Convergent adaptation of true crabs (Decapoda: Brachyura) to a gradient of terrestrial environments.

For much of terrestrial biodiversity, the evolutionary pathways of adaptation from marine ancestors are poorly understood, and have usually been viewed as a binary trait. True crabs, the decapod crustacean infraorder Brachyura, comprise over 7,600 species representing a striking diversity of morphology and ecology, including repeated adaptation to non-marine habitats. Here, we reconstruct the evolutionary history of Brachyura using new and published sequences of 10 genes for 344 tips spanning 88 of 109 brachyuran families. Using 36 newly vetted fossil calibrations, we infer that brachyurans most likely diverged in the Triassic, with family-level splits in the late Cretaceous and early Paleogene. By contrast, the root age is underestimated with automated sampling of 328 fossil occurrences explicitly incorporated into the tree prior, suggesting such models are a poor fit under heterogeneous fossil preservation. We apply recently defined trait-by-environment associations to classify a gradient of transitions from marine to terrestrial lifestyles. We estimate that crabs left the marine environment at least seven and up to 17 times convergently, and returned to the sea from non-marine environments at least twice. Although the most highly terrestrial- and many freshwater-adapted crabs are concentrated in Thoracotremata, Bayesian threshold models of ancestral state reconstruction fail to identify shifts to higher terrestrial grades due to the degree of underlying change required. Lineages throughout our tree inhabit intertidal and marginal marine environments, corroborating the inference that the early stages of terrestrial adaptation have a lower threshold to evolve. Our framework and extensive new fossil and natural history datasets will enable future comparisons of non-marine adaptation at the morphological and molecular level. Crabs provide an important window into the early processes of adaptation to novel environments, and different degrees of evolutionary constraint that might help predict these pathways.

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.

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.

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.

Comprehending the allergen repertoire of shrimp for precision molecular diagnosis of shrimp allergy.

BACKGROUND: Clinical management of shrimp allergy is hampered by the lack of accurate tests. Molecular diagnosis has been shown to more accurately reflect the clinical reactivity but the full spectrum of shrimp allergens and their clinical relevance are yet to be established. We therefore sought to comprehend the allergen repertoire of shrimp, investigate and compare the sensitization pattern and diagnostic value of the allergens in allergic subjects of two distinct populations. METHODS: Sera were collected from 85 subjects with challenge-proven or doctor-diagnosed shrimp allergy in Hong Kong and Thailand. The IgE-binding proteins of Penaeus monodon were probed by Western blotting and identified by mass spectrometry. Recombinant shrimp allergens were synthesized and analyzed for IgE sensitization by ELISA. RESULTS: Ten IgE-binding proteins were identified, and a comprehensive panel of 11 recombinant shrimp allergens was generated. The major shrimp allergens among Hong Kong subjects were troponin C (Pen m 6) and glycogen phosphorylase (Pen m 14, 47.1%), tropomyosin (Pen m 1, 41.2%) and sarcoplasmic-calcium binding protein (Pen m 4, 35.3%), while those among Thai subjects were Pen m 1 (68.8%), Pen m 6 (50.0%) and fatty acid-binding protein (Pen m 13, 37.5%). Component-based tests yielded significantly higher area under curve values (0.77-0.96) than shrimp extract-IgE test (0.70-0.75). Yet the best component test differed between populations; Pen m 1-IgE test added diagnostic value only in the Thai cohort, whereas sensitizations to other components were better predictors of shrimp allergy in Hong Kong patients. CONCLUSION: Pen m 14 was identified as a novel shrimp allergen predictive of challenge outcome. Molecular diagnosis better predicts shrimp allergy than conventional tests, but the relevant component is population dependent.

Comparative genomics of the coconut crab and other decapod crustaceans: exploring the molecular basis of terrestrial adaptation.

BACKGROUND: The complex life cycle of the coconut crab, Birgus latro, begins when an obligate terrestrial adult female visits the intertidal to hatch zoea larvae into the surf. After drifting for several weeks in the ocean, the post-larval glaucothoes settle in the shallow subtidal zone, undergo metamorphosis, and the early juveniles then subsequently make their way to land where they undergo further physiological changes that prevent them from ever entering the sea again. Here, we sequenced, assembled and analyzed the coconut crab genome to shed light on its adaptation to terrestrial life. For comparison, we also assembled the genomes of the long-tailed marine-living ornate spiny lobster, Panulirus ornatus, and the short-tailed marine-living red king crab, Paralithodes camtschaticus. Our selection of the latter two organisms furthermore allowed us to explore parallel evolution of the crab-like form in anomurans. RESULTS: All three assembled genomes are large, repeat-rich and AT-rich. Functional analysis reveals that the coconut crab has undergone proliferation of genes involved in the visual, respiratory, olfactory and cytoskeletal systems. Given that the coconut crab has atypical mitochondrial DNA compared to other anomurans, we argue that an abundance of kif22 and other significantly proliferated genes annotated with mitochondrial and microtubule functions, point to unique mechanisms involved in providing cellular energy via nuclear protein-coding genes supplementing mitochondrial and microtubule function. We furthermore detected in the coconut crab a significantly proliferated HOX gene, caudal, that has been associated with posterior development in Drosophila, but we could not definitively associate this gene with carcinization in the Anomura since it is also significantly proliferated in the ornate spiny lobster. However, a cuticle-associated coatomer gene, gammacop, that is significantly proliferated in the coconut crab, may play a role in hardening of the adult coconut crab abdomen in order to mitigate desiccation in terrestrial environments. CONCLUSION: The abundance of genomic features in the three assembled genomes serve as a source of hypotheses for future studies of anomuran environmental adaptations such as shell-utilization, perception of visual and olfactory cues in terrestrial environments, and cuticle sclerotization. We hypothesize that the coconut crab exhibits gene proliferation in lieu of alternative splicing as a terrestrial adaptation mechanism and propose life-stage transcriptomic assays to test this hypothesis.

Micro-RNA Clusters Integrate Evolutionary Constraints on Expression and Target Affinities: The miR-6/5/4/286/3/309 Cluster in Drosophila.

A striking feature of micro-RNAs is that they are often clustered in the genomes of animals. The functional and evolutionary consequences of this clustering remain obscure. Here, we investigated a micro-RNA cluster miR-6/5/4/286/3/309 that is conserved across drosophilid lineages. Small RNA sequencing revealed expression of this micro-RNA cluster in Drosophila melanogaster leg discs, and conditional overexpression of the whole cluster resulted in leg appendage shortening. Transgenic overexpression lines expressing different combinations of micro-RNA cluster members were also constructed. Expression of individual micro-RNAs from the cluster resulted in a normal wild-type phenotype, but either the expression of several ancient micro-RNAs together (miR-5/4/286/3/309) or more recently evolved clustered micro-RNAs (miR-6-1/2/3) can recapitulate the phenotypes generated by the whole-cluster overexpression. Screening of transgenic fly lines revealed downregulation of leg-patterning gene cassettes in generation of the leg-shortening phenotype. Furthermore, cell transfection with different combinations of micro-RNA cluster members revealed a suite of downstream genes targeted by all cluster members, as well as complements of targets that are unique for distinct micro-RNAs. Considered together, the micro-RNA targets and the evolutionary ages of each micro-RNA in the cluster demonstrate the importance of micro-RNA clustering, where new members can reinforce and modify the selection forces on both the cluster regulation and the gene regulatory network of existing micro-RNAs. Key words: micro-RNA, cluster, evolution.

Gut Microbiota in Decapod Shrimps: Evidence of Phylosymbiosis.

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

ENPD - A Database of Eukaryotic Nucleic Acid Binding Proteins: Linking Gene Regulations to Proteins.

Eukaryotic nucleic acid binding protein database (ENPD, http://qinlab.sls.cuhk.edu.hk/ENPD/) is a library of nucleic acid binding proteins (NBPs) and their functional information. NBPs such as DNA binding proteins (DBPs), RNA binding proteins (RBPs), and DNA and RNA binding proteins (DRBPs) are involved in every stage of gene regulation through their interactions with DNA and RNA. Due to the importance of NBPs, the database was constructed based on manual curation and a newly developed pipeline utilizing both sequenced transcriptomes and genomes. In total the database has recorded 2.8 million of NBPs and their binding motifs from 662 NBP families and 2423 species, constituting the largest NBP database. ENPD covers evolutionarily important lineages which have never been included in the previous NBP databases, while lineage-specific NBP family expansions were also found. ENPD also focuses on the involvements of DBPs, RBPs and DRBPs in non-coding RNA (ncRNA) mediated gene regulation. The predicted and experimentally validated targets of NBPs have both been recorded and manually curated in ENPD, linking the interactions between ncRNAs, DNA regulatory elements and NBPs in gene regulation. This database provides key resources for the scientific community, laying a solid foundation for future gene regulatory studies from both functional and evolutionary perspectives.

A crustacean annotated transcriptome (CAT) database.

BACKGROUND: Decapods are an order of crustaceans which includes shrimps, crabs, lobsters and crayfish. They occur worldwide and are of great scientific interest as well as being of ecological and economic importance in fisheries and aquaculture. However, our knowledge of their biology mainly comes from the group which is most closely related to crustaceans - insects. Here we produce a de novo transcriptome database, crustacean annotated transcriptome (CAT) database, spanning multiple tissues and the life stages of seven crustaceans. DESCRIPTION: A total of 71 transcriptome assemblies from six decapod species and a stomatopod species, including the coral shrimp Stenopus hispidus, the cherry shrimp Neocaridina davidi, the redclaw crayfish Cherax quadricarinatus, the spiny lobster Panulirus ornatus, the red king crab Paralithodes camtschaticus, the coconut crab Birgus latro, and the zebra mantis shrimp Lysiosquillina maculata, were generated. Differential gene expression analyses within species were generated as a reference and included in a graphical user interface database at http://cat.sls.cuhk.edu.hk/. Users can carry out gene name searches and also access gene sequences based on a sequence query using the BLAST search function. CONCLUSIONS: The data generated and deposited in this database offers a valuable resource for the further study of these crustaceans, as well as being of use in aquaculture development.

Overcoming Shellfish Allergy: How Far Have We Come?

Shellfish allergy caused by undesirable immunological responses upon ingestion of crustaceans and mollusks is a common cause of food allergy, especially in the Asia-Pacific region. While the prevalence of shellfish allergy is increasing, the mainstay of clinical diagnosis for these patients includes extract-based skin prick test and specific IgE measurement while clinical management consists of food avoidance and as-needed use of adrenaline autoinjector should they develop severe allergic reactions. Such a standard of care is unsatisfactory to both patients and healthcare practitioners. There is a pressing need to introduce more specific diagnostic methods, as well as effective and safe therapies for patients with shellfish allergy. Knowledge gained on the identifications and defining the immuno-molecular features of different shellfish allergens over the past two decades have gradually translated into the design of new diagnostic and treatment options for shellfish allergy. In this review, we will discuss the epidemiology, the molecular identification of shellfish allergens, recent progress in various diagnostic methods, as well as current development in immunotherapeutic approaches including the use of unmodified allergens, hypoallergens, immunoregulatory peptides and DNA vaccines for the prevention and treatment of shellfish allergy. The prospect of a "cure "for shellfish allergy is within reach.

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

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

Systematic analysis of the caridean shrimp superfamily Pandaloidea (Crustacea: Decapoda) based on molecular and morphological evidence.

One of the systematically controversial superfamilies in Caridea is the predominately deep-sea or cold water Pandaloidea, largely because this species-rich group of nearly 200 species in 25 genera exhibits a very high diversity of body forms and ecology. Although the relationships amongst the taxa within Pandaloidea have been repeatedly discussed based on morphology, no comprehensive molecular phylogeny exists. In this study, we present the first molecular phylogeny of the group, based on a combined dataset of two mitochondrial (12S and 16S rRNA) and six nuclear (ATP synthase ß-subunit, enolase, glyceraldehyde-3-phosphate dehydrogenase, histone 3, phosphoenolpyruvate carboxykinase and sodium-potassium ATPase α-subunit) markers, based on 62 species (about 1/3 of known biodiversity) in 22 genera (88% of genera) of two pandaloid families (Pandalidae, Thalassocarididae) and outgroups from seven other caridean families. With generally high support, the relationships within the clade are fully resolved. Pandalidae is shown to be paraphyletic with Thalassocarididae deeply nested within as a monophyletic group, and the latter is herein considered to be a synonym of Pandalidae. Five major clades are recovered, with the shallow water genera Anachlorocurtis, Chlorocurtis, Chlorotocella and Miropandalus forming a sister clade to the remaining genera. At the genus level, the phylogeny indicates Plesionika, Heterocarpus and Pandalus to be not monophyletic. The validity of Pandalopsis, Stylopandalus and Calipandalus is challenged and these genera are considered herein to be junior synonyms of Pandalus (Pandalopsis) and Plesionika (Stylopandalus and Calipandalus). Although not fully resolved, some evidence potentially considers Nothocaris to be a valid genus. Ancestral State Reconstruction successfully recovered 15 synapomorphies for the major clades, with 11 of them reported to be of systematic significance for the first time.

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.

CrusTF: a comprehensive resource of transcriptomes for evolutionary and functional studies of crustacean transcription factors.

BACKGROUND: Crustacea, the second largest subphylum of Arthropoda, includes species of major ecological and economic importance, such as crabs, lobsters, crayfishes, shrimps, and barnacles. With the rapid development of crustacean aquaculture and biodiversity loss, understanding the gene regulatory mechanisms of growth, reproduction, and development of crustaceans is crucial to both aquaculture development and biodiversity conservation of this group of organisms. In these biological processes, transcription factors (TFs) play a vital role in regulating gene expression. However, crustacean transcription factors are still largely unknown, because the lack of complete genome sequences of most crustacean species hampers the studies on their transcriptional regulation on a system-wide scale. Thus, the current TF databases derived from genome sequences contain TF information for only a few crustacean species and are insufficient to elucidate the transcriptional diversity of such a large animal group. RESULTS: Our database CrusTF ( http://qinlab.sls.cuhk.edu.hk/CrusTF ) provides comprehensive information for evolutionary and functional studies on the crustacean transcriptional regulatory system. CrusTF fills the knowledge gap of transcriptional regulation in crustaceans by exploring publicly available and newly sequenced transcriptomes of 170 crustacean species and identifying 131,941 TFs within 63 TF families. CrusTF features three categories of information: sequence, function, and evolution of crustacean TFs. The database enables searching, browsing and downloading of crustacean TF sequences. CrusTF infers DNA binding motifs of crustacean TFs, thus facilitating the users to predict potential downstream TF targets. The database also presents evolutionary analyses of crustacean TFs, which improve our understanding of the evolution of transcriptional regulatory systems in crustaceans. CONCLUSIONS: Given the importance of TF information in evolutionary and functional studies on transcriptional regulatory systems of crustaceans, this database will constitute a key resource for the research community of crustacean biology and evolutionary biology. Moreover, CrusTF serves as a model for the construction of TF database derived from transcriptome data. A similar approach could be applied to other groups of organisms, for which transcriptomes are more readily available than genomes.

MicroRNAs regulate the sesquiterpenoid hormonal pathway in Drosophila and other arthropods.

Arthropods comprise the majority of all described animal species, and understanding their evolution is a central question in biology. Their developmental processes are under the precise control of distinct hormonal regulators, including the sesquiterpenoids juvenile hormone (JH) and methyl farnesoate. The control of the synthesis and mode of action of these hormones played important roles in the evolution of arthropods and their adaptation to diverse habitats. However, the precise roles of non-coding RNAs, such as microRNAs (miRNAs), controlling arthropod hormonal pathways are unknown. Here, we investigated the miRNA regulation of the expression of the juvenile hormone acid methyltransferase gene (JHAMT), which encodes a rate-determining sesquiterpenoid biosynthetic enzyme. Loss of function of the miRNA bantam in the fly Drosophila melanogaster increased JHAMT expression, while overexpression of the bantam repressed JHAMT expression and resulted in pupal lethality. The male genital organs of the pupae were malformed, and exogenous sesquiterpenoid application partially rescued the genital deformities. The role of the bantam in the regulation of sesquiterpenoid biosynthesis was validated by transcriptomic, qPCR and hormone titre (JHB3 and JH III) analyses. In addition, we found a conserved set of miRNAs that interacted with JHAMT, and the sesquiterpenoid receptor methoprene-tolerant (Met) in different arthropod lineages, including insects (fly, mosquito and beetle), crustaceans (water flea and shrimp), myriapod (centipede) and chelicerate (horseshoe crab). This suggests that these miRNAs might have conserved roles in the post-transcriptional regulation of genes in sesquiterpenoid pathways across the Panarthropoda. Some of the identified lineage-specific miRNAs are potential targets for the development of new strategies in aquaculture and agricultural pest control.

Molecular phylogeny of Pasiphaeidae (Crustacea, Decapoda, Caridea) reveals systematic incongruence of the current classification.

Caridean shrimps constitute one of the most diverse groups of decapod crustaceans, notwithstanding their poorly resolved infraordinal relationships. One of the systematically controversial families in Caridea is the predominantly pelagic Pasiphaeidae, comprises 101 species in seven genera. Pasiphaeidae species exhibit high morphological disparity, as well as ecological niche width, inhabiting shallow to very deep waters (>4000m). The present work presents the first molecular phylogeny of the family, based on a combined dataset of six mitochondrial and nuclear gene markers (12S rDNA, 16S rDNA, histone 3, sodium-potassium ATPase α-subunit, enolase and ATP synthase ß-subunit) from 33 species belonged to six genera of Pasiphaeidae with 19 species from 12 other caridean families as outgroup taxa. Maximum likelihood and Bayesian inference analyses conducted on the concatenated dataset of 2265bp suggest the family Pasiphaeidae is not monophyletic, with Psathyrocaris more closely related to other carideans than to the other five pasiphaeid genera included in this analysis. Leptochela occupies a sister position to the remaining genera and is genetically quite distant from them. At the generic level, the analysis supports the monophyly of Pasiphaea, Leptochela and Psathyrocaris, while Eupasiphae is shown to be paraphyletic, closely related to Parapasiphae and Glyphus. The present molecular result strongly implies that certain morphological characters used in the present systematic delineation within Pasiphaeidae may not be synapomorphies and the classification within the family needs to be urgently revised.

Low-Dose Allergen-Specific Immunotherapy Induces Tolerance in a Murine Model of Shrimp Allergy.

BACKGROUND: The efficacy and safety of allergen-specific immunotherapy (AIT) are highly dose-dependent. METHODS: We investigated the dosage effects of AIT and the underlying mechanisms in a murine model of shrimp hypersensitivity. BALB/c mice were sensitized with recombinant shrimp allergen rMet e 1 and challenged orally with a high dose of rMet e 1 to elicit an allergic response. These sensitized mice were then treated with a low (0.01 mg), medium (0.05 mg), or high dosage (0.1 mg) of rMet e 1 intraperitoneally before receiving a second oral challenge. The allergic responses and immunological changes in the gut were compared between animals receiving different dosages. RESULTS: We found that all sensitized mice that received rMet e 1 immunotherapy were desensitized, regardless of the dosage, and protected at the second oral challenge. Nevertheless, the mice in the high-dosage group experienced severe systemic reactions during the treatment phase. In contrast, regulatory T (Treg) cell-associated genes were upregulated only in the low- and medium-dosage groups, and Foxp3+ cells were more abundant in the gut lymphoid tissues than in the high-dosage group. CONCLUSIONS: Our results demonstrate that low-dosage immunotherapy favors the induction of local Foxp3+ Treg cells and the upregulation of regulatory cytokines. The safety advantages and long-term efficacy of low-dosage immunotherapy should be taken into consideration when developing immunotherapy dose schedules.

Multilocus approach reveals cryptic lineages in the goby Rhinogobius duospilus in Hong Kong streams: Role of paleodrainage systems in shaping marked population differentiation in a city.

Drainage history is a well-demonstrated factor that influences the population structure of freshwater inhabitants over a broad geographic scale. However, there has been little research undertaken on such a relationship with freshwater fish on a small geographical scale, especially in Asia. In this study, we investigated the role of local, small drainage systems in affecting the population genetic structure of a freshwater goby, Rhinogobius duospilus, in Hong Kong streams using a multilocus approach. Analyses on nine genetic markers (2 mitochondrial and 7 nuclear markers, including 5 microsatellite markers) reveal prominent and intensive genetic structuring (2.1-5.4% mtDNA sequence divergence) in R. duospilus in Hong Kong. The lineages and clusters recovered from mtDNA data and assignment analysis of nuclear markers coincide with the paleodrainage networks. Furthermore, marked population subdivision between streams located on different side branches (<20km apart) within the same paleodrainage area is observed and gene flow occurs only between closely situated streams that share common paleodrainage confluences. In an extreme case, gene flow is limited between streams that are less than 5km apart. Apparently, such an intensive population structure is attributed to the regional paleodrainage pattern, together with the highly sedentary life style of R. duospilus, which reduces contemporary gene flow and dispersal between populations in neighbouring streams.