J. G. C. Hamilton

Identification of a male-produced aggregation pheromone in the western flower thrips Frankliniella occidentalis.

Two major components have been detected in the headspace volatiles of adult male Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) that are not present in the headspace volatiles of adult females. The compounds were identified as (R)-lavandulyl acetate and neryl (S)-2-methylbutanoate by comparison with synthetic standards using gas chromatography (GC), GC–mass spectrometry (MS), and chiral GC. Field trials were conducted with synthetic compounds in naturally infested crops of sweet pepper grown in large plastic greenhouses in Spain. The catch of adult females and males on blue sticky traps was increased by neryl (S)-2-methylbutanoate alone or by a 1:1 blend of (R)-lavandulyl acetate and neryl (S)-2-methylbutanoate, but (R)-lavandulyl acetate was not active alone. This is the first identification of an aggregation pheromone in the order Thysanoptera. The possible role of (R)-lavandulyl acetate is discussed.

9-Methylgermacrene-B; proposed structure for novel homosesquiterpene from the sex pheromone glands ofLutzomyia longipalpis (Diptera: Psychodidae) from Lapinha, Brazil

The principal volatile component (99 + %) of the sex pheromone glands ofLutzomyia longipalpis from Lapinha, Brazil, has been isolated and characterized as a novel homosesquiterpene with the specific structure proposed as 9-methylgermacrene-B, (E,E)-8-(1-methylethylidenyl)-1,5,10-trimethyl-1,5-cyclodecadiene.

Analysis of the sex pheromone extract of individual male Lutzomyia longipalpis sandflies from six regions in Brazil.

Abstract.  Although the phlebotomine sandfly Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae) is generally accepted to be a species complex, it is unclear how many members there are, how they are related and which are the main vectors of leishmaniasis. The vectorial capacity of each sibling species is likely to differ, thus a means of identifying the most important vector species is of critical importance to the epidemiology and control of this debilitating disease in South and Central America. In Brazil four chemotypes have been distinguished by sex pheromone analysis. In this study the sex pheromone extracts of L. longipalpis from six regions of Brazil were analysed in detail. Samples included the sympatric 1‐spot, 2‐spot and intermediate spot morphotypes from Sobral, Ceará State. The results strongly suggest that members of the complex that produce different sex pheromones are reproductively isolated, thus strengthening the argument that the different chemotypes represent true sibling species. The study also found significant differences in morphology and the amounts of sex pheromone produced by members of each chemotype from different parts of Brazil, which suggests population substructuring that has not previously been recognized. Evidence of a fifth chemotype in Brazil is also presented.

Genetic identification of two sibling species of Lutzomyia longipalpis (Diptera: Psychodidae) that produce distinct male sex pheromones in Sobral, Ceará State, Brazil.

Lutzomyia longipalpis, the main sandfly vector for New World visceral leishmaniasis is a complex of an as yet undefined number of sibling species. At present, there is no consensus on the status (single species vs. species complex) of Brazilian populations. We applied five microsatellite loci to test the hypothesis that L. longipalpis occurs as two sympatric cryptic species in Sobral, Ceará State, Brazil as predicted by male sex pheromone chemotypes described previously for field specimens from this site [S‐9‐methyl‐germacrene‐B (9MGB) and a cembrene compound]. Abdominal spot morphology corresponds with pheromone type at this locality (9MGB in ‘1 spot’ males and cembrene in ‘2 spot’ males). Genotype data from 190 wild‐caught L. longipalpis specimens collected in October 1999 and April 2001 were used to estimate genetic differentiation between the two sex pheromone populations and sampling dates. No significant (P > 0.05) genetic differences were found between the 1999 and 2001 9MGB samples (θ = 0.018; RST = −0.005), and genetic differentiation was low between the cembrene collections (θ = 0.037, P < 0.05; RST = −0.043, P > 0.05). By contrast, highly divergent allelic frequencies (largely at two microsatellite loci) corresponded to significant (P > 0.05) genetic differentiation (θ = 0.221; RST = 0.215) for all comparisons between samples with different pheromones. When pheromone samples were pooled across sample date, genetic differentiation was high (θ = 0.229; P < 0.001; Nem = 0.84). The allele frequency distribution at each of the five microsatellite loci was similar for males and females from the two collection years. Two of these loci showed highly divergent allele frequencies in the two sex pheromone populations. This was reflected in the highly significant genetic differentiation obtained from the male genotypes, between populations producing different pheromones (θ = 0.229–0.268; P < 0.0001 for the 2001 and θ = 0.254–0.558; P < 0.0001 for the 1999 collections, respectively). Similar results were obtained when the females, assigned to a pheromone type, were included in the analysis. Both a Bayesian analysis of the data set and a population assignment test provided strong evidence for two distinct populations corresponding to pheromone type. Given its genotype, the probability of assigning a 9MGB male to the original 9MGB population was 100% once the two years’ collections were pooled. For cembrene‐producing ‘2 spot’ males this probability although still high, was lower than for 9MGB males, at 86%. This microsatellite data together with previously reported reproductive isolation between the two Sobral populations confirm that premating barriers are important in speciation of L. longipalpis.

3-Methyl-α-himachalene: Proposed structure for novel homosesquiterpene sex pheromone ofLutzomyia longipalpis (diptera: Psychodidae) from Jacobina, Brazil

The principal behaviorally active volatile component (ca. 90% +) of the sex pheromone glands ofLutzomyia longipalpis from Jacobina. Brazil, has been isolated and characterized as a novel homosesquiterpene with the structure 3-methyl-α-himachalene (C16H26). A minor component (ca. 10%) of the gland extract has also been identified as the sesquiterpeneα-himachalene (C15H24). This work confirms that there are at least 3 members of theL. longipalpis species complex.

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.

A role for pheromones in mate choice in a lekking sandfly.

There is increasing evidence to suggest that pheromone-mediated communication may play an important role in mate choice in many insects. We investigated the possible role of pheromones in mate choice in the lekking sandfly, Lutzomyia longipalpis, and explored whether males or females display any behavioural traits that predict patterns of male mating success. Pairs of virgin males were introduced into a small mating chamber and allowed to interact for approximately 10 min prior to the introduction of a single virgin female. The trial continued until the female copulated with one of the males. We recorded trials on video for later analysis. After a successful copulation, we determined the quantity of pheromone remaining in the glands of both males and measured the size of the pheromone glands and tergites. We corrected behaviours and measurements for body size (tergite width). Mated males had significantly more pheromone present in their glands after a trial than unmated males. Males differed in how much time they invested in wing fanning. Members of each pair of males spent similar amounts of time wing fanning but mated males and males with more residual pheromone spent more time wing fanning than their counterparts throughout the trial. The time spent wing fanning and the amount of residual pheromone were not correlated, however. A males ability to win fights did not relate to his reproductive success. Finally, a female was equally likely to wing-fan directly at, or walk away from, either male in a given pair. The possible relationships between the amount of pheromone released and the residual pheromones left in the glands, and the level of male wing fanning and pheromone productivity, are discussed. Copyright 1998 The Association for the Study of Animal Behaviour.

Synthetic Sex Pheromone Attracts the Leishmaniasis Vector Lutzomyia longipalpis (Diptera: Psychodidae) to Traps in the Field

ABSTRACT Improving vector control remains a key goal in reducing the worlds burden of infectious diseases. More cost-effective approaches to vector control are urgently needed, particularly because vaccines are unavailable and treatment is prohibitively expensive. The causative agent of American visceral leishmaniasis (AVL), Leishmania chagasi, Cunha and Chagas (Kinetoplastida: Trypanosomatidae), is transmitted between animal and human hosts by blood-feeding female sand flies attracted to mating aggregations formed on or above host animals by male-produced sex pheromones. Our results show the potential of using synthetic pheromones to control populations of Lutzomyia longipalpis Lutz and Neiva (Diptera: Psychodidae), the sand fly vector of one of the worlds most important neglected diseases, AVL. We showed that a synthetic pheromone, (±) -9-methylger-macrene-B, produced from a low-cost plant intermediate, attracted females in the laboratory. By formulating dispensers that released this pheromone at a rate similar to that released by aggregating males, we were able to attract flies of both sexes to traps in the field. These dispensers worked equally well when deployed with mechanical light traps and inexpensive sticky traps. If deployed effectively, pheromone-based traps could be used to decrease AVL transmission rates through specific targeting and reduction of L. longipalpis populations. This is the first study to show attraction of a human disease-transmitting insect to a synthetic pheromone in the field, showing the general applicability of this novel approach for developing new tools for use in vector control.

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 real-time PCR assay to estimate Leishmania chagasi load in its natural sand fly vector Lutzomyia longipalpis.

Summary Leishmania chagasi, transmitted mainly by Lutzomyia longipalpis sand flies, causes visceral leishmaniasis and atypical cutaneous leishmaniasis in Latin America. Successful vector control depends upon determining vectorial capacity and understanding Leishmania transmission by sand flies. As microscopic detection of Leishmania in dissected sand fly guts is laborious and time-consuming, highly specific, sensitive, rapid and robust Leishmania PCR assays have attracted epidemiologists’ attention. Real-time PCR is faster than qualitative PCR and yields quantitative data amenable to statistical analyses. A highly reproducible Leishmania DNA polymerase gene-based TaqMan real-time PCR assay was adapted to quantify Leishmania in sand flies, showing intra-assay and inter-assay coefficient variations lower than 1 and 1.7%, respectively, and sensitivity to 10 pg Leishmania DNA (∼120 parasites) in as much as 100 ng sand fly DNA. Data obtained for experimentally infected sand flies yielded parasite loads within the range of counts obtained by microscopy for the same sand fly cohort or that were around five times higher than microscopy counts, depending on the method used for data analysis. These results highlight the potential of quantitative PCR for Leishmania transmission studies, and the need to understand factors affecting its sensitivity and specificity.