Modulation of fatty acid elongation in cockroaches sustains sexually dimorphic hydrocarbons and female attractiveness.

Insect cuticular hydrocarbons (CHCs) serve as important intersexual signaling chemicals and generally show variation between the sexes, but little is known about the generation of sexually dimorphic hydrocarbons (SDHCs) in insects. In this study, we report the molecular mechanism and biological significance that underlie the generation of SDHC in the German cockroach Blattella germanica. Sexually mature females possess more C29 CHCs, especially the contact sex pheromone precursor 3,11-DimeC29. RNA interference (RNAi) screen against the fatty acid elongase family members combined with heterologous expression of the genes in yeast revealed that both BgElo12 and BgElo24 were involved in hydrocarbon (HC) production, but BgElo24 is of wide catalytic activities and is able to provide substrates for BgElo12, and only the female-enriched BgElo12 is responsible for sustaining female-specific HC profile. Repressing BgElo12 masculinized the female CHC profile, decreased contact sex pheromone level, and consequently reduced the sexual attractiveness of female cockroaches. Moreover, the asymmetric expression of BgElo12 between the sexes is modulated by sex differentiation cascade. Specifically, male-specific BgDsx represses the transcription of BgElo12 in males, while BgTra is able to remove this effect in females. Our study reveals a novel molecular mechanism responsible for the formation of SDHCs and also provide evidences on shaping of the SDHCs by sexual selection, as females use them to generate high levels of contact sex pheromone.

Genetic Underpinnings of Cuticular Hydrocarbon Biosynthesis in the German Cockroach, Blattella germanica (L.): Progress and Perspectives.

Insect cuticular hydrocarbons (CHCs) serve as important waterproofing barriers and as signals and cues in chemical communication. Over the past 30 years, numerous studies on CHCs have been conducted in the German cockroach, Blattella germanica, leading to substantial progress in the field. However, there has not been a systematic review of CHC studies in this species in recent years. This review aims to provide a concise overview of the chemical composition, storage, transport, and physical properties of different CHCs in B. germanica. Additionally, we focus on the biosynthetic pathway and the genetic regulation of HC biosynthesis in this species. A considerable amount of biochemical evidence regarding the biosynthetic pathway of insect CHCs has been gathered from studies conducted in B. germanica. In recent years, there has also been an improved understanding of the molecular mechanisms that underlie CHC production in this insect. In this article, we summarize the biosynthesis of different classes of CHCs in B. germanica. Then, we review CHCs reaction to various environmental conditions and stressors and internal physiological states. Additionally, we review a body of work showing that in B. germanica, CHC profiles exhibit significant sexual dimorphism, specific CHCs act as essential precursors for female contact sex pheromone components, and we summarize the molecular regulatory mechanisms that underlie sexual dimorphism of CHC profiles. Finally, we highlight future directions and challenges in research on the biosynthesis and regulatory mechanisms of CHCs in B. germanica, and also identify potential applications of CHC studies in the pest control.

Cuticular Hydrocarbon Plasticity in Three Rice Planthopper Species.

Insect cuticular hydrocarbons (CHCs) are organic compounds of the surface lipid layer, which function as a barrier against water loss and xenobiotic penetration, while also serving as chemical signals. Plasticity of CHC profiles can vary depending upon numerous biological and environmental factors. Here, we investigated potential sources of variation in CHC profiles of Nilaparvata lugens, Laodelphax striatellus and Sogatella furcifera, which are considered to be the most important rice pests in Asia. CHC profiles were quantified by GC/MS, and factors associated with variations were explored by conducting principal component analysis (PCA). Transcriptomes were further compared under different environmental conditions. The results demonstrated that CHC profiles differ among three species and change with different developmental stages, sexes, temperature, humidity and host plants. Genes involved in cuticular lipid biosynthesis pathways are modulated, which might explain why CHC profiles vary among species under different environments. Our study illustrates some biological and ecological variations in modifying CHC profiles, and the underlying molecular regulation mechanisms of the planthoppers in coping with changes of environmental conditions, which is of great importance for identifying potential vulnerabilities relating to pest ecology and developing novel pest management strategies.

Hyperlipidemia induces vascular smooth muscle cell proliferation involving Wnt/ß-catenin signaling.

Hyperlipidemia has been shown to stimulate vascular smooth muscle cell (VSMC) proliferation. Wnt signaling pathway plays a critical role in embryonic development and cell proliferation. In this study, Sprague-Dawley rats fed with high-fat or normal diet for 12 weeks were sacrificed, and the thoracic aorta was harvested to determine wnt3a, ß-catenin, T-cell factor 4 (TCF4), and cyclin D1 expressions. VSMC proliferation within thoracic aorta and lipid accumulation within VSMCs were detected. Rat aortic VSMCs were cultured in serum from rats with hyperlipidemia or DKK-1; Wnt3a, ß-catenin, TCF4, and cyclin D1 expressions, and cell cycle distribution were determined. The findings demonstrated that increased number of VSMCs, lipid droplets, and vacuoles within thoracic aorta in the high-fat-fed group. Compared with controls, VSMCs from high-fat-fed rats showed higher mRNA expressions of wnt3a, ß-catenin, TCF4, and cyclin D1, as well as in VSMCs cultured with hyperlipidemic serum. After 24 h, VSMCs stimulated with hyperlipidemic serum showed significantly increased cell number and S-phase entry compared with cells exposed to normolipidemic serum. These effects were blocked by DKK-1. These results suggest that Wnt/ß-catenin signaling plays an important role in hyperlipidemia-induced VSMC proliferation.

The ATP-binding cassette transporter subfamily G member 4 mediates cuticular hydrocarbon transport to regulate drought tolerance in Acyrthosiphon pisum.

ABC transporters are a highly conserved membrane protein class that promote the transport of substances across membranes. Under drought conditions, insects primarily regulate the content of cuticular hydrocarbon (CHC) to retain water and prevent evaporative loss. Involvement of ABC transporter protein G (ABCG) subfamily genes in insect CHC transport has been relatively understudied. In this study, we demonstrated that ABCG4 gene in Acyrthosiphon pisum (ApABCG4) is involved in CHC transport and affects drought tolerance by regulating CHC accumulation. ApABCG4 is strongly expressed in the abdominal cuticle and embryonic stages of A. pisum. Effective silencing of ApABCG4 was achieved using RNAi, and the silencing duration was analyzed. ApABCG4 silencing resulted in a significant decrease in the total and component contents of the CHC and cuticular waxy coatings of A. pisum. Nevertheless, the internal hydrocarbon content remained unchanged. The lack of cuticular hydrocarbons significantly reduced the drought tolerance of A. pisum, shortening its survival time under drought stress. Drought stress caused significant upregulation of ApABCG4. Molecular docking showed that ApABCG4 has a high binding affinity for nine n-alkanes of CHC through electrostatic interactions. These results indicate that ApABCG4 is a novel RNAi target with key applications in aphid biological control.

Proteome Analysis of Male Accessory Gland Secretions in the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae).

In insects, male accessory gland proteins (ACPs) are important reproductive proteins secreted by male accessory glands (MAGs) of the internal male reproductive system. During mating, ACPs are transferred along with sperms inside female bodies and have a significant impact on the post-mating physiology changes of the females. Under sexual selection pressures, the ACPs exhibit remarkably rapid and divergent evolution and vary from species to species. The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a major insect pest of cruciferous vegetables worldwide. Mating has a profound impact on the females' behavior and physiology in this species. It is still unclear what the ACPs are in this species. In this study, two different proteomic methods were used to identify ACPs in P. xylostella. The proteins of MAGs were compared immediately before and after mating by using a tandem mass tags (TMT) quantitative proteomic analysis. The proteomes of copulatory bursas (CB) in mated females shortly after mating were also analyzed by the shotgun LC-MS/MS technique. In total, we identified 123 putative secreted ACPs. Comparing P. xylostella with other four insect ACPs, trypsins were the only ACPs detected in all insect species. We also identified some new insect ACPs, including proteins with chitin binding Peritrophin-A domain, PMP-22/ EMP/ MP20/ Claudin tight junction domain-containing protein, netrin-1, type II inositol 1,4,5-trisphosphate 5-phosphatase, two spaetzles, allatostatin-CC, and cuticular protein. This is the first time that ACPs have been identified and analyzed in P. xylostella. Our results have provided an important list of putative secreted ACPs, and have set the stage for further exploration of the functions of these putative proteins in P. xylostella reproduction.

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 cuticle. 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 interference (RNAi), we found that knockdown of BgTH caused molting obstacles in some cockroaches, with the survivors showing pale color and softer integuments, while knockdown 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 penetration. These results demonstrate that BgTH and BgDdc function in body pigmentation and affect the waterproofing ability of the cuticle, and the reduction of cuticular permeability 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.

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.

A single gene integrates sex and hormone regulators into sexual attractiveness.

Sex differentiation and hormones are essential for the development of sexual signals in animals, and the regulation of sexual signals involves complex gene networks. However, it is unknown whether a core gene is able to connect the upstream regulators for controlling sexual signal outputs and behavioural consequences. Here, we identify a single gene that integrates both sex differentiation and hormone signalling with sexual attractiveness in an insect model. CYP4PC1 in the German cockroach, Blattella germanica, controls the rate-limiting step in producing female-specific contact sex pheromone (CSP) that stimulates male courtship. As revealed by behavioural, biochemical, molecular, genetic and bioinformatic approaches, in sexually mature females, CYP4PC1 expression and CSP production are coordinately induced by sex differentiation genes and juvenile hormone (JH) signalling. In adult males, direct inhibition of CYP4PC1 expression by doublesexM binding in gene promoter and lack of the gonadotropic hormone JH prevent CSP production, thus avoiding male-male attraction. By manipulating the upstream regulators, we show that wild-type males prefer to court cockroaches with higher CYP4PC1 expression and CSP production in a dose-dependent manner, regardless of their sex. These findings shed light on how sex-specific and high sexual attractiveness is conferred in insects.

A new pest management strategy: transforming a non-host plant into a dead-end trap crop for the diamondback moth Plutella xylostella L.

BACKGROUND: The use of trap crops can reduce the egg production of female Plutella xylostella in cruciferous vegetables and is an effective method for controlling this pest. To date, most of the trap plants that have been studied are cruciferous plants containing high concentrations of glucosinolates, which are more attractive to P. xylostella female adults. However, the application of these trap plants also has some limitations. Studies have shown that aqueous extracts of cruciferous plants can attract P. xylostella to lay eggs. In this study, we utilized the extract of Chinese kale to treat a non-host plant, the faba bean, and evaluated the possibility of using it as a dead-end trap plant for P. xylostella control. RESULTS: Plutella xylostella females laid significantly more eggs on faba beans that had been sprayed with the extract of Chinese kale rather than on Chinese kale itself. The first instar larvae of P. xylostella failed to survive on faba beans. Notably, the faba beans with the Chinese kale extract had the strongest attraction effect on P. xylostella females when placed 3 m away from the Chinese kale. Moreover, this attraction effect of faba beans on P. xylostella for oviposition lasted for up to 15 days. CONCLUSION: Faba bean plants sprayed with the aqueous extract of Chinese kale represent a potential dead-end trap plant for P. xylostella adults and their oviposition while being invariably deadly for their offspring. The present study provides a new proof of concept of using a non-cruciferous trap plant for P. xylostella management.

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.

Involvement of integument-rich CYP4G19 in hydrocarbon biosynthesis and cuticular penetration resistance in Blattella germanica (L.).

BACKGROUND: Cuticle penetration plays an important role as a mechanism of insecticide resistance, but the underlying molecular mechanism remains poorly understood. In Blattella germanica, the cytochrome P450 gene, CYP4G19, is overexpressed in a pyrethroid-resistant strain. Here, we investigated whether CYP4G19 is involved in the biosynthesis of hydrocarbons and further contributes to cuticular penetration resistance in B. germanica. RESULTS: Compared with the susceptible strain, pyrethroid-resistant cockroaches showed lower cuticular permeability with Eosin Y staining. Removal of epicuticular lipids, mainly nonpolar hydrocarbons, with a hexane wash intensified the cuticular permeability and decreased the resistance index of the resistant strain. CYP4G19 was predominately expressed in the abdominal integument and could be upregulated by desiccation stress or short exposure to beta-cypermethrin. Overexpression of CYP4G19 in the resistant strain was positively correlated with a higher level of cuticular hydrocarbons (CHCs). RNAi-mediated knockdown of CYP4G19 significantly decreased its expression and caused a reduction in CHCs. Meanwhile, CYP4G19 suppression resulted in a non-uniform array of the lipid layer, enhanced cuticle permeability, and compromised insecticide tolerance. CONCLUSION: Our findings confirm that CYP4G19 is involved in hydrocarbon production and appears to contribute to hydrocarbon-based penetration resistance in B. germanica. This study highlights the lipid-based penetration resistance, advancing our understanding of the molecular mechanism underlying P450-mediated cuticular penetration resistance in insects. © 2019 Society of Chemical Industry.

Involvement of apolipoprotein D in desiccation tolerance and adult fecundity of Acyrthosiphon pisum.

Apolipoprotein D (ApoD) is a lipocalin superfamily member that plays important roles in the transport of small hydrophobic molecules, lipid metabolism, and stress resistance. Cuticular hydrocarbons are the principal components of the epicuticular lipid layer and play a critical role in water retention against environmental desiccation stress; however, the mechanism underlying the role of ApoD in insect desiccation tolerance has not yet been elucidated. Here, we report the molecular constitution, functional analysis, and phylogenetic relationship of the ApoD gene in Acyrthosiphon pisum (ApApoD). We found that ApApoD was transcribed throughout the life cycle of A. pisum, but was prominently expressed in the embryonic period and abdominal cuticle. In addition, we optimized the dose and silencing duration of RNAi, observing that RNAi against ApApoD significantly reduced the levels of both internal and cuticular hydrocarbons and adult fecundity. Moreover, cuticular hydrocarbon deficiency increased the sensitivity of aphids to desiccation stress and reduced their survival time, while desiccation stress significantly increased ApApoD expression. Together, it is confirmed that ApApoD participates in regulating cuticular hydrocarbon content of aphids under desiccation stress and is crucial for aphid reproduction. Therefore, the ApApoD gene of A. pisum may be a potential target for RNAi-based insect pest control due to its involvement in cuticular hydrocarbon accumulation and reproduction.

Role of Biogenic Amines in Oviposition by the Diamondback Moth, Plutella xylostella L.

Oviposition is an important reproductive behavior that is triggered by mating in insects, and biogenic amines might be involved in its regulation. The effects of biogenic amines on oviposition have only been studied in a few insect species, and the findings to date have not been conclusive. In addition, there are few studies on the effects of biogenic amines on oviposition of the diamondback moth, Plutella xylostella L. Here, we tested how mating and biogenic amines regulate oviposition of P. xylostella by injecting amines and amine receptor antagonists into virgin and mated females and counting the number of eggs laid afterward. Biogenic amines of octopamine and tyramine could induce virgin adults of P. xylostella to lay eggs, while dopamine and serotonin had no such effect on oviposition. Furthermore, the octopamine antagonists mianserin, epinastine, and phentolamine inhibited oviposition by mated females. The tyramine antagonist yohimbine, dopamine antagonist SCH23390, and serotonin antagonist ketanserin did not block oviposition by mated females, and octopamine and tyramine-inducing oviposition by virgin females could be inhibited by the octopamine antagonists mianserin and epinastine instead of the tyramine antagonist yohimbine. We conclude that octopamine and its receptors are involved in mating-triggered oviposition in P. xylostella, while tyramine acts as a subsidiary. Further, the inducing effect of tyramine on oviposition is achieved via octopamine receptors instead of tyramine receptors. This experiment is helpful to further understand the role of biogenic amines in mating regulation and to provide a new strategy for controlling P. xylostella.

Both LmCYP4G genes function in decreasing cuticular penetration of insecticides in Locusta migratoria.

BACKGROUND: Cuticular hydrocarbons (CHCs) have a critical role in preventing desiccation and penetration of xenobiotics in insects. Previous studies have shown that cytochrome P450 subfamily 4G (CYP4G) enzymes are oxidative decarbonylases, essential for CHC biosynthesis. However, it is unclear whether there are functional differences between the two CYP4G genes in most insects. In Locusta migratoria, we identified two CYP4G genes (LmCYP4G62 and LmCYP4G102). LmCYP4G102 plays a critical role in the synthesis of CHCs, but the function of LmCYP4G62 is unknown. RESULTS: We identified, characterized, and compared two LmCYP4G genes, based on L. migratoria transcriptomic and genomic databases. RT-qPCR showed that both were highly expressed in tissues with which oenocytes are associated, the integument and fat body. Immunostaining indicated that LmCYP4G62 and LmCYP4G102 were highly abundant in oenocytes in these tissues. However, the two enzymes had a different subcellular distribution, with LmCYP4G62 localized on the plasma membrane and LmCYP4G102 dispersed throughout the oenocyte cytoplasm, presumably on the endoplasmic reticulum. RNA interference-mediated gene silencing against each of the two genes resulted in reduced CHC contents, in all classes for LmCYP4G102, but mostly shorter chain CHCs for LmCYP4G62. Silencing of both genes resulted in increased insecticide penetration through the cuticle, and increased locust susceptibility to desiccation and insecticides. CONCLUSION: Our studies suggest that both LmCYP4G62 and LmCYP4G102 contribute to hydrocarbon biosynthesis and play key roles in protecting locusts from water loss and insecticide penetration, but they are not fully redundant. Further, the two LmCYP4G genes might be used as new targets for insect pest management. © 2020 Society of Chemical Industry.

BgFas1: A fatty acid synthase gene required for both hydrocarbon and cuticular fatty acid biosynthesis in the German cockroach, Blattella germanica (L.).

Insect cuticular hydrocarbons (CHCs), the evolutionary products of aquatic hexapod ancestors expanding to terrestrial environment, are deposited on the surface of insect integument and originally functioned primarily as waterproofing agents. CHCs are derived from the conserved fatty acid synthesis pathway in insects. However, the pivotal fatty acid synthase (FAS) involved in hydrocarbon (HC) biosynthesis remains unknown in many insect orders including the primitive Blattodea. Here, we investigated functional FAS genes that modulate cuticular lipid biogenesis in the German cockroach, Blattella germanica (L.). Based on our full-length transcriptomic data and the available genomic data, seven FAS genes (BgFas1-7) were identified from B. germanica. Tissue-specific expression analysis revealed that BgFas1, BgFas3, BgFas4 and BgFas7 were highly expressed in the integument, whereas BgFas2 was dominantly expressed in the fat body. BgFas5/6 mRNA was almost negligible in the tested tissues. Systemic RNAi screen was performed against BgFas1-7, we found that only RNAi knockdown of BgFas1 caused a dramatic reduction of methyl-branched HCs (mbHCs) and a slight decrease of straight-chain HCs (scHCs) for both internal and external HCs. Significant reduction of cuticular free fatty acids (cFFAs) was also detected within BgFas1-repressed cockroaches, while repression of CYP4G19 resulted in dramatic increase of cFFAs. Moreover, we found that BgFas1 mRNA levels were correlated with insect molting cycles, and could be induced by long-term mild dryness treatment. Furthermore, desiccation assay revealed that BgFas1 suppression accelerated water loss and led to early death of cockroaches under desiccation. Our results indicate that BgFas1 is necessary for both HC and cFFA biosynthesis in B. germanica. In addition, our study also confirms that cuticular lipids, particularly mbCHCs, are critical for desiccation resistance in B. germanica.

Solid-phase microextraction-based cuticular hydrocarbon profiling for intraspecific delimitation in Acyrthosiphon pisum.

Insect cuticular hydrocarbons (CHCs) play critical roles in reducing water loss and chemical communication. Species-specific CHC profiles have been used increasingly as an excellent character for species classification. However, considerably less is known about their potential for population delimitation within species. The aims of this study were to develop a solid-phase microextraction (SPME)-based CHC collection method and to investigate whether CHC profiles could serve as potential chemotaxonomic tools for intraspecific delimitation in Acyrthosiphon pisum. Optimization of fibers for SPME sampling revealed that 7 µm polydimethylsiloxane (PDMS) demonstrated the most efficient adsorption of CHCs among five different tested fibers. SPME sampling showed good reproducibility with repeated collections of CHCs from a single aphid. Validation of SPME was performed by comparing CHC profiles with those from conventional hexane extractions. The two methods showed no qualitative differences in CHCs, although SPME appeared to extract relatively fewer short-chained CHCs. While CHC profiles of a given population differed among developmental stages, wing dimorphism types, and host plants, wingless adult aphids showed very low variance in relative proportions of individual CHC components. Reproducibility of CHC profiles was explored further to classify wingless adult morphs of A. pisum from five different geographic regions that showed no variation in mitochondrial COI gene sequences. Our results demonstrate that CHC profiles are useful in intraspecific delimitation in the field of insect chemotaxonomy.

Cytochrome P450 gene, CYP4G51, modulates hydrocarbon production in the pea aphid, Acyrthosiphon pisum.

Terrestrial insects deposit a layer of hydrocarbons (HCs) as waterproofing agents on their epicuticle. The insect-specific CYP4G genes, subfamily members of P450, have been found in all insects with sequenced genomes to date. They are critical for HC biosynthesis in Drosophila; however, their functional roles in other insects including the piercing-sucking hemipterous aphids remain unknown. In this study, we presented the molecular characterization and a functional study of the CYP4G51 gene in the pea aphid, Acyrthosiphon pisum (Harris). CYP4G51 transcript was detectable across the whole life cycle of A. pisum, and was prominently expressed in the aphid head and abdominal cuticle. Up-regulation of CYP4G51 under desiccation stress was more significant in the third instar nymphs compared with the adults. Also, up-regulation of CYP4G51 was observed when the aphids fed on an artificial diet compared with those fed on the broad bean plant, and was positively correlated with a high level of cuticular HCs (CHCs). RNAi knockdown of CYP4G51 significantly reduced its expression and caused reductions in both internal and external HCs. A deficiency in CHCs resulted in aphids being more susceptible to desiccation, with increased mortality under desiccation stress. The current results confirm that CYP4G51 modulates HC biosynthesis to protect aphids from desiccation. Moreover, our data also indicate that saturated and straight-chain HCs play a major role in cuticular waterproofing in the pea aphid. A. pisum CYP4G51 could be considered as a novel RNAi target in the field of insect pest management.

Nitrogen hurdle of host alternation for a polyphagous aphid and the associated changes of endosymbionts.

Low proportion of essential amino acids (EAAs) is one of the barriers for animals to use phloem as a diet. Endosymbionts with EAAs synthesis functions are considered crucial for ameliorating the lack of EAAs in insects' diets. In this study, we transferred the insects from a cabbage-reared Myzus persicae population onto 3 new plant species including eggplant, tobacco and spinach. The performance on these plants was evaluated and the dynamics of endosymbionts in relation to this host alternation were recorded. We found that the EAAs ratio in phloem was largely determined by the concentrations of non-essential amino acids and the higher proportion of EAAs seemed to favor the population establishment on new plant species and the growth of primary endosymbionts inside insects, which indicated that nitrogen quality was an important factor for aphids to infest and spread on new plant hosts.