The objective of this research was to determine the effects of acidification, a change in water pH, on the grass shrimp Palaemonetes pugio. The null hypothesis is that when water temperature, salinity and pH change, the heart rate of the grass shrimp in beats per minute will not change. The dependent variable was heart rate as a measure of metabolic oxygen consumption. The independent variables were salinity, water temperature, pH and the length of each P. pugio.
The size in length of each P. pugio was inversely correlated with heart rate and beats per millimeter of length. The average heart rate of P. pugio at a pH of 6.5 was significantly lower than at a pH of 7.5. The heart rate of P. pugio did not vary significantly at salinities of 10, 15, 20 and 25 parts per thousand. The average heart rate of the prawn at 10°C was significantly lower than that at a water temperature of 25°C.
Arrow pointing to the location of heart in Palaemonetes pugio. Photo courtesy of David Remsen.
The null hypothesis that the heart rate of P. pugio would not vary with a change in pH was rejected; the average heart rate at 6.5 was significantly lower than at 7.5. The null hypothesis that heart rate would not vary with a change in salinity was accepted; the average heart rate did not vary significantly with changes in salinity. The null hypothesis that heart rate would not vary with a change in water temperature was rejected.
This is the first study to show that as the length of the prawn increases, its heart rate declines, regardless of variations in pH, water temperature and salinity. Based on the inverse relationship between the length of P. pugio and both heart rate and beats/mm, it is recommended that scientists in future studies measure the organism's length to calculate the beats/mm of P. pugio and other species of Palaemonetes.
Introduction
Acidification in the Chesapeake Bay and the oceans is caused by CO2 dissolving in water (H2O + CO2→ H2CO3-), which results in a declining pH through the formation of carbonic acid. No studies have yet been conducted on the effects of declining pH on grass shrimp species of Palaemonetes. Two species of grass shrimp live in Chesapeake Bay. Palaemonetes pugio prefers aquatic macrophyte and shell substrate habitats, and tolerates a wide range of salinities (Anderson 1985). Palaemonetes vulgaris can be found in low, sandy marsh creeks and can tolerate high salinities but not low ones (Knowlton et al. 1994).
Much research has been done on Palaemonetes species and the Chesapeake Bay. Knowlton and Kirby (1984) tested salinity tolerance and sodium balance in the prawn P. pugio and figured out that P. pugio can regulate its blood sodium level within its tolerable range of salinity. Knowlton (1965) tested the effects of some environmental factors on the larval development of P. vulgaris and found that larval variation is probably enhanced in nature by differences in the amount and kind of food present. Reinsel et al. (2001) tested the effects of food availability on the survival, growth and reproduction of P. pugio. Welsh (1975) observed the role of grass shrimp in a tidal marsh ecosystem, and found that the shrimp macerated detritus into a heterogeneous assortment of uneaten particles by plucking away the cellular matrix from the surfaces of large detrital fragments. Arguin et al. (2002) tested water salinity and substrate as factors affecting the seasonal abundance and spatial distribution ofPalaemonetes species. Knowlton et al. (1994) observed that when P. pugio and P. vulgaris were exposed to an array of 32 cubicles, each provided with one of four substrates (wood, mud, sand and shells), both species showed a significant preference for wood, regardless of salinity. Floyd (1977) tested the effects of temperature and salinity on the larval development of P. pugio. Results indicated that temperature, salinity and their interaction have a significant effect on survival, larval duration, number of molts and intermolt duration. Anderson (1985) observed that because populations of grass shrimp may produce more than two broods a year, length-to-frequency distributions may be polymodal and growth rates difficult to characterize.
Although many studies had investigated the life history of Palaemonetes species and their tolerance for salinity and temperature changes, there were no investigations that looked at the effect of water acidification on Palaemonetes. The objective of my research was to determine the effect of changes in pH, water temperature and salinity on the heart rate of Palaemonetes pugio.
The null hypothesis was that changes in the water temperature, salinity and pH would not have effects on the prawns' metabolism. The dependant variable was the prawns' heart rate, a marker of metabolic oxygen consumption (Knowlton, personal communication). The independent variables were salinity, water temperature, pH level and the size of the prawn.
Methods and Materials
A seine is used to collect grass shrimp.
George collects water for testing.
Specimens of P. pugio were collected from lower portions of the York River in Virginia from 4 to 7:14 p.m. on 11 September 2010. The elevation was one meter above sea level, salinity was 22 parts per thousand, the water temperature was 23°C, and the pH was 8.0. The prawns were collected using dip nets and a 1.2 meter-by-4.2 meter seine with a mesh size of 0.32 centimeters. Specimens were put into a five-gallon plastic bucket equipped with an aerator, and then transported to a home laboratory, where they were maintained in the water from which they had been collected.
The specimens are transferred to petri dishes.
Specimens were put in a five gallon bucket equipped with an aerator.
The prawn specimens were acclimated to different test conditions for at least 24 hours. Water temperature, pH, salinity and the prawns' heart rates were measured and recorded during the experiments.
Procedure for pH Experiments
In the lab, the pH of the water was lowered by adding small amounts of HCl until the desired pH levels were reached (6.5, 7.0 and 7.5). The river water in which the specimens were collected was used for the pH 8.0 test. Five specimens of P. pugio were put into holding tanks at different pH concentrations and held for a 24-hour period before measuring their heart rate. Then the specimens were transferred one at a time to a petri dish. Their heart rate was measured by counting the number of heartbeats per minute under a dissecting microscope using a stopwatch (Table 1). The heart rate was measured for pHs of 6.5, 7.0, 7.5 and 8.0 at a constant water temperature of 24°C and salinity of 22 parts per thousand, comparable to the temperature and salinity of the water from which the specimens were collected.
Procedure for Salinity Experiments
Specimens were maintained at a constant temperature and pH while the salinity of the water was varied (Table 1). Salinity was increased to 25 parts per thousand using Instant Ocean. Salinity was lowered to 10, 15 and 20 parts per thousand by adding freshwater from the James River that had been collected in a five-gallon jug. The heart rate of each specimen was measured using the same procedure described for the pH experiments.
Procedure for Water Temperature Experiments
Specimens were maintained at a constant pH and salinity while the water temperature was varied (Table 1). Specimens for 10°C were placed for 24 hours at the bottom of a refrigerator where the actual temperature was 10°C. The heart rate of each specimen at 10°C was measured immediately after it was placed in a petri dish. Those for the 15°C experiments were placed on the top shelf of the refrigerator for 24 hours, where the temperature was 12°C, and then removed and allowed to warm to 15°C, when their heart rates were measured. Specimens at 20°C were maintained in an air-conditioned room, and those at 25°C in an un-airconditioned room.
Data Analysis
George measures the heart rate of each specimen.
The average heart rate was calculated for the five specimens of P. pugio at each pH level, the five specimens at each salinity level, and the five specimens at each temperature level. After each test, the total length of each specimen was measured from rostrum to telson and used to calculate heartbeats per millimeter. Average heart rates and beats/mm for each treatment were compared using a multiple range test. Correlation analysis was used to determine the relationships between heart rate, beats/mm, pH, salinity and water temperature. Regression analysis was used to determine the relationship of heart rate and beats/mm to body length as well as the salinity, temperature and pH of the water.
Results
Table 1. Correlation analyses of salinity (ppt), water temperature (C), pH, and length (mm), heart rate (beats/minute), and beats/mm of Palaemonetes pugio.
The length of P. pugio specimens was inversely correlated with heart rate (R=-0.39583, P= 0.0016).
The length of P. pugio was inversely correlated with beats/mm (R=-0.89194, P=0.0001).
The heart rate of P. pugio was positively correlated with temperature (R=0.6068, P=> 0.0001).
Beats/mm of P. pugio was positively correlated with temperature (R=0.30583, P=> 0.0165).
The pH of the water was inversely correlated with temperature (R=-0.44476, P=0.0003).
The heart rate of P. pugio was inversely correlated with salinity (R=-0.21823, P = 0.0911).
No specimens died in any of the pH, salinity or temperature tests during the course of the experiments.
Table 2. Mean, std. dev., minimum and maximum values of heart rate (beats/minute) and beats/mm of Palaemonetes pugio at varying levels of pH, salinity (ppt), and water temperature (C).
The average shrimp heart rate at pH 6.5 was significantly lower than at pH 7.5. Average beats/mm at pH 6.5 was lower than at pH 7.0 to 8.0 (Table 2). However, there was no statistical difference between the heart rates of P. pugio at pH 6.5, 7.9, 7.5 and 8.0 (Table 3).
The heart rate of P. pugio did not vary significantly at salinities of 10, 15, 20 and 25 parts per thousand. Similarly, the beats/mm of P. pugio did not vary significantly at salinities of 10, 15, 20 and 25 parts per thousand.
Table 3. Results of Duncan's Multiple Range tests for average heart rate (beats/minute) and beats/mm total length of Palaemonetes pugio at varying pH, water temperature (C), and salinity (ppt). Underscored means do not vary significantly at p=0.05.
The average prawn heart rate at 10°C was significantly lower than at 25°C (Tables 2 and 3). Although the average beats/mm at 10°C was lower than at 15° to 25°C, it was not significantly different (Table 3).
Regression analyses (Table 4) indicated that heart rate (beats/minute) of P. pugio can be modeled with salinity, pH, water temperature and body size as follows: Heart rate = 0.24418 + (salinity) * -1.96 + (pH) * 26.50 + (water temperature) * 7.18 + (length) * -1.80
Regression analyses (Table 5) indicated that the average beats/mm of P. pugio can be modeled with salinity, pH, water temperature and body size as follows: Beats/mm = 17.65 + (salinity) * -0.167 + (pH) * 1.7 + (water temperature) * 0.32 + (length) * -0.91
Table 4. Regression analyses of salinity, pH, water temperature, and length and heart rate (beats/minute) of Palaemonetes pugio.
Discussion and Conclusions
The average prawn heart rate at pH 6.5 was lower than at pH 7.5; the null hypothesis for pH is rejected. The average prawn heart rate and beats/mm did not vary significantly with salinity, so the null hypothesis for salinity is accepted. The average prawn heart rate varied significantly with changes in temperature, so the null hypothesis for water temperature is rejected. There was a direct correlation between increasing temperature and increasing heart rate.
Table 5. Regression analyses of salinity, pH, water temperature, and length (mm) and beats/mm of body length of Palaemonetes pugio.
This is the first study to determine that as the length of P. pugio increases, its heart rate declines, regardless of variations in pH, water temperature and salinity. Similarly, as the length of P. pugio increases, beats/mm decline regardless of variations in pH, water temperature and salinity. Previous researchers (Anderson 1985, Floyd 1977, Knowlton and Kirby 1984, Knowlton and Schoen 1984, Knowlton 1965) didn't consider variations in length as a variable that could account for a variation in heart rate in their studies. Based on the inverse relationship between length of P. pugio and both heart rate and beats/mm, it is recommended that scientists in future studies measure the length of grass shrimp to calculate the beats/mm of P. pugio and other species of Palaemonetes. This will serve to remove the effects of length on heart rate when varying other parameters such as water temperature, salinity and pH.
Kirby and Knowlton (1984) showed that adult P. pugio can tolerate a wide range of salinity (1 to 35 parts per thousand), with relatively low mortality at higher salinities because the levels of sodium in their blood remained relatively constant over the range of salinity tested. This shows the ability of P. pugio to osmoregulate. The heart rate of the shrimp in the present study did not vary significantly with changes in salinity, and no specimens died during the course of the study. These results reflect the ability of P. pugio to withstand changes in salinity without having to increase its heart rate to osmoregulate. Heart rate is a marker of metabolism related to oxygen consumption and salt balance.
P. pugio is eurythermal and thrives at temperatures of 5° to 38°C. However, its optimum temperature range is 18° to 25°C (Anderson 1985). The results of the present study, in which prawn heart rates were lower at a low temperature (10°C) and greater at a higher temperature (25°C), supports the findings of temperature effects on P. pugio in studies by Anderson (1985), Knowlton and Kirby (1984) and Knowlton (1965).
The present study indicates that shrimp heart rates at pH 6.5 were significantly lower than that at pH 7.5. Specimens at pH 6.5 appeared slow and sluggish, which probably reflects their low heart rate. McCulloch (1990) studied the metabolic response of the grass shrimp Palaemonetes kadiakensis to sublethal changes in pH in Texas near the Gulf of Mexico. He determined that pH had an effect on critical oxygen concentration, which was significantly higher at a pH of 6.5 than the control (7.8), and significantly lower at a pH of 9.0 than the control. The present study, in which prawn heart rates were slow at a pH of 6.5, is contrary to the results of McCulloch, in which oxygen consumption was higher at a pH of 6.5. The reason for the difference could be related to physiological and genetic differences between the two species (P. kadiakensis and P. pugio), and the variation in salinities and temperatures between the two geographic locations in which the two species live.
I sincerely thank Dr. Robert Knowlton of George Washington University for giving me personal advice on how to conduct my experiment, and Dr. Eugene Maurakis of the University of Richmond for guiding me through the experiment and showing me how to get stuff done and run the statistical analyses.
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