Identification of acid phosphatase (ShACP) from the freshwater crab Sinopotamon henanense and its expression pattern changes in response to cadmium.

Acid phosphatase(ACP) is an important immune enzyme in crustacean humoral immunity. At present, the research on ACP mainly focuses on the biochemical properties of the enzyme, while few studies on gene expression. In this study, ShACP was cloned and the effect of cadmium stress on the expression and function of ShACP in the freshwater crab Sinopotamon henanense was studied. Analysis of the ShACP sequence and tissue distribution results showed that the cDNA sequence of ShACP was 1629 bp, including 48 bp 5' untranslated region, 1209 bp open reading frame region, and 372 bp 3' untranslated region, encoding 402 amino acids. ShACP contained multiple phosphorylation sites and mainly played a role in the hemolymph. Under low-concentration cadmium stress, the body improved immunity by enhancing the expression of ShACP, while high-concentration cadmium stress inhibited the expression of ShACP. ShACP can promote the phagocytosis of hemocytes, while cadmium stress reduced the phagocytosis of hemocytes. This study provides a theoretical basis for further research on the immune system of crabs and is of great significance for the study of crustacean immune responses under heavy metal stress.

Cloning, identification, and functional characterization of novel prophenoloxidases (ShproPO) from the freshwater crab Sinopotamon henanense in response to cadmium exposure and Aeromonas hydrophila infection.

Prophenoloxidase (proPO) is essential in the prophenoloxidase-activating system (proPO-AS) which is important for defense against foreign infection in crustaceans. However, most studies have focused on expression in the presence of a single pathogenic bacterium, and very few have addressed the presence of environmental contaminants simultaneously, such as cadmium (Cd) and Aeromonas hydrophila. Our study aimed to investigate the function of proPO in the freshwater crab Sinopotamon henanense and the changes in its expression by Cd and infection of A. hydrophila. A novel proPO from the hemocytes of S. henanense (ShproPO) was found in this research, the full-length cDNA of ShproPO was 2620 bp of encoding a protein of 678 amino acids containing three typical hemocyanin domains. The ShproPO protein could be found in both the granular (GHc) and the semi-granular hemocytes (SGHc). The ShproPO mRNA was found to be abundantly expressed in hemocytes and could be influenced by A. hydrophila infection. These results indicate that ShproPO could be involved in the antibacterial process. Further research found that low concentrations of Cd could promote its expression after infection with A. hydrophila. Therefore, it was hypothesized that Cd disrupted the response of crabs to A. hydrophila infection. Subsequently, PO enzyme activity was found to be significantly reduced through in vivo RNA interference with ShproPO, and the results suggested that ShproPO is likely to be a key enzyme in the melanization response. Finally, ShproPO was found to significantly enhance the phagocytosis of A. hydrophila-infected hemocytes by in vitro recombination, confirming that ShproPO is involved in hemocyte-mediated melanization and phagocytosis. Our findings reveal completely new insight into the immunotoxicity of Cd and the immune function of ShproPO in S. henanense.

Molecular phylogeny of Thoracotremata crabs (Decapoda, Brachyura): Toward adopting monophyletic superfamilies, invasion history into terrestrial habitats and multiple origins of symbiosis.

The Thoracotremata is a large and successful group of "true" crabs (Decapoda, Brachyura, Eubrachyura) with a great diversity of lifestyles and well-known intertidal representatives. The group represents the largest brachyuran radiation into terrestrial and semi-terrestrial environments and comprises multiple lineages of obligate symbiotic species. In consequence, they exhibit very diverse physiological and morphological adaptations. Our understanding of their evolution is, however, largely obscured by their confused classification. Here, we resolve interfamilial relationships of Thoracotremata, using 10 molecular markers and exemplars from all nominal families in order to reconstruct the pathways of lifestyle transition and to prepare a new taxonomy corresponding to phylogenetic relationships. The results confirm the polyphyly of three superfamilies as currently defined (Grapsoidea, Ocypodoidea and Pinnotheroidea). At the family level, Dotillidae, Macrophthalmidae, and Varunidae are not monophyletic. Ancestral state reconstruction analyses and divergent time estimations indicate that the common ancestor of thoracotremes already thrived in intertidal environments in the Late Cretaceous and terrestrialization became a major driver of thoracotreme diversification. Multiple semi-terrestrial and terrestrial lineages originated and radiated in the Early Eocene, coinciding with the global warming event at the Paleocene-Eocene Thermal Maximum (PETM). Secondary invasions into subtidal regions and colonizations of freshwater habitats occurred independently through multiple semi-terrestrial and terrestrial lineages. Obligate symbiosis between thoracotremes and other marine macro-invertebrates evolved at least twice. On the basis of the current molecular phylogenetic hypothesis, it will be necessary in the future to revise and recognize seven monophyletic superfamilies and revisit the morphological character states which define them.

Detoxification and recovery after cadmium exposure in the freshwater crab Sinopotamon henanense.

Cadmium (Cd) is a common pollutant in the aquatic environment, which puts the health and safety of aquatic organisms and humans at risk. In the present study, the freshwater crab Sinopotamon henanense was exposed to Cd (0, 50, 100, and 500 µg·L-1) for 14 d (0-14th d), followed by 21 d (14-35th d) of depuration. The changes in Cd bioaccumulation, microstructure, biomacromolecules (polysaccharides, neutral lipids, DNA and total proteins), and biochemical parameters (SOD, CAT, GR, TrxR, MDA and AChE) in the gills and hepatopancreas were tested. The injured microstructure, activated antioxidant system, increased MDA, and inhibited AChE of the gills and hepatopancreas responded with progressive bioaccumulation of Cd. Meanwhile, the polysaccharides and neutral lipids in the hepatopancreas reduced and DNA synthesis enhanced. During depuration, more than 58.80 ± 8.53% and 13.84 ± 12.11% of Cd was excreted from the gills and hepatopancreas, respectively. Recovery of microstructure and biomacromolecules as well as alleviated oxidative damage and neurotoxicity were also found in these two organs. Additionally, based on PCA, Ihis, GR and MDA were identified as the optimal biomarkers indicating the health status of crabs. In conclusion, S. henanense could resist Cd stress through antioxidant defence and self-detoxification.

T-Cell Epitope Immunotherapy in Mouse Models of Food Allergy.

Food allergy has been rising in prevalence over the last two decades, affecting more than 10% of the world population. Current management of IgE-mediated food allergy relies on avoidance and rescue medications; research into treatments that are safer and providing guaranteed and durable curative effects is, therefore, essential. T-cell epitope-based immunotherapy holds the potential for modulating food allergic responses without IgE cross-linking. In this chapter, we describe the methods in evaluating the therapeutic capacities of immunodominant T-cell epitopes in animal models of food allergy. Moreover, we explain in detail the methods to measure the allergen-specific antibody levels, prepare single-cell suspension from spleen, and prepare small intestine for immunohistochemical analysis of eosinophils and Foxp3+ cells.

Modulating Shrimp Tropomyosin-Mediated Allergy: Hypoallergen DNA Vaccines Induce Regulatory T Cells to Reduce Hypersensitivity in Mouse Model.

Shellfish allergy is one of the most common food allergies, with tropomyosin as the major cross-reactive allergen. However, no allergen-specific immunotherapy is clinically available. Recently, we designed two shrimp hypoallergens MEM49 and MED171. This study aimed to examine and compare the efficacy of the MEM49- and MED171-based DNA vaccines (pMEM49 and pMED171) in modulating shrimp allergy in a murine model of shrimp tropomyosin sensitivity. Intradermal immunization of BALB/c mice with pMEM49 or pMED171 effectively down-modulated allergic symptoms, tropomyosin-specific IgE levels, intestinal Th2 cytokines expression, and inflammatory cell infiltration. Both pMEM49 and pMED171 increased the frequency of regulatory T cells, but to a greater extent by pMED171 with upregulation of gut-homing molecules integrin-α4ß7. The functionality of the pMED171-induced Treg cells was further illustrated by anti-CD25-mediated depletion of Treg cells and the adoptive transfer of CD4+CD25+Foxp3+Treg cells. Collectively, the data demonstrate that intradermal administration of pMED171 leads to the priming, activation, and migration of dermal dendritic cells which subsequently induce Treg cells, both locally and systemically, to downregulate the allergic responses to tropomyosin. This study is the first to demonstrate the potency of hypoallergen-encoding DNA vaccines as a therapeutic strategy for human shellfish allergy via the vigorous induction of functional Treg cells.

Contrasting population genetic structure in three aggregating groupers (Percoidei: Epinephelidae) in the Indo-West Pacific: the importance of reproductive mode.

BACKGROUND: Understanding the factors shaping population genetic structure is important for evolutionary considerations as well as for management and conservation. While studies have revealed the importance of palaeogeographic changes in shaping phylogeographic patterns in multiple marine fauna, the role of reproductive behaviour is rarely considered in reef fishes. We investigated the population genetics of three commercially important aggregating grouper species in the Indo-West Pacific, namely the camouflage grouper Epinephelus polyphekadion, the squaretail coral grouper Plectropomus areolatus, and the common coral trout P. leopardus, with similar life histories but distinct spatio-temporal characteristics in their patterns of forming spawning aggregations. RESULTS: By examining their mitochondrial control region and 9-11 microsatellite markers, we found an overarching influence of palaeogeographic events in the population structure of all species, with genetic breaks largely coinciding with major biogeographic barriers. The divergence time of major lineages in these species coincide with the Pleistocene glaciations. Higher connectivity is evident in E. polyphekadion and P. areolatus that assemble in larger numbers at fewer spawning aggregations and in distinctive offshore locations than in P. leopardus which has multiple small, shelf platform aggregations. CONCLUSIONS: While palaeogeographic events played an important role in shaping the population structure of the target species, the disparity in population connectivity detected may be partly attributable to differences in their reproductive behaviour, highlighting the need for more investigations on this characteristic and the need to consider reproductive mode in studies of connectivity and population genetics.

Screening and identification of mimotopes of the major shrimp allergen tropomyosin using one-bead-one-compound peptide libraries.

The one-bead-one-compound (OBOC) combinatorial peptide library is a powerful tool to identify ligand and receptor interactions. Here, we applied the OBOC library technology to identify mimotopes specific to the immunoglobulin E (IgE) epitopes of the major shellfish allergen tropomyosin. OBOC peptide libraries with 8-12 amino acid residues were screened with serum samples from patients with shellfish allergy for IgE mimotopes of tropomyosin. Twenty-five mimotopes were identified from the screening and their binding reactivity to tropomyosin-specific IgE was confirmed by peptide ELISA. These mimotopes could be divided into seven clusters based on sequence homology, and epitope mapping by EpiSearch of the clustered mimotopes was performed to characterize and confirm the validity of mimotopes. Five out of six of the predicted epitopes were found to overlap with previously identified epitopes of tropomyosin. To further confirm the mimicry potential of mimotopes, BALB/c mice were immunized with mimotopes conjugated to keyhole limpet hemocyanin and assayed for their capacity to induce tropomyosin-specific antibodies. BALB/c mice that received mimotope immunization were found to have an elevated level of tropomyosin-specific immunoglobulin G, but not mice that received an irrelevant mimotope. This study pioneers the successful application of the OBOC libraries using whole sera to screen and identify multiple shrimp allergen mimotopes and validates their mimicry potential using in vitro, in vivo, and in silico methods.Cellular & Molecular Immunology advance online publication, 14 september 2015; doi:10.1038/cmi.2015.83.

Gastrointestinal Immune Response to the Shrimp Allergen Tropomyosin: Histological and Immunological Analysis in an Animal Model of Shrimp Tropomyosin Hypersensitivity.

BACKGROUND: Shellfish hypersensitivity is among the most common food allergies. A murine model of IgE-mediated shrimp allergy has been established in our laboratory. The aim of this study is to determine the intestinal histological changes and cytokine expression profile of this model sensitized with the major shellfish allergen tropomyosin. METHODS: Female Balb/c mice orally sensitized and challenged with recombinant tropomyosin were sacrificed. Continuous sections of duodenum, jejunum and ileum were prepared using the Swiss roll technique for histological and immunological analysis. Duodenal epithelial cell apoptosis and migration were examined. mRNA expression of IL-4, IL-6, IL-10, IL-13, IL-18 and IFN-γ in intestinal tissue was measured via RT-PCR. RESULTS: In tropomyosin-sensitized and challenged mice, an increased number of eosinophils, mast cells and goblet cells was found 24 h after challenge. There were also increased mast cell and goblet cell numbers at 72 h after challenge, but the level of eosinophils decreased. Differences compared with control mice are most prominent at the duodenum compared to the distal regions. In addition, TUNEL assay indicates a significantly higher apoptosis rate in sensitized mice sacrificed 72 h after challenge, and mRNA expression showed a biased Th2/Th1 cytokine profile and a higher level of murine mast cell protease 1. CONCLUSIONS: This study documented a multitude of histological and immunological changes in the gut in a murine model of shrimp allergy. Even without repetitive intragastric challenge, shrimp tropomyosin induces an increase in the number of inflammatory cells to varying degrees within the small intestine. This model provides an important tool for testing new therapeutic interventions.

Molecular phylogeny of the acorn barnacle family Tetraclitidae (Cirripedia: Balanomorpha: Tetraclitoidea): validity of shell morphology and arthropodal characteristics in the systematics of Tetraclitid barnacles.

Shell structure is a crucial aspect of barnacle systematics. Within Tetraclitidae, the diametric and monometric growth patterns and number of rows of parietal tubes in the shells are key characteristics used to infer evolutionary trends. We used molecular analysis based on seven genes (mitochondrial COI, 16S and 12S rRNA, and nuclear EF1, RPII, H3, and 18S rRNA) to test two traditional phylogenetic hypothesis: (1) Tetraclitid barnacles are divided into two major lineages, which are distinguished according to monometric and diametric shell growth patterns, and (2) the evolutionary trend in shell parietal development began with a solid shell, which developed into a single tubiferous shell, which then developed into multitubiferous shells. The results indicated that Tetraclitinae and Newmanellinae are not monophyletic, but that Austrobalaninae and Tetraclitellinae are. The phylogram based on the genetic data suggested that Bathylasmatidae is nested within the Tetraclitidae, forming a sister relationship with the Austrobalaninae and Tetraclitinae/Newmanellinae clade. Within the Tetraclitinae/Newmanellinae clade, the genera Tetraclita (multitubiferous shell), Tesseropora (single tubiferous shell), and Yamaguchiella (multitubiferous shell) are polyphyletic. The results suggested that shell morphology and growth patterns do not reflect the evolutionary history of Tetraclitidae, whereas the arthropodal characteristics are informative.

Phylogenetic relationships among genera of the Periclimenes complex (Crustacea: Decapoda: Pontoniinae) based on mitochondrial and nuclear DNA.

The genus Periclimenes Costa, 1844 is the most species-rich genus in the subfamily Pontoniinae. Recent studies have suggested that it might be a polyphyletic taxon and could be further subdivided. In this study, three protein-coding nuclear genes and one mitochondrial ribosomal gene were used to analyze the phylogenetic relationships among the genera of the Periclimenes complex, includes the genus Periclimenes and 15 related genera, viz. Ancylomenes, Brucecaris, Crinotonia, Cuapetes, Harpiliopsis, Harpilius, Laomenes, Leptomenaeus, Manipontonia, Palaemonella, Periclimenella, Philarius, Phycomenes, Unguicaris and Vir. Based on both independent and combined data analyses, the results support that the genus Periclimenes is a polyphyletic group. Furthermore, the studied Periclimenes species could be divided into several independent groups, and the taxonomic status of P. commensalis, P. brevicarpalis and P. digitalis may need to be reconsidered. Besides, the majority of the related genera of Periclimenes are suggested to be monophyletic. Our analyses also reveal that these genera approximately form two main clades, despite some deep relationships are still obscure.

Evolution and phylogeny of the mud shrimps (Crustacea: Decapoda) revealed from complete mitochondrial genomes.

BACKGROUND: The evolutionary history and relationships of the mud shrimps (Crustacea: Decapoda: Gebiidea and Axiidea) are contentious, with previous attempts revealing mixed results. The mud shrimps were once classified in the infraorder Thalassinidea. Recent molecular phylogenetic analyses, however, suggest separation of the group into two individual infraorders, Gebiidea and Axiidea. Mitochondrial (mt) genome sequence and structure can be especially powerful in resolving higher systematic relationships that may offer new insights into the phylogeny of the mud shrimps and the other decapod infraorders, and test the hypothesis of dividing the mud shrimps into two infraorders. RESULTS: We present the complete mitochondrial genome sequences of five mud shrimps, Austinogebia edulis, Upogebia major, Thalassina kelanang (Gebiidea), Nihonotrypaea thermophilus and Neaxius glyptocercus (Axiidea). All five genomes encode a standard set of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and a putative control region. Except for T. kelanang, mud shrimp mitochondrial genomes exhibited rearrangements and novel patterns compared to the pancrustacean ground pattern. Each of the two Gebiidea species (A. edulis and U. major) and two Axiidea species (N. glyptocercus and N. thermophiles) share unique gene order specific to their infraorders and analyses further suggest these two derived gene orders have evolved independently. Phylogenetic analyses based on the concatenated nucleotide and amino acid sequences of 13 protein-coding genes indicate the possible polyphyly of mud shrimps, supporting the division of the group into two infraorders. However, the infraordinal relationships among the Gebiidea and Axiidea, and other reptants are poorly resolved. The inclusion of mt genome from more taxa, in particular the reptant infraorders Polychelida and Glypheidea is required in further analysis. CONCLUSIONS: Phylogenetic analyses on the mt genome sequences and the distinct gene orders provide further evidences for the divergence between the two mud shrimp infraorders, Gebiidea and Axiidea, corroborating previous molecular phylogeny and justifying their infraordinal status. Mitochondrial genome sequences appear to be promising markers for resolving phylogenetic issues concerning decapod crustaceans that warrant further investigations and our present study has also provided further information concerning the mt genome evolution of the Decapoda.

Characterization of heat shock protein 90 in the shrimp Metapenaeus ensis: Evidence for its role in the regulation of vitellogenin synthesis.

Estrogen hormones play a vital role in the regulation of female reproductive maturation. In oviparous vertebrates, the synthesis of vitellogenin (VTG) is tightly controlled by estrogen hormone signal transduction pathway, which is mediated by estrogen receptor and heat shock protein 90 (Hsp90). In order to investigate whether a similar mechanism exists in crustaceans, the Hsp90 gene was cloned and isolated from the shrimp Metapenaeus ensis by homology cloning strategy. The Hsp90 is 2,524 bp in length, containing an open reading frame of 2,163 bp that encodes a 720 amino acid polypeptide (83 kD). The Hsp90-coding region is interrupted by four introns. MeHsp90 is differentially expressed in eyestalk, ovary, and hepatopancreas at different ovarian maturation stages, and consistently expressed in other tissues including heart, gill, gut, muscle, and central nervous system. In vitro ovary explant assay reveals that MeHsp90 expression in immature ovary can be induced by the addition of exogenous estradiol-17beta, but expression in fully mature ovary exhibits no response to estradiol-17beta treatment. In situ hybridization shows that MeHsp90 is highly expressed in previtellogenic oocytes and its expression decreases with the progress of maturation, and finally stops in late-vitellogenic oocytes. Our results indicate a strong correlation between estrogen hormones and Hsp90 expression in shrimp, suggesting that the expression of VTG may be under the regulation of estrogen hormones through a mechanism similar to that in vertebrates. The result provides insights on the control of vitellogenesis in invertebrates.

Molecular coordinated regulation of gene expression during ovarian development in the penaeid shrimp.

To understand the molecular events of ovarian development in penaeid shrimp, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed genes during ovarian maturation in Metapenaeus ensis. From a screening of 700 clones in a cDNA library of the shrimp ovary by the products of RAP-PCR of different maturation stages, 91 fragments with differentially expressed pattern as revealed by dot-blot hybridization were isolated and sequenced. Forty-two of these fragments show significant sequence similarity to known gene products and the differentially expressed pattern of 10 putative genes were further characterized via Northern hybridization. Putative glyceraldehyde-3-phosphate dehydrogenase and arginine kinase are related to provision of energy for active cellular function in oocyte development. Translationally controlled tumor protein, actin, and keratin are related to the organization of cytoskeleton to accomplish growth and development of oocytes. High mobility group protein DSP1, heat shock protein 70, and nucleoside diphosphate kinase may act as repressors before the onset of ovarian maturation. Peptidyl-prolyl cis-trans isomerase and glutathione peroxidase are related to the stabilization of proteins and oocytes. This study provides new insights on the molecular events in the ovarian development in the shrimp.

Crustacean neuropeptide genes of the CHH/MIH/GIH family: implications from molecular studies.

The crustacean eyestalk CHH/MIH/GIH gene family represents a unique group of neuropeptide originally identified in crustaceans. These neuropeptides shared a high degree of amino acid identity, and the conservation of cysteine residues at the same relative positions. Based on their biological, biochemical, and molecular properties, they can be divided into the CHH and MIH subtypes with two major members in each subtype. In the shrimp, the CHH-subtypes can be divided into two forms (CHH-A and CHH-B). The CHH-A gene also comprises several isoforms which shared a high overall sequence identity. Although the MIH subtypes are postulated to have evolved from the CHH subtypes, the number of major MIH subtypes in each species has yet to be confirmed. While most of the genes consist of the basic plan of three exons and two introns, other alternative spliced variants have recently been described. Moreover, these alternative forms are usually expressed in non-eyestalk tissues. These findings suggest that these neuropeptides may have a broader spectrum of functions in crustaceans. The results from phylogenetic analysis suggest that the evolution of this group of neuropeptides occurs in a manner similar is to the gene duplication and mutation events hypothesized for the origin of the prolactin and growth hormone gene family of the vertebrate pituitary system.