Putative bloodmeal sources in Glossina austeni tsetse fly of Arabuko Sokoke National Reserve in Kenya.

Tsetse flies, the sole biological vectors of trypanosomiasis, are predominantly controlled using visual traps and targets baited with attractant lures. Formulation of the lures is informed by compositions of odors from vertebrate hosts preferred by specific tsetse species. However, there are no effective lures for Glossina austeni, a major vector of trypanosomiasis along eastern-coastal region of Africa. Formulation of the lure can be informed by knowledge of G. austeni, preferred vertebrate hosts. We thus sought to understand these hosts by assessment of putative bloodmeal sources of this tsetse fly in Arabuko Sokoke National Reserve where this species is naturally present. We sampled tsetse flies using NGU traps, isolated non-teneral G. austeni flies based on their feeding status, and identified vertebrate source of bloodmeals in their midgut contents using vertebrate 16S rRNA-PCR High-Resolution Melting analysis. We analyzed the relative vertebrate species frequencies in the bloodmeals using Fisher's exact tests. Overall, we trapped 122 flies, most of which (66.39%) were non-teneral, among which we successfully identified the vertebrate bloodmeals in 30 samples. Specifically, we detected putative suni antelope (Neotragus moschatus), harnessed bushbuck (Tragelaphus scriptus), buffalo (Syncerus caffer) and cattle (Bos taurus) derived bloodmeals. Putative suni antelope bloodmeals were significantly more frequent (63.22%), than those of the harnessed bushbuck (23.33%), buffalo (10.00%) or cattle (3.33%) (p < 0.05 Fisher's exact tests) among the samples analyzed. Suni antelope thus appears to predominate vertebrate bloodmeal source for G. austeni in the reserve, coincident with findings reported elsewhere, and is therefore a viable candidate for bioprospecting for G. austeni responsive attractants.

Expression of the Fab enzymes (Fab I and Fab Z) from Plasmodium falciparum after exposure to Artemisia afra plant extracts and drugs screening.

The emergence and spread of drug resistance of the malaria parasite to the main treatment emphasize the need to develop new antimalarial drugs. In this context, the fatty acid biosynthesis (FAS_II) pathway of the malaria parasite is one of the ideal targets due to its crucial role in parasite survival. In this study, we report the expression and the affinity binding of Fab_I and Fab_Z after exposure to the parasite with different extracts of the Artemisia afra. The parasites were exposed for 2 days to different extracts. Gene expression was done to determine the level of expression of the fab enzymes after treatments. A GCMS was run to determine the different compounds of the plant extracts, followed by a virtual screening between the fab enzymes and the active compounds using Pyrex. The results showed different expression patterns of the Fab enzymes. Fab_I expression was downregulated in the W2 and D6 strains by the ethanolic extract but was increased by Hexane and DCM extracts. A different expression pattern was observed for Fab_Z. It was all upregulated except in the D6 strain when exposed to the ethanolic and hexane extracts. Virtual screening showed an affinity with many compounds. Hits compounds with high binding energy were detected. 11alphaHydroxyprogesterone and Aspidospermidin-17-ol were found to have high binding energy with Fab_I respectively (- 10.7 kcal/mol; - 10.2 kcal/mol). Fab_Z shows also high affinity with 11alpha-Hydroxyprogesterone (- 10 kcal/mol) and Thiourea (- 8.4 kcal/mol). This study shows the potential of A. afra to be used as a new source of novel antimalarial compounds.

Bloodmeal host identities among sympatric Glossina austeni and Glossina pallidipes tsetse flies in Shimba Hills National Reserve, Kwale, Kenya.

Odor from preferred/non-preferred tsetse fly vertebrate hosts have been exploited in R&D of attractants/repellents of the fly for human and livestock protection. Odors from vertebrate hosts of Glossina austeni and Glossina pallidipes tsetse flies can facilitate formulation of novel attractants effective against G. austeni or improvement of existing attractant blends for G. pallidipes. We compared vertebrate blood meal sources of both fly species at Shimba Hills National Reserve, Kenya, to establish putative preferred host of either species, hence potential source of G. austeni or G. pallidipes specific odors. We trapped sympatric adult flies in 2021 and 2022 using NGU traps/sticky panels baited with POCA, collected their blood meals and characterize the meals using HRM vertebrate 16S rRNA- PCR (for host identification), and compared host profiles using GLM and Fisher's exact tests. We collected 168 and 62 sympatric G. pallidipes and G. austeni with bloodmeal, respectively in 2021 and, 230 and 142 respectively in 2022. In 2021, we identified putative hosts of 65.48 and 69.35 % of the G. pallidipes and G. austeni respectively and 82.61 and 80.28%, respectively in 2022. In 2021, we detected harnessed bushbuck, buffalo, common warthog and cattle putative host bloodmeals, and additionally bushpig and suni antelope bloodmeals in 2022. Putative vertebrate bloodmeal sources were significantly different by tsetse fly species (χ2(1, N=457) = 43.215, p < 0.001) and sampling year (χ2(1, N=457) = 8.044, p = 0.005). Frequency of common warthog bloodmeals was higher in G. pallidipes (65.79 %) than G. austeni (38.60%), and that of suni antelope and harnessed bushbuck putative bloodmeals higher in G. austeni (21.05-28.07%) than in G. pallidipes (6.84 - 17.37%) in 2022. There was an apparent change in putative feeding preference/host choices in both fly species between 2021 and 2022. Host bloodmeals in G. pallidipes or G. austeni predominantly from putative harnessed bushbuck, suni antelope or common warthog reveal these vertebrates with potential odors that can be harnessed and formulated into appropriate attractants for respective species and integrated into routine control regiment for G. pallidipes and/or G. austeni.

Annotations of novel antennae-expressed genes in male Glossina morsitans morsitans tsetse flies.

Tsetse flies use antennal expressed genes to navigate their environment. While most canonical genes associated with chemoreception are annotated, potential gaps with important antennal genes are uncharacterized in Glossina morsitans morsitans. We generated antennae-specific transcriptomes from adult male G. m. morsitans flies fed/unfed on bloodmeal and/or exposed to an attractant (ε-nonalactone), a repellant (δ-nonalactone) or paraffin diluent. Using bioinformatics approach, we mapped raw reads onto G. m. morsitans gene-set from VectorBase and collected un-mapped reads (constituting the gaps in annotation). We de novo assembled these reads (un-mapped) into transcript and identified corresponding genes of the transcripts in G. m. morsitans gene-set and protein homologs in UniProt protein database to further annotate the gaps. We predicted potential protein-coding gene regions associated with these transcripts in G. m. morsitans genome, annotated/curated these genes and identified their putative annotated orthologs/homologs in Drosophila melanogaster, Musca domestica or Anopheles gambiae genomes. We finally evaluated differential expression of the novel genes in relation to odor exposures relative to no-odor control (unfed flies). About 45.21% of the sequenced reads had no corresponding transcripts within G. m. morsitans gene-set, corresponding to the gap in existing annotation of the tsetse fly genome. The total reads assembled into 72,428 unique transcripts, most (74.43%) of which had no corresponding genes in the UniProt database. We annotated/curated 592 genes from these transcripts, among which 202 were novel while 390 were improvements of existing genes in the G. m. morsitans genome. Among the novel genes, 94 had orthologs in D. melanogaster, M. domestica or An. gambiae while 88 had homologs in UniProt. These orthologs were putatively associated with oxidative regulation, protein synthesis, transcriptional and/or translational regulation, detoxification and metal ion binding, thus providing insight into their specific roles in antennal physiological processes in male G. m. morsitans. A novel gene (GMOY014237.R1396) was differentially expressed in response to the attractant. We thus established significant gaps in G. m. morsitans genome annotation and identified novel male antennae-expressed genes in the genome, among which > 53% (108) are potentially G. m. morsitans specific.

Electroantennogram and machine learning reveal a volatile blend mediating avoidance behavior by Tuta absoluta females to a wild tomato plant.

Tomato cultivation is threatened by the infestation of the nocturnal invasive tomato pinworm, Tuta absoluta. This study was based on field observations that a wild tomato plant, Solanum lycopersicum var. cerasiforme, grown in the Mount Kenya region, Kenya, is less attacked by T. absoluta, unlike the cultivated tomato plants like S. lycopersicum (var. Rambo F1). We hypothesized that the wild tomato plant may be actively avoided by gravid T. absoluta females because of the emission of repellent allelochemical constituents. Therefore, we compared infestation levels by the pest in field monocrops and intercrops of the two tomato genotypes, characterized the headspace volatiles, then determined the compounds detectable by the insect through gas chromatography-linked electroantennography (GC-EAG), and finally performed bioassays using a blend of four EAG-active compounds unique to the wild tomato. We found significant reductions in infestation levels in the monocrop of the wild tomato, and intercrops of wild and cultivated tomato plants compared to the monocrop of the cultivated tomato plant. Quantitative and qualitative differences were noted between volatiles of the wild and cultivated tomato plants, and between day and night volatile collections. The most discriminating compounds between the volatile treatments varied with the variable selection or machine learning methods used. In GC-EAG recordings, 16 compounds including hexanal, (Z)-3-hexenol, α-pinene, ß-myrcene, α-phellandrene, ß-phellandrene, (E)-ß-ocimene, terpinolene, limonene oxide, camphor, citronellal, methyl salicylate, (E)-ß-caryophyllene, and others tentatively identified as 3,7,7-Trimethyl-1,3,5-cycloheptatriene, germacrene D and cis-carvenone oxide were detected by antennae of T. absoluta females. Among these EAG-active compounds, (Z)-3-hexenol, α-pinene, α-phellandrene, limonene oxide, camphor, citronellal, (E)-ß-caryophyllene and ß-phellandrene are in the top 5 discriminating compounds highlighted by the machine learning methods. A blend of (Z)-3-hexenol, camphor, citronellal and limonene oxide detected only in the wild tomato showed dose-dependent repellence to T. absoluta females in wind tunnel. This study provides some groundwork for exploiting the allelochemicals of the wild tomato in the development of novel integrated pest management approaches against T. absoluta.

Perspectives on Odor-Based Control of Tsetse Flies in Africa.

Tsetse-transmitted trypanosomiases are among the most neglected tropical diseases in sub-Sahara Africa. Although all tsetse species are susceptible to trypanosome infections, their differential attraction/feeding preferences for different wildlife, domestic animals, and/or humans constitute critical determinants of trypanosomes species they predominantly transmit. Artificial bait technologies, based on long-range tsetse olfactory responses to natural cues emitted by preferred hosts and blends of synthetic versions that mimic these cues, have successfully been applied in attractant-odor-based ("pull" tactic) reduction of field populations of some tsetse species. Olfactory attribute associated with active avoidance of tsetse-refractory non-hosts has similarly been exploited in design of repellent-odor-based ("push" tactic) protection of livestock. These tactics have opened possibility of spatially strategic deployment of the two sets of odor baits in "push-pull" tactics. Possibility of developing blends with enhanced attraction and repellence compared with those associated with savannah tsetse fly hosts and non-hosts, respectively, have been explored, where structure activity and blends of different components generated two novel blends. The studies evaluated structure activity and blends of different components. One based on attractive constituents associated with buffalo (Syncerus caffer) comprised of ε-nonalactone, nonanoic acid, 2-nonanone (in 1:3:2 proportion) delivered together with acetone, which showed significantly better attractancy on savannah tsetse fly than the standard blend comprised of 3-propylphenol, octenol, p-cresol, and acetone (POCA). The other blend comprised of δ-nonalactone, heptanoic acid, 4-methylguaiacol and geranylacetone (in 6:4:2:1 proportion) was significantly more repellent than previously characterized blend based on tsetse fly refractory waterbuck (Kobus defassa) constituents (δ-octalactone, pentanoic acid, guaiacol and geranylacetone). So far, no effective attractants or repellents of riverine tsetse fly species have been characterized. Optimized attractant and repellent blends for savannah tsetse flies lay down useful groundwork for future development of the "push-pull" deployment tactic for area-wide control of tsetse flies. Better understanding of the physiological, cellular, and molecular basis of response in the tsetse fly to odors can potentially augment the current tsetse fly-control interventions.

Mosquitoes Larvicidal Activity of Ocimum kilimandscharicum Oil Formulation under Laboratory and Field-Simulated Conditions.

Mosquitoes are vectors of many severe diseases, including malaria, yellow as well as dengue fever, and lymphatic filariasis. The use of synthetic chemical insecticides for mosquito control has been associated with resistance development and detrimental human, and ecological effects. For a safer alternative, the emulsified Ocimum kilimandscharicum oil formulation was evaluated for its larvicidal activity. The oil was analyzed by GC and GC/MS. The formulations were evaluated against third instar mosquito larvae in the laboratory and later compared with Bacillus thuringiensis subsp. israelensis against An. gambiae under field-simulated conditions. Thirty-nine compounds were identified in the oil, the main ones being D-camphor (36.6%) and limonene (18.6%). The formulation showed significant larval mortalities against An. gambiae and An. arabiensis larvae with LC50 of 0.07 and 0.31 ppm, respectively, at 24 h. Under the field-simulated trial, within 24 h, the formulation showed 98% mortality while Bti had achieved 54%. On day three, it caused 100% mortality while Bti achieved 76.5%. The high bioactivity and sublethal toxic effects to offspring of treated mosquito larvae, in terms of disruption of larval morphological aspects, suggest the high potential of the formulation as a botanical larvicide. The formulation, thus, may provide a valuable alternative for the effective and eco-friendly control of disease vectors.

Semiochemical signatures associated with differential attraction of Anopheles gambiae to human feet.

BACKGROUND: Several human-produced volatiles have been reported to mediate the host-seeking process under laboratory conditions, yet no effective lure or repellent has been developed for field application. Previously, we found a gradation of the attractiveness of foot odors of different malaria free individuals to Anopheles gambiae sensu stricto Giles. In this study, foot odor of the individual with the most attractive 'smelly' feet to the An. gambiae was collected, analyzed and attractive blend components identified. METHODS: The foot odor of the individual with the most attractive 'smelly' feet to the An. gambiae was trapped on Porapak Q and analyzed by gas chromatography-linked mass spectrometry (GC-MS). Specific constituents perceived by the insect olfactory system were then identified by GC-linked to electro-antennography detector (GC-EAD) and characterized by GC-MS. The contribution of each constituent to the behavioral response of An. gambiae was assessed through subtractive assays under semi-field conditions in a screen-house using Counter Flow Geometry (CFG traps) baited with (i) the blend of all the EAD-active and (ii) other blends containing all components with exclusion of one component at a time. The number of mosquitoes trapped in the baited CFG traps were compared with those in the control traps. RESULTS: Eleven major and minor constituents: 2 carboxylic acids, six aldehydes, two ketones and one phenolic compound, were confirmed to be EAD-active. The contribution of each constituent to the behavioral response of An. gambiae was assessed through subtractive assays under semi- field conditions. Exclusion/ subtraction of one of the following compounds: i-butyric acid, i-valeric acid, n-octanal, n-nonanal, n-decanal, n-dodecanal, undecanal or n-tridecanal, from each blend led to reduction in the attractiveness of all the resulting blends, suggesting that all of them are critical/important for the attractiveness of the foot odor to An. gambiae mosquitoes. However, exclusion/subtraction of 4-ethoxyacetophenone, 4-ethylacetophenone and/or 2-methylphenol, led to significant enhancements in the attractiveness of the resulting blends, suggesting that each of these compounds had repellent effect on An. gambiae ss. Undecanal exhibited kairomonal activity at low natural concentrations under semi-field conditions but repellent activity at high unnatural conditions in the laboratory. Furthermore, the comparison of the mean mosquito catches in traps baited with the nine-component blend without 4-ethoxyacetophenone, 4-ethylacetophenone and the complete foot odor collection revealed that the former is significantly more attractive and confirmed the repellent effect of the two carbonyl compounds at low natural concentration levels. CONCLUSION: These results suggest that differential attractiveness of An. gambiae to human feet is due to qualitative and/or qualitative differences in the chemical compositions of the foot odors from individual human beings and relative proportions of the two chemical signatures (attractants versus repellents) as observed from the ratios of the bioactive components in the foot odors of the most attractive and least attractive individuals. Chemical signature means the ensemble of the compounds released by the organism in a specific physiological state. The chemical signature is emitter-dependent, but does not depend on receiver response. Thus, there is only one chemical signature for one individual or species that may eventually include inactive, attractive and repellent components for another organism. The nine-component attractive blend has a potential as an effective field bait for trapping of malaria vectors in human dwellings.

Characterization of a composite with enhanced attraction to savannah tsetse flies from constituents or analogues of tsetse refractory waterbuck (Kobus defassa) body odor.

Savannah tsetse flies avoid flying toward tsetse fly-refractory waterbuck (Kobus defassa) mediated by a repellent blend of volatile compounds in their body odor comprised of δ-octalactone, geranyl acetone, phenols (guaiacol and carvacrol), and homologues of carboxylic acids (C5-C10) and 2-alkanones (C8-C13). However, although the blends of carboxylic acids and that of 2-alkanones contributed incrementally to the repellency of the waterbuck odor to savannah tsetse flies, some waterbuck constituents (particularly, nonanoic acid and 2-nonanone) showed significant attractive properties. In another study, increasing the ring size of δ-octalactone from six to seven membered ring changed the activity of the resulting molecule (ε-nonalactone) on the savannah tsetse flies from repellency to attraction. In the present study, we first compared the effect of blending ε-nonalactone, nonanoic acid and 2-nonanone in 1:1 binary and 1:1:1 ternary combination on responses of Glossina pallidipes and Glossina morsitans morsitans tsetse flies in a two-choice wind tunnel. The compounds showed clear synergistic effects in the blends, with the ternary blend demonstrating higher attraction than the binary blends and individual compounds. Our follow up laboratory comparisons of tsetse fly responses to ternary combinations with different relative proportions of the three components showed that the blend in 1:3:2 proportion was most attractive relative to fermented cow urine (FCU) to both tsetse species. In our field experiments at Shimba Hills game reserve in Kenya, where G. pallidipes are dominant, the pattern of tsetse catches we obtained with different proportions of the three compounds were similar to those we observed in the laboratory. Interestingly, the three-component blend in 1:3:2 proportion when released at optimized rate of 13.71mg/h was 235% more attractive to G. pallidipes than a combination of POCA (3-n-Propylphenol, 1-Octen-3-ol, 4-Cresol, and Acetone) and fermented cattle urine (FCU). This constitutes a novel finding with potential for downstream deployment in bait technologies for more effective control of G. pallidipes, G. m. morsitans, and perhaps other savannah tsetse fly species, in 'pull' and 'pull-push' tactics.

Transcriptomic response of Anopheles gambiae sensu stricto mosquito larvae to Curry tree (Murraya koenigii) phytochemicals.

BACKGROUND: Insect growth regulators (IGRs) can control insect vector populations by disrupting growth and development in juvenile stages of the vectors. We previously identified and described the curry tree (Murraya koenigii (L.) Spreng) phytochemical leaf extract composition (neplanocin A, 3-(1-naphthyl)-L-alanine, lumiflavine, terezine C, agelaspongin and murrayazolinol), which disrupted growth and development in Anopheles gambiae sensu stricto mosquito larvae by inducing morphogenetic abnormalities, reducing locomotion and delaying pupation in the mosquito. Here, we attempted to establish the transcriptional process in the larvae that underpins these phenotypes in the mosquito. METHODS: We first exposed third-fourth instar larvae of the mosquito to the leaf extract and consequently the inherent phytochemicals (and corresponding non-exposed controls) in two independent biological replicates. We collected the larvae for our experiments sampled 24 h before peak pupation, which was 7 and 18 days post-exposure for controls and exposed larvae, respectively. The differences in duration to peak pupation were due to extract-induced growth delay in the larvae. The two study groups (exposed vs control) were consequently not age-matched. We then sequentially (i) isolated RNA (whole larvae) from each replicate treatment, (ii) sequenced the RNA on Illumina HiSeq platform, (iii) performed differential bioinformatics analyses between libraries (exposed vs control) and (iv) independently validated the transcriptome expression profiles through RT-qPCR. RESULTS: Our analyses revealed significant induction of transcripts predominantly associated with hard cuticular proteins, juvenile hormone esterases, immunity and detoxification in the larvae samples exposed to the extract relative to the non-exposed control samples. Our analysis also revealed alteration of pathways functionally associated with putrescine metabolism and structural constituents of the cuticle in the extract-exposed larvae relative to the non-exposed control, putatively linked to the exoskeleton and immune response in the larvae. The extract-exposed larvae also appeared to have suppressed pathways functionally associated with molting, cell division and growth in the larvae. However, given the age mismatch between the extract-exposed and non-exposed larvae, we can attribute the modulation of innate immune, detoxification, cuticular and associated transcripts and pathways we observed to effects of age differences among the larvae samples (exposed vs control) and to exposures of the larvae to the extract. CONCLUSIONS: The exposure treatment appears to disrupt cuticular development, immune response and oxidative stress pathways in Anopheles gambiae s.s larvae. These pathways can potentially be targeted in development of more efficacious curry tree phytochemical-based IGRs against An. gambiae s.s mosquito larvae.

Antifeedant and ovicidal activities of a new cassane and other compounds from Caesalpinia welwitschiana Oliv. and Caesalpinia bonduc L. against Tuta absoluta (Lepidoptera: Gelechiidae).

Methanolic extracts of liana of Caesalpinia welwitschiana and leaves of C. bonduc were found to possess moderate antifeedant and ovicidal activities against Tuta absoluta. Bioassay-guided isolation of constituents from the most active fraction of C. welwitschiana led to the identification of four known compounds [isobonducellin 1a and bonducellin 1 b, intricatinol 2, (-)-epigallocatechin-3-O-gallate 4] and one new constituent [welwitschianic acid 3]. The most active fraction of C. bonduc afforded two known constituents neocaesalpin L 5 and neocaesalpin A 6. The isolated structures were elucidated on the basis of their MS, UV, IR and 1 & 2 D NMR spectra and by comparison with literature data. Compounds 2, 4-6 were showed antifeedant and ovicidal properties against T. absoluta, some comparable to that of azadirachtin at 50, 100 and 200 ng/µl. Overall, the present study, conclude that the two species of the plant could be a promising source of eco-friendly botanical constituents.

Cyst nematode bio-communication with plants: implications for novel management approaches.

Bio-communication occurs when living organisms interact with each other, facilitated by the exchange of signals including visual, auditory, tactile and chemical. The most common form of bio-communication between organisms is mediated by chemical signals, commonly referred to as 'semiochemicals', and it involves an emitter releasing the chemical signal that is detected by a receiver leading to a phenotypic response in the latter organism. The quality and quantity of the chemical signal released may be influenced by abiotic and biotic factors. Bio-communication has been reported to occur in both above- and below-ground interactions and it can be exploited for the management of pests, such as cyst nematodes, which are pervasive soil-borne pests that cause significant crop production losses worldwide. Cyst nematode hatching and successful infection of hosts are biological processes that are largely influenced by semiochemicals including hatching stimulators, hatching inhibitors, attractants and repellents. These semiochemicals can be used to disrupt interactions between host plants and cyst nematodes. Advances in RNAi techniques such as host-induced gene silencing to interfere with cyst nematode hatching and host location can also be exploited for development of synthetic resistant host cultivars. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Repellence and fumigant toxicity of essential oils of Ocimum gratissimum and Ocimum kilimandscharicum on Tuta absoluta (Lepidoptera: Gelechiidae).

Tuta absoluta Meyrick originates in South America and is now one of the most important insect pests of Solanaceae in different parts of the world, including Africa. Its control has relied primarily on chemical insecticides, which are associated with negative ecological effects. In the present study, essential oils of Ocimum gratissimum and O. kilimandscharicum were tested for repellence and fumigant toxicity on the adult stages under laboratory conditions. The oil of O. gratissimum was more repellent, but its toxicity was comparable with that of O. kilimandscharicum. The major constituents of O. gratissimum were methyl eugenol (39.5%) and eugenol (29.7%). Those of O. kilimandscharicum were camphor (47.1%) and 1.8-cineole (19.3%). Eugenol (LC50 of 0.24 µl/ml, 83.3%, RI50 = 0.15) and camphor (LC50 of 0.23 µl/ml, 89.5%, RI50 = 0.13) were more toxic (at 1 µl/ml for 24 h) and repellent than the other constituents. The results show potential of the essential oils for use in integrated management of the tomato pest.

Mediation of Potato-Potato Cyst Nematode, G. rostochiensis Interaction by Specific Root Exudate Compounds.

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.

Expansions of chemosensory gene orthologs among selected tsetse fly species and their expressions in Glossina morsitans morsitans tsetse fly.

Tsetse fly exhibit species-specific olfactory uniqueness potentially underpinned by differences in their chemosensory protein repertoire. We assessed 1) expansions of chemosensory protein orthologs in Glossina morsitans morsitans, Glossina pallidipes, Glossina austeni, Glossina palpalis gambiensis, Glossina fuscipes fuscipes and Glossina brevipalpis tsetse fly species using Café analysis (to identify species-specific expansions) and 2) differential expressions of the orthologs and associated proteins in male G. m. morsitans antennae and head tissues using RNA-Seq approaches (to establish associated functional molecular pathways). We established accelerated and significant (P<0.05, λ = 2.60452e-7) expansions of gene families in G. m. morsitans Odorant receptor (Or)71a, Or46a, Ir75a,d, Ionotropic receptor (Ir) 31a, Ir84a, Ir64a and Odorant binding protein (Obp) 83a-b), G. pallidipes Or67a,c, Or49a, Or92a, Or85b-c,f and Obp73a, G. f. fuscipes Ir21a, Gustatory receptor (Gr) 21a and Gr63a), G. p. gambiensis clumsy, Ir25a and Ir8a, and G. brevipalpis Ir68a and missing orthologs in each tsetse fly species. Most abundantly expressed transcripts in male G. m. morsitans included specific Or (Orco, Or56a, 65a-c, Or47b, Or67b, GMOY012254, GMOY009475, and GMOY006265), Gr (Gr21a, Gr63a, GMOY013297 and GMOY013298), Ir (Ir8a, Ir25a and Ir41a) and Obp (Obp19a, lush, Obp28a, Obp83a-b Obp44a, GMOY012275 and GMOY013254) orthologs. Most enriched biological processes in the head were associated with vision, muscle activity and neuropeptide regulations, amino acid/nucleotide metabolism and circulatory system processes. Antennal enrichments (>90% of chemosensory transcripts) included cilium-associated mechanoreceptors, chemo-sensation, neuronal controlled growth/differentiation and regeneration/responses to stress. The expanded and tsetse fly species specific orthologs includes those associated with known tsetse fly responsive ligands (4-methyl phenol, 4-propyl phenol, acetic acid, butanol and carbon dioxide) and potential tsetse fly species-specific responsive ligands (2-oxopentanoic acid, phenylacetaldehyde, hydroxycinnamic acid, 2-heptanone, caffeine, geosmin, DEET and (cVA) pheromone). Some of the orthologs can potentially modulate several tsetse fly species-specific behavioral (male-male courtship, hunger/host seeking, cool avoidance, hygrosensory and feeding) phenotypes. The putative tsetse fly specific chemosensory gene orthologs and their respective ligands provide candidate gene targets and kairomones for respective downstream functional genomic and field evaluations that can effectively expand toolbox of species-specific tsetse fly attractants, repellents and other tsetse fly behavioral modulators.

Blending studies with selected waterbuck odor constituents or analogues in the development of a potent repellent blend against savannah tsetse.

Previous comparison of the body odors of tsetse-refractory waterbuck and those of tsetse-attractive ox and buffalo showed that a blend of 15 EAG-active compounds specific to waterbuck, including C5-C10 straight chain carboxylic acid homologues, methyl ketones (C8-C12 straight chain homologues and geranyl acetone), phenols (guaiacol and carvacrol) and δ-octalactone, was repellent to tsetse. A blend of four components selected from each class of compounds (δ-octalactone, pentanoic acid, guaiacol, and geranylacetone) showed repellence that is comparable to that of the 15 components blend and can provide substantial protection to cattle (more than 80%) from tsetse bites and trypanosome infections. Structure-activity studies with the lactone and phenol analogues showed that δ-nonalactone and 4-methylguaiacol are significantly more repellent than δ-octalactone and guaiacol, respectively. In the present study, we compared the responses of Glossina pallidipes and Glossina morsitans to i) blends comprising of various combinations of the most active analogues from each class of compounds, and ii) a four-component blend of δ-nonalactone, heptanoic acid, 4-methylguaiacol and geranyl acetone in different ratios in a two-choice wind-tunnel, followed by a field study with G. pallidipes population in a completely randomized Latin Square Design set ups. In the wind tunnel experiments, the blend of the four compounds in 6:4:2:1 ratio was found to be significantly more repellent (94.53%) than that in 1:1:1:1 proportion and those in other ratios. G. m. morsitans also showed a similar pattern of results. In field experiments with G. pallidipes population, the 6:4:2:1 blend of the four compounds also gave similar results. The results lay down useful groundwork in the large-scale development of more effective 'push' and 'push-pull' control tactics of the tsetse flies.

16S rRNA gene profiling of bacterial communities mediating production of tsetse attractive phenols in mammalian urine.

Several types of odours are involved in the location of host animals by tsetse (Diptera: Glossinidae), a vector of animal African trypanosomiasis. Host animals' ageing urine has been shown to be the source of a phenolic blend attractive to the tsetse. Nevertheless, limited research has been performed on the microbial communities' role in the production of phenols. This study aimed at profiling bacterial communities mediating the production of tsetse attractive phenols in mammalian urine. Urine samples were collected from African buffalo (Syncerus caffer), cattle (Bos taurus) and eland (Taurotragus oryx) at Kongoni Game Valley Ranch and Kenyatta University in Kenya. Urine samples, of each animal species, were pooled and left open to age in ambient conditions. Bacteriological and phenols analyses were then carried out, at 4 days ageing intervals, for 24 days. Phenols analysis revealed nine volatile phenols: 4-cresol, ortho-cresol, 3-cresol, phenol, 3-ethylphenol, 3-propylphenol, 2-methyloxyphenol, 4-ethylphenol and 4-propylphenol. Eight out of 19 bacterial isolates from the ageing urine revealed the potential to mediate production of phenols. 16S rRNA gene characterisation of the isolates closely resembled Enterococcus faecalis KUB3006, Psychrobacter alimentarius PAMC 27887, Streptococcus agalactiae 2603V, Morganella morganii sub.sp. morganii KT, Micrococcus luteus NCTC2665, Planococcus massiliensis strain ES2, Ochrobactrum pituitosum AA2 and Enterococcus faecalis OGIRF. This study established that some of the phenols emitted from mammalian urine, which influence the tsetse's host-seeking behaviour, are well characterised by certain bacteria. These results may allow the development of biotechnological models in vector control that combines the use of these bacteria in the controlled release of semiochemicals.

Structure-activity studies on analogues of 4-methylguaiacol, a cattle anal odour constituent repellent to the brown ear tick (Rhipicephalus appendiculatus).

Previously, 4-methylguaiacol, a major constituent of cattle anal odour, was found to have a high repellency on Rhipicephalus appendiculatus. In the present study, 10 structural analogues of the phenol were tested for repellency against R. appendiculatus in order to assess the effects of (i) absence or presence of the 4-alkyl group of varying length, (ii) inclusion of a double bond in the 4-alkyl chain, (iii) linking the two phenolic oxygen in a methylenedioxy bridge, (iv) replacement of the OCH3 with CH3 and inclusion of another CH3 at position 6, and (v) presence of an additional OCH3 group at position 6. The analogues comprised of 2-methoxyphenol (guaiacol), 4-ethyl-2-methoxyphenol, 4-propyl-2-methoxyphenol, 4-allyl-2-methoxyphenol (eugenol), 3,4-methylenedioxytoluene, 2,4-dimethylphenol, 4-ethyl-2-methylphenol, 2,4,6-trimethylphenol, 4-propyl-2,6-dimethoxy-phenol and 4-allyl-2,6-dimethoxyphenol, which were compared at different concentrations in a two-choice climbing assay set up. Each analogue showed either increased or reduced repellency compared with 4- methylguaiacol. The structural feature that was associated with the highest repellency was 4-propyl moiety in the guaiacol unit (RD75 = 0.031 for 4-propyl-2-methoxyphenol; that of 4-methylguaiacol = 0.564). Effects of blending selected analogues with high repellency were also compared. However, none of the blends showed incremental increases in repellency compared with that of 4-propyl-2-methoxyphenol. We are currently evaluating the effects of controlled release of this compound at different sites on cattle on the behavior and success of R. appendiculatus to locate their predilection for feeding sites.

Growth-disrupting Murraya koenigii leaf extracts on Anopheles gambiae larvae and identification of associated candidate bioactive constituents.

Plant-based constituents have been proposed as eco-friendly alternatives to synthetic insecticides for control of mosquito vectors of malaria. In this study, we first screened the effects of methanolic leaf extracts of curry tree (Murraya koenigii) growing in tropical (Mombasa, Malindi) and semi-arid (Kibwezi, and Makindu) ecological zones of Kenya on third instar An. gambiae s.s. larvae. Extracts of the plant from the semi-arid region, and particularly from Kibwezi, led to high mortality of the larvae. Bioassay-guided fractionation of the methanolic extract of the leaves of the plants from Kibwezi was then undertaken and the most active fraction (20 fold more potent than the crude extract) was then analyzed by Liquid chromatography quadruple time of flight coupled with mass spectrometry (LC-QtoF-MS) and a number of constituents were identified, including a major alkaloid constituent, Neplanocin A (5). Exposure of the third instar larvae to a sub-lethal dose (4.43 ppm) of this fraction over 7-day periods induced gross morphogenetic abnormalities in the larvae, with reduced locomotion, and delayed pupation. Moreover, the few adults that emerged from some pupae failed to fly from the water surface, unlike in the untreated control group. These results demonstrate subtle growth-disrupting effects of the phytochemical blend from M. koenigii leaves on aquatic stages An. gambiae mosquito. The study lays down some useful groundwork for the downstream development of phytochemical blends that can be evaluated for integration into eco-friendly control of An. gambiae vector population targeting the often overlooked but important immature stages of the malaria vector.

Characterisation of cattle anal odour constituents associated with the repellency of Rhipicephalus appendiculatus.

Adults of the Brown Ear Tick (Rhipicephalus appendiculatus) have a predilection for feeding inside and around the ears of cattle and other hosts. A previous study has shown that the tick locates the host ears by 'push-pull' deployment of a repellent blend emitted at the anal region and an attractant blend emitted at the ears. Interestingly, the two odours play reverse roles with Rhipicephalus evertsi, which prefer to feed around the anal region. The present study was undertaken to characterize the major constituents of the cattle anal odour and to evaluate their repellence to R. appendiculatus. The anal odour was trapped with reverse-phase C18-bonded silica, Porapak Q and Super Q placed in an oven bag attached at the anal region of the cattle for 6 h. The adsorbents were then removed and extracted with dichloromethane, and the extracted compounds analyzed by linked gas chromatography-mass spectrometry (GC-MS). The major constituents of the odour were o-xylene, 4-hydroxy-4-methyl-2-pentanone, 4-methyl-2-methoxyphenol, ethylbenzene, 2,6,6-trimethyl-[1S(1α,ß,5α)]bicycloheptanes, 5-ethoxydihydro-2(3H)-furanone, 3-methylene-2-pentanone, 5-methyl-2-phenyl-1H-indole, and 3-pentanone. The repellency of the available compounds (o-xylene, 4-hydroxy-4-methyl-2-pentanone, 4-methyl-2-methoxyphenol, ethyl benzene, 3-methylene-2-pentanone, and 3-pentanone) and blends was evaluated using a dual choice tick climbing assay at different doses. The anal odour showed repellence with RD75 of 0.39. Of the compounds tested, 4-methyl-2-methoxyphenol was found to be most repellent (RD75 = 0.56) and 3-pentanone least repellent (RD75 = 622.7). The blend of the six constituents showed RD75 of 0.34, comparable to that of the crude anal odour blend. A series of subtractive bioassays with one constituent of the 6-component blend missing was also carried out. Subtraction of 3-methylpentanone gave the most repellent blend (RD75 = 0.097), whereas subtraction of 4-methylguaiacol gave the least repellent blend (RD75 = 160.7) consistent with the high individual activity of this phenol. The study lays down useful groundwork for on-host deployment of controlled-release of a selected repellent or blend to disrupt the tick's ability to locate its preferred feeding site.