Intraspecific differentiation of sandflies specimens by optical spectroscopy and multivariate analysis

Lutzomyia longipalpis and Lutzomyia cruzi are the main sandflies species involved in the transmission of Leishmania infantum protozoan in Brazil. The morphological characteristics can be used for species identification of males specimens, while females are indistinguishable. Although, sandflies identification is essential to understand vectorial capacity, and susceptibility to infectious agents or insecticides, there is a lack of new strategies for specimen identification. In this study, Fourier transform infrared photoacoustic spectroscopy combined with multivariate analysis identified intraspecific differences between Lutzomyia populations. Successfully group clustering was achieved by principal component analysis. The main differences observed can be related to the protein content of the specimens. A classification with 100% accuracy was obtained using machine learning approach, allowing the identification of sandflies specimens.     

Geometric morphometrics for the taxonomy of 11 species of Anopheles (Nyssorhynchus) mosquitoes

The subgenus Anopheles (Nyssorhynchus) (Diptera: Culicidae) includes the primary vectors of Plasmodium spp. in Colombia. Most adult females of this subgenus are difficult to identify in the field using the available keys. With the objective of further investigating the discriminatory power of modern morphometrics, both landmark‐based and outline‐based approaches were explored using the wing venation geometry of 11 Anopheles (Nyssorhynchus) species. Wing shape was able to separate the closest species of the subgenus. When the 11 species were analysed together, validated classification scores on average 5.3–8.6 times higher than those expected by chance were observed. These scores computed from the total sample of 11 species were not satisfactory for the recognition of Anopheles benarrochi B, Anopheles oswaldoi s.l. and Anopheles strodei. These sibling species were captured in sympatry. To improve the identification power of the morphometric tool, it was necessary to analyse these species separately from the remaining species. The best classification scores were obtained using a combination of 12 landmarks collected not only on the intersections of wing veins, but also on spots. An outline approach also gave excellent reclassification scores. Another pair of sibling species, collected in allopatry, Anopheles nuneztovari and Anopheles rangeli, also showed high classification scores.     

Molecular signatures of sexual communication in the phlebotomine sand flies

Phlebotomine sand flies employ an elaborate system of pheromone communication wherein males produce pheromones that attract other males to leks (thus acting as an aggregation pheromone) and females to the lekking males (sex pheromone). In addition, the type of pheromone produced varies among populations. Despite the numerous studies on sand fly chemical communication, little is known of their chemosensory genome. Chemoreceptors interact with chemicals in an organism’s environment to elicit essential behaviors such as the identification of suitable mates and food sources. Thus, they play important roles during adaptation and speciation. Major chemoreceptor gene families, odorant receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs) together detect and discriminate the chemical landscape. Here, we annotated the chemoreceptor repertoire in the genomes of Lutzomyia longipalpis and Phlebotomus papatasi, major phlebotomine vectors in the New World and Old World, respectively. Comparison with other sequenced Diptera revealed a large and unique expansion where over 80% of the ~140 ORs belong to a single, taxonomically restricted clade. We next conducted a comprehensive analysis of the chemoreceptors in 63 L. longipalpis individuals from four different locations in Brazil representing allopatric and sympatric populations and three sex-aggregation pheromone types (chemotypes). Population structure based on single nucleotide polymorphisms (SNPs) and gene copy number in the chemoreceptors corresponded with their putative chemotypes, and corroborate previous studies that identified multiple populations. Our work provides genomic insights into the underlying behavioral evolution of sexual communication in the L. longipalpis species complex in Brazil, and highlights the importance of accounting for the ongoing speciation in central and South American Lutzomyia that could have important implications for vectorial capacity.     

Genetic Divergence in the <Emphasis Type="Italic">cacophony</Emphasis> IVS6 Intron Among Five BrazilianPopulations of <Emphasis Type="Italic">Lutzomyia longipalpis</Emphasis>

Genes involved in the reproductive isolation are particularly useful as molecular markers in speciation studies. Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae), a putative species complex, is a vector of visceral leishmaniasis in Latin America. We isolated from this species a fragment homologous to cacophony, a Drosophila gene that encodes features of the lovesong, an acoustic signal that is important in the sexual isolation of closely related species and known to vary considerably among L. longipalpis putative siblings species. Using an intron of the sandfly cacophony as a marker, we analyzed the molecular variation and sequence divergence among five populations of L. longipalpis from Brazil, three allopatric (Jacobina, Lapinha and Natal) and two putative sympatric sibling species from the locality of Sobral. A high level of polymorphism was found and analysis of the data indicates that very little gene flow is occurring among the populations of Jacobina, Lapinha, and Natal. A high level of differentiation was also observed between the two putative sympatric species of Sobral, one of which seems to be the same sibling species found in Natal, while the other is somewhat more related to Jacobina and Lapinha. However, the amount of estimated gene flow among the Sobral siblings is about seven times higher than the previously estimated for period, another lovesong gene, perhaps indicating that introgression might be affecting cacophony more than period. The results suggest that L. longipalpis is not a single species in Brazil, but it is yet not clear whether the different populations studied deserve species status rather than representing an incipient speciation process.     

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.     

Ecological interactions among phlebotomines (Diptera: Psychodidae) in an agroforestry environment of northeast Brazil

Phlebotomine vectors transmit parasites and can cause visceral leishmaniasis (VL) or cutaneous leishmaniasis (TL). Phlebotomine females are hematophagous but need to ingest carbohydrates, possibly promoting the development of protozoan parasites in their digestive tract. The present study evaluated the species composition and abundance across several habitats in a metropolitan landscape, as well as associations among phlebotomines, plants, and local climatic parameters. Three consecutive monthly collections were carried out in an Atlantic Forest fragment, using CDC light traps in peridomestic areas and cashew, coconut, and mango tree. plantations. Eight species of phlebotomine were captured: Evandromyia evandroi, Lutzomyia longipalpis, Psathyromyia shannoni, Sciopemyia sordellii, Evandromyia walkeri, Psychodopygus wellcomei, Nyssomyia whitmani, and Nyssomyia intermedia, primarily from the forest environment. L. longipalpis was confirmed as a species adapted to anthropic environments, while P. wellcomei was shown to be predominately forest‐dwelling. Phlebotomines exhibited diversified food consumption patterns in relation to carbohydrate sources. They fed on both native and exotic species of arboreal and shrubby vegetables and gramineous plants.     

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.     

Inferring distributional shifts of epidemiologically important North and Central American sandflies from Pleistocene to future scenarios

Nine sandfly species (Diptera: Psychodidae) are suspected or proven vectors of Leishmania spp. in the North and Central America region. The ecological niches for these nine species were modelled in three time periods and the overlaps for all time periods of the geographic predictions (G space), and of ecological dimensions using pairwise comparisons of equivalent niches (E space), were calculated. Two Nearctic, six Neotropical and one species in both bioregions occupied a reduced number of distribution areas. The ecological niche projections for most sandfly species other than Lutzomyia shannoni and Lutzomyia ovallesi have not expanded significantly since the Pleistocene. Only three species increase significantly to 2050, whereas all others remain stable. Lutzomyia longipalpis shared a similar ecological niche with more species than any other, although both L. longipalpis and Lutzomyia olmeca olmeca had conserved distributions over time. Climate change, at both regional and local levels, will play a significant role in the temporal and spatial distributions of sandfly species.     

Molecular and Behavioral Differentiation among Brazilian Populations of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae)

Background

Lutzomyia longipalpis is the primary vector of American visceral leishmaniasis. There is strong evidence that L. longipalpis is a species complex, but until recently the existence of sibling species among Brazilian populations was considered a controversial issue. In addition, there is still no consensus regarding the number of species occurring in this complex.

Methodology/Principal Findings

Using period, a gene that controls circadian rhythms and affects interpulse interval periodicity of the male courtship songs in Drosophila melanogaster and close relatives, we analyzed the molecular polymorphism in a number of L. longipalpis samples from different regions in Brazil and compared the results with our previously published data using the same marker. We also studied the male copulation songs and pheromones from some of these populations. The results obtained so far suggest the existence of two main groups of populations in Brazil, one group representing a single species with males producing Burst-type copulation songs and cembrene-1 pheromones; and a second group that is more heterogeneous and probably represents a number of incipient species producing different combinations of Pulse-type songs and pheromones.

Conclusions/Significance

Our results reveal a high level of complexity in the divergence and gene-flow among Brazilian populations of the L. longipalpis species complex. This raises important questions concerning the epidemiological consequences of this incipient speciation process.     

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).     

Divergence of genomic DNA in species of the subgenus Camptochironomus (Diptera, Chironomidae) differing in their cytogenetic similarity levels

The variation and divergence of genomic DNA in four species of the subgenus Camptochironomus (C. tentans, C. dilutus, C. pallidivittatus, and C. setivalva) differing in the level of their cytological similarity were analyzed using the RAPD (Randomly Amplified Polymorphic DNA) method. A high level of variation in the RAPD markers was found in the species studied. Genetic distances (GD) were assessed between natural C. tentans populations, between different species of the camptochironomus sibling species group (C. tentans, C. dilutus, and C. pallidivittatus), and between these species and C. setivalva which is outside this sibling species group. The GD values obtained characterize the levels of genomic differentiation among natural populations (GD = 0.248), among sibling species (GD = 0.635), and between incipient species (GD = 0.784) of the subgenus Camptochironomus. The degree of genomic DNA divergence between sibling and incipient species in the subgenus Camptochironomus was found to be lower than that in the genus Chironomus. The rate of genomic DNA divergence appears to be lower than the rate of chromosomal divergence in species of the subgenus Camptochironomus.     

Attraction of the sandfly Lutzomyia longipalpis to possible biomarker compounds from dogs infected with Leishmania infantum

Lutzomyia longipalpis (Diptera: Psychodidae) is the primary vector of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in the Americas. Studies have been carried out to identify new alternatives for monitoring and controlling this sandfly species, particularly with the use of chemical baits. The attractiveness of odours emitted by foxes and alcohols found in some plants has already been demonstrated in laboratory tests with Lu. longipalpis. However, no studies have evaluated the responses of these insects to volatile organic compounds (VOCs) emitted by dogs. The present study was carried out to investigate the effects on Lu. longipalpis of individual and blends of VOCs identified in hair from dogs infected with L. infantum. Effects in male and female Lu. longipalpis were assessed using wind tunnel methodology. Individual compounds including octanal, nonanal, decanal and heptadecane showed capacity for activating and/or attracting male Lu. longipalpis. Only decanal and nonanal showed effects on females. The combination of octanal, decanal and heptadecane increased activation and attraction behaviour in male sandflies, as did the blend of octanal and decanal. These findings indicate that VOCs emitted by dogs may be an interesting source of new attractants of sandflies.     

Interactions of the Males and Females of Three Sympatric Drosophila virilis-Group Species, D. montana, D. littoralis, and D. lummei, (Diptera: Drosophilidae) in Intra- and Interspecific Courtships in the Wild and in the Laboratory

Four species of the Drosophila virilis group, D. montana, D. littoralis, D. lummei, and D. ezoana, occur sympatrically in several locations in northern Europe. Courtship interactions between the flies of the three first-mentioned species were observed at malt baits in Kemi, northern Finland, to find out how the flies of different species recognize conspecific individuals and how interspecific courtships differ from intraspecific ones in the wild. Intraspecific courtships (including females of different reproductive stages) and interspecific courtships were also videotaped and analyzed in laboratory. In the wild the males courted both conspecific and allospecific females, even though the species varied in how much the males were attracted to females of different species. Interspecific courtships usually broke off when the male touched the female or when the male and/or the female vibrated his/her wings, producing acoustic cues. In the laboratory males courted conspecific females irrespective of the reproductive stage of the female, even though the courtships directed toward immature and fertilized females usually included only orienting and touching (no licking and singing). D. littoralis, and very rarely D. montana and D. lummei, males courted also allospecific females. In the few interspecific courtships between these three species, where the male proceeded to singing, females responded to male singing by vibrating their wings. This ended the courtship. It is suggested that both the chemical cues affecting female attractivity and the acoustic signals of males and females, which are produced by wing vibration, function in maintaining sexual isolation between these three species.     

Survey of Sand Flies (Diptera: Psychodidae) in an Environmentally Protected Area in Brazil

Brazil is one of the most important endemic areas for leishmaniasis worldwide. Protected areas that are tourist attractions likely present an important risk of transmission of cutaneous leishmaniasis (CL). Furthermore, with the geographical expansion of visceral leishmaniasis (VL), several studies have recorded the occurrence of its vector, Lutzomyia longipalpis, and cases of human and canine VL in such tourist areas. The Parque Estadual do Sumidouro is an environmentally protected area located in the Brazilian Cerrado biome and in an important area endemic for leishmaniasis in the state of Minas Gerais. The purpose of this study was to monitor the sand fly fauna in areas of tourist activity in the park. Sampling was performed every month, from September 2011 to August 2013, using CDC light traps at six sites of differing environmental characteristics. Sampled specimens were identified following Galati (2003), and females were submitted to molecular techniques for the detection and identification of Leishmania DNA. A total of 4,675 sand fly specimens of 25 species belonging to nine genera were collected. The most abundant species were Micropygomyia quinquefer, Lutzomyia renei and Pintomyia pessoai, although only Pi. pessoai is implicated in the transmission of Leishmania braziliensis. The species accumulation curve reached saturation on the 16th sampling event. Species richness, diversity and evenness differed among the sampled areas. The seasonal curve was not determined by a single unique species, and no single species was the most abundant in all environments sampled. The main vector of Leishmania (Leishmania) infantum, Lutzomyia longipalpis, accounted for only 5.35% of the specimens collected. Proven or suspected vectors of Leishmania (Viannia) braziliensis were recorded, and one female of the cortellezzii complex tested positive for Le. braziliensis DNA. Even with a low infection rate (0.62%), these data indicate the circulation of the parasite and reinforce the need for entomological and epidemiological surveillance in the park and its surroundings.     

Fisherian flies: benefits of female choice in a lekking sandfly

We experimentally investigated the fitness consequences of female mate choice in order to test the relative importance of three competing but non-exclusive hypotheses for the maintenance of pronounced female mating preferences on leks: that females benefit directly; that they gain indirect Fisherian benefits by producing more attractive sons; or that they benefit indirectly because preferred males possess ''good genes'' that confer increased viability on their sons and daughters. We allowed lekking female sandflies, Lutzomyia longipalpis, to choose between males of varying attractiveness to females, and monitored the consequences for their own survival and reproductive success as well as for their offspring. In contrast to the predictions of the direct-benefits model, we found no clear sire effect on the fecundity or survival of the females themselves; females mating with more attractive males did survive longer after oviposition, but never long enough to undertake a second batch of egg laying. We also found no evidence that females gained good-genes benefits in terms of enhanced offspring survival. However, we did find that generally attractive males fathered sons who were then chosen when they in turn formed leks. Although not completely precluding other benefits, our results indicate that Fisherian benefits are at least partly responsible for maintaining female choice at L. longipalpis leks. These findings indicate the importance of testing all putative benefits concurrently in exploring the maintenance of female mate choice.     

Effect of Leishmania spp infection on the survival,life expectancy,fecundity and fertility of Lutzomyia longipalpis s.l. and Lutzomyia pseudolongipalpis

We evaluated the effects of Leishmania spp infection on several population parameters of Lutzomyia longipalpis sensu lato andLutzomyia pseudolongipalpis, vectors of visceral leishmaniasis in Venezuela, under experimental conditions during the first post-feeding period. Females of both species were allowed to feed and engorge on a suspension of fresh washed human red blood cells in foetal calf serum. These blood cells were either non-infected or infected with one of the fourLeishmania spp strains and were offered through a chicken skin membrane. The longevity, life expectancy and the fecundity of uninfected flies were similar in both species, but the fertility was significantly lower in uninfected Lu. longipalpis females. In all cases, the infection of Lu. longipalpis and Lu. pseudolongipalpis by the Leishmania strains resulted in significant detrimental effects, which exerted a fitness cost expressed by reduced survival and life expectancy, as well as decreased fertility and fecundity compared with the control groups. Nevertheless, differences in these parameters were observed between these vector species depending on whether they were infected with the autochthonous Venezuelan Leishmania infantum strain (NESA) or the Brazilian reference strain (PP75). The experimental data obtained agree with field data on the natural infection of these vector species and the significance of this scenario is discussed.     

Polytene chromosomes of the sandfly Lutzomyia longipalpis and the cytogenetics of Psychodidae in relation to other Diptera

Polytene giant chromosomes of the Phlebotomine sandfly Lutzomyia longipalpis from two localities in Brazil were examined. They are preliminarily described and illustrated from larval salivary gland preparations. Polytenes in other tissues of larvae, pupae and adult flies were found to be unsuitable for detailed study. Salivary chromosome arms were long but thin (‘oligotene’) and given to much ectopic pairing. Despite their fragility and cross-linkage, patterning on these chromosomes was sufficiently well differentiated to be cytotaxonomically useful. The normal mitotic complement comprised 3 pairs of metacentrics and 1 pair of subacrocentrics. None of the chromosome pairs was sexually dimorphic. Meiotic chiasmata were seen in males and females. Characteristics of giant chromosomes in Phlebotominae and Psychodinae are compared and discussed in relation to other Diptera. Cytogenetic evidence supports inclusion of the Psychodidae with the Simuliidae, Ceratopogonidae, Culicidae and Chironomidae in the superfamily Culicoidea.     

Phlebotomine fauna,natural infection rate and feeding habits of Lutzomyia cruzi in Jaciara,state of Mato Grosso, Brazil

Visceral leishmaniasis (VL) in Brazil is transmitted by the phlebotomine Lutzomyia longipalpis and in some midwestern regions by Lutzomyia cruzi. Studies of the phlebotomine fauna, feeding habits and natural infection rate by Leishmania contribute to increased understanding of the epidemiological chain of leishmaniases and their vectorial capacity. Collections were performed in Jaciara, state of Mato Grosso from 2010-2013, during which time 2,011 phlebotomines (23 species) were captured (68.70% Lu. cruzi and 20.52% Lutzomyia whitmani). Lu. cruzi females were identified by observing the shapes of the cibarium (a portion of the mouthpart) and spermatheca, from which samples were obtained for polymerase chain reaction to determine the rates of natural infection. Engorged phlebotomines were assessed to identify the blood-meal host by ELISA. A moderate correlation was discovered between the number of Lu. cruzi and the temperature and the minimum rate of infection was 6.10%. Twenty-two females were reactive to the antisera of bird (28%), dog (3.30%) and skunk (1.60%). We conclude that Lu. cruzi and Lu. whitmani have adapted to the urban environment in this region and that Lu. cruzi is the most likely vector of VL in Jaciara. Moreover, maintenance of Leishmania in the environment is likely aided by the presence of birds and domestic and synanthropic animals.     

Multilocus Analysis of Divergence and Introgression in Sympatric and Allopatric Sibling Species of the Lutzomyia longipalpis Complex in Brazil

Background

Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Latin America, is a complex of sibling species. In Brazil, a number of very closely related sibling species have been revealed by the analyses of copulation songs, sex pheromones and molecular markers. However, the level of divergence and gene flow between the sibling species remains unclear. Brazilian populations of this vector can be divided in two main groups: one producing Burst-type songs and the Cembrene-1 pheromone and a second more diverse group producing various Pulse song subtypes and different pheromones.

Methodology/Principal Findings

We analyzed 21 nuclear loci in two pairs of Brazilian populations: two sympatric populations from the Sobral locality (1S and 2S) in northeastern Brazil and two allopatric populations from the Lapinha and Pancas localities in southeastern Brazil. Pancas and Sobral 2S are populations of the Burst/Cembrene-1 species while Lapinha and Sobral 1S are two putative incipient species producing the same pheromone and similar Pulse song subtypes. The multilocus analysis strongly suggests the occurrence of gene flow during the divergence between the sibling species, with different levels of introgression between loci. Moreover, this differential introgression is asymmetrical, with estimated gene flow being higher in the direction of the Burst/Cembrene-1 species.

Conclusions/Significance

The results indicate that introgressive hybridization has been a crucial phenomenon in shaping the genome of the L. longipalpis complex. This has possible epidemiological implications and is particularly interesting considering the potential for increased introgression caused by man-made environmental changes and the current trend of leishmaniasis urbanization in Brazil.