Crustacean Asellus Aquaticus Research Articles

Tolerance to environmental pollution in the freshwater crustacean Asellus aquaticus: A role for the microbiome.

Freshwater habitats are frequently contaminated by diverse chemicals of anthropogenic origin, collectively referred to as micropollutants, that can have detrimental effects on aquatic life. The animals' tolerance to micropollutants may be mediated by their microbiome. If polluted aquatic environments select for contaminant-degrading microbes, the acquisition of such microbes by the host may increase its tolerance to pollution. Here we tested for the potential effects of the host microbiome on the growth and survival of juvenile Asellus aquaticus, a widespread freshwater crustacean. Using faecal microbiome transplants, we provided newly hatched juveniles with the microbiome isolated from donor adults reared in either clean or micropollutant-contaminated water and, after transplantation, recipient juveniles were reared in water with and without micropollutants. The experiment revealed a significant negative effect of the micropollutants on the survival of juvenile isopods regardless of the received faecal microbiome. The micropollutants had altered the composition of the bacterial component of the donors' microbiome, which in turn influenced the microbiome of juvenile recipients. Hence, we show that relatively high environmental concentrations of micropollutants reduce survival and alter the microbiome composition of juvenile A. aquaticus, but we have no evidence that tolerance to micropollutants is modulated by their microbiome.

Exploratory behaviour divergence between surface populations, cave colonists and a cave population in the water louse, Asellus aquaticus

Behaviour is considered among the most important factors in colonising newhabitats. While population divergence in behaviour is well-documented, intraspecificvariation in exploratory behaviour in species with populations successfully colonising andadapting to extreme (compared to the ‘typical’) habitats is less understood. Here, by studyingsurface- vs. cave-adapted populations of water louse (Asellus aquaticus), we tested whether (i)adaptation to the special, ecologically isolated cave habitat includes a decrease inexplorativeness and (ii) recent, surface-type cave colonists are more explorative than theirsurface conspecifics from the source population. We repeatedly tested dispersal related novelarea exploration and dispersal speed in both the presence and absence of light. We found thatsurface populations showed higher behavioural activity in dark than in light, and they weremore explorative and dispersed faster than their cave conspecifics. Recent colonists showed atrend of higher dispersal speed compared to their source surface population. We suggest thatextreme and isolated habitats like caves might work as ‘dispersal traps’ following successfulcolonisation, because adaptation to these habitats includes the reduction of explorativeness.Furthermore, we suggest that individuals with higher explorativeness are likely tocolonise markedly new environments. Finally, we provide experimental evidence aboutsurface A. aquaticus moving more in dark than in light.Significance statementEnvironmental conditions in caves are differing drastically from those of the surface. Consequently, animals colonising subterranean habitats are subject to different selective forces than those experienced by the ancestral surface-living population. Behaviour is believed to be a key factor in successful colonisation to novel habitats; however, intraspecific behavioural variation in species with both surface- and cave-adapted populations is less known. Here, we compared dispersal related novel area exploration and dispersal speed across surface and cave-adapted populations of the freshwater crustacean Asellus aquaticus. Our results show that cave-adapted A. aquaticus are significantly less explorative and disperse slower than surface-type populations, indicating that caves may act as ‘dispersal traps’, where adaptation includes the loss of explorativeness. Also, recent cave colonists show a trend to be faster dispersers than peers from the surface source population, suggesting that individuals with higher explorativeness are likely to colonise markedly different new environments.

Embryonic origin and genetic basis of cave associated phenotypes in the isopod crustacean Asellus aquaticus

Characteristics common to animals living in subterranean environments include the reduction or absence of eyes, lessened pigmentation and enhanced sensory systems. How these characteristics have evolved is poorly understood for the majority of cave dwelling species. In order to understand the evolution of these changes, this study uses an invertebrate model system, the freshwater isopod crustacean, Asellus aquaticus, to examine whether adult differences between cave and surface dwelling individuals first appear during embryonic development. We hypothesized that antennal elaboration, as well as eye reduction and pigment loss, would be apparent during embryonic development. We found that differences in pigmentation, eye formation, and number of segments of antenna II were all present by the end of embryonic development. In addition, we found that cave and surface hatchlings do not significantly differ in the relative size of antenna II and the duration of embryonic development. To investigate whether the regions responsible for eye and pigment differences could be genetically linked to differences in article number, we genotyped F2 hybrids for the four previously mapped genomic regions associated with eye and pigment differences and phenotyped these F2 hybrids for antenna II article number. We found that the region previously known to be responsible for both presence versus absence of pigment and eye size also was significantly associated with article number. Future experiments will address whether pleiotropy and/or genetic linkage play a role in the evolution of cave characteristics in Asellus aquaticus.

Common Genetic Basis of Eye and Pigment Loss in Two Distinct Cave Populations of the Isopod Crustacean Asellus aquaticus.

Repeated evolution of similar phenotypes is a widespread phenomenon found throughout the living world and it can proceed through the same or different genetic mechanisms. Cave animals with their convergent traits such as eye and pigment loss, as well as elongated appendages, are a striking example of the evolution of similar phenotypes. Yet, few cave species are amenable to genetic crossing and mapping techniques making it challenging to determine the genetic mechanisms causing their similar phenotypes. To address this limitation, we have been developing Asellus aquaticus, a freshwater isopod crustacean, as a genetic model. Many of its cave populations originate from separate colonization events and thus independently evolved their similar cave-related phenotypes which differ from the still existent ancestral-like surface populations. In our prior work, we identified genomic regions responsible for eye and pigment loss in a single cave population from Slovenia. In this study we examined another, independently evolved cave population, also from Slovenia, and asked whether the same or different genomic regions are responsible for eye and pigment loss in the two cave populations. We generated F2 and backcross hybrids with a surface population, genotyped them for the previously identified genomic regions, and performed a complementation test by crossing individuals from the two cave populations. We found out that the same genomic regions are responsible for eye and pigment loss and that at least one of the genes causing pigment loss is the same in both cave populations. Future studies will identify the actual genes and mutations, as well as examine additional cave populations to see if the same genes are commonly associated with eye and pigment loss in this species.

Reproduction in the freshwater crustacean Asellus aquaticus along a gradient of radionuclide contamination at Chernobyl

Nuclear accidents such as Chernobyl and Fukushima have led to contamination of the environment that will persist for many years. The consequences of chronic low-dose radiation exposure for non-human organisms inhabiting contaminated environments remain unclear. In radioecology, crustaceans are important model organisms for the development of environmental radioprotection. Previous laboratory studies have demonstrated deleterious effects of radiation exposure on crustacean reproduction. However, no studies have documented the effects of chronic radiation exposure on the reproduction of natural crustacean populations. Based on data from laboratory exposures, we hypothesised that populations of the freshwater isopod Asellus aquaticus exposed to radiation for thirty years at Chernobyl would display reduced reproductive output and altered timing of reproduction. To test this hypothesis, A. aquaticus was collected from six lakes at Chernobyl over two years with total dose rates ranging from 0.06–27.1μGy/h. No significant differences in the fecundity, mass of broods or proportion of reproducing female A. aquaticus were recorded. Significant differences in the body mass of gravid females were recorded suggesting different timings of reproduction, however this was not related to radiation contamination. No significant effect of a range of environmental parameters on A. aquaticus reproduction was recorded. Our data suggests current dose rates at Chernobyl are not causing discernible effects on the reproductive output of A. aquaticus. This study is the first to assess the effects of chronic low-dose radiation exposure on the reproductive output of an aquatic invertebrate at Chernobyl. These findings are consistent with proposed radiological protection benchmarks for the maintenance of wildlife populations and will assist in management of environments impacted by radiation.

Comprehensive characterization of the acute and chronic toxicity of the neonicotinoid insecticide thiamethoxam to a suite of aquatic primary producers, invertebrates, and fish.

Thiamethoxam is a neonicotinoid insecticide used widely in agriculture to control a broad spectrum of chewing and sucking insect pests. Recent detection of thiamethoxam in surface waters has raised interest in characterizing the potential impacts of this insecticide to aquatic organisms. We report the results of toxicity testing (acute and chronic) conducted under good laboratory practices for more than 30 freshwater species (insects, molluscs, crustaceans, algae, macrophytes, and fish) and 4 marine species (an alga, a mollusc, a crustacean, and a fish). As would be anticipated for a neonicotinoid, aquatic primary producers and fish were the least sensitive organisms tested, with acute median lethal and effect concentrations (LC50/EC50) observed to be ≥80 mg/L in all cases, which far exceeds surface water exposure concentrations. Tested molluscs, worms, and rotifers were similarly insensitive (EC50 ≥ 100 mg/L), except for Lumbriculus sp., with an EC50 of 7.7 mg/L. In general, insects were the most sensitive group in the study, with most acute EC50 values < 1 mg/L. However, the crustaceans Asellus aquaticus and Ostracoda exhibited a sensitivity similar to that of insects (acute EC50 < 1 mg/L), and the midge larvae Chaoborus sp. were relatively insensitive compared with other insects (EC50 > 5.5 mg/L). The most sensitive chronic response was for Chironomus riparius, with a 30-d no-observed-effect concentration (NOEC; emergence) of 0.01 mg/L. Observed toxicity to the tested marine organisms was comparable to that of freshwater species. We used the reported data to construct species sensitivity distributions for thiamethoxam, to calculate 5% hazard concentrations (HC5s) for acute data (freshwater invertebrates), and compared these with measured concentrations from relevant North American surface waters. Overall, based on acute toxicity endpoints, the potential acute risk to freshwater organisms was found to be minimal (likelihood of exceeding HC5s < 1%). Environ Toxicol Chem 2017;36:2838-2848. © 2017 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Evolution and development in cave animals: from fish to crustaceans

Cave animals are excellent models to study the general principles of evolution as well as the mechanisms of adaptation to a novel environment: the perpetual darkness of caves. In this article, two of the major model systems used to study the evolution and development (evo-devo) of cave animals are described: the teleost fish Astyanax mexicanus and the isopod crustacean Asellus aquaticus. The ways in which these animals match the major attributes expected of an evo-devo cave animal model system are described. For both species, we enumerate the regressive and constructive troglomorphic traits that have evolved during their adaptation to cave life, the developmental and genetic basis of these traits, the possible evolutionary forces responsible for them, and potential new areas in which these model systems could be used for further exploration of the evolution of cave animals. Furthermore, we compare the two model cave animals to investigate the mechanisms of troglomorphic evolution. Finally, we propose a few other cave animal systems that would be suitable for development as additional models to obtain a more comprehensive understanding of the developmental and genetic mechanisms involved in troglomorphic evolution.

Combined Effect of Invertebrate Predation and Sublethal Pesticide Exposure on the Behavior and Survival of Asellus aquaticus (Crustacea; Isopoda)

Invertebrate communities of lentic habitats comprise, amongst others, the crustacean Asellus aquaticus (Isopoda) and the turbellarian Dendrocoelum lacteum (Tricladida). Because D. lacteum preferentially preys on A. aquaticus, contaminants introduced into the aquatic environment may affect this predator-prey interaction, finally influencing the performance of the predator. However, no studies investigating implications of organic pollutants on this food web subsystem currently exist. Hence, the present study assessed short-term implications of pesticides with different modes of action, namely, the triazole fungicide tebuconazole and the pyrethroid insecticide lambda-cyhalothrin, during a 72 h trial. The experiments for tebuconazole showed a statistically significant decrease in predatory success of D. lacteum. Lambda-cyhalothrin, in contrast, increased predation success by 40%, which is, however, not statistically significant. Both the decrease and the increase in predation seemed to be primarily driven by an altered activity of the prey A. aquaticus. This may be hypothesized because any shift in the prey's activity influenced its probability to stick to mucus, a viscous substance released by D. lacteum, or to encounter the predator directly.

DNA methylation, histone H3 methylation, and histone H4 acetylation in the genome of a crustacean

In this work, we used antibodies against histone H3 trimethylated at lysine 9 (H3K9m3); against histone H4 acetylated at lysines 5, 8, 12, and 16 (H4ac); and against DNA methylated at 5C cytosine (m5C) to study the presence and distribution of these markers in the genome of the isopod crustacean Asellus aquaticus. The use of these 3 antibodies to immunolabel spermatogonial metaphases yields reproducible patterns on the chromosomes of this crustacean. The X and Y chromosomes present an identical banding pattern with each of the antibodies. The heterochromatic telomeric regions and the centromeric regions are rich in H3K9m3, but depleted in m5C and H4ac. Thus, m5C does not seem to be required to stabilize the silence of these regions in this organism.

The colonization of Europe by the freshwater crustaceanAsellus aquaticus(Crustacea: Isopoda) proceeded from ancient refugia and was directed by habitat connectivity

Recent continental-scale phylogeographic studies have demonstrated that not all freshwater fauna colonized Europe from the classic Mediterranean peninsular refugia, and that northern or central parts of the continent were occupied before, and remained inhabited throughout the Pleistocene. The colonization history of the ubiquitous aquatic isopod crustacean Asellus aquaticus was assessed using mitochondrial COI and a variable part of nuclear 28S rDNA sequences. Phylogeographic analysis of the former suggested that dispersion proceeded possibly during late Miocene from the western part of the Pannonian basin. Several areas colonized from here have served as secondary refugia and/or origins of dispersion, well before the beginning of the Pleistocene. Postglacial large-scale range expansion was coupled with numerous separate local dispersions from different refugial areas. Connectivity of the freshwater habitat has played an important role in shaping the current distribution of genetic diversity, which was highest in large rivers. The importance of hydrographic connections for the maintenance of genetic contact was underscored by a discordant pattern of mtDNA and nuclear rDNA differentiation. Individuals from all over Europe, differing in their mtDNA to a level normally found between species or even genera (maximal within population nucleotide divergence reached 0.16 +/- 0.018), shared the same 28S rRNA gene sequence. Only populations from hydrographically isolated karst water systems in the northwestern Dinaric Karst had distinct 28S sequences. Here isolation seemed to be strong enough to prevent homogenization of the rRNA gene family, whereas across the rest of Europe genetic contact was sufficient for concerted evolution to act.

Effect of some monocyclic aromatic hydrocarbons on freshwater invertebrates.

Acute toxicity of 10 aromatic hydrocarbons was tested during 96 hr. We wanted to find out how toxicity depends upon the position of different radicals on the benzene ring. We studied toxic influence of the mentioned compounds on four freshwater invertebrates, crustaceans Asellus aquaticus L. and Gammarus fossarum Koch. and snails Amphimelania holandri Fer. and Viviparus viviparus L., inhabiting freshwaters throughout Europe.

Random amplified polymorphic DNA diversity among surface and subterranean populations of Asellus aquaticus (Crustacea: Isopoda).

The ecological and evolutionary processes leading to isolation and adaptation of cave animals compared to their surface ancestors are not yet unequivocally understood. In this study the genetic relations of four cave and three surface population of the freshwater crustacean Asellus aquaticus in the Karst region of SW Slovenia and NE Italy were assessed using RAPDs as genetic markers. The results suggest that specialized populations from two caves invaded their subterranean habitat independently, and that their morphological similarity is a result of convergent evolution. Another, less specialized cave population seems to originate from a later colonization of a cave system already inhabited by a more specialized population, but the two populations do not interbreed. This series of temporally and spatially independent invasions has generated a diversity hotspot of non-interbreeding populations of a ubiquitous freshwater crustacean, which is uniform over most of its range. Genetic variability estimated by the percentage of polymorphic RAPD fragments was similar (86-91%) in most cave and surface populations. Substantially lower values (as low as 49%) were found in two cave populations affected by heavy pollution. Two a priori groupings of populations, traditional subspecies and hydrologically connected groups, were rejected as not significant by means of nested analysis of molecular variance (AMOVA). On the other hand, groupings revealed by UPGMA clustering displayed a significant component of among-group variance. An analysis of gene flow between populations using estimated migration rates confirmed these findings.

5S ribosomal and U1 small nuclear RNA genes: A new linkage type in the genome of a crustacean that has three different tandemly repeated units containing 5S ribosomal DNA sequences

We investigated the 5S ribosomal RNA (rRNA) genes of the isopod crustacean Asellus aquaticus. Using PCR amplification, three different tandemly repeated units containing 5S rDNA were identified. Two of the three sequences were cloned and sequenced. One of them was 1842 bp and presented a 5S rRNA gene and a U1 small nuclear RNA (snRNA) gene. This type of linkage had never been observed before. The other repeat consisted of 477 bp and contained only an incomplete 5S rRNA gene lacking the first eight nucleotides and a spacer sequence. The third sequence was 6553 bp long and contained a 5S rRNA gene and the four core histone genes. The PCR products were used as probes in fluorescent in situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus. The possible evolutionary origin of the three repeated units is discussed.Key words: Asellus, isopoda, crustacea, 5S rDNA, U1 snDNA.

The cement apparatus of larval and adult Acanthocephalus anguillae (Acanthocephala), with notes on the copulatory cap and origin of gland secretion.

Light and electron microscopy were used to investigate the ultrastructure of the cement apparatus, namely cement glands and cement ducts, of mature specimens of the parasite Acanthocephalus anguillae (Muller, 1780) Luhe, 1911 recovered from the alimentary canal of fish Leuciscus cephalus (Risso, 1826). In addition, the cement apparatus of immature A. anguillae found within the body cavity of the crustacean Asellus aquaticus (L.) was examined. In immature and mature males of Acanthocephalus anguillae, there are six round cement glands and each of them has an outer cytoplasmic layer containing nuclei and surrounds a space for storage of the cement. The cytoplasmic layer produces round, membrane-bound secretory granules approximately 1 microm in diameter. Nuclei and other cellular organelles surrounded by secretory granules were noticed inside the luminal part of the gland of adult males. In some female Acanthocephalus anguillae, within the attached copulatory cap, eggs and spermatozoa were observed. A protein of about 23 kDa appeared to be the major component of proteins of isolated cement glands, as well as in detached copulatory caps.

5S ribosomal and U1 small nuclear RNA genes: a new linkage type in the genome of a crustacean that has three different tandemly repeated units containing 5S ribosomal DNA sequences.

We investigated the 5S ribosomal RNA (rRNA) genes of the isopod crustacean Asellus aquaticus. Using PCR amplification, three different tandemly repeated units containing 5S rDNA were identified. Two of the three sequences were cloned and sequenced. One of them was 1842 bp and presented a 5S rRNA gene and a U1 small nuclear RNA (snRNA) gene. This type of linkage had never been observed before. The other repeat consisted of 477 bp and contained only an incomplete 5S rRNA gene lacking the first eight nucleotides and a spacer sequence. The third sequence was 6553 bp long and contained a 5S rRNA gene and the four core histone genes. The PCR products were used as probes in fluorescent in situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus. The possible evolutionary origin of the three repeated units is discussed.

Organization, nucleotide sequence, and chromosomal mapping of a tandemly repeated unit containing the four core histone genes and a 5S rRNA gene in an isopod crustacean species.

A tandemly repeated unit of 6553 bp containing a copy of the four core histone genes H2B, H2A, H3, and H4, and also a 5S rRNA gene, was amplified by PCR from genomic DNA of the isopod crustacean Asellus aquaticus. The linkage between 5S rRNA genes and histone genes has been so far observed in only one other organism, the anostrac crustacean Artemia salina. The gene cluster was cloned and sequenced. The histone genes, in their 3' flanking region, have the interesting feature of possessing two different mRNA termination signals, the stem-loop structure and the AATAAA sequence. A part of the PCR product was used as a probe in FISH experiments to locate the gene cluster on an inter-individually variable number of chromosomes from 6 to 12 per diploid cell, always in a terminal position and never associated with the heterochromatic areas. Fluorescence in situ hybridization (FISH) was also performed on preparations of released chromatin and the reiteration level of the gene cluster was determined as approximately 200-300 copies per haploid genome.

Sex chromosome differentiation revealed by genomic in-situ hybridization.

In this work, genomic in-situ hybridization (GISH) was used to study the sex chromosome molecular differentiation on chromosomes of male and female individuals of the isopod crustacean Asellus aquaticus. As a composite hybridization probe, we contemporaneously used male and female whole genomic DNA differently labelled in the presence of an excess of unlabelled DNA of the female homogametic sex. The karyotype of A. aquaticius normally displays eight homomorphic chromosome pairs, but a heteromorphic sex chromosome pair is present in about a quarter of the males of a natural population previously identified by us. GISH did not reveal any sex chromosome molecular differentiation on the male and female homomorphic sex chromosome pair, and the karyotypes of these individuals were equally labelled by the male- and female-derived probe, while the heteromorphic Y chromosome showed a differentially labelled region only with the male-derived probe. This region evidently contains male-specific sequences but, because no similar hybridized region is observed on the male homomorphic chromosome pair, they are probably not important for sex determination but represent a molecular differentiation acquired from the Y chromosome.

Accumulation of aluminium by the freshwater crustacean Asellus aquaticus in neutral water

This study examined the accumulation of aluminium (Al), mostly as the insoluble (Al(OH)3) species, by the freshwater crustacean Asellus aquaticus at neutral pH. Animals were exposed to a range of Al concentrations (5–356 μg l−1) in three experiments. The first two were of 30 and 50 days duration, respectively, followed by transfer of the A. aquaticus to water containing no Al for 20 days. The third used live and dead animals in order to investigate the contribution made by surface adsorption of Al to the total accumulated. Significant accumulation of Al in the whole tissues occurred by day 10 in all animals in the 30- and 50- day experiment. Peak concentrations of Al were measured in animals between days 10 and 20 with high concentration factors ranging from 1.4×104 to 5.5×103. By day 30, accumulated Al had fallen but was still significantly greater than the control in the 50- day exposure experiment. This 30- day increase followed by decreased accumulation of Al was repeated over the remaining exposure period (i.e. 30–50 days) although rates of uptake and loss and peak tissue levels of Al were higher. Proportionality between environmental (water) and tissue concentrations of Al occurred at day 20 but not at day 45. Significantly more Al was accumulated by dead animals than live animals at all Al exposure concentrations. These results suggest that Al is available to the crustacean at neutral pH and that the cuticle may provide an important site of uptake.

An analysis of temperature-induced Fluctuating Asymmetry in Asellus aquaticus (Linn.)

This study examines Fluctuating Asymmetry (FA) in the isopod Crustacean Asellus aquaticus (Linn.). FA occurs in bilaterally symmetrical traits not closely related to survival or fecundity and is the presence of small random deviations from perfect symmetry caused by environmental or genetic stress. These stresses perturb the developmental programme of the organism involved, resulting in asymmetry. FA was induced in the laboratory with temperature stress. Increasing levels of FA were found with increasing environmental temperature, but alteration between extremes of temperature was not found to be as stressful as simple increases in temperature. Extreme temperatures are, therefore, inferred to be an environmental stress for A. aquaticus.

Effects of cadmium on the predator-prey interaction between the turbellarian Dendrocoelum lacteum (M�ller, 1774) and the isopod crustacean Asellus aquaticus (L.)

The toxicity of cadmium to the freshwater triclad turbellarian Dendrocoelum lacteum and the isopod crustacean Asellus aquaticus was determined for each species when maintained individually (96 h LC50 for D. lacteum, 23.22 mg Cd/L, and for A. aquaticus, 0.16 mg Cd/L) and when kept together. When exposed together over a concentration range of 20–100 μg Cd/L, the effect of this predator-prey system was, as expected, i.e., direct toxicity to A. aquaticus, but there was also a reduction in the predation rate by D. lacteum. Both responses increased with increasing toxicant concentration. The possible causes of these observed effects, including the diet of A. aquaticus, cannibalism by A. aquaticus, and speed of the predator, are discussed. The possible ecological consequences of removing either predator or prey from a freshwater ecosystem as a result of a pollution incident are also considered.