In this paper we review the natural history of pheromone communication and the current diversity of aggregation-sex pheromones in the sand fly Lutzomyia longipalpis. This species complex is the main vector of Leishmania infantum, the agent of visceral leishmaniasis in the Americas. The identification of variation in pheromone chemotypes combined with molecular and sound analyses have all contributed to our understanding of the extent of divergence among cryptic members of this complex. The importance of chemical signals as pre-mating barriers and drivers of speciation is discussed. Moreover, the importance of aggregation-sex pheromones as sexually selected signals is highlighted with evidence from the literature suggesting their potential role in species and mate recognition as well as mate assessment. The distinct evolutionary forces possibly involved are briefly reviewed and discussed in the context of this intriguing insect.

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Although the discipline of cell biology (CB) is part of the curricula of predoctoral dental schools, students often fail to recognize its practical relevance. The aim of this study was to assess the effectiveness of a practical-theoretical project-based course in closing the gaps among CB, scientific research, and dentistry for dental students. A project-based learning course was developed with nine sequential lessons to evaluate 108 undergraduate dental students enrolled in CB classes of a Brazilian school of dentistry during 2013-16. To highlight the relevance of in vitro studies in the preclinical evaluation of dental materials at the cellular level, the students were challenged to complete the process of drafting a protocol and performing a cytocompatibility assay for a bone substitute used in dentistry. Class activities included small group discussions, scientific database search and article presentations, protocol development, lab experimentation, and writing of a final scientific report. A control group of 31 students attended only one laboratory class on the same theme, and the final reports were compared between the two groups. The results showed that the project-based learning students had superior outcomes in acknowledging the relevance of in vitro methods during biocompatibility testing. Moreover, they produced scientifically sound reports with more content on methodological issues, the relationship with dentistry, and the scientific literature than the control group (p<0.05). The project-based learning students also recognized a higher relevance of scientific research and CB to dental practice. These results suggest that a project-based approach can help contextualize scientific research in dental curricula.

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The sand fly Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae: Phlebotominae) is the main vector of American visceral leishmaniasis. Adult males produce a terpenoid sex pheromone that in some cases also acts as male aggregation pheromone. We have analyzed the correlation between male pheromone production levels and pheromone gland cell morphogenesis after adult emergence from pupae. The abdominal tergites of L. longipalpis males were dissected and fixed in glutaraldehyde for transmission electron microscopy, or the pheromone was extracted in analytical grade hexane. Pheromone chemical analysis was carried out at 3- to 6-h intervals during the first 24 h after emergence and continued daily until the seventh day. All extracts were analyzed by gas chromatography. For the morphological analysis, we used insects collected at 0-6, 9-12, 12-14, and 96 h after emergence. Ultrastructural data from 0- to 6-h-old adult males revealed smaller pheromone gland cells with small microvilli at the end apparatus. Lipid droplets and peroxisomes were absent or very rare, but a large number of mitochondria could be seen. Lipid droplets started to appear in the gland cells cytoplasm approximately 9 h after adult emergence, and their number and size increased with age, together with the presence of several peroxisomes, suggesting a role for these organelles in pheromone biosynthesis. At 12-15 h after emergence, the lipid droplets were mainly distributed near the microvilli but were smaller than those in mature older males (4 d old). Pheromone biosynthesis started around 12 h after emergence and increased continuously during the first 3 d, stabilizing thereafter, coinciding with the period when males are more able to attract females.

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.

The typology, number, size, and distribution pattern of labellar and palpal sensilla of male and female Lutzomyia longipalpis (Lutz and Neiva, 1912) sand flies were analyzed by scanning electron and light microscopy, contributing to the construction of a gustatory and olfactory map in this important vector species. About 35 trichoid sensilla, sorted into three different classes according to their lengths, are present on the two labellar lobes. Five distinct structures are observed in the maxillary palps, which could be classified as noninnervated (microtrichia and scales) or the following innervated structures: 1) campaniform sensilla, found near the distal end of palpal segments II and III; 2) multiporous capitate peg sensilla, found in reduced number (1-6) on segment II, but located in a group of 15-25 on the distal third part of segment III; and 3) trichoid sensilla, distributed linearly on segment IV and spirally on segment V. Light microscopy analysis of silver-stained specimens indicated the presence of pores in the trichoid sensilla and their chemosensory function is discussed, as well as the possible function of the other sensilla.