Synthetic Sex Pheromone in a Long-Lasting Lure Attracts the Visceral Leishmaniasis Vector,Lutzomyia longipalpis,for up to 12 Weeks in Brazil

Current control methodologies have not prevented the spread of visceral leishmaniasis (VL) across Brazil. Here, we describe the development of a new tool for controlling the sand fly vector of the disease: a long-lasting lure, which releases a synthetic male sex pheromone, attractive to both sexes of Lutzomyia longipalpis. This device could be used to improve the effectiveness of residual insecticide spraying as a means of sand fly control, attracting L. longipalpis to insecticide-treated animal houses, where they could be killed in potentially large numbers over a number of weeks. Different lure designs releasing the synthetic pheromone (±)-9-methylgermacrene-B (CAS 183158-38-5) were field-tested in Araçatuba, São Paulo (SP). Experiments compared numbers of sand flies caught overnight in experimental chicken sheds with pheromone lures, to numbers caught in control sheds without pheromone. Prototype lures, designed to last one night, were first used to confirm the attractiveness of the pheromone in SP, and shown to attract significantly more flies to test sheds than controls. Longer-lasting lures were tested when new, and at fortnightly intervals. Lures loaded with 1 mg of pheromone did not attract sand flies for more than two weeks. However, lures loaded with 10 mg of pheromone, with a releasing surface of 15 cm² or 7.5 cm², attracted female L. longipalpis for up to ten weeks, and males for up to twelve weeks. Approximately five times more sand flies were caught with 7.5 cm² 10 mg lures when first used than occurred naturally in non-experimental chicken resting sites. These results demonstrate that these lures are suitably long-lasting and attractive for use in sand fly control programmes in SP. To our knowledge, this is the first sex pheromone-based technology targeting an insect vector of a neglected human disease. Further studies should explore the general applicability of this approach for combating other insect-borne diseases.     

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.     

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.     

Examination of the interior of sand fly (Diptera: Psychodidae) abdomen reveals novel cuticular structures involved in pheromone release: Discovering the manifold

The males of many species of New World Phlebotomines produce volatile terpenoid chemicals, shown in Lutzomyia longipalpis s.l. to be sex/aggregation pheromones. Pheromone is produced by secretory cells which surround a cuticular reservoir which collects the pheromone and passes it through a cuticular duct to the surface of the insect. The pheromone then passes through specialised cuticular structures on the abdominal surface prior to evaporation. The shape and distribution of the specialised structures are highly diverse and differ according to species. In this study we used SEM to examine the interior cuticular pheromone collection and transport structures of 3 members of the Lu. longipalpis s.l. species complex and Migonemyia migonei. We found a new structure which we have called the manifold which appears to be a substantial extension of the interior tergal cuticle connected in-line with the cuticular duct and reservoir. The manifold of the Campo Grande member of the complex is longer and wider than the Jacobina member whereas the manifold of the Sobral member was shorter than both other members of the complex. Overall, the secretory apparatus of the Sobral member was smaller than the other two. The manifold of M. migonei was very different to those found in Lu. longipalpis s.l. and was positioned in a pit-like structure within the tergal cuticle. The secretory reservoir was connected by a short duct to the manifold. Differences in the size and shape of the manifold may be related to the chemical structure of the pheromone and may have taxonomic value. Examination of the interior cuticle by SEM may help to locate the secretory apparatus of vector species where pheromonal activity has been inferred from behavioural studies but the external secretory structures or pheromones have not yet been found.     

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.     

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.     

Assortative Mating in Two Pheromone Strains of the Cabbage Looper Moth, Trichoplusia ni

The evolution of animal communication systems is an integral part of speciation. In moths, species specificity of the communication channel is largely a result of unique sex pheromone blends produced by females and corresponding specificity of male behavioral response. Insights into the process of speciation may result from studies of pheromone strains within a species in which reproductive isolation is not complete. Toward this end we investigated assortative mating based on female pheromone phenotypes and male response specificity between mutant and normal colonies of the cabbage looper moth, Trichoplusia ni. There was no evidence of assortative mating in small cages in which the density of moths was high. In larger cages with lower densities of moths, assortative mating was evident. In these larger cages, matings between normal males and normal females and mutant males and mutant females were more frequent than interstrain matings. Wind tunnel tests indicated that normal males responded preferentially to pheromone released by normal females, whereas mutant males did not discriminate between normal and mutant pheromone blends. In large field cages, pheromone traps baited with normal females caught equal numbers of mutant and normal males, while pheromone traps baited with mutant females caught primarily mutant males. The overall pattern of assortative mating could be explained primarily based on the normal males' preference for the pheromone blend released by normal females.     

Community deployment of a synthetic pheromone of the sand fly Lutzomyia longipalpis co-located with insecticide reduces vector abundance in treated and neighbouring untreated houses: Implications for control of Leishmania infantum

BackgroundThe rising incidence of visceral leishmaniasis due to Leishmania infantum requires novel methods to control transmission by the sand fly vector. Indoor residual spraying of insecticide (IRS) against these largely exophilic / exophagic vectors may not be the most effective method. A synthetic copy of the male sex-aggregation pheromone of the key vector species Lutzomyia longipalpis in the Americas, was co-located with residual pyrethroid insecticide, and tested for its effects on vector abundance, hence potential transmission, in a Brazilian community study.MethodsHouses within eight defined semi-urban blocks in an endemic municipality in Brazil were randomised to synthetic pheromone + insecticide or to placebo treatments. A similar number of houses located >100m from each block were placebo treated and considered as “True Controls” (thus, analysed as three trial arms). Insecticide was sprayed on a 2.6m² surface area of the property boundary or outbuilding wall, co-located within one metre of 50mg synthetic pheromone in controlled-release dispensers. Vector numbers captured in nearby CDC light traps were recorded at monthly intervals over 3 months post intervention. Recruited sentinel houses under True Control and pheromone + insecticide treatments were similarly monitored at 7–9 day intervals. The intervention effects were estimated by mixed effects negative binomial models compared to the True Control group.ResultsDose-response field assays using 50mg of the synthetic pheromone captured a mean 4.8 (95% C.L.: 3.91, 5.80) to 6.3 (95% C.L.: 3.24, 12.11) times more vectors (female Lu. longipalpis) than using 10mg of synthetic pheromone. The intervention reduced household female vector abundance by 59% (C.L.: 48.7, 66.7%) (IRR = 0.41) estimated by the cross-sectional community study, and by 70% (C.L.: 56.7%, 78.8%) estimated by the longitudinal sentinel study. Similar reductions in male Lu. longipalpis were observed. Beneficial spill-over intervention effects were also observed at nearby untreated households with a mean reduction of 24% (95% C.L.: 0.050%, 39.8%) in female vectors. The spill-over effect in untreated houses was 44% (95% C.L.: 29.7%, 56.1%) as effective as the intervention in pheromone-treated houses. Ownership of chickens increased the intervention effects in both treated and untreated houses, attributed to the suspected synergistic attraction of the synthetic pheromone and chicken kairomones. The variation in IRR between study blocks was not associated with inter-household distances, household densities, or coverage (proportion of total households treated).ConclusionsThe study confirms the entomological efficacy of the lure-and-kill method to reduce the abundance of this important sand fly vector in treated and untreated homesteads. The outcomes were achieved by low coverage and using only 1–2% of the quantity of insecticide as normally required for IRS, indicating the potential cost-effectiveness of this method. Implications for programmatic deployment of this vector control method are discussed.     

Broadly and narrowly tuned odorant receptors are involved in female sex pheromone reception in Ostrinia moths

Mate-finding communication in many moths is mediated by sex pheromones produced by females. Since the differentiation of sex pheromones is often associated with speciation, it is intriguing to elucidate how the changes in sex pheromones are tracked by the pheromone recognition system of the males. Moths of the genus Ostrinia, which show distinct differentiation in female sex pheromones, are good models to study this. The present study was initiated with the aim of identifying ORs from Ostrinia scapulalis that respond to its own pheromone components, (E)-11- and (Z)-11-tetradecenyl acetates. We isolated six OR gene candidates (OscaOR3–8) from O. scapulalis. The same set of genes homologous to OscaOR3–8 were conserved in all (eight) Ostrinia species examined in addition to the previously reported OscaOR1 (tuned to (E)-11-tetradecenol) and the Or83b homologue OscaOR2. OscaOR3 not only responded to (E)-11- and (Z)-11-tetradecenyl acetates, but also to the pheromone components of the congeners, (Z)-9-, (E)-12-, and (Z)-12-tetradecenyl acetates. OscaOR4 responded with a relatively high specificity to (E)-11-tetradecenyl acetate. While OscaOR5 responded only marginally to a few pheromone components, OscaOR6–8 did not respond to any of the compounds tested. A few conserved ORs, including a unique one with very broad responsiveness, appear to be involved in the sex pheromone reception in O. scapulalis. The findings of the present study are discussed with reference to knowledge on electrophysiological response profiles of olfactory receptor neurons in Ostrinia moths.     

Larval Breeding Sites of Lutzomyia longipalpis (Diptera: Psychodidae) in Visceral Leishmaniasis Endemic Urban Areas in Southeastern Brazil

Background

The scarcity of information on the immature stages of sand flies and their preferred breeding sites has resulted in the focus of vectorial control on the adult stage using residual insecticide house-spraying. This strategy, along with the treatment of human cases and the euthanasia of infected dogs, has proven inefficient and visceral leishmaniasis continues to expand in Brazil. Identifying the breeding sites of sand flies is essential to the understanding of the vector''s population dynamic and could be used to develop novel control strategies.

Methodology/Principal finding

In the present study, an intensive search for the breeding sites of Lutzomyia longipalpis was conducted in urban and peri-urban areas of two municipalities, Promissão and Dracena, which are endemic for visceral leishmaniasis in São Paulo State, Brazil. During an exploratory period, a total of 962 soil emergence traps were used to investigate possible peridomiciliary breeding site microhabitats such as: leaf litter under tree, chicken sheds, other animal sheds and uncovered debris. A total of 160 sand flies were collected and 148 (92.5%) were L. longipalpis. In Promissão the proportion of chicken sheds positive was significantly higher than in leaf litter under trees. Chicken shed microhabitats presented the highest density of L. longipalpis in both municipalities: 17.29 and 5.71 individuals per square meter sampled in Promissão and Dracena respectively. A contagious spatial distribution pattern of L. longipalpis was identified in the emergence traps located in the chicken sheds.

Conclusion

The results indicate that chicken sheds are the preferential breeding site for L. longipalpis in the present study areas. Thus, control measures targeting the immature stages in chicken sheds could have a great effect on reducing the number of adult flies and consequently the transmission rate of Leishmania (Leishmania) infantum chagasi.     

Distinct evolutionary patterns between chemoreceptors of 2 vertebrate olfactory systems and the differential tuning hypothesis

Most tetrapod vertebrates have 2 olfactory systems, the main olfactory system (MOS) and the vomeronasal system (VNS). According to the dual olfactory hypothesis, the MOS detects environmental odorants, whereas the VNS recognizes intraspecific pheromonal cues. However, this strict functional distinction has been blurred by recent reports that both systems can perceive both types of signals. Studies of a limited number of receptors suggest that MOS receptors are broadly tuned generalists, whereas VNS receptors are narrowly tuned specialists. However, whether this distinction applies to all MOS and VNS receptors remains unknown. The differential tuning hypothesis predicts that generalist MOS receptors detect an overlapping set of ligands and thus are more likely to be conserved over evolutionary time than specialist VNS receptors, which would evolve in a more lineage-specific manner. Here we test this prediction for all olfactory chemoreceptors by examining the evolutionary patterns of MOS-expressed odorant receptors (ORs) and trace amine-associated receptors (TAARs) and VNS-expressed vomeronasal type 1 receptors (V1Rs) and vomeronasal type 2 receptors (V2Rs) in 7 tetrapods (mouse, rat, dog, opossum, platypus, chicken, and frog). The phylogenies of V1Rs and V2Rs show abundant lineage-specific gene gains/losses and virtually no one-to-one orthologs between species. Opposite patterns are found for ORs and TAARs. Analysis of functional data and ligand-binding sites of ORs confirms that paralogous chemoreceptors are more likely than orthologs to have different ligands and that functional divergence between paralogous chemoreceptors is established relatively quickly following gene duplication. Together, these results strongly suggest that the functional profile of the VNS chemoreceptor repertoire evolves much faster than that of the MOS chemoreceptor repertoire and that the differential tuning hypothesis applies to the majority, if not all, of MOS and VNS receptors.     

Odorant receptor phylogeny confirms conserved channels for sex pheromone and host plant signals in tortricid moths

The search for mates and food is mediated by volatile chemicals. Insects sense food odorants and sex pheromones through odorant receptors (ORs) and pheromone receptors (PRs), which are expressed in olfactory sensory neurons. Molecular phylogenetics of ORs, informed by behavioral and functional data, generates sound hypotheses for the identification of semiochemicals driving olfactory behavior. Studying orthologous receptors and their ligands across taxa affords insights into the role of chemical communication in reproductive isolation and phylogenetic divergence. The female sex pheromone of green budworm moth Hedya nubiferana (Lepidoptera, Totricidae) is a blend of two unsaturated acetates, only a blend of both elicits male attraction. Females produce in addition codlemone, which is the sex pheromone of another tortricid, codling moth Cydia pomonella. Codlemone also attracts green budworm moth males. Concomitantly, green budworm and codling moth males are attracted to the host plant volatile pear ester. A congruent behavioral response to the same pheromone and plant volatile in two tortricid species suggests co‐occurrence of dedicated olfactory channels. In codling moth, one PR is tuned to both compounds, the sex pheromone codlemone and the plant volatile pear ester. Our phylogenetic analysis finds that green budworm moth expresses an orthologous PR gene. Shared ancestry, and high levels of amino acid identity and sequence similarity, in codling and green budworm moth PRs offer an explanation for parallel attraction of both species to the same compounds. A conserved olfactory channel for a sex pheromone and a host plant volatile substantiates the alliance of social and habitat signals in insect chemical communication. Field attraction assays confirm that in silico investigations of ORs afford powerful predictions for an efficient identification of behavior‐modifying semiochemicals, for an improved understanding of the mechanisms of host plant attraction in insect herbivores and for the further development of sustainable insect control.     

Focus: Zoonotic Disease: Eco-epidemiological Aspects of Visceral Leishmaniasis in the Municipality of Diamantina,Jequitinhonha Valley (Minas Gerais State, Brazil)

Background: The present study was carried out in the rural and urban area of Diamantina/Minas Gerais (MG), an endemic municipality for visceral leishmaniasis (VL) in Brazil. Methods: Patient notification records, canine prevalence, and phlebotomine fauna were evaluated. Results: In the period from 2016 to 2018, eight human cases were confirmed, with three deaths, predominantly in males. In the same period, a total of 1,388 dogs resided in the rural and urban area of the municipality were submitted to the DPP® and ELISA, with a percentage of confirmed canine cases of 29.9% and 29.4%, respectively. The entomological study conducted in the municipality revealed the presence of 10 species of sand flies, with a predominance of Lutzomyia longipalpis (55.75%), mainly in the rural area. Conclusions: Unlike what is happening in urban centers, the results of this study suggest that the VL in Diamantina is in the process of urbanization, given the high percentage of confirmed canine cases and the high density of Lu. longipalpis in the rural area of the municipality. These risk factors warn about the need for continuous surveillance and the need to control actions of VL in this area.     

Caste-Specific and Sex-Specific Expression of Chemoreceptor Genes in a Termite

The sophisticated colony organization of eusocial insects is primarily maintained through the utilization of pheromones. The regulation of these complex social interactions requires intricate chemoreception systems. The recent publication of the genome of Zootermopsis nevadensis opened a new avenue to study molecular basis of termite caste systems. Although there has been a growing interest in the termite chemoreception system that regulates their sophisticated caste system, the relationship between division of labor and expression of chemoreceptor genes remains to be explored. Using high-throughput mRNA sequencing (RNA-seq), we found several chemoreceptors that are differentially expressed among castes and between sexes in a subterranean termite Reticulitermes speratus. In total, 53 chemoreception-related genes were annotated, including 22 odorant receptors, 7 gustatory receptors, 12 ionotropic receptors, 9 odorant-binding proteins, and 3 chemosensory proteins. Most of the chemoreception-related genes had caste-related and sex-related expression patterns; in particular, some chemoreception genes showed king-biased or queen-biased expression patterns. Moreover, more than half of the genes showed significant age-dependent differences in their expression in female and/or male reproductives. These results reveal a strong relationship between the evolution of the division of labor and the regulation of chemoreceptor gene expression, thereby demonstrating the chemical communication and underlining chemoreception mechanism in social insects.     

A male-specific odorant receptor conserved through the evolution of sex pheromones in Ostrinia moth species

In many moths, mate-finding communication is mediated by the female sex pheromones. Since differentiation of sex pheromones is often associated with speciation, it is intriguing to know how the changes in female sex pheromone have been tracked by the pheromone recognition system of the males. A male-specific odorant receptor was found to have been conserved through the evolution of sex pheromone communication systems in the genus Ostrinia (Lepidoptera: Crambidae). In an effort to characterize pheromone receptors of O. scapulalis, which uses a mixture of (E)-11- and (Z)-11-tetradecenyl acetates as a sex pheromone, we cloned a gene (OscaOR1) encoding a male-specific odorant receptor. In addition, we cloned a gene of the Or83b family (OscaOR2). Functional assays using Xenopus oocytes co-expressing OscaOR1 and OscaOR2 have shown that OscaOR1 is, unexpectedly, a receptor of (E)-11-tetradecenol (E11-14:OH), a single pheromone component of a congener O. latipennis. Subsequent studies on O. latipennis showed that this species indeed has a gene orthologous to OscaOR1 (OlatOR1), a functional assay of which confirmed it to be a gene encoding the receptor of E11-14:OH. Furthermore, investigations of six other Ostrinia species have revealed that all of them have a gene orthologous to OscaOR1, although none of these species, except O. ovalipennis, a species most closely related to O. latipennis, uses E11-14:OH as the pheromone component. The present findings suggest that the male-specific receptor of E11-14:OH was acquired before the divergence of the genus Ostrinia, and functionally retained through the evolution of this genus.     

Odorant Receptors of the New Zealand Endemic Leafroller Moth Species Planotortrix octo and P. excessana

Moths use their sense of smell to find food sources, mating partners and oviposition sites. For this they possess a family of odorant receptors (ORs). Some ORs are used by both sexes whereas others have sex-specific roles. For example, male moths possess ORs specifically tuned to sex pheromones produced by conspecific females. Here we identify sets of ORs from the antennae of New Zealand endemic leafroller moths Planotortrix octo (48 ORs) and P. excessana (47 ORs) using an RNA-Seq approach. Two orthologous ORs show male-biased expression in the adult antennae of both species (OR7 and OR30) and one other OR in each species was female-biased in its expression (PoctOR25, PexcOR14) by qPCR. PAML analysis conducted on male-biased ORs indicated positive selection acting on the male-biased OR7. The fact that OR7 is likely under positive selection, that it is male-biased in its expression and that its orthologue in C. obliquana, CoblOR7, responds to sex pheromone components also utilised by Planotortrix species, suggests that this receptor may also be important in sex pheromone reception in Planotortrix species.     

Molecular detection and phylogenetic analyses of Arsenophonus endosymbiont in wild specimens of phlebotomine sand flies from Colombia

Endosymbionts have gained prominence as a potential tool for biological control strategies in reducing vector-borne diseases. This study aimed to evaluate the presence of Arsenophonus, Spiroplasma, and Rickettsia endosymbionts in wild specimens of phlebotomine sand flies, as well as in culicids collected in different regions of Colombia. Analyses were conducted through conventional PCR, Sanger sequencing of the 16S rRNA gene, and phylogenetic analyses. Individuals from among 946 phlebotomine sand flies and 143 mosquitoes were selected for taxonomic identification confirmed through the analysis of the cytochrome oxidase subunit I gene sequences. Results showed the presence of Arsenophonus bacteria in samples of Lutzomyia longipalpis, Psychodopygus panamensis, and Pintomyia evansi. Arsenophonus sequences associated with Lu. longipalpis and Ps. panamensis are phylogenetically located near to sequences of louse flies, with K2P genetic distances of 0.006. In contrast, sequences obtained from Pi. evansi are phylogenetically located near Arsenophonus nasoniae (K2P 0.001–0.014). Other sequences of endosymbionts similar to Arsenophonus with high K2P genetic distances (0.056–0.097), when compared to different reference strains of this endosymbiont, were also found in other samples of Lu. longipalpis and Ae. aegypti. To the best of our knowledge, this is the first successful attempt to detect and elucidate the phylogenetic relationship of Arsenophonus in phlebotomine sand flies, yet its role within these insect vectors remains to be fully determined; therefore, the importance of entomological surveys that help better understand its behavior and potential use as a control agent is required to enable the proactive reduction of sand fly populations.