Plate 1. Anatomy of a crayfish image from http://www.biographixmedia.com/biology/crayfish-anatomy.html

Osmoregulation: Signal Crayfish ( Pacifasstacus leniusculus ) response to changes in environmental salinity

Abstract

Introduction

Since the 1800s Natural England has recorded that over 492 species have become extinct within the UK (Natural England 2010). One of the most significant drivers for extinction within the UK is the increasing abundance of invasive species which outcompete native or endemic species (Lowe et al 2000). Almost half of the world’s crayfish species are at risk of significant population decline and extinction, due in part to the introduction of non-native species (Taylor 2002). The white clawed crayfish (Austropotamobius pallipes) has been assessed as endangered by the IUCN red list due to its rapid population decline within the UK since the 1970s (Füreder et al 2010). The major cause for population decline in this species is due to the introduction of non-native crayfish species which were farmed throughout the 1970s. One such invasive species is the Signal Crayfish (Pacifasstacus leniusculus) which is believed to be responsible for a crayfish plague caused by the fungus Aphanomyces astaci which spread rapidly throughout English rivers, devastating native crayfish species (JNCC 2015; Holdich & Reeve 1991). Signal Crayfish are more aggressive and subsequently outcompete the white clawed crayfish for niche’s in which they both occupy; consequently producing more offspring and becoming well established within waterways across the UK (Peay et al 2010). The environmental agency now enforces strict regulations for the protection of native species and the eradication of invasive species; as it is an offence for any non-native crayfish species to be released without a licence (Environmental Agency 2006).

Signal crayfish have thrived within UK rivers and lakes due to their efficiency in osmoregulation; in addition to their aggressive nature (Bubb et al 2004) . As the species is euryhaline they have the ability to regulate their haemolymph salinity concentration by maintaining it as hyperosmotic in freshwater. They do this by regulation of sodium and chloride via uptake of ions across the gills where it is then controlled by amino acid concentrations within tissue fluid (Susanto & Charmantier 2001). Though the species rarely encounter brackish water, the osmotic pressure it causes triggers and osmoregulatory response in the crayfish. The signal crayfish can regulate intracellular fluid content to prevent tissue damage and dehydration; by control of the dilation of ammonia excretions within their urine. If the crayfish is faced with hyperosmotic waters, the amount of urine will decrease however salt excretions will be in higher concentrations allowing for retention of water but release of excess salt due to the higher external salinity (Wheatly & McMahon 1982). The excess

salt is released in the form of ammonia via the green gland located at the base of the antennae (Plate 1), which is opened and closed via movement of the antennae (Wilmer et al 2005).

This investigation aims to identify via what mechanism and how efficiently signal crayfish control haemolymph pH and sodium concentrations via ammonia excretions across varying salinity concentrations; to assess the species ability to survive within changing freshwater and brackish environments.

Method

This experiment followed the experimental design outlined in Wheatly & McMahon (1982) in their investigation into responses to hypersaline exposure in the euryhaline crayfish Pacifastacus leniusculus .

Animals

Twenty Signal Crayfish where contained separately within tanks of mixed freshwater and seawater at four concentrations of salinity (0%, 25%, 50%, 75%). Two crayfish were used in behavioural observations, Wriggles (Male, 44g) and Bandit (Female, 27g). Their mean weight (60g) was used for haemolymph analysis.

Experimental protocol

Individual crayfish were housed in varying salinity concentration for three days each, progressing on concentration strength from 0% to 75%. After each three day interval a sample of the crayfishes blood (haemolymph) was taken and a sample of the tank water was taken to analyse the water ammonia content. Behavioural observations were make of Wriggles and Bandit by video recording antennal movements as they progressed through the varying salinity concentrations.

Analytical procedures

A flame photometer was used to measure the sodium concentration in the haemolymph samples taken. Before measurements were taken, a calibration curve was produced to an accurate range for the sodium levels identified in the haemolymph. The calibration was

produced via measuring three known concentrations of Na+ (Sodium) (200, 250, 300 mMoles) which were then used as a reference point for the haemolymph readings.The haemolymph samples were then given a flame photometer reading for sodium content across the three salinity concentrations (0%, 25%, 50%, 75%). These reading were then plotted against the known sodium calibration curve to determine the sodium content of the haemolymph.

A spectrophotometer was used to measure the ammonia concentration of the tank water across the three salinity concentrations. The spectrophotometer was first zeroed using 10ml of distilled water combined with 1ml sodium potassium tartrate and 0.5ml Nessler’s solution.

A reading was then taken at 425nm after five minutes and zeroed to that amount. A calibration curve was then produced using known amounts at 0.15, 0.125, 0.1, 0.075 and 0.05 mMol. 10ml of each sample were individually measured by the spectrophotometer in combination with 1ml of sodium potassium tartrate and 0.5ml of Nessler’s reagent; at 425nm after five minutes. The readings taken were then used to produce a calibration curve.The four tank water samples at increasing salinity were then individually combined with distilled water until 10ml of solution could be combined with 1ml of sodium potassium tartrate and 0.5ml of Nessler’s reagent. Readings were then taken from the spectrophotometer after 5 minutes at 425nm, for each salinity concentration. The readings were then plot against the known concentration calibration curve to determine the tank water ammonia content in mMol.

Behavioural observations

Wiggles and Bandit movements were observed and recorded to determine activity level (low, medium and high) and antennal movements throughout one minutes periods; across the increasing salinity levels (0%, 25%, 50%, 75%). Antennal movements were used as an indication of urination and ammonia excretion.

Results

Sodium and Ammonia values

Figure 2 demonstrates that the samples taken for Sodium were all parametric, however all the samples taken for Ammonia were non-parametric. Results were obtained using a Sharipo-Wilks normality test.Due to some of the data being non-parametric, a Spearman’s rank correlation was produced. Though Spearman’s indicated there was a negative correlation between all Na+ (Sodium) concentration throughout each salinity level and all NH3 (Ammonia) concentrations across each salinity level; only one of these correlations was significant. There was a significant negative correlation between Sodium concentrations at 25% FW and Ammonia concentrations at 100% FW with a P value = -0.732. This indicates that if one of the solutes is highly concentrated in 100% FW, then the other will be decreased in 25% FW; and vice versa.

Figure 2. Mean ± Std Dev for Sodium and Ammonia samples taken across increasing concentrations of salinity. P = normality P value, n = number of samples tested. FW = Freshwater

Solute 100 % FW 75 % FW 50% FW 25 % FW

Sodium (NA+ mMol)

214.87 ± 5.33 P= 0.837 n= 21

227.70 ± 9.00 P= 0.383 n=21

241.26 ± 6.81 P= 0.716 n= 21

273.17 ± 5.81 P= 0.386 n=21

Ammonia (NH3 mMol)

0.182 ± 0.18 P= 0.000 n= 21

0.240 ± 0.19 P= 0.000 n=21

0.325 ± 0.21 P= 0.002 n= 21

0.349 ± 0.244 P=0.001 n=21

Figure 3. Sodium concentrations (Na+ mMol) recorded within haemolymph samples taken at increasing salinity concentrations

Figure 4. Ammonia concentrations (NH3 mMol) recorded within tank water samples taken at increasing salinity concentrations

Sodium and Ammonia comparisons

Figure 3 demonstrates that as salinity increases, so

too does the concentration of Sodium in the haemolymph of the Signal Crayfish. This strongly supports the suggestion that the crayfish is conducting osmoregulatory activities via uptake of sodium due to osmotic pressure from the hyperosmotic solution it is contained within.

Figure 4 demonstrates that as salinity increases, so too does the concentration of Ammonia found within the tank water. This also supports the suggestion that the Signal Crayfish are regulating their haemolymph pH via excretion of excess NH3 (Ammonia) in increasing concentrations within urine as salinity level increasing. This is another osmoregulatory activity.

Behavioural observations

Subject Observation 100% FW 75% FW 50% FW 25% FWWriggles Activity levels Medium High low low

Antennal movements per minute 5 12 0 0

Bandit Activity levels Medium High Low MediumAntennal movements per minute 5 16 3 11

Figure 5 indicates that as salinity increased, both subjects demonstrated reduced activity levels in 50% and 25% FW. This could be due to the high energy cost of osmoregulation and the Crayfish reduction in movement is a method of energy preservation for increased efficiency in the metabolic processes required for osmoregulation. Antennal movement’s also reduced in Wriggles at high salinity levels, further supporting efficient osmoregulatory

% (FW) freshwater concentration across increasing salinity

0

50

100

150

200

250

300

Average Sodium concentration of haemolymph across increasing salinity levels

100%FW75% FW50%FW25%FW

mM

ol N

a+

% (FW) freshwater concen-tration across increasing

salinity

0

0.1

0.2

0.3

0.4

Average Ammonia concentration of tank water across increasing salinity levels

100%FW75% FW50%FW25%FW

mM

ol N

H3

Figure 5. Recorded behavioural observations from two Signal Crayfish subjects across increasing salinity levels.

activities by retention of water in reduced urination, but containing higher concentrations of Ammonia. However Bandit, who also reduced its antennal movements notably at 50% FW, still maintained relatively high antennal movements at 25% FW. This could be an indication that Bandit not only had an excess of Ammonia for excretion at 25% FW, but also a slight water excess that too required excretion.

Discussion

The findings of this investigation strongly support findings such as those produced by Wheatly & McMahon (1982) and Wheatly & Henry (1987) which identify Signal Crayfish as osmoregulators. Data collected and tested in this investigation identified that as salinity concentration increased and freshwater concentration decreased, there was a notable increase in Sodium content within each crayfish haemolymph indicating that Signal Crayfish were capable of responding to osmotic pressure being applied by the hyperosmotic solution it was contained within, by attempting to achieve isosmotic balance by excretion of water and uptake of Sodium ions via ion regulation. Due to this the null hypothesis that “there is no change in Sodium concentration within haemolymph as salinity increases” must be rejected and the alternative hypothesis that “there is a change in Sodium concentration within haemolymph as salinity increases” must be accepted.Ammonia concentrations within the tank water also increased when salinity increased, supporting findings by Usio et al (2006) that urination by crayfish decreases with increased salinity, but Ammonia concentrations within the urine increase. The null hypothesis of “no change in Ammonia concentration across increasing salinity” must be rejected and the alternative of “there is a change in Ammonia concentration across increasing salinity” must be accepted.The Signal Crayfish behavioural response to increasing salinity demonstrates efficient energy conservation for prolonged osmoregulatory activities under high hyperosmotic conditions. This would indicate that the species is highly adaptive to brackish waters and can efficiently osmoregulate sodium levels and pH within its haemolymph to thrive within some of the more extreme environments of the UK’s waterways. To further improve this study, a similar investigation as to the osomoreglatory efficiency of interspecific competitors such as the white-clawed crayfish would be of great benefit in determining preferential salinity concentrations during niche selection between the two species.

References

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