Aldehyde reductase activity in the antennae of Helicoverpa armigera

In the present study, we identified two aldehyde reductase activities in the antennae of Helicoverpa species, NADH and NADPH‐dependent activity. We expressed one of these proteins of H. armigera, aldo‐keto reductase (AKR), which bears 56% identity to bovine aldose reductase, displays a NADPH‐dependent activity and is mainly expressed in the antennae of adults. Whole‐mount immunostaining showed that the enzyme is concentrated in the cells at the base of chemosensilla and in the nerves. The enzyme activity of H. armigera AKR is markedly different from those of mammalian enzymes. The best substrates are linear aliphatic aldehydes of 8–10 carbon atoms, but not hydroxyaldehydes. Both pheromone components of H. armigera, which are unsaturated aldehydes of 16 carbons, are very poor substrates. Unlike mammalian AKRs, the H. armigera enzyme is weakly affected by common inhibitors and exhibits a different behaviour from the action of thiols. A model of the enzyme suggests that the four cysteines are in their reduced form, as are the seven cysteines of mammalian enzymes. The occurrence of orthologous proteins in other insect species, that do not use aldehydes as pheromones, excludes the possibility of classifying this enzyme among the pheromone‐degrading enzymes, as has been previously described in other insect species.     

Modular cis‐regulatory logic of yellow gene expression in silkmoth larvae

Colour patterns in butterflies and moths are crucial traits for adaptation. Previous investigations have highlighted genes responsible for pigmentation (ie yellow and ebony). However, the mechanisms by which these genes are regulated in lepidopteran insects remain poorly understood. To elucidate this, molecular studies involving dipterans have largely analysed the cis‐regulatory regions of pigmentation genes and have revealed cis‐regulatory modularity. Here, we used well‐developed transgenic techniques in Bombyx mori and demonstrated that cis‐regulatory modularity controls tissue‐specific expression of the yellow gene. We first identified which body parts are regulated by the yellow gene via black pigmentation. We then isolated three discrete regulatory elements driving tissue‐specific gene expression in three regions of B. mori larvae. Finally, we found that there is no apparent sequence conservation of cis‐regulatory regions between B. mori and Drosophila melanogaster, and no expression driven by the regulatory regions of one species when introduced into the other species. Therefore, the trans‐regulatory landscapes of the yellow gene differ significantly between the two taxa. The results of this study confirm that lepidopteran species use cis‐regulatory modules to control gene expression related to pigmentation, and represent a powerful cadre of transgenic tools for studying evolutionary developmental mechanisms.     

Expression patterns and colocalization of two sensory neurone membrane proteins in Ectropis obliqua Prout,a geometrid moth pest that uses Type‐II sex pheromones

Sensory neurone membrane proteins (SNMPs) function as essential cofactors for insect sex pheromone detection. In this study, we report two SNMPs in Ectropis obliqua Prout, a serious geometrid pest that produces typical Type‐II sex pheromones. Sequence alignments and phylogenetic analyses showed that EoblSNMP1 and EoblSNMP2 belong to two distinct SNMP subfamilies. Quantitative real‐time PCR suggested that EoblSNMP1 was male antennae‐biased, whereas EoblSNMP2 was highly expressed on male antennae but was also expressed on female antennae and other chemosensory tissues. Additionally, EoblSNMP1 and EoblSNMP2 differed in their developmental expression profiles. In situ hybridization revealed that EoblSNMP1 was sensilla trichodea I specific, whereas EoblSNMP2 was expressed in sensilla trichodea I and the sensilla basiconica; furthermore, EoblSNMP1 and EoblSNMP2 were co‐expressed in sensilla trichodea I but in different cells. This study suggests that EoblSNMP1 is functionally distinct from EoblSNMP2 in E. obliqua; EoblSNMP1 may specifically contribute to the recognition of sex pheromones, whereas EoblSNMP2 exhibits multiple olfactory roles. Our findings comprehensively reveal the expression patterns of SNMPs in a lepidopteran species that uses Type‐II sex pheromones, providing new insights into the functional evolution of SNMPs from lepidopteran moths with Type‐I sex pheromones to those with Type‐II sex pheromones.     

Functional analysis of female‐biased odorant binding protein 6 for volatile and nonvolatile host compounds in Adelphocoris lineolatus (Goeze)

The polyphagous mirid bug Adelphocoris lineolatus relies heavily on olfactory cues to track suitable host plants. Thus, a better understanding of the molecular basis of its olfactory detection could contribute to the development of effective pest management strategies. In the present study, we report the expression profile of the odorant binding protein gene A. lineolatus odorant binding protein 6 (AlinOBP6). Quantitative real‐time PCR experiments suggest that AlinOBP6 is female adult antennae‐biased. Cellular immunolocalization analyses show that AlinOBP6 is highly expressed in the lymph of both multiporous sensilla basiconica and uniporous sensilla chaetica. A ligand binding analysis showed that recombinant AlinOBP6 not only bound tightly to host plant volatile compounds but also to nonvolatile compounds. Homology modelling and molecular docking analyses confirmed these unusual ligand binding profiles and revealed that the amino acid residues involved in the recognition of volatile and nonvolatile compounds are distinct. The results of our study are the first to suggest that an antenna‐ and female‐biased OBP in an hemipteran insect is expressed in both olfactory and gustatory sensilla as a mechanism to respond to volatile and nonvolatile host compounds. These findings warrant further research into the molecular mechanisms of chemosensation for mirid bugs in responsive to host plant location.     

A case‐control study and meta‐analysis reveal the association between COX‐2 G‐765C polymorphism and primary end‐stage hip and knee osteoarthritis

Background

Osteoarthritis (OA) is a popular arthrosis featured as pain, limited joint activity, and deformity. Cyclooxygenase‐2 (COX‐2) has been reported to be up‐regulated in arthritic tissues and is integral to the progression of osteoarthritis (OA). Previous studies showed the COX‐2 promoter G‐765C polymorphism could influence COX‐2 expression. However, the relationship between the variant and OA risk is contrasting.

Methods

We conducted a case‐control study with 196 primary end‐stage hip and knee OA cases and 196 controls in a Chinese Han population. Subsequently, we integrated this case‐control study in a meta‐analysis to acquire greater statistical power. The results from our case‐control study using MassARRAY genotyping technology and binary logistic regression statistical methods.

Results

The variant carriers in the Chinese Han population had a lower primary end‐stage hip and knee OA susceptibility (C vs G: OR = 0.350, 95%CI: 0.154‐0.797, P = .012; GC vs GG: adjusted OR = 0.282, 95%CI: 0.118‐0.676, P = .005). Stratification studies indicated that a higher GC frequency in women decreased not only knee OA susceptibility but also unilateral knee OA risk. The meta‐analysis showed that the variant exhibited a significantly decreased OA risk through comparisons involving allelic, homozygous, heterozygous, and dominant models.

Conclusion

Our findings suggest that the COX‐2 G‐765C polymorphism exerts a protective effect against primary end‐stage knee osteoarthritis in a female Chinese Han population.

     

A cytochrome P450 gene plays a role in the recognition of sex pheromones in the tobacco cutworm,Spodoptera litura

Cytochrome P450 (P450 or CYP) genes are involved in fundamental physiological functions, and might be also associated with the olfactory recognition of sex pheromones in beetles and moths. A P450 gene, Spodoptera litura CYP4L4 (SlituCYP4L4), was cloned for the first time from the antennae of S. litura. SlituCYP4L4 was almost exclusively expressed in the adult stage and predominantly expressed in the adult antennae. In situ hybridization showed that SlituCYP4L4 localized mainly at the base of the long sensilla trichoidea, which responds to sex pheromone components. Pretreatment with an S. litura sex pheromone significantly reduced the expression levels of SlituCYP4L4, consistent with other genes involved in sex pheromone recognition. The expression level of SlituCYP4L4 was different in moths collected with different ratios of sex pheromone lures and collected in different geographical locations. After gene knockdown of SlituCYP4L4 in the antennae, the electroantennogram (EAG) responses of male and female moths to (9Z,11E)‐tetradecadienyl acetate or (9Z,12E)‐tetradecadienyl acetate were significantly decreased. In contrast, EAG responses to plant volatiles and sex pheromones of other moth species were not significantly influenced in these moths. SlituCYP4L4 was also expressed in the gustatory tissues and sensilla, which suggests that SlituCYP4L4 may have other functions in the chemosensory system. Our results have shown for the first time the function of a CYP gene with appendage‐specific expression in insect sex pheromone recognition, especially in adult moths.     

Functional characterization of ultraspiracle in Leptinotarsa decemlineata using RNA interference assay

A heterodimer of ultraspiracle (USP) and ecdysone receptor (EcR) mediates 20‐hydroxyecdysone (20E) signalling cascade to regulate insect moulting and metamorphosis. However, at least two questions remain to be addressed in terms of the molecular importance of USP in insect species. First, is USP involved in both regulation of ecdysteroidogenesis and mediation of 20E signalling in non‐drosophilid insects, as in Drosophila melanogaster? Second, does USP play any role in larval metamorphosis except as the partner of heterodimeric receptor to activate the downstream 20E signalling genes? In this paper, we found that RNA interference (RNAi) of LdUSP in the final (fourth) instar larvae reduced the messenger RNA levels of four ecdysteroidogenesis genes (Ldspo, Ldphm, Lddib and Ldsad) and 20E titre, and repressed the expression of five 20E signal genes (EcRA, HR3, HR4, E74 and E75) in Leptinotarsa decemlineata. The LdUSP RNAi larvae remained as prepupae, with developing antennae, legs and discs of forewings and hindwings. Dietary supplement with 20E restored the expression of the five 20E signal genes, but only partially alleviated the decreased pupation rate in LdUSP RNAi beetles. Knockdown of LdUSP at the penultimate (third) instar larvae did not affect third–fourth instar moulting. However, silencing LdUSP caused similar but less severe impairments on pupation. Accordingly, we propose that USP is undoubtedly necessary for ecdysteroidogenesis, for mediation of 20E signalling and for initiation of metamorphosis in L. decemlineata.     

Larval sensilla of the moth Heliothis virescens respond to sex pheromone components

Female‐released sex pheromones orchestrate the mating behaviour of moths. Recent studies have shown that sex pheromones not only attract adult males but also caterpillars. Single sensillum recordings revealed that larval antennal sensilla of the moth Heliothis virescens respond to specific sex pheromone components. In search for the molecular basis of pheromone detection in larvae, we found that olfactory sensilla on the larval antennae are equipped with the same molecular elements that mediate sex pheromone detection in adult male moths, including the Heliothis virescens receptors 6 (HR6) and HR13, as well as sensory neurone membrane protein 1 (SNMP1). Thirty‐eight olfactory sensory neurones were identified in three large sensilla basiconica; six of these are considered as candidate pheromone responsive cells based on the expression of SNMP1. The pheromone receptor HR6 was found to be expressed in two cells and the receptor HR13 in three cells. These putative pheromone responsive neurones were accompanied by cells expressing pheromone‐binding protein 1 (PBP1) and PBP2. The results indicate that the responsiveness of larval sensilla to female‐emitted sex pheromones is based on the same molecular machinery as in the antennae of adult males.     

Multiple functions of miR‐8‐3p in the development and metamorphosis of the red flour beetle,Tribolium castaneum

The microRNA miR‐8‐3p is conserved among insects and closely involved in development and immunity, but its functions in vivo are unexplored in the red flour beetle, Tribolium castaneum. Here, we show that miR‐8‐3p was highly expressed in late larva and early adult stages, as determined by quantitative real‐time PCR. It was enriched in the fat body and cuticle in late larval tissues and abundant in the head and cuticle in early adult tissues, indicating this microRNA plays important roles during T. castaneum development. Specific inhibition of miR‐8‐3p in late larvae led to metamorphosis defects in the development of wings, eyes, legs and embryo. Moreover, a series of genes related to organism development were identified as miR‐8‐3p targets by computational prediction and microRNA–messenger RNA interaction validation, including Wingless, Eyg, Fpps and Sema‐1a. These genes were critical for the regulation of the larva‐to‐adult transition. Eyg, as a functional target of miR‐8‐3p, participates in eye development, which was further confirmed by luciferase assay and loss‐of‐function analyses. In brief, miR‐8‐3p is broadly involved in the development of wings, eyes and legs through its target genes and has extensive regulatory roles during T. castaneum development.     

Identification of an E‐box DNA binding protein,activated protein 4, and its function in regulating the expression of the gene encoding diapause hormone and pheromone biosynthesis‐activating neuropeptide in Helicoverpa armigera

Activated protein 4 (AP‐4), an E‐box DNA‐binding protein, was cloned from the cotton bollworm, Helicoverpa armigera (Har). The expression of Har‐AP‐4 mRNA and the protein that it encodes are significantly higher in nondiapause pupae than in diapause pupae. In vitro‐translated Har‐AP‐4 can bind specifically to the E‐box motif on the promoter of the diapause hormone and pheromone biosynthesis‐activating neuropeptide (DH‐PBAN). Har‐AP‐4, fused with the green fluorescent protein (GFP), is localized to the nucleus, and overexpression of Har‐AP‐4 can significantly activate the promoter of the DH‐PBAN gene that is involved in nondiapause pupal development in H. armigera. These results suggest that Har‐AP‐4, which binds to the promoter of DH‐PBAN, may play a role in regulating pupal development in H. armigera.     

Isoform specific roles of Broad‐Complex in larval development in Leptinotarsa decemlineata

Broad‐Complex (BrC) is a downstream target of both 20‐hydroxyecdysone and juvenile hormone signalling. BrC regulates morphogenetic changes between nymphal instars in hemimetabolans, whereas it controls pupal commitment, pupal morphogenesis and inhibits adult differentiation in holometabolans. Among five BrC cDNAs (Z1–Z4 and Z6) identified in the Colorado potato beetle, we found in this work that Z1, Z2 and Z6 were mainly expressed at the last (fourth) instar and prepupal stages, whereas the levels of Z3 and Z4 increased during the penultimate (third) instar stage, peaked at the last instar larval phase and gradually decreased at the prepupal and pupal periods. When knocking down all BrC isoforms by RNA interference (RNAi) at the penultimate instar stage, around 20% of the resultant larvae remained as moribund beetles. These moribund BrC RNAi larvae were completely or partially wrapped in old cuticle. Likewise, a portion of larvae treated for a single double‐stranded RNA of Z3, Z4 or Z6 displayed a degree of similar aberrancies, increasing in the order of isoforms Z6 < Z3 < Z4. When silencing all BrC isoforms at the last instar period, most of the RNAi larvae did not normally pupate or emerge as adults. Separately silencing each of the five zinc finger domains revealed that approximately 70% of the Z1 RNAi larvae remained as prepupae, around 60% of the Z6 RNAi specimens formed aberrant prepupae or pupae and about 60% of the Z2 RNAi beetles became deformed pupae. After removal of the old exuviae, these deformed larvae in which either Z1, Z2 or Z6 was depleted possessed adult prothorax and mesothorax, developing antenna, mouthparts and wing discs. Moreover, less than 50% of the resultant pupae finally emerged as adults when either of Z1, Z2 or Z6 was knocked down. Therefore, our findings reveal, for the first time, that the two roles of BrC in insect groups (ie directing morphogenetic changes during juvenile development and regulating larval–pupal–adult metamorphosis) are played by different BrC isoforms in Leptinotarsa decemlineata.     

Two classic OBPs modulate the responses of female Holotrichia oblita to three major ester host plant volatiles

Insects possess a fairly sophisticated olfactory system in their antennae to detect odorants essential for their survival and reproduction. Among them, insect first perceives odour sources by odorant-binding proteins (OBPs) to locate host-plants. Methyl salicylate, (Z)-3-hexenyl acetate and dibutyl phthalate are major volatile components of Ulmus pumila and Ricinus communis and elicit strong responses of the scarab beetle Holotrichia oblita adults. However, olfactory perception of the scarab beetle to these odorant compounds is unclear. In the current study, we cloned the OBP6 and OBP7 of H. oblita. The expression pattern shows that the two genes were highly expressed in the antennae of female beetles. Binding assays verified that the HoblOBP6 had a better binding affinity to methyl salicylate, and so did HoblOBP7 to (Z)-3-hexenyl acetate and dibutyl phthalate. The effect on the responses of female beetles to the three compounds was decreased significantly after these two genes were silenced by RNA interference. These results indicate that HoblOBP6 and HoblOBP7 are essential for female H. oblita perception of methyl salicylate, (Z)-3-hexenyl acetate and dibutyl phthalate. Our study provides important insights into the olfactory mechanism of female H. oblita to ester plant volatiles and could facilitate the development of potential pest control strategies in the field.     

Structural and biochemical evaluation of Ceratitis capitata odorant‐binding protein 22 affinity for odorants involved in intersex communication

In insects, odorant‐binding proteins (OBPs) connect the peripheral sensory system to receptors of olfactory organs. Medfly Ceratitis capitata CcapObp22 shows 37% identity and close phylogenetic affinities with Drosophila melanogaster OBP69a/pheromone‐binding protein related protein 1. The CcapObp22 gene is transcribed in the antennae and maxillary palps, suggesting an active role in olfaction. Here, we recombinantly produced CcapObp22, obtaining a 13.5 kDa protein capable of binding multiple strongly hydrophobic terpene compounds, including medfly male pheromone components. The highest binding affinity [half maximal effective concentration (EC50) = 0.48 µM] was to (E,E)‐α‐farnesene, one of the most abundant compounds in the male pheromone blend. This odorant was used in cocrystallization experiments, yielding the structure of CcapOBP22. The monomeric structure shows the typical OBP folding, constituted by six α‐helical elements interconnected by three disulphide bridges. A C‐terminal seventh α‐helix constitutes the wall of a deep, L‐shaped hydrophobic cavity. Analysis of the electron density in this cavity suggested trapping of farnesene in the crystal structure, although with partial occupancy. Superposition of the CcapOBP22 structure with related seven‐helical OBPs highlights striking similarity in the organization of the C‐terminal segment of these proteins. Collectively, our molecular and physiological data on medfly CcapOBP22 suggest its involvement in intersex olfactory communication.