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Land hermit crabs are a group of shell-carrying crabs that have evolved remarkable terrestrial adaptations in behavior, morphology, physiology, and biochemistry. However, the genetic mechanisms underlying these adaptations remain unclear. In addition, usually it is very difficult to get good genome assemblies for crustaceans. In this study, we managed to assemble the first chromosome-level genome for a land hermit crab (Coenobita brevimanus) with careful manual curation. The final assembly spans 4.74 Gb, with the contig N50 of 1.75 Mb and scaffold N50 of 42.95 Mb, encompassing 117 chromosomes that account for 96.54% of the genome. The evaluations including genome BUSCO (95.26%), Merqury qv (35.88) and the mapping ratio of pair-end short reads (99.48%) showed the high-continuity of C. brevimanus genome assembly, making it the genome with the highest quality in crustaceans with genome size bigger than 3 Gb. The availability of this chromosome-scale genome of crustaceans represents a valuable resource for the land hermit crab, which represents an independent water-to-land transition evolutionary event in the animal kingdom., (© 2024. The Author(s).)

The family Diogenidae Ortmann, 1892 is a diverse and abundance group of hermit crabs, but their systematics and phylogenetic relationships are highly complex and unresolved. Herein, we gathered nucleotide sequence data from two mitochondrial (16S rRNA and COI) and two nuclear (NaK and PEPCK) genes for a total of 2,308 bp in length across 38 species from six extant diogenid genera. Molecular data were combined with 41 morphological characters to estimate the largest phylogeny of diogenid hermit crabs to date with the aim of testing the proposed taxonomic scheme of Diogenidae and addressing intergeneric relationships within this family. Despite conflicts between mitochondrial and nuclear DNA trees, the combined-data tree reflects the contributions of each dataset, and improves tree resolution and support for internal nodes. Contrary to traditional classification, our total evidence revealed a paraphyletic Diogenidae based on internally nested representatives of Coenobitidae Dana, 1851. Within Diogenidae, the studied diogenid hermit crabs were split between two clades with high support, which contradicts recent morphological classification scheme for Diogenidae sensu lato based on fossil records. The genus Diogenes Dana, 1851 was found nested inside Paguristes Dana, 1851, which formed a clade being separated from the remainder, pointing towards paraphyly in Paguristes . In another clade, Dardanus Paulson, 1875 occupied a basal position relative to the other diogenids, while Calcinus Dana, 1851 and Clibanarius Dana, 1852 showed sister relationships and formed a cluster with Ciliopagurus Forest, 1995. Among the morphological characters examined, carapace shield and telson were identified as phylogenetically significant for grouping diogenid genera, while phylogenetic insignificance of gill number was evidenced by its mosaic pattern in diogenid phylogeny. The present study sheds light on the controversial generic phylogeny of Diogenidae and highlights the necessity for thorough taxonomic revisions of this family as well as some genera ( e.g ., Paguristes ) to reconcile current classifications with phylogenetic relationships., Competing Interests: The authors declare that they have no competing interests., (© 2024 Cheng et al.)

Crabs are a large subtaxon of the Arthropoda, the most diverse and species-rich metazoan group. Several outstanding questions remain regarding crab diversification, including about the genomic capacitors of physiological and morphological adaptation, that cannot be answered with available genomic resources. Physiologically and ecologically diverse Anomuran porcelain crabs offer a valuable model for investigating these questions and hence genomic resources of these crabs would be particularly useful. Here, we present the first two genome assemblies of congeneric and sympatric Anomuran porcelain crabs, Petrolisthes cinctipes and Petrolisthes manimaculis from different microhabitats. Pacific Biosciences high-fidelity sequencing led to genome assemblies of 1.5 and 0.9 Gb, with N50s of 706.7 and 218.9 Kb, respectively. Their assembly length difference can largely be attributed to the different levels of interspersed repeats in their assemblies: The larger genome of P. cinctipes has more repeats (1.12 Gb) than the smaller genome of P. manimaculis (0.54 Gb). For obtaining high-quality annotations of 44,543 and 40,315 protein-coding genes in P. cinctipes and P. manimaculis, respectively, we used RNA-seq as part of a larger annotation pipeline. Contrarily to the large-scale differences in repeat content, divergence levels between the two species as estimated from orthologous protein-coding genes are moderate. These two high-quality genome assemblies allow future studies to examine the role of environmental regulation of gene expression in the two focal species to better understand physiological response to climate change, and provide the foundation for studies in fine-scale genome evolution and diversification of crabs., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Mysids are small crustaceans that are closely related to shrimp/prawns and crabs but not subject to food allergen labeling requirements for raw materials. In the past, a processed food that contained Japanese smelt (wakasagi) was suspected of producing a false-positive result in shrimp/prawn and crab allergen test because of the presence of consumed mysids. However, there was no reported methods to confirm mysid presence. Therefore, we developed a PCR method to detect mysids. The developed PCR method had high specificity for a mysid species, with no amplification observed from samples of shrimp, crab, krill, mantis shrimp, or the meat of Japanese smelt. In addition, DNA extracted from the internal organs of Japanese smelt was amplified by this PCR method, and sequencing revealed mysid DNA. This confirmed that mysids remained in the internal organs of Japanese smelt following consumption. This PCR method for mysid detection even amplified Japanese smelt-containing processed food samples that were suspected to have produced a false-positive result in shrimp/prawn and crab ELISA. Thus, this PCR method would enable to detect such false positives are caused by mysid contamination.

Human impacts on the environment affect organisms at all levels of biological organisation and ultimately can change their phenotype. Over time, phenotypic change may arise due to selection but individual phenotypes are also subject to change via genotype × environment interactions. In animals, behaviour is the most flexible aspect of phenotype, and hence the most liable to change across environmental gradients including exposure to pollution. Here we review current knowledge on the impacts of pollution, broadly defined to include the release of substances, energy, and the effects of carbon emissions, on the behaviour of a highly studied group, the globally distributed hermit crabs. We first show how their obligate association with empty gastropod shells underpins their use as model organisms for the study of resource-assessment, contest, and risk-coping behaviours. Intense study of hermit crabs has advanced our understanding of how animals use information, and we discuss the ways in which pollutants can disrupt the cognitive processes involved. We then highlight current studies of hermit crabs, which paint a clear picture of behavioural changes due to multiple pollutants. Impacts on behaviour vary across pollutants and entire suites of behaviours can be influenced by a single pollutant, with the potential for interactive and cascade effects. Hermit crabs offer the opportunity for detailed behavioural analysis, including application of the repeated measures animal-personality framework, and they are highly amenable to experimental manipulations. As such, we show how they now provide a model system for studying the impacts of pollution on behaviour, yielding insights broadly applicable across animal diversity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This study aimed to apply the comet assay on Aegla platensis crabs as a suitable non-destructive approach for in situ monitoring of freshwater genotoxicity. Animals were captured during four sampling periods in a stream under minor anthropogenic impacts in Southern Brazil. Crabs were captured with a hand net, then the hemolymph samples were collected, and the animals were released into the stream after a 20-min recovery time. Hemolymph samples were transported to the laboratory and used to perform the alkaline comet assay. Results showed an intermediate level in the DNA damage index (range 107.3-165.0 arbitrary unit). No significant differences were observed among the different sampling periods. Hemolymph was successfully used as a non-lethal source of biological samples, and the comet assay using A. platensis proved to be a feasible approach for genotoxicity studies., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A complete mitochondrial genome (mitogenome) can provide important information for gene rearrangement, molecular evolution and phylogenetic analysis. Currently, only a few mitogenomes of hermit crabs (superfamily Paguridae) in the infraorder Anomura have been reported. This study reports the first complete mitogenome of the hermit crab Diogenes edwardsii assembled using high-throughput sequencing. The mitogenome of Diogenes edwardsii is 19,858 bp in length and comprises 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. There are 28 and six genes observed on the heavy and light strands, respectively. The genome composition was highly A + T biased (72.16%), and exhibited a negative AT-skew (-0.110) and positive GC-skew (0.233). Phylogenetic analyses based on the nucleotide dataset of 16 Anomura species indicated that D. edwardsii was closest related to Clibanarius infraspinatus in the same family, Diogenidae. Positive selection analysis showed that two residues located in cox1 and cox2 were identified as positively selected sites with high BEB value (>95%), indicating that these two genes are under positive selection pressure. This is the first complete mitogenome of the genus Diogenes , and this finding helps us to represent a new genomic resource for hermit crab species and provide data for further evolutionary status of Diogenidae in Anomura.

The complete mitochondrial genome (mitogenome) contributed crucial information, which could improve the better understanding of molecular phylogenetic analysis, evolution, and gene rearrangements. However, only a few Coenobitidae and Albuneidae mitogenomes have been described, and controversies about the Anomuran phylogeny remain. Here, we determined three Coenobitidae species (Coenobita perlatus, Coenobita rugosus, Coenobita clypeatus) and one Albuneidae species (Emerita talpoida). As the representative species of genera or families, and even the first species of the genus, supplementing these four species is essential to study the phylogenetic relationships within Anomura. Most of the four novel mitogenomes have the typical 37 genes, except for E. talpoida, which lacked trnF. Through combining these new data with 31 Anomuran mitogenomes from Genbank, different gene rearrangement patterns and internal phylogenetic relationships of Anomura were investigated. In our phylogeny analysis, all the Anomuran species were clustered into one group, and the polyphyly of Paguroidea was well supported. Moreover, various peculiar mitochondrial gene orders (MGOs) were summarized among Anomura and preliminary determined their rearrangement mechanisms through CREx. Twenty different MGOs were suggested in our research, and we were focused seven MGO patterns (Pattern A-J) in the process of discussing the gene rearrangement mechanism. Currently making full use of the large taxon sampling, our results provide valuable information on the evolutionary status of Anomuran species and are available for systematic rearrangement and phylogenetic analyses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The infraorder Anomura is a species-rich clade of decapod crustaceans recognized by its remarkable disparity in terms of morphology, anatomy, ecology, physiology, and behavior. This study assembled and characterized the complete mitochondrial genomes of two anomuran species, the hermit crab Coenobita clypeatus and the mole crab Emerita talpoida. The AT-rich mitochondrial genomes of C. clypeatus and E. talpoida are 16,469 bp and 15,810 bp long, respectively, and are composed of 13 protein-coding genes (PCGs), two ribosomal RNA genes, and 22 transfer RNA genes. A 1,390 bp and 553 bp long intergenic space is assumed to be the D-loop in C. clypeatus and E. talpoida, respectively. Mitochondrial synteny in C. clypeatus is identical to that reported in other congeneric hermit crabs while synteny in E. talpoida is identical to that described for the confamilial mole crab Stemonopa insignis. No major differences occur between the studied species and their respective congeneric / cofamilial species in terms of nucleotide composition and codon usage profiles of PCGs. Selective pressure analysis in PCGs, rarely conducted in anomuran crabs, indicate that all these mitochondrial PCGs experience purifying selection and that this purifying selection is stronger in some (i.e., cox family genes and cob) compared to other PCGs (e.g., atp8). Most of the tRNA genes exhibited a typical 'cloverleaf' secondary structure with few exceptions in the two studied species. In C. clypeatus, tRNA-Ser1 lacks the thymine pseudouracil cytosine (TΨC) loop while tRNA-Phe and tRNA-Tyr each exhibit a deletion of the dihydroxyuridine (DHU) loop but not the arm. In turn, in E. talpoida, tRNA-Phe and tRNA-Arg exhibit a deletion of the DHU loop but not the arm while tRNA-Ser1 lacks the TΨC arm. A phylogenomic analysis based on translated PCGs confirms the monophyly of the infraorder Anomura and retrieves most/all relationships at the superfamily and family level previously reported for anomurans. The analysis supports the monophyletic status of the families Albuneidae, Lithodidae, Coenobitidae, and Porcellanidae. In turn, the superfamily Paguroidea, and the families Paguridae and Diogenidae are polyphyletic., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Crustacean hyaluronidases are poorly understood both in terms of their enzymatic properties and in terms of their structural features. In this work, we show that the hepatopancreas homogenate of the red king crab has a hyaluronidase activity that is an order of magnitude higher than its commercial counterpart. Zymography revealed that the molecular weight of a protein with hyalorunidase activity is 40-50 kDa. Analysis of the hepatopancreas transcriptome and results of cloning and sequencing of cDNA revealed a hyaluronidase sequence with an expected molecular weight of 42.5 kDa. Further analysis showed that hyaluronat enzymatic cleavage follows the [Formula: see text]-elimination mechanism, which is well known for bacterial hyaluronidases. The results of ion-exchange chromatography showed that the final product of hyaluronate degradation is unsaturated tetrasaccharide. Thus, we identified a new hyaluronidase of higher eukaryotes, which is not integrated into the modern classification of hyaluronidases., (© 2021. The Author(s).)

Complete mitochondrial genomes (mitogenomes) can provide useful information for phylogenetic relationships, gene rearrangement, and molecular evolution. In this study, the complete mitogenomes of two hermit crabs, Dardanus arrosor and Dardanus aspersus, were sequenced for the first time and compared with other published mitogenomes of Paguroidea. Each of the two mitogenomes contains an entire set of 37 genes and a putative control region, but they display different gene arrangements. The different arrangements of the two mitogenomes might be the result of transposition, reversal, and tandem duplication/random loss events from the ancestral pancrustacean pattern. Genome sequence similarity analysis reveals the gene rearrangement in 15 Paguroidea mitogenomes. After synteny analysis between the 15 Paguroidea mitogenomes, an obvious rearranged region is found in D. aspersus mitogenome. Across the 13 protein-coding genes (PCGs) tested, COI has the least and ND6 has the largest genetic distances among the 15 hermit crabs, indicating varied evolution rates of PCGs. In addition, the dN/dS ratio analysis shows that all PCGs are evolving under purifying selection. The phylogenetic analyses based on both gene order and sequence data present the monophyly of three families (Paguridae, Coenobitidae, and Pylochelidae) and the paraphyly of the family Diogenidae. Meanwhile, the phylogenetic tree based on the nucleotide sequences of 13 PCGs shows that two Dardanus species formed a sister group with five Coenobitidae species. These findings help to better understand the gene rearrangement and phylogeny of Paguroidea, as well as provide new insights into the usefulness of mitochondrial gene order as a phylogenetic marker., (© 2021. The Author(s).)

A new species of freshwater anomuran crab, Aegla buenoi n. sp., is described. The new taxon was collected from two streams within the Cinzas River basin, Paran state, Brazil. We used morphological and molecular data (COI mtDNA) to distinguish the new species from its congeners. Aegla buenoi n. sp. is differentiated by morphological diagnostic features of the cephalothorax, chelipeds, second abdominal epimeron, and uropods. Molecular results confirm the separation of A. buenoi n. sp. from closely related species (A. castro Schmitt, 1942, A. lata Bond-Buckup Buckup, 1994, and A. jacutinga Marl Teixeira, 2020). Hence, our study increases the known diversity of aeglids and reports the first species of Aegla from the Cinzas River basin.

Morphological characters, as presently applied to describe members of the Paguristes tortugae Schmitt, 1933 species complex, appear to be of limited value in inferring phylogenetic relationships within the genus, and may have similarly misinformed understanding of relationships between members of this complex and those presently assigned to the related genera Areopaguristes Rahayu McLaughlin, 2010 and Pseudopaguristes McLaughlin, 2002. Previously undocumented observations of similarities and differences in color patterns among populations additionally suggest genetic divergences within some species, or alternatively seem to support phylogenetic groupings of some species. In the present study, a Maximum Likelihood (ML) phylogenetic analysis was undertaken based on the H3, 12S mtDNA, and 16S mtDNA sequences of 148 individuals, primarily representatives of paguroid species from the western Atlantic. This molecular analysis supported a polyphyletic Diogenidae Ortmann, 1892, although incomplete taxonomic sampling among the genera of Diogenidae limits the utility of this finding for resolving family level relationships. Several hypotheses regarding the evolutionary relationships among hermit crab genera were refuted by the Kishino-Hasegawa (KH). Shimodaira-Hasegawa (SH) and Approximately Unbiased (AU) tree topology tests, among them the hypothesis that Areopaguristes is monophyletic. A lack of support for the monophyly of Areopaguristes calls into question the phylogenetic validity of gill number for the differentiation of Paguristes, Areopaguristes, and Pseudopaguristes. The study was inconclusive with regard to the relationships among these three genera, but previously unknown diversity within both Paguristes and Areopaguristes was demonstrated. Existence of an undescribed species confounded under the name Paguristes tortugae Schmitt, 1933 was supported by genetics, morphology, and coloration. A second undescribed species with remarkable similarity to Areopaguristes hummi Wass, 1955 was discovered based on genetics and coloration.

The blue king crab, Paralithodes platypus, which belongs to the family Lithodidae, is a commercially and ecologically important species. However, a high-quality reference genome for the king crab has not yet been reported. Here, we assembled the first chromosome-level blue king crab genome, which contains 104 chromosomes and an N50 length of 51.15 Mb. Furthermore, we determined that the large genome size can be attributed to the insertion of long interspersed nuclear elements and long tandem repeats. Genome assembly assessment showed that 96.54% of the assembled transcripts could be aligned to the assembled genome. Phylogenetic analysis showed the blue king crab to have a close relationship with the Eubrachyura crabs, from which it diverged 272.5 million years ago. Population history analyses indicated that the effective population of the blue king crab declined sharply and then gradually increased from the Cretaceous and Neogene periods, respectively. Furthermore, gene families related to developmental pathways, steroid and thyroid hormone synthesis, and inflammatory regulation were expanded in the genome, suggesting that these genes contributed substantially to the environmental adaptation and unique body plan evolution of the blue king crab. The high-quality reference genome reported here provides a solid molecular basis for further study of the blue king crab's development and environmental adaptation., (© 2020 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Endogenous antimicrobial peptides (AMPs) are evolutionarily ancient factors of innate immunity, which are produced by all multicellular organisms and play a key role in their protection against infection. Red king crab (Paralithodes camtschaticus), also called Kamchatka crab, is widely distributed and the best known species of all king crabs belonging to the family Lithodidae. Despite their economic importance, the genetic resources of king crabs are scarcely known and no full-genome sequences are available to date. Therefore, analysis of the red king crab transcriptome and identification and characterization of its AMPs could potentially contribute to the development of novel antimicrobial drug candidates when antibiotic resistance has become a global health threat. In this study, we sequenced the P. camtschaticus transcriptomes from carapace, tail flap and leg tissues using an Illumina NGS platform. Libraries were systematically analyzed for gene expression profiles along with AMP prediction. By an in silico approach using public databases we defined 49 cDNAs encoding for AMP candidates belonging to diverse families and functional classes, including buforins, crustins, paralithocins, and ALFs (anti-lipopolysaccharide factors). We analyzed expression patterns of 27 AMP genes. The highest expression was found for Paralithocin 1 and Crustin 3, with more than 8,000 reads. Other paralithocins, ALFs, crustins and ubiquicidins were among medium expressed genes. This transcriptome data set and AMPs provide a solid baseline for further functional analysis in P. camtschaticus. Results from the current study contribute also to the future application of red king crab as a bio-resource in addition to its being a known seafood delicacy.

The complete mitochondrial genomes (mitogenomes) can indicate phylogenetic relationships among organisms, as well as useful information about the process of molecular evolution and gene rearrangement mechanisms. However, knowledge on the complete mitogenome of Coenobitidae (Decapoda: Anomura) is quite scarce. Here, we describe in detail the complete mitogenome of Coenobita brevimanus, which is 16,393 bp in length, and contains 13 protein-coding genes, two ribosomal RNA, 22 transfer RNA genes, as well as a putative control region. The genome composition shows a moderate A + T bias (65.0%), and exhibited a negative AT-skew (-0.148) and a positive GC-skew (0.183). Five gene clusters (or genes) involving eleven tRNAs and two PCGs were found to have rearranged with respect to the pancrustacean ground pattern gene order. Duplication-random loss and recombination models were determined as most likely to explain the observed large-scale gene rearrangements. Phylogenetic analysis placed all Coenobitidae species into one clade. The polyphyly of Paguroidea was well supported, whereas the non-monophyly of Galatheoidea was inconsistence with previous findings on Anomura. Taken together, our results help to better understand gene rearrangement process and the evolutionary status of C. brevimanus and lay a foundation for further phylogenetic studies of Anomura., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Hydrothermal vents and cold seeps are typical deep-sea chemosynthetically-driven ecosystems that allow high abundance of specialized macro-benthos. To gather knowledge about the genetic basis of adaptation to these extreme environments, species shared between different habitats, especially for the dominant species, are of particular interest. The galatheid squat lobster, Shinkaia crosnieri Baba and Williams, 1998, is one of the few dominant species inhabiting both deep-sea hydrothermal vents and cold seeps. In this study, we performed transcriptome analyses of S. crosnieri collected from the Iheya North hydrothermal vent (HV) and a cold seep in the South China Sea (CS) to provide insights into how this species has evolved to thrive in different deep-sea chemosynthetic ecosystems., Results: We analyzed 5347 orthologs between HV and CS to identify genes under positive selection through the maximum likelihood approach. A total of 82 genes were identified to be positively selected and covered diverse functional categories, potentially indicating their importance for S. crosnieri to cope with environmental heterogeneity between deep-sea vents and seeps. Among 39,806 annotated unigenes, a large number of differentially expressed genes (DEGs) were identified between HV and CS, including 339 and 206 genes significantly up-regulated in HV and CS, respectively. Most of the DEGs associated with stress response and immunity were up-regulated in HV, possibly allowing S. crosnieri to increase its capability to manage more environmental stresses in the hydrothermal vents., Conclusions: We provide the first comprehensive transcriptomic resource for the deep-sea squat lobster, S. crosnieri, inhabiting both hydrothermal vents and cold seeps. A number of stress response and immune-related genes were positively selected and/or differentially expressed, potentially indicating their important roles for S. crosnieri to thrive in both deep-sea vents and cold seeps. Our results indicated that genetic adaptation of S. crosnieri to different deep-sea chemosynthetic environments might be mediated by adaptive evolution of functional genes related to stress response and immunity, and alterations in their gene expression that lead to different stress resistance. However, further work is required to test these proposed hypotheses. All results can constitute important baseline data for further studies towards elucidating the adaptive mechanisms in deep-sea crustaceans.

Complete mitochondrial genome (mitogenome) provides important information for better understanding of gene rearrangement, molecular evolution and phylogenetic analysis. Currently, only a few Paguridae mitogenomes have been reported. Herein, we described the complete mitogenome of hermit crab Pagurus nigrofascia. The total length was 15,423 bp, containing 13 protein-coding genes (PCGs), two ribosomal RNA, 22 transfer RNA genes, as well as an AT-rich region. The genome composition was highly A + T biased (71.4%), and exhibited a negative AT-skew (-0.006) and GC-skew (-0.138). Eight tRNA genes, two PCGs and an AT-rich region found to be rearranged with respect to the pancrustacean ground pattern gene order. Duplication-random loss and recombination model were adopted to explain the large-scale gene rearrangement events. Two phylogenetic trees of Anomura involving 12 families were constructed. The results showed that all Paguridae species were clustered into one clade except Pagurus longicarpus, which for the first time imposed raises doubt about the morphological taxonomy of this species. Furthermore, the present study found that higher- level phylogenetic relationships within Anomura were controversial, compared with the previous studies. Our results help to better understand gene rearrangements and the evolutionary status of P. nigrofascia and lay foundation for further phylogenetic study of Anomura., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Munida isos is a deep sea squat lobster species that is widely distributed across the New Zealand and east Australian region, and is often associated with deep sea vulnerable marine ecosystems. To investigate its population genetic structure and patterns of regional connectivity, microsatellite loci were developed for M. isos from two genomic libraries using the Illumina HiSeq 2500 sequencing platform. Twenty-six loci amplified consistently in M. isos from the Tasman Sea, among which 20 were polymorphic and selectively neutral. Evidence of null alleles was observed at eight loci. Most loci exhibited moderate to high levels of polymorphism, with an average polymorphic information content value of 0.482. The mean number of alleles per locus was 7.45, with a mean expected heterozygosity of 0.520. Thirteen loci exhibited significant deviation from Hardy-Weinberg equilibrium, while only one locus pair was in linkage disequilibrium after false discovery rate correction for multiple testing ( P < 0.05). Cross-species amplification tests revealed that the transferability of 14 loci (70%) was positive for the two congeners M. endeavourae and M. gracilis . The accessibility to new polymorphic microsatellite loci will facilitate population genetic studies and aid in developing conservation and management strategies for vulnerable marine ecosystems.

The infraorder Anomura consists of a morphologically and ecologically heterogeneous group of decapod crustaceans, and has attracted interest from taxonomists for decades attempting to find some order out of the seemingly chaotic diversity within the group. Species-level diversity within the Anomura runs the gamut from the "hairy" spindly-legged yeti crab found in deep-sea hydrothermal vent environments to the largest known terrestrial invertebrate, the robust coconut or robber crab. Owing to a well-developed capacity for parallel evolution, as evidenced by the occurrence of multiple independent carcinization events, Anomura has long tested the patience and skill of both taxonomists attempting to find order, and phylogeneticists trying to establish stable hypotheses of evolutionary inter-relationships. In this study, we performed genome skimming to recover the mitogenome sequences of 12 anomuran species including the world's largest extant invertebrate, the robber crab (Birgus latro), thereby over doubling these resources for this group, together with 8 new brachyuran mitogenomes. Maximum-likelihood (ML) and Bayesian-inferred (BI) phylogenetic reconstructions based on amino acid sequences from mitogenome protein-coding genes provided strong support for the monophyly of the Anomura and Brachyura and their sister relationship, consistent with previous studies. The majority of relationships within families were supported and were largely consistent with current taxonomic classifications, whereas many relationships at higher taxonomic levels were unresolved. Nevertheless, we have strong support for a polyphyletic Paguroidea and recovered a well-supported clade of a subset of paguroids (Diogenidae + Coenobitidae) basal to all other anomurans, though this requires further testing with greater taxonomic sampling. We also introduce a new feature to the MitoPhAST bioinformatics pipeline (https://github.com/mht85/MitoPhAST) that enables the extraction of mitochondrial gene order (MGO) information directly from GenBank files and clusters groups based on common MGOs. Using this tool, we compared MGOs across the Anomura and Brachyura, identifying Anomura as a taxonomic "hot spot" with high variability in MGOs among congeneric species from multiple families while noting the broad association of highly-rearranged MGOs with several anomuran lineages inhabiting extreme niches. We also demonstrate the value of MGOs as a source of novel synapomorphies for independently reinforcing tree-based relationships and for shedding light on relationships among challenging groups such as the Aegloidea and Lomisoidea that were unresolved in phylogenetic reconstructions. Overall, this study contributes a substantial amount of new genetic material for Anomura and attempts to further resolve anomuran evolutionary relationships where possible based on a combination of sequence and MGO information. The new feature in MitoPhAST adds to the relatively limited number of bioinformatics tools available for MGO analyses, which can be utilized widely across animal groups., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Serpins are a family of serine protease inhibitors that are involved in numerous physiological processes and are known to regulate innate immunity pathways. To advance our understanding of their role in P. camtschaticus, a commercially significant species, we cloned and characterized a serpin from this species, designated serpin PC, that has anticoagulant and anticomplement effects on human blood. We found that serpin PC is a secreted protein with a typical serpin-like primary structure that is similar to other known crustacean serpins. Recombinant serpin PC was found to have inhibitory activity against R/K-specific bovine cationic trypsin. The reaction proceeds through the formation of a stable covalent complex of peptidase with P1 residue R383 of serpin PC. This interaction is characterized by a relatively high overall inhibition constant k ass =(2.3 ± 0.7) × 10 6  M -1 s -1 and an SI of 4.7 ± 0.8. Protein localization by western blotting showed that serpin PC is present in the muscles and, to a lesser extent, the heart, whereas it is transcribed predominantly in hemocytes and the heart. Through peptidase activity profiling of hemocytes and plasma, we found that serpin PC inhibits at least two R/K-specific activities and showed that it inhibits phenoloxidase (PO) activity induction in hemocytes., (Copyright © 2018. Published by Elsevier Ltd.)

A new species of hermit crab, Pagurus fraserorum n. sp. (family Paguridae) is described from rocky subtidal reefs off KwaZulu-Natal, South Africa, and illustrated using both conventional drawings and colour photographs, and via three-dimensional (3D) X-ray micro-computed tomography (μCT). Because of the limitation μCT has in detecting very fine and soft structures, a novel approach of manually drawing setation and spinulation onto the two-dimensional images of the 3D visualizations was used to illustrate the pereopods. In addition, an interactive figure and rotation movie clips in the supplement section complement the species description, and the 3D raw data of the 3D type data are downloadable from the Gigascience Database repository. The new species is the sixth species of Pagurus Fabricius, 1775 reported from South Africa and is closely allied to the Indo-Pacific P. boriaustraliensis Morgen, 1990 and P. pitagsaleei McLaughlin, 2002, from which it differs by its shorter ocular peduncles, by the armature of the carpus of the right cheliped, and also in colouration. This study presents the first description of a hermit crab in which a majority of taxonomic details are illustrated through 3D volume-rendered illustrations. In addition, colour photographs and COI molecular barcodes are provided, and the latter compared to COI sequences of specimens from Western Australia previously identified as P. boriaustraliensis and of specimens of P. pitagsaleei from Taiwan, as well as to three additional South African members of the genus. The South African taxon was confirmed to be genetically distinct from all species tested., Competing Interests: The authors have declared that they have no competing interests.

Background: DNA barcoding has demonstrated that many discrete phenotypes are in fact genetically distinct (pseudo)cryptic species. Genetically identical, isogenic individuals, however, can also express similarly different phenotypes in response to a trigger condition, e.g. in the environment. This alternative explanation to cryptic speciation often remains untested because it requires considerable effort to reject the hypothesis that the observed underlying genetic homogeneity of the different phenotypes may be trivially caused by too slowly evolving molecular markers. The widespread squat lobster Munida gregaria comprises two discrete ecotypes, gregaria s. str. and subrugosa, which were long regarded as different species due to marked differences in morphological, ecological and behavioral traits. We studied the morphometry and genetics of M. gregaria s. l. and tested (1) whether the phenotypic differences remain stable after continental-scale sampling and inclusion of different life stages, (2) and whether each phenotype is underpinned by a specific genotype., Results: A total number of 219 gregaria s. str. and subrugosa individuals from 25 stations encompassing almost entire range in South America were included in morphological and genetic analyses using nine unlinked hypervariable microsatellites and new COI sequences. Results from the PCA and using discriminant functions demonstrated that the morphology of the two forms remains discrete. The mitochondrial data showed a shallow, star-like haplotype network and complete overlap of genetic distances within and among ecotypes. Coalescent-based species delimitation methods, PTP and GMYC, coherently suggested that haplotypes of both ecotypes forms a single species. Although all microsatellite markers possess sufficient genetic variation, AMOVA, PCoA and Bayesian clustering approaches revealed no genetic clusters corresponding to ecotypes or geographic units across the entire South-American distribution. No evidence of isolation-by-distance could be detected for this species in South America., Conclusions: Despite their pronounced bimodal morphologies and different lifestyles, the gregaria s. str. and subrugosa ecotypes form a single, dimorphic species M. gregaria s. l.. Based on adequate geographic coverage and multiple independent polymorphic loci, there is no indication that each phenotype may have a unique genetic basis, leaving phenotypic plasticity or localized genomic islands of speciation as possible explanations.

The taxonomy of the Brazilian aeglid species Aegla paulensis Schmitt, 1942 from two disjunct hydrographic basins is revised using morphological and molecular data. Results show that six disjunctive populations of Aegla paulensis form a species complex. Aegla paulensis sensu stricto is redescribed and Aegla rosanae Campos Jr., 1998 is revalidated. The four remaining populations previously assigned to Aegla paulensis are now recognized as different species, namely Aegla  vanini n. sp., Aegla japi n. sp., Aegla jaragua n. sp. and Aegla jundiai n. sp. All new species are described and illustrated and are well supported by both morphological and molecular data. Aegla lancinhas Bond-Buckup & Buckup in Santos et al., 2015, which until recently was confounded with Aegla paulensis s. str., is supported as a valid species. A key to all members of the A. paulensis species complex is provided, and their phylogenetic and biogeographic relationships to other closely related species is discussed.

The complete mitochondrial genome of the hermit crab Clibanarius infraspinatus was recovered by genome skimming using Next-Gen sequencing. The Clibanarius infraspinatus mitogenome has 16,504 base pairs (67.94% A + T content) made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs and a putative 1500 bp non-coding AT-rich region. The Clibanarius infraspinatus mitogenome sequence is the first for the family Diogenidae and the second for the superfamily Paguroidea and exhibits a translocation of the ND3 gene not previously reported for the Decapoda.

The mitochondrial genome sequence of the porcellanid crab, Petrolisthes haswelli is provided, making it the second for the family Porcellanidae and the third for the superfamily Galatheoidea. Petrolisthes haswelli has a mitogenome of 15,348 bp consisting of 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The base composition of the P. haswelli mitogenome is 35.66% for T, 18.65% for C, 34.35% for A and 11.34% for G, with an AT bias of 70.01%. The mitogenome gene order is identical to the mitogenome of Neopetrolisthes maculatus, the only other species of the family with a sequenced mitogenome.

Hydrothermal vents in the Southern Ocean are the physiologically most isolated chemosynthetic environments known. Here, we describe Kiwa tyleri sp. nov., the first species of yeti crab known from the Southern Ocean. Kiwa tyleri belongs to the family Kiwaidae and is the visually dominant macrofauna of two known vent sites situated on the northern and southern segments of the East Scotia Ridge (ESR). The species is known to depend on primary productivity by chemosynthetic bacteria and resides at the warm-eurythermal vent environment for most of its life; its short-range distribution away from vents (few metres) is physiologically constrained by the stable, cold waters of the surrounding Southern Ocean. Kiwa tylerihas been shown to present differential life history adaptations in response to this contrasting thermal environment. Morphological adaptations specific to life in warm-eurythermal waters, as found on - or in close proximity of - vent chimneys, are discussed in comparison with adaptations seen in the other two known members of the family (K. hirsuta, K. puravida), which show a preference for low temperature chemosynthetic environments.

The complete larval development of the hermit crab Pagurus maculosus, is described and illustrated based on specimens reared in the laboratory at 15 °C and 33-35 PSU. Newly hatched larvae invariably passed through a short prezoeal stage (10 minutes to 2 hours), four zoeal stages (each of 7 days,) and one megalopal stage (14 days). Distinct morphological features of each larval stage of the present study are compared with other closely related species in Japanese waters, and we found many differences in morphology and the duration of zoeal stages between them. We mentioned significant diagnostic characters separating this species from other congeners in Japanese waters that include the presence of red-yellowish chromatophores on the maxillipeds. This is the first report of complete larvae development of Pagurus maculosus.

Squat lobsters from Madagascar, Vanuatu, Papua New Guinea, Fiji, eastern Australia and French Polynesia belonging to the Agononida incerta (Henderson, 1888) species complex are described as four new species: A. madagascerta, A. polycerta, A. tasmancerta and A. vanuacerta. This brings to ten the number of species in this complex. All species are morphologically distinguishable only on the basis of the shape of the anterolateral margin of the telson and setation of the dactyli of pereopods 2-4. The morphological delineation of nine of the species and their taxonomic status are robustly supported by phylogenetic analysis of the partial 16S rDNA gene and the partial mitochondrial cytochrome oxidase subunit 1 genes, and in some cases by colour. A phylogenetic analysis of the nine species for which molecular data are available grouped the species in two clades, one of four species with facial spines on the upper surface of pereopod 4 and the other of five species lacking facial spines.

Studies of genetic connectivity and population structure in deep-sea chemosynthetic ecosystems often focus on endosymbiont-hosting species that are directly dependent on chemical energy extracted from vent effluent for survival. Relatively little attention has been paid to vent-associated species that are not exclusively dependent on chemosynthetic ecosystems. Here we assess connectivity and population structure of two vent-associated invertebrates--the shrimp Chorocaris sp. 2 and the squat lobster Munidopsis lauensis--that are common at deep-sea hydrothermal vents in the western Pacific. While Chorocaris sp. 2 has only been observed at hydrothermal vent sites, M. lauensis can be found throughout the deep sea but occurs in higher abundance around the periphery of active vents We sequenced mitochondrial COI genes and deployed nuclear microsatellite markers for both species at three sites in Manus Basin and either North Fiji Basin (Chorocaris sp. 2) or Lau Basin (Munidopsis lauensis). We assessed genetic differentiation across a range of spatial scales, from approximately 2.5 km to more than 3000 km. Population structure for Chorocaris sp. 2 was comparable to that of the vent-associated snail Ifremeria nautilei, with a single seemingly well-mixed population within Manus Basin that is genetically differentiated from conspecifics in North Fiji Basin. Population structure for Munidopsis lauensis was more complex, with two genetically differentiated populations in Manus Basin and a third well-differentiated population in Lau Basin. The unexpectedly high level of genetic differentiation between M. lauensis populations in Manus Basin deserves further study since it has implications for conservation and management of diversity in deep-sea hydrothermal vent ecosystems.

Study of hermit crabs from Alboran Sea has allowed recognition of two different morphological forms under what had been understood as Pagurus forbesii. Based on morphological observations with various species of Pagurus, and molecular studies, a new species is described as P. pseudosculptimanus. An overview of species of Pagurus from the eastern Atlantic and Mediterranean Sea is provided.

The morphological transformation of hermit crabs into crab-like king crabs in the evolution of decapod crustaceans represents a remarkable case of carcinization or evolutionary shaping into a crab-like form. In this study, we focus on internal organs such as the hemolymph vascular system and adjacent anatomical structures of several Recent hermit crab (Paguridae) and king crab (Lithodidae) species. There are various correspondences in the morphology of the arterial systems in the dorsal cephalothorax of the two taxa, especially with regard to the anterior aorta, anterior lateral arteries, and hepatic arteries. In the pleon, the posterior aorta in both taxa displays a proximal bifurcation and follows an asymmetrical course. The ventral vessel system, on the other hand, which mainly supplies the limbs, differs significantly between the taxa, with pagurids displaying the plesiomorphic condition. The pattern of the ventral vessel system in Lithodidae is influenced by morphological transformations of integumental structures during carcinization. One of these transformations was the broadening of the sternites, which resulted in a widening of the space between the endosternites. In addition, changes in the morphology of the endophragmal skeleton in Lithodidae led to an increase in the potential for intraspecific variability and interspecific variation in the arterial branching pattern., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: The infraorder Anomura has long captivated the attention of evolutionary biologists due to its impressive morphological diversity and ecological adaptations. To date, 2500 extant species have been described but phylogenetic relationships at high taxonomic levels remain unresolved. Here, we reconstruct the evolutionary history-phylogeny, divergence times, character evolution and diversification-of this speciose clade. For this purpose, we sequenced two mitochondrial (16S and 12S) and three nuclear (H3, 18S and 28S) markers for 19 of the 20 extant families, using traditional Sanger and next-generation 454 sequencing methods. Molecular data were combined with 156 morphological characters in order to estimate the largest anomuran phylogeny to date. The anomuran fossil record allowed us to incorporate 31 fossils for divergence time analyses., Results: Our best phylogenetic hypothesis (morphological + molecular data) supports most anomuran superfamilies and families as monophyletic. However, three families and eleven genera are recovered as para- and polyphyletic. Divergence time analysis dates the origin of Anomura to the Late Permian ~259 (224-296) MYA with many of the present day families radiating during the Jurassic and Early Cretaceous. Ancestral state reconstruction suggests that carcinization occurred independently 3 times within the group. The invasion of freshwater and terrestrial environments both occurred between the Late Cretaceous and Tertiary. Diversification analyses found the speciation rate to be low across Anomura, and we identify 2 major changes in the tempo of diversification; the most significant at the base of a clade that includes the squat-lobster family Chirostylidae., Conclusions: Our findings are compared against current classifications and previous hypotheses of anomuran relationships. Many families and genera appear to be poly- or paraphyletic suggesting a need for further taxonomic revisions at these levels. A divergence time analysis provides key insights into the origins of major lineages and events and the timing of morphological (body form) and ecological (habitat) transitions. Living anomuran biodiversity is the product of 2 major changes in the tempo of diversification; our initial insights suggest that the acquisition of a crab-like form did not act as a key innovation.

The phylogeny of the superfamily Chirostyloidea (Decapoda: Anomura) has been poorly understood owing to limited taxon sampling and discordance between different genes. We present a nine-gene dataset across 15 chirostyloids, including all known yeti crabs (Kiwaidae), to improve the resolution of phylogenetic affinities within and between the different families, and to date key divergences using fossil calibrations. This study supports the monophyly of Chirostyloidea and, within this, a basal split between Eumunididae and a Kiwaidae-Chirostylidae clade. All three families originated in the Mid-Cretaceous, but extant kiwaids and most chirostylids radiated from the Eocene onwards. Within Kiwaidae, the basal split between the seep-endemic Kiwa puravida and a vent clade comprising Kiwa hirsuta and Kiwa spp. found on the East Scotia and Southwest Indian ridges is compatible with a hypothesized seep-to-vent evolutionary trajectory. A divergence date estimate of 13.4-25.9 Ma between the Pacific and non-Pacific lineages is consistent with Kiwaidae spreading into the Atlantic sector of the Southern Ocean via the newly opened Drake Passage. The recent radiation of Kiwaidae adds to the list of chemosynthetic fauna that appear to have diversified after the Palaeocene/Eocene Thermal Maximum, a period of possibly widespread anoxia/dysoxia in deep-sea basins.

The genus Lauriea belongs to the family Galatheidae and is easily differentiated from other genera of the family by the endopod of the uropod being much wider than long and the dactyli of the walking legs being curved and strongly biunguiculate. Examination of many specimens collected during recent expeditions from Madagascar to French Polynesia and using morphological and molecular data revealed the existence of six species, five of them new, that are genetically distinct yet morphologically very similar. Furthermore, another new species, having a triunguiculate P2-4 dactyli, represents a new genus, Triodonthea.

The systematics of the hermit crab genus Pagurus is complex, with a long history of taxonomic rearrangements. This group, with worldwide distribution, encompasses a large number of morphologically heterogeneous species. A total of 173 currently recognized species have been morphologically organized into 11 informal groups, in attempts to establish internal stability. This arrangement and the relationships among species of each informal group have remained obscure and have never been evaluated from a molecular phylogenetic viewpoint. Attempts to resolve these high-level classification problems have been restricted to morphological studies, with no satisfactory results because of the extensive overlap of the characters used. We evaluated the phylogenetic position of some of these informal groups of species of Pagurus occurring along the Pacific and Atlantic coasts of South America, using partial fragments of two molecular markers (16S mtDNA and Histone 3 nDNA). Our results indicated a clear polyphyletic arrangement of Pagurus; at the same time, the internal clades fit perfectly with pre-established informal morphological groups from South America, which may warrant a formal taxonomic subdivision of the genus. Additionally, the species belonging to the "provenzanoi group" clearly fit within a different taxon and should be reviewed. Also, the presence of the genus Propagurus in the ingroup could be interpreted by non-exclusive hypotheses: from one point of view, the genus Propagurus is nested within Pagurus, and therefore Pagurus is not valid as currently composed; or possibly, that more than one genus should be recognized among the South American species presently assigned to Pagurus. This latter perspective accords with the proposal to restrict Pagurus to only a few species.

The small ubiquitin-like modifier (SUMO) pathway in eukaryotes is an essential post-translational modification required for a variety of cellular processes, development and organelle biogenesis. SUMO-conjugating enzyme (Ubc9) is an important conjunction enzyme in the SUMO pathway. SUMO-1 and Ubc9 have been found in vertebrates; however, their expression in crustaceans was poorly characterized. In this study, the SUMO-1 and Ubc9 genes were identified in the developing testis and ovary of Chinese mitten crab, Eriocheir sinensis, and designated EsSUMO-1 and EsUbc9, respectively. Quantitative real-time PCR demonstrated the expression level of both mRNAs varied significantly during testis and ovary development. In the testis, EsSUMO-1 and EsUbc9 were expressed at moderate levels in stage II-1, increased at stage II-2, and then gradually declined in stage IV. In the ovary, EsSUMO-1 and EsUbc9 expression were low in the early stage, reach the highest level at stage III-2, and then gradually decreased in stage IV. Transcripts from both genes were detected using in situ hybridization throughout the testis and ovary, in the spermatids and oocytes. The pattern of EsSUMO-1 and EsUbc9 expression in the testis and ovary suggests that SUMOylation may play an important role in spermatogenesis and oogenesis in E. sinensis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Intertidal zone organisms can experience transient freezing temperatures during winter low tides, but their extreme cold tolerance mechanisms are not known. Petrolisthes cinctipes is a temperate mid-high intertidal zone crab species that can experience wintertime habitat temperatures below the freezing point of seawater. We examined how cold tolerance changed during the initial phase of thermal acclimation to cold and warm temperatures, as well as the persistence of cold tolerance during long-term thermal acclimation. Thermal acclimation for as little as 6 h at 8°C enhanced cold tolerance during a 1 h exposure to -2°C relative to crabs acclimated to 18°C. Potential mechanisms for this enhanced tolerance were elucidated using cDNA microarrays to probe for differences in gene expression in cardiac tissue of warm- and cold-acclimated crabs during the first day of thermal acclimation. No changes in gene expression were detected until 12 h of thermal acclimation. Genes strongly upregulated in warm-acclimated crabs represented immune response and extracellular/intercellular processes, suggesting that warm-acclimated crabs had a generalized stress response and may have been remodelling tissues or altering intercellular processes. Genes strongly upregulated in cold-acclimated crabs included many that are involved in glucose production, suggesting that cold acclimation involves increasing intracellular glucose as a cryoprotectant. Structural cytoskeletal proteins were also strongly represented among the genes upregulated in only cold-acclimated crabs. There were no consistent changes in composition or the level of unsaturation of membrane phospholipid fatty acids with cold acclimation, which suggests that neither short- nor long-term changes in cold tolerance are mediated by changes in membrane fatty acid composition. Overall, our study demonstrates that initial changes in cold tolerance are likely not regulated by transcriptomic responses, but that gene-expression-related changes in homeostasis begin within 12 h, the length of a tidal cycle.

Background: Multiple loci and population genetic methods were employed to study the phylogeographic history of the Patagonian freshwater crab Aegla neuquensis (Aeglidae: Decopoda). This taxon occurs in two large river systems in the Patagonian Steppe, from the foothills of the Andes Mountains east to the Atlantic Ocean., Methodology/principal Findings: A nuclear phylogeny and multilocus nested clade phylogeographic analysis detected a fragmentation event between the Negro and Chico-Chubut river systems. This event occurred approximately 137 thousand years ago. An isolation-with-migration analysis and maximum-likelihood estimates of gene flow showed asymmetrical exchange of genetic material between these two river systems exclusively in their headwaters. We used information theory to determine the best-fit demographic history between these two river systems under an isolation-with-migration model. The best-fit model suggests that the Negro and the ancestral populations have the same effective population sizes; whereas the Chico-Chubut population is smaller and shows that gene flow from the Chico-Chubut into the Negro is four times higher than in the reverse direction. Much of the Chico-Chubut system appears to have only been recently colonized while the Negro populations appear to have been in place for most of the evolutionary history of this taxon., Conclusions/significance: Due to mitochondrial introgression, three nuclear loci provided different phylogeographic resolution than the three mitochondrial genes for an ancient fragmentation event observed in the nuclear phylogeny. However, the mitochondrial locus provided greater resolution on more recent evolutionary events. Our study, therefore, demonstrates the need to include both nuclear and mitochondrial loci for a more complete understanding of evolutionary histories and associated phylogeographic events. Our results suggest that gene flow between these systems, before and after fragmentation was through periodic paleolakes that formed in the headwaters region. Fragmentation between the Negro and Chico-Chubut systems was driven by the disappearance of these paleolakes during the Patagonian Glaciation.

Species with a broad distribution rarely have the same genetic make-up throughout their entire range. In some cases, they may constitute a cryptic complex consisting of a few species, each with a narrow distribution, instead of a single-, widely distributed species. These differences can have profound impacts for biodiversity conservation planning. The genetic differentiation of four populations of Aegla longirostri, a freshwater crab found in two geographically isolated basins in Rio Grande do Sul State, Brazil, was investigated by analyzing pentanucleotide multi-loci microsatellites in a heteroduplex assay. Although no morphological differences were evident, we found significant genetic differentiation among the four populations, based on F(ST) values and clustering analysis. This high level of differentiation may be indicative of cryptic species in these populations. If this hypothesis is correct, then the species occurring in the Ibicuí-Mirim River, at the southern limit of the Atlantic Rain Forest, would be under threat, considering its very restricted distribution.

The Anomura presents the greatest degree of morphological disparity in the decapod Crustacea, with body forms ranging from the symmetrical and asymmetrical hermit crabs to squat lobsters and king crabs. The phylogeny of the anomurans has been fraught with controversy. Recent debate has focused primarily on the phenomenon of carcinization, the evolution of crab-like form from a non-crab-like ancestor, focused chiefly on derivation of king crabs from asymmetrical hermit crabs--the "hermit to king" hypothesis. We show by phylogenetic analysis of five nuclear protein-coding gene sequences that hermit crabs have a single origin, but surprisingly, that almost all other major clades and body forms within the Anomura, are derived from within the hermit crabs. The crab-like form and squat lobster form have each evolved at least twice from separate symmetrical hermit crab ancestors. In each case, a carcinization trend can be posited via a transition series from the initial symmetrical long-tailed hermit crab form, through the intermediate squat lobster or asymmetrical hermit crab form, to the final crab-like form. Adaptation to dextral shell habitation evolved at least twice, once in an exclusively deep-water clade and once in the common ancestor of all other asymmetrical hermit crabs (from which king crabs are derived). These remarkable cases of parallelism suggest considerable phenotypic flexibility within the hermit crab ground plan, with a general tendency toward carcinization. Rather than having a separate origin from other major clades, hermit crabs have given rise to most other major anomuran body types.

The monophyletic status of the squat lobster superfamily Galatheoidea has come under increasing doubt by studies using evidence as diverse as larval and adult somatic morphology, sperm ultrastructure, and molecular data. Here we synthesize phylogenetic data from these diverse strands, with the addition of new molecular and morphological data to examine the phylogeny of the squat lobsters and assess the status of the Galatheoidea. A total of 64 species from 16 of the 17 currently recognised anomuran families are included. Results support previous work pointing towards polyphyly in the superfamily Galatheoidea and Paguroidea, specifically, suggesting independent origins of the Galatheidae+Porcellanidae and the Chirostylidae+Kiwaidae. Morphological characters are selected that support clades resolved in the combined analysis and the taxonomic status of Galatheoidea sensu lato is revised. Results indicate that Chirostylidae are more closely related to an assemblage including Aegloidea, Lomisoidea and Paguroidea than to the remaining Galatheoidea and are referred to the superfamily Chirostyloidea to include the Chirostylidae and Kiwaidae. A considerable amount of research highlighting morphological differences supporting this split is discussed. The Galatheoidea sensu stricto is restricted to the families Galatheidae and Porcellanidae, and diagnoses for both Chirostyloidea and Galatheoidea are provided. Present results highlight the need for a detailed revision of a number of taxa, challenge some currently used morphological synapomorphies, and emphasise the need for integrated studies with wide taxon sampling and multiple data sources to resolve complex phylogenetic questions., (Copyright © 2010 Elsevier Inc. All rights reserved.)

A well-recognized obstacle to efficient high-throughput analysis of cDNA libraries is the differential abundance of various transcripts in any particular cell type. Decreasing the prevalence of clones representing abundant transcripts before sequencing, using cDNA normalization, may significantly increase the efficacy of random sequencing and is essential for rare gene discovery. Duplex-specific nuclease (DSN) normalization allows the generation of normalized full-length-enriched cDNA libraries to permit a high gene discovery rate. The method is based on the unique properties of DSN from the Kamchatka crab and involves denaturation-reassociation of cDNA, degradation of the ds-fraction formed by abundant transcripts by DSN, and PCR amplification of the remaining ss-DNA fraction. The method has been evaluated in various plant and animal models.

Introduction: In spite of contemporary morphological taxonomy appraisals, apparent high morphological similarity raises uncertainty about the species status of certain Pagurus hermit crabs. This is exemplified between two European species, Pagurus excavatus (Herbst, 1791) and Pagurus alatus (Fabricius 1775), whose species status is still difficult to resolve using morphological criteria alone., Methodology/principal Findings: To address such ambiguities, we used combinations of Maximum Likelihood (ML) and Bayesian Inference (BI) methods to delineate species boundaries of P. alatus and P. excavatus and formulate an intermediate Pagurus phylogenetic hypothesis, based upon single and concatenated mitochondrial (cytochrome oxidase I [COI]) and nuclear (16S and 28s ribosomal RNA) gene partitions. The molecular data supported the species status of P. excavatus and P. alatus and also clearly resolved two divergent clades within hermit crabs from the Northeast Atlantic Ocean and the Mediterranean Sea., Conclusions/significance: Despite the abundance and prominent ecological role of hermit crabs, Pagurus, in North East Atlantic Ocean and Mediterranean Sea ecosystems, many important aspects of their taxonomy, biology, systematics and evolution remain poorly explored. The topologies presented here should be regarded as hypotheses that can be incorporated into the robust and integrated understanding of the systematic relationships within and between species of the genus Pagurus inhabiting the Northeast Atlantic Ocean and the Mediterranean Sea.

The diversity on coral reefs has long captivated observers. We examine the mechanisms of speciation, role of ecology in speciation, and patterns of species distribution in a typical reef-associated clade-the diverse and colorful Calcinus hermit crabs-to address the origin of tropical marine diversity. We sequenced COI, 16S, and H3 gene regions for approximately 90% of 56 putative species, including nine undescribed, "cryptic" taxa, and mapped their distributions. Speciation in Calcinus is largely peripatric at remote locations. Allopatric species pairs are younger than sympatric ones, and molecular clock analyses suggest that >2 million years are needed for secondary sympatry. Substantial niche conservatism is evident within clades, as well as a few major ecological shifts between sister species. Color patterns follow species boundaries and evolve rapidly, suggesting a role in species recognition. Most species prefer and several are restricted to oceanic areas, suggesting great dispersal abilities and giving rise to an ocean-centric diversity pattern. Calcinus diversity patterns are atypical in that the diversity peaks in the west-central oceanic Pacific rather than in the Indo-Malayan "diversity center." Calcinus speciation patterns do not match well-worn models put forth to explain the origin of Indo-West Pacific diversity, but underscore the complexity of marine diversification.

Mutations or environmental factors that result in reversal of conspicuous left-right asymmetries provide an opportunity to study developmental mechanisms. They may also provide insight into evolutionary changes in asymmetry states within and between species. King crabs (family Lithodidae) have a larger right claw and females typically exhibit a dextrally offset abdomen. Nevertheless, I observed a high incidence of left handedness in laboratory reared box crabs (Lopholithodes foraminatus) and captured the first known egg-bearing female lithodid to exhibit reversed asymmetry. This provided a unique opportunity to characterize the reversed phenotype and to compare the incidence of reversed asymmetry in the offspring of normal and reversed females. Asymmetry of the chelae became apparent in the first postzoeal stage (glaucothoe) and handedness was maintained through subsequent instars. Females with larger left claws developed reversed abdominal asymmetry by the fourth crab stage. No reversed asymmetry was observed in the mandibles of zoea larvae or juveniles of either handedness. The incidence of reversed asymmetry in glaucothoe reared from one reversed and three normal females was high (between 20% and 30%), and independent of maternity (P=0.67). Removal of the right cheliped of fourth stage zoeae, and the major cheliped of glaucothoe, did not reverse the direction of asymmetry. Elevated larval rearing temperature also did not affect the frequency of reversed individuals. This lack of evidence for either heritability or induction of handedness is enigmatic. Further investigation of reversed asymmetry in lithodid crabs may provide valuable insights into the development and evolution of bilateral asymmetries.

Central predictions of climate warming models include increased climate variability and increased severity of heat waves. Physiological acclimatization in populations across large-scale ecological gradients in habitat temperature fluctuation is an important factor to consider in detecting responses to climate change related increases in thermal fluctuation. We measured in vivo cardiac thermal maxima and used microarrays to profile transcriptome heat and cold stress responses in cardiac tissue of intertidal zone porcelain crabs across biogeographic and seasonal gradients in habitat temperature fluctuation. We observed acclimatization dependent induction of heat shock proteins, as well as unknown genes with heat shock protein-like expression profiles. Thermal acclimatization had the largest effect on heat stress responses of extensin-like, beta tubulin, and unknown genes. For these genes, crabs acclimatized to thermally variable sites had higher constitutive expression than specimens from low variability sites, but heat stress dramatically induced expression in specimens from low variability sites and repressed expression in specimens from highly variable sites. Our application of ecological transcriptomics has yielded new biomarkers that may represent sensitive indicators of acclimatization to habitat temperature fluctuation. Our study also has identified novel genes whose further description may yield novel understanding of cellular responses to thermal acclimatization or thermal stress.

We assessed the conservation priority of 18 freshwater ecoregions in southern South America on the basis of Aegla (genus of freshwater crabs) genetic diversity and distribution. Geographical distributions for 66 Aegla species were taken from the literature and plotted against ecoregions and main river basins of southern South America. Species richness and number of threatened and endemic species were calculated for each area. To assess taxonomic and phylogenetic diversity, we generated a molecular phylogeny based on DNA sequences for one nuclear (28S) and 4 mitochondrial (12S, 16S, COI, and COII) genes. All species richness and phylogenetic methods agreed, to a large extent, in their rankings of the importance of conservation areas, as indicated by the Spearman's rank correlation coefficient (p < 0.01); nonetheless, some of the lowest correlations were observed between taxonomic and phylogenetic diversity indices. The 5 ecoregions of the Laguna dos Patos Basin (Eastern Brazil), Central Chile, South Brazilian Coast, Chilean Lakes, and Subtropical Potamic Axis (northern Argentina and southern Uruguay and Paraguay) had the highest biodiversity scores. Conservation of these regions will preserve the largest number of species and the greatest amount of genetic diversity within the South American freshwater Aegla fauna. Biodiversity across rivers and within areas was heterogeneously distributed in the ecoregions of Upper Paraná, Ribeira do Iguape, Upper Uruguay, and South Brazilian Coast (i.e., one river showed significantly more biodiversity than any other river from the same ecoregion), but homogeneously distributed in the other ecoregions. Hence, conservation plans in the former regions will potentially require less effort than plans in the latter regions., (©2009 Society for Conservation Biology.)

Crustins are distributed across the decapods and are believed to play a significant part in the humoral defense system of their host. In this study, two crustin isoforms from Hyas araneus hemocytes were purified and tested for antimicrobial activity against selected microorganisms. They show both antibacterial and antifungal activity, with highest activity against the Gram-positive bacteria Corynebacterium glutamicum. Sequencing of the transcripts showed them to have a mature peptide of 90 amino acids and differing in three positions in the mature peptide. They were named CruHa1 and CruHa2. Real-time RT-PCR revealed that they mainly are expressed in hemocytes. Screening a cDNA library detected a crustin sequence in Paralithodes camtschaticus hemocytes, coding for a mature peptide of 98 amino acids. It was named CruPc. Based on phylogenetic inference and primary structure, CruHa1 and CruHa2 were placed within the Type I group of crustins, while CruPc belongs to the Type II.

A novel DSN-depletion method allows elimination of selected sequences from full-length-enriched cDNA libraries. Depleted cDNA can be applied for subsequent EST sequencing, expression cloning, and functional screening approaches. The method employs specific features of the kamchatka crab duplex-specific nuclease (DSN). This thermostable enzyme is specific for double-stranded (ds) DNA, and is thus used for selective degradation of ds DNA in complex nucleic acids. DSN depletion is performed prior to library cloning, and includes the following steps: target cDNA is mixed with excess driver DNA (representing fragments of the genes to be eliminated), denatured, and allowed to hybridize. During hybridization, driver molecules form hybrids with the target sequences, leading to their removal from the ss DNA fraction. Next, the ds DNA fraction is hydrolyzed by DSN, and the ss fraction is amplified using long-distance PCR. DSN depletion has been tested in model experiments.