J OURNAL OF C RUSTACEAN B IOLOGY, 36(2), 189-197, 2016
EXOSKELETON CALCIFICATION IN NORWEGIAN POPULATIONS OF THE
CRAYFISH ASTACUS ASTACUS (LINNAEUS, 1758) (DECAPODA: ASTACIDAE)
VARIES WITH SIZE, GENDER, AND AMBIENT CALCIUM CONCENTRATION
Svein Birger Wærvågen 1,∗ , Tom Andersen 2 , and Trond Taugbøl 3
1 Department
of Natural Sciences and Technology, Hedmark University of Applied Sciences, Hamar, Norway
2 Department of Biosciences, University of Oslo, Oslo, Norway
3 Glommen & Laagens Brukseierforening, Lillehammer, Norway
ABSTRACT
(Ca2+ )
Declining ambient calcium
concentrations in boreal, soft-water lakes of North America and Europe is one of many threats facing
their biotic assemblages such as crayfish populations. We examined the specific exoskeleton calcium (Ca) concentration in Astacus astacus
(Linnaeus, 1758) populations from a wide range of ambient Ca2+ concentrations to determine a possible correlation between the amount
of Ca accumulated in their carapaces and the ambient Ca2+ concentrations. Exoskeleton Ca was the major constituent of the crayfish A.
astacus carapaces in this survey (21.2 to 25.8% Ca of dry weight (DW)), whereas magnesium (Mg) displayed a disproportionately low
constituent. The strong correlation between mineral contents of dry weight (DW) and ash weight (AW) (r = 0.98) allowed us to refer
mineral contents consequently to DW. A linear model using gender, length and ambient Ca2+ concentration (log transformed) explained
82% of the variation in carapace Ca content (as % DW). Astacus astacus females were slightly more calcified than males (0.4% of DW,
when adjusted for ambient Ca2+ and body length). Large-bodied populations were slightly, but significantly more heavily calcified than
those with smaller bodies: carapace Ca content increased by 0.2% DW for each cm increase in body length. The strong logarithmic effect
of ambient Ca2+ implies that carapace Ca content increases by 1.7 × log(2) = 1.2% DW for every doubling of the Ca2+ concentration in
the water.
K EY W ORDS: base cations, calcium decline, climate change, intermoult calcification, magnesium
DOI: 10.1163/1937240X-00002406
I NTRODUCTION
Most freshwater crayfishes belong to three families, Astacidae and Cambaridae in the Northern Hemisphere (Europe and North America, respectively), and Parastacidae in
the Southern Hemisphere (Australasia) (Hobbs, 1988). The
genus Astacus has a natural widespread distribution in Europe (Cukerzis, 1988; Skurdal and Taugbøl, 2002), and the
most common species is the cold-water noble crayfish Astacus astacus (Linnaeus, 1758) present in 39 European countries (Souty-Grosset et al., 2006). In Norway it is mainly
restricted to the south-eastern geographical region, and has
been stocked in most water bodies due to its commercial
value (Skurdal et al., 1999).
Crayfishes moult throughout life, and grow by shedding
the exoskeleton. Adult females and males of A. astacus
normally shed their exoskeleton once and twice per year,
respectively, with main moulting periods in early summer
and early autumn (Skurdal and Qvenild, 1986; Huner et al.,
1991). Moult timing and frequency, as well as length increment per moult can vary, both between populations and annually within the same locality, depending on climatic conditions, population size, and food availability. The crayfish
moult cycle consists of several stages termed A-E. Stage C
displays the longest duration, occupying nearly 65% of the
moult cycle, of which the intermoult stage C4 is by far the
∗ Corresponding
longest and most heavily calcified substage (Drach, 1939,
1944; Travis, 1965; Stevenson, 1968; Lowery, 1988; Aiken
and Waddy, 1992). Moulting itself takes a few hours, remineralisation is almost complete within 2 to 4 days, but it
takes at least two weeks for the crayfish exoskeleton to completely harden (Welinder, 1975; Huner et al., 1978; Taugbøl
et al., 1997). Significant differences in exoskeleton calcification between moult stages have been documented (Huner et
al., 1990).
Bicarbonate (or Ca2+ ) has been suggested as the major determinant to distribution and success for calciumdemanding invertebrates such as crayfishes and several other
crustaceans (Yan et al., 1989; Hessen et al., 1991; Wheatly,
1996; Wærvågen et al., 2002; Jeziorski et al., 2008; Edwards
et al., 2009). In general, freshwater crayfishes possess heavily calcified exoskeleton. This exoskeleton calcification is
mainly restricted to the endocuticle (Travis, 1965), and is
termed the calcified zone (Drach, 1939). The crayfish exoskeleton comprises about 50% of total body dry weight
(Wheatly and Ayers, 1995) with almost 90% of whole-body
mineral contained in the intermoult cuticle (Huner et al.,
1976). CaCO3 normally accounts for most of the inorganic
matter (Welinder, 1974, 1975; Huner and Lindqvist, 1985;
Huner et al., 1990; Taugbøl et al., 1997). The rigidity of the
exoskeleton is important as a barrier to parasites (Söderhäll
author; e-mail: [email protected]
© The Crustacean Society, 2016. Published by Brill NV, Leiden
DOI:10.1163/1937240X-00002406
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 36, NO. 2, 2016
and Cerenius, 1992) and also provides significant protection against cannibalism and predation (France, 1987). Each
individual faces a critical period at each moulting, and establishment of a proper exoskeleton in crayfishes is of crucial importance for individual survival and sustaining vital
populations. Crayfish populations in soft water, at insufficient environmental Ca2+ levels, could therefore produce a
less rigid exoskeleton and are probably more susceptible to
several negative environmental factors.
Freshwater crayfishes have a highly efficient branchial
uptake of minerals, even from low ambient concentrations
(Greenaway, 1974a, 1985, 1993). The branchial Ca2+ uptake
is, however, reduced when pH is lowered from neutral
towards 5.5, and as a result acidification has a negative
impact on crayfish populations (Malley, 1980; France, 1987,
1993). Low ambient pH and Ca2+ has been shown to erode
calcium from the crayfish exoskeleton (Wood and Rogano,
1986). With high Ca2+ (35 mg Ca2+ l−1 ) but still low
pH (4), Morgan and Mc Mahon (1982) found increased
haemolymph concentration of Ca2+ in Procambarus clarkii
(Girard, 1852), which is probably also an indication of
losing calcium from the exoskeleton. A large amount of Ca
is also lost by ecdysis (Greenaway, 1985), but the actual
loss is probably also dependent on ambient Ca2+ (Wheatly
and Ayers, 1995; Wheatly, 1996). The latter is reinforced
by the fact that post-moult Ca2+ uptake is primarily ionic
(Wheatly, 1996). Some intermoult Ca is retained in the
paired gastrolith used in postmoult calcification (Willig
and Keller, 1973), and dietary calcium could also to some
extent compensate for low ambient Ca2+ (Greenaway, 1985;
Hessen et al., 1991).
There are contradictory reports on the correlation between
Ca content in the exoskeleton and the ambient Ca2+ concentration (Chaisemartin, 1967; Mills and Lake, 1976; Huner
and Lindqvist, 1985; Lahti, 1988; Jussila et al., 1995; Taugbøl et al., 1997). In recent years, however, declining Ca2+
concentrations in boreal soft-water lakes of North America
and Europe following climate change have become a major threat towards calcium-demanding freshwater crayfishes
(Jeziorski et al., 2008; Cairns and Yan, 2009; and ref. in
both). Decreased Ca2+ levels in surface waters follow the
decrease in the exchangeable base cation pool in forest soils
(Watmough and Aherne, 2008). The causes for this depletion
of forest soils include logging and subsequent re-growth of
forests (Watmough et al., 2003), reductions in atmospheric
Ca2+ inputs (Likens et al., 1996), and from long term atmospheric acidic deposition (Skjelkvåle et al., 2005). If
Ca2+ concentrations in surface waters fall below thresholds
where freshwater crayfishes and other crustacean species
can sustain vital populations, the ecological impacts could
be widespread and pronounced (Taugbøl, 2004; Jeziorski et
al., 2008; Yan et al., 2008). Over 50% of the world’s crayfish species are consequently suggested to be threatened by
population decline or extinction (Taylor, 2002).
In this study, the specific Ca concentration in the intermoult (C4 ) carapaces were examined in 16 different populations of A. astacus from localities exhibiting a wide range
of ambient Ca2+ concentrations. These sites form part of the
distribution of A. astacus in Europe. The aim was to determine if there is a correlation between the amount of Ca accu-
mulated in the carapaces of these crayfishes and the ambient
Ca2+ concentrations in their environments, a question of significant ecological importance.
M ATERIALS AND M ETHODS
Localities for obtaining samples were chosen from a greatest possible
span in the degree of water hardness to include populations from softwater to hard-water habitats (Table 1). Localities were also chosen to
include a span in altitude, natural and limed (addition of calcite, primarily
CaCO3 ) sites, together with both stocked and wildstock populations. Four
of the investigated lakes were limed from early-mid 1990s and onwards,
whereas two lakes were limed for less than a decade (Table 1). Crayfish
were collected from 16 different sites in the southeastern part of Norway,
mainly in 1998 and partly during 1996 and 1997 (Tables 1 and 2). Samples
were collected using crayfish pots and handpicked on a few occasions
while scuba diving and immediately frozen. The crayfish data set consisted
of 376 individuals (Table 2), of which 37.5% were females. The largest
sampling effort was in Lake Einavann (72 samples), whereas two locations
(Lake Bergsvannet and River Akerselva) had only 6 individuals each.
Intermoult crayfishes are in negative calcium balance in most freshwaters
due to low external electrolyte concentrations, and Ca2+ is slowly lost into
the environment. Since calcium loss has been reported during winter and
spring (Greenaway, 1972; Wheatly and Gannon, 1995), all samples in this
study were collected exclusively in September and October (Table 2) and
consequently represent intermoult stage C4 . The sex of each individual was
determined, and total body length was measured from tip of the rostrum to
the end of the tail fan (Table 2).
Water samples were taken in the lakes’ outlet, and results are given as
mean of spring (May) and fall (October) (Table 1). pH was measured on
a Metrohm 691 with electrode Metrohm type 6.02219.100, conductivity
was measured on a Metrohm 644 with electrode type 6.0901.110 with a
cell constant of 0.75. Alkalinity was determined by a titration to pH 4.5
using the pH-meter mentioned above. Water colour (Pt) was measured
spectrophotometrically on a Shimadzu UV-1201at 410 nm on water filtered
through a 0.45 μm Millipore membrane filter. Ca2+ and Mg2+ were
measured on an atomic absorption spectrophotometer (AAS) Perkin-Elmer
460 at 422.7 nm.
Carapace samples were taken (immediately upon thawing in 1998) from
the branchiostegites lateral side of the carapace with an 11 mm diameter
cork borer (0.95 cm2 ). Each sample was freed from adhering tissue, washed
in two separate containers with distilled water, air dried for approximately
30 min. and weighed for wet weight (WW). Samples were further dried
for at least 12 h at 110°C to obtain dry weight (DW), followed by burning
at 550°C for at least 12 h in a muffle furnace (Carbolite CWF 1100) to
get ash weight (AW), which is similar to total mineral contents. Each
burned exoskeleton piece was cooled in a vacuum exicator until AW was
measured, then transferred to an acid-washed container and digested with
concentrated Scanpure HNO3 (65%). After dilution, La(NO3 )3 solution
(77.9 g La(NO3 )3 l−1 + 25 ml conc. HCl l−1 ) was added to the samples
to make up 10% of final volume. Standards were treated the same way, and
the Ca2+ and Mg2+ concentrations were measured on an AAS as described
above.
The main gradients in water chemistry were identified by principal
component analysis with all variables except pH log-transformed. In order
to simplify the analysis and avoid pseudo-replication, we chose to compute
means across individuals grouped by location and sex, and used these
aggregated data in the rest of the analysis. Regression models used to
predict mean carapace Ca content were fitted by using ambient Ca2+ and
water colour (both log-transformed), sex, and body length as independent
variables. Minimal, adequate models were found by a backward selection
procedure using the Bayesian Information Criterion (BIC) (Johnson and
Omland, 2004), which is often found to give a good balance between
goodness of fit and model robustness.
R ESULTS
The investigated locations spanned a 7-fold range in Ca2+
concentrations from 2.3 mg Ca2+ l−1 in Lake Krøderen
to 16.1 mg Ca2+ l−1 in Lake Steinsfjorden, respectively
(Table 1). The buffer capacity expressed as alkalinity (ALK)
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WÆRVÅGEN ET AL.: EXOSKELETON CALCIFICATION IN ASTACUS ASTACUS
Table 1. Altitudes and water chemistry parameters of investigated sites as mean of spring and fall 1998. In liming history: NL, not limed, year of first
liming in running programs, and liming period for closed programs.
Lake (L)/river (R)
Altitude
(m)
pH
K25
(μS cm−1 )
ALK
(mmol l−1 )
Pt-colour
(mg l−1 )
Ca2+
(mg l−1 )
Mg2+
(mg l−1 )
L Krøderen
R Ådalselva
L Harasjøen
L Bæreia
R Akerselva
L Søndre Øyungen
L Digeren
L Nessjøen
L Rokosjøen
L Moensvatn
L Søndre Billingen
L Lyseren
L Hemnessjøen
L Bergsvannet
L Einavann
L Steinsfjorden
133
100
280
231
140
194
236
132
215
243
182
162
133
36
398
63
6.30
6.37
6.09
6.28
6.43
6.06
6.43
6.32
6.25
6.61
6.54
6.58
6.90
6.94
7.31
7.48
20
22
29
31
32
31
31
43
31
43
36
55
72
74
90
113
0.113
0.120
0.118
0.133
0.125
0.108
0.144
0.163
0.155
0.150
0.178
0.210
0.310
0.400
0.495
0.785
17
17
62
21
17
85
42
43
96
47
43
14
19
28
23
8
2.3
2.4
3.0
3.2
3.3
3.3
3.6
3.7
4.0
4.0
4.4
4.6
5.9
8.3
12.9
16.1
0.4
0.5
0.6
0.6
0.5
0.7
0.6
1.2
0.8
0.5
0.7
1.3
2.0
1.7
1.2
1.9
covered an 8-fold range from 0.1 mmol l−1 in soft-water sites
to almost 0.8 mmol l−1 in the hard-water Lake Steinsfjorden.
Measured values of pH varied from close to 6 in the
lowest alkalinity locations, to a maximum of 7.5 in Lake
Steinsfjorden.
Most of the water-chemistry variables were strongly
correlated, reflecting that the first two principal component
analysis (PCA) axes explained more than 95% of the total
variation. All variables except water colour (Pt) had strong
loadings on the first axis, such that the two major gradients
Liming
history
NL
NL
NL
1991
NL
1994
1994-2000
NL
1994
1994-2003
1992
NL
NL
NL
NL
NL
can be identified as base-cation richness (PC1) and organic
carbon content (PC2), as illustrated by the biplot in Fig. 1.
The strong correlations of the PC1 variables means that any
of the base cation indicators (pH, conductivity, alkalinity,
Ca2+ , or Mg2+ ) could serve as a predictor variable, but also
that one cannot discriminate between the effects of ionic
strength, pH, buffer capacity, and other factors on dependent
variables. On the other hand, water colour (Pt) is so weakly
correlated with the other water chemistry variables that it can
be used as an independent predictor variable.
Table 2. Astacus astacus population characteristics as means from 16 different sites collected in 1996, 1997, and 1998. Population size can be either small
(S), middle (M), or large (L).
Lake (L)/river (R)
Mean
body
length
+
(mm)
Carapace
density
+
(mg DW
cm−2 )
Crayfish
population
size
Total
number of
crayfish
collected
and
analysed
L Krøderen
R Ådalselva
L Harasjøen
L Bæreia
R Akerselva
L Søndre Øyungen
L Digeren
L Nessjøen
L Rokosjøen
L Moensvatn
L Søndre Billingen
L Lyseren
L Hemnessjøen
L Bergsvannet
L Einavann
87.5
93.8
87.4
76.2
82.3
89.5
59.0
79.3
102.1
81.2
74.1
102.9
108.1
83.5
89.7
26.5
22.9
24.0
23.5
22.8
24.8
19.2
22.4
29.0
23.5
22.6
31.2
31.5
29.9
30.7
L
L
L
M
L
S
S
M
M
M
M
L
L
L
L
33
32
44
32
6
13
14
12
10
23
11
15
17
6
72
87.8
30.4
L
36
L Steinsfjorden
Collection date (Sample number (N))
16 October 1997 (18), 08 October 1998 (15)
13 September 1996 (16), 10 September 1998 (16)
15 September 1998 (16), 14 October 1998 (28)
23 October 1997 (13), 13 October 1998 (19)
15 October 1998 (6)
18 September 1998 (13)
08 September 1998 (14)
13 October 1998 (12)
13 October 1998 (10)
30 September 1998 (23)
13 October 1998 (11)
15 September 1998 (15)
15 September 1998 (17)
05 October 1998 (6)
17 October 1996 (13), 12 September 1997 (13), 11 October
1997 (17), 15 September 1998 (15), 08 October 1998 (14)
24 September 1996 (16), 16 September 1997 (10),
22 September 1998 (10)
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 36, NO. 2, 2016
Fig. 1. Principal components, where PC1 and PC2 are the first two PCA axes, of water chemistry visualized as a biplot with variable loadings as red arrows
and sampling locations in black positioned by their principal component scores.
The scatterplot matrix in Fig. 2 shows the relationships
between mean values aggregated across A. astacus individuals by lake and gender. Carapace calcium content is highly
correlated with ash content of the carapace (r = 0.976) such
that either of them could be used as an indicator of calcification. Magnesium content of the carapace is not correlated with any of the other variables (r = −0.21 to 0.38, all
with p > 0.05). This contrast is also reflected by the average Ca2+ :Mg2+ ratio being 5.7 in lake water, compared
to ratio 138 in the carapaces. Carapace density is significantly correlated with length as well as ash and Ca content
(r = 0.66-0.74, all with p < 0.01), but not with Mg content (r = 0.30, p = 0.101). None of the variables in Fig. 2
show any strong systematic relationships with gender. Based
on this, we chose carapace Ca content as the dependent variable in our regression models, while we used gender and
body length as covariates to adjust for systematic differences
between populations and sexes.
Population-wise mean Ca contents varied from 21.2 to
25.8% Ca of DW in Lake S. Øyungen and Lake Steinsfjorden, respectively (Table 3). Ash content explained 95% of
the variation in carapace Ca content, with a slope of 0.44 ±
0.02 (standard error). The 44% Ca content in ash is therefore quite similar to that of pure calcite (Ca:CaCO3 = 40%).
Carapace density, expressed as mg dry weight per cm2 , increases significantly with length (Pearson correlation coefficient r = 0.74, p = 2.2E−6), meaning that larger individuals have more robust exoskeletons. As carapace density
correlates positively with both ash and Ca content (r = 0.66
and 0.71, respectively, or p = 5.5E−5 and 8.8E−6), the exoskeleton becomes denser by increased calcification. It is
noteworthy that Ca content is poorly correlated with body
length (r = 0.30, p = 0.10) but well correlated with carapace density (r = 0.74), which means that body length is
probably a more suitable covariate in regression models for
carapace Ca content than carapace density.
The Bayesian Information Criterion (BIC) selection for
the full data set resulted in a model with log(Ca2+ ), log(Pt),
and sex as independent variables. Residual analyses showed
that this model was strongly dependent on a single observation on males from Lake S. Øyungen, which, by far, had
lower Ca contents in their carapaces than any other population. Leaving this observation out gave a final model where
body length was retained, but not log(Pt). We interpret this
as water colour not having an effect on calcification per se,
but just serving as a proxy for the unusually low Ca content in Lake S. Øyungen males. This model explained 82%
of the total variance in carapace Ca content, with ambient
Ca2+ contributing 65%, sex 14%, and body length < 3% of
the total variance. Diagnostic plots of the residuals of this
model revealed no striking deviations from the standard assumptions of general linear models.
Carapace Ca content generally increased linearly with
the logarithm of ambient Ca2+ concentration, as Fig. 3
illustrates. Females have, on average, 0.4% higher Ca
content than males when adjusted for ambient Ca2+ and
body length, whereas Ca content increases by 0.2% per cm
of body length when adjusted for ambient Ca2+ and sex.
WÆRVÅGEN ET AL.: EXOSKELETON CALCIFICATION IN ASTACUS ASTACUS
193
Fig. 2. Scatterplot matrix of Astacus astacus body lengths (mm) and carapace properties: mean values grouped by location and sex, where red and blue
dots represent females and males, respectively. Crayfish total body length (mm), Ash/DW (% Ash of DW), Ca/DW (% Ca of DW), Mg/DW (% Mg of DW),
and DW/cm2 is carapace density (DW in mg).
D ISCUSSION
Exoskeleton calcification in crayfishes depend on several
variables such as climatic conditions, population size and
competition, sex, food availability, predation, and ambient
concentrations of water mineral. Samples from the anterior
A. astacus branchiostegites very well represent mean content
values from different body regions (Huner and Lindqvist,
1985; Lahti, 1988; Huner et al., 1990), and might be the best
single representative sample for the total exoskeleton. Our
study examined 16 different populations of A. astacus covering a wide range of habitats in Norway. The relationship
between Ca content in the crayfish exoskeleton and ambient
Ca2+ concentration has been the subject of numerous studies (Chaisemartin, 1967; Mills and Lake, 1976; Huner and
Lindqvist, 1985; Lahti, 1988; Jussila et al., 1995; Taugbøl et
al., 1997). The calcification of crayfish exoskeletons in relation to such a wide range of environmental conditions as
documented herein has never before been reported in a single study, and could provide new information on adaption
for several other crayfish species within their natural habitats and ambient Ca2+ levels.
We found carapace Ca content increases by 0.2% of
DW for each cm increase in body length, whereas Stein
and Murphy (1976) found that inorganic content was a
linear function of exoskeleton length. This study shows
that carapace Ca content is poorly correlated with body
length, which implies that body length is probably a more
suitable covariate in regression models for carapace Ca
content than carapace density. On the other hand, we found
that carapace density was significantly correlated with body
length as well as ash and Ca content, and also carapace Ca
content to be well correlated with its density (thickness).
The latter was also found by Jussila et al. (1995), and
these findings could be comparable to the reported directly
proportional relationship between total body calcium and
fresh body weight (Greenaway, 1974a). The exoskeleton
density was found to increase with size (age) in three species
of Cambaridae (Huner et al., 1976). Large-size crayfish
species were reported to be more heavily calcified than
smaller size species in some studies (Chaisemartin, 1962;
Mills and Lake, 1976). Exoskeleton calcification in astacid
and cambarid crayfishes are affected by sexual maturity
and the sexually active life stage with consequent lower
moulting frequencies, especially in females, allowing more
time for calcification (Huner and Lindqvist, 1985). The
studied populations of the cool-water species A. astacus
revealed no significant inter-annual variation in the percent
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 36, NO. 2, 2016
Table 3. Ambient Ca2+ values from the different sites, total minerals (ash), and Ca content of adult Astacus astacus as mg and %, all samples from anterior
branchiostegites. Population status at sampling time 1996-1998 and recent trends until 2010 (Johnsen, 2010), where population status is ranged in increasing
productivity as; vulnerable, viable, and vital.
Lake Ca2+
(mg l−1 )
% ash
(DW)
mg Ca mg−1
(DW)
% Ca
(DW)
% Ca
(AW)
Population status in Norway at sample times
and 2010
L Krøderen
2.3
62.15
0.225
22.48
36.16
R Ådalselva
2.4
62.20
0.222
22.17
35.64
L Harasjøen
3.0
63.56
0.226
22.64
35.62
L Bæreia
3.2
63.72
0.230
23.03
36.14
R Akerselva
3.3
64.92
0.236
23.64
36.41
L Søndre Øyungen
3.3
59.75
0.212
21.24
35.52
L Digeren
3.6
63.70
0.227
22.73
35.71
L Nessjøen
3.7
62.73
0.222
22.22
35.43
L Rokosjøen
4.0
64.43
0.236
23.60
36.64
L Moensvatn
4.0
65.25
0.237
23.70
36.32
L Søndre Billingen
4.4
63.99
0.230
23.03
35.99
L Lyseren
4.6
65.89
0.245
24.54
37.24
L Hemnessjøen
5.9
68.30
0.250
24.98
36.56
L Bergsvannet
8.3
68.15
0.253
25.30
37.12
L Einavann
12.9
67.66
0.250
25.07
37.04
L Steinsfjorden
16.1
70.96
0.258
25.77
36.31
Reestablished 1958, 1997-1998 viable,
2010 some decline
Introduced 1960s, 1996-1998 viable, 2010
still viable
Viable 1998, 2010 declined Ca and pH with
vulnerable population
Limed since 1991, 1997-1998 viable, 2010
small increase
Viable 1998, 2010 still viable river
population
Vulnerable population 1998, 2010 acid
episodes and still vulnerable
Vulnerable 1998, 2010 declined Ca and pH
with vulnerable population
Viable in 1998, 2010 increased population
after crayfish plague end
Viable in 1998, 2010 vulnerable population
due to acid episodes
Introduced in 1938, 1998 viable, 2010 still
viable population
Viable 1998, 2010 declined population as
also ambient Ca and pH
Vital 1998, 2010 declined population as also
ambient Ca and pH
Viable 1998, 2010 vital population and
increased productivity
Introduced 1931, 1998 vital, 2010 still vital
and good productivity
Vital 1996-1998, 2010 still vital with high
productivity
Vital 1996-1998, 2010 still vital with high
productivity
5.3
64.84
0.235
23.50
36.24
Lake (L)/river (R)
Total means
Ca of dry weight in any sites when compared at the same
time of year.
Since males normally moult twice per year, one might
expect them to have less calcified exoskeletons than females.
We found that females had, on average, 0.4% higher Ca
content than males when adjusted for ambient Ca2+ and
body length. This is in accordance with the findings of
Jussila et al. (1995), indicating (albeit not significantly,
maybe due to small sample size) that females have the
most dense exoskeletons. Other studies have found no sex
differences in exoskeleton calcification (Greenaway, 1974a),
and Huner et al. (1990) found no such differences between
sexes for any of the several moult stages. Furthermore,
no significant differences in Ca concentrations between
different regions of the exoskeletons and between sexes
have been observed (Lahti, 1988). Sexually active, mature
males have larger chelae and are more heavily calcified than
immature males or females (Wheatly and Gannon, 1995).
Calcium content in intermoult exoskeleton approximates
whole-body calcium content (mineral mass) and thus reflects
the relationship between crayfish calcification as a function
of environmental Ca2+ levels (Wheatly and Ayers, 1995).
In our study, crayfish harbouring sites with ambient
Ca2+ exceeding 5 mg l−1 were characterised by vital
populations. We also found a few viable populations at
ambient Ca2+ concentrations as low as 2.4 mg l−1 , whereas
some of the other soft-water lake crayfish populations
are vulnerable and unstable due to low Ca2+ and pH
(Table 3). Natural populations of A. astacus, however, have
been reported to sustain ambient Ca2+ concentrations as
low as 3 mg Ca2+ l−1 (Huner and Lindqvist, 1985) or
even 2 mg Ca2+ l−1 (Jussila et al., 1995; Taugbøl et al.,
1997), as also reported by France (1987) for Orconectes.
Successful moulting has been observed at ambient Ca2+
concentrations below 5 mg l−1 , but Ca2+ in excess of
5 mg l−1 was found by Lowery (1988) to be necessary
for proper exoskeleton calcification in Cherax tenuimanus
(Smith, 1912). Rukke (2002) revealed decreased survival
and growth at ambient Ca2+ concentrations < 5 mg Ca2+ l−1
in A. astacus in experimental studies. Lower Ca2+ thresholds
for the distribution of populations of astacids and species of
WÆRVÅGEN ET AL.: EXOSKELETON CALCIFICATION IN ASTACUS ASTACUS
195
Fig. 3. Model predictions from a linear model for mean Astacus astacus carapace Ca content using ambient calcium, sex, and body length as independent
variables. Observation coloured according to sex (red for females, blue for males) and scaled according to mean body length. Prediction lines have different
colours for the sexes, and different transparencies according to body length in 10 mm steps (labels next to lines). The one deviating observation (males from
Lake S. Øyungen), not included in the model, is also shown for comparison.
Austropotamobius have been reported to be approximately
5 mg Ca2+ l−1 (Chaisemartin, 1967; Greenaway, 1974a, b;
Jay and Holdich, 1977, 1981; Wheatly and Gannon, 1995).
The strong logarithmic effect of ambient Ca2+ observed
in our study implies that carapace Ca increases by 1.7 ×
log(2) = 1.2% of DW for every doubling of the Ca2+
concentration in the water. A positive correlation between
calcium concentrations in lakes and in crayfish exoskeletons
was also found by Mills and Lake (1976). In our study, Ca
was the most important carapace mineral, with Mg content
a disproportionately scarce component, as also reported in
several other studies (Welinder, 1975; Huner et al., 1976;
Huner and Lindquist, 1985; Jussila et al., 1995). We found
mean lake water Ca2+ :Mg2+ ratio to be 5.7 while mean
carapace Ca:Mg ratio was 138, indicating that exoskeleton
calcification is selective for Ca2+ relative to other divalent
base cations. Crayfishes have less robust exoskeletons in
lakes with low ambient Ca2+ than in lakes with more
readily available Ca2+ , while still being able to establish
successfully populations (Jussila et al., 1995). The ability
of crayfishes to calcify their exoskeletons is species-specific
and depends on environmental mineral levels (Greenaway,
1985). Huner and Lindqvist (1985) found significantly
greater values for mineral matter in the intermoult (C4 )
exoskeletons of warmwater (77-78%) than in coolwater (7174%) species. Exoskeletons of cambarids were found to be
more heavily calcified, with both Ca and Mg, than those of
astacids (Jussila, 1997).
Carapace Ca content generally increased linearly with the
logarithm within the entire ambient Ca2+ concentration span
of 2.3 to 16.1 mg l−1 , with mean carapace Ca contents
ranging from 21.2 to 25.8% Ca of DW (Fig. 3). Jussila et
al. (1995) found very similar levels, showing the linearity to
our study in three A. astacus populations between ambient
Ca2+ at 2.5, 14.7 and 27.4 mg l−1 and % carapace Ca of
DW of 22.9, 25.2, and 27.9, respectively. Crayfish in Lake S.
Øyungen were in an early unstable re-establishment stage at
sampling time in 1998. This could be an explanation for the
extreme low calcification values observed for males in Lake
S. Øyungen, an effect that could be reinforced by a possible
delayed second moulting male period close to sampling
(Fig. 3). Crayfish exoskeleton Ca content seems to reflect
the haemolymph concentration of Ca2+ (Wheatly and Ayers,
1995), whereas the mean population carapace Ca contents
found in this study reflect the ambient Ca2+ concentrations
and might possibly also indicate ambient Ca2+ shortage in
this species.
Low ambient pH affects ion regulation and the balance
between haemolymph and the surrounding water in A. astacus (Appelberg, 1985). Crayfishes exposed to low pH will
loose calcium from the exoskeleton to the environment, but
increased haemolymph Ca2+ also indicates an exoskeleton
196
JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 36, NO. 2, 2016
buffering effect which could prevent some negative effects of
acidification (Wood and Rogano, 1986). Postmoult crayfish
exoskeleton calcification depends largely upon the uptake of
ambient Ca2+ because food supply and gastroliths are insufficient (Agdeboye et al., 1975). Low pH inhibits Ca2+ uptake, amongst other processes, and thus also inhibits growth
(Wheatly, 1996). Increasing acidity and low Ca2+ could also
have increased effects on calcium metabolism of the exoskeleton (Lahti, 1988). Low pH levels inhibit Ca2+ uptake
mechanisms at moult (Borgstrøm and Hendrey, 1976), and
Ca2+ uptake is completely inhibited at about pH 4 (Malley,
1980). Furthermore, high aluminium levels are generally followed by death in the pre-moult or moulting stages (Fjeld et
al., 1988). All these negative processes could affect commercial interests, and liming management has been carried out
for decades in Norway and elsewhere to protect and restore
many crayfish populations (Appelberg, 1992).
Crayfishes constitute the most threatened group of freshwater animals in the world, with an alarming rate of decline for populations as well as species (Watmough and Aherne, 2008). Regarding the on-going conspicuous Ca2+ decline observed in soft-water lakes following climate change
(Skjelkvåle et al., 2005; Jeziorski et al., 2008; Yan et al.,
2008; Cairns and Yan, 2009), knowledge about calcification
of crayfishes is important because it might reflect ecological
impacts, population success, and dispersal of these groups.
Among the studied soft-water sites harbouring populations
with lowest degree of calcification (Table 3), recent results
also indicate declines in ambient Ca2+ and crayfish populations (Johnsen, 2010). Further analyses are required to determine the role of minor minerals, especially in soft-water
lakes when Ca2+ shortage could have some physiological
limitations (Jussila et al., 1995). Reduced crayfish exoskeleton calcification, following ambient Ca2+ decline, could be
critical for the survival of many of their populations in softwater lakes. Such future scenario in the lakes investigated
and many soft-water sites in general, could be critical for
crayfish populations in locations with low buffering capacity
with or without decline in base-cations. Many of these lakes
are now being limed, and thus underline the importance of
liming in sites exhibiting critically poor alkalinity.
ACKNOWLEDGEMENTS
The authors are grateful to Georg Gjøstein, Bjørn Reidar Hansen, Rolf
Hoff, and Rune Nordeide for collecting crayfish specimens. We are further
grateful to Stein I. Johnsen and Jens Petter Nilssen for valuable information
and discussions, and to Anne Gro Brodshaug for help with the references.
This work was partially supported by the RCN/Miljø 2015 project 243907
“Targeted strategies for safeguarding the noble crayfish against alien and
emerging threats.” We also thank Robert Wilson for proofreading and
editing of the manuscript, and to the anonymous referees for their generous
clarifying inputs.
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ACCEPTED: 16 December 2015.
AVAILABLE ONLINE: 28 January 2016.