Combined effects of pentachlorophenol and nano-TiO2 with different sizes on antioxidant, digestive, and immune responses of the swimming crab Portunus trituberculatus.

Marine nano-titanium dioxide (nano-TiO2) and pentachlorophenol (PCP) pollution are escalating concerns in coastal areas. This study investigated the combined effects of continuous exposure to nano-TiO2 (25 nm, 100 nm) and PCP (0, 1, 10 µg/L) for 28 days on the antioxidant, digestive, and immune abilities of the swimming crab Portunus trituberculatus. Compared with the control group, the interaction between nano-TiO2 and PCP was significantly higher than exposure to a single stressor, with a pronounced decrease in amylase activity observed due to the reducing nano-TiO2 particle sizes. Resulting in increased MDA and SOD activity. The expression levels of Toll4, CSP3, and SER genes in crab hemolymph showed perturbations following exposure to nano-TiO2 and PCP. In summary, according to the results of CAT, GPX, PES and AMS enzyme activities, it was concluded that compared to the larger particle size (100 nm), the single stress of nano-TiO2 at a smaller particle size (25 nm) and co-stress with PCP have more significant impacts on P. trituberculatus. However, the potential physiological regulation mechanism of the interaction between these pollutants remains elusive and requires further study.

Comparative Transcriptome Analysis Reveals the Light Spectra Affect the Growth and Molting of Scylla paramamosain by Changing the Chitin Metabolism.

Light is an essential ecological factor that has been demonstrated to affect aquatic animals' behavior, growth performance, and energy metabolism. Our previous study found that the full-spectrum light and cyan light could promote growth performance and molting frequency of Scylla paramamosain while it was suppressed by violet light. Hence, the purpose of this study is to investigate the underlying molecular mechanism that influences light spectral composition on the growth performance and molting of S. paramamosain. RNA-seq analysis and qPCR were employed to assess the differentially expressed genes (DEGs) of eyestalks from S. paramamosain reared under full-spectrum light (FL), violet light (VL), and cyan light (CL) conditions after 8 weeks trial. The results showed that there are 5024 DEGs in FL vs. VL, 3398 DEGs in FL vs. CL, and 3559 DEGs in VL vs. CL observed. GO analysis showed that the DEGs enriched in the molecular function category involved in chitin binding, structural molecular activity, and structural constituent of cuticle. In addition, the DEGs in FL vs. VL were mainly enriched in the ribosome, amino sugar and nucleotide sugar metabolism, lysosome, apoptosis, and antigen processing and presentation pathways by KEGG pathway analysis. Similarly, ribosome, lysosome, and antigen processing and presentation pathways were major terms that enriched in FL vs. CL group. However, only the ribosome pathway was significantly enriched in up-regulated DEGs in VL vs. CL group. Furthermore, five genes were randomly selected from DEGs for qPCR analysis to validate the RNA-seq data, and the result showed that there was high consistency between the RNA-seq and qPCR. Taken together, violet light exposure may affect the growth performance of S. paramamosain by reducing the ability of immunity and protein biosynthesis, and chitin metabolism.

Exploring the Exopolysaccharide Production Potential of Bacterial Strains Isolated from Tunisian Blue Crab Portunus segnis Microbiota.

The blue crab (BC) Portunus segnis is considered an invasive species colonizing Tunisian coasts since 2014. This work aims to explore its associated bacteria potential to produce anionic exopolysaccharides (EPSs) in order to open up new ways of valorization. In this study, different BC samples were collected from the coastal area of Sfax, Tunisia. First, bacterial DNA was extracted from seven different fractions (flesh, gills, viscera, carapace scraping water, and three wastewaters from the production plant) and then sequenced using the metabarcoding approach targeting the V3-V4 region of the 16S rDNA to describe their microbiota composition. Metabarcoding data showed that the dominant bacterial genera were mainly Psychrobacter, Vagococcus, and Vibrio. In parallel, plate counting assays were performed on different culture media, and about 250 bacterial strains were isolated and identified by sequencing the 16S rDNA. EPS production by this new bacterial diversity was assessed to identify new compounds of biotechnological interest. The identification of the bacterial strains in the collection confirmed the dominance of Psychrobacter spp. strains. Among them, 43 were identified as EPS producers, as revealed by Stains-all dye in agarose gel electrophoresis. A Buttiauxella strain produced an EPS rich in both neutral sugars including rare sugars such as rhamnose and fucose and uronic acids. This original composition allows us to assume its potential for biotechnological applications and, more particularly, for developing innovative therapeutics. This study highlights bacterial strains associated with BC; they are a new untapped source for discovering innovative bioactive compounds for health and cosmetic applications, such as anionic EPS.

A DM9-containing protein from crab Eriocheir sinensis functions as a novel multipotent pattern recognition receptor.

DM9-containing protein in invertebrates functions as pattern recognition receptor (PRR) to play significant roles in innate immunity. In the present study, a novel DM9-containg protein (defined as EsDM9CP-1) was identified from the Chinese mitten crab Eriocheir sinensis. EsDM9CP-1 is composed of 330 amino acids containing a Methyltransf_FA domain and two tandem DM9 repeats. The deduced amino acid sequence of EsDM9CP-1 shared low similarity with the previously identified DM9CPs from other species, and it was closely clustered with Platyhelminthes DM9CPs and then assigned into the branch of invertebrate DM9CPs in the unrooted phylogenetic tree. The mRNA transcripts of EsDM9CP-1 were highly expressed in haemocytes, gill, and heart. After Aeromonas hydrophila stimulation, the expression levels of EsDM9CP-1 mRNA in haemocytes increased significantly at 3 h (3.88-fold, p < 0.05) and 6 h (2.71-fold, p < 0.05), compared with that of PBS group, respectively. EsDM9CP-1 protein was mainly distributed in the cytoplasm and membrane of haemocytes. The recombinant EsDM9CP-1 protein (rEsDM9CP-1) exhibited binding affinity to MAN, PGN, LPS and Poly (I:C), and also to Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli, A. hydrophila and Vibrio splendidus) and fungi (Pichia pastoris and Metschnikowia bicuspidata) in a Ca2+-dependent manner. It was able to agglutinate A. hydrophila, S. aureus, M. luteus, M. bicuspidata and P. pastoris, and inhibit the growth of A. hydrophila and M. bicuspidate. These results suggested that EsDM9CP-1 in crab not only functioned as a PRR, but also agglutinated and inhibited the growth of microbes.

Cellular localization and potential ligands of a novel scavenger receptor class B/CD36 protein homolog (Pt-SRB2) identified in the marine crab, Portunustrituberculatus.

The scavenger receptor class B family proteins (SRB) are multiligand membrane receptor proteins. Herein, a novel SRB homolog (Pt-SRB2) was identified in Portunus trituberculatus. The open reading frame of Pt-SRB2 was predicted to encode 520 amino acid residues comprising a typical CD36 domain. Phylogenetic analysis showed that Pt-SRB2 distinctly clustered with the SRB homologs of most crustaceans and Drosophila but was separate from all vertebrate CD36/SRB. Semi-quantitative and Real-time quantitative PCR revealed that the abundance of Pt-SRB2 transcripts was the highest in hepatopancreas than in other tested tissues. Overexpressed Pt-SRB2 was distributed primarily in the cell membrane and cytoplasm of HEK293T or Drosophila Schneider 2 cells. In crab hemocytes, Pt-SRB2 was distributed primarily in the cell membrane by immunofluorescence staining. In addition, the immunofluorescence staining showed that green fluorescence signals were mainly located in the inner lumen membrane of the hepatopancreatic tubules. Moreover, solid-phase enzyme-linked immunosorbent assay revealed that rPt-SRB2-L exhibited relative high affinity with lipopolysaccharides, and relative moderate binding affinity with lipoteichoic acid or peptidoglycan. Of note, rPt-SRB2-L showed high binding affinity with eicosapentaenoic acid among a series of long-chain polyunsaturated fatty acids. Taken together, this study provided valuable data for understanding the functions of the crab CD36/SRB.

E93 gene in the swimming crab, Portunus trituberculatus: Responsiveness to 20-hydroxyecdysone and methyl farnesoate and role on regulating ecdysteroid synthesis.

Ecdysone-induced protein 93 (E93) is a metamorphic determinant involved in crosstalk between 20-hydroxyecdysone (20E) and juvenile hormone (JH) during the insect molting process. The present study identified the E93 gene from the swimming crab, P. trituberculatus, and found it was widely distributed in adult tissues. PtE93 mRNA levels in Y-organ and epidermis fluctuated during the molt cycle, suggesting its involvement in juvenile molting. In vitro and in vivo treatments with 20E led to an induction of PtE93 expression in Y-organ and epidermis, while we found the opposite effect for methyl farnesoate (MF) treatments, a crustacean equivalent of insect JH. We also observed that two genes for ecdysteroid biosynthesis, Spook (Spo) and Shadow (Sad), were suppressed by 20E and induced by MF, showing a negative correlation between PtE93 and ecdysteroid biosynthesis. PtE93 RNA interference (RNAi) induced Spo and Sad expression levels, elevated ecdysteroid content in culture medium, and relieved the 20E inhibitory effect on ecdysteroid synthesis, indicating an inhibitory role of PtE93 on ecdysteroid synthesis. Overall, our results suggest that E93 may be involved in the crosstalk between 20E and MF during crustacean molting, and its presence in Y-organ is closely related to ecdysteroid synthesis.

Identification of transient receptor potential channel genes from the swimming crab, Portunus Trituberculatus, and their expression profiles under acute temperature stress.

BACKGROUND: Temperature is an important environment factor that is critical to the survival and growth of crustaceans. However, the mechanisms by which crustaceans detect changes in temperature are still unclear. The transient receptor potential (TRP) channels are non-selective cation channels well known for properties in temperature sensation. However, comprehensive understandings on TRP channels as well as their temperature sensing functions are still lacking in crustaceans. RESULTS: In this study, a total of 26 TRP genes were identified in the swimming crab, Portunus trituberculatus, which can be classified into TRPA, TRPC, TRPP, TRPM, TRPML, TRPN and TRPV. Tissue expression analysis revealed a wide distribution of these TRP genes in P. trituberculatus, and antennules, neural tissues, and ovaries were the most commonly expressed tissues. To investigate the responsiveness of TRP genes to the temperature change, 18 TRPs were selected to detect their expression after high and low temperature stress. The results showed that 12 TRPs showed induced gene expression in both high and low temperature groups, while 3 were down-regulated in the low temperature group, and 3 showed no change in expression in either group. CONCLUSIONS: This study characterized the TRP family genes in P. trituberculatus, and explored their involvement in response to temperature stress. Our results will enhance overall understanding of crustacean TRP channels and their possible functions.

Temporal and Spatial Signatures of Scylla paramamosain Transcriptome Reveal Mechanistic Insights into Endogenous Ovarian Maturation under Risk of Starvation.

Variability in food availability leads to condition-dependent investments in reproduction. This study is aimed at understanding the metabolic response and regulatory mechanism of female Scylla paramamosain in response to starvation in a temporal- and tissue-specific manner. The mud crabs were starved for 7 (control), 14, 28, and 40 days for histological and biochemical analysis in the hepatopancreas, ovary, and serum, as well as for RNA sequencing on the hepatopancreas and ovary. We further highlighted candidate gene modules highly linked to physiological traits. Collectively, our observations suggested that starvation triggered endogenous ovarian maturation at the expense of hepatopancreas mass, with both metabolic adjustments to optimize energy and fatty acid supply from hepatopancreas to ovary in the early phase, followed by the activation of autophagy-related pathways in both organs over prolonged starvation. These specific adaptive responses might be considered efficient strategies to stimulate ovarian maturation of Scylla paramamosain under fasting stress, which improves the nutritional value of female mud crabs and other economically important crustaceans.

Molecular mechanism of the NOS/NOX regulation of antibacterial activity in Eriocheir sinensis.

To elucidate the role of nitric oxide synthase (NOS), which produces the free radical nitric oxide (NO), and nicotinamide adenine dinucleotide phosphate oxidase (NOX), which produces the superoxide anion (O2-), in the innate immunity of Eriocheir sinensis, the full lengths of the NOS and NOX genes were cloned via rapid amplification of the cDNA ends and then expressed in the prokaryotic form to obtain the recombinant proteins, NOS-HIS and NOX-HIS. Through bacterial binding and stimulation experiments, the molecular mechanisms of NOS and NOX in the innate immunity of E. sinensis were explored. Based on the results, NOS and NOX were 5900 bp and 4504 bp long, respectively, and were evolutionarily conserved. Quantitative real-time PCR revealed that NOS and NOX were expressed in all studied tissues, and both were expressed in the highest amounts in hemocytes. NOS-HIS and NOX-HIS could bind to bacteria with different binding powers; their binding ability to gram-positive bacteria was higher than that of binding to gram-negative bacteria. After stimulation with Aeromonas hydrophila, NOS expression was significantly up-regulated at 3, 6, and 48 h, and NOX expression was significantly down-regulated at 3, 12, 24, and 48 h. After bacterial stimulation, the NOS enzyme activity in the serum of E. sinensis was also significantly up-regulated at 6 and 48 h, and the NOX enzyme activity was significantly down-regulated at 12 and 48 h, aligning with the gene expression trend. Moreover, the related free radical molecules, NO, O2-, and H2O2, tended to decrease after bacterial stimulation. Overall, the gene expression and enzyme activity of NOS and NOX had been changed respectively, and the contents of a series of free radical molecules (NO, O2- and H2O2) were induced in E. sinensis after bacterial stimulation, which then exert antibacterial immunity.

Diversification of freshwater crabs on the sky islands in the Hengduan Mountains Region, China.

The numerous naturally-fragmented sky islands (SIs) in the Hengduan Mountains Region (HMR) of southwestern China constitute discontinuous landscapes where montane habitats are isolated by dry-hot valleys which have fostered exceptional species diversification and endemicity. However, studies documenting the crucial role of SI on the speciation dynamics of native freshwater organisms are scarce. Here we used a novel set of comprehensive genetic markers (24 nuclear DNA sequences and complete mitogenomes), morphological characters, and biogeographical information to reveal the evolutionary history and speciation mechanisms of a group of small-bodied montane potamids in the genus Tenuipotamon. Our results provide a robustly supported phylogeny, and suggest that the vicariance events of these montane crabs correlate well with the emergence of SIs due to the uplift of the HMR during the Late Oligocene. Furthermore, ancestrally, mountain ridges provided corridors for the dispersal of these montane crabs that led to the colonization of moist montane-specific habitats, aided by past climatic conditions that were the crucial determinants of their evolutionary history. The present results illustrated that the mechanisms isolating SIs are reinforced by the harsh-dry isolating climatic features of dry-hot valleys separating SIs and continue to affect local diversification. This offers insights into the causes of the high biodiversity and endemism shown by the freshwater crabs of the HMR-SIs in southwestern China.

The comparison of morphology and transcriptome in the inner membrane reveals the potential mechanism of the heritable carapace color of the Chinese mitten crab Eriocheir sinensis.

Carapace color plays an important role in the communication, reproduction, and self-defense of crustaceans, which is also related to their economic value. Chinese mitten crab (Eriocheir sinensis) is an important aquaculture species in China, and there are different strains with heritable carapace colors, i.e. Green, White, and Red. However, there is a lack of research on the formation mechanism of carapace color of this species. This study was conducted to compare the histology and transcriptome in the inner membrane of three carapace color strains of E. sinensis. Histological comparisons revealed that the inner membrane of green and red carapace crabs contained more melanin, appearing in clusters, and had a higher presence of yellow or orange pigments. In contrast, the inner membrane of white carapace crabs had smaller and fewer melanin particles, as well as a lower presence of yellow or orange pigments. Observation under an electron microscope showed that the inner membrane of E. sinensis contained a large number of collagen fibers and various types of cells, including fibroblasts, melanocytes, and other tissue cells, which exhibited different levels of activity. Transcriptome analysis showed that the Green, Red, and White strains of E. sinensis had approximately 80.3 K, 81.6 K and 80.3 K expressed unigenes in their inner membranes, respectively. When comparing Green and Red crabs, there were 2, 850 upregulated genes and 2, 240 downregulated genes. In the comparison between Red and White crabs, there were 2, 853 upregulated genes and 2, 583 downregulated genes. Furthermore, there were 2, 336 upregulated genes and 2, 738 downregulated genes in the inner membranes between White and Green crabs. Among these genes, some members of the solute carriers family, which are involved in carotenoid transportation, showed differential expression among the three carapace color strains. Additionally, significant differences were observed in the expression of genes related to melanin synthesis, including wingless/integrate, tyrosinase, guanine nucleotide-binding protein inhibitory subunit, cell adhesion molecule, adenylyl cyclase, and creb-binding protein. there were no differences in the gene expression levels of the crustacyanin family. In conclusion, this study identified several candidate genes associated with carapace color in the inner membrane of E. sinensis, suggesting a close relationship between the heritable carapace colors and the transport of the carotenoids as well as the synthesis of melanin.

Evolution and expression analysis of carotenoid cleavage oxygenase gene family in Chinese mitten crab Eriocheir sinensis.

Carotenoid cleavage oxygenase (CCO) plays a pivotal role in various biological activities, including antioxidant and immune functions in animals. This paper investigates the evolution and expression of CCO genes based on three chordates and 27 arthropods. Aquatic animals exhibit a higher abundance of CCO genes. Despite this, research on CCO in crustaceans has been notably limited, with a complete absence of any previous studies on the CCO genes for the Chinese mitten crab (Eriocheir sinensis). In this study, six CCO genes were identified in the E. sinensis genome database. Results reveal that the evolution of the CCO gene family in Crustacea is primarily characterized by purifying selection, with a preference for employing similar codons. EsCCO1 and EsCCO3 were mainly expressed in the epidermal layer, and EsCCO4 was mainly expressed in the hindgut. Meanwhile, EsCCO5 and EsCCO6 were mainly expressed in the hepatopancreas and endometrium. A notable detail that different EsCCO genes demonstrate distinct expression patterns within various tissues of E. sinensis. The findings of this study offer fundamental insights that could serve as a basis for further exploration into the functions and regulatory mechanisms of CCO genes in crustacean species.

Effects of hypo-osmotic shock on osmoregulatory responses and expression levels of selected ion transport-related genes in the sesarmid crab Episesarma mederi (H. Milne Edwards, 1853).

This study examined the osmoregulatory responses to hypo-osmotic shock in the commercially and ecologically important crab Episesarma mederi (H. Milne Edwards, 1853). After the acclimation for one week at a salinity of 25 PSU, Adult males E. mederi were immediately exposed to salinities of 5 PSU and 25 PSU (the control group). The time course of changes in haemolymph osmolality, gill Na+/K+ ATPase (NKA) activity, oxygen uptake rates, and mRNA expression levels of ion-transport related genes, including the NKA-α subunit, V-type H+ATPase (VT) and Na+/K+/2Cl-(NKCC), were determined. The results showed that E. mederi was a strong hyperosmoregulator after exposure to 5 PSU, achieved by modulations of NKA activity in their posterior gills rather than the anterior gills. The crabs acclimated to 5 PSU increased oxygen uptake, especially during the initial exposure, reflecting increased energetic costs for osmotic stress responses. In the posterior gills, the NKA activities of the crabs acclimated to 5 PSU at 3, 72 and 168 h were significantly higher than those in the control group. Elevated NKA-α subunit expression levels were detected at 6 h and 12 h. Increased expression levels of VT and NKCC were identified at 6 h and 12 h, respectively. Our results indicate that elevated gill NKA activity at 3 h could result from enzyme activity and kinetic alterations. On the other hand, the gill NKA activity at 72 and 168 h was sustained by elevated NKA-α subunit expression. Hence, these adaptive responses in osmoregulation enable the crabs to withstand hypo-osmotic challenges and thrive in areas of fluctuating salinity in mangroves and estuaries.

Differential molecular responses of hemolymph and hepatopancreas of swimming crab, Portunus trituberculatus, infected with Ameson portunus (Microsporidia).

Ameson portunus (Microsporidia) has caused serious economic losses to the aquaculture industry of swimming crab, Portunus trituberculatus. The hemolymph and hepatopancreas are the main immune organs of P. trituberculatus, and the main sites of A. portunus infection. Elucidating the response characteristics of hemolymph and hepatopancreas to microsporidian infection facilitates the development of microsporidiosis prevention and control strategy. This study performed comparative transcriptomic analysis of hemolymph (PTX/PTXA) and hepatopancreas (PTG/PTGA) of P. trituberculatus uninfected and infected with A. portunus. The results showed that there were 223 and 1309 differentially expressed genes (DEGs) in PTX/PTXA and PTG/PTGA, respectively. The lysosome pathway was significantly enriched after the invasion of the hemolymph by A. portunus. Also, immune-related genes were all significantly up-regulated in the hemolymph and hepatopancreas, suggesting that the invasion by A. portunus may activate host immune responses. Unlike hemolymph, antioxidant and detoxification-related genes were also significantly up-regulated in the hepatopancreas. Moreover, metabolism-related genes were significantly down-regulated in the hepatopancreas, suggesting that energy synthesis, resistance to pathogens, and regulation of oxidative stress were suppressed in the hepatopancreas. Hemolymph and hepatopancreas have similarity and tissue specificity to microsporidian infection. The differential genes and pathways identified in this study can provide references for the prevention and control of microsporidiosis.

Transcriptome and metabolomics analysis of adaptive mechanism of Chinese mitten crab (Eriocheir sinensis) to aflatoxin B1.

Aflatoxin B1 (AFB1), with the strong toxicity and carcinogenicity, has been reported to great toxicity to the liver and other organs of animals. It cause huge economic losses to breeding industry, including the aquaculture industry. Chinese mitten crabs (Eriocheir sinensis), as one of important species of freshwater aquaculture in China, are deeply disturbed by it. However, the molecular and metabolic mechanisms of hepatopancreas and ovary in crabs underlying coping ability are still unclear. Hence, we conducted targeted injection experiment with or without AFB1, and comprehensively analyzed transcriptome and metabolomics of hepatopancreas and ovary. As a result, 210 and 250 DEGs were identified in the L-C vs. L-30 m and L-C vs. L-60 m comparison, among which 14 common DEGs were related to six major functional categories, including antibacterial and detoxification, ATP energy reaction, redox reaction, nerve reaction, liver injury repair and immune reaction. A total of 228 and 401 DAMs in the ML-C vs. ML-30 m and ML-C vs. ML-60 m comparison both enriched 12 pathways, with clear functions of cutin, suberine and wax biosynthesis, tyrosine metabolism, purine metabolism, nucleotide metabolism, glycine, serine and threonine metabolism, ABC transporters and tryptophan metabolism. Integrated analysis of metabolomics and transcriptome in hepatopancreas discovered three Co-enriched pathways, including steroid biosynthesis, glycine, serine and threonine metabolism, and sphingolipid metabolism. In summary, the expression levels and functions of related genes and metabolites reveal the regulatory mechanism of Chinese mitten crab (Eriocheir sinensis) adaptability to the Aflatoxin B1, and the findings contribute to a new perspective for understanding Aflatoxin B1 and provide some ideas for dealing with it.

[Sequence analysis of Paragonimus internal transcribed spacer 2 and cyclooxygenase 1 genes in freshwater crabs in Henan Province].

OBJECTIVE: To investigate the sequences of internal transcribed spacer 2 (ITS2) and cyclooxygenase 1 (COX1) genes of Paragonimus metacercariae in freshwater crabs in Henan Province, identify the species of Paragonimus and evaluate its genetic relationships with Paragonimus isolates from other provinces in China. METHODS: Freshwater crabs were collected from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province from 2016 to 2021, and Paragonimus metacercariae were detected in freshwater crabs. Genomic DNA was extracted from Paragonimus metacercariae, and the ITS2 and COX1 genes were amplified using PCR assay, followed by sequencing of PCR amplification products. The gene sequences were spliced and aligned using the software DNASTAR, and aligned with the sequences of Paragonimus genes in the GenBank. Phylogenetic trees were created using the MEGA6 software with the Neighbor-Joining method based on ITS2 and COX1 gene sequences, with Fasciola hepatica as the outgroup. RESULTS: The detection rates of Paragonimus metacercariae were 6.83% (11/161), 50.82% (31/61), 18.52% (5/26), 8.76% (12/137), 14.29% (9/63), 17.76% (19/105), 18.50% (32/173) and 42.71% (41/96) in freshwater crabs from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province, with a mean detection rate of 19.46% (160/822), and a mean infection intensity of 0.57 metacercariae/g. The amplified ITS2 and COX1 gene fragments of Paragonimus were approximately 500 bp and 450 bp in lengths, respectively. The ITS2 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (99.8% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: MW960209.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with P. skrjabini from Sichuan Province (GenBank accession number: AY618747.1), Guangxi Zhuang Autonomous Region (GenBank accession number: AY618729.1) and Hubei Province (GenBank accession number: AY618751.1), and P. miyazaki from Fujian Province (GenBank accession number: AY618741.1) and Japan (GenBank accession number: AB713405.1). The COX1 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (90.0% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: AY618798.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with all P. skrjabini and clustered into the same sub-clade with P. skrjabini from Hubei Province (GenBank accession numbers: AY618782.1 and AY618764.1). CONCLUSIONS: Paragonimus species from freshwater crabs in Henan Province were all characterized as P. skrjabini, and the ITS2 and COX1 gene sequences had the highest homology to those of P. skrjabini from Hubei Province. The results provide insights into study of Paragonimus in Henan Province and China.

Phylogenetics and Population Genetics of the Petrolisthes lamarckii-P. haswelli Complex in China: Old Lineage and New Species.

Petrolisthes lamarckii (Leach, 1821) and P. haswelli Miers, 1884 are a pair of sister species of porcelain crabs, both of which are common in the intertidal zone of southern China, typically found under rocks and in the crevices of coral reefs. However, the distribution, genetic relationship and diversity of the two species in China have not been rigorously studied. Meanwhile, P. lamarckii is considered as a complex of cryptic species due to their diverse morphological features. In this study, we identified 127 specimens of the P. lamarckii-P. haswelli complex (LH complex) and recognised a new species through morphological and molecular analysis. Furthermore, we constructed a time-calibrated phylogeny of the LH complex using three mitochondrial and two nuclear genes from all three species, finding that the divergence of the LH complex can be traced back to the Miocene epoch, and that the genetic diversity increased during the Mid-Pleistocene transition period. Glacial refugia formed during the Pleistocene climatic oscillations has been regarded as one of the contributing factors to the diversification of marine organisms in the north-western Pacific. Petrolisthes haswelli demonstrates a wide distribution along the southern coast of China, while other lineages display more restricted distributions. The research on the demographic history and gene flow of P. haswelli revealed that the Chinese coastal populations experienced an expansion event approximately 12.5 thousand years ago (Kya) and the asymmetrical gene flows were observed between the two sides of the Taiwan Strait and Qiongzhou Strait, respectively, which is likely influenced by the restriction of ocean currents.

The analyses of the complete mitochondrial genomes of three crabs revealed novel gene rearrangements and phylogenetic relationships of Brachyura.

BACKGROUND: Brachyura crab is the largest branch of Decapoda crustacean. Phylogenetic relationships within Brachyura remain controversial to be investigated. The mitochondrial genome (mitogenome) is an important molecular marker for studying the phylogenetic relationships of Brachyura. METHODS AND RESULTS: To understand the phylogeny of Brachyura, the three complete mitogenomes from Charybdis annulata, Leptodius exaratus, and Spider crab were sequenced and annotated. Their full length was 15,747, 15,716, and 16,608 bp long, respectively. The first two crabs both contained 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a control region. However, Spider crab contained 13 PCGs, two rRNA genes, 25 tRNA genes and a control region. The mitogenomes of each of the three crabs exhibited high AT content (67.8%, 69.1%, and 70.8%), with negative AT skews (-0.014, - 0.028, and - 0.017) and GC skews (-0.269, - 0.286, and - 0.341). The gene order of C. annulata was identical to the ancestor of Brachyura. Compared with the ancestor of Brachyura, L. exaratus exhibited the gene rearrangements of Val (V)-rrnS-control region, and Spider crab had the four copies of Lys (K). Phylogenetic analyses indicated that C. annulata belonged to Portunidae family, Portunoidea superfamilies, L. exaratus belonged to Xanthidae family, Xanthoidea superfamilies, and Spider crab belonged to Mithracidae family, Majoidea superfamilies. Phylogenetic analyses showed that the two species (Somanniathelphusa boyangensis and Huananpotamon lichuanense) belonging to the Potamoidea were sister groups to the Thoracotremata, thus supporting the conclusion that Heterotremata is polyphyletic. CONCLUSION: The results of this study enriched the crab mitogenome database and enabled us to better understand the phylogenetic relationships of Brachyura.

Genomics and transcriptomics of the Chinese mitten crabs (Eriocheir sinensis).

To gain a deeper understanding of the genetic factors influencing the growth and development of Eriocheir sinensis, a well-known species of hairy crab found in Yangcheng Lake, this study focused on the de novo genome and full-length transcriptome information of the selected subjects. Specifically, Yangcheng Lake hairy crabs were chosen as the experimental samples. Initially, a genome analysis was performed, resulting in the identification of gene fragments with a combined length of 1266,092,319 bp. Subsequently, a transcriptome analysis was conducted on a mixture of tissues from four different sites, namely muscle, brain, eye, and heart, to further investigate the genetic characteristics at the transcriptome level. The Pacific Biosciences (Pacio) single-molecule real-time sequencing system generated a total of 36.93 G sub-fragments and 175,90041 effective inserts. This research contributes to the indirect comprehension of genetic variations underlying individual traits. Furthermore, a comparison of the obtained data with relevant literature emphasizes the advantages of this study and establishes a basis for further investigations on the Chinese mitten crab.

Phylomitogenomics elucidates the evolution of symbiosis in Thoracotremata (Decapoda: Cryptochiridae, Pinnotheridae, Varunidae).

Background: Thoracotremata belong to the large group of "true" crabs (infraorder Brachyura), and they exhibit a wide range of physiological and morphological adaptations to living in terrestrial, freshwater and marine habitats. Moreover, the clade comprises various symbiotic taxa (Aphanodactylidae, Cryptochiridae, Pinnotheridae, some Varunidae) that are specialised in living with invertebrate hosts, but the evolutionary history of these symbiotic crabs is still partially unresolved. Methods: Here we assembled and characterised the complete mitochondrial genomes (hereafter mitogenomes) of three gall crab species (Cryptochiridae): Kroppcarcinus siderastreicola, Opecarcinus hypostegus and Troglocarcinus corallicola. A phylogenetic tree of the Thoracotremata was reconstructed using 13 protein-coding genes and two ribosomal RNA genes retrieved from three new gall crab mitogenomes and a further 72 available thoracotreme mitogenomes. Furthermore, we applied a comparative analysis to characterise mitochondrial gene order arrangement, and performed a selection analysis to test for selective pressure of the protein-coding genes in symbiotic Cryptochiridae, Pinnotheridae, and Varunidae (Asthenognathus inaequipes and Tritodynamia horvathi). Results: The results of the phylogenetic reconstruction confirm the monophyly of Cryptochiridae, which clustered separately from the Pinnotheridae. The latter clustered at the base of the tree with robust branch values. The symbiotic varunids A. inaequipes and T. horvathi clustered together in a clade with free-living Varunidae species, highlighting that symbiosis in the Thoracotremata evolved independently on multiple occasions. Different gene orders were detected in symbionts and free-living species when compared with the ancestral brachyuran gene order. Lastly, the selective pressure analysis detected two positively selected sites in the nad6 gene of Cryptochiridae, but the evidence for positive selection in Pinnotheridae and A. inaequipes and T. horvathi was weak. Adaptive evolution of mitochondrial protein-coding genes is perhaps related to the presumably higher energetic demands of a symbiotic lifestyle.