Use of species delimitation approaches to tackle the cryptic diversity of an assemblage of high Andean butterflies (Lepidoptera: Papilionoidea).

Cryptic biological diversity has generated ambiguity in taxonomic and evolutionary studies. Single-locus methods and other approaches for species delimitation are useful for addressing this challenge, enabling the practical processing of large numbers of samples for identification and inventory purposes. This study analyzed an assemblage of high Andean butterflies using DNA barcoding and compared the identifications based on the current morphological taxonomy with three methods of species delimitation (automatic barcode gap discovery, generalized mixed Yule coalescent model, and Poisson tree processes). Sixteen potential cryptic species were recognized using these three methods, representing a net richness increase of 11.3% in the assemblage. A well-studied taxon of the genus Vanessa, which has a wide geographical distribution, appeared with the potential cryptic species that had a higher genetic differentiation at the local level than at the continental level. The analyses were useful for identifying the potential cryptic species in Pedaliodes and Forsterinaria complexes, which also show differentiation along altitudinal and latitudinal gradients. This genetic assessment of an entire assemblage of high Andean butterflies (Papilionoidea) provides baseline information for future research in a region characterized by high rates of endemism and population isolation.

In Vitro Assessment of Early Bacterial Activity on Micro/Nanostructured Ti6Al4V Surfaces.

It is imperative to understand and systematically compare the initial interactions between bacteria genre and surface properties. Thus, we fabricated a flat, anodized with 80 nm TiO2 nanotubes (NTs), and a rough Ti6Al4V surface. The materials were characterized using field-emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM). We cultured in vitro Staphylococcus epidermidis (S. epidermidis) and Pseudomonas aeruginosa (P. aeruginosa) to evaluate the bacterial-surface behavior by FE-SEM and viability calculation. In addition, the initial effects of human osteoblasts were tested on the materials. Gram-negative bacteria showed promoted adherence and viability over the flat and rough surface, while NTs displayed opposite activity with altered morphology. Gram-positive bacteria illustrated similar cellular architecture over the surfaces but with promoted surface adhesion bonds on the flat alloy. Rough surfaces supported S. epidermidis viability, whilst NTs exhibited lower vitality. NTs advocated promoted better osteoblast organization with enhanced vitality. Gram-positive bacteria suggested preferred adhesion capability over flat and carbon-rich surfaces. Gram-negative bacteria were strongly disturbed by NTs but largely stimulated by flat and rough materials. Our work proposed that the chemical profile of the material surface and the bacterial cell wall characteristics might play an important role in the bacteria-surface interactions.

Into the Andes: multiple independent colonizations drive montane diversity in the Neotropical clearwing butterflies Godyridina.

Understanding why species richness peaks along the Andes is a fundamental question in the study of Neotropical biodiversity. Several biogeographic and diversification scenarios have been proposed in the literature, but there is confusion about the processes underlying each scenario, and assessing their relative contribution is not straightforward. Here, we propose to refine these scenarios into a framework which evaluates four evolutionary mechanisms: higher speciation rate in the Andes, lower extinction rates in the Andes, older colonization times and higher colonization rates of the Andes from adjacent areas. We apply this framework to a species-rich subtribe of Neotropical butterflies whose diversity peaks in the Andes, the Godyridina (Nymphalidae: Ithomiini). We generated a time-calibrated phylogeny of the Godyridina and fitted time-dependent diversification models. Using trait-dependent diversification models and ancestral state reconstruction methods we then compared different biogeographic scenarios. We found strong evidence that the rates of colonization into the Andes were higher than the other way round. Those colonizations and the subsequent local diversification at equal rates in the Andes and in non-Andean regions mechanically increased the species richness of Andean regions compared to that of non-Andean regions ('species-attractor' hypothesis). We also found support for increasing speciation rates associated with Andean lineages. Our work highlights the importance of the Andean slopes in repeatedly attracting non-Andean lineages, most likely as a result of the diversity of habitats and/or host plants. Applying this analytical framework to other clades will bring important insights into the evolutionary mechanisms underlying the most species-rich biodiversity hotspot on the planet.

Molecular detection of flaviviruses and alphaviruses in mosquitoes (Diptera: Culicidae) from coastal ecosystems in the Colombian Caribbean.

Arboviruses belonging to the genera Flavivirus and Alphavirus were detected in mosquitoes in a rural area of San Bernardo del Viento (Córdoba, Colombia). A total of 22,180 mosquitoes were collected, sorted into 2,102 pools, and tested by generic/nested reverse transcription-polymerase chain reaction. Venezuelan equine encephalitis virus, dengue virus, West Nile virus, St. Louis encephalitis virus, yellow fever virus, and Culex flavivirus were detected and identified by sequencing. The detection of arboviral pathogens in this zone represents possible circulation and indicates a human health risk, demonstrating the importance of virological surveillance activities.

The Sabethines of Northern Andean Coffee-Growing Regions of Colombia.

Sampling for sabethine mosquitoes occurred intermittently from September 2007 to April 2013 in 17 municipalities, located in 5 departments (divisions) in the northern Andean coffee-growing regions of Colombia. Of the 9 genera within the Sabethini tribe known to occur in the Neotropical region, 6 were encountered including 15 species: Jonhbelkinia ulopus, Limatus durhamii, Sabethes ignotus, Sa. luxodens, Sa. undosus, Shannoniana fluviatilis, Trichoprosopon compressum, Tr. digitatum, Tr. evansae, Tr. pallidiventer s.l., Tr. pallidiventer s.s., Wyeomyia arthrostigma, Wy. oblita, Wy. ulocoma, and Wy. undulata. The species Sa. luxodens and Wy. undulata constitute new records for Colombia. These records broaden the knowledge of this important group that includes some important species related to the arbovirus transmission. Records are from the northern Colombian Andes, a region noted for coffee cultivation and ecotourism.

Improving the COI DNA barcoding library for Neotropical phlebotomine sand flies (Diptera: Psychodidae).

Sand fly species are traditionally identified using morphological traits, though this method is hampered by the presence of cryptic species. DNA barcoding is a widely used tool in the case of insects of medical importance, where it is necessary to know quickly which species are present in a transmission area. Here, we assess the usefulness of mitochondrial cytochrome c oxidase subunit I (COI) DNA barcoding as a practical tool for species identification, correct assignment of isomorphic females, and to evaluate the detection of cryptic diversity that occurs in the same species. A fragment of the COI gene was used to generate 156 new barcode sequences for sand flies from different countries of the Neotropical region, mainly Colombia, which had been identified morphologically as 43 species. The sequencing of the COI gene allowed the detection of cryptic diversity within species and correctly associated isomorphic females with males identified by morphology. The maximum intraspecific genetic distances ranged from 0 to 8.32% and 0 to 8.92% using uncorrected p distances and the Kimura 2-parameter (K2P) model, respectively. The minimum interspecific distance (nearest neighbor) for each species ranged from 1.5 to 14.14% and 1.51 to 15.7% using p and K2P distances, respectively. Three species had more than 3% maximum intraspecific distance: Psychodopygus panamensis, Micropygomyia cayennensis cayennensis, and Pintomyia evansi. They also were split into at least two molecular operational taxonomic units (MOTUs) each, using different species delimitation algorithms. Regarding interspecific genetic distances, the species of the genera Nyssomyia and Trichophoromyia generated values lower than 3% (except Nyssomyia ylephiletor and Ny. trapidoi). However, the maximum intraspecific distances did not exceed these values, indicating the presence of a barcode gap despite their proximity. Also, nine sand fly species were DNA barcoded for the first time: Evandromyia georgii, Lutzomyia sherlocki, Ny. ylephiletor, Ny. yuilli pajoti, Psathyromyia punctigeniculata, Sciopemyia preclara, Trichopygomyia triramula, Trichophoromyia howardi, and Th. velezbernali. The COI DNA barcode analysis enabled the correct delimitation of several Neotropical sand fly species from South and Central America and raised questions about the presence of cryptic species for some taxa, which should be further assessed.

Exochorion in the tribe Nymphalini (Lepidoptera: Nymphalidae): the genus Hypanartia Hbner, [1821] and comparison with related genera.

We describe and compare the exochorion of six species of Hypanartia (H. paullus, H. bella, H. lethe, H. godmanii, H. dione disjuncta, and H. trimaculata autumna) using specimens collected in the field and deposited in entomological collections. We used a standard staining technique and scanning electron microscopy to visualize and describe the main exochorionic characters, especially the ridges and the differentiation in the micropylar, perimicropylar, and transition zones in the apical region. We included plates with photographs, drawings, and schemes for clearer visualization of these structures, which are tabulated for comparison. For our characterization, we considered the ridges in colonnade as the main exochorionic feature in the Nymphalini. Thus, we made brief comparisons of these features among several species of the tribe, particularly with the genus Antanartia. The exochorionic characters agree with the separation of groups (paullus group and dione group). By examining the characteristics of H. paullus, the basal species of the paullus group, we found that ridges with conspicuous columns (e.g., in one section only) appears to be the plesiomorphic state, while those with elongated shafts and walls only in the intercolumn represent the apomorphic state.

Atherosclerotic-Derived Endothelial Cell Response Conducted by Titanium Oxide Nanotubes.

Atherosclerosis lesions are described as the formation of an occlusive wall-vessel plaque that can exacerbate infarctions, strokes, and even death. Furthermore, atherosclerosis damages the endothelium integrity, avoiding proper regeneration after stent implantation. Therefore, we investigate the beneficial effects of TiO2 nanotubes (NTs) in promoting the initial response of detrimental human atherosclerotic-derived endothelial cells (AThEC). We synthesized and characterized NTs on Ti6Al4V by anodization. We isolated AThEC and tested the adhesion long-lasting proliferation activity, and the modulation of focal adhesions conducted on the materials. Moreover, ultrastructural cell-surface contact at the nanoscale and membrane roughness were evaluated to explain the results. Our findings depicted improved filopodia and focal adhesions stimulated by the NTs. Similarly, the NTs harbored long-lasting proliferative metabolism after 5 days, explained by overcoming cell-contact interactions at the nanoscale. Furthermore, the senescent activity detected in the AThEC could be mitigated by the modified membrane roughness and cellular stretch orchestrated by the NTs. Importantly, the NTs stimulate the initial endothelial anchorage and metabolic recovery required to regenerate the endothelial monolayer. Despite the dysfunctional status of the AThEC, our study brings new evidence for the potential application of nano-configured biomaterials for innovation in stent technologies.

Reduced dengue incidence following city-wide wMel Wolbachia mosquito releases throughout three Colombian cities: Interrupted time series analysis and a prospective case-control study.

BACKGROUND: The introduction of Wolbachia (wMel strain) into Aedes aegypti mosquitoes reduces their capacity to transmit dengue and other arboviruses. Randomised and non-randomised studies in multiple countries have shown significant reductions in dengue incidence following field releases of wMel-infected Ae. aegypti. We report the public health outcomes from phased, large-scale releases of wMel-Ae. aegypti mosquitoes throughout three contiguous cities in the Aburrá Valley, Colombia. METHODOLOGY/PRINCIPAL FINDINGS: Following pilot releases in 2015-2016, staged city-wide wMel-Ae. aegypti deployments were undertaken in the cities of Bello, Medellín and Itagüí (3.3 million people) between October 2016 and April 2022. The impact of the Wolbachia intervention on dengue incidence was evaluated in two parallel studies. A quasi-experimental study using interrupted time series analysis showed notified dengue case incidence was reduced by 95% in Bello and Medellín and 97% in Itagüí, following establishment of wMel at ≥60% prevalence, compared to the pre-intervention period and after adjusting for seasonal trends. A concurrent clinic-based case-control study with a test-negative design was unable to attain the target sample size of 63 enrolled virologically-confirmed dengue (VCD) cases between May 2019 and December 2021, consistent with low dengue incidence throughout the Aburrá Valley following wMel deployments. Nevertheless, VCD incidence was 45% lower (OR 0.55 [95% CI 0.25, 1.17]) and combined VCD/presumptive dengue incidence was 47% lower (OR 0.53 [95% CI 0.30, 0.93]) among participants resident in wMel-treated versus untreated neighbourhoods. CONCLUSIONS/SIGNIFICANCE: Stable introduction of wMel into local Ae. aegypti populations was associated with a significant and sustained reduction in dengue incidence across three Colombian cities. These results from the largest contiguous Wolbachia releases to-date demonstrate the real-world effectiveness of the method across large urban populations and, alongside previously published results, support the reproducibility of this effectiveness across different ecological settings. TRIAL REGISTRATION: NCT03631719.

Large-scale releases and establishment of wMel Wolbachia in Aedes aegypti mosquitoes throughout the Cities of Bello, Medellín and Itagüí, Colombia.

BACKGROUND: The wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and has been shown to reduce the transmission of dengue and other Aedes-borne viruses. Here we report the entomological results from phased, large-scale releases of Wolbachia infected Ae. aegypti mosquitoes throughout three contiguous cities located in the Aburrá Valley, Colombia. METHODOLOGY/PRINCIPAL FINDINGS: Local wMel Wolbachia-infected Ae. aegypti mosquitoes were generated and then released in an initial release pilot area in 2015-2016, which resulted in the establishment of Wolbachia in the local mosquito populations. Subsequent large-scale releases, mainly involving vehicle-based releases of adult mosquitoes along publicly accessible roads and streets, were undertaken across 29 comunas throughout Bello, Medellín and Itagüí Colombia between 2017-2022. In 9 comunas these were supplemented by egg releases that were undertaken by staff or community members. By the most recent monitoring, Wolbachia was found to be stable and established at consistent levels in local mosquito populations (>60% prevalence) in the majority (67%) of areas. CONCLUSION: These results, from the largest contiguous releases of wMel Wolbachia mosquitoes to date, highlight the operational feasibility of implementing the method in large urban settings. Based on results from previous studies, we expect that Wolbachia establishment will be sustained long term. Ongoing monitoring will confirm Wolbachia persistence in local mosquito populations and track its establishment in the remaining areas.

New records of Sabethini (Diptera: Culicidae) from Colombia.

BACKGROUND: In the Neotropical Region, the mosquitoes, grouped in the tribe Sabethini (Arthropoda, Insecta, Diptera: Culicidae) are considered of medical importance by the role that some species may have in arbovirus transmission; also, because they are good bioindicators. More than 400 species are currently recognised and are mainly associated with forest areas. The tribe Sabethini is poorly studied and the information about diversity and distribution for species relating to it is scarce. In Colombia, 54 species of the tribe are known; however, several geographical areas have not been included in the studies for this group and data for recent field collections are not available; therefore, the records are outdated. NEW INFORMATION: This study presents the species list of the Sabethini tribe in Colombia, based on a review of previous publications and recent unpublished data. The list includes 68 species of nine genera and 16 subgenera. The genus Wyeomyia has the highest species number (39), followed by Sabethes (14). A total of 29 new records are registered and actualized information related to the local distribution in some Departments is presented, including geographic coordinates. In this paper, the distribution records of Sabethini for Colombia are updated, revealing the high diversity of this group in the country and providing some useful information for species that may need surveillance or control.

Recovering Osteoblast Functionality on TiO2 Nanotube Surfaces Under Diabetic Conditions.

Introduction: Titanium (Ti) and its alloys (eg, Ti6Al4V) are exceptional treatments for replacing or repairing bones and damaged surrounding tissues. Although Ti-based implants exhibit excellent osteoconductive performance under healthy conditions, the effectiveness and successful clinical achievements are negatively altered in diabetic patients. Concernedly, diabetes mellitus (DM) contributes to osteoblastic dysfunctionality, altering efficient osseointegration. This work investigates the beneficial osteogenic activity conducted by nanostructured TiO2 under detrimental microenvironment conditions, simulated by human diabetic serum. Methods: We evaluated the bone-forming functional properties of osteoblasts on synthesized TiO2 nanotubes (NTs) by anodization and Ti6Al4V non-modified alloy surfaces under detrimental diabetic conditions. To simulate the detrimental environment, MC3T3E-1 preosteoblasts were cultured under human diabetic serum (DS) of two diagnosed and metabolically controlled patients. Normal human serum (HS) was used to mimic health conditions and fetal bovine serum (FBS) as the control culture environment. We characterized the matrix mineralization under the detrimental conditions on the control alloy and the NTs. Moreover, we applied immunofluorescence of osteoblasts differentiation markers on the NTs to understand the bone-expression stimulated by the biochemical medium conditions. Results: The diabetic conditions depressed the initial osteoblast growth ability, as evidenced by altered early cell adhesion and reduced proliferation. Nonetheless, after three days, the diabetic damage was suppressed by the NTs, enhancing the osteoblast activity. Therefore, the osteogenic markers of bone formation and the differentiation of osteoblasts were reactivated by the nanoconfigured surfaces. Far more importantly, collagen secretion and bone-matrix mineralization were stimulated and conducted to levels similar to those of the control of FBS conditions, in comparison to the control alloy, which was not able to reach similar levels of bone functionality than the NTs. Conclusion: Our study brings knowledge for the potential application of nanostructured biomaterials to work as an integrative platform under the detrimental metabolic status present in diabetic conditions.

DNA barcoding of Lutzomyia longipalpis species complex (Diptera: Psychodidae), suggests the existence of 8 candidate species.

The sand fly Lutzomyia (L.) longipalpis has been implicated as the primary vector of Leishmania infantum, the causative agent of visceral leishmaniasis VL. In addition, it has been associated with atypical cutaneous leishmaniasis transmission in the Neotropic and Central America, respectively. The existence of a L. longipalpis complex species has been suggested with important implications for leishmaniasis epidemiology; however, the delimitation of species conforming it remains a topic of controversy. The DNA Barcoding Initiative based on cox1 sequence variation was used to identify the MOTUs in L. longipalpis including previously described L. pseudolongipalpis. The genetic variation was analyzed based on tree and distance methods. Fifty-five haplotypes were obtained from 103 sequences which were assigned to MOTUs, with a clear separation and a high correspondence of individuals to the groups. Maximum likelihood and Bayesian phylogenetic analysis showed eight MOTUs (100% bootstrap) with high genetic divergence (12.6%). Data obtained in the present study suggest that L. longipalpis complex consists of at least 8 lineages that may represent species. It would be desirable perform additional morphological and molecular analysis of L. longipalpis from Colosó (Caribbean ecoregion) considering that specimens from that area were grouped with L. pseudolongipalpis one of the complex species previously described from Venezuela, which has not been registered in Colombia.

Micropylar and Perimicropylar Regions of the Egg Exochorion in Six Genera of Dismorphiinae (Lepidoptera: Pieridae).

Using scanning electron microscopy (SEM), we examine and compare the micropylar and perimicropylar regions of the egg exochorion in six genera of the subfamily Dismorphiinae (Pieridae): Leptidea Billberg, Enantia Hübner, Pseudopieris Godman Salvin, Lieinix Gray, Dismorphia Hübner, and Moschoneura Butler. We provide the first descriptions of the morphological structures of the chorion in these genera. Leptidea is the most primitive genus, and Dismorphia represents the most diverse. We examined the jethys complex of Enantia to study diversity of characters within the genus. We conclude that the exochorion of Dismorphiinae is the most plesiomorphic in relation to Coliadinae and Pierinae, owing to its simple morphology without a transition zone surrounding the wreath (perimicropylar region). Leptidea (Leptideini) shows the least derived characters of the subfamily, followed by Enantia (Dismorphiini). The latter genus exhibits several typical characters of the tribe that are distinctive from other more specialized genera. Exochorionic similarities among the four species of the jethys complex support their close relationship; E. mazai exhibits the most derived features among the species of the complex. Pseudopieris and Moschoneura exhibit several plesiomorphies, such as a large number of openings (micro-grid), but they also show some derived features, such as three semi-rings in the wreath. Dismorphia and Lieinix exhibit characters in their most derived states, such as a striated micro-grid (most conspicuous in Lieinix) and basal aeropyles (most conspicuous in Dismorphia), a character shared with Moschoneura. The purpose of this work is exploratory and descriptive and would be improved by a larger sample size. This contribution is the third in a series of studies on the chorion of Dismorphiinae eggs.

Toward standards in practices and techniques on ootaxonomy in the Pieridae (Lepidoptera: Papilionoidea).

We present a proposal on the standards used on ootaxonomy practices and techniques in the butterfly family Pieridae Duponchel (Lepidoptera: Papilionoidea) in five stages: 1) getting the specimens, 2) integration into a collection, 3) dissections to recover the exochorion, 4) elaboration of images of it, and 5) the preparation of its description with the necessary diagrams and tabulations. Also, we present the detailed techniques applied in observation and graphic representation, based on the methylene blue staining techniques and those required for the use with the scanning electron microscope (SEM). We compare the result of the standardized techniques with those from macro photography, drawings, and photographs with SEM-all of them found in books with descriptions and images of eggs of the Pieridae. We present a glossary and general aspects of the exochorion in the Pieridae as an Appendix to this article. Standardized techniques show more accurate and extensive character retrieval for systematics. For the scale in which they work, these techniques recovered more information than those present from oviposited eggs, where the exochorionic base is not seen. Also, the descriptions contain detailed data on more structures-which are comparable to each other-than are absent in the references mentioned. We present the recovered characters with the techniques found in the literature as three synthetically supplementary materials.

Description of Lutzomyia velezi, a new species of phlebotomine sand fly (Diptera: Psychodidae) from the Department of Antioquia, Colombia.

The phlebotomine sand fly Lutzomyia velezi sp.nov. was described and illustrated from male specimens collected by light trap in the Reserva Natural Cañon del Río Claro in the Central Cordillera of the Colombian Andes. The new species belongs to the series sanguinaria of the subgenus Helcocyrtomyia, which is represented in Colombia by Lutzomyia cirrita, Lutzomyia hartmanni, Lutzomyia sanguinaria, Lutzomyia scorzai, Lutzomyia sp. of Pichindé and Lutzomyia tortura. The new species can be differentiated from others of the subgenus by the combination of the following characteristics: long antennal ascoids, reaching level of the papilla, coxite with a single basal seta and fifth palpomere longer than or equal to the sum of the lengths of the third and fourth palpomeres.

Pine plantations and five decades of land use change in central Chile.

The expansion of forest plantations is cause for concern because of their environmental effects, and the loss of native forests and agricultural land. Our goal was to quantify the increase in pine plantation, and concomitant loss of native forests, in central Chile since ca. 1960, and to identify in which settings native forests were lost most rapidly. We analyzed aerial photographs from 1955 and 1961, Landsat images from 1975 and 1998, and Google Earth high-resolution satellite images from 2014. To ensure high classification accuracy, we visually interpreted images for a systematic 3-km grid and assigned each point as either 'pine plantation', 'native forest', 'agricultural-livestock lands', or 'other'. We also calculated latitude, longitude, slope, Euclidean distance to the nearest road and to the nearest pulp mill, and the frequency of land use surrounding each point as potential variables to explain observed land use changes. Pine plantations expansion started even before 1960, when 12% of all points were already pine plantations, was particularly rapid from 1975 (18% of sample points) to 1998 (38%), and stabilized thereafter (37% by 2014). From 1975 to 1998 alone, 40% of native forests were replaced by pine plantations, and agricultural-livestock lands declined by 0.7%, 0.9%, 1% per year before 1975, from 1975 to 1998, and after 1998 respectively. Native forests that were surrounded by pine plantations, were most likely to be converted to plantations, and from 1960 to 1975, also native forests near pulp mills. The probability of change from agricultural-livestock lands to pine plantations was mainly influenced by slope, with most agricultural-livestock lands remaining in areas with low slopes.

DNA barcodes, morphology and geographic distribution confirm a new butterfly species in the genus Rhamma (Lepidoptera: Lycaenidae).

We diagnose a new butterfly species from the Belmira paramo in the central cordillera of the Colombian Andes. We infer from the barcoding analysis, wing pattern, morphology and distribution that this entity is not a geographical variation or subspecies of any named lycaenid, and it is described herein as Rhamma eleonorae sp. nov. Adult specimens and female genitalia are illustrated and compared with R. arria (Hewitson, 1870) and R. oxida (Hewitson, 1870), the most closely related taxa based on similarities of wing pattern and COI sequences.

Chorion exploration in the tribe Anthocharidini (Lepidoptera: Pieridae) and their possible importance in its systematics.

We describe and compare the morphology of the chorion in nine species belonging to five genera of the tribe Anthocharidini (Pieridae: Pierinae), from a sample of 12 females with mature eggs, the bibliographic record of oviposited eggs, and photographs of scanning electron microscopy (SEM). The eggs examined come from Mexico, Spain, Brazil and Costa Rica. Its characterization was made considering the main structural features of the chorion in this tribe; it includes a distinction of structures in the apical zone and differentiation between the chorionic regions. We attached to this work sheets, diagrams, and terminology to understand and clarify the descriptions. Our results agree with the proposal of Anthocharidini as the least derived tribe of the Pierinae, considering that Hebomoia, a specialized genus, is not part of it. Tribes such as Leptosiaini, Elodinini or Nepheroniini also have more chorionic characteristics related to more derived tribes of the Pierinae.

Wild specimens of sand fly phlebotomine Lutzomyia evansi, vector of leishmaniasis, show high abundance of Methylobacterium and natural carriage of Wolbachia and Cardinium types in the midgut microbiome.

Phlebotomine sand flies are remarkable vectors of several etiologic agents (virus, bacterial, trypanosomatid Leishmania), posing a heavy health burden for human populations mainly located at developing countries. Their intestinal microbiota is involved in a wide range of biological and physiological processes, and could exclude or facilitate such transmission of pathogens. In this study, we investigated the Eubacterial microbiome from digestive tracts of Lu. evansi adults structure using 16S rRNA gene sequence amplicon high throughput sequencing (Illumina MiSeq) obtained from digestive tracts of Lu. evansi adults. The samples were collected at two locations with high incidence of the disease in humans: peri-urban and forest ecosystems from the department of Sucre, Colombia. 289,068 quality-filtered reads of V4 region of 16S rRNA gene were obtained and clustered into 1,762 operational taxonomic units (OTUs) with 97% similarity. Regarding eubacterial diversity, 14 bacterial phyla and 2 new candidate phyla were found to be consistently associated with the gut microbiome content. Proteobacteria, Firmicutes, and Bacteroidetes were the most abundant phyla in all the samples and the core microbiome was particularly dominated by Methylobacterium genus. Methylobacterium species, are known to have mutualistic relationships with some plants and are involved in shaping the microbial community in the phyllosphere. As a remarkable feature, OTUs classified as Wolbachia spp. were found abundant on peri-urban ecosystem samples, in adult male (OTUs n = 776) and unfed female (OTUs n = 324). Furthermore, our results provide evidence of OTUs classified as Cardinium endosymbiont in relative abundance, notably higher with respect to Wolbachia. The variation in insect gut microbiota may be determined by the environment as also for the type of feeding. Our findings increase the richness of the microbiota associated with Lu. evansi. In this study, OTUs of Methylobacterium found in Lu. evansi was higher in engorged females, suggesting that there are interactions between microbes from plant sources, blood nutrients and the parasites they transmit during the blood intake.