Redescription of Euastacus clydensis Riek, 1969 (Crustacea: Parastacidae), a valid species of spiny crayfish from southern New South Wales, Australia.

The Giant Sydney Crayfish (Euastacus spinifer (Heller, 1865)) was thought to have a wide range in New South Wales, Australia, spanning some 600 km north-south. A recent extensive molecular phylogenetic and population genomic analysis of E. spinifer across its geographical range revealed strong population structure corresponding to several major geographically correlated clades, the southernmost clade being the most genetically divergent and clearly a separate species. This southern clade corresponds to the junior synonym E. clydensis Riek, 1969 and is sister to the clade comprising the remaining populations of E. spinifer and Euastacus vesper. We formally remove E. clydensis from the synonymy of E. spinifer, increasing the recognised number of species of Euastacus to 54. Euastacus clydensis is redescribed based on type and other material, and is distinguished from E. spinifer by differences in abdominal spination and the form of the antennal scaphocerite. Euastacus clydensis has a restricted southern New South Wales range in the Shoalhaven and Jervis Bay-Clyde River catchments, from Moss Vale south to the vicinity of Clyde Mountain; much of the known range of E. clydensis was burnt in the 2019-2020 eastern Australian megafires.

Genetic diversity, phylogenetic position and morphometric analysis of Astacus colchicus (Decapoda, Astacidae): a new insight into Eastern European crayfish fauna.

The phylogeny of European crayfish fauna, especially with respect to Eastern European species, is still far from being completely resolved. To fill this gap, we analyzed most of the European crayfish species focusing on the phylogenetic position of the endemic crayfish Astacus colchicus, inhabiting Georgia. Three mitochondrial and one nuclear marker were used to study evolutionary relationships among European crayfish species, resulting in the unique phylogenetic position of A. colchicus indicating independent species status to A. astacus. Phylogenetic analyses revealed a deep molecular divergence of A. colchicus in comparison to A. astacus (6.5-10.9% in mtDNA and 1.1% in nDNA) as well as to Pontastacus leptodactylus and P. pachypus (5.5-10.0% in mtDNA and 1.4-2.4% in nDNA). Absent ventral process on second male pleopod and abdominal somites II and III with pleura rounded lacking prominent spines clearly indicate taxonomic assignment to the genus Astacus; however, the species is distributed almost in the middle of Ponto-Caspian area typical by occurrence of the genus Pontastacus. Several morphological indices linked to head length, carapace, and total body length and width were found to demonstrate apparent differences between A. colchicus and A. astacus. Although this study provides a novel insight into European crayfish phylogeography, we also point out the gaps in comprehensive study of the P. leptodactylus species complex, which could reveal details about the potential species status of particular species and subspecies within this genus.

The future of endangered crayfish in light of protected areas and habitat fragmentation.

The long-term survival of a species requires, among other things, gene flow between populations. Approaches for the evaluation of fragmentation in the frame of freshwater habitats consider only a small amount of the information that combined demography and geography are currently able to provide. This study addresses two species of Austropotamobius crayfish in the light of population genetics, spatial ecology and protected areas of the Carpathians. Advancing the classical approaches, we defined ecological distances upon the rasterised river network as a surrogate of habitat resistance to migration, quantifying the deviations from the species´ suitability range for a set of relevant geospatial variables in each cell of the network. Molecular analyses revealed the populations of the two Austropotamobius crayfish species are clearly distinct, lacking hybridisation. Comparing pairs of populations, we found, in some cases, a strong disagreement regarding genetic and ecological distances, potentially due to human-mediated translocations or the geophysical phenomena of regressive erosion, which may have led to unexpected colonisation routes. Protected areas were found to offer appropriate local habitat conditions but failed to ensure connectivity. The methodology applied in this study allowed us to quantify the contribution of each geospatial (environmental) variable to the overall effect of fragmentation, and we found that water quality was the most important variable. A multilevel approach proved to reveal a better understanding of drivers behind the distribution patterns, which can lead to more adequate conservation measures.

Do hydroelectric dams affect the abundance and population structure of freshwater shrimps?

Freshwater shrimps play an important role in many ecological processes since they are epibenthic detritivores but also prey on other invertebrates and are predated by fishes. The knowledge about their biology allow the development of management strategies to improve the use of natural resources by avoiding overfishing and enhancing productivity. Here we evaluated the population structure of the shrimp Macrobrachium amazonicum in the river Rio Grande, upstream of the Água Vermelha hydroelectric dam. They were captured monthly from October 2017 until March 2018, in six sites along the river. The first site was ∿1000 m distant from the dam and the sixth was ∿4000 m distant. A sac-like sieve and traps were used to capture the shrimps. The sieve was dragged three times for 180o. The traps were left by the river margins for six hours. In the laboratory, the shrimps were sexed and measured (carapace length; CL). In total, 6455 individuals were captured: 4499 females (294 breeding), 1445 males, and 217 juveniles. Female mean size was 7.50 ± 3.47 mm, male mean size was 7.44 ± 2.40 mm, and juvenile mean size was 3.24 ± 1.10 mm. The smallest individual was found in site III and it was a juvenile of 1.08 mm. The largest had 20 mm and was found in site II. The smallest breeding female had 3.8 mm CL. The largest individuals were more abundant in the sites near the dam, whereas the smallest were more abundant in the farthest sites. The highest abundance was seen in sites III and IV, and the lowest, in site VI. Unlike males and juveniles, females (both breeding and non-breeding) were more abundant (Anova, p < 0.01) near the dam. By knowing the population structure of M. amazonicum it is possible to understand how they are being affected by the environmental changes caused by the hydroelectric dam

Comparison of the novel dipstick DNA extraction technique with two established techniques for use in biological barcoding.

The novel dipstick DNA extraction method was tested for its reliability and usability for biological barcoding in comparison to a commercial kit and to a simplified isopropanol precipitation method using crayfish gill tissue. Following DNA extraction, the mitochondrial COI-gene was amplified in a PCR-reaction using a standard set of universal invertebrate primers. All three extraction techniques resulted in successful amplifications. With the dipstick method, PCR immediately follows the very brief DNA extraction technique. We suggest that the dipstick method is an affordable, efficient, and reliable DNA extraction method uniquely suited for biological barcoding that results in reliable and reproducible amplification for downstream applications such as sequencing. Additional tests on crayfish with primers for different parts of the mitochondrial genome and on fish using specific fish COI-primers confirmed these findings. Due to the few steps involved in the DNA extraction procedure the dipstick technique is also highly recommended for high school and university biology courses.

Involvement of gamma interferon inducible lysosomal thiol reductase in the innate immune responses of red swamp crayfish, Procambarus clarkii.

The Gamma interferon inducible lysosomal thiol reductase (GILT) plays a key biological role in the immune responses and involves in the processing of class II MHC-restricted antigen by stimulating disulfide bond reduction in mammals. To determine the biological function of GILT in the innate immune system of crustaceans, we sequenced and cloned GILT gene from red swamp crayfish, Procambarus clarkii (Pc-GILT). The deduced amino acid sequence of Pc-GILT contained the putative conserved structures of the GILT family proteins: the GILT signature (CQHGX2ECX2NX4C) sequence and the active site (CXXS) motif. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis suggested that a recombinant Pc-GILT protein was successfully expressed in Escherichia coli (E. coli). Quantitative real-time PCR analysis showed that Pc-GILT transcript level was highest in the hepatopancreas followed by the gut, heart and muscles. Additionally, we analyzed the transcription level of Pc-GILT gene in hepatopancreas of red swamp crayfish under biotic stress conditions. The expression of Pc-GILT gene upregulated after viral (poly I:C) and bacterial (peptidoglycan, lipopolysaccharide) infection. The suppression of Pc-GILT by double stranded RNA influenced the transcript levels of various immune-related genes. These observations indicate that the Pc-GILT probably plays a key biological role in the innate immune responses of red swamp crayfish, since it modulates the expression of genes associated with immune pathways.

Investigating the genetic and epigenetic basis of big biological questions with the parthenogenetic marbled crayfish: A review and perspectives.

In the last 15 years, considerable attempts have been undertaken to develop the obligately parthenogenetic marbled crayfish Procambarus virginalis as a new model in biology. Its main advantage is the production of large numbers of offspring that are genetically identical to the mother, making this crustacean particularly suitable for research in epigenetics. Now, a draft genome, transcriptome and genome-wide methylome are available opening new windows for research. In this article, I summarize the biological advantages and genomic and epigenetic features of marbled crayfish and, based on first promising data, discuss what this new model could contribute to answering of ''big'' biological questions. Genome mining is expected to reveal new insights into the genetic specificities of decapod crustaceans, the genetic basis of arthropod reproduction, moulting and immunity, and more general topics such as the genetic underpinning of adaptation to fresh water, omnivory, biomineralization, sexual system change, behavioural variation, clonal genome evolution, and resistance to cancer. Epigenetic investigations with the marbled crayfish can help clarifying the role of epigenetic mechanisms in gene regulation, tissue specification, adult stem cell regulation, cell ageing, organ regeneration and disease susceptibility. Marbled crayfish is further suitable to elucidate the relationship between genetic and epigenetic variation, the transgenerational inheritance of epigenetic signatures and the contribution of epigenetic phenotype variation to the establishment of social hierarchies, environmental adaptation and speciation. These issues can be tackled by experiments with highly standardized laboratory lineages, comparison of differently adapted wild populations and the generation of genetically and epigenetically edited strains.

CPAP3 proteins in the mineralized cuticle of a decapod crustacean.

The pancrustacean theory groups crustaceans and hexapods (once thought to comprise separate clades within the Arthropoda) into a single clade. A key feature common to all pancrustaceans is their chitinous exoskeleton, with a major contribution by cuticular proteins. Among these, are the CPAP3's, a family of cuticular proteins, first identified in the hexapod Drosophila melanogaster and characterized by an N-terminal signaling peptide and three chitin-binding domains. In this study, CPAP3 proteins were mined from a transcriptomic library of a decapod crustacean, the crayfish Cherax quadricarinatus. Phylogenetic analysis of other CPAP3 proteins from hexapods and other crustaceans showed a high degree of conservation. Characterization of the crayfish proteins, designated CqCPAP3's, suggested a major role for CPAP3'sin cuticle formation. Loss-of-function experiments using RNAi supported such a notion by demonstrating crucial roles for several CqCPAP3 proteins during molting. A putative mode of action for the CqCPAP3 proteins -theoretically binding three chitin strands- was suggested by the structural data obtained from a representative recombinant CqCPAP3. The similarities between the CqCPAP3 proteins and their hexapod homologues further demonstrated common genetic and proteinaceous features of cuticle formation in pancrustaceans, thereby reinforcing the linkage between these two highly important phylogenetic groups.

More evolution underground: Accelerated mitochondrial substitution rate in Australian burrowing freshwater crayfishes (Decapoda: Parastacidae).

To further understand the evolutionary history and mitogenomic features of Australia's highly distinctive freshwater crayfish fauna, we utilized a recently described rapid mitogenome sequencing pipeline to generate 24 new crayfish mitogenomes including a diversity of burrowing crayfish species and the first for Astacopsis gouldi, the world's largest freshwater invertebrate. Whole mitogenome-based phylogeny estimates using both Bayesian and Maximum Likelihood methods substantially strengthen existing hypotheses for systematic relationships among Australian freshwater crayfish with evidence of pervasive diversifying selection and accelerated mitochondrial substitution rate among the members of the clade representing strongly burrowing crayfish that may reflect selection pressures for increased energy requirement for adaptation to terrestrial environment and a burrowing lifestyle. Further, gene rearrangements are prevalent in the burrowing crayfish mitogenomes involving both tRNA and protein coding genes. In addition, duplicated control regions were observed in two closely related Engaeus species, together with evidence for concerted evolution. This study significantly adds to the understanding of Australian freshwater crayfish evolutionary relationships and suggests a link between mitogenome evolution and adaptation to terrestrial environments and a burrowing lifestyle in freshwater crayfish.

Phylogenetic evidence from freshwater crayfishes that cave adaptation is not an evolutionary dead-end.

Caves are perceived as isolated, extreme habitats with a uniquely specialized biota, which long ago led to the idea that caves are "evolutionary dead-ends." This implies that cave-adapted taxa may be doomed for extinction before they can diversify or transition to a more stable state. However, this hypothesis has not been explicitly tested in a phylogenetic framework with multiple independently evolved cave-dwelling groups. Here, we use the freshwater crayfish, a group with dozens of cave-dwelling species in multiple lineages, as a system to test this hypothesis. We consider historical patterns of lineage diversification and habitat transition as well as current patterns of geographic range size. We find that while cave-dwelling lineages have small relative range sizes and rarely transition back to the surface, they exhibit remarkably similar diversification patterns to those of other habitat types and appear to be able to maintain a diversity of lineages through time. This suggests that cave adaptation is not a "dead-end" for freshwater crayfish, which has positive implications for our understanding of biodiversity and conservation in cave habitats.

Microsatellite evidence of dispersal mechanism of red swamp crayfish (Procambarus clarkii) in the Pearl River basin and implications for its management.

Discerning the dispersal patterns of invasive species is critically important for the design of effective management strategies and the development of appropriate theoretical models predicting the spatial expansion of introduced populations. Post-introduction dispersal may occur naturally or via human transport, but for many organisms, assessing the relative contribution of each of these factors is difficult using traditional methods. Here, we explored the genetic patterns associated with the spread of red swamp crayfish (Procambarus clarkii) among 21 populations in the Pearl River basin and 2 peripheral populations in the Yangtze River basin. We found the genetic diversity of P. clarkii in the Pearl River basin was somewhat lower than in the Yangtze River basin. We also found (1) there was significant genetic differentiation between populations, (2) genetic differentiation was not related to geographic distance (i.e., isolation by distance), and (3) a Bayesian assignment analysis revealed three distinct genetic clusters and genetic admixture. Our results therefore provide evidence that human-mediated multiple introductions occurred in the Pearl River basin. Anthropogenic activities such as commercial transportation were likely responsible for the long-distance dispersal of P. clarkii. This study provides useful information for developing management strategies.

Microevolution of the noble crayfish (Astacus astacus) in the Southern Balkan Peninsula.

BACKGROUND: The noble crayfish (Astacus astacus) displays a complex historical and contemporary genetic status in Europe. The species divergence has been shaped by geological events (i.e. Pleistocene glaciations) and humanly induced impacts (i.e. translocations, pollution, etc.) on its populations due to species commercial value and its niche degradation. Until now, limited genetic information has been procured for the Balkan area and especially for the southernmost distribution of this species (i.e. Greece). It is well known that the rich habitat diversity of the Balkan Peninsula offers suitable conditions for genetically diversified populations. Thus, the present manuscript revisits the phylogenetic relationships of the noble crayfish in Europe and identifies the genetic make-up and the biogeographical patterns of the species in its southern range limit. RESULTS: Mitochondrial markers (i.e. COI and 16S) were used in order to elucidate the genetic structure and diversity of the noble crayfish in Europe. Two of the six European haplotypic lineages, were found exclusively in Greece. These two lineages exhibited greater haplotypic richness when compared with the rest four (of "Central European" origin) while they showed high genetic diversity. Divergence time analysis identified that the majority of this divergence was captured through Pleistocene, suggesting a southern glacial refugium (Greece, southern Balkans). Furthermore, six microsatellite markers were used in order to define the factors affecting the genetic structure and demographic history of the species in Greece. The population structure analysis revealed six to nine genetic clusters and eight putative genetic barriers. Evidence of bottleneck effects in the last ~5000 years (due to climatic and geological events and human activities) is also afforded. Findings from several other research fields (e.g. life sciences, geology or even archaeology) have been utilized to perceive the genetic make-up of the noble crayfish. CONCLUSIONS: The southernmost part of Balkans has played a major role as a glacial refugium for A. astacus. Such refugia have served as centres of expansion to northern regions. Recent history of the noble crayfish in southern Balkans reveals the influence of environmental (climate, geology and/or topology) and anthropogenic factors.

Transcriptomic characterization and curation of candidate neuropeptides regulating reproduction in the eyestalk ganglia of the Australian crayfish, Cherax quadricarinatus.

The Australian redclaw crayfish (Cherax quadricarinatus) has recently received attention as an emerging candidate for sustainable aquaculture production in Australia and worldwide. More importantly, C. quadricarinatus serves as a good model organism for the commercially important group of decapod crustaceans as it is distributed worldwide, easy to maintain in the laboratory and its reproductive cycle has been well documented. In order to better understand the key reproduction and development regulating mechanisms in decapod crustaceans, the molecular toolkit available for model organisms such as C. quadricarinatus must be expanded. However, there has been no study undertaken to establish the C. quadricarinatus neuropeptidome. Here we report a comprehensive study of the neuropeptide genes expressed in the eyestalk in the Australian crayfish C. quadricarinatus. We characterised 53 putative neuropeptide-encoding transcripts based on key features of neuropeptides as characterised in other species. Of those, 14 neuropeptides implicated in reproduction regulation were chosen for assessment of their tissue distribution using RT-PCR. Further insights are discussed in relation to current knowledge of neuropeptides in other species and potential follow up studies. Overall, the resulting data lays the foundation for future gene-based neuroendocrinology studies in C. quadricarinatus.

A new species of crayfish in the genus Orconectes (Decapoda: Cambaridae) from the Tennessee River Drainage with comments on and key to members of the O. juvenilis Species Complex.

The crayfish subgenus Procericambarus (genus Orconectes) found in the Eastern Highlands of the United States represents a rich assemblage of stream-dwelling macroinvertebrates. While the taxonomic status and distribution of its members were clarified in a previous study, newly diagnosed characters and newly collected specimens warranted a revision to previous hypotheses of Procericambarus membership in the southeastern United States. Using morphological characters, we describe herein a new species of crayfish occurring in the Tennessee River drainage of northern Alabama and southwestern Tennessee. We also provide a revised key for the identification of members of O. juvenilis Species Complex.

Cambarus (Depressicambarus) clairitae, a new species of crayfish (Decapoda: Cambaridae) from Alabama with a review of the halli Group in the subgenus Depressicambarus.

Cambarus (Depressicambarus) clairitae, new species, is an epigean crayfish from two drainages of the Locust Fork system in Blount and Jefferson counties, Alabama. It belongs to the halli Group in the subgenus Depressicambarus. The new species is morphologically most similar to Cambarus (Depressicambarus) englishi. They differ in a several morphological characters. Cambarus englishi has a more strongly recurved central projection, a wider areola, and a more distinct and set off rostral acumen than the new species. It also has light gray to white antennae while the antennae of the new species are brown. In addition to the description of the new species, the halli Group in Alabama is discussed.

Two new species of South American freshwater crayfish genus Parastacus Huxley, 1879 (Crustacea: Decapoda: Parastacidae).

Two new species of Parastacus Huxley, 1879 are described from material collected in the state of Rio Grande do Sul, southern Brazil: Parastacus fluviatilis sp. nov. from highland streams and Parastacus caeruleodactylus sp. nov. from wetlands. Parastacus fluviatilis sp. nov. is distinguished mainly by large chelipeds with dense setae cover on the cutting edge of fingers, telson subtriangular with two lateral blunt spines and strongly concave ventral surface of lateral process of thoracic sternites 6 and 7. Parastacus caeruleodactylus sp. nov. is distinguished mainly by blue cheliped fingers and a large gap between them, reduced abdomen, dorsal and ventral margins of dactylus, propodus and carpus of second pair of pereiopods with tufts of long setae and mid-dorsal carina of exopod of uropods unarmed. According to IUCN Red List criteria both species are considered endangered. Habitat characterization and a method for defining the shape of second abdominal pleura are also provided.

Cambarus (Jugicambarus) adustus, a new species of crayfish from northeastern Kentucky delimited from the Cambarus (J.) aff. dubius species complex.

A new species of burrowing crayfish, Cambarus (Jugicambarus) adustus, is described from Lewis County in northeastern Kentucky, USA. The new species is most similar morphologically to C. dubius. Cambarus adustus coloration differs from C. dubius by lacking red, orange and blue hues, and instead is brown over the entire body surface. Morphological differences between C. dubius and C. adustus exist in the form I male gonopod, with C. adustus possessing a caudal knob, while C. dubius does not. In addition, the lateral carapace of C. adustus is distinctly tuberculate, whereas in C. dubius the carapace lacks extensive tuberculation. Cambarus (J.) adustus appears to have an extremely small geographic range (~19.5 km2), and as such we suggest its consideration for both state and federal levels of protection.

PcToll2 positively regulates the expression of antimicrobial peptides by promoting PcATF4 translocation into the nucleus.

Drosophila Toll and mammalian Toll-like receptors (TLRs) are a family of evolutionarily conserved immune receptors that play a crucial role in the first-line defense against intruded pathogens. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in TLR signaling and other physiological processes. However, in crustaceans, whether ATF4 homologs were involved in TLR signaling remains unclear. In the current study, we identified a Toll homolog PcToll2 and a novel ATF4 homolog PcATF4 from Procambarus clarkii, and analyzed the likely regulatory activity of PcATF4 in PcToll2 signaling. The complete cDNA sequence of PcToll2 was 4175 bp long containing an open reading frame of 2820 bp encoding a 939-amino acid protein, and the cDNA sequence of PcATF4 was 2027 bp long with an open reading frame of 1296 bp encoding a 431-amino acid protein. PcToll2 and human TLR4 shared the high identity and they were grouped into a cluster. Furthermore, PcToll2 had a close relationship with other shrimp TLRs that possessed potential antibacterial activity. PcToll2 was highly expressed in the hemocytes, heart and gills, while PcATF4 mainly distributed in gills. Upon challenge with Vibrio parahemolyticus, PcToll2 and PcATF4 together with the antimicrobial peptides of ALF1 and ALF2 were significantly up-regulated in the hemocytes, and the PcATF4 was translocated into the nucleus. After PcToll2 silencing and challenge with Vibrio, the translocation of PcATF4 into the nucleus was inhibited and the expression of ALF1 and ALF2 was reduced, but the expression of PcDorsal and PcSTAT was not affected. Furthermore, after PcATF4 knockdown and challenge with or without Vibrio, the expression of ALF1 and ALF2 was also decreased while the expression of PcToll2 was upregulated. These results suggested that PcToll2 might regulate the expression of ALF1 and ALF2 by promoting the import of PcATF4, instead of the routine transcription factor PcDorsal, into the nucleus participating in the immune defense against Gram-negative bacteria.

Characterization and function of a cathepsin B in red crayfish (Procambarus clarkii) following lipopolysaccharide challenge.

Cathepsin B is a lysosomal cysteine protease of the papain-like enzyme family with multiple biological functions. In the present study, a cathepsin B gene (named PcCTSB) was cloned and characterized from the red crayfish, Procambarus clarkii. The cDNA fragments of PcCTSB was 990 bp in length. It encoded a putative protein of 329 amino acid residues with predicted molecular weight of 36.4 kDa and isoelectric point of 7.020. Sequence alignment revealed that PcCTSB protein is 53.6%-80.4% identical with those from other 10 species. The predicted tertiary structure of PcCTSB protein was highly similar to that of animals. The results of the phylogenetic analysis indicated that the PcCTSB protein could be clustered with the Eriocheir sinensis cathepsin B protein. The recombinant protein of PcCTSB was expressed successfully in Escherichia coli cells. The mRNA expressions of PcCTSB were detected in all tested tissues, particularly high in the hepatopancreas. After lipopolysaccharide (LPS) challenge, the expression levels of PcCTSB were up-regulated significantly at different time points compared with control. Our results suggested that the PcCTSB might play an important role in defending against the pathogenes infection.

A New Crayfish of the Genus Cambarus (Decapoda: Cambaridae) From the Flint River Drainage in Northern Alabama and South Central Tennessee, U.S.A.

Examination of specimens of the crayfish species Cambarus (Hiaticambarus) longirostris and two recently described crayfish revealed the existence of an undescribed species from the Flint River watershed in northern Alabama and southern Tennessee. Cambarus (Hiaticambarus) lentiginosus differs from C. longirostris, C. (Hiaticambarus) andersoni, and C. (Hiaticambarus) diupalma in aspects of carapace and chela morphology and pigmentation pattern. Cambarus (H.) lentiginosus possesses a median carina on the dorsal surface of the acumen of the rostrum, a distinctly enlarged tubercle near the base of the opposable surface of the dactyl, and a speckled pigmentation pattern over a light base color; these characters are lacking in other known Hiaticambarus species. The known range of the species is restricted to the Flint River watershed. Cambarus (H.) lentiginosus is considered Endangered using American Fisheries Society conservation categorization.