Proteomic and transcriptomic analyses of saliva and salivary glands from the Asian citrus psyllid, Diaphorina citri.

Salivary secretions play critical roles in interactions among insects, insect-vectored pathogens, and host plants. The Asian citrus psyllid Diaphorina citri is a sap-sucking Hemipteran that serves as a vector for Candidatus Liberibacter asiaticus, the causal agent of citrus greening disease ("Huanglongbing" or HLB). D. citri continuously injects saliva into host plants using specialized stylets so as to feed and transmit the HLB pathogen. Knowledge on the composition and function of salivary proteins of this pest is very limited. In this study, proteomic and transcriptomic approaches were adopted to characterize the protein composition of the saliva and salivary glands in D. citri. A total of 246 and 483 proteins were identified in saliva and dissected salivary glands, respectively, via LC-MS/MS analyses. Comparative analyses of the identified proteins were performed between D. citri and other reported Hemipteran insect species. Transcription levels of the genes coding for the identified proteins were determined via RNA-sequencing among different tissues including salivary glands and other digestive tissues. Identification of putative effectors that are expressed exclusively or abundantly in salivary glands provides the foundation for future functional studies towards the understanding of their roles in interactions among D. citri, HLB pathogen, and their citrus host. BIOLOGICAL SIGNIFICANCE: This is a systematic analysis on proteins in saliva and dissected salivary glands. A high percentage of novel proteins have been identified due to the large amounts of samples collected. This report gives a more comprehensive repertoire of potential effector proteins that may be possibly involved in modulating host defense, altering nutrient metabolism, and facilitating Ca. L. asiaticus transmission.

Analyses of chemosensory genes provide insight into the evolution of behavioral differences to phytochemicals in Bactrocera species.

Insect olfactory systems have evolved to recognize phytochemicals and respond to olfactory-triggered cues that vary depending on needs. Several Bactrocera species are attracted explicitly to particular phytochemicals, including methyl eugenol (ME) and cue lure/raspberry ketone (CL/RK). The attraction of Bactrocerans to ME/RK is primarily driven by olfaction. Therefore, the divergent behavioral phenotypes are due to the differences in genes expressed in antennae. High quality transcriptomes were generated with mRNA from dissected antennae, to analyze the differences in olfaction-related genes of three ME-responders B. dorsalis, B. papayae and B. correcta and two RK-responders B. cucurbitae and B. tau. Many distinct quantitative and qualitative differences were identified in their respective chemosensory repertoires. Tissue- and sex-specific expression analyses identified antennae-predominant and sex-biased chemosensory genes. Sequence comparison revealed variations among family members of odorant-binding proteins and odorant-receptors between ME-responders and RK-responders. Differences in composition, expression levels, and sequence of proteins encoded by olfactory-related genes were identified between ME-responders and RK-responders. Some of the differences might contribute to the divergence in response to plant-derived odorants. Taken together, our results provide insights into the evolution of an olfactory system at molecular level in Bactrocera ME- and CL/RK-responding species.

Distinct microbial communities among different tissues of citrus tree Citrus reticulata cv. Chachiensis.

Plant microbiota colonize all organs of a plant and play crucial roles including supplying nutrients to plants, stimulating seed germination, promoting plant growth, and defending plants against biotic and abiotic stress. Because of the economic importance, interactions between citrus and microbes have been studied relatively extensively, especially citrus-pathogen interactions. However, the spatial distribution of microbial taxa in citrus trees remains under-studied. In this study, Citrus reticulata cv. Chachiensis was examined for the spatial distribution of microbes by sequencing 16S rRNA genes. More than 2.5 million sequences were obtained from 60 samples collected from soil, roots, leaves, and phloem. The dominant microbial phyla from all samples were Proteobacteria, Actinobacteria and Acidobacteria. The composition and structure of microbial communities in different samples were analyzed by PCoA, CAP, Anosim and MRPP methods. Variation in microbial species between samples were analyzed and the indicator microbes of each sample group were identified. Our results suggested that the microbial communities from different tissues varied significantly and the microenvironments of tree tissues could affect the composition of its microbial community.

Comparative transcriptomic analyses revealed genes and pathways responsive to heat stress in Diaphorina citri.

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a serious pest that feeds on plant phloem sap of citrus trees, and transmits Candidatus Liberibacter asiaticus, a bacterium that induces the destructive disease called Huanglongbing. Increasing evidence suggests that high temperatures could affect various biological traits, including size, longevity, mortality, behavior and metabolism of D. citri. However, the relevant mechanisms of heat stress remain unclear. In this study, a large set of transcriptomic data derived from D. citri adults were generated and differentially expressed genes (DEGs) after heat stress were identified by RNA sequencing. A total of 118, 399 unigenes were obtained, from which 37, 665 were mapped to sequences from at least one database. Seven hundreds and twenty-two unigenes were affected by high temperature of 40 °C for 4 h, in which 486 up-regulated and 236 down-regulated, and part of heat shock proteins, antioxidant and detoxification genes and cathepsins were identified as the DEGs. KEGG pathway enrichment analysis demonstrated that part of genes involved in protein synthesis and processing, metabolism, immunity, and signal transduction were differentially expressed under heat stress. Furthermore, the mRNA expression levels of 20 DEGs were confirmed by qRT-PCR, which verified the accuracy of high-throughput sequencing. Our results revealed that the response of D. citri adults to high temperatures is associated with a range of changes involved in various physiological and biochemical processes. Our data provide a basis for future research to improve our understanding on the molecular mechanism for heat responses in D. citri adults.

Identification and expression analysis of odorant binding proteins and chemosensory proteins from dissected antennae and mouthparts of the rice bug Leptocorisa acuta.

The rice bug, Leptocorisa acuta (Tunberg) (Hemiptera: Alydidae), is a notorious pest in Asia, and it is significantly attracted by the volatiles derived from host plants. However, it remains unknown how L. acuta recognizes host volatile compounds at the molecular level. Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are thought to be responsible for the initial biochemical recognition during olfactory perception. Here, we followed the RNA Sequencing (RNA-Seq) approach to identify candidate genes encoding OBPs and CSPs from dissected antennae and mouthparts of L. acuta. In total, 26 unigenes were identified coding for OBPs (22 Classic OBPs and four Plus-C OBPs), and 17 unigenes coding for CSPs. Real-time quantitative PCR (RT-qPCR) revealed that 11 OBPs (LacuOBP1, 5, 6, 7, 9, 11, 13, 14, 17, 20 and 23) and nine CSPs (LacuCSP2, 3, 4, 5, 6, 8, 9, 10 and 12) were predominantly expressed in antennae, indicating that they might be essential for detection of general odorants and pheromones. Among these antennae-predominantly expressed genes, LacuOBP11 and LacuOBP13 showed male-biased expression and therefore may play crucial roles in the detection of sex pheromones. Seven LacuOBPs (LacuOBP4, 8, 10, 12, 21, 25 and 26) and two CSPs (LacuCSP7 and LacuOBP11) were predominantly expressed in mouthparts, suggesting that these genes might be involved in taste perception. Our work provides a starting point to facilitate functional study of these OBPs and CSPs in L. acuta at the molecular level in the future.

Candidates for chemosensory genes identified in the Chinese citrus fly, Bactrocera minax, through a transcriptomic analysis.

BACKGROUND: The males of many Bactrocera species (Diptera: Tephritidae) respond strongly to plant-derived chemicals (male lures) and can be divided into cue lure/raspberry ketone (CL/RK) responders, methyl eugenol (ME) responders and non-responders. Representing a non-responders, Bactrocera minax display unique olfactory sensory characteristics compared with other Bactrocera species. The chemical senses of insects mediate behaviors that are associated with survival and reproduction. Here, we report the generation of transcriptomes from antennae and the rectal glands of both male and female adults of B. minax using Illumina sequencing technology, and annotated gene families potentially responsible for chemosensory. RESULTS: We developed four transcriptomes from different tissues of B. minax and identified a set of candidate genes potentially responsible for chemosensory by analyzing the transcriptomic data. The candidates included 40 unigenes coding for odorant receptors (ORs), 30 for ionotropic receptors (IRs), 17 for gustatory receptors (GRs), three for sensory neuron membrane proteins (SNMPs), 33 for odorant-binding proteins (OBPs), four for chemosensory proteins (CSPs). Sex- and tissue-specific expression profiles for candidate chemosensory genes were analyzed via transcriptomic data analyses, and expression profiles of all ORs and antennal IRs were investigated by real-time quantitative PCR (RT-qPCR). Phylogenetic analyses were also conducted on gene families and paralogs from other insect species together. CONCLUSIONS: A large number of chemosensory genes were identified from transcriptomic data. Identification of these candidate genes and their expression profiles in various tissues provide useful information for future studies towards revealing their function in B. minax.

Candidate genes coding for odorant binding proteins and chemosensory proteins identified from dissected antennae and mouthparts of the southern green stink bug Nezara viridula.

The southern green stink bug Nezara viridula (Hemiptera: Pentatomidae) is a highly polyphagous pest that can significantly impact many major crops worldwide. Insect odorant binding proteins (OBPs) and chemosensory proteins (CSPs) transport chemicals and play critical roles in chemoreception. Studies on N. viridula OBPs and CSPs should increase our overall understandings on chemosensory systems and chemical ecology of stink bugs, which may lead to improved pest control. In this study, we identified candidate genes encoding putative OBPs and CSPs in N. viridula by generating transcriptomes from dissected antennae and mouthparts. In total, the 42 unigenes were identified coding for OBPs (34 Classic OBPs and eight Plus-C OBPs) and 13 unigenes coding for CSPs. Expression profiles of OBP- and CSP -encoding genes were compared between antennae and mouthparts based on FKPM values. Candidates for antenna-predominant OBPs and CSPs were selected for real-time quantitative PCR analyses. Analyses of tissue expression profiles revealed that 17 OBP-encoding genes, and four CSP genes were primarily expressed in antennae, suggesting their putative roles in perception of volatiles. The sex-biased expression patterns of these antenna-predominant genes suggested that they may have important functions in reproduction of the insect. This is a systematic analysis on OBPs and CSPs in a stink bug, providing a comprehensive resource for future functional studies not only for N. viridula, but also for other stink bugs as well.

Candidate genes involved in spiroacetal biosynthesis in the oriental fruit fly, Bactrocera dorsalis.

Spiroacetals are widespread in nature as components of volatile semiochemical secretions from many insect species. The general pathway for spiroacetal biosynthesis in Bactrocera sp. is preliminarily established, but many genes involved in this pathway remain to be characterized. By analyzing transcriptomes of the rectal glands (RGs) from immature and mature females of the oriental fruit fly, Bactrocera dorsalis, a set of genes encoding two acetyl-CoA carboxylases (ACCs), two fatty acid synthases (FASs), eight desaturases (DESs), twelve fatty acyl-CoA reductases (FARs), seventy-two cytochrome P450 enzymes (CYPs), and twenty-three odorant binding proteins (OBPs) were identified. We investigated the expression of candidate genes in immature and mature stages based on the RNA-seq data and Real-time quantitative PCR. Expression profiling revealed that some of these genes were primarily expressed in female rectal glands among different tissues, and were up-regulated in mature females. Semi-quantitative RT-PCR assays were also adapted to examine tissue-specific expression of selected candidate genes. Additionally, their putative functions in spiroacetal synthesis and transportation are proposed. Our study provided large-scale sequence information for further functional studies on spiroacetal biosynthetic pathways.

Antennal transcriptome and expression analyses of olfactory genes in the sweetpotato weevil Cylas formicarius.

The sweetpotato weevil, Cylas formicarius (Fabricius), is a serious pest of sweetpotato. Olfaction-based approaches, such as use of synthetic sex pheromones to monitor populations and the bait-and-kill method to eliminate males, have been applied successfully for population management of C. formicarius. However, the molecular basis of olfaction in C. formicarius remains unknown. In this study, we produced antennal transcriptomes from males and females of C. formicarius using high-throughput sequencing to identify gene families associated with odorant detection. A total of 54 odorant receptors (ORs), 11 gustatory receptors (GRs), 15 ionotropic receptors (IRs), 3 sensory neuron membrane proteins (SNMPs), 33 odorant binding proteins (OBPs), and 12 chemosensory proteins (CSPs) were identified. Tissue-specific expression patterns revealed that all 54 ORs and 11 antennal IRs, one SNMP, and three OBPs were primarily expressed in antennae, suggesting their putative roles in olfaction. Sex-specific expression patterns of these antenna-predominant genes suggest that they have potential functions in sexual behaviors. This study provides a framework for understanding olfaction in coleopterans as well as future strategies for controlling the sweetpotato weevil pest.

Transcriptome sequencing of Tessaratoma papillosa antennae to identify and analyze expression patterns of putative olfaction genes.

Studies on insect olfaction have increased our understanding of insect's chemosensory system and chemical ecology, and have improved pest control strategies based on insect behavior. In this study, we assembled the antennal transcriptomes of the lychee giant stink bug, Tessaratoma papillosa, by using next generation sequencing to identify the major olfaction gene families in this species. In total, 59 odorant receptors, 14 ionotropic receptors (8 antennal IRs), and 33 odorant binding proteins (28 classic OBPs and 5 plus-C OBPs) were identified from the male and female antennal transcriptomes. Analyses of tissue expression profiles revealed that all 59 OR transcripts, 2 of the 8 antennal IRs, and 6 of the 33 OBPs were primarily expressed in the antennae, suggesting their putative role in olfaction. The sex-biased expression patterns of these antenna-predominant genes suggested that they may have important functions in the reproductive behavior of these insects. This is the first report that provides a comprehensive resource to future studies on olfaction in the lychee giant stink bug.

Antennal and abdominal transcriptomes reveal chemosensory gene families in the coconut hispine beetle, Brontispa longissima.

Antennal and abdominal transcriptomes of males and females of the coconut hispine beetle Brontispa longissima were sequenced to identify and compare the expression patterns of genes involved in odorant reception and detection. Representative proteins from the chemosensory gene families likely essential for insect olfaction were identified. These include 48 odorant receptors (ORs), 19 ionotropic receptors (IRs), 4 sensory neuron membrane proteins (SNMPs), 34 odorant binding proteins (OBPs) and 16 chemosensory proteins (CSPs). Phylogenetic analysis revealed the evolutionary relationship of these proteins with homologs from Coleopterans or other insects, and led to the identification of putative aggregation pheromone receptors in B. longissima. Comparative expression analysis performed by calculating FPKM values were also validated using quantitative real time-PCR (qPCR). The results revealed that all ORs and antennal IRs, two IR co-receptors (BlonIR8a and BlonIR25a) and one SNMP (BlonSNMP1a) were predominantly expressed in antennae when compared to abdomens, and approximately half of the OBPs (19) and CSPs (7) were enriched in antennae. These findings for the first time reveal the identification of key molecular components in B. longissima olfaction and provide a valuable resource for future functional analyses of olfaction, and identification of potential targets to control this quarantine pest.