Minimal Rickettsial Infection Rates and Distribution of Ticks in Uganda: An Assessment of the Seasonal Effects and Relevance to Tick-Borne Disease Risk in East Africa.

Tick-borne diseases (TBDs) pose a significant risk to humans and represent one of the major factors influencing readiness within the United States' military worldwide. Additionally, ticks and TBDs constitute major animal health problems leading to economic losses at multiple levels affecting low- and middle-income countries the hardest. Tick control is frequently hampered by issues ranging from acaricide resistance to lack of data on tick distribution and infection rates. We conducted a cross-sectional study to assess tick species distribution, host use, and rickettsial pathogen infection rate of ticks in different areas of the Uganda Cattle Corridor. We identified 4,425 hard ticks (Ixodida: Ixodidae) comprised of seven species by morphological characters with 3,315 ticks collected from four locations during the dry season and 1,110 ticks from one location during the wet season. Rickettsial pathogen prevalence was assessed in ticks collected from two districts to determine the minimum infection rate compared across seasons, village location, and tick species. We found statistically significant differences in the abundance and distribution of tick species among districts in the dry season, host animal species, and the proportion of rickettsial positive pools between villages. Seasonality, village location, and tick species do not affect the minimum infection rate of rickettsial pathogens of ticks in Uganda, but village location affects the proportion of positive tick pools. These results indicate geographical and seasonal differences among pathogen-harboring ticks contributing to our understanding of the current distribution of ticks and TBDs in Uganda.

Defining the mechanisms of action and mosquito larva midgut response to a yeast-encapsulated orange oil larvicide.

BACKGROUND: Yeast-encapsulated orange oil (YEOO) is a novel, ingestible larvicide that combines the benefits of a low-cost essential oil with yeast, an attractive food source for mosquito larvae. In this work, we investigated the underlying mechanisms of action associated with YEOO ingestion by Aedes aegypti larvae. METHODS: Aedes aegypti third-stage larvae (L3) were treated with sublethal or lethal concentrations of YEOO. Genes associated with apoptosis, autophagy and innate immune responses were investigated by RT-qPCR in guts and carcasses dissected from treated and control larvae. Differential expression of cytochrome P450 genes in the CYP6 and CYP9 families were also investigated. Confocal and transmission electron microscopy were used to assess damage caused by YEOO throughout the larval alimentary canal. TUNEL was used to assess apoptosis via DNA fragmentation. RESULTS: The apoptosis genes IAP1 and IAP2 in larvae displayed opposing effects following exposure to lethal doses of YEOO, with a 26-fold induction of IAP1 at 8 h post YEOO ingestion. The effector caspase CASPS8 displayed a 6.7-fold induction in the gut and concomitant 70-fold induction in the carcass at 8 h post YEOO ingestion. The midgut epithelia regenerator, Vein, had an 11-fold induction in the gut after 4 h and was repressed 7.6-fold in the carcass at 24 h. Sublethal concentrations (< LC50) led to significant differential expression of CYP6 and CYP9 genes. Midgut epithelial damage was highlighted by the destruction of microvilli, vacuolization of midgut cells and damage to cell junctions and basal lamina as early as 30 min. Larval type 2 peritrophic matrix structural integrity and porosity remain unchanged. CONCLUSION: Our results strongly suggest that the robust larvicidal activity of YEOO is due to a generalized broad-acting mechanism combining epithelial damage and apoptosis, with concomitant expression of multiple innate response genes involved in epithelial regeneration and detoxification. YEOO's amenability for use as part of an integrated vector management program makes this novel larvicide a practical approach for mosquito larval control in the future.

Risk Assessment of the Role of the Ecotones in the Transmission of Zoonotic Cutaneous Leishmaniasis in Central Tunisia.

Zoonotic cutaneous leishmaniasis (ZCL), endemic in Central and Southern Tunisia, is caused by Leishmania major (Kinetoplastida: Trypanosomatidae), which is transmitted by the sand fly Phlebotomus papatasi. In Tunisia, the fat sand rat Psammomys obesus and the desert jird Meriones shawi are the principal reservoir hosts of L. major. The presence of the P. papatasi vector of the L. major etiologic agent of ZCL was assessed in the vicinity of villages in endemic areas of Central Tunisia. The study was performed from September through October 2019, a period corresponding to the main peak of activity of P. papatasi. Sand flies were collected from rodent burrows located at the ecotone level, which is the transition zone between the natural environment and human settlement. Sand flies were identified to species level and tested for the presence of L. major by PCR. Our entomological survey showed that P. papatasi is the most abundant sand fly species associated with rodent burrows, and this abundance is even higher in ecotones primarily occupied by P. obesus in comparison to ecotones occupied by M. shawi. Infections with Leishmania major were detected only in P. papatasi, with an overall minimum infection rate (MIR) of 2.64%. No significant difference was observed between the MIRs in ecotones of P. obesus and of M. shawi. Incidence of ZCL in the studied areas ranged from 200 to 700 cases per 100,000 inhabitants, with a mean incidence of 385.41 per 100,000. Higher ZCL incidence was identified in ecotones of M. shawi compared to ecotones of P. obesus. ZCL cases are positively correlated with the MIRs. Considering the short flight range of P. papatasi, increases in its densities associated with burrows of P. obesus or M. shawi at the ecotone level expand the overlap of infected vectors with communities and subsequently increase ZCL incidence. Therefore, control measures should target P. papatasi populations at the ecotones.

High larvicidal efficacy of yeast-encapsulated orange oil against Aedes aegypti strains from Brazil.

BACKGROUND: Botanical substances such as essential oils (EOs) have demonstrated insecticidal properties and are a valid option for vector control. However, free EOs are unreliable as mosquito larvicides due their easy degradation by environmental exposure to ultraviolet light and higher temperatures. Here, we assessed the efficacy of a mosquito larvicide based on orange oil in a yeast-based delivery system against Aedes aegypti strains with different resistance status towards chemical neurotoxic insecticides. This larvicide preparation was physicochemically characterized in a previous report. METHODS: Larvae of four Ae. aegypti strains from different regions of Brazil and different resistance profiles for deltamethrin (pyrethroid) and temephos (organophosphate) were tested against yeast-encapsulated orange oil (YEOO) in laboratory conditions for measurement of LC50 and LC90 values. The same assays were performed with the Belo Horizonte strain under environmental conditions (natural light and temperature). The resistance profiles of these strains were compared to the Rockefeller reference strain in all conditions. RESULTS: YEOO was found to be a highly active larvicide (LC50 < 50 mg/L) against all Ae. aegypti strains tested in both laboratory conditions (LC50 = 8.1-24.7 mg/L) and environmental conditions with natural light and temperature fluctuation (LC50 = 20.0-49.9 mg/L). Moreover, all strains were considered susceptible (RR < 5) to YEOO, considering resistance ratios calculated based on the Rockefeller strain. The resistance ratios were only higher than 2.5 for LC90-95 of Belo Horizonte in the laboratory, probably due the higher heterogeneity associated with older egg papers (> 5 months). CONCLUSION: YEOO demonstrates high larvicidal activity against Ae. aegypti strains with resistant phenotypes for deltamethrin (PY) and temephos (OP). This larvicidal activity suggests the potential for the development of YEOO as an alternative intervention to synthetic insecticides in integrated vector management programs, for populations with resistance to commonly used insecticides.

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

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.

Transcriptional profiling and physiological roles of Aedes aegypti spermathecal-related genes.

BACKGROUND: Successful mating of female mosquitoes typically occurs once, with the male sperm being stored in the female spermatheca for every subsequent oviposition event. The female spermatheca is responsible for the maintenance, nourishment, and protection of the male sperm against damage during storage. Aedes aegypti is a major vector of arboviruses, including Yellow Fever, Dengue, Chikungunya, and Zika. Vector control is difficult due to this mosquito high reproductive capacity. RESULTS: Following comparative RNA-seq analyses of spermathecae obtained from virgin and inseminated females, eight transcripts were selected based on their putative roles in sperm maintenance and survival, including energy metabolism, chitin components, transcriptional regulation, hormonal signaling, enzymatic activity, antimicrobial activity, and ionic homeostasis. In situ RNA hybridization confirmed tissue-specific expression of the eight transcripts. Following RNA interference (RNAi), observed outcomes varied between targeted transcripts, affecting mosquito survival, egg morphology, fecundity, and sperm motility within the spermathecae. CONCLUSIONS: This study identified spermatheca-specific transcripts associated with sperm storage in Ae. aegypti. Using RNAi we characterized the role of eight spermathecal transcripts on various aspects of female fecundity and offspring survival. RNAi-induced knockdown of transcript AeSigP-66,427, coding for a Na+/Ca2+ protein exchanger, specifically interfered with egg production and reduced sperm motility. Our results bring new insights into the molecular basis of sperm storage and identify potential targets for Ae. aegypti control.

Yeast-encapsulated essential oils: a new perspective as an environmentally friendly larvicide.

BACKGROUND: Effective mosquito control approaches incorporate both adult and larval stages. For the latter, physical, biological, and chemical control have been used with varying results. Successful control of larvae has been demonstrated using larvicides including insect growth regulators, e.g. the organophosphate temephos, as well as various entomopathogenic microbial species. However, a variety of health and environmental issues are associated with some of these. Laboratory trials of essential oils (EO) have established the larvicidal activity of these substances, but there are currently no commercially available EO-based larvicides. Here we report on the development of a new approach to mosquito larval control using a novel, yeast-based delivery system for EO. METHODS: Food-grade orange oil (OO) was encapsulated into yeast cells following an established protocol. To prevent environmental contamination, a proprietary washing strategy was developed to remove excess EO that is adsorbed to the cell exterior during the encapsulation process. The OO-loaded yeast particles were then characterized for OO loading, and tested for efficacy against Aedes aegypti larvae. RESULTS: The composition of encapsulated OO extracted from the yeast microparticles was demonstrated not to differ from that of un-encapsulated EO when analyzed by high performance liquid chromatography. After lyophilization, the oil in the larvicide comprised 26-30 percentage weight (wt%), and is consistent with the 60-65% reduction in weight observed after the drying process. Quantitative bioassays carried with Liverpool and Rockefeller Ae. aegypti strains in three different laboratories presented LD50 of 5.1 (95% CI: 4.6-5.6) to 27.6 (95% CI: 26.4-28.8) mg/l, for L1 and L3/L4 mosquito larvae, respectively. LD90 ranged between 18.9 (95% CI: 16.4-21.7) mg/l (L1 larvae) to 76.7 (95% CI: 69.7-84.3) mg/l (L3/L4 larvae). CONCLUSIONS: The larvicide based on OO encapsulated in yeast was shown to be highly active (LD50 < 50 mg/l) against all larval stages of Ae. aegypti. These results demonstrate its potential for incorporation in an integrated approach to larval source management of Ae. aegypti. This novel approach can enable development of affordable control strategies that may have significant impact on global health.

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

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 ST 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 ST values fall in the range 0.024-0.648. CONCLUSION: The clustering patterns and pairwise F ST 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.

Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases

BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases.

BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

Midgut morphological changes and autophagy during metamorphosis in sand flies.

During metamorphosis, holometabolous insects undergo significant remodeling of their midgut and become able to cope with changes in dietary requirements between larval and adult stages. At this stage, insects must be able to manage and recycle available food resources in order to develop fully into adults, especially when no nutrients are acquired from the environment. Autophagy has been previously suggested to play a crucial role during metamorphosis of the mosquito. Here, we investigate the overall morphological changes of the midgut of the sand fly during metamorphosis and assess the expression profiles of the autophagy-related genes ATG1, ATG6, and ATG8, which are associated with various steps of the autophagic process. Morphological changes in the midgut start during the fourth larval instar, with epithelial degeneration followed by remodeling via the differentiation of regenerative cells in pre-pupal and pupal stages. The changes in the midgut epithelium are paired with the up-regulation of ATG1, ATG6 and ATG8 during the larva-adult transition. Vein, a putative epidermal growth factor involved in regulating epithelial midgut regeneration, is also up-regulated. Autophagy has further been confirmed in sand flies via the presence of autophagosomes residing within the cytoplasmic compartment of the pupal stages. An understanding of the underlying mechanisms of this process should aid the future management of this neglected tropical vector.

An integrated overview of the midgut bacterial flora composition of Phlebotomus perniciosus, a vector of zoonotic visceral leishmaniasis in the Western Mediterranean Basin.

BACKGROUND: The Leishmania developmental life cycle within its sand fly vector occurs exclusively in the lumen of the insect's digestive tract in the presence of symbiotic bacteria. The composition of the gut microbiota and the factors that influence its composition are currently poorly understood. A set of factors, including the host and its environment, may influence this composition. It has been demonstrated that the insect gut microbiota influences the development of several human pathogens, such as Plasmodium falciparum. For sand flies and Leishmania, understanding the interactions between the parasite and the microbial environment of the vector midgut can provide new tools to control Leishmania transmission. METHODOLOGY/PRINCIPAL FINDINGS: The midguts of female Phlebotomus perniciosus from laboratory colonies or from the field were collected during the months of July, September and October 2011 and dissected. The midguts were analyzed by culture-dependent and culture-independent methods. A total of 441 and 115 cultivable isolates were assigned to 30 and 11 phylotypes from field-collected and colonized P. perniciosus, respectively. Analysis of monthly variations in microbiota composition shows a species diversity decline in October, which is to the end of the Leishmania infantum transmission period. In parallel, a compilation and a meta-analysis of all available data concerning the microbiota of two Psychodidae genera, namely Phlebotomus and Lutzomyia, was performed and compared to P. perniciosus, data obtained herein. This integrated analysis did not reveal any substantial divergences between Old and New world sand flies with regards to the midgut bacterial phyla and genera diversity. But clearly, most bacterial species (>76%) are sparsely distributed between Phlebotominae species. CONCLUSION/SIGNIFICANCE: Our results pinpoint the need for a more exhaustive understanding of the bacterial richness and abundance at the species level in Phlebotominae sand flies in order to capture the role of midgut bacteria during Leishmania development and transmission. The occurrence of Bacillus subtilis in P. perniciosus and at least two other sand fly species studied so far suggests that this bacterial species is a potential candidate for paratransgenic or biolological approaches for the control of sand fly populations in order to prevent Leishmania transmission.

Zoonotic visceral leishmaniasis transmission: modeling, backward bifurcation, and optimal control.

Visceral leishmaniasis (VL), a vector-borne disease caused by protozoan flagellates of the genus Leishmania, is transmitted by sand flies. After malaria, VL is the second-largest parasitic killer, responsible for an estimated 500,000 infections and 51,000 deaths annually worldwide. Mathematical models proposed for VL have included the impact of dogs versus wild canids in disease dissemination and models developed to assist in control approaches. However, quantitative conditions that are required to control or eradicate VL transmission are not provided and there are no mathematical methods proposed to quantitatively calculate optimal control strategies for VL transmission. The research objective of this work was to model VL disease transmission system (specifically Zoonotic VL), perform bifurcation analysis to discuss control conditions, and calculate optimal control strategies. Three time-dependent control strategies involving dog populations, sand fly population, and humans are mainly discussed. Another strategy sometimes used in attempts to control zoonotic VL transmission, dog culling, is also evaluated in this paper.

Changes of Sand Fly Populations and Leishmania infantum Infection Rates in an Irrigated Village Located in Arid Central Tunisia.

The current spread of zoonotic visceral leishmaniasis (ZVL) throughout arid areas of Central Tunisia is a major public health concern. The main objective of this study is to investigate whether the development of irrigation in arid bio-geographical areas in Central Tunisia have led to the establishment of a stable cycle involving sand flies of the subgenus Larroussius and Leishmania infantum, and subsequently to the emergence of ZVL. Sand flies were collected from the village of Saddaguia, a highly irrigated zone located within an arid bio-geographical area of Central Tunisia by using modified Centers for Diseases Control (CDC) light traps. Morphological keys were used to identify sand flies. Collected sand flies were pooled with up to 30 specimens per pool according to date and tested by nested Polymerase Chain Reaction (PCR) DNA sequencing from positive pools was used to identify Leishmania spp. A total of 4915 sand flies (2422 females and 2493 males) were collected from Saddaguia in September and in October 2014. Morphological identification confirmed sand flies of the subgenus Larroussius to be predominant. PCR analysis followed by DNA sequencing indicated that 15 pools were infected with L. infantum yielding an overall infection rate of 0.6%. The majority of the infected pools were of sand fly species belonging to subgenus Larroussius. Intense irrigation applied to the arid bio-geographical areas in Central Tunisia is at the origin of the development of an environment capable of sustaining important populations of sand flies of the subgenus Larroussius. This has led to the establishment of stable transmission cycles of L. infantum and subsequently to the emergence of ZVL.

Effects of specific antisera targeting peritrophic matrix-associated proteins in the sand fly vector Phlebotomus papatasi.

In many hematophagous insects, the peritrophic matrix (PM) is formed soon after a blood meal (PBM) to compartmentalize the food bolus. The PM is an important component of vector competence, functioning as a barrier to the development of many pathogens including parasites of the genus Leishmania transmitted by sand flies. PM morphology and permeability are associated with the proteins that are part of the PM scaffolding, including several peritrophins, and chitin fibers. Here, we assessed the effects of specific antisera targeting proteins thought to be an integral part of the PM scaffolding and its process of maturation and degradation. Phlebotomus papatasi sand flies were fed with red blood cells reconstituted with antisera targeting the chitinase PpChit1, and the peritrophin PpPer2. Sand fly midguts were dissected at different time points and processed for light microscopy (LM), confocal and transmission electron (TEM) microscopies (24, 42-46, 48 and 72h PBM), scanning electron (SEM) (48h PBM) and atomic force (AFM) (30h PBM) microscopies. TEM and WGA-FITC staining indicate PM degradation was significantly delayed following feeding of flies on anti-PpChit1. AFM analysis at 30h PBM point to an increase in roughness' amplitude of the PM of flies that fed on either anti-PpChit1 or anti-PpPer2. Collective, our data suggest that antibodies targeting PM-associated proteins affects the kinetics of PM maturation, delaying its degradation and disruption and are potential targets on transmission-blocking vaccines strategies.

Vectorborne Transmission of Leishmania infantum from Hounds, United States.

Leishmaniasis is a zoonotic disease caused by predominantly vectorborne Leishmania spp. In the United States, canine visceral leishmaniasis is common among hounds, and L. infantum vertical transmission among hounds has been confirmed. We found that L. infantum from hounds remains infective in sandflies, underscoring the risk for human exposure by vectorborne transmission.

Bacterial Infection and Immune Responses in Lutzomyia longipalpis Sand Fly Larvae Midgut.

The midgut microbial community in insect vectors of disease is crucial for an effective immune response against infection with various human and animal pathogens. Depending on the aspects of their development, insects can acquire microbes present in soil, water, and plants. Sand flies are major vectors of leishmaniasis, and shown to harbor a wide variety of Gram-negative and Gram-positive bacteria. Sand fly larval stages acquire microorganisms from the soil, and the abundance and distribution of these microorganisms may vary depending on the sand fly species or the breeding site. Here, we assess the distribution of two bacteria commonly found within the gut of sand flies, Pantoea agglomerans and Bacillus subtilis. We demonstrate that these bacteria are able to differentially infect the larval digestive tract, and regulate the immune response in sand fly larvae. Moreover, bacterial distribution, and likely the ability to colonize the gut, is driven, at least in part, by a gradient of pH present in the gut.

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

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.

Genetic structuring and fixed polymorphisms in the gene period among natural populations of Lutzomyia longipalpis in Brazil.

BACKGROUND: Even one hundred years after being originally identified, aspects of the taxonomy of the sand fly Lutzomyia longipalpis, the principal vector of Leishmania infantum in the Americas, remain unresolved for Brazilian populations of this vector. The diversity of morphological, behavioral, biochemical, and ethological characters, as well as the genetic variability detected by molecular markers are indicative of the presence of a complex of species. METHODS: In this study, a 525 bp fragment of the period gene was used to evaluate sympatric populations of L. longipalpis. A combination of probabilistic methods such as maximum likelihood and genetic assignment approach to investigate sympatric species of L. longipalpis were applied in three populations of Northeast Brazil. RESULTS: Fixed polymorphisms in geographically isolated populations of L. longipalpis from two localities in the state of Ceará and one in the state of Pernambuco, Brazil, was identified in a 525 bp fragment of the gene period (per). Our results suggest a direct relationship between the number of spots found in males' tergites and the genetic variation in cryptic species of L. longipalpis. The fragment used in this study revealed the nature of the ancestral morphotype 1S. CONCLUSION: New polymorphisms were identified in the gene per which can be used as a genetic barcode to sympatric taxonomy of L. longipalpis. The per gene fragment confirmed the presence of two siblings species of L. longipalpis in Sobral and showed that these same species are present in two other localities, representing an expansion within the L. longipalpis species complex with regards to the states of Ceará and Pernambuco.

The flagellar protein FLAG1/SMP1 is a candidate for Leishmania-sand fly interaction.

Leishmaniasis is a serious problem that affects mostly poor countries. Various species of Leishmania are the agents of the disease, which take different clinical manifestations. The parasite is transmitted by sandflies, predominantly from the Phlebotomus genus in the Old World and Lutzomyia in the New World. During development in the gut, Leishmania must survive various challenges, which include avoiding being expelled with blood remnants after digestion. It is believed that attachment to the gut epithelium is a necessary step for vector infection, and molecules from parasites and sand flies have been implicated in this attachment. In previous work, monoclonal antibodies were produced against Leishmania. Among these an antibody was obtained against Leishmania braziliensis flagella, which blocked the attachment of Leishmania panamensis flagella to Phlebotomus papatasi guts. The protein recognized by this antibody was identified and named FLAG1, and the complete FLAG1 gene sequence was obtained. This protein was later independently identified as a small, myristoylated protein and called SMP1, so from now on it will be denominated FLAG1/SMP1. The FLAG1/SMP1 gene is expressed in all developmental stages of the parasite, but has higher expression in promastigotes. The anti-FLAG1/SMP1 antibody recognized the flagellum of all Leishmania species tested and generated the expected band by western blots. This antibody was used in attachment and infection blocking experiments. Using the New World vector Lutzomyia longipalpis and Leishmania infantum chagasi, no inhibition of attachment ex vivo or infection in vivo was seen. On the other hand, when the Old World vectors P. papatasi and Leishmania major were used, a significant decrease of both attachment and infection were seen in the presence of the antibody. We propose that FLAG1/SMP1 is involved in the attachment/infection of Leishmania in the strict vector P. papatasi and not the permissive vector L. longipalpis.