1. Plate 1.Anatomyofa crayfishimagefrom
http://www.biographixmedia.com/biology/crayfis
h-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

2. 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

3. 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.
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 2. Mean ±Std Devfor Sodium andAmmonia samples takenacross increasing concentrations
ofsalinity. P =normality P value, n =number ofsamples tested. FW =Freshwater

4. 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 takenatincreasing salinity
concentrations
0
50
100
150
200
250
300
% (FW) freshwater concentration
across increasing salinity
mMolNa+
Average Sodium concentration ofhaemolymph
acrossincreasing salinity levels
100%FW
75% FW
50%FW
25%FW
0
0.1
0.2
0.3
0.4
0.5
% (FW) freshwater concentration
across increasing salinity
mMolNH3
Average Ammoniaconcentrationoftank water
acrossincreasing salinity levels
100%FW
75% FW
50%FW
25%FW
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% FW
Wriggles Activity levels Medium High low low
Antennal
movements
per minute 5 12 0 0
Bandit Activity levels Medium High Low Medium
Antennal
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
activities by retention of water in reduced urination, but containing higher concentrations of
Figure 5. Recorded behavioural observations from two Signal Crayfish
subjects across increasing salinity levels.

5. 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.

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