Comparative transcriptomic analysis of salivary glands between the zoophytophagous Cyrtorhinus lividipennis and the phytozoophagous Apolygus lucorum.

BACKGROUND: Saliva plays a crucial role in shaping the feeding behavior of insects, involving processes such as food digestion and the regulation of interactions between insects and their hosts. Cyrtorhinus lividipennis serves as a predominant natural enemy of rice pests, while Apolygus lucorum, exhibiting phytozoophagous feeding behavior, is a destructive agricultural pest. In this study, a comparative transcriptome analysis, incorporating the published genomes of C.lividipennis and A.lucorum, was conducted to reveal the role of salivary secretion in host adaptation. RESULTS: In contrast to A.lucorum, C.lividipennis is a zoophytophagous insect. A de novo genome analysis of C.lividipennis yielded 19,706 unigenes, including 16,217 annotated ones. On the other hand, A.lucorum had altogether 20,111 annotated genes, as obtained from the published official gene set (20,353 unigenes). Functional analysis of the top 1,000 salivary gland (SG)-abundant genes in both insects revealed that the SG was a dynamically active tissue engaged in protein synthesis and secretion. Predictions of other tissues and signal peptides were compared. As a result, 94 and 157 salivary proteins were identified in C.lividipennis and A.lucorum, respectively, and were categorized into 68 and 81 orthogroups. Among them, 26 orthogroups were shared, potentially playing common roles in digestion and detoxification, including several venom serine proteases. Furthermore, 42 and 55 orthogroups were exclusive in C.lividipennis and A.lucorum, respectively, which were exemplified by a hyaluronidase in C.lividipennis that was associated with predation, while polygalacturonases in A.lucorum were involved in mesophyll-feeding patterns. CONCLUSIONS: Findings in this study provide a comprehensive insight into saliva secretions in C.lividipennis and A.lucorum via a transcriptome approach, reflecting the intricate connections between saliva secretions and feeding behaviors. It is found that conserved salivary secretions are involved in shaping the overlapping feeding patterns, while a plethora of unique salivary secretions may drive the evolution of specific feeding behaviors crucial for their survival. These results enhance our understanding of the feeding mechanisms in different insects from the perspective of saliva and contribute to future environmentally friendly pest control by utilizing predatory insects.

Antennal transcriptome analysis of odorant-binding proteins and characterization of GOBP2 in the variegated cutworm Peridroma saucia.

Odorant-binding proteins (OBPs) are expressed at extremely high concentrations in the chemo-sensilla lymph of insects and have long been thought to be crucial for delivering the semiochemicals to the odorant receptors. They are represented by multiple classes: general odorant-binding proteins (GOBP1 and GOBP2) and pheromone-binding proteins. In the current study, we identified a total of 35 OBPs in the antennal transcriptome of Peridroma saucia, a worldwide pest that causes serious damage to various crops. A gene expression value (TPM, transcripts per million) analysis revealed that seven OBPs (PsauPBP1/2/3, PsauGOBP1/2, PsauOBP6, and PsauOBP8) were highly abundant in the antennae. Next, we focused on the expression and functional characterization of PsauGOBP2. Real-time quantitative-PCR analysis demonstrated that PsauGOBP2 was predominantly expressed in the antennae of both sexes. Fluorescence binding assays showed that the recombinant PsauGOBP2 strongly binds to the female sex pheromone components Z11-16: Ac (Ki = 4.2 µM) and Z9-14: Ac (Ki = 4.9 µM) and binds moderately (6 µM ≤ Ki ≤ 13 µM) to the host plant volatiles phenylethyl acetate, ß-myrcene, and dodecanol. Further 3D structural modeling and molecular docking revealed that several crucial amino acid residues are involved in ligand binding. The results not only increase our understanding of the olfactory system of P. saucia but also provide insights into the function of PsauGOBP2 that has implications for developing sustainable approaches for P. saucia management.

A mouthpart transcriptome for Spodoptera frugiperda adults: identification of candidate chemoreceptors and investigation of expression patterns.

Moth mouthparts, consisting of labial palps and proboscis, not only are the feeding device but also are chemosensory organs for the detection of chemical signals from surrounding environment. Up to now, the chemosensory systems in the mouthpart of moths are largely unknown. Here, we performed systematic analyses of the mouthpart transcriptome of adult Spodoptera frugiperda (Lepidoptera: Noctuidae), a notorious pest that spreads worldwide. A total of 48 chemoreceptors, including 29 odorant receptors (ORs), 9 gustatory receptors (GRs), and 10 ionotropic receptors (IRs), were annotated. Further phylogenetic analyses with these genes and homologs from other insect species determined that specific genes, including ORco, carbon dioxide receptors, pheromone receptor, IR co-receptors, and sugar receptors, were transcribed in the mouthpart of S. frugiperda adults. Subsequently, expression profiling in different chemosensory tissues demonstrated that the annotated ORs and IRs were mainly expressed in S. frugiperda antennae, but one IR was also highly expressed in the mouthparts. In comparison, SfruGRs were mainly expressed in the mouthparts, but 3 GRs were also highly expressed in the antennae or the legs. Further comparison of the mouthpart-biased chemoreceptors using RT-qPCR revealed that the expression of these genes varied significantly between labial palps and proboscises. This study provides the first large-scale description of chemoreceptors in the mouthpart of adult S. frugiperda and provides a foundation for further functional studies of chemoreceptors in the mouthpart of S. frugiperda as well as of other moth species.

Identification and expression analysis of chemosensory receptors in the tarsi of fall armyworm, Spodoptera frugiperda (J. E. Smith).

Chemosensation of tarsi provides moths with the ability to detect chemical signals which are important for food recognition. However, molecular mechanisms underlying the chemosensory roles of tarsi are still unknown. The fall armyworm Spodoptera frugiperda is a serious moth pest that can damage many plants worldwide. In the current study, we conducted transcriptome sequencing with total RNA extracted from S. frugiperda tarsi. Through sequence assembly and gene annotation, 23 odorant receptors 10 gustatory receptors and 10 inotropic receptors (IRs) were identified. Further phylogenetic analysis with these genes and homologs from other insect species indicated specific genes, including ORco, carbon dioxide receptors, fructose receptor, IR co-receptors, and sugar receptors were expressed in the tarsi of S. frugiperda. Expression profiling with RT-qPCR in different tissues of adult S. frugiperda showed that most annotated SfruORs and SfruIRs were mainly expressed in the antennae, and most SfruGRs were mainly expressed in the proboscises. However, SfruOR30, SfruGR9, SfruIR60a, SfruIR64a, SfruIR75d, and SfruIR76b were also highly enriched in the tarsi of S. frugiperda. Especially SfruGR9, the putative fructose receptor, was predominantly expressed in the tarsi, and with its levels significantly higher in the female tarsi than in the male ones. Moreover, SfruIR60a was also found to be expressed with higher levels in the tarsi than in other tissues. This study not only improves our insight into the tarsal chemoreception systems of S. frugiperda but also provides useful information for further functional studies of chemosensory receptors in S. frugiperda tarsi.

Molecule characterization of chemosensory and metabolism-related genes in the proboscis of Athetis lepigone.

Introduction: The moth species Athetis lepigone (Möschler) (Lepidoptera: Noctuidae), which has recently been identified as a pest of summer maize (Zea mays L.) in China, has demonstrated a rapid proliferation with in the Huang-Huai-Hai Plain region since its initial discovery in Hebei Province in 2005. It has become a prevalent pest of corn crops, and its ability to adapt quickly to its surroundings is currently being investigated. One of the key characteristics of its siphoning mouthparts is not only the feeding apparatus itself but also the chemosensory organs that enable the detection of chemical signals from the surrounding environment. However, there is a lack of comprehensive research on the genes responsible for chemosensory and metabolic mechanisms in the proboscises of male and female A. lepigone adults. Methods: In this study, we utilized transcriptome analysis to identify a total of fifty chemosensory genes from six distinct families, including 19 odorant-binding proteins (OBPs), 22 chemosensory proteins (CSPs), one co-receptor (Orco), six odorant receptors (ORs), four ionotropic receptors (IRs), and two sensory neuron membrane proteins (SNMPs) in the proboscis. Notably, seven OBPs, two CSPs, and one OR were discovered for the first time. Additionally, fourteen genes related to metabolism, including cytochrome P450 (CYPs) and carboxylesterases (CXEs), were also identified. Furthermore, a qualitative analysis was conducted on the relative transcript levels of eight related genes. The expression of 21 annotated chemosensory and metabolic genes was compared between A. lepigone adults and larvae using qRT-PCR, revealing tissue specificity. The majority of genes exhibited predominant expression in the antennae and proboscis during the adult stage, while showing slight expression in the combination of sixth-instar larval head oral appendages (maxilla, labium, and antenna) and pheromone gland-ovipositors of female adults. Results/discussion: Our study points to a new pest control strategies that these newly discovered genes have the potential to serve as targets for enhancing future pest control, including mating disruption and the use of food attractants. And it would be advantageous to ascertain the distribution of chemosensory gene expression and gain insights into the functionalities of these genes, thereby establishing a novel theoretical framework for the advancement of eco-friendly pesticides and efficient pest management strategies in the future.

Candidate chemosensory receptors in the antennae and maxillae of Spodoptera frugiperda (J. E. Smith) larvae.

Although most of the damage caused by lepidopteran insects to plants is caused by the larval stage, chemosensory systems have been investigated much more frequently for lepidopteran adults than for larvae. The fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is a polyphagous and worldwide pest. To understand the larval chemosensory system in S. frugiperda, we sequenced and assembled the antennae and maxillae transcriptome of larvae in the sixth instar (larval a-m) using the Illumina platform. A total of 30 putative chemosensory receptor genes were identified, and these receptors included 11 odorant receptors (ORs), 4 gustatory receptors (GRs), and 15 ionotropic receptors/ionotropic glutamate receptors (IRs/iGluRs). Phylogeny tests with the candidate receptors and homologs from other insect species revealed some specific genes, including a fructose receptor, a pheromone receptor, IR co-receptors, CO2 receptors, and the OR co-receptor. Comparison of the expression of annotated genes between S. frugiperda adults and larvae (larval a-m) using RT-qPCR showed that most of the annotated OR and GR genes were predominantly expressed in the adult stage, but that 2 ORs and 1 GR were highly expressed in both the adult antennae and the larval a-m. Although most of the tested IR/iGluR genes were mainly expressed in adult antennae, transcripts of 3 iGluRs were significantly more abundant in the larval a-m than in the adult antennae of both sexes. Comparison of the expression levels of larval a-m expressed chemosensory receptors among the first, fourth, and sixth instars revealed that the expression of some of the genes varied significantly among different larval stages. These results increase our understanding of the chemosensory systems of S. frugiperda larvae and provide a basis for future functional studies aimed at the development of novel strategies to manage this pest.

Identification and Characterization of Chemosensory Receptors in the Pheromone Gland-Ovipositor of Spodoptera frugiperda (J. E. Smith).

Chemoreception by moth ovipositors has long been suggested, but underlying molecular mechanisms are mostly unknown. To reveal such chemosensory systems in the current study, we sequenced and assembled the pheromone gland-ovipositor (PG-OV) transcriptome of females of the fall armyworm, Spodoptera frugiperda, a pest of many crops. We annotated a total of 26 candidate chemosensory receptor genes, including 12 odorant receptors (ORs), 4 gustatory receptors (GRs), and 10 ionotropic receptors (IRs). The relatedness of these chemosensory receptors with those from other insect species was predicted by phylogenetic analyses, and specific genes, including pheromone receptors, ORco, CO2 receptors, sugar receptors, and IR co-receptors, were reported. Although real-time quantitative-PCR analyses of annotated genes revealed that OR and IR genes were mainly expressed in S. frugiperda antennae, two ORs and two IRs expressed in antennae were also highly expressed in the PG-OV. Similarly, GR genes were mainly expressed in the proboscis, but two were also highly expressed in the PG-OV. Our study provides the first large-scale description of chemosensory receptors in the PG-OV of S. frugiperda and provides a foundation for exploring the chemoreception mechanisms of PG-OV in S. frugiperda and in other moth species.

Bombyx mori nucleopolyhedrovirus ORF54, a viral desmoplakin gene, is associated with the infectivity of budded virions.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF54 (Bm54), a member of the viral desmoplakin N-terminus superfamily, is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF66, which is required for the efficient egress of nucleocapsids from the nucleus and occlusion body formation. In this paper, we generated a bacmid with the Bm54 gene deleted via homologous recombination in Escherichia coli and characterized the mutant virus using a transfection-infection assay and transmission electron microscopy analysis. Our results demonstrated that the cells transfected with viral DNA lacking Bm54 produced non-infectious budded viruses (BVs). Electron microscopy showed that although the deletion of Bm54 did not affect assembly and release of nucleocapsids, it severely affected polyhedron formation. In conclusion, deletion of Bm54 resulted in non-infectious BV and defective polyhedra. Although the sequences of Bm54 and Ac66 are very similar, the two genes function quite differently in the regulation of viral life cycle.

Gene expression profiling of resistant and susceptible Bombyx mori strains reveals nucleopolyhedrovirus-associated variations in host gene transcript levels.

We investigated variations in the gene expression of Bombyx mori following infection with a nucleopolyhedrovirus (BmNPV). Two B. mori strains, KN and 306, which are highly resistant and susceptible to BmNPV infection, respectively, were used in this study. The infection profiles of BmNPV in the B. mori KN and 306 larvae revealed that the virus invaded the midguts of both these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, two cDNA libraries were constructed in order to compare the BmNPV responsive gene expressions between the two silkworm lines. In total, 62 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that eight genes were significantly up-regulated in the midgut of the KN strain following BmNPV infection. Our results imply that these up-regulated genes may be involved in the B. mori immune response against BmNPV infection.

Involvement of Bombyx mori nucleopolyhedrovirus ORF41 (Bm41) in BV production and ODV envelopment.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF41 (Bm41), homologous to Ac52, is a gene present in most lepidopteran nucleopolyhedroviruses. Bm41 transcripts and encoded protein in BmNPV-infected cells can be detected from 3 and 6 h post-infection, respectively. Immunoassays have shown that Bm41 is not a viral structural protein and is detected in both the nuclei and cytoplasm of infected cells. A Bm41-disrupted virus (vBm(De)) and a repaired virus (vBm(Re)) were generated to investigate the function of Bm41. The results showed that Bm41 was essential for viral replication, and the disruption of Bm41 resulted in a much lower viral titer. Transmission electron microscopy revealed that disruption of Bm41 affected normal nucleocapsid envelopment and polyhedra formation in the nucleus. The disruption of Bm41 might severely affect odv-ec27 and polyhedrin expression. The disrupted virus reduced BmNPV infectivity in an LD(50) bioassay and took 18-23 h longer to kill larvae than wild-type virus in an LT(50) bioassay.

Characterization of an early gene orf122 from Bombyx mori nucleopolyhedrovirus.

The open reading frame 122 of Bombyx mori nucleopolyhedrovirus (BmNPV) (Bm122) has been observed to be a conserved gene in the lepidopteran baculoviruses that have been completely sequenced so far. Its transcript was detected at 3 h post infection (h p.i.) and remained detectable at up to 96 h p.i. Temporal transcription analysis indicated that Bm122 is transcribed by host RNA polymerase. The size of the translational product of the Bm122 gene in Tn5B-1-4 cells was approximately 23 kDa, which is in agreement with the predicted value of 22.9 kDa, suggesting that no major posttranslational modification occurred in the primary protein product. The subcellular localization of Bm122 was studied using EGFP-Bm122, which revealed that Bm122 protein was accumulated within the nuclear region of virus-infected BmN cells. All these results suggest that Bm122 is an early gene encoding a protein that functions in the nucleus.

Bombyx mori nucleopolyhedrovirus ORF9 is a gene involved in the budded virus production and infectivity.

The ORF9 of Bombyx mori nucleopolyhedrovirus (BmNPV) (Bm9) is conserved in all completely sequenced lepidopteran nucleopolyhedroviruses. RT-PCR analysis demonstrated that Bm9 is an early and late transcribed gene that is initiated at 3 h post-infection, and immunofluorescence microscopy showed that Bm9 is localized mainly in the cytoplasm of infected cells. To determine the role of Bm9 during virus infection, Bm9 was knocked out by recombination in a BmNPV genome propagated as a bacmid in Escherichia coli. The budded virus (BV) production of Bm9-deleted bacmids was reduced more than 10-fold compared with wild-type (wt) bacmid; however, the kinetics of viral DNA replication were unaffected. The defect in BV production was recovered by the Bm9 rescue bacmid. In addition, electron microscope observations revealed that polyhedra formation was not affected by the deletion of Bm9. Bioassays showed that the Bm9-deleted bacmid took approximately 14-22 h longer to kill fifth instar B. mori larvae than wt bacmid, and the LD(50) was about 15 times higher than that of the wt bacmid. In conclusion, Bm9 is an important but not essential factor in virus production and infectivity in vivo and in vitro.

Characterization of a Bombyx mori nucleopolyhedrovirus with Bmvp80 disruption.

A BmNPV Bacmid with the Bmvp80 gene disrupted was constructed using the ET-recombination system in Escherichia coli to investigate the role of Bmvp80 during the baculovirus life cycle. Disruption of Bmvp80 resulted in single cell infection phenotype, whereas a rescue BmBacmid restored budded virus titers to wild type levels; however, the homologous gene Ac104 (Acvp80) from AcMNPV could not complement the BmBacmid lacking a functional Bmvp80 gene. Electron microscopy of cells transfected with BmNPV lacking functional Bmvp80 revealed that the number of nucleocapsids was markedly lower. These results suggest that Bmvp80 is essential for normal budded virus production and nucleocapsid maturation, and is functionally divergent between baculovirus species.

Bombyx mori nucleopolyhedrovirus ORF56 encodes an occlusion-derived virus protein and is not essential for budded virus production.

Bombyx mori nucleopolyhedrovirus ORF56 (Bm56) is a baculovirus core gene that is highly conserved in all baculoviruses that have had their genomes sequenced to date. Its transcripts in BmNPV-infected cells could be detected from 12 h post-infection (p.i.) and the encoded protein could be detected at 16 h p.i. by using a polyclonal antibody against glutathione S-transferase-Bm56 fusion protein. Western blot analysis showed that Bm56 is a structural component of the occlusion-derived virus nucleocapsid. Subsequent confocal microscopy revealed that Bm56 was distributed in the outer nuclear membrane and the intranuclear region of infected cells. To investigate the role of Bm56 in virus replication, a Bm56-knockout bacmid of BmNPV was constructed via homologous recombination in Escherichia coli. The Bm56 deletion had no effect on budded virus (BV) production in cultured cells; however, the deletion affected occlusion-body morphogenesis. A larval bioassay demonstrated that the Bm56 deletion did not reduce infectivity, whereas it resulted in a 50 % lethal time that was 16-18 h longer than that of the wild-type bacmid at every dose used in this study. These results indicate that Bm56 facilitates efficient virus production in vivo; however, it is not essential for BV production in vitro.