Effects of Functional Depletion of Doublesex on Male Development in the Sawfly, Athalia rosae

The doublesex (dsx) gene, which encodes a transcription factor, regulates sexual differentiation in insects. Sex-specific splicing of dsx occurs to yield male- and female-specific isoforms, which promote male and female development, respectively. Thus, functional disruption of dsx leads to an intersexual phenotype in both sexes. We previously identified a dsx ortholog in the sawfly, Athalia rosae. Similar to dsx in other insects, dsx in the sawfly yields different isoforms in males and females as a result of alternative splicing. The sawfly exploits a haplodiploid mode of reproduction, in which fertilized eggs develop into diploid females, whereas unfertilized eggs parthenogenetically develop into haploid males. In the present study, we knocked down the A. rosae ortholog of dsx (Ardsx) during several developmental stages with repeated double-stranded RNA (dsRNA) injections. Knockdown of Ardsx via parental RNA interference (RNAi), which enables knockdown of genes in offspring embryos, led to a lack of internal and external genitalia in haploid male progeny. Additional injection of dsRNA targeting Ardsx in these animals caused almost complete male-to-female sex reversal, but the resulting eggs were infertile. Notably, the same knockdown approach using diploid males obtained by sib-crossing caused complete male-to-female sex reversal; they were morphologically and behaviorally females. The same RNAi treatment did not affect female differentiation. These results indicate that dsx in the sawfly is essential for male development and its depletion caused complete male-to-female sex reversal. This is the first demonstration of functional depletion of dsx not causing intersexuality but inducing total sex reversal in males instead.

Repat33 Acts as a Downstream Component of Eicosanoid Signaling Pathway Mediating Immune Responses of Spodoptera exigua, a Lepidopteran Insect

Repat (=response to pathogen) is proposed for an immune-associated gene family from Spodoptera exigua, a lepidopteran insect. In this gene family, 46 members (Repat1–Repat46) have been identified. They show marked variations in their inducible expression patterns in response to infections by different microbial pathogens. However, their physiological functions in specific immune responses and their interactions with other immune signaling pathways remain unclear. Repat33 is a gene highly inducible by bacterial infections. The objective of this study was to analyze the physiological functions of Repat33 in mediating cellular and humoral immune responses. Results showed that Repat33 was expressed in all developmental stages and induced in immune-associated tissues such as hemocytes and the fat body. RNA interference (RNAi) of Repat33 expression inhibited the hemocyte-spreading behavior which impaired nodule formation of hemocytes against bacterial infections. Such RNAi treatment also down-regulated expression levels of some antimicrobial genes. Interestingly, Repat33 expression was controlled by eicosanoids. Inhibition of eicosanoid biosynthesis by RNAi against a phospholipase A2 (PLA2) gene suppressed Repat33 expression while an addition of arachidonic acid (a catalytic product of PLA2) to RNAi treatment recovered such suppression of Repat33 expression. These results suggest that Repat33 is a downstream component of eicosanoids in mediating immune responses of S. exigua.

Efficacy of double-stranded RNA for the large-scale control and prevention of sacbrood virus in Apis cerana (Hymenoptera: Apidae) apiaries

Sacbrood virus (SBV) infection in Apis cerana has caused tremendous damage in India, Thailand, Vietnam, and China since the 1970s. The disease caused by this virus results in colony collapse disorder in A. cerana and is also a devastating disease affecting A. cerana in South Korean apiaries. It has almost resulted in the elimination of the species. Therefore, control measures for this emerging threat are urgently needed. SBV RNA interference (RNAi) targeting VP1 was prepared to test the safety and efficacy of protection and treatment in artificially infected larvae and in infected colonies in South Korean apiaries. The efficacy of VP1 double-stranded RNA (dsRNA) was confirmed for the protection and treatment of infected larvae by increasing the survival rate in comparison with that in untreated larvae. Furthermore, an optimal application procedure was established for the large-scale RNAi treatment of SBV in apiaries. The protection of healthy colonies from SBV by RNAi was demonstrated in 100% of apiaries, and the treatment results showed that after five administrations, the SBV in infected colonies was mitigated to a safe level at which no symptoms of the disease were observed. Importantly, the low cost of dsRNA production in this study enables its application as a specific drug in large scale in South Korean apiculture.

Molecular Characteristics of Fat Body Protein 1 in the Oriental Fruit Fly, Bactrocera dorsalis

Bactrocera dorsails fat body protein 1 (Bdfbp1) cDNA was cloned (GenBank accession no. MT514270), and the complete 3,749-bp cDNA encoded a 1,152-amino acid protein. The phylogenetic relationship of dipteran fbp1s was analyzed. The sequence XP_028900815 from the insect genome project for Zeugodacus cucurbitae (LOC105219342) was proposed that two fbp1 genes were present in the sequence. The developmental transcriptional expression profiles were determined. In the larval stages, Bdfbp1 mRNA had significantly higher expression in the late third instar larvae compared with first, second, and early third instar larvae. In the pupal stages, the highest expression of Bdfbp1 mRNA was found in the newly pupated pupae and then decreased with age. In the fat body of female adults, Bdfbp1 was highly expressed in newly emerged samples and decreased rapidly over the following three days. In the fat body of male adults, Bdfbp1 was highly expressed in newly eclosed samples. RNAi treatment decreased the expression level of Bdfbp1 without statistical difference. However, RNAi treatment significantly decreased the rate of eclosion. These results suggest that Bdfbp1 may function as a storage protein and be associated with adult eclosion.

Molecular Identification and Immunity Functional Characterization of Lmserpin1 in Locusta migratoria manilensis

Serine protease inhibitors (Serpins) are a broadly distributed superfamily of proteins that exist in organisms with the role of immune responses. Lmserpin1 gene was cloned firstly from Locusta migratoria manilensis and then was detected in all tested stages from eggs to adults and six different tissues through qRT-PCR analysis. The expression was significantly higher in the 3rd instars and within integument. After RNAi treatment, the expression of Lmserpin1 was significantly down-regulated at four different time points. Moreover, it dropped significantly in the fat body and hemolymph at 24 h after treatment. The bioassay results indicated that the mortality of L. migratoria manilensis treated with dsSerpin1 + Metarhizium was significantly higher than the other three treatments. Furthermore, the immune-related genes (PPAE, PPO, and defensin) treated by dsSerpin1 + Metarhizium were significantly down-regulated compared with the Metarhizium treatment, but the activities of phenoloxidase (PO), peroxidase (POD), superoxide dismutase (SOD), glutathione S-transferase (GST), and multifunctional oxidase (MFO) were fluctuating. Our results suggest that Lmserpin1 plays a crucial role in the innate immunity of L. migratoria manilensis. Lmserpin1 probably took part in regulation of melanization and promoted the synthesis of antimicrobial peptides (AMPs).

Prostaglandin catabolism in Spodoptera exigua, a lepidopteran insect

ABSTRACTSeveral prostaglandins (PGs) and PG-synthesizing enzymes have been identified from insects. PGs mediate cellular and humoral immune responses. However, uncontrolled and prolonged immune responses might have adverse effects on survival. PG catabolism in insects has not been reported. Here, using a transcriptomic analysis, we predicted the presence of two PG-degrading enzymes, PG dehydrogenase (SePGDH) and PG reductase (SePGR), in Spodoptera exigua, a lepidopteran insect. SePGDH and SePGR expression levels were upregulated after immune challenge. However, their expression peaks occurred after those of PG biosynthesis genes, such as those encoding PGE2 synthase or PGD2 synthase. SePGDH and SePGR expression levels were upregulated after injection with PGE2 or PGD2. In contrast, such upregulated expression was not detected after injection with leukotriene B4, an eicosanoid inflammatory mediator. RNA interference (RNAi) using double-stranded RNAs specific to SePGDH or SePGR suppressed their expression levels. The RNAi treatment resulted in an excessive and fatal melanization of larvae even after a non-pathogenic bacterial infection. Phenoloxidase (PO) activity mediating the melanization in larval plasma was induced by bacterial challenge or PGE2 injection. Although the induced PO activity decreased after 8 h in control larvae, those treated with dsRNAs specific to PG-degrading enzyme genes kept a high PO activity for a longer period. These results suggest that SePGDH and SePGR are responsible for PG degradation at a late phase of the immune response.

Prostaglandin catabolism in Spodoptera exigua, a lepidopteran insect

ABSTRACTSeveral prostaglandins (PGs) and PG-synthesizing enzymes have been identified from insects. PGs can mediate cellular and humoral immune responses. However, uncontrolled and prolonged immune responses might have adverse effects on survival. PG catabolism in insects has not been reported. Here, using a transcriptomic analysis, we predicted two PG-degrading enzymes, PG dehydrogenase (SePGDH) and PG reductase (SePGR), in Spodoptera exigua, a lepidopteran insect. SePGDH and SePGR expression levels were upregulated after immune challenge. However, their expression peaks occurred after those of PG biosynthesis genes such as PGE2 synthase or PGD2 synthase. Indeed, SePGDH and SePGR expression levels were upregulated after injection with PGE2 or PGD2. In contrast, such upregulated expression was not detected after injection with leukotriene B4, an eicosanoid inflammatory mediator. RNA interference (RNAi) using double-stranded RNAs specific to SePGDH or SePGR suppressed their expression levels. The RNAi treatment resulted in an excessive and fatal melanization of larvae even after a non-pathogenic bacterial infection. Phenoloxidase (PO) activity mediating the melanization in larval plasma was induced by bacterial challenge or PGE2 injection. Although the induced PO activity decreased after 8 h in control, larvae treated with dsRNAs specific to PG-degrading enzyme genes kept the high PO activities for a longer period compared to control larvae. These results suggest that SePGDH and SePGR are responsible for PG degradation at a late phase of immune responses.

RNAi analysis of Doublesex1 expression in Daphnia pulex Leydig, 1860 (Cladocera)

To explore the role of the Doublesex gene (Dsx1) in the switching between reproductive modes in Daphnia pulex Leydig, 1860, we performed gene silencing using RNA interference (RNAi). We also investigated the expression of gfp (Green Fluorescent Protein) by quantitative-PCR, and the expression of Dsx1 mRNA after RNAi by real-time PCR. Dsx1 expression was significantly greater in males than in females, and was down-regulated in experimental groups compared with the control group. The decrease was more significant in females (35% of controls) than males (47% of controls). The gene fragment of Dsx1 amplified by PCR was ligated to the pEASY-Blunt vector to obtain the recombinant plasmid expressed recombinant protein induced by isopropyl-β-D-thiogalactoside (IPTG). After having been purified by Ni-column affinity chromatography, the recombinant protein as antigen was used to immune rabbits. The antiserum was successfully purified by the protein A method to obtain the Dsx1 polyclonal antibody, an IgG concentration of 2.65 mg/ml, titer 240 000. Western blotting showed that RNAi treatment significantly reduced DSX1 protein levels. Whole-mount immunofluorescence analysis revealed that the fluorescence intensity of appendages was significantly lower following RNAi treatment than the negative control group, and comparable with the blank control group, further confirming the decrease in Dsx1 expression after RNAi treatment. The number of offspring in all experimental groups was measured, and was significantly greater in the RNAi-treated group compared with the control group. These results provide a foundation for further work on the molecular basis of switching between the two modes of reproduction in cladocerans. The findings also further increase our understanding of the regulatory effects of Dsx1 in the differing sexes in these crustaceans.