A survey on the off-target effects of insecticidal double-stranded RNA targeting the Hvβ'COPI gene in the crop pest Henosepilachna vigintioctopunctata through RNA-seq

The specificity of the double-stranded RNA(dsRNA) used in the RNA interference(RNAi) technique is crucial for the success of sequence-specific gene silencing. Currently, RNAi-mediated insect control is a trending research topic.However, the off-target effects of the dsRNA in RNAi are a major concern. In this study, the ds Hvβ’COPI(coat protein complex I, β’ subunit)-treated and untreated transcriptomes of the 28-spotted potato lady beetle(Henosepilachna vigintioctopunctata) were compared to understand its off-target gene silencing effects. The RNA-seq results revealed that 63 and 44 differentially expressed genes(DEGs) were upregulated and downregulated, respectively, in the ds Hvβ’COPI treated group as compared with the control. Validation of the differential expressions of some selected DEGs via reverse transcription-quantitative PCR(RT-qPCR) analysis confirmed the reliability of the transcriptome analysis results. Further downstream analysis revealed that there were no genes homologous with Hvβ’COPI in H. vigintioctopunctata. Additionally,no genes with a >11 bp continuous match with ds Hvβ’COPI were found in the H. vigintioctopunctata transcriptome. Six genes(Hvcitron, Hvhelicase, Hvtransposase, Hvserine, Hvdynein, and Hv E3 ubiquitin) were selected to examine the offtarget activity of ds Hvβ’COPI based on their potential involvement in various H. vigintioctopunctata metabolic pathways.The severity of silencing these six off-target genes was evaluated by employing RNAi. The RNAi results confirmed the downregulation of the expression of all six genes, although there was no significant lethality. The findings of this study will be helpful in the risk analysis of future RNAi-mediated pest control experiments.

The production relationship of destruxins and blastospores of Metarhizium anisopliae with virulence against Plutella xylostella

Metarhizium anisopliae as an essential entomopathogenic fungus has been known to produce destruxins （a kind of cyclo-peptidic mycotoxins） and blastospores in submerged culture. Blastospores and destruxins are candidates for in- secticides, but the relations of both productions and the impact factors are unclear yet. In this study, we investigated the effects of inoculums, rotation, dissolved oxygen （DO） on the productions of blastospores and destruxins A and B （DA and DB） in submerged culture of M. anisopliae strain MaQ01. The results indicated that DO levels were regulated by inoculum amounts and rotation speeds, meanwhile, the productions of DA, DB and blastospores were also closely influenced by those factors. Totally, when DO value was more than 40%, the higher productions of destruxins and blastospores were achieved, by contrast, lower than 40% of DO values resulted in lower productions. The regression analysis suggested that the productions of DA, DB and blastospores were positively correlated with the DO levels. Meanwhile, the positive correlations between the productions of DA or DB and blastospores were also found. Briefly, when the rotation is 150 r min-1 and the inoculum is 1.0xl06 spore mL-1, the DA, DB and blastospores achieved the best production of 61.81 mg mL-1, 24.74 mg mL-1 and 5.73x 108 spore mL-1, respectively. In addition, the pathogenicities of blastospores and conidia against Plutella xylostella were bioassayed. The higher mortalities of P. xylostella were totally recorded in blastospore treatments than in conidia treatments, especially in lower dosages and earlier periods. Our research will give some new insights to production of destruxins and blastospores by using M. anisopliae.

Transcriptome Analysis of Female and Male Henosepilachna vigintioctopunctata(Coleoptera:Coccinellidae)Identifies Sex-related Genes

Henosepilachna vigintioctopunctata is an emerging horticultural pest of Solanaceae plants in Asia.Here,we employed transcriptome sequencing of three female and three male H.vigintioctopunctata libraries to identify sex-related genes.We recorded 281599294 bp of clean reads,and de novo assembly generated 85206 unigenes,with N_(50) of 1311 bp and average length of 763 bp.We found 5002 genes highly expressed in males and 2179 genes highly expressed in females.Expression profiles of six unigenes specific to females,six unigenes specific to males,and ten unigenes common to both sexes were verified using semi-quantitive RT-PCR;results showed expression levels of these 22 unigenes were relatively consistent with the transcriptome analysis.Using all unigenes as references,we identified 6657 putative simple sequence repeats.The transcriptome sequences and gene expression profiles of female and male H.vigintioctopunctata help explore genetic mechanisms of sexual dimorphism.

Effects of Aschersonia aleyrodis on the life table and demographic parameters of Bemisia tabaci

The present study reports the sublethal effects of the entomopathogenic fungus, Aschersonia aleyrodis（Webber） on Bemisia tabaci（Gennadius）（Homoptera： Aleyrodidae）. A fungal suspension of A. aleyrodis isolate Aa005 containing 1×107 conidia mL^-1 was sprayed against B. tabaci on eggplant leaves under greenhouse conditions. The effects of fungal application on survival as well as life table parameters of the whitefly were observed at different post inoculation periods. The results indicated that A. aleyrodis can significantly affect the survival of 1st, 2nd, and 3rd nymphal instars of B. tabaci. Developmental periods of different instar nymphs were not affected by fungal application. A. aleyrodis spores persisted well and significantly affected the survivorship of young instar nymphs of B. tabaci at different post incubation periods. Life table results suggested that A. aleyrodis has no impact on general fecundity and longevity of B. tabaci. When the pathogen was exposed to the open environment and maintained for a relatively longer period, a reduction in the reproductive rate and intrinsic rate of increase was caused by the fungal spores, though the sublethal effects were not as good as the control treatment. The results suggest that the ability of spores to suppress an increase in whitefly population persists well after incubation of spores to the external environment（up to 9 days）.

Insulin peptides and their receptors regulate ovarian development and oviposition behavior in Diaphorina citri

Diaphorina citri is an important vector of Citrus Huanglongbing(HLB)disease.After feeding on young host plant shoots,the population of D.citri can increase significantly.Females also only lay eggs on young shoots.However,there are few studies on the mechanism of this phenomenon.Exogenous nutrient signals can affect the insulin signaling system of D.citri after feeding on young shoots.In this study,the expression of upstreamfactors DclLPl,DclLP2,and DclR in the insulin signaling system of D.citri was upregulated after feeding on young shoots.After being silenced by RNA interference technology,the results showed that the number of oviposited eggs of D.citri was significantly decreased and the ovarian development was inhibited with severe vacuolation.In addition,detection using quantitative reverse transcription-polymerase chain reaction showed that the upstream regulatory gene DcRheb of the target of rapamycin(TOR)pathway and the downstream reproduction-related DcVg gene were also significantly downregulated.These results suggest that feeding upon young shoots may upregulate the expression levels of upstream factors DclLPl,DclLP2,and DclR in the insulin signaling system.The signal will be through upregulating the expression of DcRheb,an upstream gene of the TOR signaling pathway.This in turn influences yolk metabolism,which eventually causes the ovaries of female D.citri to mature and therefore initiate oviposition behavior.

RNA interference-mediated silencing of vATPase subunits A and E affect survival and development of the 28-spotted ladybeetle,Henosepilachna vigintioctopunctata

RNA interference(RNAi)has emerged as a powerful tool for developing novel management strategies for controlling insect pests.The 28-spotted ladybeetle,Henosepilachna vigintioctopunctata is one of the most important pests attacking solanaceous plants in Asia.In this study,the potential of dietary RNAi to manage H.vigintioctopunctata was investigated using both in vitro synthesized and bacterially expressed double-stranded RNAs(dsRNAs)of HvvATPase A and HvvATPase E.The expression levels of HvvATPase A and HvvATPase E were higher in Malpighian tubules than in other tissue types.The silencing of HvvATPase A and HvvATPase E led to significant mortality in H.vigintioctopunctata larvae.In addition,the ingestion of HvvATPase A and HvvATPase E significantly deterred feeding behavior and subsequently arrested the development of H.vigintioctopunctata.Notably,the bacterially expressed dsRNAs consistently caused higher mortality in larvae and adults.Finally,the nontarget effects of the dsRNAs of H.vigintioctopunctata on the predatory ladybeetle Propylaea japonica were evaluated.P.japonica 1st instar larvae were administered vATPase A and vATPase E dsRNAs from H.vigintioctopunctata and P.japonica under the worst-case scenario,in which dsGFP served as negative control.There were significant effects of dsHvvATPase A on P.japonica at the transcriptional level but not at the organismal level,whereas dsHvvATPase E did not effect P.japonica at either the transcriptional or the organismal level.Collectively,the results of the study suggest that HvvATPase A and HvvATPase E can act as novel molecular targets for the control of H.vigintioctopunctata.

Antagonistic interaction between male-killing and cytoplasmic incompatibility induced by Cardinium and Wolbachia in the whitefly,Bemisia tabaci

Cardinium and Wolbachia are maternally inherited bacterial symbionts of arthropods that can manipulate host reproduction by increasing the fitness of infected females.Here,we report that Cardinium and Wolbachia coinfection induced male-killing and cytoplasmic incompatibility(CI)when they coexisted in a cryptic species of whitefly,Bemisia tabaci Asia II7.Cardinium and Wolbachia symbionts were either singly or simul-taneously localized in the bacteriocytes placed in the abdomen of B.tabaci nymphs and adults.Cardinium-Wolbachia coinfection induced male-killing and resulted in a higher female sex ratio in the intraspecific amphigenetic progeny of Asia II7 IcwH and IcwL lines;interestingly,male-illing induction was enhanced with increased Cardinium titer.Moreover,single infection of Wolbachia induced partial CI in the Asia II7 Iw line and resulted in reduced fecundity,higher embryonic mortality,and lower female sex ratio.The uninfected Asia II7 Iu line had significantly higher fecundity,lower embryonic and nymphal mortalities,and a lower level of CI than both the Wolbachia infected Asia II7 Iw line and the Cardinium--Wolbachia-coinfected Asia II7 IcwH line.Our findings indicate that Cardinium-Wolbachia coinfection induced male-killing,which may have had antag-onistic effects on Wolbachia-induced CI in the Asia II7 whiteflies.For the first time,our study revealed that B.tabaci Asia II7 reproduction is co-manipulated by Cardinium and Wolbachia endosymbionts.

Oral delivery of dsHvlwr is a feasible method for managing the pest Henosepilachna vigintioctopunctata(Coleoptera:Coccinellidae)

RNA interference(RNAi)techniques have emerged as powerful tools that facilitate development of novel management strategies for insect pests,such as Henosepilachna vigintioctopunctata(Coleoptera:Coccinellidae),which is a major pest of solanaceous plants in Asia.In this study,the potential of oral delivery of in vvYro-synthesized and bacterially expressed double-stranded H.vigintioctopunctata lesswright(Iwr)gene(dsHvlwr)to manage of H.vigintioctopunctata was investigated.Our results showed that the gene Hvlwr had a 480-bp open reading frame and encoded a 160-amino acid protein.Hvlwr expression levels were greater in the fat body than other tissue types.Hvlwr silencing led to greater H.vigintioctopunctata mortality rates and appeared to be time-and partially dose-dependent,likely as a result of the number of hemocytes increasing with dsRNA concentration,but decreasing with time.Bacterially expressed dsHvlwr that was applied to leaf discs caused 88%,66%,and 36%mortality in 1st instars,3rd instars,and adults after 10,10,and 14 d,respectively;when applied to living plants,there was greater mortality in 1 st and 3rd instars,but there was no effect on adults.Furthermore,dsHvlwr led to improved plant protection against H.vigintioctopunctata.Our study shows an effective dietary RNAi response in H.vigintioctopunctata and that Hvlwr is a promising RNAi target gene for control of this pest species.

Role of digestive protease enzymes and related genes in host plant adaptation of a polyphagous pest,Spodoptera frugiperda

The evolutionary success of phytophagous insects depends on their ability toefficiently exploit plants as a source of energy for survival.Herbivorous insects largelydepend on the efficiency,flexibility,and diversity of their digestive physiology and sophistication of their detoxification system to use chemically diverse host plants as foodsources.The fall armyworm,Spodoptera frugiperda(J.E.Smith),is a polyphagous pest ofmany commercially important crops.To elucidate the ability of this insect pest to adaptto host plant mechanisms,we evaluated the impact of primary(corn)and alternate(rice)host plants after 1l generations on gut digestive enzymatic activity and expression profiles of related genes.Results indicated that the total protease and classspecific trypsinand chymotrypsinlike protease activity of S.frugiperda significantly differed among hostplant treatments.The classspecifiq protease profiles greatly differed in S.frugiperdamidguts upon larval exposure to different treatments with inhibitors compared with treatments without inhibitors.Similarly,the single and cumulative effects of the enzymespecific inhibitors TLCK,TPCK,and E64 significantly increased larval mortality and reduced larval growth/mass across different plant treatments.Furthermore,the quantitativereverse transcription polymerase chain reaction results revealed increased transcription oftwo trypsin(SfTry3,SfTry7)and one chymotrypsin gene(Sfchym9),which indicatedthat they have roles in host plant adaptation.Knockdown of these genes resulted in significantly reduced mRNA expression levels of the trypsin genes.This was related to theincreased mortality observed in treatments compared with the dsRED control.This resultindicates possible roles of S.frugiperda gut digestive enzymes and related genes in hostplant adaptation.