Candidate Chemosensory Genes Identified in the Adult Antennae of Sympiezomias velatus and Binding Property of Odorant-Binding Protein 15.

Chemosensory genes play important roles in insect behaviors and have thus become potential molecular targets for pest control based on the manipulation of chemoreception-driven behaviors. The great gray weevil Sympiezomias velatus (Chevrolat) (Coleoptera: Curculionidae) is an important agricultural pest that causes serious economic losses to many crops in China, but its chemosensory genes have not been reported. Here we assembled the antennal transcriptomes of female and male adult S. velatus and revealed the major chemosensory genes necessary for olfaction. A total of 138 candidate chemosensory genes in six families were identified, including 41 encoding odorant-binding proteins (OBPs), 11 encoding chemosensory proteins (CSPs), 62 encoding odorant receptors (ORs), 15 encoding gustatory receptors (GRs), six encoding ionotropic receptors (IRs), and three encoding sensory neuron membrane proteins (SNMPs). We analyzed their phylogenetic relationship based on the amino acid sequences of these chemosensory-related protein families in S. velatus and other insects, and the expression profiles based on their antennal transcriptomes. Chemosensory genes that show antenna-abundant/specific or sex-biased expression were observed, suggesting that these genes might have functions in olfaction. Furthermore, we chose an antenna-abundant OBP belonging to ABPX subfamily, SvelOBP15, to investigate its binding property. The results showed that among 33 tested compounds, SvelOBP15 displayed high binding affinities (Ki = 7.36-12.94 µmol/L) with farnesol, nerolidol, limonene and diisobutyl phthalate, indicating that SvelOBP15 plays olfactory roles by binding and transporting specific plant volatiles. These findings will help us better understand the olfactory systems of S. velatus, and provide a basis for functional elucidation of these chemosensory genes.

Effect of Photoperiod on Longevity, Food Consumption, and Reproduction of Holotrichia oblita (Coleoptera: Scarabaeidae).

Holotrichia oblita (Faldermann) (Coleoptera: Scarabaeidae) is a major soil insect pest that damages forest trees, crops, and lawns. Adults of H. oblita fly, forage, and mate at night but remain underground during the day. We studied the effect of photoperiod on H. oblita reproduction. H. oblita females laid more eggs at 8:16 (L:D) h and 0:24 (L:D) h than other photoperiods. As the scotophase increased, the preoviposition period decreased and the oviposition period increased. Female longevity exceeded that of males at all photoperiods, and both males and females at 0:24 (L:D) h had the shortest longevity. The number of eggs laid per female increased with increasing food consumption. Females at 8:16 (L:D) h had the greatest food consumption and laid the most eggs, while females at 24:0 (L:D) h had the lowest food consumption and laid few eggs. The food intake of adults increased gradually and decreased slowly after reaching a peak. Females began to lay eggs when their food consumption reached a maximum. These results indicate that a scotophase is necessary for the reproduction of H. oblita. A long scotophase promotes greater oviposition. The effect of photoperiod on reproduction is affected by food intake.

Involvement of chemosensory proteins in host plant searching in the bird cherry-oat aphid.

Chemosensory systems are considered to play an important role in host plant selection in herbivorous insects. However, few studies have focused on chemosensory proteins (CSPs) for aphid host-location mechanisms. The roles of CSPs in searching for different Poaceae species (wheat, barley, triticale, maize and sorghum) were tested in Rhopalosiphum padi, an important cereal pest. The olfactometer assays showed that R. padi responds to plant odors. Seven R. padi CSP genes were identified. Influence of aphid morph, tissue and starvation state on expression patterns of CSPs was evaluated. Expression levels of CSP1, CSP4, CSP5 and CSP6 in winged aphids were significantly higher than those in wingless ones. Transcription levels of four genes (CSP1, CSP4, CSP5 and CSP6) were relatively higher in the head with antennae, and the four genes tended to be upregulated following starvation. Silencing of three CSPs (CSP4, CSP5 and CSP6) altered aphid host-location behavior in response to the five different host plants tested. Three volatile compounds of host plants (octanal, [E]-2-hexenol and linalool) have significant attraction to winged R. padi according to the four-arm olfactometer tests. Molecular docking predicted hydrogen bonding sites which played key roles in the binding of CSP4, CSP5 and CSP6 with volatile compounds. Knockdown of CSP4 or CSP5 significantly decreased the staying time of R. padi in the arms with octanal. However, knockdown of CSP6 could not affect the response of R. padi to octanal. These results bring evidence for the involvement of three CSPs in R. padi host-location behavior.

Identification and Characterization of Neuropeptides and Their G Protein-Coupled Receptors (GPCRs) in the Cowpea Aphid Aphis craccivora.

Neuropeptides are the most abundant and diverse signal molecules in insects. They act as neurohormones and neuromodulators to regulate the physiology and behavior of insects. The majority of neuropeptides initiate downstream signaling pathways through binding to G protein-coupled receptors (GPCRs) on the cell surface. In this study, RNA-seq technology and bioinformatics were used to search for genes encoding neuropeptides and their GPCRs in the cowpea aphid Aphis craccivora. And the expression of these genes at different developmental stages of A. craccivora was analyzed by quantitative real-time PCR (qRT-PCR). A total of 40 candidate genes encoding neuropeptide precursors were identified from the transcriptome data, which is roughly equivalent to the number of neuropeptide genes that have been reported in other insects. On this basis, software analysis combined with homologous prediction estimated that there could be more than 60 mature neuropeptides with biological activity. In addition, 46 neuropeptide GPCRs were obtained, of which 40 belong to rhodopsin-like receptors (A-family GPCRs), including 21 families of neuropeptide receptors and 7 orphan receptors, and 6 belong to secretin-like receptors (B-family GPCRs), including receptors for diuretic hormone 31, diuretic hormone 44 and pigment-dispersing factor (PDF). Compared with holometabolous insects such as Drosophila melanogaster, the coding genes for sulfakinin, corazonin, arginine vasopressin-like peptide (AVLP), and trissin and the corresponding receptors were not found in A. craccivora. It is speculated that A. craccivora likely lacks the above neuropeptide signaling pathways, which is consistent with Acyrthosiphon pisum and that the loss of these pathways may be a common feature of aphids. In addition, expression profiling revealed neuropeptide genes and their GPCR genes that are differentially expressed at different developmental stages and in different wing morphs. This study will help to deepen our understanding of the neuropeptide signaling systems in aphids, thus laying the foundation for the development of new methods for aphid control targeting these signaling systems.

Two Odorant-Binding Proteins of the Dark Black Chafer (Holotrichia parallela) Display Preferential Binding to Biologically Active Host Plant Volatiles.

The dark black chafer (DBC), Holotrichia parallela, is an important pest of multiple crops. Insect host-searching behaviors are regulated by host plant volatiles. Therefore, a better understanding of the mechanism linking the chemosensory system to plant volatiles at the molecular level will benefit DBC control strategies. Based on antenna transcriptome data, two highly expressed antenna-specific odorant-binding proteins (HparOBP20 and 49) were selected to identify novel DBC attractants using reverse chemical ecology methods. We expressed these proteins, mapped their binding specificity, and tested the activity of the plant volatiles in the field. The ligands used in the binding specificity assays included 31 host-plant-associated volatiles and two sex pheromone components. The results showed that (1) HparOBP20 and 49 are involved in odor recognition; (2) these proteins bind attractive plant volatiles strongly and can therefore be employed to develop environmentally friendly DBC management strategies; and (3) the green-leaf volatile (Z)-3-hexenyl acetate shows a high binding affinity to HparOBP20 (Ki = 18.51 µM) and HparOBP49 (Ki = 39.65 µM) and is highly attractive to DBC adults, especially females. In the field test, a (Z)-3-hexenyl acetate trap caught an average of 13 ± 1.202 females per day, which was significantly greater than the corresponding male catch (F2,6 = 74.18, P < 0.0001). (Z)-3-Hexenyl acetate may represent a useful supplement to the known sex pheromone for DBC attraction. In the present study, the binding characteristics of two HparOBPs with host plant volatiles were screened, providing behaviourally active compounds that might be useful for DBC control, based on reverse chemical ecology.

Expression of Neuropeptide F Gene and Its Regulation of Feeding Behavior in the Pea Aphid, Acyrthosiphon pisum.

Neuropeptide F (NPF) signaling systems are widespread and highly evolutionarily conserved from vertebrates to invertebrates. In fact, NPF has been identified in many insect species and plays regulatory roles in diverse physiological processes, such as feeding, learning, reproduction and stress responses. NPF operates by interacting with the NPF receptor (NPFR). Here, we characterized and determined the presumed role of NPF signaling in the wingless parthenogenetic pea aphid, Acyrthosiphon pisum. Quantitative real-time reverse transcription-PCR (qRT-PCR) revealed that the expression levels of both NPF and NPFR transcripts varied across developmental stages, which implies that the NPF signaling system might participate in the developmental regulation of aphid physiological processes or behaviors. The NPF transcript was mainly detected in the head but not in the gut, whereas the NPFR transcript was mainly detected in both the gut and head. In addition, the NPF transcript levels were markedly up-regulated in starved aphids compared with satiated aphids, and the transcript levels recovered after re-feeding. In contrast, the NPFR transcript levels remained stable in starved and re-fed aphids. Furthermore, RNAi knockdown by the injection of NPF dsRNA into wingless adult aphids significantly reduced their food intake. Further analysis of the modification of aphid feeding behavior on broad bean plants using electrical penetration graphs (EPGs) revealed that both the probing time and the total duration of phloem activity decreased significantly in the NPF treatment group. These results indicated a lower appetite for food after NPF knockdown, which could explain the reduction in aphid food intake. NPF silencing was also shown to reduce reproduction but not survival in aphids. Overall, the results of these experiments suggest that NPF plays an important role in regulation of feeding in A. pisum.

Plant Volatiles Increase Sex Pheromone Attraction of Holotrichia parallela (Coleoptera: Scarabaeoidea).

Holotrichia parallela (Coleoptera: Scarabaeoidea) is a notorious pest of many crops. To improve the effectiveness of its female-produced sex pheromone (L-isoleucine methyl ester:(R)-(-)-linalool = 6:1), 14 plant volatiles, including dodecanoic acid, dodecanal, farnesol, α-farnesene, (Z)-3-hexen-1-ol, (E)-2-hexen-1-ol, (Z)-3-hexenyl acetate, (E)-2-hexenyl acetate, (R)-(+)-limonene, α-phellandrene, α-pinene, ocimene, methyl benzoate, and benzaldehyde, were individually evaluated using electroantennography and olfactometer assays. (E)-2-Hexenyl acetate and (Z)-3-hexenyl acetate were found to elicit the strongest responses in both males and females. Further testing of these two compounds in mixtures with the sex pheromone indicated that (E)-2-hexenyl acetate had a stronger synergistic effect than (Z)-3-hexenyl acetate. Field evaluations showed that mixtures of (E)-2-hexenyl acetate and the sex pheromone resulted in significantly higher catches than the sex pheromone alone. Using a 5:1 mixture of the sex pheromone and (E)-2-hexenyl acetate, the maximum number of females per trap per day was 14, showing a synergistic effect of a factor of four. For males, a 3:1 mixture of the sex pheromone and (E)-2-hexenyl acetate yielded a maximum number of 310 individuals per trap per day, equivalent to a synergistic effect of 175%. These results may provide the basis for the development of efficient pest management systems against H. parallela using plant volatiles and insect sex pheromones.

A gustatory receptor tuned to D-fructose in antennal sensilla chaetica of Helicoverpa armigera.

Insect gustatory systems play important roles in food selection and feeding behaviors. In spite of the enormous progress in understanding gustation in Drosophila, for other insects one of the key elements in gustatory signaling, the gustatory receptor (GR), is still elusive. In this study, we report that fructose elicits behavioral and physiological responses in Helicoverpa armigera (Harm) to fructose and identify the gustatory receptor for this sugar. Using the proboscis extension reflex (PER) assays we found that females respond to fructose following stimulation of the distal part of the antenna, where we have identified contact chemosensilla tuned to fructose in tip recording experiments. We isolated three full-length cDNAs encoding candidate HarmGRs based on comparison with orthologous GR sequences in Heliothis virescens and functionally characterized the responses of HarmGR4 to 15 chemicals when this receptor was expressed in Xenopus oocytes with two-electrode voltage-clamp recording. Among the tastants tested, the oocytes dose-dependently responded only to D-fructose (EC50 = 0.045 M). By combining behavioral, electrophysiological and molecular approaches, these results provide basic knowledge for further research on the molecular mechanisms of gustatory reception.

Transcriptome and tissue-specific expression analysis of Obp and Csp genes in the dark black chafer.

The dark black chafer, Holotrichia parallela, is an economically important pest in China and worldwide. Traps based on chemical communication are being developed as an alternative control measure to pesticides for this pest, and studies to reveal chemical communication mechanisms in this pest are highly desirable. To systematically analyze genes potentially involved in chemical communication in this pest, we generated a comprehensive transcriptome with combined samples derived from multiple tissues and developmental stages. A total of 43,967 nonredundant sequences (unigenes) with average length of 806 bp were obtained. These unigenes were annotated into different pathways using gene ontology analysis and cluster analysis of orthologous groups of proteins, and kyoto encyclopedia of genes and genomes. In total, 25 transcripts encoding odorant-binding proteins (OBPs) and 16 transcripts encoding chemosensory proteins (CSPs) were identified based on homology searches. Tissue-specific expression profile indicates that OBP17 and CSP7 are likely responsible for male sex pheromone recognition, whereas OBP1-4, OBP9, OBP13-14, OBP17-18, OBP20, OBP22, OBP25, CSP1-7, CSP11, and CSP12-15 are likely responsible for chemical communication between the beetle and environments. Our data shall provide a foundation for further research on the molecular aspects of chemical communication of this insect, and for comparative genomic studies with other species.

Molecular and biochemical characterization of two odorant-binding proteins from dark black chafer, Holotrichia parallela.

The dark black chafer, Holotrichia parallela Motschulsky, is an economically important pest worldwide. Odorant-based lures and traps are being developed as a key kind of alternative control measures for this pest, and studies to reveal the mechanisms for chemotaxis in this pest are necessary. Two full-length cDNAs encoding different odorant-binding proteins (OBPs) were cloned. The predicted proteins were found to have the functional domains characteristic of typical OBPs and share a high degree of sequence similarity with OBP1 and OBP2 from other insects and were therefore designated as H. parallela OBP-1 and H. parallela OBP-2 (HparOBP-1 and HparOBP-2, respectively). These two OBPs were specifically expressed in antennae. The binding affinity of two purified proteins indicated that HparOBP-1 and HparOBP-2 could selectively interact with various volatiles emitted from host plants and pheromone components. Among the 10 chemicals tested, HparOBP-1 could bind to six of the tested compounds with a dissociation concentration (Ki) less than 20, and HparOBP-2 could bind to three of the compounds. The two OBPs are probably involved in chemotaxis of the dark black chafer. This discovery should accelerate research on chemical communications of this pest, which could potentially lead to the improvement of control measures based on lures and traps.

Mutations in acetylcholinesterase genes of Rhopalosiphum padi resistant to organophosphate and carbamate insecticides.

Apple grain aphid, Rhopalosiphum padi (Linnaeus), is an important wheat pest. In China, it has been reported that R. padi has developed high resistance to carbamate and organophosphate insecticides. Previous work cloned from this aphid 2 different genes encoding acetylcholinesterase (AChE), which is the target enzyme for carbamate and organophosphate insecticides, and its insensitive alteration has been proven to be an important mechanism for insecticide resistance in other insects. In this study, both resistant and susceptible strains of R, padi were developed, and their AChEs were compared to determine whether resistance resulted from this mechanism and whether these 2 genes both play a role in resistance. Bioassays showed that the resistant strain used was highly or moderately resistant to pirimicarb, omethoate, and monocrotophos (resistance ratio, 263.8, 53.8, and 17.5, respectively), and showed little resistance to deltamethrin or thiodicarb (resistance ratio, 5.2 and 3.4, respectively). Correspondingly, biochemistry analysis found that AChE from resistant aphids was very insensitive to the first 3 insecticides (I50 increased 43.0-, 15.2-, and 8.8-fold, respectively), but not to thiodicarb (I50 increased 1.1-fold). Enzyme kinetics tests showed that resistant and susceptible strains had different AChEs. Sequence analysis of the 2 AChE genes cloned from resistant and susceptible aphids revealed that 2 mutations in Ace2 and 1 in Ace1 were consistently associated with resistance. Mutation F368(290)L in Ace2 localized at the same position as a previously proven resistance mutation site in other insects. The other 2 mutations, S329(228)P in Ace1 and V435(356)A in Ace2, were also found to affect the enzyme structure. These findings indicate that resistance in this aphid is mainly the result of insensistive AChE alteration, that the 3 mutations found might contribute to resistance, and that the AChEs encoded by both genes could serve as targets of insecticides.