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.

Influence of Host-Plant Surface Chemicals on the Oviposition of the Cereal Stemborer Busseola Fusca.

The chemical composition of plant surfaces plays a role in selection of host plants by herbivorous insects. Once the insect reaches the plant, these cues determine host acceptance. Laboratory studies have shown that the stem borer Busseola fusca (Lepidoptera: Noctuidae), an important pest of sorghum and maize in sub-Saharan Africa, is able to differentiate between host and non-host plant species. However, no information is available on the cues used by this insect to seek and accept the host plant. Thus, the role of surface phytochemical stimuli on host selection and oviposition by B. fusca was studied in the laboratory using two host plants, sorghum, Sorghum bicolor, and maize, Zea mays, and one non-host plant, Napier grass, Pennisetum purpureum. The numbers of eggs and egg masses deposited on the three plant species were compared first under no-choice and choice conditions. In both cases, more eggs and egg masses were laid on maize and sorghum than on the non-host. Artificial surrogate stems treated with a water or chloroform surface extract of each plant were then compared with surrogate stems treated with, respectively, water or chloroform as controls, under similar conditions. Surrogate stems treated with plant water extracts did not show an increase in oviposition when compared to controls, indicating that the major compounds in these extracts, i.e., simple sugars and free amino acids, are not significantly responsible for the oviposition preference. By contrast, a chloroform extract of sorghum enhanced oviposition on the surrogate stems compared to the control, while those of maize and Napier grass showed no significant effects. Analysis of the chloroform extract of sorghum showed higher amounts of α-amyrin, ß-amyrin, and n-nonacosane compared to those of maize and Napier grass. A blend of the three chemicals significantly increased oviposition compared to the chloroform-treated control, indicating that these compounds are part of the surface chemical signature of the plant responsible for host recognition and oviposition by B. fusca.

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.

Anti-protozoal activity of aporphine and protoberberine alkaloids from Annickia kummeriae (Engl. & Diels) Setten & Maas (Annonaceae).

BACKGROUND: Malaria, trypanosomiasis and leishmaniasis have an overwhelming impact in the poorest countries in the world due to their prevalence, virulence and drug resistance ability. Currently, there is inadequate armory of drugs for the treatment of malaria, trypanosomiasis and leishmaniasis. This underscores the continuing need for the discovery and development of new anti-protozoal drugs. Consequently, there is an urgent need for research aimed at the discovery and development of new effective and safe anti-plasmodial, anti-trypanosomal and anti-leishmanial drugs. METHODS: Bioassay-guided chromatographic fractionation was employed for the isolation and purification of antiprotozoal alkaloids. RESULTS: The methanol extract from the leaves of Annickia kummeriae from Tanzania exhibited a strong anti-plasmodial activity against the multi-drug resistant Plasmodium falciparum K1 strain (IC50 0.12 ± 0.01 µg/ml, selectivity index (SI) of 250, moderate activity against Trypanosoma brucei rhodesiense STIB 900 strain (IC50 2.50 ± 0.19 µg/ml, SI 12) and mild activity against Leishmania donovani axenic MHOM-ET-67/82 strain (IC50 9.25 ± 0.54 µg/ml, SI 3.2). Bioassay-guided chromatographic fractionation led to the isolation of four pure alkaloids, lysicamine (1), trivalvone (2), palmatine (3), jatrorrhizine (4) and two sets of mixtures of jatrorrhizine (4) with columbamine (5) and palmatine (3) with (-)-tetrahydropalmatine (6). The alkaloids showed low cytotoxicity activity (CC50 30 - >90 µg/ml), strong to moderate anti-plasmodial activity (IC50 0.08 ± 0.001 - 2.4 ± 0.642 µg/ml, SI 1.5-1,154), moderate to weak anti-trypanosomal (IC50 2.80 ± 0.001 - 14.3 ± 0.001 µg/ml, SI 2.3-28.1) and anti-leishmanial activity IC50 2.7 ± 0.001 - 20.4 ± 0.003 µg/ml, SI 1.7-15.6). CONCLUSION: The strong anti-plasmodial activity makes these alkaloids good lead structures for drug development programs.

Zanthoxoaporphines A-C: Three new larvicidal dibenzo[de,g]quinolin-7-one alkaloids from Zanthoxylum paracanthum (Rutaceae).

The bioassay-guided purification of Zanthoxylum paracanthum (Rutaceae) extracts led to the isolation of three new alkaloids, namely 1-hydroxy-10-methoxy-7H-dibenzo[de,g]quinolin-7-one (zanthoxoaporphine A, 2), 1-hydroxy-7H-dibenzo[de,g]quinolin-7-one (zanthoxoaporphine B, 3) and 1,8-dihydroxy-9-methoxy-7H-dibenzo[de,g]quinolin-7-one (zanthoxaporphine C, 4), and a known lignan identified as sesamin (1). Isolation and purification of the constituent compounds was achieved through conventional chromatographic methods. The chemical structures of the isolated compounds were determined on the basis of UV, IR, NMR and MS data, and confirmed by comparison with those reported in the literature. The larvicidal activity of some of the isolated compounds was investigated by using third-instar Anopheles gambiae larvae.

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.

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.

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.

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.

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.

Comparison of volatile blends and gene sequences of two isolates of Metarhizium anisopliae of different virulence and repellency toward the termite Macrotermes michaelseni.

Previously, we reported an interesting relationship between virulence and repellency of different isolates of the fungus Metarhizium anisopliae towards the termite Macrotermes michaelseni: the higher the virulence of a given isolate, the greater its repellency. In the present study, we compared the volatile profiles of two isolates, one that was more virulent (and repellent) and one that was less virulent (and repellent) to the termite. The prominent components of the two blends were characterized by gas chromatography-mass spectrometry and authenticated by gas chromatography co-injections with synthetic standards. There were both qualitative and quantitative differences between the two blends. The repellencies of synthetic blends of 10 prominent constituents of the volatiles of the two isolates were compared and that of the more virulent isolate was found to be significantly more repellent. Subtractive bioassays were carried out with one of the constituents of each of the two 10-component blends missing at a time to determine its relative contribution to the overall repellency. The results indicated that the repellency of the volatiles of each isolate was primarily due to synergistic effects of a smaller number of constituents. Intraspecific differences between the two isolates were also reflected in their nucleotide sequences.

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.

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.

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.

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.

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.

Arm-in-cage testing of natural human-derived mosquito repellents.

BACKGROUND: Individual human subjects are differentially attractive to mosquitoes and other biting insects. Previous investigations have demonstrated that this can be attributed partly to enhanced production of natural repellent chemicals by those individuals that attract few mosquitoes in the laboratory. The most important compounds in this respect include three aldehydes, octanal, nonanal and decanal, and two ketones, 6-methyl-5-hepten-2-one and geranylacetone [(E)-6,10-dimethylundeca-5,9-dien-2-one]. In olfactometer trials, these compounds interfered with attraction of mosquitoes to a host and consequently show promise as novel mosquito repellents. METHODS: To test whether these chemicals could provide protection against mosquitoes, laboratory repellency trials were carried out to test the chemicals individually at different concentrations and in different mixtures and ratios with three major disease vectors: Anopheles gambiae, Culex quinquefasciatus and Aedes aegypti. RESULTS: Up to 100% repellency was achieved depending on the type of repellent compound tested, the concentration and the relative composition of the mixture. The greatest effect was observed by mixing together two compounds, 6-methyl-5-hepten-2-one and geranylacetone in a 1:1 ratio. This mixture exceeded the repellency of DEET when presented at low concentrations. The repellent effect of this mixture was maintained over several hours. Altering the ratio of these compounds significantly affected the behavioural response of the mosquitoes, providing evidence for the ability of mosquitoes to detect and respond to specific mixtures and ratios of natural repellent compounds that are associated with host location. CONCLUSION: The optimum mixture of 6-methyl-5-hepten-2-one and geranylacetone was a 1:1 ratio and this provided the most effective protection against all species of mosquito tested. With further improvements in formulation, selected blends of these compounds have the potential to be exploited and developed as human-derived novel repellents for personal protection.

Expression of metallothionein and alpha-tubulin in heavy metal-tolerant Anopheles gambiae sensu stricto (Diptera: Culicidae).

Anopheles mosquitoes have been shown to adapt to heavy metals in their natural habitats. In this study we explored the possibility of using Anopheles gambiae sensu stricto as bio-reporters for environmental heavy metal pollution through expressions of their metal-responsive metallothionein and alpha-tubulin genes. The study was undertaken with third instar larvae after selection by cadmium, copper, or lead at LC(30) through five successive generations. Expression levels were determined in the 5th generation by semi-quantitative RT-PCR on the experimental and control populations. The data were analyzed using one-way ANOVA. The highest metallothionein (F(3,11)=4.574, P=0.038) and alpha-tubulin (F(3,11)=12.961, P=0.002) responses were observed in cadmium-tolerant treatments. There was significantly higher expression of metallothionein in cadmium or copper treatments relative to the control (P=0.012), and in cadmium than in lead treatments (P=0.044). Expressions of alpha-tubulin were significantly higher in cadmium than in control treatments (P=0.008). These results demonstrate the capacity of An. gambiae s.s. to develop tolerance to increased levels of heavy metal challenge. The results also confirm the potential of heavy metal-responsive genes in mosquitoes as possible bio-indicators of heavy metal environmental pollution. How the tolerance and expressions relate to An. gambiae s.s. fitness and vectorial capacity in the environment remains to be elucidated.

Constituents of the essential oil of Suregada zanzibariensis leaves are repellent to the mosquito, Anopheles gambiae s.s.

In traditional African communities, repellent volatiles from certain plants generated by direct burning or by thermal expulsion have played an important role in protecting households against vectors of malaria and other diseases. Previous research on volatile constituents of plants has shown that some are good sources of potent mosquito repellents. In this bioprospecting initiative, the essential oil of leaves of the tree, Suregada zanzibariensis Verdc. (Angiospermae: Euphobiaceae) was tested against the mosquito, Anopheles gambiae s.s. Giles (Diptera: Culicidae) and found to be repellent. Gas chromatography (GC), GC-linked mass spectrometry (GC-MS) and, where possible, GC-co-injections with authentic compounds, led to the identification of about 34 compounds in the essential oil. About 56% of the constituents were terpenoid ketones, mostly methyl ketones. Phenylacetaldehyde (14.4%), artemisia ketone (10.1%), (1S)-(-)-verbenone (12.1%) and geranyl acetone (9.4%) were the main constituents. Apart from phenylacetaldehyde, repellent activities of the other main constituents were higher than that of the essential oil. The blends of the main constituents in proportions found in the essential oil were more repellent to An. gambiae s.s. than was the parent oil (p < 0.05), and the presence of artemisia ketone in the blend caused a significant increase in the repellency of the resulting blend. These results suggested that blends of some terpenoid ketones can serve as effective An. gambiae s.s. mosquito repellents.