Behavior analysis.

<p><b>(A)</b> Latency to start copula in virgin females (first copula) and already-mated females (second copula). Two-tailed t test, p<0.01. <b>(B)</b> Duration of copula of virgin females (first copula) and already-mated females (second copula). Two-tailed t test, p<0.01.</p

<i>L. longipalpis</i> male reproductive system: seminal vesicle, general aspects and the external epithelium.

<p><b>A)</b> LM longitudinal section through the seminal vesicle shows that it is surrounded by muscle cells (M) and formed by three consecutive distinct layers of cells: the outer epithelial seminal vesicle wall (SVW), the denser, invaginated epithelial wall of the seminal vesicle and the inner circular epithelial layer of the <i>vas deferens</i> (VD). Note the basal localization of the nuclei (N) and the different granule types inside the cells at the inner and outer seminal vesicle wall (black and white arrows, respectively). <b>B)</b> LM transversal section of the seminal vesicle showing the insertion of the terminal endings of the <i>vasa deferentia</i> (VD), forming two lobes. Note the folded seminal vesicle wall (SVW) around the <i>vasa deferentia</i>; C: Cuticle; Hg: Hindgut. <b>C)</b> LM section showing the different spherical granules (arrows) in the cytoplasm of the cells of the seminal vesicle wall (SVW), and the basal location of the nuclei (N); Sp: spermatozoon; VD: <i>vas deferens</i>. <b>D)</b> TEM showing the cells at the outer seminal vesicle wall (SVW) with several cytoplasmic granules (white arrows) with different electron-density as compared to that of granules in the invaginated seminal vesicle wall (black arrows). Note the basal location of the nuclei (N) and the inner <i>vas deferens</i> (VD). <b>E)</b> TEM image showing the muscle fibers (M) and the fat body (FB) that envelope the seminal vesicle. The arrows indicate the dense granules; N: nucleus; Mi: mitochondria; SVW seminal vesicle wall. <b>F)</b> Detail by TEM showing the large nucleus (N) and mitochondria (Mi) located at the basal portion of cells at the peripheral epithelium of the seminal vesicle wall. The cytoplasmic granules are not membrane bound and appear to contain lipid droplets of different densities (arrows).</p

<i>L. longipalpis</i> male reproductive system: <i>vas deferens</i> and spermatozoa.

<p><b>A)</b> LM section of the fifth abdominal segment, showing the testis (Te) and the <i>vas deferens</i> (VD) that conducts the sperm from the testis to the seminal vesicle (SV). Note the germ cells inside the testis with different shapes. A well-developed fat body (FB) surrounds the testis and the seminal vesicle; MT: Malpighian tubule. <b>B)</b> TEM of the tubular <i>vas deferens</i> packed with mature spermatozoa (Sp). It is enveloped by a thin basal membrane and a layer of interdigitated epithelial cells; N: nucleus of the epithelial cell. <b>C)</b> Detail of spermatozoa by TEM showing the flagelar axoneme (Ax), which presents a 9+0 array of microtubules; Mi: mitochondrion. A higher magnification of the axoneme is also shown in the inset. <b>D)</b> Longitudinal section by TEM of spermatozoa showing flagellum (F), capping perforatorium (P) and the thin acrosomal complex (Ac); PM: plasma membrane; N: meshwork of compact DNA at the spermatozoon head.</p

Schematic view of the male reproductive system of <i>Lutzomia longipalpis</i>.

<p>A pair of testes (Te) is linked via <i>vasa deferentia</i> (VD) to a pear-shaped seminal vesicle (SV). An ejaculatory duct (ED) then connects the seminal vesicle to the ejaculatory pump (EP). The <i>vasa deferentia</i> are inserted (a) into invaginations (b) of the seminal vesicle wall (c). Cells at layers “a”, “b” and “c” present different types of cytoplasmic granules; SP: mature spermatozoa. Modified from Valdez (2001) and Fausto et al. 2000.</p

<i>L. longipalpis</i> male reproductive system: inner seminal vesicle wall and <i>vas deferens.</i>

<p>A<b>)</b> TEM image showing cells from the inner seminal vesicle wall. They present well-developed endoplasmic reticulum (ER) and the cytoplasmic granules (arrows) are more compact than those of cells from the outer layer; the cells are joined by septate junctions (white arrowhead); N: Nucleus; Mi: Mitochondria. B) Detail by TEM showing the dense granules (arrows) of cells from the inner seminal vesicle wall: they are membrane bound and contain several electron dense vesicles; Mi: mitochondria. A detail of a septate junction (white arrowhead) is shown in the inset. <b>C)</b> TEM image showing cells at the inner <i>vas deferens</i> (VD). They present well-developed endoplasmic reticulum (ER), cytoplasmic granules (arrows) and a large ovoid nucleus (N) at the basal portion; Mi: Mitochondria. <b>D)</b> Detail by TEM of cells at the inner <i>vas deferens</i>: They contain mitochondria (Mi), Golgi complex (G), large amount of swollen endoplasmic reticulum cisternae (ER) and present membrane-bound cytoplasmic granules that contain one to several vesicles, each presenting peripheral electron-density (arrows); the <i>vas deferens</i> wall is enveloped by a thin layer of muscle cells (M); N: nucleus.</p

Mating frequency of virgin and mated females.

<p>Occurrence of copula in <i>L. longipalpis</i> for virgin (1° copula) and mated (2° copula) females. A single pair (1 male - 1 female) was used in each trial. X² = 10.071 and p<0.01.</p

<i>L. longipalpis</i> male reproductive system: the ejaculatory pump.

<p><b>A)</b> LM detail of the cuticular ejaculatory pump (EP) and the two aedeagal filaments (AF). Note the external genitalia, or terminalia (Tn), formed by modification of the eighth, ninth and tenth abdominal segments. <b>B)</b> LM transversal section of the ejaculatory pump (EP) showing the central cuticular duct (CD); HG: Hind gut; SV: seminal vesicle. <b>C)</b> LM longitudinal section of the ejaculatory pump (EP) showing well-developed muscle fibers (M) surrounding a central cuticular duct (CD). Below is a longitudinal section through the seminal vesicle (SV) at the point of insertion of a <i>vas deferens</i> (to the right), showing the two consecutive seminal vesicle cell layers with different granule types (black and white arrows). <b>D)</b> TEM transversal section of the ejaculatory pump (EP) showing the muscle fibers (M) with mitochondria (Mi) surrounding the cuticular duct (CD) <b>E)</b> TEM detail of the muscle fibers (M) with mitochondria (Mi). <b>F)</b> TEM detail showing the disposition of the muscle fibers (M); The thin outer sheet is disposed transversally. Mi: mitochondria.</p

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

<div><p>Background</p><p><i>Lutzomyia longipalpis</i>, 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.</p><p>Methodology/Principal Findings</p><p>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.</p><p>Conclusions/Significance</p><p>The results indicate that introgressive hybridization has been a crucial phenomenon in shaping the genome of the <i>L. longipalpis</i> 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.</p></div

Haplotype networks from sympatric and allopatric comparisons of selected loci with extreme <i>F</i>_ST values.

<p>(A) The network of the loci <i>ζcop</i> and <i>rpL17A</i> from the sympatric species of the <i>Lutzomyia longipalpis</i> complex shows mixed a haplotype distribution, unlike the two well-separated cluster for the allopatric species. This is in agreement with the low degree of divergence between sympatric and high divergence between allopatric species. (B) The loci <i>per</i>, <i>sec22</i>, <i>up</i> and <i>para</i> presented sympatric and allopatric networks with haplotypes separated by species group, which also corroborates the high values of pairwise <i>F</i>_ST (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002495#pntd-0002495-t002" target="_blank">Table 2</a>). Each <i>L. longipalpis</i> population is represented by one color: Sobral 1S (orange), Sobral 2S (green), Lapinha (red) and Pancas (light green). Colored circles represent unique haplotypes, and their sizes are proportional to their frequencies. Black circles are hypothetical haplotypes. Curved lines represent alternative branching between haplotypes.</p

Tajima's neutrality test of 21 loci for the four siblings of <i>L. longipalpis</i> from Brazil.

<p>S1S, Sobral 1S; S2S, Sobral 2S; Lap, Lapinha; Pan, Pancas.</p