Response of female Lutzomyia longipalpis to host odour kairomones from human skin

Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae) is the vector of Leishmania chagasi, the aetiologic agent of visceral leishmaniasis in the New World. In the present study, the response of female sandflies from Jacobina, Brazil, to human odours from six different volunteers was investigated. Glass Petri dishes were handled by different volunteers and then exposed to female sandflies. There was a significant difference between subjects in that some individuals were more attractive or less repellent to sandflies. Response of flies to handled Petri dishes was higher during the first minutes of observation, suggesting the presence of volatile compounds in hand odours. Extracts of glass Petri dishes that had been handled by the volunteers were made with organic solvents such as acetone, methanol, pentane and ether. These were then concentrated and tested for sandfly response. Only extracts carried out with non-polar solvents such as pentane and ether were able to transfer odours from handled glass Petri dishes onto clean dishes. The attractivity of male and female human subjects was monitored for 80 days, and minor fluctuations in attractiveness were observed.     

Genetic inherence of the response to human kairomones by two allopatric members of the Lutzomyia longipalpis complex

Abstract.  The sandfly Lutzomyia longipalpis (Lutz & Neiva) is the main vector of Leishmania infantum in the New World. Several studies show that Lu. longipalpis is a species complex of at least three members. The feeding habits among the members of the complex vary from one geographical location to another. These differences in feeding habits may be related to differences between different members of the complex. The present study investigates differences in the response to human kairomones by two members of the complex, as well as the possibility that differences in the response have a genetic basis. One of the members used in this study is from Jacobina Bahia State, Brazil. Males from this population are known to produce a sex pheromone characterized by a C16 molecule identified as 3-methyl-α-himachelene. The other member is from a population originating in Marajó Island, Pará State, Brazil. Males from this location secrete a sex pheromone characterized by a C20 molecule whose structure remains to be elucidated, but is known to be a diterpene type. Our findings indicate that Jacobina females are significantly more responsive to human odours than Marajo females. When Jacobina and Marajó populations of Lu. longipalpis complex are cross-mated, the response of F1 females to the human odours is found to be genetically controlled.     

Semiochemical mediation of oviposition by the phlebotomine sandfly Lutzomyia longipalpis

Abstract. Extracts of rabbit food, hay and rabbit faeces elicited a positive oviposition response from gravid female Lutzomyia longipalpis sandflies (Diptera: Phlebotominae). Combined extract of rabbit food and oviposition pheromone had a synergistic effect on sandfly egg-laying, greatly increasing the number of eggs laid and resulting in a highly targeted response. Individually tubed flies, exposed to the combined extract, were shown to be 3.5 times more likely to survive oviposition and laid 2.5 times more eggs than control flies. A laboratory oviposition trap baited with the combined extract was tested in a cage and caught 31 (62%) of 50 gravid L.longipalpis over a 72h period.     

The effect of male sandfly pheromone and host factors as attractants for female Lutzomyia longipalpis (Diptera: Psychodidae)

Abstract Male Lutzomyia longipalpis (Lutz & Neiva) pheromone extracted from tergal glands combined with heat, carbon dioxide gas and hamster urine was found to be attractive to virgin female sandflies. The host factors if offered in the absence of pheromone or a heat source were, however, unattractive to female flies. Pheromone stored for 6 days and then combined with the same host components remained attractive to female flies but storage failed to enhance its attractiveness. The effects of carbon dioxide gas, hamster urine, chicken uropygial gland extract and octenol plus acetone on the responses of females were inconsistent. A ceramic battery powered heat source was tested with pheromone and host attractants and showed potential for field application in developing a pheromone trap.     

Behavioural and electrophysiological responses of the phlebotomine sandfly Lutzomyia longipalpis (Diptera: Psychodidae) when exposed to canid host odour kairomones

Compounds from the odour-producing glands of the fox Vulpes vulpes were collected. This complex mixture of compounds was used to stimulate the ‘ascoid’ olfactory organs of female sandflies in single sensillum and gas chromatography-linked single sensillum recordings. Sixteen of these compounds were identified using gas chromatography-linked mass spectrometry and amounts present were determined. The compounds fell into four organic classes: ketones, carboxylic acids, alcohols and aldehydes. Specific neurones present in the ascoid sensillum that responded to each of these classes of compound were characterized. A bioassay chamber was developed that gave female sandflies the choice of two odour sources. Female sandflies were attracted upwind by fox odour and were trapped in closer proximity to the fox odour port than the control port. Synthetic compounds were recombined in appropriate quantities to mimic the fox odour. In this bioassay, the synthetic blend attracted sandflies upwind, and again they were caught closer to the test port than the control port. Furthermore, the synthetic fox odour induced an electrophysiological response from neurones in the ascoid sensillum that was very similar to that induced by natural fox odour. No synthetic compound alone induced the same behavioural response from sandflies as did whole fox odour. However, benzaldehyde, 4-hydroxy-4-methyl-2-pentanone and 4-methyl-2-pentanone alone did cause sandflies to fly upwind and to be caught closer to the test port than the control.     

Constitutive and blood meal-induced trypsin genes in Lutzomyia longipalpis

Trypsins constitute some of the most abundant midgut digestive proteases expressed by hematophagous insects upon blood feeding. In addition to their role in the digestion of the blood meal, these proteases also have been implicated in the ability of certain pathogens to infect their natural vector. In sand flies, digestive proteases including trypsins were associated with early killing of Leishmania and are believed to play a role in the species-specificity dictating sand fly vectorial capacity. Our group is involved in studies of midgut digestive proteases in the sand fly Lutzomyia longipalpis, the principal vector of visceral leishmaniasis in Brazil. Here we report on the identification of two cDNAs, Lltryp1 and Lltryp2, which code for putative midgut trypsins in L. longipalpis. Analyses of RNA abundance using semi-quantitative RT-PCR show a different pattern of expression between the two genes. Lltryp1 expression remains undetected until blood feeding and reaches a peak at 12 h post-blood meal (PBM), returning to pre-blood meal levels at 72 h PBM. Additionally, Lltryp1 expression is undetected during larval development. Lltryp2, on the other hand, is constitutively expressed as high levels in the non-blood fed female, but is reduced upon blood feeding. At the end of the digestive cycle, Lltryp2 regains its pre-blood meal levels. This cDNA also is present in all developmental stages and in adult males. This pattern of expression is reminiscent of what is seen in mosquitoes and Old World sand flies, but has characteristics that are unique to L. longipalpis.     

Isolation of oviposition pheromone from the eggs of the sandfly Lutzomyia longipalpis

Abstract. Semiochemical components of eggs of the sandfly Lutzomyia longipalpis (Diptera: Psychodidae) were separated by high performance liquid chromatography. HPLC fractions were examined quantitatively and qualitatively by gas chromatography (GC). A bioassay was used to determine the oviposition attraction of gravid L. longipalpis to each of the fractions separately and a peak responsible for the semiochemical activity was identified. Gravid flies were placed in individual oviposition tubes to determine if the peak of interest was an oviposition stimulant. The active semiochemical fraction attracted gravid flies for oviposition. Furthermore, egg laying was enhanced: gravid flies exposed to the pheromone oviposited earlier and laid more eggs than control flies. GC analysis indicated that 1200 eggs (2 days old) gave a yield of 12.75 ug of active pheromone. This fraction had similar HPLC and GC retention times to caryophyllene oxide, suggesting comparable polarity and molecular weight.     

Response of the sandfly Lutzomyia longipalpis to an oviposition pheromone associated with conspecific eggs

Response of female Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae) to an oviposition attractant and/or stimulant associated with conspecific eggs was investigated in the laboratory. Females of two populations laid significantly higher mean number of eggs on sites with 160 eggs already present than on bare control sites. This response was lost when eggs placed on test sites were previously washed in organic solvents and distilled water. Age of eggs placed on test sites, 1-6 days after being laid, did not seem to affect the oviposition response of the females. Comparing effects of different numbers of eggs, positive responses were obtained when 80, 160 or 320 eggs were placed on test sites. No significant differences between mean egg numbers laid on test sites and controls were detected when batches of only 20 or 40 eggs were used.     

Laboratory response of female Lutzomyia longipalpis sandflies to a host and male pheromone source over distance

A nylon net cage (260 x 40 x 40 cm) provided with unidirectional air flow was used to demonstrate the response of female sandflies, Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae) to a host and extract of male pheromone glands over a distance of 240 cm. Females responded more rapidly and in greater numbers to the host if male pheromone was present. Results suggest that one function of the pheromone may be to attract females from some distance to a common mating site, on or near the host.     

Life cycle differences among Brazilian sandflies of the Lutzomyia longipalpis sibling species complex

The developmental cycles of five Brazilian populations of the Lutzomyia longipalpis Lutz & Neiva species complex (Diptera: Psychodidae) were compared under laboratory conditions. Three of the populations were derived from insects collected in allopatric sites at Natal (Rio Grande do Norte State), Jacobina (Bahia State) and Lapinha Cave (Minas Gerais State). The other two originated from Sobral (Ceará State), where the males of two sympatric species can be distinguished by the presence of one (1S) or two (2S) pairs of abdominal spots. The results of the present study clearly show that all three populations whose males produce C₁₆ pheromones and use pulse-type copulation songs (Jacobina, Lapinha Cave and Sobral 1S) are more easily adapted to the colonization conditions used in our laboratory, producing larger egg batches, with higher survival and an overall faster developmental cycle. This contrasts with populations producing C₂₀ male pheromones and using burst-type copulation songs (Natal and Sobral 2S) that produce smaller egg batches, have higher oviposition mortality and a slower rate of development under identical laboratory conditions. In conclusion, these phenological differences are a further indication of the differentiation of the siblings within the Lu. longipalpis species complex.     

Deltamethrin-impregnated bednets reduce human landing rates of sandfly vector Lutzomyia longipalpis in Amazon households

The entomological efficacy of using 25% deltamethrin EC insecticide-treated bednets (ITNs) was evaluated against the sandfly Lutzomyia longipalpis Lutz and Neiva (Diptera: Psychodidae), the principal vector of zoonotic visceral leishmaniasis (ZVL) in Latin America. A crossover field study in Amazon Brazil (Marajó Island, Pará State) demonstrated that, compared with untreated nets, the insecticide increased the barrier effect of nets by 39% (95% confidence interval [CI] 34-44%), reduced human landing rates by 80% (95% CI 62-90%) and increased the 24-h mortality rate from 0% to 98% (95% CI 93-99%) inside ITNs. The presence of an ITN also reduced the human landing rate on unprotected persons outside the net in the same room by 56% (95% CI 52-59%), and increased 24-h mortality to 68% (95% CI 62-73%) compared to 0.4% (0.1-2.0%) in untreated houses. The reduction in human landing rates in ITN rooms was associated with a doubling in the proportion of sandflies alighting on walls compared with that in untreated rooms, which was attributed to insecticide-induced excito-repellency. There was no evidence that sandflies were diverted onto unprotected hosts. Human landing catches inside houses peaked between 19.00 hours and 23.00 hours and declined steadily to zero at 02.00 hours and thereafter. House-to-house questionnaires established that only 34% of households owned at least one net (median two, range 1-8), only 20% of the population slept under a net (33% of 0-5-year-old children), and the majority (73%) of the population slept in hammocks. Combined data pertaining to sleeping times for children and sandfly activity period indicate that > 50% of sandfly bites inside houses, and substantially more outside houses, were taken before a third of children were potentially protected by a net. This study demonstrates the clear entomological efficacy of ITNs against Lu. longipalpis in this endemic region. The effectiveness of ITNs at preventing ZVL infection and disease has still to be evaluated.     

The sandfly Lutzomyia longipalpis shows specific humoral responses to bacterial challenge

Abstract The presence of immune molecules induced by microorganisms in the haemolymph of Lutzomyia longipalpis sandflies has been investigated. Injections of Escherichia coli and Micrococcus luteus into female sandflies induced anti-bacterial activity in the haemolymph. Inhibition zone assays showed that haemolymph from E.coli and M.luteus injected sandflies differentially inhibited M.luteus growth. This differential effect was specific to M. luteus infection since anti-Zs. coli activity was similar in haemolymph from both E.coli oxM.luteus injected sandflies. Haemolymph following injection of either bacteria showed the induction of a 4kDa peptide. Haemolymph from M.luteus injected sandflies also contained a 33 kDa polypeptide which was absent in haemolymph from E.coli and control uninfected insects. Sandflies, in common with other insects, were shown to possess general and specific humoral immune responses to the presence of microorganisms.     

Host preferences of the phlebotomine sandfly Lutzomyia longipalpis in Amazonian Brazil

Experiments were undertaken to determine the relative attractiveness of humans, dogs and chickens to Lutzomyia longipalpis, the principal vector of Leishmania chagasi causing American visceral leishmaniasis. Field experiments in two villages on Marajó Island, Pará State, Brazil, showed that one boy attracted significantly more flies than one dog or chicken, and slightly fewer flies than a group of six chickens. Experiments with laboratory-bred female flies showed that a significantly greater number of flies engorged on a single human than on either a single dog or chicken, and man-biting catches demonstrated the willingness of flies to bite in the field. It appears that Lu.longipalpis has catholic feeding habits, the attractiveness of different hosts being largely a function of their relative sizes. These results are discussed with reference to the epidemiology of visceral leishmaniasis in Brazil.     

Polymorphic dinucleotide microsatellite loci in the sandfly Lutzomyia longipalpis (Diptera: Phlebotominae)

The sandfly Lutzomyia longipalpis, an important vector of visceral leishmaniasis in the New World, is believed to be a species complex. In an effort to better understand population dynamics and speciation in this vector we developed a panel of dinucleotide — (CA)_n— microsatellite loci using an enrichment technique. Eleven polymorphic loci that produced consistent allelic banding patterns were characterized using a laboratory population of L. longipalpis. These dinucleotide microsatellite loci were more polymorphic than trinucleotide microsatellites characterized in wild‐caught samples of two other sandfly species; the variability of these loci was unexpected because the laboratory flies were believed to be inbred.     

Courtship behaviour in the sandfly Lutzomyia longipalpis, the New World vector of visceral leishmaniasis

Courtship behaviour in Lutzomyia longipalpis Lutz and Neiva (Diptera: Psychodidae) was examined to determine the sequence of behaviours that occur prior to copulation. Courtship consisted of a series of male and female touching and wing-flapping behaviours, with males performing a greater variety of wing-flapping behaviours than previously described. Occurrence of male approach-flapping, semi-circling and female stationary-flapping were all predictors of eventual copulation, and may coincide with the dispersal of pheromones or the production of auditory signals important to courtship. Touching occurred in the majority of observations, with contact most often made with the tips of the legs and antennae. This behaviour, not previously described in sandflies, was initiated by males and females, and may indicate the use of contact pheromones, a form of communication previously overlooked in L. longipalpis. Future studies are required to separate the auditory and chemical signals associated with wing-flapping, and to confirm whether L. longipalpis possesses cuticular hydrocarbons capable of inducing behavioural responses. The identification of signals that inhibit sexual behaviour during courtship may be particularly relevant to developing mating disruption techniques against L. longipalpis.     

Differential application of lambda-cyhalothrin to control the sandfly Lutzomyia longipalpis

Abstract. To study the impact of residual pyrethroid insecticide on the abundance and distribution of peridomestic Lutzomyia longipalpis , the sandfly vector of visceral leishmaniasis in Brazil, lambda-cyhalothrin was applied at 20 mg a.i.m^-2 in the following interventions: (i) spraying of all animal pens in a village (blanket coverage); (ii) treatment of a subset of animal pens, either by spraying, or by installation of insecticide-impregnated 1 m² cotton sheets as 'targets' (focal coverage).
By sampling with CDC light traps, and using a novel analytical approach, we detected a 90% reduction in Lu. longipalpis abundance in sprayed sheds of the focal intervention. However, there was no discernible effect on the abundance of other phlebotomines trapped in sheds, or on the abundance of Lu. longipalpis in untreated dining-huts and houses. This differential impact on Ludongipalpis abundance is explained in terms of the disruption of male pheromone production. Treated targets were approximately half as effective as residual spraying in reducing the abundance of Lu.longipalpis in sheds.
Following blanket intervention, the abundance of Lu.longipalpis in traps fell by only 45% (not significant): catches at untreated dining-huts actually increased, possibly because the blanket coverage diverted Lu. longipalpis away from major aggregation sites at animal pens. It is recommended that care be taken during vector control programmes to ensure that all potential aggregation sites are treated. The possible consequences of leaving some sites untreated include poor control of peridomestic sandfly abundance and an increase in the biting rate on dogs and humans.     

Sugar meal sources for the phlebotomine sandfly Lutzomyia longipalpis in Ceará State, Brazil

Abstract. In the Brazilian village of BoqueiraTo do Renato Parente, Ceará State, where visceral leishmaniasis is endemic, significant variation in the abundance of phlebotomine sandflies between different types of vegetation was demonstrated by castor oil sticky trap collections. Population densities of Lutzomyia longipalpis (Lutz & Neiva) sandflies were higher in beans and maize crops than in the surrounding open and tree scrub. Significant relationships were detected between the abundances of sandflies and aphids, suggesting aphid honey dew as a potential food source. Sugar meal analysis, using high-performance anion exchange (HPAE) chromatography with pulsed amperometric detection (PAD) and the cold anthrone test, was used to demonstrate that significantly more sandflies fed on bean plants contaminated with aphids and honeydew than on uncontaminated beans. Furthermore, higher concentrations of sugars were detected in flies which fed on aphid/honeydew-contaminated beans, suggesting that sugar was more easily available and/or that honeydew/aphids act as phagostimulants for sandflies. Amongst wild sandflies collected from a bean field, significantly more female sandflies were 'sugar-positive' than males, though the sugar concentrations in positive sandflies were similar for both sexes. The concentrations of di- and trisaccharides in sandfly homogenates, honeydew extracts and phloem exudates were very low.     

Metaphase karyotypes and G-banding in sandflies of the Lutzomyia longipalpis complex

Mitotic metaphase chromosomes (2n = 8) from brain cells of fourth instar sandfly larvae of four geographical strains of the Lutzomyia longipaplis complex were examined microscopically, with bright-field illumination, after staining by a new G-banding technique involving exposure of air-dried chromosome preparations to quinacrine and ultraviolet light. Differences of G-banding and/or position of the centromere on chromosome 4 (the smallest chromosome pair) distinguished four putative sibling species from Costa Rica, Colombia and Brazil (distinctive populations from Jacobina and Lapinha Caves). The karyotype of the population from Jacobina, Brazil, showed an apparently plesiomorphic pattern of G-banding. On the basis of their recognizably different mitotic karyotypes, cytogenetic identification of separate taxa in the L. longipalpis complex should be useful for specific female vector competence and ecology studies.