Stability and suitability of housekeeping genes in phlebotomine sand flies.

We investigated gene expression patterns in Lutzomyia and Phlebotomus sand fly vectors of leishmaniases. Using quantitative PCR, we assessed the expression stability of potential endogenous control genes commonly used in dipterans. We analyzed Lutzomyia longipalpis and Phlebotomus papatasi samples from L3 and L4 larval stages, adult sand flies of different sexes, diets, dsRNA injection, and Leishmania infection. Six genes were evaluated: actin, α-tubulin, GAPDH, 60 S ribosomal proteins L8 and L32 (RiboL8 and RiboL32), and elongation factor 1-α (EF1-α). EF1-α was among the most stably expressed along with RiboL8 in L. longipalpis larvae and RiboL32 in adults. In P. papatasi, EF1-α and RiboL32 were the top in larvae, while EF1-α and actin were the most stable in adults. RiboL8 and actin were the most stable genes in dissected tissues and infected guts. Additionally, five primer pairs designed for L. longipalpis or P. papatasi were effective in PCR with Lutzomyia migonei, Phlebotomus duboscqi, Phlebotomus perniciosus, and Sergentomyia schwetzi cDNA. Furthermore, L. longipalpis RiboL32 and P. papatasi α-tubulin primers were suitable for qPCR with cDNA from the other four species. Our research provides tools to enhance relative gene expression studies in sand flies, facilitating the selection of endogenous control for qPCR.

Entomological aspects of leishmaniasis transmission in the urban area of Teófilo Otoni, a municipality endemic for the disease in the Brazilian state of Minas Gerais.

Brazil is endemic for both visceral (VL) and cutaneous (CL) clinical forms of leishmaniasis, poverty-associated diseases with worldwide distribution. Leishmania parasites are the etiological agents of leishmaniases, which are transmitted to humans through the bites of infected phlebotomine sand flies. From 2018 to 2023, 15 cases of VL and 129 cases of CL were reported in Téofilo Otoni, an important economic center in the Brazilian state of Minas Gerais. Owing to the lack of data on the entomological fauna, the present study aimed to clarify this main aspect of leishmaniasis. From May, 2021 to April, 2023, entomological captures were performed monthly in ten neighborhoods in Teófilo Otoni. The influence of bioclimatic variables on insect populations was evaluated, and natural infection by Leishmania spp. was investigated using molecular methods. A total of 306 specimens of 12 species of phlebotomine sand fly were collected. The majority (91.6%) were proven or putative vectors of leishmaniasis agents. The population of insects tended to increase during the cooler and drier months. Although Leishmania infection was not detected in any of the samples, the presence of vectors provides conditions for the maintenance and expansion of the transmission cycle of leishmaniasis in Teófilo Otoni.

Phlebotomine (Diptera: Psychodidae) species and their blood meal sources in a new leishmaniasis focus in Los Montes de María, Bolívar, in northern Colombia / Especies de flebotomíneos y sus fuentes de ingesta sanguínea en un nuevo foco de leishmaniasis en Los Montes de María (Bolívar) al norte de Colombia.

Introduction. El Alférez, a village in Los Montes de María (Bolívar, Colombia) and a macro-focus of leishmaniasis, recorded its first case in 2018, evidencing changes in the distribution and eco-epidemiology of the disease, although interactions between vectors and local fauna remain unknown. Objective. To evaluate the diversity of sandflies and their blood meal sources in the community of El Alférez in the municipality of El Carmen de Bolívar (Bolívar, Colombia). Materials and methods. In 2018, sandflies were collected using LED-based light traps in domestic, peridomestic, and sylvatic ecotopes and identified at the species level. Multiplex polymerase chain reaction targeting the mitochondrial cytochrome B gene was used to analyze blood from the digestive tract. Results. Lutzomyia evansi was the most abundant species (71.85%; n = 485/675), followed by Lu. panamensis, Lu. gomezi, Lu. trinidadensis, Lu. dubitans, Lu. abonnenci, and Lu.aclydifera. Twenty-five percent of the species had blood meals from Canis familiaris (36.00%; n = 9/25), Ovis aries (36.00%; n=9:/25), Bos taurus (24.00%; n = 6/25), Sus scrofa (20.00%; n = 5/25), and Homo sapiens (8.00%; n = 2/25). Lutzomyia evansi registered the highest feeding frequency (68.00%; n = 17/25), predominantly on a single (44.00%; n = 11/25) or multiple species (24.00%; n = 6/25). Conclusion. Results indicate a eclectic feeding behavior in Lu. evansi, implying potential reservoir hosts for Leishmania spp. and increasing transmission risk. This study is a first step towards understanding the diversity of mammalian blood sources used by sandflies, that may be crucial for vector identification and formulation of effective control measures.

Introducción. En 2018, en la vereda El Alférez de Los Montes de María (Bolívar, Colombia), un macrofoco de leishmaniasis, se reportó el primer caso y se evidenciaron cambios en la distribución y ecoepidemiología de la enfermedad. No obstante, las interacciones entre vectores y fauna local aún son desconocidas. Objetivo. Evaluar la diversidad de flebotomíneos y sus fuentes de alimentación sanguínea en la comunidad de El Alférez del municipio de El Carmen de Bolívar (Bolívar, Colombia). Materiales y métodos. En el 2018, se recolectaron flebotomíneos mediante trampas de luz led ubicadas en el domicilio, el peridomicilio y en el área silvestre, y se identificaron a nivel de especie. Se utilizó la reacción en cadena de la polimerasa múltiple dirigida al gen mitocondrial citocromo B para analizar la sangre del aparato digestivo. Resultados. Lutzomyia evansi fue la especie más abundante (71,85 %; n = 485/675), seguida por Lu. panamensis, Lu. gomezi, Lu. trinidadensis, Lu. dubitans, Lu. abonnenci y Lu. aclydifera. El 25 % (n = 25/100) de las especies analizadas tuvieron como fuentes de ingesta sanguínea a Canis familiaris (36 %; n = 9/25), Ovis aries (36 %; n = 9/25), Bos taurus (24 %; n = 6/25), Sus scrofa (20 %; n = 5/25) y Homo sapiens (8 %; n = 2/25). Lutzomyia evansi fue la especie con la mayor frecuencia de alimentación (68 %; n = 17/25), predominantemente de una sola especie (44 %; n = 11/25) o de varias (24 %; n = 6/25).

Sand fly blood meal volumes and their relation to female body weight under experimental conditions.

BACKGROUND: Sand fly females require a blood meal to develop eggs. The size of the blood meal is crucial for fecundity and affects the dose of pathogens acquired by females when feeding on infected hosts or during experimental membrane-feeding. METHODS: Under standard laboratory conditions, we compared blood meal volumes taken by females of ten sand fly species from four genera: Phlebotomus, Lutzomyia, Migonomyia, and Sergentomyia. The amount of ingested blood was determined using a haemoglobin assay. Additionally, we weighed unfed sand flies to calculate the ratio between body weight and blood meal weight. RESULTS: The mean blood meal volume ingested by sand fly females ranged from 0.47 to 1.01 µl. Five species, Phlebotomus papatasi, P. duboscqi, Lutzomyia longipalpis, Sergentomyia minuta, and S. schwetzi, consumed about double the blood meal size compared to Migonomyia migonei. The mean body weight of females ranged from 0.183 mg in S. minuta to 0.369 mg in P. duboscqi. In males, the mean body weight ranged from 0.106 mg in M. migonei to 0.242 mg in P. duboscqi. Males were always lighter than females, with the male-to-female weight ratio ranging from 75% (in Phlebotomus argentipes) to 52% (in Phlebotomus tobbi). CONCLUSIONS: Females of most species took a blood meal 2.25-3.05 times their body weight. Notably, the relatively tiny females of P. argentipes consumed blood meals 3.34 times their body weight. The highest (Mbl/Mf) ratios were found in both Sergentomyia species studied; females of S. minuta and S. schwetzi took blood meals 4.5-5 times their body weight. This parameter is substantially higher than that reported for mosquitoes and biting midges.

Ecological determinants of leishmaniasis vector, Lutzomyia spp.: A scoping review.

Leishmania spp. are zoonotic parasites transmitted by phlebotomine sand flies, including those of the Lutzomyia genus, which can cause leishmaniases in both humans and dogs. Lutzomyia spp. are established in many countries in South and Central America and some areas of the southern United States, with suspected potential of these vectors to undergo further range expansion due to climate change. A scoping review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extensions for Scoping Reviews (PRISMA-ScR) guidelines to describe the current state of knowledge on the key ecological factors associated with Lutzomyia spp. survival, reproduction and establishment. The following electronic databases were searched for eligible studies published from 1 January 1990, to the date of search, 26 April 2023: CAB Direct (CABI), MEDLINE (via Ovid), Biological Sciences Database and Environmental Sciences Database. Primary research articles that were available in English and focused on ecological factors associated with Lutzomyia spp., such as climatic and habitat factors, geographic range, seasonality and temporality, and host abundance, were eligible for inclusion in the study. Following de-duplication, a total of 167 studies were included in Level 1 screening, 64 studies were included in Level 2 screening and 31 studies met the criteria for data extraction. Study locations included Argentina, Brazil, Colombia, Peru, Venezuela, the United States, Mexico and Canada, with some studies including multiple regions. A total of 31 different Lutzomyia spp. were assessed across these studies, with most (51.6%) of the studies focused on Lutzomyia longipalpis. Eligible studies investigated factors such as seasonality (n = 5), temperature (n = 19), precipitation (n = 13), humidity (n = 2), vegetation presence or requirements (n = 13), ecotypes (n = 7), and/or community type (i.e., urban, suburban, rural) (n = 5). Lutzomyia spp. activity was found to be higher during the rainy season, and peak when temperatures were between 20 and 25°C. Lutzomyia spp. were also found to preferentially reside in tropical or subtropical forests, which are characterised by their lack of a distinct dry season and high precipitation. This scoping review summarised the current state of the literature on the ecological factors associated with the survival, activity and reproduction of Lutzomyia spp. While there appears to be some consensus in the literature regarding some ecological requirements (such as seasonality, temperature and habitat features), overall, there is a lack of published research in this topic. This poses a significant challenge for future studies, which aim to predict the future distribution of Lutzomyia spp. in the context of climate and land use changes. Additional ecological research is urgently needed on Lutzomyia spp. given their relevance to both human and animal health.

Abundance of Leishmania (Kinetoplastida: Trypanosomatidae) vectors in Baldim, a municipality in the Espinhaço Range Biosphere Reserve in Brazil.

Leishmaniasis is a zoonoses caused by protozoan parasites belonging to the Leishmania genus that is transmitted to humans through the bite of previously infected phlebotomine sandflies (Diptera:Psychodidae:Phlebotominae). In Brazil, 2 types of leishmaniasis are endemic, tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL). In Baldim, a municipality in the Espinhaço Range Biospere Reserve (ERBR), 5 autochthonous cases of TL, and one autochthonous case of VL were reported from 2017 to 2022. Owing to the lack of data on entomological fauna, we surveyed the occurrence and distribution of phlebotomine sandflies that could act as Leishmania vectors, as well as natural Leishmania infection. During 1 year, 918 sandfly specimens belonging to 12 species were collected. The predominant species was Lutzomyia longipalpis (Lutz & Neiva, 1912) (32.0%), a proven VL agent, followed by Evandromyia evandroi (Costa Lima & Antunes, 1936) (20.7%), Evandromyia cortelezzii (Brèthes, 1923) (17.0%), Nyssomyia whitmani (Antunes & Coutinho, 1939) (12.2%), and Nyssomyia intermedia (Lutz & Neiva, 1912) (10.5%), all putative or proven TL agents. Seasonal fluctuations and endo-exophilic behavior were delineated for the main phlebotomine sandflies. The population of Lu. longipalpis was higher during the coolest and driest months, although different profiles were noted for the other species. Natural Leishmania infection was not detected in any of the 197 phlebotomine sandfly females analyzed. The results were compared with those previously obtained in other municipalities belonging to the ERBR. The presence of Leishmania vectors and human cases of leishmaniasis indicate a risk of Leishmania transmission in the region.

Phlebotomine sand flies (Diptera: Psychodidae, Phlebotominae): Diversity of potential Leishmania vectors in northern Colombia.

Phlebotomine sand flies are critical vectors of Leishmania parasites, impacting public health significantly. This study focused on assessing the diversity of sand flies in a rural area of El Carmen de Bolívar Municipality, northern Colombia, employing rarefaction curves and Hill numbers to understand potential vector communities and inform environmental management. From January 2018 to April 2019 (five samplings), sand flies were collected using CDC light traps with blue LED in domestic/peridomestic/sylvatic ecotopes, identifying species per Young and Duncan (1994) and Galati (2003). Hill numbers provided diversity estimates across samples, while Principal Component Analysis correlated with environmental factors with phlebotomine species presence and abundance. 8,784 phlebotomine individuals were collected; 56.4 % females and 43.6% males (ratio 3:2). These individuals belonged to eight species: Pintomyia evansi, Psychodopygus panamensis, Lutzomyia gomezi, Micropygomyia cayennensis, Evandromyia dubitans, Psathyromyia aclydifera, Pintomyia serrana, and Pintomyia rangeliana; with Pi. evansi being the most abundant species (74.39 %; 6,530 exemplars). The ANOVA showed no significant differences between phlebotomine sand flies abundances across ecotopes (p = 0.018). Species of epidemiological relevance as Pi. evansi and Lu. gomezi not show a positive correlation with environmental variables evaluated, only Ps. panamensis was positively correlated with precipitation. However, the study emphasizes the need for a continuous sand fly monitoring and research to enhance leishmaniasis control strategies, highlighting the necessity to expand knowledge on phlebotomine diversity and environmental interactions to understand vector ecology and disease dynamics better.

Humidity and temperature preference in two Neotropical species of sand flies.

BACKGROUND: Arthropods vector a multitude of human disease-causing organisms, and their geographic ranges are shifting rapidly in response to changing climatic conditions. This is, in turn, altering the landscape of disease risk for human populations that are brought into novel contact with the vectors and the diseases they carry. Sand flies in the genera Lutzomyia and Pintomyia are vectors of serious disease-causing agents such as Leishmania (the etiological agent of leishmaniasis) and may be expanding their range in the face of climate change. Understanding the climatic conditions that vector species both tolerate physiologically and prefer behaviorally is critical to predicting the direction and magnitude of range expansions and the resulting impacts on human health. Temperature and humidity are key factors that determine the geographic extent of many arthropods, including vector species. METHODS: We characterized the habitat of two species of sand flies, Lutzomyia longipalpis and Pintomyia evansi. Additionally, we studied two behavioral factors of thermal fitness-thermal and humidity preference in two species of sand flies alongside a key aspect of physiological tolerance-desiccation resistance. RESULTS: We found that Lu. longipalpis is found at cooler and drier conditions than Pi. evansi. Our results also show significant interspecific differences in both behavioral traits, with Pi. evansi preferring warmer, more humid conditions than Lu. longipalpis. Finally, we found that Lu. longipalpis shows greater tolerance to extreme low humidity, and that this is especially pronounced in males of the species. CONCLUSIONS: Taken together, our results suggest that temperature and humidity conditions are key aspects of the climatic niche of Lutzomyia and Pintomyia sand flies and underscore the value of integrative studies of climatic tolerance and preference in vector biology.

Pathogen-associated molecular patterns (PAMPs) derived from Leishmania and bacteria increase gene expression of antimicrobial peptides and gut surface proteins in sand flies.

The interaction between pathogens and vectors' physiology can impact parasite transmission. Studying this interaction at the molecular level can help in developing control strategies. We study leishmaniases, diseases caused by Leishmania parasites transmitted by sand fly vectors, posing a significant global public health concern. Lipophosphoglycan (LPG), the major surface glycoconjugate of Leishmania, has been described to have several roles throughout the parasite's life cycle, both in the insect and vertebrate hosts. In addition, the sand fly midgut possesses a rich microbiota expressing lipopolysaccharides (LPS). However, the effect of LPG and LPS on the gene expression of sand fly midgut proteins or immunity effectors has not yet been documented. We experimentally fed Lutzomyia longipalpis and Phlebotomus papatasi sand flies with blood containing purified LPG from Leishmania infantum, Leishmania major, or LPS from Escherichia coli. The effect on the expression of genes encoding gut proteins galectin and mucin, digestive enzymes trypsin and chymotrypsin, and antimicrobial peptides (AMPs) attacin and defensins was assessed by quantitative PCR (qPCR). The gene expression of a mucin-like protein in L. longipalpis was increased by L. infantum LPG and E. coli LPS. The gene expression of a galectin was increased in L. longipalpis by L. major LPG, and in P. papatasi by E. coli LPS. Nevertheless, the gene expression of trypsins and chymotrypsins did not significantly change. On the other hand, both L. infantum and L. major LPG significantly enhanced expression of the AMP attacin in both sand fly species and defensin in L. longipalpis. In addition, E. coli LPS increased the expression of attacin and defensin in L. longipalpis. Our study showed that Leishmania LPG and E. coli LPS differentially modulate the expression of sand fly genes involved in gut maintenance and defence. This suggests that the glycoconjugates from microbiota or Leishmania may increase the vector's immune response and the gene expression of a gut coating protein in a permissive vector.

Experimental Susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis (Psychodidae: Phlebotominae) to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi (Trypanosomatidae: Leishmaniinae).

The present work assessed the experimental susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi. A L. (Leishmania) chagasi-Lu. longipalpis combination was used as a susceptible control. Wild-caught Ny. antunesi and laboratory-bred Lu. longipalpis were membrane-fed on blood with a 5 × 106/mL log-phase promastigote culture suspension and dissected on days 2 and 8 post-blood meal (pbm) for analysis focused on the assessment of parasitoses, as well as placement and promastigote morphotyping. Survival curves were constructed. In all combinations, promastigotes were observed on day 8 pbm. For both Leishmania species, in Lu. longipalpis, the presence of parasites was observed up to the stomodeal valve, while in Ny. antunesi, the presence of parasites was observed up to the cardia. There were no significant differences in parasitosis between L. (V.) lainsoni and L. (V.) lindenbergi in either Ny. antunesi or Lu. longipalpis. Six morphological promastigote forms were distinguished in Giemsa-stained gut smears. The survival curves of all combinations decreased and were affected differently by several Lu. longipalpis-parasite combinations, as well with Lu. longipalpis-uninfected blood. These findings stress Lu. longipalpis as experimentally susceptible to Leishmania spp. and suggest the putative susceptibility of Ny. antunesi to L. (V.) lainsoni and L. (V.) lindenbergi.

Canine leishmaniasis in the Americas: etiology, distribution, and clinical and zoonotic importance.

Canine leishmaniasis is a widespread disease on the American continent, with cases reported from Uruguay to the USA and Canada. While numerous Leishmania spp. have been reported in dogs in this region, Leishmania infantum and Leishmania braziliensis are the most common etiological agents of canine leishmaniasis from a continental perspective. Nonetheless, other species may predominate locally in some countries. The participation of dogs in the transmission cycle of various Leishmania spp. has long been speculated, but evidence indicates that their role as reservoirs of species other than L. infantum is negligible. Various native wildlife (e.g., small rodents, marsupials, sloths, and monkeys) are, in fact, the primary hosts of Leishmania spp. in the Americas. In this review, an updated list of Leishmania spp. infecting dogs in the Americas is presented, along with their distribution and clinical and zoonotic importance.

Rhabdoviral Endogenous Sequences Identified in the Leishmaniasis Vector Lutzomyia longipalpis Are Widespread in Sandflies from South America.

Sandflies are known vectors of leishmaniasis. In the Old World, sandflies are also vectors of viruses while little is known about the capacity of New World insects to transmit viruses to humans. Here, we relate the identification of RNA sequences with homology to rhabdovirus nucleocapsids (NcPs) genes, initially in the Lutzomyia longipalpis LL5 cell lineage, named NcP1.1 and NcP2. The Rhabdoviridae family never retrotranscribes its RNA genome to DNA. The sequences here described were identified in cDNA and DNA from LL-5 cells and in adult insects indicating that they are transcribed endogenous viral elements (EVEs). The presence of NcP1.1 and NcP2 in the L. longipalpis genome was confirmed in silico. In addition to showing the genomic location of NcP1.1 and NcP2, we identified another rhabdoviral insertion named NcP1.2. Analysis of small RNA molecules derived from these sequences showed that NcP1.1 and NcP1.2 present a profile consistent with elements targeted by primary piRNAs, while NcP2 was restricted to the degradation profile. The presence of NcP1.1 and NcP2 was investigated in sandfly populations from South America and the Old World. These EVEs are shared by different sandfly populations in South America while none of the Old World species studied presented the insertions.

Age, sex, and mating status discrimination in the sand fly Lutzomyia longipalpis using near infra-red spectroscopy (NIRS).

BACKGROUND: Understanding aspects related to the physiology and capacity of vectors is essential for effectively controlling vector-borne diseases. The sand fly Lutzomyia longipalpis has great importance in medical entomology for disseminating Leishmania parasites, the causative agent of Leishmaniasis, one of the main neglected diseases listed by the World Health Organization (WHO). In this respect, it is necessary to evaluate the transmission potential of this species and the success of vector control interventions. Near-infrared spectroscopy (NIRS) has been used to estimate the age of mosquitoes in different conditions (laboratory, semi-field, and conservation), taxonomic analysis, and infection detection. However, no studies are using NIRS for sand flies. METHODS: In this study, we developed analytic models to estimate the age of L. longipalpis adults under laboratory conditions, identify their copulation state, and evaluate their gonotrophic cycle and diet. RESULTS: Sand flies were classified with an accuracy of 58-82% in 3 age groups and 82-92% when separating them into young (<8 days) or old (>8 days) insects. The classification between mated and non-mated sandflies was 98-100% accurate, while the percentage of hits of females that had already passed the first gonotrophic cycle was only 59%. CONCLUSIONS: We consider the age and copula estimation results very promising, as they provide essential aspects of vector capacity assessment, which can be obtained quickly and at a lower cost with NIRS.

Establishment of a laboratory colony of Pressatia choti (Diptera: Psychodidae), a suspected vector of Leishmania braziliensis

ABSTRACT Background: Pressatia choti is a common sand fly found in the Atlantic Forest of Brazil, which is suspected to be involved in the transmission of Leishmania braziliensis. Herein, we aimed to establish a Pr. choti laboratory colony. Methods: Wild-caught female sand flies were blood fed on hamsters and maintained under controlled conditions (temperature: 26 °C; relative humidity: 70%). Results: Of the 301 collected female sandflies, 288 were identified as Pr. choti. The life cycle duration ranged from 31 to 56 days. Conclusions: We successfully established a Pr. choti colony, whose biological parameters were similar to those of other neotropical sand flies.

Semiochemical delivery systems based on natural polymers to attract sand flies (Diptera: Psychodidae).

BACKGROUND: The successful use of semiochemicals to attract insects to traps is based on research on the most suitable compounds and their release profiles over time. Based on the group's promising results, matrices with a more adequate release profile and more eco-friendly properties for the release of 1-hexanol were developed. To use a more suitable prototype in the field, the most promising systems were added to a capsule and evaluated in a wind tunnel. Behavioral experiments were performed using the sand fly species, Lutzomyia longipalpis, to evaluate the efficacy of the proposed system. METHODS: Different delivery systems were developed by varying the polymer (gellan gum and pectin) ratio, crosslinker (aluminum chloride) concentration, and glutaraldehyde removal.The delivery systems were loaded with 1-hexanol, and their release profiles were evaluated using gravimetric analysis under ambient and high-humidity conditions. When the matrix system was placed inside a plastic container, modulations in the active release profile were observed and the system could be reused. Actid attraction behaviors of the sand fly species, Lu. longipalpis, were evaluated in a wind tunnel when exposed to 1-hexanol-loaded release systems at different times. RESULTS: Among the four formulations evaluated, System 2 (gellan gum and pectin in a 1:1 ratio with 5% aluminum chloride) exhibited the most promising release profile, with greater uniformity and longer compound release time. The maximum 1-hexanol release uniformity was achieved over a longer time, mainly every 24 h, under both ambient and high-humidity conditions. System 2 can be reused at least once with the same structure. The wind tunnel trials exhibited efficient activation and attraction of Lu. longipalpis to 1-hexanol after 24, 48, and 72 h in System 2 placed inside the capsules. CONCLUSIONS: The polymeric matrix supplemented with 1-hexanol and introduced in plastic capsules showed promising results in attracting sand flies. This system can be used as a solution for other attractive compounds as well as in other applications where their release needs to be controlled or prolonged.

Effects of host species on microbiota composition in Phlebotomus and Lutzomyia sand flies.

BACKGROUND: Blood-sucking phlebotomine sand flies are vectors of the protozoan parasites Leishmania spp. Although the intestinal microbiota is involved in a wide range of biological and physiological processes and has the potential to alter vector competence, little is known about the factors that modify the gut microbiota composition of sand flies. As a key step toward addressing this issue, we investigated the impact of host species on the gut bacterial composition in Phlebotomus and Lutzomyia sand flies reared under the same conditions. METHODS: Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing were used to characterize the overall bacterial composition of three laboratory-reared sandflies: Phlebotomus papatasi, Ph. duboscqi, and Lutzomyia longipalpis. RESULTS: Our results showed that the larvae of the three sand fly species harbored almost the same microbes but had different relative abundances. Adult Ph. papatasi and Ph. duboscqi revealed similar microbiome compositions, which were distinct from that of adult Lu. longipalpis. Furthermore, we showed that Ph. papatasi and Ph. duboscqi are hosts for different bacterial genera. The experiment was repeated twice to improve accuracy and increase reliability of the data, and the same results were obtained even when a distinct composition of the microbiome among the same species was identified probably because of the use of different larvae food batch. CONCLUSIONS: The present study provides key insights into the role of host species in the gut microbial content of different sand fly species reared under the same conditions, which may influence their susceptibility to Leishmania infection.

Survey of phlebotomine sand fly fauna in a public Zoo in Brazil: Species diversity, seasonality, and host variety.

Leishmaniasis is a dynamic disease in which transmission conditions change due to environmental and human behavioral factors. Epidemiological analyses have shown modifications in the spread profile and growing urbanization of the disease, justifying the expansion of endemic areas and increasing number of cases in dogs and humans. In the city of Belo Horizonte, located in the southeastern state of Minas Gerais (Brazil), visceral leishmaniasis (VL) is endemic, with a typical urban transmission pattern, but with different regional prevalence. This study was conducted at the Zoo of the Foundation of Municipal Parks and Zoobotany of Belo Horizonte (FPMZB-BH), located in the Pampulha region, which is among the areas most severely affected by VL. This study aimed to determine the taxonomic diversity of native phlebotomine sand flies (Diptera: Psychodidae), identify climatic variables that potentially affect the phenology of these insects, and determine the blood meal sources for female phlebotomine sand flies. To achieve this, 10 mammal enclosures in the zoo were selected using the presence of possible leishmaniasis reservoirs as a selection criterion, and sampled using light traps between August 2019 and August 2021. A total of 6034 phlebotomine sand flies were collected, indicating nine species, with Lutzomyia longipalpis being the very abundant species (65.35% of the total). Of the 108 engorged phlebotomine collected females, seven samples (6.5%) were positive for blood meals from humans, marsupials, canids, and birds. Relative humidity and rainfall increased the phenology of phlebotomine sand flies, with population increases in the hottest and wettest months. The data obtained will provide guidelines for competent health agencies to implement vector control measures to reduce the risk of leishmaniasis transmission in the FPMZB-BH.

The Program for the Control of Visceral Leishmaniasis in Brazil: The Effect of the Systematic Euthanasia of Seropositive Dogs as a Single Control Action in Porteirinha, a Brazilian City with an Intense Transmission of Visceral Leishmaniasis.

BACKGROUND: Porteirinha is endemic for visceral leishmaniasis (VL), with intense disease transmission of the disease. We evaluated the impact of canine euthanasia as a single control measure on the incidence of VL in humans and canines. METHODS: A prospective observational cohort study was carried out over four years (1998-2002) in 8 of the 12 neighborhoods of the city. The dynamics of canine visceral leishmaniasis (CVL) transmission were evaluated for 2 years, before beginning the screening-culling intervention. The comparative morbidity index (CMI) was used to stratify areas with the greatest risk of CVL, and the spatial distribution of human and canine VL cases was compared using univariate and bivariate K-functions. RESULTS: Human cases conglomerated in three neighborhoods. Spatial clusters were detected for CVL in 1998, 2000, and 2001, but not in 1999, when greater spatial dispersion occurred. The screening and culling intervention reduced the number of human VL cases and decreased the incidence of CVL, mainly in neighborhoods with a high CMI. CONCLUSIONS: The systematic euthanasia of seropositive dogs was shown to be an effective control action of the Program for Control of Visceral Leishmaniasis (PCLV) in Brazil. The fundamental role of domestic dogs in the epidemiological chain of VL was reaffirmed.

Molecular detection of Wolbachia and Bartonella as part of the microbiome of phlebotomine sand flies from Chiapas, Mexico.

Phlebotomine sand flies are dipterans of relevance due to their role as vectors of several pathogens worldwide. Bacteria in the gut of sand flies possibly affect their vectorial capacity and competence to transmit parasites. A retrospective study was performed in sand fly specimens that had previously been collected in four localities of the state of Chiapas during the period 2009-2011 to detect Wolbachia and Bartonella and their possible coinfection with Leishmania. For the molecular detection of bacteria, we used primers and conditions that had previously been reported. A total of 531 sand fly specimens of 10 species were analyzed. Four Wolbachia strains were detected in five sand fly species, showing a prevalence of 8.6%. All the Wolbachia strains had previously been reported in other taxa. In one sand fly species, we also detected a new lineage of Bartonella evidenced by a phylogenetic analysis. No sand fly specimens showed coinfections of these bacteria and Leishmania. The bacteria found in the phlebotomine sand flies are possibly transmitted by plant-mediated horizontal transmission and during blood meal feeding.

Experimental Colonization of Sand Flies (Lutzomyia longipalpis; Diptera: Psychodidae) by Bartonella ancashensis.

Background: Bartonella ancashensis is a recently described Bartonella species endemic to Peru, where it causes verruga peruana in humans. While the arthropod vector of B. ancashensis transmission is unknown, human coinfections with Bartonella bacilliformis suggest that phlebotomine sand flies are a vector. Materials and Methods: To address the hypothesis that sand flies are involved in the bacterium's transmission, Lutzomyia longipalpis sand flies were used as an infection model, together with green fluorescent protein-expressing B. ancashensis. Results: Results showed that bacterial infections were clearly established, limited to the anterior midgut of the female fly, and maintained for roughly 7 days. At 3-7 days postinfection, a prominent microcolony of aggregated bacteria was observed in the anterior midgut, immediately distal to the stomodeal valve of the esophagus. In contrast, eggs, diuretic fluid, feces, and other tissues were not infected. Conclusion: These results suggest that certain sand fly species within the endemic zone for B. ancashensis may play a role in the bacterium's maintenance and possibly in its transmission to humans.