Regulation of early spermatogenesis in the giant prawn Macrobrachium rosenbergii by a GCL homolog†.

The germ cell-less gene is crucial for gonad development in various organisms. Early interventions in its expression suggested a regulatory role at the mitotic stages of spermatogenesis, and its early knockout resulted in complete sterility in Drosophila. Genomic and transcriptomic data available for the catadromous giant prawn Macrobrachium rosenbergii enabled the identification of a germ cell-less homolog for this species, which we termed MroGCL (mRNA accession number OQ533056). An open reading frame containing 494 amino acids and a typical evolutionarily conserved BTB/POZ domain suggests possible protein-protein interaction functions in keeping with the Drosophila germ cell-less protein. Genomic mapping of MroGCL showed a full length of 120 896 bases. Analysis of the temporal expression of MroGCL showed constant expression in early prawn embryonic and larval stages, but a significant increase 10 days after metamorphosis when crucial sexual differentiation processes occur in prawns. In adult animals, high expression was detected in the gonads compared to the somatic tissues. RNAi-based knock-down experiments showed that both the silenced and control groups reached advanced spermatogenic stages, but that there was a significant decrease in the yield of spermatozoa in about half of the silenced animals. This finding supports our hypothesis that MroGCL is crucial for mitosis during early stage spermatogenesis. In conclusion, this study contributes to the understanding of crustacean gonad development and provides a stepping stone in the development of environmentally valuable sterile crustacean populations.

Molecular Phylogeny of Thoracotreme Crabs Including Nine Newly Determined Mitochondrial Genomes.

Mitochondrial genomes are used widely for the molecular phylogenetic analysis of animals. Although phylogenetic analyses based on the mitogenomes of brachyurans often yield well-resolved phylogenies, most interfamilial phylogenetic relationships in Thoracotremata remain unclear. We determined nine new mitogenomes of Thoracotremata, including mitogenomes of Camptandriidae (Deiratonotus japonicus), Dotillidae (Ilyoplax integra, Ilyoplax pusilla, and Tmethypocoelis choreutes), Macrophthalmidae (Ilyograpsus nodulosus), Pinnotheridae (Arcotheres sp. and Indopinnixa haematosticta), Plagusiidae (Guinusia dentipes), and Percnidae (Percnon planissimum). Interestingly, Percnon planissimum (Percnidae) was found to possess ≥ 19 repeated sequences in the control region. The gene orders of Il. nodulosus, Arcotheres sp., and In. haematosticta were revealed to be unique among thoracotreme crabs. Although the results of Bayesian and maximum likelihood (ML) phylogenetic analyses of three datasets were incongruent, highly supported clades (PP ≥ 0.99 or BS ≥ 99%) were not contradictory among the analyses. All analyses suggested the paraphyly of Grapsoidea and Ocypodoidea, corroborating the findings of previous studies based on molecular phylogenies of thoracotreme crabs. The phylogenetic positions of symbiotic thoracotreme crabs, Pinnotheridae and Cryptochiridae, were highly supported (Pinnotheridae + Ocypodidae and Cryptochiridae + Grapsidae, respectively) for the Bayesian analyses but not for the ML analyses. Analyses of more thoracotreme species' mitogenome sequences in additional studies will further strengthen the framework for thoracotreme evolution.

Vibrio harveyi in a Caribbean spiny lobster (Panulirus argus) with hepatopancreas necrosis.

A Caribbean spiny lobster, Panulirus argus, was submitted for necropsy after a number of species-specific fatalities in a public aquarium. The hemolymph was opaque and did not clot and the hepatopancreas had multiple foci of necrosis centered on gram-negative bacteria. Pure cultures of Vibrio harveyi were isolated, identified initially by matrix laser desorption ionization-time of flight mass spectrometry, and confirmed by multilocus sequencing of the gyrB, recA, rpoA, and pyrH genes. As Caribbean spiny lobsters continue to be used for consumption and displayed in public aquariums, chronicling potential pathogens is warranted to inform differential diagnoses and to develop management strategies to reduce incidence of infectious disease in captive populations.

Microsporidia Ameson earli sp. n. and A. michaelis (Sprague 1965) infecting blue crabs Callinectes sapidus from shedding facilities in Louisiana.

During a survey for pathogens and commensals of blue crabs in commercial soft shell shedding facilities in Louisiana, we discovered an occurrence of microsporidiosis in two of forty examined crabs. Judging from spore shape and size, tissue tropism and external signs of muscle pathology, the causative agent of infections was identified as Ameson michaelis, a muscle-infecting species that has been repeatedly detected in populations of Callinectes sapidus in Louisiana since 1965. However, retrospective ultrastructural examination revealed that in one of Ameson-infected crabs, infection was caused by a parasite with ultrastructural characters not completely compliant with the ones of A. michaelis. The major difference was the absence of microtubule-like appendages attached to the exospore, typical of A. michaelis and other Ameson spp. SSUrDNA-inferred pairwise evolutionary distances between the novel species and other Ameson spp. ranged from 0.006 to 0.051; it was 0.039 in the case of A. michaelis. Hence, we describe here a new species in the genus Ameson, and name it after Prof. Earl Weidner, our colleague and friend, an outstanding microsporidiologist and the author of pioneer papers on the ultrastructure and physiology of A. michaelis.

A comprehensive molecular phylogeny of the brachyuran crab superfamily Xanthoidea provides novel insights into its systematics and evolutionary history.

Maximum likelihood and Bayesian phylogenies for the brachyuran crab superfamily Xanthoidea were estimated based on three mitochondrial and four nuclear genes to infer phylogenetic relationships and inform taxonomy. Habitat data was then used in conjunction with several diversification rates analyses (BAMM, BiSSE, HiSSE, and FiSSE) to test evolutionary hypotheses regarding the diversification of xanthoid crabs. The phylogenies presented are the most comprehensive to date in terms of global diversity as they include all four constituent families (Xanthidae, Panopeidae, Pseudorhombilidae, and Linnaeoxanthidae) spanning all oceans in which xanthoid crabs occur. Six Xanthoidea families are recognised. Panopeidae and Xanthidae sensu stricto are the two largest family-level clades, which are reciprocally monophyletic. Pseudorhombilidae is nested within and is here treated as a subfamily of Panopeidae. Former subfamilies or tribes of Xanthidae sensu lato are basally positioned clades in Xanthoidea and are here assigned family-level ranks: Garthiellidae, Linnaeoxanthidae, Antrocarcinidae, and Nanocassiopidae. The genera Linnaeoxantho and Melybia were recovered in separate clades with Linnaeoxantho being sister to the family Antrocarcinidae, while Melybia was recovered within the family Panopeidae. The existing subfamily classification of Xanthidae and Panopeidae is drastically restructured with 20 xanthid and four panopeid subfamilies provisionally recognised. Diversification-time analyses inferred the origin of Xanthoidea and Garthiellidae in the Eocene, while the other families originated during the Oligocene. The majority of genus- and species-level diversification took place during the Miocene. Ancestral state reconstruction based on depth of occurrence (shallow vs. deep water) shows some ambiguity for the most recent common ancestor of Xanthoidea and Nanocassiopidae. The most recent common ancestors of Antrocarcinidae and Panopeidae were likely deep-water species, while those of Garthiellidae and Xanthidae were probably shallow-water species. Several shifts in net diversification rates were detected but they were not associated with depth-related habitat transitions.

Patterns of genome size variation in caridean shrimps: new estimates for non-gambarelloides Synalpheus species.

Genome size (GS) or DNA nuclear content is considered a useful index for making inferences about evolutionary models and life history in animals, including taxonomic, biogeographical, and ecological scenarios. However, patterns of GS variation and their causes in crustaceans are still poorly understood. This study aimed to describe the GS of five Neotropical Synalpheus non-gambarelloides shrimps (S. apioceros, S. minus, S. brevicarpus, S. fritzmueller, and S. scaphoceris) and compare the C-values of all Caridea infraorder in terms of geography and phylogenetics. All animals were sampled in the coast of São Paulo State, Brazil, and GS was assessed by flow cytometry analysis (FCA). The C-values ranged from 7.89 pg in S. apioceros to 12.24 pg in S. scaphoceris. Caridean shrimps had higher GS than other Decapoda crustaceans. The results reveal a tendency of obtaining larger genomes in species with direct development in Synalpheus shrimps. In addition, a tendency of positive biogeographical (latitudinal) correlation with Caridea infraorder was also observed. This study provides remarkable and new protocol for FCA (using gating strategy for the analysis), which led to the discovery of new information regarding GS of caridean shrimps, especially for Neotropical Synalpheus, which represents the second-largest group in the Caridea infraorder.

Sacculina carcini impact on energy content of the shore crab Carcinus maenas L.

The impact of Sacculina carcini infection on the nutritional status of the shore crab Carcinus maenas was investigated in the western Dutch Wadden Sea for a period of 20 months. About 3.3% of the population was sacculinized, i.e. externally infected with S. carcini and only 0.7% presented scars of previous infection. The results of mixed linear models showed that sacculinized and non-sacculinized crabs had similar morphometric condition, while the energy density of parasitized crabs (externa excluded) was significantly reduced by about 4.3% overall, and by up to 5.8% in crabs under 40 mm carapace width. However, when Sacculina externa was included in the energy determinations, the difference in energy density decreased to 1.2%, while total energy content of the pair infected crab-parasite including externa was 30.8% higher than non-sacculinized crabs of similar size. The total energy content of ovigerous females (eggs included) was even higher, near doubling the energy of similar-sized crabs. The same way, total energy content of Sacculina externa was about 4 times lower than total energy of egg mass. The results suggest that the rhizocephalan parasite is efficient in consuming the energy that the host may allocate for growth and maintenance, but require future studies to disentangle the impact of the degree of internal infection and the implications for the dynamics of the population.

Haplosporidium acetes n. sp. infecting the hepatopancreas of jelly prawns Acetes sibogae australis from Moreton Bay, Australia.

Wild Acetes sibogae australis from northern Moreton Bay, Australia displaying opacity of the hepatopancreas were sampled and examined histologically, revealing infection by multinucleate plasmodia of a haplosporidian-like parasite in the epithelial cells of the hepatopancreas. A morphological and phylogenetic investigation identified the parasite as a novel species of the order Haplosporida, and the parasite is described as Haplosporidium acetes n. sp. This is the first report of disease caused by a haplosporidian in wild Australian decapod crustaceans, and the first record of haplosporidiosis in sergestid shrimp. Infections of H. acetes were observed in all cell types (R, B, F and E) within the hepatopancreas. Infected epithelial cells became hypertrophied as they filled with haplosporidian parasites and, in heavy infections, caused almost complete displacement of normal hepatopancreas tissue. Although sporulation was not observed, infected jelly prawns appeared terminally diseased. Infections became grossly evident in around 5% of wild prawns during early autumn at a time of year when jelly prawn populations decline rapidly with decreasing water temperatures, however histopathology indicated at least 13% of apparently normal jelly prawns were also infected. Further studies are required in order to determine if this parasite influences jelly prawn population dynamics. In addition, we report co-infection of a novel microsporidian parasite in the Enterocytozoon Group Microsporidia (EGM) infecting nuclei of hepatopancreatic epithelial cells. The microsporidian was phylogenetically distinct from Enterocytozoon hepatopenaei (EHP) known to infect penaeid shrimp in Asia.

The pet trade as a source of non-native decapods: the case of crayfish and shrimps in a thermal waterbody in Hungary.

Ornamental aquaculture and the related pet industry are known to be important sources of non-native species worldwide. In the temperate zone, thermal waterbodies are attractive places for irresponsible owners to release unwanted freshwater pets including decapod crustaceans. Several non-native ornamental species have been reported in the thermal locality of Miskolctapolca (a suburb of Miskolc, Hungary). So we surveyed this site in March 2019-November 2021 to update local occurrence records and detect potentially newly released species. A well-established population of Neocaridina denticulata and the occurrence of Caridina cf. babaulti had previously been noted. However, for the first time at this site, we found the shrimps Atyopsis moluccensis, Caridina gracilirostris and C. multidentata, as well as the crayfish Procambarus virginalis, P. clarkii, Cherax quadricarinatus, C. boesemani and C. snowden, and several formally undescribed Cherax species originating from New Guinea. Furthermore, in most species, gravid females carrying eggs were also noticed. Three shrimps, A. moluccensis, C. gracilirostris and C. multidentata, were recorded for the first time in European wild. Further monitoring of this locality and better education of the general public regarding the risks associated with the release of non-native species are strongly recommended.

Dietary preferences of brachyuran crabs from Taiwan for marine or terrestrial food sources: evidence based on fatty acid trophic markers.

BACKGROUND: Trophic interactions are key processes, which determine the ecological function and performance of organisms. Many decapod crustaceans feed on plant material as a source for essential nutrients, e.g. polyunsaturated fatty acids. Strictly herbivorous feeding appears only occasionally in marine decapods but is common in land crabs. To verify food preferences and to establish trophic markers, we studied the lipid and fatty acid composition of the midgut glands of two marine crab species (Grapsus albolineatus and Percnon affine), one semi-terrestrial species (Orisarma intermedium, formerly Sesarmops intermedius), and one terrestrial species (Geothelphusa albogilva) from Taiwan. RESULTS: All species showed a wide span of total lipid levels ranging from 4 to 42% of the dry mass (%DM) in the marine P. affine and from 3 to 25%DM in the terrestrial G. albogilva. Triacylglycerols (TAG) were the major storage lipid compound. The fatty acids 16:0, 18:1(n-9), and 20:4(n-6) prevailed in all species. Essential fatty acids such as 20:4(n-6) originated from the diet. Terrestrial species also showed relatively high amounts of 18:2(n-6), which is a trophic marker for vascular plants. The fatty acid compositions of the four species allow to clearly distinguish between marine and terrestrial herbivorous feeding due to significantly different amounts of 16:0, 18:1(n-9), and 18:2(n-6). CONCLUSIONS: Based on the fatty acid composition, marine/terrestrial herbivory indices were defined and compared with regard to their resolution and differentiating capacity. These indices can help to reveal trophic preferences of unexplored species, particularly in habitats of border regions like mangrove intertidal flats and estuaries.

Gut Microbiota in Decapod Shrimps: Evidence of Phylosymbiosis.

Gut microbiota have long attracted the interest of scientists due to their profound impact on the well-being of animals. A non-random pattern of microbial assembly that results in a parallelism between host phylogeny and microbial similarity is described as phylosymbiosis. Phylosymbiosis has been consistently observed in different clades of animal hosts, but there have been no studies on crustaceans. In this study, we investigated whether host phylogeny has an impact on the gut microbiota assemblages in decapod shrimps. We examined the gut microbial communities in 20 shrimp species from three families inhabiting distinct environments, using metabarcoding analyses of the V1-V3 hypervariable region of the 16S rRNA gene. Gut microbial communities varied within each shrimp group but were generally dominated by Proteobacteria. A prevalent phylosymbiotic pattern in shrimps was evidenced for the first time by the observations of (1) the distinguishability of microbial communities among species within each group, (2) a significantly lower intraspecific than interspecific gut microbial beta diversity across shrimp groups, (3) topological congruence between host phylogenetic trees and gut microbiota dendrograms, and (4) a correlation between host genetic distances and microbial dissimilarities. Consistent signals of phylosymbiosis were observed across all groups in dendrograms based on the unweighted UniFrac distances at 99% operational taxonomic units (OTUs) level and in Mantel tests based on the weighted UniFrac distances based on 97% OTUs and amplicon sequence variants. Penaeids exhibited phylosymbiosis in most tests, while phylosymbiotic signals in atyids and pandalids were only detected in fewer than half of the tests. A weak phylogenetic signal was detected in the predicted functions of the penaeid gut microbiota. However, the functional diversities of the two caridean groups were not significantly related to host phylogeny. Our observations of a parallelism in the taxonomy of the gut microbiota with host phylogeny for all shrimp groups examined and in the predicted functions for the penaeid shrimps indicate a tight host-microbial relationship during evolution.

Comparative study of the hemolymph microbiome between live and recently dead American lobsters Homarus americanus.

Lobsters and other crustaceans do not have sterile hemolymph. Despite this, little is known about the microbiome in the hemolymph of the lobster Homarus americanus. The purpose of this study was to characterize the hemolymph microbiome in lobsters. The lobsters were part of a larger study on the effect of temperature on epizootic shell disease, and several died during the course of the study, providing an opportunity to examine differences in the microbiomes between live and recently dead (1-24 h) animals. The hemolymph microbiomes of live lobsters was different from those in dead animals and both were different from the tank microbiome in which the animals had been held. The microbiomes of live lobsters were more diverse and had a different suite of bacteria than those from dead animals. The dominant taxa in live lobsters belonged to Flavobacteriaceae and Rhodobacteraceae, whereas Vibrionaceae and Enterobacteriaceae were predominant in the dead lobsters. Although aquarium microbiomes overlapped with the hemolymph microbiomes, there was less overlap and lower abundance of taxa in comparison with hemolymph from live lobsters. Previous studies reporting bacteria in the digestive tract of lobsters suggested that Vibrionaceae and Enterobacteriaceae had invaded the hemolymph via the gut. Our study suggests that hemolymph bacteria abundant in live lobsters do not originate from the tank milieu and comprise a rich, natural, or native background of bacterial constituents.

Cytopathology and immune response in the hepatopancreas of decapod crustaceans.

The hepatopancreas of decapod crustaceans is used as an example to illustrate the range of cytopathologies, detoxification mechanisms, and immune responses that environmental toxicants and pathogens can induce in a single organ. The hepatopancreas is the central metabolic organ of decapods and consists of hundreds of blindly-ending tubules and intertubular spaces. The tubular epithelium contains 5 structurally and functionally different cell types, and the interstitium contains haemolymph, haemocytes, connective tissue, and fixed phagocytes. Some physiological conditions such as moulting and starvation cause marked but reversible ultrastructural alterations of the epithelial cells. Environmental toxicants induce either detoxification mechanisms or structural damage in cells, depending on toxicant and concentration. The hepatopancreas is also a main target organ for pathogens, mainly viruses, bacteria, and protists that enter the body via the digestive tract and gills and replicate in the hepatopancreatocytes. The cytopathologies caused by toxicants and pathogens affect single cell types specifically or, more often, several cell types simultaneously. Pathogenesis often begins in a certain cell organelle such as the nucleus, mitochondrion, or endoplasmic reticulum, spreads to other organelles, and ends with death of the infected cell. Fixed phagocytes in the interstitium capture and degrade pathogens that move from the infected tubules into the intertubular spaces or enter the hepatopancreas via circulation. Relatively few disease agents elicit the melanisation and encapsulation reaction that encloses infected tubules by a rigid melanised capsule and kills the entrapped pathogens.

A phylogenomic framework, evolutionary timeline and genomic resources for comparative studies of decapod crustaceans.

Comprising over 15 000 living species, decapods (crabs, shrimp and lobsters) are the most instantly recognizable crustaceans, representing a considerable global food source. Although decapod systematics have received much study, limitations of morphological and Sanger sequence data have yet to produce a consensus for higher-level relationships. Here, we introduce a new anchored hybrid enrichment kit for decapod phylogenetics designed from genomic and transcriptomic sequences that we used to capture new high-throughput sequence data from 94 species, including 58 of 179 extant decapod families, and 11 of 12 major lineages. The enrichment kit yields 410 loci (greater than 86 000 bp) conserved across all lineages of Decapoda, more clade-specific molecular data than any prior study. Phylogenomic analyses recover a robust decapod tree of life strongly supporting the monophyly of all infraorders, and monophyly of each of the reptant, 'lobster' and 'crab' groups, with some results supporting pleocyemate monophyly. We show that crown decapods diverged in the Late Ordovician and most crown lineages diverged in the Triassic-Jurassic, highlighting a cryptic Palaeozoic history, and post-extinction diversification. New insights into decapod relationships provide a phylogenomic window into morphology and behaviour, and a basis to rapidly and cheaply expand sampling in this economically and ecologically significant invertebrate clade.

Structure, function and development of the digestive system in malacostracan crustaceans and adaptation to different lifestyles.

The digestive system of the malacostracan crustaceans, namely the decapods, isopods, amphipods and mysids, is among the most complex organ systems of the animal kingdom serving multiple functions such as food processing, absorption and storage of nutrients, synthesis of digestive enzymes and blood proteins, detoxification of xenobiotics and osmoregulation. It is rather well investigated compared to other invertebrates because the Malacostraca include many ecological keystone species and food items for humans. The Decapoda and Peracarida share food processing with chewing and filtering structures of the stomach but differ with respect to morphology and ultrastructure of the digestive glands. In the Peracarida, the digestive glands are composed of few, relatively large lateral caeca, whereas in the Decapoda, hundreds to thousands of blindly ending tubules form a voluminous hepatopancreas. Morphogenesis and onset of functionality of the digestive system strongly depend on the mode of development. The digestive system is early developed in species with feeding planktonic larvae and appears late in species with direct lecithotrophic development. Some structures of the digestive system like the stomach ossicles are rather constant in higher taxa and are of taxonomic value, whereas others like the chewing structures are to some degree adapted to the feeding strategy. The nutrient absorbing and storing cells of the digestive glands show considerable ultrastructural variation during moult cycle, vitellogenesis and starvation. Some of the various functions of the digestive system are already assigned to specific sections of the digestive tract and cell types, but others still await precise localization.

Phylogenomic analyses of brachyuran crabs support early divergence of primary freshwater crabs.

Recent fossil calibrated molecular phylogenies have revealed that the Brachyura underwent rapid radiation during the Cretaceous and Early Tertiary, resulting in many early diverging lineages separated by short internodes that remain difficult to resolve. Here we present the first phylogenomic analyses of Brachyura using transcriptome data from 30 brachyuran species and 22 families. Analyses were carried out on a dataset containing 372 putative homologous loci (246,590 bps) and included data from 21 newly generated transcriptomes. With minor exceptions, all phylogenetic analyses recovered a congruent, highly resolved and well supported brachyuran phylogeny. Consistent with previous work, this phylogeny suggests that primary freshwater crabs diverged early in brachyuran evolution, falling sister to Thoracotremata, thus supporting recent proposal for establishment of subsection Potamoida for primary freshwater crabs. The interfamilial relationships among heterotremes were well resolved in our analyses but those within Thoracotremata remained problematic. Phylogenomic analyses clearly provide a powerful means for resolving brachyuran relationships, but future studies would benefit greatly from increased taxon sampling of transcriptome data.

Isolation and characterisation of 14 novel microsatellite markers through Next Generation Sequencing for the commercial Atlantic seabob shrimp Xiphopenaeus kroyeri.

Assessing population genetic structure is a crucial step to support fisheries and conservation management. DNA microsatellite molecular markers are a widely used tool in population genotyping. In the present study, we characterised and developed 14 novel polymorphic microsatellite markers for a decapod crustacean, the Atlantic seabob shrimp Xiphopenaeus kroyeri (Heller, 1862), through rapid and cost-effective Illumina shotgun sequencing and a Galaxy-based bioinformatic pipeline. We genotyped 60 individuals from 2 populations with the newly developed microsatellites, resulting in the detection of 3 to 29 alleles per locus. Four loci deviated from Hardy-Weinberg equilibrium. Cross-amplification in a cryptic congeneric species was successful for eight loci (57%). The microsatellite loci developed in this study will be highly relevant for genetic and evolutionary studies of X. kroyeri, and for the stock management of this commercially exploited species.

Osmo-ionic regulation and carbonic anhydrase, Na+/K+-ATPase and V-H+-ATPase activities in gills of the ancient freshwater crustacean Aegla schmitti (Anomura) exposed to high salinities.

Aeglidae anomuran crabs originated in the sea, but invaded and diversified in southern South American freshwater (FW) streams. We here aimed at examining their tolerance of increased salinity, after a long time of evolution in FW (~33 million years). Aegla schmitti were exposed to FW and dilute seawater of salinities 15, 20, and 25‰ for 1, 5 and 10 days. Mortality in 35‰ was also assessed. Hemolymph osmolality, Na+, K+, Cl-, and Mg2+ ions, and hydration levels of the abdominal muscle were assayed. The activities of the Carbonic Anhydrase (CA), Na+/K+-ATPase (NKA) and V-H+-ATPase (VHA) were also assayed in the gills. A. schmitti preserves osmoregulatory mechanisms of its marine ancestors. It is able to survive in high salinities (25‰) for at least 10 days. Mortality in 35‰ was of 56% after 1 day, and of 100% after 7 days. In 25‰, NaCl is apparently hyporegulated at all times, while hemolymph osmolality rises after 5 days. CA and NKA activities remained unchanged in all experimental conditions, while VHA activity decreased after 10 days in 25‰. Hemolymph NaCl data was compatible with either hyporegulation and/or putative influx of NaCl into cells for regulatory volume increase (RVI). Further studies should deepen the understanding of the roles of low permeabilities and saturation of high affinity uptake systems in truly FW decapods, in their responses to high salinities. Moreover, the fate of extracellular NaCl as secretion in true hypo-regulation and/or influx into cells for RVI should also be investigated.

ORDER within the chaos: Insights into phylogenetic relationships within the Anomura (Crustacea: Decapoda) from mitochondrial sequences and gene order rearrangements.

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.

Molecular phylogeny of ten intertidal hermit crabs of the genus Pagurus inferred from multiple mitochondrial genes, with special emphasis on the evolutionary relationship of Pagurus lanuginosus and Pagurus maculosus.

The hermit crab genus Pagurus exhibits high species diversity and a wide geological distribution. Despite the high species diversity of hermit crabs in the western Pacific coast of Japan, molecular phylogenetic studies of these species have yet to be conducted. To investigate their molecular phylogeny and genetic diversity, we obtained nearly complete mitochondrial genome sequences for ten Pagurus species found along the Pacific coast of Japan by next-generation sequencing, which were compared to other congeners deposited in the GenBank database. The genomes ranged from 13,458 to 16,401 base pairs in length, possessing 13 protein-coding genes, 2 rRNAs, and 22 tRNA genes. Based on the reconstructed phylogeny, we found that (1) Japanese Pagurus species separated into three groups, nested within the Northern Pacific species. (2) Pagurus lanuginosus and Pagurus maculosus, showed high morphological similarities, implying close kinship. Indeed, these two species were genetically closest to each other, compared to the remaining species studied. (3) An unspecified specimen sampled from the deep sea, which morphologically resembled Pagurus, might be a member of the Pagurus genus, but is genetically distant from the other Japanese Pagurus species. The novel data reported here may provide new perspectives for systematic studies of hermit crabs; these results provide important information that will facilitate population-level research and identifying intraspecific variation of these non-model, but ecologically important, decapod species.