Minimum Evolution tree of sequences from Brazilian populations of <i>Lu. longipalpis</i> producing Burst-type (dark green circles) and Pulse-type songs (red circles), <i>Lu. cruzi</i> (light green circles) and the more distant sibling species <i>Lu. pseudolongipalpis</i> (open circles) used as outgroup.

<p>The sequences E1S8A and E1S8B are the only red circles that cluster with the Burst-type sequences. Bootstrap values based on 1000 replications (values below 50% are not shown).</p

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

<div><p>The sand fly <em>Lutzomyia longipalpis</em> (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 <em>paralytic</em> (<em>para</em>) gene, a locus involved in the control of courtship songs in <em>Drosophila</em>, among a number of <em>Lu. longipalpis</em> 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 <em>Lu. longipalpis</em> with a very closely related species, <em>Lutzomyia cruzi</em>, which produces Burst-type songs. The results indicated a higher number of fixed differences between <em>Lu. cruzi</em> and the Pulse-type populations of <em>Lu. longipalpis</em> than with those producing Burst-type songs. The data confirmed our previous assumptions that the presence of different sibling species of the <em>Lu. longipalpis</em> 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 <em>para</em> 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.</p> </div

Polymorphism summaries of the <i>para</i> gene fragment from populations of <i>Lu. longipalpis</i> and <i>Lu. cruzi</i>.

<p><b>B.</b> Burst type song; <b>P.</b> Pulse type song; <b>n</b>. number of sequences; <b>S</b>. number of segregating sites; <b>ð</b>. nucleotide diversity; <b>è</b>. neutral parameter based on the segregating sites; <b>D_T</b>. Tajima test of neutrality. <b>D*</b> and <b>F*</b>. Fu and Li's tests of neutrality. Numbers in parentheses represent the analysis of nucleotide diversity considering the regions with gaps. *p<0.05.</p

Map of Brazil with the approximate location of the studied samples.

<p>Map of Brazil with the approximate location of the studied samples.</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

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: 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

<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

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: <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