order to ensurereproducibility within and between all plates All valuesare expressed as means plusmn standard error (SE) Percentagedata (hatching success) and microbial abundance data (copynumber) were arcsine- and log-transformed respectivelybefore analysis with parametric statistics Mean grain sizewas converted from phi units (120601) to mm for presentationThe number of 16S18S copies ulminus1 of template was convertedto a number of 16S18S copies gminus1 of nest sand to allowfor comparison across samples All analyses were tested forstatistical significance at 120572 lt 005
3 Results
The sample arribada occurring in August 2012 (estimated at72152 egg-laying females) and another arribada (September2012 estimated at 211954 egg-laying females) occurringbefore nest excavations were carried out in the middleof October An earthquake measuring 76 on the Richterscale occurred on September 5 2012 the epicenter locatedapproximately 50 km south of Ostional which caused visiblestructural changes to the beach topography Nest density forthe entire beach at the time of excavation was 36 plusmn 04 nestsmminus2 and overall hatching success for nests laid on the mainnesting beach during the August arribada was 34 plusmn 54 (RValverde unpublished data) At the time of excavation thenest densities in the high-nest-density tidal-wash and low-nest-density areas were 52 plusmn 04 20 plusmn 06 and 30 plusmn 05 nestsmminus2 respectively Two nests were excluded from the analysisdue to unforeseeable factors that likely caused a change inthe variables that we attempted to control in this study (ietemperature and pO
2of the nest) Specifically we excluded a
nest in the high-density nesting area that was partially shadedby a palm tree and another in the tidal-wash nesting area thatremained permanently inundated by the nearby estuary afterthe earthquake Hatching success in the high-nest-densityarea (06) was significantly lower than in the low-nest-density (682) and tidal-wash (547) areas of the beach(119875 lt 0001 Figure 1)
The pO2within egg clutches in the high-nest-density area
was significantly lower than that in other areas of the beachin both the first (154 plusmn 09 kPa 119875 lt 0001) and second halfof incubation (115 plusmn 05 kPa 119875 lt 0001 Figure 2) The meanminimumpO
2among nests in the high-nest-density area was
97plusmn 03 kPa with one clutch exhibiting pO2as low as 87 kPa
Nest pO2varied significantly with beach area and incubation
day with a significant interaction occurring between thesetwo factors (119865
was positively correlatedwith pO2in both the first and second
half of the incubation period (119903 = 0872 119875 lt 00001 and119903 = 0591 119875 = 0020 resp Table 1)
Journal of Marine Biology 5
100
80
60
40
20
0
Hat
chin
g su
cces
s (
)
Low-density Tidal-wash High-densityBeach area
A
B
Figure 1 Mean (plusmn SE) hatching success of nests located in differentnesting areas of the beach (119899 = 5) Different letters denote statisticaldifference Hatching success in the high-nest-density area (06)was significantly lower than in the low-nest-density (682) andtidal-wash (547) areas of the beach (119875 lt 0001)
20
18
16
14
12
10
pO2
(kPa
)
Low-densityTidal-wash
High-density
0 10 20 30 40 50
Incubation day
Figure 2 Mean (plusmn SE) partial pressure of oxygen (pO2 kPa) in
nests throughout the incubation period for nests located in thehigh-density tidal-wash and low-density nesting areas of the beach(119899 = 5) The pO
2within nests in the high-nest-density area was
significantly lower than that in other areas of the beach in both thefirst and the second half of incubation (119875 lt 0001 and 119875 lt 00001resp)
Nest temperatures were significantly lower in the tidal-wash area in the first and secondhalf of incubation (119875 = 0001and 119875 = 0001 resp Figure 3) Temperatures in the high-nest-density area of the beach were the highest in the secondhalf of incubation (357plusmn02∘C) and remained above the lethallimit (35∘C [38]) for a significantly longer duration of timein comparison to other areas of the beach (170 plusmn 07 d 119875 =0009) Nest temperature varied significantly with beach areaand incubation day with a significant interaction occurringbetween these two factors (119865
Table 1 Relationships between hatching success and partial pres-sure of oxygen (pO2) and temperature in the first (1st) and second(2nd) half of incubation bacterial and fungal abundance andorganic matter content for all study nests Copy number data fromseveral nests fell outside the range of detection for the presentanalysis and were therefore left out (see Section 3 for more details)Hatching success was positively correlated with nest pO2 in both thefirst and second half of incubation and negatively correlatedwith thebacterial abundance in nest sand
Variable Half of incubationperiod
Hatching success119903 119899 119875
pO21st 0872 13 lt00012nd 0591 13 0020
Temperature 1st minus0025 13 09292nd minus0207 13 0459
Figure 3 Mean nest temperature throughout the incubation periodin nests located in the high-density tidal-wash and low-densitynesting areas of the beach (119899 = 5) The field lethal incubationtemperature is indicated with a horizontal grey dashed line (35∘CValverde et al 2010) Nest temperatures were significantly lower inthe tidal-wash area in the first and the second half of incubation(119875 = 0001 and 119875 = 0001 resp)
However hatching success was not significantly correlatedwith nest temperatures in the first or second half of incuba-tion (Table 1)
The organic matter content of nest sand was significantlyhigher in the high-nest-density area (40) than in the low-nest-density (32) and tidal-wash (30) areas of the beach(119875 = 0015 and 119875 = 0003 resp) The sand from nestsin the low-nest-density area had a significantly larger meangrain size (0366mm) than in both the high-nest-density(0282mm) and tidal-wash (0283mm) areas of the beach
6 Journal of Marine Biology
(119875 = 0020 for both comparisons) Additionally the sandfrom all nests in the high-nest-density tidal-wash and low-nest-density areas of the beach wasmoderately sorted (120590
12060110
101 and 098 resp) and finely skewed (Sk120601035 036 and
103 resp)There was no significant difference in the slope of the
standard curve across assays for bacteria (119864 = 10223plusmn1411198772= 099 slope = minus327plusmn003 intercept = 3683plusmn056 CV =
341) or fungi (119864 = 8455 plusmn 225 1198772 = 100 plusmn 000 slope =minus376plusmn007 intercept = 4852plusmn080 CV = 168) indicatingthat copy numbers were comparable across assays (119875 = 0856and 119875 = 0088 resp) Microscopy counts (range 572 times 106ndash743 times 107 cells gminus1 of nest sand) were closely correlated withqPCR results for 16S rRNA gene copy number gminus1 of sand forall nests (119903 = 0404 119875 = 0013) Both the 16S and 18S copynumbers for one nest in the low-nest-density area as well asthe 18S copy number for two nests in the tidal-wash area werebelow the detection limit of the present analysis and weretherefore omitted
There was a significantly higher abundance of bacteriain nest sand from the high-nest-density area in comparisonto other areas of the beach (119875 = 0029) There was also ahigher abundance of fungi in nest sand from the high-nest-density area of the beach though this difference wasmarginal(119875 = 0063) While there was a negative correlation betweenhatching success and bacterial abundance for all nests thecorrelation between hatching success and fungal abundancewas marginal (119903 = minus0585 119875 = 0046 and 119903 = minus0607119875 = 0063 resp Table 1 and Figure 4) There was a negativecorrelation between the number of bacteria and egg clutchpO2in both the first and second half of incubation (119903 =
minus0741 119875 = 0006 and 119903 = minus0797 119875 = 0002 resp) aswell as a positive correlation between the number of bacteriaand nest temperatures in the second half of incubation (119903 =0818 119875 = 0001 Table 2) Furthermore there was a positivecorrelation between bacterial abundance and organic mattercontent in nest sand across all areas of the beach (119903 = 0636119875 = 0026)
4 Discussion
In this study we monitored nest conditions throughout theincubation period and quantified the microbial abundanceof nest sand in association with sea turtle hatching successWe found a negative relationship between the microbialabundance of nest sand and hatching success and observedsignificantly low pO
2in egg clutches located in the high-
nest-density area of the beach Egg clutches located in thetidal-wash area of the beach had higher hatching successand pO
2as well as lower organic matter content than those
located in the high-nest-density area of the beach Theseresults are consistent with previous reports that seawatermay have a positive effect on hatching success [39 40]presumably by decreasing microbial abundance incubationtemperature andor organic matter content Our results alsosupport our hypothesis that the low pO
2associated with
high microbial abundance in nest sand is responsible for thelower hatching rates observed at Ostional beach Collectively
105
106
107
108
109
1010
1011
BacteriaFungi
100
80
60
40
20
0
Hat
chin
g su
cces
s (
)
Log copy number gminus1 nest sand
Figure 4 Relationship between hatching success and microbialabundance (bacteria and fungi) for nests located in each nesting areaof the beach (119899 = 12 and 119899 = 10 resp) Regression lines for bacteria(solid) and fungi (dashed) are included for visualization purposesWhile therewas a negative correlation between hatching success andbacterial abundance for all nests the correlation between hatchingsuccess and fungal abundance was marginal (119875 = 0046 and119875 = 0063 resp) For Spearmanrsquos rank correlation coefficients seeTable 1
these data suggest a nonpathogenic mechanism for sea turtleembryonic mortality resulting from high rates of microbialdecomposition altering the nest environment beyond theoptimal range for embryonic development
Because all nests in this study incubated during the sametime period we are fairly confident that a majority of factorsaffecting embryonic development and hatching success werereasonably controlled However there may be factors that wedid notmonitor that caused incongruences in nest conditionsand could have influenced hatching success in this studyAdditionally an earthquake of large magnitude such as theone that occurred during this study could have feasiblycaused movement-induced mortality of sea turtle embryosHowever the earthquake occurred on day 25 of incubationafter the critical period (12 h to 14 d) during whichmovementsignificantly reduced hatching success [41] and thereforelikely had no adverse effects on the egg clutches in this study
41 Hatching Success Hatching success was highest in nestslocated in the low-nest-density area High-nest-densitieshave previously been observed to have direct physiologicaleffects (ie lower pO
2andhigher pCO
2within the egg clutch)
that negatively impacted hatching success [4] Nest densitiesof up to 5 nests mminus2 are unlikely to have a significant effect onhatching success (ge56) while densities of 9 nests mminus2 maysignificantly reduce hatching success to 30 [4]However thenests in this studywere subject to relatively low-nest-densities(2ndash5 nests mminus2) which likely were not high enough to causethe significant drop in hatching success observed
Journal of Marine Biology 7
Table 2 Relationships between bacterial and fungal abundance and the partial pressure of oxygen (pO2) and temperature in the first (1st)and second (2nd) half of incubation for all study nests There was a negative correlation between bacterial abundance and nest pO2 in boththe first and second half of incubation and a positive correlation with nest temperatures in the second half of incubation There was also anegative correlation between fungal abundance and nest pO2 in the first half of incubation
42 Nest Oxygen Content pO2for the low-nest-density and
tidal-wash areas of the beach was similar to that previouslyobserved in other studies on olive ridley sea turtles [4 8]Those studies also observed a change in the pO
2of nests
over the incubation period with pO2decreasing later in
the incubation period as a result of embryo respiration Thesignificant interaction effect of beach area and incubation dayon egg clutch pO
2indicates that the pO
2within egg clutches
changed differently across the areas of the beachOn the other hand the pO
2observed in egg clutches
located in the high-nest-density area in this study is lowerthan that previously observed in other studies [8 22 42]Specifically the drop in pO
2that we observed early in
the incubation period is uncharacteristic of sea turtle eggclutches given that the metabolic activity of the embryos istypically negligible at this time [42] Clutch pO
2typically
remains constant through the first half of incubation andthen decreases and tends to be negatively correlated with thenumber ofmetabolizing embryos per clutch [8 22] Howeverthis was not the case for clutches located in the high-nest-density area of the beach where pO
2began to drop from the
start of incubation and was lower in both the first and secondhalf of the incubation period in comparison to other areas ofthe beach
The negative correlation between microbial abundance(of both bacteria and fungi) and clutch pO
2at Ostional
suggests that microbial decomposition was responsible forthese decreases in pO
2 In particular our observations of high
abundance of bacteria and organic matter in nest sand fromthe high-nest-density area of the beach in association withlow pO
2further support this hypothesis Additionally if this
decrease in pO2resulted from the decomposition of embryos
killed by pathogens in these nests one would expect a lagtime (ie time for infection) and an abrupt change in oxy-gen and temperature following embryo death Furthermorewhile nest density alone affects the oxygen content of neststhe minimum pO
2observed in the high-nest-density area
(97 kPa) in this study was lower than previously observedat similar and even higher nest densities (approximately145ndash170 kPa) [4 8] Given that these previous studies inldquocleanrdquo sand (with presumably low organic matter content)did not find such discrepancies in the pO
2of egg clutches
at similar densities it is likely that microbial decompositionis responsible for the differences observed here Howeverfurther studies that include a controlled measure of pO
2in
beach sand outside of egg clutches and within egg clutches
at controlled densities are necessary to properly test thishypothesis
Because sea turtle embryos have a relatively low toleranceto hypoxia early in the incubation period [43] it is likelythat the drop in pO
2that we observed in the high-nest-
density area of the beach led to total nest failure While thecritical oxygen tension of olive ridley sea turtle eggs hasnot been studied this value was as high as 165 kPa on day22 of incubation for loggerhead sea turtle (Caretta caretta)eggs which are comparable in size [43] Assuming that thecritical oxygen tension of olive ridley sea turtle eggs is similarand given that egg clutch pO
2fell below 165 kPa on day 15
of incubation in the high-nest-density area it is likely thatany eggs located here were exposed to pO
2below a lethal
threshold
43 Nest Temperature The nest temperatures observed inthis study are similar to those observed in other studieson olive ridley sea turtles in this region [4 8 38] Ingeneral mean incubation temperatures were above the meanpivotal temperature of 305∘C for olive ridleys [44 45]indicating that the hatchling sex ratio could be female-biasedat Ostional as previously suggested [38] Previous studieshave also observed a change in nest temperatures throughoutthe incubation period with temperatures increasing laterin incubation in conjunction with increasing embryonicmetabolism [4 8] Nest temperatures were fairly consistentacross the different areas of the beach throughout the incu-bation period with the exception of nests located in thetidal-wash area where lower nest temperatures likely resultedfrom higher water content in the sandThe incubation periodwas 2 to 3 days longer in this group of nests and is likely aresult of slower embryonic development due to lower nesttemperatures [46]
While most nest temperatures fell within the wide rangeof tolerance for sea turtle embryological development (25ndash35∘C [39]) early in the incubation period the slightly highertemperatures observed in the high-nest-density area of thebeach later in incubation may have exceeded this optimalrange long enough to affect hatching success Though thefield lethal limit for olive ridley sea turtles at this beach isapproximately 35∘C embryos may be capable of recoveringfrom short-term exposure to high temperatures while longerdurations are lethal [38] Results from this study supportthis notion in that nests with lower hatching success (high-nest-density area) were also exposed to temperatures above
8 Journal of Marine Biology
the lethal limit for a longer duration of time The differentcombination of heat released from embryonic metabolismandor microbial decomposition is likely responsible for theobserved differences in temperature later in incubation
44 Characteristics of Nest Sand The mean grain size andsorting coefficient of sand from nests in this study werecomparable to those previously observed at a variety ofsea turtle nesting beaches [47] Given the relatively smallrange of mean grain size observed across study nests (0282ndash0366mm) granulometry likely did not drive the differencesin hatching success across the areas of the beach
The high amounts of organic matter in the sand fromthis study in comparison to other sea turtle nesting beaches[47] are not surprising as this is likely a result of the highrates of nest destruction due to the large number of nestingfemales and high-nest-density at this beach [9 19 48] Theassociation of high organic matter content with a higherabundance of bacteria in the sand from the high-nest-densityarea in this study supports the hypothesis that high organicmatter is associated with elevated microbial proliferation anddecomposition at Ostional and may also explain the low eggclutch pO
2in comparison to previous studies [7 8]
45 Microbial Abundance The number of bacterial 16S andfungal 18S rRNA gene copies in this study ranged from 105to 1010 copies gminus1 of sea turtle nest sand A previous studyfound that the sand at an arribada beach contained six ordersof magnitude more bacterial colonies gminus1 in comparison toa neighboring beach although no specific abundance datawere provided [10 14] Previous studies that have quantified16S or 18S rRNA gene copies in similar sediments have foundbetween 106 and 109 copies gminus1 [26 49]
The negative relationship between bacterial abundanceand hatching success that we observed supports the hypoth-esis that the microbial abundance in nest sand at Ostionalis high enough to adversely affect embryonic developmentSpecifically we suggest that the mechanism behind thisrelationship is not pathogenic but rather a result of high ratesof microbial decomposition of organic matter altering thenest environment beyond the optimal range for embryonicdevelopment Studies on underground nesting species ofbirds and reptiles suggest similar influences of microbialdecomposition on nest pO
2and temperature [50 51] It is
possible that the relationship between microbial activity andhatching success is nonlinear with a threshold beyond whichhigh rates of microbial respiration disrupt the diffusion ofoxygen to the nest to the point of impeding embryonic devel-opment For example the relationship between microbialabundance and hatching success appears to reach a thresholdat sim1 times 109ndash1010 16S18S copies gminus1 nest sand Furtherstudies that span a broader range in microbial abundanceare required to confirm the true nature of the relationshipbetween the microbial abundance of nest sand and sea turtlehatching success The coinciding occurrence of lower pO
2
higher temperatures higher organicmatter and low hatchingsuccess in the high-nest-density area of the beach serves asan extreme example of the adverse conditions imposed bymicrobial decomposition at Ostional
In addition to the indirect effect of microbial abundanceon the nest environment there are likely other effects ofmicrobial presence on sea turtle hatching success that wedid not consider because they were not within the scope ofthe present study For example we did not explore speciescomposition or diversity nor did we evaluate the presence orabsence of pathogens The potential presence of pathogenicmicrobes in the sand at Ostional cannot be ruled out Severalspecies of fungi in the Fusarium solani species complex havebeen identified as pathogens to sea turtle eggs and are knownto occur at nesting beaches around the world [15 16] It isalso possible that the effects of microbial abundance andthe nest environment on hatching success are synergisticwith the effects of lethal pathogens [15] For example onceembryos are killed due to the adverse effects of microbialoxygen depletion on the nest environment this may resultin the proliferation of fungi which could then spread toadjacent eggs Further studies should therefore focus onsampling from several components of the nest environment(ie cloacal fluid nest sand eggshell unhatched embryosand live hatchlings) as well as assessing the abundancediversity and activity of both fungi and bacteria to providea more complete understanding of the relationship betweenmicrobial activity and the reproductive success of sea turtles
Based on our comparative study it appears that tidalwashing decreases the organic matter and microbial abun-dance in the sand thus increasing hatching success Alter-natively the high humidity in the sand resulting fromtidal washing may also keep incubation temperatures fromreaching lethal temperaturesTherefore seawater inundationmay be critical in maintaining the population of sea turtlesat Ostional Using these natural mechanisms as a model forconservation management practices could be adopted topromote improved embryo survival and hatchling produc-tion For example seawater could be used to decrease organicmatter and microbial abundance in hatchery sand or byperiodically irrigating the beachwith seawaterThe treatmentof sand or relocation of hatcheries on a regular basis is a con-ventional management practice in conservation programs toavoid organic matter build-up as well as microbial and larvalinfestations In fact the leatherback sea turtle conservationprogram at Ostional successfully increased hatching successin the leatherback hatchery by using sand from below thehigh tide line [40]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank G Bran Y Arguello-GomezM Gomez S Steele and all the employees and volunteers atRNVS Ostional for their help in the field The authors wouldalso like to thank S Berthrong A Oporta McCarthy and RLeisen for their contributions to themolecular and laboratoryanalyses This material is based upon work supported by the
Journal of Marine Biology 9
National Science Foundation Graduate Research Fellowshipunder Grant no 511182 Additional funds to support the laband field research for this study were provided by the PADIFoundation National Geographic Young Explorers Grant(C220-12) USFWS Marine Turtle Conservation Act (96200-0-G037) and internal grants from the College of CharlestonThis research was approved by the College of CharlestonIACUC and permits were granted by MINAET (Resolucionno ACT-OR-DR-078) and CONAGEBio (R-020-2012-OT-CONAGEBIO) of Costa Rica
References
[1] A Abreu-Grobois and P T Plotkin ldquoIUCNSMTSG (2008) Lep-idochelys olivaceardquo The IUCN Red List of Threatened SpeciesVersion 20142 httpwwwiucnredlistorg
[2] R A Valverde C M Orrego M T Tordoir et al ldquoOlive ridleymass nesting ecology and egg harvest at ostional beach CostaRicardquoChelonian Conservation and Biology vol 11 no 1 pp 1ndash112012
[3] S E Cornelius R Arauz J Fretey et al ldquoEffect of land basedharvest of Lepidochelysrdquo in The Biology and Conservation ofRidley Sea Turtles P T Plotkin Ed pp 231ndash251 Johns HopkinsUniversity Press Baltimore Md USA 2007
[4] S Honarvar M P OrsquoConnor and J R Spotila ldquoDensity-dependent effects on hatching success of the olive ridley turtleLepidochelys olivaceardquo Oecologia vol 157 no 2 pp 221ndash2302008
[5] V S Beezy and R A Valverde ldquoA comparison ofmethodologiesfor estimating the nest density of olive ridley arribadas atOstional Costa Ricardquo in Proceedings of the 31st Annual Sympo-sium on Sea Turtle Biology andConservation T T Jones andB PWallace Eds NOAA Technical Memorandum NMFS-SEFSC-631 pp 194ndash195 2012
[6] S E Cornelius M A Ulloa J C Castro M Mata del Valleand D C Robinson ldquoManagement of olive ridley sea turtles(Lepidochelys olivacea) nesting at Playas Nancite and OstionalCosta Ricardquo in Neotropical Wildlife Use and Conservation JRobinson and K Redford Eds vol 1 pp 111ndash135 Universityof Chicago Press Chicago Ill USA 1991
[7] R A Valverde S E Cornelius and C L Mo ldquoDecline of theolive ridley sea turtle (Lepidochelys olivacea) nesting assemblageat Nancite Beach Santa Rosa National Park Costa RicardquoChelonian Conservation and Biology vol 3 pp 58ndash63 1998
[8] S Clusella Trullas and F V Paladino ldquoMicro-environment ofolive ridley turtle nests deposited during an aggregated nestingeventrdquo Journal of Zoology vol 272 no 4 pp 367ndash376 2007
[9] D McPherson and D Kibler ldquoEcological impact of olive ridleynesting at Ostional Costa Ricardquo in Proceedings of the 25thAnnual Symposium on Sea Turtle Biology and ConservationH Kalb A Rohde K Gayheart and K Shanker Eds NOAATechnical Memorandum NMFS-SEFSC-582 p 204 2008
[10] C L Mo I Salas and M Caballero ldquoAre fungi and bacteriaresponsible for olive ridleyrsquos egg lossrdquo in Proceedings of the 10thAnnual Workshop on Sea Turtle Biology and Conservation T HRichardson J I Richardson and M Donnelly Eds pp 249ndash252 NOAA Techn 1990
[11] S Honarvar J R Spotila andM P OrsquoConnor ldquoMicrobial com-munity structure in sand on two olive ridley arribada nestingbeaches Playa La Flor Nicaragua and Playa Nancite Costa
Ricardquo Journal of Experimental Marine Biology and Ecology vol409 no 1-2 pp 339ndash344 2011
[12] A D Phillott and C J Parmenter ldquoThe distribution of failedeggs and the appearance of fungi in artificial nests of green(Chelonia mydas) and loggerhead (Caretta caretta) sea turtlesrdquoAustralian Journal of Zoology vol 49 no 6 pp 713ndash718 2001
[13] J Wyneken T J Burke M Salmon and D K Pedersen ldquoEggfailure in natural and relocated sea turtle nestsrdquo Journal ofHerpetology vol 22 no 1 pp 88ndash96 1988
[14] C L Mo M Caballero and I Salas ldquoMicroorganism infectionof olive ridley eggsrdquo in Proceedings of the 12th Annual Workshopon Sea Turtle Biology and Conservation J I Richardson and TH Richardson Eds NOAA Technical Memorandum NMFS-SEFSC-361274 pp 81ndash84 1995
[15] J M Sarmiento-Ramırez E Abella M P Martın et alldquoFusarium solaniis responsible for mass mortalities in nests ofloggerhead sea turtle Caretta caretta in Boavista Cape VerderdquoFEMS Microbiology Letters vol 312 no 2 pp 192ndash200 2010
[16] J M Sarmiento-Ramırez E Abella-Perez A D Phillott etal ldquoGlobal distribution of two fungal pathogens threateningendangered sea turtlesrdquo PLoS ONE vol 9 Article ID e858532014
[17] J Patino-Martinez A Marco L Quinones E Abella R MAbad and J Dieguez-Uribeondo ldquoHow do hatcheries influenceembryonic development of sea turtle eggs Experimental anal-ysis and isolation of microorganisms in leatherback turtle eggsrdquoJournal of Experimental Zoology Part A Ecological Genetics andPhysiology vol 317 no 1 pp 47ndash54 2012
[18] A D Phillott and C J Parmenter ldquoFungal colonization of greensea turtle (Chelonia mydas) nests is unlikely to affect hatchlingconditionrdquo Herpetological Conservation amp Biology vol 9 pp297ndash301 2014
[19] MOcanaMHarfush-Melendez and SHeppell ldquoMass nestingof olive ridley sea turtles Lepidochelys olivacea at La EscobillaMexico linking nest density and rates of destructionrdquo Endan-gered Species Research vol 16 pp 45ndash54 2012
[20] DMadden J Ballestero C Calvo R Carlson E Christians andE Madden ldquoSea turtle nesting as a process influencing a sandybeach ecosystemrdquo Biotropica vol 40 no 6 pp 758ndash765 2008
[21] R Valverde and C Gates ldquoPopulation surveys on mass nestingbeachesrdquo in Research and Management Techniques for theConservation of Sea Turtles K L Eckert K A Bjorndal FA Abreu-Gobrois and M Donnelly Eds IUCNSSC MarineTurtle Specialist Group Publication No 4 1999
[22] B P Wallace P R Sotherland J R Spotila R D ReinaB F Franks and F V Paladino ldquoBiotic and abiotic factorsaffect the nest environment of embryonic leatherback turtlesDermochelys coriaceardquo Physiological and Biochemical Zoologyvol 77 no 3 pp 423ndash432 2004
[23] J D Miller ldquoDetermining clutch size and hatching successrdquo inResearch and Management Techniques for the Conservation ofSea Turtles K L Eckert K A Bjorndal F A Abreu-Groboisand M Donnelly Eds IUCNSSC Marine Turtle SpecialistGroup Publication No 4 1999
[24] S J Blott and K Pye ldquoGradistat a grain size distribution andstatistics package for the analysis of unconsolidated sedimentsrdquoEarth Surface Processes and Landforms vol 26 no 11 pp 1237ndash1248 2001
[25] W Krumbein and F Pettijohn Manual of Sedimentary Petrog-raphy Appleton-Century-Crofts New York NY USA 1938
10 Journal of Marine Biology
[26] N C Prevost-Boure R Christen S Dequiedt et al ldquoValidationand application of a PCR primer set to quantify fungal commu-nities in the soil environment by real-time quantitative PCRrdquoPLoS ONE vol 6 no 9 Article ID e24166 2011
[27] N Fierer J A Jackson R Vilgalys and R B Jackson ldquoAssess-ment of soil microbial community structure by use of taxon-specific quantitative PCR assaysrdquo Applied and EnvironmentalMicrobiology vol 71 no 7 pp 4117ndash4120 2005
[28] A Guidot J-C Debaud and R Marmeisse ldquoSpatial distribu-tion of the below-ground mycelia of an ectomycorrhizal fungusinferred from specific quantification of its DNA in soil samplesrdquoFEMS Microbiology Ecology vol 42 no 3 pp 477ndash486 2002
[29] T B de Gregoris N Aldred A S Clare and J G BurgessldquoImprovement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxardquo Journal of Microbio-logical Methods vol 86 no 3 pp 351ndash356 2011
[30] E J Vainio and J Hantula ldquoDirect analysis of wood-inhabitingfungi using denaturing gradient gel electrophoresis of amplifiedribosomal DNArdquoMycological Research vol 104 no 8 pp 927ndash936 2000
[31] S Turner K M Pryer V P W Miao and J D Palmer ldquoInves-tigating deep phylogenetic relationships among cyanobacteriaand plastids by small subunit rRNA sequence analysisrdquo TheJournal of Eukaryotic Microbiology vol 46 no 4 pp 327ndash3381999
[32] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash174 John Wiley amp Sons Chichester UK1991
[33] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications pp 315ndash322 1990
[34] N Uemura KMakimuraM Onozaki et al ldquoDevelopment of aloop-mediated isothermal amplificationmethod for diagnosingPneumocystis pneumoniardquo Journal of Medical Microbiology vol57 no 1 pp 50ndash57 2008
[35] C J Smith D B Nedwell L F Dong and A M OsbornldquoEvaluation of quantitative polymerase chain reaction-basedapproaches for determining gene copy and gene transcriptnumbers in environmental samplesrdquo Environmental Microbiol-ogy vol 8 no 5 pp 804ndash815 2006
[36] C J Smith and A M Osborn ldquoAdvantages and limitationsof quantitative PCR (Q-PCR)-based approaches in microbialecologyrdquo FEMS Microbiology Ecology vol 67 no 1 pp 6ndash202009
[37] R A Ackerman ldquoOxygen consumption by sea turtle (CheloniaCaretta) eggs during developmentrdquo Physiological Zoology vol54 pp 316ndash324 1981
[38] R A Valverde S Wingard F Gomez M T Tordoir and C MOrrego ldquoField lethal incubation temperature of olive ridley seaturtle Lepidochelys olivacea embryos at a mass nesting rookeryrdquoEndangered Species Research vol 12 no 1 pp 77ndash86 2010
[39] L G Fonseca W N Villachica R E Matarrita and R AValverde ldquoReporte final de la anidacioon de tortuga lora(Lepidochelys olivacea) Playa Nancite Parque Nacional SantaRosa Costa Rica (Temporada 2010ndash2011)rdquo Reporte Final deTemporada al USFWS 2011
[40] C Figgener A Castillo-MacCarthy J Mora-Sandoval YArguelo-Gomez and W Quiros-Pereira ldquoRaising the hatchingsuccess of leatherback nests at the olive ridley mass-nesting
beach inOstional Guanacaste Costa Ricardquo in Proceedings of the34thAnnual Symposiumon SeaTurtle Biology andConservation2014
[41] C Limpus V Baker and J Miller ldquoMovement induced mortal-ity of loggerhead eggsrdquoHerpetologica vol 35 pp 335ndash338 1979
[42] R A Ackerman ldquoThe respiratory gas exchange of sea turtlenests (Chelonia Caretta)rdquo Respiration Physiology vol 31 no 1pp 19ndash38 1977
[43] Y-C Kam ldquoPhysiological effects of hypoxia onmetabolism andgrowth of turtle embryosrdquo Respiration Physiology vol 92 no 2pp 127ndash138 1993
[44] TWibbels D Rostal andR Byles ldquoHigh pivotal temperature inthe sex determination of the olive ridley sea turtle Lepidochelysolivacea from Playa Nancite Costa Ricardquo Copeia no 4 pp1086ndash1088 1998
[45] C J McCoy R C Vogt and E J Censky ldquoTemperature-controlled sex determination in the sea turtle Lepidochelysolivaceardquo Journal of Herpetology vol 17 no 4 pp 404ndash4061983
[46] N A Miller ldquoPO2in loggerhead sea turtle (Caretta caretta)
[47] J A Mortimer ldquoThe influence of beach sand characteristicson the nesting behavior and clutch survival of green turtles(Chelonia mydas)rdquo Copeia vol 1990 no 3 pp 802ndash817 1990
[48] S E Cornelius The Sea Turtles of Santa Rosa National ParkFundacion de Parques Nacionales San Jose Costa Rica 1986
[49] X Chen E Peltier B S M Sturm and C B Young ldquoNitrogenremoval and nitrifying and denitrifying bacteria quantificationin a stormwater bioretention systemrdquo Water Research vol 47no 4 pp 1691ndash1700 2013
[50] R S Seymour and R A Ackerman ldquoAdaptations to under-ground nesting in birds and reptilesrdquo American Zoologist vol20 no 2 pp 437ndash447 1980
[51] R S Seymour D Vleck and C M Vleck ldquoGas exchange in theincubationmounds of megapode birdsrdquo Journal of ComparativePhysiology B vol 156 no 6 pp 773ndash782 1986
following PCR conditions 4min at 95∘C followed by 30cycles of 95∘C for 1min 2min at the annealing temperatureof 55∘C and 72∘C for 15min with a final elongation step of10min at 72∘C [33 34] Each 25 120583L PCR reaction containedthe following 5 120583L of buffer (5x colorless GoTaq Flexi BufferPromega) 3 120583L of MgCl
2(25mM Promega) 025 120583L bovine
serum albumin (10 120583g 120583Lminus1 Promega) 05120583L dNTP (10mMThermo Scientific) 05120583L of each primer (5120583M IntegratedDNA Technologies) 01 120583L of DNA polymerase (5U120583Lminus1GoTaq Flexi DNA polymerase Promega) 1315 120583L H
2O and
2 120583L template DNA Bacterial and fungal PCR products werethen run on a 05 agarose gel purified with a QIAquickGel Extraction Kit (Qiagen) and eluted in 30 120583L at the finalstep Standards were quantified using the Qubit dsDNA BRAssay Kit (Invitrogen Life Technologies) and a Qubit 20Fluorometer (Qubit Systems) to determine the quantity ofDNA in the final sample The amplicon length was thenused to calculate the number of copies per 120583L under theassumption that the molecular weight of each bp is 650A standard curve was generated using 10-fold dilutions ofthese standards across 8 orders of magnitude thereforestandardizing qPCR results by copy number
Each plate included triplicate reactions per DNA sampleand for the standard curve as well as a no-template control tocheck for contamination Amelt curve (1min at 95∘C 1min at55∘C +05∘C 10 sminus1 to 95∘C) was used at the end of each qPCRrun to ensure the fluorescence signal resulted from specificityto the PCR product rather than from primer dimers orother nonspecific products The fluorescein dye included inthe master mix allowed for standard normalization of erroracross samples Threshold cycles (Ct) were automaticallycalculated by the software based on the average backgroundnoise Samples with less than one order of magnitude ofseparation (33 Ct value) from the no-template control wereexcluded given that this was outside of the detection limit ofthe present analysis [35 36] These samples were thereforereanalyzed using the same standard curve with a less dilutetemplate in order to ensure accurate quantification Samplesthat fell below the detection limit regardless of dilution wereexcluded from the analysis
210 Statistical Analysis Statistical analyses were conductedusing JMP 10 (SAS Institute httpwwwjmpcom) Hatch-ing success temperature pO
2 bacterial and fungal abun-
dance organic matter content and mean grain size data wereall analyzed using ANOVA When assumptions of normalityand homogeneity of variance were not met nonparametrictests were used (WilcoxonKruskal-Wallis) Statistically sig-nificant results were further testedwith Tukeyrsquos HSDpost hoctest to determine significant differences between beach areasRelationships between variables (hatching success pO
2 tem-
perature grain size organic matter content and bacterialand fungal abundance) were evaluated using Spearmanrsquos rankcorrelation coefficients
Because the metabolic demand of sea turtle embryos islow within the first half of incubation given their limiteddevelopment differences in oxygen content and temperaturein the egg clutch within this time period can presumablybe attributed to microbial activity [8 37] Mean nest pO
2
and temperature were therefore analyzed for the first andsecond half of incubation separately Repeatedmeasurementsof temperature and oxygen were also analyzed with repeatedmeasures MANOVA To allow for sample comparison acrossqPCR plates an ANCOVA was used to ensure there was nosignificant difference between standard curves from differentruns We also used a positive control on each plate tocalculate a coefficient of variation (CV) in order to ensurereproducibility within and between all plates All valuesare expressed as means plusmn standard error (SE) Percentagedata (hatching success) and microbial abundance data (copynumber) were arcsine- and log-transformed respectivelybefore analysis with parametric statistics Mean grain sizewas converted from phi units (120601) to mm for presentationThe number of 16S18S copies ulminus1 of template was convertedto a number of 16S18S copies gminus1 of nest sand to allowfor comparison across samples All analyses were tested forstatistical significance at 120572 lt 005
3 Results
The sample arribada occurring in August 2012 (estimated at72152 egg-laying females) and another arribada (September2012 estimated at 211954 egg-laying females) occurringbefore nest excavations were carried out in the middleof October An earthquake measuring 76 on the Richterscale occurred on September 5 2012 the epicenter locatedapproximately 50 km south of Ostional which caused visiblestructural changes to the beach topography Nest density forthe entire beach at the time of excavation was 36 plusmn 04 nestsmminus2 and overall hatching success for nests laid on the mainnesting beach during the August arribada was 34 plusmn 54 (RValverde unpublished data) At the time of excavation thenest densities in the high-nest-density tidal-wash and low-nest-density areas were 52 plusmn 04 20 plusmn 06 and 30 plusmn 05 nestsmminus2 respectively Two nests were excluded from the analysisdue to unforeseeable factors that likely caused a change inthe variables that we attempted to control in this study (ietemperature and pO
2of the nest) Specifically we excluded a
nest in the high-density nesting area that was partially shadedby a palm tree and another in the tidal-wash nesting area thatremained permanently inundated by the nearby estuary afterthe earthquake Hatching success in the high-nest-densityarea (06) was significantly lower than in the low-nest-density (682) and tidal-wash (547) areas of the beach(119875 lt 0001 Figure 1)
The pO2within egg clutches in the high-nest-density area
was significantly lower than that in other areas of the beachin both the first (154 plusmn 09 kPa 119875 lt 0001) and second halfof incubation (115 plusmn 05 kPa 119875 lt 0001 Figure 2) The meanminimumpO
2among nests in the high-nest-density area was
97plusmn 03 kPa with one clutch exhibiting pO2as low as 87 kPa
Nest pO2varied significantly with beach area and incubation
day with a significant interaction occurring between thesetwo factors (119865
was positively correlatedwith pO2in both the first and second
half of the incubation period (119903 = 0872 119875 lt 00001 and119903 = 0591 119875 = 0020 resp Table 1)
Journal of Marine Biology 5
100
80
60
40
20
0
Hat
chin
g su
cces
s (
)
Low-density Tidal-wash High-densityBeach area
A
B
Figure 1 Mean (plusmn SE) hatching success of nests located in differentnesting areas of the beach (119899 = 5) Different letters denote statisticaldifference Hatching success in the high-nest-density area (06)was significantly lower than in the low-nest-density (682) andtidal-wash (547) areas of the beach (119875 lt 0001)
20
18
16
14
12
10
pO2
(kPa
)
Low-densityTidal-wash
High-density
0 10 20 30 40 50
Incubation day
Figure 2 Mean (plusmn SE) partial pressure of oxygen (pO2 kPa) in
nests throughout the incubation period for nests located in thehigh-density tidal-wash and low-density nesting areas of the beach(119899 = 5) The pO
2within nests in the high-nest-density area was
significantly lower than that in other areas of the beach in both thefirst and the second half of incubation (119875 lt 0001 and 119875 lt 00001resp)
Nest temperatures were significantly lower in the tidal-wash area in the first and secondhalf of incubation (119875 = 0001and 119875 = 0001 resp Figure 3) Temperatures in the high-nest-density area of the beach were the highest in the secondhalf of incubation (357plusmn02∘C) and remained above the lethallimit (35∘C [38]) for a significantly longer duration of timein comparison to other areas of the beach (170 plusmn 07 d 119875 =0009) Nest temperature varied significantly with beach areaand incubation day with a significant interaction occurringbetween these two factors (119865
Table 1 Relationships between hatching success and partial pres-sure of oxygen (pO2) and temperature in the first (1st) and second(2nd) half of incubation bacterial and fungal abundance andorganic matter content for all study nests Copy number data fromseveral nests fell outside the range of detection for the presentanalysis and were therefore left out (see Section 3 for more details)Hatching success was positively correlated with nest pO2 in both thefirst and second half of incubation and negatively correlatedwith thebacterial abundance in nest sand
Variable Half of incubationperiod
Hatching success119903 119899 119875
pO21st 0872 13 lt00012nd 0591 13 0020
Temperature 1st minus0025 13 09292nd minus0207 13 0459
Figure 3 Mean nest temperature throughout the incubation periodin nests located in the high-density tidal-wash and low-densitynesting areas of the beach (119899 = 5) The field lethal incubationtemperature is indicated with a horizontal grey dashed line (35∘CValverde et al 2010) Nest temperatures were significantly lower inthe tidal-wash area in the first and the second half of incubation(119875 = 0001 and 119875 = 0001 resp)
However hatching success was not significantly correlatedwith nest temperatures in the first or second half of incuba-tion (Table 1)
The organic matter content of nest sand was significantlyhigher in the high-nest-density area (40) than in the low-nest-density (32) and tidal-wash (30) areas of the beach(119875 = 0015 and 119875 = 0003 resp) The sand from nestsin the low-nest-density area had a significantly larger meangrain size (0366mm) than in both the high-nest-density(0282mm) and tidal-wash (0283mm) areas of the beach
6 Journal of Marine Biology
(119875 = 0020 for both comparisons) Additionally the sandfrom all nests in the high-nest-density tidal-wash and low-nest-density areas of the beach wasmoderately sorted (120590
12060110
101 and 098 resp) and finely skewed (Sk120601035 036 and
103 resp)There was no significant difference in the slope of the
standard curve across assays for bacteria (119864 = 10223plusmn1411198772= 099 slope = minus327plusmn003 intercept = 3683plusmn056 CV =
341) or fungi (119864 = 8455 plusmn 225 1198772 = 100 plusmn 000 slope =minus376plusmn007 intercept = 4852plusmn080 CV = 168) indicatingthat copy numbers were comparable across assays (119875 = 0856and 119875 = 0088 resp) Microscopy counts (range 572 times 106ndash743 times 107 cells gminus1 of nest sand) were closely correlated withqPCR results for 16S rRNA gene copy number gminus1 of sand forall nests (119903 = 0404 119875 = 0013) Both the 16S and 18S copynumbers for one nest in the low-nest-density area as well asthe 18S copy number for two nests in the tidal-wash area werebelow the detection limit of the present analysis and weretherefore omitted
There was a significantly higher abundance of bacteriain nest sand from the high-nest-density area in comparisonto other areas of the beach (119875 = 0029) There was also ahigher abundance of fungi in nest sand from the high-nest-density area of the beach though this difference wasmarginal(119875 = 0063) While there was a negative correlation betweenhatching success and bacterial abundance for all nests thecorrelation between hatching success and fungal abundancewas marginal (119903 = minus0585 119875 = 0046 and 119903 = minus0607119875 = 0063 resp Table 1 and Figure 4) There was a negativecorrelation between the number of bacteria and egg clutchpO2in both the first and second half of incubation (119903 =
minus0741 119875 = 0006 and 119903 = minus0797 119875 = 0002 resp) aswell as a positive correlation between the number of bacteriaand nest temperatures in the second half of incubation (119903 =0818 119875 = 0001 Table 2) Furthermore there was a positivecorrelation between bacterial abundance and organic mattercontent in nest sand across all areas of the beach (119903 = 0636119875 = 0026)
4 Discussion
In this study we monitored nest conditions throughout theincubation period and quantified the microbial abundanceof nest sand in association with sea turtle hatching successWe found a negative relationship between the microbialabundance of nest sand and hatching success and observedsignificantly low pO
2in egg clutches located in the high-
nest-density area of the beach Egg clutches located in thetidal-wash area of the beach had higher hatching successand pO
2as well as lower organic matter content than those
located in the high-nest-density area of the beach Theseresults are consistent with previous reports that seawatermay have a positive effect on hatching success [39 40]presumably by decreasing microbial abundance incubationtemperature andor organic matter content Our results alsosupport our hypothesis that the low pO
2associated with
high microbial abundance in nest sand is responsible for thelower hatching rates observed at Ostional beach Collectively
105
106
107
108
109
1010
1011
BacteriaFungi
100
80
60
40
20
0
Hat
chin
g su
cces
s (
)
Log copy number gminus1 nest sand
Figure 4 Relationship between hatching success and microbialabundance (bacteria and fungi) for nests located in each nesting areaof the beach (119899 = 12 and 119899 = 10 resp) Regression lines for bacteria(solid) and fungi (dashed) are included for visualization purposesWhile therewas a negative correlation between hatching success andbacterial abundance for all nests the correlation between hatchingsuccess and fungal abundance was marginal (119875 = 0046 and119875 = 0063 resp) For Spearmanrsquos rank correlation coefficients seeTable 1
these data suggest a nonpathogenic mechanism for sea turtleembryonic mortality resulting from high rates of microbialdecomposition altering the nest environment beyond theoptimal range for embryonic development
Because all nests in this study incubated during the sametime period we are fairly confident that a majority of factorsaffecting embryonic development and hatching success werereasonably controlled However there may be factors that wedid notmonitor that caused incongruences in nest conditionsand could have influenced hatching success in this studyAdditionally an earthquake of large magnitude such as theone that occurred during this study could have feasiblycaused movement-induced mortality of sea turtle embryosHowever the earthquake occurred on day 25 of incubationafter the critical period (12 h to 14 d) during whichmovementsignificantly reduced hatching success [41] and thereforelikely had no adverse effects on the egg clutches in this study
41 Hatching Success Hatching success was highest in nestslocated in the low-nest-density area High-nest-densitieshave previously been observed to have direct physiologicaleffects (ie lower pO
2andhigher pCO
2within the egg clutch)
that negatively impacted hatching success [4] Nest densitiesof up to 5 nests mminus2 are unlikely to have a significant effect onhatching success (ge56) while densities of 9 nests mminus2 maysignificantly reduce hatching success to 30 [4]However thenests in this studywere subject to relatively low-nest-densities(2ndash5 nests mminus2) which likely were not high enough to causethe significant drop in hatching success observed
Journal of Marine Biology 7
Table 2 Relationships between bacterial and fungal abundance and the partial pressure of oxygen (pO2) and temperature in the first (1st)and second (2nd) half of incubation for all study nests There was a negative correlation between bacterial abundance and nest pO2 in boththe first and second half of incubation and a positive correlation with nest temperatures in the second half of incubation There was also anegative correlation between fungal abundance and nest pO2 in the first half of incubation
42 Nest Oxygen Content pO2for the low-nest-density and
tidal-wash areas of the beach was similar to that previouslyobserved in other studies on olive ridley sea turtles [4 8]Those studies also observed a change in the pO
2of nests
over the incubation period with pO2decreasing later in
the incubation period as a result of embryo respiration Thesignificant interaction effect of beach area and incubation dayon egg clutch pO
2indicates that the pO
2within egg clutches
changed differently across the areas of the beachOn the other hand the pO
2observed in egg clutches
located in the high-nest-density area in this study is lowerthan that previously observed in other studies [8 22 42]Specifically the drop in pO
2that we observed early in
the incubation period is uncharacteristic of sea turtle eggclutches given that the metabolic activity of the embryos istypically negligible at this time [42] Clutch pO
2typically
remains constant through the first half of incubation andthen decreases and tends to be negatively correlated with thenumber ofmetabolizing embryos per clutch [8 22] Howeverthis was not the case for clutches located in the high-nest-density area of the beach where pO
2began to drop from the
start of incubation and was lower in both the first and secondhalf of the incubation period in comparison to other areas ofthe beach
The negative correlation between microbial abundance(of both bacteria and fungi) and clutch pO
2at Ostional
suggests that microbial decomposition was responsible forthese decreases in pO
2 In particular our observations of high
abundance of bacteria and organic matter in nest sand fromthe high-nest-density area of the beach in association withlow pO
2further support this hypothesis Additionally if this
decrease in pO2resulted from the decomposition of embryos
killed by pathogens in these nests one would expect a lagtime (ie time for infection) and an abrupt change in oxy-gen and temperature following embryo death Furthermorewhile nest density alone affects the oxygen content of neststhe minimum pO
2observed in the high-nest-density area
(97 kPa) in this study was lower than previously observedat similar and even higher nest densities (approximately145ndash170 kPa) [4 8] Given that these previous studies inldquocleanrdquo sand (with presumably low organic matter content)did not find such discrepancies in the pO
2of egg clutches
at similar densities it is likely that microbial decompositionis responsible for the differences observed here Howeverfurther studies that include a controlled measure of pO
2in
beach sand outside of egg clutches and within egg clutches
at controlled densities are necessary to properly test thishypothesis
Because sea turtle embryos have a relatively low toleranceto hypoxia early in the incubation period [43] it is likelythat the drop in pO
2that we observed in the high-nest-
density area of the beach led to total nest failure While thecritical oxygen tension of olive ridley sea turtle eggs hasnot been studied this value was as high as 165 kPa on day22 of incubation for loggerhead sea turtle (Caretta caretta)eggs which are comparable in size [43] Assuming that thecritical oxygen tension of olive ridley sea turtle eggs is similarand given that egg clutch pO
2fell below 165 kPa on day 15
of incubation in the high-nest-density area it is likely thatany eggs located here were exposed to pO
2below a lethal
threshold
43 Nest Temperature The nest temperatures observed inthis study are similar to those observed in other studieson olive ridley sea turtles in this region [4 8 38] Ingeneral mean incubation temperatures were above the meanpivotal temperature of 305∘C for olive ridleys [44 45]indicating that the hatchling sex ratio could be female-biasedat Ostional as previously suggested [38] Previous studieshave also observed a change in nest temperatures throughoutthe incubation period with temperatures increasing laterin incubation in conjunction with increasing embryonicmetabolism [4 8] Nest temperatures were fairly consistentacross the different areas of the beach throughout the incu-bation period with the exception of nests located in thetidal-wash area where lower nest temperatures likely resultedfrom higher water content in the sandThe incubation periodwas 2 to 3 days longer in this group of nests and is likely aresult of slower embryonic development due to lower nesttemperatures [46]
While most nest temperatures fell within the wide rangeof tolerance for sea turtle embryological development (25ndash35∘C [39]) early in the incubation period the slightly highertemperatures observed in the high-nest-density area of thebeach later in incubation may have exceeded this optimalrange long enough to affect hatching success Though thefield lethal limit for olive ridley sea turtles at this beach isapproximately 35∘C embryos may be capable of recoveringfrom short-term exposure to high temperatures while longerdurations are lethal [38] Results from this study supportthis notion in that nests with lower hatching success (high-nest-density area) were also exposed to temperatures above
8 Journal of Marine Biology
the lethal limit for a longer duration of time The differentcombination of heat released from embryonic metabolismandor microbial decomposition is likely responsible for theobserved differences in temperature later in incubation
44 Characteristics of Nest Sand The mean grain size andsorting coefficient of sand from nests in this study werecomparable to those previously observed at a variety ofsea turtle nesting beaches [47] Given the relatively smallrange of mean grain size observed across study nests (0282ndash0366mm) granulometry likely did not drive the differencesin hatching success across the areas of the beach
The high amounts of organic matter in the sand fromthis study in comparison to other sea turtle nesting beaches[47] are not surprising as this is likely a result of the highrates of nest destruction due to the large number of nestingfemales and high-nest-density at this beach [9 19 48] Theassociation of high organic matter content with a higherabundance of bacteria in the sand from the high-nest-densityarea in this study supports the hypothesis that high organicmatter is associated with elevated microbial proliferation anddecomposition at Ostional and may also explain the low eggclutch pO
2in comparison to previous studies [7 8]
45 Microbial Abundance The number of bacterial 16S andfungal 18S rRNA gene copies in this study ranged from 105to 1010 copies gminus1 of sea turtle nest sand A previous studyfound that the sand at an arribada beach contained six ordersof magnitude more bacterial colonies gminus1 in comparison toa neighboring beach although no specific abundance datawere provided [10 14] Previous studies that have quantified16S or 18S rRNA gene copies in similar sediments have foundbetween 106 and 109 copies gminus1 [26 49]
The negative relationship between bacterial abundanceand hatching success that we observed supports the hypoth-esis that the microbial abundance in nest sand at Ostionalis high enough to adversely affect embryonic developmentSpecifically we suggest that the mechanism behind thisrelationship is not pathogenic but rather a result of high ratesof microbial decomposition of organic matter altering thenest environment beyond the optimal range for embryonicdevelopment Studies on underground nesting species ofbirds and reptiles suggest similar influences of microbialdecomposition on nest pO
2and temperature [50 51] It is
possible that the relationship between microbial activity andhatching success is nonlinear with a threshold beyond whichhigh rates of microbial respiration disrupt the diffusion ofoxygen to the nest to the point of impeding embryonic devel-opment For example the relationship between microbialabundance and hatching success appears to reach a thresholdat sim1 times 109ndash1010 16S18S copies gminus1 nest sand Furtherstudies that span a broader range in microbial abundanceare required to confirm the true nature of the relationshipbetween the microbial abundance of nest sand and sea turtlehatching success The coinciding occurrence of lower pO
2
higher temperatures higher organicmatter and low hatchingsuccess in the high-nest-density area of the beach serves asan extreme example of the adverse conditions imposed bymicrobial decomposition at Ostional
In addition to the indirect effect of microbial abundanceon the nest environment there are likely other effects ofmicrobial presence on sea turtle hatching success that wedid not consider because they were not within the scope ofthe present study For example we did not explore speciescomposition or diversity nor did we evaluate the presence orabsence of pathogens The potential presence of pathogenicmicrobes in the sand at Ostional cannot be ruled out Severalspecies of fungi in the Fusarium solani species complex havebeen identified as pathogens to sea turtle eggs and are knownto occur at nesting beaches around the world [15 16] It isalso possible that the effects of microbial abundance andthe nest environment on hatching success are synergisticwith the effects of lethal pathogens [15] For example onceembryos are killed due to the adverse effects of microbialoxygen depletion on the nest environment this may resultin the proliferation of fungi which could then spread toadjacent eggs Further studies should therefore focus onsampling from several components of the nest environment(ie cloacal fluid nest sand eggshell unhatched embryosand live hatchlings) as well as assessing the abundancediversity and activity of both fungi and bacteria to providea more complete understanding of the relationship betweenmicrobial activity and the reproductive success of sea turtles
Based on our comparative study it appears that tidalwashing decreases the organic matter and microbial abun-dance in the sand thus increasing hatching success Alter-natively the high humidity in the sand resulting fromtidal washing may also keep incubation temperatures fromreaching lethal temperaturesTherefore seawater inundationmay be critical in maintaining the population of sea turtlesat Ostional Using these natural mechanisms as a model forconservation management practices could be adopted topromote improved embryo survival and hatchling produc-tion For example seawater could be used to decrease organicmatter and microbial abundance in hatchery sand or byperiodically irrigating the beachwith seawaterThe treatmentof sand or relocation of hatcheries on a regular basis is a con-ventional management practice in conservation programs toavoid organic matter build-up as well as microbial and larvalinfestations In fact the leatherback sea turtle conservationprogram at Ostional successfully increased hatching successin the leatherback hatchery by using sand from below thehigh tide line [40]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank G Bran Y Arguello-GomezM Gomez S Steele and all the employees and volunteers atRNVS Ostional for their help in the field The authors wouldalso like to thank S Berthrong A Oporta McCarthy and RLeisen for their contributions to themolecular and laboratoryanalyses This material is based upon work supported by the
Journal of Marine Biology 9
National Science Foundation Graduate Research Fellowshipunder Grant no 511182 Additional funds to support the laband field research for this study were provided by the PADIFoundation National Geographic Young Explorers Grant(C220-12) USFWS Marine Turtle Conservation Act (96200-0-G037) and internal grants from the College of CharlestonThis research was approved by the College of CharlestonIACUC and permits were granted by MINAET (Resolucionno ACT-OR-DR-078) and CONAGEBio (R-020-2012-OT-CONAGEBIO) of Costa Rica
References
[1] A Abreu-Grobois and P T Plotkin ldquoIUCNSMTSG (2008) Lep-idochelys olivaceardquo The IUCN Red List of Threatened SpeciesVersion 20142 httpwwwiucnredlistorg
[2] R A Valverde C M Orrego M T Tordoir et al ldquoOlive ridleymass nesting ecology and egg harvest at ostional beach CostaRicardquoChelonian Conservation and Biology vol 11 no 1 pp 1ndash112012
[3] S E Cornelius R Arauz J Fretey et al ldquoEffect of land basedharvest of Lepidochelysrdquo in The Biology and Conservation ofRidley Sea Turtles P T Plotkin Ed pp 231ndash251 Johns HopkinsUniversity Press Baltimore Md USA 2007
[4] S Honarvar M P OrsquoConnor and J R Spotila ldquoDensity-dependent effects on hatching success of the olive ridley turtleLepidochelys olivaceardquo Oecologia vol 157 no 2 pp 221ndash2302008
[5] V S Beezy and R A Valverde ldquoA comparison ofmethodologiesfor estimating the nest density of olive ridley arribadas atOstional Costa Ricardquo in Proceedings of the 31st Annual Sympo-sium on Sea Turtle Biology andConservation T T Jones andB PWallace Eds NOAA Technical Memorandum NMFS-SEFSC-631 pp 194ndash195 2012
[6] S E Cornelius M A Ulloa J C Castro M Mata del Valleand D C Robinson ldquoManagement of olive ridley sea turtles(Lepidochelys olivacea) nesting at Playas Nancite and OstionalCosta Ricardquo in Neotropical Wildlife Use and Conservation JRobinson and K Redford Eds vol 1 pp 111ndash135 Universityof Chicago Press Chicago Ill USA 1991
[7] R A Valverde S E Cornelius and C L Mo ldquoDecline of theolive ridley sea turtle (Lepidochelys olivacea) nesting assemblageat Nancite Beach Santa Rosa National Park Costa RicardquoChelonian Conservation and Biology vol 3 pp 58ndash63 1998
[8] S Clusella Trullas and F V Paladino ldquoMicro-environment ofolive ridley turtle nests deposited during an aggregated nestingeventrdquo Journal of Zoology vol 272 no 4 pp 367ndash376 2007
[9] D McPherson and D Kibler ldquoEcological impact of olive ridleynesting at Ostional Costa Ricardquo in Proceedings of the 25thAnnual Symposium on Sea Turtle Biology and ConservationH Kalb A Rohde K Gayheart and K Shanker Eds NOAATechnical Memorandum NMFS-SEFSC-582 p 204 2008
[10] C L Mo I Salas and M Caballero ldquoAre fungi and bacteriaresponsible for olive ridleyrsquos egg lossrdquo in Proceedings of the 10thAnnual Workshop on Sea Turtle Biology and Conservation T HRichardson J I Richardson and M Donnelly Eds pp 249ndash252 NOAA Techn 1990
[11] S Honarvar J R Spotila andM P OrsquoConnor ldquoMicrobial com-munity structure in sand on two olive ridley arribada nestingbeaches Playa La Flor Nicaragua and Playa Nancite Costa
Ricardquo Journal of Experimental Marine Biology and Ecology vol409 no 1-2 pp 339ndash344 2011
[12] A D Phillott and C J Parmenter ldquoThe distribution of failedeggs and the appearance of fungi in artificial nests of green(Chelonia mydas) and loggerhead (Caretta caretta) sea turtlesrdquoAustralian Journal of Zoology vol 49 no 6 pp 713ndash718 2001
[13] J Wyneken T J Burke M Salmon and D K Pedersen ldquoEggfailure in natural and relocated sea turtle nestsrdquo Journal ofHerpetology vol 22 no 1 pp 88ndash96 1988
[14] C L Mo M Caballero and I Salas ldquoMicroorganism infectionof olive ridley eggsrdquo in Proceedings of the 12th Annual Workshopon Sea Turtle Biology and Conservation J I Richardson and TH Richardson Eds NOAA Technical Memorandum NMFS-SEFSC-361274 pp 81ndash84 1995
[15] J M Sarmiento-Ramırez E Abella M P Martın et alldquoFusarium solaniis responsible for mass mortalities in nests ofloggerhead sea turtle Caretta caretta in Boavista Cape VerderdquoFEMS Microbiology Letters vol 312 no 2 pp 192ndash200 2010
[16] J M Sarmiento-Ramırez E Abella-Perez A D Phillott etal ldquoGlobal distribution of two fungal pathogens threateningendangered sea turtlesrdquo PLoS ONE vol 9 Article ID e858532014
[17] J Patino-Martinez A Marco L Quinones E Abella R MAbad and J Dieguez-Uribeondo ldquoHow do hatcheries influenceembryonic development of sea turtle eggs Experimental anal-ysis and isolation of microorganisms in leatherback turtle eggsrdquoJournal of Experimental Zoology Part A Ecological Genetics andPhysiology vol 317 no 1 pp 47ndash54 2012
[18] A D Phillott and C J Parmenter ldquoFungal colonization of greensea turtle (Chelonia mydas) nests is unlikely to affect hatchlingconditionrdquo Herpetological Conservation amp Biology vol 9 pp297ndash301 2014
[19] MOcanaMHarfush-Melendez and SHeppell ldquoMass nestingof olive ridley sea turtles Lepidochelys olivacea at La EscobillaMexico linking nest density and rates of destructionrdquo Endan-gered Species Research vol 16 pp 45ndash54 2012
[20] DMadden J Ballestero C Calvo R Carlson E Christians andE Madden ldquoSea turtle nesting as a process influencing a sandybeach ecosystemrdquo Biotropica vol 40 no 6 pp 758ndash765 2008
[21] R Valverde and C Gates ldquoPopulation surveys on mass nestingbeachesrdquo in Research and Management Techniques for theConservation of Sea Turtles K L Eckert K A Bjorndal FA Abreu-Gobrois and M Donnelly Eds IUCNSSC MarineTurtle Specialist Group Publication No 4 1999
[22] B P Wallace P R Sotherland J R Spotila R D ReinaB F Franks and F V Paladino ldquoBiotic and abiotic factorsaffect the nest environment of embryonic leatherback turtlesDermochelys coriaceardquo Physiological and Biochemical Zoologyvol 77 no 3 pp 423ndash432 2004
[23] J D Miller ldquoDetermining clutch size and hatching successrdquo inResearch and Management Techniques for the Conservation ofSea Turtles K L Eckert K A Bjorndal F A Abreu-Groboisand M Donnelly Eds IUCNSSC Marine Turtle SpecialistGroup Publication No 4 1999
[24] S J Blott and K Pye ldquoGradistat a grain size distribution andstatistics package for the analysis of unconsolidated sedimentsrdquoEarth Surface Processes and Landforms vol 26 no 11 pp 1237ndash1248 2001
[25] W Krumbein and F Pettijohn Manual of Sedimentary Petrog-raphy Appleton-Century-Crofts New York NY USA 1938
10 Journal of Marine Biology
[26] N C Prevost-Boure R Christen S Dequiedt et al ldquoValidationand application of a PCR primer set to quantify fungal commu-nities in the soil environment by real-time quantitative PCRrdquoPLoS ONE vol 6 no 9 Article ID e24166 2011
[27] N Fierer J A Jackson R Vilgalys and R B Jackson ldquoAssess-ment of soil microbial community structure by use of taxon-specific quantitative PCR assaysrdquo Applied and EnvironmentalMicrobiology vol 71 no 7 pp 4117ndash4120 2005
[28] A Guidot J-C Debaud and R Marmeisse ldquoSpatial distribu-tion of the below-ground mycelia of an ectomycorrhizal fungusinferred from specific quantification of its DNA in soil samplesrdquoFEMS Microbiology Ecology vol 42 no 3 pp 477ndash486 2002
[29] T B de Gregoris N Aldred A S Clare and J G BurgessldquoImprovement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxardquo Journal of Microbio-logical Methods vol 86 no 3 pp 351ndash356 2011
[30] E J Vainio and J Hantula ldquoDirect analysis of wood-inhabitingfungi using denaturing gradient gel electrophoresis of amplifiedribosomal DNArdquoMycological Research vol 104 no 8 pp 927ndash936 2000
[31] S Turner K M Pryer V P W Miao and J D Palmer ldquoInves-tigating deep phylogenetic relationships among cyanobacteriaand plastids by small subunit rRNA sequence analysisrdquo TheJournal of Eukaryotic Microbiology vol 46 no 4 pp 327ndash3381999
[32] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash174 John Wiley amp Sons Chichester UK1991
[33] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications pp 315ndash322 1990
[34] N Uemura KMakimuraM Onozaki et al ldquoDevelopment of aloop-mediated isothermal amplificationmethod for diagnosingPneumocystis pneumoniardquo Journal of Medical Microbiology vol57 no 1 pp 50ndash57 2008
[35] C J Smith D B Nedwell L F Dong and A M OsbornldquoEvaluation of quantitative polymerase chain reaction-basedapproaches for determining gene copy and gene transcriptnumbers in environmental samplesrdquo Environmental Microbiol-ogy vol 8 no 5 pp 804ndash815 2006
[36] C J Smith and A M Osborn ldquoAdvantages and limitationsof quantitative PCR (Q-PCR)-based approaches in microbialecologyrdquo FEMS Microbiology Ecology vol 67 no 1 pp 6ndash202009
[37] R A Ackerman ldquoOxygen consumption by sea turtle (CheloniaCaretta) eggs during developmentrdquo Physiological Zoology vol54 pp 316ndash324 1981
[38] R A Valverde S Wingard F Gomez M T Tordoir and C MOrrego ldquoField lethal incubation temperature of olive ridley seaturtle Lepidochelys olivacea embryos at a mass nesting rookeryrdquoEndangered Species Research vol 12 no 1 pp 77ndash86 2010
[39] L G Fonseca W N Villachica R E Matarrita and R AValverde ldquoReporte final de la anidacioon de tortuga lora(Lepidochelys olivacea) Playa Nancite Parque Nacional SantaRosa Costa Rica (Temporada 2010ndash2011)rdquo Reporte Final deTemporada al USFWS 2011
[40] C Figgener A Castillo-MacCarthy J Mora-Sandoval YArguelo-Gomez and W Quiros-Pereira ldquoRaising the hatchingsuccess of leatherback nests at the olive ridley mass-nesting
beach inOstional Guanacaste Costa Ricardquo in Proceedings of the34thAnnual Symposiumon SeaTurtle Biology andConservation2014
[41] C Limpus V Baker and J Miller ldquoMovement induced mortal-ity of loggerhead eggsrdquoHerpetologica vol 35 pp 335ndash338 1979
[42] R A Ackerman ldquoThe respiratory gas exchange of sea turtlenests (Chelonia Caretta)rdquo Respiration Physiology vol 31 no 1pp 19ndash38 1977
[43] Y-C Kam ldquoPhysiological effects of hypoxia onmetabolism andgrowth of turtle embryosrdquo Respiration Physiology vol 92 no 2pp 127ndash138 1993
[44] TWibbels D Rostal andR Byles ldquoHigh pivotal temperature inthe sex determination of the olive ridley sea turtle Lepidochelysolivacea from Playa Nancite Costa Ricardquo Copeia no 4 pp1086ndash1088 1998
[45] C J McCoy R C Vogt and E J Censky ldquoTemperature-controlled sex determination in the sea turtle Lepidochelysolivaceardquo Journal of Herpetology vol 17 no 4 pp 404ndash4061983
[46] N A Miller ldquoPO2in loggerhead sea turtle (Caretta caretta)
[47] J A Mortimer ldquoThe influence of beach sand characteristicson the nesting behavior and clutch survival of green turtles(Chelonia mydas)rdquo Copeia vol 1990 no 3 pp 802ndash817 1990
[48] S E Cornelius The Sea Turtles of Santa Rosa National ParkFundacion de Parques Nacionales San Jose Costa Rica 1986
[49] X Chen E Peltier B S M Sturm and C B Young ldquoNitrogenremoval and nitrifying and denitrifying bacteria quantificationin a stormwater bioretention systemrdquo Water Research vol 47no 4 pp 1691ndash1700 2013
[50] R S Seymour and R A Ackerman ldquoAdaptations to under-ground nesting in birds and reptilesrdquo American Zoologist vol20 no 2 pp 437ndash447 1980
[51] R S Seymour D Vleck and C M Vleck ldquoGas exchange in theincubationmounds of megapode birdsrdquo Journal of ComparativePhysiology B vol 156 no 6 pp 773ndash782 1986
Figure 1 Mean (plusmn SE) hatching success of nests located in differentnesting areas of the beach (119899 = 5) Different letters denote statisticaldifference Hatching success in the high-nest-density area (06)was significantly lower than in the low-nest-density (682) andtidal-wash (547) areas of the beach (119875 lt 0001)
20
18
16
14
12
10
pO2
(kPa
)
Low-densityTidal-wash
High-density
0 10 20 30 40 50
Incubation day
Figure 2 Mean (plusmn SE) partial pressure of oxygen (pO2 kPa) in
nests throughout the incubation period for nests located in thehigh-density tidal-wash and low-density nesting areas of the beach(119899 = 5) The pO
2within nests in the high-nest-density area was
significantly lower than that in other areas of the beach in both thefirst and the second half of incubation (119875 lt 0001 and 119875 lt 00001resp)
Nest temperatures were significantly lower in the tidal-wash area in the first and secondhalf of incubation (119875 = 0001and 119875 = 0001 resp Figure 3) Temperatures in the high-nest-density area of the beach were the highest in the secondhalf of incubation (357plusmn02∘C) and remained above the lethallimit (35∘C [38]) for a significantly longer duration of timein comparison to other areas of the beach (170 plusmn 07 d 119875 =0009) Nest temperature varied significantly with beach areaand incubation day with a significant interaction occurringbetween these two factors (119865
Table 1 Relationships between hatching success and partial pres-sure of oxygen (pO2) and temperature in the first (1st) and second(2nd) half of incubation bacterial and fungal abundance andorganic matter content for all study nests Copy number data fromseveral nests fell outside the range of detection for the presentanalysis and were therefore left out (see Section 3 for more details)Hatching success was positively correlated with nest pO2 in both thefirst and second half of incubation and negatively correlatedwith thebacterial abundance in nest sand
Variable Half of incubationperiod
Hatching success119903 119899 119875
pO21st 0872 13 lt00012nd 0591 13 0020
Temperature 1st minus0025 13 09292nd minus0207 13 0459
Figure 3 Mean nest temperature throughout the incubation periodin nests located in the high-density tidal-wash and low-densitynesting areas of the beach (119899 = 5) The field lethal incubationtemperature is indicated with a horizontal grey dashed line (35∘CValverde et al 2010) Nest temperatures were significantly lower inthe tidal-wash area in the first and the second half of incubation(119875 = 0001 and 119875 = 0001 resp)
However hatching success was not significantly correlatedwith nest temperatures in the first or second half of incuba-tion (Table 1)
The organic matter content of nest sand was significantlyhigher in the high-nest-density area (40) than in the low-nest-density (32) and tidal-wash (30) areas of the beach(119875 = 0015 and 119875 = 0003 resp) The sand from nestsin the low-nest-density area had a significantly larger meangrain size (0366mm) than in both the high-nest-density(0282mm) and tidal-wash (0283mm) areas of the beach
6 Journal of Marine Biology
(119875 = 0020 for both comparisons) Additionally the sandfrom all nests in the high-nest-density tidal-wash and low-nest-density areas of the beach wasmoderately sorted (120590
12060110
101 and 098 resp) and finely skewed (Sk120601035 036 and
103 resp)There was no significant difference in the slope of the
standard curve across assays for bacteria (119864 = 10223plusmn1411198772= 099 slope = minus327plusmn003 intercept = 3683plusmn056 CV =
341) or fungi (119864 = 8455 plusmn 225 1198772 = 100 plusmn 000 slope =minus376plusmn007 intercept = 4852plusmn080 CV = 168) indicatingthat copy numbers were comparable across assays (119875 = 0856and 119875 = 0088 resp) Microscopy counts (range 572 times 106ndash743 times 107 cells gminus1 of nest sand) were closely correlated withqPCR results for 16S rRNA gene copy number gminus1 of sand forall nests (119903 = 0404 119875 = 0013) Both the 16S and 18S copynumbers for one nest in the low-nest-density area as well asthe 18S copy number for two nests in the tidal-wash area werebelow the detection limit of the present analysis and weretherefore omitted
There was a significantly higher abundance of bacteriain nest sand from the high-nest-density area in comparisonto other areas of the beach (119875 = 0029) There was also ahigher abundance of fungi in nest sand from the high-nest-density area of the beach though this difference wasmarginal(119875 = 0063) While there was a negative correlation betweenhatching success and bacterial abundance for all nests thecorrelation between hatching success and fungal abundancewas marginal (119903 = minus0585 119875 = 0046 and 119903 = minus0607119875 = 0063 resp Table 1 and Figure 4) There was a negativecorrelation between the number of bacteria and egg clutchpO2in both the first and second half of incubation (119903 =
minus0741 119875 = 0006 and 119903 = minus0797 119875 = 0002 resp) aswell as a positive correlation between the number of bacteriaand nest temperatures in the second half of incubation (119903 =0818 119875 = 0001 Table 2) Furthermore there was a positivecorrelation between bacterial abundance and organic mattercontent in nest sand across all areas of the beach (119903 = 0636119875 = 0026)
4 Discussion
In this study we monitored nest conditions throughout theincubation period and quantified the microbial abundanceof nest sand in association with sea turtle hatching successWe found a negative relationship between the microbialabundance of nest sand and hatching success and observedsignificantly low pO
2in egg clutches located in the high-
nest-density area of the beach Egg clutches located in thetidal-wash area of the beach had higher hatching successand pO
2as well as lower organic matter content than those
located in the high-nest-density area of the beach Theseresults are consistent with previous reports that seawatermay have a positive effect on hatching success [39 40]presumably by decreasing microbial abundance incubationtemperature andor organic matter content Our results alsosupport our hypothesis that the low pO
2associated with
high microbial abundance in nest sand is responsible for thelower hatching rates observed at Ostional beach Collectively
105
106
107
108
109
1010
1011
BacteriaFungi
100
80
60
40
20
0
Hat
chin
g su
cces
s (
)
Log copy number gminus1 nest sand
Figure 4 Relationship between hatching success and microbialabundance (bacteria and fungi) for nests located in each nesting areaof the beach (119899 = 12 and 119899 = 10 resp) Regression lines for bacteria(solid) and fungi (dashed) are included for visualization purposesWhile therewas a negative correlation between hatching success andbacterial abundance for all nests the correlation between hatchingsuccess and fungal abundance was marginal (119875 = 0046 and119875 = 0063 resp) For Spearmanrsquos rank correlation coefficients seeTable 1
these data suggest a nonpathogenic mechanism for sea turtleembryonic mortality resulting from high rates of microbialdecomposition altering the nest environment beyond theoptimal range for embryonic development
Because all nests in this study incubated during the sametime period we are fairly confident that a majority of factorsaffecting embryonic development and hatching success werereasonably controlled However there may be factors that wedid notmonitor that caused incongruences in nest conditionsand could have influenced hatching success in this studyAdditionally an earthquake of large magnitude such as theone that occurred during this study could have feasiblycaused movement-induced mortality of sea turtle embryosHowever the earthquake occurred on day 25 of incubationafter the critical period (12 h to 14 d) during whichmovementsignificantly reduced hatching success [41] and thereforelikely had no adverse effects on the egg clutches in this study
41 Hatching Success Hatching success was highest in nestslocated in the low-nest-density area High-nest-densitieshave previously been observed to have direct physiologicaleffects (ie lower pO
2andhigher pCO
2within the egg clutch)
that negatively impacted hatching success [4] Nest densitiesof up to 5 nests mminus2 are unlikely to have a significant effect onhatching success (ge56) while densities of 9 nests mminus2 maysignificantly reduce hatching success to 30 [4]However thenests in this studywere subject to relatively low-nest-densities(2ndash5 nests mminus2) which likely were not high enough to causethe significant drop in hatching success observed
Journal of Marine Biology 7
Table 2 Relationships between bacterial and fungal abundance and the partial pressure of oxygen (pO2) and temperature in the first (1st)and second (2nd) half of incubation for all study nests There was a negative correlation between bacterial abundance and nest pO2 in boththe first and second half of incubation and a positive correlation with nest temperatures in the second half of incubation There was also anegative correlation between fungal abundance and nest pO2 in the first half of incubation
42 Nest Oxygen Content pO2for the low-nest-density and
tidal-wash areas of the beach was similar to that previouslyobserved in other studies on olive ridley sea turtles [4 8]Those studies also observed a change in the pO
2of nests
over the incubation period with pO2decreasing later in
the incubation period as a result of embryo respiration Thesignificant interaction effect of beach area and incubation dayon egg clutch pO
2indicates that the pO
2within egg clutches
changed differently across the areas of the beachOn the other hand the pO
2observed in egg clutches
located in the high-nest-density area in this study is lowerthan that previously observed in other studies [8 22 42]Specifically the drop in pO
2that we observed early in
the incubation period is uncharacteristic of sea turtle eggclutches given that the metabolic activity of the embryos istypically negligible at this time [42] Clutch pO
2typically
remains constant through the first half of incubation andthen decreases and tends to be negatively correlated with thenumber ofmetabolizing embryos per clutch [8 22] Howeverthis was not the case for clutches located in the high-nest-density area of the beach where pO
2began to drop from the
start of incubation and was lower in both the first and secondhalf of the incubation period in comparison to other areas ofthe beach
The negative correlation between microbial abundance(of both bacteria and fungi) and clutch pO
2at Ostional
suggests that microbial decomposition was responsible forthese decreases in pO
2 In particular our observations of high
abundance of bacteria and organic matter in nest sand fromthe high-nest-density area of the beach in association withlow pO
2further support this hypothesis Additionally if this
decrease in pO2resulted from the decomposition of embryos
killed by pathogens in these nests one would expect a lagtime (ie time for infection) and an abrupt change in oxy-gen and temperature following embryo death Furthermorewhile nest density alone affects the oxygen content of neststhe minimum pO
2observed in the high-nest-density area
(97 kPa) in this study was lower than previously observedat similar and even higher nest densities (approximately145ndash170 kPa) [4 8] Given that these previous studies inldquocleanrdquo sand (with presumably low organic matter content)did not find such discrepancies in the pO
2of egg clutches
at similar densities it is likely that microbial decompositionis responsible for the differences observed here Howeverfurther studies that include a controlled measure of pO
2in
beach sand outside of egg clutches and within egg clutches
at controlled densities are necessary to properly test thishypothesis
Because sea turtle embryos have a relatively low toleranceto hypoxia early in the incubation period [43] it is likelythat the drop in pO
2that we observed in the high-nest-
density area of the beach led to total nest failure While thecritical oxygen tension of olive ridley sea turtle eggs hasnot been studied this value was as high as 165 kPa on day22 of incubation for loggerhead sea turtle (Caretta caretta)eggs which are comparable in size [43] Assuming that thecritical oxygen tension of olive ridley sea turtle eggs is similarand given that egg clutch pO
2fell below 165 kPa on day 15
of incubation in the high-nest-density area it is likely thatany eggs located here were exposed to pO
2below a lethal
threshold
43 Nest Temperature The nest temperatures observed inthis study are similar to those observed in other studieson olive ridley sea turtles in this region [4 8 38] Ingeneral mean incubation temperatures were above the meanpivotal temperature of 305∘C for olive ridleys [44 45]indicating that the hatchling sex ratio could be female-biasedat Ostional as previously suggested [38] Previous studieshave also observed a change in nest temperatures throughoutthe incubation period with temperatures increasing laterin incubation in conjunction with increasing embryonicmetabolism [4 8] Nest temperatures were fairly consistentacross the different areas of the beach throughout the incu-bation period with the exception of nests located in thetidal-wash area where lower nest temperatures likely resultedfrom higher water content in the sandThe incubation periodwas 2 to 3 days longer in this group of nests and is likely aresult of slower embryonic development due to lower nesttemperatures [46]
While most nest temperatures fell within the wide rangeof tolerance for sea turtle embryological development (25ndash35∘C [39]) early in the incubation period the slightly highertemperatures observed in the high-nest-density area of thebeach later in incubation may have exceeded this optimalrange long enough to affect hatching success Though thefield lethal limit for olive ridley sea turtles at this beach isapproximately 35∘C embryos may be capable of recoveringfrom short-term exposure to high temperatures while longerdurations are lethal [38] Results from this study supportthis notion in that nests with lower hatching success (high-nest-density area) were also exposed to temperatures above
8 Journal of Marine Biology
the lethal limit for a longer duration of time The differentcombination of heat released from embryonic metabolismandor microbial decomposition is likely responsible for theobserved differences in temperature later in incubation
44 Characteristics of Nest Sand The mean grain size andsorting coefficient of sand from nests in this study werecomparable to those previously observed at a variety ofsea turtle nesting beaches [47] Given the relatively smallrange of mean grain size observed across study nests (0282ndash0366mm) granulometry likely did not drive the differencesin hatching success across the areas of the beach
The high amounts of organic matter in the sand fromthis study in comparison to other sea turtle nesting beaches[47] are not surprising as this is likely a result of the highrates of nest destruction due to the large number of nestingfemales and high-nest-density at this beach [9 19 48] Theassociation of high organic matter content with a higherabundance of bacteria in the sand from the high-nest-densityarea in this study supports the hypothesis that high organicmatter is associated with elevated microbial proliferation anddecomposition at Ostional and may also explain the low eggclutch pO
2in comparison to previous studies [7 8]
45 Microbial Abundance The number of bacterial 16S andfungal 18S rRNA gene copies in this study ranged from 105to 1010 copies gminus1 of sea turtle nest sand A previous studyfound that the sand at an arribada beach contained six ordersof magnitude more bacterial colonies gminus1 in comparison toa neighboring beach although no specific abundance datawere provided [10 14] Previous studies that have quantified16S or 18S rRNA gene copies in similar sediments have foundbetween 106 and 109 copies gminus1 [26 49]
The negative relationship between bacterial abundanceand hatching success that we observed supports the hypoth-esis that the microbial abundance in nest sand at Ostionalis high enough to adversely affect embryonic developmentSpecifically we suggest that the mechanism behind thisrelationship is not pathogenic but rather a result of high ratesof microbial decomposition of organic matter altering thenest environment beyond the optimal range for embryonicdevelopment Studies on underground nesting species ofbirds and reptiles suggest similar influences of microbialdecomposition on nest pO
2and temperature [50 51] It is
possible that the relationship between microbial activity andhatching success is nonlinear with a threshold beyond whichhigh rates of microbial respiration disrupt the diffusion ofoxygen to the nest to the point of impeding embryonic devel-opment For example the relationship between microbialabundance and hatching success appears to reach a thresholdat sim1 times 109ndash1010 16S18S copies gminus1 nest sand Furtherstudies that span a broader range in microbial abundanceare required to confirm the true nature of the relationshipbetween the microbial abundance of nest sand and sea turtlehatching success The coinciding occurrence of lower pO
2
higher temperatures higher organicmatter and low hatchingsuccess in the high-nest-density area of the beach serves asan extreme example of the adverse conditions imposed bymicrobial decomposition at Ostional
In addition to the indirect effect of microbial abundanceon the nest environment there are likely other effects ofmicrobial presence on sea turtle hatching success that wedid not consider because they were not within the scope ofthe present study For example we did not explore speciescomposition or diversity nor did we evaluate the presence orabsence of pathogens The potential presence of pathogenicmicrobes in the sand at Ostional cannot be ruled out Severalspecies of fungi in the Fusarium solani species complex havebeen identified as pathogens to sea turtle eggs and are knownto occur at nesting beaches around the world [15 16] It isalso possible that the effects of microbial abundance andthe nest environment on hatching success are synergisticwith the effects of lethal pathogens [15] For example onceembryos are killed due to the adverse effects of microbialoxygen depletion on the nest environment this may resultin the proliferation of fungi which could then spread toadjacent eggs Further studies should therefore focus onsampling from several components of the nest environment(ie cloacal fluid nest sand eggshell unhatched embryosand live hatchlings) as well as assessing the abundancediversity and activity of both fungi and bacteria to providea more complete understanding of the relationship betweenmicrobial activity and the reproductive success of sea turtles
Based on our comparative study it appears that tidalwashing decreases the organic matter and microbial abun-dance in the sand thus increasing hatching success Alter-natively the high humidity in the sand resulting fromtidal washing may also keep incubation temperatures fromreaching lethal temperaturesTherefore seawater inundationmay be critical in maintaining the population of sea turtlesat Ostional Using these natural mechanisms as a model forconservation management practices could be adopted topromote improved embryo survival and hatchling produc-tion For example seawater could be used to decrease organicmatter and microbial abundance in hatchery sand or byperiodically irrigating the beachwith seawaterThe treatmentof sand or relocation of hatcheries on a regular basis is a con-ventional management practice in conservation programs toavoid organic matter build-up as well as microbial and larvalinfestations In fact the leatherback sea turtle conservationprogram at Ostional successfully increased hatching successin the leatherback hatchery by using sand from below thehigh tide line [40]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank G Bran Y Arguello-GomezM Gomez S Steele and all the employees and volunteers atRNVS Ostional for their help in the field The authors wouldalso like to thank S Berthrong A Oporta McCarthy and RLeisen for their contributions to themolecular and laboratoryanalyses This material is based upon work supported by the
Journal of Marine Biology 9
National Science Foundation Graduate Research Fellowshipunder Grant no 511182 Additional funds to support the laband field research for this study were provided by the PADIFoundation National Geographic Young Explorers Grant(C220-12) USFWS Marine Turtle Conservation Act (96200-0-G037) and internal grants from the College of CharlestonThis research was approved by the College of CharlestonIACUC and permits were granted by MINAET (Resolucionno ACT-OR-DR-078) and CONAGEBio (R-020-2012-OT-CONAGEBIO) of Costa Rica
References
[1] A Abreu-Grobois and P T Plotkin ldquoIUCNSMTSG (2008) Lep-idochelys olivaceardquo The IUCN Red List of Threatened SpeciesVersion 20142 httpwwwiucnredlistorg
[2] R A Valverde C M Orrego M T Tordoir et al ldquoOlive ridleymass nesting ecology and egg harvest at ostional beach CostaRicardquoChelonian Conservation and Biology vol 11 no 1 pp 1ndash112012
[3] S E Cornelius R Arauz J Fretey et al ldquoEffect of land basedharvest of Lepidochelysrdquo in The Biology and Conservation ofRidley Sea Turtles P T Plotkin Ed pp 231ndash251 Johns HopkinsUniversity Press Baltimore Md USA 2007
[4] S Honarvar M P OrsquoConnor and J R Spotila ldquoDensity-dependent effects on hatching success of the olive ridley turtleLepidochelys olivaceardquo Oecologia vol 157 no 2 pp 221ndash2302008
[5] V S Beezy and R A Valverde ldquoA comparison ofmethodologiesfor estimating the nest density of olive ridley arribadas atOstional Costa Ricardquo in Proceedings of the 31st Annual Sympo-sium on Sea Turtle Biology andConservation T T Jones andB PWallace Eds NOAA Technical Memorandum NMFS-SEFSC-631 pp 194ndash195 2012
[6] S E Cornelius M A Ulloa J C Castro M Mata del Valleand D C Robinson ldquoManagement of olive ridley sea turtles(Lepidochelys olivacea) nesting at Playas Nancite and OstionalCosta Ricardquo in Neotropical Wildlife Use and Conservation JRobinson and K Redford Eds vol 1 pp 111ndash135 Universityof Chicago Press Chicago Ill USA 1991
[7] R A Valverde S E Cornelius and C L Mo ldquoDecline of theolive ridley sea turtle (Lepidochelys olivacea) nesting assemblageat Nancite Beach Santa Rosa National Park Costa RicardquoChelonian Conservation and Biology vol 3 pp 58ndash63 1998
[8] S Clusella Trullas and F V Paladino ldquoMicro-environment ofolive ridley turtle nests deposited during an aggregated nestingeventrdquo Journal of Zoology vol 272 no 4 pp 367ndash376 2007
[9] D McPherson and D Kibler ldquoEcological impact of olive ridleynesting at Ostional Costa Ricardquo in Proceedings of the 25thAnnual Symposium on Sea Turtle Biology and ConservationH Kalb A Rohde K Gayheart and K Shanker Eds NOAATechnical Memorandum NMFS-SEFSC-582 p 204 2008
[10] C L Mo I Salas and M Caballero ldquoAre fungi and bacteriaresponsible for olive ridleyrsquos egg lossrdquo in Proceedings of the 10thAnnual Workshop on Sea Turtle Biology and Conservation T HRichardson J I Richardson and M Donnelly Eds pp 249ndash252 NOAA Techn 1990
[11] S Honarvar J R Spotila andM P OrsquoConnor ldquoMicrobial com-munity structure in sand on two olive ridley arribada nestingbeaches Playa La Flor Nicaragua and Playa Nancite Costa
Ricardquo Journal of Experimental Marine Biology and Ecology vol409 no 1-2 pp 339ndash344 2011
[12] A D Phillott and C J Parmenter ldquoThe distribution of failedeggs and the appearance of fungi in artificial nests of green(Chelonia mydas) and loggerhead (Caretta caretta) sea turtlesrdquoAustralian Journal of Zoology vol 49 no 6 pp 713ndash718 2001
[13] J Wyneken T J Burke M Salmon and D K Pedersen ldquoEggfailure in natural and relocated sea turtle nestsrdquo Journal ofHerpetology vol 22 no 1 pp 88ndash96 1988
[14] C L Mo M Caballero and I Salas ldquoMicroorganism infectionof olive ridley eggsrdquo in Proceedings of the 12th Annual Workshopon Sea Turtle Biology and Conservation J I Richardson and TH Richardson Eds NOAA Technical Memorandum NMFS-SEFSC-361274 pp 81ndash84 1995
[15] J M Sarmiento-Ramırez E Abella M P Martın et alldquoFusarium solaniis responsible for mass mortalities in nests ofloggerhead sea turtle Caretta caretta in Boavista Cape VerderdquoFEMS Microbiology Letters vol 312 no 2 pp 192ndash200 2010
[16] J M Sarmiento-Ramırez E Abella-Perez A D Phillott etal ldquoGlobal distribution of two fungal pathogens threateningendangered sea turtlesrdquo PLoS ONE vol 9 Article ID e858532014
[17] J Patino-Martinez A Marco L Quinones E Abella R MAbad and J Dieguez-Uribeondo ldquoHow do hatcheries influenceembryonic development of sea turtle eggs Experimental anal-ysis and isolation of microorganisms in leatherback turtle eggsrdquoJournal of Experimental Zoology Part A Ecological Genetics andPhysiology vol 317 no 1 pp 47ndash54 2012
[18] A D Phillott and C J Parmenter ldquoFungal colonization of greensea turtle (Chelonia mydas) nests is unlikely to affect hatchlingconditionrdquo Herpetological Conservation amp Biology vol 9 pp297ndash301 2014
[19] MOcanaMHarfush-Melendez and SHeppell ldquoMass nestingof olive ridley sea turtles Lepidochelys olivacea at La EscobillaMexico linking nest density and rates of destructionrdquo Endan-gered Species Research vol 16 pp 45ndash54 2012
[20] DMadden J Ballestero C Calvo R Carlson E Christians andE Madden ldquoSea turtle nesting as a process influencing a sandybeach ecosystemrdquo Biotropica vol 40 no 6 pp 758ndash765 2008
[21] R Valverde and C Gates ldquoPopulation surveys on mass nestingbeachesrdquo in Research and Management Techniques for theConservation of Sea Turtles K L Eckert K A Bjorndal FA Abreu-Gobrois and M Donnelly Eds IUCNSSC MarineTurtle Specialist Group Publication No 4 1999
[22] B P Wallace P R Sotherland J R Spotila R D ReinaB F Franks and F V Paladino ldquoBiotic and abiotic factorsaffect the nest environment of embryonic leatherback turtlesDermochelys coriaceardquo Physiological and Biochemical Zoologyvol 77 no 3 pp 423ndash432 2004
[23] J D Miller ldquoDetermining clutch size and hatching successrdquo inResearch and Management Techniques for the Conservation ofSea Turtles K L Eckert K A Bjorndal F A Abreu-Groboisand M Donnelly Eds IUCNSSC Marine Turtle SpecialistGroup Publication No 4 1999
[24] S J Blott and K Pye ldquoGradistat a grain size distribution andstatistics package for the analysis of unconsolidated sedimentsrdquoEarth Surface Processes and Landforms vol 26 no 11 pp 1237ndash1248 2001
[25] W Krumbein and F Pettijohn Manual of Sedimentary Petrog-raphy Appleton-Century-Crofts New York NY USA 1938
10 Journal of Marine Biology
[26] N C Prevost-Boure R Christen S Dequiedt et al ldquoValidationand application of a PCR primer set to quantify fungal commu-nities in the soil environment by real-time quantitative PCRrdquoPLoS ONE vol 6 no 9 Article ID e24166 2011
[27] N Fierer J A Jackson R Vilgalys and R B Jackson ldquoAssess-ment of soil microbial community structure by use of taxon-specific quantitative PCR assaysrdquo Applied and EnvironmentalMicrobiology vol 71 no 7 pp 4117ndash4120 2005
[28] A Guidot J-C Debaud and R Marmeisse ldquoSpatial distribu-tion of the below-ground mycelia of an ectomycorrhizal fungusinferred from specific quantification of its DNA in soil samplesrdquoFEMS Microbiology Ecology vol 42 no 3 pp 477ndash486 2002
[29] T B de Gregoris N Aldred A S Clare and J G BurgessldquoImprovement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxardquo Journal of Microbio-logical Methods vol 86 no 3 pp 351ndash356 2011
[30] E J Vainio and J Hantula ldquoDirect analysis of wood-inhabitingfungi using denaturing gradient gel electrophoresis of amplifiedribosomal DNArdquoMycological Research vol 104 no 8 pp 927ndash936 2000
[31] S Turner K M Pryer V P W Miao and J D Palmer ldquoInves-tigating deep phylogenetic relationships among cyanobacteriaand plastids by small subunit rRNA sequence analysisrdquo TheJournal of Eukaryotic Microbiology vol 46 no 4 pp 327ndash3381999
[32] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash174 John Wiley amp Sons Chichester UK1991
[33] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications pp 315ndash322 1990
[34] N Uemura KMakimuraM Onozaki et al ldquoDevelopment of aloop-mediated isothermal amplificationmethod for diagnosingPneumocystis pneumoniardquo Journal of Medical Microbiology vol57 no 1 pp 50ndash57 2008
[35] C J Smith D B Nedwell L F Dong and A M OsbornldquoEvaluation of quantitative polymerase chain reaction-basedapproaches for determining gene copy and gene transcriptnumbers in environmental samplesrdquo Environmental Microbiol-ogy vol 8 no 5 pp 804ndash815 2006
[36] C J Smith and A M Osborn ldquoAdvantages and limitationsof quantitative PCR (Q-PCR)-based approaches in microbialecologyrdquo FEMS Microbiology Ecology vol 67 no 1 pp 6ndash202009
[37] R A Ackerman ldquoOxygen consumption by sea turtle (CheloniaCaretta) eggs during developmentrdquo Physiological Zoology vol54 pp 316ndash324 1981
[38] R A Valverde S Wingard F Gomez M T Tordoir and C MOrrego ldquoField lethal incubation temperature of olive ridley seaturtle Lepidochelys olivacea embryos at a mass nesting rookeryrdquoEndangered Species Research vol 12 no 1 pp 77ndash86 2010
[39] L G Fonseca W N Villachica R E Matarrita and R AValverde ldquoReporte final de la anidacioon de tortuga lora(Lepidochelys olivacea) Playa Nancite Parque Nacional SantaRosa Costa Rica (Temporada 2010ndash2011)rdquo Reporte Final deTemporada al USFWS 2011
[40] C Figgener A Castillo-MacCarthy J Mora-Sandoval YArguelo-Gomez and W Quiros-Pereira ldquoRaising the hatchingsuccess of leatherback nests at the olive ridley mass-nesting
beach inOstional Guanacaste Costa Ricardquo in Proceedings of the34thAnnual Symposiumon SeaTurtle Biology andConservation2014
[41] C Limpus V Baker and J Miller ldquoMovement induced mortal-ity of loggerhead eggsrdquoHerpetologica vol 35 pp 335ndash338 1979
[42] R A Ackerman ldquoThe respiratory gas exchange of sea turtlenests (Chelonia Caretta)rdquo Respiration Physiology vol 31 no 1pp 19ndash38 1977
[43] Y-C Kam ldquoPhysiological effects of hypoxia onmetabolism andgrowth of turtle embryosrdquo Respiration Physiology vol 92 no 2pp 127ndash138 1993
[44] TWibbels D Rostal andR Byles ldquoHigh pivotal temperature inthe sex determination of the olive ridley sea turtle Lepidochelysolivacea from Playa Nancite Costa Ricardquo Copeia no 4 pp1086ndash1088 1998
[45] C J McCoy R C Vogt and E J Censky ldquoTemperature-controlled sex determination in the sea turtle Lepidochelysolivaceardquo Journal of Herpetology vol 17 no 4 pp 404ndash4061983
[46] N A Miller ldquoPO2in loggerhead sea turtle (Caretta caretta)
[47] J A Mortimer ldquoThe influence of beach sand characteristicson the nesting behavior and clutch survival of green turtles(Chelonia mydas)rdquo Copeia vol 1990 no 3 pp 802ndash817 1990
[48] S E Cornelius The Sea Turtles of Santa Rosa National ParkFundacion de Parques Nacionales San Jose Costa Rica 1986
[49] X Chen E Peltier B S M Sturm and C B Young ldquoNitrogenremoval and nitrifying and denitrifying bacteria quantificationin a stormwater bioretention systemrdquo Water Research vol 47no 4 pp 1691ndash1700 2013
[50] R S Seymour and R A Ackerman ldquoAdaptations to under-ground nesting in birds and reptilesrdquo American Zoologist vol20 no 2 pp 437ndash447 1980
[51] R S Seymour D Vleck and C M Vleck ldquoGas exchange in theincubationmounds of megapode birdsrdquo Journal of ComparativePhysiology B vol 156 no 6 pp 773ndash782 1986
(119875 = 0020 for both comparisons) Additionally the sandfrom all nests in the high-nest-density tidal-wash and low-nest-density areas of the beach wasmoderately sorted (120590
12060110
101 and 098 resp) and finely skewed (Sk120601035 036 and
103 resp)There was no significant difference in the slope of the
standard curve across assays for bacteria (119864 = 10223plusmn1411198772= 099 slope = minus327plusmn003 intercept = 3683plusmn056 CV =
341) or fungi (119864 = 8455 plusmn 225 1198772 = 100 plusmn 000 slope =minus376plusmn007 intercept = 4852plusmn080 CV = 168) indicatingthat copy numbers were comparable across assays (119875 = 0856and 119875 = 0088 resp) Microscopy counts (range 572 times 106ndash743 times 107 cells gminus1 of nest sand) were closely correlated withqPCR results for 16S rRNA gene copy number gminus1 of sand forall nests (119903 = 0404 119875 = 0013) Both the 16S and 18S copynumbers for one nest in the low-nest-density area as well asthe 18S copy number for two nests in the tidal-wash area werebelow the detection limit of the present analysis and weretherefore omitted
There was a significantly higher abundance of bacteriain nest sand from the high-nest-density area in comparisonto other areas of the beach (119875 = 0029) There was also ahigher abundance of fungi in nest sand from the high-nest-density area of the beach though this difference wasmarginal(119875 = 0063) While there was a negative correlation betweenhatching success and bacterial abundance for all nests thecorrelation between hatching success and fungal abundancewas marginal (119903 = minus0585 119875 = 0046 and 119903 = minus0607119875 = 0063 resp Table 1 and Figure 4) There was a negativecorrelation between the number of bacteria and egg clutchpO2in both the first and second half of incubation (119903 =
minus0741 119875 = 0006 and 119903 = minus0797 119875 = 0002 resp) aswell as a positive correlation between the number of bacteriaand nest temperatures in the second half of incubation (119903 =0818 119875 = 0001 Table 2) Furthermore there was a positivecorrelation between bacterial abundance and organic mattercontent in nest sand across all areas of the beach (119903 = 0636119875 = 0026)
4 Discussion
In this study we monitored nest conditions throughout theincubation period and quantified the microbial abundanceof nest sand in association with sea turtle hatching successWe found a negative relationship between the microbialabundance of nest sand and hatching success and observedsignificantly low pO
2in egg clutches located in the high-
nest-density area of the beach Egg clutches located in thetidal-wash area of the beach had higher hatching successand pO
2as well as lower organic matter content than those
located in the high-nest-density area of the beach Theseresults are consistent with previous reports that seawatermay have a positive effect on hatching success [39 40]presumably by decreasing microbial abundance incubationtemperature andor organic matter content Our results alsosupport our hypothesis that the low pO
2associated with
high microbial abundance in nest sand is responsible for thelower hatching rates observed at Ostional beach Collectively
105
106
107
108
109
1010
1011
BacteriaFungi
100
80
60
40
20
0
Hat
chin
g su
cces
s (
)
Log copy number gminus1 nest sand
Figure 4 Relationship between hatching success and microbialabundance (bacteria and fungi) for nests located in each nesting areaof the beach (119899 = 12 and 119899 = 10 resp) Regression lines for bacteria(solid) and fungi (dashed) are included for visualization purposesWhile therewas a negative correlation between hatching success andbacterial abundance for all nests the correlation between hatchingsuccess and fungal abundance was marginal (119875 = 0046 and119875 = 0063 resp) For Spearmanrsquos rank correlation coefficients seeTable 1
these data suggest a nonpathogenic mechanism for sea turtleembryonic mortality resulting from high rates of microbialdecomposition altering the nest environment beyond theoptimal range for embryonic development
Because all nests in this study incubated during the sametime period we are fairly confident that a majority of factorsaffecting embryonic development and hatching success werereasonably controlled However there may be factors that wedid notmonitor that caused incongruences in nest conditionsand could have influenced hatching success in this studyAdditionally an earthquake of large magnitude such as theone that occurred during this study could have feasiblycaused movement-induced mortality of sea turtle embryosHowever the earthquake occurred on day 25 of incubationafter the critical period (12 h to 14 d) during whichmovementsignificantly reduced hatching success [41] and thereforelikely had no adverse effects on the egg clutches in this study
41 Hatching Success Hatching success was highest in nestslocated in the low-nest-density area High-nest-densitieshave previously been observed to have direct physiologicaleffects (ie lower pO
2andhigher pCO
2within the egg clutch)
that negatively impacted hatching success [4] Nest densitiesof up to 5 nests mminus2 are unlikely to have a significant effect onhatching success (ge56) while densities of 9 nests mminus2 maysignificantly reduce hatching success to 30 [4]However thenests in this studywere subject to relatively low-nest-densities(2ndash5 nests mminus2) which likely were not high enough to causethe significant drop in hatching success observed
Journal of Marine Biology 7
Table 2 Relationships between bacterial and fungal abundance and the partial pressure of oxygen (pO2) and temperature in the first (1st)and second (2nd) half of incubation for all study nests There was a negative correlation between bacterial abundance and nest pO2 in boththe first and second half of incubation and a positive correlation with nest temperatures in the second half of incubation There was also anegative correlation between fungal abundance and nest pO2 in the first half of incubation
42 Nest Oxygen Content pO2for the low-nest-density and
tidal-wash areas of the beach was similar to that previouslyobserved in other studies on olive ridley sea turtles [4 8]Those studies also observed a change in the pO
2of nests
over the incubation period with pO2decreasing later in
the incubation period as a result of embryo respiration Thesignificant interaction effect of beach area and incubation dayon egg clutch pO
2indicates that the pO
2within egg clutches
changed differently across the areas of the beachOn the other hand the pO
2observed in egg clutches
located in the high-nest-density area in this study is lowerthan that previously observed in other studies [8 22 42]Specifically the drop in pO
2that we observed early in
the incubation period is uncharacteristic of sea turtle eggclutches given that the metabolic activity of the embryos istypically negligible at this time [42] Clutch pO
2typically
remains constant through the first half of incubation andthen decreases and tends to be negatively correlated with thenumber ofmetabolizing embryos per clutch [8 22] Howeverthis was not the case for clutches located in the high-nest-density area of the beach where pO
2began to drop from the
start of incubation and was lower in both the first and secondhalf of the incubation period in comparison to other areas ofthe beach
The negative correlation between microbial abundance(of both bacteria and fungi) and clutch pO
2at Ostional
suggests that microbial decomposition was responsible forthese decreases in pO
2 In particular our observations of high
abundance of bacteria and organic matter in nest sand fromthe high-nest-density area of the beach in association withlow pO
2further support this hypothesis Additionally if this
decrease in pO2resulted from the decomposition of embryos
killed by pathogens in these nests one would expect a lagtime (ie time for infection) and an abrupt change in oxy-gen and temperature following embryo death Furthermorewhile nest density alone affects the oxygen content of neststhe minimum pO
2observed in the high-nest-density area
(97 kPa) in this study was lower than previously observedat similar and even higher nest densities (approximately145ndash170 kPa) [4 8] Given that these previous studies inldquocleanrdquo sand (with presumably low organic matter content)did not find such discrepancies in the pO
2of egg clutches
at similar densities it is likely that microbial decompositionis responsible for the differences observed here Howeverfurther studies that include a controlled measure of pO
2in
beach sand outside of egg clutches and within egg clutches
at controlled densities are necessary to properly test thishypothesis
Because sea turtle embryos have a relatively low toleranceto hypoxia early in the incubation period [43] it is likelythat the drop in pO
2that we observed in the high-nest-
density area of the beach led to total nest failure While thecritical oxygen tension of olive ridley sea turtle eggs hasnot been studied this value was as high as 165 kPa on day22 of incubation for loggerhead sea turtle (Caretta caretta)eggs which are comparable in size [43] Assuming that thecritical oxygen tension of olive ridley sea turtle eggs is similarand given that egg clutch pO
2fell below 165 kPa on day 15
of incubation in the high-nest-density area it is likely thatany eggs located here were exposed to pO
2below a lethal
threshold
43 Nest Temperature The nest temperatures observed inthis study are similar to those observed in other studieson olive ridley sea turtles in this region [4 8 38] Ingeneral mean incubation temperatures were above the meanpivotal temperature of 305∘C for olive ridleys [44 45]indicating that the hatchling sex ratio could be female-biasedat Ostional as previously suggested [38] Previous studieshave also observed a change in nest temperatures throughoutthe incubation period with temperatures increasing laterin incubation in conjunction with increasing embryonicmetabolism [4 8] Nest temperatures were fairly consistentacross the different areas of the beach throughout the incu-bation period with the exception of nests located in thetidal-wash area where lower nest temperatures likely resultedfrom higher water content in the sandThe incubation periodwas 2 to 3 days longer in this group of nests and is likely aresult of slower embryonic development due to lower nesttemperatures [46]
While most nest temperatures fell within the wide rangeof tolerance for sea turtle embryological development (25ndash35∘C [39]) early in the incubation period the slightly highertemperatures observed in the high-nest-density area of thebeach later in incubation may have exceeded this optimalrange long enough to affect hatching success Though thefield lethal limit for olive ridley sea turtles at this beach isapproximately 35∘C embryos may be capable of recoveringfrom short-term exposure to high temperatures while longerdurations are lethal [38] Results from this study supportthis notion in that nests with lower hatching success (high-nest-density area) were also exposed to temperatures above
8 Journal of Marine Biology
the lethal limit for a longer duration of time The differentcombination of heat released from embryonic metabolismandor microbial decomposition is likely responsible for theobserved differences in temperature later in incubation
44 Characteristics of Nest Sand The mean grain size andsorting coefficient of sand from nests in this study werecomparable to those previously observed at a variety ofsea turtle nesting beaches [47] Given the relatively smallrange of mean grain size observed across study nests (0282ndash0366mm) granulometry likely did not drive the differencesin hatching success across the areas of the beach
The high amounts of organic matter in the sand fromthis study in comparison to other sea turtle nesting beaches[47] are not surprising as this is likely a result of the highrates of nest destruction due to the large number of nestingfemales and high-nest-density at this beach [9 19 48] Theassociation of high organic matter content with a higherabundance of bacteria in the sand from the high-nest-densityarea in this study supports the hypothesis that high organicmatter is associated with elevated microbial proliferation anddecomposition at Ostional and may also explain the low eggclutch pO
2in comparison to previous studies [7 8]
45 Microbial Abundance The number of bacterial 16S andfungal 18S rRNA gene copies in this study ranged from 105to 1010 copies gminus1 of sea turtle nest sand A previous studyfound that the sand at an arribada beach contained six ordersof magnitude more bacterial colonies gminus1 in comparison toa neighboring beach although no specific abundance datawere provided [10 14] Previous studies that have quantified16S or 18S rRNA gene copies in similar sediments have foundbetween 106 and 109 copies gminus1 [26 49]
The negative relationship between bacterial abundanceand hatching success that we observed supports the hypoth-esis that the microbial abundance in nest sand at Ostionalis high enough to adversely affect embryonic developmentSpecifically we suggest that the mechanism behind thisrelationship is not pathogenic but rather a result of high ratesof microbial decomposition of organic matter altering thenest environment beyond the optimal range for embryonicdevelopment Studies on underground nesting species ofbirds and reptiles suggest similar influences of microbialdecomposition on nest pO
2and temperature [50 51] It is
possible that the relationship between microbial activity andhatching success is nonlinear with a threshold beyond whichhigh rates of microbial respiration disrupt the diffusion ofoxygen to the nest to the point of impeding embryonic devel-opment For example the relationship between microbialabundance and hatching success appears to reach a thresholdat sim1 times 109ndash1010 16S18S copies gminus1 nest sand Furtherstudies that span a broader range in microbial abundanceare required to confirm the true nature of the relationshipbetween the microbial abundance of nest sand and sea turtlehatching success The coinciding occurrence of lower pO
2
higher temperatures higher organicmatter and low hatchingsuccess in the high-nest-density area of the beach serves asan extreme example of the adverse conditions imposed bymicrobial decomposition at Ostional
In addition to the indirect effect of microbial abundanceon the nest environment there are likely other effects ofmicrobial presence on sea turtle hatching success that wedid not consider because they were not within the scope ofthe present study For example we did not explore speciescomposition or diversity nor did we evaluate the presence orabsence of pathogens The potential presence of pathogenicmicrobes in the sand at Ostional cannot be ruled out Severalspecies of fungi in the Fusarium solani species complex havebeen identified as pathogens to sea turtle eggs and are knownto occur at nesting beaches around the world [15 16] It isalso possible that the effects of microbial abundance andthe nest environment on hatching success are synergisticwith the effects of lethal pathogens [15] For example onceembryos are killed due to the adverse effects of microbialoxygen depletion on the nest environment this may resultin the proliferation of fungi which could then spread toadjacent eggs Further studies should therefore focus onsampling from several components of the nest environment(ie cloacal fluid nest sand eggshell unhatched embryosand live hatchlings) as well as assessing the abundancediversity and activity of both fungi and bacteria to providea more complete understanding of the relationship betweenmicrobial activity and the reproductive success of sea turtles
Based on our comparative study it appears that tidalwashing decreases the organic matter and microbial abun-dance in the sand thus increasing hatching success Alter-natively the high humidity in the sand resulting fromtidal washing may also keep incubation temperatures fromreaching lethal temperaturesTherefore seawater inundationmay be critical in maintaining the population of sea turtlesat Ostional Using these natural mechanisms as a model forconservation management practices could be adopted topromote improved embryo survival and hatchling produc-tion For example seawater could be used to decrease organicmatter and microbial abundance in hatchery sand or byperiodically irrigating the beachwith seawaterThe treatmentof sand or relocation of hatcheries on a regular basis is a con-ventional management practice in conservation programs toavoid organic matter build-up as well as microbial and larvalinfestations In fact the leatherback sea turtle conservationprogram at Ostional successfully increased hatching successin the leatherback hatchery by using sand from below thehigh tide line [40]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank G Bran Y Arguello-GomezM Gomez S Steele and all the employees and volunteers atRNVS Ostional for their help in the field The authors wouldalso like to thank S Berthrong A Oporta McCarthy and RLeisen for their contributions to themolecular and laboratoryanalyses This material is based upon work supported by the
Journal of Marine Biology 9
National Science Foundation Graduate Research Fellowshipunder Grant no 511182 Additional funds to support the laband field research for this study were provided by the PADIFoundation National Geographic Young Explorers Grant(C220-12) USFWS Marine Turtle Conservation Act (96200-0-G037) and internal grants from the College of CharlestonThis research was approved by the College of CharlestonIACUC and permits were granted by MINAET (Resolucionno ACT-OR-DR-078) and CONAGEBio (R-020-2012-OT-CONAGEBIO) of Costa Rica
References
[1] A Abreu-Grobois and P T Plotkin ldquoIUCNSMTSG (2008) Lep-idochelys olivaceardquo The IUCN Red List of Threatened SpeciesVersion 20142 httpwwwiucnredlistorg
[2] R A Valverde C M Orrego M T Tordoir et al ldquoOlive ridleymass nesting ecology and egg harvest at ostional beach CostaRicardquoChelonian Conservation and Biology vol 11 no 1 pp 1ndash112012
[3] S E Cornelius R Arauz J Fretey et al ldquoEffect of land basedharvest of Lepidochelysrdquo in The Biology and Conservation ofRidley Sea Turtles P T Plotkin Ed pp 231ndash251 Johns HopkinsUniversity Press Baltimore Md USA 2007
[4] S Honarvar M P OrsquoConnor and J R Spotila ldquoDensity-dependent effects on hatching success of the olive ridley turtleLepidochelys olivaceardquo Oecologia vol 157 no 2 pp 221ndash2302008
[5] V S Beezy and R A Valverde ldquoA comparison ofmethodologiesfor estimating the nest density of olive ridley arribadas atOstional Costa Ricardquo in Proceedings of the 31st Annual Sympo-sium on Sea Turtle Biology andConservation T T Jones andB PWallace Eds NOAA Technical Memorandum NMFS-SEFSC-631 pp 194ndash195 2012
[6] S E Cornelius M A Ulloa J C Castro M Mata del Valleand D C Robinson ldquoManagement of olive ridley sea turtles(Lepidochelys olivacea) nesting at Playas Nancite and OstionalCosta Ricardquo in Neotropical Wildlife Use and Conservation JRobinson and K Redford Eds vol 1 pp 111ndash135 Universityof Chicago Press Chicago Ill USA 1991
[7] R A Valverde S E Cornelius and C L Mo ldquoDecline of theolive ridley sea turtle (Lepidochelys olivacea) nesting assemblageat Nancite Beach Santa Rosa National Park Costa RicardquoChelonian Conservation and Biology vol 3 pp 58ndash63 1998
[8] S Clusella Trullas and F V Paladino ldquoMicro-environment ofolive ridley turtle nests deposited during an aggregated nestingeventrdquo Journal of Zoology vol 272 no 4 pp 367ndash376 2007
[9] D McPherson and D Kibler ldquoEcological impact of olive ridleynesting at Ostional Costa Ricardquo in Proceedings of the 25thAnnual Symposium on Sea Turtle Biology and ConservationH Kalb A Rohde K Gayheart and K Shanker Eds NOAATechnical Memorandum NMFS-SEFSC-582 p 204 2008
[10] C L Mo I Salas and M Caballero ldquoAre fungi and bacteriaresponsible for olive ridleyrsquos egg lossrdquo in Proceedings of the 10thAnnual Workshop on Sea Turtle Biology and Conservation T HRichardson J I Richardson and M Donnelly Eds pp 249ndash252 NOAA Techn 1990
[11] S Honarvar J R Spotila andM P OrsquoConnor ldquoMicrobial com-munity structure in sand on two olive ridley arribada nestingbeaches Playa La Flor Nicaragua and Playa Nancite Costa
Ricardquo Journal of Experimental Marine Biology and Ecology vol409 no 1-2 pp 339ndash344 2011
[12] A D Phillott and C J Parmenter ldquoThe distribution of failedeggs and the appearance of fungi in artificial nests of green(Chelonia mydas) and loggerhead (Caretta caretta) sea turtlesrdquoAustralian Journal of Zoology vol 49 no 6 pp 713ndash718 2001
[13] J Wyneken T J Burke M Salmon and D K Pedersen ldquoEggfailure in natural and relocated sea turtle nestsrdquo Journal ofHerpetology vol 22 no 1 pp 88ndash96 1988
[14] C L Mo M Caballero and I Salas ldquoMicroorganism infectionof olive ridley eggsrdquo in Proceedings of the 12th Annual Workshopon Sea Turtle Biology and Conservation J I Richardson and TH Richardson Eds NOAA Technical Memorandum NMFS-SEFSC-361274 pp 81ndash84 1995
[15] J M Sarmiento-Ramırez E Abella M P Martın et alldquoFusarium solaniis responsible for mass mortalities in nests ofloggerhead sea turtle Caretta caretta in Boavista Cape VerderdquoFEMS Microbiology Letters vol 312 no 2 pp 192ndash200 2010
[16] J M Sarmiento-Ramırez E Abella-Perez A D Phillott etal ldquoGlobal distribution of two fungal pathogens threateningendangered sea turtlesrdquo PLoS ONE vol 9 Article ID e858532014
[17] J Patino-Martinez A Marco L Quinones E Abella R MAbad and J Dieguez-Uribeondo ldquoHow do hatcheries influenceembryonic development of sea turtle eggs Experimental anal-ysis and isolation of microorganisms in leatherback turtle eggsrdquoJournal of Experimental Zoology Part A Ecological Genetics andPhysiology vol 317 no 1 pp 47ndash54 2012
[18] A D Phillott and C J Parmenter ldquoFungal colonization of greensea turtle (Chelonia mydas) nests is unlikely to affect hatchlingconditionrdquo Herpetological Conservation amp Biology vol 9 pp297ndash301 2014
[19] MOcanaMHarfush-Melendez and SHeppell ldquoMass nestingof olive ridley sea turtles Lepidochelys olivacea at La EscobillaMexico linking nest density and rates of destructionrdquo Endan-gered Species Research vol 16 pp 45ndash54 2012
[20] DMadden J Ballestero C Calvo R Carlson E Christians andE Madden ldquoSea turtle nesting as a process influencing a sandybeach ecosystemrdquo Biotropica vol 40 no 6 pp 758ndash765 2008
[21] R Valverde and C Gates ldquoPopulation surveys on mass nestingbeachesrdquo in Research and Management Techniques for theConservation of Sea Turtles K L Eckert K A Bjorndal FA Abreu-Gobrois and M Donnelly Eds IUCNSSC MarineTurtle Specialist Group Publication No 4 1999
[22] B P Wallace P R Sotherland J R Spotila R D ReinaB F Franks and F V Paladino ldquoBiotic and abiotic factorsaffect the nest environment of embryonic leatherback turtlesDermochelys coriaceardquo Physiological and Biochemical Zoologyvol 77 no 3 pp 423ndash432 2004
[23] J D Miller ldquoDetermining clutch size and hatching successrdquo inResearch and Management Techniques for the Conservation ofSea Turtles K L Eckert K A Bjorndal F A Abreu-Groboisand M Donnelly Eds IUCNSSC Marine Turtle SpecialistGroup Publication No 4 1999
[24] S J Blott and K Pye ldquoGradistat a grain size distribution andstatistics package for the analysis of unconsolidated sedimentsrdquoEarth Surface Processes and Landforms vol 26 no 11 pp 1237ndash1248 2001
[25] W Krumbein and F Pettijohn Manual of Sedimentary Petrog-raphy Appleton-Century-Crofts New York NY USA 1938
10 Journal of Marine Biology
[26] N C Prevost-Boure R Christen S Dequiedt et al ldquoValidationand application of a PCR primer set to quantify fungal commu-nities in the soil environment by real-time quantitative PCRrdquoPLoS ONE vol 6 no 9 Article ID e24166 2011
[27] N Fierer J A Jackson R Vilgalys and R B Jackson ldquoAssess-ment of soil microbial community structure by use of taxon-specific quantitative PCR assaysrdquo Applied and EnvironmentalMicrobiology vol 71 no 7 pp 4117ndash4120 2005
[28] A Guidot J-C Debaud and R Marmeisse ldquoSpatial distribu-tion of the below-ground mycelia of an ectomycorrhizal fungusinferred from specific quantification of its DNA in soil samplesrdquoFEMS Microbiology Ecology vol 42 no 3 pp 477ndash486 2002
[29] T B de Gregoris N Aldred A S Clare and J G BurgessldquoImprovement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxardquo Journal of Microbio-logical Methods vol 86 no 3 pp 351ndash356 2011
[30] E J Vainio and J Hantula ldquoDirect analysis of wood-inhabitingfungi using denaturing gradient gel electrophoresis of amplifiedribosomal DNArdquoMycological Research vol 104 no 8 pp 927ndash936 2000
[31] S Turner K M Pryer V P W Miao and J D Palmer ldquoInves-tigating deep phylogenetic relationships among cyanobacteriaand plastids by small subunit rRNA sequence analysisrdquo TheJournal of Eukaryotic Microbiology vol 46 no 4 pp 327ndash3381999
[32] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash174 John Wiley amp Sons Chichester UK1991
[33] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications pp 315ndash322 1990
[34] N Uemura KMakimuraM Onozaki et al ldquoDevelopment of aloop-mediated isothermal amplificationmethod for diagnosingPneumocystis pneumoniardquo Journal of Medical Microbiology vol57 no 1 pp 50ndash57 2008
[35] C J Smith D B Nedwell L F Dong and A M OsbornldquoEvaluation of quantitative polymerase chain reaction-basedapproaches for determining gene copy and gene transcriptnumbers in environmental samplesrdquo Environmental Microbiol-ogy vol 8 no 5 pp 804ndash815 2006
[36] C J Smith and A M Osborn ldquoAdvantages and limitationsof quantitative PCR (Q-PCR)-based approaches in microbialecologyrdquo FEMS Microbiology Ecology vol 67 no 1 pp 6ndash202009
[37] R A Ackerman ldquoOxygen consumption by sea turtle (CheloniaCaretta) eggs during developmentrdquo Physiological Zoology vol54 pp 316ndash324 1981
[38] R A Valverde S Wingard F Gomez M T Tordoir and C MOrrego ldquoField lethal incubation temperature of olive ridley seaturtle Lepidochelys olivacea embryos at a mass nesting rookeryrdquoEndangered Species Research vol 12 no 1 pp 77ndash86 2010
[39] L G Fonseca W N Villachica R E Matarrita and R AValverde ldquoReporte final de la anidacioon de tortuga lora(Lepidochelys olivacea) Playa Nancite Parque Nacional SantaRosa Costa Rica (Temporada 2010ndash2011)rdquo Reporte Final deTemporada al USFWS 2011
[40] C Figgener A Castillo-MacCarthy J Mora-Sandoval YArguelo-Gomez and W Quiros-Pereira ldquoRaising the hatchingsuccess of leatherback nests at the olive ridley mass-nesting
beach inOstional Guanacaste Costa Ricardquo in Proceedings of the34thAnnual Symposiumon SeaTurtle Biology andConservation2014
[41] C Limpus V Baker and J Miller ldquoMovement induced mortal-ity of loggerhead eggsrdquoHerpetologica vol 35 pp 335ndash338 1979
[42] R A Ackerman ldquoThe respiratory gas exchange of sea turtlenests (Chelonia Caretta)rdquo Respiration Physiology vol 31 no 1pp 19ndash38 1977
[43] Y-C Kam ldquoPhysiological effects of hypoxia onmetabolism andgrowth of turtle embryosrdquo Respiration Physiology vol 92 no 2pp 127ndash138 1993
[44] TWibbels D Rostal andR Byles ldquoHigh pivotal temperature inthe sex determination of the olive ridley sea turtle Lepidochelysolivacea from Playa Nancite Costa Ricardquo Copeia no 4 pp1086ndash1088 1998
[45] C J McCoy R C Vogt and E J Censky ldquoTemperature-controlled sex determination in the sea turtle Lepidochelysolivaceardquo Journal of Herpetology vol 17 no 4 pp 404ndash4061983
[46] N A Miller ldquoPO2in loggerhead sea turtle (Caretta caretta)
[47] J A Mortimer ldquoThe influence of beach sand characteristicson the nesting behavior and clutch survival of green turtles(Chelonia mydas)rdquo Copeia vol 1990 no 3 pp 802ndash817 1990
[48] S E Cornelius The Sea Turtles of Santa Rosa National ParkFundacion de Parques Nacionales San Jose Costa Rica 1986
[49] X Chen E Peltier B S M Sturm and C B Young ldquoNitrogenremoval and nitrifying and denitrifying bacteria quantificationin a stormwater bioretention systemrdquo Water Research vol 47no 4 pp 1691ndash1700 2013
[50] R S Seymour and R A Ackerman ldquoAdaptations to under-ground nesting in birds and reptilesrdquo American Zoologist vol20 no 2 pp 437ndash447 1980
[51] R S Seymour D Vleck and C M Vleck ldquoGas exchange in theincubationmounds of megapode birdsrdquo Journal of ComparativePhysiology B vol 156 no 6 pp 773ndash782 1986
Table 2 Relationships between bacterial and fungal abundance and the partial pressure of oxygen (pO2) and temperature in the first (1st)and second (2nd) half of incubation for all study nests There was a negative correlation between bacterial abundance and nest pO2 in boththe first and second half of incubation and a positive correlation with nest temperatures in the second half of incubation There was also anegative correlation between fungal abundance and nest pO2 in the first half of incubation
42 Nest Oxygen Content pO2for the low-nest-density and
tidal-wash areas of the beach was similar to that previouslyobserved in other studies on olive ridley sea turtles [4 8]Those studies also observed a change in the pO
2of nests
over the incubation period with pO2decreasing later in
the incubation period as a result of embryo respiration Thesignificant interaction effect of beach area and incubation dayon egg clutch pO
2indicates that the pO
2within egg clutches
changed differently across the areas of the beachOn the other hand the pO
2observed in egg clutches
located in the high-nest-density area in this study is lowerthan that previously observed in other studies [8 22 42]Specifically the drop in pO
2that we observed early in
the incubation period is uncharacteristic of sea turtle eggclutches given that the metabolic activity of the embryos istypically negligible at this time [42] Clutch pO
2typically
remains constant through the first half of incubation andthen decreases and tends to be negatively correlated with thenumber ofmetabolizing embryos per clutch [8 22] Howeverthis was not the case for clutches located in the high-nest-density area of the beach where pO
2began to drop from the
start of incubation and was lower in both the first and secondhalf of the incubation period in comparison to other areas ofthe beach
The negative correlation between microbial abundance(of both bacteria and fungi) and clutch pO
2at Ostional
suggests that microbial decomposition was responsible forthese decreases in pO
2 In particular our observations of high
abundance of bacteria and organic matter in nest sand fromthe high-nest-density area of the beach in association withlow pO
2further support this hypothesis Additionally if this
decrease in pO2resulted from the decomposition of embryos
killed by pathogens in these nests one would expect a lagtime (ie time for infection) and an abrupt change in oxy-gen and temperature following embryo death Furthermorewhile nest density alone affects the oxygen content of neststhe minimum pO
2observed in the high-nest-density area
(97 kPa) in this study was lower than previously observedat similar and even higher nest densities (approximately145ndash170 kPa) [4 8] Given that these previous studies inldquocleanrdquo sand (with presumably low organic matter content)did not find such discrepancies in the pO
2of egg clutches
at similar densities it is likely that microbial decompositionis responsible for the differences observed here Howeverfurther studies that include a controlled measure of pO
2in
beach sand outside of egg clutches and within egg clutches
at controlled densities are necessary to properly test thishypothesis
Because sea turtle embryos have a relatively low toleranceto hypoxia early in the incubation period [43] it is likelythat the drop in pO
2that we observed in the high-nest-
density area of the beach led to total nest failure While thecritical oxygen tension of olive ridley sea turtle eggs hasnot been studied this value was as high as 165 kPa on day22 of incubation for loggerhead sea turtle (Caretta caretta)eggs which are comparable in size [43] Assuming that thecritical oxygen tension of olive ridley sea turtle eggs is similarand given that egg clutch pO
2fell below 165 kPa on day 15
of incubation in the high-nest-density area it is likely thatany eggs located here were exposed to pO
2below a lethal
threshold
43 Nest Temperature The nest temperatures observed inthis study are similar to those observed in other studieson olive ridley sea turtles in this region [4 8 38] Ingeneral mean incubation temperatures were above the meanpivotal temperature of 305∘C for olive ridleys [44 45]indicating that the hatchling sex ratio could be female-biasedat Ostional as previously suggested [38] Previous studieshave also observed a change in nest temperatures throughoutthe incubation period with temperatures increasing laterin incubation in conjunction with increasing embryonicmetabolism [4 8] Nest temperatures were fairly consistentacross the different areas of the beach throughout the incu-bation period with the exception of nests located in thetidal-wash area where lower nest temperatures likely resultedfrom higher water content in the sandThe incubation periodwas 2 to 3 days longer in this group of nests and is likely aresult of slower embryonic development due to lower nesttemperatures [46]
While most nest temperatures fell within the wide rangeof tolerance for sea turtle embryological development (25ndash35∘C [39]) early in the incubation period the slightly highertemperatures observed in the high-nest-density area of thebeach later in incubation may have exceeded this optimalrange long enough to affect hatching success Though thefield lethal limit for olive ridley sea turtles at this beach isapproximately 35∘C embryos may be capable of recoveringfrom short-term exposure to high temperatures while longerdurations are lethal [38] Results from this study supportthis notion in that nests with lower hatching success (high-nest-density area) were also exposed to temperatures above
8 Journal of Marine Biology
the lethal limit for a longer duration of time The differentcombination of heat released from embryonic metabolismandor microbial decomposition is likely responsible for theobserved differences in temperature later in incubation
44 Characteristics of Nest Sand The mean grain size andsorting coefficient of sand from nests in this study werecomparable to those previously observed at a variety ofsea turtle nesting beaches [47] Given the relatively smallrange of mean grain size observed across study nests (0282ndash0366mm) granulometry likely did not drive the differencesin hatching success across the areas of the beach
The high amounts of organic matter in the sand fromthis study in comparison to other sea turtle nesting beaches[47] are not surprising as this is likely a result of the highrates of nest destruction due to the large number of nestingfemales and high-nest-density at this beach [9 19 48] Theassociation of high organic matter content with a higherabundance of bacteria in the sand from the high-nest-densityarea in this study supports the hypothesis that high organicmatter is associated with elevated microbial proliferation anddecomposition at Ostional and may also explain the low eggclutch pO
2in comparison to previous studies [7 8]
45 Microbial Abundance The number of bacterial 16S andfungal 18S rRNA gene copies in this study ranged from 105to 1010 copies gminus1 of sea turtle nest sand A previous studyfound that the sand at an arribada beach contained six ordersof magnitude more bacterial colonies gminus1 in comparison toa neighboring beach although no specific abundance datawere provided [10 14] Previous studies that have quantified16S or 18S rRNA gene copies in similar sediments have foundbetween 106 and 109 copies gminus1 [26 49]
The negative relationship between bacterial abundanceand hatching success that we observed supports the hypoth-esis that the microbial abundance in nest sand at Ostionalis high enough to adversely affect embryonic developmentSpecifically we suggest that the mechanism behind thisrelationship is not pathogenic but rather a result of high ratesof microbial decomposition of organic matter altering thenest environment beyond the optimal range for embryonicdevelopment Studies on underground nesting species ofbirds and reptiles suggest similar influences of microbialdecomposition on nest pO
2and temperature [50 51] It is
possible that the relationship between microbial activity andhatching success is nonlinear with a threshold beyond whichhigh rates of microbial respiration disrupt the diffusion ofoxygen to the nest to the point of impeding embryonic devel-opment For example the relationship between microbialabundance and hatching success appears to reach a thresholdat sim1 times 109ndash1010 16S18S copies gminus1 nest sand Furtherstudies that span a broader range in microbial abundanceare required to confirm the true nature of the relationshipbetween the microbial abundance of nest sand and sea turtlehatching success The coinciding occurrence of lower pO
2
higher temperatures higher organicmatter and low hatchingsuccess in the high-nest-density area of the beach serves asan extreme example of the adverse conditions imposed bymicrobial decomposition at Ostional
In addition to the indirect effect of microbial abundanceon the nest environment there are likely other effects ofmicrobial presence on sea turtle hatching success that wedid not consider because they were not within the scope ofthe present study For example we did not explore speciescomposition or diversity nor did we evaluate the presence orabsence of pathogens The potential presence of pathogenicmicrobes in the sand at Ostional cannot be ruled out Severalspecies of fungi in the Fusarium solani species complex havebeen identified as pathogens to sea turtle eggs and are knownto occur at nesting beaches around the world [15 16] It isalso possible that the effects of microbial abundance andthe nest environment on hatching success are synergisticwith the effects of lethal pathogens [15] For example onceembryos are killed due to the adverse effects of microbialoxygen depletion on the nest environment this may resultin the proliferation of fungi which could then spread toadjacent eggs Further studies should therefore focus onsampling from several components of the nest environment(ie cloacal fluid nest sand eggshell unhatched embryosand live hatchlings) as well as assessing the abundancediversity and activity of both fungi and bacteria to providea more complete understanding of the relationship betweenmicrobial activity and the reproductive success of sea turtles
Based on our comparative study it appears that tidalwashing decreases the organic matter and microbial abun-dance in the sand thus increasing hatching success Alter-natively the high humidity in the sand resulting fromtidal washing may also keep incubation temperatures fromreaching lethal temperaturesTherefore seawater inundationmay be critical in maintaining the population of sea turtlesat Ostional Using these natural mechanisms as a model forconservation management practices could be adopted topromote improved embryo survival and hatchling produc-tion For example seawater could be used to decrease organicmatter and microbial abundance in hatchery sand or byperiodically irrigating the beachwith seawaterThe treatmentof sand or relocation of hatcheries on a regular basis is a con-ventional management practice in conservation programs toavoid organic matter build-up as well as microbial and larvalinfestations In fact the leatherback sea turtle conservationprogram at Ostional successfully increased hatching successin the leatherback hatchery by using sand from below thehigh tide line [40]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank G Bran Y Arguello-GomezM Gomez S Steele and all the employees and volunteers atRNVS Ostional for their help in the field The authors wouldalso like to thank S Berthrong A Oporta McCarthy and RLeisen for their contributions to themolecular and laboratoryanalyses This material is based upon work supported by the
Journal of Marine Biology 9
National Science Foundation Graduate Research Fellowshipunder Grant no 511182 Additional funds to support the laband field research for this study were provided by the PADIFoundation National Geographic Young Explorers Grant(C220-12) USFWS Marine Turtle Conservation Act (96200-0-G037) and internal grants from the College of CharlestonThis research was approved by the College of CharlestonIACUC and permits were granted by MINAET (Resolucionno ACT-OR-DR-078) and CONAGEBio (R-020-2012-OT-CONAGEBIO) of Costa Rica
References
[1] A Abreu-Grobois and P T Plotkin ldquoIUCNSMTSG (2008) Lep-idochelys olivaceardquo The IUCN Red List of Threatened SpeciesVersion 20142 httpwwwiucnredlistorg
[2] R A Valverde C M Orrego M T Tordoir et al ldquoOlive ridleymass nesting ecology and egg harvest at ostional beach CostaRicardquoChelonian Conservation and Biology vol 11 no 1 pp 1ndash112012
[3] S E Cornelius R Arauz J Fretey et al ldquoEffect of land basedharvest of Lepidochelysrdquo in The Biology and Conservation ofRidley Sea Turtles P T Plotkin Ed pp 231ndash251 Johns HopkinsUniversity Press Baltimore Md USA 2007
[4] S Honarvar M P OrsquoConnor and J R Spotila ldquoDensity-dependent effects on hatching success of the olive ridley turtleLepidochelys olivaceardquo Oecologia vol 157 no 2 pp 221ndash2302008
[5] V S Beezy and R A Valverde ldquoA comparison ofmethodologiesfor estimating the nest density of olive ridley arribadas atOstional Costa Ricardquo in Proceedings of the 31st Annual Sympo-sium on Sea Turtle Biology andConservation T T Jones andB PWallace Eds NOAA Technical Memorandum NMFS-SEFSC-631 pp 194ndash195 2012
[6] S E Cornelius M A Ulloa J C Castro M Mata del Valleand D C Robinson ldquoManagement of olive ridley sea turtles(Lepidochelys olivacea) nesting at Playas Nancite and OstionalCosta Ricardquo in Neotropical Wildlife Use and Conservation JRobinson and K Redford Eds vol 1 pp 111ndash135 Universityof Chicago Press Chicago Ill USA 1991
[7] R A Valverde S E Cornelius and C L Mo ldquoDecline of theolive ridley sea turtle (Lepidochelys olivacea) nesting assemblageat Nancite Beach Santa Rosa National Park Costa RicardquoChelonian Conservation and Biology vol 3 pp 58ndash63 1998
[8] S Clusella Trullas and F V Paladino ldquoMicro-environment ofolive ridley turtle nests deposited during an aggregated nestingeventrdquo Journal of Zoology vol 272 no 4 pp 367ndash376 2007
[9] D McPherson and D Kibler ldquoEcological impact of olive ridleynesting at Ostional Costa Ricardquo in Proceedings of the 25thAnnual Symposium on Sea Turtle Biology and ConservationH Kalb A Rohde K Gayheart and K Shanker Eds NOAATechnical Memorandum NMFS-SEFSC-582 p 204 2008
[10] C L Mo I Salas and M Caballero ldquoAre fungi and bacteriaresponsible for olive ridleyrsquos egg lossrdquo in Proceedings of the 10thAnnual Workshop on Sea Turtle Biology and Conservation T HRichardson J I Richardson and M Donnelly Eds pp 249ndash252 NOAA Techn 1990
[11] S Honarvar J R Spotila andM P OrsquoConnor ldquoMicrobial com-munity structure in sand on two olive ridley arribada nestingbeaches Playa La Flor Nicaragua and Playa Nancite Costa
Ricardquo Journal of Experimental Marine Biology and Ecology vol409 no 1-2 pp 339ndash344 2011
[12] A D Phillott and C J Parmenter ldquoThe distribution of failedeggs and the appearance of fungi in artificial nests of green(Chelonia mydas) and loggerhead (Caretta caretta) sea turtlesrdquoAustralian Journal of Zoology vol 49 no 6 pp 713ndash718 2001
[13] J Wyneken T J Burke M Salmon and D K Pedersen ldquoEggfailure in natural and relocated sea turtle nestsrdquo Journal ofHerpetology vol 22 no 1 pp 88ndash96 1988
[14] C L Mo M Caballero and I Salas ldquoMicroorganism infectionof olive ridley eggsrdquo in Proceedings of the 12th Annual Workshopon Sea Turtle Biology and Conservation J I Richardson and TH Richardson Eds NOAA Technical Memorandum NMFS-SEFSC-361274 pp 81ndash84 1995
[15] J M Sarmiento-Ramırez E Abella M P Martın et alldquoFusarium solaniis responsible for mass mortalities in nests ofloggerhead sea turtle Caretta caretta in Boavista Cape VerderdquoFEMS Microbiology Letters vol 312 no 2 pp 192ndash200 2010
[16] J M Sarmiento-Ramırez E Abella-Perez A D Phillott etal ldquoGlobal distribution of two fungal pathogens threateningendangered sea turtlesrdquo PLoS ONE vol 9 Article ID e858532014
[17] J Patino-Martinez A Marco L Quinones E Abella R MAbad and J Dieguez-Uribeondo ldquoHow do hatcheries influenceembryonic development of sea turtle eggs Experimental anal-ysis and isolation of microorganisms in leatherback turtle eggsrdquoJournal of Experimental Zoology Part A Ecological Genetics andPhysiology vol 317 no 1 pp 47ndash54 2012
[18] A D Phillott and C J Parmenter ldquoFungal colonization of greensea turtle (Chelonia mydas) nests is unlikely to affect hatchlingconditionrdquo Herpetological Conservation amp Biology vol 9 pp297ndash301 2014
[19] MOcanaMHarfush-Melendez and SHeppell ldquoMass nestingof olive ridley sea turtles Lepidochelys olivacea at La EscobillaMexico linking nest density and rates of destructionrdquo Endan-gered Species Research vol 16 pp 45ndash54 2012
[20] DMadden J Ballestero C Calvo R Carlson E Christians andE Madden ldquoSea turtle nesting as a process influencing a sandybeach ecosystemrdquo Biotropica vol 40 no 6 pp 758ndash765 2008
[21] R Valverde and C Gates ldquoPopulation surveys on mass nestingbeachesrdquo in Research and Management Techniques for theConservation of Sea Turtles K L Eckert K A Bjorndal FA Abreu-Gobrois and M Donnelly Eds IUCNSSC MarineTurtle Specialist Group Publication No 4 1999
[22] B P Wallace P R Sotherland J R Spotila R D ReinaB F Franks and F V Paladino ldquoBiotic and abiotic factorsaffect the nest environment of embryonic leatherback turtlesDermochelys coriaceardquo Physiological and Biochemical Zoologyvol 77 no 3 pp 423ndash432 2004
[23] J D Miller ldquoDetermining clutch size and hatching successrdquo inResearch and Management Techniques for the Conservation ofSea Turtles K L Eckert K A Bjorndal F A Abreu-Groboisand M Donnelly Eds IUCNSSC Marine Turtle SpecialistGroup Publication No 4 1999
[24] S J Blott and K Pye ldquoGradistat a grain size distribution andstatistics package for the analysis of unconsolidated sedimentsrdquoEarth Surface Processes and Landforms vol 26 no 11 pp 1237ndash1248 2001
[25] W Krumbein and F Pettijohn Manual of Sedimentary Petrog-raphy Appleton-Century-Crofts New York NY USA 1938
10 Journal of Marine Biology
[26] N C Prevost-Boure R Christen S Dequiedt et al ldquoValidationand application of a PCR primer set to quantify fungal commu-nities in the soil environment by real-time quantitative PCRrdquoPLoS ONE vol 6 no 9 Article ID e24166 2011
[27] N Fierer J A Jackson R Vilgalys and R B Jackson ldquoAssess-ment of soil microbial community structure by use of taxon-specific quantitative PCR assaysrdquo Applied and EnvironmentalMicrobiology vol 71 no 7 pp 4117ndash4120 2005
[28] A Guidot J-C Debaud and R Marmeisse ldquoSpatial distribu-tion of the below-ground mycelia of an ectomycorrhizal fungusinferred from specific quantification of its DNA in soil samplesrdquoFEMS Microbiology Ecology vol 42 no 3 pp 477ndash486 2002
[29] T B de Gregoris N Aldred A S Clare and J G BurgessldquoImprovement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxardquo Journal of Microbio-logical Methods vol 86 no 3 pp 351ndash356 2011
[30] E J Vainio and J Hantula ldquoDirect analysis of wood-inhabitingfungi using denaturing gradient gel electrophoresis of amplifiedribosomal DNArdquoMycological Research vol 104 no 8 pp 927ndash936 2000
[31] S Turner K M Pryer V P W Miao and J D Palmer ldquoInves-tigating deep phylogenetic relationships among cyanobacteriaand plastids by small subunit rRNA sequence analysisrdquo TheJournal of Eukaryotic Microbiology vol 46 no 4 pp 327ndash3381999
[32] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash174 John Wiley amp Sons Chichester UK1991
[33] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications pp 315ndash322 1990
[34] N Uemura KMakimuraM Onozaki et al ldquoDevelopment of aloop-mediated isothermal amplificationmethod for diagnosingPneumocystis pneumoniardquo Journal of Medical Microbiology vol57 no 1 pp 50ndash57 2008
[35] C J Smith D B Nedwell L F Dong and A M OsbornldquoEvaluation of quantitative polymerase chain reaction-basedapproaches for determining gene copy and gene transcriptnumbers in environmental samplesrdquo Environmental Microbiol-ogy vol 8 no 5 pp 804ndash815 2006
[36] C J Smith and A M Osborn ldquoAdvantages and limitationsof quantitative PCR (Q-PCR)-based approaches in microbialecologyrdquo FEMS Microbiology Ecology vol 67 no 1 pp 6ndash202009
[37] R A Ackerman ldquoOxygen consumption by sea turtle (CheloniaCaretta) eggs during developmentrdquo Physiological Zoology vol54 pp 316ndash324 1981
[38] R A Valverde S Wingard F Gomez M T Tordoir and C MOrrego ldquoField lethal incubation temperature of olive ridley seaturtle Lepidochelys olivacea embryos at a mass nesting rookeryrdquoEndangered Species Research vol 12 no 1 pp 77ndash86 2010
[39] L G Fonseca W N Villachica R E Matarrita and R AValverde ldquoReporte final de la anidacioon de tortuga lora(Lepidochelys olivacea) Playa Nancite Parque Nacional SantaRosa Costa Rica (Temporada 2010ndash2011)rdquo Reporte Final deTemporada al USFWS 2011
[40] C Figgener A Castillo-MacCarthy J Mora-Sandoval YArguelo-Gomez and W Quiros-Pereira ldquoRaising the hatchingsuccess of leatherback nests at the olive ridley mass-nesting
beach inOstional Guanacaste Costa Ricardquo in Proceedings of the34thAnnual Symposiumon SeaTurtle Biology andConservation2014
[41] C Limpus V Baker and J Miller ldquoMovement induced mortal-ity of loggerhead eggsrdquoHerpetologica vol 35 pp 335ndash338 1979
[42] R A Ackerman ldquoThe respiratory gas exchange of sea turtlenests (Chelonia Caretta)rdquo Respiration Physiology vol 31 no 1pp 19ndash38 1977
[43] Y-C Kam ldquoPhysiological effects of hypoxia onmetabolism andgrowth of turtle embryosrdquo Respiration Physiology vol 92 no 2pp 127ndash138 1993
[44] TWibbels D Rostal andR Byles ldquoHigh pivotal temperature inthe sex determination of the olive ridley sea turtle Lepidochelysolivacea from Playa Nancite Costa Ricardquo Copeia no 4 pp1086ndash1088 1998
[45] C J McCoy R C Vogt and E J Censky ldquoTemperature-controlled sex determination in the sea turtle Lepidochelysolivaceardquo Journal of Herpetology vol 17 no 4 pp 404ndash4061983
[46] N A Miller ldquoPO2in loggerhead sea turtle (Caretta caretta)
[47] J A Mortimer ldquoThe influence of beach sand characteristicson the nesting behavior and clutch survival of green turtles(Chelonia mydas)rdquo Copeia vol 1990 no 3 pp 802ndash817 1990
[48] S E Cornelius The Sea Turtles of Santa Rosa National ParkFundacion de Parques Nacionales San Jose Costa Rica 1986
[49] X Chen E Peltier B S M Sturm and C B Young ldquoNitrogenremoval and nitrifying and denitrifying bacteria quantificationin a stormwater bioretention systemrdquo Water Research vol 47no 4 pp 1691ndash1700 2013
[50] R S Seymour and R A Ackerman ldquoAdaptations to under-ground nesting in birds and reptilesrdquo American Zoologist vol20 no 2 pp 437ndash447 1980
[51] R S Seymour D Vleck and C M Vleck ldquoGas exchange in theincubationmounds of megapode birdsrdquo Journal of ComparativePhysiology B vol 156 no 6 pp 773ndash782 1986
the lethal limit for a longer duration of time The differentcombination of heat released from embryonic metabolismandor microbial decomposition is likely responsible for theobserved differences in temperature later in incubation
44 Characteristics of Nest Sand The mean grain size andsorting coefficient of sand from nests in this study werecomparable to those previously observed at a variety ofsea turtle nesting beaches [47] Given the relatively smallrange of mean grain size observed across study nests (0282ndash0366mm) granulometry likely did not drive the differencesin hatching success across the areas of the beach
The high amounts of organic matter in the sand fromthis study in comparison to other sea turtle nesting beaches[47] are not surprising as this is likely a result of the highrates of nest destruction due to the large number of nestingfemales and high-nest-density at this beach [9 19 48] Theassociation of high organic matter content with a higherabundance of bacteria in the sand from the high-nest-densityarea in this study supports the hypothesis that high organicmatter is associated with elevated microbial proliferation anddecomposition at Ostional and may also explain the low eggclutch pO
2in comparison to previous studies [7 8]
45 Microbial Abundance The number of bacterial 16S andfungal 18S rRNA gene copies in this study ranged from 105to 1010 copies gminus1 of sea turtle nest sand A previous studyfound that the sand at an arribada beach contained six ordersof magnitude more bacterial colonies gminus1 in comparison toa neighboring beach although no specific abundance datawere provided [10 14] Previous studies that have quantified16S or 18S rRNA gene copies in similar sediments have foundbetween 106 and 109 copies gminus1 [26 49]
The negative relationship between bacterial abundanceand hatching success that we observed supports the hypoth-esis that the microbial abundance in nest sand at Ostionalis high enough to adversely affect embryonic developmentSpecifically we suggest that the mechanism behind thisrelationship is not pathogenic but rather a result of high ratesof microbial decomposition of organic matter altering thenest environment beyond the optimal range for embryonicdevelopment Studies on underground nesting species ofbirds and reptiles suggest similar influences of microbialdecomposition on nest pO
2and temperature [50 51] It is
possible that the relationship between microbial activity andhatching success is nonlinear with a threshold beyond whichhigh rates of microbial respiration disrupt the diffusion ofoxygen to the nest to the point of impeding embryonic devel-opment For example the relationship between microbialabundance and hatching success appears to reach a thresholdat sim1 times 109ndash1010 16S18S copies gminus1 nest sand Furtherstudies that span a broader range in microbial abundanceare required to confirm the true nature of the relationshipbetween the microbial abundance of nest sand and sea turtlehatching success The coinciding occurrence of lower pO
2
higher temperatures higher organicmatter and low hatchingsuccess in the high-nest-density area of the beach serves asan extreme example of the adverse conditions imposed bymicrobial decomposition at Ostional
In addition to the indirect effect of microbial abundanceon the nest environment there are likely other effects ofmicrobial presence on sea turtle hatching success that wedid not consider because they were not within the scope ofthe present study For example we did not explore speciescomposition or diversity nor did we evaluate the presence orabsence of pathogens The potential presence of pathogenicmicrobes in the sand at Ostional cannot be ruled out Severalspecies of fungi in the Fusarium solani species complex havebeen identified as pathogens to sea turtle eggs and are knownto occur at nesting beaches around the world [15 16] It isalso possible that the effects of microbial abundance andthe nest environment on hatching success are synergisticwith the effects of lethal pathogens [15] For example onceembryos are killed due to the adverse effects of microbialoxygen depletion on the nest environment this may resultin the proliferation of fungi which could then spread toadjacent eggs Further studies should therefore focus onsampling from several components of the nest environment(ie cloacal fluid nest sand eggshell unhatched embryosand live hatchlings) as well as assessing the abundancediversity and activity of both fungi and bacteria to providea more complete understanding of the relationship betweenmicrobial activity and the reproductive success of sea turtles
Based on our comparative study it appears that tidalwashing decreases the organic matter and microbial abun-dance in the sand thus increasing hatching success Alter-natively the high humidity in the sand resulting fromtidal washing may also keep incubation temperatures fromreaching lethal temperaturesTherefore seawater inundationmay be critical in maintaining the population of sea turtlesat Ostional Using these natural mechanisms as a model forconservation management practices could be adopted topromote improved embryo survival and hatchling produc-tion For example seawater could be used to decrease organicmatter and microbial abundance in hatchery sand or byperiodically irrigating the beachwith seawaterThe treatmentof sand or relocation of hatcheries on a regular basis is a con-ventional management practice in conservation programs toavoid organic matter build-up as well as microbial and larvalinfestations In fact the leatherback sea turtle conservationprogram at Ostional successfully increased hatching successin the leatherback hatchery by using sand from below thehigh tide line [40]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank G Bran Y Arguello-GomezM Gomez S Steele and all the employees and volunteers atRNVS Ostional for their help in the field The authors wouldalso like to thank S Berthrong A Oporta McCarthy and RLeisen for their contributions to themolecular and laboratoryanalyses This material is based upon work supported by the
Journal of Marine Biology 9
National Science Foundation Graduate Research Fellowshipunder Grant no 511182 Additional funds to support the laband field research for this study were provided by the PADIFoundation National Geographic Young Explorers Grant(C220-12) USFWS Marine Turtle Conservation Act (96200-0-G037) and internal grants from the College of CharlestonThis research was approved by the College of CharlestonIACUC and permits were granted by MINAET (Resolucionno ACT-OR-DR-078) and CONAGEBio (R-020-2012-OT-CONAGEBIO) of Costa Rica
References
[1] A Abreu-Grobois and P T Plotkin ldquoIUCNSMTSG (2008) Lep-idochelys olivaceardquo The IUCN Red List of Threatened SpeciesVersion 20142 httpwwwiucnredlistorg
[2] R A Valverde C M Orrego M T Tordoir et al ldquoOlive ridleymass nesting ecology and egg harvest at ostional beach CostaRicardquoChelonian Conservation and Biology vol 11 no 1 pp 1ndash112012
[3] S E Cornelius R Arauz J Fretey et al ldquoEffect of land basedharvest of Lepidochelysrdquo in The Biology and Conservation ofRidley Sea Turtles P T Plotkin Ed pp 231ndash251 Johns HopkinsUniversity Press Baltimore Md USA 2007
[4] S Honarvar M P OrsquoConnor and J R Spotila ldquoDensity-dependent effects on hatching success of the olive ridley turtleLepidochelys olivaceardquo Oecologia vol 157 no 2 pp 221ndash2302008
[5] V S Beezy and R A Valverde ldquoA comparison ofmethodologiesfor estimating the nest density of olive ridley arribadas atOstional Costa Ricardquo in Proceedings of the 31st Annual Sympo-sium on Sea Turtle Biology andConservation T T Jones andB PWallace Eds NOAA Technical Memorandum NMFS-SEFSC-631 pp 194ndash195 2012
[6] S E Cornelius M A Ulloa J C Castro M Mata del Valleand D C Robinson ldquoManagement of olive ridley sea turtles(Lepidochelys olivacea) nesting at Playas Nancite and OstionalCosta Ricardquo in Neotropical Wildlife Use and Conservation JRobinson and K Redford Eds vol 1 pp 111ndash135 Universityof Chicago Press Chicago Ill USA 1991
[7] R A Valverde S E Cornelius and C L Mo ldquoDecline of theolive ridley sea turtle (Lepidochelys olivacea) nesting assemblageat Nancite Beach Santa Rosa National Park Costa RicardquoChelonian Conservation and Biology vol 3 pp 58ndash63 1998
[8] S Clusella Trullas and F V Paladino ldquoMicro-environment ofolive ridley turtle nests deposited during an aggregated nestingeventrdquo Journal of Zoology vol 272 no 4 pp 367ndash376 2007
[9] D McPherson and D Kibler ldquoEcological impact of olive ridleynesting at Ostional Costa Ricardquo in Proceedings of the 25thAnnual Symposium on Sea Turtle Biology and ConservationH Kalb A Rohde K Gayheart and K Shanker Eds NOAATechnical Memorandum NMFS-SEFSC-582 p 204 2008
[10] C L Mo I Salas and M Caballero ldquoAre fungi and bacteriaresponsible for olive ridleyrsquos egg lossrdquo in Proceedings of the 10thAnnual Workshop on Sea Turtle Biology and Conservation T HRichardson J I Richardson and M Donnelly Eds pp 249ndash252 NOAA Techn 1990
[11] S Honarvar J R Spotila andM P OrsquoConnor ldquoMicrobial com-munity structure in sand on two olive ridley arribada nestingbeaches Playa La Flor Nicaragua and Playa Nancite Costa
Ricardquo Journal of Experimental Marine Biology and Ecology vol409 no 1-2 pp 339ndash344 2011
[12] A D Phillott and C J Parmenter ldquoThe distribution of failedeggs and the appearance of fungi in artificial nests of green(Chelonia mydas) and loggerhead (Caretta caretta) sea turtlesrdquoAustralian Journal of Zoology vol 49 no 6 pp 713ndash718 2001
[13] J Wyneken T J Burke M Salmon and D K Pedersen ldquoEggfailure in natural and relocated sea turtle nestsrdquo Journal ofHerpetology vol 22 no 1 pp 88ndash96 1988
[14] C L Mo M Caballero and I Salas ldquoMicroorganism infectionof olive ridley eggsrdquo in Proceedings of the 12th Annual Workshopon Sea Turtle Biology and Conservation J I Richardson and TH Richardson Eds NOAA Technical Memorandum NMFS-SEFSC-361274 pp 81ndash84 1995
[15] J M Sarmiento-Ramırez E Abella M P Martın et alldquoFusarium solaniis responsible for mass mortalities in nests ofloggerhead sea turtle Caretta caretta in Boavista Cape VerderdquoFEMS Microbiology Letters vol 312 no 2 pp 192ndash200 2010
[16] J M Sarmiento-Ramırez E Abella-Perez A D Phillott etal ldquoGlobal distribution of two fungal pathogens threateningendangered sea turtlesrdquo PLoS ONE vol 9 Article ID e858532014
[17] J Patino-Martinez A Marco L Quinones E Abella R MAbad and J Dieguez-Uribeondo ldquoHow do hatcheries influenceembryonic development of sea turtle eggs Experimental anal-ysis and isolation of microorganisms in leatherback turtle eggsrdquoJournal of Experimental Zoology Part A Ecological Genetics andPhysiology vol 317 no 1 pp 47ndash54 2012
[18] A D Phillott and C J Parmenter ldquoFungal colonization of greensea turtle (Chelonia mydas) nests is unlikely to affect hatchlingconditionrdquo Herpetological Conservation amp Biology vol 9 pp297ndash301 2014
[19] MOcanaMHarfush-Melendez and SHeppell ldquoMass nestingof olive ridley sea turtles Lepidochelys olivacea at La EscobillaMexico linking nest density and rates of destructionrdquo Endan-gered Species Research vol 16 pp 45ndash54 2012
[20] DMadden J Ballestero C Calvo R Carlson E Christians andE Madden ldquoSea turtle nesting as a process influencing a sandybeach ecosystemrdquo Biotropica vol 40 no 6 pp 758ndash765 2008
[21] R Valverde and C Gates ldquoPopulation surveys on mass nestingbeachesrdquo in Research and Management Techniques for theConservation of Sea Turtles K L Eckert K A Bjorndal FA Abreu-Gobrois and M Donnelly Eds IUCNSSC MarineTurtle Specialist Group Publication No 4 1999
[22] B P Wallace P R Sotherland J R Spotila R D ReinaB F Franks and F V Paladino ldquoBiotic and abiotic factorsaffect the nest environment of embryonic leatherback turtlesDermochelys coriaceardquo Physiological and Biochemical Zoologyvol 77 no 3 pp 423ndash432 2004
[23] J D Miller ldquoDetermining clutch size and hatching successrdquo inResearch and Management Techniques for the Conservation ofSea Turtles K L Eckert K A Bjorndal F A Abreu-Groboisand M Donnelly Eds IUCNSSC Marine Turtle SpecialistGroup Publication No 4 1999
[24] S J Blott and K Pye ldquoGradistat a grain size distribution andstatistics package for the analysis of unconsolidated sedimentsrdquoEarth Surface Processes and Landforms vol 26 no 11 pp 1237ndash1248 2001
[25] W Krumbein and F Pettijohn Manual of Sedimentary Petrog-raphy Appleton-Century-Crofts New York NY USA 1938
10 Journal of Marine Biology
[26] N C Prevost-Boure R Christen S Dequiedt et al ldquoValidationand application of a PCR primer set to quantify fungal commu-nities in the soil environment by real-time quantitative PCRrdquoPLoS ONE vol 6 no 9 Article ID e24166 2011
[27] N Fierer J A Jackson R Vilgalys and R B Jackson ldquoAssess-ment of soil microbial community structure by use of taxon-specific quantitative PCR assaysrdquo Applied and EnvironmentalMicrobiology vol 71 no 7 pp 4117ndash4120 2005
[28] A Guidot J-C Debaud and R Marmeisse ldquoSpatial distribu-tion of the below-ground mycelia of an ectomycorrhizal fungusinferred from specific quantification of its DNA in soil samplesrdquoFEMS Microbiology Ecology vol 42 no 3 pp 477ndash486 2002
[29] T B de Gregoris N Aldred A S Clare and J G BurgessldquoImprovement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxardquo Journal of Microbio-logical Methods vol 86 no 3 pp 351ndash356 2011
[30] E J Vainio and J Hantula ldquoDirect analysis of wood-inhabitingfungi using denaturing gradient gel electrophoresis of amplifiedribosomal DNArdquoMycological Research vol 104 no 8 pp 927ndash936 2000
[31] S Turner K M Pryer V P W Miao and J D Palmer ldquoInves-tigating deep phylogenetic relationships among cyanobacteriaand plastids by small subunit rRNA sequence analysisrdquo TheJournal of Eukaryotic Microbiology vol 46 no 4 pp 327ndash3381999
[32] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash174 John Wiley amp Sons Chichester UK1991
[33] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications pp 315ndash322 1990
[34] N Uemura KMakimuraM Onozaki et al ldquoDevelopment of aloop-mediated isothermal amplificationmethod for diagnosingPneumocystis pneumoniardquo Journal of Medical Microbiology vol57 no 1 pp 50ndash57 2008
[35] C J Smith D B Nedwell L F Dong and A M OsbornldquoEvaluation of quantitative polymerase chain reaction-basedapproaches for determining gene copy and gene transcriptnumbers in environmental samplesrdquo Environmental Microbiol-ogy vol 8 no 5 pp 804ndash815 2006
[36] C J Smith and A M Osborn ldquoAdvantages and limitationsof quantitative PCR (Q-PCR)-based approaches in microbialecologyrdquo FEMS Microbiology Ecology vol 67 no 1 pp 6ndash202009
[37] R A Ackerman ldquoOxygen consumption by sea turtle (CheloniaCaretta) eggs during developmentrdquo Physiological Zoology vol54 pp 316ndash324 1981
[38] R A Valverde S Wingard F Gomez M T Tordoir and C MOrrego ldquoField lethal incubation temperature of olive ridley seaturtle Lepidochelys olivacea embryos at a mass nesting rookeryrdquoEndangered Species Research vol 12 no 1 pp 77ndash86 2010
[39] L G Fonseca W N Villachica R E Matarrita and R AValverde ldquoReporte final de la anidacioon de tortuga lora(Lepidochelys olivacea) Playa Nancite Parque Nacional SantaRosa Costa Rica (Temporada 2010ndash2011)rdquo Reporte Final deTemporada al USFWS 2011
[40] C Figgener A Castillo-MacCarthy J Mora-Sandoval YArguelo-Gomez and W Quiros-Pereira ldquoRaising the hatchingsuccess of leatherback nests at the olive ridley mass-nesting
beach inOstional Guanacaste Costa Ricardquo in Proceedings of the34thAnnual Symposiumon SeaTurtle Biology andConservation2014
[41] C Limpus V Baker and J Miller ldquoMovement induced mortal-ity of loggerhead eggsrdquoHerpetologica vol 35 pp 335ndash338 1979
[42] R A Ackerman ldquoThe respiratory gas exchange of sea turtlenests (Chelonia Caretta)rdquo Respiration Physiology vol 31 no 1pp 19ndash38 1977
[43] Y-C Kam ldquoPhysiological effects of hypoxia onmetabolism andgrowth of turtle embryosrdquo Respiration Physiology vol 92 no 2pp 127ndash138 1993
[44] TWibbels D Rostal andR Byles ldquoHigh pivotal temperature inthe sex determination of the olive ridley sea turtle Lepidochelysolivacea from Playa Nancite Costa Ricardquo Copeia no 4 pp1086ndash1088 1998
[45] C J McCoy R C Vogt and E J Censky ldquoTemperature-controlled sex determination in the sea turtle Lepidochelysolivaceardquo Journal of Herpetology vol 17 no 4 pp 404ndash4061983
[46] N A Miller ldquoPO2in loggerhead sea turtle (Caretta caretta)
[47] J A Mortimer ldquoThe influence of beach sand characteristicson the nesting behavior and clutch survival of green turtles(Chelonia mydas)rdquo Copeia vol 1990 no 3 pp 802ndash817 1990
[48] S E Cornelius The Sea Turtles of Santa Rosa National ParkFundacion de Parques Nacionales San Jose Costa Rica 1986
[49] X Chen E Peltier B S M Sturm and C B Young ldquoNitrogenremoval and nitrifying and denitrifying bacteria quantificationin a stormwater bioretention systemrdquo Water Research vol 47no 4 pp 1691ndash1700 2013
[50] R S Seymour and R A Ackerman ldquoAdaptations to under-ground nesting in birds and reptilesrdquo American Zoologist vol20 no 2 pp 437ndash447 1980
[51] R S Seymour D Vleck and C M Vleck ldquoGas exchange in theincubationmounds of megapode birdsrdquo Journal of ComparativePhysiology B vol 156 no 6 pp 773ndash782 1986
National Science Foundation Graduate Research Fellowshipunder Grant no 511182 Additional funds to support the laband field research for this study were provided by the PADIFoundation National Geographic Young Explorers Grant(C220-12) USFWS Marine Turtle Conservation Act (96200-0-G037) and internal grants from the College of CharlestonThis research was approved by the College of CharlestonIACUC and permits were granted by MINAET (Resolucionno ACT-OR-DR-078) and CONAGEBio (R-020-2012-OT-CONAGEBIO) of Costa Rica
References
[1] A Abreu-Grobois and P T Plotkin ldquoIUCNSMTSG (2008) Lep-idochelys olivaceardquo The IUCN Red List of Threatened SpeciesVersion 20142 httpwwwiucnredlistorg
[2] R A Valverde C M Orrego M T Tordoir et al ldquoOlive ridleymass nesting ecology and egg harvest at ostional beach CostaRicardquoChelonian Conservation and Biology vol 11 no 1 pp 1ndash112012
[3] S E Cornelius R Arauz J Fretey et al ldquoEffect of land basedharvest of Lepidochelysrdquo in The Biology and Conservation ofRidley Sea Turtles P T Plotkin Ed pp 231ndash251 Johns HopkinsUniversity Press Baltimore Md USA 2007
[4] S Honarvar M P OrsquoConnor and J R Spotila ldquoDensity-dependent effects on hatching success of the olive ridley turtleLepidochelys olivaceardquo Oecologia vol 157 no 2 pp 221ndash2302008
[5] V S Beezy and R A Valverde ldquoA comparison ofmethodologiesfor estimating the nest density of olive ridley arribadas atOstional Costa Ricardquo in Proceedings of the 31st Annual Sympo-sium on Sea Turtle Biology andConservation T T Jones andB PWallace Eds NOAA Technical Memorandum NMFS-SEFSC-631 pp 194ndash195 2012
[6] S E Cornelius M A Ulloa J C Castro M Mata del Valleand D C Robinson ldquoManagement of olive ridley sea turtles(Lepidochelys olivacea) nesting at Playas Nancite and OstionalCosta Ricardquo in Neotropical Wildlife Use and Conservation JRobinson and K Redford Eds vol 1 pp 111ndash135 Universityof Chicago Press Chicago Ill USA 1991
[7] R A Valverde S E Cornelius and C L Mo ldquoDecline of theolive ridley sea turtle (Lepidochelys olivacea) nesting assemblageat Nancite Beach Santa Rosa National Park Costa RicardquoChelonian Conservation and Biology vol 3 pp 58ndash63 1998
[8] S Clusella Trullas and F V Paladino ldquoMicro-environment ofolive ridley turtle nests deposited during an aggregated nestingeventrdquo Journal of Zoology vol 272 no 4 pp 367ndash376 2007
[9] D McPherson and D Kibler ldquoEcological impact of olive ridleynesting at Ostional Costa Ricardquo in Proceedings of the 25thAnnual Symposium on Sea Turtle Biology and ConservationH Kalb A Rohde K Gayheart and K Shanker Eds NOAATechnical Memorandum NMFS-SEFSC-582 p 204 2008
[10] C L Mo I Salas and M Caballero ldquoAre fungi and bacteriaresponsible for olive ridleyrsquos egg lossrdquo in Proceedings of the 10thAnnual Workshop on Sea Turtle Biology and Conservation T HRichardson J I Richardson and M Donnelly Eds pp 249ndash252 NOAA Techn 1990
[11] S Honarvar J R Spotila andM P OrsquoConnor ldquoMicrobial com-munity structure in sand on two olive ridley arribada nestingbeaches Playa La Flor Nicaragua and Playa Nancite Costa
Ricardquo Journal of Experimental Marine Biology and Ecology vol409 no 1-2 pp 339ndash344 2011
[12] A D Phillott and C J Parmenter ldquoThe distribution of failedeggs and the appearance of fungi in artificial nests of green(Chelonia mydas) and loggerhead (Caretta caretta) sea turtlesrdquoAustralian Journal of Zoology vol 49 no 6 pp 713ndash718 2001
[13] J Wyneken T J Burke M Salmon and D K Pedersen ldquoEggfailure in natural and relocated sea turtle nestsrdquo Journal ofHerpetology vol 22 no 1 pp 88ndash96 1988
[14] C L Mo M Caballero and I Salas ldquoMicroorganism infectionof olive ridley eggsrdquo in Proceedings of the 12th Annual Workshopon Sea Turtle Biology and Conservation J I Richardson and TH Richardson Eds NOAA Technical Memorandum NMFS-SEFSC-361274 pp 81ndash84 1995
[15] J M Sarmiento-Ramırez E Abella M P Martın et alldquoFusarium solaniis responsible for mass mortalities in nests ofloggerhead sea turtle Caretta caretta in Boavista Cape VerderdquoFEMS Microbiology Letters vol 312 no 2 pp 192ndash200 2010
[16] J M Sarmiento-Ramırez E Abella-Perez A D Phillott etal ldquoGlobal distribution of two fungal pathogens threateningendangered sea turtlesrdquo PLoS ONE vol 9 Article ID e858532014
[17] J Patino-Martinez A Marco L Quinones E Abella R MAbad and J Dieguez-Uribeondo ldquoHow do hatcheries influenceembryonic development of sea turtle eggs Experimental anal-ysis and isolation of microorganisms in leatherback turtle eggsrdquoJournal of Experimental Zoology Part A Ecological Genetics andPhysiology vol 317 no 1 pp 47ndash54 2012
[18] A D Phillott and C J Parmenter ldquoFungal colonization of greensea turtle (Chelonia mydas) nests is unlikely to affect hatchlingconditionrdquo Herpetological Conservation amp Biology vol 9 pp297ndash301 2014
[19] MOcanaMHarfush-Melendez and SHeppell ldquoMass nestingof olive ridley sea turtles Lepidochelys olivacea at La EscobillaMexico linking nest density and rates of destructionrdquo Endan-gered Species Research vol 16 pp 45ndash54 2012
[20] DMadden J Ballestero C Calvo R Carlson E Christians andE Madden ldquoSea turtle nesting as a process influencing a sandybeach ecosystemrdquo Biotropica vol 40 no 6 pp 758ndash765 2008
[21] R Valverde and C Gates ldquoPopulation surveys on mass nestingbeachesrdquo in Research and Management Techniques for theConservation of Sea Turtles K L Eckert K A Bjorndal FA Abreu-Gobrois and M Donnelly Eds IUCNSSC MarineTurtle Specialist Group Publication No 4 1999
[22] B P Wallace P R Sotherland J R Spotila R D ReinaB F Franks and F V Paladino ldquoBiotic and abiotic factorsaffect the nest environment of embryonic leatherback turtlesDermochelys coriaceardquo Physiological and Biochemical Zoologyvol 77 no 3 pp 423ndash432 2004
[23] J D Miller ldquoDetermining clutch size and hatching successrdquo inResearch and Management Techniques for the Conservation ofSea Turtles K L Eckert K A Bjorndal F A Abreu-Groboisand M Donnelly Eds IUCNSSC Marine Turtle SpecialistGroup Publication No 4 1999
[24] S J Blott and K Pye ldquoGradistat a grain size distribution andstatistics package for the analysis of unconsolidated sedimentsrdquoEarth Surface Processes and Landforms vol 26 no 11 pp 1237ndash1248 2001
[25] W Krumbein and F Pettijohn Manual of Sedimentary Petrog-raphy Appleton-Century-Crofts New York NY USA 1938
10 Journal of Marine Biology
[26] N C Prevost-Boure R Christen S Dequiedt et al ldquoValidationand application of a PCR primer set to quantify fungal commu-nities in the soil environment by real-time quantitative PCRrdquoPLoS ONE vol 6 no 9 Article ID e24166 2011
[27] N Fierer J A Jackson R Vilgalys and R B Jackson ldquoAssess-ment of soil microbial community structure by use of taxon-specific quantitative PCR assaysrdquo Applied and EnvironmentalMicrobiology vol 71 no 7 pp 4117ndash4120 2005
[28] A Guidot J-C Debaud and R Marmeisse ldquoSpatial distribu-tion of the below-ground mycelia of an ectomycorrhizal fungusinferred from specific quantification of its DNA in soil samplesrdquoFEMS Microbiology Ecology vol 42 no 3 pp 477ndash486 2002
[29] T B de Gregoris N Aldred A S Clare and J G BurgessldquoImprovement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxardquo Journal of Microbio-logical Methods vol 86 no 3 pp 351ndash356 2011
[30] E J Vainio and J Hantula ldquoDirect analysis of wood-inhabitingfungi using denaturing gradient gel electrophoresis of amplifiedribosomal DNArdquoMycological Research vol 104 no 8 pp 927ndash936 2000
[31] S Turner K M Pryer V P W Miao and J D Palmer ldquoInves-tigating deep phylogenetic relationships among cyanobacteriaand plastids by small subunit rRNA sequence analysisrdquo TheJournal of Eukaryotic Microbiology vol 46 no 4 pp 327ndash3381999
[32] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash174 John Wiley amp Sons Chichester UK1991
[33] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications pp 315ndash322 1990
[34] N Uemura KMakimuraM Onozaki et al ldquoDevelopment of aloop-mediated isothermal amplificationmethod for diagnosingPneumocystis pneumoniardquo Journal of Medical Microbiology vol57 no 1 pp 50ndash57 2008
[35] C J Smith D B Nedwell L F Dong and A M OsbornldquoEvaluation of quantitative polymerase chain reaction-basedapproaches for determining gene copy and gene transcriptnumbers in environmental samplesrdquo Environmental Microbiol-ogy vol 8 no 5 pp 804ndash815 2006
[36] C J Smith and A M Osborn ldquoAdvantages and limitationsof quantitative PCR (Q-PCR)-based approaches in microbialecologyrdquo FEMS Microbiology Ecology vol 67 no 1 pp 6ndash202009
[37] R A Ackerman ldquoOxygen consumption by sea turtle (CheloniaCaretta) eggs during developmentrdquo Physiological Zoology vol54 pp 316ndash324 1981
[38] R A Valverde S Wingard F Gomez M T Tordoir and C MOrrego ldquoField lethal incubation temperature of olive ridley seaturtle Lepidochelys olivacea embryos at a mass nesting rookeryrdquoEndangered Species Research vol 12 no 1 pp 77ndash86 2010
[39] L G Fonseca W N Villachica R E Matarrita and R AValverde ldquoReporte final de la anidacioon de tortuga lora(Lepidochelys olivacea) Playa Nancite Parque Nacional SantaRosa Costa Rica (Temporada 2010ndash2011)rdquo Reporte Final deTemporada al USFWS 2011
[40] C Figgener A Castillo-MacCarthy J Mora-Sandoval YArguelo-Gomez and W Quiros-Pereira ldquoRaising the hatchingsuccess of leatherback nests at the olive ridley mass-nesting
beach inOstional Guanacaste Costa Ricardquo in Proceedings of the34thAnnual Symposiumon SeaTurtle Biology andConservation2014
[41] C Limpus V Baker and J Miller ldquoMovement induced mortal-ity of loggerhead eggsrdquoHerpetologica vol 35 pp 335ndash338 1979
[42] R A Ackerman ldquoThe respiratory gas exchange of sea turtlenests (Chelonia Caretta)rdquo Respiration Physiology vol 31 no 1pp 19ndash38 1977
[43] Y-C Kam ldquoPhysiological effects of hypoxia onmetabolism andgrowth of turtle embryosrdquo Respiration Physiology vol 92 no 2pp 127ndash138 1993
[44] TWibbels D Rostal andR Byles ldquoHigh pivotal temperature inthe sex determination of the olive ridley sea turtle Lepidochelysolivacea from Playa Nancite Costa Ricardquo Copeia no 4 pp1086ndash1088 1998
[45] C J McCoy R C Vogt and E J Censky ldquoTemperature-controlled sex determination in the sea turtle Lepidochelysolivaceardquo Journal of Herpetology vol 17 no 4 pp 404ndash4061983
[46] N A Miller ldquoPO2in loggerhead sea turtle (Caretta caretta)
[47] J A Mortimer ldquoThe influence of beach sand characteristicson the nesting behavior and clutch survival of green turtles(Chelonia mydas)rdquo Copeia vol 1990 no 3 pp 802ndash817 1990
[48] S E Cornelius The Sea Turtles of Santa Rosa National ParkFundacion de Parques Nacionales San Jose Costa Rica 1986
[49] X Chen E Peltier B S M Sturm and C B Young ldquoNitrogenremoval and nitrifying and denitrifying bacteria quantificationin a stormwater bioretention systemrdquo Water Research vol 47no 4 pp 1691ndash1700 2013
[50] R S Seymour and R A Ackerman ldquoAdaptations to under-ground nesting in birds and reptilesrdquo American Zoologist vol20 no 2 pp 437ndash447 1980
[51] R S Seymour D Vleck and C M Vleck ldquoGas exchange in theincubationmounds of megapode birdsrdquo Journal of ComparativePhysiology B vol 156 no 6 pp 773ndash782 1986
[26] N C Prevost-Boure R Christen S Dequiedt et al ldquoValidationand application of a PCR primer set to quantify fungal commu-nities in the soil environment by real-time quantitative PCRrdquoPLoS ONE vol 6 no 9 Article ID e24166 2011
[27] N Fierer J A Jackson R Vilgalys and R B Jackson ldquoAssess-ment of soil microbial community structure by use of taxon-specific quantitative PCR assaysrdquo Applied and EnvironmentalMicrobiology vol 71 no 7 pp 4117ndash4120 2005
[28] A Guidot J-C Debaud and R Marmeisse ldquoSpatial distribu-tion of the below-ground mycelia of an ectomycorrhizal fungusinferred from specific quantification of its DNA in soil samplesrdquoFEMS Microbiology Ecology vol 42 no 3 pp 477ndash486 2002
[29] T B de Gregoris N Aldred A S Clare and J G BurgessldquoImprovement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxardquo Journal of Microbio-logical Methods vol 86 no 3 pp 351ndash356 2011
[30] E J Vainio and J Hantula ldquoDirect analysis of wood-inhabitingfungi using denaturing gradient gel electrophoresis of amplifiedribosomal DNArdquoMycological Research vol 104 no 8 pp 927ndash936 2000
[31] S Turner K M Pryer V P W Miao and J D Palmer ldquoInves-tigating deep phylogenetic relationships among cyanobacteriaand plastids by small subunit rRNA sequence analysisrdquo TheJournal of Eukaryotic Microbiology vol 46 no 4 pp 327ndash3381999
[32] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic acid Tech-niques in Bacterial Systematics E Stackebrandt and M Good-fellow Eds pp 115ndash174 John Wiley amp Sons Chichester UK1991
[33] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications pp 315ndash322 1990
[34] N Uemura KMakimuraM Onozaki et al ldquoDevelopment of aloop-mediated isothermal amplificationmethod for diagnosingPneumocystis pneumoniardquo Journal of Medical Microbiology vol57 no 1 pp 50ndash57 2008
[35] C J Smith D B Nedwell L F Dong and A M OsbornldquoEvaluation of quantitative polymerase chain reaction-basedapproaches for determining gene copy and gene transcriptnumbers in environmental samplesrdquo Environmental Microbiol-ogy vol 8 no 5 pp 804ndash815 2006
[36] C J Smith and A M Osborn ldquoAdvantages and limitationsof quantitative PCR (Q-PCR)-based approaches in microbialecologyrdquo FEMS Microbiology Ecology vol 67 no 1 pp 6ndash202009
[37] R A Ackerman ldquoOxygen consumption by sea turtle (CheloniaCaretta) eggs during developmentrdquo Physiological Zoology vol54 pp 316ndash324 1981
[38] R A Valverde S Wingard F Gomez M T Tordoir and C MOrrego ldquoField lethal incubation temperature of olive ridley seaturtle Lepidochelys olivacea embryos at a mass nesting rookeryrdquoEndangered Species Research vol 12 no 1 pp 77ndash86 2010
[39] L G Fonseca W N Villachica R E Matarrita and R AValverde ldquoReporte final de la anidacioon de tortuga lora(Lepidochelys olivacea) Playa Nancite Parque Nacional SantaRosa Costa Rica (Temporada 2010ndash2011)rdquo Reporte Final deTemporada al USFWS 2011
[40] C Figgener A Castillo-MacCarthy J Mora-Sandoval YArguelo-Gomez and W Quiros-Pereira ldquoRaising the hatchingsuccess of leatherback nests at the olive ridley mass-nesting
beach inOstional Guanacaste Costa Ricardquo in Proceedings of the34thAnnual Symposiumon SeaTurtle Biology andConservation2014
[41] C Limpus V Baker and J Miller ldquoMovement induced mortal-ity of loggerhead eggsrdquoHerpetologica vol 35 pp 335ndash338 1979
[42] R A Ackerman ldquoThe respiratory gas exchange of sea turtlenests (Chelonia Caretta)rdquo Respiration Physiology vol 31 no 1pp 19ndash38 1977
[43] Y-C Kam ldquoPhysiological effects of hypoxia onmetabolism andgrowth of turtle embryosrdquo Respiration Physiology vol 92 no 2pp 127ndash138 1993
[44] TWibbels D Rostal andR Byles ldquoHigh pivotal temperature inthe sex determination of the olive ridley sea turtle Lepidochelysolivacea from Playa Nancite Costa Ricardquo Copeia no 4 pp1086ndash1088 1998
[45] C J McCoy R C Vogt and E J Censky ldquoTemperature-controlled sex determination in the sea turtle Lepidochelysolivaceardquo Journal of Herpetology vol 17 no 4 pp 404ndash4061983
[46] N A Miller ldquoPO2in loggerhead sea turtle (Caretta caretta)
[47] J A Mortimer ldquoThe influence of beach sand characteristicson the nesting behavior and clutch survival of green turtles(Chelonia mydas)rdquo Copeia vol 1990 no 3 pp 802ndash817 1990
[48] S E Cornelius The Sea Turtles of Santa Rosa National ParkFundacion de Parques Nacionales San Jose Costa Rica 1986
[49] X Chen E Peltier B S M Sturm and C B Young ldquoNitrogenremoval and nitrifying and denitrifying bacteria quantificationin a stormwater bioretention systemrdquo Water Research vol 47no 4 pp 1691ndash1700 2013
[50] R S Seymour and R A Ackerman ldquoAdaptations to under-ground nesting in birds and reptilesrdquo American Zoologist vol20 no 2 pp 437ndash447 1980
[51] R S Seymour D Vleck and C M Vleck ldquoGas exchange in theincubationmounds of megapode birdsrdquo Journal of ComparativePhysiology B vol 156 no 6 pp 773ndash782 1986
