Positive Chemotaxis of the Entomopathogenic Nematode Steinernema australe (Panagrolaimorpha: Steinenematidae) towards High-Bush Blueberry (Vaccinium corymbosum) Root Volatiles.

The foraging behavior of the infective juveniles (IJs) of entomopathogenic nematodes (EPNs) relies on host-derived compounds, but in a tri-trophic context, herbivore-induced root volatiles act as signals enhancing the biological control of insect pests by recruiting EPNs. In southern Chile, the EPN Steinernema australe exhibits the potential to control the raspberry weevil, Aegorhinus superciliosus, a key pest of blueberry Vaccinium corymbosum. However, there is no information on the quality of the blueberry root volatile plume or the S. australe response to these chemicals as putative attractants. Here, we describe the root volatile profile of blueberries and the chemotaxis behavior of S. australe towards the volatiles identified from Vaccinium corymbosum roots, infested or uninfested with A. superciliosus larvae. Among others, we found linalool, α-terpineol, limonene, eucalyptol, 2-carene, 1-nonine, 10-undecyn-1-ol, and methyl salicylate in root volatiles and, depending on the level of the emissions, they were selected for bioassays. In the dose-response tests, S. australe was attracted to all five tested concentrations of methyl salicylate, 1-nonine, α-terpineol, and 2-carene, as well as to 100 µg mL-1 of 10-undecyn-1-ol, 0.1 and 100 µg mL-1 of linalool, and 100 µg mL-1 of limonene, whereas eucalyptol elicited no attraction or repellency. These results suggest that some volatiles released from damaged roots attract S. australe and may have implications for the biocontrol of subterranean pests.

Structural investigation of selective binding dynamics for the pheromone-binding protein 1 of the grapevine moth, Lobesia botrana.

The European grapevine moth, Lobesia botrana (Denis & Schiffermüller), is a serious pest in vineyards in North and South America. Mating disruption techniques have been used to control and monitor L. botrana on the basis of its sexual communication. This needs a well-tuned olfactory system, in which it is believed that pheromone-binding proteins (PBPs) are key players that transport pheromones in the antennae of moths. In this study, the selectivity of a PBP, named as LbotPBP1, was tested by fluorescence binding assays against 11 sex pheromone components and 6 host plant volatiles. In addition, its binding mechanism was predicted on the basis of structural analyses by molecular docking and complex and steered molecular dynamics (SMD). Our results indicate that LbotPBP1 binds selectively to sex pheromone components over certain host plant volatiles, according to both in vitro and in silico tests. Thus, chain length (14 carbon atoms) and functional groups (i.e., alcohol and ester) appear to be key features for stable binding. Likewise, residues such as Phe12, Phe36, and Phe118 could participate in unspecific binding processes, whilst Ser9, Ser56, and Trp114 could participate in the specific recognition and stabilization of sex pheromones instead of host plant volatiles. Moreover, our SMD approach supported 11-dodecenyl acetate as the best ligand for LbotPBP1. Overall, the dynamics simulations, contact frequency analysis and SMD shed light on the binding mechanism of LbotPBP1 and could overcome the imprecision of molecular docking, supporting the in vitro binding assays. Finally, the role of LbotPBP1 in the chemical ecology of L. botrana is discussed.

Virtual Screening of Plant Volatile Compounds Reveals a High Affinity of Hylamorpha elegans (Coleoptera: Scarabaeidae) Odorant-Binding Proteins for Sesquiterpenes From Its Native Host.

Hylamorpha elegans(Burmeister) is a native Chilean scarab beetle considered to be a relevant agricultural pest to pasture and cereal and small fruit crops. Because of their cryptic habits, control with conventional methods is difficult; therefore, alternative and environmentally friendly control strategies are highly desirable. The study of proteins that participate in the recognition of odorants, such as odorant-binding proteins (OBPs), offers interesting opportunities to identify new compounds with the potential to modify pest behavior and computational screening of compounds, which is commonly used in drug discovery, may help to accelerate the discovery of new semiochemicals. Here, we report the discovery of four OBPs inH. elegans as well as six new volatiles released by its native host Nothofagus obliqua(Mirbel). Molecular docking performed between OBPs and new and previously reported volatiles from N. oblique revealed the best binding energy values for sesquiterpenic compounds. Despite remarkable divergence at the amino acid level, three of the four OBPs evaluated exhibited the best interaction energy for the same ligands. Molecular dynamics investigation reinforced the importance of sesquiterpenes, showing that hydrophobic residues of the OBPs interacted most frequently with the tested ligands, and binding free energy calculations demonstrated van der Waals and hydrophobic interactions to be the most important. Altogether, the results suggest that sesquiterpenes are interesting candidates for in vitro and in vivo assays to assess their potential application in pest management strategies.

Comparative transcriptomic analysis of chemoreceptors in two sympatric scarab beetles, Hylamorpha elegans and Brachysternus prasinus.

Chemoreception through odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs) represents the functions of key proteins in the chemical ecology of insects. Recent studies have identified chemoreceptors in coleopterans, facilitating the evolutionary analysis of not only ORs but also IRs and GRs. Thus, Cerambycidae, Tenebrionidae and Curculionidae have received increased attention. However, knowledge of the chemoreceptors from Scarabaeidae is still limited, particularly for those that are sympatric. Considering the roles of chemoreceptors, this analysis could shed light on evolutionary processes in the context of sympatry. Therefore, the aim of this study was to identify and compare the repertoires of ORs, GRs and IRs between two sympatric scarab beetles, Hylamorpha elegans and Brachysternus prasinus. Here, construction of the antennal transcriptomes of both scarab beetle species and analyses of their phylogeny, molecular evolution and relative expression were performed. Thus, 119 new candidate chemoreceptors were identified for the first time, including 17 transcripts for B. prasinus (1 GR, 3 IRs and 13 ORs) and 102 for H. elegans (22 GRs, 14 IRs and 66 ORs). Orthologs between the two scarab beetle species were found, revealing specific expansions as well as absence in some clades. Purifying selection appears to have occurred on H. elegans and B. prasinus ORs. Further efforts will be focused on target identification to characterize kairomone and/or pheromone receptors.

A mutation increases the specificity to plant compounds in an insect chemosensory protein.

Host plant recognition are highly dependent on chemosensory perception, which involves chemosensory proteins (CSPs) that bind key chemical compounds the host plants. In this work, we hypothesize that two closely related aphid taxa, which differ in diet breadth, also differ in their CSPs. We detected a non-synonymous difference (lysine for asparagine) between M. persicae sensu stricto (Mpp) and the subspecies M. p. nicotianae (Mpn) in the sequence of a CSP (CSP5). We modeled in silico the binding capacity of both CSP5s variants with 163 different potential ligands from their host plants (120 unique from tobacco, 29 unique from peach, and 14 common ligands). After docking analysis with all ligands, we selected the three best ligands for each variant to perform molecular dynamics (tobacco: 2-cyclopentene-1,4-dione, salicylaldehyde, and benzoic acid; peach: phenol, valeric acid, and benzonitrile). The binding energy of the MpnCSP5 model to the studied ligands was, in all cases, lower than with the MppCSP5 model. The ligands from the host plants showed more stable binding with MpnCSP5 than with MppCSP5. This result suggests that the set of CSPs studied among M. persicae s. str. and M. p. nicotianae are very similar, but focusing on the CSP5 protein, we found a single key mutation that increases affinities for host compounds for M. p. nicotianae, which might have contributed to the specialization to tobacco. This study provides new insights into an evolutionary trend toward specificity in a binding protein.

Analysis of glutathione-S-transferases from larvae of Galleria mellonella (Lepidoptera, Pyralidae) with potential alkaloid detoxification function.

The greater wax moth, Galleria mellonella, is a global pest for beehives, doing damage in the larval stage. Although a significant number of studies have reported on larvae and adults, to date no effective pest control has been implemented. In this study, we tested larval resistance to alkaloids from Berberis microphylla, and the objective was to identify enzymes that participate in alkaloid detoxification through enzymatic assays, bioinformatics analysis and qRT-PCR. Findings suggest glutathione-S-transferases (GSTs), from an increased metabolic mechanism, are responsible for alkaloid detoxification rather than cytochrome P450 (CYP), carboxylesterases (CarE). A bioinformatics analysis from transcriptome data revealed 22 GSTs present in both G. mellonella larvae and adults. The qRT-PCR experiments corroborated the presence of the 22 GSTs in larvae, where GST8 and GST20 stood out with the highest expression after berberine treatment. Structural information around GST8 and GST20 suggests that GST8 could bind berberine stronger than GST20. These findings represent an important advance in the study of detoxification enzymes in G. mellonella, expanding the role of delta-class GSTs towards alkaloids. Likewise, GST inhibition by alkaloid analogs is proposed in the framework of integrated pest management strategies.

Sex- and tissue-specific expression of odorant-binding proteins and chemosensory proteins in adults of the scarab beetle Hylamorpha elegans (Burmeister) (Coleoptera: Scarabaeidae).

In this study, we addressed the sex- and tissue-specific expression patterns of odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) in Hylamorpha elegans (Burmeister), an important native scarab beetle pest species from Chile. Similar to other members of its family, this scarab beetle exhibit habits that make difficult to control the pest by conventional methods. Hence, alternative ways to manage the pest populations based on chemical communication and signaling (such as disrupting mating or host finding process) are highly desirable. However, developing pest-control methods based on chemical communication requires to understand the molecular basis for pheromone recognition/chemical perception in this species. Thus, with the aim of discovering olfaction-related genes, we obtained the first reference transcriptome assembly of H. elegans. We used different tissues of adult beetles from males and females: antennae and maxillary palps, which are well known for embedded sensory organs. Then, the expression of predicted odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) was analyzed by qRT-PCR. In total, 165 transcripts related to chemoperception were predicted. Of these, 16 OBPs, including one pheromone-binding protein (PBP), and four CSPs were successfully amplified by qRT-PCR. All of these genes were differentially expressed in the sensory tissues with respect to the tibial tissue that was used as a control. The single predicted PBP found was highly expressed in the antennal tissues, particularly in males, while several OBPs and one CSP showed male-biased expression patterns, suggesting that these proteins may participate in sexual recognition process. In addition, a single CSP was expressed at higher levels in female palps than in any other studied condition, suggesting that this CSP would participate in oviposition process. Finally, all four CSPs exhibited palp-biased expression while mixed results were obtained for the expression of the OBPs, which were more abundant in the palps than in the antennae. These results suggest that these chemoperception proteins would be interesting novel targets for control of H. elegans, thus providing a theoretical basis for further studies involving new pest control methods.

Analysis of the grapevine moth Lobesia botrana antennal transcriptome and expression of odorant-binding and chemosensory proteins.

The grapevine moth, Lobesia botrana, is considered a harmful pest for vineyards in Chile as well as in North America and Europe. Currently, monitoring and control methods of L. botrana are based on its main sex pheromone component, being effective for low population densities. In order to improve control methods, antennal olfactory proteins in moths, such as odorant-binding proteins (OBPs) and odorant receptors (ORs) have been studied as promising targets for the discovery of new potent semiochemicals, which have not been reported for L. botrana. Therefore, the objective of this study was to identify the repertoire of proteins related to chemoreception in L. botrana by antennal transcriptome and analyze the relative expression of OBPs and CSPs in male and female antennae. Through next-generation sequencing of the antennal transcriptome by Ilumina HiSeq2500 we identified a total of 118 chemoreceptors, from which 61, 42 and 15 transcripts are related to ORs, ionotropic receptors (IRs) and gustatory receptors (GRs), respectively. Furthermore, RNA-Seq data revealed 35 transcripts for OBPs and 18 for chemosensory proteins (CSPs). Analysis by qRT-PCR showed 20 OBPs significantly expressed in female antennae, while 5 were more expressed in males. Similarly, most of the CSPs were significantly expressed in female than male antennae. All the olfactory-related sequences were compared with homologs and their phylogenetic relationships elucidated. Finally, our findings in relation to the improvement of L. botrana management are discussed.