Significance of eggshell morphology as an additional tool to distinguish species of sand flies (Diptera: Psychodidae: Phlebotominae)

Morphological characteristics of eggshells are important in sand fly ootaxonomy. In this study, eggshells from Phlebotomus stantoni Newstead, Sergentomyia khawi (Raynal), and Grassomyia indica (Theodor) sand flies collected in Chiang Mai province, Thailand were examined and characterized using light microscopy (LM) and scanning electron microscopy (SEM). Then, eggshell morphology of these three species was described for the first time. Each gravid female was forced to lay eggs by decapitation and the eggs were collected for SEM analysis. Egg laying females were identified by morphological examination and molecular typing using cytochrome b (Cytb) as a molecular marker. The chorionic sculpturing of Ph. stantoni eggs combines two patterns on the same egg: unconnected parallel ridges and reticular patterns. Sergentomyia khawi and Gr. indica have similar chorionic polygonal patterns, but their exochorionic morphology and aeropylar area are different. Results indicate that eggshell morphological characteristics such as chorionic pattern, exochorionic morphology, inter-ridge/boundary area, aeropylar area (including the number of aeropyles) and basal layer, can be useful to develop morphological identification keys of eggs. These can serve as an additional tool to distinguish species of sand flies. In addition, the chorionic sculpturing of the eggs of the three species of sand flies observed by LM is useful for species identification in gravid females with spermathecae obscured by eggs.


Introduction
Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are hematophagous insects. Some species are vectors of several viral, bacterial, and protozoal diseases, especially leishmaniasis. In the last few decades, new proposals for classification and identification of Phlebotominae sand flies have been based primarily on adult morphology [1]. However, morphological

Sand fly collection and experimental design
Sand flies were collected on a weekly basis in August 2020 (four nights in total) in Doi Saket, Chiang Mai province, Thailand (E 18.892554, S 99.120376). Three CDC light-traps were set up in a cowshed from 6 PM to 6 AM. Collected sand flies were transferred to the laboratory of the Department of Parasitology, Faculty of Medicine, Chiang Mai University, for species identification and examination of eggs.
The experimental design used for species identification of female sand flies and morphological examination and characterization of sand fly eggs is shown in Fig 1. Gravid females with similar morphology were separated into two groups. In Group 1, females were dissected individually for morphological identification using LM. Their carcasses were molecularly identified through DNA sequencing of the cytochrome b (Cytb) gene. LM analysis of the chorionic sculpturing pattern of eggs in each gravid female was also performed. In Group 2, each gravid female was forced to lay eggs by decapitation on wet Whatman filter papers under a dissecting microscope. After laying eggs, DNA was extracted from the female carcass. DNA sequencing of the Cytb gene for molecular identification of species was carried out through polymerase chain reaction (PCR). The eggs were left on the wet papers in a container for 24 h and subjected to SEM for measurements and morphological characterization including chorionic pattern, exochorionic morphology, inter-ridge/boundary area, aeropylar area, and basal layer.

Light microscopy
The female samples in Group 1 were dissected in Phosphate Buffer Saline (PBS; 10 mM sodium phosphate, 145 mM sodium chloride, pH 7.2) under a dissecting binocular microscope. Only the female heads and abdominal segments 8-11 were detached and mounted in Hoyer's medium. The morphological characteristics of the sand flies (cibarium, including armature and pigment patch; spermathecae) and eggs (chorionic sculpturing pattern) were examined and photographed using the LEICA DM1000 microscopy camera (Leica microsystems, Wetzlar, Germany). Species were identified using the adult morphological keys of Lewis [38,39] as well as original descriptions and a recent taxonomical study by Depaquit and colleagues [28].

Scanning electron microscopy and measurements of eggs
Eggs from individual females were fixed with 2.5% glutaraldehyde in 0.1 cacodylate buffer (pH 7.2) for 12 h at 4˚C and washed twice for 10 min with PBS (pH 7.2). Next, samples were fixed with 1% osmium tetroxide in PBS for 1 h, and then dehydrated in a graded series of ethanol (50%, 70%, 90%, 95% for 10 min each and then twice with 100% ethanol for 30 min each), followed by critical point drying in liquid CO 2 and coating with gold particles in a sputter-coating apparatus. The gold-coated preparations were examined under a scanning electron microscope, JEOL JSM-6610LV (JEOL, Tokyo, Japan), at 25-30 kV. SEM images of 30 eggs from at least three females in each species were used for egg measurements, including: length and width of egg, width and height of ridge, diameter and height of palisade unit, and diameter of tubercle. The values were presented as mean ± SD.

DNA extraction, PCR, DNA sequencing and phylogenetic analysis
Each female carcass was transferred into 1.5 ml microcentrifuge tubes containing 200 μl of lysis buffer for gDNA extraction using a tissue DNA extraction kit (GeneJet Genomic DNA Purification kit, Thermo scientific, USA) following the manufacturer's instructions. Genomic DNA was stored at 4˚C until use. PCR of the Cytb gene was performed using the N1N-PDR (5'-CAY-ATT-CAA-CCW-GAA-TGA-TA -3') and C3B-PDR (5'-GGT-AYW-TTG-CCT-CGA-WTT-CGW-TAT-GA -3') primers [40,41]. The reaction was carried out in a final volume of 25 µl including 2.5 µl PCR buffer, 2.5 µl MgCl 2 , 2.5 µl dNTPs, 0.2 µl Taq polymerase, 0.4 µl forward, 0.4 µl reverse primers, 10.5 µl distilled water and 6 µl DNA template. PCR condition was performed with an initial denaturation step at 94˚C for 3 min for 1 cycle, followed by 5 cycles: denaturation at 94˚C for 30 sec, annealing at 40˚C for 60 sec, extension at 68˚C for 60 sec; and then 35 cycles of denaturation at 94˚C for 60 sec, annealing at 45˚C for 60 sec, extension at 68˚C for 60 sec and a final extension step at 68˚C for 10 min. PCR products were analyzed using electrophoresis on 1.5% agarose/1X TAE buffer gels, strained with ethidium bromide, and visualized under a UV transilluminator. PCRs were purified using the Gen-eJet PCR purification kit (Thermo Fisher scientific, USA) following the manufacturer's instructions. The purified PCRs were sent to Macrogen Inc., Seoul, South Korea, for sequencing. A phylogenetic tree was built using the maximum likelihood method with the Tamura 3-parameter distance model in MEGA X [42].

Species identification
Twenty-two (22) gravid female sand flies were collected from the study area. They were examined and molecularly identified according to species. The Cytb sequences of each sample were deposited in GenBank as listed in Table 1. Phylogenetic analysis revealed three species of sand flies, i.e., Ph. stantoni, Se. khawi, and Gr. indica (Fig 2).

LM and SEM analyses
The eggshell structures of the three sand fly species, Ph. stantoni, Se. khawi, and Gr. indica, were examined and described. In general, all eggs were elongated, elipsoidal in shape with one side slightly flattened and rounded polar regions. In this study, only an aeropyle of Ph. stantoni was observed at the anterior pole of eggs.
Two chorionic patterns of eggs were found: (1) an unconnected parallel ridges pattern combined with a reticular pattern in Ph. stantoni and (2) a polygonal pattern in Se. khawi and Gr. indica. The details of the eggshell structures of the three sand fly species are described below.
Phlebotomus stantoni. Phlebotomus stantoni has a cibarium with approximately 15 pointed denticles of various lengths, which are irregularly arranged. The two or three median denticles are longer and stouter than the others and without a pigment patch ( Fig 3A). The species has a fusiform spermatheca with 15 or 16 rings. The neck is thick and short, the head more or less oblong. The duct is striated and the common duct is very long, about 1.5 times the length of the individual ones, with a thick wall sclerotized at its basal part ( Fig 3B). LM analysis of eggs in Ph. stantoni gravid females showed two patterns: unconnected parallel ridges and a reticular pattern on the same egg ( Fig 3C).
Exochorionic morphology. Ridges are formed by double non-columnar palisades that project from the surface of the eggshell. In some areas, the ridges are fused, forming an unconnected parallel ridges pattern. In other areas, the ridges exist separately thereby forming a reticular pattern. Each ridge is 3.37 ± 0.79 μm in width and 5.88 ± 0.35 μm in height (Fig 4C-4F).
Inter-ridge/boundary area. Fibrous material in the inter-ridge area is coarsely arranged with minute protuberances covering the basal layer (Fig 4E and 4F).
Aeropylar area. One aeropyle opening is present. The aeropyle is surrounded by randomly distributed protuberances and covered by very small dots (Fig 4G and 4H). The aeropylar area is delimited by a single non-columnar circular ridge.
Basal layer. A wide reticular mesh conformation of the basal layer near the aeropyle opening is present ( Fig 4I).
Sergentomyia khawi. Sergentomyia khawi has a cibarium containing 10-12 hind teeth with two or three rows of fore teeth with a light pigment patch ( Fig 5A). It has a narrow shaped spermatheca with a few wrinkles, with a knob in a deep narrow pit ( Fig 5C). LM analysis of eggs in Se. khawi gravid females reveals polygonal (rectangular, pentagonal, or hexagonal) patterns of the chorion (Fig 5B-5D).
Exochorionic morphology. Ridges are formed by the intersection of a single or double parallel series of rounded palisade units that project from the surface of eggshell. The diameter and height of each palisade unit are 1.26 ± 0.21 μm and 0.8 ± 0.14 μm, respectively. Almost all palisade units are linked or united at the top (Fig 6B and 6C).
Inter-ridge/boundary area. Finely arranged fibrous material as a compact coat covering the basal layer of the egg is present in the inter-ridge area (Fig 6D).
Aeropylar area. A wide reticular mesh conformation is present at the pole ( Fig 6E). Grassomyia indica. Grassomyia indica has a cibarium with 29-30 teeth along a convex curved line and a dark brown pigment patch with a straight anterior margin (Fig 7A). It has a round finely speculate capsule spermatheca with minute projections at its tip ( Fig 7C). LM analysis of eggs inside Gr. indica females reveals polygonal patterns (rectangular, pentagonal, or hexagonal) of the chorionic sculpturing (Fig 7B-7D).
Exochorionic morphology. Boundaries of the polygonal shapes are formed by the intersection of 2-5 lines of small randomly arranged tubercles. Some tubercles project from the surface of the eggshell. The average of diameter of the tubercles is 0.68 ± 0.14 μm. No tubercles are linked or united at the top (Fig 8C and 8D).
Inter-ridge/boundary area. Fibrous material in the inter-ridge area is finely arranged as a compact coat covering the basal layer (Fig 8D).
Aeropylar area. A thick coat material is present at the pole (Fig 8C). Table 2 summarizes the important details of ultrastructural morphology of Old World sand fly eggs examined by SEM.

PLOS ONE
Eggshell morphology of sand flies as an additional tool to distinguish species have been previously examined by SEM. In these past studies, the sand fly eggs were obtained from females reared in laboratories (laboratory strains) [22,23,25] or collected in the field. The females were allowed to feed on animal blood in the laboratory to favor egg development [20,24,25] but no molecular methods for species identification were mentioned. In the case of field-caught female sand flies, sibling species which are morphologically indistinguishable but reproductively isolated may be obtained together. Rogo and colleagues [24] observed that eggs laid by an individual female of Ph. martini were identical but those laid by different females of the same species (identified by morphology) were not. In addition, eggs of Ph. martini females collected from different origins, such as Kenya and Ethiopia, showed different chorionic patterns [20,21]. Therefore, in this study, both morphological identification keys of female sand flies and DNA sequences of the Cytb gene were used for species identification and confirmation. In addition, a systematic collection of egg batches from individual females was performed by decapitating the gravid females. It is the first time this method was used in sand flies from Thailand and it allowed us to obtain synchronized eggs for SEM analysis. The species of the females and eggs were reliable and consistent by using these different approaches.
Our results show that the egg morphology of the three confirmed species, Ph. stantoni, Se. khawi and Gr. indica, followed the shape of eggs of all the previously described species: they have a long and oval shape with one side slightly flattened and rounded polar regions. Five details in the ultrastructural morphology of sand fly eggs including chorionic pattern, exochorionic morphology, inter-ridge/boundary area, aeropylar area and basal layer were examined and characterized by SEM. However, only complete details of Ph. stantoni eggs were obtained. The chorionic sculpturing of Ph. stantoni (subgenus Anaphlebotomus) eggs differs from that of sand flies in other subgenera. We demonstrated for the first time that the chorionic pattern of Ph. stantoni eggs combines "unconnected parallel ridges" and a "reticular" pattern on the same egg. In Ph. argentipes (subgenus Euphlebotomus) and Ph. papatasi (subgenus Phlebotomus), although the chorionic pattern of eggs has been grouped under "connected parallel ridges", a reticular pattern was observed in some areas on the eggs [25]. A combination of double parallel connected ridges and reticular pattern has also been reported in Lu. verrucarum [8]. Moreover, a variation of the chorionic sculpturing pattern among sand flies in the genus Phlebotomus has been reported. For example, in the subgenus Larroussius, the pattern of Ph. perniciosus and Ph. perfiliewi eggs is "unconnected parallel ridges" [23] whereas various patterns (polygonal, beehive cell, and parallel ridges) have been reported in Ph. aculeatus [24]. Furthermore, in the subgenus Synphlebotomus, the chorionic pattern of Ph. celiae eggs is "reticular" while Ph. martini sand flies from different countries (Kenya and Ethiopia) have different chorionic patterns  of eggs, such as "connected parallel ridges", "reticular", "mountain-like", irregular patterns [20,21,24]. In the genus Sergentomyia, a polygonal pattern is quite common, however, a "volcano-like" pattern and "parallel ridges" have been reported in Se. kirki (subgenus Rondanomyia) and Se. garnhami (subgenus Sergentomyia), respectively [20]. The chorionic pattern of Se. khawi and Gr. indica from Thailand is polygonal but their exochorionic morphology and aeropylar area are different. The exochorionic morphology of Gr. indica is unique. Boundaries of the polygonal pattern formed by 2-5 lines of small randomly arranged tubercles in Gr. indica were noted and described for the first time. The aeropylar area of Se. khawi is covered by a wide reticular mesh conformation whereas the aeropylar area of Gr. indica is covered by a thick coat material. Differences in exochorionic morphology, the inter-ridge/boundary area, the aeropylar area (including the number of aeropyles) and basal layer of some sand fly species in the genus Phlebotomus in the New World have been reported [10, 12, 15-19, 22, 23, 25]. Therefore, the chorionic pattern together with other important details of the ultrastructural morphology may be useful for developing morphological identification keys of eggs as an additional tool to distinguish species of sand flies. More information on egg ultrastructural morphology of sand flies in the Old World and the New World with correct molecular genetic information are required for further analysis to ascertain their taxonomic values before any conclusion can be drawn.
Although the use of SEM has significantly improved the characterization of the ultrastructural morphology of sand fly eggs, in this study, the chorionic sculpturing of the eggs of Ph. stantoni, Se. khawi, and Gr. indica, were observed by LM. We demonstrate that in the case of gravid females (whose spermathecae is obscured by eggs), the chorionic sculpturing of eggs is useful to identify these three species. In this study, the developmental stages of eggs in the gravid female sand flies could not be estimated since these samples were taken from females caught in the wild. Our results showed that in LM, the chorionic pattern of eggs inside the females of each species was the same as shown in SEM. The egg samples for SEM were allowed to fully melanise for 24 h before SEM processing. This suggests that the chorionic patterns of fully mature eggs were present in the abdomens of gravid females of these three sand fly species. In Lu. longipalpis, fully melanised eggs have been observed in the abdomens of gravid sand flies. It highlights that sand fly eggs can fully melanise prior to oviposition [43]. Therefore, it is interesting to further investigate chorionic patterns in each developmental stages of sand fly eggs of each species. In addition, further studies on eggshell morphology of sand flies from other areas and other species are needed to compare eggshell patterns as the environments of oviposition sites influence the exochorion of sand fly eggs [4]. At least two species in the same genus in the Old World, i.e., Phlebotomus, Sergentomyia, Grassomyia, Idiophlebotomus, Chinius, Parvidens, Spelaomyia and Spelaeophlebotomus should be investigated to evaluate their informativity for classification of sand flies.
In conclusion, the ultrastructural morphology of the eggshells of Ph. stantoni, Se. khawi, and Gr. indica sand flies was described for the first time. Our findings indicate that the chorionic sculpturing pattern together with other important details of the ultrastructural morphology can be used to develop morphological identification keys of eggs. These can serve as an additional tool to distinguish species of sand flies. The systematic collection of egg batches from individually gravid females by decapitating allowed us to obtain synchronized eggs for SEM analysis. In addition, insect species were confirmed using both Cytb sequencing as well as adult morphological identification keys. Together, these methods provided useful information for sand fly ootaxonomy. Furthermore, the chorionic sculpturing of the eggs of these three species observed by LM is useful for species identification in the case of gravid females with spermathecae obscured by eggs.