Injection Of dsRNA Research Articles

Candidate target genes of the male-specific expressed Doublesex in the termite Reticulitermes speratus.

Eusocial insects such as termites, ants, bees, and wasps exhibit a reproductive division of labor. The developmental regulation of reproductive organ (ovaries and testes) is crucial for distinguishing between reproductive and sterile castes. The development of reproductive organ in insects is regulated by sex-determination pathways. The sex determination gene Doublesex (Dsx), encoding transcription factors, plays an important role in this pathway. Therefore, clarifying the function of Dsx in the developmental regulation of sexual traits is important to understand the social evolution of eusocial insects. However, no studies have reported the function of Dsx in hemimetabolous eusocial group termites. In this study, we searched for binding sites and candidate target genes of Dsx in species with available genome information as the first step in clarifying the function of Dsx in termites. First, we focused on the Reticulitermes speratus genome and identified 101 candidate target genes of Dsx. Using a similar method, we obtained 112, 39, and 76 candidate Dsx target genes in Reticulitermes lucifugus, Coptotermes formosanus, and Macrotermes natalensis, respectively. Second, we compared the candidate Dsx target genes between species and identified 37 common genes between R. speratus and R. lucifugus. These included several genes probably involved in spermatogenesis and longevity. However, only a few common target genes were identified between R. speratus and the other two species. Finally, Dsx dsRNA injection resulted in the differential expression of several target genes, including piwi-like protein and B-box type zinc finger protein ncl-1 in R. speratus. These results provide valuable resource data for future functional analyses of Dsx in termites.

Silencing of crustacean hyperglycemic hormone gene expression reveals the characteristic energy and metabolic changes in the gills and epidermis of crayfish Procambarus clarkii.

The crustacean hyperglycemic hormone (CHH) is a multifaceted neuropeptide instrumental in regulating carbohydrate and lipid metabolism, reproduction, osmoregulation, molting, and metamorphosis. Despite its significance, there is a dearth of research on its metabolic impact on the gills and epidermis-key organs in osmoregulation and molting processes. This study employed CHH dsRNA injections to silence CHH gene expression in Procambarus clarkii, followed by a metabolomic analysis of the gills and epidermis using nuclear magnetic resonance spectroscopy. Metabolic profiling through principal component analysis revealed the most pronounced changes at 24h post-injection (hpi) in the epidermis and at 48hpi in the gills. At 24hpi, the epidermis exhibited significant modulation in 25 enrichment sets and 20 KEGG pathways, while at 48hpi, 5 metabolite sets and 6 KEGG pathways were prominently regulated. Notably, pathways associated with amino acid metabolism, carbohydrate metabolism, and cofactor and vitamin metabolism were affected. A marked decrease in glucose and other carbohydrates suggested a compromised carbohydrate supply, whereas increased levels of citrate cycle intermediates implied a potential boost in energy provision. The silencing of CHH gene expression hampered the carbohydrate supply, which was possibly the main energy derived substrates. Conversely, the gills displayed significant alterations in 15 metabolite sets and 16 KEGG pathways at 48 hpi, with no significant changes at 24hpi. These changes encompassed amino acid, carbohydrate, and lipid metabolism pathways. The decline in TCA cycle intermediates pointed to a potential downregulation of the cycle, whereas a decrease in ketone bodies indicated a shift towards lipid metabolism for energy production. Additionally, increased levels of nicotinate, nicotinamide, and quinolinate were observed in both organs. Overall, CHH's impact on the epidermis was prominent at 24hpi and diminished thereafter, whereas its influence on metabolism in gills was delayed but intensified at 48hpi. This differential CHH effect between gills and epidermis in P. clarkii provides new insights into the organ-specific regulatory mechanisms of CHH on energy metabolism and osmoregulation, warranting further comparative studies to elucidate the distinct roles of CHH in these organs.

The nuclear receptor gene E75 plays a key role in regulating the molting process of the spider mite, Tetranychus urticae.

The nuclear receptor gene Ecdysone-induced protein 75 (E75), as the component of ecdysone response genes in the ecdysone signaling pathway, has important regulatory function for insect molting. However, the regulatory function of E75 during the molting process of spider mites is not yet clear. In this study, the expression pattern of E75 in the molting process of the spider mite Tetranychus urticae was analyzed. The results showed that there was a peak at 8h post-molting, followed by a decline 8h after entering each respective quiescent stage across various developmental stages. During the deutonymph stage, the expression dynamics of E75, observed at 4-h intervals, indicated that the transcript levels of TuE75 peaked at 24h, coinciding with the onset of molting in the mites. To investigate the function of TuE75 during the molting process, silencing TuE75 through dsRNA injection into deutonymph mites at the age of 8h yielded a notable outcome: 78% of the deutonymph mites were unable to progress to the adult stage. Among these phenotypic mites, 37% were incapable of transitioning into the quiescent state and eventually succumbed after a certain period. An additional 41% of the mites successfully entered the quiescent state but encountered difficulties in shedding the old epidermis, leading to eventual mortality. In summary, these results suggested that TuE75 plays a key role in the molting process of T. urticae.

Combined analysis of the proteome and metabolome provides insight into microRNA-1174 function in Aedes aegypti mosquitoes

BackgroundPathogenic viruses can be transmitted by female Aedes aegypti (Ae. aegypti) mosquitoes during blood-meal acquisition from vertebrates. Silencing of mosquito- and midgut-specific microRNA (miRNA) 1174 (miR-1174) impairs blood intake and increases mortality. Determining the identity of the proteins and metabolites that respond to miR-1174 depletion will increase our understanding of the molecular mechanisms of this miRNA in controlling blood-feeding and nutrient metabolism of mosquitoes.MethodsAntisense oligonucleotides (antagomirs [Ant]) Ant-1174 and Ant-Ct were injected into female Ae. aegypti mosquitoes at 12–20 h posteclosion, and depletion of miR-1174 was confirmed by reverse transcription quantitative real-time PCR (RT-qPCR). Ant-1174-injected and control mosquitoes were collected before the blood meal at 72 h post-injection for tandem mass tag-based proteomic analysis and liquid chromatography-tandom mass spectrometry non-target metabolomic analysis to identify differentially expressed proteins and metabolites, respectively. RNA interference (RNAi) using double-stranded RNA (dsRNA) injection was applied to investigate the biological roles of these differentially expressed genes. The RNAi effect was verified by RT-qPCR and western blotting assays. Triglyceride content and ATP levels were measured using the appropriate assay kits, following the manufacturers’ instructions. Statistical analyses were conducted with GraphPad7 software using the Student’s t-test.ResultsUpon depletion of mosquito- and midgut-specific miR-1174, a total of 383 differentially expressed proteins (DEPs) were identified, among which 258 were upregulated and 125 were downregulated. Functional analysis of these DEPs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment suggested that miR-1174 plays important regulatory roles in amino acid metabolism, nucleotide metabolism, fatty acid metabolism and sugar metabolism pathways. A total of 292 differential metabolites were identified, of which 141 were upregulated and 151 were downregulated. Integrative analysis showed that the associated differential proteins and metabolites were mainly enriched in a variety of metabolic pathways, including glycolysis, citrate cycle, oxidative phosphorylation and amino acid metabolism. Specifically, the gene of one upregulated protein in miR-1174-depleted mosquitoes, purine nucleoside phosphorylase (PNP; AAEL002269), was associated with the purine, pyrimidine and niacin-nicotinamide metabolism pathways. PNP knockdown seriously inhibited blood digestion and ovary development and increased adult mortality. Mechanically, PNP depletion led to a significant downregulation of the vitellogenin gene (Vg); in addition, some important genes in the ecdysone signaling and insulin-like peptide signaling pathways related to ovary development were affected.ConclusionsThis study demonstrates differential accumulation of proteins and metabolites in miR-1174-depleted Ae. aegypti mosquitoes using proteomic and metabolomic techniques. The results provide functional evidence for the role of the upregulated gene PNP in gut physiological activities. Our findings highlight key molecular changes in miR-1174-depleted Ae. aegypti mosquitoes and thus provide a basis and novel insights for increased understanding of the molecular mechanism involved in a lineage-specific miRNA in mosquito vectors.Graphical

The genome-wide characterization and associated cold-tolerance function of the superoxide dismutase in the cold response of the tick Haemaphysalis longicornis

Accumulating evidence suggests that superoxide dismutase (SOD) is the first line of antioxidant defense in organisms and plays an important role in scavenging reactive oxygen species produced during environmental stress. However, limited information is available regarding the response of SOD genes to cold stress in ticks. Therefore, in the present study, SOD genes were cloned and identified from the genome of Haemaphysalis longicornis, and the function of SOD during the cold response was further explored. Seven SOD genes were characterized: HlCCS1, HlCCS2, HlMSD, HlCSD1, HlCSD2, HlCSD3, and HlCSD4. Bioinformatics analysis showed that HlCCS1 and HlCCS2 are copper chaperones of SODs. HlCSD1–HlCSD4 belong to the Cu/Zn SOD, whereas HlMSD belongs to the Mn SOD gene family. Fluorescence quantitative PCR showed that the expression of HlCCS2, HlMSD, and HlCSD1–3 was upregulated, whereas HlCCS1 and HlCSD4 were downregulated during the cold response of H. longicornis. Western blotting confirmed changes in the relative expression of HlCSD3 and HlMSD in H. longicornis after cold treatment. Mortality of H. longicornis increased significantly after dsRNA injection of HlCCS2, HlMSD, HlCSD1, and HlCSD3. The above results show that SODs have different regulatory functions during the cold response in H. longicornis, and there might be an interaction between treatment temperature and duration. Furthermore, the results lay a foundation for subsequent research on the molecular mechanism of cold tolerance in H. longicornis and shed light on the population distribution and diffusion limit of ticks.

ZSCAN4 Regulates Zygotic Genome Activation and Telomere Elongation in Porcine Parthenogenetic Embryos.

Zinc finger and SCAN domain-containing 4 (ZSCAN4), a DNA-binding protein, maintains telomere length and plays a key role in critical aspects of mouse embryonic stem cells, including maintaining genomic stability and defying cellular senescence. However, the effect of ZSCAN4 in porcine parthenogenetic embryos remains unclear. To investigate the function of ZSCAN4 and the underlying mechanism in porcine embryo development, ZSCAN4 was knocked down via dsRNA injection in the one-cell stage. ZSCAN4 was highly expressed in the four- and five- to eight-cell stages in porcine embryos. The percentage of four-cell stage embryos, five- to eight-cell stage embryos, and blastocysts was lower in the ZSCAN4 knockdown group than in the control group. Notably, depletion of ZSCAN4 induced the protein expression of DNMT1 and 5-Methylcytosine (5mC, a methylated form of the DNA base cytosine) in the four-cell stage. The H3K27ac level and ZGA genes expression decreased following ZSCAN4 knockdown. Furthermore, ZSCAN4 knockdown led to DNA damage and shortened telomere compared with the control. Additionally, DNMT1-dsRNA was injected to reduce DNA hypermethylation in ZSCAN4 knockdown embryos. DNMT1 knockdown rescued telomere shortening and developmental defects caused by ZSCAN4 knockdown. In conclusion, ZSCAN4 is involved in the regulation of transcriptional activity and is essential for maintaining telomere length by regulating DNMT1 expression in porcine ZGA.

Mn-XRN1 Has an Inhibitory Effect on Ovarian Reproduction in Macrobrachium nipponense.

XRN1 is an exoribonuclease that degrades mRNA in the cytoplasm along the 5'-3' direction. A previous study indicated that it may be involved in the reproduction of Macrobrachium nipponense. Quantitative real-time PCR was used to detect the spatiotemporal expression pattern of Mn-XRN1. At the tissue level, Mn-XRN1 was significantly expressed in the ovary. During development, Mn-XRN1 was significantly expressed at the CS stage of the embryo, on the 10th day post-larval and in the O2 stage of ovarian reproduction. The in situ hybridization results showed the location of Mn-XRN1 in the ovary. The expression of Mn-VASA was significantly increased after in vivo injection of Mn-XRN1 dsRNA. This suggests that Mn-XRN1 negatively regulates the expression of Mn-VASA. Furthermore, we counted the number of M. nipponense at various stages of ovarian reproduction on different days after RNAi. The results showed that ovarian development was significantly accelerated. In general, the results of the present study indicate that Mn-XRN1 has an inhibitory effect on the ovarian maturation of M. nipponense. The inhibitory effect might be through negative regulation of Mn-VASA.

Differential responses to double-stranded RNA injection and feeding in Mormon cricket (Orthoptera: Tettigoniidae).

The Mormon cricket, Anabrus simplex, is a flightless katydid, one of the major devastating rangeland pests in several states of the western United States. During the past few years, their sudden and periodic outbreaks into massive migratory bands caused significant economic losses to the rangeland forage and agricultural crops, particularly grain crops. Current population management methods rely heavily on broad-spectrum chemical insecticides, which could be toxic to nontargets, and even the targeted species might develop resistance in the long run. Therefore, we assessed the potential of RNA interference (RNAi)-based alternative management strategies that could supplement the current methods. In insects, RNAi efficiency varies with the method of double-stranded RNA (dsRNA) delivery. We tested 2 different methods of dsRNA delivery: injection and oral feeding of dsRNA. The results showed that Mormon crickets are sensitive to injection of dsRNA in a dose-dependent manner, but refractory to the oral feeding of dsRNA. Further, we confirmed the high nuclease activity in the insect midgut. In order to protect the dsRNA from the dsRNase activity and facilitate its uptake in the midgut, we encapsulated dsRNA inside poly lactic-co-glycolic acid (PLGA) nanoparticles and studied its release kinetics and RNAi efficiency by oral feeding. The release kinetics clearly suggested that the PLGA nanoparticle permeates from the insect digestive system to the hemolymph; however, it failed to induce an efficient RNAi response of the targeted genes. In conclusion, our findings suggest the different responses to dsRNA delivery methods in Mormon crickets, and further investigations involving dsRNA stability and its uptake mechanism are required to use RNAi as an alternative Mormon cricket population management strategy.

Functional roles of two novel P450 genes in the adaptability of Conogethes punctiferalis to three commonly used pesticides.

Introduction: Insect cytochrome P450 (CYP450) genes play important roles in the detoxification and metabolism of xenobiotics, such as plant allelochemicals, mycotoxins and pesticides. The polyphagous Conogethes punctiferalis is a serious economic pest of fruit trees and agricultural crops, and it shows high adaptability to different living environments. Methods: The two novel P450 genes CYP6CV1 and CYP6AB51 were identified and characterized. Quantitative real-time PCR (qRT-PCR) technology was used to study the expression patterns of the two target genes in different larval developmental stages and tissues of C. punctiferalis. Furthermore, RNA interference (RNAi) technology was used to study the potential functions of the two P450 genes by treating RNAi-silenced larvae with three commonly used pesticides. Results: The CYP6CV1 and CYP6AB51 genes were expressed throughout various C. punctiferalis larval stages and in different tissues. Their expression levels increased along with larval development, and expression levels of the two target genes in the midgut were significantly higher than in other tissues. The toxicity bioassay results showed that the LC50 values of chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin on C. punctiferalis larvae were 0.2028μg/g, 0.0683μg/g and 0.6110mg/L, respectively. After treating with different concentrations of chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin (LC10, LC30, LC50), independently, the relative expressions of the two genes CYP6CV1 and CYP6AB51 were significantly induced. After the dsRNA injection, the expression profiles of the two CYP genes were reduced 72.91% and 70.94%, respectively, and the mortality rates of the larvae significantly increased when treated with the three insecticides independently at LC10 values. Discussion: In the summary, after interfering with the CYP6CV1 and CYP6AB51 in C. punctiferalis, respectively, the sensitivity of C. punctiferalis to chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin was significantly increased, indicating that the two CYP6 genes were responsible for the adaptability of C. punctiferalis to the three chemical insecticides in C. punctiferalis. The results from this study demonstrated that CYP6CV1 and CYP6AB51 in C. punctiferalis play crucial roles in the detoxification of chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin.

RNA Interference-Mediated Knockdown of Tryptophan 2,3-Dioxygenase and Kynurenine-3-Monooxygenase in Monochamus Alternatus: Implications for Insect Control

Monochamus alternatus Hope (Coleoptera: Cerambycidae), an invasive beetle that has caused billions of dollars in economic losses, is a serious pest of Pinus massoniana in many Asian countries. Clarifying the eye pigment gene and its knockdown phenotype of M. alternatus can provide functional gene identification and a marker for screening of gene editing, as well as help develop new control ideas. In this study, we first screened the transcriptome and found one homologous gene of tryptophan 2,3-dioxygenase (TDO) and one of kynurenine-3-monooxygenase (KMO). By measuring the expression levels of TDO and KMO in different developmental periods, it was indicated that TDO and KMO were expressed in various stages of M. alternatus. The gene expression of MaKMO was higher than MaTDO, showing high expression after pupation and decreasing at the beginning of eclosion. MaTDO and MaKMO were knocked down using RNA interference technology in different periods of expression, and the temporal expression changes were obtained using RT-qPCR technology. The results showed that the expressions of MaTDO and MaKMO were significantly inhibited by the injection of dsRNA; the expressions of MaTDO at 48 h, 96 h and after pupation were 21.9%, 32.3% and 59.2%, respectively, meanwhile, those of KMO were 23.4%, 25.0% and 69.7%, respectively. There was a significant change in eye color, and the beetles were able to pupate normally without their activity being affected. Therefore, both MaTDO and MaKMO can be used as tag genes for M. alternatus. A dominant marker system based on eye color can be developed for the genetic manipulation and control of M. alternatus.

FOXO-like Gene Is Involved in the Regulation of 20E Pathway through mTOR in Eriocheir sinensis

The Forkhead Box O (FOXO) gene plays a key role in various biological processes, such as growth, metabolism, development, immunity and longevity. Molting is an essential process for crustacean growth, which is mainly regulated by 20-hydroxyecdysone (20E) and molt-inhibiting hormone (MIH). Although the role of FOXO in regulating the immune response of crustaceans is well documented, its involvement in controlling crustacean molting remains unclear. In this study, a FOXO-like gene (designed as EsFOXO-like) was identified in Eriocheir sinensis, and the regulation of the 20E pathway by EsFOXO-like was also investigated. The coding sequence of EsFOXO-like was 852 bp, which consisted of 283 amino acids including a conserved Forkhead (FH) domain. EsFOXO-like shared high similarity with FOXO genes from other crustaceans, and the mRNA expression levels of the EsFOXO-like gene were highest in the hepatopancreas and lowest in the hemocytes. However, transcription and protein expression of the EsFOXO-like gene were found to be up-regulated only during the pre-molt stage in the hepatopancreas, with lower expression levels observed at the post-molt stage. To explore the role of EsFOXO-like in the 20E pathway, EsFOXO-like was firstly inhibited by a specific FOXO inhibitor (AS1842856) and then through an EsFOXO-like dsRNA injection, respectively, and the results showed that the relative expression levels of EsFOXO-like were notably decreased in the hepatopancreas after both the inhibitor and dsRNA treatments. The 20E concentration, the mRNA expression levels of the 20E receptors including the ecdysone receptor (EcR) and the retinoid-X receptor (RXR) and EsmTOR transcription in the AS1842856 group or the EsFOXO-RNAi group were all significantly higher than that in the control group, while the mRNA expression level of EsMIH was significantly decreased after EsFOXO-like inhibition. To further investigate whether the EsFOXO-like acts through mTOR or not, Rapamycin was administered to inhibit mTOR activity in EsFOXO-like inhibited crabs. The results revealed a significant reduction in the concentration of 20E and the expression level of EsMIH in the AS1842856 + Rapamycin group compared to the AS1842856 + DMSO group, accompanied by an increase in EsEcR and EsRXR expression. These findings collectively suggest that EsFOXO-like regulates the 20E pathway through mTOR, which offered valuable insights into the understanding of the molting process in crustaceans.

Functional importance of groups I and II chitinases in cuticle chitin turnover during molting in a wood-boring beetle, Monochamus alternatus

Insects must periodically replace their old cuticle/exoskeleton with a new one in a process called molting or ecdysis to allow for continuous growth through sequential developmental stages. Many RNA interference (RNAi) studies have demonstrated that certain chitinases (CHTs) play roles in this vital physiological event because knockdown of these CHT genes resulted in developmental arrest during the ensuing molting period in several insect species. In this research we analyzed the functions of group I (MaCHT5) and group II (MaCHT10) CHT genes in molting of the Japanese pine sawyer, Monochamus alternatus, an important forest pest known as a major vector of the pinewood nematode. Real-time qPCR revealed that these two CHT genes differ in their expression patterns during late stages of development. Depletion of either MaCHT5 or MaCHT10 transcripts by RNAi resulted in lethal larval-pupal and pupal-adult molting defects depending on the double-stranded RNA (dsRNA) injection timing during development. The insects were unable to shed their old cuticle and died. Furthermore, transmission electron microscopic analysis revealed that, unlike dsEGFP-treated controls, dsMaCHT5- and dsMaCHT10-treated pharate adults exhibited a failure of degradation of the endocuticular layer of their old pupal cuticle, retaining nearly intact horizontal chitinous laminae and vertical pore canal fibers. Both enzymes were indispensable for complete turnover of the chitinous old endocuticle, which is critical for insect molting. The possible functions of two spliced variants of MaCHT10, namely, MaCHT10a and MaCHT10b, are also discussed. Our results add to the knowledge base for further functional studies of insect chitin catabolism by revealing the relative importance of both MaCHT5 and MaCHT10 in chitin turnover with subtle differences in their action. These essential genes and their encoded proteins are potential targets to manipulate for controlling populations of M. alternatus and other pest insects.

Gene cloning, protein expression, and enzymatic characterization of a double-stranded RNA degrading enzyme in Apolygus lucorum.

RNA interference (RNAi) is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells. However, silencing efficacy varies greatly among different insect species. Recently, we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection. The disappearance of double-stranded RNA (dsRNA) could be a potential factor that restricts RNAi efficiency. Here, we found that dsRNA can be degraded in midgut fluids, and a dsRNase of A. lucorum (AldsRNase) was identified and characterized. Sequence alignment indicated that its 6 key amino acid residues and the Mg2+ -binding site were similar to those of other insects' dsRNases. The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase. AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle, with peaks at the 4th instar ecdysis in the whole body. The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA. When comparing the substrate specificity of AldsRNase, 3 specific substrates (dsRNA, small interfering RNA, and dsDNA) were all degraded, and the most efficient degradation is dsRNA. Subsequently, immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells. Through cloning and functional study of AldsRNase, the enzyme activity and substrate specificity of the recombinant protein, as well as the subcellular localization of nuclease, the reason for the disappearance of dsRNA was explained, which was useful in improving RNAi efficiency in A. lucorum and related species.

A Cartilage Matrix Protein Regulates Collagen Synthesis in Mantle of Magallana gigas (Crassostrea gigas) under Ocean Acidification

The shell biosynthesis of oysters plays a critical role in protection against environmental stress, in which cartilage matrix proteins (CMPs) determine the mineralogical and crystallographic properties of the shell. In the present study, a cartilage matrix protein (designated as MgCMP1) was identified from the Pacific oyster Magallana gigas (Crassostrea gigas) with the objective of understanding its possible role in shell formation. The open reading frame (ORF) of MgCMP1 was 1815 bp, encoding a polypeptide of 605 amino acids with two von Willebrand factor (VWA) domains. The mRNA transcript of MgCMP1 was expressed constitutively in all examined tissues with a higher level in the mantle, especially highest in the middle fold (MF) of the three folds of the mantle. In addition, the interaction between recombinant protein MgCMP1 (rMgCMP1) and recombinant protein bone morphogenesis protein 7 (rMgBMP7) was identified in vitro. After injection of dsRNA to inhibit the expression of MgCMP1, the mRNA expression level of Mgcollagen I and Mgcollagen X in the MF of the mantle significantly decreased. After pre-puncturing and acidification treatment (pH 7.8), the thickness and length of the new formation shells were lower than those in control group (pH 8.1), and the positive hybridization signals of the MgCMP1 mRNA transcript in the three mantle folds were obviously weakened, especially in the MF, whereas the mRNA expression level of MgCMP1, Mgcollagen I and Mgcollagen X in the MF of mantle decreased significantly. These results suggested that MgCMP1 was involved in regulating the expression of Mgcollagen I and Mgcollagen X in the MF of the mantle in response to ocean acidification (OA).

Characterization and transcriptomic analysis of a native fungal pathogen against the rice pest Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens, is one of the most destructive pests of rice. Given the threats posed by insecticide resistance to its control, eco-friendly strategies based on microbial pathogens emerged as a promising biocontrol alternative. In the present study, we isolated a native fungal pathogen against BPH from infected BPH cadavers and preliminarily identified as a strain of Aspergillus fumigatus based on morphological and molecular methods. Laboratory bioassay revealed that this fungal strain was highly virulent to BPH both at nymphal and adult stages, with the median lethal times (LT50) of 7.5 and 5.8 days under high conidial concentration of 1 × 109 conidia mL-1. A genome-wide view of gene expressions in BPH against fungal attack was analyzed by transcriptomic sequencing and consequently a large number of differentially expressed genes that mainly involved in host immune defense and cell detoxification were found. RNAi-mediated knockdown of an upregulated gene encoding a serine protease (NlSPN) could cause a significant decrease in BPH survival. Combination of dsRNA injection and fungal infection showed an additive effect on BPH mortality, which provided clues to develop new pest management strategies against BPH.

Efficient RNA interference method during caste differentiation with hormone treatment in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae).

Unveiling the proximate mechanism of caste differentiation is crucial for understanding insect social evolution, and gene function analysis is an important tool in this endeavor. The RNA interference (RNAi) technique is useful in termites, but its knockdown effects may differ among species. One of the most important model species in the field of termite sociogenomics is Reticulitermes speratus Kolbe (Isoptera: Rhinotermitidae). Presoldier and worker differentiation of this species can be artificially induced by juvenile hormone and 20-hydroxyecdysone application, respectively. However, appropriate RNAi technique of genes expressed during caste differentiation has never been considered. To clarify this issue, first, we injected nine different volumes of nuclease-free water (NFW, 0-404.8 nL) into workers and found that survival and caste differentiation rates were strongly reduced by the application of the top three largest volumes. Second, we injected double-stranded (ds) RNA of ecdysone receptor homolog (RsEcR) (2.0 µg/151.8 nL NFW) into workers with hormone treatments. The expression levels of RsEcR were significantly reduced at 9 days after dsRNA injection. RsEcR RNAi strongly affected both molting events during presoldier and worker differentiation induced by hormone treatments. The present results highlight the need for caution regarding injection volumes for RNAi experiments using hormone treatments. We suggest that the injection of dsRNA solution (2 µg; approximately 100-200 nL) is suitable for RNAi experiments during caste differentiation induced by hormone application in R. speratus.

An ecdysteroid-regulated 16-kDa protein homolog participates in the immune response of the crayfish Procambarus clarkii

An ecdysteroid-regulated 16-kDa protein homolog (named Pc-E16), encoding 150 amino acid residues with a conserved MD-2-related lipid-recognition domain, was first identified in Procambarus clarkii. Phylogenetic analyses indicated similarity between Pc-E16 and 16-kDa proteins from Aplysia californica and insects. Recombinant Pc-E16 protein was successfully expressed in BL21 (DE3) Escherichia coli cells, and polyclonal antibodies against purified Pc-E16 proteins were prepared. In comparison with other tissues, Pc-E16 was highly expressed in the intestine; real-time PCR and Western blotting results indicated that Pc-E16 expression was significantly induced by lipopolysaccharides in hepatopancreas and hemocytes. Pc-E16-mediated signaling pathways were investigated by digital gene expression analysis following RNA interference targeting Pc-E16. A total of 6103 differentially expressed genes (DEGs) were identified, of which 3318 were up- and 2785 were downregulated. Many DEGs were involved in binding and catalytic activity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that DEGs were clustered into 225 pathways, and 15 significantly enriched pathways were identified at the immune system level. In addition, the expression level of Pc-E16 in hemocytes and hepatopancreas was obviously downregulated at 48 h after dsRNA injection, and Pc-E16-RNAi treatment affected the expression levels of immune-related genes. Altogether, our results suggest that Pc-E16 is involved in the innate immune response of P. clarkii.

Examination of the effects of Nanos gene knockdown on fecundity and reproductive modes in the brine shrimp, Artemia parthenogenetica

Artemia is not only a crucial live food source in fish and crustacean larviculture but also an ideal experimental organism for biological studies. Female Artemia subjects release either diapause-encysted embryos (cysts) via oviparous reproduction or nauplii via ovoviviparous reproduction. As a maternal effector gene, nanos is involved in the differentiation and migration of primordial germ cells. In the present study, the open reading frame (ORF) of the nanos gene of Artemia parthenogenetica was screened and analyzed. Nanos cDNA contained a 933-bp ORF, encoding 310 amino acids with a conserved zinc-finger motif. The expression patterns of nanos in various tissues and during embryo development in Artemia were analyzed by qRT-PCR. The results showed that nanos was specifically expressed in the ovary, with the highest expression being observed during early ovary development. Moreover, nanos knockdown induced by dsRNA injection significantly decreased the gene and protein expression levels of nanos. In addition, knockdown of nanos resulted in an obvious shift from ovoviviparity to oviparity and a reduced number of offspring, corresponding to irregular oocyte shape, cell membrane damage, and cytoplasmic vacuolization. Our study suggests that nanos not only regulates the ovary development and reproductive performance of Artemia but also play a critical role in determining its reproductive mode. This study will improve our knowledge of the role of nanos in the reproductive process of Artemia and the molecular mechanisms underlying reproduction in this shrimp.

Mercury-Modulated Immune Responses in Arctic Barnacle Goslings (Branta leucopsis) upon a Viral-Like Immune Challenge.

Historical mining activities in Svalbard (79°N/12°E) have caused local mercury (Hg) contamination. To address the potential immunomodulatory effects of environmental Hg on Arctic organisms, we collected newborn barnacle goslings (Branta leucopsis) and herded them in either a control or mining site, differing in Hg levels. An additional group at the mining site was exposed to extra inorganic Hg(II) via supplementary feed. Hepatic total Hg concentrations differed significantly between the control (0.011 ± 0.002 mg/kg dw), mine (0.043 ± 0.011 mg/kg dw), and supplementary feed (0.713 ± 0.137 mg/kg dw) gosling groups (average ± standard deviation). Upon immune challenge with double-stranded RNA (dsRNA) injection, endpoints for immune responses and oxidative stress were measured after 24 h. Our results indicated that Hg exposure modulated the immune responses in Arctic barnacle goslings upon a viral-like immune challenge. Increased exposure to both environmental as well as supplemental Hg reduced the level of natural antibodies, suggesting impaired humoral immunity. Hg exposure upregulated the expression of proinflammatory genes in the spleen, including inducible nitric oxide synthase (iNOS) and interleukin 18 (IL18), suggesting Hg-induced inflammatory effects. Exposure to Hg also oxidized glutathione (GSH) to glutathione disulfide (GSSG); however, goslings were capable of maintaining the redox balance by de novo synthesis of GSH. These adverse effects on the immune responses indicated that even exposure to low, environmentally relevant levels of Hg might affect immune competence at the individual level and might even increase the susceptibility of the population to infections.

Molecular identification of an insulin-like peptide from the swimming crab Portunus trituberculatus and evidence for its glucoregulation function

Insulin-like peptides (ILPs) are essential to the animal kingdom for regulating growth and development, reproduction, behavior, metabolism, and lifespan. In crustaceans, the most well-known ILP is insulin-like androgenic gland hormone, a key hormone in regulating sex differentiation and reproduction. Identification of other ILPs and their functions are still limited. In this study, an insulin-like peptide gene of the swimming crab Portunus trituberculatus was cloned and characterized. Its transcripts were mainly found in nerve tissues and expression could be induced by glucose, implying a putative role in glucoregulation. After depletion of endogenous ILP, injection of ILP dsRNA (dsILP) significantly elevated blood glucose levels and recombinant ILP (rILP) decreased hemolymph glucose levels, further clarifying the involvement of acquired ILP in hemolymph glucose regulation. Injection of dsILP decreased PtAkt, PtGS, PtPFK and increased PtGSK and PtPEPCK gene expression. The opposite profile was observed after glucose and rILP injection, indicating that PtILP might negatively regulate hemolymph glucose levels via the IIS (insulin/IGF-1 signaling) pathway by inhibiting gluconeogenesis and promoting glycogen synthesis and glycolysis. This study has refined the mechanism of glucose regulation in crustaceans and laid the foundation for further studies on ILP function.