Analysis of Rhodopsin G Protein-Coupled Receptor Orthologs Reveals Semiochemical Peptides for Parasite (Schistosoma Mansoni) and Host (Biomphalaria Glabrata) Interplay

Background: Schistosomiasis is a medically significant disease caused by helminth parasites of the genus Schistosoma. The schistosome life cycle requires chemically mediated interactions with an intermediate (aquatic snail) and definitive (human) host. Blocking parasite development within the snail stage requires improved understanding of the interactions between the snail host and the Schistosoma water-borne free-living form (miracidium). Innovations in snail genomics and aquatic chemical communication provide an ideal opportunity to explore snail-parasite coevolution at the molecular level. Rhodopsin G protein-coupled receptors (GPCRs) are of particular interest in studying how trematode parasites navigate towards their snail hosts. The potential role of GPCRs in parasites makes them candidate targets for new antihelminthics that disrupt the intermediate host life-cycle stages, thus preventing subsequent human infections.Results: A genomic-bioinformatic approach was used to identify GPCR orthologs between the snail Biomphalaria glabrata and miracidia of its obligate parasite Schistosoma mansoni. We show that 8 S. mansoni rhodopsin GPCRs expressed within the miracidial stage share overall amino acid similarity with 8 different B. glabrata rhodopsin GPCRs, particularly within transmembrane domains, suggesting conserved structural features. These GPCRs include an orphan peptide receptor as well as several with strong sequence homologies with rhabdomeric opsin receptors, a serotonin receptor, a sulfakinin (SK) receptor, an allatostatin-A (buccalin) receptor and an FMRFamide receptor. Buccalin and FMRFa peptides were identified in water conditioned by B. glabrata, and we show synthetic buccalin and FMRFa can stimulate significant rates of change of direction and turn-back responses in S. mansoni miracidia.Conclusions: Ortholog GPCRs were identified in S. mansoni miracidia and B. glabrata. These GPCRs may detect similar ligands, including snail-derived odorants that could facilitate miracidial host finding. These results lay the foundation for future research elucidating the mechanisms by which GPCRs mediate host finding which can lead to the potential development of novel anti-schistosome interventions.

Identification and expression of odorant binding proteins in the egg-parasitoid Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae, Telenominae)

Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae) is an egg-parasitoid of the southern green stink bug, Nezara viridula (Linneaus) (Hemiptera, Pentatomidae). Many behaviors associated with female T. basalis host-finding and acceptance are mediated by chemosensory pathways, for which olfactory, gustatory and ionotropic receptors have been previously identified. Odorant binding proteins (OBPs) are small, globular proteins, one of the functions of which is the transport of odorant ligands through the aqueous lymph of chemosensory sensilla to these receptors. We identified 18 classical OBP sequences in the T. basalis genome and transcriptomes sharing an average 26.8% pairwise identity. Gene tree analyses suggest very limited lineage-specific expansion and identify potential orthologs among other scelionids and Hymenoptera. Transcriptome mapping and qPCR comparison of expression levels in antennae and bodies of both sexes determine that at least five TbOBPs are preferentially expressed in the female antennae. These are, therefore, prime candidates for further study to determine their role in detecting host-produced semiochemicals.

Intercropping With Peppermint Increases Ground Dwelling Insect and Pollinator Abundance and Decreases Drosophila suzukii in Fruit

Intercropping can be used to reduce pest insects within agricultural systems, e.g., through deterring pests directly or by increasing habitat for their natural enemies. For example, plant produced volatile organic compounds (VOCs) can deter or confuse host-finding by insects through olfactory disruption. Drosophila suzukii is an invasive fruit fly of agricultural concern as it can lay its eggs in both ripening and fresh fruits and, uses olfactory cues to identify its wide range of host plants. Peppermint plants (Mentha × piperita) produce high levels of VOCs while growing and may, therefore, be suitable as an intercrop to reduce D. suzukii infestations in the field, as peppermint essential oil VOCs have previously been shown to deter D. suzukii in olfactory trials. We conducted a field intercropping experiment to evaluate the effectiveness of peppermint plants compared to traditional ryegrass/clover mixes in reducing D. suzukii oviposition in the field, and the effect of peppermint intercrops on other invertebrates. In the field, we monitored sentinel fruit baits weekly for D. suzukii infestation. Additionally, we monitored intercropping effects on the invertebrate community through weekly pitfall trap collection and through a pollinator point survey. We monitored for local, farm level presence of D. suzukii through apple cider vinegar traps within crop fields and along hedgerows and found high abundance of D. suzukii (>3,000 individuals trapped). Peppermint intercrops had fewer D. suzukii emerge from fruit baits and supported greater beneficial insect abundance (predators and pollinators) compared to ryegrass/clover. However, levels of D. suzukii were low across both intercrop types. Overall, we found that peppermint intercrops could be a potential aromatic intercrop used to reduce D. suzukii adult emergence from fruit compared to conventional ryegrass/clover mixes, however this trial should be replicated over multiple growing seasons, geographic locations, and host fruits. Furthermore, further study should determine the effects of the intercrop on the focal crop of interest.

Characterization of Anopheles stephensi Odorant Receptor 8, an Abundant Component of the Mouthpart Chemosensory Transcriptome

Several mosquito species within the genus Anopheles are vectors for human malaria, and the spread of this disease is driven by the propensity of certain species to feed preferentially on humans. The study of olfaction in mosquitoes is important to understand dynamics of host-seeking and host-selection; however, the majority of these studies focus on Anopheles gambiae or An. coluzzii, both vectors of malaria in Sub-Saharan Africa. Other malaria vectors may recognize different chemical cues from potential hosts; therefore, in this study, we investigated An. stephensi, the south Asian malaria mosquito. We specifically focused on the mouthparts (primarily the maxillary palp and labella) that have been much less investigated compared to the antennae but are also important for host-seeking. To provide a broad view of chemoreceptor expression, RNAseq was used to examine the transcriptomes from the mouthparts of host-seeking females, blood-fed females, and males. Notably, AsOr8 had a high transcript abundance in all transcriptomes and was, therefore, cloned and expressed in the Drosophila empty neuron system. This permitted characterization with a panel of odorants that were selected, in part, for their presence in the human odor profile. The responsiveness of AsOr8 to odorants was highly similar to An. gambiae Or8 (AgOr8), except for sulcatone, which was detected by AsOr8 but not AgOr8. Subtle differences in the receptor sensitivity to specific odorants may provide clues to species- or strain-specific approaches to host-seeking and host selection. Further exploration of the profile of An. stephensi chemosensory proteins may yield a better understanding of how different malaria vectors navigate host-finding and host-choice.

FMRFamide-Like Peptide 22 Influences the Head Movement, Host Finding, and Infection of Heterodera glycines

The FMRFamide-like peptides (FLPs) represent the largest family of nematode neuropeptides and are involved in multiple parasitic activities. The immunoreactivity to FMRFamide within the nervous system of Heterodera glycines, the most economically damaging parasite of soybean [Glycine max L. (Merr)], has been reported in previous research. However, the family of genes encoding FLPs of H. glycines were not identified and functionally characterized. In this study, an FLP encoding gene Hg-flp-22 was cloned from H. glycines, and its functional characterization was uncovered by using in vitro RNA interference and application of synthetic peptides. Bioinformatics analysis showed that flp-22 is widely expressed in multiple nematode species, where they encode the highly conserved KWMRFamide motifs. Quantitative real-time (qRT)-PCR results revealed that Hg-flp-22 was highly expressed in the infective second-stage juveniles (J2s) and adult males. Silencing of Hg-flp-22 resulted in the reduced movement of J2s to the host root and reduced penetration ability, as well as a reduction in their subsequent number of females. Behavior and infection assays demonstrated that application of synthetic peptides Hg-FLP-22b (TPQGKWMRFa) and Hg-FLP-22c (KMAIEGGKWVRFa) significantly increased the head movement frequency and host invasion abilities in H. glycines but not in Meloidogyne incognita. In addition, the number of H. glycines females on the host roots was found to be significantly higher in Hg-FLP-22b treated nematodes than the ddH2O-treated control J2s. These results presented in this study elucidated that Hg-flp-22 plays a role in regulating locomotion and infection of H. glycines. This suggests the potential of FLP signaling as putative control targets for H. glycines in soybean production.

Neural and behavioural responses of the pollen-specialist bee Andrena vaga to Salix odours

An effective means of finding food is crucial for organisms. Whereas specialized animals select a small number of potentially available food sources, generalists use a broader range. Specialist (oligolectic) bees forage on a small range of flowering plants for pollen and use primarily olfactory and visual cues to locate their host flowers. So far, however, little is known about the specific cues oligoleges use to discriminate between hosts and non-hosts and how floral scent compounds of hosts and non-hosts are processed in the bees’ olfactory system. In this study, we recorded physiological responses of the antennae (electroantennographic detection coupled to gas chromatography GC-EAD) and in the brain (optical imaging, GC-imaging), and studied host-finding behaviour of oligolectic Andrena vaga bees, a specialist on Salix plants. In total, we detected 37 physiologically active compounds in host and non-host scents. 4-Oxoisophorone, a common constituent in the scent of many Salix species, evoked strong responses in the antennal lobe glomeruli of A. vaga, but not the generalist honeybee Apis mellifera. The specific glomerular responses to 4-oxoisophorone in natural Salix scents reveals a high degree of specialization in A. vaga for this typical Salix odorant component. In behavioural experiments, we found olfactory cues to be the key attractants for A. vaga to Salix hosts, which are also used to discriminate between hosts and non-hosts, and demonstrated a behavioural activity for 4-oxoisophorone. A high sensitivity to floral scents enables the specialized bees to effectively find flowers and it appears that A. vaga bees are highly tuned to 4-oxoisophorone at a very low concentration.

Disruption of the odorant coreceptor Orco impairs foraging and host finding behaviors in the New World screwworm fly

The evolution of obligate ectoparasitism in blowflies (Diptera: Calliphoridae) has intrigued scientists for over a century, and surprisingly, the genetics underlying this lifestyle remain largely unknown. Blowflies use odors to locate food and oviposition sites; therefore, olfaction might have played a central role in niche specialization within the group. In insects, the coreceptor Orco is a required partner for all odorant receptors (ORs), a major gene family involved in olfactory-evoked behaviors. Hence, we characterized the Orco gene in the New World screwworm, Cochliomyia hominivorax, a blowfly that is an obligate ectoparasite of warm-blooded animals. In contrast, most of the closely related blowflies are scavengers that lay their eggs on dead animals. We show that the screwworm Orco orthologue (ChomOrco) is highly conserved within Diptera, showing signals of strong purifying selection. Expression of ChomOrco is broadly detectable in chemosensory appendages, and is related to morphological, developmental, and behavioral aspects of the screwworm biology. We used CRISPR/Cas9 to disrupt ChomOrco and evaluate the consequences of losing the OR function on screwworm behavior. In two-choice assays, Orco mutants displayed an impaired response to floral-like and animal host-associated odors, suggesting that OR-mediated olfaction is involved in foraging and host-seeking behaviors in C. hominivorax. These results broaden our understanding of the chemoreception basis of niche occupancy by blowflies.

An Assessment of Steinernema rarum as a Biocontrol Agent in Sugarcane with Focus on Sphenophorus levis, Host-Finding Ability, Compatibility with Vinasse and Field Efficacy

In Brazil, countless insect species attack and damage sugarcane (Saccharum officinarum L.), which is an extremely important crop since it is planted on more than 10 million hectares. Among these insects, the sugarcane billbug, Sphenophorus levis (Coleoptera: Curculionidae), is of great importance as the larvae open tunnels in the rhizome of the plant, causing high damage and losses. This insect is attracted mainly to vinasse, which is the liquid fraction generated from the alcohol production and discarded onto the sugarcane fields for fertigation. Toward a novel control method for S. levis, the native entomopathogenic nematode Steinernema rarum (Pam 25) was compared with S. carpocapsae (IL 1) and Heterorhabditis bacteriophora (HBEN01) in respect to their ability to search for larvae of two insect hosts (Galleria mellonella and S. levis) within the cane rhizome. The selected nematode S. rarum was also assessed for rate effects, its survival in vinasse and field efficacy to control sugarcane pests S. levis, Hyponeuma taltula (Lepidoptera, Erebidae) and Leucothyreus alvarengai (Coleoptera: Melolonthidae). Steinernema rarum exhibited superior virulence to G. mellonella and S. levis larvae inserted into the cane rhizomes (75–78% mortality) compared to S. carpocapsae (30–53%) and H. bacteriophora (18–28%). Vinasse affected S. rarum when infective juveniles were suspended in the liquid compost for more than 6 h but did not affect the nematode when kept on the straw and soil treated with the compost. Steinernema rarum tested at 1–3 × 108 infective juveniles/ha on the sugarcane field caused 74.1, 56.3 and 50.6% control of S. levis, L. alvarengai and H. taltula, respectively.