Publications by authors named "Iliano V Coutinho-Abreu"

29 Publications

  • Page 1 of 1

Binding of Leishmania infantum Lipophosphoglycan to the Midgut Is Not Sufficient To Define Vector Competence in Sand Flies.

mSphere 2020 09 9;5(5). Epub 2020 Sep 9.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA

The major surface lipophosphoglycan (LPG) of parasites is critical to vector competence in restrictive sand fly vectors in mediating attachment to the midgut epithelium, considered essential to parasite survival and development. However, the relevance of LPG for sand flies that harbor multiple species of remains elusive. We tested binding of wild-type (WT), LPG-defective (Δ mutants), and add-back (Δ+) lines to sand fly midguts and their survival in sand flies WT parasites attached to the midgut , with late-stage parasites binding to midguts in significantly higher numbers than were seen with early-stage promastigotes. Δ mutants did not bind to midguts, and this was rescued in the Δ+ lines, indicating that midgut binding is mediated by LPG. When sand flies were infected with the WT or Δ or Δ+ line of the BH46 or BA262 strains, the BH46 Δ mutant, but not the BA262 Δ mutant, survived and grew to numbers similar to those seen with the WT and Δ+ lines. Exposure of BH46 and BA262 Δ mutants to blood-engorged midgut extracts led to mortality of the BA262 Δ but not the BH46 Δ parasites. These findings suggest that LPG protects parasites on a strain-specific basis early in infection, likely against toxic components of blood digestion, but that it is not necessary to prevent evacuation along with the feces in the permissive vector It is well established that the presence of LPG is sufficient to define the vector competence of restrictive sand fly vectors with respect to parasites. However, the permissiveness of other sand flies with respect to multiple species suggests that other factors might define vector competence for these vectors. In this study, we investigated the underpinnings of survival and development in its natural vector, We found that LPG-mediated midgut binding persists in late-stage parasites. This observation is of relevance for the understanding of vector-parasite molecular interactions and suggests that only a subset of infective metacyclic-stage parasites (metacyclics) lose their ability to attach to the midgut, with implications for parasite transmission dynamics. However, our data also demonstrate that LPG is not a determining factor in retention in the midgut of , a permissive vector. Rather, LPG appears to be more important in protecting some parasite strains from the toxic environment generated during blood meal digestion in the insect gut. Thus, the relevance of LPG in parasite development in permissive vectors appears to be a complex issue and should be investigated on a strain-specific basis.
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http://dx.doi.org/10.1128/mSphere.00594-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485685PMC
September 2020

Leishmania infection induces a limited differential gene expression in the sand fly midgut.

BMC Genomics 2020 Sep 4;21(1):608. Epub 2020 Sep 4.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.

Background: Sand flies are the vectors of Leishmania parasites. To develop in the sand fly midgut, Leishmania multiplies and undergoes various stage differentiations giving rise to the infective form, the metacyclic promastigotes. To determine the changes in sand fly midgut gene expression caused by the presence of Leishmania, we performed RNA-Seq of uninfected and Leishmania infantum-infected Lutzomyia longipalpis midguts from seven different libraries corresponding to time points which cover the various Leishmania developmental stages.

Results: The combined transcriptomes resulted in the de novo assembly of 13,841 sand fly midgut transcripts. Importantly, only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Further, we observed distinct differentially expressed sand fly midgut transcripts corresponding to the presence of each of the various Leishmania stages suggesting that each parasite stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> 32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes (< 32 fold). The molecular functions of these genes have been associated with the metabolism of lipids and detoxification of xenobiotics.

Conclusion: Overall, our data suggest that the presence of Leishmania produces a limited change in the midgut transcript expression profile in sand flies. Further, Leishmania modulates sand fly gene expression early on in the developmental cycle in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.
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http://dx.doi.org/10.1186/s12864-020-07025-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487717PMC
September 2020

Phlebotomus papatasi sand fly predicted salivary protein diversity and immune response potential based on in silico prediction in Egypt and Jordan populations.

PLoS Negl Trop Dis 2020 07 13;14(7):e0007489. Epub 2020 Jul 13.

Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame, Notre Dame, Indiana, United States of America.

Phlebotomus papatasi sand flies inject their hosts with a myriad of pharmacologically active salivary proteins to assist with blood feeding and to modulate host defenses. In addition, salivary proteins can influence cutaneous leishmaniasis disease outcome, highlighting the potential of the salivary components to be used as a vaccine. Variability of vaccine targets in natural populations influences antigen choice for vaccine development. Therefore, the objective of this study was to investigate the variability in the predicted protein sequences of nine of the most abundantly expressed salivary proteins from field populations, testing the hypothesis that salivary proteins appropriate to target for vaccination strategies will be possible. PpSP12, PpSP14, PpSP28, PpSP29, PpSP30, PpSP32, PpSP36, PpSP42, and PpSP44 mature cDNAs from field collected P. papatasi from three distinct ecotopes in the Middle East and North Africa were amplified, sequenced, and in silico translated to assess the predicted amino acid variability. Two of the predicted sequences, PpSP12 and PpSP14, demonstrated low genetic variability across the three geographic isolated sand fly populations, with conserved multiple predicted MHCII epitope binding sites suggestive of their potential application in vaccination approaches. The other seven predicted salivary proteins revealed greater allelic variation across the same sand fly populations, possibly precluding their use as vaccine targets.
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http://dx.doi.org/10.1371/journal.pntd.0007489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377520PMC
July 2020

A second generation leishmanization vaccine with a markerless attenuated Leishmania major strain using CRISPR gene editing.

Nat Commun 2020 07 10;11(1):3461. Epub 2020 Jul 10.

Division of Emerging and Transfusion Transmitted Diseases, CBER, FDA, Silver Spring, MD, 20993, USA.

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen). Notably, LmCen is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen immunization results in protection and an immune response comparable to leishmanization. LmCen is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.
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http://dx.doi.org/10.1038/s41467-020-17154-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351751PMC
July 2020

Safety and immunogenicity of a mosquito saliva peptide-based vaccine: a randomised, placebo-controlled, double-blind, phase 1 trial.

Lancet 2020 06 11;395(10242):1998-2007. Epub 2020 Jun 11.

LID Clinical Studies Unit, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

Background: In animal models, immunity to mosquito salivary proteins protects animals against mosquito-borne disease. These findings provide a rationale to vaccinate against mosquito saliva instead of the pathogen itself. To our knowledge, no vector salivary protein-based vaccine has been tested for safety and immunogenicity in humans. We aimed to assess the safety and immunogenicity of Anopheles gambiae saliva vaccine (AGS-v), a peptide-based vaccine derived from four A gambiae salivary proteins, in humans.

Methods: In this randomised, placebo-controlled, double-blind, phase 1 trial, participants were enrolled at the National Institutes of Health Clinical Center in Bethesda, MD, USA. Participants were eligible if they were healthy adults, aged 18-50 years with no history of severe allergic reactions to mosquito bites. Participants were randomly assigned (1:1:1), using block randomisation and a computer-generated randomisation sequence, to treatment with either 200 nmol of AGS-v vaccine alone, 200 nmol of AGS-v with adjuvant (Montanide ISA 51), or sterile water as placebo. Participants and clinicians were masked to treatment assignment. Participants were given a subcutaneous injection of their allocated treatment at day 0 and day 21, followed by exposure to feeding by an uninfected Aedes aegypti mosquito at day 42 to assess subsequent risk to mosquito bites in a controlled setting. The primary endpoints were safety and immunogenicity at day 42 after the first immunisation. Participants who were given at least one dose of assigned treatment were assessed for the primary endpoints and analysis was by intention to treat. The trial was registered with ClinicalTrials.gov, NCT03055000, and is closed for accrual.

Findings: Between Feb 15 and Sept 10, 2017, we enrolled and randomly assigned 49 healthy adult participants to the adjuvanted vaccine (n=17), vaccine alone (n=16), or placebo group (n=16). Five participants did not complete the two-injection regimen with mosquito feeding at day 42, but were included in the safety analyses. No systemic safety concerns were identified; however, one participant in the adjuvanted vaccine group developed a grade 3 erythematous rash at the injection site. Pain, swelling, erythema, and itching were the most commonly reported local symptoms and were significantly increased in the adjuvanted vaccine group compared with both other treatment groups (nine [53%] of 17 participants in the adjuvanted vaccine group, two [13%] of 16 in the vaccine only group, and one [6%] of 16 in the placebo group; p=0·004). By day 42, participants who were given the adjuvanted vaccine had a significant increase in vaccine-specific total IgG antibodies compared with at baseline than did participants who were give vaccine only (absolute difference of log-fold change of 0·64 [95% CI 0·39 to 0·89]; p=0·0002) and who were given placebo (0·62 [0·34 to 0·91]; p=0·0001). We saw a significant increase in IFN-γ production by peripheral blood mononuclear cells at day 42 in the adjuvanted vaccine group compared with in the placebo group (absolute difference of log ratio of vaccine peptide-stimulated vs negative control 0·17 [95% CI 0·061 to 0·27]; p=0·009) but we saw no difference between the IFN-γ production in the vaccine only group compared with the placebo group (0·022 [-0·072 to 0·116]; p=0·63).

Interpretation: AGS-v was well tolerated, and, when adjuvanted, immunogenic. These findings suggest that vector-targeted vaccine administration in humans is safe and could be a viable option for the increasing burden of vector-borne disease.

Funding: Office of the Director and the Division of Intramural Research at the National Institute of Allergy and Infectious Diseases, and National Institutes of Health.
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http://dx.doi.org/10.1016/S0140-6736(20)31048-5DOI Listing
June 2020

Distinct gene expression patterns in vector-residing Leishmania infantum identify parasite stage-enriched markers.

PLoS Negl Trop Dis 2020 03 3;14(3):e0008014. Epub 2020 Mar 3.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America.

Background: Leishmaniasis is a vector-borne neglected disease. Inside the natural sand fly vector, the promastigote forms of Leishmania undergo a series of extracellular developmental stages to reach the infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking.

Methodology/principal Findings: We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis separated the transcripts corresponding to the different Leishmania promastigote stages, the procyclic, nectomonad, leptomonad and metacyclics. Importantly, there were a significant number of differentially expressed genes when comparing the sequential development of the various Leishmania stages in the sand fly. There were 836 differentially expressed (DE) genes between procyclic and long nectomonad promastigotes; 113 DE genes between nectomonad and leptomonad promastigotes; and 302 DE genes between leptomonad and metacyclic promastigotes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each Leishmania stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple potential stage-specific markers for L. infantum.

Conclusions: Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of potential stage-specific markers for further development as molecular tools for epidemiological studies.
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http://dx.doi.org/10.1371/journal.pntd.0008014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053709PMC
March 2020

Odorant ligands for the CO receptor in two Anopheles vectors of malaria.

Sci Rep 2019 02 22;9(1):2549. Epub 2019 Feb 22.

Department of Molecular Cell and Systems Biology, University of California Riverside, Riverside, CA, 92521, USA.

Exhaled CO is an important host-seeking cue for Anopheles mosquitoes, which is detected by a highly conserved heteromeric receptor consisting of three 7-transmembrane proteins Gr22, Gr23, and Gr24. The CO receptor neuron has been shown to also respond sensitively to a variety of odorants in Aedes aegypti. The detection of CO is important for upwind navigation and for enhancing the attraction to body heat as well as to skin odorants. The orthologs of the CO receptor proteins are present in malaria-transmitting mosquitoes like Anopheles coluzzii and Anopheles sinensis. Activators and inhibitors of the CO-neuron were tested on the maxillary palps in these two species by single-sensillum electrophysiology. The electrophysiological testing of three prolonged-activator odorants identified originally in Aedes aegypti also showed varying ability to reduce the CO-ellicited increase in spikes. These findings provide a foundation for comparing the functional conservation with the evolutionary conservation of an important class of odorant receptor. The identification of a suite of natural odorants that can be used to modify the CO-detection pathway may also contribute to odor-blends that can alter the behavior of these disease transmitting mosquitoes.
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http://dx.doi.org/10.1038/s41598-019-39099-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385339PMC
February 2019

Comparative Evolution of Sand Fly Salivary Protein Families and Implications for Biomarkers of Vector Exposure and Salivary Vaccine Candidates.

Front Cell Infect Microbiol 2018 29;8:290. Epub 2018 Aug 29.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, United States.

Sand fly salivary proteins that produce a specific antibody response in humans and animal reservoirs have been shown to be promising biomarkers of sand fly exposure. Furthermore, immunity to sand fly salivary proteins were shown to protect rodents and non-human primates against infection. We are missing critical information regarding the divergence amongst sand fly salivary proteins from different sand fly vectors, a knowledge that will support the search of broad or specific salivary biomarkers of vector exposure and those for vaccines components against leishmaniasis. Here, we compare the molecular evolution of the salivary protein families in New World and Old World sand flies from 14 different sand fly vectors. We found that the protein families unique to OW sand flies are more conserved than those unique to NW sand flies regarding both sequence polymorphisms and copy number variation. In addition, the protein families unique to OW sand flies do not display as many conserved cysteine residues as the one unique to the NW group (28.5% in OW vs. 62.5% in NW). Moreover, the expression of specific protein families is restricted to the salivary glands of unique sand fly taxon. For instance, the ParSP15 family is unique to the Larroussius subgenus whereas phospholipase A2 is only expressed in member of Larroussius and Adlerius subgenera. The SP2.5-like family is only expressed in members of the Phlebotomus and Paraphlebotomus subgenera. The sequences shared between OW and NW sand flies have diverged at similar rates (38.7 and 45.3% amino acid divergence, respectively), yet differences in gene copy number were evident across protein families and sand fly species. Overall, this comparative analysis sheds light on the different modes of sand fly salivary protein family divergence. Also, it informs which protein families are unique and conserved within taxon for the choice of taxon-specific biomarkers of vector exposure, as well as those families more conserved across taxa to be used as pan-specific vaccines for leishmaniasis.
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http://dx.doi.org/10.3389/fcimb.2018.00290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123390PMC
August 2019

Population genetics analysis of Phlebotomus papatasi sand flies from Egypt and Jordan based on mitochondrial cytochrome b haplotypes.

Parasit Vectors 2018 03 27;11(1):214. Epub 2018 Mar 27.

Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, 46556, USA.

Background: Phlebotomus papatasi sand flies are major vectors of Leishmania major and phlebovirus infection in North Africa and across the Middle East to the Indian subcontinent. Population genetics is a valuable tool in understanding the level of genetic variability present in vector populations, vector competence, and the development of novel control strategies. This study investigated the genetic differentiation between P. papatasi populations in Egypt and Jordan that inhabit distinct ecotopes and compared this structure to P. papatasi populations from a broader geographical range.

Methods: A 461 base pair (bp) fragment from the mtDNA cytochrome b (cyt b) gene was PCR amplified and sequenced from 116 individual female sand flies from Aswan and North Sinai, Egypt, as well as Swaimeh and Malka, Jordan. Haplotypes were identified and used to generate a median-joining network, F values and isolation-by-distance were also evaluated. Additional sand fly individuals from Afghanistan, Iran, Israel, Jordan, Libya, Tunisia and Turkey were included as well as previously published haplotypes to provide a geographically broad genetic variation analysis.

Results: Thirteen haplotypes displaying nine variant sites were identified from P. papatasi collected in Egypt and Jordan. No private haplotypes were identified from samples in North Sinai, Egypt, two were observed in Aswan, Egypt, four from Swaimeh, Jordan and two in Malka, Jordan. The Jordan populations clustered separately from the Egypt populations and produced more private haplotypes than those from Egypt. Pairwise F values fall in the range 0.024-0.648.

Conclusion: The clustering patterns and pairwise F values indicate a strong differentiation between Egyptian and Jordanian populations, although this population structure is not due to isolation-by-distance. Other factors, such as environmental influences and the genetic variability in the circulating Le. major parasites, could possibly contribute to this heterogeneity. The present study aligns with previous reports in that pockets of genetic differentiation exists between populations of this widely dispersed species but, overall, the species remains relatively homogeneous.
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http://dx.doi.org/10.1186/s13071-018-2785-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5872541PMC
March 2018

Sequential blood meals promote Leishmania replication and reverse metacyclogenesis augmenting vector infectivity.

Nat Microbiol 2018 05 19;3(5):548-555. Epub 2018 Mar 19.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.

Sand flies, similar to most vectors, take multiple blood meals during their lifetime. The effect of subsequent blood meals on pathogens developing in the vector and their impact on disease transmission have never been examined. Here, we show that ingestion of a second uninfected blood meal by Leishmania-infected sand flies triggers dedifferentiation of metacyclic promastigotes, considered a terminally differentiated stage inside the vector , to a leptomonad-like stage, the retroleptomonad promastigote. Reverse metacyclogenesis occurs after every subsequent blood meal where retroleptomonad promastigotes rapidly multiply and differentiate to metacyclic promastigotes enhancing sand fly infectiousness. Importantly, a subsequent blood meal amplifies the few Leishmania parasites acquired by feeding on infected hosts by 125-fold, and increases lesion frequency by fourfold, in twice-fed compared with single-fed flies. These findings place readily available blood sources as a critical element in transmission and propagation of vector-borne pathogens.
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http://dx.doi.org/10.1038/s41564-018-0125-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6007031PMC
May 2018

Gut Microbes Egested during Bites of Infected Sand Flies Augment Severity of Leishmaniasis via Inflammasome-Derived IL-1β.

Cell Host Microbe 2018 Jan 28;23(1):134-143.e6. Epub 2017 Dec 28.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA. Electronic address:

Leishmania donovani parasites are the cause of visceral leishmaniasis and are transmitted by bites from phlebotomine sand flies. A prominent feature of vector-transmitted Leishmania is the persistence of neutrophils at bite sites, where they protect captured parasites, leading to enhanced disease. Here, we demonstrate that gut microbes from the sand fly are egested into host skin alongside Leishmania parasites. The egested microbes trigger the inflammasome, leading to a rapid production of interleukin-1β (IL-1β), which sustains neutrophil infiltration. Reducing midgut microbiota by pretreatment of Leishmania-infected sand flies with antibiotics or neutralizing the effect of IL-1β in bitten mice abrogates neutrophil recruitment. These early events are associated with impairment of parasite visceralization, indicating that both gut microbiota and IL-1β are important for the establishment of Leishmania infections. Considering that arthropods harbor a rich microbiota, its potential egestion after bites may be a shared mechanism that contributes to severity of vector-borne disease.
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http://dx.doi.org/10.1016/j.chom.2017.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832060PMC
January 2018

Molecular Diversity between Salivary Proteins from New World and Old World Sand Flies with Emphasis on Bichromomyia olmeca, the Sand Fly Vector of Leishmania mexicana in Mesoamerica.

PLoS Negl Trop Dis 2016 07 13;10(7):e0004771. Epub 2016 Jul 13.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America.

Background: Sand fly saliva has been shown to have proteins with potent biological activities, salivary proteins that can be used as biomarkers of vector exposure, and salivary proteins that are candidate vaccines against different forms of leishmaniasis. Sand fly salivary gland transcriptomic approach has contributed significantly to the identification and characterization of many of these salivary proteins from important Leishmania vectors; however, sand fly vectors in some regions of the world are still neglected, as Bichromomyia olmeca (formerly known as Lutzomyia olmeca olmeca), a proven vector of Leishmania mexicana in Mexico and Central America. Despite the importance of this vector in transmitting Leishmania parasite in Mesoamerica there is no information on the repertoire of B. olmeca salivary proteins and their relationship to salivary proteins from other sand fly species.

Methods And Findings: A cDNA library of the salivary glands of wild-caught B. olmeca was constructed, sequenced, and analyzed. We identified transcripts encoding for novel salivary proteins from this sand fly species and performed a comparative analysis between B. olmeca salivary proteins and those from other sand fly species. With this new information we present an updated catalog of the salivary proteins specific to New World sand flies and salivary proteins common to all sand fly species. We also report in this work the anti-Factor Xa activity of Lofaxin, a salivary anticoagulant protein present in this sand fly species.

Conclusions: This study provides information on the first transcriptome of a sand fly from Mesoamerica and adds information to the limited repertoire of salivary transcriptomes from the Americas. This comparative analysis also shows a fast degree of evolution in salivary proteins from New World sand flies as compared with Old World sand flies.
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http://dx.doi.org/10.1371/journal.pntd.0004771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943706PMC
July 2016

VSG overcomes an early barrier to survival of African trypanosomes in tsetse flies.

Proc Natl Acad Sci U S A 2016 06 10;113(25):6821-3. Epub 2016 Jun 10.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852.

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http://dx.doi.org/10.1073/pnas.1607008113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922182PMC
June 2016

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Sci Rep 2016 Jan 13;6:19300. Epub 2016 Jan 13.

Vector Molecular Biology Section, LMVR, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD.

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.
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http://dx.doi.org/10.1038/srep19300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725370PMC
January 2016

Impact of Insect Salivary Proteins in Blood Feeding, Host Immunity, Disease, and in the Development of Biomarkers for Vector Exposure.

Curr Opin Insect Sci 2015 Aug;10:98-103

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, 20878.

Functional genomic approaches based on expression of recombinant proteins linked to biochemical and disease model approaches resulted in the discovery of novel biological activities and the role some of these proteins play in disease transmission. Importantly, the expression of salivary proteins was recently shown to be affected by environmental factors and by the presence of the pathogen in the salivary gland. A practical application resulting from insect saliva research is the use of insect antigenic salivary protein as biomarkers of vector exposure in humans and animal reservoirs, an approach that is yielding interesting results in the field.
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http://dx.doi.org/10.1016/j.cois.2015.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4553692PMC
August 2015

Impact of insect salivary proteins in blood feeding, host immunity, disease, and in the development of biomarkers for vector exposure.

Curr Opin Insect Sci 2015 Aug 29;10:98-103. Epub 2015 Apr 29.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20878, USA. Electronic address:

Functional genomic approaches based on expression of recombinant proteins linked to biochemical and disease model approaches resulted in the discovery of novel biological activities and the role some of these proteins play in disease transmission. Importantly, the expression of salivary proteins was recently shown to be affected by environmental factors and by the presence of the pathogen in the salivary gland. A practical application resulting from insect saliva research is the use of insect antigenic salivary protein as biomarkers of vector exposure in humans and animal reservoirs, an approach that is yielding interesting results in the field.
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http://dx.doi.org/10.1016/j.cois.2015.04.014DOI Listing
August 2015

Phlebotomus papatasi SP15: mRNA expression variability and amino acid sequence polymorphisms of field populations.

Parasit Vectors 2015 May 29;8:298. Epub 2015 May 29.

Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, 46556, USA.

Background: The Phlebotomus papatasi salivary protein PpSP15 was shown to protect mice against Leishmania major, suggesting that incorporation of salivary molecules in multi-component vaccines may be a viable strategy for anti-Leishmania vaccines.

Methods: Here, we investigated PpSP15 predicted amino acid sequence variability and mRNA profile of P. papatasi field populations from the Middle East. In addition, predicted MHC class II T-cell epitopes were obtained and compared to areas of amino acid sequence variability within the secreted protein.

Results: The analysis of PpSP15 expression from field populations revealed significant intra- and interpopulation variation.. In spite of the variability detected for P. papatasi populations, common epitopes for MHC class II binding are still present and may potentially be used to boost the response against Le. major infections.

Conclusions: Conserved epitopes of PpSP15 could potentially be used in the development of a salivary gland antigen-based vaccine.
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http://dx.doi.org/10.1186/s13071-015-0914-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472253PMC
May 2015

Odorants for surveillance and control of the Asian Citrus Psyllid (Diaphorina citri).

PLoS One 2014 27;9(10):e109236. Epub 2014 Oct 27.

Department of Entomology, University of California Riverside, Riverside, California, United States of America; Center for Disease Vector Research, University of California Riverside, Riverside, California, United States of America.

Background: The Asian Citrus Psyllid (ACP), Diaphorina citri, can transmit the bacterium Candidatus Liberibacter while feeding on citrus flush shoots. This bacterium causes Huanglongbing (HLB), a major disease of citrus cultivation worldwide necessitating the development of new tools for ACP surveillance and control. The olfactory system of ACP is sensitive to variety of odorants released by citrus plants and offers an opportunity to develop new attractants and repellents.

Results: In this study, we performed single-unit electrophysiology to identify odorants that are strong activators, inhibitors, and prolonged activators of ACP odorant receptor neurons (ORNs). We identified a suite of odorants that activated the ORNs with high specificity and sensitivity, which may be useful in eliciting behavior such as attraction. In separate experiments, we also identified odorants that evoked prolonged ORN responses and antagonistic odorants able to suppress neuronal responses to activators, both of which can be useful in lowering attraction to hosts. In field trials, we tested the electrophysiologically identified activating odorants and identified a 3-odor blend that enhances trap catches by ∼230%.

Conclusion: These findings provide a set of odorants that can be used to develop affordable and safe odor-based surveillance and masking strategies for this dangerous pest insect.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109236PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4209970PMC
June 2015

Odor coding in a disease-transmitting herbivorous insect, the Asian citrus psyllid.

Chem Senses 2014 Jul 5;39(6):539-49. Epub 2014 Jun 5.

Department of Entomology, 3401 Watkins Drive, University of California, Riverside, CA 92521, USA.

Olfactory systems discriminate odorants very efficiently and herbivorous insects use them to find hosts in confounding and complex odor landscapes. The Asian citrus psyllid (ACP), Diaphorina citri, feeds on citrus flush and transmits Candidatus Liberibacter that causes citrus greening disease globally. Here, we perform a systematic analysis of odor detection in the ACP antenna using single-unit electrophysiology of rhinarial plate sensilla to a large panel of odorants from plants. We identify neurons that respond strongly to odorants found in the host citrus plants. Comparisons with the generalist yeast-feeding Drosophila melanogaster and specialist anthropophilic Anopheles gambiae reveal differences in odor-coding strategies for the citrus-seeking ACP. These findings provide a foundation for understanding host-odor coding in herbivorous insects.
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http://dx.doi.org/10.1093/chemse/bju023DOI Listing
July 2014

Profiling of human acquired immunity against the salivary proteins of Phlebotomus papatasi reveals clusters of differential immunoreactivity.

Am J Trop Med Hyg 2014 May 10;90(5):923-938. Epub 2014 Mar 10.

Phlebotomus papatasi sand flies are among the primary vectors of Leishmania major parasites from Morocco to the Indian subcontinent and from southern Europe to central and eastern Africa. Antibody-based immunity to sand fly salivary gland proteins in human populations remains a complex contextual problem that is not yet fully understood. We profiled the immunoreactivities of plasma antibodies to sand fly salivary gland sonicates (SGSs) from 229 human blood donors residing in different regions of sand fly endemicity throughout Jordan and Egypt as well as 69 US military personnel, who were differentially exposed to P. papatasi bites and L. major infections in Iraq. Compared with plasma from control region donors, antibodies were significantly immunoreactive to five salivary proteins (12, 26, 30, 38, and 44 kDa) among Jordanian and Egyptian donors, with immunoglobulin G4 being the dominant anti-SGS isotype. US personnel were significantly immunoreactive to only two salivary proteins (38 and 14 kDa). Using k-means clustering, donors were segregated into four clusters distinguished by unique immunoreactivity profiles to varying combinations of the significantly immunogenic salivary proteins. SGS-induced cellular proliferation was diminished among donors residing in sand fly-endemic regions. These data provide a clearer picture of human immune responses to sand fly vector salivary constituents.
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http://dx.doi.org/10.4269/ajtmh.13-0130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015589PMC
May 2014

Characterization of Phlebotomus papatasi peritrophins, and the role of PpPer1 in Leishmania major survival in its natural vector.

PLoS Negl Trop Dis 2013 14;7(3):e2132. Epub 2013 Mar 14.

Department of Entomology, Kansas State University, Manhattan, Kansas, USA.

The peritrophic matrix (PM) plays a key role in compartmentalization of the blood meal and as barrier to pathogens in many disease vectors. To establish an infection in sand flies, Leishmania must escape from the endoperitrophic space to prevent excretion with remnants of the blood meal digestion. In spite of the role played regarding Leishmania survival, little is known about sand fly PM molecular components and structural organization. We characterized three peritrophins (PpPer1, PpPer2, and PpPer3) from Phlebotomus papatasi. PpPer1 and PpPer2 display, respectively, four and one chitin-binding domains (CBDs). PpPer3 on the other hand has two CBDs, one mucin-like domain, and a putative domain with hallmarks of a CBD, but with changes in key amino acids. Temporal and spatial expression analyses show that PpPer1 is expressed specifically in the female midgut after blood feeding. PpPer2 and PpPer3 mRNAs were constitutively expressed in midgut and hindgut, with PpPer3 also being expressed in Malpighian tubules. PpPer2 was the only gene expressed in developmental stages. Interestingly, PpPer1 and PpPer3 expression are regulated by Le. major infection. Recombinant PpPer1, PpPer2 and PpPer3 were obtained and shown to display similar biochemical profiles as the native; we also show that PpPer1 and PpPer2 are able to bind chitin. Knockdown of PpPer1 led to a 44% reduction in protein, which in spite of producing an effect on the percentage of infected sand flies, resulted in a 39% increase of parasite load at 48 h. Our data suggest that PpPer1 is a component for the P. papatasi PM and likely involved in the PM role as barrier against Le. major infection.
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http://dx.doi.org/10.1371/journal.pntd.0002132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597473PMC
August 2013

Expression plasticity of Phlebotomus papatasi salivary gland genes in distinct ecotopes through the sand fly season.

BMC Ecol 2011 Oct 10;11:24. Epub 2011 Oct 10.

The Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA.

Background: Sand fly saliva can drive the outcome of Leishmania infection in animal models, and salivary components have been postulated as vaccine candidates against leishmaniasis. In the sand fly Phlebotomus papatasi, natural sugar-sources modulate the activity of proteins involved in meal digestion, and possibly influence vectorial capacity. However, only a handful of studies have assessed the variability of salivary components in sand flies, focusing on the effects of environmental factors in natural habitats. In order to better understand such interactions, we compared the expression profiles of nine P. papatasi salivary gland genes of specimens inhabiting different ecological habitats in Egypt and Jordan and throughout the sand fly season in each habitat.

Results: The majority of investigated genes were up-regulated in specimens from Swaymeh late in the season, when the availability of sugar sources is reduced due to water deprivation. On the other hand, these genes were not up-regulated in specimens collected from Aswan, an irrigated area less susceptible to drought effects.

Conclusion: Expression plasticity of genes involved with vectorial capacity in disease vectors may play an important epidemiological role in the establishment of diseases in natural habitats.
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http://dx.doi.org/10.1186/1472-6785-11-24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209445PMC
October 2011

Ecological genomics of sand fly salivary gland genes: an overview.

J Vector Ecol 2011 Mar;36 Suppl 1:S58-63

Department of Entomology, Kansas State University, 123 W. Waters Hall, Manhattan, KS 66506, U.S.A.

Sand fly saliva contains an array of bioactive molecules that facilitate blood feeding and also function as modulators of the vertebrate immune response. Such a complex of biologically active molecules was shown to be both conserved and divergent among sand fly species. It is likely that expression of sand fly salivary molecules could be modulated by environmental factors, both biotic and abiotic, that ultimately dictate the quality, and possibly quantity, of the secreted saliva. Carbohydrates are an integral part of the sand fly diet, and sugar-sources found in natural habitats are potentially involved in defining the profile of sand fly saliva, and may influence vectorial capacity. Saliva can drive the outcome of Leishmania infection in animal models, and salivary molecules are potential targets for development of vaccines to control Leishmania infection. Thus, identifying what environmental factors effectively modulate sand fly saliva in the field is a critical step towards the development of meaningful protection strategies against leishmaniasis that are based on salivary compounds from sand fly vectors.
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http://dx.doi.org/10.1111/j.1948-7134.2011.00112.xDOI Listing
March 2011

Differential expression of salivary gland genes in the female sand fly Phlebotomus papatasi (Diptera: Psychodidae).

J Med Entomol 2010 Nov;47(6):1146-55

Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame, IN 46556, USA.

Saliva from blood-sucking arthropods modulates host homostasis and immunity, making salivary components potential candidates to be used against pathogens transmitted by these biting insects. Functional characterization of salivary molecules is fundamental to gain a better understanding into their roles during blood feeding and to determine under which conditions such molecules are expressed in the insect saliva. In the current study, we investigated the expression profile of 10 salivary genes from the sand fly Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae), a principal vector of Leishmania major. Our analyses using quantitative polymerase chain reaction were aimed at defining whether diet or senescence influences the expression of P. papatasi salivary gland-expressed genes in laboratory-reared female sand flies. Our results demonstrate that at least one of the most abundant salivary transcripts, SP44, is consistently modulated by either senescence or diet. In contrast, another abundant transcript, SP32, was expressed without any influence from the diet received or the age of the sand fly. Differential expression of the other eight transcripts was not consistently regulated by either diet or age, suggesting that other factors may have a greater influence on differential expression of these salivary gland proteins.
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http://dx.doi.org/10.1603/me10072DOI Listing
November 2010

Targeting the midgut secreted PpChit1 reduces Leishmania major development in its natural vector, the sand fly Phlebotomus papatasi.

PLoS Negl Trop Dis 2010 Nov 30;4(11):e901. Epub 2010 Nov 30.

Department of Entomology, Kansas State University, Manhattan, Kansas, United States of America.

Background: During its developmental cycle within the sand fly vector, Leishmania must survive an early proteolytic attack, escape the peritrophic matrix, and then adhere to the midgut epithelia in order to prevent excretion with remnants of the blood meal. These three steps are critical for the establishment of an infection within the vector and are linked to interactions controlling species-specific vector competence. PpChit1 is a midgut-specific chitinase from Phlebotomus papatasi presumably involved in maturation and degradation of the peritrophic matrix. Sand fly midgut chitinases, such as PpChit1, whether acting independently or in a synergistic manner with Leishmania-secreted chitinase, possibly play a role in the Leishmania escape from the endoperitrophic space. Thus, we predicted that silencing of sand fly chitinase will lead to reduction or elimination of Leishmania within the gut of the sand fly vector.

Methodology/principal Findings: We used injection of dsRNA to induce knock down of PpChit1 transcripts (dsPpChit1) and assessed the effect on protein levels post blood meal (PBM) and on Leishmania major development within P. papatasi. Injection of dsPpChit1 led to a significant reduction of PpChit1 transcripts from 24 hours to 96 hours PBM. More importantly, dsPpChit1 led to a significant reduction in protein levels and in the number of Le. major present in the midgut of infected P. papatasi following a infective blood meal.

Conclusion/significance: Our data supports targeting PpChit1 as a potential transmission blocking vaccine candidate against leishmaniasis.
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http://dx.doi.org/10.1371/journal.pntd.0000901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994919PMC
November 2010

Transmission blocking vaccines to control insect-borne diseases: a review.

Mem Inst Oswaldo Cruz 2010 Feb;105(1):1-12

Biology of Disease Vectors Laboratory, Department of Entomology, Kansas State University, Manhattan, KS 66506, USA.

Insect-borne diseases are responsible for severe mortality and morbidity worldwide. As control of insect vector populations relies primarily on the use of insecticides, the emergence of insecticide resistance as well to unintended consequences of insecticide use pose significant challenges to their continued application. Novel approaches to reduce pathogen transmission by disease vectors are been attempted, including transmission-blocking vaccines (TBVs) thought to be a feasible strategy to reduce pathogen burden in endemic areas. TBVs aim at preventing the transmission of pathogens from infected to uninfected vertebrate host by targeting molecule(s) expressed on the surface of pathogens during their developmental phase within the insect vector or by targeting molecules expressed by the vectors. For pathogen-based molecules, the majority of the TBV candidates selected as well as most of the data available regarding the effectiveness of this approach come from studies using malaria parasites. However, TBV candidates also have been identified from midgut tissues of mosquitoes and sand flies. In spite of the successes achieved in the potential application of TBVs against insect-borne diseases, many significant barriers remain. In this review, many of the TBV strategies against insect-borne pathogens and their respective ramification with regards to the immune response of the vertebrate host are discussed.
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http://dx.doi.org/10.1590/s0074-02762010000100001DOI Listing
February 2010

Transgenesis and paratransgenesis to control insect-borne diseases: current status and future challenges.

Parasitol Int 2010 Mar 9;59(1):1-8. Epub 2009 Oct 9.

Biology of Disease Vectors Laboratory, Department of Entomology, Kansas State University, Manhattan, KS, USA.

Insect-borne diseases cause significant human morbidity and mortality. Current control and preventive methods against vector-borne diseases rely mainly on insecticides. The emergence of insecticide resistance in many disease vectors highlights the necessity to develop new strategies to control these insects. Vector transgenesis and paratransgenesis are novel strategies that aim at reducing insect vectorial capacity, or seek to eliminate transmission of pathogens such as Plasmodium sp., Trypanosoma sp., and Dengue virus currently being developed. Vector transgenesis relies on direct genetic manipulation of disease vectors making them incapable of functioning as vectors of a given pathogen. Paratransgenesis focuses on utilizing genetically modified insect symbionts to express molecules within the vector that are deleterious to pathogens they transmit. Despite the many successes achieved in developing such techniques in the last several years, many significant barriers remain and need to be overcome prior to any of these approaches become a reality. Here, we highlight the current status of these strategies, pointing out advantages and constraints, and also explore issues that need to be resolved before the establishment of transgenesis and paratransgenesis as tools to prevent vector-borne diseases.
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http://dx.doi.org/10.1016/j.parint.2009.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824031PMC
March 2010

Lutzomyia longipalpis s.l. in Brazil and the impact of the Sao Francisco River in the speciation of this sand fly vector.

Parasit Vectors 2008 Oct 3;1(1):37. Epub 2008 Oct 3.

Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA.

In our recently published article "Lutzomyia longipalpis s.l. in Brazil and the impact of the Sao Francisco River in the speciation of this sand fly vector" by Iliano V. Coutinho-Abreu et al. a sentence located in paragraph 8 in the Discussion section had its meaning altered due to the improper insertion of three words.
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http://dx.doi.org/10.1186/1756-3305-1-37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2572594PMC
October 2008

Lutzomyia longipalpis s.l. in Brazil and the impact of the Sao Francisco River in the speciation of this sand fly vector.

Parasit Vectors 2008 06 12;1(1):16. Epub 2008 Jun 12.

Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA.

Lutzomyia longipalpis s.l. (Diptera: Psychodidae) is the principal vector of Leishmania infantum chagasi in the Americas, and constitutes a complex of species. Various studies have suggested an incipient speciation process based on behavioral isolation driven by the chemotype of male sexual pheromones. It is well known that natural barriers, such as mountains and rivers can directly influence population divergence in several organisms, including insects. In this work we investigated the potential role played by the Sao Francisco River in eastern Brazil in defining the current distribution of Lu. longipalpis s.l. Our studies were based on analyses of polymorphisms of the cytochrome b gene (cyt b) sequences from Lu. longipalpis s.l. available in public databases, and from additional field-caught individuals. Altogether, 9 distinct populations and 89 haplotypes were represented in the analyses. Lu. longipalpis s.l. populations were grouped according to their distribution in regards to the 10 degrees S parallel: north of 10 degrees S (<10 degrees S); and south of 10 degrees S (>10 degrees S). Our results suggest that although no polymorphisms were fixed, moderate genetic divergences were observed between the groups analyzed (i.e., FST = 0.184; and Nm = 2.22), and were mostly driven by genetic drift. The population divergence time estimated between the sand fly groups was about 0.45 million years (MY), coinciding with the time of the change in the course of the Sao Francisco River, during the Mindel glaciation. Overall, the polymorphisms on the cyt b haplotypes and the current speciation process detected in Lu. longipalpis s.l. with regards to the distribution of male sexual pheromones suggest a role of the Sao Francisco River as a significant geographical barrier in this process.
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http://dx.doi.org/10.1186/1756-3305-1-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2474595PMC
June 2008