Embryonic development and metabolic costs in Gulf killifishFundulus grandis exposed to varying environmentalsalinities

Charles A. Brown • Fernando Galvez •

Christopher C. Green

Received: 9 March 2011 / Accepted: 16 December 2011 / Published online: 18 January 2012

� Springer Science+Business Media B.V. 2012

Abstract The Gulf killifish (Fundulus grandis) is a

euryhaline fish found in coastal marsh along the entire

of Gulf of Mexico and southern Atlantic of coast of the

United States. The objective of this study was to

investigate the effects of salinity on embryogenesis

in the Gulf killifish. Four recirculation systems at

salinities of 0.4, 7, 15, and 30 g/L were maintained at a

static temperature with flow-through trays, containing

embryos (n = 39) placed in triplicate into each

system. Throughout embryogenesis, the rate of devel-

opment, ammonia and urea excretion, and heart rate

were monitored. Percent hatch was recorded, and

morphological parameters were measured for larvae at

hatch. As salinity was increased, the rate of embryo-

genesis decreased. Salinity significantly affected

percent hatch with an 80.0% ± 2.6% for 7 g/L and

39.1 ± 4.3, 45.4 ± 4.5, and 36.3% ± 12.0% for 0.4,

15, and 30 g/L, respectively. Salinity and stage of

development significantly affected production of

ammonia and urea. As salinity increased, the dominate

metabolite end product changed from urea to

ammonia. However, the 15 g/L salinity treatment

had the two highest levels of urea recorded. Heart rate

was unaffected by salinity but increased throughout

embryogenesis and remained constant once embryos

reached stages where hatching has been recorded.

While mean total length was not affected by salinity,

embryos incubated in 30 g/L produced larvae with

significantly thicker body depth at hatch. The 0.4, 7,

and 15 g/L salinity treatments all had similar mean

hours to hatch. The 30 g/L treatment resulted in a

significantly longer mean time to hatch and smaller

body cavity area at hatch.

Keywords Fundulus � Embryogenesis � Salinity �Metabolite � Development

Introduction

Embryonic osmoregulation is important in salt marsh

species of teleosts that produce sessile eggs. The Gulf

killifish (Fundulus grandis) is a euryhaline cyprin-

odontid native to the coastal salt marshes of the Gulf of

Mexico and southern Atlantic coast of the United

States (Nordlie 2000; Oesterling et al. 2004). This

species is a fractional spawner with a protracted

spawning period, peaking in the spring and fall months

(Nordlie 2000). Throughout the year, most popula-

tions of Gulf killifish reside and spawn in marshes

where salinities range from 5 to 39 g/L, with some

C. A. Brown � C. C. Green (&)

Louisiana State University Agricultural Center,

Aquaculture Research Station, 2410 Ben Hur Road,

Baton Rouge, LA 70820, USA

e-mail: cgreen@agcenter.lsu.edu

F. Galvez

Department of Biological Sciences, Louisiana State

University, 216 Life Sciences Building, Baton Rouge,

LA 70803, USA

123

Fish Physiol Biochem (2012) 38:1071–1082

DOI 10.1007/s10695-011-9591-z

populations found in entirely freshwater conditions

(Simpson and Gunter 1956; Nordlie 2000, 2006).

While adults have been shown to relocate to more

favorable salinities depending on temperature,

embryos continue to develop throughout a wide range

of salinities (Greeley and MacGregor 1983; Miller

et al. 1983).

Recent work on ion regulation and gas exchange at

early stages of life-history has demonstrated that the

gills of embryonic and larval fish play a more critical

role in ion regulation than they do in gas exchange (for

review see Rombough 2007). Developing embryos

can rely solely on passive diffusion of oxygen across

their body, when the external surface area to biomass

ratio is high, though the development of a functioning

heart and circulatory system occur before it is needed

to address respiratory demand of embryos (Pelster and

Burggren 1996; Kranenbarg et al. 2000). Osmoregu-

lation is also performed on the body surface due to the

localization of ion-transporting cells during early

development. Embryonic mummichog (Fundulus het-

eroclitus), a Gulf killifish sister species found along

the Atlantic coast, use mitochondrion-rich cells

located in the basolateral membrane of the yolk sac

to actively osmoregulate in hypertonic environments

until the gills form and mitochondrion-rich cells begin

to emerge on the gill surface of embryos immediately

before hatch (Evans et al. 1999; Katoh et al. 2000;

Burnett et al. 2007). Despite active regulation of ionic

movements, salinity has not been found to influence

oxygen demand of mummichog and larval striped

mullet (Mugil cephalus; Walsh et al. 1989; Kidder

et al. 2006a). Prior to spawning, eggs retain maternal

osmotic conditions but rapidly adjust to the surround-

ing osmotic pressure by absorption of water into the

perivitelline space upon fertilization (Holliday and

Blaxter 1960; Davenport et al. 1981; Finn 2007).

Water permeability and passive transfer of osmolytes

between the embryo and its environment are mini-

mized by the chorion and perivitelline space after

water hardening (Alderdice 1988; Finn 2007).

Metabolic by-products in the form of ammonia and

urea are produced by physiological processes includ-

ing growth, cellular maintenance, and osmoregulation.

Ammonia is the primary metabolite produced in adult

fish, which despite its high toxicity, can be excreted

passively and continually into surrounding water

under most conditions (Mommsen and Walsh 1992;

Walsh 1998; Randall and Tsui 2002). In contrast,

many embryonic fish are capable of producing urea as

their primary nitrogenous waste, facilitating storage of

this much less toxic substance rather than passive

diffusion of ammonia across the chorion (Depeche

et al. 1979; Wright and Land 1998). Several pathways

of urea production and transport exist in teleosts,

including arginine catabolism, purine catabolism, and

the ornithine-urea cycle (OUC; Walsh 1998). The

OUC is the most active pathway of ammonia conver-

sion to urea for embryonic and larval teleosts (Griffith

1991), but becomes inactive in most fish species later

in development. Urea production via the OUC is

energetically costly but may be critical to survival

in situations where ammonia may not be readily

expelled, such as environments that are alkaline with

high buffering capacities or with high external

ammonia concentrations (Wood et al. 1989; Walsh

1998). Urea production is also a product of growth due

to its liberation by arginase during catabolism of

endogenous free amino acids (FAAs; Watford 2003).

Arginase catabolism produces ornithine, which can

subsequently be diverted into polyamine biosynthesis

through the coordinated action of ornithine decarbox-

ylase (Pegg 2006; Montanez et al. 2007). Polyamines

are a family of highly cationic molecules implicated in

numerous physiological processes including cell pro-

liferation, growth, and apoptosis. Ornithine decarbox-

ylase has been shown to be among the most highly

differentially expressed transcripts following hypoos-

motic transfer in a wide array of biota (Davis and

Ristow 1995; Watts et al. 1996; Mitchell et al. 1998;

Henry and Watts 2001) including fishes (Hascilowicz

et al. 2002; Whitehead et al. 2010, 2011). Hypersaline

conditions have also been shown to influence urea

excretion in adult mangrove killifish (Rivulus marmo-

ratus) as urea, and non-essential FAAs are retained for

osmoregulatory purposes at the cost of decreased rates

of FAA catabolism (Frick and Wright 2002).

Embryogenesis has been shown to be influenced by

environmental salinity in euryhaline teleosts. Though

adult California killifish (Fundulus parvipinnis) have

been found in isolated pools at salinities of up to

70 g/L, embryos display high rates of mortality due to

sensitivity of high osmotic pressure prior to gastrula-

tion (Rao 1974). Upper salinity tolerances among

larval teleosts reflect environmental salinities typi-

cally encountered during development (see review in

Bunn et al. 2000). Annual killifish (Austrofundulus

limnaeus) embryos typically develop in hypersaline

1072 Fish Physiol Biochem (2012) 38:1071–1082

123

ephemeral pools at salinities in excess of 50 g/L and

maintain a physiological osmolarity of 290 mOsmol/

kg H2O due to the low chorionic permeability to salts

and water (Machado and Podrabsky 2007). Salinities

outside of the optimal osmoregulatory ranges of

embryos have negative impacts on embryos during

incubation, which are usually manifested in the forms

of decreased growth and rate of development (Dushk-

ina 1973; Bunn et al. 2000; Boeuf and Payan 2001;

Sampaio and Bianchini 2002).

The purpose of this study was to determine the

influence of salinity on embryogenesis in Gulf killi-

fish. This study determined to what degree environ-

mentally relevant salinities ranging from 0.4 to 30 g/L

influence embryogenesis (Simpson and Gunter 1956;

Nordlie 2000, 2006). To test this hypothesis, we

measured the rates of embryogenesis, time to hatch,

percent hatch, morphological development at hatch,

heart rate, and production of ammonia and urea for

developing embryos. In particular, this investigation

tested how the osmotic stress from a range of salinities

manifested itself during Gulf killifish embryogenesis.

Specifically, this study tested the interaction of salinity

and stage of embryonic development on heart rate, as a

functioning circulatory system has been shown not to

be needed in the transport and deliver of oxygen in

embryonic Gulf killifish until well after hatch has

occurred. The influence of the interaction between

salinity and stage of embryogenesis on the concentra-

tions of nitrogenous end products, ammonia and urea,

were also be tested in this investigation.

Materials and methods

Water quality, sourcing of gametes,

and experimental design

A broodstock of Gulf killifish was obtained from

Grand Isle, Louisiana and held in outdoor pools

maintained at the Louisiana State University AgCen-

ter’s Aquaculture Research Station (Baton Rouge, LA,

USA) at 29.0 ± 0.1�C and a salinity of 7.6 g/L. Initial

alkalinity and hardness (reported as mg/L CaCO3) for

each treatment were determined by titration using

water quality testing kits (Hach Co., Loveland, CO,

USA). Salinity, temperature, and DO were measured

with a YSI 85 (YSI Inc., Yellow Springs, OH, USA).

An Accumet� Basic AB15 pH meter (Fisher

Scientific, Pittsburgh, PA, USA) was used to measure

pH. Total ammonia nitrogen and nitrite nitrogen were

measured with a Hach� DR 4000 Spectrophotometer

using the salicylate (Hach� Method 10023) and

diazotization (Hach� Method 8507) methods,

respectively.

To obtain embryos for this study, 40 females and 5

males were collected from the broodstock population

and manually strip spawned. Eggs were fertilized as

described by Brown et al. (2011). Newly fertilized

eggs were soaked in a 2 g/L solution of sodium sulfite

for *15 min to decrease adhesion (Stone and Ludwig

1993). Embryos were then enumerated and divided

into 12 baskets with mesh screen ports (39 embryos

per basket) in a flow-through recirculating system in

the Life Sciences Aquatic’s Facility in the Department

of Biological Sciences (LSU). Baskets were placed in

triplicate into the four independent recirculating

systems. Four salinity treatments were previously

mixed to nominal salinities of 0.4, 7, 15, and 30 g/L

using Instant Ocean� Salt Mix (Aquarium Systems

Inc., Mentor, OH, USA). A photoperiod of 12:12

light:dark cycle was maintained for the treatment

systems. Each system was outfitted with a LogTag

Temperature Recorder (LogTag Recorders, Auckland,

New Zealand). Twenty-nine additional newly fertil-

ized eggs were placed in triplicate into static trays at

7 g/L to obtain a baseline fertilization rate. Percent

hatch was not recorded because embryos were killed

throughout this study for a separate experiment.

A second experiment was performed to examine the

influence of salinity on percent hatch. Six females and

seven males were obtained from the same brood stock

population to obtain embryos for this study. Forty-four

eggs were placed in triplicate into four recirculating

systems, each set at 0.4, 7, 15, and 30 g/L salinity.

Eggs were allowed to develop until hatch, and percent

hatch was calculated. Baseline fertilization for this

experiment was determined by placing 40, 41, and 54

eggs into three static trays at a salinity of 7 g/L.

Staging for embryogenesis

The duration of time from fertilization through five

distinct stages of development was recorded to

determine the rate of embryogenesis among salinity

treatments. Staging was based on embryological

development described by Armstrong and Child

(1965) for the mummichog. Relevant stages were

Fish Physiol Biochem (2012) 38:1071–1082 1073

123

selected based on ease of visual confirmation and

significance to metabolite processing in the forms of

ammonia and urea. Stage 15 was characterized by the

earliest formation of the germ ring around the

blastoderm. Stage 19 was characterized by formation

of eye buds on the embryonic keel, which had formed

definitive margins. Stage 25 was characterized by the

onset of circulation, although blood cells did not

appear to be pigmented at this stage. Ocular structures

appeared to be fully formed but lacked any pigmen-

tation. Melanophores were present but had not fully

expanded. Stage 28 was characterized by retinal

pigmentation. Blood cells had developed pigmenta-

tion, and melanophores had begun to expand. Pro-

nephros was reported to be active from stage 27. Stage

34 was characterized by a fully formed and open lower

jaw. Hatching can occur at this stage due to the

liberation of chorionase from hatching glands located

in the gill and buccal cavity of mummichog (DiMic-

hele and Taylor 1981). Occasional movements of the

jaw and pectoral fins occurred at stage 34. The caudal

fin was also well developed with blood vessels

radiating parallel to the rays of the fin. Stage 35 was

characterized by the extension of the head, with eyes

that moved and responded to light stimuli. Five

embryos were randomly selected from each replicate

salinity treatment throughout each day of the exper-

iment to determine developmental stage. When 80%

of embryos from a replicate were at a target stage, they

were subsampled for ammonia, urea, and heart rate.

Metabolite sampling

Water ammonia and urea were determined as a

measure of their whole-body production and excretion

rates from four embryos per treatment replicate in

1.5 mL of sampling medium in triplicate. A fourth

well was filled with 1.5 mL of sample water without

embryos to serve as a blank. Embryos remained in the

sample water for 4 h at which time they were placed

back into their respective treatment and replicate

systems. Sample water was stored at -20�C prior to

analysis. To test samples for urea, a portion of sample

water was lyophilized with a Lyph-Lock18� (Labco-

noco�, Kansas City, MO, USA) to concentrate the

sample and reconstituted with 500 lL of de-ionized

water. The remaining original sample was tested for

ammonia with the spectrophotometric technique

described by Verdouw et al. (1978). Standard curves

were prepared for each salinity treatment at concen-

trations of 0, 25, 50, 75, 100, 125, and 150 lmol/L

(NH4)2SO4 and were prepared according to Verdouw

et al. (1978). Standard curves for each salinity

treatment ammonia assay had r2 values of 0.97 or

greater. Water samples for urea analysis were prepared

in triplicate for spectrophotometric assay with a

QuaniChromTM Urea Assay Kit as per manufacturer’s

instructions (BioAssay Systems, Hayward, CA, USA;

Jung et al. 1975). Standard curves were prepared for

each salinity treatment via serial dilution at concen-

trations of 0, 0.02, 0.26, 1.30, 2.60, 13.00, and

26.01 lmol/L CH4N2O and were prepared according

to QuaniChromTM Urea Assay Kit instructions. Stan-

dard curves for each salinity treatment urea assay had

r2 values of 0.99 or greater. Spectrophotometric assays

were read with a BioTek� Synergy 2 Multi-Purpose

Microplate Reader (BioTek�, Winooski, VT, USA)

and interpreted using Gen5TM analysis software

(BioTek�, Winooski, VT, USA). Assuming that

ammonia and urea excretion rates were the primary

forms of nitrogenous waste excretion, we calculated

total nitrogen (TN; lmol/L/h) excretion using the

formula.

TN ¼ ðð2U) + AÞ ð1Þ

where U equals urea concentrations (lmol/L/h) and A

equals ammonia concentrations (lmol/L/h).

Heart rate sampling

From the onset of circulation (stage 25), heart rates of

embryos (n = 3) were recorded for each treatment

replicate (n = 9 total) at target stages for salinity

treatments and reported as beats per minute (BPM).

Heart rate was recorded by videotaping the hearts of

individual embryos for a minute with a digital video

camera (Sony Digital Handycam, NY, USA) inter-

faced with a dissecting microscope at a magnification

of 209.

Size at hatch

Treatment groups were monitored twice daily for

hatched larvae. Larvae were preserved in 10%

buffered formalin, and time-to-hatch (TH) from

fertilization was recorded. Total length (TL), body-

depth-at-vent (BD), and body cavity area (BCA) were

determined for these larvae with techniques described

1074 Fish Physiol Biochem (2012) 38:1071–1082

123

by Brown et al. (2011). BCA was measured instead of

yolk volume due to the inability to distinguish yolk

from the abdominal cavity.

Statistical analysis

A general linearized model analysis of variance

(ANOVA) in randomized block design was performed

to test for significant differences in embryo morpho-

metrics, TH from fertilization, and urea and ammonia

excretion among salinities, stage, and the interaction

of salinity and stage. The Ryan–Einot–Gabriel–Wel-

sch (REGWQ) post hoc test was used for multiple

comparisons among treatment groups. Logistic regres-

sion analysis was used to determine the dependence of

hatch on salinity. Hatch percentages were arcsine-

transformed and analyzed with a general linearized

model ANOVA. Multivariate analysis of variance

(MANOVA) was used to determine morphometric

variance among salinity treatments. Principal compo-

nent analysis was used to test for multicollinearity

among yolk morphometric measurements. All tests

were performed with Statistical Analysis Software,

Version 9.1 of the SAS System for Windows (SAS

Institute Inc., Cary, NC, USA). Results are shown

as means ± standard error of the mean (SEM). All

hypotheses were tested at a significance level of

a = 0.05.

Results

Percent fertilization and percent hatch

Mean fertilization was 69.7% ± 3.2% at 7 g/L salin-

ity. Salinity significantly affected percent hatch with

mean hatch percentages of 39.1 ± 2.5, 80.0 ± 1.5,

45.4 ± 2.6, and 36.3% ± 6.9% for salinity trials of

0.4, 7, 15, and 30 g/L, respectively (P B 0.05). Mean

water quality parameters throughout the embryo

incubations are presented in Table 1.

Time-to-hatch and rate of development

General linearized model ANOVA indicated that

salinity had a significant effect on TH (P B 0.05).

First appearance hatch was about 245 h after fertil-

ization for the 0.4, 7, and 15 g/L treatments and 287 h

for the 30 g/L treatment (Fig. 1). The shortest mean

TH was 292.3 ± 29.3 h post-fertilization (hpf) for the

0.4 g/L treatment. Post hoc analysis determined that

mean TH was significantly longer for 30 g/L at

368.2 ± 30.6 hpf. Mean TH for the remaining treat-

ments is reported in Table 2. No statistical inference

could be established for the rate of development due

to the lack of variance among replicates in their

treatment response (Fig. 2).

Table 1 Mean water quality parameters ± standard error of the mean for the salinity trials

Water quality Nominal salinity (g/L)

0.4 7 15 30

Salinity (g/L) 0.53 ± 0.01 7.13 ± 0.05 14.95 ± 0.11 27.85 ± 0.20

DO (mg/L)a 7.29 ± 0.08 7.02 ± 0.07 6.62 ± 0.06 6.07 ± 0.05

Temperature (�C) 25.12 ± 0.09 24.51 ± 0.07 24.59 ± 0.08 24.61 ± 0.08

pH 7.70 ± 0.04 7.62 ± 0.02 7.81 ± 0.02 7.89 ± 0.01

TAN (mg/L)b NDd ND ND ND

Nitrite (mg/L) ND ND ND ND

Alkalinity (mg/L)c 40 68 80 118

Hardness (mg/L)c 92 1,610 2,990 5,300

a Dissolved Oxygenb Total Ammonia Nitrogenc Reported as CaCO3, at initiation of studyd Not detectible

Fish Physiol Biochem (2012) 38:1071–1082 1075

123

Total length, body-depth-at-vent, and body cavity

area

A statistically significant effect of salinity on TL at

hatch was determined (P B 0.05), and mean TL for the

15 g/L treatment was significantly shorter than the

other salinity groups (Table 2). Salinity had a statis-

tically significant effect on BD (P B 0.05), with BD

generally demonstrated to increase as salinity

increased (Table 2). A PCA of the size at hatch data

found a principal component that accounted for 80% of

the variance of the data. The first principal component

is related to the length of the yolk sac, depth of the yolk

sac, and BCA. A statistically significant effect of

salinity on BCA was determined (P B 0.05). Post hoc

analysis found that BCA at hatch was significantly

smaller for the 30 g/L treatment than the remaining

salinities. MANOVA results indicated a significant

effect of salinity on morphometric parameters (P B

0.05), with salinity explaining 58% of the variation

among morphometric variables.

Heart rate

Salinity did not have a significant effect on heart rate

(P C 0.05); however, stage of development did have a

significant effect on heart rate (P B 0.05). BPM

increased as stage increased for all salinity treatments

except 15 g/L. Post hoc analysis determined a signif-

icant increase in mean heart rate at stage 28, followed

by a significant decrease in BPM at stage 34 for the

15 g/L salinity treatment (Fig. 3).

Stage and metabolite excretion

Ammonia excretion was significantly affected by the

interaction between salinity and stage (P B 0.05).

Ammonia excretion was high at stage 15 among all 4

salinity treatments, but decreased significantly as

embryogenesis progressed (Fig. 4a). Significant main

and interaction effects for salinity and stage were

detected for urea excretion (P B 0.05). Urea excretion

remained low throughout embryogenesis for the two

lowest salinity treatments of 0.4 and 7 g/L, except at

Fig. 1 Duration of hatch for the nominal incubation salinities.

Mean percent duration of hatch (h) post-fertilization exposed to

treatment salinities. Lines depict incubation salinities: 0.4, 7, 15,

and 30 g/L

Table 2 Morphometric parameters at hatch and mean time to hatch (hours ± standard error of the mean) for embryos reared in

varying salinities

Salinity (g/L) Larvae (n) Length (mm) Body-depth-at-vent (mm) Body cavity area (m2) Mean time to hatch (h)

0.4 27 6.57 ± 0.11a 0.63 ± 0.01b 0.71 ± 0.04a,b 292.3 ± 29.3b

7 21 6.55 ± 0.12a 0.64 ± 0.01ab 0.66 ± 0.04b 312.2 ± 29.6b

15 33 6.18 ± 0.11b 0.60 ± 0.01c 0.79 ± 0.04a 301.7 ± 29.1b

30 15 6.72 ± 0.12a 0.66 ± 0.01a 0.49 ± 0.05c 368.2 ± 30.6a

Columns with different superscript are significantly different (Ryan–Enoit–Gabriel–Welsch-studentized range; P \ 0.05)

Fig. 2 Hours post-fertilization required for Gulf killifish

embryos to reach stages 15, 19, 25, 28, 34, and 35 according

to the characterization by Armstrong and Child (1965). Columnsdepict incubation salinities: 0.4, 7, 15, and 30 g/L

1076 Fish Physiol Biochem (2012) 38:1071–1082

123

stages 25 and 28 when excretion significantly

increased (Fig. 4b). Urea excretion varied throughout

the 15 g/L incubation treatment and reached the

highest recorded levels among treatments. No urea

was detected throughout the 30 g/L treatment except

at stage 25. These combined effects of salinity on urea

and ammonia led to a significant effect of saintly on

total nitrogen excretion (P \ 0.05).

Discussion

The effect of salinity on the rate of embryogenesis

has been studied in many teleost species, with an

overall observation that ontogenic responses and the

extent of these responses are species-specific, indi-

cating that species have adapted to native salinity

ranges (Laurence and Rogers 1976; Fonds 1979;

Imsland et al. 2001; Cook et al. 2005). In the present

study, the rate of embryogenesis was most affected

by salinity at stage 25, marked by the onset of

circulation, and beyond, tending to decrease as

salinity was increased beyond isotonic levels

(Fig. 2). When examining the effect of salinity on

mummichog embryogenesis, Tay and Garside (1975)

did not find a change in the rate of development in

salinities ranging from 0 to 30 g/L. However, they

reported that hypersaline conditions (60 g/L) began

to slow embryogenesis at stages 25 and above

(Fig. 2). Perschbacher et al. (1990) demonstrated

that mean TH of Gulf killifish did not statistically

differ between salinities of 0 to 30 g/L. In the current

experiment, only the 30 g/L treatment had signifi-

cantly longer mean TH. Salinities outside optimal

osmotic range, which likely represents the energet-

ically least expensive salinity, may also reduce

percent hatch. Despite a uniform mean TH for the

three lowest salinity treatments in the current study,

we observed a reduced hatch percent of 39.1% ±

4.3% for the 0.4 g/L treatment compared to

Fig. 3 Mean heart rate (beats per minute ± standard error of

the mean) for embryos at different stages of development.

Letters above columns depict significant differences among

salinity treatments at stages 25, 28, 34, and 35 of development

according to the characterization by Armstrong and Child

(1965) (Ryan–Enoit–Gabriel–Welsch-studentized range;

P \ 0.05). Columns depict incubation salinities: 0.4, 7, 15,

and 30 g/L

Fig. 4 Mean metabolites produced ± standard error of the

mean from embryos reared in four different salinities for

ammonia (a), urea (b), and total nitrogen excretion (c). Capitalletters depict statistical differences in metabolite production

among stages (Ryan–Enoit–Gabriel–Welsch-studentized range;

P \ 0.05). Lowercase letters depict statistical differences in

metabolite production among salinities (Ryan–Enoit–Gabriel–

Welsch-studentized range; P \ 0.05)

Fish Physiol Biochem (2012) 38:1071–1082 1077

123

80.0% ± 2.6% for 7 g/L. Rao (1974) determined that

California killifish embryos had a longer mean TH

and reduced hatching percentages of 33.3 and 51.4%,

respectively, in freshwater compared to 76.8 and

75.8% for 5 g/L.

Minimum osmotic effort occurs most often when

the incubation medium is isotonic with the yolk and

perivitelline fluids (Alderdice et al. 1979). At tonic-

ities outside of this optimal range, energy may be

diverted away from growth and development of

embryos (Rao 1974; Boeuf and Payan 2001; Sampaio

and Bianchini 2002). In isotonic conditions, mummi-

chog are estimated to devote less than 1% of their total

energy use to osmotic regulation compared to an

*10% energy expenditure when mummichog are

transferred to a higher salinity medium and a *1%

energy expenditure when mummichog are transferred

to a lower salinity medium (Kidder et al. 2006b); these

data also attest to the potentially more costly effects of

hypoosmoregulation for Gulf killifish. Tilapia (Ore-

ochromis mossambicus) embryos reared in saltwater

produced an increased number and size of mitochon-

drion-rich cells located on the yolk-sac membrane

compared to that observed in freshwater-reared

embryos (Ayson et al. 1994). In the current study,

the larvae in the 30 g/L treatment had a significantly

smaller BCA at hatch compared to lower salinity

treatments. Although there was a significant influence

of salinity on BCA at hatch in the current study, there

was no significant influence of salinity on TL at hatch.

Gulf killifish embryos hatched within a narrow range

of mean TL among the four salinity treatments, which

has also been demonstrated in mummichog when

incubated across a similar wide range of salinities

(Fortin et al. 2008). Though hatching with greater yolk

volume would afford larvae more time between

endogenous and exogenous feeding (Miller et al.

1988), a decrease in heterogeneity of TH and TL could

lead to decreased occurrences of intracohort canni-

balism (Baras and Jobling 2002; Fessehaye et al.

2006).

The primary form of nitrogen excretion and the

total amount of nitrogen excreted during embryogen-

esis were strongly influenced by salinity and stage of

development (Fig. 4). There are many factors that

influence the production of nitrogenous metabolites

during early development, including the processing of

yolk nutrients, cellular maintenance, growth, and

development, with the amount of energy required for

these tasks increasing proportionately to the mass of

the embryo (Rombough 2011). A pulse in total

nitrogenous waste excretion was observed in the

15 g/L treatment between stages 25 and 28, which

coincided temporally with the initiation of heart

contractions and the activation of the pronephros,

respectively (Fig. 4c). During embryogenesis, one of

the earliest functioning organs is the heart (Pelster

2002); however, the onset of heart contractions occurs

well before the need of a recirculating system to meet

oxygen demand of tissues in embryonic zebrafish

(Danio rerio), since adequate amounts of oxygen can

diffuse through the skin of embryos (Pelster and

Burggren 1996; Barrionuevo and Burggren 1999;

Rombough 2007). Blood flow mediated by the onset of

heart contractions may function alternatively to facil-

itate transport of other nutrients or to rid the body of

metabolites (Pelster and Burggren 1996).

Regardless of incubation salinity, all embryos had

comparably high levels of ammonia excretion at stage

15. In the current experiment, water hardening

occurred approximately 18–20 h prior to embryos

reaching stage 15. The pulse of ammonia observed at

this stage may be related to the formation of cross-

linking isopeptide bonds during water hardening of the

chorion (Oppen-Bersten et al. 1990). Apart from this

salinity-independent spike at stage 15, ammonia

excretion rates were relatively low except for those

in the 30 g/L-reared embryos at stage 25 and beyond.

There was no detectable ammonia excretion in

embryos reared in 0.4 g/L after the pulse at stage 15,

and only negligible excretion in embryos exposed to

7.0 g/L, between stages 19 and 35. Recent work has

suggested that in freshwater fish, ammonia is excreted

passively via a suite of rhesus (Rh) glycoproteins

expressed on the gill epithelium (Wright and Wood

2009). Ammonia is then thought to combine with H?

from a V-type H-ATPase or sodium proton exchange

mechanism to produce ammonium, which remains

functionally trapped within water. Although their role

in ammonia excretion in Gulf killifish is not known,

Rh glycoproteins are expressed during early embry-

onic development in other teleost fish, potentially

explaining the tendency of freshwater-acclimated fish

to convert from ureotelism to ammoniotelism

(Reviewed in Wright and Wood 2009). This ammonia

pump system appears most active under conditions of

low environmental sodium (Tsui et al. 2009) and not

functional in sea water due to the development of

1078 Fish Physiol Biochem (2012) 38:1071–1082

123

opposing Na? and transepithelial gradients at the gills

of marine fish (Evans 2008). This suggests that either

Rh glycoproteins are not expressed in Gulf killifish

under hypoosmotic conditions or that, if present, these

proteins function or are regulated in a unique manner.

Interestingly, euryhaline killifish species are ostensi-

bly marine teleosts, which despite the ability to

acclimate to freshwater, maintain much of their

marine gill phenotype at concentrations approaching

freshwater (i.e., at 0.4 g/L; Copeland 1950). Under

hyperosmotic conditions, a presumptive Na?/NH4?

exchange (NHE-3) mechanism is thought to exist in

the gills of marine fish and function in nitrogenous

waste excretion (Reviewed in Wright and Wood

2009). There is evidence that NHE-3 functions in this

ammonium excretory capacity under high saline

conditions of the proximal tubule of the mammalian

kidney (Reviewed in Wiener and Hamm 2007).

Interestingly, NHE-3 is localized to the apical mem-

brane of the gills of mummichog, and its expression

increases during seawater acclimation (Scott et al.

2008), supporting a possible role of this protein in

ammonium excretion and acid–base regulation under

hyperosmotic conditions. Alternatively, ammonium

may be eliminated in hyperosmotic conditions by

means of an apical Na?/NH4? exchange, in addition to

passive diffusion of NH4? through paracellular gap

junctions (Wilkie 1997, 2002). Clearly, there is a need

to identify the role of Rh glycoproteins, NHE-3, or

other pathways in ammonia excretion in marine

teleosts, including Gulf killifish, during early-life

development.

Urea excretion rates were also strongly influenced

by salinity and stage of embryonic development

(Fig. 4b). We noted that urea excretion increased

rapidly at stage 28 in 15 g/L and remained elevated

throughout stage 35 (Fig. 4b), accounting for the high

total nitrogen excretion rates in this treatment

(Fig. 4c). This is in contrast to the 0.4 and 7 g/L-

reared embryos, which excreted a lower, yet more

continual amount of urea, or the 30 g/L-reared

embryos, which excreted no urea (Fig. 4b, c). Several

studies have demonstrated a functional ornithine-urea

cycle in embryonic fish; a function that is lost in most

teleost fish shortly after hatch (Wright et al. 1995).

Urea synthesis may also occur by arginase catabolism

and polyamine production (Montanez et al. 2007).

Urea production has been found to be influenced by

the stage of embryogenesis for the guppy (Poecilia

reticulata) and rainbow trout increasing as embryo-

genesis progressed but decreasing immediately before

hatch (Depeche et al. 1979). Similarly, adult mangrove

killifish increased physiological FAA levels and

retained more urea during transfer to increasing

salinities (Frick and Wright 2002), although urea

excretion decreased in three adult euryhaline species,

including striped bass (Morone saxatilis), rainbow

trout, and brown trout (Salmo trutta) at elevated

salinities (Altinok and Grizzle 2004). Frick and

Wright (2002) found that although urea excretion for

the mangrove killifish was significantly lower for fish

acclimated to hypersaline conditions (45 g/L) com-

pared to fish acclimated to salinities of 0, 15, and

30 g/L, total nitrogen excretion (ammonia and urea)

was not significantly different among salinity treat-

ments. Regardless, it is unlikely that the ureotelic

behavior of the 15 g/L-reared animals is a mechanism

to avoid ammonia toxicity during embryonic devel-

opment since there was no ammonia excretion in the

30 g/L-exposed animals, which had the longest TH of

all treatments.

One inconsistent aspect of the current study was the

15 g/L treatment, which produced the smallest larvae

at hatch in addition to erratic heart rates and urea levels

throughout the study. Mean TH of the 15 g/L treat-

ment was about the same as both 0.4 and 7 g/L

salinities, but had significantly smaller TL and BD

than the other three salinity treatments. Hatching is

independent of stage of embryogenesis once develop-

ment of tissues that release hatching enzymes is

completed in mummichog (DiMichele and Taylor

1981); external influences such as hypoxic conditions

and frequent agitation to eggs can induce premature

hatching in a broad range of species (Warkentin 2007).

DO levels in the 15 g/L treatment did not deviate from

other treatments (Table 1), and embryos from the

15 g/L treatment were handled similarly. While DO

concentrations remained above critical levels in all

treatment salinities throughout the course of the

experiment, abiotic factors such as salinity, pH,

alkalinity, and hardness may have influenced the rate

of embryogenesis. Fluctuations in heart rate observed

at stages 28 and onward may be a cause or result of the

urea levels that varied in concert with heart rate. The

highest recorded urea levels were in the 15 g/L salinity

at stages 28 and 35 (Fig. 4b). Cortisol has been shown

to increase expression of glutamine synthetase, an

enzyme involved in the ornithine-urea cycle (OUC),

Fish Physiol Biochem (2012) 38:1071–1082 1079

123

and urea transporter proteins in Gulf toadfish (Momm-

sen et al. 1999; McDonald et al. 2000). The influence

of cortisol in this experiment is only speculative as

concentrations of cortisol were not tested in either

embryos or maternal broodstock, nor do we know

whether OUC enzymes are ever expressed during

embryogenesis in Gulf killifish. While the influence of

external cues are well documented as triggers for

hatching, physiological triggers, such as internal

ammonia concentrations, are poorly understood and

do not appear to trigger hatch (Yasumasu et al. 1992,

1996; Wright and Fyhn 2001).

This experiment demonstrated the embryological

responses of Gulf killifish to salinities typically

encountered in the wild. Hatching within a concise

mean TH range and at similar sizes reduces the effects

of cannibalism; however, reduced hatch percentages in

0.4 and 30 g/L and a protracted mean TH for 30 g/L

indicate the peripheral ranges of salinity tolerance

during embryogenesis. Salinity and stage of develop-

ment were found to significantly influence ammonia

and urea excretion for embryonic Gulf killifish.

Increased ammonia released at higher salinities may

be a result of higher concentrations of Na? available

for ion exchanges (Wilkie 1997), resulting in a

decreased need for urea production as a method of

ammonia detoxification. Future studies comparing

changes in plasma ion content across a range of

salinities, genetic regulation of enzymes involved in

the OUC, and urea transporter proteins would prove

beneficial in determining the magnitude of urea’s

role in ion and metabolite regulation during

embryogenesis.

Acknowledgments This work was supported in part by

funding from the National Science Foundation to Fernando

Galvez and Louisiana Department of Wildlife and Fisheries to

Christopher Green. A special thanks to Yanling Meng and Ying

Guan for their assistance with embryo staging. The assistance of

Kurt Svoboda and Andrew Whitehead for the use of laboratory

materials is greatly appreciated. This manuscript was approved

by the Director of the Louisiana Agricultural Experiment

Station as number 2011-244-6308.

References

Alderdice DF (1988) Osmotic and ionic regulation in teleost

eggs and larvae. In: Hoar WS, Randall DJ (eds) Fish

physiology, vol 11A. Academic Press, Inc, San Diego,

pp 163–252

Alderdice DF, Rao T, Rosenthal H (1979) Osmotic responses of

eggs and larvae of the Pacific herring to salinity and cad-

mium. Helgol Mar Res 32:508–538

Altinok I, Grizzle JM (2004) Excretion of ammonia and urea by

phylogenetically diverse fish species in low salinities.

Aquaculture 238:499–507

Armstrong PB, Child JS (1965) Stages in the normal develop-

ment of Fundulus heteroclitus. Biol Bull 128:143–168

Ayson FG, Kaneko T, Hasegawa S, Hirano T (1994) Develop-

ment of mitochondrion-rich cells in the yolk-sac membrane

of embryos and larvae of tilapia, Oreochromis mossambi-cus, in fresh water and seawater. J Exp Zoo 270:129–135

Baras E, Jobling M (2002) Dynamics of intracohort cannibalism

in cultured fish. Aquac Res 33:461–479

Barrionuevo WR, Burggren WW (1999) O-2 consumption and

heart rate in developing zebrafish (Danio rerio): influence

of temperature and ambient O-2. Am J Physiol-Reg I

276:R505–R513

Boeuf G, Payan P (2001) How should salinity influence fish

growth? Comp Biochem Physiol C 130:411–423

Brown CA, Gothreaux CT, Green CC (2011) Effects of tem-

perature and salinity during incubation on hatching and

yolk utilization of Gulf Killifish Fundulus grandis embryos.

Aquaculture 315:335–339

Bunn NA, Fox CJ, Webb T (2000) A literature review of studies

on fish egg mortality: implications for the estimation of

spawning stock biomass by the annual egg production

method. Sci Ser Tech Rep CEFAS 111:1–37

Burnett KG, Bain LJ, Baldwin WS et al (2007) Fundulus as the

premier teleost model in environmental biology: opportu-

nities for new insights using genomics. Comp Biochem

Physiol D 2:257–286

Cook MA, Guthrie KM, Rust MB, Plesha PD (2005) Effects of

salinity and temperature during incubation on hatching and

development of lingcod Ophiodon elongatus Girard,

embryos. Aquac Res 36:1298–1303

Copeland DE (1950) Adaptive behavior of the chloride cell in

the gill of fundulus heteroclitus. J Morphol 87:369–379

Davenport J, Lønning S, Kjørsvik E (1981) Osmotic and

structural changes during early development of eggs and

larvae of the cod, Gadus morhua L. J Fish Biol 19:317–331

Davis RH, Ristow JL (1995) Osmotic effects on the polyamine

pathway of Neurospora crassa. Exp Mycol 19:314–319

Depeche J, Gilles R, Daufresne S, Chiapello H (1979) Urea

content and urea production via the ornithine-urea cycle

pathway during the ontogenic development of two teleost

fishes. Comp Biochem Physiol A 63:51–56

DiMichele L, Taylor MH (1981) The mechanism of hatching in

Fundulus heteroclitus- development and physiology. J Exp

Zool 217:73–79

Dushkina LA (1973) Influence of salinity on eggs, sperm and

larvae of low-vertebral herring reproducing in coastal

waters of Soviet Union. Mar Biol 19:210–223

Evans DH (2008) Teleost fish osmoregulation: what have we

learned since August Krogh, Homer Smith, and Ancel

Keys? Am J Physiol 295:R705–R713

Evans DH, Piermarini PM, Potts WTW (1999) Ionic transport in

the fish gill epithelium. J Exp Zool 283:641–652

Fessehaye Y, Kabir A, Bovenhuis H, Komen H (2006) Predic-

tion of cannibalism in juvenile Oreochromis niloticus

1080 Fish Physiol Biochem (2012) 38:1071–1082

123

based on predator to prey weight ratio, and effects of age

and stocking density. Aquaculture 255:314–322

Finn RN (2007) The physiology and toxicology of salmonid

eggs and larvae in relation to water quality criteria. Aquat

Toxicol 81:337–354

Fonds M (1979) Laboratory observations on the influence of

temperature and salinity on development of the eggs and

growth of the larvae of Solea solea (Pisces). Mar Ecol Prog

Ser 1:91–99

Fortin MG, Couillard CM, Pellerin J, Lebeuf M (2008) Effects of

salinity on sublethal toxicity of atrazine to mummichog

(Fundulus heteroclitus) larvae. Mar Environ Res 65:

158–170

Frick NT, Wright PA (2002) Nitrogen metabolism and excretion

in the mangrove killifish Rivulus marmoratus I. The

influence of environmental salinity and external ammonia.

J Exp Biol 205:79–89

Greeley MS, MacGregor R (1983) Annual and semilunar

reproductive cycles of the Gulf killifish, Fundulus grandis,

on the Alabama Gulf coast. Copeia 1983:711–718

Griffith RW (1991) Guppies, toadfish, lungfish, coelacanths and

frogs—a scenario for the evolution of urea retention in

fishes. Environ Biol Fish 32:199–218

Hascilowicz T, Murai N, Matsufuji S, Murakami Y (2002)

Regulation of ornithine decarboxylase by antizymes and

antizyme inhibitor in zebrafish (Danio rerio). BBA-Gene

Struct Expr 1578:21–28

Henry RP, Watts SA (2001) Early carbonic anhydrase induction

in the gills of the blue crab, Callinectes sapidus, during low

salinity acclimation is independent of ornithine decarbox-

ylase activity. J Exp Zool 289:350–358

Holliday FGT, Blaxter JHS (1960) The effects of salinity on the

developing eggs and larvae of the herring. J Mar Biol Assoc

UK 39:591–603

Imsland AK, Foss A, Gunnarsson S, Berntssen MHG, Fitz-

Gerald R, Bonga SW, Ham E, Nævdal G, Stefansson SO

(2001) The interaction of temperature and salinity on

growth and food conversion in juvenile turbot (Scoph-thalmus maximus). Aquaculture 198:353–367

Jung D, Biggs H, Erickson J, Ledyard PU (1975) New colori-

metric reaction for end-point, continuous-flow, and kinetic

measurement of urea. Clin Chem 21:1136–1140

Katoh F, Shimizu A, Uchida K, Kaneko T (2000) Shift of

chloride cell distribution during early life stages in sea-

water-adapted killifish, Fundulus heteroclitus. Zool Sci

17:11–18

Kidder GW, Petersen CW, Preston RL (2006a) Energetics of

osmoregulation: I. Oxygen consumption by Fundulusheteroclitus. J Exp Zool A 305A:309–317

Kidder GW, Petersen CW, Preston RL (2006b) Energetics of

osmoregulation: II. Water flux and osmoregulatory work in

the euryhaline fish, Fundulus heteroclitus. J Exp Zool A

305A:318–327

Kranenbarg S, Muller M, Gielen JLW, Verhagen JHG (2000)

Physical constraints on body size in teleost embryos.

J Theor Biol 204:113–133

Laurence GC, Rogers CA (1976) Effects of temperature and

salinity on comparative embryo development and mortality

of Atlantic cod (Gadus morhua L.) and haddock (Melan-ogrammus aeglefinus L.). J Conseil 36:220–228

Machado B, Podrabsky J (2007) Salinity tolerance in diapausing

embryos of the annual killifish Austrofundulus limnaeus is

supported by exceptionally low water and ion permeability.

J Comp Physiol B 177:809–820

McDonald M, Wood C, Wang Y, Walsh P (2000) Differential

branchial and renal handling of urea, acetamide and thio-

urea in the gulf toadfish Opsanus beta: evidence for two

transporters. J Exp Biol 203:1027–1037

Miller CA, Fivizzani AJ, Meier AH (1983) Water temperature

influences salinity selection in the Gulf killifish, Fundulusgrandis. Can J Zool 61:1265–1269

Miller TJ, Crowder LB, Rice JA, Marschall EA (1988) Larval

size and recruitment mechanisms in fishes: toward a con-

ceptual framework. Can J Fish Aquat Sci 45:1657–1670

Mitchell JLA, Judd GG, Leyser A, Choe CY (1998) Osmotic

stress induces variation in cellular levels of ornithine

decarboxylase antizyme. Biochem J 329:453–459

Mommsen TP, Walsh PJ (1992) Biochemical and environ-

mental perspectives on nitrogen-metabolism in fishes.

Experientia 48:583–593

Mommsen TP, Vijayan MM, Moon TW (1999) Cortisol in tel-

eosts: dynamics, mechanisms of action, and metabolic

regulation. Rev Fish Biol Fish 9:211–268

Montanez R, Sanchez-Jimenez F, Aldana-Montes JF, Medina

MA (2007) Polyamines: metabolism to systems biology

and beyond. Amino Acids 33:283–289

Nordlie FG (2000) Patterns of reproduction and development of

selected resident teleosts of Florida salt marshes. Hydro-

biologia 434:165–182

Nordlie FG (2006) Physicochemical environments and toler-ances of cyprinodontoid fishes found in estuaries and salt

marshes of eastern North America. Rev Fish Biol Fish

16:51–106

Oesterling MJ, Adams CM, Lazur AM (2004) Marine baitfish

culture: workshop report on candidate species and con-

siderations for commercial culture in the southeast US, vol

VSG-04-12. Virginia Sea Grant, Gloucester Point

Oppen-Bersten DO, Helvik JV, Walther BT (1990) The major

structural proteins of cod (Gadus morhua) eggshell and

protein crosslinking during teleost egg hardening. Dev Biol

137:258–265

Pegg AE (2006) Regulation of ornithine decarboxylase. J Bio-

chem 281:14529–14532

Pelster B (2002) Developmental plasticity in the cardiovascular

system of fish, with special reference to the zebrafish.

Comp Biochem Physiol A Mol Integr Physiol 133:547–553

Pelster B, Burggren WW (1996) Disruption of hemoglobin

oxygen transport does not impact oxygen-dependent

Physiological processes in developing embryos of zebra

fish (Danio rerio). Circ Res 79:358–362

Perschbacher PW, Aldrich DV, Strawn K (1990) Survival and

growth of the early stages of Gulf killifish in various

salinities. Prog Fish Cult 52:109–111

Randall DJ, Tsui TKN (2002) Ammonia toxicity in fish. Mar

Pollut Bull 45:17–23

Rao TR (1974) Influence of salinity on the eggs and larvae of the

California killifish Fundulus parvipinnis. Mar Biol 24:155–162

Rombough P (2007) The functional ontogeny of the teleost gill:

which comes first, gas or ion exchange? Comp Biochem

Physiol A 148:732–742

Fish Physiol Biochem (2012) 38:1071–1082 1081

123

Rombough P (2011) The energetics of embryonic growth.

Respir Physiol Neurobiol 178:22–29

Sampaio LA, Bianchini A (2002) Salinity effects on osmoreg-

ulation and growth of the euryhaline flounder Paralichthys

orbignyanus. J Exp Mar Biol Ecol 269:187–196

Scott GR, Baker DW, Schulte PM, Wood CM (2008) Physio-

logical and molecular mechanisms of osmoregulatory

plasticity in killifish after seawater transfer. J Exp Biol

211:2450–2459

Simpson DG, Gunter G (1956) Notes on the habitats, systematic

characters, and life histories of Texas salt water Cyprin-odontes. Tulane Stud Zool 4:115–134

Stone N, Ludwig GM (1993) Technical notes: estimating

numbers of golden shiner eggs on spawning mats. Prog

Fish Cult 55:53–54

Tay KL, Garside ET (1975) Some embryogenic responses of

mummichog, Fundulus heteroclitus (L) (Cyprinodonti-dae), to continuous incubation in various combinations of

temperature and salinity. Can J Zool 53:920–933

Tsui TKN, Hung CYC, Nawata CM, Wilson JM, Wright PA,

Wood CM (2009) Ammonia transport in cultured gill

epithelium of freshwater rainbow trout: the importance of

Rhesus glycoproteins and the presence of an apical Na?/

NH4? exchange complex. J Exp Biol 212:878–892

Verdouw H, Van Echteld CJA, Dekkers EMJ (1978) Ammonia

determination based on indophenol formation with sodium

salicylate. Wat Res 12:399–402

Walsh PJ (1998) Nitrogen excretion and metabolism. In: Evans

DH (ed) The physiology of fishes, 2nd edn. CRC Press,

Boca Raton, pp 199–214

Walsh WA, Swanson C, Lee CS, Banno JE, Eda H (1989)

Oxygen consumption by eggs and larvae of striped mullet,

Mugil cephalus, in relation to development, salinity and

temperature. J Fish Biol 35:347–358

Warkentin KM (2007) Oxygen, gills, and embryo behavior:

mechanisms of adaptive plasticity in hatching. Comp

Biochem Physiol A 148:720–731

Watford M (2003) The urea cycle: teaching intermediary

metabolism in a physiological setting. J Biochem Mol Biol

Educ 31:289–297

Watts SA, Yeh EW, Henry RP (1996) Hypoosmotic stimulation

of ornithine decarboxylase activity in the brine shrimp

Artemia franciscana. J Exp Zool 274:15–22

Whitehead A, Galvez F, Zhang S, Oleksiak M (2010) Functional

genomics of physiological plasticity and local adaptation in

killifish: an emerging model in environmental genomics.

J Hered. doi:10.1093/jhered/esq077

Whitehead A, Roach JL, Zhang S, Galvez F (2011) Genomic

mechanisms of evolved physiological plasticity in killifish

distributed along an environmental salinity gradient. Proc

Natl Acad Sci 108:6193–6198

Wiener ID, Hamm LL (2007) Molecular mechanisms of renal

ammonia transport. Annu Rev Physiol 69:317–340

Wilkie MP (1997) Mechanisms of ammonia excretion across

fish gills. Comp Biochem Physiol 118:39–50

Wilkie MP (2002) Ammonia excretion and urea handling by fish

gills: present understanding and future research challenges.

J Exp Zool 293:284–301

Wood CM, Perry SF, Wright PA, Bergman HL, Randall DJ

(1989) Ammonia and urea dynamics in the Lake Magadi

tilapia, a ureotelic teleost fish adapted to an extremely

alkaline environment. Respir Physiol Neurobiol 77:1–20

Wright PA, Fyhn HJ (2001) Ontogeny of nitrogen metabolism

and excretion. In: Wright PA, Anderson PM (eds) Fish

Physiology. Academic Press, San Diego, pp 149–200

Wright PA, Land MD (1998) Urea Production and transport in

teleost fishes. Comp Biochem Physiol A Mol Integr

Physiol 119:47–54

Wright PA, Wood CM (2009) A new paradigm for ammonia

excretion in aquatic animals: role of Rhesus (Rh) glyco-

proteins. J Exp Biol 212:2303–2312

Wright PA, Felskie A, Anderson P (1995) Induction of orni-

thine-urea cycle enzymes and nitrogen metabolism and

excretion in rainbow trout (Oncorhynchus mykiss) during

early life stages. J Exp Biol 198:127–135

Yasumasu S, Yamada K, Akasaka K, Mitsunaga K, Iuchi I,

Shimada H, Yamagami K (1992) Isolation of cDNAs for

LCE and HCE, two constituent proteases of the hatching

enzyme of Oryzias latipes, and concurrent expression of

their mRNAs during development. Dev Biol 153:752–758

Yasumasu S, Shimada II, Inohaya K, Iuchi I, Yasumasu I,

Yamagami K (1996) Different exon-intron organizations

of the genes for two astacin-like proteases, high choriolytic

enzyme (choriolysin H) and low choriolytic enzyme

(chorioly L), the constituents of the fish hatching enzyme.

Eur J Biochem 237:752–758

1082 Fish Physiol Biochem (2012) 38:1071–1082

123