Exochorionic pattern of ten sand fly species (Diptera: Psychodidae: Phlebotominae) from Mexico

Background Phlebotomine sand flies are of biological importance because of their role as vectors of several patho‑ gens. Morphological identification faces challenges to separate related species; therefore, the study of immature stages, as the egg and its exochorion sculpturing pattern could provide useful characters for taxonomic and phyloge‑ netic studies. In the Americas, morphological information of the egg exochorion obtained through scanning electron microscopy has become a complementary tool for taxonomic classification of sand fly species. The aim of this study was to examine and describe the exochorion pattern of eggs obtained from the abdomen of gravid wild females of 10 sand fly species collected in different areas of Mexico. Results We describe the chorionic pattern of 10 sand fly species collected during the period 1997–2023, which was classified as (1) polygonal, (2) connected and unconnected parallel ridges, (3) verrucose, (4) volcano‑like and (5) disperse, being the polygonal pattern the most common among several species of the genera Dampfomyia , Micropy-gomia and Lutzomyia . Conclusions This study describes the exochorion pattern of eggs directly obtained from the abdomen of preserved female specimens, supporting that extraction of eggs directly from an abdomen of a collection specimen might be a viable alternative to gather information of taxonomic value. We describe the eggshell of Bichromomyia olmeca olmeca , Dampfomyia deleoni , Micropygomyia cayennensis , Micropygomyia chiapanensis , Micropygomyia vindicator , Micropygomyia durani, Lutzomyia cruciata , Psathyromyia maya , Psathyromyia texana and Trichopygomyia triramula . We detected differences with respect to previous descriptions of Micropygomyia chiapanensis , Lutzomyia cruciata , and Psathyromyia texana , providing new reference information.

In the Americas, since 1975 the morphological description of the exochorion sculpturing of eggs through scanning electron microscopy (SEM) has resulted in a complementary tool for taxonomic classification of sand fly species both at generic and specific levels (Alencar & Scarpassa, 2018;Jariyapan et al., 2022;Ward & Ready, 1975).The study of the exochorion pattern is important because it is a layer that confers protection from the environment to the embryo, preventing desiccation and regulating gaseous exchange, which in several insect groups has taxonomic significance, besides that it conforms a character that reflects functional adaptations to different types of environment where sand flies oviposit, and could reflect phylogenetic relationships among sand fly species (Jariyapan et al., 2022;Pérez & Ogusuku, 1997;Ward & Ready, 1975).Although some species show a similar pattern, it is generally possible to observe specific details in the microanatomy of the eggs which allow to differentiate sibling species (Bahia et al., 2007).

Egg collection
Phlebotomine sand flies revised and analyzed were previously collected by Instituto de Diagnóstico y Referencia Epidemiológicos "Dr.Manuel Martínez Báez" (InDRE), during the period 1997-2023 using CDC (U.S. Centers for Disease Control) light trap, and Shannon traps, and were preserved in 70% alcohol at − 20 °C.Sand flies were collected from several localities endemic of Leishmaniasis in the states of Guerrero, Nayarit, Nuevo León, Oaxaca, Quintana Roo, and Veracruz, Mexico.We separated and analyzed female sand flies that showed eggs in their abdomen (gravid females).Head and last segments of the abdomen of all selected female sand flies were dissected for morphological identification, and remaining parts of the abdomen were dissected for obtaining the eggs, which were individualized and preserved in ethanol 70%.Eggs were separated carefully as they are compacted within the abdomen.Inside the abdomen, eggs were coated with a sticky layer that holds the eggs together.This sticky coating makes it difficult to separate each egg, so not all available egg were recovered in several specimens.

Taxonomic identification
For taxonomic identification we performed a semipermanent mounting (Lozano-Sardaneta et al., 2023), and used the taxonomic keys of Galati ( 2019) and the abbreviation system proposed by Marcondes (2007).We followed the summarized information of exochorionic sculpture patterns proposed by Alencar and Scarpassa (2018).

SEM procedures
Eggs were treated for SEM examination at Laboratorio de Microscopía y Fotograf ía de la Biodiversidad of Instituto de Biología, Universidad Nacional Autónoma de México (UNAM).The eggs were cleaned in ethanol 70%, then were dehydrated in an ascending series of ethanol (80% OH, 90% OH and 100% OH), and subjected to critical point drying in carbon dioxide (CO 2 ) in a machine Emitech k850.Eggs were then mounted on an aluminum support and coated with gold plated (QUORUM Q150R ES).

Data analysis
The eggs were observed and photographed in a Hitachi SU1510 scanning electron microscope (Hitachi, Japan).Pictures obtained were visualized and measured through the program Image Pro Plus-3D suite version 7.0 (Media Cybernetics, Maryland, USA), and then edited in Adobe Photoshop CS5 software.

Sand flies analyzed
A total of 24 sand fly specimens, belonging to seven genera and 10 species were analyzed to determine chorionic pattern of the eggs.Generally, eggs obtained of the sand fly abdomens are elongate-ellipsoidal with rounded edges, as described for other species.Only Bichromomyia olmeca olmeca showed a different shape, since it was a rounder and smaller egg.In none of the eggs analyzed was it possible to observe the micropyle.
In the case of Bi. olmeca olmeca, Mi. chiapanensis and Pa.maya we provided information of the eggs size and shape, however achieving only a partial description of the exochorion pattern, as eggs were apparently damaged during processing and it is likely the exochorion is incomplete.Therefore, complementary studies are necessary in these species.

Chorionic pattern
Detailed descriptions of the eggs of the ten sand fly species examined are presented below.

Discussion
Studies of the egg exochorion until now have described nine morphological chorionic patterns for specimens of the subfamily Phlebotominae in the Americas.We present the exochorion pattern of ten sand fly species from different regions in Mexico.For the species Mi. chiapanensis and Lu.cruciata there are previously described patterns in Mexico, while for Pa.texana a pattern is known from the United States, besides this species having a wide distribution in Mexico.Chorionic patterns in sand flies have been little studied because it is difficult to obtain eggs in natural conditions or to establish laboratory colonies (Noguera et al., 2003).In this study, we corroborate that it is possible to obtain an egg chorionic pattern without laying the female to spawn, instead dissecting the abdomen of a gravid female to extract the eggs directly (Jariyapan et al., 2022).Although this method is efficient and economic, yielding morphological information of sand fly eggs, in some cases eggs are difficult to separate, probably because the chorion, as a secretion of the ovarian follicular cells, is viscous, in order to foster adherence of egg to a substrate when laid.Apparently, within the female, this viscous layer joins the egg, which makes it difficult to separate one egg from another and in some cases made it difficult to visualize the chorionic pattern.The chorion consists of three layers: an inner thin layer, an intermediate thick porous layer and an outer layer bearing a sculptured pattern (Pérez & Ogusuku, 1997).However, we observed and additional layer that covers the pattern, in some cases preventing these areas to be observed in the eggs of Lu. cruciata, Pa.maya and Mi.vindicator.We were unable to observe micropyles on eggs directly obtained from a female abdomen.However, previous studies state that this structure is usually surrounded by an ornamented area that could be used as species-specific characters for sand fly ootaxonomy (Fausto et al., 2001;Pérez & Ogusuku, 1997).
We observed five chorionic patterns, with the polygonal pattern as the most common between the species analyzed.This pattern has been previously recorded in at least 29 species, presenting cells with different forms such as rectangular, pentagonal, and hexagonal, with morphology varying according to genus or species (Alencar & Scarpassa, 2018; Montes de Oca-Aguilar et al., 2014, 2016).Variation in chorionic pattern may also contribute to understanding adaptations to different types of environments of oviposition sites, since differences in the size of the ridges (high or smooth) has been recorded depending on the need for water storage (Jariyapan et al., 2022;Ward & Ready, 1975).Therefore, complementary environmental information is needed to allow establishing oviposition sites according to variation of chorionic pattern.
In the present study, species with a polygonal pattern were Da. deleoni, Mi. cayennensis, Mi. durani, Mi. vindicator and Pa. texana. Previously, Endris et al. (1987) stated that Pa.texana presents a polygonal pattern, yet they did not provide evidence nor a description, therefore we do not know whether the chorionic pattern in the Mexican specimens is similar to the previously recorded one from United States.However, we observed that the ridges show thin lines with square-like shapes, with the rough area between each ridge showing small protuberances.
In the sand fly genus Dampfomyia a pattern of parallel ridges (connected and unconnected) has been previously recorded in the species Da. anthophora (Endris et al., 1987) and Da.beltrani (Montes de Oca-Aguilar et al., 2014).However, in Da. deleoni collected from Quintana Roo, we observed a polygonal pattern characterized by pentagonal and rectangular cells.It is common for species of the same genus to show a similar pattern, however different patterns may be present within the same genus, for instance in the genus Nyssomyia some species present a polygonal pattern (e.g., Ni. antunesi, Ni. yuilli) and other parallel ridges (e.g., Ni. whitmani, Ni. intemedia) (Alencar & Scarpassa, 2018).
Regarding the species of Micropygomyia analyzed (Mi.cayennensis, Mi. durani, Mi. vindicator), they showed a polygonal pattern with clear differences between them; these three descriptions conform new exochorion records for this genus.In the species Mi. trinidadensis and Mi.vexator it has also been recorded a polygonal pattern, but the elliptical (Mi.venezuelensis) and reticular (Mi.absonodonta) patterns also have been described (Alencar & Scarpassa, 2018).We observed a different pattern for Mi.chiapanensis, besides that a previous study described the eggs of this species with a polygonal pattern with square, pentagon and hexagon forms, in specimens collected from the locality Actopan, Veracruz (Montes de Oca-Aguilar et al., 2016).The specimens analyzed in this study, from Ignacio de la Llave, Veracruz, showed a verrucose pattern in some areas of the eggs.Since it has been recorded that species of Micropygomyia generally show a polygonal pattern, we believe likely that the eggs lost this third layer during sample processing, turning out that only the verrucose pattern of the intermediate layer was observed.Complementary studies are necessary to explore the variability in the morphology of the eggs of these species.
The chorionic pattern of unconnected and connected parallel ridges of Lu. cruciata eggs has been previously recorded (Endris et al., 1987;Montes de Oca-Aguilar et al., 2017).According to its distribution, this species may present variation in characteristics of its chorionic pattern.In Mexico, three exochorionic patterns related to Lu. cruciata exist until now, which are classified into three morphotypes: (1) TACH (from Chiapas) with polygonal ridges, (2) AVER (from Veracruz) with connected parallel and longitudinal ridges, and (3) HOYU (from Yucatán) showing ridges with weak connections.Based on the patterns observed in our samples, we propose a new morphotype named Nayarit (XANAY), characterized by connected parallel granular ridges on the ventral side and unconnected parallel granular ridges on laterals of the egg.Since Lu. cruciata is widely distributed in Mexico and plays an important role in the transmission of Leishmania, this new morphological information may prove useful for the classification of this species.
The volcano-like pattern has been previously described in species of Bichromomyia and Psychodopygus, and it has been suggested that this pattern could be associated to species that deposit their eggs in damp microhabitats (Alencar & Scarpassa, 2018;Fausto et al., 2001;Ward & Ready, 1975).Now, such pattern is also described for the first time in Trichopygomyia, although unlike the other genera, eggs of Tr. triramula are smaller than the other species.In relation to scales observed in the exochorion we are unsure whether they are part of the ornamentation or not, these being the first time these structures are recorded.In Mexico, Tr. triramula has been only recorded from Chiapas and Veracruz (Ibáñez-Bernal & Durán-Luz, 2022), and now we record it from Oaxaca for the first time.This species is not considered of epidemiological relevance in the transmission of Leishmania in Mexico.
We were unable to determine a pattern in Bi. olmeca olmeca, yet we observed that the first layer showed a polygonal pattern, although in the middle layer some areas of the egg have a rugose pattern that appears similar to the volcano-like pattern, so complementary information would help confirm this finding.A previous study has determined that the egg of the species Bichromomyia olmeca nociva and Bichromomyia flaviscutellata has the volcano-like pattern.However, from our study, size and shape of the egg provide the most significant differences, since unlike the other species, in Bi. olmeca olmeca the egg showed an oval shape and is smaller (156 × 83 µm) compared to the other species which report it twice as long (392-410 µm) but with a similar width (82-86 µm) (Alencar & Scarpassa, 2018).The species Bi. olmeca is morphologically classified into the subspecies Bi. olmeca olmeca, Bi. olmeca bicolor, and Bi.olmeca nociva.Recently, it has been suggested that Bi. olmeca olmeca should be raised to species status because its genetic differences with other subspecies (Lozano-Sardaneta et al., 2023).We encourage efforts to explore sculpturing pattern and distinguishing traits of the egg in species of the genus Bichromomyia, which include species of high significance in the transmission of Leishmania spp. in the Americas (Lozano-Sardaneta et al., 2023).
We provide evidence for the first time on the exochorionic pattern of Pa.maya, which we classified as disperse.However, this classification should be considered preliminary, as in our SEM image we observed an apparent extra outer layer, given the possibility that the egg may contain another layer with another pattern that was lost during processing of the material, so complementary information is necessary to elucidate the actual pattern.

Conclusions
This study includes the description of the exochorion pattern of eggs directly obtained from the abdomen of female specimens from Mexico, supporting that extraction of eggs directly from an abdomen might be an option to complement taxonomic descriptions.We performed a morphological description of the eggshell of 10 species using this method, including Bi. olmeca olmeca, Da. deleoni, Mi. cayennensis, Mi. chiapanensis, Mi. vindicator, Mi. durani, Lu. cruciata, Pa. maya, Pa. texana and Tr. triramula.Notably, we detected differences with respect to previous descriptions in species such as Mi.chiapanensis, Lu. cruciata, and Pa.texana, which provide additional information to the classification of immature stages.Further studies on exochorion patterns would enrich morphological and phylogenetic knowledge of sand fly species.

Fig. 1
Fig. 1 Exochorion pattern of Bichromomyia olmeca olmeca: A, B Complete egg.C Polygonal pattern.D Zoom to middle layer

Fig. 6 Fig. 7
Fig. 6 Exochorion pattern of Micropygomyia durani.A Complete egg; B, C Zoom to the polygonal area

Fig. 8
Fig. 8 Exochorion pattern of Psathyromyia maya.A Complete egg; B Disperse pattern; C, D Zoom to the disperse area

Fig. 10
Fig. 10 Exochorion pattern of Trichopygomyia triramula.A, B Complete egg, C Zoom to the volcano-like pattern