On abnormal Phlebotomus perniciosus (Diptera: Psychodidae: Phlebotominae) from the center of Tunisia.

Phlebotomus perniciosus is the most important vector of Leishmania infantum in the Western part of the Mediterranean basin. Atypical specimens of Ph. perniciosus called (pna) with a parameral sheath simply curved, not bifurcated, have been reported in many locations. In this study, we describe abnormal Ph. perniciosus male specimens. Sand flies were collected in center Tunisia and identified morphologically. Cytochrome b PCR-sequencing was carried out for abnormal Ph. perniciosus male specimens in order to confirm the morphological identification and assess the intraspecific genetic polymorphism. Abnormal Ph. perniciosus specimens were characterized by a multifurcated parameral sheath. A parsimonious haplotype network based on cyt b locus analysis showed that typical and abnormal Ph. perniciosus described in our investigation were grouped together in the same branch. Thus, genetic outcomes confirmed that the new phenotype is only an original morphotype of Ph. perniciosus.

Phlebotomus neglectus (Diptera: Psychodidae): New Insights on Its Presence in Iran Based on Three Independent Genetic Loci.

The idea of the existence of Phlebotomus (Larroussius) neglectus (Diptera: Psychodidae) Tonnoir, 1921 in Iran and the skepticism about the existence of Phlebotomus major s.str. Annandale, 1910 had been grown recently in the country. This study reports a combined analysis of mitochondrial and ribosomal DNA target regions of P. major s.l.Annandale, 1910, specimens collected from different parts of Iran. Two different morphotypes were found among the collected samples based on the shape of the aedeagus, ventrally located hairs of the coxite, and parameral sheets. One morphotype seemed similar to P. neglectus Tonnoir 1921 or P. major krimensis Perfiliv1966 (called here MI.N.K.); the other one was similar to P. neglectus and to some extent to P. notus Artemiev & Neronov 1984 (here called MII.N.NO). Cytochrome B, elongation factor 1-alpha, and internal transcribed spacer II loci were amplified, sequenced, and characterized. High sequence homology (98-100%) was observed between P. neglectus and these morphotypes, and phylogenetic analysis was also concordant. Phlebotomus neglectus sequences available in GenBank are located as the sister group of sequences here, particularly near to morphotype MII.N.NO. Moreover, ITS2 locus provides the maximum resolution for differentiation of two morphotypes. Based on achieving results, although a strong support for the presence of P. neglectus was provided, but it is too early to say that P. major s.str. does/does not exist in Iran. This question could be resolved by studying more samples and, most importantly, by comparing the topotypes of P. neglectus and P. major s. str. if possible in the future.

Sand fly fauna of South-Eastern Romania, with the description of Phlebotomus (Transphlebotomus) simonahalepae n. sp. (Diptera: Psychodidae).

BACKGROUND: An entomological study was conducted in the Canaraua Fetii Special Protection Area in the Dobrogea region, South-Eastern Romania. Four sand fly species were recorded at this location between 1968 and 1970: Phlebotomus neglectus, Ph. balcanicus, Ph. sergenti and Sergentomyia minuta. The most abundant sand fly species recorded at that time were Ph. balcanicus and Se. minuta. In the context of a countrywide study to update the sand fly species diversity, we surveyed the same area, recording also a previously unknown Ph. (Transphlebotomus) sp., for which we provide a formal description here. METHODS: Sand flies were collected between July and August in 2018 and 2019 in three sites from Canaraua Fetii, Dobrogea region, Romania. The general aspect of the landscape is of a canyon (vertical, narrow walls and deep valleys). Species identification was done using both morphological and molecular analyses. RESULTS: Out of 645 collected sand flies, 644 (99.8%) were morphologically identified as Ph. neglectus, while one female specimen (0.2%) was assigned to a previously unknown species, belonging to the subgenus Transphlebotomus. The morphological and molecular examination of this specimen showed that it is a previously unknown species which we formally describe here as Phlebotomus (Transphlebotomus) simonahalepae n. sp. Also, Ph. balcanicus, Ph. sergenti, and Se. minuta (previously recorded in this location) were not present. CONCLUSIONS: The study revealed for the first time the presence of sand flies of the subgenus Transphlebotomus in Romania. Moreover, a new species, Ph. simonahalepae n. sp., was described based on a female specimen, raising the number of species in this subgenus to six. In the investigated natural habitat, the predominant species was Ph. neglectus instead of Ph. balcanicus and Se. minuta (recorded as the predominant species in 1968-1970).

Evolution, systematics and historical biogeography of sand flies of the subgenus Paraphlebotomus (Diptera, Psychodidae, Phlebotomus) inferred using restriction-site associated DNA markers.

Phlebotomine sand flies are the main natural vectors of Leishmania, which cause visceral and tegumentary tropical diseases worldwide. However, their taxonomy and evolutionary history remain poorly studied. Indeed, as for many human disease vectors, their small size is a challenge for morphological and molecular works. Here, we successfully amplified unbiased copies of whole genome to sequence thousands of restriction-site associated DNA (RAD) markers from single specimens of phlebotomines. RAD markers were used to infer a fully resolved phylogeny of the subgenus Paraphlebotomus (11 species + 5 outgroups, 32 specimens). The subgenus was not recovered as monophyletic and we describe a new subgenus Artemievus subg. nov. Depaquit for Phlebotomus alexandri. We also confirm the validity of Ph. riouxi which is reinstated as valid species. Our analyses suggest that Paraphlebotomus sensu nov. originated ca 12.9-8.5 Ma and was possibly largely distributed from peri-Mediterranean to Irano-Turanian regions. Its biogeographical history can be summarized into three phases: i) a first split between Ph. riouxi + Ph. chabaudi and other species that may have resulted from the rise of the Saharan belt ca 8.5 Ma; ii) a Messinian vicariant event (7.3-5.3 Ma) during which the prolonged drought could have resulted in the divergence of main lineages; iii) a recent radiation event (3-2 Ma) that correspond to cycles of wet and dry periods in the Middle East and the East African subregions during the Pleistocene. Interestingly these cycles are also hypothetical drivers of the diversification of rodents, in the burrows of which Paraphlebotomus larvae develop. By meeting the challenge of sequencing pangenomics markers from single, minute phlebotomines, this work opens new avenues for improving our understanding of the epidemiology of leishmaniases and possibly other human diseases transmitted by arthropod vectors.

First Findings and Molecular Data of Phlebotomus mascittii (Diptera: Psychodidae) in the Cantabrian Cornice (Northern Spain).

Phlebotomus (Transphlebotomus) mascittii Grassi, 1908 (Diptera: Psychodidae) has been found in several European countries. In Spain, sporadic records were reported in the early '80s in Catalonia (Northeast Spain), and it was never detected again. Recent entomological surveys carried out between 2004 and 2020 revealed the presence of several specimens of P. mascittii in Spain. The species identification was confirmed by both morphological and molecular analyses. The analyzed specimens belonged to the haplotype (COI_2) defined by one polymorphic site compared to other European specimens. Phlebotomus mascittii was found in low population densities in rural areas associated with livestock farms and in an urban cemetery during the summer season. This study provides the first records of this species in various localities along the Cantabrian cornice (Northern Spain) and represents its westernmost observation in the Palearctic region. The implications of the finding of this uncommon species are discussed at different levels, with emphasis on its suspected role in the transmission of leishmaniosis.

Occurrence of Phlebotomine sand flies (Diptera: Psychodidae) in the northeastern plain of Italy.

BACKGROUND: Recent climate and environmental changes have resulted in the geographical expansion of Mediterranean Leishmania infantum vectors towards northern latitudes and higher altitudes in different European countries, including Italy, where new foci of canine leishmaniasis have been observed in the northern part of the country. Northern Italy is also an endemic area for mosquito-borne diseases. During entomological surveillance for West Nile virus, mosquitoes and other hematophagous insects were collected, including Phlebotomine sand flies. In this study, we report the results of Phlebotomine sand fly identification during the entomological surveillance conducted from 2017 to 2019. METHODS: The northeastern plain of Italy was divided by a grid with a length of 15 km, and a CO2-CDC trap was placed in each geographical unit. The traps were placed ~ 15 km apart. For each sampling site, geographical coordinates were recorded. The traps were operated every two weeks, from May to November. Sand flies collected by CO2-CDC traps were identified by morphological and molecular analysis. RESULTS: From 2017 to 2019, a total of 303 sand flies belonging to the species Phlebotomus perniciosus (n = 273), Sergentomyia minuta (n = 5), P. mascittii (n = 2) and P. perfiliewi (n = 2) were collected, along with 21 unidentified specimens. The trend for P. perniciosus collected during the entomological surveillance showed two peaks, one in July and a smaller one in September. Sand flies were collected at different altitudes, from -2 m above sea level (a.s.l.) to 145 m a.s.l. No correlation was observed between altitude and sand fly abundance. CONCLUSIONS: Four Phlebotomine sand fly species are reported for the first time from the northeastern plain of Italy. Except for S. minuta, the sand fly species are competent vectors of Leishmania parasites and other arboviruses in the Mediterranean Basin. These findings demonstrate the ability of sand flies to colonize new environments previously considered unsuitable for these insects. Even though the density of the Phlebotomine sand fly population in the plain areas is consistently lower than that observed in hilly and low mountainous areas, the presence of these vectors could herald the onset of epidemic outbreaks of leishmaniasis and other arthropod-borne diseases in areas previously considered non-endemic.

TRsandflies: A Web-Based Software for the Morphometric Identification of Sand Flies in Turkey.

Sand flies are vector of several diseases, mostly cutaneous and visceral leishmaniasis (CL and VL). Also, 29 sand fly species have been identified in previous fauna studies carried out in 40 provinces of Turkey. Totally, 24 sand flies species belonging to Phlebotomus (Ph.) (Diptera: Psychodidae) genus have been proven or reported as possible vector species. This study aimed to develop a new software which could contribute to researchers' decision making about the identification of sand flies with obtained data from entomological surveys conducted before in Turkey. Developed software called TRsandflies included 35 textbox created with parameters obtained from caught sand flies specimens by the above-mentioned surveys. It also contained 130 photos and distribution maps related to 24 sand flies species. In addition, C# language and MYSQL database were used in the program. TRsandflies had three different forms (pages) allowing the user to compare the specimens and known species. In the species identification trials with three repetitions carried out in the program, except for the specimens belonging to the Transphlebotomus Artemiev & Neronov, 1984 subgenus, morphometric data of all previously collected sand fly species specimens were included. The process of running the morphometric measurement results of predetermined specimens in the program provided us with an accurate prediction rate of 86.66% in male specimens and 71.66% in female specimens. We concluded that the web-based software developed could play an important role in reducing the rate of possible errors that might be encountered by conventional identification methods.

Sand fly fauna of Crete and the description of Phlebotomus (Adlerius) creticus n. sp. (Diptera: Psychodidae).

BACKGROUND: The Greek island of Crete is endemic for both visceral leishmaniasis (VL) and recently increasing cutaneous leishmaniasis (CL). This study summarizes published data on the sand fly fauna of Crete, the results of new sand fly samplings and the description of a new sand fly species. METHODS: All published and recent samplings were carried out using CDC light traps, sticky traps or mouth aspirators. The specific status of Phlebotomus (Adlerius) creticus n. sp., was assessed by morphological analysis, cytochrome b (cytb) sequencing and MALDI-TOF protein profiling. RESULTS: Published data revealed the presence of 10 Phlebotomus spp. and 2 Sergentomyia spp. During presented field work, 608 specimens of 8 species of Phlebotomus and one species of Sergentomyia were collected. Both published data and present samplings revealed that the two most common and abundant species were Phlebotomus neglectus, a proven vector of Leishmania infantum causing VL, and Ph. similis, a suspected vector of L. tropica causing CL. In addition, the field surveys revealed the presence of a new species, Ph. (Adlerius) creticus n. sp. CONCLUSIONS: The identification of the newly described species is based on both molecular and morphological criteria, showing distinct characters of the male genitalia that differentiate it from related species of the subgenus Adlerius as well as species-specific sequence of cytb and protein spectra generated by MALDI-TOF mass spectrometry.

Morphological identification keys for adults of sand flies (Diptera: Psychodidae) in Sri Lanka.

BACKGROUND: Phlebotomine sand flies are a medically important group of insects that is responsible for the transmission of leishmaniasis. Surveillance plays a major role in vector control programmes through exploring species abundance, potential entomological risk and designing appropriate control measures. In field surveillance programmes of such nature, morphological identification of vector species is of paramount importance. However, in Sri Lanka, there is no published taxonomic key available for the identification of leishmaniasis vectors. METHOD: Identification keys for both male and females of the sand flies recorded in Sri Lanka were developed using morphological features. Main identification features were compared with the original observation of specimens collected from surveys and the use of published literature. Photographic illustrations of morphological features are included with the intention of making the keys user-friendly for non-taxonomists. RESULTS: A total of 22 sand fly species (Diptera: Psychodidae) of the genera Phlebotomus and Sergentomyia reported in Sri Lanka from 1910 to 2019 are included in the present work. CONCLUSION: This simplified key, along with photographs taken from specimens would be beneficial to the health staff, entomologists and research staff who deal with leishmaniasis control programmes and vector-related studies.

Conserved and distinct morphological aspects of the salivary glands of sand fly vectors of leishmaniasis: an anatomical and ultrastructural study.

BACKGROUND: Sand flies are vectors of Leishmania spp., the causative agents of leishmaniasis in vertebrates, including man. The sand fly saliva contains powerful pharmacologically active substances that prevent hemostasis and enhance Leishmania spp. infections. On the other hand, salivary proteins can protect vaccinated mice challenged with parasites. Therefore, sand fly salivary proteins are relevant for the epidemiology of leishmaniasis and can be a potential target for a vaccine against leishmaniasis. Despite this, studies on sand fly salivary glands (SGs) are limited. METHODS: The present study analyzes, in detail, the morphology, anatomy and ultrastructure of the SGs of sand fly vectors of the genera Lutzomyia and Phlebotomus. We used histology, transmission and scanning electron microscopy and lectin labeling associated with confocal laser microscopy. RESULTS: The SGs have conserved and distinct morphological aspects according to the distinct sand fly species. Each SG has a single rounded lobe constituting of c.100-120 secretory cells. The SG secretory cells, according to their ultrastructure and lectin binding, were classified into five different subpopulations, which may differ in secretory pathways. CONCLUSIONS: To the best of our knowledge, these morphological details of sand fly salivary glands are described for the first time. Further studies are necessary to better understand the role of these different cell types and better relate them with the production and secretion of the saliva substances, which has a fundamental role in the interaction of the sand fly vectors with Leishmania.

Two new phlebotomine sandfly species (Diptera: Psychodidae) from the Highlands of Madagascar.

The Malagasy phlebotomine sandfly fauna includes 17 species: five belong to the genus Phlebotomus, ten to the genus Sergentomyia, and two to the genus Grassomyia. The current article describes Sergentomyia brunhesi sp. nov. and Sergentomyia vistellei sp. nov. from the Malagasy Highlands. Females were described morphologically from specimens collected at Ranomafana-Ifanadiana and Andringitra. Partial molecular sequences of cyt b and 28S rDNA were retrieved for Se. vistellei sp. nov. Waiting new data, we consider these species as ungrouped Sergentomyia. Two new species of Sergentomyia are recorded at higher altitudes in Madagascar. Knowledge of local Biodiversity is increasing. New investigations have to be carried out to describe males, to understand their bionomics, and to identify other potential new species at higher altitudes.

Diel periodicity and visual cues guide oviposition behavior in Phlebotomus papatasi, vector of old-world cutaneous leishmaniasis.

BACKGROUND: Phlebotomine sand flies are vectors of human leishmaniases, important neglected tropical diseases. In this study, we investigated diel patterns of oviposition behavior, effects of visual cues on oviposition-site selection, and whether these affect the attraction of gravid Phlebotomus papatasi (Scopoli), the vector of old-world cutaneous leishmaniasis, to olfactory cues from oviposition sites. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate these questions, we conducted a series of experiments using attraction and oviposition assays within free-flight test chambers containing gravid females entrained under a 14:10 hrs light:dark photoperiod. By replacing sticky-screens or moist filter papers every three hours, we showed that oviposition site search occurs mainly in the latest part of the night whereas peak oviposition occurs during the early part of the night. Behavioral responses to olfactory oviposition cues are regulated by time-of-day and can be disrupted by transient exposure to a constant darkness photoperiod. Gravid females, but not any other stage, age, or sex, were attracted to dark, round oviposition jars, possibly resembling rodent burrow openings. This visual attraction disappeared in the absence of an illumination source. Egg deposition rate was not affected by jar color. Olfactory cues had the strongest effect when the visual cues were minimal. CONCLUSION AND SIGNIFICANCE: Our study showed, for the first time, that visual cues in the form of oviposition-site color, lighting level, and photoperiod are important in guiding the oviposition behavior of phlebotomine sand flies. Furthermore, such visual cues could modify the flies' sensitivity to olfactory oviposition cues. Our results suggest that chemosensory and visual cues are complementary, with visual cues used to orient gravid females towards oviposition sites, possibly at long- to medium-ranges during crepuscular periods, while olfactory cues are used to approach the burrow in darkness and assess its suitability at close-range. Implications to sand fly control are discussed.

Morphological and Molecular Identification of Phlebotomus mascittii Grassi, 1908 Populations From Slovenia.

The Transphlebotomus subgenus has been drawing attention in last decade due to the uncertain vector capacity and cryptic life history of the group. During a 2015 entomological survey, Phlebotomine flies were collected in western locations of Slovenia, with a total of 22 specimens of the Transphlebotomus subgenus collected. In addition to morphological identifications, we confirmed the presence of Phlebotomus mascittii Grassi, 1908 (Diptera: Psychodidae) by nucleotide sequences of the mitochondrial cytochrome b gene. During the study, we found that P. mascittii is most abundant in the area of coastal-karst region of Slovenia, while in rare occasions can also be collected in Northeastern part of the country, near the Austrian border. Vector competence of P. mascitii, like all other member species of the Transphlebotomus subgenus, is unclear, but potential to transmit Leishmania (Kinetoplastida: Trypanosomatidae) pathogens or phleboviruses is strongly suspected.

RNA-seq analysis reveals differences in transcript abundance between cultured and sand fly-derived Leishmania infantum promastigotes.

Leishmania infantum is responsible for human and canine leishmaniasis in the Mediterranean basin, where the major vector is Phlebotomus perniciosus. Because isolation of sufficient parasites from the sand fly gut is technically challenging, axenic cultivation of promastigotes is routinely used to obtain material for biochemical and genetic analyses. Here, we report the use of Spliced Leader RNA-seq (SL-seq) to compare transcript abundance in cultured promastigotes and those obtained from the whole midgut of the sand fly 5 days after infection. SL-seq allows for amplification of RNA from the parasite avoiding contamination with RNA from the gut of the insect. The study has been performed by means of a single technical replicate comparing pools of samples obtained from sand fly-derived (sfPro) and axenic culture promastigotes (acPro). Although there was a moderate correlation (R2 = 0.83) in gene expression, 793 genes showed significantly different (≥2-fold, p <0.05) mRNA levels in sand fly-derived promastigotes and in culture, of which 31 were up-regulated ≥8-fold (p < 10-8 in most cases). These included several genes that are typically up-regulated during metacyclogenesis, suggesting that sand fly-derived promastigotes contain a substantial number of metacyclics, and/or that their differentiation status as metacyclics is more advanced in these populations. Infection experiments and studies evaluating the proportion of metacyclic promastigotes in culture and within the sand fly gut, previously reported by us, support the last hypothesis.

Phlebotomus (Paraphlebotomus) chabaudi and Phlebotomus riouxi: closely related species or synonyms?

Phlebotomus riouxi Depaquit, Killick-Kendrick & Léger 1998 was described as a species closely related to Phlebotomus chabaudi Croset, Abonnenc & Rioux 1970, differing mainly by the size and number of setae of the coxite basal lobe. Molecular studies carried out on several populations from Algeria and Tunisia and based on mitochondrial genes cytochrome b (Cytb) and cytochrome oxidase I (COI) supported the typological validity of these two species. Recently, specimens from a single population in southern Tunisia were morphologically identified as Ph. riouxi, Ph. chabaudi and intermediates, but were clustered in the same clade according to their Cytb and nuclear gene elongation factor-1 α (EF-1α) sequences. These species were thus synonymized. To further explore this synonymy, we carried out a molecular study on specimens from Algeria and Tunisia using the same molecular markers and a part of 28S rDNA. We did not find any morphologically intermediate specimens in our sampling. We highlighted differences between the genetic divergence rates within and between the two species for the three markers and we identified new haplotypes. The sequence analysis did not reveal any signature of introgression in allopatric nor in sympatric populations such as in the Ghomrassen population. Phylogenetic analyses based on our specimens revealed that the two main clades are Ph. chabaudi and Ph. riouxi, in agreement with the morphological identification. These results support the validity of Ph. riouxi and Ph. chabaudi as typological species.

Wing morphology variations in a natural population of Phlebotomus tobbi Adler and Theodor 1930.

Cutaneous leishmaniasis (CL) is highly endemic in the Cukurova region, located on the crossroads of main refugee routes from the Middle East to Europe on the eastern Mediterranean part of Turkey. Our purpose was to investigate the phenotypic variation of Phlebotomus tobbi, the known vector of CL in the region, during one active season. Sand flies and microclimatic data were collected monthly from May to October, 2011, from five locations in six villages in the study area. A geometric morphometric approach was used to investigate wing morphology. Shape analyses revealed that males collected in May and June comprised one group, while specimens collected in August, September, and October formed a second group. Specimens from July were found to be distributed within these two groups. A similar distribution pattern was observed for females, but specimens from October were represented as the third district group. Significant size variation was detected for both sexes between months. Wing size and temperature were negatively correlated for females, but there was no temperature effect for males. Wing size of both sexes was increased in correlation to increasing relative humidity. Males were found to have smaller wings with increasing population density.

New records, distribution, and updated checklists of old world Phlebotomine sand flies, with emphasis on Africa, southwest Asia, and central Asia.

This article includes new records, distribution, and updated checklist of Phlebotomine sand flies (Psychodidae, Diptera) in the Old World (Africa including West Indian Ocean Islands, Southwest Asia, and Central Asia) based on specimen collections housed in different repositories worldwide. About 124 species have primary types housed in 5 repositories including holotypes (45 species, 4 subspecies), syntypes (28 species, 3 subspecies), "types" (14 species), allotypes (10 species), paratypes (36 species, 3 subspecies), lectotypes (13 species), and cotype (5 species), mounted on 671 slides. New abbreviations were proposed for 2 subgenera in the genus Phlebotomus and 6 subgenera in the genus Sergentomyia. New country records were noted in Phlebotomus (4 species in 4 subgenera in 7 countries) and Sergentomyia (10 species in 4 subgenera in 8 countries). For species diversity in the Old World, Phlebotomus includes 92 species and 7 subspecies in 9 subgenera, while Sergentomyia includes 166 species and 16 subspecies in 12 subgenera. A total of 95 species and 7 subspecies of 2 genera (Phlebotomus and Sergentomyia) were recorded in Africa while about 26 species and 16 subspecies in Southwest Asia and Central Asia.

Wing geometry of Phlebotomus stantoni and Sergentomyia hodgsoni from different geographical locations in Thailand.

Geographic populations of the two main sandflies genera present in Thailand were studied for species and population identification. Size and shape of Phlebotomus stantoni and Sergentomyia hodgsoni from different island and mainland locations were examined by landmark-based geometric morphometrics. Intraspecific and interspecific wing comparison was carried out based on 12 anatomical landmarks. The wing centroid size of P. stantoni was generally larger than that of S. hodgsoni. Within both species, wings from the continent were significantly larger than those from island populations. Size variation could be significant between geographic locations, but could also overlap between genera. The wing venation geometry showed non-overlapping differences between two species. The within-species variation of geometric shape between different geographical locations was highly significant, but it could not interfere with the interspecies difference. The lack of species overlapping in shape, and the high discrimination between geographic populations, make geometric shape a promising character for future taxonomic and epidemiological studies.

Ecology and morphological variations in wings of Phlebotomus ariasi (Diptera: Psychodidae) in the region of Roquedur (Gard, France): a geometric morphometrics approach.

BACKGROUND: Phlebotomus ariasi Tonnoir, 1921, is the predominant sand fly species in the Cevennes region and a proven vector of Leishmania infantum, which is the main pathogen of visceral and canine leishmaniasis in the south of France. Even if this species is widely present in Western Mediterranean countries, its biology and ecology remain poorly known. The main goals of this work are to investigate the phenotypic variation of P. ariasi at a local scale in a region characterized by climatic and environmental fluctuations, and to determine if slope and altitude could affect the sand fly phenotypes. RESULTS: Sand flies were captured along a 14 km-long transect in 2011 from May to October. At the same time, environmental data such as altitude and slope were also collected. Morphological analysis of P. ariasi wings was performed by a geometric morphometrics approach. We found morphological variation among local populations of P. ariasi. Strong shape and size variations were observed in the course of the season (particularly in June and July) for both genders. During June, we highlighted differences in wing phenotypes according to altitude for both sexes and to slope and station for females. CONCLUSIONS: The phenotypic variations observed in P. ariasi along the studied transect indicated these populations are subjected to environmental pressures. Nevertheless, it seems that sand flies are more sensitive to extrinsic factors in June and July, suggesting a phenotypic plasticity.

The immature stages of Micropygomyia (Coquillettimyia) chiapanensis (Dampf) (Diptera: Psychodidae, Phlebotominae).

The egg exochorion, larval instars and pupa of the phlebotomine sand fly Micropygomyia (Coquillettimyia) chiapanensis (Dampf) are described and illustrated based on specimens collected in the locality of Farallón, municipality of Actopan, Veracruz, Mexico. Morphology of fourth instar larval mouthparts, particularly the incisor lobe and molar lobe shape of mandible, could be important for species identification of immature Phlebotominae. In this work is compared the pupal chaetotaxy of Mi. chiapanensis with other species previously described. The fourth instar larva of Mi. chiapanensis is compared with other species of this genus, the most important differentiating characters being the size, shape and position of the abdominal dorsal internal seta.