Nitric oxide plays a crucial role in midgut immunity under microsporidian infection in Antheraea pernyi.

The insect gut participates in initial local immune responses by producing reactive oxygen and nitrogen species as well as anti-microbial peptides to resist pathogenic invasions. Nitric oxide (NO), a signaling and an immune effector molecule synthesized by the enzyme NO synthase (NOS), mediates an early step of the signal transduction pathway. In this study, we evaluated NO levels after Nosema pernyi infection in Antheraea pernyi gut. NOS activity was higher in the microsporidia-infected gut of A. pernyi than in that of control. Three NOS-related genes were cloned, and their spatio-temporal expression patterns were evaluated. ApNOS2 was expressed quickly in the midgut after N. pernyi infection. Sodium nitroprusside, dihydrate (SNP), or Nω-L-nitro-arginine methyl ester, hydrochloride (L-NAME), altered the NO content in A. pernyi midgut. Anti-microbial peptides (AMPs) in the groups exposed to N. pernyi plus SNP and N. pernyi plus L-NAME exhibited higher and lower expression, respectively, relative to the control. These results indicate that microsporidia infection triggers short-term activation of NO and NOS genes in the A. pernyi gut that is downregulated after 24 h. Notably, infection rates can be influenced by a NOS inhibitor. Furthermore, NO can be induced by pathogens. Similarly, NO content in the A. pernyi gut also influences AMPs in humoral immunity and some immune-related genes. Our results suggest that nitric oxide plays a vital role in A. pernyi gut immunity.

QTL mapping using microsatellite linkage reveals target-site mutations associated with high levels of resistance against three mitochondrial complex II inhibitors in Tetranychus urticae.

The acaricides cyflumetofen, cyenopyrafen, and pyflubumide act as inhibitors of the mitochondrial electron transport system at complex II (succinate dehydrogenase; SDH), a new mode of action in arthropods. The development and mechanisms of low-level resistance against cyenopyrafen and cyflumetofen have been previously reported in Tetranychus urticae. In the present study, we investigated high levels of resistance against three SDH inhibitors in T. urticae field populations and clarify the genetic basis of resistance using quantitative trait locus (QTL) analysis. First, we constructed a microsatellite linkage map comprising 64 markers assembled into three linkage groups (LGs) with total length of 683.8 cM and average marker spacing of 11.03 cM. We then used the linkage map to perform QTL mapping, and identified significant QTLs contributing to resistance to cyflumetofen (one QTL on LG1), cyenopyrafen (one QTL on LG3), and pyflubumide (two QTLs on LG1 and LG3). The QTL peaks on LG1 for cyflumetofen and pyflubumide overlapped and included the SdhB locus. For cyenopyrafen resistance, the QTLs on LG3 included the SdhC locus. For cyflumetofen resistance, we found an I260T mutation in SdhB. For pyflubumide and cyenopyrafen resistance, we detected I260V and S56L substitutions in SdhB and SdhC, respectively, by direct sequencing. Both I260 in SdhB and S56 in SdhC were present in highly conserved regions of the ubiquinone binding site formed at the interface among SdhB, SdhC, and SdhD. Mutations at these positions have been implicated in resistance against fungicides that act as Sdh inhibitors in various pathogens. Therefore, we consider these mutations to be target-site resistance mutations for these acaricidal SDH inhibitors.

RNAi of the nuclear receptor HR3 suggests a role in the molting process of the spider mite Panonychus citri.

Ecdysteroids regulate molting in arthropods by binding to heterodimers of the ecdysone receptor and retinoid-X-receptor, homologous to the ultraspiracle protein, to induce the expression of downstream signal response genes including the nuclear receptor HR3. However, the detailed expression dynamics of HR3 during molting in spider mites are not yet clear. In this study, the full length of PcHR3 was retrieved based on the genome of citrus red mite, Panonychus citri. The open reading frame is 1707 bp encoding 568 amino acids, which contains a DNA binding domain and a ligand binding domain. Then, the expression pattern of PcHR3 was analyzed throughout the development of the deutonymph by RT-qPCR. The result showed that PcHR3 was mainly transcribed in the late deutonymph stage, when the deutonymph was at least 24 h old and motionless, the critical point at which the mites started molting. Transcription reached the highest level in 32-h-old deutonymphs and decreased by 36 h, where the mites remained in a quiescent state. Further silencing of PcHR3 by leaf-disc-based delivery of dsRNA to 8-h-old deutonymph mites, resulted in retarded development and death of 58% of deutonymphs. In summary, we suggest that PcHR3 regulates the latter stages of molting in P. citri.

Synonymous SNPs of viral genes facilitate virus to escape host antiviral RNAi immunity.

Synonymous single nucleotide polymorphisms (SNPs) are involved in codon usage preference or mRNA splicing. Up to date, however, the role of synonymous SNPs in immunity remains unclear. To address this issue, the SNPs of white spot syndrome virus (WSSV) were characterized in shrimp in the present study. Our results indicated that there existed synonymous SNPs in the mRNAs of wsv151 and wsv226, two viral genes of WSSV. In the presence of SNP siRNA, wild-type siRNA, wild-type mRNA and SNP mRNA of wsv151 or wsv226, RNAi was significantly suppressed, showing that the synonymous SNPs of wsv151 and wsv226 played negative roles in host siRNA pathway due to mismatch of siRNA with its target. In insect cells, the mismatch, caused by synonymous SNPs of wsv151 or wsv226, between siRNA and its target inhibited the host RNAi. Furthermore, the data revealed that the co-injection of SNP siRNA and wild-type siRNA of wsv151 or wsv226 into WSSV-infected shrimp led to a significant increase of WSSV copies compared with that of SNP siRNA alone or wild-type siRNA alone, indicating that the synonymous SNPs of viral genes could be a strategy of virus escaping host siRNA pathway in shrimp in vivo. Therefore, our study provided novel insights into the underlying mechanism of virus escaping host antiviral RNAi immunity by synonymous SNPs of viral genes.

Novel Method for the Purification of House Dust Mite Allergen Der p 1 and Its Use in Structure-Based Chemical Design of Novel Inhibitors.

House dust mites are globally significant triggers of allergic disease. Notable among their extensive repertoire of allergens are the Group 1 cysteine peptidase allergens which function as digestive enzymes in house dust mites. Compelling evidence suggests that the proteolytic activity of these molecules plays a key role in the development and maintenance of allergic diseases through the activation of innate immune mechanisms which exploit genetic predispositions to allergy. Growing interest in this area creates a requirement for high-quality purified protein, whether natural or recombinantly expressed. It has also identified these allergens as therapeutic targets for a novel approach to allergy treatment through modulation of innate immune responses. The purpose of this chapter is to describe a new method for the purification of Der p 1 and use of the protein produced in a screening assay designed for the discovery of novel inhibitors of Group 1 house dust mite allergens.

Understanding lipopolysaccharide aggregation and its influence on activation of Factor C.

The quantification of lipopolysaccharide (LPS) shed by bacteria within aqueous samples is typically performed by binding LPS to a protein called Factor C within a lysate prepared from the blood of horseshoe crabs (Limulus amebocyte lysate (LAL)). How the state of aggregation of LPS impacts Factor C activation, however, is not understood, particularly in the presence of select salts and non-ionic surfactants that are commonly incorporated into pharmaceutical formulations. To address this open question, herein we report on the aggregation status of LPS in aqueous solution, characterized using angle-dependent static and dynamic light scattering with and without chelating salts and polysorbate surfactants, and its correlation with activation of Factor C. Because the aggregation status of LPS is kinetically controlled, care was taken to compare LPS aggregation and activity using identically prepared samples. By plotting LPS activity versus the LPS aggregate size distribution over varied solution conditions, we found a positive correlation between LPS aggregate sizes between 30 and 50 nm and LAL activity. Overall, our results support the hypothesis that activation of Factor C is dependent of LPS aggregate size, and that the modulating effects of salts and surfactants on activation of Factor C is associated with changes in the LPS aggregation.

Involvement of gamma interferon inducible lysosomal thiol reductase in the innate immune responses of red swamp crayfish, Procambarus clarkii.

The Gamma interferon inducible lysosomal thiol reductase (GILT) plays a key biological role in the immune responses and involves in the processing of class II MHC-restricted antigen by stimulating disulfide bond reduction in mammals. To determine the biological function of GILT in the innate immune system of crustaceans, we sequenced and cloned GILT gene from red swamp crayfish, Procambarus clarkii (Pc-GILT). The deduced amino acid sequence of Pc-GILT contained the putative conserved structures of the GILT family proteins: the GILT signature (CQHGX2ECX2NX4C) sequence and the active site (CXXS) motif. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis suggested that a recombinant Pc-GILT protein was successfully expressed in Escherichia coli (E. coli). Quantitative real-time PCR analysis showed that Pc-GILT transcript level was highest in the hepatopancreas followed by the gut, heart and muscles. Additionally, we analyzed the transcription level of Pc-GILT gene in hepatopancreas of red swamp crayfish under biotic stress conditions. The expression of Pc-GILT gene upregulated after viral (poly I:C) and bacterial (peptidoglycan, lipopolysaccharide) infection. The suppression of Pc-GILT by double stranded RNA influenced the transcript levels of various immune-related genes. These observations indicate that the Pc-GILT probably plays a key biological role in the innate immune responses of red swamp crayfish, since it modulates the expression of genes associated with immune pathways.

Carbonyl reductase sniffer from the model organism daphnia: Cloning, substrate determination and inhibitory sensitivity.

Carbonyl reductases (CRs) represent a fundamental enzymatic defense mechanism against oxidative stress. While commonly two carbonyl reductases (CBR1 and CBR3) are found in mammalian genomes, invertebrate model organisms like Drosophila melanogaster express no CR but a functional homolog to human CBR1, termed sniffer. The importance of sniffer could be demonstrated in D. melanogaster where it protected against age-dependent neurodegeneration. Interestingly, the microcrustacean Daphnia harbors four copies of the CR gene (CR1, CR2, CR3, CR4) in addition to one sniffer gene. Due to this unique equipment Daphnia is an ideal model organism to investigate the function of sniffer. Recombinant sniffer from D. magna und D. pules were produced in E. coli, purified by Ni-affinity chromatography and tested with a variety of aliphatic and aromatic diketones, reactive aldehydes and precursors of advanced glycation end products (AGE). The highest catalytic activities were determined for sniffer from D. pulex with the aromatic dicarbonyls 9,10-phenanthrenequinone (kcat/Km = 2.6 s-1 x µM-1) and isatin (kcat/Km = 1.5 s-1 x µM-1). While sniffer from D. magna displayed preference for the same two substances, the respective catalytic activities were noticeably lower. Kinetic constants with aliphatic diketones were generally lower than those with aromatic dicarbonyls for both sniffer enzymes. The best aliphatic diketone as substrate for sniffer from D. magna and D. pulex was hexane-3,4-dione with kcat/Km = 0.23 s-1 µM-1 and kcat/Km = 0.35 s-1 µM-1, respectively. Poor or no detectable activity of the two sniffer enzymes was seen with the aliphatic diketones 2,5-hexanedione and 3,5-heptanedione, the aldehydes butanal, hexanal, decanal, crotonaldehyde, acrolein, trans-2-hexenal, and the AGE precursors glyoxal, methylglyoxal, furfural and glyceraldehyde, indicating no physiological function in the metabolism of short-chain aldehydes. Substrate inhibition for both sniffer enzymes was observed with the quinone substrates 1,4-naphthoquinone and 2-methyl-1,4-benzoquinone. From a variety of pesticides endosulfan turned out as an effective inhibitor of the sniffer enzymes (Ki = 9.2 µM for sniffer from D. magna, Ki = 12.0 µM for sniffer from D. pulex). In conclusion, the present results on sniffer from the protein superfamily of the short-chain dehydrogenases/reductases (SDR) in Daphnia ssp. complement earlier studies on carbonyl reductases in the same species and indicate that Daphnia is an interesting model to study the overall response to carbonyl stress.

The leucokinin-like peptide receptor from the cattle fever tick, Rhipicephalus microplus, is localized in the midgut periphery and receptor silencing with validated double-stranded RNAs causes a reproductive fitness cost.

The cattle fever tick, Rhipicephalus microplus (Canestrini) (Acari: Ixodidae), is a one-host tick that infests primarily cattle in tropical and sub-tropical regions of the world. This species transmits deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. Although R. microplus was eradicated in the USA, tick populations in Mexico and South America have acquired resistance to many of the applied acaricides. Recent acaricide-resistant tick reintroductions detected in the U.S. underscore the need for novel tick control methods. The octopamine and tyramine/octopamine receptors, both G protein-coupled receptors (GPCR), are believed to be the main molecular targets of the acaricide amitraz. This provides the proof of principle that investigating tick GPCRs, especially those that are invertebrate-specific, may be a feasible strategy for discovering novel targets and subsequently new anti-tick compounds. The R. microplus leucokinin-like peptide receptor (LKR), also known as the myokinin- or kinin receptor, is such a GPCR. While the receptor was previously characterized in vitro, the function of the leucokinin signaling system in ticks remains unknown. In this work, the LKR was immunolocalized to the periphery of the female midgut and silenced through RNA interference (RNAi) in females. To optimize RNAi experiments, a dual-luciferase system was developed to determine the silencing efficiency of LKR-double stranded RNA (dsRNA) constructs prior to testing those in ticks placed on cattle. This assay identified two effective dsRNAs. Silencing of the LKR with these two validated dsRNA constructs was verified by quantitative real time PCR (qRT-PCR) of female tick dissected tissues. Silencing was significant in midguts and carcasses. Silencing caused decreases in weights of egg masses and in the percentages of eggs hatched per egg mass, as well as delays in time to oviposition and egg hatching. A role of the kinin receptor in tick reproduction is apparent.

Kinetic characterization of the gill (Na+, K+)-ATPase in a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)ATP-ase activity, but also containing other microsomal ATPases. Only a single immune-reactive (Na+, K+)-ATPase with Mr of ≈110 kDa is present that hydrolyzes ATP with VM = 130.3 ±â€¯4.8 nmol Pi min-1 mg protein-1 and K0.5 = 0.065 ±â€¯0.00162 mmol L-1, exhibiting site-site interactions. Stimulation by Na+ (VM = 127.5 ±â€¯5.3 nmol Pi min-1 mg protein-1, K0.5 = 5.3 ±â€¯0.42 mmol L-1), Mg2+ (VM = 130.6 ±â€¯6.8 nmol Pi min-1 mg protein-1, K0.5 = 0.33 ±â€¯0.042 mmol L-1), K+ (VM = 126.7 ±â€¯7.7 nmol Pi min-1 mg protein-1, K0.5 = 0.65 ±â€¯0.0079 mmol L-1) and NH4+ (VM = 134.5 ±â€¯8.6 nmol Pi min-1 mg protein-1, K0.5 = 1.28 ±â€¯0.44 mmol L-1) also obeys cooperative kinetics. Ouabain (KI = 0.18 ±â€¯0.058 mmol L-1) inhibits total ATPase activity by ≈70%. This study reveals considerable differences in the kinetic characteristics of the gill (Na+, K+)-ATPase in a hololimnetic population that appear to result from the adaptation of diadromous Macrobrachium amazonicum populations to different limnic habitats.

Identification of target genes for RNAi-mediated control of the Twospotted Spider Mite.

RNA interference (RNAi) is being developed for the management of pests that destroy crops. The twospotted Spider Mite (TSSM), Tetranychus urticae is a worldwide pest due to its unique physiological and behavioral characteristics including extraordinary ability to detoxify a wide range of pesticides and feed on many host plants. In this study, we conducted experiments to identify target genes that could be used for the development of RNAi-based methods to control TSSM. Leaf disc feeding assays revealed that knockdown in the expression genes coding for proteins involved in the biosynthesis and action of juvenile hormone (JH) and action of ecdysteroids [Methoprene-tolerant (Met), retinoid X receptor ß, farnesoic acid O-methyltransferase, and CREB-binding protein] caused 35-56% mortality. Transgenic tobacco plants expressing hairpin dsRNA targeting Met gene were generated and tested. About 48% mortality was observed in TSSM raised on transgenic tobacco plants expressing dsMet. These studies not only broaden our knowledge on understanding hormone action in TSSM but also identified target genes that could be used in RNAi-mediated control of TSSM.

Multiple combinations of RDL subunits diversify the repertoire of GABA receptors in the honey bee parasite Varroa destructor.

In insects, γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter, and GABA-gated ion channels are the target of different classes of insecticides, including fipronil. We report here the cloning of six subunits (four RDL, one LCCH3, and one GRD) that constitute the repertoire of the GABA-gated ion channel family of the Varroa mite (Varroa destructor), a honey bee ectoparasite. We also isolated a truncated GRD subunit with a premature stop codon. We found that when expressed in Xenopus laevis oocytes, three of the four RDL subunits (VdesRDL1, VdesRDL2, and VdesRDL3) formed functional, homomultimeric anionic receptors, whereas GRD and LCCH3 produced heteromultimeric cationic receptors. These receptors displayed specific sensitivities toward GABA and fipronil, and VdesRDL1 was the most resistant to the insecticide. We identified specific residues in the VdesRDL1 pore-lining region that explain its high resistance to fipronil. VdesRDL4 did not form a functional receptor when expressed alone, but it assembled with VdesRDL1 to form a heteromultimeric receptor with properties distinct from those of the VdesRDL1 homomultimeric receptor. Moreover, VdesRDL1 physically interacted with VdesRDL3, generating a heteromultimeric receptor combining properties of both subunits. On the other hand, we did not detect any functional interaction between VdesLCCH3 and the VdesRDL subunits, an observation that differed from what was previously reported for Drosophila melanogaster In conclusion, this study provides insights relevant to improve our understanding of the precise role of GABAergic signaling in insects and new tools for the development of Varroa mite-specific insecticidal agents that do not harm honey bees.

Allergen Delivery Inhibitors: Characterisation of Potent and Selective Inhibitors of Der p 1 and Their Attenuation of Airway Responses to House Dust Mite Allergens.

Group 1 allergens of house dust mites (HDM) are globally significant triggers of allergic disease. They are considered as initiator allergens because their protease activity enables the development of allergy to a spectrum of unrelated allergens from various sources. This initiator-perpetuator function identifies Group 1 HDM allergens as attractive drug design targets for the first small-molecule approach directed towards a non-human, root cause trigger of allergic disease. The purpose of this study was to: (i) identify exemplar inhibitors of these allergens using Der p 1 as a design template, and (ii) characterise the pharmacological profiles of these compounds using in vitro and in vivo models relevant to allergy. Potent inhibitors representing four different chemotypes and differentiated by mechanism of action were investigated. These compounds prevented the ab initio development of allergy to the full spectrum of HDM allergens and in established allergy they inhibited the recruitment of inflammatory cells and blunted acute allergic bronchoconstriction following aerosol challenge with the full HDM allergen repertoire. Collectively, the data obtained in these experiments demonstrate that the selective pharmacological targeting of Der p 1 achieves an attractive range of benefits against exposure to all HDM allergens, consistent with the initiator-perpetuator function of this allergen.

Dual oxidases participate in the regulation of hemolymph microbiota homeostasis in mud crab Scylla paramamosain.

Dual oxidases (DUOXs) were originally identified as NADPH oxidases (NOXs), found to be associated with the reactive oxygen species (ROS) hydrogen peroxide (H2O2) production at the plasma membrane and crucial in host biological processes. In this study, SpDUOX1 and SpDUOX2 of mud crab (Scylla paramamosain) were identified and studied. Both SpDUOX1 and SpDUOX2 are transmembrane proteins, including an N-signal peptide region and a peroxidase homology domain in the extracellular region, transmembrane regions, and three EF (calcium-binding region) domains, a FAD-binding domain, and a NAD binding domain in the intracellular region. The SpDUOXs were expressed in all tissues examined, but mainly in hepatopancreas, heart, and mid-intestine. The expression of the SpDUOXs in the hemolymph of mud crabs was up-regulated after challenge with Vibrio parahemolyticus or LPS. RNA interference (RNAi) of the SpDUOXs resulted in reduced ROS production in hemolymph. The bacterial count increased in the hemolymph of mud crabs injected with SpDUOX1 or SpDUOX2-RNAi, while the bacterial clearance ability of hemolymph significantly reduced. At the phylum level, the phyla Bacteroidetes and Actinobacteria were significantly increased, while Proteobacteria were significantly reduced following SpDUOX2 knockdown. There was a significant increase in the relative abundance of the genera Marinomonas, Pseudoalteromonas, Shewanella, and Hydrogenoph in SpDUOX2 depleted mud crabs compared with the controls. Our current findings therefore indicated that SpDUOXs might play important roles in maintaining the homeostasis in the hemolymph microbiota of mud crab.

Identification and functional analysis of transforming growth factor-ß type I receptor (TßR1) from Scylla paramamosain: The first evidence of TßR1 involved in development and innate immunity in crustaceans.

The transforming growth factor-ß (TGF-ß) receptor-mediated TGF-ß signaling cascade plays important roles in diverse cellular processes, including cell proliferation, differentiation, growth, apoptosis and inflammation in vertebrates. In the present study, the type I TGF-ß receptor (TßR1) was firstly identified and characterized in mud crab Scylla paramamosain. The full-length cDNA of SpTßR1 was 1, 986 bp with a 1, 608 bp open reading frame, which encoded a putative protein of 535 amino acids including a typical transmembrane region, a conserved glycine-serine (GS) motif and a S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Real-time PCR analysis showed that SpTßR1 was predominantly expressed at early embryonic development stage and was highly expressed at postmolt stages during molt cycle, suggesting its participation in development and growth. Moreover, the expression levels of SpTßR1 in hepatopancreas and hemocytes were positively induced after the challenges of Vibro alginolyticus and Poly (I:C), indicating the involvement of SpTßR1 in responding to both bacterial and viral infections. The in vivo RNA interference assays demonstrated that the expression levels of two NF-κB members (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF2-6) were significantly suppressed when the SpTßR1 was silenced. Additionally, the expression levels of SpTßR1, SpRelish, SpDorsal and AMPs were consistently down-regulated or up-regulated when the primary cultured hemocytes were treated with TßR1 antagonist or agonist for 24 h. These results indicated that TßR1 not only contributed to the crabs' development and growth but also played vital role in the innate immunity of S. paramamosain, and it also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.

Stress tolerance in diapausing embryos of Artemia franciscana is dependent on heat shock factor 1 (Hsf1).

Embryos of the crustacean, Artemia franciscana, may undergo oviparous development, forming encysted embryos (cysts) that are released from females and enter diapause, a state of suppressed metabolism and greatly enhanced stress tolerance. Diapause-destined embryos of A. franciscana synthesize three small heat shock proteins (sHsps), p26, ArHsp21 and ArHsp22, as well as artemin, a ferritin homologue, all lacking in embryos that develop directly into nauplii. Of these diapause-specific molecular chaperones, p26 and artemin are important contributors to the extraordinary stress tolerance of A. franciscana cysts, but how their synthesis is regulated is unknown. To address this issue, a cDNA for heat shock factor 1 (Hsf1), shown to encode a protein similar to Hsf1 from other organisms, was cloned from A. franciscana. Hsf1 was knocked down by RNA interference (RNAi) in nauplii and cysts of A. franciscana. Nauplii lacking Hsf1 died prematurely upon release from females, showing that this transcription factor is essential to the survival of nauplii. Diapause cysts with diminished amounts of Hsf1 were significantly less stress tolerant than cysts containing normal levels of Hsf1. Moreover, cysts deficient in Hsf1 possessed reduced amounts of p26, ArHsp21, ArHsp22 and artemin, revealing dependence on Hsf1 for expression of their genes and maximum stress tolerance. The results demonstrate an important role for Hsf1, likely in concert with other transcription factors, in the survival and growth of A. franciscana and in the developmentally regulated synthesis of proteins responsible for the stress tolerance of diapausing A. franciscana cysts.

Characterization, expression patterns of molt-inhibiting hormone gene of Macrobrachium nipponense and its roles in molting and growth.

The oriental river prawn, Macrobrachium nipponense, is an important commercial aquaculture resource in China. In order to overwinter, M. nipponense displays decreased physiological activity and less consumption of energy. Sudden warming would trigger molting and cause an extensive death, resulting in huge economic losses. Therefore, it is of great practical significance to study the molting mechanism of oriental river prawns. Molt-inhibiting hormone gene (MIH) plays a major role in regulating molting in crustaceans. In this study, a full length MIH cDNA of M. nipponense (Mn-MIH) was cloned from the eyestalk. The total length of the Mn-MIH was 925 bp, encoding a protein of 119 amino acids. Tissue distribution analysis showed that Mn-MIH was highly expressed in the eyestalk, and that it had relatively low expression in gill, ovary, and abdominal ganglion. Mn-MIH was detected in all developmental stages, and changed regularly in line with the molting cycle of the embryo and larva. Mn-MIH varied in response to the molting cycle, suggesting that Mn-MIH negatively regulates ecdysteroidogenesis. Mn-MIH inhibition by RNAi resulted in a significant acceleration of molting cycles in both males and females, confirming the inhibitory role of MIH in molting. After long-term RNAi males, but not females had significant weight gain, confirming that Mn-MIH plays an important role in growth of M. nipponense. Our work contributes to a better understanding of the role of Mn-MIH in crustacean molting and growth.

PI3K signaling pathways modulated white spot syndrome virus (WSSV) replication in Procambarus clarkii.

The PI3K/AKT signaling pathway is commonly exploited to regulate viral replication and affect the fate of infected cells. In the present study, a PI3K-specific inhibitor (LY294002) was employed to pretreat crayfish to evaluate the effects of PI3K/AKT signaling pathway in WSSV replication. The results showed that the WSSV copy numbers in crayfish pretreated with LY294002 were significantly lower than those in Tris-HCl pretreatment crayfish on the sixth and tenth day after WSSV infection. In semigranular cells, the apoptosis rates were up-regulated on the third day post-WSSV infection, and a significantly lower proportion of apoptosis cells were observed in LY294002-pretreatment group. The expression level of Bax, Bax inhibitor-1 and lectin mRNA in haemocytes of crayfish were increased after WSSV infection. After the secondary stimulation with Tris-HCl, the Bax expression level in LY294002-pretreatment crayfish was significantly higher than that of crayfish pretreated with Tris-HCl on the third or sixth day, but the Toll and lectin mRNA expression decreased significantly on the third, sixth and tenth day. The Bax mRNA expression levels in LY294002-WSSV group were significantly higher than those in Tris-HCl-WSSV group on the third and tenth day. The Bax inhibitor-1 mRNA expression levels in LY294002-WSSV group were significantly lower than those in Tris-HCl-WSSV crayfish on the third day. These results together indicated that the hosts PI3K/AKT signaling pathway play positive roles in WSSV replication through the balance between host cell apoptois and innate immune responses. This information is helpful to further understand the role of PI3K/AKT signaling pathway on WSSV replication in Decapoda crustaceans.

CPAP3 proteins in the mineralized cuticle of a decapod crustacean.

The pancrustacean theory groups crustaceans and hexapods (once thought to comprise separate clades within the Arthropoda) into a single clade. A key feature common to all pancrustaceans is their chitinous exoskeleton, with a major contribution by cuticular proteins. Among these, are the CPAP3's, a family of cuticular proteins, first identified in the hexapod Drosophila melanogaster and characterized by an N-terminal signaling peptide and three chitin-binding domains. In this study, CPAP3 proteins were mined from a transcriptomic library of a decapod crustacean, the crayfish Cherax quadricarinatus. Phylogenetic analysis of other CPAP3 proteins from hexapods and other crustaceans showed a high degree of conservation. Characterization of the crayfish proteins, designated CqCPAP3's, suggested a major role for CPAP3'sin cuticle formation. Loss-of-function experiments using RNAi supported such a notion by demonstrating crucial roles for several CqCPAP3 proteins during molting. A putative mode of action for the CqCPAP3 proteins -theoretically binding three chitin strands- was suggested by the structural data obtained from a representative recombinant CqCPAP3. The similarities between the CqCPAP3 proteins and their hexapod homologues further demonstrated common genetic and proteinaceous features of cuticle formation in pancrustaceans, thereby reinforcing the linkage between these two highly important phylogenetic groups.

Thrombin inhibitor from the salivary gland of the camel tick Hyalomma dromedarii.

Blood-sucking arthropods have different types of anticoagulants to allow the ingestion of a blood meal from their hosts. In this study, five anticoagulants prolonging the activated partial thromboplastin time were resolved from the salivary gland crude extract of the camel tick Hyalomma dromedarii by chromatography on diethylaminoethyl (DEAE)-cellulose column. They were designated P1, P2, P3, P4 and P5 according to their elution order. P5 was found to be a potent thrombin inhibitor and purified by ultrafiltration through two centrifugal concentrators of 50 and 30 kDa molecular weight cut-off (MWCO), respectively. The camel tick salivary gland thrombin inhibitor was purified 60.6 folds with a specific activity of 564 units/mg protein. It turned out to be homogenous on native-PAGE with molecular weight of 36 kDa as detected on 12% SDS-PAGE. It inhibits bovine thrombin competitively with K i value of 0.55 µM. A task for the future will be the elucidation of this thrombin inhibitor structure to allow its application in thrombosis treatment.