Species diversity, barcode, detection of pathogens and blood meal pattern in Phlebotominae (Diptera: Psychodidae) from northeastern Mexico.

More than 90 species of phlebotomines are vectors of parasites, bacteria, and viruses, which cause disease in animals and humans. Therefore, their study is necessary to establish prevention and control strategies. Mexico is an endemic country for leishmaniasis, mostly in the center and southern regions of the country, yet only few studies have been conducted in the northern part of the country. The present study aims to: (a) assess the alpha diversity of Phlebotominae in an annual cycle, (b) to correlate climatic variables with abundance, (c) to generate barcodes of these insects as part of the integrative taxonomy, and (d) to detect Leishmania, Wolbachia and blood sources in an area close to where a case of autochthonous leishmaniasis has been detected in Nuevo Leon, Mexico. A systematic sampling was conducted during three consecutive nights from 17:00 to 22:00 h., placing Shannon traps, CDC traps with incandescent light, and BG Sentinel 2 + BG Lure traps. A total catch effort of 660 nights/traps/hours was achieved, in which a total number of 707 phlebotomines (58% female and 42% male) of six species were collected and identified. The most abundant species were Psathyromyia cratifer (57%) and Psathyromyia shannoni sensu stricto (26%). The highest abundance (72%; 507/707) was collected during March, April and May 2021. Barcodes were generated for four species of phlebotomines, which represent new records for Mexico. For the molecular detection of microorganisms, 302 specimens were analyzed, although no specimens were positive for Leishmania spp. Wolbachia strains were detected in phlebotomines with an infection rate of 1.32% (4/302) and found in Pa. cratifer and Lu. cruciata. Likewise, human DNA was identified in female Lu. cruciata and Pa. cratifer phlebotomines. These findings indicate the presence of potential vector species of the parasite Leishmania spp. This result shows the need for further entomological surveillance to elucidate the transmission mechanisms in these northern areas of the country.

Species diversity and detection of pathogens in phlebotomine sand flies collected from forest management areas of Quintana Roo, Mexico.

Sand flies have expanded their areas of distribution, thereby increasing the risk of pathogen transmission in non-endemic areas. To establish efficient prevention and control strategies for the transmission of vector-borne pathogens, it is important to understand seasonal dynamics of their vectors. In Mexico, there are several areas where the contact between sand flies, hosts and reservoirs favours the transmission of the pathogen. We compared sand fly communities in a forest management area and a conserved area in Noh-Bec, Quintana Roo, Mexico. The analysis included species diversity, activity peaks and molecular detection of pathogens. Sand flies were collected from November to December 2021 and April to May 2022, during 84 night-traps. The conserved area showed higher numbers and greater species heterogeneity of sand flies as compared with the other sites. The ß-diversity analysis revealed that sites disturbed by logging (S1, S2, S3) had greater similarity (90%) in their sand fly species composition than a conserved area (S4) (similarity = 36%). Although none of the specimens were infected with Leishmania, we detected Wolbachia (19.4%) in all four sites, as well as Bartonella (3.25%) only in the disturbed sites. Further studies on the dynamics of sand fly populations and their association with pathogens are necessary.

Phylogenetic relations among Mexican phlebotomine sand flies (Diptera: Psychodidae) and their divergence time estimation.

Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) have biological relevance as vectors of several pathogens. To ensure periodic entomological monitoring it is necessary to have efficient and accurate tools for an adequate taxonomic identification. There are only few studies on phylogenetic analyses of phlebotomine sand flies from Neotropics, based mostly on morphological and/or molecular data, which makes the delimitation of intra- and interspecific variability of species challenging. Here we generated new molecular information on sand fly species distributed in endemic areas of leishmaniasis in Mexico, using mitochondrial and ribosomal genes, and incorporating morphological information available. Specifically, we established their phylogenetic relationships, and estimated their divergence time. Our study provides molecular information for 15 phlebotomine sand fly species from different areas of Mexico, contributing to the genetic inventory and phylogenetic relations among Neotropical species of the subfamily Phlebotominae. Mitochondrial genes proved to be suitable markers for the molecular identification of phlebotomine sand flies. However, the incorporation of additional nuclear gene information could increase the significance of phylogenetic inferences. We also provided evidence about a possible divergence time of phlebotomine sand fly species, supporting their presumable origin in the Cretaceous period.

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.

The Low Variability of Tc24 in Trypanosoma cruzi TcI as an Advantage for Chagas Disease Prophylaxis and Diagnosis in Mexico.

(1) Background: Chagas disease is the main neglected tropical disease in America. It is estimated that around 6 million people are currently infected with the parasite in Latin America, and 25 million live in endemic areas with active transmission. The disease causes an estimated economic loss of USD 24 billion dollars annually, with a loss of 75,200 working years per year of life; it is responsible for around ~12,000 deaths annually. Although Mexico is an endemic country that recorded 10,186 new cases of Chagas disease during the period of 1990-2017, few studies have evaluated the genetic diversity of genes that could be involved in the prophylaxis and/or diagnosis of the parasite. One of the possible candidates proposed as a vaccine target is the 24 kDa trypomastigote excretory-secretory protein, Tc24, whose protection is linked to the stimulation of T. cruzi-specific CD8+ immune responses. (2) Methods: The aim of the present study was to evaluate the fine-scale genetic diversity and structure of Tc24 in T. cruzi isolates from Mexico, and to compare them with other populations reported in the Americas with the aim to reconsider the potential role of Tc24 as a key candidate for the prophylaxis and improvement of the diagnosis of Chagas disease in Mexico. (3) Results: Of the 25 Mexican isolates analysed, 48% (12) were recovered from humans and 24% (6) recovered from Triatoma barberi and Triatoma dimidiata. Phylogenetic inferences revealed a polytomy in the T. cruzi clade with two defined subgroups, one formed by all sequences of the DTU I and the other formed by DTU II-VI; both subgroups had high branch support. Genetic population analysis detected a single (monomorphic) haplotype of TcI throughout the entire distribution across both Mexico and South America. This information was supported by Nei's pairwise distances, where the sequences of TcI showed no genetic differences. (4) Conclusions: Given that both previous studies and the findings of the present work confirmed that TcI is the only genotype detected from human isolates obtained from various states of Mexico, and that there is no significant genetic variability in any of them, it is possible to propose the development of in silico strategies for the production of antigens that optimise the diagnosis of Chagas disease, such as quantitative ELISA methods that use this region of Tc24.

Is Psathyromyia shannoni (Diptera: Psychodidae: Phlebotominae) a species complex? Retrospective study of genetic diversity of COI gene, pathogens and geographic distribution.

The sand fly Psathyromyia shannoni is a broadly distributed species that is relevant for the transmission of pathogens such as Leishmania, Bartonella and viruses in several countries of America. This species belongs to the Shannoni complex. Yet its identification is difficult due to morphologic intraspecific polymorphisms that make it difficult to distinguish between species, and could therefore lead to misidentification and overestimation of its distribution. The aim of this study was to perform a retrospective study on the genetic diversity of Pa. shannoni based on the Cytochrome Oxidase subunit 1 gene and considering its geographic distribution to achieve a better identification and differentiation from other species of the Shannoni complex. According to the Maximum Likelihood analysis and the data on the genetic structure, we propose a modified delimitation of Pa. shannoni species by classifying it into at least three genetic lineages, based on genetic variability and distribution. However, more genetic information on the COI gene, mainly from countries where this species has been reported, is needed to strengthen this proposal.

Detection of Wolbachia and Leishmania DNA in sand flies (Diptera: Psychodidae, Phlebotominae) from a focus of cutaneous leishmaniasis in Tabasco, Mexico.

Phlebotomine sand flies are the main vectors of Leishmania genus species worldwide; therefore, the detection of some reproductive parasites, such as Wolbachia, has been considered a possible strategy for biological control. In Mexico, leishmaniasis cases have been recorded in 25 states, yet only two sand fly species have been related to Wolbachia spp. Although the state of Tabasco has a high number of leishmaniasis cases, only few studies have been done on sand fly species. The aim of this study was to analyze the diversity of sand fly species and to detect Wolbachia spp. and/or Leishmania spp. in the captured specimens. Sand flies were collected at the locality of Huimango, Tabasco, Mexico, during October 2019, using nine light traps (CDC) and two Shannon traps per night. The specimens were identified and females were analyzed by PCR for the DNA detection for pathogens. A total of 193 sand fly specimens belonging to five species were morphologically identified. Pintomyia ovallesi was the most abundant species (76.84%), followed by Micropygomyia cayennensis (6.40%). Furthermore, first records of four sand fly species were established for the state of Tabasco, thereby increasing the species richness in the state from four to eight. We observed a natural infection rate of 9.7% (10/103) for Leishmania and 0.91% (1/103) for Wolbachia. The importance of conducting entomological surveys in endemic areas of leishmaniasis in Mexico is highlighted, to determine whether other sand fly species may be potential vectors of Leishmania spp., and if some Wolbachia strains could be relevant for the control of leishmaniasis.

Molecular detection of Rickettsia sp. cf. Rickettsia monacensis in Ixodes sp. cf. Ixodes affinis collected from white-tailed deer in Campeche, Mexico.

Deer encompass a group of large-sized vertebrates that serve as hosts for a wide variety of ectoparasites, mainly ticks. In Mexico, ticks have relevance as vectors of pathogenic microorganisms, and 20 species of hard ticks are associated with four species of deer, although only a single study has been conducted to detect bacterial agents associated with ticks from deer in the country. In February, 2019 three white-tailed deers (Odocoileus virginianus) were hunted from the locality of Chiná from the municipality of Campeche, Mexico. The sampled deers were parasitized by 26 ticks belonged to three species: Amblyomma mixtum (5♀, 1♂), Amblyomma ovale (2♀, 1♂), and Ixodes sp. cf. Ixodes affinis (15♀, 2♂). Specimens were screened individually for Anaplasma, Borrelia, Ehrlichia, and Rickettsia DNA by the amplification of several fragments of 16S rRNA, gltA, 17-kDa, and flaB genes. This study report for the first time the presence of Rickettsia sp. cf. Rickettsia monacensis in Ixodes sp. cf. Ixodes affinis in Mexico.

The genus Rickettsia in Mexico: Current knowledge and perspectives.

The genus Rickettsia encompasses 35 valid species of intracellular, coccobacilli bacteria that can infect several eukaryotic taxa, causing multiple emerging and re-emerging diseases worldwide. This work aimed to gather and summarise the current knowledge about the genus Rickettsia in Mexico, updating the taxonomy of the bacteria and their hosts by including all the records available until 2020, to elucidate host-parasite relationships and determine the geographical distribution of each Rickettsia species present in the country. Until now, 14 species of Rickettsia belonging to four groups have been recorded in Mexico. These species have been associated with 26 arthropod species (14 hard ticks, three soft ticks, two sucking lice, and seven fleas) and 17 mammal species distributed over 30 states in Mexico. This work highlights the high biological inventory of rickettsias for Mexico and reinforces the need to approach the study of this group from a One Health perspective.

Rickettsial agents detected in the genus Psathyromyia (Diptera:Phlebotominae) from a Biosphere Reserve of Veracruz, Mexico.

Phlebotomine sand flies are considered the main vectors of Leishmania, the causal agents of leishmaniasis, which is a serious emerging public health problem worldwide. The use of biological control alternatives, like endosymbiotic bacteria (Wolbachia and Rickettsia), have been proposed to decrease sand fly populations and reduce Leishmania transmissions, yet only few records on the detection of Wolbachia or Rickettsia in sand flies are available worldwide. The aim of this study was to perform the molecular detection of Rickettsial agents associated with sand flies from the last patch of a rainforest in south-eastern Mexico, where a high prevalence of Leishmania infantum has been reported. Sampling effort of sand flies covered 300 trap-nights between 2011 and 2013, and a total of 925 specimens from twelve species were morphologically identified. Using PCR techniques, we identified a new lineage of the endosymbionts Rickettsia in Psathyromyia aclydifera (prevalence of 19.54%), and Wolbachia in Psathyromyia shannoni and Lutzomyia sp. (prevalence of 25%). The detected Wolbachia lineage was similar to the wWhi strain found in Pa. shannoni from Colombia and Nyssomyia whitmani from Brazil; whereas the identified Rickettsia represents a new lineage worldwide. This is the first record of Rickettsial agents associated to sand flies from this region, yet it remains for analysed if these bacteria possibly play a role as vector control agents, capable of reducing the sand fly populations in Mexico.

Species diversity and blood meal sources of phlebotomine sand flies (Diptera: Psychodidae) from Los Tuxtlas, Veracruz, Mexico.

Phlebotomine sand flies can transmit several species of the genus Leishmania, that cause leishmaniasis, a serious neglected tropical disease worldwide. Although Mexico is an endemic country for the disease, studies on the biology, ecology, and the identification of blood meal sources of phlebotomine sand flies in some states remain unexplored. For this reason, this study aimed to evaluate the species diversity of sand flies, and identify their blood meal sources in the Biosphere Reserve Los Tuxtlas, Veracruz, an area with a high prevalence of Leishmania infantum. The cumulative sampling effort of sand flies covered 300 trap-nights between March 2011 and May 2013. For estimating species diversity, we calculated the species richness (q = 0), the diversity of the species (q = 1) and the dominant species (q = 2). To identify the blood meal sources, we amplified and sequenced a fragment of ≈400 bp of the vertebrate Cytb gene. A total of 951 specimens belonging to 15 species were collected. Psathyromyia aclydifera and Psychodopygus panamensis were the most abundant species. We were able to identify seven terrestrial vertebrate species, among which human beings were the most common source of the blood meal. In this study, relevant information on the structure of sand fly populations and their blood meal sources was obtained, providing basic and important information about the interactions between sand flies, hosts and Leishmania species.

Genetic diversity of Borrelia burgdorferi sensu stricto: Novel strains from Mexican wild rodents.

Borrelia burgdorferi s.s. is a Gram-negative spirochaete, the aetiological agent of Lyme disease, the most common vector-borne disease in the Northern hemisphere. Reports on the presence of B. burgdorferi in central Mexico have been strongly criticized, since these were based only on unspecific serological methods. Furthermore, the worldwide genetic diversity of B. burgdorferi s.s. has not been evaluated. For this reason, the aim of the present study was to confirm the presence of B. burgdorferi in the central area of Mexico and to evaluate its relationship with regard to the global genetic diversity of B. burgdorferi s.s. To achieve this, fragments of the flagellin and the outer surface protein A genes were amplified from ear biopsies of the arboreal wild endemic mice Habromys schmidlyi. With these sequences, a concatenated Bayesian analysis was performed to confirm the identity of B. burgdorferi s.s. Afterwards, the global genetic diversity of this bacterial species was evaluated using our sequences and those available in GenBank. A prevalence of 10.4% (5/48) of H. schmidlyi infected with Borrelia sp. was detected, and the phylogenetic analyses confirmed the identity of B. burgdorferi s.s. Using both genes, the genetic diversity was low. However, genetic structuring analyses revealed that populations of western United States and those from Mexico formed slightly different genetic groups, separated from the populations of the rest of the world. Our study not only confirms the presence of this bacterium in central Mexico, but also shows the most southern record of this bacterium so far. It also highlights the importance of H. schmidlyi as a new potential host of this bacterial species. Our study also provides first genetic data on an incipient process of divergence in B. burgdorferi s.s. populations of eastern United States and central Mexico.

Molecular identification of Dermatobia hominis (Diptera: Oestridae): a neglected agent causing myiasis in Mexico.

Myiasis represents a group of neglected tropical diseases caused by the infestation of vertebrate tissues by dipterous larvae. We herein report an imported case of foruncular myasis caused by Dermatobia hominis in Mexico City. The species was confirmed by DNA sequencing and phylogenetic reconstruction analysis.

Molecular detection of Leishmania infantum in sand flies (Diptera: Psychodidae: Phlebotominae) from Veracruz, Mexico.

Phlebotomine sand flies are vectors of Leishmania parasites causing different clinical forms of leishmaniases that represent a serious emerging public health problem worldwide. Mexico harbours a large diversity of sand flies, yet only six species have been considered suspected vectors of Leishmania. The disease has been recorded in 25 states, where the State of Veracruz has recorded the highest number of cases with leishmaniases, although no systematic or epidemiological surveillance studies of Leishmania vectors have been carried out in the region. For that reason, the aim of this study was to perform the molecular detection of Leishmania DNA in phlebotomine sand flies collected from a humid tropical region in Veracruz. We confirmed the presence of Leishmania DNA in eight sand fly species. Sand flies with the highest infection were Psathyromyia aclydifera and Pychodopygus panamensis. This is also the first report of Leishmania DNA in Psathyromyia aclydifera, Psathyromyia carpenteri, Dampfomyia beltrani and Brumptomyia mesai. Our findings highlight the importance of entomological surveys and epidemiological studies, since they enable to determine whether sand fly species may be potential Leishmania vectors in a given area of the Mexico, as transmission dynamics can vary in the different regions.

DNA barcoding and fauna of phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) from Los Tuxtlas, Veracruz, Mexico.

Mexico has great diversity of phlebotomine sand flies related to cases of leishmaniasis, yet few studies have dressed the molecular taxonomy of these sand fly species. The use of the cytochrome oxidase subunit 1 (COI) gene, as a DNA Barcode has facilitated the molecular identification of sand flies species worldwide. We use the DNA barcode as a useful tool for the identification of phlebotomine sand flies of the natural reserve Los Tuxtlas from Veracruz, México. A fragment of 536 bp of the COI gene was obtained from 36 individuals belonging to eight species of five genera (Dampfomyia, Lutzomyia, Psathyromyia, Psychodopygus and Brumptomyia) with coverage between 92-100%, and found similarities ranging from 93-98% with other New World phlebotomine sand flies. The NJ dendogram grouped sand flies into eight clusters according to identified species, supported by bootstrap of 97%-100%. In conclusion, all phlebotomine sand flies were correctly identified and agree with the morphological identification, also could separate genetics the isomorphic females of the genus Brumptomyia.

First Report of Bartonella SP. In Sand Flies (Diptera: Psychodidae: Phlebotominae) From Southern Mexico.

The genus Bartonella encompasses several zoonotic species that cause emerging infectious diseases in humans. These species are transmitted to humans by hematophagous arthropods. In South America, sand flies are the most important vectors of Bartonella bacilliformis, the etiological agent of Verruga peruana. Due to the importance of bartonellosis in public health, the aim of this study was to detect Bartonella in wild populations of phlebotomine sand flies in southern Mexico. In total, 29 sand flies belonging to 6 species were collected. Only 2 specimens were positive, representing a prevalence of 8.69% (2/23). This appears to be the first report of Bartonella sp. in phlebotomine sand flies outside the endemic area of Verruga peruana in South America. Further studies are needed to clarify the relation between this bacterium and sand flies in Mexico.