Identification of sex pheromones of Lutzomyia longipalpis (Lutz & Neiva, 1912) populations from the state of São Paulo, Brazil

In Brazil, four populations of Lutzomyia longipalpis each producing different sex pheromones are recognised. It has been suggested that these chemotype populations represent true sibling species. In this study we present the results of an analysis, by coupled gas chromatography-mass spectrometry, of the pheromones of males L. longipalpis from two different municipalities of the state of S?o Paulo. Our study showed that L. longipalpis from these two municipalities produced different sex pheromones from each other. This coupled with the remarkable difference between the epidemiological situation in Ara?atuba and Espírito Santo do Pinhal, suggests that the (S)-9-methylgermacrene-B and cembrene-1 populations may have different vectorial capacities.     

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.     

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 (theta = 0.018; RST = -0.005), and genetic differentiation was low between the cembrene collections (theta = 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 (theta = 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 (theta = 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 (theta = 0.229-0.268; P < 0.0001 for the 2001 and theta = 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.     

Isolation and identification of 9-methylgermacrene-B as the putative sex pheromone of Lutzomyia cruzi (Mangabeira, 1938) (Diptera: Psychodidae)

Lutzomyia (Lutzomyia) cruzi has been named as a probable vector of Leishmania chagasi in Corumbá, Mato Grosso do Sul, Brazil. Taxonomically L. cruzi is closely related to the L. longipalpis species complex. Females of L. cruzi and L. longipalpis are morphologically indistinguishable and associated males must be examined carefully to confirm identifications. Chemical analysis hexane extracts of male L. cruzi has revealed the presence of a 9-methylgermacrene-B (C16), a homosesquiterpene (mw 218) previously shown to be the sex pheromone of one of the members of the L. longipalpis species complex.     

Sex pheromone and period gene characterization of Lutzomyia longipalpis sensu lato (Lutz & Neiva) (Diptera: Psychodidae) from Posadas, Argentina

Lutzomyia longipalpis s.l. is the primary vector of Leishmania (L.) infantum in the New World. In this study, male Lutzomyia longipalpis specimens from Posadas, Argentina were characterized for two polymorphic markers: the male sex pheromone and the period (per) gene. The male sex pheromone was identified as (S)-9-methylgermacrene-B, the same compound produced by Lu. longipalpis from Paraguay and many populations from Brazil. The analysis of per gene sequences revealed that the population from Argentina is significantly differentiated from previously studied Brazilian populations. Marker studies could contribute to the understanding of the distribution and spread of urban American visceral leishmaniasis, thus aiding in the design of regional surveillance and control strategies.     

GENE FLOW IN LICHENS

Experimental studies of the evolutionary biology of lichen fungi have been hampered by massive difficulties of in vitro culture and artificial crosses are still not possible. Gene flow in these organisms is demonstrated here for the first time by the analysis of secondary products in the progeny of individuals from natural populations of mixed chemotypes of the Cladonia chlorophaea complex. All of the chemotypes in this study have been interpreted as distinct sibling species. In the Appalachian Mountains of North Carolina, however, the grayi and merochlorophaea chemotypes are found to belong to a single interbreeding populations that is reproductively isolated from the cryptochlorophaea chemotype. In the Coastal Plain, the cryptochlorophaea chemotype hybridizes with the local endemic perlomera chemotype. This study has major impact for species concept in lichens because consideration of neither morphologial tendencies nor biogenetic relationships of the secondary products could have predicted its result.     

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.     

Chemical and molecular characterization of Phomopsis and Cytospora-like endophytes from different host plants in Brazil

Phomopsis and related taxa comprise important endophytic and plant pathogenic species, and are known for the production of a diverse array of secondary metabolites. Species concepts within this group based on morphological characters and assumed host specificity do not reflect phylogenetic affinities. Additional phenotypic characters, such as profiles of secondary metabolites, are needed for practical species recognition. We investigated 36 strains of Phomopsis spp. and Cytospora-like fungi, obtained as endophytes of different host plants in Brazil, using metabolite profiling based on HPLC-UV/liquid chromatography -mass spectrometry (LC-MS) combined with cluster analysis of the results. Strains were also subjected to phylogenetic analyses based on internal transcribed spacer (ITS) rDNA. Six chemotypes were identified. Chemotypes 1-5 contained Phomopsis strains, while Cytospora-like strains formed the chemotype 6. Strains of chemotype 1 typically produced alternariols, altenusin, altenuene, cytosporones, and dothiorelones. Alternariol and seven unknown compounds were consistently produced by strains of chemotype 2. Members of chemotypes 3-5 produced poor metabolite profiles containing few chemical markers. Cytospora-like endophytes (chemotype 6) produced a characteristic set of metabolites including cytosporones and dothiorelones. Bayesian and Maximum Parsimony (MP) trees classified strains of each chemotype into single phylogenetic lineages or closely related groups. Strains of chemotypes 1 and 2 formed a monophyletic group along with Diaporthe neotheicola. The remaining Phomopsis strains formed monophyletic (chemotype 4) or polyphyletic (chemotypes 3 and 5) lineages inside a large and well supported clade. Cytospora-like strains formed a monophyletic lineage located at an intermediary position between Diaporthe/Phomopsis and Valsa/Cytospora clades. The combined results show that the production of secondary metabolites by Phomopsis and related Diaporthales may be species-specific, giving support to the use of metabolite profiling and chemical classification for phenotypic recognition and delimitation of species.     

Sex pheromone evolution is associated with differential regulation of the same desaturase gene in two genera of leafroller moths

Chemical signals are prevalent in sexual communication systems. Mate recognition has been extensively studied within the Lepidoptera, where the production and recognition of species-specific sex pheromone signals are typically the defining character. While the specific blend of compounds that makes up the sex pheromones of many species has been characterized, the molecular mechanisms underpinning the evolution of pheromone-based mate recognition systems remain largely unknown. We have focused on two sets of sibling species within the leafroller moth genera Ctenopseustis and Planotortrix that have rapidly evolved the use of distinct sex pheromone blends. The compounds within these blends differ almost exclusively in the relative position of double bonds that are introduced by desaturase enzymes. Of the six desaturase orthologs isolated from all four species, functional analyses in yeast and gene expression in pheromone glands implicate three in pheromone biosynthesis, two Δ9-desaturases, and a Δ10-desaturase, while the remaining three desaturases include a Δ6-desaturase, a terminal desaturase, and a non-functional desaturase. Comparative quantitative real-time PCR reveals that the Δ10-desaturase is differentially expressed in the pheromone glands of the two sets of sibling species, consistent with differences in the pheromone blend in both species pairs. In the pheromone glands of species that utilize (Z)-8-tetradecenyl acetate as sex pheromone component (Ctenopseustis obliquana and Planotortrix octo), the expression levels of the Δ10-desaturase are significantly higher than in the pheromone glands of their respective sibling species (C. herana and P. excessana). Our results demonstrate that interspecific sex pheromone differences are associated with differential regulation of the same desaturase gene in two genera of moths. We suggest that differential gene regulation among members of a multigene family may be an important mechanism of molecular innovation in sex pheromone evolution and speciation.     

Fixed Differences in the paralytic Gene Define Two Lineages within the Lutzomyia longipalpis Complex Producing Different Types of Courtship Songs

The sand fly Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae), the most important vector of American visceral leishmaniasis, is widely distributed in Latin America. There is currently a consensus that it represents a species complex, however, the number and distribution of the different siblings is still uncertain. Previous analyses have indicated that Brazilian populations of this vector can be divided into two main groups according to the type of courtship song (Burst vs. Pulse) males produce during copulation. Nevertheless, no diagnostic differences have been observed between these two groups with most molecular markers used to date. We analyzed the molecular divergence in a fragment of the paralytic (para) gene, a locus involved in the control of courtship songs in Drosophila, among a number of Lu. longipalpis populations from Brazil producing Burst and Pulse-type songs. Our results revealed a very high level of divergence and fixed differences between populations producing the two types of songs. We also compared Lu. longipalpis with a very closely related species, Lutzomyia cruzi, which produces Burst-type songs. The results indicated a higher number of fixed differences between Lu. cruzi and the Pulse-type populations of Lu. longipalpis than with those producing Burst-type songs. The data confirmed our previous assumptions that the presence of different sibling species of the Lu. longipalpis complex in Brazil can be divided into two main groups, one representing a single species and a second more heterogeneous group that probably represents a number of incipient species. We hypothesize that para might be one of the genes directly involved in the control of the courtship song differences between these two groups or that it is linked to other loci associated with reproductive isolation of the Brazilian species.     

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.     

RAPD based genetic relationships between populations of three chemotypes of Cunila galioides Benth.

Cunila galioides, a popular aromatic and medicinal plant of South Brazil, is one of the 12 South American species of the genus Cunila. With a center of distribution on the highlands of South Brazil, C. galioides has been separated in three chemotypes, characterized by high concentrations of citral, ocimene and menthene, respectively. Twenty populations collected at Rio Grande do Sul and Santa Catarina States, were analyzed by RAPD using 20 arbitrary primers that generated 239 scorable bands. Genetic distances were calculated using Jaccard's coefficient. Cluster analysis was carried out using the Single Linkage method. The analysis with RAPD markers revealed wide variation within C. galioides that reflected a high level of diversity within this species. Cluster and canonical discriminating analysis indicated that the populations of the citral and menthene chemotypes are more closely related to each other, and the populations of the ocimene chemotype represent a different genetic pool.     

Molecular and phenotypic descriptions of Stachybotrys chlorohalonata sp. nov. and two chemotypes of Stachybotrys chartarum found in water-damaged buildings

Twenty-five Stachybotrys isolates from two previous studies have been examined and compared, using morphological, chemical and phylogenetic methods. The results show that S. chartarum sensu lato can be segregated into two chemotypes and one new species. The new species, S. chlorohalonata, differs morphologically from S. chartarum by having smooth conidia, being more restricted in growth and producing a green extracellular pigment on the medium CYA. S. chlorohalonata and S. chartarum also have different tri5, chs1 and tub1 gene fragment sequences. The two chemotypes of S. chartarum, chemotype S and chemotype A, have similar morphology but differ in production of metabolites. Chemotype S produces macrocyclic trichothecenes, satratoxins and roridins, while chemotype A produces atranones and dolabellanes. There is no difference between the two chemotypes in the tub1 gene fragment, but there is a one nucleotide difference in each of the tri5 and the chs1 gene fragments.     

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.     

Identification of sibling species of the bryozoan Bugula neritina that produce different anticancer bryostatins and harbor distinct strains of the bacterial symbiont "Candidatus Endobugula sertula".

Although the cosmopolitan marine bryozoan Bugula neritina is recognized as a single species, natural products from this bryozoan vary among populations. B. neritina is the source of the anticancer drug candidate bryostatin 1, but it also produces other bryostatins, and different populations contain different bryostatins. We defined two chemotypes on the basis of previous studies: chemotype O contains bryostatins with an octa-2,4-dienoate substituent (including bryostatin 1), as well as other bryostatins; chemotype M lacks bryostatins with the octa-2,4-dienoate substituent. B. neritina contains a symbiotic gamma-proteobacterium "Candidatus Endobugula sertula," and it has been proposed that bryostatins may be synthesized by bacterial symbionts. In this study, B. neritina populations along the California coast were sampled for genetic variation and bryostatin content. Colonies that differ in chemotype also differ genetically by 8% in the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene; this difference is sufficient to suggest that the chemotypes represent different species. Each species contains a distinct strain of "E. sertula" that differs at four nucleotide sites in the small subunit ribosomal RNA (SSU rRNA) gene. These results indicate that the chemotypes have a genetic basis rather than an environmental cause. Gene sequences from an Atlantic sample matched sequences from the California chemotype M colonies, suggesting that this type may be cosmopolitan due to transport on boat hulls.     

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.     

The mode of pheromone evolution: evidence from bark beetles

Sex and aggregation pheromones consist of species-specific blends of chemicals. The way in which different species' blends have evolved has been the subject of some debate. Theoretical predictions suggest that differences between species have arisen not through the accruing of small changes, but through major shifts in chemical composition. Using data on the aggregation pheromones of 34 species of bark beetle from two genera, Dendroctonus and Ips, we investigated how the distributions of the chemical components of their pheromone blends mirror their phylogenetic relationships. We tested whether there were consistent patterns that could be used to help elucidate the mode of pheromone evolution. Although there were obvious differences in pheromone blends between the two genera, the differences between species within each genus followed a less clear phylogenetic pattern. In both genera, closely related species are just as different as more distantly related species. Within Dendroctonus, particularly, most chemical components were distributed randomly across the phylogeny. Indeed, for some chemicals, closely related species may actually be more different than would be expected from a random distribution of chemical components. This argues strongly against the idea of minor shifts in pheromone evolution. Instead, we suggest that, within certain phylogenetic constraints, pheromone evolution in bark beetles is characterized by large saltational shifts, resulting in sibling species being substantially phenotypically (i.e. pheromonally) different from one another, thus agreeing with theoretical predictions.     

Analysis of the activity patterns of two sympatric sandfly siblings of the Lutzomyia longipalpis species complex from Brazil

Abstract.  Lutzomyia longipalpis s.l. (Lutz & Neiva) (Diptera: Psychodidae) is the main vector of visceral leishmaniasis in Latin America. Differences in copulation songs, pheromones and molecular markers show that L.  longipalpis is a species complex in Brazil. The patterns of activity of insect vectors are important in disease transmission. In addition, differences in activity rhythms have a potential role as a temporal reproductive isolation mechanism in closely related species. We compared the activity patterns of males and females of two sympatric species of the Longipalpis complex from Sobral (Ceará State, Brazil) in controlled laboratory conditions. We observed small but significant differences between the two species in the activity phase in both males and females.     

Activity of Lutzomyia pseudolongipalpis and L. longipalpis s.l. (Diptera: Psychodidae) in Venezuela

The nocturnal activity of the phlebotomine sandfly Lutzomyia pseudolongipalpis and two populations of L. longipalpis s.l. from different American visceral leishmaniasis foci in Venezuela was studied using collection bottle rotator traps. The activity of L. pseudolongipalpis from Lara State was continuous throughout the night, while that of L. longipalpis s.l. from El Layero, Guárico State and from Santa Ana del Valle, Margarita Island, was greatest before 23:00 h. The activity of sandflies of both populations and sexes steadily decreased thereafter. These different patterns seem to correlate with genetic data that indicate the presence in Venezuela of at least two sibling species in the L. longipalpis complex. The advantages of the bottle rotator trap for this type of study are discussed.