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IntroductionPreviously, we documented that the malaria vector Anopheles gambiae responds to volatile emissions from the root exudate water of the invasive plant, Parthenium hysterophorus. However, the origin of the volatiles remains to be investigated. Here, we isolated bacteria from the root exudate water of the plant, test the influence of their volatiles in gravid An. gambiae oviposition, and examined relationships between volatile profiles and oviposition.MethodsBacteria from root exudate water of P. hysterophorus were isolated using culture on Luria Bertani medium and identified by sequencing the 16S rRNA gene. Cultures of individual isolates were evaluated for egg laying response by gravid An. gambiae and number of eggs laid compared using generalized linear models relative to those in crude bacteria-mixture. Headspace volatile emissions of the bacterial isolates were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) and relationships between volatile organic compound (VOC) profiles and gravid mosquito oviposition examined using Random Forest Analysis. Proximate analysis was performed to assess the difference in volatile chemistry among the different isolates.ResultsThree isolates were identified as Gram-negative bacteria belonging to two families: Enterobacteriaceae (Enterobacter sp. and Enterobacter mori) and Alcaligenaceae (Alcaligens aquatilis). An. gambiae laid 3-fold more eggs in cultures of A. aquatilis than in those of Enterobacter sp. In turn, approx. 4-fold more eggs were laid in cultures of E. mori than A. aquatilis. Overall, 16 VOCs were identified in the headspace of the isolates belonging to the chemical classes benzenoids, pyrazines, aldehydes, terpenes, alcohols, alkanes, and indoles. Random Forest Analysis identified 10 compounds contributing the most to the attraction of odors of the bacteria isolates to oviposition. Specifically, dodecane and indole were emitted in higher amounts in odors of Enterobacter sp than the other two species. Proximate analysis revealed differential attraction of the isolates on the gravid mosquito to be associated with their volatile profiles.ConclusionOur results provide first report of E. mori or A. aquatilis mediating attractive oviposition responses in An. gambiae in support of the important role microbes play in insect oviposition. The potential use of the microbes and associated volatiles in malaria vector management needs further investigation.

Phlebotomine sand flies are medically important as vectors of the protozoan parasites that cause leishmaniasis and other bacterial and viral pathogens. Previous work demonstrated that both sexes of certain species of sandflies are attracted to 1-octen-3-ol (octenol). Since 1-octen-3-ol exists as two enantiomeric isomers ─ ((R)-(-)- (R-form) and (S)-(+)- (S-form), we tested the hypothesis that the two enantiomeric forms and racemic mixture (R/S) attracted different sand fly species. We carried out field trials in a leishmaniasis endemic foci in Baringo County, Kenya. In a randomized design, trap captures of sandflies in CDC light traps baited with the R-, S- and racemic (R/S) forms of 1-octen-3-ol in hexane varied with the form and dose of the compound. Interestingly, of the captured species, only Phlebotomus martini, the vector of the parasite causing visceral leishmaniasis, exhibited a dose-dependent response to octenol; captures of both sexes of the species being generally 1.7-fold higher with the R- than S-form. There was no significant effect of treatment on captures of Sergentomyia species (S. schwetzi, S. antennata, S, clydei). Our findings have implications for surveillance of sandfly populations as part of leishmaniasis epidemiologic investigation.

IntroductionCulicoides biting midges (Diptera: Ceratopogonidae) are vectors of arboviral pathogens that primarily affect livestock represented by Schmallenberg virus (SBV), epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV). In Kenya, studies examining the bionomic features of Culicoides including species diversity, blood-feeding habits, and association with viruses are limited.MethodsAdult Culicoides were surveyed using CDC light traps in two semi-arid ecologies, Baringo and Kajiado counties, in Kenya. Blood-fed specimens were analysed through polymerase chain reaction (PCR) and sequencing of cytochrome oxidase subunit 1 (cox1) barcoding region. Culicoides pools were screened for virus infection by generic RT-PCR and next-generation sequencing (NGS).ResultsAnalysis of blood-fed specimens confirmed that midges had fed on cattle, goats, sheep, zebra, and birds. Cox1 barcoding of the sampled specimens revealed the presence of known vectors of BTV and epizootic hemorrhagic disease virus (EHDV) including species in the Imicola group (Culicoides imicola) and Schultzei group (C. enderleni, C. kingi, and C. chultzei). Culicoides leucostictus and a cryptic species distantly related to the Imicola group were also identified. Screening of generated pools (11,006 individuals assigned to 333 pools) by generic RT-PCR revealed presence of seven phylogenetically distinct viruses grouping in the genera Goukovirus, Pacuvirus and Orthobunyavirus. The viruses showed an overall minimum infection rate (MIR) of 7.0% (66/333, 95% confidence interval (CI) 5.5-8.9). In addition, full coding sequences of two new iflaviruses, tentatively named Oloisinyai_1 and Oloisinyai_2, were generated by next-generation sequencing (NGS) from individual homogenate of Culicoides pool.ConclusionThe results indicate a high genetic diversity of viruses in Kenyan biting midges. Further insights into host-vector-virus interactions as well as investigations on the potential clinical significance of the detected viruses are warranted.

Intraguild interactions are known to trigger competition among phytophagous insects that depend on the same food source, which may have implications on natural enemy performance. Here, we tested the hypothesis that intraguild interactions between the greenhouse whitefly, Trialeurodes vaporariorum, and the South American tomato pinworm, Phthorimaea absoluta on the host plant tomato would affect performance of the pinworm ectoparasitoid Bracon nigricans. In oviposition bioassays, P. absoluta females significantly laid more eggs and were attracted to tomato plants infested with T. vaporariorum than uninfested plants. Additionally, T. vaporariorum infestation decreased B. nigricans parasitism on P. absoluta larvae and attraction to tomato plants. In coupled gas chromatography-electroantennographic detection (GC-EAD) recordings, antennae of P. absoluta and B. nigricans females detected 18 and 15 components, respectively from the herbivore-induced volatiles, which were identified by GC-mass spectrometry (GC-MS) as green leaf volatiles (GLVs) and terpenes. In dose-response behavioral assays, P. absoluta and B. nigricans responses varied with the composition and concentration of blends and individual compounds tested. Whereas P. absoluta was attracted to GLVs and terpenes, B. nigricans avoided most of these chemicals, especially the GLV (E)-2-hexenal and terpenes δ-3-carene, and (E)-caryophyllene. The results suggest that herbivore intraguild interactions can influence the behavioral ecology and performance of natural enemies which may have evolutionary significance. Also, these specific GLVs and terpenes can be exploited in the management of P. absoluta.

The tomato leafminer, Tuta absoluta is a destructive invasive pest of cultivated tomato and other Solanaceae plants, with yield losses of 80-100%. Mirid predators are key natural enemies of T. absoluta, but they also feed on host plants in the absence of their prey. Management of T. absoluta is a challenge due to its high biotic potential, resistance to many insecticides and the absence of sufficiently adapted auxiliary fauna in its new dispersion zones. Olfaction plays an important role in the tritrophic interaction between tomato, its herbivore pest T. absoluta and its mirid predators, which can be influenced by non-host plant odors. However, how non-host odours shape this interaction is poorly understood. Previously, we had demonstrated belowground crop protection properties of certain Asteraceae plants against the root-knot nematode Meloidogyne incognita, pest of tomato and other Solanaceae plants. Additionally, Asteraceae plants impact negatively on feeding behavior of above-ground pests of Solanaceae plants, including the greenhouse whitefly (Trialeurodes vaporariorum) and green peach aphid (Myzus persicae). Here, we tested the hypothesis that foliar volatiles from some of these non-host Asteraceae plants can influence the tomato-T. absoluta-mirid predator tritrophic interaction. In olfactometer assays, T. absoluta females were attracted to volatiles of the Solanaceae host plants tomato and giant nightshade but avoided volatiles of the Asteraceae plants, blackjack and marigold, and the positive control, wild tomato, when tested alone or in combination with the host plants. Coupled gas chromatography-mass spectrometry analysis showed that host and non-host plants varied in their emission of volatiles, mainly monoterpenes and sesquiterpenes. Random forest analysis combined with behavioral assays identified monoterpenes as the host plant attractive blend to T. absoluta and its mirid predator, with sesquiterpenes identified as the non-host plant repellent blend against T. absoluta. Contrastingly, the mirid predator was indifferent to the non-host plant repellent sesquiterpenes. Our findings indicate that terpenes influence the tomato-T. absoluta-mirid predator tritrophic interaction. Further, our results emphasize the importance of studying crop protection from a holistic approach to identify companion crops that serve multi-functional roles.

Trypanosomes are important global livestock and human pathogens of public health importance. Elucidating the chemical mechanisms of trypanosome-relevant host interactions can enhance the design and development of a novel, next-generation trypanosomosis diagnostics. However, it is unknown how trypanosome infection affects livestock volatile odors. Here, we show that Trypanosoma congolense and Trypanosoma vivax infections induced dihydro-β- ionone and junenol, while abundance of dihydro-α-ionone, phenolics, p-cresol, and 3-propylphenol significantly elevated in cow urine. These biomarkers of trypanosome infection are conserved in cow breath and the urine metabolites of naturally infected cows, regardless of population, diet, or environment differences. Furthermore, treating trypanosome-infected cows reduced the levels of these indicators back to the pre-infection levels. Finally, we demonstrated that the potential of some specific biomarkers of phenolic origin may be used to detect active trypanosome infections, including low-level infections that are not detectable by microscopy. The sensitivity and specificity of biomarkers detection are suited for rapid, robust, and non-invasive trypanosomosis diagnosis under field conditions.

Insects that transmit many of the world’s deadliest animal diseases, for instance trypanosomosis, find their suitable hosts and avoid non-preferred hosts mostly through olfactory cues. The waterbuck repellent blend (WRB) comprising geranylacetone, guaiacol, pentanoic acid, and δ-octalactone derived from waterbuck skin odor is a repellent to some savannah-adapted tsetse flies and reduces trap catches of riverine species. However, the cellular and molecular mechanisms associated with detection and coding of the repellent odors remain to be elucidated. Here, we demonstrated that WRB inhibited blood feeding in both Glossina pallidipes Austen, 1903 and Glossina fuscipes fuscipes Newstead, 1910. Using the DREAM (Deorphanization of Receptors based on Expression Alterations in odorant receptor mRNA levels) technique, combined with ortholog comparison and molecular docking, we predicted the putative odorant receptors (ORs) for the WRB in G. f. fuscipes, a non-model insect. We show that exposure of G. f. fuscipes in vivo to WRB odorant resulted in up- and downregulation of mRNA transcript of several ORs. The WRB component with strong feeding inhibition altered mRNA transcript differently as compared to an attractant odor, showing these two odors of opposing valence already segregate at the cellular and molecular levels. Furthermore, molecular dynamics simulations demonstrated that the predicted ligand–OR binding pockets consisted mostly of hydrophobic residues with a few hydrogen bonds but a stable interaction. Finally, our electrophysiological response showed the olfactory sensory neurons of G. f. fuscipes tuned to the tsetse repellent components in different sensitivity and selectivity.

Potato (Solanum tuberosum) is a widely consumed staple food crop worldwide whose production is threatened by potato cyst nematodes (PCN). To infect a host, PCN eggs first need to be stimulated to hatch by chemical components in the host root exudates, yet it remains unknown how most root exudate components influence PCN behavior. Here, we evaluated the influence of eight compounds identified by LC-QqQ-MS in the root exudate of potato on the hatching response of the PCN, Globodera rostochiensis at varying doses. The eight compounds included the amino acids tyrosine, tryptophan and phenylalanine; phytohormones zeatin and methyl dihydrojasmonate; steroidal glycoalkaloids α-solanine and α-chaconine and the steroidal alkaloid solanidine. We additionally tested two other Solanaceae steroidal alkaloids, solasodine and tomatidine, previously identified in the root exudates of tomato, an alternative host for PCN. In dose-response assays with the individual compounds, the known PCN hatching factors α-chaconine and α-solanine stimulated the highest number of eggs to hatch, ∼47 and ∼42%, respectively, whereas the steroidal alkaloids (aglycones), solanidine and solasodine and potato root exudate (PRE) were intermediate, 28% each and 21%, respectively, with tomatidine eliciting the lowest hatching response 13%. However, ∼60% of the hatched juveniles failed to emerge from the cyst, which was compound- and concentration-dependent. The amino acids, phytohormones and the negative control (1% DMSO in water), however, were generally non-stimulatory. The use of steroidal glycoalkaloids and their aglycones in the suicidal hatching of PCN offers promise as an environmentally sustainable approach to manage this pest.

Oviposition site selection by gravid female insects is an important determinant in species distribution, abundance, and population dynamics. Females may assess the suitability of a potential oviposition substrate by using cues from conspecific or heterospecific individuals already present. Here, we assessed whether the presence of conspecific or heterospecific larvae and parasites influenced oviposition decisions by the stable fly, Stomoxys calcitrans (Linneaus). Using dual and multiple-choice oviposition bioassays, we found that gravid female S. calcitrans avoided substrates with conspecific larvae, the larvae of house flies, Musca domestica (Linneaus), and the mite Macrocheles muscaedomesticae (Scopoli). Avoidance of conspecific and heterospecific larvae persisted in the dark, suggesting that this behavior is mediated by chemical rather than visual cues. When we reared S. calcitrans in the presence of conspecific larvae and the larvae of house flies at different densities we found that this negatively affected emergence time, larval weight, larval survival, pupal weight, pupal survival, and adult weight. We also demonstrated that individuals of S. calcitrans developed in the presence of mites exhibited low egg hatchability, and poor larval and adult survival. Our study provides additional support for the “preference-performance” hypothesis in S. calcitrans, with gravid females preferring to lay eggs on a substrate that will enhance offspring fitness. We recommend that the chemical cues involved in avoidance by gravid female S. calcitrans of substrates with conspecific and heterospecific larvae should be elucidated. This could lead to the discovery of repellent chemicals important for S. calcitrans management.