dsRNA Interference Research Articles

Sws2 Gene Positively Regulates Melanin Production in Plectropomus leopardus Skin via Direct Regulation of the Synthesis of Retinoic Acid.

Opsins are a class of transmembrane proteins encoded by opsin genes, and they play a variety of functional roles. Short wavelength-sensitive opsin 2 (sws2), one of the five classes of visual opsin genes, mainly senses blue light. Previous research has indicated that sws2 is essential for melanocyte formation in fish; however, its specific role in skin color differentiation remains to be elucidated. Here, we identified the sws2 gene in a prized reef-dwelling fish, Plectropomus leopardus. The full-length P. leopardus sws2 gene encodes a protein consisting of 351 amino acids, and exhibits substantial homology with other fish species. The expression of the sws2 gene was widespread across P. leopardus tissues, with high expression in eye and skin tissues. Through immunohistochemistry and in situ hybridization analyses, we discovered that the sws2 gene was primarily localized in the rod and cone cells of the retina, and epidermal cells of the skin. Furthermore, dsRNA interference was used for sws2 gene knockdown in living P. leopardus to elucidate its function in skin color differentiation. Black-color-related genes, melanin contents, and tyrosinase activity in the skin significantly decreased after sws2 knockdown (p < 0.05), but red-color-related genes and carotenoid and lutein contents significantly increased (p < 0.05). Retinoic acid injection produced the opposite results. Our results suggested that the sws2 gene influences P. leopardus skin color regulation by affecting vitamin synthesis and melanin-related gene expression levels. This study establishes a foundation for elucidating the molecular mechanisms by which sws2 regulates melanocyte formation in fish skin.

Identification and function analysis of steroid hormone synthesis pathway-related gene—Hsd3b in Scylla paramamosain

Mud crab (Scylla paramamosain) has become an important mariculture crab along the southeast coast of China due to its strong adaptability, delicious taste, and rich nutrition. Several vertebrate steroid hormones and their synthesis-related genes and receptors have been found in crustaceans, but there are few reports on their synthesis process and mechanism. 3-beta-hydroxysteroid dehydrogenase (HSD3B) is a member of the Short-chain Dehydrogenase/Reductase (SDR) family, and an indispensable protein in vertebrates' steroid hormone synthesis pathway. In this study, the SpHsd3b gene sequence was obtained from the transcriptome data of S. paramamosain, and its full-length open reading frame (ORF) was cloned. The spatial and temporal expression pattern of SpHsd3b was performed by quantitative real-time PCR (qRT-PCR). SpHsd3b dsRNA interference (RNAi) and HSD3B inhibitor (trilostane) were used to analyze the function of SpHSD3B. The results showed that the SpHsd3b gene has an 1113 bp ORF encoding 370 amino acids with a 3β-HSD domain. SpHSD3B has lower homology with HSD3B of vertebrates and higher homology with HSD3B of crustaceans. SpHsd3b was expressed in all examined tissues in mature crabs, and its expression was significantly higher in the testes than in the ovaries. SpHsd3b expression level was highest in the middle stage of testicular development, while its expression was higher in the early and middle stages of ovarian development. RNAi experiment and trilostane injection results showed that SpHSD3B had regulatory effects on several genes related to gonadal development and steroid hormone synthesis. 15-day trilostane suppression could also inhibit ovarian development and progesterone level of hemolymph. According to the above results, crustaceans may have steroid hormone synthesis pathways like vertebrates, and the Hsd3b gene may be involved in the gonadal development of crabs. This study provides further insight into the function of genes involved in steroid hormone synthesis in crustaceans.

NADK-mediated proline synthesis enhances high-salinity tolerance in the razor clam

Euryhaline organisms can accumulate organic osmolytes to maintain osmotic balance between their internal and external environments. Proline is a pivotal organic small molecule and plays an important role in osmoregulation that enables marine shellfish to tolerate high-salinity conditions. During high-salinity challenge, NAD kinase (NADK) is involved in de novo synthesis of NADP(H) in living organisms, which serves as a reducing agent for the biosynthetic reactions. However, the role of shellfish NADK in proline biosynthesis remains elusive. In this study, we show the modulation of NADK on proline synthesis in the razor clam (Sinonovacula constricta) in response to osmotic stress. Under acute hypersaline conditions, gill tissues exhibited a significant increase in the expression of ScNADK. To elucidate the role of ScNADK in proline biosynthesis, we performed dsRNA interference in the expression of ScNADK in gill tissues to assess proline content and the expression levels of key enzyme genes involved in proline biosynthesis. The results indicate that the knock-down of ScNADK led to a significant decrease in proline content (P<0.01), as well as the expression levels of two proline synthetase genes P5CS and P5CR involved in the glutamate pathway. Razor clams preferred to use ornithine as substrate for proline synthesis when the glutamate pathway is blocked. Exogenous administration of proline greatly improved cell viability and mitigated cell apoptosis in gills. In conclusion, our results demonstrate the important role of ScNADK in augmenting proline production under high-salinity stress, by which the razor clam is able to accommodate salinity variations in the ecological niche.

Characterisation and functionalisation of chitosan nanoparticles as carriers for double-stranded RNA (dsRNA) molecules towards sustainable crop protection.

The need to minimise the impact of phytosanitary treatments for disease control boosted researchers to implement techniques with less environmental impact. The development of technologies using molecular mechanisms based on the modulation of metabolism by short dsRNA sequences appears promising. The intrinsic fragility of polynucleotides and the high cost of these techniques can be circumvented by nanocarriers that protect the bioactive molecule enabling high efficiency delivery to the leaf surface and extending its half-life. In this work, a specific protocol was developed aiming to assess the best methodological conditions for the synthesis of low-size chitosan nanoparticles (NPs) to be loaded with nucleotides. In particular, NPs have been functionalised with partially purified Green Fluorescent Protein dsRNAs (GFP dsRNA) and their size, surface charge and nucleotide retention capacity were analysed. Final NPs were also stained with FITC and sprayed on Nicotiana benthamiana leaves to assess, by confocal microscopy, both a distribution protocol and the fate of NPs up to 6 days after application. Finally, to confirm the ability of NPs to increase the efficacy of dsRNA interference, specific tests were performed: by means of GFP dsRNA-functionalised NPs, the nucleotide permanence during time was assessed both in vitro on detached wild-type N. benthamiana leaves and in planta; lastly, the inhibition of Botrytis cinerea on single leaves was also evaluated, using a specific fungal sequence (Bc dsRNA) as the NPs' functionalising agent. The encouraging results obtained are promising in the perspective of long-lasting application of innovative treatments based on gene silencing.

A metabotropic glutamate receptor affects the growth and development of Schistosoma japonicum.

Schistosomiasis is a zoonotic parasitic disease caused by schistosome infection that severely threatens human health. Therapy relies mainly on single drug treatment with praziquantel. Therefore, there is an urgent need to develop alternative medicines. The glutamate neurotransmitter in helminths is involved in many physiological functions by interacting with various cell-surface receptors. However, the roles and detailed regulatory mechanisms of the metabotropic glutamate receptor (mGluR) in the growth and development of Schistosoma japonicum remain poorly understood. In this study, we identified two putative mGluRs in S. japonicum and named them SjGRM7 (Sjc_001309, similar to GRM7) and SjGRM (Sjc_001163, similar to mGluR). Further validation using a calcium mobilization assay showed that SjGRM7 and SjGRM are glutamate-specific. The results of in situ hybridization showed that SjGRM is mainly located in the nerves of both males and gonads of females, and SjGRM7 is principally found in the nerves and gonads of males and females. In a RNA interference experiment, the results showed that SjGRM7 knockdown by double-stranded RNA (dsRNA) in S. japonicum caused edema, chassis detachment, and separation of paired worms in vitro. Furthermore, dsRNA interference of SjGRM7 could significantly affect the development and egg production of male and female worms in vivo and alleviate the host liver granulomas and fibrosis. Finally, we examined the molecular mechanisms underlying the regulatory function of mGluR using RNA sequencing. The data suggest that SjGRM7 propagates its signals through the G protein-coupled receptor signaling pathway to promote nervous system development in S. japonicum. In conclusion, SjGRM7 is a potential target for anti-schistosomiasis. This study enables future research on the mechanisms of action of Schistosomiasis japonica drugs.

Cloning and functional analysis of a pacifastin-like protein from the sea cucumber, Apostichopus japonicus

Pacifastin family proteins play a crucial role in regulating innate immune responses such as phagocytosis in invertebrates. However, the function of the Ajpacifastin-like counterpart in the sea cucumber Apostichopus japonicus remains elusive. In this study, the pacifastin gene of A. japonicus was cloned, characterized and named Ajpacifastin-like. The open reading frame of Ajpacifastin-like is 1497 bp in length and encodes a polypeptide containing 498 amino acid residues. Structural analysis revealed that the protein encoded by Ajpacifastin-like contains two pacifastin light chain domains (amino acids 287–322 and amino acids 376–407). Real-time reverse transcriptase PCR showed that Ajpacifastin-like mRNA is ubiquitously expressed in all tissues examined, with the highest expression in muscle. Ajpacifastin-like mRNA expression was significantly upregulated to 3.27-fold after challenge with Vibrio splendidus for 24 h. To explore the function of the Ajpacifastin-like protein in the immune response of A. japonicus, dsRNA interference with Ajpacifastin-like expression and with the expression of its postulated target gene was performed. Flow cytometry analysis showed that the rate of phagocytosis by coelomocytes increased to 1.21-fold in individuals treated with specific Ajpacifastin-like siRNA. However, rate of phagocytosis by coelomocytes decreased to 86% in individuals treated with Ajphenoloxidase siRNA. These results show that the Ajpacifastin-like gene is ubiquitously expressed in almost all tissues and that Ajpacifastin-like protein acts as an immunomodulatory factor via phenoloxidase to mediate phagocytosis by coelomocytes in pathogen-challenged A. japonicus.

Ovarian transcriptome and metabolic responses of RNAi-mediated farnesyl pyrophosphate synthase knockdown in Neocaridina denticulata sinensis

Methyl farnesoate (MF) is considered the equivalent of JH in crustaceans and plays an essential role in many crucial physiological processes. It is believed that farnesyl pyrophosphate synthase (FPPS) plays an essential role in the biosynthesis of mevalonate, which is a branch of the JH/MF pathway. The full-length cDNA of FPPS (NdFPPS) from Neocaridina denticulata sinensis was isolated and characterized, and the deduced amino acid of NdFPPS contained a polyprenyl_synt domain. In addition to its ubiquitous tissue expression, NdFPPS was significantly expressed in the ovary. In vivo gene silencing with dsRNA interference was performed to further investigate the function of NdFPPS. An ovarian transcriptomic analysis of dsNdFPPS experimental and control groups was used to compare, annotate, and classify differentially expressed genes (DEGs). A total of 9230 DEGs were identified in the experimental and control groups based on FPKM values, of which 5082 were up-regulated genes and 4148 were down-regulated genes. 761 GO terms and 102 KEGG pathways were enriched for the DEGs. Our results suggest that NdFPPS might play an important role in ovarian development, however, further functional study is needed to elucidate physiological role of NdFPPS in reproduction.

A StAR‐like gene involved in carotenoid metabolism and inner shell color in the freshwater pearl mussel, Hyriopsis cumingii

AbstractCarotenoids play key roles in organism coloration and have been found to have an effect on shell color. Steroidogenic acute regulatory protein‐like (StAR‐like) is a key gene involved in the accumulation of carotenoids. In this study, the full‐length cDNA sequence of HcStAR‐like, containing 994 nucleotides with an open reading frame encoding 307 amino acid residues, was isolated from the freshwater pearl mussel, Hyriopsis cumingii. HcStAR‐like expression levels were significantly higher in all tissues examined in golden (G‐) mussels compared with white (W‐) and purple (P‐) mussels (p &lt; .05). Moreover, although HcStAR‐like expression levels were higher in P‐mussels than W‐mussels, this difference was only significant in the hepatopancreas (p &lt; .05). Our in situ hybridization assay indicated that StAR‐like was localized to the outer fold of the mantle and the joint of the outer and middle folds of the mantle. The dsRNA interference of HcStAR‐like expression in G‐mussels was effective with an interference rate of 79.89% (p &lt; .05). The total carotenoid content (TCC) in the mantle of the RNAi group decreased by 48.63% (p &lt; .05). Our findings suggested that HcStAR‐like was involved in the accumulation of carotenoids and inner shell pigmentation in H. cumingii.

A novel Ush transcription factor involving in hematopoiesis of Eriocheir sinensis.

The FOG transcriptional factor is a co-regulator that recognizes and binds to the GATA N-terminal zinc-finger domain and participates in hemocyte production and differentiation. In this study, an FOG-like gene, Ush, was characterized from Eriocheir sinensis, which consists of an 897bp full-length open reading frame, encoding a polypeptide of 298 amino acids with four ZnF_C2H2 domains. The EsUsh mRNA transcripts were mainly expressed in the hematopoietic tissue (HPT) and hemocytes, and were significantly higher in hyalinocytes than semi-granulocytes and granulocytes, which were separated by Percoll gradient centrifugation. The transcription levels of EsUsh were found to be significantly upregulated in HPT, but downregulated in hemocytes after exsanguination. By using flow cytometry to determine the percentage of hemocyte sub-population after exsanguination, the percentage of hyalinocytes was found to significantly downregulated, while the percentage of granulocytes was significantly upregulated. Silencing EsUsh by dsRNA interference significantly decreased the percentage of hyalinocytes and small granulocytes, and increased the percentage of medium granulocytes and large granulocytes. Such findings suggest that EsUsh might be involved in hemocyte production and differentiation, especially in promoting hyalinocyte formation and limiting granulocyte generation and differentiation.

Identification and functional analysis of the estradiol 17β-dehydrogenase gene on the shell hardness of Scylla paramamosain during the molting cycle

Estradiol 17 β-dehydrogenase (17E2DH) is an oxidoreductase gene involved in the metabolism of androgen and estrogen in mammals and birds, and is also associated with molting in crustaceans. However, neither the full-length estradiol 17 β-dehydrogenase nor its molecular mechanism during molting has been reported in crabs. In this study, the cDNA encoding 17-beta-dehydrogenase was cloned from the hepatopancreas of Scylla paramamosain and named Sp17E2DH. The full length Sp17E2DH consists of 922 bp with an open reading frame encoding 255 amino acids. Phylogenetic analysis showed that the predicted Sp17E2DH protein is conserved and clustered with the 17E2DH from other crustacean species. RT-qPCR and immunohistochemistry analyses indicated that the expression level of Sp17E2DH was lowest before molting (TQ), up-regulated at 3 h after molting (TH3h), and highest at 48 h after molting (TH48h) in the hepatopancreas and muscles. An RNA interference experiment showed that the mRNA expression level of Sp17E2DH was significantly down-regulated (P < 0.05) and the hardness of crab shells was significantly decreased (P < 0.05) at TH48h in the hepatopancreas and muscles after injection of Sp17E2DH-targeted dsRNA. Genes such as gastrolith protein (P = 0.045), cuticular protein 15 (P = 0.021), chitin synthase (P = 0.047) and troponin C2 (P = 0.013) were significantly suppressed at TH48h after Sp17E2DH dsRNA injection compared to the control groups, which were potentially regulated by Sp17E2DH to control shell hardness of S. paramamosain. In summary, the results suggest that Sp17E2DH plays a vital role in the molting cycle and shell hardening of the mud crab, and the dsRNA interference of Sp17E2DH may be used to prolong the marketing shelf life of live soft-shelled mud crabs.

The transcription factor c-Fos plays a negative role in prawn immunity against WSSV infection

Members of the Fos family (c-Fos, FosB) dimerize with Jun proteins to form the activator protein 1 transcription factor complex and regulate diverse cellular processes including immunity. In the current study, a c-Fos homologue with six isoforms were identified from the oriental river prawn, Macrobrachium nipponense, and these six MnFos isoforms were produced by alternative splicing of first exon and/or exon skipping. The MnFos transcripts were detected in all tissues examined, with the highest level in the hepatopancreas. Moreover, these MnFos transcripts were significantly induced after immune stimulation by white spot syndrome virus (WSSV). Interestingly, silencing of MnFos in vivo by dsRNA interference led to a lower mortality of WSSV-infected prawns, as well as a great decrease in the expression of viral envelope protein VP28. Furthermore, silencing of MnFos induced the expression of multiple antimicrobial peptide genes in normal or WSSV-infected prawns, including crustins and anti-lipopolysaccharide factors. All these findings indicated that MnFos plays a negative role in prawn immunity against WSSV infection, possibly by its negative regulation on the expression of antimicrobial peptides. Such discovery is of great significance for further understanding of the interaction between WSSV and the immune system of prawns.

Expression of p38MAPK and its regulation of apoptosis under high temperature stress in the razor clam Sinonovacula constricta

p38MAPK is a key branch of the MAPK (mitogen-activated protein kinase) pathway that plays an important role in physiological processes such as apoptosis, cell proliferation and growth. In this experiment, we screened and identified one p38MAPK gene in the razor clam Sinonovacula constricta, which encoded 359 amino acids and was widely expressed in various adult tissues. After 24 h of high temperature stress at 34 °C, the transcript expression of p38MAPK showed significant changes in all tested tissues. In particular in the gill and hepatopancreas tissues, where the expression increased 1.81 and 7.83 times compared with the control group, respectively (P < 0.01). Furthermore, we examined the expression of the apoptosis suppressor gene Bcl-2 and pro-apoptosis gene Bax by knock-down of p38MAPK with dsRNA interference in the gill and hepatopancreas tissues. The obvious up-regulation expression of Bcl-2 and significant suppression of Bax were observed, respectively (P < 0.01). Moreover, the TUNEL staining technique was used to detect apoptosis before and after interference. The degree of apoptosis in the gill and hepatopancreas tissues was reduced after interference with p38MAPK, and the ROS content was significantly reduced (P < 0.01). The results suggested that p38MAPK had a regulatory role in the heat tolerance of razor clams.

Physiological role of CYP17A1-like in cadmium detoxification and its transcriptional regulation in the Pacific oyster, Crassostrea gigas

Cadmium (Cd) is one of the most harmful heavy metals due to its persistence and bioaccumulation through the food chains, posing health risks to human. Oysters can bioaccumulate and tolerate high concentrations of Cd, providing a great model for studying molecular mechanism of Cd detoxification. In a previous study, we identified two CYP genes, CYP17A1-like and CYP2C50, that were potentially involved in Cd detoxification in the Pacific oyster, Crassostrea gigas. In this work, we performed further investigations on their physiological roles in Cd detoxification through RNA interference (RNAi). After injection of double-stranded RNA (dsRNA) into the adductor muscle of oysters followed by Cd exposure for 7 days, we observed that the expressions of CYP17A1-like and CYP2C50 in interference group were significantly suppressed on day 3 compared with control group injected with PBS. Moreover, the mortality rate and Cd content in the CYP17A1-like dsRNA interference group (dsCYP17A1-like) was significantly higher than those of the control on day 3. Furthermore, the activities of antioxidant enzymes, including SOD, CAT, GST, were significantly increased in dsCYP17A1-like group, while were not changed in dsCYP2C50 group. More significant tissue damage was observed in gill and digestive gland of oysters in RNAi group than control group, demonstrating the critical role of CYP17A1-like in Cd detoxification. Dual luciferase reporter assay revealed three core regulatory elements of MTF-1 within promoter region of CYP17A1-like, suggesting the potential transcriptional regulation of CYP17A1-like by MTF-1 in oysters. This work demonstrated a critical role of CYP17A1-like in Cd detoxification in C. gigas and provided a new perspective toward unravelling detoxification mechanisms of bivalves under heavy metal stress.

A hexokinase from the oyster Crassostrea gigas is involved in immune recognition as a pattern recognition receptor

Hexokinase (HK) is generally recognized as a crucial enzyme participating in glycolysis. In the present study, a HK (named CgHK) was identified as a potential pattern recognition receptor (PRR) from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgHK was of 1395 bp, encoding a peptide of 464 amino acids with one Hexokinase_1 domain and one Hexokinase_2 domain. The predicted amino acid sequence of CgHK shared 17%–29% similarities with that of other known HKs. The mRNA transcripts of CgHK were constitutively detected in all the examined tissues, with relative high expression level in labial palp and haemocytes. CgHK protein was mainly observed in the cytoplasm of oyster haemocytes. The mRNA expression level of CgHK in haemocytes was significantly up-regulated and peaked at 3 h after Vibrio splendidus (7.64-fold, p < 0.001) and lipopolysaccharide (LPS) (11.86-fold, p < 0.001) stimulations. The recombinant CgHK protein (rCgHK) exhibited Mg2+-dependent adenosine triphosphate (ATP) binding activity in vitro and activity to bind D-(+)-glucose (GLU) and various pathogen-associated molecular pattern (PAMPs) such as LPS and peptidoglycan (PGN) in the absence of Mg2+. It also displayed higher binding activity towards V. splendidus and relatively lower binding activity towards Staphylococcus aureus, Escherichia coli, and Micrococcus luteus. After the mRNA expression of CgHK in haemocytes was knocked down by dsRNA interference, the expression of CgIL17-5 mRNA in haemocytes was considerably down-regulated at 3 h after the stimulation with V. splendidus (0.33-fold, p < 0.001). These results collectively indicated that CgHK was able to recognize various PAMPs and pathogenic bacteria as a PRR apart from being the enzyme to exert ATP binding activity in glycolysis, and activate the anti-bacterial immune response by promoting the expression of pro-inflammatory cytokines CgIL17-5 in oyster haemocytes.

A trypsin-like serine protease domain of masquerade gene in crayfish Procambarus clarkii could activate prophenoloxidase and inhibit bacterial growth

Masquerade (Mas) is a secreted trypsin-like serine protease (SPs) and involved in immune response in some arthropods. However, according to previous studies, Mas presents different functional activities. In the present study, the functional mechanisms of Mas in crayfish Procambarus clarkii immune defense were studied. A fragment cDNA sequence of PcMas was identified and characterized. From the structural analysis, it contains a trypsin-like serine protease domain. The highest expression level of PcMas was detected in hepatopancreas. The infection of A. hydrophila could induce the expression of PcMas, while the WSSV infection did not cause changes in the expression of PcMas. Through the prokaryotic expression system, the PcMas protein was expressed in E. coli. It was verified that PcMas can bind to bacteria in vitro and inhibit the growth of the bacteria. By dsRNA interference with the expression of PcMas, the decrease expression of PcMas led to a decrease in the activity of phenoloxidase in hemolymph and an increase of mortality caused by A. hydrophila infection. The injection of recombinant protein can enhance the activity of phenoloxidase and reduce mortality caused by A. hydrophila infections. Therefore, the present study confirmed that PcMas could improve the body's immune response to eliminate bacterial pathogens by binding with bacteria and activating the prophenoloxidase system. The results will enrich the molecular mechanisms of crustaceans immune defense.

Characterization of a gill-abundant crustin with microbiota modulating function in Litopenaeus vannamei

Gills as the respiratory tissue of crustacean encounter various threats in the environment. The immune defense in gills is essential to the health of shrimp. In the present study, we identified a novel gill-abundant crustin, LvCrustin Ⅱ-1, from the shrimp Litopenaeus vannamei. The full-length open reading frame of LvCrustin Ⅱ-1 is 522 bp, which encodes 173 amino acid residues. The amino acid sequence of LvCrustin Ⅱ-1 contained a signal peptide, a glycine rich region, a cysteine rich region and a whey acidic protein (WAP) domain. The expression level of LvCrustin Ⅱ-1 was significantly up-regulated at different time points after Vibrio parahaemolyticus immersion. When LvCrustin Ⅱ-1 was silenced by dsRNA interference, the count of bacteria colonies increased significantly in the hepatopancreas of shrimp after V. parahaemolyticus immersion, which indicated that the infection progress of pathogenic bacteria was accelerated after LvCrustin Ⅱ-1 knockdown. Compared with the microbiota of seawater, the lower proportion of aquatic bacteria and higher proportion of symbiont in the gills microbiota of shrimp indicated the bacterial colonization was modulated by the host. Knockdown of LvCrustin Ⅱ-1 changed the proportion of some potential pathogens and aquatic bacteria, which supported the idea that the new identified crustin in the gills played important roles in modulation of the microbiota community in shrimp. The present data provided new insights into the multiple functions of crustin in the immunity of shrimp.

Identification of Foxl2 in freshwater mussel Hyriopsis cumingii and its involvement in sex differentiation.

The freshwater mussel Hyriopsis cumingii, is the most important species for pearl culture in China. At present, the mechanisms underlying sex differentiation and determination remain unclear in this species. Herein the open reading frame (ORF) of Foxl2 from H. cumingii (Hc-Foxl2) was cloned, and Hc-Foxl2 expression levels in six tissues (the gonad, gill, adductor muscle, foot, mantle, and kidney) were determined. Further, we performed quantitative real-time PCR to compare expressions levels between 1 and 8months of age and 1-, 2-, and 3-year-old H. cumingii. The localization of Hc-Foxl2 expression in the ovary was analyzed by in situ hybridization, and its function was explored using RNA interference. We found that the ORF region of Hc-Foxl2 was 1215bp in length, encoded 404 amino acids, and contained conserved FH domains. Hc-Foxl2 was expressed in the male and female tissues, with the expression levels being significantly higher in the ovary than in the testis. In 1-8-month-old H. cumingii, Hc-Foxl2 was expressed at the highest level at 5months of age, and the gonads began to differentiate at the same time. Moreover, in 1-, 2-, and 3-year-old individuals, Hc-Foxl2 expression levels in the ovaries gradually decreased, but they were higher than those in the testis. Strong hybridization signals for Hc-Foxl2 were detected on the oocyte membrane in 3-year-old female mussels. We also performed double-stranded RNA (dsRNA) interference experiments using three dsRNA strands, which were injected into 5-month-old H. cumingii; the interference effects were the best at 12h and 48h post-injection. After interference with Hc-Foxl2, the expression levels of Wnt4, which has an antagonistic relationship with Foxl2 during ovarian development, were slightly increased. Thus, we speculate that Hc-Foxl2 is a female-related gene in H. cumingii and that it is involved in sex differentiation and ovarian development.

Roles of phenoloxidases in the sea cucumberApostichopus japonicusunder environmental stresses

Sea cucumber Apostichopus japonicus is an important economic species in echinoderms; however, its culture frequently suffered from diseases. Environmental stress was found to be a dominant inducer of A. japonicus diseases, and thus, discovering the characteristics of immune system under environmental stress is of great significance for disease control in A. japonicus culture. Phenoloxidase (PO) is recognized as a crucial immune-related enzyme in invertebrates. In order to preliminarily illustrate the different roles that A. japonicus PO isoforms play under environmental stresses, the transcriptional expression of three identified A. japonicus PO isoforms (named as AjPOI, AjPOII and AjPOIII respectively) after different in vivo environmental stress simulations was determined with quantitative real-time PCR method. Meanwhile, the transcriptional expression analysis of other representational immune-related factors under environmental stresses was also performed for the evaluation of relationships between AjPOs and other immune-related factors. The correlations between AjPOs and other immune-related factors were analysed using Pearson correlation coefficient, and the effects of AjPOs on other immune-related factors were then tested by dsRNA interference method. The results of transcriptional expression analysis indicated that AjPOI was involved in the resistance to low salinity, low temperature and hypoxia; AjPOII and AjPOIII were both required for the resistance to low salinity, high temperature, low temperature and hypoxia; and all three AjPOs were excluded from the response against high salinity. In addition, three AjPOs were found to take part in different periods of resistance to environmental stresses, and this property made A. japonicus PO system capable of outputting resistance activities continuously. The correlation analysis combined with dsRNA interference test revealed that AjPOI was capable of regulating AjCTSD, AjI-LYZ and AjMTF expression; AjPOII and AjPOIII both played important roles in the regulation of AjC-LYZ expression; and AjPOIII was also able to regulate AjToll expression. These findings suggested that A. japonicus PO isoforms function in innate immunity not only via catalysing the oxidative reactions, but also by broadly regulating the expression of other immune-related factors. On the whole, three AjPOs showed obvious differences among each other in the resistance to environmental stresses and the regulation of other immune-related factors, but comparatively, AjPOII and AjPOIII shared more similarity.

Comparison of effects of dsRNA and siRNA RNA interference on insulin-like androgenic gland gene (IAG) in red swamp crayfish Procambarus clarkii.

RNA interference (RNAi), which employs double-strand RNA (dsRNA) or small interference RNA (siRNA), is a popular reverse genetic manipulation tool to study gene function. Presently, there is few reports on the implementation of RNAi on the insulin-like androgenic gland gene (IAG) in red swamp crayfish Procambarus clarkii. In this study, the effective sequence of siRNA and optimal injection dose were determined, and the effects of RNAi using dsRNA, siRNA, and long-term RNAi were investigated. The results showed that the doses of 0.5 and 1 µg/g of body weight of IAG-siRNA3 produced significantly better inhibition than 0.1 µg/g. qPCR assays showed that both dsRNA and siRNA silenced the IAG expression in five tissues (brain, ventral nerve cord, androgenic gland, testis, and vas deferens) in adult P. clarkii, with the effectiveness decreasing over time, inhibiting the production of spermatid. dsRNA exhibited a longer interference effect than siRNA in adults. For long-term interference (P. clarkii juveniles were injected 7 times with 1 µg/g of body weight of IAG-dsRNA), and found that the secondary sexual characteristics of juveniles were affected, while the control group developed normally. The results of this study could lay the foundation for crayfish sex reversal with IAG RNAi, and provide the reference for those studies in which the technique of RNAi was used.

Identification and functional study of an LRR domain containing membrane protein in Litopenaeus vannamei

Leucine-rich repeat (LRR) is a vital structure in some pattern recognition receptors such as TLRs, NLRs and newly reported LRR-containing proteins. Apart from some limited reported LRR-containing proteins, most of LRR proteins, especially immune-related proteins, remain uncharacterized functionally. In the present study, a transmembrane protein containing several LRR motifs, designated as LvLRRm, was identified from the shrimp Litopenaeus vannamei. LvLRRm contained a long signal peptide, one LRRNT region, 12 LRR motifs, one LRRCT region and a transmembrane region. The transcripts of LvLRRm were widely distributed in all tested tissues of shrimp and they were responsive to Vibrio parahaemolyticus infection in several immune-related tissues including Oka, intestine, gill and hemocytes. Knockdown of LvLRRm by dsRNA interference led to a decreased survival rate of shrimp infected by Vibrio parahaemolyticus and an increased in vivo Vibrio propagation. Meanwhile, knockdown of LvLRRm also down-regulated the expression levels of genes involved in antibacterial immune signaling pathways, including the transcription factors LvDorsal and LvRelish, and several antimicrobial peptides. These data suggested that LvLRRm played important roles in shrimp against Vibrio infection, which was probably functioning through activation of antibacterial immune signaling pathways. The present study provided new evidence to elucidate the immune function of LRR-containing proteins in invertebrates.