Development and application of environmental DNA surveillance for the threatened crucian carp (Carassius carassius)

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Predator recognition and defence strategies in crucian carp, Carassius carassius

Crucian carp from populations that lack piscivores are extremely vulnerable to predation. However, in the presence of piscivores these fish develop an inducible morphological defence, a deep body. This switch from a vulnerable, shallow-bodied morph to a morphologically defended morph makes this species very suitable for investigations of anti-predator strategies, and trade-offs between morphological and behavioural defences. To address these questions, we performed eight different experiments. We found that crucian carp exhibited fright responses to chemical cues from unfamiliar predators (northern pike, perch) when these were fed prey that contained alarm substance (for northern pike: crucian carp, roach; for perch: crucian carp). Cues from small pike that were fed prey that lacked alarm substance (swordtails) caused no significant fright response whereas cues from larger pike with the same diet did. Perch on a chironomid diet elicited weaker but significant fright responses. Starved predators caused as strong fright reactions as recently fed ones did, whereas no response was exhibited towards nonpredatory fish (roach, crucian carp). Crucian carp were able to detect the presence of pike after cues had been diluted to an equivalent of 21 000 l, and larger predators elicited stronger fright responses. Prior experience of predators decreased fright responses. In particular, individuals from populations that coexisted with northern pike responded less to chemical cues from northern pike than individuals without prior experience did. Thus, crucian carp may use both alarm-substance related and predator-related cues to identify predators. Further, they were able to discriminate between large and small predators. Finally, individuals from populations that coexist with predators exhibit less pronounced fright responses. These fish have an induced morphological defence, a deep body, which most likely decreases the need for strong antipredator behaviour.     

A molecular approach to detect hybridisation between crucian carp (Carassius carassius) and non-indigenous carp species (Carassius spp. and Cyprinus carpio)

1. Releases of non‐native fish into the wild is an increasing problem posing considerable ecological and genetic threats through direct competition and hybridisation. 2. We employed six microsatellite markers to identify first generation hybrids and backcrosses between native crucian carp (Carassius carassius) and introduced goldfish (C. auratus) and common carp (Cyprinus carpio) in the U.K. We also investigated the genetic characteristics of the taxonomically controversial gibel carp (Carassius spp.) from sites across Europe. 3. Natural hybridisation between goldfish and crucian carp occurs frequently, although hybrids between all other species pairs were observed. Only 62% of British crucian carp populations (n = 21) consisted exclusively of pure crucian carp. In some populations hybrids were so frequent, that no pure crucian carp were caught, indicating a high competitive ability of hybrids. 4. Most hybrids belonged to the F1 generation but backcrossing was evident at a low frequency in goldfish × crucian carp hybrids and goldfish × common carp hybrids. Furthermore, some local populations had high frequencies of backcrosses, raising the opportunity for introgression. 5. Gibel carp from Germany and Italy belonged to two triploid clonal lineages that were genetically closely related to goldfish, whereas all individuals identified from British populations proved to be crucian carp × goldfish hybrids. 6. Our study suggests that the release of closely related exotic cyprinids not only poses a threat to the genetic integrity and associated local adaptations of native species, but may also contribute to shifts in community structure through competitive interactions.     

Anaerobic wintering of crucian carp (Carassius carassius L.)--II. Metabolic products

In long-term experimental anoxia (up to 140 days) crucian carp excreted ethanol and acetic acid. The maximum concentration of ethanol found in fish blood was ca 0.1%. The excretion rate of ethanol at temperatures from -0.5 to +5 degrees C was ca 25 micrograms/g fish wet wt/hr, but increased rapidly with increasing temperature. At 2 and 5 degrees C the ethanol excretion rate was independent of fish size, but at 12 and 18 degrees C a higher rate was observed in smaller fish. The decrease of water pH below 5 in the experimental vial during long anoxia was assumed to be due to excretion of acid compounds by the fish.     

Molecular identification of hybrids of the invasive gibel carp Carassius auratus gibelio and crucian carp Carassius carassius in Swedish waters

Both mitochondrial DNA sequence and two nuclear microsatellite markers were used to confirm the identity of the first record of Carassius auratus gibelio in the western (Swedish) Baltic Sea region. A total of 49 fishes were analysed, where 22 were from three Swedish sites connected to the Baltic Sea. The D-loop mitochondrial DNA sequences showed that 16 of 22 Swedish fishes were related to C. a. gibelio. The phylogenetic analysis of these sequences showed that these fish are probably not native, but represent different lineages of C. a. gibelio from China, Japan and Russia. All except three of these 16 fishes had microsatellite alleles suggesting hybridization with Carassius carassius. These findings suggest that a cryptic invasion of C. a. gibelio might be in progress.     

Seasonality of dihydropyridine receptor binding in the heart of an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius L.)

Prolonged anoxia tolerance of facultative anaerobes is based on metabolic depression and thus on controlled reduction of energy-utilizing processes. One proposed survival mechanism is the closing of ion channels to decrease energetic cost of ion pumping (Hochachka PW. Science 231: 234-241, 1986). To test this hypothesis, the involvement of L-type Ca2+ channels in seasonal anoxia tolerance of the vertebrate heart was examined by determining the number of [methyl-3H]PN200-110 (a ligand of L-type Ca2+ channel alpha-subunit) binding sites of the cardiac tissue and the density of Ca2+ current in ventricular myocytes of an anoxia-resistant fish species, the crucian carp. In their natural environment, the fish were exposed for > 3 mo of hypoxia (O2 < 2.5 mg/l) followed by almost 8 wk of anoxia that resulted in abrupt depletion of cardiac glycogen stores in late spring. Unexpectedly, however, the number of [methyl-3H]PN200-110 binding sites did not decline in hypoxia/anoxia as predicted by the channel arrest hypothesis but remained constant for most of the year. However, in early summer, the number of [methyl-3H]PN200-110 binding sites doubled for a period of approximately 2 mo, which functionally appeared as a 74% larger Ca2+ current density. Thus the anoxia tolerance of the carp heart cannot be based on downregulation of Ca2+ channel units in myocytes but is likely to depend on suppressed heart rate, i.e., regulation of the heart at the systemic level, and direct depressive effects of low temperature on Ca2+ current to achieve savings in cardiac work load and ion pumping. The summer peak in the number of functional Ca2+ channels indicates a short period of high cardiac activity possibly associated with reproduction and active perfusion of tissues after the winter stresses.     

Seasonal feeding activity and ontogenetic dietary shifts in crucian carp,Carassius carassius

Synopsis A pronounced seasonal cycle in the activity of the intestinal alkaline phosphatase indicated a 6 month pause in feeding by crucian carp in an anoxic pond during winter. Zero enzyme activity was not reached until ice cover of the pond was complete, although even in the preceding two months no food was found in the guts of the fish. The summer diets of four size classes of fish consisted of varying proportions of planktonic and benthic arthropods, including Odonata which were consumed exclusively by the largest size class of crucian carp. Chironomid larvae were consumed even by the smallest fish investigated, although planktonic Cladocera were preferred by this size class. Benthic Cladocera were almost totally absent in the diet of fish> 10 cm. Habitat segregation between size classes developed synchronously with the shift to benthic food. The stunting of crucian carp populations in small ponds is interpreted as resulting from limited resources.     

New cell types of the pancreatic islets in the crucian carp,Carassius carassius

Summary The pancreatic islets ofCarassius carassius have been studied by electron microscopy. 1. Besides A-, B- and D-cells, two new cell types, the fourth and the fifth, have been identified. The fourth cell type is numerous; it occurs interposed among the other types of islet cells or in small clusters. The secretory granules (90–280 mg in diameter) are round or oval and usually with much lower electron density than α- and δ-granules. The secretory granules of the fifth type of cell (approximately 140–240 mμ in diameter) contain finely granular material and an electron dense core that is round or often tetra- or hexagonal. 2. The islet cells with clear cytoplasmic matrix generally contain large numbers of fine, agranular and cored vesicles 400–680 ? in diameter. They appear, in bead-like chains, or randomly scattered throughout the cytoplasm, or often clustered in aggregates close to the secretory granules and show evidence of incorporation into the secretory granules. The two types of vesicles may be formed by constriction or pinching-off of the tubular smooth endoplasmic reticulum.     

Genetic polymorphism and isoenzyme patterns of lactate dehydrogenase in tench (Tinea tinea), crucian carp (Carassius carassius) and carp (Cyprinus carpio)

Isoenzyme patterns and the polymorphism of lactate dehydrogenase (LDH) were investigated in 3 fish species of family Cyprinidae, i.e. tench ( Tinea tinea ), crucian carp ( Carassius carassius ) and carp ( Cyprinus carpio). The isoenzyme patterns were tissue and species specific. In crucian carp subunits with different electro-phoretic mobility are present, which are genetically controlled from the B¹, B², A¹, A²and C loci, while the set of loci in carp is B¹, B², A, C¹and C²and in tench B, A, C. The locus B of LDH in tench, the locus B²in crucian carp, and the loci B¹, C¹and C²in carp are polymorphic and have two different alleles in each case. The polymorphism did not affect the total LDH activity in the tissues. All the populations investigated were in Hardy-Weinberg equilibrium. The genetic control of the polymorphism in B¹and C¹loci in carp was proved by test matings. The polymorphism in B loci tested in erythrocytes may be utilized as genetic markers in the fish breeding.     

Identification of natural hybrids of gibel carp Carassius auratus gibelio (Bloch) and crucian carp Carassius carassius (L.) from lower Dyje River floodplain (Czech Republic)

Morphometric and genetic variation were examined in parental Carassius auratus gibelio and Carassius carassius individuals, and their natural hybrids. Of meristic traits, only the number of gill rakers clearly distinguished hybrids (39·4 ± 1·4) from parental C. a. gibelio (48·3 ± 0·7) and C. carassius (28·6 ± 1·1). MtDNA sequences showed that the hybrids were descendants of female C. a. gibelio . Microsatellite analysis confirmed the presence in hybrids of variants typical of C. a. gibelio and C. carassius .     

Genetic polymorphism and isoenzyme patterns of lactate dehydrogenase in tench (Tinca tinca), crucian carp (Carassius carassius) and carp (Cyprinus carpio)

Isoenzyme patterns and the polymorphism of lactate dehydrogenase (LDH) were investigated in 3 fish species of family Cyprinidae, i.e. tench (Tinca tinca), crucian carp (Carassius carassius) and carp (Cyprinus carpio). The isoenzyme patterns were tissue and species specific. In crucian carp subunits with different electrophoretic mobility are present, which are genetically controlled from the B1, B2, A1, A2 and C loci, while the set of loci in carp is B1, B2, A, C1 and C2 and in tench B, A, C. The locus B of LDH in tench, the locus B2 in crucian carp, and the loci B1, C1 and C2 in carp are polymorphic and have two different alleles in each case. The polymorphism did not affect the total LDH activity in the tissues. All the populations investigated were in Hardy-Weinberg equilibrium. The genetic control of the polymorphism in B1 and C1 loci in carp was proved by test matings. The polymorphism in B loci tested in erythrocytes may be utilized as genetic markers in the fish breeding.     

1H-NMR study of the metabolome of an exceptionally anoxia tolerant vertebrate, the crucian carp (Carassius carassius)

The crucian carp (Carassius carassius) can tolerate anoxia for days to months, depending on the temperature. In this study, we applied ¹H-NMR-based metabolomics to polar extracts of crucian carp brain, heart, muscle and liver samples obtained from fish exposed to either control normoxic conditions, acute anoxia (24 h), chronic anoxia (1 week) or reoxygenation (for 1 week following chronic anoxia) at 5 °C. Spectra of the examined tissues revealed changes in several energy-related compounds. In particular, anoxic stress resulted in decreased concentrations of phosphocreatine (muscle, liver) and glycogen (liver) and ATP/ADP (liver, heart and muscle) and increased concentrations of lactate (brain, heart, muscle) and beta-hydroxybutyric acid (all tissues). Likewise, increased concentrations of inhibitory compounds (glycine, gamma-amino butyric acid or GABA) and decreased concentrations of excitatory metabolites (glutamate, glutamine) were confirmed in the anoxic brain extracts. Additionally, a decrease of N-acetylaspartate (NAA), an important neuronal marker, was also observed in anoxic brains. The branched-chain amino acids (BCAA) valine/isoleucine/leucine increased in all anoxic tissues. Possibly, this general tissue increase can be due to an inhibited mitochondrial function or due to protein degradation/protein synthesis inhibition. In this study, the potential and strength of the ¹H-NMR is highlighted by the detection of previously unrecognized changes in metabolites. Specifically, myo-inositol substantially decreased in the heart of anoxic crucian carp and anoxic muscle tissue displayed a decreased concentration of taurine, providing novel insights into the anoxia responses of the crucian carp.     

Regional distribution of monoamines and monoamine metabolites in the brain of the crucian carp (Carassius carassius L.)

1. Monoamines (which all demand oxygen for their synthesis) and monoamine metabolites were quantified in 6 brain regions of the extremely anoxia tolerant crucian carp (Carassius carassius L.).2. The norepinephrine levels were generally twice as high as the dopamine levels. No epinephrine was found.3. The major dopamine metabolite seemed to be homovanillic acid. No 3,4-dihydroxyphenylacetic acid was found.4. Serotonin occurred at about the same levels as dopamine. The levels of the main serotonin metabolite, 5-hydroxyindole-3-acetic acid, were about 10% of that of serotonin.5. All three monoamines had a similar distribution, with the highest concentrations in hypothalamus and the lowest in cerebellum and vagal lobes.6. The distribution and levels of monoamines agreed with that of anamniote vertebrates in general, suggesting that the crucian carp has not adapted to anoxia by abandoning or minimizing the use of monoaminergic systems.     

Body morphology of crucian carp Carassius carassius in lakes with or without piscivorous fish

Crucian carp Carassius carassius was sampled during 1993 and 1994 in 21 lakes and ponds in east Norway In 10 of these lakes and ponds no piscivorous fish species was present, and in the remaining 11 lakes and ponds predators such as perch Perca fluvtalilis. pike Esox lucius, and trout Salmo trutta were common In general, crucian carp was larger in the lakes with predators than in those without predators The relative body depth (body height/body length) of crucian carp was significantly deeper in populations sympatric with predators (mean 0 358), compared to allopatric populations (mean 0 286) The variation in relative body depth was larger among the sympatric populations than among the allopatric populations The observed difference in relative body depth may be due to 1) predator induced changes m body morphology, 2) increased growth rates in lakes containing predator due to reduced intraspecific competition, and 3) size-selective predation     

The effect of starvation on the ultrastructure of the red and white myotomal muscles of the crucian carp (Carassius carassius)

Summary The effect of 16 weeks total starvation on the ultrastructure of the red and white myotomal muscles of the crucian carp (Carassius Carassius) has been investigated. In the white fibres the amount of myofibrillar material fell from 89.6% to 70.7% of the total fibre volume whilst in the red fibres the fall was from 72.2% to 70.3%. The sarcoplasmic reticulum appeared to have become swollen during starvation in both fibre types. In the white fibres the terminal cisternae of some triads seem to have fused. The volume of the red fibres occupied by mitochondria was reduced from 16.2 % to 5.9 %. The concentration of mitochondria in the white fibres was too low to detect any quantitative changes. A marked reduction in the amount of euchromatin material was observed in most white fibre nuclei and many red fibre nuclei. Many of the ultrastructural changes noted in the present study can be correlated with biochemical changes known to occur in the red and white myotomal muscles of fish during starvation. This work was supported by a grant from the Natural Environmental Research Council.     

Differential regulation of AMP-activated kinase and AKT kinase in response to oxygen availability in crucian carp (Carassius carassius)

We investigated whether two kinases critical for survival during periods of energy deficiency in anoxia-intolerant mammalian species, AMP-activated kinase (AMPK), and protein kinase B (AKT), are equally important for hypoxic/anoxic survival in the extremely anoxia-tolerant crucian carp (Carassius carassius). We report that phosphorylation of AMPK and AKT in heart and brain showed small changes after 10 days of severe hypoxia (0.3 mg O2/l at 9 degrees C). In contrast, anoxia exposure (0.01 mg O2/l at 8 degrees C) substantially increased AMPK phosphorylation but decreased AKT phosphorylation in carp heart and brain, indicating activation of AMPK and deactivation of AKT. In agreement, blocking the activity of AMPK in anoxic fish in vivo with 20 mg/kg Compound C resulted in an elevated metabolic rate (as indicated by increased ethanol production) and tended to reduce energy charge. This is the first in vivo experiment with Compound C in a nonmammalian vertebrate, and it appears that AMPK plays a role in mediating anoxic metabolic depression in crucian carp. Real-time RT-PCR analysis of the investigated AMPK subunit revealed that the most likely composition of subunits in the carp heart is alpha2, beta1B, gamma2a, whereas a more even expression of subunits was found in the brain. In the heart, expression of the regulatory gamma2-subunit increased in the heart during anoxia. In the brain, expression of the alpha1-, alpha2-, and gamma1-subunits decreased with anoxia exposure, but expression of the gamma2-subunit remained constant. Combined, our findings suggest that AMPK and AKT may play important, but opposing roles for hypoxic/anoxic survival in the anoxia-tolerant crucian carp.     

Effects of chronic hypoxia on inward rectifier K(+) current ( I(K1)) in ventricular myocytes of crucian carp (Carassius carassius) heart

Some ectothermic vertebrates show unusually good tolerance to oxygen shortage and it is therefore assumed that they might, as a defense mechanism, decrease number or activity of ion channels in order to reduce membrane leakage and thereby ATP-dependent ion pumping in hypoxia. Although several studies have provided indirect evidence in favor of this channel arrest hypothesis, only few experiments have examined activity of ion channels directly from animals exposed to chronic hypoxia or anoxia in vivo. Here we compare the inwardly rectifying K⁺ current (I_K1), a major leak and repolarizing K⁺ pathway of the heart, in cardiac myocytes of normoxic and hypoxic crucian carp, using the whole-cell and cell-attached single-channel patch-clamp methods. Whole-cell conductance of I_K1 was 0.5 ± 0.04 nS/pF in normoxic fish and did not change during the 4 weeks hypoxic (O₂ < 0.4 mg/l; 2.68 mmHg) period, meanwhile the activity of Na⁺/K⁺ATPase decreased 33%. Single-channel conductance of the I_K1 was 20.5 ± 0.8 pS in control fish and 21.4 ± 1.1pS in hypoxic fish, and the open probability of the channel was 0.80 ± 0.03 and 0.74 ± 0.04 (P > 0.05) in control and hypoxic fish, respectively. Open and closed times also had identical distributions in normoxic and hypoxic animals. These results suggest that the density and activity of the inward rectifier K⁺ channel is not modified by chronic hypoxia in ventricular myocytes of the crucian carp heart. It is concluded that instead of channel arrest, the hypoxic fish cardiac myocytes obtain energy savings through action potential arrest due to hypoxic bradycardia.     

Distribution of aldehyde dehydrogenase and alcohol dehydrogenase in summer-acclimatized crucian carp, Carassius carassius L.

The tissue distribution of aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) in summer-acclimatized crucian carp showed almost the same exceptional pattern as previously found in winter-acclimatized specimens. There was a nearly complete spatial separation of ALDH and ADH; in other vertebrates these enzymes occur together. This exceptional enzyme distribution is probably an adaptation to the extraordinary ability of Carassius to produce ethanol as the major metabolic end product during anoxia. Since the crucian carp is less likely to encounter anoxia during the summer, the present results suggest that the crucian carp is unable to switch over to a 'normal' ALDH and ADH distribution in the summer. However, it is also possible that there is an advantage for the summer-acclimatized crucian carp in keeping ALDH and ADH separate, because of occasional anoxic periods.     

An experimental study of body size and food size relations in crucian carp,Carassius carassius

Synopsis In dense, single-species assemblages of crucian carp, competition is intense and results in populations of stunted fish. To explore mechanisms underlying this competition, we measured handling times, return rates, and prey choice for five sizes of crucian carp feeding on six sizes of a standardized food. Handling times increased with prey size and decreased with fish size. Return rates (dry mass ingested per unit handling time) increased dramatically with fish size, and generally decreased with increasing prey size, especially for small fish. Patterns of return rates among size-classes suggested that one or more size-related shifts in feeding efficiency exist for crucian carp; combined with physiological stresses related to winter anoxia, the inability of fish to make these shifts may contribute to size structures observed in high-density populations. Comparisons of relations among fish size, prey size, and return rates for crucian carp and bluegill, Lepomis macrochirus, suggest that similar intraspecific competitive relations exist between generalist species with size-structured populations. Despite differences in return rates among prey sizes, the extent of food selectivity based on prey size exhibited by crucian carp in two types of choice trials was less than predicted. Crucian carp commonly take in several items before mechanically processing food with their pharyngeal apparatus; this multiple prey processing may contribute to disparities between observed choice patterns and those predicted based on return rates for single prey.