Genomic analysis of two phlebotomine sand fly vectors of Leishmania from the New and Old World.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.

Examination of the interior of sand fly (Diptera: Psychodidae) abdomen reveals novel cuticular structures involved in pheromone release: Discovering the manifold.

The males of many species of New World Phlebotomines produce volatile terpenoid chemicals, shown in Lutzomyia longipalpis s.l. to be sex/aggregation pheromones. Pheromone is produced by secretory cells which surround a cuticular reservoir which collects the pheromone and passes it through a cuticular duct to the surface of the insect. The pheromone then passes through specialised cuticular structures on the abdominal surface prior to evaporation. The shape and distribution of the specialised structures are highly diverse and differ according to species. In this study we used SEM to examine the interior cuticular pheromone collection and transport structures of 3 members of the Lu. longipalpis s.l. species complex and Migonemyia migonei. We found a new structure which we have called the manifold which appears to be a substantial extension of the interior tergal cuticle connected in-line with the cuticular duct and reservoir. The manifold of the Campo Grande member of the complex is longer and wider than the Jacobina member whereas the manifold of the Sobral member was shorter than both other members of the complex. Overall, the secretory apparatus of the Sobral member was smaller than the other two. The manifold of M. migonei was very different to those found in Lu. longipalpis s.l. and was positioned in a pit-like structure within the tergal cuticle. The secretory reservoir was connected by a short duct to the manifold. Differences in the size and shape of the manifold may be related to the chemical structure of the pheromone and may have taxonomic value. Examination of the interior cuticle by SEM may help to locate the secretory apparatus of vector species where pheromonal activity has been inferred from behavioural studies but the external secretory structures or pheromones have not yet been found.

Correction: Enhanced attraction of sand fly vectors of Leishmania infantum to dogs infected with zoonotic visceral leishmaniasis.

[This corrects the article DOI: 10.1371/journal.pntd.0009647.].

Isolation in Natural Host Cell Lines of Wolbachia Strains wPip from the Mosquito Culex pipiens and wPap from the Sand Fly Phlebotomus papatasi.

Endosymbiotic intracellular bacteria of the genus Wolbachia are harboured by many species of invertebrates. They display a wide range of developmental, metabolic and nutritional interactions with their hosts and may impact the transmission of arboviruses and protozoan parasites. Wolbachia have occasionally been isolated during insect cell line generation. Here, we report the isolation of two strains of Wolbachia, wPip and wPap, during cell line generation from their respective hosts, the mosquito Culex pipiens and the sand fly Phlebotomus papatasi. wPip was pathogenic for both new C. pipiens cell lines, CPE/LULS50 and CLP/LULS56, requiring tetracycline treatment to rescue the lines. In contrast, wPap was tolerated by the P. papatasi cell line PPL/LULS49, although tetracycline treatment was applied to generate a Wolbachia-free subline. Both Wolbachia strains were infective for a panel of heterologous insect and tick cell lines, including two novel lines generated from the sand fly Lutzomyia longipalpis, LLE/LULS45 and LLL/LULS52. In all cases, wPip was more pathogenic for the host cells than wPap. These newly isolated Wolbachia strains, and the novel mosquito and sand fly cell lines reported here, will add to the resources available for research on host-endosymbiont relationships, as well as on C. pipiens, P. papatasi, L. longipalpis and the pathogens that they transmit.

Enhanced attraction of sand fly vectors of Leishmania infantum to dogs infected with zoonotic visceral leishmaniasis.

BACKGROUND: The sand fly Phlebotomus perniciosus is the main vector of Leishmania infantum, etiological agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin. Dogs are the main reservoir host of this disease. The main objective of this study was to determine, under both laboratory and field conditions, if dogs infected with L. infantum, were more attractive to female P. perniciosus than uninfected dogs. METHODOLOGY/PRINCIPAL FINDINGS: We carried out a series of host choice experiments and found that infected dogs were significantly more attractive to P. perniciosus than uninfected dogs in the laboratory as well as in the field. Significantly more P. perniciosus fed on infected dogs than on uninfected dogs. However, the fecundity of P. perniciosus fed on infected dogs was adversely impacted compared to uninfected dogs by lowering the number of laid eggs. Phlebotomus perfiliewi, the second most abundant sand fly species in the field site and a competent vector of L. infantum had similar trends of attractivity as P. perniciosus toward infected dogs under field conditions. CONCLUSIONS: The results strongly suggest that L. infantum causes physiological changes in the reservoir host which lead to the host becoming more attractive to both male and female P. perniciosus. These changes are likely to improve the chance of successful transmission because of increased contact with infected hosts and therefore, infected dogs should be particularly targeted in the control of zoonotic visceral leishmaniasis in North Africa.

Modelling Sand Fly Lutzomyia longipalpis Attraction to Host Odour: Synthetic Sex-Aggregation Pheromone Dominates the Response.

Zoontic visceral leishmaniasis (ZVL) due to Leishmania infantum is a potentially fatal protozoan parasitic disease of humans and dogs. In the Americas, dogs are the reservoir and the sand fly, Lutzomyia longipalpis, the principal vector. A synthetic version of the male sand fly produced sex-aggregation pheromone attracts both female and male conspecifics to co-located insecticide, reducing both reservoir infection and vector abundance. However the effect of the synthetic pheromone on the vector's "choice" of host (human, animal reservoir, or dead-end host) for blood feeding in the presence of the pheromone is less well understood. In this study, we developed a modelling framework to allow us to predict the relative attractiveness of the synthetic pheromone and potential alterations in host choice. Our analysis indicates that the synthetic pheromone can attract 53% (95% CIs: 39%-86%) of host-seeking female Lu. longipalpis and thus it out-competes competing host odours. Importantly, the results suggest that the synthetic pheromone can lure vectors away from humans and dogs, such that when co-located with insecticide, it provides protection against transmission leading to human and canine ZVL.

Odour of domestic dogs infected with Leishmania infantum is attractive to female but not male sand flies: Evidence for parasite manipulation.

Globally visceral leishmaniasis (VL) causes thousands of human deaths every year. In South America, the etiologic agent, Leishmania infantum, is transmitted from an infected canine reservoir to human hosts by the bite of the sand fly vector; predominantly Lutzomyia longipalpis. Previous evidence from model rodent systems have suggested that the odour of infected hosts is altered by the parasite making them more attractive to the vector leading to an increased biting rate and improved transmission prospects for the pathogen. However, there has been no assessment of the effect of Le infantum infection on the attractiveness of dogs, which are the natural reservoirs for human infection. Hair collected from infected and uninfected dogs residing in a VL endemic city in Brazil was entrained to collect the volatile chemical odours present in the headspace. Female and male Lu. longipalpis sand flies were offered a choice of odour entrained from infected and uninfected dogs in a series of behavioural experiments. Odour of uninfected dogs was equally attractive to male or female Lu. longipalpis when compared to a solvent control. Female Lu. longipalpis were significantly more attracted to infected dog odour than uninfected dog odour in all 15 experimental replicates (average 45.7±0.87 females attracted to infected odour; 23.9±0.82 to uninfected odour; paired T-test, P = 0.000). Male Lu. longipalpis did not significantly prefer either infected or uninfected odour (average 36.1±0.4 males to infected odour; 35.7±0.6 to uninfected odour; paired T-test, P = 0.722). A significantly greater proportion of females chose the infected dog odour compared to the males (paired T-test, P = 0.000). The results showed that the odour of dogs infected with Le. infantum was significantly more attractive to blood-seeking female sand flies than it was to male sand flies. This is strong evidence for parasite manipulation of the host odour in a natural transmission system and indicates that infected dogs may have a disproportionate significance in maintaining infection in the canine and human population.

Community deployment of a synthetic pheromone of the sand fly Lutzomyia longipalpis co-located with insecticide reduces vector abundance in treated and neighbouring untreated houses: Implications for control of Leishmania infantum.

BACKGROUND: The rising incidence of visceral leishmaniasis due to Leishmania infantum requires novel methods to control transmission by the sand fly vector. Indoor residual spraying of insecticide (IRS) against these largely exophilic / exophagic vectors may not be the most effective method. A synthetic copy of the male sex-aggregation pheromone of the key vector species Lutzomyia longipalpis in the Americas, was co-located with residual pyrethroid insecticide, and tested for its effects on vector abundance, hence potential transmission, in a Brazilian community study. METHODS: Houses within eight defined semi-urban blocks in an endemic municipality in Brazil were randomised to synthetic pheromone + insecticide or to placebo treatments. A similar number of houses located >100m from each block were placebo treated and considered as "True Controls" (thus, analysed as three trial arms). Insecticide was sprayed on a 2.6m2 surface area of the property boundary or outbuilding wall, co-located within one metre of 50mg synthetic pheromone in controlled-release dispensers. Vector numbers captured in nearby CDC light traps were recorded at monthly intervals over 3 months post intervention. Recruited sentinel houses under True Control and pheromone + insecticide treatments were similarly monitored at 7-9 day intervals. The intervention effects were estimated by mixed effects negative binomial models compared to the True Control group. RESULTS: Dose-response field assays using 50mg of the synthetic pheromone captured a mean 4.8 (95% C.L.: 3.91, 5.80) to 6.3 (95% C.L.: 3.24, 12.11) times more vectors (female Lu. longipalpis) than using 10mg of synthetic pheromone. The intervention reduced household female vector abundance by 59% (C.L.: 48.7, 66.7%) (IRR = 0.41) estimated by the cross-sectional community study, and by 70% (C.L.: 56.7%, 78.8%) estimated by the longitudinal sentinel study. Similar reductions in male Lu. longipalpis were observed. Beneficial spill-over intervention effects were also observed at nearby untreated households with a mean reduction of 24% (95% C.L.: 0.050%, 39.8%) in female vectors. The spill-over effect in untreated houses was 44% (95% C.L.: 29.7%, 56.1%) as effective as the intervention in pheromone-treated houses. Ownership of chickens increased the intervention effects in both treated and untreated houses, attributed to the suspected synergistic attraction of the synthetic pheromone and chicken kairomones. The variation in IRR between study blocks was not associated with inter-household distances, household densities, or coverage (proportion of total households treated). CONCLUSIONS: The study confirms the entomological efficacy of the lure-and-kill method to reduce the abundance of this important sand fly vector in treated and untreated homesteads. The outcomes were achieved by low coverage and using only 1-2% of the quantity of insecticide as normally required for IRS, indicating the potential cost-effectiveness of this method. Implications for programmatic deployment of this vector control method are discussed.

Insecticide-impregnated netting: A surface treatment for killing Lutzomyia longipalpis (Diptera: Psychodidae), the vector of Leishmania infantum.

The sand fly Lutzomyia longipalpis is the main vector of Leishmania infantum in Brazil. Synthetic male-produced sex/aggregation pheromone co-located with micro-encapsulated λ-cyhalothrin in chicken sheds can significantly reduce canine infection and sand fly densities in a lure-and-kill strategy. In this study, we determined if insecticide-impregnated netting (IN) could replace insecticide residual spraying (IRS). We compared numbers of Lu. longipalpis attracted and killed in experimental and real chicken sheds baited with pheromone and treated with a 1 m2 area of either insecticide spray or netting. First, we compared both treatments in experimental sheds to control mortality established from light trap captures. We then compared the long-term killing effect of insecticide spray and netting, without renewal, in experimental sheds over a period of 16 weeks. Finally, a longitudinal intervention study in real chicken sheds compared the numbers and proportions of Lu. longipalpis collected and killed before and after application of both treatments. In Experiment 1, a higher proportion of males and females captured in IRS- and IN-treated sheds were dead at 24 h compared to controls (P < 0.05). No difference was found in the proportion of females killed in sheds treated with IN or IRS (P = 0.15). A slightly higher proportion of males were killed by IRS (100%) compared to IN (98.6%; P < 0.05). In Experiment 2, IN- and IRS-treated traps were equally effective at killing females (P = 0.21) and males (P = 0.08). However, IRS killed a significantly higher proportion of females and males after 8 (P < 0.05) and 16 (P < 0.05) weeks. In Experiment 3, there was no significant difference between treatments in the proportion of females killed before (P = 0.88) or after (P = 0.29) or males killed before (P = 0.76) or after (P = 0.73) intervention. Overall, initially the IN was as effective as IRS at killing female and male Lu. longipalpis in both experimental and real chicken sheds. However, the relative lethal effect of the IN deteriorated over time when stored under prevailing environmental conditions.

Significant reduction in abundance of peridomestic mosquitoes (Culicidae) and Culicoides midges (Ceratopogonidae) after chemical intervention in western São Paulo, Brazil.

BACKGROUND: We assessed the impact of two sand fly insecticide interventions (insecticide spraying and insecticide-impregnated dog collars) on the peridomestic abundance and distribution of mosquitoes (Culicidae) and biting midges (Ceratopogonidae) in western São Paulo (Brazil) in a long-term (42-month) evaluation. Both of these dipteran groups are vectors of diseases of medical and veterinary relevance to humans and domestic animals in Brazil. METHODS: The interventions in the 3-arm stratified randomised control trial were: pheromone + insecticide (PI) (chicken roosts were sprayed with microencapsulated lambda-cyhalothrin; pheromone lure has no effect on the Diptera pests studied here); dog-collars (DC) (dogs fitted with deltamethrin-impregnated collars); and control (C) (unexposed to pyrethroids) were extended by 12 months. During that time, adult mosquitoes and midges were sampled along 280 households at three household locations (inside human dwellings, dog sleeping sites and chicken roosts). RESULTS: We collected 3145 culicids (9 genera, 87.6% Culex spp.) distributed relatively uniformly across all 3 arms: 41.9% at chicken roosts; 37.7% inside houses; and 20.3% at dog sleeping sites. We collected 11,464 Culicoides (15 species) found mostly at chicken roosting sites (84.7%) compared with dog sleeping sites (12.9%) or houses (2.4%). Mosquitoes and Culicoides were most abundant during the hot and rainy season. Increased daytime temperature was marginally associated with increased mosquito abundance (Z = 1.97, P = 0.049) and Culicoides abundance (Z = 1.71, P = 0.087). There was no significant association with daily average rainfall for either group. Household-level mosquito and midge numbers were both significantly reduced by the PI intervention 56% [incidence rate ratio, IRR = 0.54 (95% CI: 0.30-0.97), P ≤ 0.05] and 53% [IRR = 0.47 (95% CI: 0.26-0.85), P ≤ 0.05], respectively, compared to the control intervention. The abundance of both dipteran groups at dog sleeping sites was largely unaffected by the PI and DC interventions. The PI intervention significantly reduced abundance of mosquitoes inside houses (41%) and at chicken roosting sites (48%) and reduced midge abundance by 51% in chicken roosting sites. CONCLUSIONS: Sprayed insecticide at chicken roosting sites reduced the abundance of mosquitoes and midges at the peridomestic level while dog collars had no effect on numbers for any group.

Synthetic sex-aggregation pheromone of Lutzomyia longipalpis, the South American sand fly vector of Leishmania infantum, attracts males and females over long-distance.

BACKGROUND: In South America the sand fly Lutzomyia longipalpis is the predominant vector of Leishmania infantum, the parasite that causes canine and human visceral leishmaniasis. Co-location of synthetic male sex-aggregation pheromone with an insecticide provided protection against canine seroconversion, parasite infection, reduced tissue parasite loads, and female sand fly densities at households. Optimising the sex-aggregation pheromone + insecticide intervention requires information on the distance over which female and male Lu. longipalpis would be attracted to the synthetic pheromone in the field. METHODOLOGY/PRINCIPAL FINDINGS: Wild Lu. longipalpis were collected at two peridomestic study sites in Governador Valadares (Minas Gerais, Brazil). Sand flies were marked with coloured fluorescent powder using an improved protocol and then released into an existing domestic chicken shed at two independent sites, followed by recapture at synthetic-pheromone host-odour baited traps placed up to 30 metres distant from the release point. In total 1704 wild-caught Lu. longipalpis were released into the two chicken sheds. Overall 4.3% of the marked flies were recaptured in the pheromone baited experimental chicken sheds compared to no marked flies recaptured in the control sheds. At the first site, 14 specimens (10.4% of the marked and released specimens) were recaptured at 10m, 36 (14.8%) at 20m, and 15 (3.4%) at 30m. At the second site, lower recapture rates were recorded; 8 marked specimens (1.3%) were recaptured at 5 and 10m and no marked specimens were recaptured at 15m. Approximately 7x more marked males than females were recaptured although males were only 2x as common as females in the released population. 52% of the marked Lu. longipalpis were collected during the first night of sampling, 32% on the second night, and 16% on the third night. CONCLUSIONS/SIGNIFICANCE: The study established that both male and female sand flies can be attracted to the synthetic sex-aggregation pheromone in the presence of host odour over distances up to at least 30m in the field depending on local environmental and meterological conditions.

Characterization of copulatory courtship song in the Old World sand fly species Phlebotomus argentipes.

Acoustic communication in the form of courtship and mating songs are often involved in reproductive isolation between species of Diptera, such as Drosophila, mosquitoes and sand flies. The patterns of courtship songs in New World sand fly species evolve quickly under sexual selection; and therefore, represent an important trait that can be used as a marker to study the evolution of species complexes and may aid identification of sibling species with a complex. The ability to identify vector species within species complexes is of critical importance for effective and efficient vector control programs. Species-specific song patterns seems to contribute to reproductive isolation in New World sand fly species, suggesting that auditory communication signals may be widespread among these important vectors of leishmaniasis. The main goal of the present study was to characterize the copulatory courtship song of Phlebotomus argentipes, an important vector of visceral leishmaniasis in the Old World. Ph. argentipes males produce acoustic signals during copulation and two types of songs were observed. The one we called primary song is a 'pulse song' with similar length and amplitude to the previously observed 'P1' pattern recorded in Brazilian populations of Lu. longipalpis s.l. The secondary song has 'sine song' characteristics and is quite different from any song produced by New World species. The discovery of this copulation courtship songs in Ph. argentipes supports the possibility that acoustic communication in sandflies might be more widespread than previously thought, including Old World species. Our results highlight the importance of further research on acoustic communication in the Ph. argentipes species complex and other Old World vectors of leishmaniasis.

Sand fly synthetic sex-aggregation pheromone co-located with insecticide reduces the incidence of infection in the canine reservoir of visceral leishmaniasis: A stratified cluster randomised trial.

OBJECTIVE: To evaluate the efficacy of a synthetic sex-aggregation pheromone of the sand fly vector Lu. longipalpis, co-located with residual insecticide, to reduce the infection incidence of Leishmania infantum in the canine reservoir. METHODS: A stratified cluster randomised trial was designed to detect a 50% reduction in canine incident infection after 24 months in 42 recruited clusters, randomly assigned to one of three intervention arms (14 cluster each): synthetic pheromone + insecticide, insecticide-impregnated dog collars, or placebo control. Infection incidence was measured by seroconversion to anti-Leishmania serum antibody, Leishmania parasite detection and canine tissue parasite loads. Changes in relative Lu. longipalpis abundance within households were measured by setting three CDC light traps per household. RESULTS: A total 1,454 seronegative dogs were followed-up for a median 15.2 (95% C.I.s: 14.6, 16.2) months per cluster. The pheromone + insecticide intervention provided 13% (95% C.I. 0%, 44.0%) protection against anti-Leishmania antibody seroconversion, 52% (95% C.I. 6.2%, 74·9%) against parasite infection, reduced tissue parasite loads by 53% (95% C.I. 5.4%, 76.7%), and reduced household female sand fly abundance by 49% (95% C.I. 8.2%, 71.3%). Variation in the efficacy against seroconversion varied between trial strata. Equivalent protection attributed to the impregnated-collars were 36% (95% C.I. 14.4%, 51.8%), 23% (95% C.I. 0%, 57·5%), 48% (95% C.I. 0%, 73.4%) and 43% (95% C.I. 0%, 67.9%), respectively. Comparison of the two interventions showed no statistically consistent differences in their efficacies; however, the errors were broad for all outcomes. Reductions in sand fly numbers were predominant where insecticide was located (chicken and dog sleeping sites), with no evidence of insecticide-induced repellence onto humans or dogs. CONCLUSION: The synthetic pheromone co-located with insecticide provides protection particularly against canine L. infantum parasite transmission and sand fly vector abundance. The effect estimates are not dissimilar to those of the insecticide-impregnated collars, which are documented to reduce canine infection incidence, human infection and clinical VL disease incidence, in different global regions. The trialled novel lure-and-kill approach is a low-cost potential vector control tool against ZVL in the Americas.

eNose analysis of volatile chemicals from dogs naturally infected with Leishmania infantum in Brazil.

BACKGROUND: Visceral leishmaniasis (VL) in Brazil is a neglected, vector-borne, tropical parasitic disease that is responsible for several thousand human deaths every year. The transmission route involves sand flies becoming infected after feeding on infected reservoir host, mainly dogs, and then transmitting the Leishmania infantum parasites while feeding on humans. A major component of the VL control effort is the identification and euthanasia of infected dogs to remove them as a source of infection. A rapid, non-invasive, point-of-care device able to differentiate between the odours of infected and uninfected dogs may contribute towards the accurate diagnosis of canine VL. METHODOLOGY/PRINCIPAL FINDINGS: We analysed the headspace volatile chemicals from the hair of two groups of dogs collected in 2017 and 2018 using a bench-top eNose volatile organic chemical analyser. The dogs were categorised as infected or uninfected by PCR analysis of blood samples taken by venepuncture and the number of parasites per ml of blood was calculated for each dog by qPCR analysis. We demonstrated using a robust clustering analysis that the eNose data could be discriminated into infected and uninfected categories with specificity >94% and sensitivity >97%. The eNose device and data analysis were sufficiently sensitive to be able to identify infected dogs even when the Leishmania population in the circulating blood was very low. CONCLUSIONS/SIGNIFICANCE: The study illustrates the potential of the eNose to rapidly and accurately identify dogs infected with Le. infantum. Future improvements to eNose analyser sensor sensitivity, sampling methodology and portability suggest that this approach could significantly improve the diagnosis of VL infected dogs in Brazil with additional potential for effective diagnosis of VL in humans as well as for the diagnosis of other parasitic diseases.

Susceptibility of wild-caught Lutzomyia longipalpis (Diptera: Psychodidae) sand flies to insecticide after an extended period of exposure in western São Paulo, Brazil.

BACKGROUND: In Brazil, members of the sand fly species complex Lutzomyia longipalpis transmit Leishmania infantum, a protist parasite that causes visceral leishmaniasis. Male Lu. longipalpis produce a sex pheromone that is attractive to both females and males. During a cluster randomised trial, to determine the combined effect of synthetic sex-aggregation pheromone and insecticide on Le. infantum transmission Lu. longipalpis had been continuously exposed to insecticide for 30 months. The objective of this study was to determine the effect of continuous exposure to the insecticides used in the trial on the susceptibility of Lu. longipalpis population. METHODS: During the trial the sand flies had been exposed to either lambda-cyhalothrin [pheromone + residual insecticide spray (PI)], deltamethrin [dog collars (DC)] or no insecticide [control (C)], for 30 months (November 2012 to April 2015). The insecticide treatment regime was kept in place for an additional 12 months (May 2015-April 2016) during this susceptibility study. Sand flies collected from the field were exposed to WHO insecticide-impregnated papers cyhalothrin (0.05%), deltamethrin (0.5%) and control (silicone oil) in a modified WHO insecticide exposure trial to determine their susceptibility. RESULTS: We collected 788 Lu. longipalpis using CDC-light traps in 31 municipalities across the three trial arms. Probit analysis showed that the knockdown times (KDTs) of Lu. longipalpis collected from the lambda-cyhalothrin exposed PI-arm [KDT50: 31.1 min, confidence interval (CI): 29.6-32.6 and KDT90: 44.2 min, CI: 42.1-46.7] were longer than the KDTs from the non-insecticide-treated C-arm (KDT50: 26.3 min, CI: 25.1-27.6 and KDT90: 38.2, CI: 36.5-40.2) (no-overlapping 95% CIs). KDTs of Lu. longipalpis collected from the deltamethrin exposed DC-arm had similar values (KDT50: 13.7 min, CI: 10.1-16.2 and KDT90: 26.7 min, CI: 21.8-30.6) to those for the C-arm (KDT50: 13.5 min; CI: 12.2-14.8 and KDT90: 23.2 min, CI: 21.4-25.4) (overlapping CIs). The wild-caught unexposed Lu. longipalpis (C-arm), took approximately twice as long to knock down as laboratory-colonised specimens for both insecticides. CONCLUSIONS: Our study reveals slight changes in KDT, in sand flies after prolonged exposure to lambda-cyhalothrin in the presence of pheromone. These changes are not considered to have reached the reference levels indicative of resistance in sand flies suggesting that pheromone and insecticide treatment at the level indicated in this study do not constitute a significant risk of increased insecticide resistance. Prolonged exposure to deltamethrin in dog collars did not result in changes to KDT.

Characterization of Male-Produced Aggregation Pheromone of the Bean Flower Thrips Megalurothrips sjostedti (Thysanoptera: Thripidae).

Aggregation of the bean flower thrips, Megalurothrips sjostedti (Trybom) (Thysanoptera: Thripidae), has been observed on cowpea, Vigna unguiculata (L.) Walp. To understand the mechanism underpinning this behavior, we studied the responses of M. sjostedti to headspace volatiles from conspecifics in a four-arm olfactometer. Both male and female M. sjostedti were attracted to male, but not to female odor. Gas chromatography/mass spectrometry (GC/MS) analyses revealed the presence of two distinct compounds in male M. sjostedti headspace, namely (R)-lavandulyl 3-methylbutanoate (major compound) and (R)-lavandulol (minor compound); by contrast, both compounds were only present in trace amounts in female headspace collections. A behavioral assay using synthetic compounds showed that male M. sjostedti was attracted to both (R)-lavandulyl 3-methylbutanoate and (R)-lavandulol, while females responded only to (R)-lavandulyl 3-methylbutanoate. This is the first report of a male-produced aggregation pheromone in the genus Megalurothrips. The bean flower thrips is the primary pest of cowpea, which is widely grown in sub-Saharan Africa. The attraction of male and female M. sjostedti to these compounds offers an opportunity to develop ecologically sustainable management methods for M. sjostedti in Africa.

Attraction of Lutzomyia longipalpis to synthetic sex-aggregation pheromone: Effect of release rate and proximity of adjacent pheromone sources.

In South America, the Protist parasite that causes visceral leishmaniasis, a potentially fatal human disease, is transmitted by blood-feeding female Lutzomyia longipalpis sand flies. A synthetic copy of the male produced sex-aggregation pheromone offers new opportunities for vector control applications. We have previously shown that the pheromone placed in plastic sachets (lures) can attract both females and males to insecticide treated sites for up to 3 months. To use the pheromone lure in a control program we need to understand how the application of lures in the field can be optimised. In this study we investigated the effect of increasing the number of lures and their proximity to each other on their ability to attract Lu. longipalpis. Also for the first time we applied a Bayesian log-linear model rather than a classic simple (deterministic) log-linear model to fully exploit the field-collected data. We found that sand fly response to pheromone is significantly related to the quantity of pheromone and is not influenced by the proximity of other pheromone sources. Thus sand flies are attracted to the pheromone source at a non-linear rate determined by the amount of pheromone being released. This rate is independent of the proximity of other pheromone releasing traps and indicates the role of the pheromone in aggregation formation. These results have important implications for optimisation of the pheromone as a vector control tool and indicate that multiple lures placed in relatively close proximity to each other (5 m apart) are unlikely to interfere with one another.

Sobralene, a new sex-aggregation pheromone and likely shunt metabolite of the taxadiene synthase cascade, produced by a member of the sand fly Lutzomyia longipalpis species complex.

A new sex-aggregation pheromone, sobralene, produced by the sand fly Lutzomyia longipalpis from Sobral (Ceará State, Brazil) is shown to have the novel 6,12-membered ring-fused diterpene structure 3. It is proposed that sobralene is a likely shunt metabolite of the taxadiene synthase-catalysed cyclisation of geranygeranyl diphosphate.

Reduced translucency and the addition of black patterns increase the catch of the greenhouse whitefly, Trialeurodes vaporariorum, on yellow sticky traps.

The greenhouse whitefly Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) is a pest of a wide range of vegetable and ornamental crops in greenhouses around the world. Yellow sticky traps are highly attractive to flying adults and so are frequently used to monitor the pest. Our aim was to test whether changes in trap translucency or the addition of printed black patterns could increase the catch on yellow sticky traps in greenhouses. Field trials were carried out in commercial crops of strawberry and tomato under glass over three years. Reduced trap translucency increased trap catches by a factor of 1.5 to 7.0 and the catch increased significantly for both females and males. Spectrometer measurements showed that the increased catch was consistent with an increased landing stimulus from a colour opponency mechanism i.e. the ratio of energy from 500-640 nm to the energy from 300-500 nm. Printed black patterns increased trap catches on specific types of trap, by a factor of 1.4 to 2.3, and the catch increased significantly for both females and males. The patterns increased trap catch on moderately translucent traps, but decreased trap catch on less translucent traps. The evidence points to a contrast/edge effect of pattern, but laboratory experiments are needed to clarify this. Exploitation of these translucency and pattern effects could improve the efficacy of yellow traps for monitoring and mass trapping in crops.

A temporal comparison of sex-aggregation pheromone gland content and dynamics of release in three members of the Lutzomyia longipalpis (Diptera: Psychodidae) species complex.

BACKGROUND: Lutzomyia longipalpis is the South American vector of Leishmania infantum, the etiologic agent of visceral leishmaniasis (VL). Male L. longipalpis produce a sex-aggregation pheromone that is critical in mating, yet very little is known about its accumulation over time or factors involved in release. This laboratory study aimed to compare accumulation of pheromone over time and determine factors that might influence release in three members of the L. longipalpis species complex. METHODOLOGY/PRINCIPAL FINDINGS: We investigated male sex-aggregation pheromone gland content at different ages and the release rate of pheromone in the presence or absence of females under different light conditions by gas chromatography-mass spectrometry (GC-MS). Pheromone gland content was determined by extraction of whole males and pheromone release rate was determined by collection of headspace volatiles. Pheromone gland content appeared age-related and pheromone began to accumulate between 6 to 12 h post eclosion and gradually increased until males were 7-9 days old. The greatest amount was detected in 9-day old Campo Grande males ((S)-9-methylgermacrene-B; X ± SE: 203.5 ± 57.4 ng/male) followed by Sobral 2S males (diterpene; 199.9 ± 34.3) and Jacobina males ((1S,3S,7R)-3-methyl-α-himachalene; 128.8 ± 30.3) at 7 days old. Pheromone release was not continuous over time. During a 4-hour period, the greatest quantities of pheromone were released during the first hour, when wing beating activity was most intense. It was then substantially diminished for the remainder of the time. During a 24 h period, 4-5 day old male sand flies released approximately 63 ± 11% of the pheromone content of their glands, depending on the chemotype. The presence of females significantly increased pheromone release rate. The light regime under which the sand flies were held had little influence on pheromone release except on Sobral 2S chemotype. CONCLUSIONS/SIGNIFICANCE: Accumulation of pheromone appears to occur at different rates in the different chemotypes examined and results in differing amounts being present in glands over time. Release of accumulated pheromone is not passive, but depends on biotic (presence of females) and abiotic (light) circumstances. There are marked differences in content and release between the members of the complex suggesting important behavioural, biosynthetic and ecological differences between them.