Circabidian rhythm of sex pheromone reception in a scarab beetle.

Animals have endogenous clocks that regulate their behavior and physiology. These clocks rely on environmental cues (time givers) that appear approximately every 24 h due to the Earth's rotation; thus, most insects exhibit a circadian rhythm. One notable exception is the scarab beetle, Holotrichia parallela, a severe agricultural pest in China, Japan, South Korea, and India. Females emerge from the soil every other night, reach the canopy of host plants, evert an abdominal gland, and release a pheromone bouquet comprising l-isoleucine methyl ester (LIME) and l-linalool. To determine whether this circa'bi'dian rhythm affects the olfactory system, we aimed to identify H. parallela sex pheromone receptor(s) and study their expression patterns. We cloned 14 odorant receptors (ORs) and attempted de-orphanizing them in the Xenopus oocyte recording system. HparOR14 gave robust responses to LIME and smaller responses to l-linalool. Structural modeling, tissue expression profile, and RNAi treatment followed by physiological and behavioral studies support that HparOR14 is a sex pheromone receptor-the first of its kind discovered in Coleoptera. Examination of the HparOR14 transcript levels throughout the adult's life showed that on sexually active days, gene expression was significantly higher in the scotophase than in the photophase. Additionally, the HparOR14 expression profile showed a circabidian rhythm synchronized with the previously identified pattern of sex pheromone emission. 48 h of electroantennogram recordings showed that responses to LIME were abolished on non-calling nights. In contrast, responses to the green leaf volatile (Z)-3-henexyl acetate remained almost constant throughout the recording period.

Involvement of Holotrichia parallela odorant-binding protein 3 in the localization of oviposition sites.

Organic fertilizers-derived volatiles attract Holotrichia parallela during oviposition. However, the mechanisms underlying the perception of oviposition cues in H. parallela remain unclear. Here, H. parallela odorant-binding protein 3 (HparOBP3) was identified as a key OBP. Bioinformatics analysis showed that HparOBP3 clustered together with Holotrichia oblita OBP8. HparOBP3 was mainly expressed in the antennae of both sexes. Recombinant HparOBP3 exhibited distinct binding affinities towards 22 compounds released by organic fertilizers. After 48 h of RNA interference (RNAi), the expression of HparOBP3 in male and female antennae was decreased by 90.77 % and 82.30 %, respectively. In addition, silencing of HparOBP3 significantly reduced the electrophysiological responses and tropism of males to cis-3-hexen-1-ol, 1-hexanol, and (Z)-ß-ocimene as well as females to cis-3-hexen-1-ol, 1-hexanol, benzaldehyde, and (Z)-ß-ocimene. Molecular docking indicated that hydrophobic residues Leu-83, Leu-87, Phe-108, and Ile-120 of HparOBP3 were important amino acids for interacting with ligands. Mutation of the key residue, Leu-83, significantly diminished the binding ability of HparOBP3. Furthermore, acrylic plastic arena bioassays showed that the attraction and oviposition indexes of organic fertilizers to H. parallela were reduced by 55.78 % and 60.11 %, respectively, after silencing HparOBP3. These results suggest that HparOBP3 is essential in mediating the oviposition behavior of H. parallela.

PI3K/Akt/CncC signaling pathway mediates the response to EPN-Bt infection in Holotrichia parallela larvae.

BACKGROUND: Combining the entomopathogenic nematode (EPN), Heterorhabditis beicherriana LF strain, and Bacillus thuringiensis (Bt) HBF-18 strain is a practical strategy to manage the larvae of Holotrichia parallela Motschulsky (white grubs). However, the mechanisms underlying the larval defense response to this combined biocontrol strategy are unknown. RESULTS: The activities of some antioxidant enzymes (SOD, POD, CAT) and some detoxifying enzymes (AChE, P-450, CarE, GST) in grubs showed an activation-inhibition trend throughout the EPN-Bt exposure time course. Eight potentially key antioxidant and detoxifying enzyme genes in response to EPN-Bt infection were identified from the midgut of grubs through RNA sequencing. After silencing CAT, CarE18, and GSTs1, the enzyme activities were significantly decreased by 30.29%, 68.80%, and 34.63%, respectively. Meanwhile, the mortality of grubs was increased by 18.40%, 46.30%, and 42.59% after exposure to EPN-Bt for 1 day. Interestingly, the PI3K/Akt signaling pathway was significantly enriched in KEGG enrichment analysis, and the expression levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), cap 'n' collar isoform-C (CncC), kelch-like ECH-associated protein 1 (Keap1), and CarE18 were all up-regulated when exposed to EPN-Bt for 1 day. Furthermore, RNAi-mediated PI3K silencing showed a similar down-regulated trend between PI3K/Akt/CncC and CarE18. Moreover, silencing PI3K rendered grubs more susceptible to EPN-Bt and accelerated symbiotic bacteria multiplication in grubs. CONCLUSION: These results suggest that the PI3K/Akt/CncC pathway mediates the expression of CarE18 and participates in the defense response of H. parallela larvae against EPN-Bt infection. Our data provide valuable insights into the design of appropriate management strategies for this well-known agricultural pest. © 2022 Society of Chemical Industry.

Octopamine modulates insect mating and Oviposition.

The neuro-mechanisms that regulate insect reproduction are not fully understood. Biogenic amines, including octopamine, are neuromodulators that have been shown to modulate insect reproduction in various ways, e.g., promote or inhibit insect mating or oviposition. In this study, we examined the role of octopamine in regulating the reproduction behaviors of a devastating underground insect pest, the dark black chafer (Holotrichia parallela). We first measured the abundance of octopamine in different neural tissues of the adult chafer pre- and post-mating, demonstrating that octopamine decreased in the abdominal ganglia of females but increased in males post-mating. We then fed the adult H. parallela with a concentration gradient of octopamine to test the effects on insect reproductive behaviors. Compared with its antagonist mianserin, octopamine at the concentration of 2 µg/mL resulted in the highest increase in males' preference for sex pheromone and females' oviposition, whereas the mianserin-treatment increased the survival rate and prolonged the lifespan of H. parallela. In addition, we did not observe significant differences in egg hatchability between octopamine and mianserin-treated H. parallela. Our results demonstrated that octopamine promotes H. parallela mating and oviposition with a clear low dosage effect, illustrated how neural substrates modulate insect behaviors, and provided insights for applying octopamine in pest management.

Molecular characterization of sex pheromone binding proteins from Holotrichia oblita (Coleoptera: Scarabaeida).

Pheromone binding proteins (PBPs), a subfamily of the odorant binding proteins (OBPs), capture and transfer sex pheromones across the sensillum lymph to pheromone receptors and initiate insect courtship and mating. In this study, we functionally characterized ten OBPs from the black chafer, Holotrichia oblita (HoblOBPs), among which six HoblOBPs (HoblOBP2, 4, 5, 8, 9 and 24) were shown to recognize sex pheromones using electroantennography assays (EAG) and in vitro fluorescence competitive binding assays. Insect tropism to sex pheromones was significantly reduced after those genes were knocked down in vivo, e.g. HoblOBP24 RNAi reduced the tropism of H. oblita to methyl glycinate by 34%. Furthermore, molecular docking revealed key residues for the binding of the six HoblOBPs with sex pheromones. And hydrogen bonds and hydrophobic forces were shown to be the main forces in the binding of the six HoblOBPs and their sex pheromone ligands. Our study characterized six H. oblita PBPs and their binding abilities to sex pheromone ligands. The results will improve our understanding on the olfactory mechanisms that H. oblita utilizes to recognize sex pheromones, and will promote the development of novel strategies for controlling H. oblita and other insect pests.

Evidence of the Involvement of a Plus-C Odorant-Binding Protein HparOBP14 in Host Plant Selection and Oviposition of the Scarab Beetle Holotrichia parallela.

Holotrichia parallela is one of the agriculturally important scarab beetle pests in China. In this study, HparOBP14 was cloned, which is the most abundantly expressed among the OBP genes in the legs of female H. parallela adults. Sequence comparison and phylogenetic analysis showed that HparOBP14 has a Plus-C structure motif. The expression profile analysis revealed that HparOBP14 expression was the highest in the female antennae and then in the legs. The fluorescence competitive binding experiment of the recombinant HparOBP14 protein showed that HparOBP14 had an affinity with 6-methyl-5-heptene-2-one (plant volatile), 3-methylindole, p-cymene, methanol, formaldehyde, α-pinene, and geraniol (organic fertilizer volatile). Knockdown HparOBP14 expression decreased significantly the EAG response of the injected female adults to p-cymene, methanol, formaldehyde, α-pinene, and geraniol. Similarly, the injected female adults were significantly less attracted to geraniol and methanol. Therefore, HparOBP14 might bind organic matter volatiles during oviposition. These results are not only helpful to analyze the olfactory recognition mechanism of female adult H. parallela when choosing suitable oviposition sites, but also to provide target genes for green prevention and control of H. parallela in the future.

Immune-related genes of the larval Holotrichia parallela in response to entomopathogenic nematodes Heterorhabditis beicherriana LF.

BACKGROUND: Entomopathogenic nematodes (EPNs) emerge as compatible alternatives to conventional insecticides in controlling Holotrichia parallela larvae (Coleoptera: Scarabaeidae). However, the immune responses of H. parallela against EPNs infection remain unclear. RESULTS: In present research, RNA-Seq was firstly performed. A total of 89,427 and 85,741 unigenes were achieved from the midgut of H. parallela larvae treated with Heterorhabditis beicherriana LF for 24 and 72 h, respectively; 2545 and 3156 unigenes were differentially regulated, respectively. Among those differentially expressed genes (DEGs), 74 were identified potentially related to the immune response. Notably, some immune-related genes, such as peptidoglycan recognition protein SC1 (PGRP-SC1), pro-phenoloxidase activating enzyme-I (PPAE-I) and glutathione s-transferase (GST), were induced at both treatment points. Bioinformatics analysis showed that PGRP-SC1, PPAE-I and GST were all involved in anti-parasitic immune process. Quantitative real-time PCR (qRT-PCR) results showed that the three immune-related genes were expressed in all developmental stages; PGRP-SC1 and PPAE-I had higher expressions in midgut and fat body, respectively, while GST exhibited high expression in both of them. Moreover, in vivo silencing of them resulted in increased susceptibility of H. parallela larvae to H. beicherriana LF. CONCLUSION: These results suggest that H. parallela PGRP-SC1, PPAE-I and GST are involved in the immune responses to resist H. beicherriana LF infection. This study provides the first comprehensive transcriptome resource of H. parallela exposure to nematode challenge that will help to support further comparative studies on host-EPN interactions.

Flight muscles degenerate by programmed cell death after migration in the wheat aphid, Sitobion avenae.

OBJECTIVE: Previous studies showed that flight muscles degenerate after migration in some aphid species; however, the underlying molecular mechanism remains virtually unknown. In this study, using the wheat aphid, Sitobion avenae, we aim to investigate aphid flight muscle degeneration and the underlying molecular mechanism. RESULTS: Sitobion avenae started to differentiate winged or wingless morphs at the second instar, the winged aphids were fully determined at the third instar, and their wings were fully developed at the fourth instar. After migration, the aphid flight muscles degenerated via programmed cell death, which is evidenced by a Terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling assay. Then, we identified a list of differentially expressed genes before and after tethered flights using differential-display reverse transcription-PCR. One of the differentially expressed genes, ubiquitin-ribosomal S27a, was confirmed using qPCR. Ubiquitin-ribosomal S27a is drastically up regulated following the aphids' migration and before the flight muscle degeneration. Our data suggested that aphid flight muscles degenerate after migration. During flight muscle degeneration, endogenous proteins may be degraded to reallocate energy for reproduction.

Functional characterization of odorant-binding proteins from the scarab beetle Holotrichia oblita based on semiochemical-induced expression alteration and gene silencing.

With the advent of next-generation sequencing, it is now possible to rapidly identify the entire repertoire of olfactory genes likely to be involved in chemical communication of an insect species. It remains, however, a challenge to identify olfactory proteins, such as odorant receptors and odorant-binding proteins (OBPs), vis-à-vis the odorants they detect. It has been reported that exposing the olfactory system to a physiologically relevant odorant alters the transcript levels of odorant receptor(s) involved in the detection of the tested odorant. We applied this paradigm in an attempt to identify putative OBPs from the scarab beetle Holotrichia oblita involved in the reception of plant-derived kairomones. Twenty-nine OBP genes were identified in the H. oblita transcriptome, 20 of which were enriched in antennae compared with nonolfactory tissues. Of these, 2 OBP genes, HoblOBP13 and HoblOBP9, were upregulated upon exposure to one of the female attractants (E)-2-hexenol and phenethyl alcohol; none of the OBP transcripts changed upon exposure to methyl anthranilate, which does not attract H. oblita females. Binding assays showed that HoblOBP13 and HoblOBP9 have high affinity for (E)-2-hexenol and phenethyl alcohol, respectively. RNAi treatment showed that transcripts of both HoblOBP13 and HoblOBP9 declined in a time-course manner 24-72 h postinjection. OBP-dsRNA-treated female beetles showed significantly lower attraction to (E)-2-hexenol and phenethyl alcohol than did water-injected beetles and those treated with GFP-dsRNA. We, therefore, concluded that HoblOBP13 and HoblOBP9 are essential for H. oblita reception of the plant-derived kairomones (E)-2-hexenol and phenethyl alcohol.

Identification and comparison of candidate odorant receptor genes in the olfactory and non-olfactory organs of Holotrichia oblita Faldermann by transcriptome analysis.

A sophisticated olfactory system is part of the explanation for the prominence of insects among animals because of the essential roles of the olfactory system in foraging, host seeking, mating, ovipositing and avoiding toxic substances. In this study, we sequenced and analysed the transcriptomes of olfactory tissue (antennae) and non-olfactory tissue (legs) of the scarab beetle, Holotrichia oblita Faldermann, which is a serious underground pest in China. We obtained approximately 80.2 million 150bp reads that were assembled into 61,038 unigenes with an average length of 890bp. Among the transcripts, 70% of the unigenes were annotated. A total of 44 odorant receptors (ORs) and 9 ionotropic receptors (IRs) were identified based on homology searches. Then, quantitative real-time PCR experiments were performed to investigate the expression patterns of 32 putative chemosensory genes. The results showed that these genes were highly expressed in olfactory organs (antennae) and might play a key role in the olfaction-related behaviours in H. oblita. Based on the results of our phylogenetic analysis and the detailed tissue and sex-biased expression characteristics, the different roles of the receptor proteins in the olfactory system were also indicated. The results of this study will provide the foundation for further understanding of the olfactory odorant receptors of H. oblita at the molecular level and ultimately help to develop novel targets for manipulating this pest.

Identification of candidate chemosensory genes by transcriptome analysis in Loxostege sticticalis Linnaeus.

Loxostege sticticalis Linnaeus is an economically important agricultural pest, and the larvae cause great damage to crops, especially in Northern China. However, effective and environmentally friendly chemical methods for controlling this pest have not been discovered to date. In the present study, we performed HiSeq2500 sequencing of transcriptomes of the male and female adult antennae, adult legs and third instar larvae, and we identified 54 candidate odorant receptors (ORs), including 1 odorant receptor coreceptor (Orco) and 5 pheromone receptors (PRs), 18 ionotropic receptors (IRs), 13 gustatory receptors (GRs), 34 odorant binding proteins (OBPs), including 1 general odorant binding protein (GOBP1) and 3 pheromone binding proteins (PBPs), 10 chemosensory proteins (CSPs) and 2 sensory neuron membrane proteins (SNMPs). The results of RNA-Seq and RT-qPCR analyses showed the expression levels of most genes in the antennae were higher than that in the legs and larvae. Furthermore, PR4, OR1-4, 7-11, 13-15, 23, 29-32, 34, 41, 43, 47/IR7d.2/GR5b, 45, 7/PBP2-3, GOBP1, OBP3, 8 showed female antennae-biased expression, while PR1/OBP2, 7/IR75d/CSP2 showed male antennae-biased expression. However, IR1, 7d.3, 68a/OBP11, 20-22, 28/CSP9 had larvae enriched expression, and OBP15, 17, 25, 29/CSP5 were mainly expressed in the legs. The results shown above indicated that these genes might play a key role in foraging, seeking mates and host recognition in the L. sticticalis. Our findings will provide the basic knowledge for further studies on the molecular mechanisms of the olfactory system of L. sticticalis and potential novel targets for pest control strategies.

Identification and molecular characterization of a chitin-binding protein from the beet webworm, Loxostege sticticalis L.

As the first crucial barrier in the midgut of insects, the peritrophic membrane (PM) plays an important role in preventing external invasion. PM proteins, as the major components of the PM, determine the structure and function of this membrane. A new PM protein, named LstiCBP, from the PM of Loxostege sticticalis larvae was identified using cDNA library screening. The full cDNA of LstiCBP is 2606 bp in length and contains a 2403 bp ORF that encodes an 808-amino acid preprotein with a 15-amino acid as signal peptide. The deduced protein sequence of the cDNA contains 8 cysteine-rich chitin-binding domains (CBDs). Recombinant LstiCBP was successfully expressed in BL21 cells using recombinant plasmid DNA and showed high chitin-binding activity. LstiCBP expression was detected in the midgut at both the transcriptional and translational levels; however, the biochemical and physiological functions of LstiCBP in L. sticticalis require further investigation.

Functional characterization of chemosensory proteins in the scarab beetle, Holotrichia oblita Faldermann (Coleoptera: Scarabaeida).

Chemosensory proteins (CSPs) play important roles in chemical communication by insects, as they recognize and transport environmental chemical signals to receptors within sensilla. In this study, we identified HoblCSP1 and HoblCSP2 from a cDNA library of Holotrichia oblita antennae, successfully expressed them in E. coli and purified them by Ni ion affinity chromatography. We then measured the ligand-binding specificities of HoblCSP1 and HoblCSP2 to 50 selected ligands in a competitive binding assay. These results demonstrated that HoblCSP1 and HoblCSP2 have similar ligand-binding spectra. Both proteins displayed the highest affinity for ß-ionone, α-ionone and cinnamaldehyde, indicating that they prefer binding to odorants other than sex pheromones. Additionally, immuno-localization revealed that HoblCSP1 is highly concentrated in sensilla basiconica, while HoblCSP2 is specifically localized to sensilla placodea. In conclusion, HoblCSP1 and HoblCSP2 are responsible for binding to general odorants with slightly different specificities due to their different in vivo environments.

Potential cooperations between odorant-binding proteins of the scarab beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae).

It was previously thought that the odorant binding proteins (OBPs) in the sensillum lymph might serve as carriers, which could carry lipophilic odorant molecules to olfactory receptors. In this study, two novel OBP genes of the scarab beetle Holotrichia oblita were screened using an antennal cDNA library. The full cDNA of HoblOBP3 and HoblOBP4 was cloned using reverse transcription PCR and rapid amplification of the cDNA ends. Homology modeling of both OBPs was performed using SWISS-MODEL on-line tools. Next, the two OBPs were expressed in Escherichia coli and purified using Ni ion affinity chromatography. The ligand-binding properties of HoblOBP3 and HoblOBP4 in 42 ligands respectively were measured using the fluorescence probe N-phenyl-naphthylamine (1-NPN). The results obtained from competitive binding assays demonstrated that HoblOBP4 showed a broader range of binding affinities to the test compounds, while HoblOBP3 displays more specific binding affinity. Furthermore, other OBPs and CSPs were expressed in Escherichia coli and purified using Ni ion affinity chromatography. Binding curves were measured for binary mixtures of OBPs and CSPs using 1-NPN, and the Scatchard plots exhibited "J"-like nonlinear correlation trends in some samples. In addition, competitive binding assays of the HoblOBP1 and HoblOBP2 mixtures and of the HoblOBP2 and HoblOBP4 mixtures with representative compounds unexpectedly demonstrated good affinity, which revealed extreme differences that were only obtained using the individual proteins. In the immunocytochemical analysis, colocalization of HoblOBP1 and HoblOBP2, and of HoblOBP2 and HoblOBP4, was detected in the sensilla basiconica and sensilla placodea, respectively. All of these results suggested that HoblOBP1 and HoblOBP2, as well as HoblOBP2 and HoblOBP4, may serve as heterodimers in the sensillum lymph.

Functional analysis of general odorant binding protein 2 from the meadow moth, Loxostege sticticalis L. (Lepidoptera: Pyralidae).

Odorant binding proteins play a crucial role in transporting semiochemicals across the sensillum lymph to olfactory receptors within the insect antennal sensilla. In this study, the general odorant binding protein 2 gene was cloned from the antennae of Loxostege sticticalis, using reverse transcription PCR and rapid amplification of cDNA ends. Recombinant LstiGOBP2 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR assays indicated that LstiGOBP2 mRNA is expressed mainly in adult antennae, with expression levels differing with developmental age. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the LstiGOBP2 protein has binding affinity to a broad range of odorants. Most importantly, trans-11-tetradecen-1-yl acetate, the pheromone component of Loxostege sticticalis, and trans-2-hexenal and cis-3-hexen-1-ol, the most abundant plant volatiles in essential oils extracted from host plants, had high binding affinities to LstiGOBP2 and elicited strong electrophysiological responses from the antennae of adults.

Fluorescence competition assay for the assessment of green leaf volatiles and trans-ß-farnesene bound to three odorant-binding proteins in the wheat aphid Sitobion avenae (Fabricius).

Odorant-binding proteins (OBPs) are important parts of insect olfactory systems, and sensitive olfaction is vital for phytophagous insects in host foraging. Electrophysiological studies are helpful in understanding olfactory sensing in Sitobion avenae (Hemiptera: Aphididae), but the functions of odorant-binding proteins in this insect are poorly understood. In this study, we used fluorescence competition assays to measure the binding specificities of SaveOBPs. The results showed that both SaveOBP2 and SaveOBP3 were superior to SaveOBP7 in binding green leaf volatiles. It was unexpected that SaveOBP7 bound trans-ß-farnesene strongly, which was known as alarm pheromone of this species. Host volatiles were recognized much more easily by SaveOBP2, and the observed binding activity of SaveOBP2 equaled for tested green leaf volatiles. Our results imply that SaveOBP7 might play a more important role in aphid alarm pheromone discrimination.

Function and immunocytochemical localization of two novel odorant-binding proteins in olfactory sensilla of the scarab beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae).

Odorant-binding proteins (OBPs) are found in both insects and vertebrates, and it is believed that they are involved in chemical communication. In this study, we identify and express 2 OBPs from the scarab beetle, Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae). HoblOBP1 shows more similarities with other scarab beetle OBPs, whereas HoblOBP2 is more diverse. N-phenyl-1-naphthylamine (1-NPN) is used as a fluorescent probe in ligand-binding experiment, and results indicate that both HoblOBPs prefer plant volatiles to putative H. oblita sex pheromones. HoblOBP1 shows binding affinity to a wider range of test compounds, but HoblOBP2 displays more specific binding affinity. Cinnamaldehyde and 2,4-di-tert-butylphenol bind to HoblOBP1 can elicit strong electrophysiological responses of the antennae from female H. oblita adults, respectively. Methyl salicylate also shows good affinity to HoblOBP2 and it can elicit moderate electrophysiological responses. Although, ß-ionone is one of the ligands of the strongest binding, it elicits a weak electrophysiological response. In the immunocytochemical analysis, we observe that HoblOBP1 and HoblOBP2 are coexpressed in sensilla basiconica and placodea in both sexes.

Complete mitochondrial genome of Chilo suppressalis (Walker) (Lepidoptera: Crambidae).

The complete sequence of the mitochondrial genome (mitogenome) of the rice stem borer Chilo suppressalis (Walker) (Lepidoptera: Crambidae) was determined to be 15,465 bp. It contains 13 protein-coding genes (PCGs), 22 tRNA genes, the large and small rRNA genes, and an A+T-rich region. The nucleotide composition of the mitogenome of C. suppressalis is highly A+T biased, accounting for 79.70% in whole mitogenome, 77.74% in PCGs, 84.70% in tRNAs, 81.20% in rRNAs and 94.19% in A+T-rich region, respectively. The PCGs have typical ATN start codons, except for cox1, which contains the unusual CGA. The C. suppressalis A+T-rich region contains a conserved structure combining the motif ATAGA and a 19-bp poly-T stretch, but absence of the 9-bp poly-A element upstream trnM.

Mitochondrial genome of the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) and comparison with other Lepidopterans.

We present the complete sequence of the mitochondrial genome (mitogenome) of the cotton bollworm Helicoverpa armigera. The 15,347-bp mitogenome of H. armigera was arranged in the same order described for all other sequenced lepidopterans, which differs from the most common type found in insects, due to the movement of trnM to a position 5'-upstream of trnI. The gene overlap in the H. armigera mitogenome is totally 23 bp in six locations. The H. armigera mitogenome has a total of 175 bp of intergenic spacer sequences spread over 14 regions ranging in size from 1 to 45 bp. The nucleotide composition of the whole mitogenome of H. armigera is highly A+T biased, accounting for 80.97%, with a slightly positive AT skewness and negative GC skewness, indicating the occurrence of more A than T, C more than G. The protein-encoding genes have typical mitochondrial start codons, except for cox1, which contains the unusual CGA. The cox1, cox2, and nad4 genes have incomplete stop codons (T). The lrRNA and srRNA genes are 1395 and 794-bp long, respectively. All tRNAs have a typical cloverleaf structure of mitochondrial tRNAs, except for trnS1(AGN), the dihydrouridine arm of which could not form a stable stem-loop structure. The H. armigera A+T-rich region contains a conserved structure combining the motif ATAGA and a 19-bp poly-T stretch, but absence of the 9-bp poly-A element upstream of trnM.

Identification and molecular characterization of a new member of the peritrophic membrane proteins from the meadow moth, loxostege sticticalis.

The peritrophic membrane (PM) plays an important role in protecting insects. The PM proteins are important to determinate the formation and function of the PM. A new PM protein, named Lsti99, was identified from the PM of Loxostege sticticalis larvae by cDNA library screening. The full cDNA of Lsti99 is 1392 bp in length, contains an open reading frame (ORF) of 1245 bp that encodes a preprotein of 415 amino acid residues with a 17-amino acid signal peptide. The sequence of Lsti99 showed no homology to other known PM proteins. The recombinant Lsti99 was successfully expressed in insect cells (Sf9) using recombinant baculoviruses and was used to isolate the antibodies to Lsti99 from the polyclonal antiserum. Lsti99 was expressed mainly in the PM, but weaker bands could be detected in the head and integument as well. The Lsti99 protein could be separated from the PM complex by chitinase in vitro, but M2R did not show effect in vitro confirming the chitin-binding activity of Lsti99. The biochemical and physiological functions of Lsti99 in L. sticticalis require further investigation.