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In this paper, a novel cascade reaction of caesium carbonate-promoted Michael addition and lactonization for the one-pot synthesis of 3-alkyl-3-N-substituted aminobenzofuran-2(3H)-one derivatives has been established based on the screening of the alkaline reagents and optimization of reaction conditions, in which the N-substituted (ortho-hydroxy)aryl glycine esters were used as the Michael donors to react with different α, β-unsaturated carbonyl compounds. In the case of using the asymmetric starting material, the epimers could be successfully separated by conventional chromatography. In addition, plausible mechanisms were suggested and the absolute configuration of the epimer was analysed. All the chemical structures of unreported benzofuran-2(3H)-one derivatives were characterized by 1H nuclear magnetic resonance (NMR), 13C NMR, IR and high-resolution mass spectrometry (HRMS).

Abstract Background The continuously developing pesticide resistance is a great threat to agriculture and human health. Understanding the mechanisms of insecticide resistance is a key step in dealing with the phenomenon. Insect cuticle is recently documented to delay xenobiotic penetration which breaks the previous stereotype that cuticle is useless in insecticide resistance, while the underlying mechanism remains scarce. Results Here, we find the integument contributes over 40.0% to insecticide resistance via different insecticide delivery strategies in oriental fruit fly. A negative relationship exists between cuticle thickening and insecticide penetration in resistant/susceptible, also in field strains of oriental fruit fly which is a reason for integument-mediated resistance. Our investigations uncover a regulator of insecticide penetration that miR-994 mimic treatment causes cuticle thinning and increases susceptibility to malathion, whereas miR-994 inhibitor results in opposite phenotypes. The target of miR-994 is a most abundant cuticle protein (CPCFC) in resistant/susceptible integument expression profile, which possesses capability of chitin-binding and influences the cuticle thickness-mediated insecticide penetration. Our analyses find an upstream transcriptional regulatory signal of miR-994 cascade, long noncoding RNA (lnc19419), that indirectly upregulates CPCFC in cuticle of the resistant strain by sponging miR-994. Thus, we elucidate the mechanism of cuticular competing endogenous RNAs for regulating insecticide penetration and demonstrate it also exists in field strain of oriental fruit fly. Conclusions We unveil a regulatory axis of lnc19419 ~ miR-994 ~ CPCFC on the cuticle thickness that leads to insecticide penetration resistance. These findings indicate that competing endogenous RNAs regulate insecticide resistance by modulating the cuticle thickness and provide insight into the resistance mechanism in insects.

Abstract Background Citrus fruits are economically and nutritionally important but have been severely affected by Huanglongbing disease (HLB), its natural spread is mainly by the Asian Citrus Psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). Chemicals are often used to control this pest, but this is not sustainable. Meanwhile, there are few environmentally friendly bioinsecticides to control D. citri in China. Results In this study, an entomofungal pathogen wz-1 was isolated from a D. citri carcass in the field, which resulted in a cumulative mortality rate of 75.27% in adult D. citri 7 days after inoculation of the spore suspension. It was identified as Cladosporium subuliforme based on morphological analysis as well as sequence analysis of several molecular markers (Internal Transcribed Spacers, Translation Elongation Factor 1-α and Actin). Remarkably, the lethality rate of adult D. citri reached 53.13%, 48 h after treatment with the aqueous phase extracts of wz-1. Hydroxyquinoline and phytosphingosine in the extracts were identified as potentially active metabolites using LC–MS. Conclusions The entomopathogenicity and bioinsecticidal potential of C. subuliforme were previously unknown. Obtained results showed that both spores and extracts of wz-1 can effectively kill adult D. citri, providing an available fungal resource and a theoretical basis for biocontrol of the HLB insect vector D. citri.

ABSTRACT Parasitoid wasps are a diverse group of parasitoid insects that play a crucial role in biological control and integrated pest management programs due to their wide range of host species and complex behaviors. Previous studies have reported that select RNA viruses in parasitoid wasps can affect either the host-parasitoid wasp or the pest host parasitized by the wasps. Therefore, a study of the dynamics of RNA viruses in parasitoid wasps is essential for artificial breeding programs and improving their parasitic capabilities. In this study, we detected the presence of nine novel and three known RNA viruses with complete genomes in three parasitoid wasp species: Diachasmimorpha longicaudata, Fopius arisanus, and Spalangia endius. These wasps are parasitoids that can parasitel tephritid flies, one of the most important pest groups of fruits and vegetables. In D. longicaudata, the small RNAs derived from the viruses exhibited distinct peaks at 22 nt and were symmetrically distributed across the viral genome. These findings indicate that these viruses can activate the host’s RNAi immune response. PCR detection further revealed that DlNaLV efficiently infects female individuals of D. longicaudata with 100% efficiency, and ZcNLV is capable of infecting both D. longicaudata and its host, Zeugodacus cucurbitae. Our findings contribute to a better understanding of RNA viruses in parasitoid wasps and lay the foundation for future research on the triple-level trophic relationships between tephritid flies, parasitoid wasps, and RNA viruses. IMPORTANCE Parasitoid wasp populations have developed persistent beneficial symbiotic relationships with several viruses through repeated evolution. However, there have been limited reports on RNA viruses in parasitoid wasps of tephritid flies, a significant pest group affecting fruits and vegetables. This study explores the diversity of RNA viruses in three parasitoid wasps of tephritid flies and highlights the potential biological significance of specific viruses in Diachasmimorpha longicaudata. These findings have important implications for the development of sustainable pest management strategies and the enhancement of artificial rearing techniques for parasitoid wasps.

Gravid female fruit flies show a strong preference to 1-octen-3-ol, a volatile compound from mango. Two odorant receptors are shown to regulate 1- octen-3-ol induced oviposition behaviour.

Endosymbionts play crucial roles in various physiological activities within insect hosts. The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is an important vector for Candidatus Liberibacter asiaticus (CLas), a fatal pathogenic bacterial agent causing the disease Huanglongbing in the citrus industry. This study combines high-throughput sequencing of 16S ribosomal RNA amplicons to explore how CLas affects the bacterial community in different color morphs (blue, gray), genders, and tissues (cuticle, gut, mycetome, Malpighian tubule, ovary, and testis) of ACP. We found that there was no significant differences in the bacterial community diversity and CLas acquired ratio between the different color morphs and genders of ACP adults. However, acquiring CLas could promote the adult bacterial community’s diversity and richness more than in the uninfected condition. The presence of CLas could increase the Wolbachia and unclassified_Enterobacteriaceae proportions more than in the uninfected condition. The bacterial community diversity in the CLas infected tissues of ovary and cuticle, was lower than the uninfected condition, but the richness of all tissues was not different between the infected and uninfected conditions. CLas could also change the bacterial structure in different tissues and make the bacterial relationship network simpler than it is in an uninfected condition. Furthermore, we used quantitative real-time PCR to assess the dynamic changes of Wolbachia in CLas uninfected and infected color morphs and tissues of ACP. The results showed that Wolbachia titers were significantly higher in CLas infected adults than in uninfected adults. In different tissues, the Wolbachia titers in the testis, ovary, and Malpighian tubule were higher than their uninfected counterparts. Our results provide essential knowledge for understanding the symbionts of the ACP and how CLas affects the bacterial community of the ACP.

Fecundity is arguably one of the most important life history traits, as it is closely tied to fitness. Most arthropods are recognized for their extreme reproductive capacity. For example, a single female of the oriental fruit fly Bactrocera dorsalis, a highly invasive species that is one of the most destructive agricultural pests worldwide, can lay more than 3000 eggs during its life span. The ovary is crucial for insect reproduction and its development requires further investigation at the molecular level. We report here that miR-309a is a regulator of ovarian development in B. dorsalis. Our bioinformatics and molecular studies have revealed that miR-309a binds the transcription factor pannier (GATA-binding factor A/pnr), and this activates yolk vitellogenin 2 (Vg 2) and vitellogenin receptor (VgR) advancing ovarian development. We further show that miR-309a is under the control of juvenile hormone (JH) and independent from 20-hydroxyecdysone. Thus, we identified a JH-controlled miR-309a/pnr axis that regulates Vg2 and VgR to control the ovarian development. This study has further enhanced our understanding of molecular mechanisms governing ovarian development and insect reproduction. It provides a background for identifying targets for controlling important Dipteran pests.

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) is one of the broad host ranges and economically-important insect pests in tropical and subtropical areas. A wide range of hosts means they have strong adaptation ability to changes in dietary macronutrients (e.g., sucrose and protein). However, the effects of dietary conditions on the phenotypes and genotypes of B. dorsalis are still unclear. In this study, we aimed to investigate the effects of larval dietary sucrose on the life history traits and stress tolerance of B. dorsalis, and its defense response at the molecular level. The results showed that low-sucrose (LS) induced decreased body size, shortened developmental duration, and enhanced sensitivity to beta-cypermethrin. Otherwise, high-sucrose (HS) diet increased developmental duration, adult fecundity, and tolerance to malathion. Based on transcriptome data, 258 and 904 differentially expressed genes (DEGs) were identified in the NS (control) versus LS groups, and NS versus HS groups, respectively. These yielded DEGs were relevant to multiple specific metabolisms, hormone synthesis and signaling, and immune-related pathways. Our study will provide biological and molecular perspective to understand phenotypic adjustments to diets and the strong host adaptability in oriental fruit flies.

Abstract Background Olfactory systems take on important tasks to distinguish salient information from a complex olfactory environment, such as locating hosts, mating, aggression, selecting oviposition sites, and avoiding predators. The olfactory system of an adult insect consists of two pairs of main olfactory appendages on the head, the antennae, and the palps, which are covered with sensilla. Benzothiazole and 1-octen-3-ol could elicit oviposition behavior in gravid B. dorsalis are regarded as oviposition stimulants. However, the mechanism for how B. dorsalis percepts benzothiazole and 1-octen-3-ol still remains unknown. Results We conducted a comparative analysis of the antennal transcriptomes in different genders of B. dorsalis using Illumina RNA sequencing (RNA-seq). We identified a total of 1571 differentially expressed genes (DEGs) among the two sexes, including 450 female-biased genes and 1121 male-biased genes. Among these DEGs, we screened out 24 olfaction-related genes and validated them by qRT-PCR. The expression patterns of these genes in different body parts were further determined. In addition, we detected the expression profiles of the screened female-biased chemosensory genes in virgin and mated female flies. Furthermore, the oviposition stimulants-induced expression profilings were used to identify chemosensory genes potentially responsible for benzothiazole and 1-octen-3-ol perception in this fly. Conclusions The results from this study provided fundamental data of chemosensory DEGs in the B. dorsalis antenna. The odorant exposure assays we employed lay a solid foundation for the further research regarding the molecular mechanism of benzothiazole and 1-octen-3-ol mediated oviposition behavior in B. dorsalis.

The innate immune system of insects is the front line of self-defense against pathogen invasion. Peptidoglycan recognition proteins (PGRPs) are important components and play key roles in insect immune systems by recognizing peptidoglycan (PGN) in bacterial cell walls. We characterized two isoforms of the PGRP-LC gene, BdPGRP-LCa and BdPGRP-LCb, from Bactrocera dorsalis (Hendel), an important fruit and vegetable pest worldwide. These two isoforms contain an open reading frames of 1 668 bp and 1 731 bp, encoding a protein of 555 and 576 amino acids, respectively. Quantitative real-time PCR results showed that both transcripts were prominently expressed in midgut and fat body of B. dorsalis adult. Inoculation of pathogens showed that both isoforms actively responded to Escherichia coli PGN. We also observed a light response to Staphylococcus aureus PGN. Upon Beauveria bassiana inoculation, the expression of BdPGRP-LCa was enhanced, but the expression of BdPGRP-LCb was suppressed. Suppression of both transcripts by RNA interference led to increased mortality of flies challenged by E. coli, indicating that the two isoforms are involved in sensing Gram-negative bacterial infections.

Abstract Background Long non-coding RNAs (lncRNAs) are involved in many fundamental biological processes, such as transcription regulation, protein degradation, and cell differentiation. Information on lncRNA in the melon fly, Zeugodacus cucurbitae (Coquillett) is currently limited. Results We constructed 24 RNA-seq libraries from eight tissues (midgut, Malpighian tubules, fat body, ovary, and testis) of Z. cucurbitae adults. A total of 3124 lncRNA transcripts were identified. Among those, 1464 were lincRNAs, 1037 were intronic lncRNAs, 301 were anti-sense lncRNAs, and 322 were sense lncRNAs. The majority of lncRNAs contained two exons and one isoform. Differentially expressed lncRNAs were analyzed between tissues, and Malpighian tubules versus testis had the largest number. Some lncRNAs exhibited strong tissue specificity. Specifically expressed lncRNAs were identified and filtered in tissues of female and male Z. cucurbitae based on their expression levels. Four midgut-specific lncRNAs were validated by quantitative real-time polymerase chain reaction (RT-qPCR), and the data were consistent with RNA-seq data. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of targets of midgut-specific lncRNAs indicated an enrichment of the metabolic process. Conclusions This was the first systematic identification of lncRNA in the melon fly. Expressions of lncRNAs in multiple adult tissues were evaluated by quantitative transcriptomic analysis. These qualitative and quantitative analyses of lncRNAs, especially the tissue-specific lncRNAs in Z. cucurbitae, provide useful data for further functional studies.

Vitellogenin (Vg) genes encode the major egg yolk protein precursor in arthropods. In this study, four Vgs were identified in Zeugodacus cucurbitae (Coquillett). Sequence analysis showed that four ZcVgs had the conserved Vg domain. Phylogenetic analysis indicated that four ZcVgs were homologous to the Vgs of Tephritidae insects. The temporal and spatial expression patterns of ZcVgs were analyzed by quantitative real-time polymerase chain reaction (RT-qPCR), and the four ZcVgs showed high expression levels in female adults, especially in the fat body. The expression of ZcVg1 and ZcVg3 was down-regulated by a low dosage (0.5 μg) of 20-hydroxyecdysone (20E), and ZcVg2, ZcVg3, and ZcVg4 were up-regulated by a high dosage (1.0 and 2.0 μg) of 20E. The expression of ZcVg1 and ZcVg2 was up-regulated by 5 μg of juvenile hormone (JH), while all of the ZcVgs were down-regulated by a low and high dosage of JH. Expression of ZcVgs was down-regulated after 24 h of starvation and recovered to normal after nutritional supplementation. After micro-injection of the gene-specific double-stranded RNA, the ZcVgs’ expression was significantly suppressed, and ovarian development was delayed in Z. cucurbitae females. The results indicate that RNA interference of reproduction-related genes is a potential pest control method that works by manipulating female fertility.

Barklice in the genus Lepinotus (Psocoptera: Trogiidae) are small, soft-bodied stored-product pests that are difficult to control. We sequenced and annotated the mitochondrial (mt) genome of Lepinotus sp. The mt genome of Lepinotus sp. is 16,299 bp in size with 74.4% A + T content. The gene order was highly conserved in some of the Trogimorpha barklice. Two types of tandem repeat units were identified in CR of Lepinotus sp. The phylogenetic analysis showed that Trogiidae species was the sister group to Lepidopsocidae barklice, and the suborder Troctomorpha was polyphyletic.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the principal vector of the Candidatus Liberibacter asiaticus (CLas) bacterium that causes Huanglongbing (HLB) disease. The D. citri salivary glands (SG) is an important barrier to the transmission of CLas. Despite its importance, the transcriptome and proteome of SG defense against CLas are unstudied in D. citri. In the present study, we generated a comparative transcriptome dataset of the SG in infected and uninfected D. citri using an Illumina RNA-Seq technology. We obtained 407 differentially expressed genes (DEGs), including 159 upregulated DEGs and 248 downregulated DEGs. Functional categories showed that many DEGs were associated with the ribosome, the insecticide resistance, the immune response and the digestion in comparison with CLas-infected SG and CLas-free SG. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases confirmed that metabolism and immunity were important functions in the SG. Among the DEGs, 68 genes (35 upregulated and 33 downregulated) encoding putative-secreted proteins were obtained with a signal peptide, suggesting that these genes may play important roles in CLas infection. A total of 673 SG proteins were identified in uninfected D. citri by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, and 30 DEGs (15 upregulated and 15 downregulated) were found using the local tBLASTP programs. Among the 30 DEGs, many DEGs mainly involved in the metabolism and cellular processes pathways. This study provides basic transcriptome and proteome information for the SG in D. citri, and helps illuminate the molecular interactions between CLas and D. citri.

Esterases (ESTs) play important roles in metabolizing various physiologically endogenous and exogenous compounds, and various environmental xenobiotics in insects. The psocid, Liposcelis bostrychophila is a major pest of stored products worldwide and rapidly develops resistance to commonly insecticides. However, the involvement of ESTs in insecticide metabolization and the application of RNAi approach in psocids have not been well elucidated. In this study, we characterized four LbEST genes and investigated the transcriptional levels of these genes at different developmental stages and under different insecticides exposures to assess their potential roles in response to insecticides. The four LbESTs contain a catalytic triad (Ser-His-Glu) linked to an oxyanion hole and acyl pocket involved in substrate stabilization during its hydrolysis. Synergism observed with the esterase-inhibitor DEF suggests the involvement of esterases in malathion detoxification. LbESTs were expressed during the whole of developmental stages, but predominant abundance in the first nymphal instar and adult stage. The mRNA level of three LbEST genes (except for LbEST4) was induced (1.29- to 5.60 fold) in response to malathion or deltamethrin exposures, indicating that these esterases are involved in the detoxification process. Silencing of LbEST1, LbEST2 or LbEST3 through dsRNA feeding led to a higher mortality of psocids upon the malathion treatment compared to controls (1.83 to 2.69-fold), demonstrating that these esterase genes play roles in malathion detoxification in L. bostrychophila. Our study provides new evidence for understanding of the function and regulation mechanism of esterases in L. bostrychophila in insecticide detoxification. The current study also suggests that the present RNAi method could be applied for gene functional studies in psocids.

Insect antennae are important olfactory organs that house high concentrations of odorant-binding proteins (OBPs) in the sensillum lymph. Previous studies in other insects have shown that OBPs play important roles in transporting odorants and enhancing the sensitivity of the olfactory system. However, the functions of OBPs in the oriental fruit fly, Bactrocera dorsalis, especially those specifically expressed in antennae, have not been fully elucidated. In this study, cDNA libraries were constructed from both the male and female antennal transcriptome, and twenty OBPs were identified in total. The expression profiles of these OBPs were examined in the adult antenna, head, thorax, leg, and abdomen of both sexes. Seven of the identified OBP genes had significantly higher expression in both the male and female antennae than in other tissues, while the transcript levels of the remaining OBPs varied across different tissues. Regarding the function of antenna-specific OBPs, we targeted Bdorsobp2 as a representative for further RNA interference (RNAi) and identified via electrophysiology a decrease in detection of a potential species-specific a potent attractant, methyl eugenol. Moreover, subsequent behavioral assay data showed that the behavioral response of B. dorsalis toward this odorant decreased when Bdorobp2 was silenced with injection of double-stranded RNA (dsRNA). Combined, these results support our initial hypothesis that antennae-specific OBPs are of critical importance for insect odorant detection, sensitivity, and behavior.

Abstract Background The oriental fruit fly, Bactrocera dorsalis (Hendel) has been considered to be one of the most important agricultural pest around the world. As a holometabolous insect, larvae must go through a metamorphosis process with dramatic morphological and structural changes to complete their development. To better understand the molecular mechanisms of these changes, RNA-seq of B. dorsalis from wandering stage (WS), late wandering stage (LWS) and white puparium stage (WPS) were performed. Results In total, 11,721 transcripts were obtained, out of which 1914 genes (578 up-regulated and 1336 down-regulated) and 2047 genes (655 up-regulated and 1392 down-regulated) were found to be differentially expressed between WS and LWS, as well as between WS and WPS, respectively. Of these DEGs, 1862 and 1996 genes were successfully annotated in various databases. The analysis of RNA-seq data together with qRT-PCR validation indicated that during this transition, the genes in the oxidative phosphorylation pathway, and genes encoding P450s, serine protease inhibitor, and cuticular proteins were down-regulated, while the serine protease genes were up-regulated. Moreover, we found some 20-hydroxyecdysone (20E) biosynthesis and signaling pathway genes had a higher expression in the WS, while the genes responsible for juvenile hormone (JH) synthesis, degradation, signaling and transporter pathways were down-regulated, suggesting these genes might be involved in the process of larval pupariation in B. dorsalis. For the chitinolytic enzymes, the genes encoding chitinases (chitinase 2, chitinase 5, chitinase 8, and chitinase 10) and chitin deacetylase might play the crucial role in the degradation of insect chitin with their expressions significantly increased during the transition. Here, we also found that chitin synthase 1A might be involved in the chitin synthesis of cuticles during the metamorphosis in B. dorsalis. Conclusions Significant changes at transcriptional level were identified during the larval pupariation of B. dorsalis. Importantly, we also obtained a vast quantity of RNA-seq data and identified metamorphosis associated genes, which would all help us to better understand the molecular mechanism of metamorphosis process in B. dorsalis.

Carotenoids play many crucial roles in organisms. Recently, the de novo synthesis of carotenoids has been reported in pea aphid (Acyrthosiphon pisum) through horizontally transferred genes. However, their upstream pathway in the pea aphid is poorly understood. Geranylgeranyl diphosphate synthase (GGPPS) is the functional enzyme in the synthesis of geranylgeranyl diphosphate (GGPP) which is a precursor for the biosynthesis of many biological metabolites, including carotenoid synthesis. In this study, we performed a series of experiments to characterize GGPPS gene and its association with carotenoid biosynthesis. (1) determining the transcript abundance and carotenoid content in two geographical strain with red and green morphs, and (2) examining the abundance of carotenoid related genes and carotenoid levels after silencing of GGPPS in both red and green morphs. We observed that GGPPS was more highly expressed in the green morph than in the red morph of two strains of the pea aphid. The total level of carotenoids was also higher in green morphs than in red morphs in both strains. In addition to the total carotenoid difference, the carotenoids found in the two morphs also differed. There were α-carotene, β-carotene, and γ-carotene in the green morphs, but three additional carotenoids, including cis-torulene∗, trans-torulene∗, and 3,4-didehydrolycopene∗, were present in the red morphs. Silencing the GGPPS by RNAi in both the red and green morphs decreased the expression of some carotenoid biosynthesis-related genes, including carotenoid synthase/cyclase genes and carotenoid desaturase genes in green morphs. Carotenoid levels were decreased in both green and red morphs. However, the specific carotenoids present were not changed after silencing GGPPS. These results demonstrated that GGPPS may act as the upstream enzyme to influence the synthesis of the total amount of carotenoids. The present study provided important molecular evidence for the conserved roles of GGPPS associated with carotenoids biosynthesis and will enhance further investigation on the mechanisms of carotenoid biosynthesis in pea aphid.

Abstract Phormicins belong to defensin family, which are important antimicrobial peptides (AMPs) in insects. These AMPs are inducible upon challenging by immune triggers. In the present study, we identified the cDNA of a phormicin gene (BdPho) in the oriental fruit fly, Bactrocera dorsalis (Hendel), a ruinous agricultural pest causing great economic losses to fruits and vegetables. The cDNA of BdPho contains a 282 bp open reading frame encoding 93 amino acid residues, and the predicted molecular weight and isoelectric point of BdPho peptide were 9.83 kDa and 7.54, respectively. Quantitative real-time PCR analyses showed that the transcription level of BdPho was the highest in adult during different developmental stages and was the highest in abdomen among adult tagmata. Moreover, BdPho was highly expressed in fat body among different tissues, both in female and male adult. The mRNA level of BdPho was significantly up-regulated to 7.46- and 14.53-fold at 3 and 6 h after the insects were challenged with peptidoglycans from Escherichia coli (PGN-EB), respectively, suggesting its antimicrobial activity against Gram-negative microorganisms. Furthermore, the expression level of BdPho was significantly up-regulated to 3.83-fold after mating, suggesting that female adults might enhance their immunity by up-regulating the expression level of BdPho during mating. These results firstly describe the basic properties of the phormicin gene from B. dorsalis, and lay the foundation for investigating functional properties of AMPs and exploring the molecular mechanisms in the immune system.

The Asian citrus psyllid, D. citri Kuwayama is the primary vector for Candidatus Liberibacter asiaticus (CLas), which causes a destructive disease in citrus plants. Bacterial symbionts are important determinants of insect physiology, and they can be impacted by many external factors. Temperature is an important abiotic factor affecting insect physiology, and it is also known that differences in symbiont proportions may vary in different insect genders. To date, it is unclear how the symbionts of D. citri are affected by temperature and gender. This study used high-throughput sequencing of 16S ribosomal RNA amplicons to determine how temperature and gender affect the bacterial communities present in D. citri. We identified 27 amplicon sequence variants (ASVs) belonging to 10 orders, seven classes, and five phyla. The dominant phylum was Proteobacteria (99.93%). Other phyla, including Firmicutes, Bacteroidota, Deinococcota, Cyanobacteria, and Actinobacteriota, were less abundant (Profftella (71.77–81.59%) and Wolbachia (18.39–28.22%) were the predominant taxa in all samples. Under high-temperature treatment, Profftella was more common in females, while Wolbachia had a higher abundance in males. In males, Profftella was more abundant under low-temperature treatments than under high-temperature treatments. In contrast, Wolbachia showed a higher abundance under high-temperature treatments than under low-temperature treatments. An RT-qPCR (quantitative real-time PCR) approach confirmed the results obtained with high-throughput DNA sequencing. Our results provide a basis for understanding the co-adaptation of D. citri and its symbionts to environmental temperature stress.

The neurohormone octopamine regulates many crucial physiological processes in insects and exerts its activity via typical G-protein coupled receptors. The roles of octopamine receptors in regulating behavior and physiology in Coleoptera (beetles) need better understanding. We used the red flour beetle, Tribolium castaneum, as a model species to study the contribution of the octopamine receptor to behavior and physiology. We cloned the cDNA of a β-adrenergic-like octopamine receptor (TcOctβ2R). This was heterologously expressed in human embryonic kidney (HEK) 293 cells and was demonstrated to be functional using an in vitro cyclic AMP assay. In an RNAi assay, injection of dsRNA demonstrated that TcOctβ2R modulates beetle locomotion, mating duration, and fertility. These data present some roles of the octopaminergic signaling system in T. castaneum. Our findings will also help to elucidate the potential functions of individual octopamine receptors in other insects.

Abstract Background Diet composition (yeast:carbohydrate ratio) is an important determinant of growth, development, and reproduction. Recent studies have shown that decreased yeast intake elicits numerous transcriptomic changes and enhances somatic maintenance and lifespan, which in turn reduces reproduction in various insects. However, our understanding of the responses leading to a decrease in yeast ratio to 0% is limited. Results In the present study, we investigated the effects of a sugar-only diet (SD) on the gene expression patterns of the oriental fruit fly, Bactrocera dorsalis (Hendel), one of the most economically important pests in the family Tephritidae. RNA sequencing analyses showed that flies reared on an SD induced significant changes in the expression levels of genes associated with specific metabolic as well as cell growth and death pathways. Moreover, the observed upregulated genes in energy production and downregulated genes associated with reproduction suggested that SD affects somatic maintenance and reproduction in B. dorsalis. As expected, we observed that SD altered B. dorsalis phenotypes by significantly increasing stress (starvation and desiccation) resistance, decreasing reproduction, but did not extend lifespan compared to those that received a normal diet (ND) regime. In addition, administration of an SD resulted in a reduction in antioxidant enzyme activities and an increase in MDA concentrations, thereby suggesting that antioxidants cannot keep up with the increase in oxidative damage induced by SD regime. Conclusions The application of an SD diet induces changes in phenotypes, antioxidant responses, and gene expressions in B. dorsalis. Previous studies have associated extended lifespan with reduced fecundity. The current study did not observe a prolongation of lifespan in B. dorsalis, which instead incurred oxidative damage. The findings of the present study improve our understanding of the molecular, biochemical, and phenotypic response of B. dorsalis to an SD diet.

Abstract In this study, we investigated the effects of dietary restriction (DR) and variable diets on phenotypes and gene expression in oriental fruit fly, Bactrocera dorsalis (Hendel), one of the most economically important pests in the family Tephritidae around the world. As expected, we found that DR altered the B. dorsalis phenotypes by significantly increasing stress resistance and lifespan, but reduced egg production when compared with the control diet. The results suggested a trade-off between reproduction versus somatic maintenance (stress resistance) and lifespan in B. dorsalis. Diet also had a significant effect on hatchability, and DR could increase the egg hatching success of B. dorsalis. Furthermore, DR up-regulated metabolic pathways involved in energy homeostasis and down-regulated pathways in egg production, which might mediate trade-offs between somatic maintenance and reproduction under DR regimes. The gene expression profiles in response to the acute dietary switches indicated that the digestive and metabolic pathways maybe involved in the adaptability of flies to variable dietary resources. In summary, the research facilitates a better understanding of the molecular mechanisms responsible for the B. dorsalis’ phenotypic adjustments to the different qualities of the available diets.

Pesticide resistance is a serious problem that poses a major challenge to pest control. One of the most potent resistance mechanisms is the overexpression of genes coding for detoxification enzymes. The expression of detoxification genes is regulated by a series of transcription factors. Previous studies have revealed that the increased expression of detoxification genes contributes to the insecticide tolerance of Bactrocera dorsalis. Our objective was thus to identify the transcription factors involved in this process. Temporal expression profiles showed that the transcription factor MafB and detoxification genes were expressed highly in the fat body. Further analysis showed that the expression of MafB, GSTz2, and CYP473A3 was induced by abamectin. Disruption of the MafB transcription factor through RNA interference decreased the transcript levels of GSTz2 and CYP473A3 and increased the susceptibility to abamectin significantly. Direct silencing of the expression of GSTz2 also increased susceptibility to abamectin, while CYP473A3 did not. In conclusion, these results suggest that the expression of GSTz2 and CYP473A3 was regulated by the transcription factor MafB, and the up-regulation of GSTz2 via MafB decreased the susceptibility of B. dorsalis to abamectin.

Ecdysis triggering hormone (ETH), released by the Inka cells, is a master hormone in regulating the ecdysis process in insect. Here we investigated the presence and role of the ETH signaling in the female adult of the oriental fruit fly, Bactrocera dorsalis (Hendel) that is one of the most important invasive pest insects in agriculture worldwide. In the female adult, ETH was confirmed in the Inka cells at the tracheae by immunostaining and also in vitro exposure to ETH stimulated the isolated corpora allata of adult in activity. Then we prepared cDNA of females at 0, 5, 10, 15, and 20 days after adult eclosion, and RT-qPCR showed that the expression pattern of ETH and its receptor ETHR-B started from a peak at the day of adult eclosion (day 0), then dropped to basal levels and increased again between day 10 and 15 which is also the period corresponding to ovary growth. In contrast, ETHR-A was absent with Ct values of >33. The expression patterns of the ecdysteroid-producing Halloween genes Spook and Shade, and the vitellogenin genes Vg1, Vg2, and Vg3 co-occurred with peak levels at days 10–15, and also juvenile hormone acid methyltransferase (JHAMT) showed increased levels on day 15. Further in RNAi assays to better understand the role of ETH and ETHR, dsRNA was injected to adult and this led to a respective decrease in expression of 62 and 56% for ETH and ETHR-B, while ETHR-A stayed absent with Ct values of 33. In these RNAi-females, there was an apparently decreased expression for JHAMT and Vg2, together with a significant decrease of the JH titer and egg production. Injection of the JH mimetic methoprene could rescue Vg2 expression and egg production. Upstream, in dsETH/dsETHR-injected females, 20-hydroxyecdysone (20E) injection rescued the transcriptions of ETH and ETHR and also egg production. In summary, our results shed more light on the pivotal role that the ETH peptide hormone and its receptor ETHR-B play an essential role in the reproduction of the female adult of B. dorsalis, via the regulation of JH and vitellogenin, which are controlled by a pulse of 20E.

The pea aphid, Acyrthosiphon pisum, is an important agricultural pest and biological model organism, and RNA interference (RNAi) is an important tool for functional genomics and for insect pest management. However, the efficiency of RNAi in pea aphids is variable, limiting its application in aphids. In this study, we present optimized conditions for inducing and increasing the gene silencing efficiency of RNAi in pea aphids. The optimal gene silencing of the target Aphunchback gene was achieved by injecting 600 ng double-stranded (ds) RNA, and the highest mRNA depletion rate (74%) was detected at 36 h after injection. Moreover, the same gene silencing conditions were used to achieve transcript silencing for nine different genes in the pea aphid, although the silencing efficiencies for the different genes varied. Furthermore, the pre-exposure of aphids to dsRNA (600 ng dsGFP) led to significant hunchback silencing following a secondary exposure to 60 ng of dshunchback, a dose which did not lead to gene silencing when independently injected. The information presented here can be exploited to develop more efficient RNAi bioassays for pea aphids, both as gene functional study tools and an insect pest control strategy.

The ATP-binding cassette (ABC) transporter is a protein superfamily that transports specific substrate molecules across lipid membranes in all living species. In insects, ABC transporter is one of the major transmembrane protein families involved in the development of xenobiotic resistance. Here, we report 49 ABC transporter genes divided into eight subfamilies (ABCA-ABCH), including seven ABCAs, seven ABCBs, 10 ABCCs, two ABCDs, one ABCE, three ABCFs, 16 ABCGs, and three ABCHs according to phylogenetic analysis in Zeugodacus cucurbitae, a highly destructive insect pest of cucurbitaceous and other related crops. The expressions level of 49 ABC transporters throughout various developmental stages and within different tissues were evaluated by quantitative transcriptomic analysis, and their expressions in response to three different insecticides were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). These ABC transporter genes were widely expressed at developmental stages but most highly expressed in tissues of the midgut, fat body and Malpighian tube. When challenged by exposure to three insecticides, abamectin, β-cypermethrin, and dinotefuran, the expressions of ZcABCB7 and ZcABCC2 were significantly up-regulated. ZcABCB1, ZcABCB6, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, and ZcABCC7 were significantly up-regulated in the fat body at 24 h after β-cypermethrin exposure. These data suggest that ZcABCB7 and ZcABCC2 might play key roles in xenobiotic metabolism in Z. cucurbitae. Collectively, these data provide a foundation for further analysis of ABCs in Z. cucurbitae.

The oriental fruit fly (Bactrocera dorsalis) is a pest that causes large economic losses in the fruit and vegetable industry, so its control is a major challenge. Nuclear receptors (NRs) are a superfamily of ligand-dependent transcription factors that directly combine with DNA to regulate the expression of downstream target genes. NRs are closely associated with multiple physiological processes such as metabolism, reproduction, and development. Through sequence searches and analysis, we identified 21 B. dorsalis NR genes, all of which contained at least one of the two characteristic binding domains. On the basis of the conserved sequences and phylogenetic relationships, we divided the 21 NR genes into seven subfamilies. All members of the NR0 subfamily and BdHR83, which belonged to the NR2E group, lacked ligand-binding domains. The BdDSF and BdHR51, which also belonged to the NR2Egroup, and BdE78 (which belonged to the NR1E group) all lacked DNA-binding domains. The BdDSF and BdHR83 sequences were incomplete, and were not successfully amplified. Development- and tissue-specific expression profiling demonstrated that the transcript levels of the 19 NR genes varied considerably among eggs, larva, pupae, and adults, as well as among larval and adult male and female tissues. Our results will contribute to a better understanding of NR evolution and expand our knowledge of B. dorsalis physiology.

The pea aphid is an important pest of vegetables and causes serious losses worldwide. RNA interference (RNAi) is an effective pest control tool, and three sub-pathways have been described: The miRNA pathway, siRNA pathway, and piRNA pathway. A large number of genes in miRNA pathway and piRNA pathway are found to be expanded. To study the roles of these genes, the expression of 25 core RNAi genes was screened in spatiotemporal samples, artificially synthesized dsRNA and miRNA treated samples. The 25 genes were all expressed during different development stages and in different tissues. In dsRNA-treated samples and miRNA-treated samples, the expressions of genes in these three pathways were induced, especially the expanded genes. This suggests a complex network of RNAi core genes in the three sub-pathways. Treatment of miRNA seems to induce gene expression in a dosage-dependent manner. These results increase our knowledge of the siRNA pathway and related factors from RNAi pathway in aphids and promote the use of RNAi for the control of aphid pests.

NADPH-cytochrome P450 reductase (CPR) plays an essential role in the cytochrome P450 enzyme system, which aids in the metabolism of endogenous and exogenous compounds including the detoxification of insecticides. In this study, the CPR transcript in Aphis (Toxoptera) citricidus (Kirkaldy) was cloned, and the deduced amino acid sequence contained an N-terminal membrane anchor, three conserved binding domains (flavin mononucleotide, flavin adeninedinucleotide, and nicotinamide adenine dinucleotide phosphate), a flavin adeninedinucleotide-binding motif, and catalytic residues. Based on phylogenetic analysis, AcCPR was grouped in the hemipteran branch. AcCPR was ubiquitously expressed at all developmental stages and was most abundant in the adults and least abundant in third instar nymphs. Compared with other tested tissues of adults, the expression level of AcCPR was significantly high in the gut. Feeding double-stranded RNA of AcCPR reduced the AcCPR mRNA level and the activity of AcCPR in aphids, and the treated insects exhibited higher susceptibility to abamectin than the control group. Furthermore, the heterologous overexpression of AcCPR in Sf9 cells resulted in a greater viability than control cells when treated with abamectin. All results demonstrated that AcCPR may contribute to the resistance of A.citricidus to abamectin, and CPR may be a potential target for novel insecticide design or a new factor in the development of insecticide resistance.

Corazonin (Crz) is a widely distributed neuropeptide (or neurohormone) in insects with diverse physiological functions. The present study aimed to reveal the functions of Crz and its receptor (CrzR) in the regulation of sexual behavior and fertility in male Bactrocera dorsalis. Tissue-specific expression analyses showed that the BdCrz transcript was most abundant in the central nervous system (CNS), and the BdCrzR transcript was most abundant in both the fat body and CNS. Immunochemical localization confirmed that three pairs of Crz-immunoreactive neurons are located in the dorsolateral protocerebrum region of male adult brain. Importantly, RNAi-mediated Crz knockdown lengthened mating duration in males, and knockdown of Crz or CrzR strongly decreased male fertility in the following 3 days, while the courtship behavior and mating efficiency were not affected. The reduced number of sperm in the reproductive organs of mated females indicated that Crz knockdown in males reduced sperm transfer. The findings of this study indicate that Crz contributes to the reproductive physiology of the oriental fruit fly B. dorsalis by regulating sperm transfer in male adults.

Juvenile hormone (JH) prevents metamorphosis during insect larval stages and promotes adult reproductive processes. Krüppel-homolog 1 (Kr-h1), a zinc finger transcription factor assumed to be induced by JH via the JH receptor methoprene-tolerant (Met), mediates the antimetamorphic effect of JH in insects, but its function in JH-mediated reproductive processes has not been fully explored. In this study, Met and Kr-h1 involved in the JH signaling pathway were first cloned and identified from the oriental fruit fly, Bactrocera dorsalis, an important pest infesting fruit and vegetables worldwide. Subsequent spatiotemporal expression analysis revealed that Met and Kr-h1 were both highly expressed in 7-day-old adults and fat body of female adults, respectively. Treatment with a JH analog (methoprene) significantly induced the expression of JH signaling and vitellogenin (Vg) genes and accelerated ovary development. RNA interference (RNAi) further revealed that either Met or Kr-h1 depletion at the adult stage of B. dorsalis impeded ovary development, with significantly lower egg production noted as well. In addition, rescue through methoprene application after RNAi stimulated the expression of JH signaling and Vg genes. Although there were still differences in ovary phenotype between rescued insects and the pre-RNAi control, ovary redevelopment with a larger surface area was observed, consistent with the spatiotemporal expression and phenotypes recorded in the original methoprene experiment. Our data reveal the involvement of Met and Kr-h1 in insect vitellogenesis and egg production, thus indicating the crucial role of the JH signaling pathway in insect reproduction.

The citrus red mite, Panonychus citri, a major citrus pest distributed worldwide, has evolved severe resistance to various classes of chemical acaricides/insecticides including pyrethroids. It is well known that the resistance to pyrethroids is mainly caused by point mutations of voltage-gated sodium channel gene in a wide range of pests. However, increasing number of evidences support that pyrethroids resistance might also be resulted from the integrated mechanisms including metabolic mechanisms. In this study, firstly, comparative analysis of RNA-seq data showed that multiple detoxification genes, including a GSTs gene PcGSTd1, were up-regulated in a fenpropathrin-resistant population compared with the susceptible strain (SS). Quantitative real time-PCR results showed that the exposure of fenpropathrin had an induction effect on the transcription of PcGSTd1 in a time-dependent manner. In vitro inhibition and metabolic assay of recombinant PcGSTd1 found that fenpropathrin might not be metabolized directly by this protein. However, its antioxidant role in alleviating the oxidative stress caused by fenpropathrin was demonstrated via the reversely genetic experiment. Our results provide a list of candidate genes which may contribute to a multiple metabolic mechanisms implicated in the evolution of fenpropathrin resistance in the field population of P. citri. Furthermore, during the detoxification process, PcGSTd1 plays an antioxidant role by detoxifying lipid peroxidation products induced by fenpropathrin.

Natalisins (NTLs) are conservative neuropeptides, which are only found in arthropods and are documented to regulate reproductive behaviors in insects. In our previous study, we have confirmed that NTLs regulate the reproductive process in an important agricultural pest, Bactrocera dorsalis (Hendel). Hence, in this study, to further confirm the in vivo function of NTL receptor (NTLR) and assess the potential of NTLR as an insecticide target, RNA interference targeting NTLR mRNA was performed. We found that mating frequencies of both males and females were reduced by RNAi-mediated knockdown of the NTLR transcript, while there was no effect on mating duration. Moreover, we functionally expressed the B. dorsalis NTLR in Chinese Hamster Ovary (CHO) cells and was co-transfected with an aequorin reporter to measure ligand activities. A total of 13 biostable multi-Aib analogs were tested for agonistic and antagonistic activities. While most of these NTL analogs did not show strong activity, one analog (NLFQV[Aib]DPFF[Aib]TRamide) had moderate antagonistic activity. Taken together, we provided evidence for the important roles of NTLR in regulating mating frequencies of both male and female in this fly and also provided in vitro data on mimetic analogs that serve as leading structures for the development of agonists and antagonists to disrupt the NTL signaling pathway.

The vitellogenin receptor (VgR) functions as an essential component in uptaking and transporting vitellogenin (Vg) in female adults, which is involved in ovary development and oviposition. This study aimed to clarify the molecular characteristics and function of VgR in the oriental fruit fly Bactrocera dorsalis (Hendel). Here, we identified the full-length of BdVgR (GenBank Accession No. JX469118), encoding a 1925 residue (aa) protein with a 214.72 kDa molecular mass and several typical motifs of low-density lipoprotein receptor superfamily (LDLR). Phylogenic analysis suggested that BdVgR was evolutionary conserved with other Dipteran VgRs. The expression of BdVgR was exclusively detected in the ovaries rather than head, thorax or other tissues. The developmental expression patterns showed that the signal of BdVgR was detectable in very beginning of adult stage, and positively correlated with the growth rate of ovaries and the expression levels of its ligands. In addition, we also demonstrated that the expression level of BdVgR, and ovary development were significantly suppressed after being injected with BdVgR-targeted dsRNA. Together, all of these results indicated that BdVgR was critical for yolk protein absorption and ovary maturation in B. dorsalis, playing a vital role in female reproduction.

Ryanodine receptors (RyRs) play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR), an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif) and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action.

The production and uptake of yolk protein play an important role in the reproduction of all oviparous organisms. Vitellogenin (Vg) is the precursor of vitellin (Vn), which is the major egg storage protein, and vitellogenin receptor (VgR) is a necessary protein for the uptake of Vg into developing oocytes. In this paper, we characterize the full-length Vg and VgR, PcVg1 and PcVgR, respectively, of the citrus red mite Panonychus citri (McGregor). The PcVg1 cDNA is 5748 nucleotides (nt) with a 5553-nt open reading frame (ORF) coding for 1851 amino acids (aa), and the PcVgR is 6090 nt, containing an intact ORF of 5673 nt coding an expected protein of 1891 aa. The PcVg1 aa sequence shows a typical GLCG domain and several K/RXXR cleavage sites, and PcVgR comprises two ligand-binding domains, two epidermal growth factor (EGF)-like regions containing YWTD motifs, a transmembrane domain, and a cytoplasmic domain. An analysis of the aa sequences and phylogenetics implied that both genes were genetically distinct from those of ticks and insects. The transcriptional profiles determined by real-time quantitative PCR in different developmental stages showed that both genes present the same expressional tendencies in eggs, larvae, nymphs, and adults. This suggested that the biosynthesis and uptake of PcVg occurs coordinately. The strong reproductive capacity of P. citri has been hypothesized as an important factor in its resistance; consequently, understanding the molecular mechanisms regulating Vg and VgR are fundamental for mite control.

Insects must undergo ecdysis for successful development and growth, and the ecdysis triggering hormone (ETH), released by the Inka cells, is a master hormone in this process. In this study, we determined the sequence of the ETH precursor and receptors in an agriculturally important pest insect, the oriental fruit fly Bactrocera dorsalis (Hendel). We identified two functionally distinct splice receptor isoforms: BdETH-R-A and BdETH-R-B, and when expressed in Chinese hamster ovary (CHO-WTA11) cells, they exhibited a high sensitivity to the two mature peptides BdETH1 and BdETH2. The BdETH transcript was detected in the tracheal tissue of the larvae. Inka cells were identified with immunohistochemical antibody staining against Drosophila melanogaster ETH1, and in situ hybridization with specific DNA probes. Selective RNA silencing of BdETH or BdETH-R-A, but not of BdETH-R-B, caused developmental failure at ecdysis. The dsRNA-treated larvae displayed tracheal defects and could not shed the old cuticle followed by death. Our results demonstrated that BdETH, via activation of BdETH-R-A but not ETH-R-B, plays an essential role in regulating the process of larva-larva ecdysis in B. dorsalis.

The oriental fruit fly Bactrocera dorsalis (Hendel) causes devastating losses to agricultural crops world-wide and is considered to be an economically important pest. Little is known about the digestive enzymes such as serine proteases (SPs) in B. dorsalis, which are important both for energy supply and mitigation of fitness cost associated with insecticide tolerance. In this study, we identified five SP genes in the midgut of B. dorsalis, and the alignments of their deduced amino acid sequences revealed the presence of motifs conserved in the SP superfamily. Phylogenetic analyses with known SPs from other insect species suggested that three of them were trypsin-like proteases. Analyses of the expression profiles among the different developmental stages showed that all five genes were most abundant in larvae than in other stages. When larvae were continuously fed on diet containing 0.33 μg/g β-Cypermethrin, expression of all five genes were upregulated in the midgut but the larval development was delayed. Biochemical assays were consistent with the increased protease activity exhibited by SPs in the midgut after treatment with β-Cypermethrin. Taken together, these findings provide evidence for the hypothesis that enhanced SP activity may play an indirect role in relieving the toxicity stress of insecticide in B. dorsalis.

Chitin synthase synthesizes chitin, which is critical for the arthropod exoskeleton. In this study, we cloned the cDNA sequences of a chitin synthase 1 gene, PcCHS1, in the citrus red mite, Panonychus citri (McGregor), which is one of the most economically important pests of citrus worldwide. The full-length cDNA of PcCHS1 contains an open reading frame of 4605 bp of nucleotides, which encodes a protein of 1535 amino acid residues with a predicted molecular mass of 175.0 kDa. A phylogenetic analysis showed that PcCHS1 was most closely related to CHS1 from Tetranychus urticae. During P. citri development, PcCHS1 was constantly expressed in all stages but highly expressed in the egg stage (114.8-fold higher than in the adult). When larvae were exposed to diflubenzuron (DFB) for 6 h, the mite had a significantly high mortality rate, and the mRNA expression levels of PcCHS1 were significantly enhanced. These results indicate a promising use of DFB to control P. citri, by possibly acting as an inhibitor in chitin synthesis as indicated by the up-regulation of PcCHS1 after exposure to DFB.

Intrapuparial development is a special pattern of metamorphosis in cyclorrhaphous flies, in which the pupa forms in an opaque, barrel-like puparium. This has been well studied in forensic insects for age estimations. In this study, the intrapuparial development of a quarantine agricultural pest, Bactrocera dorsalis (Hendel), was studied under a constant temperature of 27 ± 1 °C and 70 ± 5% relative humidity. Results showed that intrapuparial development could be divided into five stages: Larval-pupal apolysis, cryptocephalic pupa, phanerocephalic pupa, pharate adult, and emergent adult. It lays a morphology-based foundation for molecular mechanism studies and enhances the understanding of the physiological basis for changes in intrapuparial development. More importantly, the chronology of intrapuparial development can be used to predict the emergence time of tephritid flies, indicating when to spray insecticides to control these phytophagous agricultural pests. This may be an effective approach to reduce the use of insecticides and slow down the evolution of insecticidal resistance.

Glutamine synthetase (GS) is a key enzyme in glutamine synthesis and is associated with multiple physiological processes in insects, such as embryonic development, heat shock response, and fecundity regulation. However, little is known about the influence of GS on female fecundity in the oriental fruit fly, Bactrocera dorsalis. Based on the cloning of BdGSs, mitochondrial BdGSm and cytoplasmic BdGSc, we determined their expressions in the tissues of adult B. dorsalis. BdGSm was highly expressed in the fat body, while BdGSc was highly expressed in the head and midgut. Gene silencing by RNA interference against two BdGSs isoforms suppressed target gene expression at the transcriptional level, leading to a reduced ovarian size and lower egg production. The specific inhibitor L-methionine S-sulfoximine suppressed enzyme activity, but only the gene expression of BdGSm was suppressed. A similar phenotype of delayed ovarian development occurred in the inhibitor bioassay. Significantly lower expression of vitellogenin and vitellogenin receptor was observed when GS enzyme activity was suppressed. These data illustrate the effects of two GS genes on adult fecundity by regulating vitellogenin synthesis in different ways.

Peptidoglycans (PGNs) are major bacterial components recognized by the immune systems of insects and mammals. PGN recognition proteins (PGRPs) are widely distributed and highly conserved in vertebrates and invertebrates. PGRPs are a family of pattern recognition receptors that recognize peptidoglycan and regulate immune responses. In this study, we cloned two PGRP genes (BdPGRP-SA and BdPGRP-SD) from Bactrocera dorsalis (Hendel), which encode 192 and 196 amino acid residues, respectively. Both genes were highly expressed in adults, especially in the fat body and midgut. These two genes were up-regulated when challenged by the immune triggers, PGN-EB (Escherichia coli O111:B4) and PGN-SA (Staphylococcus aureus). The suppression of transcriptional expression of either gene by RNA interference (RNAi) resulted in increased sensitivities to Gram-negative E. coli and Gram-positive S. aureus PGNs. Suppression of BdPGRP-SA and -SD expression by RNAi resulted in weak expressions of four antimicrobial peptides (AMPs) upon injected with E. coli or S. aureus. BdPGRP-SA and -SD are involved in recognizing both Gram-negative and Gram-positive bacteria independently to activate the downstream AMP’s response to bacterial infection.

The citrus red mite, Panonychus citri (McGregor), is a global citrus pest, and has developed severe resistance to several types of acaricides. However, the molecular mechanisms of resistance in this mite remain unknown. In this study, seven full-length cDNAs encoding glutathione S-transferases (GSTs) genes were identified and characterized in P. citri. The effects of pyridaben and fenpropathrin exposure on the expression of these genes were also investigated. Phylogenetic analysis revealed that the seven GSTs genes in P. citri cloned in this study belong to three different cytosolic classes, including four in mu, two in delta and one in zeta. Among these seven GSTs genes, the relative expression level of PcGSTm1 was significantly higher in adult than in the other life stages (egg, larvae and nymph). Compared with the control, the mRNA levels of the seven GST genes did not change significantly following exposure to pyridaben at LC10. However, RT-qPCR results showed that, when exposed to LC10 of fenpropathrin, six GSTs gene (PcGSTm1, PcGSTm3, PcGSTm4, PcGSTd1, PcGSTd2 and PcGSTz1) transcripts increased in a time-dependent manner. This is the first insight into the molecular characteristics of GSTs gene cDNAs in P. citri. The elevated GSTs gene transcripts following exposure to fenpropathrin might be one of the mechanisms involved in detoxification of this acaricide.

Insect chitinases are hydrolytic enzymes that are required for the degradation of glycosidic bonds of chitin. In this study, we identified and characterized a full-length cDNA of the chitinase gene (BdCht2) in the oriental fruit fly, Bactrocera dorsalis. The cDNA contains an open reading frame (ORF) of 1449 bp that encodes 483 amino acid residues and 126- and 296-bp non-coding regions at the 5'- and 3'-ends, respectively. The BdCht2 genome has four exons and three introns. The predicted molecular mass of the deduced BdCht2 is approximately 54.3 kDa, with an isoelectric point of 5.97. The 977 bp 5' flanking region was identified and the transcription factor binding sites were predicted. Bioinformatic analyses showed that the deduced amino acid sequence of BdCht2 had 34%–66% identity to that of chitinases identified in other insect species. Quantitative real-time PCR (qPCR) analyses indicated that BdCht2 was mainly expressed during the larval-pupal and pupal-adult transitions. The tissue-specific expression showed that the highest expression was in the integument, followed by the fat body and other tissues. Moreover, the expression of BdCht2 was upregulated significantly upon 20-hydroxyecdysone (20E) at different dose injections after 8 h compared to that of the control. Starvation also increased the expression of BdCht2 in the third-instar larvae and was suppressed again by re-feeding the insects. These results suggest that BdCht2 plays an important role in the molting process of B. dorsalis larvae and can be regulated by 20E.

Methiopyrisulfuron is a novel sulfonylurea herbicide with good activity for annual broadleaf and gramineal weeds control. Present study was to investigate the effects of organic amendments (including peat (PE), sewage sludge (SS), and humic acid (HA)) on adsorption, desorption and leaching of methiopyrisulfuron in soils. The batch equilibration technique was applied for adsorption-desorption experiments and the leaching was tested through soil column simulated experiments under laboratory conditions. The Freundlich model may well describe adsorption-desorption of methiopyrisulfuron on organic amendments, the natural soil, and amended soils. Organic amendments could not only greatly increase the adsorption capacity of methiopyrisulfuron, but also significantly enhance the hysteresis of desorption of methiopyrisulfuron. The correlations between Kf-ads and organic matter content of amended soils were significant, and the correlations between H and soil organic matter in amended soils with PE, SS, and HA were significant too. The results of soil column experiments indicated that organic amendments greatly decreased leaching of methiopyrisulfuron. This study suggested that PE, SS, and HA could greatly influence environmental behavior of methiopyrisulfuron in soils. Use of organic amendments might be an effective management practice for controlling potential pollution of methiopyrisulfuron to environment.

Chitin synthase (CHS), a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2) was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis.

The oriental fruit fly (Bactrocera dorsalis) is a species of tephritid fruit fly, endemic to Southeast Asia but also introduced to many regions of the US, and it is one of the major pest species with a broad host range of cultivated and wild fruits. Although males of B. dorsalis respond strongly to methyl eugenol and this is used for monitoring and estimating populations, the molecular mechanism of the oriental fruit fly olfaction has not been elucidated yet. Therefore, in this project, using next generation sequencing technologies, we sequenced the transcriptome of the antennae of male and female adults of B. dorsalis. We identified a total of 20 candidate odorant binding proteins (OBPs), 5 candidate chemosensory proteins (CSPs), 35 candidate odorant receptors (ORs), 12 candidate ionotropic receptors (IRs) and 4 candidate sensory neuron membrane proteins (SNMPs). The sex-specific expression of these genes was determined and a subset of 9 OR genes was further characterized by qPCR with male and female antenna, head, thorax, abdomen, leg and wing samples. In the male antennae, 595 genes showed a higher expression, while 128 genes demonstrated a higher expression in the female antennae. Interestingly, 2 ORs (BdorOR13 and BdorOR14) were highly and specifically expressed in the antennae of males, and 4 ORs (BdorOR13, BdorOR16, BdorOR18 and BdorOR35) clustered with DmOR677, suggesting pheromone reception. We believe this study with these antennae-enriched OBPs, CSPs, ORs, IRs and SNMPs can play an important role in the detection of pheromones and general odorants, and so in turn our data improve our current understanding of insect olfaction at the molecular level and provide important information for disrupting the behavior of the oriental fruit fly using chemical communication methods.

Biogenic amines perform many kinds of important physiological functions in the central nervous system (CNS) of insects, acting as neuromodulators, neurotransmitters, and neurohormones. The five most abundant types of biogenic amines in invertebrates are dopamine, histamine, serotonin, tyramine, and octopamine (OA). However, in beetles, an important group of model and pest insects, the role of tyramine β-hydroxylase (TβH) in the OA biosynthesis pathway and the regulation of behavior remains unknown so far. We therefore investigated the molecular characterization and spatiotemporal expression profiles of TβH in red flour beetles (Triboliun castaneum). Most importantly, we detected the production of OA and measured the crawling speed of beetles after dsTcTβH injection. We concluded that TcTβH controls the biosynthesis amount of OA in the CNS, and this in turn modulates the mobility of the beetles. Our new results provided basic information about the key genes in the OA biosynthesis pathway of the beetles, and expanded our knowledge on the physiological functions of OA in insects.

Insulin receptors play key roles in growth, development, and polymorphism in insects. Here, we report two insulin receptor genes (AcInR1 and AcInR2) from the brown citrus aphid, Aphis (Toxoptera) citricidus. Transcriptional analyses showed that AcInR1 increased during the nymph–adult transition in alate aphids, while AcInR2 had the highest expression level in second instar nymphs. AcInR1 is important in aphid development from fourth instar nymphs to adults as verified by dsRNA feeding mediated RNAi. The silencing of AcInR1 or/and AcInR2 produced a variety of phenotypes including adults with normal wings, malformed wings, under-developed wings, and aphids failing to develop beyond the nymphal stages. Silencing of AcInR1 or AcInR2 alone, and co-silencing of both genes, resulted in 73% or 60%, and 87% of aphids with problems in the transition from nymph to normal adult. The co-silencing of AcInR1 and AcInR2 resulted in 62% dead nymphs, but no mortality occurred by silencing of AcInR1 or AcInR2 alone. Phenotypes of adults in the dsInR1 and dsInR2 were similar. The results demonstrate that AcInR1 and AcInR2 are essential for successful nymph–adult transition in alate aphids and show that RNAi methods may be useful for the management of this pest.