Two putative fatty acid synthetic genes of BgFas3 and BgElo1 are responsible for respiratory waterproofing in Blattella germanica

Water retention is critical for physiological homeostasis and survival in terrestrial insects. While deposition of hydrocarbons on insect cuticles as a key measure for water conservation has been extensively investigated, we know little about other mechanisms for preventing water loss in insects. Here, we report two fatty acid synthetic genes that are independent of hydrocarbon production but crucial for water retention in the German cockroach Blattella germanica (L.). First, an integument enriched fatty acid elongase gene (BgElo1) was identified as a critical gene for desiccation resistance in B. germanica;however, knockdown of BgElo1 surprisingly failed to cause a decline in cuticular lipids. In addition, RNA interference (RNAi)-knockdown of an upstream fatty acid synthase gene (BgFas3) showed a similar phenotype, and transmission electron microscopy analysis revealed that BgFas3- or BgElo1-RNAi did not affect cuticle architecture. Bodyweight loss test showed that repression of BgFas3 and BgElo1 significantly increased the weight loss rate, but the difference disappeared when the respiration was closed by freeze killing the cockroaches. A water immersion test was performed, and we found that BgFas3- and BgElo1-RNAi made it difficult for cockroaches to recover from drowning, which was supported by the upregulation of hypoxia-related genes after a 10-h recovery from drowning. Moreover, a dyeing assay with water-soluble Eosin Y showed that this was caused by the entry of water into the respiratory system. Our research suggests that BgFas3 and BgElo1 are required for both inward and outward waterproofing of the respiratory system. This study benefits the understanding of water retention mechanisms in insects.

Lipophorin receptor regulates the cuticular hydrocarbon accumulation and adult fecundity of the pea aphid Acyrthosiphon pisum

Cuticular hydrocarbons form a barrier that protects terrestrial insects from water loss via the epicuticle.Lipophorin loads and transports lipids,including hydrocarbons,from one tissue to another.In some insects,the lipophorin receptor(LpR),which binds to lipophorin and accepts its lipid cargo,is essential for female fecundity because it mediates the incorporation of lipophorin by developing oocytes.However,it is unclear whether LpR is involved in the accumulation of cuticular hydrocarbons and its precise role in aphid reproduction remains unknown.We herein present the results of our molecular characterization,phylogenetic analysis,and functional annotation of the pea aphid(Acyrthosiphon pisum)LpR gene(ApLpR).This gene was transcribed throughout the A.pisum life cycle,but especially during the embryonic stage and in the abdominal cuticle.Furthermore,we optimized the RHA interference(RNAi)parameters by determining the ideal dose and duration for gene silencing in the pea aphid.We observed that the RNAi-based ApLpR suppression significantly decreased the internal and cuticular hydrocarbon contents as well as adult fecundity.Additionally,a deficiency in cuticular hydrocarbons increased the susceptibility of aphids to desiccation stress,with decreased survival rates under simulated drought conditions.Moreover,ApLpR expression levels significantly increased in response to the desiccation treatment.These results confirm that ApLpR is involved in transporting hydrocarbons and protecting aphids from desiccation stress.Furthermore,this gene is vital for aphid reproduction.Therefore,the ApLpR gene of A.pisum may be a novel RNAi target relevant for insect pest management.

Melanin synthesis genes BgTH and BgDdc affect body color and cuticle permeability in Blattella germanica

Melanin is involved in cuticle pigmentation and sclerotization of insects,which is critical for maintaining structural integrity and functional completeness of insect cuti-cle.The 2 key enzymes of tyrosine hydroxylase(TH)and dopa decarboxylase(DDC)predicted in melanin biosynthesis are usually conserved in insects.However,it is unclear whether their function is related to epidermal permeability.In this study,we identified and cloned the gene sequences of BgTH and BgDdc from Blattella germanica,and revealed that they both showed a high expression at the molting,and BgTH was abundant in the head and integument while BgDdc was expressed highest in the fat body.Using RNA in-terference(RNAi),we found that knockdown of BgTH caused molting obstacles in some cockroaches,with the survivors showing pale color and softer integuments,while knock-down of BgDdc was viable and generated an abnormal light brown body color.Desiccation assay showed that the dsBgTH-injected adults died earlier than control groups under a dry atmosphere,but dsBgDdc-injected cockroaches did not.In contrast,when dsRNA-treated cockroaches were reared under a high humidity condition,almost no cockroaches died in all treatments.Furthermore,with eosin Y staining assay,we found that BgTH-RNAi resulted in a higher cuticular permeability,and BgDdc-RNAi also caused slight dye pen-etration.These results demonstrate that BgTH and BgDdc function in body pigmentation and affect the waterproofing ability of the cuticle,and the reduction of cuticular perme-ability may be achieved through cuticle melanization.

Downregulation of NADPH-cytochrome P450 reductase via RNA interference increases the susceptibility of Acyrthosiphon pisum to desiccation and insecticides

Nicotinamide adenine dinucleotide phosphate(NADPH)-cytochrome P450 reductase(CPR)is involved in the metabolism of endogenous and exogenous substances,and detoxification of insecticides.RNA interference(RNAi)of CPR in certain insects causes developmental defects and enhanced susceptibility to insecticides.However,the CPR of Acyrthosiphon pisum has not been characterized,and its function is still not understood.In this study,we investigated the biochemical functions of A.pisum CPR(ApCPR).ApCPR was found to be transcribed in all developmental stages and was abundant in the embryo stage,and in the gut,head,and abdominal cuticle.After optimizing the dose and silencing duration of RNAi for downregulating ApCPR,we found that ApCPR suppression resulted in a significant decrease in the production of cuticular and internal hydrocarbon contents,and of cuticular waxy coatings.Deficiency in cuticular hydrocarbons(CHCs)decreased the survival rate of A.pisum under desiccation stress and increased its susceptibility to contact insecticides.Moreover,desiccation stress induced a significant increase in ApCPR mRNA levels.We further confirmed that ApCPR participates in CHC production.These results indicate that ApCPR modulates CHC production,desiccation tolerance,and insecticide susceptibility in A.pisum,and presents a novel target for pest control.