We describe and compare the morphology and morphometry of the egg exochorion for five species and two morphotypes of Mexican triatomines with scanning electron microscopy. The results show differences in egg ornamentation for each species, including between morphotypes. Polygonal ornamentation was observed in each species, including pentagons, octagons and, in certain cases, a majority of hexagons. We observed small perforations in T. protracta protracta, small spheres on the T. lecticularia polygons, a crown with festoons in T. barberi, and less complex ornamentation in T. mexicana. Through morphometric analysis, significant differences in egg dimensions were determined for the studied species.
Pinto (1924) conducted the first study of triatomine eggs, describing the egg morphology and color of T. brasiliensis and differentiating the eggs from other Triatomines in Brazil (Costa et al. 1997). Galliard (1935) demonstrated that insect eggs have a characteristic chorionic structure and performed comparative morphological studies on eggs from certain triatomine species using an optical microscope. Galliard concluded that the differences in egg ornamentation may be used for species differentiation (Visciarelli et al. 2004). Lent and Wygodzinsky (1979) showed that the characteristic exochorion is species-specific for different triatomine species. Barata (1981) used scanning electron microscopy (SEM) on eggs from ten species in the genus Rhodnius, demonstrating species differentiation and elaborating a dichotomous key to diagnose species. Triatomine egg macroscopic exochorial structures were studied by Barata (1998) to demonstrate relevance. In this study, nine of the 15 known genera were analyzed with the morphometry among the species examined and compared between genera and species.
Obara et al. (2007b) studied six species in the genus Triatoma and concluded that morphological characteristics of eggs compose a new parameter that may aid in creating future dichotomous keys and support the determination of a role for each vector species. For the species studied, the surface of the exochorion or egg bodies has a polygonal cell-based ornamentation. The predominant cell shape is hexagonal except in T. barberi, which does not have this ornamentation. The operculum, chorial rim, and opercular border are also relevant structures in egg morphology. The presence and absence of other structures such as the neck, collar, and lateral flattening may be used to distinguish species (Barata 1998, Rivas et al. 2009). The purpose of this study was to discern the egg morphology and morphometry for the five species and two morphotypes, which are significant vectors. We also propose to use such characteristics as taxonomic patterns for identifying Mexican triatomines.
MATERIALS AND METHODS
We studied egg morphology for the following Mexican species in the Triatoma genus: T. barberi, T. dimidiata, T. lecticularia, T. mexicana, T. rubida, T. protracta protracta, and T. protracta nahuatlae. Studied specimens of these species were obtained from the triatomine colony at the Entomology Laboratory (ENCB-IPN). The colony was maintained at 28 ± 1° C/ 60% RH (Ryckman 1952). Each week, the colony was fed on New Zealand rabbit. After oviposition, the eggs were harvested and placed in separate vials.
Ten eggs, ten exochorions, and ten opercules for each species were observed with optical microscopy (OM) using a Nikon SMZ1500 stereoscopic microscope. Images were collected for each structure using a Nikon Sight DS-Fil camera and were used as general reference.
Scanning Electron Microscopy (SEM)
For SEM analysis, nine eggs from each species were used; they were mounted on a sample-holder using two-sided carbon tape and then coated in gold for 500 s in a Denton Vacuum DESKII ionizer. The eggs were then observed using a JSM-5800LV model JEOL® Scanning Electron Microscope, and micrographs were digitally captured using the Inter Video MISPVS software.Triatoma barberi eggs were treated in two ways: five were washed with a 10% NaOH solution for 90 min before SEM mounting; the remaining four were mounted directly without washing.
An ocular micrometer was used to measure 50 eggs and 50 opercula from each species. Exochorion length and width as well as operculum diameter were also measured. Data were analyzed for outliers using the criteria of 1.5 IQR (Interquartile Range), followed by Canonical Discriminant Analysis (CDA). The Mahalanobis distance was used to determine statistically significant differences among species for the set of three measured variables. Class means values on canonical variables were visualized through Cartesian coordinates; the direction and orientation represented the level of association between each variable and species. We used SAS version 9 statistical software.
The eggs from each species studied via optical microscopy had an oval shape and an operculum. Egg color shifted from white, to orange or pink over time; this color shift in the eggs was due to embryo development because the egg shell was always white.
Triatoma barberi– The body was an ellipsoid and narrowed toward the operculum (Figure 1A). The eggs were coated with a cementing substance that facilitates surface adherence, and this substance covered the entire chorion surface. Observed under optical and electron microscopes, the polygonal ornamentation was not apparent, but a rough and striated surface was observed (Figure 2A). When the eggs were washed with 10% NaOH, the polygonal ornamentation became apparent and hexagons were dominant (Figure 2B). A neck was not observed; a thick chorial rim with faint polygonal cells and small, dispersed granules was observed (Figure 3A). The operculum showed a collar comprised of a series of connected projections that marked the operculum outer edge. In the center of this structure, a smaller number of projections were observed; the projections located in the middle were longer and arranged in two concentric circles separated by a clearly defined groove with no polygonal ornamentation (Figures 4A, 5A).
Triatoma dimidiata– This species had an oval-shaped body that was more globular than other Triatoma species (Figure 1B). The chorion showed a faint polygonal ornamentation that varied from pentagons to heptagons with predominantly smooth-surfaced hexagons. The grooves that delineate one polygon from another were deep and wide compared with other species studied and were clearly separated primarily in the vertices (Figure 2C). The connection between the chorial and opercular borders formed a faint triangular elevation. In polygons that were proximal to this area, the vertices were rounder, and incomplete polygons were observed at the far end (Figure 3B). Unlike the body, the operculum had clear polygonal ornamentation without a single dominant shape although four-, five- and six-sided structures were evident. The latter were longer near the opercular border. At the edge of the opercular border, incomplete polygons with a rough surface texture and scattered pores were observed (Figures 4B, 5B).
Triatoma lecticularia– This species had an ellipsoid body with a prominent collar and narrow base (Figure 1C). The chorion surface comprised polygonal cells joined as small spherical shapes in a straight line that covered the egg. The inner portion of the polygons was empty, and ornamentation was easier to observe (Figure 2D). A narrow neck was observed at the base, which was wider at the operculum connection space. A well-defined seam between the chorial and opercular borders was observed, which met at a prominent fold (Figure 3C). Just beyond the opercular border, the operculum had a clear concentric circle and faint ornamentation without spherical shapes. The remaining operculum showed the same morphology as the body, consisting of a cluster of rounded shapes that covered the surface as polygons. These polygons were narrower at the operculum center when this formation was observed under greater magnification. In certain cases, the shapes in the operculum center did not appear (Figures 4C, 5C).
Triatoma mexicana– Eggs of this species had an oval-shaped body and operculum without a neck (Figure 1D). The chorion had polygonal ornamentation, which comprised primarily pentagons and hexagons. The cells were smooth and slightly elevated (Figure 2E); a neck was not observed. The operculum directly contacted the body, forming a deep and evident groove between the chorial and opercular borders. The opercular border was thicker than the chorial rim (Figure 3D). The operculum exhibited polygonal ornamentation without a dominant shape but with more padded cells than the body. The groove between each cell was wider and deeper than in the body. Fused cells were observed at the center of the operculum, resulting in a rough surface; the fused cells at the edge were incomplete. Along the outer edge of the operculum, we observed an area that retained the width through the circumference and had a rough surface but no polygons (Figures 4D, 5D).
Triatoma rubida– The body of eggs of this species was slightly elongated and oval-shaped (Figure 1E) with polygonal ornamentation on the chorion and cells with four to eight sides. We observed certain areas with predominantly pentagonal shapes (Figure 2F). The neck was absent. The chorial and opercular borders formed a slightly elevated triangular fold with a dividing groove (Figure 3E). The operculum comprised heterogeneous polygonal ornamentation that covered the entire surface; the shapes were slightly padded compared with the body (Figures 4E, 5E).
Triatoma protracta nahuatlae– Eggs of this species had an oval-shaped body (Figure 1F) and perfectly defined polygonal ornamentation. The borders of each hexagon were clearly defined, and each polygon was padded with a slightly rough surface. Each hexagon vertex was well defined with a pore-like perforation (Figure 2G). The chorial rim was wide and separated from the opercular border by an unpronounced groove (Figure 3F). The operculum had three areas of slightly different morphology. The area closest to the opercular border had a series of cylindrical projections with the same length. The second area contained an additional series of projections that were longer and more irregular than the first, and the third area at the operculum center had large, irregular projections. The projections in the third area were more diffuse compared with the first two areas so that the space between them was evident, facilitating observation of small spherical particles that adhered to the operculum (Figures 4F, 5F).
Triatoma protracta protracta– The egg body had predominantly hexagonal polygonal ornamentation with visible pore-like perforations at each polygon vertex. The polygons were outlined with faint grooves along each edge, which were slightly elevated or slightly cushioned compared with T. protracta nahuatlae, which had a rough surface (Figures 1G, 2H). There was a slightly thicker opercular border, and there was a faint groove at the connection point for this border and the chorial rim (Figure 3G). The operculum morphology contained festoons and was grouped into three primary areas. The first area was the edge of the operculum with a series of slightly perceptible festoons (because they were almost fused) with faint grooves between them. The second area had more elevated projections compared with the first area, which were tightly grouped in certain areas. In the second area, the small and large projections were interleaved. There were larger, more diffuse projections in the third area at the operculum center; the projections of the third area were much wider and longer than those in the other two areas, which allowed us to observe a very rough surface at the operculum center (Figures 4G, 5G).
Table 1 shows means and standard error of mean for the species and variables studied. Morphometric analysis (CDA) demonstrated significant differences for Mahalanobis distances (p < 0.0001) among the species and T. protracta morphotypes studied. Figure 6 shows the distinction between taxa in three canonical axes.
Table 1. Mean and standard error of mean (SEM) of length, width, and opercular diameter (μm) of eggs of studied species.
T. protracta protracta
T. protracta nahuatlae
The importance of triatomine egg ornamentation was highlighted by Pinto (1924) when describing T. brasiliensis eggs (Neiva 1911). Pinto differentiated these eggs from other triatomines in Brazil; since then eggs have been continuously studied although with little depth and only with South American species. Even though Mexico has the greatest number of species (Salazar-Schettino 2003), little research had been done on triatomine eggs. The purpose of this study was to use optical and scanning electron microscopy to elucidate exchorial characteristics, and morphometrically analyze eggs from five species and two morphotypes in the genus Triatoma.
The SEM data for the eggs showed that the triatomine species had polygonal ornamentation with pentagons, hexagons, heptagons, and octagons on the body. T. barberi eggs were coated with a cementing substance and had a rough, non-ornamented surface, as described by Barata (1998). However, when the small eggs were washed with a low-concentration alkaline solution, we observed polygonal ornamentation dominated with hexagons. Evangelista-Martínez et al. (2010) studied ornamentation in T. barberi eggs from the state of Hidalgo using 0.1 M cacodylic acid washes and later processed samples for SEM.
We found significant morphological variations between the eggs from the species studied herein and grouped them in accordance with Barata (1998). The first group comprised T. mexicana, T. dimidiata, and T. rubida; the common characteristic was polygons on the exochorion in the body and operculum. The second group comprised T. lecticularia, T. protracta protracta, and T. protracta nahuatlae, which do not have clear ornamentation even though the exochoria had polygonal cells. As previously described, these cells comprised a variety of arrangements and structures. T. barberi would be in the first group if ornamentation was considered. On the other hand, if egg ornamentation was covered, T. barberi would be grouped in the second category.
Although the ornamentation in the first group was similar, they were clearly distinguishable based on shape and size. It is worth mentioning that T. dimidiata has a similar egg ornamentation compared to Meccus pallidipennis Stal 1872, M. picturatus Usinger 1939, M. phyllosomus (Burmeister 1835), and M. longipennis Usinger 1939, which were studied by Obara et al. (2007a), However, the eggs of T. dimidiata were smaller than eggs of these four species. It also had a more rounded form as determined using the length-width proportions; the width was almost three-fourths as much as the length. For polygon morphology, species in the Meccus genus had neither a deep grove at each polygon vertex nor small perforations in the polygons distal to the operculum.
Triatoma rubida eggs had ornamentation similar to those in T. tibiamaculata (Obara et al. 2007b) with variability in polygon type and surface texture. The major difference between the species was the opercular border, which was thick in T. tibiamaculata eggs compared with those of T. rubida.
Triatoma mexicana was clearly differentiated from the previous two species based on egg dimensions and from T. dimidiata through the shallow grooves between the polygons. Small pores were not observed in the outer portion of the operculum. T. mexicana was differentiated from T. rubida because it had no polygons on the egg body, which had more than six sides.
The descriptions herein are the first available from Mexico on T. rubida and T. mexicana in terms of egg ornamentation, while T. dimidiata eggs have been described for a Veracruz population (Mello et al. 2009). It is worth stressing that there is a strong morphological and chromatic variation between populations of this species, and egg morphology is a useful trait given its wide distribution. For the second group, the common characteristic among the three species was the presence of projections in the operculum that were particular for each species. T. lecticularia did not have projections, but its polygons pattern was unique compared with previously studied species. Barata (1998) referred to this phenomenon as a “complex ornamentation.”
The case of T. protracta protracta and T. protracta nahuatlae morphotypes requires some particular consideration. It is worth noting that Ryckman (1962) proposed separating this species into five subspecies, but Villalobos et al. (2012) recently conducted a detailed study on the morphology of eggs and genital plates for these morphotypes and suggested that they are in the process of advanced differentiation that may result in two recognizable species.
Current studies highlight egg morphology as a useful tool for differentiating closely related species in the Triatominae subfamily (Obara et al. 2007b). The characteristics observed here, coupled with a thorough comparison with previous studies, lead us to conclude that egg ornamentation in the triatomine species observed via OM and SEM is an efficient tool for identifying species in this group. It is important to note that eggs may be observed at any stage through embryonic development considering that egg morphology does not change (Lizaraso 1957, Chaves and Añez 2003).
Also relevant is the fact that these characteristics have been used to resolve taxonomic issues in triatomines, including T. maculata and T. pseudomaculata, which were in synonymia for many years until a comprehensive study on their morphology, geographical distribution, and egg structure was conducted (Gonçalves et al. 1985). Barata (1981) used egg ornamentation via SEM to identify species and proposed a dichotomous key for identifying ten species in the Rhodnius genus. Our results show that OM and SEM are important instruments that may aid in species identification of Triatominae. Further research on structures that are unique to this group may support the techniques used herein and aid in group systematics.