Holotrichia Oblita Faldermann Research Articles

The mechanism underlying OBP heterodimer formation and the recognition of odors in Holotrichia oblita Faldermann

Insect odorant-binding proteins (OBPs) play key roles in transport odors to receptors and contribute to insect survival. The cooperative interaction of HoblOBP1 and HoblOBP2 in Holotrichia oblita Faldermann (Coleoptera: Melolonthidae) could increase their binding capacity for ligands. In present study, molecular docking results showed that OBP1/OBP2 complex formed a large binding pocket and interacted with the ligands by hydrogen bonds and hydrophobic interactions. Then, nine amino acids for single site mutations, three paired for double sites, and negative control were mutated into alanine successfully by site-directed mutagenesis. Finally, fluorescence binding assays of these mutants showed that breaking one or two pairs of hydrogen bonds between HoblOBP1 and HoblOBP2 or formed with the ligands significantly decrease the binding affinity with the ligands. However, hydrophobic site mutants still showed slight binding affinity to the ligands. Therefore, the three pairs of hydrogen bonds involved in heterodimer formation and the five hydrogen bonding sites in binding pocket played a key role in response to odors in H. oblita. Our findings may promote further understanding of the mechanisms underlying OBP dimer formation and the role of OBP dimers in odor perception and discrimination.

Molecular and functional characterization of odorant-binding protein genes in Holotrichia oblita Faldermann

Holotrichia oblita is one of the nastiest pests in China. In present research, four full-length cDNA encoding of HoblOBP genes were cloned and sequenced from H. oblita. The mRNA of HoblOBPs were predominantly expressed in antenna. The recombinant HoblOBPs proteins were obtained for fluorescence binding assays. Four of HoblOBPs could mediate the response of H. oblita to organic fertilizers-derived attractants, including HoblOBP5 binding to skatole; HoblOBP8 binding to p-cresol, indole and skatole; HoblOBP9 binding to indole and 4-allylanisole; and HoblOBP24 binding to p-cresol, indole and 4-ethylphenol. Further, RNA interference demonstrated that transcripts of HoblOBP5, 8, 9, and 24 decreased in a time-dependent manner after dsRNA-injection. Knockdown of HoblOBP5, 8, 9, and 24 by injection of dsRNA successfully interfered with behavioral responses towards the target compounds in beetles. Our results showed that HoblOBP5, HoblOBP8, HoblOBP9 and HoblOBP24 are essential in mediating the approach behavior of H. oblita.

The Mitochondrial Genomes of Phytophagous Scarab Beetles and Systematic Implications.

In this study, we newly sequenced five mitogenomes of representatives of phytophagous scarab beetles (Coleoptera: Scarabaeidae) by using next-generation sequencing technology. Two species have complete (or nearly complete) mitogenome sequences, namely Popillia mutans Newman (Coleoptera: Scarabaeidae) and Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae). The remaining three species have the partial mitogenomes, and the missing genes are mainly located adjacent to the control region. The complete (or nearly complete) mitogenomes have the same genome structure as most of the existing Scarabaeidae mitogenomes. We conducted phylogenetic analyses together with 24 published mitogenomes of Scarabaeoidea. The results supported a basal split of coprophagous and phytophagous Scarabaeidae. The subfamily Sericinae was recovered as sister to all other phytophagous scarab beetles. All analyses supported a non-monophyletic Melolonthinae, which included two different non-sister clades. The Cetoniinae was recovered as sister to a clade including Rutelinae and Dynastinae. Although the Rutelinae was rendered paraphyletic by Dynastinae in the Bayesian trees inferred under the site-heterogeneous CAT-GTR or CAT-MTART model, discordant patterns were given in some of ML trees estimated using the homogeneous GTR model.

Identification and comparison of candidate odorant receptor genes in the olfactory and non-olfactory organs of Holotrichia oblita Faldermann by transcriptome analysis

A sophisticated olfactory system is part of the explanation for the prominence of insects among animals because of the essential roles of the olfactory system in foraging, host seeking, mating, ovipositing and avoiding toxic substances. In this study, we sequenced and analysed the transcriptomes of olfactory tissue (antennae) and non-olfactory tissue (legs) of the scarab beetle, Holotrichia oblita Faldermann, which is a serious underground pest in China. We obtained approximately 80.2 million 150bp reads that were assembled into 61,038 unigenes with an average length of 890bp. Among the transcripts, 70% of the unigenes were annotated. A total of 44 odorant receptors (ORs) and 9 ionotropic receptors (IRs) were identified based on homology searches. Then, quantitative real-time PCR experiments were performed to investigate the expression patterns of 32 putative chemosensory genes. The results showed that these genes were highly expressed in olfactory organs (antennae) and might play a key role in the olfaction-related behaviours in H. oblita. Based on the results of our phylogenetic analysis and the detailed tissue and sex-biased expression characteristics, the different roles of the receptor proteins in the olfactory system were also indicated. The results of this study will provide the foundation for further understanding of the olfactory odorant receptors of H. oblita at the molecular level and ultimately help to develop novel targets for manipulating this pest.

Increased Efficacy of Entomopathogenic Nematode-Insecticide Combinations Against Holotrichia oblita (Coleoptera: Scarabaeidae).

Combinations of entomopathogenic nematodes Steinernema longicaudum X-7 and Heterorhabditis bacteriophora H06 with chlorantraniliprole, diflubenzuron, and imidacloprid insecticides at different rates for the control of the white grub, Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae), were evaluated both in the laboratory and in peanut fields. In the laboratory, the combinations had a synergistic or additive effect on the second-instar larvae of H. oblita and caused faster mortality than one nematode species or insecticide alone. Heterorhabditis bacteriophora-chlorantraniliprole and H. bacteriophora-imidacloprid showed synergistic effects on the larvae. When higher concentrations of the insecticides were combined with nematodes, the stronger synergistic effects were found. In peanut fields, S. longicaudum-imidacloprid, H. bacteriophora-imidacloprid, or H. bacteriophora-chlorantraniliprole also showed synergistic effects against the larvae. The three nematode-insecticide combinations produced similar percentage reductions of the grub larvae and less percentages of injured legumes, compared with the chlorpyrifos treatment. Cost-benefit analysis showed that H. bacteriophora 5.0 × 103 infective juveniles (IJs) per plant (equal to 7.5 × 108 IJ ha-1) combined with imidacloprid at the recommended concentration is a practical strategy for the practitioner to manage the white grubs in the peanut production.

Influence of Constant Temperature on Reproductive Parameters of Holotrichia oblita (Coleoptera: Scarabaeidae).

Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae) is a major pest both in field crops and forests because the larvae could eat the roots of most crops in the field, and the adults damage the leaves of trees and field crops. In this study, we focused on the effects of temperature on H. oblita reproductive parameters. The results indicated H. oblita female adults at 25°C could lay more eggs (84.0 eggs per female) and have the shortest preoviposition period (19.1 d), the greatest oviposition rate (2.8 eggs per female per 3 d), and largest percentage of life span spent in oviposition (59.5%). The longevity and the time to 50% egg laying decreased with increasing temperature, and female longevity was always longer than male longevity. The preoviposition and postoviposition period decreased with increasing temperature from 15 to 25°C and then increased when the temperature increased from 25 to 30°C. These results show that 25°C is the optimal temperature for reproduction of H. oblita.

Efficacy of entomopathogenic Steinernema and Heterorhabditis nematodes against Holotrichia oblita

The white grub Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae) causes serious damage in peanut fields in China. The development of an environmentally friendly control method for this pest is urgently needed. The efficacy of entomopathogenic nematodes against this pest in the laboratory and peanut fields was evaluated in this study. Both Steinernema longicaudum Shen and Wang and Heterorhabditis bacteriophora Poinar (Rhabditida: Steinernematidae and Heterorhabditidae) had promising control efficacy against H. oblita larvae in the laboratory. S. longicaudum X-7 and H. bacteriophora H06 at 1.0 × 104 and 5.0 × 103 IJs per plant caused a similar percentage reduction of the grub larvae and percentage of the injured legumes to chlorpyrifos. The peanut yields from the nematode-treated plots at 5.0 × 103 IJs per plant were at least 55 and 15 % higher than those from the water control and the chlorpyrifos-treated plots. No significant differences were found in the percentage reductions of the grub larvae at different larval stage applications. The peanut yields in plots treated by S. longicaudum X-7 and H. bacteriophora H06 were also not significantly different. The cost-benefit analysis showed that S. longicaudum X-7 and H. bacteriophora H06 are promising agents for H. oblita larvae control in peanut fields. Our findings indicate that entomopathogenic nematodes have good potential for safe management of H. oblita in peanut production.

Cultivable gut bacteria of scarabs (Coleoptera: Scarabaeidae) inhibit Bacillus thuringiensis multiplication.

The entomopathogen Bacillus thuringiensis is used to control various pest species of scarab beetle but is not particularly effective. Gut bacteria have diverse ecological and evolutionary effects on their hosts, but whether gut bacteria can protect scarabs from B. thuringiensis infection remains poorly understood. To investigate this, we isolated 32 cultivable gut bacteria from Holotrichia oblita Faldermann, Holotrichia parallela Motschulsky, and Anomala corpulenta Motschulsky, and analyzed their effect on B. thuringiensis multiplication and Cry toxin stability. 16S rDNA analysis indicated that these gut bacteria belong to the Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes phyla. A confrontation culture analyses of the 32 isolates against three scarab-specific B. thuringiensis strains showed that the majority of the scarab gut bacteria had antibacterial activity against the B. thuringiensis strains. The Cry toxin stability analysis results showed that while several strains produced proteases capable of processing the scarab-specific toxin Cry8Ea, none were able to completely degrade it. These results suggest that gut bacteria can potentially affect the susceptibility of scarabs to B. thuringiensis and that this should be considered when considering future control measures.

Prediction of the key binding site of odorant‐binding protein of Holotrichia oblita Faldermann (Coleoptera: Scarabaeida)

The scarab beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae) is a predominant underground pest in the northern parts of China, and its larvae (grubs) cause great economic losses because of its wide range of host plants and covert habitats. Environmentally friendly strategies for controlling adults would have novel and broad potential applications. One potential pest management measure is the regulation of olfactory chemoreception to control target insect pests. In the process of olfactory recognition, odorant-binding proteins (OBPs) are believed to carry hydrophobic odorants from the environment to the surface of olfactory receptor neurons. To obtain a better understanding of the relationship between OBP structures and their ligands, homology modelling and molecular docking have been conducted on the interaction between HoblOBP1 and hexyl benzoate in the present study. Based on the results, site-directed mutagenesis and binding experiments were combined to describe the binding sites of HoblOBP1 and to explore its ligand-binding mechanism. After homology modelling of HoblOBP1, it was found that the three-dimensional structure of HoblOBP1 consists of six α-helices and three disulphide bridges that connect the helices, and the hydrophobic pockets are both composed of five helices. Based on the docking study, we found that van der Waals interactions and hydrophobic interactions are both important in the bonding between HoblOBP1 and hexyl benzoate. Intramolecular residues formed the hydrogen bonds in the C terminus of the protein and the bonds are crucial for the ligand-binding specificity. Finally, MET48, ILE80 and TYR111 are binding sites predicted for HoblOBP1. Using site-directed mutagenesis and fluorescence assays, it was found that ligands could not be recognized by mutant of Tyr111. A possible explanation is that the compound could not be recognized by the mutant, and remains in the binding cavity because of the loss of the intramolecular hydrogen bonding that acts as a holder. So we believe that Tyr111 of HoblOBP1 is a key binding site. We also believe that Ile80A is a very important binding site, especially to some ligands.

Establishment and characterization of three new cell lines from the embryonic tissue of Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae)

The establishment of new insect cell lines plays important roles in the researches of insect pathology, insect toxicology, insecticide screening and activity assay, etc. Using embryos of Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae) as materials, this study describes the establishment of three cell lines designated as QAU-Ho-E-3 (Ho-3), QAU-Ho-E-4 (Ho-4), and QAU-Ho-E-6 (Ho-6), respectively. Currently, the three cell lines have been passaged more than 50 times in the TNM-FH insect cell medium containing 10% fetal bovine serum (FBS). All of them showed adherent growth. The majority of Ho-3 cells are spindle-shaped, with a size of 24.35 ± 5.29 × 11.56 ± 1.67 μm. The Ho-4 cells were either spindle-shaped or oblong, with a size of 38.07 ± 8.57 × 17.62 ± 2.48 μm. The Ho-6 cells were primarily round in shape with a diameter of 14.54 ± 1.96 μm. The Ho-3 and Ho-4 cell lines contained 20 chromosomes (i.e., diploid, 2n = 20) at passages 14 and 45. The Ho-6 cell line contained 20 chromosomes (i.e., diploid, 2n = 20) at passage 14 but 40 chromosomes (i.e., polyploidy, 4n = 40) at passage 45. The results of random amplified polymorphic DNA (RAPD) analysis showed that the RAPD fingerprint of the three cell lines was consistent with that of H. oblita eggs, but clearly different from that of BTI-Tn5B1-4 and Sf-9 cells, demonstrating that the three cell lines Ho-3, Ho-4, and Ho-6 are H. oblita cell lines. The results of the growth curve test showed that the population doubling times of Ho-3, Ho-4, and Ho-6 were 101.1, 105.2, and 83.6 h, respectively. The viral infection assay indicated that these H. oblita cell lines were not permissive to infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or Bombyx mori nucleopolyhedrovirus (BmNPV).

Potential Cooperations between Odorant-Binding Proteins of the Scarab Beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae)

It was previously thought that the odorant binding proteins (OBPs) in the sensillum lymph might serve as carriers, which could carry lipophilic odorant molecules to olfactory receptors. In this study, two novel OBP genes of the scarab beetle Holotrichia oblita were screened using an antennal cDNA library. The full cDNA of HoblOBP3 and HoblOBP4 was cloned using reverse transcription PCR and rapid amplification of the cDNA ends. Homology modeling of both OBPs was performed using SWISS-MODEL on-line tools. Next, the two OBPs were expressed in Escherichia coli and purified using Ni ion affinity chromatography. The ligand-binding properties of HoblOBP3 and HoblOBP4 in 42 ligands respectively were measured using the fluorescence probe N-phenyl-naphthylamine (1-NPN). The results obtained from competitive binding assays demonstrated that HoblOBP4 showed a broader range of binding affinities to the test compounds, while HoblOBP3 displays more specific binding affinity. Furthermore, other OBPs and CSPs were expressed in Escherichia coli and purified using Ni ion affinity chromatography. Binding curves were measured for binary mixtures of OBPs and CSPs using 1-NPN, and the Scatchard plots exhibited “J”-like nonlinear correlation trends in some samples. In addition, competitive binding assays of the HoblOBP1 and HoblOBP2 mixtures and of the HoblOBP2 and HoblOBP4 mixtures with representative compounds unexpectedly demonstrated good affinity, which revealed extreme differences that were only obtained using the individual proteins. In the immunocytochemical analysis, colocalization of HoblOBP1 and HoblOBP2, and of HoblOBP2 and HoblOBP4, was detected in the sensilla basiconica and sensilla placodea, respectively. All of these results suggested that HoblOBP1 and HoblOBP2, as well as HoblOBP2 and HoblOBP4, may serve as heterodimers in the sensillum lymph.

Function and Immunocytochemical Localization of Two Novel Odorant-Binding Proteins in Olfactory Sensilla of the Scarab Beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae)

Odorant-binding proteins (OBPs) are found in both insects and vertebrates, and it is believed that they are involved in chemical communication. In this study, we identify and express 2 OBPs from the scarab beetle, Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae). HoblOBP1 shows more similarities with other scarab beetle OBPs, whereas HoblOBP2 is more diverse. N-phenyl-1-naphthylamine (1-NPN) is used as a fluorescent probe in ligand-binding experiment, and results indicate that both HoblOBPs prefer plant volatiles to putative H. oblita sex pheromones. HoblOBP1 shows binding affinity to a wider range of test compounds, but HoblOBP2 displays more specific binding affinity. Cinnamaldehyde and 2,4-di-tert-butylphenol bind to HoblOBP1 can elicit strong electrophysiological responses of the antennae from female H. oblita adults, respectively. Methyl salicylate also shows good affinity to HoblOBP2 and it can elicit moderate electrophysiological responses. Although, β-ionone is one of the ligands of the strongest binding, it elicits a weak electrophysiological response. In the immunocytochemical analysis, we observe that HoblOBP1 and HoblOBP2 are coexpressed in sensilla basiconica and placodea in both sexes.