RESPIRATORY RESPONSES IN THE FRESHWATER SNAIL (Pomacea bridgesii) ARE

DIFFERENTIALLY AFFECTED BY TEMPERATURE, BODY MASS

AND OXYGEN AVAILABILITY

Wenasa Frifer, B.S.

Thesis Prepared for the Degree of

MASTER OF SCIENCE

UNIVERSITY OF NORTH TEXAS

August 2016

APPROVED:

Warren Burggren, Major Professor Dzialowski Dzialowski, Committee Member Pamela Padilla, Committee Member David Holdeman, Dean of the College of

Arts and Sciences Victor Prybutok, Vice Provost of the

Toulouse Graduate School

Frifer, Wenasa. Respiratory Responses in the Freshwater Snail (Pomacea bridgesii) are

Differentially Affected by Temperature, Body Mass and Oxygen Availability. Master of Science

(Biology), August 2016, 52 pp., 2 tables, 6 figures, references, 95 titles.

Pomacea bridgesii is a snail species native to tropical and sub-tropical regions, where it

usually faces variability in water, temperature and oxygen level. This study of the effect of

temperature on mass-specific oxygen consumption (ṀO2) and its relation to body weight

shows that the ṀO2 of juvenile snails in normoxia (18-21 kPa) acclimated at temperature of

25°C ranged from 5 to 58 µMol O2/g/h, with a mean of 41.4 ± 18.3 µMol O2/g/h (n=7). Adult

snails in normoxia at 25°C show less variation, ranging from 13 to 23 µMol O2/g/h , with a

mean of 24.4± 6.1 µMol O2/g/h (n=12). The Q10 value for juvenile snails was higher in the

interval 25-30°C (Q 10=5.74) than in the interval 20-25°C (Q10= 0.286). In adult snails, Q10 was

higher in the interval 20-25°C (Q10=3.19). ṀO2 of P. bridgesii in relation to body weight showed

a negative linear correlation between metabolic rate and body weight with b values between

0.23 and 0.76. Also, both juvenile and adult snails exhibited weak O2 regulation. In general,

the different respiratory characteristics between juvenile and adult snails might be related to

the differences of individual life history, which caused them to perform differently in face of

temperatures change. Additionally, Pomacean snails species originated in tropical habitats where

there is a lack of thermal fluctuation. For this reason, Pomacean snails may be less likely to have

evolved effective thermal acclimation capabilities.

Copyright 2016

by

Wenasa Frifer

ii

ACKNOWLEDGEMENTS

I am using this opportunity to express my gratitude to everyone who supported me

throughout my thesis project. I am thankful for their aspiring guidance, invaluable constructive

criticism and friendly advice during the project work. I am sincerely grateful to them for sharing

their truthful and illuminating views on a number of issues related to the project.

I express my warm thanks to Dr. Warren Burggren and the members of my committee,

including Dr. Ed Dzialowski and Dr. Pamela Padilla, for their support and guidance. Benjamin

Dubansky deserves particular acknowledgment for his assistance with my experimental

methods with this project. I thank all my friends and family for their patience and support

during the entire my MSc work. This accomplishment would not have been possible without

them.

Thank you.

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iv

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...................................................................................................................iii LIST OF TABLES ................................................................................................................................ vi LIST OF FIGURES ............................................................................................................................. vii CHAPTER 1. INTRODUCTION ........................................................................................................... 1

1.1 Taxonomy of Pomacean Snails ............................................................................... 1

1.1.1 The Family Ampullariidae ........................................................................... 2

1.1.2 The Genus Pomacea ................................................................................... 2

1.2 Biology of Pomacean Snails .................................................................................... 2

1.2.1 Ecology and Invasive Nature ....................................................................... 2

1.2.2 Morphology ................................................................................................. 4

1.2.3 Food and Feeding ........................................................................................ 4

1.2.4 Reproduction .............................................................................................. 6

1.2.5 Growth and Longevity ................................................................................. 7

1.2.6 Physiology ................................................................................................... 8

1.3 Choice of Research Animal, Pomacea bridgesii .................................................... 13

1.4 Objectives and Hypothesis .................................................................................... 16 CHAPTER 2. METHODS AND MATERIALS ...................................................................................... 18

2.1 Animal Husbandry ................................................................................................. 18

2.2 Oxygen Consumption Determination ................................................................... 18

2.3 Effects of Temperature on Oxygen Consumption ................................................ 19

2.4 Oxygen Consumption Calculation ......................................................................... 20

2.5 Statistical Analysis ................................................................................................. 21 CHAPTER 3. RESULTS ..................................................................................................................... 22

3.1 Effect of Development on ṀO2 in Normoxia ........................................................ 22

3.2 Effect of Temperature on ṀO2 Normoxia ............................................................ 22

3.3 Effects of Oxygen Partial Pressure on ṀO2 .......................................................... 26

v

3.4 Effect of Body Mass .............................................................................................. 26 CHAPTER 4. DISCUSSION ............................................................................................................... 31

4.1 Critique of the Methods........................................................................................ 31

4.2 Physiological Findings ........................................................................................... 32

4.2.1 Temperature and Oxygen Consumption .................................................. 32

4.2.2 Oxygen Level and Oxygen Consumption .................................................. 36

4.2.3 Body Mass and Oxygen Consumption ...................................................... 38

4.3 Implications of Metabolic Rate to the Distribution of Pomacea bridgesii ........... 39

4.4 Future Experiments ............................................................................................... 40 REFERENCES .................................................................................................................................. 41

LIST OF TABLES

Table 3.1

The temperature coefficient (Q10) in normoxia (21-18 KPa) over 5°C intervals in juvenile

and adult snails………………………………………………………………………………….………………………..24

Table 3.2

Intercept, slope values of the regression lines of the three exposure groups, F value and

the significant level of the difference between the metabolic rates in three exposure

groups ……………………………………………………………………………………………………………..……......29

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LIST OF FIGURES

Figure 1.1 Shell morphology of Pomacean snails …………………………………………………………….…..5

Figure 1.2 Shell features of the shell of Pomacea bridgessii……………….………………………………..15

Figure 3.1 The effect of temperature exposure on ṀO2 (µMol O2/g/h) in normoxia

At three different exposure temperatures….……………………………….……………………………25

Figure 3.2 Effect of acute changes in PO2 on ṀO2 of juvenile apple snails acclimated to

20°C, 25°C and 30°C. ………………………………………………………………………………………………....27

Figure 3.3 Effect of PO2 on ṀO2 of adult apple snails acclimated to 20 °C, 25°C and

30°C……………………………………………………………………………………………………………………………..28

Figure 3.4 Scatter plot showing the effect of body mass on mass-specific ṀO2 of the

apple snail, Pomacea bridgesii, at 20 °C, 25°C and 30°C…………………………….…………………30

vii

CHAPTER 1

INTRODUCTION

1.1 Taxonomy of Pomacean Snails

1.1.1 The Family Ampullariidae

The family Ampullariidae comprises a family of large freshwater snails, including species

of freshwater amphibious snails (Cowie and Theingo, 2003) (Table 1). Freshwater snails of this

family are typically distributed in humid tropical and sub-tropical habitats in South and Central

America, Africa, and Southeast Asia (Cowie and Theingo, 2003). This family includes the largest

of all freshwater snails, Pomacea maculate, which can be greater than 155 mm in size (Pain,

1960) and P. urceus, which can reach a shell length of 145 mm (Burky, 1974). This family in its

native habitats usually constitutes a major portion of the freshwater Mollusca fauna (Cowie and

Theingo, 2003).

The family Ampullaridea includes nine genera and over 150 named species (Hayes et al.,

2009). The two genera containing the most species are Pomacea perry with 50 species and Pila

roding with 30 species (Berthold, 1991; Cowie and Thiengo, 2003). Snails of those two genera

are typically called “apple snails” due to their big size, round shape, and the green color of their

shells (Cowie, 1997). Ampullariidae are an economically important species because they can be

agricultural pests in their native region, and because even more significant negative economic

and ecological impacts occur from their introduction into new habitats (Cowie, 2006; Joshi and

Sebatain, 2006).

1

1.1.2 The Genus Pomacea

The genus Pomacea consists of two subgenera, Pomacea sensu stricto and Pomacea

effuse jousseume (Cazzaniga, 2002). Pomacea are endemic throughout South and Central

America and the Caribbean, with one species, P. palludosa, spread throughout the

southeastern United States (Howells, 2001b). Many species of Pomacea are successful invaders

that have impacted commercial crops (e.g., rice), and native vegetation in south Asia and the

United States (Burlakova et al., 2009; Morrison and Hay, 2010).

1.2 Biology of Pomacean Snails

1.2.1 Ecology and Invasive Nature

Pomacean snails inhabit a wide range of ecosystems, ranging from slowly moving or

sluggish water in lowland swamps, ditches, ponds and lakes to flowing rivers (Andrews, 1965a;

Keawjam, 1986; Perera and Walla, 1996). Some species have adapted to living in artificial,

agriculture habitats such as rice paddies and taro patches (Andrews, 1965b; Keawjam, 1986;

Louda and McKaye, 1982; Cowie, 2006). Preferred habitats vary among closely related species;

for instance, P. canaliculata has been found in relatively still water, while P. insularm inhabits

flowing rivers (Cowie, 2006).

Pomacean snails are popular aquarium pets due to their attractive color and their use

for cleaning algae from aquariums (Cowie, 2006; Howells et al., 2006). P. paludosa is a native

species in the United States, located in the south east of the United States (Howells, 2002;

Cowie, 2006). However, there are at least five exotic species that have established themselves

following introduction into North America, including P. bridgesii, P. canaliculata, P. harustrum,

P. insularum, and Marisa cornuarietis (Rawlings et a., 2007; Howells et al., 2006). The

2

introductions of these species, primarily from release and escape from aquariums, have

resulted in the relocation of native species (Britton, 1999; Howells, 2001a, b). Significant

introductions in the United States started in the 1950s and 1960s (Howells et al., 2006).

P. paludosa has a native population in Florida, Alabama, and Georgia, where no

significant impacts have been reported to the aquatic macrophytes due to relocation from their

range habitat (Rawlings et al., 2007; Howells et al., 2006). However, three of five exotic species

(P. insularum, P. canaliculata, and Marisa cornuarietis) are invasive pests. In fact, P.

canaliculata is on the list of the world’s worst 100 invasive alien species, due to their potential

effect on the ecosystem and huge economic losses especially in Asia (Lowe et al., 2000). This

species has been confirmed in Arizona and California and has spread into the rice-growing area

in California, where it might cause potential damage (Rawlings et al., 2007). P. insularum also

caused environmental impacts to the ecosystem and serious damage to agriculture in Texas

and other parts of the United States including Alabama, Mississippi, and Louisiana

(Ramakrishnan, 2007). Marisa cornuarietis has been introduced in Florida to control aquatic

plant nuisances, but has caused negative impacts on native plant species and animals that

depend on these plants (Simberlof and Stiling, 1996). In contrast to three species, P. bridgesii

and P. haustrum seem to be non-invasive species, while P. haustrum has been in the United

States for over 30 years with minimal impact. P. bridgesii consumes algae with only a small

impact on aquatic plants (Howells, 2002).

Due to the potential impacts of Pomacean snails on agriculture and commercial crops

described above, the U.S Department of Agriculture (USDA) and Texas Parks and Wildlife

Department (TPWA) have banned both the importation of apple snails into United States and

3

all interstate movement, with the exception of P. bridgesii larger than 20 mm (Howells et al.,

2006).

1.2.2 Morphology

Pomacea have indented and sutured shells that, depending on the species, range from

30-110 mm (Perera and Walls, 1996). The shells of Pomacean snails are oval and sub-globosely

or globosely conic (Keawajam and Upatham, 1990; Perera and Walls, 1996). Shell color varies

from white, yellowish to brown, greenish to brown (Thiengo, 1987; Keawajam and Upatham,

1990; Thiengo et al., 1993). The surface of the shell can be smooth with the external surface

exhibiting minute transverse striae (growth lines) that have formed at slower growth rates

(Keawajam and Upatham, 1990). The operculum is thick and corneous and marked by

concentric growth with the nucleus near the center of the shell (Keawajam and Upatham, 1990;

Perera and Walls, 1996) (figure 1.1).

1.2.3 Food and Feeding

Pomacean snails are mainly macrophytophagous and feed on a variety of aquatic plants

(Howells, 2002). For instance, Pomacea canaliculata feed voraciously on macrophytic

vegetation, and high population densities can result in the consumption of the entire

macrophyte plant within very short periods (Lach et al., 2000). This snail can detect food

sources from some distance by using chemical cues in the water. Its growth rates correlate with

its ability to feed on its preferred plant (Estebenet, 1995; Lach et al., 2000). Due to the ability of

Pomacean snails to consume a wide variety of plants, they are successful aquatic invaders (Lach

et al., 2000).

4

Figure 1.1 Shell morphology of Pomacean snails (from Ghesquiere, 2003)

5

1.2.4 Reproduction

Pomacean snails are dioecious, have internal fertilizing, and are oviparous (Cowie, 2002). The

beginning of breeding is seasonal and varies with temperature, humidity, food availability, and

geographic location (Andrews, 1964). Some species, such as Pomacea canaliculata, reproduce

throughout the whole year in tropical and subtropical climates. In temperate climates, the

reproductive season starts in March and ends in October (Estebent and Cazzaniga, 1992;

Estebent and Martin, 2002; Howells et al., 2006). These snails show high fecundities, producing

eggs that are oviposited at the waterline on exposed hard surfaces such as rocks and vegetation

(Snyder and Snyder, 1971). This behavior might be an adaption to protect eggs from aquatic

predators and to provide high levels of oxygen for developing embryos, as their aquatic habitats

are often hypoxic (Snyder and Snyder, 1971). The egg clutches have a calcareous covering,

which might be used as a source of calcium for developing embryo (Andrews, 1964; Tampa,

1980; Turner and McCabe, 1990). In most Pomacean snails, oviposition happens during the

night or early in the morning (Andrews, 1964; Chang, 1985; Halwart, 1994; Schnorbach, 1995;

Albrecht et al., 1996) due to the risk of predation and dehydration when crawling above the

water line (Albrecht et al., 1996; Estebenet and Martin, 2002). Incubation lasts from 7 to 34

days, and varies among different species of Pomacea (Perera and Walla, 1996; Ramnaraine,

2003). After hatching, new juveniles fall or crawl into water (Cowie, 2006).

The eggs of Pomacean snails have bright colors. For instance, P. bridgesii, P.

canaliculata, P. lineata, P. insularm, and P. palludosa produce eggs that are pink, red, or orange.

However, P. glauca produce eggs that are green. Other species of Pomacea such as, P.

flagellate, P. falconensis, P. fasciate, P.gossei, and P. capine produce eggs that are white

6

(Snyder and Snyder, 1971; Keawjam and Upatham, 1990; Thiengo et al., 1993; Perera and

Walls, 1996; Cowie2002). The number of eggs per egg mass is different among Pomacean

snails. For instance, females of P. urceus produce between 50-200 eggs per egg mass (Burky et

al., 1972), while females of P.canaliculata lay between 200-700 eggs per mass (Fujio and Brand

1990).

1.2.5 Growth and Longevity

Growth rates are affected by abiotic factors such as ambient temperature, photoperiod,

and season (Estebent and Cazzaning, 1992; Estebenet and Martin, 2002). Pomacean snails may

reach maturity within 60-80 days (de la Crus et al., 2001) and live over 3 years if given favorable

temperatures (Estenbent and Cazzangia 1992). Temperature affects longevity, maturity, and

reproduction in Pomacean snails (Cowie, 2006). For instance, at a constant temperature 25°C,

P. canaliculata can reach maturity in 7 months, then breed throughout a 4 month breeding

season, for a total life span of 13.5 months. However, while under seasonal conditions when

temperatures fluctuated from 7°C to 28°C, snails took 2 years to reach maturity and the life

span is up to 4 years, allowing them to breed for two annual seasons (Cowie, 2006; Scott, 1954;

Estebent and Cazzaning, 1992).

Food availability also influences the growth rate of Pomacean snails. The growth rate of

P. delioides depends on food availability, the water level, and the duration of the dry season

(Cowie, 2006). Maximum size varies among populations (Keawjam, 1986; Estebent and

Cazzaning, 1992; Donny and Beissinger, 1993) and might be influenced by environmental

factors such as habitat size (Johnson, 1958), microclimatic difference, water regimes (Donny

and Beissinger, 1993), and population density (Williamson et al., 1976). For example,

7

P.canaliculata reaches 30mm in length in Hawaii, while in Asia it has variously been reported to

be 65mm long (Schnorbach,1995) or 90mm long (Heidenriedch et al.,1997).

1.2.6 Physiology

1.2.6.1 Temperature Tolerance

Temperature is an important factor that affects the distribution of aquatic animals and

influences biological factors such as respiration and growth rate (Prosser and Health, 1991;

Brown, 2001). Pomacean snails tend to be stenothermal, living within a narrow range of

temperatures (Segal, 1961). The actual upper and lower lethal temperature varies among

Pomacean species due to their adaptation to their natural climatic environment (Cowie, 2006).

For instance, the upper lethal temperature of P. insularum is 37°C, while the lower lethal

temperature is 15°C (Ramakrishnan, 2007). On the other hand, the upper lethal temperature

for P. paludosa is 40°C when snails are exposed for 1-4 hours, while this species can survive at

5°C (Freiburge and Hazelwood,1977).

1.2.6.2 Salinity Tolerance

Salinity is a major factor that can affect the distribution of aquatic animals and their

physiological functions (Remane and Schlieper, 1971; Jacobsen and Forbes, 1977). Even though

Pomacean snails exhibit some tolerance of salinity, they primarily live in fresh water (Cowie,

2006). P. bridgesii exhibits tolerance to salinity levels ranging from 0-6.8 ‰ ppt in which

probability of survival was higher than 80% after 3 days of exposure (Jordan and Deaton, 1999).

The salinity range of P. insularum also ranges from 0-6.8‰ppt n which snails’ survival was

greater than 90% after 28 days of exposure (Ramakrishnan, 2007).

8

1.2.6.3. Aestivation

Aestivation is the state of slowing of activity and aerobic dormancy during hot, dry

seasons such as summer (Storey, 2012). Pomacean snails aestivate during the dry season of

their habitat. When the dry season starts, snails start estivation by burrowing into the mud

(Cowie, 2006). Some species such as P. ureus bury superficially, keeping the dorsal portion of

the shell remaining above the mud (Burky et al., 1972). Other species bury up to 1 m deep in

the ground. The length of aestivation differs between laboratory experiments and the wild.

Some species have the ability to extend the aestivation period in the lab for longer than has

been reported in nature. Aestivation length also varies among species, being 18 months in P.

lineata (Little, 1968), 17 months in P. urceus (Burky et al., 1972) and only 3 months in P.

canaliculata (Schnorbach, 1995). During the aestivation period, Pomacean snails are able to

resist the loss of soft tissue weight. For example, P. lineata can withstand up to 50% of soft

tissue weight loss (Little, 1968), while P. urceus up to 62% of soft tissue weight loss (Burky et al.,

1972). During aestivation, the metabolism of some species is anaerobic, such as in Pomacea

globose, (Aldrige, 1983) while the metabolism is aerobic in other species such as in P. urceus

(Burky et al., 1972). In fact, P. globosa bury deeply in the ground, so their anaerobic metabolism

might be an adaptation to this behavior, while aerobic metabolism in P. urceus may still be

possible due to their superficial burying in mud (Cowie, 2006).

1.2.6.4 Respiration

Pomacea snails are amphibious (Cowie, 2006). They have in their mantle cavity both a

ctenidium (gill) on the right side of the mantle cavity and the lung (pulmonary sack) on the left

side (Andrews, 1965 b). The lung is used for aerial gas exchange while the gill is used for aquatic

9

exchange (Thieng, 1987). This ability to use both lung and gill allow Pomacean snails to survive

in conditions of aerial exposure and during low oxygen conditions (Cowie, 2006).

The ctenidium consists of a number of gill filaments or lamellae which are triangular and

hang freely in to the mantle cavity (Kotpal, 2012; Demian, 1965). The pulmonary sac is a large

bag that hangs into the mantle cavity and occupies a large portion of the pulmonary chamber.

The lung can be opened and closed by a sphincter when the snails are underwater (Demian,

1965). The membranes of the pulmonary sac are very richly vascularized to exchange oxygen

and carbon dioxide. The siphon is formed of a fold of the mantle cavity (left nuchal lobe) which

is shaped into a respiratory tube (Demian, 1965). Pomacean snails are well adapted for three

different modes of respiration: branchial respiration, siphonal pulmonary respiration and direct

pulmonary respiration (Demian, 1965). Branchial respiration is achieved while the snails are

submerged by a continuous flow of water inside the mantle cavity, mainly created by the

epitaenia (ciliated organ) (Demian, 1965; Santos et al., 1987). Water crosses over the

osphradius, the organ that is located in the mantel cavity in front of the lung and uses to detect

chemicals in the water, then flows over the gills and leaves the mantle cavity through the

siphon (Demian, 1965). Siphonal respiration is achieved when the snail rolls up its left nuchal

lobe into the siphon and estends its siphon to the water surface, while the pneumstoma (lung

opening) is open and moved to fit at the base of the siphon (Demian, 1965; Santos et al., 1986).

The snail makes 5-20 pumping movements with its head in and out of the shell, which allows air

to flow in and out of the lung cavity in a tidal fashion (Demian, 1965). Direct pulmonary

respiration occurs when the snail leaves the water. The lung remains open most of the time to

allow airflow into the lung, but there is no respiratory siphon formed (Santos et al., 19876). In

10

Pomacean snails, lung ventilation is obligatory, so they regularly come to the surface to take a

breath (Cowie, 2006). The degree of dependence on aerial respiration varies between species.

For example, P. urceus can survive submergence for 5 days in aerated tanks (Burky and Burky,

1977). The lack of aerial respiration negatively affects feeding, survival, and activity and their

access to the air is an important defining factor for predicting distribution and abundance of

snails (Seuffert and Martín, 2010). For instance, when Pomacea canaliculata are submerged

without the ability to access air, all snails frequently die (Seuffert and Martín, 2010).

Temperature affects the degree of dependence on aerial respiration. P. canaliculata

usually connects to the air by its siphon to ventilate through the lung, and the length of time

interval between emersions decreases with temperature (Seuffert and Martín, 2009). Lung

ventilation increases with the decrease of dissolved oxygen in the water (McClary, 1964; Burky

and Burky, 1977). In P. canaliculata, for example, when dissolved oxygen levels are low (1-

2ppm), the siphon is extended to the water surface to breathe air, but when dissolved oxygen

levels in the water are high (5-6 ppm), they stay under the water and use their ctenidium for

gas exchange (San Martins et al., 2009).

1.2.6.5 Oxygen Consumption and Responses to Environmental Change

In aquatic environments, the availability of oxygen is inversely proportional to

temperature, salinity and the level of hypoxia (Aldridge, 1983). Temperature increases the

oxygen demand in freshwater gastropods by typically 2-3 times with each 10°C increase of

temperature – the so-called Q10 for metabolism1 (McMahon 1983, Prosser and Health, 1991).

1 The temperature coefficient Q10 is the factor by which physiological rates increase when the body temperature increases by 10°C rise in temperature.

11

The metabolic rates of aquatic animals, evident in their oxygen consumption, are affected by

temperature and ambient oxygen tension (Hawkins and Ultsch, 1978). Ampullariid snails exhibit

amphibious behaviors and have gills to perform aquatic respiration and a well-developed lung

for aerial respiration (Andrews, 1965; Berthold, 1991; McClary, 1964; Seuffert and Martin,

2009).

The oxygen consumption rates for P. lineata have been reported to increase with rising

temperatures and be highest between 10-15°C, and the oxygen consumption (ṀO2) increases

slightly at 35°C. The temperature sensitivity of oxygen consumption of this species, as defined

by the Q10, doubled from 5°C to 20°C but then actually declined from 20°C to 25°C (Santos et

al., 1986). P. palludosa show higher oxygen consumption rates in air than in water. They also

show metabolic temperature sensitivity, with Q₁₀ values 2.58 and 2.97 between 10-20°C

(Freiberg and Hazelwood, 1977). In juvenile P. insularum, the metabolic rate increased

significantly with increases in temperature between 20°C and 35°C. However, metabolic rate

was depressed at 40°C – the lethal temperature - and Q10 increases with temperature between

25°C -35°C with Q₁₀ values between 1.31 and 1.39 (Ramakrishnan, 2007). Marisa cornuarietis

exhibit increased oxygen consumption with increases of temperature between 10°C to 35°C.

Also, �̇�𝑀O2 declined slightly at 40°C that was the lethal temperature and the Q10 increased with

size at 10°C-20 °C and 20°C-30°C (Akelund, 1969; Freiberg and Hazelwwod, 1977)

1.2.6.6 Influence of Body Size on Oxygen Consumption

A number of studies have investigated the relationship between body weight and

oxygen uptake, and these studies have indicated the expected increase in oxygen consumption

with increasing body mass, and a decrease in mass-specific oxygen consumption with the

12

increasing body mass (Von Brand et al., 1948; Berg and Ockelmann, 1959; Wright, 1971;

Akerlund, 1974; Calow, 1975; Fitch, 1975; Freiburg and Hazelwood, 1977). For example,

Pomacea lineata displayed an increase in oxygen consumption with increase in body mass, with

the slope of log-log b=0.76, and a decreased in mass-specific oxygen consumption with weight

under normoxic condition (Santos et al., 1986).

1.3 Choice of Research Animal: Pomacea bridgesii

This study has chosen Pomacea bridgesii as the experimental animal for investigating

oxygen consumption rates. Knowing the ability of this species to sustain their oxygen needs is

very essential to predict their distribution in Texas and the rest of the United States, given their

potential threat as an invasive species. Some specific information on this species is now

provided.

The common name of Pomacea bridgesii is the spike top apple snail, and it is native

South America (Perera and Walls, 1996). P. bridgesii was introduced to Florida in the early

1960s due to aquarium trade release and from tropical fish farms (Clench, 1966; Howelles et al.,

2006). The snail shell varies in color from golden , dark greenish to brown with dark striped

bands (Perera and Walls,1996) .The shell differs in size from 40-50 mm wide and 45-65 mm in

length (Putnam,2011). The spire is sharp and high, and has about 5 to 6 whorls (Putnam, 2011).

The shell has square shoulders and the sutures are at a 90° angle (Putnam, 2011) (Figure 1.2).

P. bridgesii feed on algae and dead plants (Aditya and Raut, 2001). Due to these food

13

preferences, they are attractive for ornamental use and for cleaning aquariums of algae

(Howelles, 2002). P. bridgesii is dioecious, with internal fertilization, and oviparous (Chang,

1985). Sexual dimorphism is not visible, though females tend to be larger in size than males

(Estebenet and Cazzaniga, 1998; Tanaka et al., 1999; Estebenet and Martin, 2002).

P. bridgesii lay eggs about 6-8 cm above the water line (Snyder and Snyder, 1971). The

eggs are pink in color and the embryos within have a calcareous shell (Andrews, 1964; Tompa,

1980; Turner and McCabe, 1990). The hatching period is around 15 to 24 days at 23 ± 1°C and

the hatching success rate is high (89 to 100%)( Coelho et al., 2012). The juveniles require

minimal care due to their similar behavior patterns to adults (Coelho et al., 2012).

Juveniles and adults of P. bridgesii use the lung for aerial respiration and the gill for

aquatic respiration (Thieng, 1987). This ability to use both lung and gills allows survival under

dry and low oxygen conditions and allows them to inhabit waters with low dissolved oxygen

(Coelho et al., 2012).

14

Figure 1.2 The shell morphology of pomacea bridgesii. From Blume, 1957. Scale: 1cm. Photo by

R.H. Cowie.

15

1.4 Objectives and Hypotheses

Pomacea bridgesii is a native species in tropical and sub-tropical regions where both

aquatic hypoxia and temperature variability is common. Knowing their metabolic responses to

oxygen and temperature is critical from ecological and physiological perspectives, and helps us

understand the reasons that can allow Pomacea bridgesii to be a successful invader in more

temperate climates. Moreover, metabolic behavior plays critical roles in growth, biomass, and

buoyancy (Burky, 1974), so it is essential to investigate the effect of temperature and water

regimes on oxygen consumption in juvenile and large adult apple snails. Temperature

influences the relationship between metabolic rate and body weight. This, I hypotheses this for

Pomacea bridgesii:

1) Pomacea bridgesii under control conditions in the lab will respond to low oxygen

partial pressures and increase of temperature differently depend on previous life

history

2) Oxygen consumption increases with increasing temperature and oxygen

consumption decreases with increasing body mass, and

3) Oxygen consumption will allow the snail to cope to temperature change, potentially

aiding its invasion of temperate climates with potentially high temperature

variability during the winter due to climate change.

These three hypotheses will be tested by determining:

1) The impact of temperature on oxygen consumption of Pomacea bridgesii

2) The impact of body size on oxygen consumption of Pomacea bridgesii

3) The temperature sensitivity of oxygen consumption, as expressed by the Q10 for

16

Oxygen consumption.

4) The critical oxygen partial pressure for oxygen consumption.

17

CHAPTER 2

MATERIAL AND METHODS

2.1 Animal Husbandry

Apple snails (Pomacea bridgesii) were obtained from a local aquarium store (Fish and

Chirps, Denton, TX). Adult snails were maintained in the laboratory in 37.8 L glass aquaria at a

density of 1 snail per 3L of water. Water temperature was maintained at 25°C, except for in

experimental situations as noted. Aquarium water was continuously filtered to maintain water

quality. Water in the aquariums was constantly oxygenated with ambient air and the aquaria

were exposed to a 12h light/dark photoperiod.

Calcium requirements for developing snails are crucial. Consequently, moderately hard

water was formulated with deionized water (NaHCO3 =0.096 g/L, KCl =0.004 g/L, MgSO4,

=0.06g/L and CaSO4 =0.06 g/L). P. bridgesii deposits its eggs above the waterline, so the tank

was filled with 30.2 L of prepared water leaving 10 cm of dry, exposed glass between the water

line and aquarium covers for egg deposition. Juvenile P. bridgesii were held with adults after

hatching. Tanks were covered with a lid to keep eggs moist and prevent snails from escaping.

Tanks were monitored daily, and any dead snails were removed from the tanks. All snails were

fed 5 times a week with algae pellets (29% protein) from Tetra Veggie Company. Water changes

and filter cleaning was conducted every two weeks.

2.2 Oxygen Consumption Determination

Oxygen partial pressure in glass respirometry chambers was measured using fiber optic

oxygen optodes (PreSens) interfaced to a computer using AutoResp aquatic respirometry

18

software (Loligo, Inc.). Metabolic rates were measured as oxygen consumption (ṀO2),

expressed as µMol O2/hour/gram animal (µMol O2/g/h).

Four respiratory chambers were submerged in a tank filled with 60-63 L of formulated

water. The water was aerated and circulated through a filter to remove particulate matter. The

tank was connected to a water bath (Isotemp™, Fisher Scientific) to maintain the desired

temperature. The opening and the closing of the flush pump, as well as oxygen PO2

measurements and the temperature, were automatically controlled by a software system

(Loligo System). For juvenile snails, ṀO2 was measured using closed respirometry that consists

of sealed chamber of known volume, while for adult snails, intermittent respirometry using the

flush and refill cycle was employed.

Oxygen concentration in the water bath was monitored by a galvanic oxygen sensor,

connected to the DAQ-M. Also, temperature was monitored by a temperature sensor

connected to the computer Loligo OXY-REG device. Each chamber was connected to two pumps

to refresh the chamber water.

For all measurements, the four chambers of the Loligo system were submerged in a 10 L

tank filled with water to ensure temperature stability.

2.3 Effects of Temperature on Oxygen Consumption

Oxygen consumption response to acute temperature change was recorded for one

animal in each chamber for three exposure groups of juvenile snails, while for adult snails the

replicate animals’ number was (n=2) for each of three exposure groups. Both juvenile and adult

snails were maintained at 20°C, 25°C, or 30°C for a minimum of 24h and each group of snails

was chosen randomly and kept in the separated tank prior to the experiment. At the beginning

19

of the experiment, the ṀO2 measurement cycle was 15-20 min, which was then followed by a

2-3 min wait time to avoid any reading mistake, and then was finally followed by 5-7 min of

flush to renew the oxygen partial pressure of the water in the chambers.

For juvenile snails, the PO2 was recorded from near full air saturation (PO2=21.3Kpa) to

a low level of oxygen partial pressure (PO2= 1 KPa), a decline of 1 KPa taking approximately 10

min on average. The oxygen partial pressure decline inside the closed chamber was measured

every 5 min over ~6 h, depending on the snail’s activity.

For adult snails, the PO2 decline in the chamber was recorded four times from a high

oxygen partial pressure to a low level of oxygen partial pressure (PO2= 20 KPa to 16 KPa), then

from (PO2= 16KPa to 12kPa ), then from (PO2=12 KPa to 8 KPa) and then finally from

(PO2=8KPa to 4 KPa) for 20 min in each loop cycle, for a total of 6 loops cycles for each

different PO2 . The duration of the whole experiment was approximately 10 hours for each

complete respirometry run. Immediately after the completion of ṀO2 determination, an empty

water-filled chamber was run as a control (blank) to ensure that there was no bacterial growth

within the chambers.

2.4 Oxygen Consumption Calculation

The determination of mass-specific rate of oxygen consumption ṀO2 (µMol O2/g/h) for

each animal was estimated by using the following equation:

�̇�𝑀O2 = O2 solubility (ml/l/mmHg) * ∆PO2 (mmHg) * chamber volume (l) * 1 /mass (g) * 1

/time (min) * conversion factor from ml O2/g/h to µmol of O2/g/h

The Q10 was calculated from the Van’t Hoff equation:

20

Q10 = (MR2/MR1) 10/ (T2 –T1)

Where MR1 is the metabolic rate (as oxygen consumption) at a lower temperature T 1and MR2

is the metabolic rates at a higher temperature T2.

The critical ambient oxygen partial pressure, P crit, is the PO2 value below which an

animal is not able to regulate its oxygen uptake and becomes an oxygen conformer (Bayne,

1976; Mangum and Van Winkle, 1973). P crit was determined by looking for the graphs visually.

2.5 Statistical Analysis

Two way ANOVA was used to analyze the effect of temperature and development of

snails on metabolic rate and confirm the effect of acute oxygen partial pressure, with Holm-

Sidak tests used to test for significant differences among the exposure groups. To determine

the influence of temperature on relation between oxygen consumption and body weight, linear

regressions were created, with associated correlation coefficients and significance levels. All

data are presented as means ± 1 standard error. A significance level of 0.05 was adopted for all

statistical tests.

21

CHAPTER 3

RESULTS

3.1 Effect of Development on ṀO2 in Normoxia

The ṀO2 of individual juvenile snails in normoxia (18-21 kPa) exposure to their

preferred temperature of 25°C ranged from 5 to 58 µMol O2/g/h, with a mean of 41.4 ± 18.3

µMol O2/g/h (n=7). The ṀO2 of individual adult snails in normoxia at their preferred

temperature of 25°C was lower and with less variation, ranging from 13 to 23 µMol O2/g/h,

with a mean of 24.4± 6.1 µMol O2/g/h (n=12) (Figure 3.1). Yet, Holm-Sidak’s multiple

comparison tests indicated that the ṀO2 of juveniles exposure to 25°C was not significantly

different (p<0.05) in juvenile and adult snails.

3.2 Effect of Temperature on ṀO2 in Normoxia

While the ṀO2 of juvenile and adult snails was not significantly different at 25°C,

exposure temperature had a significant and complex effect on ṀO2. In juvenile snails, ṀO2 was

significantly (P<0.05) higher at 30°C as expected, ranging from ranged from 78 to 107 µMol

OR2R/g/h with a mean of 99.4± 11 µMol OR2R/g/h (n=8). Paradoxically, however, ṀOR2 Rof juveniles

at 20°C was also significantly higher than in juveniles at 25°C, ranging from 64 to 88 µMol

OR2R/g/h with a mean of 77.4± 7.2 µMol OR2R/g/h (n=8) (Figure 3.1)

In contrast to the juveniles, adult snails showed the expected lower ṀO2 at 20°C (7 to

21 µMol O2/g/h with a mean of 14±4.2 µMol O2/g/h (n=8) than the ṀO2 in adults at 25°C

22

(range 13 to 33 µMol O2/g/h with a mean of 24.4±6 µMol O2/g/h(n= 12 ). ṀO2 at 30°C ranged

from 14 to 20 µMol O2/g/h with a mean of 17.3 ± 3.2 µMol O2/g/h (n=4) (Figure 3.1).

Two way ANOVA revealed that the exposure temperature alone did not significantly

impact ṀO2 (F=3.11, p=0.0550), but ṀO2 was highly significantly impacted by age (juvenile and

adult) (F=43.2, p<0.0001). There was also a significant interaction between exposure

temperature and development (age) (F=5.5, p=0.0057). Holm-Sidak’s multiple comparison tests

indicated that the exposure groups at 20°C and 30°C were significantly (p<0.05) different in

both of juvenile and adult snails.

Temperature sensitivity for ṀO2, determined from calculation of Q10 values, depended

upon age of the snails. In normoxia, the Q10 value for juvenile snails was higher in the interval

25-30°C (Q 10=5.74) than in the interval 20-25°C (Q10= 0.286). In the adult snails, however, Q10

was higher in the interval 20-25°C (Q10=3.19) while Q10 in the interval 25-30°C it was lower

(Q10= 0.50) (Table 3.1).

23

Table 3.1. The temperature coefficient (Q10) in normoxia(21-18 KPa) over 5°C intervals in

juvenile and adult snails.

Juvenile snails (0.32 g ± 0.07 g) Adult snails (16.4 g ± 0.47 g)

Temperature (°C)

ṀO2 (µMol O2/g/h)

Q10 Temperature (°C)

ṀO2 (µMol O2/g/h)

Q10

20°C

25°C

30°C

77.4±7.2

41.4±18.3

99.4±10.8

0.286

(20-25°C)

5.74

(25-30°C)

20°C

25°C

30°C

13.6±4.2

24.4±6.08

17.3±3.2

3.19

(20-25°C)

0.50

(25-30°C)

24

Figure 3.1 The effect of temperature exposure on ṀO2 (µMol O2/g/h) in normoxia at three

different exposure temperatures groups in both juvenile and adult snails. Capitalized letters

above each bar indicate their significance relationships separately within juvenile and within

adult populations. All adult values were significantly different from all juvenile values.

25

3.3 Effects of Acute Oxygen Partial Pressure on ṀO2

ṀO2 of juvenile snails declined with a drop in ambient PO2 of just 1-3 kPa, especially at

the warmest temperature, and continued to fall through moderate and then severe hypoxia

(Figure 3.2.A). Two way ANOVA indicated that ṀO2 was significantly affected by oxygen partial

pressure PO2 (F=50.4, p<0.0001) as well as the exposure temperature (F=47.6, p<0.0001). The

slopes of a linear regression through the means of each population were highly

significantly different in three exposure groups(p<0.0001) (Figure.3.2.B).

Generally, the ṀO2 of adult snails was less affected by acutely declining PO2 than in

juvenile snails at all three exposure temperatures groups. Two way ANOVA shows that while

ṀO2 was not impacted by PO2 (F=1.8, p=0.1301), it was significantly impacted by exposure

temperature (F= 15.6, p<0.0001) (Figure 3.3.A). Linear regression analysis indicated that the

ṀO2 for three groups of adults was not significantly different (p=0.214) between the exposure

groups. Similarly, the differences between the slopes were not significant while the

differences between the elevations are extremely significant (Figure 3.3.B).

3.4 Effect of Body Mass

The mass-specific ṀO2 of P. bridgesii decreased with increasing body weight, as

anticipated (Figure 3.4). The correlation coefficients r2 were high and significant (P<0.001) in

the 20°C population, 0.81, and in the 30°C population, 0.74. However, r2 was low 0.15 at 25°

C (p>0.10)(Table 3.2).

26

Figure 3.2 Effect of acute changes in PO2 on ṀO2 of juvenile apple snails exposure to 20°C, 25°C

and 30°C. A) Means ± SE for the three exposure temperatures as a function of PO2. A box

indicates no significant different among the groups. B) Linear regressions through the means

are presented. P value indicates the probability that the slopes of the lines are different from

zero.

27

Figure 3.3 Effect of PO2 on ṀO2 of adult apple snails exposure to 20 °C, 25°C and 30°C. A)

Means ± SE for the three exposure temperatures as a function of PO2. A box indicates no

significant different among the groups. B) Linear regressions through the means in presented

in A. are shown. P value indicates the probability that the slopes of the lines are different from

zero.

28

Table 3.2. Intercept (a), slope values (b) of the regression lines, F value and the significant level (p value) for mass specific ṀO2 of the three exposure groups(n=47).

Temperature(°C) n Intercept(a) Slope (b) F r P

20°C 16 -2.45 51.9 59.8 0.81 <0.0001

25°C 19 -0.409 29.11 2.99 0.15 0.1014

30°C 12 -2.87 65.4 28.31 0.74 0.0003

29

Figure.3.4. Scatter plot showing the effect of body mass on mass-specific ṀO2 of the apple

snail, Pomacea bridgesii, at 20°C (n=15), 25°C (n=20) and 30°C (n=12). Note that body mass is

plotted on a semi-log scale.

30

CHAPTER 4

DISCUSION

4.1 Critique of the Methods

The purpose of the study was to determine the effect of temperature, body weight, and

level of oxygen on oxygen consumption of Pomacea bridgesii. Juvenile and adult snails were

used to address the question of whether Pomacea bridgesii would be able to maintain its

oxygen consumption in face of temperature change and oxygen tension decline. Individuals

were randomly chosen from a large sample of snails (47 individuals). This availability of a large

number of individuals allowed for comprehensive statistical analysis.

For measuring oxygen consumption, closed-system respirometry was used for juvenile

snails while intermittent respirometry was used for adult snails. Closed respirometry is closed

(sealed) chamber where is the animal placed to measure the decline of oxygen concentration

over the time (Svendsen et al., 2016). On the other hand, intermittent respirometry is a

combination of short term series of closed respirometry cycle flowing by flushing intervals to

refresh the water inside the chamber (Svendsen et al., 2016).

Both methods are subject to criticism. One objection to using closed respirometry for

the juvenile snails is that, due to the decline of oxygen concentration in the respirometer during

the measurement cycle. Thus, it could be argued that the measurements of “normoxia”

actually represent a range of high but not fully air-saturated conditions.

Another criticism of the closed respirometry method is that the snails in closed

respirometers experienced declining levels of oxygen concentration over short period of time.

This contrasts to the natural environment, where the decline of oxygen occurs over a relatively

31

long period of time, which allows the animal to potentially acclimate to the low level of oxygen.

In addition to possible hypoxia during the measurements, there is another challenge for

animals to face during the experiment which is the effect of potentially high levels of CO2 and

nitrogen waste inside the chamber (Sheldon and walker,1989; Svendsen et al., 2016). Finally, it

should be consider that the presence of gas bubbles might skew the results. All these

circumstances could cause problems of unreliability with the collected data. In summary, for

juvenile snails the reader should be aware that the present results should be consider with

some caution that the conditions inside the closed chamber may not reflect the real natural life.

Yet, in the present experiments every attempt was made to minimize these possible negative

factors in oxygen consumption measurement by closed respirometry.

For adult snails, intermittent flow respirometry displays the best features to measure

oxygen consumption. The combination of the elements from two methods (closed respirometry

and flow respirometry) eliminated the problems associated with both methods (Svendsen et

al., 2016).

Another important consideration for all animals in this study is that the life histories of

experimental animals (and their parents, considering epigenetic inheritance) were unknown as

these animals were obtained from pet store, and could contribute to the variation in collected

data (Burggren, 2014) and also there is a genetic variation between individual animals.

4.2 Physiological Findings

4.2.1 Temperature and Oxygen Consumption

Temperature is important environmental parameter which influences the rate of most

physiology and biochemical activities (Precht et al., 1973). For aquatic organisms to overcome

32

temperature fluctuations they should be able to physiological and metabolically compensate

with any normal variation of environmental temperature (Aldridge, 1983). This change in rate

of physiological activities occurring in response to the temperature change is referred to as

thermal acclimation (Yanzeel, 1998). Most studies of metabolic rate and temperature

acclimation have been focused on temperate euthermal species - few published studies are

available for tropical and sub-tropical steno thermal species (Ramakrishnan, 2007)

In Mollusca, metabolic rate is significantly influenced by both acute experimental and

longer-term acclimation temperatures (Al-Khateeb, 2002). The temperatures at which

poikilothermic animals such as Molluscs show a maximum rate differ within a species and

depend on each animal’s recent thermal history (Al-Khateeb, 2002). In fact, pulmonate

freshwater species tend to show metabolic rate acclimation in both field and laboratory

conditions (McMahon, 1983). Stenothermal tropical species such as Pomacean snails exposed

to a narrow range of temperature change tend to have their metabolic rate less influenced by

temperature acclimation as there is no advantage of that adjustment (Segal, 1961). Some

researchers suggest that freshwater gastropods from stenothermal tropical habitats might be

less influenced by the selection pressure for evolution of thermal acclimation of oxygen

consumption compared to eurythermal temperate habitats (McMahon, 1979; Ramakrishnan,

2007). In this study, ṀO2 of juvenile snails in normoxia (18-21 kPa) acclimated at temperature

of 25°C ranged from 5 to 58 µMol O2/g/h while dult snails in normoxia at 25°C ṀO2 ranging

from 13 to 23 µMol O2/g/h . ṀO2 of Juvenile snails at 30°C, ranged from 78 to 107 µMol O2/g/h

while adult snails ṀO2 ranged from 14 to 20 µMol O2/g/h. ṀO2 of juveniles at 20°Cranging

from 64 to 88 µMol O2/g/h while adult snails showed the expected lower ṀO2 at 20°C (7 to 21

33

µMol O2/g/h)

In contrast, P. insularum adjusted mean ṀO2 at 20°C 44.77 µ L O/animal(mg) /h, while

at 25°C 59.37 µ L O/animal(mg) /h, at 30°C 68.06 µ L O/animal(mg) /h (Ramakrishnan, 2007).

The present study showed that there were strong developmental effects on the

temperature-metabolism relationship in P. bridgesii. The metabolic rate in juvenile snails

significantly differed at 20°C, 25°C and 30°C, as expected given that they are poikilotherms and

will respond to ambient temperature changes accordingly. Unexpectedly, however, in juvenile

snails there was a higher rate of oxygen consumption at 30°C and 20°C and lower rate at 25°C.

In contrast, adult snails displayed higher metabolic rates at 20°C and 25°C but a decreased rate

at 30°C. Under normoxic conditions (18-21 kPa), the ṀO2 of individual juvenile snails at their

preferred temperature of 25°C was higher than in adult snails at that same temperature, as

would be expected if this species conformed to metabolic scaling relationships based on body

size. While at 20°C ṀO2 was higher and highest at 30°C.

One explanation for these unexpected temperature-metabolism relationships is that

short term (acute) exposure to the change of temperature, such as in the present experiments,

provided an insufficient acclimation time to observe the acclimation response, since a

significant acclimation period is required for full metabolic changes to be expressed (Hill et al,

2004). However, as poikilotherms, these animals should have shown at least basic metabolic

rate changes associated with increase or decrease in temperature. For example, under acute

temperature exposure, P. lineata showed increased of oxygen consumption with raising

temperature between 5°C and 40°C and also showed some tolerance to high temperature. In

contrast, the oxygen consumption of P. insularum increased with temperature between 20°C

34

and 35°C while at 40°C showed highly depressed (Sanatos et al., 1987; Ramakrishnan, 2007).

Marisa cornuarietis also showed increased oxygen consumption with increase temperature

from 10°C to 35°C, but was reduced slightly at 35°C (Freiberg and Hazelwood, 1977). Another

possible point to consider in explanation is that the individuals in the present study were

obtained from a commercial fish store, so the thermal history of these animals was unknown.

Knowing the individual history of the experimental animals is important, as the physiological

responses can be significantly influenced by their recent history (AlKhateeb, 2002; Hill et al,

2004; Burggren, 2014). For example, Mytilus californianus is a species of mussel living on the

West Coast of the United States. This species was collected at three different altitudes and

each group was exposed to different temperatures and the pumping rateP1F

2P determined for each

group. The results indicated that the group that was collected at high altitude had a higher

pumping rate at 9°C in comparing to others (Hill et al, 2004). This illustrates that individual

history is important point to consider when study poikilotherms animals as the physiological

responses depends on the life history of animal (Hill et al, 2004)

Another point to consider is that as is increasingly epigenetic effects reflecting parental

experiences which cause offspring to response differently to physiological change. As an

example, in the water flea Daphnia magna, groups whose mothers had been exposed to

hypoxia (~4KPa) showed higher rate of V�O2 in comparison to the other groups whose mothers

had not been experienced hypoxic condition (Andrewartha and Burggren, 2012). As another

example, in the emu (Dromaius novaehollandiae), the maternal effect on egg size leads to high

embryonic metabolic rate in larger eggs more than in smaller ones (Dzialowski and Sotherland,

2 Is the rate at which animals pumped water across their gills.

35

2004)

These examples suggest that perhaps some combination of thermal and oxygen history

may have contributed to the unpredicted findings on metabolic rate and temperature reported

in the present study.

Further insight into the metabolism-temperature relationship of P. bridgesii may come

from examining the Q10 values for oxygen consumption in this species (Table 2) as well as other

species for which it has been reported. The Q10 values of P. bridgesii in this study were higher

in juvenile snails than in larger adult snails. The higher Q10 at 25-30°C for juvenile might be

related to locomotory activity (Akerlund, 1969). In freshwater pulmonates and probranches,

Q10 decreased as the animal reached their upper thermal limits (Ramakrishnan, 2007). P.

lineata shows doubled Q 10 values from 5°C to 20°C and then reduced values from 20°C to

25°C (Santos et al., 1986), a pattern similar to that recorded in the present study for adult P.

bridgesii. However, in P. palludosa the Q₁₀ values were higher (2.58 and 2.97) between 10-20

°C than between 20-30°C (Freiberg and Hazelwood, 1977). In juvenile P. insularum, the Q10

increases with temperature between 25°C -35°C with Q₁₀ values between 1.31 and 1.39

(Ramakrishnan, 2007), while in Marisa cornuarietis Q10 increased with size at 10°C-20°Cand

20°C -30°C (Akelund,1969; Freiberg and Hazelwwod,1977). The reason for the complex

temperature-metabolism relationships in P. bridgesii has not been resolved in the present

study. However, at a minimum we can conclude that the decline in oxygen consumption at

high temperatures in the adults indicates that 30°C is towards their upper thermal limit.

4.2.2 Oxygen Level and Oxygen Consumption

Oxygen availability is another important environmental parameter which determines

36

the distribution of aquatic animals, especially Pomacean snails (Hanley and Ultsch, 1999;

Seuffert and Martín, 2010). Pomacea snails are amphibious (Cowie, 2006). They use a lung for

aerial gas exchange while the gill is used for aquatic exchange (Thieng, 1987). The dependence

on air breathing increases with declining dissolved oxygen, water temperature increase and a

high level of fouling of water (Aldridge,1983). Pomacean snails have the ability to maintain

normal levels of oxygen consumption down to dissolved oxygen saturation levels of 21-50% of

full saturation by employing branchial respiration (Hanley and Ultsch, 1991; Ramakrishnan,

2007).

Aquatic animals can be classified into one of two groups, oxyconformers or

oxyregulators, based on their response to a decrease of oxygen partial pressure

PO2.Oxyconformers are animals that reduce their oxygen consumption (V�O2) with decreasing

of oxygen tension (PO2), while oxyregulators maintain their oxygen consumption constant over

a wide range of deceasing oxygen tension (PO2) until a critical PO2 is reached, below which

ṀO2 declines directly with further decreases of PO2 (Sloman et al., 2006; Ramakrishnan, 2007).

Among Pomacean snails, juvenile of P.insularum shows poor O2 regulation (oxyconformation)

while P. lineata exhibited effective O2 regulation in the face of declining of oxygen partial

pressure (Santos et al., 1986; Ramakrishnan, 2007).

In the present study, visual inspection of the graphs (Figures 2.1, 3.1) show that juvenile

P. bridgesii were oxyconfromers, since ṀO2 began to decline more or less immediately with

falling PO2. In fact, the decline began with the first decrease in PO2, indicating critical ambient

oxygen tension (P crit,) of at or just below normoxic levels. Adult P. bridgesii, on the other hand,

were oxyconfromers at 20°C and 30°C but at 25 °C showed poor regulation with declining PO2 .

37

In the current experiment, both juveniles and adults have not previously experienced

any hypoxic condition, as all the tanks were aerated. However, as indicated above for

temperature, the oxygenation history of the parents of the snails could not be verified, and may

have led to epigenetic transgenerational influences (Burggren, 2014).

4.2.3 Body Mass and Oxygen Consumption

In general, metabolic rate decreases with increasing body weight in molluscs (Santos

and Mendes, 1981), which conforms to general allometric relationships in almost all animals

irrespective of whether they are ectotherms or endotherms, oxyconformers or oxyregulators

and usually described by this function, R = a M b where is R is the metabolic rate, a is the

intercept in a log-log plot, M is body mass, and b is the slope of a log-log plot ( b value is less

than 1)( Glazier, 2015). In prosobranch snails, the scaling exponent, b, in the allometric relating

oxygen consumption and body size are between 0.67 and 0.95. However, in pulmonate snails

the b-values are between 0.45 and 1 (Berg and Ockelmann, 1959). For example, Pomacea

insularum had b-values ranging from 0.9 to 1 at test temperatures ranging from 20°C to 30°C

(Ramakrishnan, 2007). In Maisa cornuariets , at test temperatures of 20°C -35 °C b had values

ranging from 0.49 to 0.78 (Akerlund, 1974)

The present experiments revealed that the oxygen consumption of P. bridgesii

decreased with increasing body weight and showed a negative linear correlation between

metabolic rate and body weight with b values between 0.23 and 0.76. The wide variation

observed in the metabolic rate of juvenile and adult of P. bridgesii could be explained by

interspecific variation in the b value which may be vary by seasons (Berg and Ocklmann, 1959).

Also, the results of the present experiments on metabolic rate show that mass-specific oxygen

38

consumption is higher in small snails than in big snails. The explanations for that difference

mass-specific oxygen consumption in small individuals as compared with large one is still not

clear (Al-Khateeb, 2009). It might be that big animals have a larger proportion of tissue with low

metabolic rate, such as connective tissue and shell components. Small individuals have large

number of mitochondria which probably utilized for more energy production to support the

growth process (Al-Khateeb, 2009).

4.3 Implications of Metabolic Rate to the Distribution of Pomacea bridgesii

The Ampullariid snail (Pomacea bridgesii) is endemic to South American habitats (Perera

and Walls, 1996). The introduction of this species in the USA started in the 1960s, in Florida

(Howells et al., 2006). In Texas, P. bridgesii has been reported in the Brazos River, Waco,

McClenna County in 2004 (Howells et al., 2006). There is apparently not any natural predator

for this species in these invasive habitats, so, there is a possibility for this species to spread out

and displace the native species Pomacea paludosa in Florida (Warren, 1997; Howells et al.,

2006) and in Texas. Also, P. bridgesii might have negative impacts on native plant species,

leading to system imbalances in the ecosystem. In Florida, everglades kits which feed on native

species P. paludosa, had difficulty to extract the meat from Pomacean snails as result to the

thickness of operculum .Due to that, P. bridgesii and other Pomacean species might replacing a

native species P. paludosa in their habitat (Warren, 1997; Howells et al., 2006).

Knowing the physiological characteristics of this species is essential to predicting their

distribution and determining the risk of assessment for their invasion of new habitats, such as

aquatic habitats in Texas. In fact, temperature plays a critical role in all life functions of

39

organism and animal distribution (Hanley and Ultsch,1999; Sokolova and Pörtner, 2003;). The

optimum ambient temperature of Pomacean snails ranges from 20°C to 30°C and 25°C consider

as optimal temperature for growth and reproduction (Estebenet and Martin, 2002). Due to

climate change, the mean annual air temperatures has been estimated to increase by >1°C

every 50 years in the USA (NOAA, 2007a, b). As a result of climate change, Pomacean snails

may experience an enhanced ability to invade warm temperate habitat such as Southern region

of the USA (Perera and Walls, 1996; Ramakrishnan, 2007). On the other hand, the present

experiments indicate that adult snails show reduced ṀO2 at higher temperatures. Thus, it may

become too warm for this species in the most southern regions, but at the same time the

middle regions of the country could be ripe for invasion by P. bridgesii.

4.4 Future Experiments

According to my experiments, knowing the recent life history of animal may be

important to interpretation of the results. Epigenetic changes may lead to the change on

physiological response on organism. Consequently, I suggest that in future experiments:

• Individuals be collected from the known environments

• Animals be study deeply for the effect of temperature, PH, oxygen level on

respiratory rate on two different generations.

• Attention be paid to epigenetic effects - whether maternal or paternal – by exposing

animals to physiological stress such as hypoxia or salinity to show physiological and

histological changes in their offspring that might take place through epigenetic

mechanisms.

40

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