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.     

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.     

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.     

Studies on populations of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in Brazil

Studies were performed on five Brazilian populations of Lutzomyia longipalpis: Salvaterra (PA), S?o José do Ribamar (MA), Canindé (CE), Natal (RN) and Gruta da Lapinha, Lagoa Santa (MG). No morphological differences were observed that could distinguish between these populations. Homogeneity tests showed that the allopatric populations display a certain heterogeneity and that the sympatric populations, with different patterns of spots, are homogeneous. The Student-Newman-Keuls test, represented by Euler-Venn diagrams, showed a disjunction between the populations from the north/northeast and the one from Gruta da Lapinha. Genetic distances between the four populations (excluding the Canindé population) were within the range of intrapopulational differences. The Gruta da Lapinha population displayed a heterozygotic deficiency that could be a consequence of high levels of inbreeding due to cryptic habits of living in a small cave. These results do not favor the hypothesis of a L. longipalpis species complex in Brazil, and the species should be considered high polymorphic.     

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.     

Attraction of Lutzomyia longipalpis to human skin odours

Abstract. Male and female Lutzomyia longipalpis sandflies showed attraction to human skin emanations placed on warmed glass Petri dishes. Unfed virgin females were more strongly attracted than males, which also showed attraction. Four human subjects were tested and significant variation was found between the numbers of sandflies attracted to their skin emanations. This suggests that some individuals were more attractive than others. There was a significant difference between the response shown by sandflies from the Jacobina and Lapinha regions of Brazil, suggesting that sandflies from the Jacobina region were more anthropophilic. In addition, sandflies from Jacobina had a significantly higher level of activity than those from Lapinha. The role of sandfly attraction to humans as a risk factor in Leishmania transmission is considered.     

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.     

Crop colonisation, feeding, and reproduction by the predatory beetle, Hippodamia convergens, as indicated by stable carbon isotope analysis

Abstract.  1. Habitat management to enhance natural enemy populations in agricultural systems may help regulate levels of crop pests, but little research addresses the behaviour of immigrating beneficial insects.
2. Stable carbon isotopes were used in complementary laboratory and field studies to examine colonisation behaviour of an ephemeral agricultural habitat by the lady beetle, Hippodamia convergens Guérin-Méneville.
3. Under laboratory conditions, H. convergens carbon isotope ratios, δ¹³C, changed after its food supply was shifted from a C₄- to a C₃-based diet of aphids produced on grain sorghum or cotton respectively. Final isotope ratios of adult H. convergens were closer to that of the new C₃-based diet, with most change in δ¹³C occurring within 3 days after the diet shift.
4. The carbon isotope ratios of lady beetle adults collected in cotton fields suggested that grain sorghum was a continuous source for H. convergens until many nearby sorghum fields matured and senesced.
5. When cotton aphid ( Aphis gossypii Glover) prey were absent, carbon isotope ratios of beetle populations did not change over time and virtually no egg production by H. convergens was detected. This indicates that beetles were feeding little on non-aphid resources originating in cotton.
6. With cotton aphids present, beetle isotope ratios decreased towards the carbon isotope ratio of cotton, indicating adult feeding in cotton. As a result, egg masses produced had carbon isotope ratios in the C₃ range of values.
7. The results suggest that some predator species may be retained in habitats without large prey populations, a quality essential in controlling pests in agricultural systems.     

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.     

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.     

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.     

Alkane composition diversity among populations of dwarf forms of Juniperus communis L.: comparison between western Europe and northern American populations

The cuticular wax composition of leaves has been analysed in three western European populations (Corsica, central Pyrenees, northern Alps) of Juniperus communis var. saxatilis Pall. (=  J. nana Willd., nom illeg.) and in one population of J. communis L. var. depressa Pursh. from North America (Sierra Nevada). Gas chromatography shows the presence of 13 alkanes in all samples ranging from C₂₃ to C₃₅ with important intraspecific polymorphism in alkane content. The dominant alkanes range from C₃₃ to C₃₅. Alkanes C₂₁ and C₂₂ were found only in Corsica and Sierra Nevada populations. Canonical discriminant analysis separated the J. communis L. var. depressa Pursh. of the population of Sierra Nevada from other populations of J. communis var. saxatilis Pall. on the basis of their higher C₃₁ content and the constant presence of C₂₁ and C₂₂ alkanes. J. communis var. saxatilis Pall. populations from the Pyrenees are close to northern Alps populations characterized by high concentrations of C₃₃, C₃₄ and C₃₅ alkanes. This paper confirms the existence of Juniperus var. saxatilis Pall. in the Pyrenees (France).  © The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 140 , 165–168.     

Reproductive isolation between sympatric and allopatric Brazilian populations of Lutzomyia longipalpis s.l. (Diptera: Psychodidae)

Lutzomyia longipalpis s.l., the main vector of Leishmania chagasi in Latin America, is a species complex although the exact number of siblings is yet unknown. In Brazil, the siblings differ in male copulatory courtship songs and pheromones that most certainly act as pre-zygotic reproductive barriers. Here we analysed the reproductive isolation between three allopatric and two sympatric populations of Lu. longipalpis s.l. from Brazil. The results indicate a strong copulatory and pre-mating isolation between the three allopatric populations. In addition, the results also indicate a stronger pre-mating isolation between the two sympatric siblings than between the three allopatric ones, suggesting a role for reinforcement in the speciation of the Lu. longipalpis s.l. complex.     

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

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.     

Distribution of C3 and C4 grasses along an altitudinal gradient in Central Argentina

The distribution pattern of C₃ and C₄ grasses was studied in eight sites located between 350 m and 2100 m along an altitudinal gradient in Central Argentina. Of 139 taxa fifty-nine are C₃ and eighty C₄. Species of the C₃ tribes (Stipeae, Poeae, Meliceae, Aveneae, Bromeae and Triticeae) and C₃ Paniceae species increase in number at higher elevations; only one C₃ species was found below 650 m. C₄ Aristideae, Pappophoreae, Eragrostideae, Cynodonteae, Andropogoneae and Paniceae increase at lower altitudes. The floristic crossover point is at about 1500 m; the ground cover cross-over point is at about 1000 m. Analysis of the relationships between % C₄ species along the gradient and nine climatic and environmental variables showed the highest correlation with July mean temperature, but all temperature variables show highly significant correlations with % C₄. Correlation with annual rainfall is lower but also significant. These results are consistent with previous research showing the relative importance of C₄ grasses as temperature increases. C₃ species make a high contribution to relative grass coverage below the C₃/C₄ floristic crossover point but are rare below 1000 m.     

C4 photosynthesis at low temperatures

Abstract. C₄ plants grown in optimum conditions are, by comparison to C₃, capable of higher maximum dry-matter yields and greater efficiencies of water and nitrogen use, yet they are rare outside the subtropics. Both latitudinal and altitudinal limits of C₄ distributions correlate most closely with a mean minimum temperature of 8-10°C during the period of active growth. The possibility that the C₄ process is inherently incapable of functioning at low temperatures is examined. The reversible effects of chilling on the quantum efficiency of C₄ photosynthesis and the functioning of the individual steps in the C₄ cycle are examined. Chilling also produces an irreversible loss of capacity to assimilate CO₂ which is directly proportional to the light received during chilling. It is suggested that the reversible reduction in capacity to assimilate CO₂ and the lack of an alternative pathway for the utilization of lightgenerated reducing power may make C₄ species more prone to chilling-dependent photoinhibition. Laboratory studies and limited field observations suggest that this damage would be most likely to occur during photosynthetic induction at the temperatures and light levels encountered on clear, cool mornings during the spring and early summer in cool climates. Even those C₄ species occurring naturally in cool climates do not appear fully capable of tolerating these conditions; indeed their growth patterns suggest that they may be adapted by avoiding 'rather than enduring' such conditions.     

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.     

Seasonal differences in photosynthesis between the C3 and C4 subspecies of Alloteropsis semialata are offset by frost and drought

The regional abundance of C₄ grasses is strongly controlled by temperature, however, the role of precipitation is less clear. Progress in elucidating the direct effects of photosynthetic pathway on these climate relationships is hindered by the significant genetic divergence between major C₃ and C₄ grass lineages. We addressed this problem by examining seasonal climate responses of photosynthesis in Alloteropsis semialata , a unique grass species with both C₃ and C₄ subspecies. Experimental manipulation of rainfall in a common garden in South Africa tested the hypotheses that: (1) photosynthesis is greater in the C₄ than C₃ subspecies under high summer temperatures, but this pattern is reversed at low winter temperatures; and (2) the photosynthetic advantage of C₄ plants is enhanced during drought events. Measurements of leaf gas exchange over 2 years showed a significant photosynthetic advantage for the C₄ subspecies under irrigated conditions from spring through autumn. However, the C₄ leaves were killed by winter frost, while photosynthesis continued in the C₃ plants. Unexpectedly, the C₄ subspecies also lost its photosynthetic advantage during natural drought events, despite greater water-use efficiency under irrigated conditions. This study highlights previously unrecognized roles for climatic extremes in determining the ecological success of C₃ and C₄ grasses.     

Expression of Modes of Photosynthesis (C3, CAM) in Clusia criuva Camb. in a CerradolGallery Forest Transect

Abstract: The large majority of the Ca . 150 species of the neotropical shrub and tree genus Clusia have the potential to perform Crassulacean acid metabolism (CAM). They are either obligate CAM plants or C₃/CAM intermediate plants. Only a very small percentage of the plants studied so far are apparently obligate C₃ species. Among these was C. criuva , until recent laboratory studies showed that it may also have a certain CAM capacity under artificial stress conditions. Measurements of stomatal conductance (porometry) and chlorophyll fluorescence variables of C. criuva occurring along a transect from deep shade inside a gallery forest across the semi-shaded ecotone towards a cerrado and into the exposed cerrado itself in central Brazil now show that it can adapt its photosynthetic apparatus to effective performance of C₃ photosynthesis under highly different photosynthetic photon flux densities. In addition, however, it does have a certain potential for CAM and cannot be considered as a strictly obligate C₃ plant. Should a basic capacity for performing CAM be a general property of the genus, the quest for CAM traits in other remaining putatively obligate C₃ species of the genus ought to be pursued.