Correlates of the peridomestic abundance of Lutzomyia longipalpis (Diptera: Psychodidae) in Amazonian Brazil

Abstract. Abundance of the sandfly Lutzomyia longipalpis , the vector of Leishmania chagasi , was surveyed using CDC light-suction traps in fifteen villages and 180 homesteads on Marajo island, Para State, Brazil. Flies were most abundant in cultivated areas, secondary growth and open woodland, away from savanna grassland and forest. Within homesteads, the abundance of flies in animal sheds was weakly associated with the number of hosts kept therein, and inversely related to the number elsewhere, both human and animal. Houses harboured more flies if dogs were present and if the house had: a roof of thatch rather than of tiles or tin; a thatched roof which had not been treated with insecticide; relatively more holes in walls and roof. The sex ratio of flies was more male-biased at higher densities. We weigh the importance of these variables as risk factors for Le.chagasi infection.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

A comparison of molecular markers to detect Lutzomyia longipalpis naturally infected with Leishmania (Leishmania) infantum

The aim of the present study was to detect natural infection by Leishmania (Leishmania) infantum in Lutzomyia longipalpis captured in Barcarena, state of Pará, Brazil, through the use of three primer sets. With this approach, it is unnecessary to previously dissect the sandfly specimens. DNA of 280 Lu. longipalpis female specimens were extracted from the whole insects. PCR primers for kinetoplast minicircle DNA (kDNA), the mini-exon gene and the small subunit ribosomal RNA (SSU-rRNA) gene of Leishmania were used, generating fragments of 400 bp, 780 bp and 603 bp, respectively. Infection by the parasite was found with the kDNA primer in 8.6% of the cases, with the mini-exon gene primer in 7.1% of the cases and with the SSU-rRNA gene primer in 5.3% of the cases. These data show the importance of polymerase chain reaction as a tool for investigating the molecular epidemiology of visceral leishmaniasis by estimating the risk of disease transmission in endemic areas, with the kDNA primer representing the most reliable marker for the parasite.     

The first record of Lutzomyia longipalpis in the Argentine northwest

In 2004, the urban presence of Lutzomyia longipalpis was recorded for the first time in Formosa province. In 2006, the first autochthonous case of human urban visceral leishmaniasis (VL) was recorded in Misiones in the presence of the vector, along with some canine VL cases. After this first case, the vector began to spread primarily in northeast Argentina. Between 2008-2011, three human VL cases were reported in Salta province, but the presence of Lu. longipalpis was not recorded. Captures of Phlebotominae were made in Tartagal, Salta, in 2013, and the presence of Lu. longipalpis was first recorded in northwest Argentina at that time. Systematic sampling is recommended to observe the distribution and dispersion patterns of Lu. longipalpis and consider the risk of VL transmission in the region.     

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.     

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.     

Isozymic and metric variation in the Lutzomyia longipalpis complex

Abstract Male Lutzomyia longipalpis of two types from Bolivia were compared using isozyme electrophoresis and wing morphometry. One sample ( ex Chiflonkaka Cave, alt. 2800 m at Toro Toro, Charcas Province, Potosi Department) was 'two-spot' phenotype males (i.e. tergites III and IV with paired pale patches of pheromone glands), whereas two other locality samples (Apa Apa and Imanaco, Sud Yungas Province, La Paz Department) were one-spot male phenotype (only tergite IV with paired pale patches). Multilocus enzyme electrophoresis (using ACON, aGPD, GPI, IDH, MDH, ME, 6PGD, PGM, LAP and PEPB) found no difference between samples from adjacent hen houses at Apa Apa. Nei's standard genetic distance between one-spot samples from Apa Apa and Imanaco (5 km apart, 1500m alt.) was 0.001-0.002, whereas the two-spot males from Toro Toro (800 km away) showed a genetic distance of 0.081 from the one-spot males (Apa Apa and Imanaco). This genetic distance is commensurate with speciation, but may simply be intraspecific differentiation due to 'isolation by distance'.
For comparative wing morphometry, we included additional material of one-spot males from Bolivia (Guyabal, Sud Yungas, La Paz), Brazil, Colombia and Nicaragua. These three other country samples were assumed to be different sibling species in the complex Lutzomyia longipalpis (Lanzaro et al , 1993). Statistics were based on univariate and multivariate analysis. The comparison between size-in and size-free canonical variate analysis (CVA) indicated that the wing morphometric divergence between one-spot and two-spot Bolivian phenotypes was not size dependent and could have taxonomic significance.     

Polymerase chain reaction‐based assay for the detection and identification of sand fly gregarines in Lutzomyia longipalpis,a vector of visceral leishmaniasis

Gregarines that parasitise phlebotomine sand flies belong to the genus Psychodiella and, even though they are highly host‐specific, only five species have been described to date. Their most outstanding features include the unique localisation of the oocysts in the accessory glands of the female host, which ensures contamination of the egg surface during oviposition, and the fact that they naturally parasitise the vectors of Leishmania, causal agent of leishmaniasis. The type species, Ps. chagasi, was first described in Lutzomyia longipalpis, vector of visceral leishmaniasis (VL), from Brazil. We recently reported Ps. chagasi sequences in Lu. longipalpis from Posadas (Misiones, Argentina), an endemic VL location where this gregarine had not been previously recorded. In order to analyse the incidence of Ps. chagasi infections in Lu. longipalpis from this location, the aim of this study was to develop a diagnostic assay for sand fly gregarine parasites in Lu. longipalpis. For this, we designed primers using the Ps. chagasi sequences we previously identified and performed an in vitro validation by PCR amplification of the original sand fly samples. Their specificity and sensitivity as diagnostic primers were subsequently confirmed by PCR reactions using total DNA extracted from naturally infected Lu. longipalpis from the same location (Posadas, Argentina).     

Predicting the geographic distribution of Lutzomyia longipalpis (Diptera: Psychodidae) and visceral leishmaniasis in the state of Mato Grosso do Sul,Brazil

To understand the geographic distribution of visceral leishmaniasis (VL) in the state of Mato Grosso do Sul (MS), Brazil, both the climatic niches of Lutzomyia longipalpis and VL cases were analysed. Distributional data were obtained from 55 of the 79 counties of MS between 2003-2012. Ecological niche models (ENM) of Lu. longipalpis and VL cases were produced using the maximum entropy algorithm based on eight climatic variables. Lu. longipalpis showed a wide distribution in MS. The highest climatic suitability for Lu. longipalpis was observed in southern MS. Temperature seasonality and annual mean precipitation were the variables that most influenced these models. Two areas of high climatic suitability for the occurrence of VL cases were predicted: one near Aquidauana and another encompassing several municipalities in the southeast region of MS. As expected, a large overlap between the models for Lu. longipalpis and VL cases was detected. Northern and northwestern areas of MS were suitable for the occurrence of cases, but did not show high climatic suitability for Lu. longipalpis . ENM of vectors and human cases provided a greater understanding of the geographic distribution of VL in MS, which can be applied to the development of future surveillance strategies.     

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.     

Molecular detection of Wolbachia pipientis in natural populations of sandfly vectors of Leishmania infantum in endemic areas: first detection in Lutzomyia longipalpis

A polymerase chain reaction‐based method was used to screen sandflies for infection with Wolbachia (Rickettsiales: Rickettsiaceae), an intracellular bacterial endosymbiont found in many arthropods and filarial hosts. Positive results were obtained in five of 200 field‐collected sandflies and were confirmed by sequencing. All sandflies were Lutzomyia longipalpis (Diptera: Psychodidae) captured in a region endemic for visceral leishmaniasis in Brazil. This is the first study to identify Wolbachia infection in this Lutzomyia species, which is the main vector of leishmaniasis in the study area. The low infection rate found in this study (2.5%), together with the lack of detection of Wolbachia in previous studies and the diversity found in the sequences analysed, suggests horizontal transmission to these sandflies.