Effects of the entomopathogenic fungus Clonostachys rosea on mortality rates and gene expression profiles in Diaphorina citri adults.

Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a serious pest of citrus. The insect also transmits Candidatus Liberibacter asiaticus, the pathogen of a devastating citrus disease called Huanglongbing. Clonostachys rosea is a versatile fungus that possesses nematicidal and insecticidal activities. The effect of C. rosea against D. citri remains unclear. Here we examined the pathogenicity of C. rosea against D. citri adults. A mortality rate of 46.67% was observed in adults treated with 1 × 108 conidia/mL spore suspension. Comparative transcriptomic analyses identified 259 differentially-expressed genes (DEGs) between controls and samples treated with fungi. Among the DEGs, 183 were up-regulated and 76 down-regulated. Genes with altered expression included those involved in immunity, apoptosis and cuticle formation. Our preliminary observation indicated that C. rosea is virulent against ACP adults and has the potential as a biological control agent for ACP management in the field.

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.

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.

Aligned Li+ Tunnels in Core-Shell Li(NixMnyCoz)O2@LiFePO4 Enhances Its High Voltage Cycling Stability as Li-ion Battery Cathode.

Layered transition-metal oxides (Li[NixMnyCoz]O2, NMC, or NMCxyz) due to their poor stability when cycled at a high operating voltage (>4.5 V) have limited their practical applications in industry. Earlier researches have identified Mn(II)-dissolution and some parasitic reactions between NMC surface and electrolyte, especially when NMC is charged to a high potential, as primarily factors responsible for the fading. In our previous work, we have achieved a capacity of NMC active material close to theoretical value and optimized its cycling performance by a depolarized carbon nanotubes (CNTs) network and an unique "pre-lithiation process" that generates an in situ organic coating (∼40 nm) to prevent Mn(II) dissolution and minimize the parasitic reactions. Unfortunately, this organic coating is not durable enough during a long-term cycling when the cathode operates at a high potential (>4.5 V). This work attempts to improve the surface protection of the NMC532 particles by applying an active inorganic coating consisting of nanosized- and crystal-orientated LiFePO4 (LFP) (about 50 nm, exposed (010) face) to generate a core-shell nanostructure of Li(NixMnyCoz)O2@LiFePO4. Transmission electron microscopy (TEM) and etching X-ray photoelectron spectroscopy have confirmed an intimate contact coating (about 50 nm) between the original structure of NMC and LFP single-particle with atomic interdiffusion at the core-shell interface, and an array of interconnected aligned Li+ tunnels are observed at the interface by cross-sectional high-resolution TEM, which were formed by ball-milling and then strictly controlling the temperature below 100 °C. Batteries based on this modified NMC cathode material show a high reversible capacity when cycled between 3.0 and 4.6 V during a long-term cycling.

Prelithiation Activates Li(Ni0.5Mn0.3Co0.2)O2 for High Capacity and Excellent Cycling Stability.

Transition metal oxide materials Li(NixMnyCoz)O2 (NMC) based on layered structures are expected to replace LiFePO4 in automotive Li-ion batteries because of their higher specific capacity and operating potential. However, the actual usable capacity is much lower than the promised theoretical value [Uchaker, E.; Cao, G. Nano Today 2014, 9, 499-524; Tarascon, J.-M.; Armand, M. Nature 2001, 414, 359-367], in addition to the often poor cycling performance and the first-cycle Coulombic efficiency, for which Mn(II)-dissolution, its immobilization in solid electrolyte interface (SEI), oxidation of electrolytes by Ni, and other parasitic process thereat have been held responsible [Zhan, C., et al. Nat. Commun. 2013, 4, 2437; Wang, L., et al. J. Solid State Electrochem. 2009, 13, 1157-1164; Lin, F., et al. Nat. Commun. 2014, 5, 4529]. Previously, we reported a composite Li(Ni0.5Mn0.3Co0.2)O2 (NMC532) depolarized by the embedded carbon nanotube (CNT) and achieved capacity close to the theoretical limit [Wu, Z., et al. Nano. Lett. 2014, 14, 4700-4706]; unfortunately, this high capacity failed to be maintained in long-term cycling due to the degrading contacts between the active ingredient and CNT network. On the basis of that NMC532/CNT composite, the present work proposes a unique "prelithiation process", which brought the cathode to low potentials before regular cycling and led to an interphase that is normally formed only on anode surfaces. The complete coverage of cathode surface by this ∼40 nm thick interphase effectively prevented Mn(II) dissolution and minimized the side reactions of Ni, Co, and Mn at the NMC interface during the subsequent cycling process. More importantly, such a "prelithiation" process activated a structure containing two Li layers near the surface of NMC532 particles, as verified by XRD and first principle calculation. Hence, a new cathode material of both high capacity with depolarized structure and excellent cycling performance was generated. This new structure can be incorporated in essentially all the NMC-based layered cathode materials, providing us with an effective tool to tailor-design future new cathode materials for lithium batteries.

Kinetics Tuning of Li-Ion Diffusion in Layered Li(NixMnyCoz)O2.

Using ab initio calculations combined with experiments, we clarified how the kinetics of Li-ion diffusion can be tuned in LiNixMnyCozO2 (NMC, x + y + z = 1) materials. It is found that Li-ions tend to choose oxygen dumbbell hopping (ODH) at the early stage of charging (delithiation), and tetrahedral site hopping (TSH) begins to dominate when more than 1/3 Li-ions are extracted. In both ODH and TSH, the Li-ions surrounded by nickel (especially with low valence state) are more likely to diffuse with low activation energy and form an advantageous path. The Li slab space, which also contributes to the effective diffusion barriers, is found to be closely associated with the delithiation process (Ni oxidation) and the contents of Ni, Co, and Mn.

Depolarized and fully active cathode based on Li(Ni0.5Co0.2Mn0.3)O2 embedded in carbon nanotube network for advanced batteries.

Improving battery capacity and power is a daunting challenge, and in Li-ion batteries positive electrodes often set the limitation on both properties. Layered transition-metal oxides have served as the mainstream cathode materials for high-energy batteries due to their large theoretical capacity (∼ 280 mAh/g). Here we report a significant enhancement of cathode capacity utilization through a novel concept of material design. By embedding Li(Ni0.5Co0.2Mn0.3)O2 in the single wall carbon nanotube (CNT) network, we created a composite in which all components are electrochemically active. Long-term charge/discharge stability was obtained between 3.0 and 4.8 V, and both Li(Ni0.5Co0.2Mn0.3)O2 and CNT contribute to the overall reversible capacity by 250 and 50 mAh/g, respectively. The observed improvement causes significant depolarization within the electrodes through the CNT network system. Additionally, the depolarization provides the ideal template to understand the solid reaction mechanism of Li(Ni0.5Co0.2Mn0.3)O2 by demonstrating well-defined two-stage delithiation kinetics consistent with first-principle calculations, which would be otherwise impossible. These results deliver new insights on both practical designs and fundamental understandings of battery cathodes.

Sequence analysis of mitochondrial ND1 gene can reveal the genetic structure and origin of Bactrocera dorsalis s.s.

BACKGROUND: The oriental fruit fly, Bactrocera dorsalis s.s., is one of the most important quarantine pests in many countries, including China. Although the oriental fruit fly has been investigated extensively, its origins and genetic structure remain disputed. In this study, the NADH dehydrogenase subunit 1 (ND1) gene was used as a genetic marker to examine the genetic diversity, population structure, and gene flow of B. dorsalis s.s. throughout its range in China and southeast Asia. RESULTS: Haplotype networks and phylogenetic analysis indicated two distinguishable lineages of the fly population but provided no strong support for geographical subdivision in B. philippinensis. Demographic analysis revealed rapid expansion of B. dorsalis s.s. populations in China and Southeast Asia in the recent years. The greatest amount of genetic diversity was observed in Manila, Pattaya, and Bangkok, and asymmetric migration patterns were observed in different parts of China. The data collected here further show that B. dorsalis s.s. in Yunnan, Guangdong, and Fujian Provinces, and in Taiwan might have different origins within southeast Asia. CONCLUSIONS: Using the mitochondrial ND1 gene, the results of the present study showed B. dorsalis s.s. from different parts of China to have different genetic structures and origins. B. dorsalis s.s. in China and southeast Asia was found to have experienced rapid expansion in recent years. Data further support the existence of two distinguishable lineages of B. dorsalis s.s. in China and indicate genetic diversity and gene flow from multiple origins.The sequences in this paper have been deposited in GenBank/NCBI under accession numbers KC413034-KC413367.

Host plant-induced changes in metabolism and osmotic regulation gene expression in Diaphorina citri adults.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a worldwide citrus pest. It transmits the pathogen Candidatus Liberibacter spp. of Huanglongbing (HLB), causing severe economic losses to the citrus industry. Severalgenera of plants in the Rutaceae family are the hosts of D. citri. However, the impact of these hosts on the metabolism and osmotic regulation gene expression of the pest remains unexplored. In this study, the contents of total sugars, sucrose, fructose, and glucose in young shoots, old leaves, and young leaves of 'Shatangju' mandarin and Murraya exotica were analyzed. Metabolomic analysis found that sucrose and trehalose were more abundant in the gut samples of D. citri adults fed on M. exotica when compared to what's in 'Shatangju' mandarin. A total of six aquaporin genes were identified in D. citri through the genome and transcriptome data. Subsequently, the expression patterns of these genes were investigated with respect to their developmental stage and tissue specificity. Additionally, the expression levels of osmotic regulation and trehalose metabolism genes in adults fed on different plants were evaluated. Our results provide useful information on the transfer of sugar between plants and D. citri. Our results preliminary revealed the sugar metabolism regulation mechanism in D. citri adults.

Bamboo-like dual-phase nanostructured copper composite strengthened by amorphous boron framework.

Grain boundary engineering is a versatile tool for strengthening materials by tuning the composition and bonding structure at the interface of neighboring crystallites, and this method holds special significance for materials composed of small nanograins where the ultimate strength is dominated by grain boundary instead of dislocation motion. Here, we report a large strengthening of a nanocolumnar copper film that comprises columnar nanograins embedded in a bamboo-like boron framework synthesized by magnetron sputtering co-deposition, reaching the high nanoindentation hardness of 10.8 GPa among copper alloys. The boron framework surrounding copper nanograins stabilizes and strengthens the nanocolumnar copper film under indentation, benefiting from the high strength of the amorphous boron framework and the constrained deformation of copper nanocolumns confined by the boron grain boundary. These findings open a new avenue for strengthening metals via construction of dual-phase nanocomposites comprising metal nanograins embedded in a strong and confining light-element grain boundary framework.

Genome-wide identification, characterization and functional analysis of the chitianse and chitinase-like gene family in Diaphorina citri.

BACKGROUND: Insect chitinases play vital roles in postembryonic development, especially during the molting process, and are potential targets for the RNA interference (RNAi)-based insecticidal strategy. Systematic functional analyses of chitinase genes have already been conducted on numerous insect pests, but similar analyses have not been carried out on Diaphorina citri. RESULTS: Eleven chitinase/chitinase-like genes and one endo-ß-N-acetylglucosaminidase (ENGase) gene were identified in the Diaphorina citri genome using various bioinformatic tools. Transcriptomes of the integument and midgut from fifth-instar nymphs and freshly-emerged adults of Diaphorina citri were generated and sequenced. Potential functions of 12 chitinase/chitinase-like genes were examined during nymph-adult transitions. Four chitinase genes, including DcCht5, DcCht7, DcCht10-1 and DcCht10-2, were mainly expressed in the integument of fifth-instar nymphs. These four genes were also up-regulated significantly under 20-hydroxyecdysone (20E) treatments. RNAi-mediated knockdown of these four genes suggests that they are essential for nymph-adult transition. CONCLUSION: Our results demonstrated essential roles of the chitinase/chitinase-like genes during the nymph-adult transition in Diaphorina citri, which are potentially useful targets for controlling the Diaphorina citri pest. © 2022 Society of Chemical Industry.

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.

Identification and Expression Profiling of Chemosensory Genes in Hermetia illucens via a Transcriptomic Analysis.

The black soldier fly, Hermetia illucens, is a cosmopolitan insect of the family Stratiomyidae (Diptera). Chemosensory genes encode proteins involved directly in the detection of odorants. In this study, we sequenced the antennal transcriptome of H. illucens adults to identify chemosensory genes. Putative unigenes encoding 27 odorant binding proteins (OBPs), five chemosensory proteins (CSPs), 70 odorant receptors (ORs), 25 ionotropic receptors (IRs), 10 gustatory receptors (GRs) and two sensory neuron membrane proteins (SNMPs) were identified. Tissue-specific expression profiles of the identified OBPs, CSPs and SNMPs were investigated using RT-PCR. Eight OBPs (HillOBP1-2, 9, 11-14, and 17), one CSP (HillCSP5) and one SNMP (HillSNMP1) were predominantly expressed in antennae. Further real-time quantitative PCR analyses revealed that the antennae-enriched unigenes also exhibited significant differences in expression between males and females. Among the sex-biased unigenes, six ORs showed female-biased expression, suggesting that these genes might participate in female-specific behaviors such as oviposition site searching. Sixteen ORs and two OBPs showed male-biased expression, indicating that they may play key roles in the detection of female sex pheromones. Our study is the first attempt to delineate the molecular basis of chemoreception in H. illucens. Our data provide useful information for comparative studies on the differentiation and evolution of Dipteran chemosensory gene families.

Transcriptome analysis of putative detoxification genes in the Asian citrus psyllid, Diaphorina citri.

BACKGROUND: The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a notorious pest that transmits the causal agent of huanglongbing (also called citrus greening disease). Resistance to insecticide in this destructive pest poses a serious threat to the citrus industry. To date, no systemic studies on genes coding for detoxification enzymes has been carried out on D. citri. RESULTS: Multiple transcriptomes were generated through deep sequencing of RNA libraries. Candidate genes potentially involved in detoxification including cytochrome P450 monooxygenases (CYPs), glutathione S-transferases (GSTs), and esterases (ESTs) were systematically identified by searching the transcriptomes and a draft genome assembly. A total of 49, 14 and 20 genes were found encoding CYPs, GSTs, and ESTs, respectively, in D. citri. The total numbers of candidate detoxification genes were much smaller than the counterparts reported in other insect species, which may reflect the strict oligophagy of this insect species. Developmental stage- and tissue-specific expression patterns of the identified genes as well as their responses to insecticide treatments identified a small set of genes that could participate in detoxifying plant secondary metabolites and insecticides. CONCLUSION: Our studies represent the most comprehensive investigation to date on identification, characterization and expression profiling of detoxification genes in D. citri. The information revealed in this study shall be useful in designing strategies to manage this important insect pest. © 2020 Society of Chemical Industry.

Odorant-binding proteins and chemosensory proteins potentially involved in host plant recognition in the Asian citrus psyllid, Diaphorina citri.

BACKGROUND: Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are two families of small water-soluble proteins involved in odor detection and subsequent signal transmission. Determination of their binding mechanisms and specificity towards different odorants is important for developing OBPs/CSPs as targets in pest control management. RESULTS: We re-annotated genes encoding putative OBPs and CSPs in the Asian citrus psyllid (Diaphorina citri) draft genome using various bioinformatic tools. Genes encoding nine OBPs (seven Classic and two Plus-C) and 12 CSPs were identified, consistent with our previous transcriptomic results. Tissue-specific and developmental expression analyses suggested that genes encoding six OBPs and four CSPs were predominantly expressed in antennae, and displayed various expression patterns in different development stages, suggesting potential involvement in olfactory perception. Competitive fluorescence binding assays with 13 candidate ligands, including known host plant volatiles, sex pheromone components and repellents, showed that DcitOBP3 could bind to various odorants, whereas DcitOBP6, 8 and 9 bound specifically to host plant terpenoids. DcitCSP1 and 12 could also bind to certain terpenoids with high binding specificity. CONCLUSION: OBP- and CSP-encoding genes were systematically identified by annotating the draft D. citri genome and those potentially involved in odorant detection and signal transmission were identified by analyzing their tissue-expression profiles and odorant-binding affinities, particularly to the peripheral molecular perception of host plant terpenoids. The identified genes may provide potential targets for efficient pest control. © 2020 Society of Chemical Industry.

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.

Identification and Expression Analyses of Olfactory Gene Families in the Rice Grasshopper, Oxya chinensis, From Antennal Transcriptomes.

The rice grasshopper Oxya chinensis is an important agricultural pest of rice and other gramineous plants. Chemosensory genes are crucial factors in direct interactions with odorants in the olfactory process. Here we identified genes encoding 18 odorant-binding proteins (OBPs), 13 chemosensory proteins (CSPs), 94 olfactory receptors (ORs), 12 ionotropic receptors (IRs), and two sensory neuron membrane proteins (SNMPs) from O. chinensis using an transcriptomic approach. Semi-quantitative RT-PCR assays revealed that six OBP-encoding genes (OchiOBP4, 5, 8, 9, 10, and 14), one CSP gene (OchiOBP10) and two IR genes (OchiIR28 and 29) were exclusively expressed in antennae, suggesting their roles in olfaction. Real-time quantitative PCR analyses revealed that genes expressed exclusively or predominantly in antennae also displayed significant differences in expression levels between males and females. Among the differentially expressed genes, 17 OR-encoding genes, one CSP- and one SNMP-gene showed female-biased expression, suggesting that they may be involved in some female-specific behaviors such as seeking oviposition site; whereas the three remaining OR-encoding genes showed male-biased expression, indicating their possible roles in sensing female sex pheromones. Our results laid a solid foundation for future studies to reveal olfactory mechanisms as well as designing strategies for controlling this rice pest.

Selection of Reference Genes for Optimal Normalization of Quantitative Real-Time Polymerase Chain Reaction Results for Diaphorina citri Adults.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), can cause direct damage to citrus trees and is the main vector for the devastating disease, citrus greening disease or huanglongbing. Most molecular studies on this important insect pest use real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR) to quantify gene expression, including analyzing molecular basis for insecticide resistance in field populations. One critical factor to cause inaccuracy in RT-qPCR results is the lack of appropriate internal reference genes for optimal data normalization. In this study, the expression levels of 10 selected reference genes were evaluated in different tissue samples of psyllid adults and in the insects treated with different temperatures and insecticides. Data were analyzed using different computational algorithms, including Delta Ct, BestKeeper, NormFinder, geNorm, and RefFinder. According to our results, at least two reference genes should be used for the normalization of RT-qPCR data in this insect. The best choices of reference genes for different samples are as follows: ACT1 and Ferritin for different tissue samples, RPS20 and Ferritin for samples treated with different temperatures, TBP and EF1α for samples treated with imidacloprid, and Ferritin and TBP for samples treated with beta-cypermethrin. The reference genes identified in this study should be useful for future studies to analyze the expression patterns of target genes, especially for genes linked with temperature adaptability and insecticide resistance in this insect species in the future.

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.