Characterization and ecological risk assessment of microplastics accumulated in sea water, sand, sediment, shell water and selected tissues of hermit crab of Sundarban Biosphere Reserve.

Sundarban, a Ramsar site of India, has been encountering an ecological threat due to the presence of microplastic (MP) wastes generated from different anthropogenic sources. Clibanarius longitarsus, an intertidal hermit crab of Sundarban Biosphere Reserve, resides within the abandoned shell of a gastropod mollusc, Telescopium telescopium. We characterized and estimated the MP in the gills and gut of hermit crab, as well as in the water present in its occupied gastropod shell. The average microplastic abundance in sea water, sand and sediment were 0.175 ± 0.145 MP L-1, 42 ± 15.03 MP kg-1 and 67.63 ± 24.13 MP kg-1 respectively. The average microplastic load in hermit crab was 1.94 ± 0.59 MP crab-1, with 33.89 % and 66.11 % in gills and gut respectively. Gastropod shell water exhibited accumulation of 1.69 ± 1.43 MP L-1. Transparent and fibrous microplastics were documented as the dominant polymers of water, sand and sediment. Shell water exhibited the prevalence of green microplastics followed by transparent ones. Microscopic examination revealed microplastics with 100-300 µm size categories were dominant across all abiotic compartments. ATR-FTIR and Raman spectroscopy confirmed polyethylene and polypropylene as the prevalent polymers among the five identified polymers of biotic and abiotic components. The target group index indicated green and black as the preferable microplastics of crab. The ecological risk analysis indicated a considerable level of environmental pollution risk in Sundarban and its inhabiting organisms. This important information base may facilitate in developing a strategy of mitigation to limit the MP induced ecological risk at Sundarban Biosphere Reserve.

Review of the concept of Profilicollis Meyer, 1931 with a description of Profilicollis rancoensis n. sp. (Acanthocephala: Polymorphidae) from the freshwater crab, Aegla abtao Schmitt, 1942 (Decapoda: Anomura) in Chile, with a key to congeneric species.

Profilicollis rancoensis n. sp. is the tenth species of Profilicollis Meyer, 1931 which includes 9 other species mostly known from marine decapod crabs and shore birds. Cystacanths of P. rancoensis are described from the dominant freshwater crab Aegla abtao in Ranco Lake, Chile and are morphologically distinguished from cystacanths of the 9 other species based on a combination of 4 characters. These are body size, number of proboscis hook rows, number of hooks per row, and length of the largest anterior 2-4 hooks. Male and female cystacanths of P. rancoensis are 2.10-3.33 mm long having an ovoid proboscis with 14 rows of 6-7 hooks per row, with the largest anterior 2-4 hooks being 105-110 micrometers long; the anterior trunk has many small spines in 70-80 concentric rings, each with 50-60 spines around them; hook roots are simple, directed posteriorly, about as long as the blades anteriorly with unremarkable anterior manubria; the cephalic ganglion are in mid-receptacle just anterior to the level of the anterior trunk; the lemnisci are long and slender; the testes are in the anterior trunk, posterior trunk, or one in each; the primordia of 2 tubular cement glands are evident; strong bundles of fibers link the anterior and posterior trunk; and the posterior trunk has a corrugated surface cuticula. Molecular analysis (COI and 18S) sequences coincided with the morphology and support its taxonomy. The phylogenetic profile revealed that P. rancoensis n. sp. fell into the Profilicollis clade. Both sequences showed low genetic variation, and three different haplotypes were found. The new species was more closely related to P. botulus (Van Cleave, 1916) Witenberg, 1932 than to other Profilicollis species.

Title: Révision du concept de Profilicollis Meyer, 1931 avec la description de Profilicollis rancoensis n. sp. (Acanthocephala, Polymorphidae) du crabe d'eau douce Aegla abtao Schmitt, 1942 (Decapoda, Anomura) au Chili, avec une clé des espèces congénères. Abstract: Profilicollis rancoensis n. sp. est la dixième espèce de Profilicollis Meyer, 1931 qui comprend neuf autres espèces principalement connues de crabes décapodes marins et d'oiseaux de rivage. Les cystacanthes de P. rancoensis sont décrits chez le crabe d'eau douce dominant Aegla abtao dans le lac Ranco, au Chili et se distinguent morphologiquement des cystacanthes des neuf autres espèces sur la base d'une combinaison de quatre caractères. Il s'agit de la taille du corps, du nombre de rangées de crochets du proboscis, du nombre de crochets par rangée et de la longueur des 2 à 4 crochets antérieurs les plus grands. Les cystacanthes mâles et femelles de P. rancoensis mesurent de 2,10 à 3,33 mm de long et ont une trompe ovoïde avec 14 rangées de 6 à 7 crochets par rangée, les 2 à 4 crochets antérieurs les plus grands mesurant 105 à 110 micromètres de long ; le tronc antérieur a de nombreuses petites épines en 70-80 anneaux concentriques chacun avec 50-60 épines ; les racines des crochets sont simples, dirigées vers l'arrière, à peu près aussi longues que les lames vers l'avant avec une manubrie antérieure sans particularité ; les ganglions céphaliques sont au milieu du réceptacle juste en avant du niveau du tronc antérieur ; les lemnisques sont longs et minces ; les testicules sont dans le tronc antérieur, le tronc postérieur ou un dans chacun ; les ébauches des 2 glandes cémentaires tubulaires sont évidentes ; de solides faisceaux de fibres relient le tronc antérieur et postérieur ; le tronc postérieur a une cuticule à surface ondulée. Les séquences d'analyse moléculaire (COI et 18S) coïncidaient avec la morphologie et confirmaient sa taxonomie. Le profil phylogénétique a révélé que P. rancoensis n. sp. appartient au clade Profilicollis. Les deux séquences ont montré une faible variation génétique et trois haplotypes différents ont été trouvés. La nouvelle espèce était plus proche de P. botulus (Van Cleave, 1916) Witenberg, 1932 que des autres espèces de Profilicollis.

A study of allergen detection panel in Guangzhou, southern China based on real-world data from the past 7 years.

This study aims to reduce the cost of allergen testing for Guangzhou, China by limiting the number of allergens for which patients are tested, and provide a testing panel to improve diagnostic and therapeutic efficiency. This retrospective study of real-world data from 2012 to 2019 included 39,570 patients with suspected allergies in Guangzhou, southern China. All the patients were tested for one or more of the following allergens serum specific immunoglobulin E (sIgE): Dermatophagoides pteronyssinus, Dermatophagoides farinae, cat dander, dog dander, Artemisia vulgaris, Aspergillus fumigatus, Alternaria alternata, Blattella germanica, egg whites, milk, wheat, peanuts, soybeans, Cancer pagurus, and Penaeus monodon by PhadiaCAP 1000. Totally, only the positive rates of allergens sIgE in D. farinae, D. pteronyssinus, milk, egg whites, B. germanica, C. pagurus, A. alternata, and P. monodon were > 10%, the other allergens were between 4-7%. Moreover, among the allergic diseases, dust mites exhibited the overall highest positive rate, followed by milk and B. germanica. In children, milk was the main allergen, whereas in adults, mites, cockroaches, shrimp, and crab allergens had higher positive rates. The optimal scale analysis shows that the multiple sensitization classification of patients can be divided into three categories: I D. farinae and D. pteronyssinus; II. C. pagurus, P. monodon, and B. germanica; III. Milk and egg whites. Generally, a panel including 4 allergens can detect > 90% of the potential allergy in this local population. In Guangzhou, southern China, D. farinae, milk, B. germanica, and A. alternata as a panel screening allergy for suspected allergic patients was suggested base on this study.

Taxonomic status and phylogenetic relationship of Anomura (Crustacea: Decapoda) based on mitochondrial sequences and gene order rearrangements.

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.

Mitochondrial genomes of the land hermit crab Coenobita clypeatus (Anomura: Paguroidea) and the mole crab Emerita talpoida (Anomura: Hippoidea) with insights into phylogenetic relationships in the Anomura (Crustacea: Decapoda).

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.

Carcinoecium-Forming Sea Anemone Stylobates calcifer sp. nov. (Cnidaria, Actiniaria, Actiniidae) from the Japanese Deep-Sea Floor: A Taxonomical Description with Its Ecological Observations.

Here we describe Stylobates calcifer sp. nov. (Cnidaria, Actiniaria, Actiniidae), a new carcinoecium-forming sea anemone from the deep-sea floor of Japan. Stylobates produces a carcinoecium that thinly covers the snail shells inhabited by host hermit crabs Pagurodofleinia doederleini. The new species is distinct from other species by the shape of the marginal sphincter muscle, the distribution of cnidae, the direction of the oral disk, and host association. The species' novelty is supported by the data of its mitochondrial genes 12S, 16S, and COIII and nuclear genes 18S and 28S. Also, we conducted behavioral observation of this new species, focusing on the feeding behavior and interaction with the specific host hermit crab. Our observations suggest that this sea anemone potentially feeds on the suspended particulate organic matter from the water column or the food residuals of hermit crabs. When the host's shell changed, intensive manipulation for transference of S. calcifer sp. nov. was recorded. However, although the hermit crab detached and transferred the sea anemone to the new shell after shell change, the sea anemone did not exhibit active or cooperative participation. Our data suggest that the sea anemone may not produce a carcinoecium synchronously to its host's growth, contrary to the anecdotal assumption about carcinoecium-forming sea anemones. Conversely, the host hermit crab's growth may not depend entirely on the carcinoecium produced by the sea anemone. This study is perhaps the first observation of the behavioral interaction of the rarely studied carcinoecium-forming mutualism in the deep sea.

A comparative assessment of the morphology of Profilicollis altmani (Acanthocephala, Polymorphidae) from crustaceans and shore birds in Peru, with special notes on hook elemental analysis (EDXA), SEM imaging, histopathology, and molecular profile.

The morphology of cystacanths and adults of Profilicollis altmani (Perry, 1942) Van Cleave, 1947 (Polymorphidae) were studied from the Pacific mole crab Emerita analoga (Stimpson) (Crustacea, Hippidae) and Belcher's gull Larus belcheri (Vigors) (Aves, Laridae), respectively, in Peru. Comparative morphometrics with accounts of other populations of P. altmani from elsewhere off the Pacific and Atlantic coasts of North and South America revealed marked intraspecific population variations. We report scanning electron micrographs (SEM) of new features, not before noted or captured in line drawings by earlier observers. We further present microscope images that reveal internal details not previously reported or possible to see with SEM. Energy dispersive X-ray analysis (EDXA) revealed unusual patterns in the chemistry of proboscis hooks especially the high sulfur and diminished phosphorous and calcium in hook tips and low sulfur and high levels of phosphorous and calcium at mid hooks. The size and shape of all hooks of the cystacanths are reported for the first time. Histopathological studies in L. belcheri from Peru are also included. Cystacanths of P. altmani from California were also analyzed for molecular patterns and compared with other sequences reported from other locations. The molecular data and the analysis of our new sequences of cytochrome oxidase I (COI) showed that haplotypes of P. altmani had low genetic variation; the species is not geographically structured, and within its clade no monophyletic group is formed.

TITLE: Évaluation comparative de la morphologie de Profilicollis altmani (Acanthocephala, Polymorphidae) de crustacés et d'oiseaux de rivage au Pérou, en particulier l'analyse élémentaire des crochet (EDXA), l'imagerie SEM, l'histopathologie et le profil moléculaire. ABSTRACT: La morphologie des cystacanthes et des adultes de Profilicollis altmani (Perry, 1942) Van Cleave, 1947 (Polymorphidae) a été étudiée, respectivement, à partir du crustacé Emerita analoga (Stimpson) (Crustacea, Hippidae) et du Goéland Siméon Larus belcheri (Vigors) (Aves, Laridae), au Pérou. La morphométrie comparative avec des données d'autres populations de P. altmani d'autres localités au large des côtes du Pacifique et de l'Atlantique de l'Amérique du Nord et du Sud a révélé des variations intraspécifiques marquées des populations. Nous rapportons des nouvelles observations de microscopie électronique à balayage, non notées auparavant ou non capturées dans des dessins au trait par des observateurs antérieurs. Nous présentons en outre des images au microscope qui révèlent des détails internes qui n'ont pas été signalés auparavant ni possibles à voir avec MET. L'analyse aux rayons X à dispersion d'énergie (EDXA) a révélé des modèles inhabituels dans la chimie des crochets de la trompe, en particulier la teneur élevée en soufre et une diminution du phosphore et du calcium dans les pointes des crochets et des niveaux faibles en soufre et en phosphore et en calcium au milieu des crochets. La taille et la forme de tous les crochets des cystacanthes sont décrites pour la première fois. Des études histopathologiques sur L. belcheri du Pérou sont également incluses. Les cystacanthes de P. altmani de Californie ont également été analysés pour les modèles moléculaires et comparés à d'autres séquences rapportées d'autres endroits. Les données moléculaires et l'analyse de nos nouvelles séquences de cytochrome oxydase I (COI) ont montré que les haplotypes de P. altmani avaient une faible variation génétique. L'espèce n'est pas structurée géographiquement, et au sein de son clade aucun groupe monophylétique n'est formé.

Multiple metals and agricultural use affects oxidative stress biomarkers in freshwater Aegla crabs / Presença de múltiplos metais e agricultura afetam biomarcadores de estresse oxidativo em caranguejos de água doce (Aegla)

Metals and agrochemicals are among the main aquatic contaminants, being able to trigger oxidative stress in exposed organisms. The objective of this work was to evaluate the correlation between the level of oxidative stress biomarkers in Aegla crabs (Crustacea, Anomura) with (i) the set of metals present in the streams sediment and (ii) with land uses of three hydrographic basins. The study was carried out in streams (≤ 2nd order) of hydrographic basins in southern Brazil (Basins of Rio Suzana, Rio Ligeirinho-Leãozinho and Rio Dourado). In these streams were quantified the land uses and Cu, Cr, Cd, Fe, Mn and Zn concentrations in the sediment. The enzymes Catalase (CAT) and Glutathione Reductase (GR), as well as the level of membrane lipid peroxidation (TBARS), were analyzed in adult females. The PCA analysis showed that the distribution of metals was different between the basins. Cd, Cr and Fe were correlated positively with CAT and negatively with TBARS and GR. The Dourado basin had the lowest concentrations of these three metals and the highest levels of TBARS. However, in Dourado basin there is predominance of agriculture land use, and TBARS was positively correlated with agricultural land use. Besides in Dourado basin, GR activity was higher than in the others basins, indicating a compensatory response in relation to CAT inhibition. The basins of Suzana and Ligeirinho-Leãozinho rivers had lower TBARS values, which may be due to the induction of CAT in response to metals accumulated in sediment. In summary, this work indicates that in the basins with a higher concentration of toxic metals there is an adaptive response of CAT induction, which reduces TBARS in Aegla. On the other hand, in the basin with lower metallic contamination, TBARS occurrence was primarily influenced by agricultural land use.

Os metais e agroquímicos estão entre os principais contaminantes aquáticos, podendo desencadear estresse oxidativo em organismos expostos. O objetivo deste trabalho foi avaliar uma possível correlação entre o nível de biomarcadores de estresse oxidativo em Aegla (Crustacea, Anomura) com (i) o conjunto de metais presentes no sedimento e (ii) com os usos da terra, em três bacias hidrográficas distintas. O estudo foi realizado em riachos (≤ 2ª ordem) de bacias hidrográficas do Sul do Brasil (Bacias do Rio Suzana, do Rio Ligeirinho-Leãozinho e do Rio Dourado), as quais foram caracterizadas em função do percentual de usos da terra e do nível de Cu, Cr, Cd, Fe, Mn e Zn no sedimento. As enzimas Catalase (CAT) e Glutationa Redutase (GR), bem como o nível de peroxidação lipídica das membranas (TBARS), foram analisadas em fêmeas adultas. Uma análise de PCA mostrou que a distribuição de metais foi distinta entre as bacias. Cd, Cr e Fe no sedimento correlacionaram positivamente com a CAT e negativamente com TBARS e GR. Entretanto, a bacia do Dourado apresentou os menores níveis destes três metais e os maiores níveis de TBARS, o que pode ser justificado pelo predomínio da agricultura nesta bacia, já que o TBARS correlacionou positivamente com o percentual de uso agrícola. Nesta bacia, a atividade da GR foi mais alta do que nas outras, indicando uma resposta compensatória em relação a inibição da CAT. As bacias do rio Suzana e rio Ligeirinho-Leãozinho apresentaram valores menores de TBARS, o que pode decorrer da indução da CAT em função dos metais acumulados no sedimento. Em síntese, este trabalho indica que nas bacias com maior concentração de metais tóxicos ocorre uma resposta adaptativa de indução da CAT, o que reduz os níveis de TBARS em Aegla. Por outro lado, na bacia com menor contaminação metálica os níveis de TBARS foram primariamente influenciados pelo uso agrícola.

Molecular phylogenetic analysis of the Paguristes tortugae Schmitt, 1933 complex and selected other Paguroidea (Crustacea: Decapoda: Anomura).

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.

Hemocytic sarcoma of the body wall in a California king crab Paralithodes californiensis.

Neoplasia is rarely reported in decapod crustaceans, and sarcoma has not been previously reported in any crab species. A California king crab Paralithodes californiensis with a recent history of autotomy (4 legs lost) and anorexia was found dead. Grossly, the crab had a pigmented ulcer on the right cheliped merus. Necropsy tissue samples were placed in 10% neutral buffered formalin and processed routinely for histology. Both histochemical (i.e. Brown and Brenn Gram, Fite-Faraco acid fast, Fontana-Masson, Giemsa, hematoxylin and eosin, Masson's trichrome, periodic acid-Schiff [PAS], phosphotungstic acid-hematoxylin, and von Kossa) and immunohistochemical (i.e. cytokeratin, vimentin, and lysozyme) stains were performed. The body wall (presumably of the right cheliped merus) was ulcerated and subtended by a densely cellular, unencapsulated, invasive neoplasm composed of spindle cells arranged in intersecting streams and bundles embedded in a small to moderate amount of fibromatous stroma. Neoplastic cells were oval to elongate with fibrillar, pale eosinophilic cytoplasm that occasionally contained moderate numbers of small, spherical, brightly eosinophilic granules that were highlighted with PAS and Giemsa stains. Neoplastic cells had mild atypia and no evident mitoses. Immunohistochemical stains were noncontributory. This neoplasm is consistent with hemocytic sarcoma of semi-granulocytic origin. Decapod crustaceans have 3 types of hemocytes: hyalinocytes, granulocytes, and semi-granulocytes. Neoplastic cells had PAS- and Giemsa-positive granules, which are present in both semi-granulocytes and granulocytes. Semi-granulocytes can elongate and are associated with deposition of extracellular matrix during some immune responses. Neoplastic cells were elongate and associated with deposition of matrix. These findings suggest neoplastic cells were of semi-granulocytic origin.

Influence of pesticides and abiotic conditions on biochemical biomarkers in Aegla aff. longirostri (crustacea, anomura): Implications for conservation.

Freshwater ecosystems are constantly threatened by the advance of agricultural activities. Abiotic variables (such as temperature, ammonia, and nitrite) and contaminants (e.g. pesticides) can potentially interact, increasing metabolism and the absorption of toxic substances, which can alter the ability of organisms to establish adequate stress responses. This study aimed to verify which pesticides were most frequently found and in the greatest quantities in low-order streams, and whether the combination of these pesticides with the abiotic variables altered the biological metabolism of aeglids. These freshwater crustaceans are important shredders that inhabit low-order streams and are sensitive to disturbances and/or abrupt environmental variations. The animals were exposed in situ in four streams (reference site and sites 1, 2, and 3). The reference site is a preserved stream with no apparent anthropogenic interference where aeglids still occur, while the other sites no longer exhibit populations of these animals and are influenced by agricultural activities. The exposure was performed bimonthly from November 2017 to September 2018 and lasted 96 h. Measured abiotic data and water samples were collected through all days of exposure. The analyzed biochemical parameters were acetylcholinesterase activity in muscle; and glutathione S-transferase, lipid peroxidation, protein carbonylation, non-protein thiols, antioxidant capacity against peroxides, and reactive oxygen species (ROS) in muscle, gills, and hepatopancreas. We found 24 active principles of pesticides, the most frequently being clomazone, atrazine, and propoxur. Bentazone was present at the highest amounts. The parameters evaluated in this study, including biochemical biomarkers and abiotic factors measured from the water, provided a separation of the months as a function of environmental conditions. There was a difference in activity and biomarker levels throughout the year within the same site and in some months between sites. The greater concentration or variety of pesticides associated with extreme abiotic (very high temperatures) data generated increased oxidative stress, with high levels of protein damage and considerable lipid damage in all tissues, as well as elevation in ROS, even with high levels of antioxidant capacity and non-protein thiols. With these data, we intend to warn about the risks of exposure to these environmental conditions by trying to contribute to the preservation of limnic fauna, especially aeglid crabs, because most species are under some degree of threat.

Origin and evolution of the symbiosis between sea anemones (Cnidaria, Anthozoa, Actiniaria) and hermit crabs, with additional notes on anemone-gastropod associations.

The anemone-crab mutualism is ubiquitous in temperate and tropical marine environments. In this symbiosis, one or more anemones live on a shell inhabited by a hermit crab and reciprocal phoretic, trophic, and defensive benefits are exchanged between the partners. Sea anemone-hermit crab symbionts belong to three families: Hormathiidae (Calliactis and Paracalliactis), Sagartiidae (Carcinactis and Verrillactis), and Actiniidae (Stylobates). Hermit crabs establish most partnerships by detaching anemones and placing them on their shell; sea anemones can also mount shells unaided, triggered by a mollusc-derived substance in the periostracum of the shell. At least partial cooperation by the anemones is necessary for successful establishment of the symbiosis. Here, we expand the evolutionary framework for hormathiid symbionts by generating a phylogeny with at least one member of each actiniarian symbiotic genus with hermit crabs using five molecular markers (16S, 12S, 18S, 28S, CO3). We not only corroborated the results from a previous study by finding two origins of hermit crab symbiosis within Hormathiidae, but also found additional origins for hermit crab symbiosis within Actiniaria. We provide for the first time evidence of a close relationship between symbionts Carcinactis dolosa and V. paguri. The ability to secrete chitin by the ectoderm of the column is inferred to be broadly convergent within Actiniaria whereas the secretion of a chitinous carcinoecium by the pedal disc is a distinct but convergent morphological adaptation of several lineages within Actiniaria. Our finding of multiple origins for both the hermit crab and gastropod symbioses suggests that the shell-mounting behavior might only have been the precursor of the hermit crab association among Calliactis spp.

Novel gene rearrangement in the mitochondrial genome of Coenobita brevimanus (Anomura: Coenobitidae) and phylogenetic implications for Anomura.

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.

Optimisation of bioimpedance measurements of neuronal activity with an ex vivo preparation of Cancer pagurus peripheral nerves.

BACKGROUND: In mammals, fast neural Electrical Impedance Tomography (EIT) can image the myelinated component of the compound action potentials (CAP) using a nerve cuff. If applied to unmyelinated fibres this has great potential to improve selective neuromodulation ("electroceuticals") to avoid off-target effects. Previously, bioimpedance recordings were averaged from unmyelinated crab leg nerve fibres, but the signal to noise ratio (SNR) needs improving. NEW METHOD: Currently, functional non-invasive neuronal imaging is accomplished through surface electrodes or genetically expressed indicators that provide good spatial, but poor temporal, resolution. Here is an improved method for bioimpedance measurements from a model of unmyelinated fibres to enable optimisation through improvement of the 1) signal processing measurement paradigm, 2) neurophysiology, and 3) electrode-nerve interface. RESULTS: For bioimpedance recordings, the recruitment and necessity of the CAP was quantified and saline significantly improved the SNR. An improved protocol resulted in averaging not being required, as sequentially recorded traces produced bioimpedance changes of -0.232 ± 0.064% that did not show phase or timing related artefacts. COMPARISON WITH EXISTING METHOD: Here, two bioimpedance traces displayed an SNR of ≥3:1, while previously over >100 averages were required with greater inter-experimental variability. 10 paired traces were averaged for an SNR of ≥9:1, or near real-time measurement. CONCLUSIONS: This method facilitates further studies aiming to enable non-invasive localization of fascicular activity in unmyelinated fibres within peripheral nerves. This technique could ultimately produce the first 3-D tomographic images to help guide selective neuromodulation using bioelectric devices.

1-O-alkylglycerols from the hepatopancreas of the crab Paralithodes camtschaticus, liver of the squid Berryteuthis magister, and liver of the skate Bathyraja parmifera, and their anticancer activity on human melanoma cells.

1-O-alkylglycerols (AKG) are a class of natural ether lipids derived from 1-O-alkyl-2,3-diacyl-sn-glycerols by deacylation. In this study, 1-O-alkylglycerol (AKG) composition was investigated in the hepatopancreas lipids of the crab Paralithodes camtschaticus and the liver lipids of the squid Berryteuthis magister and the skate  Bathyraja parmifera. One of the principal AKG in marine organisms was 1-O-hexadecyl-sn-glycerol (AKG 16:0). To assess AKG influence on melanoma, we evaluated the cytotoxicity and antiproliferative actions of natural AKG 16:0 and synthetic 1-O-octyl-sn-glycerol (AKG 8:0) on three human melanoma cell lines SK-Mel-5, SK-Mel-28, and RPMI-7951. Natural AKG 16:0 in concentration up to 20 µM was not toxic to all cell lines. AKG 8:0 showed no toxicity to cells SK-Mel-5 and SK-Mel-28 in concentrations up to 20 µM but had moderate cytotoxicity to RPMI-7951 cells with an IC50 of 13 µM. Both investigated substances inhibited the proliferation, formation, and growth of cell colonies of RPMI-7951. PRACTICAL APPLICATIONS: AKG exhibit a variety of biological activities, including anticancer effects. In this study, the liver lipids of the skate B. parmifera and the hepatopancreas lipids of crab P. camtschaticus were shown to be sources of AKG. Our data showed that AKG can be used to prevent the formation of new colonies of malignant cells in combination therapy against melanoma. The results will be useful for future studies involving marine ether lipids and the examination of their anticancer properties against malignant cells.

Short- and long-term salinity challenge, osmoregulatory ability, and (Na+, K+)-ATPase kinetics and α-subunit mRNA expression in the gills of the thinstripe hermit crab Clibanarius symmetricus (Anomura, Diogenidae).

The evolutionary history of the Crustacea reveals ample adaptive radiation and the subsequent occupation of many osmotic niches resulting from physiological plasticity in their osmoregulatory mechanisms. We evaluate osmoregulatory ability in the intertidal, thinstripe hermit crab Clibanarius symmetricus after short-term exposure (6 h) or long-term acclimation (10 days) to a wide salinity range, also analyzing kinetic behavior and α-subunit mRNA expression of the gill (Na+, K+)-ATPase. The crab strongly hyper-regulates its hemolymph at 5 and 15‰S (Salinity, g L-1) but weakly hyper-regulates up to ≈27‰S. After 6 h exposure to 35‰S and 45‰S, C. symmetricus slightly hypo-regulates its hemolymph, becoming isosmotic after 10 days acclimation to these salinities. (Na+, K+)-ATPase specific activity decreases with increasing salinity for both exposure periods, reflecting physiological adjustment to isosmoticity. At low salinities, the gill enzyme exhibits a single, low affinity ATP binding site. However, at elevated salinities, a second, high affinity, ATP binding site appears, independently of exposure time. (Na+, K+)-ATPase α-subunit mRNA expression increases only after 10 days acclimation to 5‰S. Our findings suggest that hemolymph hyper-regulation is effected by alterations in enzyme activity during short-term exposure, but is sustained by increased mRNA expression during long-term acclimation. The decrease in gill (Na+, K+)-ATPase activity seen as a consequence of increasing salinity appears to underlie biochemical adjustments to hemolymph isosmoticity as hypo-regulatory ability diminishes.

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.

Triple Signal Amplification Strategy for Ultrasensitive Determination of miRNA Based on Duplex Specific Nuclease and Bridge DNA-Gold Nanoparticles.

Abnormal expression of miRNAs always occurs in solid tumors. Thus, it is critical to sensitively and selectively detect such biomarkers for the diagnosis and prognosis of diseases. Here, we report a biosensing scheme for the determination of miRNA with triple signal amplification based on target-triggered cyclic duplex specific nuclease digestion and bridge DNA-gold nanoparticles. Electrochemical signals are recorded to present initial levels of miRNA. This method is ultrasensitive with a wide linear range of 10-17 to 10-11 M. The limit of detection is down to 6.8 aM. Moreover, the overexpression of miR-21 is confirmed in lung cancer patients by the proposed method, which is in good accordance with qRT-PCR results. In addition, the developed biosensor does not need a reverse transcription process or any thermal cycling processes. Its performance satisfies the requirement for convenient, rapid, sensitive, and specific early diagnosis of cancers. Therefore, it may have great potential utility in the near future.

Paralithocins, Antimicrobial Peptides with Unusual Disulfide Connectivity from the Red King Crab, Paralithodes camtschaticus.

As part of an ongoing exploration of marine invertebrates as a source of new antimicrobial peptides, hemocyte extracts from the red king crab, Paralithodes camtschaticus, were studied. Three cationic cysteine (Cys)-rich peptides, named paralithocins 1-3, were isolated by bioassay-guided purification, and their amino acid sequences determined by Edman degradation and expressed sequences tag analysis. Disulfide bond mapping was performed by high-resolution tandem mass spectrometry. The peptides (38-51 amino acids in length) share a unique Cys motif composed of eight Cys, forming four disulfide bridges with a bond connectivity of (Cys relative position) Cys1-Cys8, Cys2-Cys6, Cys3-Cys5, and Cys4-Cys7, a disulfide arrangement that has not been previously reported among antimicrobial peptides. Thus, paralithocins 1-3 may be assigned to a previously unknown family of antimicrobial peptides within the group of Cys-rich antimicrobial peptides. Although none of the isolated peptides displayed antimicrobial activity against the target strains Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus, they inhibited the growth of several marine bacterial strains with minimal inhibitory concentrations in the 12.5-100 µM range. These findings corroborate the hypothesis that marine organisms are a valuable source for discovering bioactive peptides with new structural motifs.

Metabolic suppression in the pelagic crab, Pleuroncodes planipes, in oxygen minimum zones.

The pelagic red crab, Pleuroncodes planipes, is abundant throughout the Eastern Tropical Pacific in both benthic and pelagic environments to depths of several hundred meters. The oxygen minimum zones in this region reaches oxygen levels as low as 0.1kPa at depths within the crabs vertical range. Crabs maintain aerobic metabolism to a critical PO2 of ~0.27±0.2kPa (10°C), in part by increasing ventilation as oxygen declines. At subcritical oxygen levels, they enhance anaerobic ATP production slightly as indicated by modest increases in lactate levels. However, hypoxia tolerance is primarily mediated via a pronounced suppression of aerobic metabolism (~70%). Metabolic suppression is achieved, primarily, via reduced protein synthesis, which is a major sink for metabolic energy. Posttranslational modifications on histone H3 suggest a condensed chromatin state and, hence, decreased transcription. Under hypoxia, p-H3S10, Ac-H3K9, Ac-H3K14 were 39, 68, and 36% of control values, respectively. We also report a net decrease in protein translation. In particular, eEF2 activity is reduced due to a ~5-fold increase in inhibitory phosphorylation and a significant decrease in protein level. Elevated heat shock proteins suggest that, despite impressive tolerance, the cellular stress response is triggered during hypoxia. We discuss the implications for pelagic ecology and biogeochemical cycles.