A chromosome-scale genome assembly of the nipa palm hispid beetle Octodonta nipae.

Nipa palm hispid beetle (Octodonta nipae) is an insect species that is native to Malaysia but has spread to southern China and beyond, seriously threatening palm production. A lack of high-quality genome resources has hindered understanding of the insect's invasive characteristics and ecological adaptations. Here, we combined Illumina short read, PacBio long-read, and high-throughput chromosome conformation capture (Hi-C) sequencing technologies to generate a high-quality, chromosome-scale genome assembly of nipa palm hispid beetle. The genome assembly was 1.31 Gb in size, consisting of nine chromosomes. The contig and scaffold N50 values were 1.022 Mb and 148.6 Mb, respectively. The genome assembly completeness was estimated at 99.1%. Annotation revealed 16,305 protein-coding genes and 62.16% repeat sequences. This high-quality genome assembly is a valuable resource that will contribute to understanding of the genetic factors underlying the invasive characteristics of nipa palm hispid beetle, ultimately promoting development of efficient control policies.

Establishment of RNAi-Mediated Pest Control Method for Red Imported Fire Ant, Solenopsis invicta.

RNAi plays a crucial role in insect gene function research and pest control field. Nonetheless, the variable efficiency of RNAi across diverse insects and off-target effects also limited its further application. In this study, we cloned six essential housekeeping genes from Solenopsis invicta and conducted RNAi experiments by orally administering dsRNA. Then, we found that mixing with liposomes significantly enhanced the RNAi efficiency by targeting for SiV-ATPaseE. Additionally, we observed a certain lethal effect of this dsRNA on queens by our established RNAi system. Furthermore, no strict sequence-related off-target effects were detected. Finally, the RNAi effect of large-scale bacteria expressing dsRNA was successfully confirmed for controlling S. invicta. In summary, this study established an RNAi system for S. invicta and provided a research template for the future development of nucleic acid drugs based on RNAi.

Histone Acetylation Enhancing Host Melanization in Response to Parasitism by an Endoparasitoid Wasp.

Endoparasitoids are insects that develop within other insects, employing unique strategies to enhance their offspring's survival. They inject polydnavirus and/or venom into their hosts along with eggs, effectively suppressing the host's immune system. Polydnavirus from Braconidae and Ichneumonidae wasps can integrate into the host's genome to express viral genes using the host's transcription systems. However, the ability of parasitoids without polydnavirus to manipulate host gene expression remains unclear. Lysine acetylation (LysAc), a post-translational modification critical for gene regulation, is hypothesized to be used by endoparasitoids lacking polydnavirus. We utilized the Chalcidoidea wasp Tetrastichus brontispae, which lacks polydnavirus, as an idiobiont endoparasitoid model to test this hypothesis, with pupae of the nipa palm hispid beetle Octodonta nipae as the host. Parasitism by T. brontispae resulted in the reduced expression of histone deacetylase Rpd3 and elevated levels of LysAc modification at histones H3.3K9 and H3.3K14 through proteomics and LysAc modification omics. The knockdown of Rpd3 increased the expression level of OnPPAF1 and OnPPO involved in the phenoloxidase cascade, leading to melanization in the host body whereby it resembled a mummified parasitized pupa and ultimately causing pupa death. This study enhances our understanding of how endoparasitoids employ histone acetylation to regulate immunity-related genes, offering valuable insights into their survival strategies.

The Cellular Immunological Responses and Developmental Differences between Two Hosts Parasitized by Asecodes hispinarum.

This study aims to investigate the developmental interactions of Asecodes hispinarum Boucek on Brontispa longissima Gestro and Octodonta nipae Maulik, as well as the cellular immune responses of B. longissima and O. nipae larvae in response to parasitism by A. hispinarum, with the hope of determining the reason for the difference in larval breeding of A. hispinarum in B. longissima and O. nipae. The effects of parasitism by A. hispinarum on the larval development, hemocyte count, and proportion of the hemocyte composition of the two hosts were carried out through selective assay and non-selective assay using statistical analysis and anatomical imaging. There was no significant difference in parasitic selection for A. hispinarum on the larvae of these two beetles; however, more eggs were laid to B. longissima larvae than to O. nipae larvae after parasitism by A. hispinarum. The eggs of A. hispinarum were able to grow and develop normally inside the larvae of B. longissima, and the parasitism caused the larvae of B. longissima become rigid within 7 d, with a high larval mortality rate of 98.88%. In contrast, the eggs of A. hispinarum were not able to develop normally inside the O. nipae larvae, with a high encapsulation rate of 99.05%. In addition, the parasitism by A. hispinarum caused a 15.31% mortality rate in O. nipae larvae and prolonged the larval stage by 5 d and the pupal stage by 1 d. The number of hemocytes during the 12, 24, 48, 72, and 96 h of the four instars from O. nipae larvae was 6.08 times higher than from B. longissima larvae of the same age. After 24 h of being parasitized by A. hispinarum, the total number of hemocytes and granulocyte proportion of B. longissima larvae increased significantly. However, the total number of hemocytes and plasmatocyte proportion of O. nipae increased significantly after 24, 72, and 96 h, and the proportion of granulocytes increased significantly after 12 h post-parasitism. The results in the present study indicated that A. hispinarum was unable to successfully reproduce offspring in O. nipae, but its spawning behavior had an adverse effect on the larval development of its host. In addition, the adequate number of hemocytes and more pronounced changes in the hemocyte count and hemocyte composition ratio in the larvae after parasitization may be important factors for the successful encapsulation in O. nipae larvae.

The first complete genome sequence of an iflavirus from the endoparasitoid wasp Tetrastichus brontispae.

The complete genome sequence of a novel iflavirus isolated from the gregarious and koinobiont endoparasitoid Tetrastichus brontispae, tentatively named "Tetrastichus brontispae RNA virus 3" (TbRV-3), was determined by total RNA and Sanger sequencing. The complete genome is 9998 nucleotides in length, 8934 nt of which encodes a putative polyprotein of 2978 amino acids. TbRV-3 was found to have a similar genome organization and to contain conserved domains and motifs found in other iflaviruses, with some variations. Phylogenetic analysis based on deduced amino acid sequences of the RdRp domain showed that TbRV-3 clustered with Dinocampus coccinellae paralysis virus (DcPV). However, the percent amino acid sequence identity of the putative capsid proteins of TbRV-3 and DcPV determined using BLASTp was below the species demarcation threshold (90%), suggesting that TbRV-3 is a new iflavirus. This is the first virus of the family Iflaviridae to be isolated from a wasp of the family Eulophidae.

Immune function differences between two color morphs of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) at different life stages.

Several studies demonstrated that in insects cuticle melanism is interrelated with pathogen resistance, as melanin-based coloration and innate immunity possess similar physiological pathways. For some insects, higher pathogen resistance was observed in darker individuals than in individuals with lighter cuticular coloration. Here, we investigated the difference in immune response between two color morphs (black and red) and between the life stages (pupa and adult) of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae). Here in this study, cuticle thickness, microbial test (antimicrobial activity, phenoloxidase activity, and hemocyte density), and immune-related gene expression were evaluated at different stages of RPW. Study results revealed that cuticle thickness of black phenotype was thicker than red phenotype at old-pupa stage, while no significant difference found at adult stage. These results may relate to the development processes of epidermis in different stages of RPW. The results of antimicrobial activity, phenoloxidase (PO) activity, and hemocyte density analyses showed that adults with a red phenotype had stronger pathogen resistance than those with a black phenotype. In addition to antimicrobial activity and PO activity, we tested relative gene expression in the fat body of old pupae. The results of hemolymph antimicrobial analysis showed that old pupae with a red phenotype were significantly different from those with a black phenotype at 12 hr after Staphylococcus aureus injection, suggesting that red phenotype pupae were more sensitive to S. aureus. Examination of gene expression in the fat body also revealed that the red phenotype had a higher immune response than the black phenotype. Our results were inconsistent with the previous conclusion that dark insects had increased immune function, suggesting that the relationship between cuticle pigmentation and immune function in insects was not a direct link. Additional possible factors that are associated with the immune response, such as life-history, developmental, physiological factors also need to be considered.

Variation in Parasitoid Virulence of Tetrastichus brontispae during the Targeting of Two Host Beetles.

In host-parasitoid interactions, antagonistic relationship drives parasitoids to vary in virulence in facing different hosts, which makes these systems excellent models for stress-induced evolutionary studies. Venom compositions varied between two strains of Tetrastichus brontispae, Tb-Bl and Tb-On. Tb-Bl targets Brontispa longissima pupae as hosts, and Tb-On is a sub-population of Tb-Bl, which has been experimentally adapted to a new host, Octodonta nipae. Aiming to examine variation in parasitoid virulence of the two strains toward two hosts, we used reciprocal injection experiments to compare effect of venom/ovarian fluids from the two strains on cytotoxicity, inhibition of immunity and fat body lysis of the two hosts. We found that Tb-Onvenom was more virulent towards plasmatocyte spreading, granulocyte function and phenoloxidase activity than Tb-Blvenom. Tb-Blovary was able to suppress encapsulation and phagocytosis in both hosts; however, Tb-Onovary inhibition targeted only B. longissima. Our data suggest that the venom undergoes rapid evolution when facing different hosts, and that the wasp has good evolutionary plasticity.

Cloning and Immunosuppressive Properties of an Acyl-Activating Enzyme from the Venom Apparatus of Tetrastichus brontispae (Hymenoptera: Eulophidae).

Venom injected into the host plays vital roles in facilitating successful parasitization and development for parasitoid wasps, especially those devoid of polydnavirus, and the abundant venom proteins appear to be most likely involved in parasitization success. Previously, we found the four most abundant venom proteins, including 4-coumarate:CoA ligase-like 4 (4CL4-like), in the Tetrastichus brontispae (Hymenoptera: Eulophidae) venom apparatus. In this study, we cloned, expressed T. brontispae 4CL4-like (Tb4CL4-like) in Escherichia coli, and investigated its immunosuppressive properties. The deduced amino acid sequence for Tb4CL4-like shares high identity at conserved amino acids associated with the acyl-activating enzyme (AAE) consensus motif but shows only <40% identity with the members in the AAE superfamily. mRNA abundance analysis indicated that Tb4CL4-like was transcribed mainly in the venom apparatus. Recombinant Tb4CL4-like inhibited Octodonta nipae (Coleoptera: Chrysomelidae) pupal cellular encapsulation and spreading by targeting the hemocyte cytoskeleton and reduced the hemocyte-mediated phagocytosis of E. coli in vivo. Moreover, Tb4CL4-like exhibited greater affinity to palmitic acid and linolenic acid based on the molecular docking assay and is hypothesized to be involved in fatty acid metabolism. In conclusion, our results suggest that Tb4CL4-like may be an immunity-related AAE protein that is involved in the regulation of host immunity through fatty acid metabolism-derived signaling pathways.

Functional conservation and division of two single-carbohydrate-recognition domain C-type lectins from the nipa palm hispid beetle Octodonta nipae (Maulik).

As an invasive pest, the complete and effective innate immune system is crucial for the nipa palm hispid beetle Octodonta nipae (Maulik) to adjust to new environments. C-type lectins (CTLs) are large families of carbohydrate-binding proteins that possess one or more characteristic carbohydrate-recognition domains (CRD) and function as pattern-recognition receptors, which play important roles in mediating humoral and cellular immunity. In the present study, for the first time, we report two CTL-Ss (single-CRD CTLs) from O. nipae (Maulik) (designated OnCTL1 and OnCTL2). The two CTL-Ss share high identity at conserved amino acids associated with conserved carbohydrate binding sites Gln-Pro-Asp (QPD) motifs and clearly show a 1:1 orthologous relationship in insects, which endow them with functional conservation and diversification. mRNA abundance analysis showed that OnCTL1 was upregulated upon Staphylococcus aureus and Escherichia coli challenge at 6 and 12 h, while OnCTL2 underwent no changes upon E. coli challenge and was even downregulated after S. aureus infection. Knockdown of OnCTL1 significantly decreased the transcripts of two key serine proteases (prophenoloxidase activating factors), OnPPAF1 and OnPPAF3, followed by the reduction of haemolymph phenoloxidase activity; it also increased the expression of Defensin2B. In contrast, silencing of OnCTL2 significantly decreased the expression of Defensin2B and Attacin3C, the encapsulation index, and the phagocytosis rate compared to the dsEGFP group. The spreading results showed that more irregularly shaped plasmatocytes and lower levels of aggregation were found in OnCTL2-silenced pupae than in the dsOnCTL1 and dsEGFP groups. We can infer from the results of this study that the two OnCTLs play important roles in the immune system and generate a functional division: OnCTL1 seems to function more in humoral immunity including mediating bacterial recognition and activating the phenoloxidase cascade, and OnCTL2 plays a greater role in enhancing cellular immunity. These observations could replenish information on the functional diversification of insect CTLs, and also provide valuable information to unravel the immunity in O. nipae.

The First Complete Genome Sequence of a Novel Tetrastichus brontispae RNA Virus-1 (TbRV-1).

The complete sequence of a novel RNA virus isolated from Tetrastichus brontispae (TbRV-1) was determined to be 12,239 nucleotides in length with five non-overlapping, linearly arranged coding sequences (CDS), potentially encoding nucleoproteins, hypothetical proteins, matrix proteins, glycoproteins, and RNA-dependent RNA polymerases. Sequence analysis indicated that the RNA-dependent RNA polymerase of TbRV-1 shares a 65% nucleotide and 67% amino acid sequence identity with Hubei dimarhabdovirus 2, suggesting that TbRV-1 is a member of the dimarhabdovirus supergroup. This corresponded to the result of the phylogenetic analysis. The affiliation of TbRV-1 with members of the family Rhabdoviridae was further validated by similar transcription termination motifs (GGAACUUUUUUU) to the Drosophila sigmavirus. The prevalence of TbRV-1 in all tissues suggested that the virus was constitutive of, and not specific to, any wasp tissue. To our knowledge, this is the first report on the complete genome sequence of a dimarhabdovirus in parasitoids.

Combination of label-free quantitative proteomics and transcriptomics reveals intraspecific venom variation between the two strains of Tetrastichus brontispae, a parasitoid of two invasive beetles.

The venom apparatus is a conserved organ in parasitoids that shows adaptations correlated with life-style diversification. Combining transcriptomics and label-free quantitative proteomics, here we explored the venom apparatus components of the endoparasitoid Tetrastichus brontispae (Eulophidae), and provide a comparison of the venom apparatus proteomes between its two closely related strains, T. brontispae-Octodonta nipae (Tb-On) and T. brontispae-Brontispa longissima (Tb-Bl). Tb-Bl targets the B. longissima pupa as its habitual host. However, Tb-On is an experimental derivative of Tb-Bl, which has been exposed to the O. nipae pupa as host consecutively for over 40 generation. Results showed that approximately 1505 venom proteins were identified in the T. brontispae venom apparatus. The extracts contained novel venom proteins, such as 4-coumarate-CoA ligase 4. A comparative venom proteome analysis revealed that significant quantitative and qualitative differences in venom composition exist between the two strains; although the most abundant venom proteins were shared between them. The differentially produced proteins were mainly enriched in fatty acid biosynthesis and melanotic encapsulation response. Six of these enriched proteins presented increased levels in Tb-On, and this result was validated by parallel reaction monitoring (PRM) analysis. Overall, our data reveal that venom composition can evolve quickly and respond to host selection.

Effects of ovarian fluid, venom and egg surface characteristics of Tetrastichus brontispae (Hymenoptera: Eulophidae) on the immune response of Octodonta nipae (Coleoptera: Chrysomelidae).

Although the importance of parasitoids as biocontrol agents has long been recognized, systematic studies of the physiological mechanisms are scarce, especially in those parasitoids that are able to successfully invade their hosts by activating host immune responses. This study explored this phenomenon by investigating the effects of ovarian fluid, venom and egg surface characteristics of Tetrastichus brontispae (Hymenoptera: Eulophidae) on host immunity. The results showed that the injection of venom alone induced higher phenoloxidase activity, while a mixture of ovarian plus venom fluids provoked higher granulocyte and plasmatocyte spreading ratios, highlighting the role that egg surface characteristics may play in successful parasitism. After thorough investigation, the presence of a hemomucin homologue was documented on the egg surface (which was named Tetrastichus brontispae adipocyte plasma membrane associated protein-like, TbAPMAP-like), while the absence of polydnaviruses, fibrous layers and virus-like filaments was confirmed. The higher encapsulation index of eggs incubated with TbAPMAP-like polyclonal antibody demonstrated the protection of the protein against encapsulation. These results contribute to our understanding of the mechanisms used by endoparasitoids to evade encapsulation during the early parasitism stage while enriching our knowledge of local active regulatory mechanisms. It is likely that this is the first study to determine the egg protective properties of TbAPMAP-like in host-parasite systems.

Identification of three prophenoloxidase-activating factors (PPAFs) from an invasive beetle Octodonta nipae Maulik (Coleoptera: Chrysomelidae) and their roles in the prophenoloxidase activation.

A typical characteristic of the insect innate immune system is the activation of the serine protease cascade in the hemolymph. As being the terminal component of the extracellular serine protease cascade in the prophenoloxidase (proPO) activating system, proPO-activating factors (PPAFs) activated by the upstream cascade may generate active phenoloxidase, which then induces downstream melanization. In the present study, we reported three PPAFs from the nipa palm hispid beetle Octodonta nipae (Maulik) (designated as OnPPAF1, OnPPAF2, OnPPAF3). All three OnPPAFs contained a single clip domain at the amino-terminus followed by a trypsin-like serine protease domain at the carboxyl-terminus, except the Ser in the active sites of OnPPAF2 and OnPPAF3 was substituted with Gly. Transcript expression analysis revealed that all OnPPAFs were highly expressed in hemolymph, whereas OnPPAF2 showed an extremely low mRNA abundance compared with that of OnPPAF1 and OnPPAF3, and that the abundance of all three OnPPAFs was dramatically increased upon bacterial challenge. Knockdown of OnPPAF1 or OnPPAF3 resulted in a reduction of hemolymph phenoloxidase activity and an inhibition of hemolymph melanization, whereas the knockdown of OnPPAF2 did not affect the proPO cascade. Our work thus implies that the three OnPPAFs may have different functions and regulation during immune responses in O. nipae.

Molecular cloning and expression of the vitellogenin gene and its correlation with ovarian development in an invasive pest Octodonta nipae on two host plants.

There is an ongoing relationship between host plants and herbivores. The nutrient substances and secondary compounds found in the host plant can not only impact the growth and development process of herbivores, but, more importantly, may also affect their survival and reproductive fitness. Vitellogenesis is the core process of reproductive regulation and is generally considered as a reliable indicator for evaluating the degree of ovarian development in females. Vitellogenin (Vg) plays a critical role in the synthesis and secretion of yolk protein. In this study, the full-length cDNA of the Vg gene in an alien invasive species, the nipa palm hispid beetle Octodonta nipae Maulik (Coleoptera: Chrysomelidae) (OnVg) was cloned and, the effect of host plant on the OnVg expression level and ovarian development was investigated. The results revealed that the OnVg was highly and exclusively expressed in adult females, but barely detectable in larvae, pupae and adult males. The relative expression level of OnVg and egg hatchability were much higher in females fed on Phoenix canariensis (their preferred host) than those fed on Phoenix roebelenii. A positive correlation relationship between OnVg expression and egg hatchability was also detected. Additionally, the anatomy of the female reproductive system showed that the ovaries of individuals fed on P. canariensis were considerably more developed than in females fed on P. roebelenii. The results may be applicable to many pest management situations through reproductive disturbance by alternating host plant species or varieties or by reproductive regulation through vitellogenesis mediated by specific endocrine hormones.

Altered immune function of Octodonta nipae (Maulik) to its pupal endoparasitoid, Tetrastichus brontispae Ferrière.

Most studies on the contribution of the altered immune response by endoparasitoid have been restricted to the interactions between Ichneumonoidea and their hosts, while effects of parasitism by Chalcidoidea on the hosts have rarely been characterized except some wasps such as Pteromalidae. Endoparasitoid Tetrastichus brontispae Ferrière, belonging to Eulophidae (Hymenoptera), has a great potential to control some Coleopteran beetles such as Octodonta nipae, one invasive species in southern China. However, the physiological mechanism underlying the escape from the melanotic encapsulation in O. nipae pupae has not been demonstrated. In the present study, effects of parasitism on the immune function of its pupal host O. nipae were investigated. The combining results that granulocytes and plasmatocytes could phagocytize bacteria from 2 to 48h and granulocytes, plasmatocytes and oenocytoids were prophenoloxidase/phenoloxidase positive hemocytes indicated that granulocytes, plasmatocytes and oenocytoids were the main immunocompetent hemocytes in O. nipae pupae. Parasitism by T. brontispae resulted in a significant increase in the percentage of hemocytes viability and spreading at 96h, growing percentage of granulocytes at 24h but no effects on the total hemocyte counts, and an enhanced phenoloxidase activity only at 12 and 72h while a significantly longer melanization time of the hemolymph at 96h following parasitism. These results indicate that mixtures of systemic active and local active regulation are used for T. brontispae to escape host encapsulation in O. nipae pupae. The present study contributes to the understanding of the diversity of virulence strategies used by parasitoids.

Transcriptome immune analysis of the invasive beetle Octodonta nipae (Maulik) (Coleoptera: Chrysomelidae) parasitized by Tetrastichus brontispae Ferrière (Hymenoptera: Eulophidae).

The beetle Octodonta nipae (Maulik) (Coleoptera: Chrysomelidae) is a serious invasive insect pest of palm plants in southern China, and the endoparasitoid Tetrastichus brontispae Ferrière (Hymenoptera: Eulophidae) is a natural enemy of this pest that exhibits great ability in the biocontrol of O. nipae. For successful parasitism, endoparasitoids often introduce or secrete various virulence factors to suppress host immunity. To investigate the effects of parasitization by T. brontispae on the O. nipae immune system, the transcriptome of O. nipae pupae was analyzed with a focus on immune-related genes through Illumina sequencing. De novo assembly generated 49,919 unigenes with a mean length of 598 bp. Of these genes, 27,490 unigenes (55.1% of all unigenes) exhibited clear homology to known genes in the NCBI nr database. Parasitization had significant effects on the transcriptome profile of O. nipae pupae, and most of these differentially expressed genes were down-regulated. Importantly, the expression profiles of immune-related genes were significantly regulated after parasitization. Taken together, these transcriptome sequencing efforts shed valuable light on the host (O. nipae) manipulation mechanisms induced by T. brontispae, which will pave the way for the development of novel immune defense-based management strategies of O. nipae, and provide a springboard for further molecular analyses, particularly of O. nipae invasion.

Different toxicity of the novel Bacillus thuringiensis (Bacillales: Bacillaceae) strain LLP29 against Aedes albopictus and Culex quinquefasciatus (Diptera: Culicidae).

Bacillus thuringiensis (Bt) (Berliner) strain LLP29 produces a crystal protein Cyt1Aa6 toxic to mosquito vectors of human diseases. However, the susceptibility of Culex quinquefasciatus (Say) in the current study was 8.25 times higher than that of Aedes albopictus (Skuse) with this single protein Cyt1Aa6 purified from LLP29. To understand the mechanism of the novel mosquitocidal protein, the binding characteristic of brush border membrane vesicles from the two tested mosquitoes was investigated. Enzyme-linked immunosorbent assay showed that Cyt1Aa6 bound to the two mosquitoes' brush border membrane vesicles. However, the titer of Ae. albopictus was a little higher than that of Cx. quinquefasciatus, with 3.21 and 2.91, respectively. Ligand Western blot analysis showed Cyt1Aa6 toxin specifically bound to the same three proteins (i.e., 68, 54, and 26 kDa) in the two mosquitoes, but one another protein, approximately to 37 kDa, could just be detected in Cx. quinquefasciatus. However, little difference was found in the test of immunohistochemistry. Cyt1Aa6 was detected in the midguts of both mosquitoes with histopathological changes. It would of great importance to the knowledge of the novel toxin against to Cx. quinquefasciatus and Ae. albopictus.

Cloning, ligand-binding, and temporal expression of ecdysteroid receptors in the diamondback moth, Plutella xylostella.

BACKGROUND: The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a devastating pest of cruciferous crops worldwide, and has developed resistance to a wide range of insecticides, including diacylhydrazine-based ecdysone agonists, a highly selective group of molt-accelerating biopesticides targeting the ecdysone receptors. RESULT: In this study, we cloned and characterized the ecdysone receptors from P. xylostella, including the two isoforms of EcR and a USP. Sequence comparison and phylogenetic analysis showed striking conservations among insect ecdysone receptors, especially between P. xylostella and other lepidopterans. The binding affinity of ecdysteroids to in vitro-translated receptor proteins indicated that PxEcRB isoform bound specifically to ponasterone A, and the binding affinity was enhanced by co-incubation with PxUSP (Kd =3.0±1.7 nM). In contrast, PxEcRA did not bind to ponasterone A, even in the presence of PxUSP. The expression of PxEcRB were consistently higher than that of PxEcRA across each and every developmental stage, while the pattern of PxUSP expression is more or less ubiquitous. CONCLUSIONS: Target site insensitivity, in which the altered binding of insecticides (ecdysone agonists) to their targets (ecdysone receptors) leads to an adaptive response (resistance), is one of the underlying mechanisms of diacylhydrazine resistance. Given the distinct differences at expression level and the ligand-binding capacity, we hypothesis that PxEcRB is the ecdysone receptor that controls the remodeling events during metamorphosis. More importantly, PxEcRB is the potential target site which is modified in the ecdysone agonist-resistant P. xylostella.

Cloning, characterisation and expression profiling of the cDNA encoding the ryanodine receptor in diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae).

BACKGROUND: The rynodine receptors (RyRs) are the main targets of diamide insecticides such as chlorantraniliprole. To provide the basis for a good understanding of the molecular mechanisms of diamide insecticide resistance, an RyR gene from Plutella xylostella was cloned and characterised in the present paper. RESULTS: A full-length cDNA sequence of RyR was cloned from P. xylostella through RT-PCR and rapid amplification of cDNA ends (RACE). The gene (named PxRyR1) is 15 753 bp long, with an open reading frame of 15 354 bp, encoding a predicted RyR of 5117 amino acids. An alternative splicing of the PxRyR1 was also cloned and named PxRyR2. The PxRyR1 shares 77-93% identity with other insect RyRs. Quantitative real-time PCR analysis showed that the PxRyR was expressed at a high level in second-instar larvae and adults, at a low level in prepupae and pupae and abundantly in the body wall muscle and head (respectively 6.00 and 3.12 times the expression in the gut). Western blot analysis with anti-RyR antibodies showed that the RyR was mainly present in the body wall muscle and head, but barely present in the haemocyte and gut. CONCLUSIONS: There are at least two alternative splices of PxRyR expressed in all developmental stages and tissues in P. xylostella at various levels. The results provided the basis for further understanding of the mechanisms of resistance to diamide insecticides in P. xylostella.

Mosquito cell line C6/36 shows resistance to Cyt1Aa6.

This study aimed to investigate the resistance mechanism of C6/36 cells to Cyt1Aa6 protein under selection pressure. Receptor binding properties of Cyt1Aa6 toward sensitive and resistant C6/36 cells were investigated. More sensitive cells were detected with goat-anti-rabbit-FITC-labeled antibody, and the quantity of in vitro activated Cyt1Aa6 toxin bound to resistant cells was greatly reduced. Ligand western blot assays showed that disappearance of the 26 kDa protein and weakness of the positive bands of 68 kDa from resistant cells might lead to the resistance of C6/36 cells to Cyt1Aa6 toxin. The resistance of C6/36 cells was detected under selection in vitro-activated Cytl1Aa6 toxin. Receptor binding demonstrated that reduced Cyt1Aa6 bound to resistant cells, which might be closely related to the disappearance and weakness of some proteins. The results presented here are the first to demonstrate that Cyt1Aa protein, a uniquely characteristic toxin, induced resistance at the cellular level. It might be attributed to the change of receptors.