The embryos of K. scorpioides can be distinguished from other species by their phenotypic pattern of pigmentation, characteristics of the longitudinal keel of the carapace, dermal shields of the exoskeleton, formed by the junction of the carapace with the plastron. Comparisons on the development between chelonian species is still complex, due to differences in size and time involved in embryonic phases [22]. The use of measures of body size, chronological age and morphological stage are tools used to standardize these interspecific comparisons [23]. Traditionally, two “test models” have been used for Testudines, those of [6, 10]. They serve as a basis for studies that have described the embryology of turtle development, and there are differences in development between species.
The appearance of the chelonian carapace is a variable element between species, both in the stages of development, as well as in morphology and color. At 42 days (Stage VI) the carapace of the K. scorpioides embryo is defined, with the beginning of the formation of the plastron, in a characteristic pattern of the turtles. These findings are consistent with other species of turtles, such as the marine Chrysemys picta and Chelonia mydas, in studies similar to this one, appearing between 22 and 28 days of incubation, which is shorter, but assigned to the shorter incubation time [24].
The initial formation of the eyes is initiated by the appearance of the optic vesicle, highlighting the visual apparatus in a marked way after the development of the retina, iris, pupil and lower and upper eyelids. The optic vesicle is clearly visible in P. expansa in stage 6 (6 days), while the optic vesicle is visible in stage 7 (9 days) in Mauremys japonica [9] and C. serpentina [10] in stage 8 (4 days) in Pelodiscus sinensis [12].
In the red turtle (Emydura subglobosa), the lower eyelid appears early, around the 13th day (stage 5) and already begins to overlap the eye, reaching the lens level [13]. In this aspect, in the C. serpentina turtle these characteristics were observed at 30 days of development and at 20 days in P. sinensis [10, 12]. In M. japonica (stone turtle), the caruncle was initially evidenced as a small white process under the nostril (stage 17), showing a sharp point in stage 26, a peculiar characteristic of the species [9]. At 63 days (Stage IX), the ranfoteca is observed covering the branches of the mandible together with the formation of the barbel in the ventral region of the mandible (Fig. 2A and 4D). It helps animals to select, shatter and ingest their food to replace teeth. It has its own characteristics that vary according to the diet, and can therefore be used to identify different species of turtles [25].
Differently from the description by [8], for Trachemys scripta, which was based only on the characteristics of the thoracic members as a criterion for delimiting the stages. In K. scorpioides we have deepened the characterization of the various stages of development starting from the initial stage until the hatching. For [8] the morphology of the thoracic limbs is the main criterion for delimiting the stages, as it is easily analyzed until the second half of development, and is highly conserved among most turtles. The tendency in the development of the limbs is that the thoracic ones precede the pelvic ones, an event described in the species (Chelydra serpentina, Pelodiscus sinensis, Emydura subglobosa and M. japonica), or both manifest simultaneously and synchronously (Podecnemis expansa and Chrysemys picta) [12].
The sexual dimorphism of the adults of K. scorpioides is striking, with males showing long tail and concave plastron and females with short tail and straight plastron [26]. In newly hatched cubs the tail is the same size, still short and the plastron straight. Another important structure in sexual differentiation is the urogenital papilla, [14] describes that it is the precursor of the cloaca in the Amazon turtle (Podocnemis expansa), appearing at 30–35 days (Stage 19); in Emydura subglobosa, this structure becomes noticeable at 28 days (Stage 9) [13].
The growth rates of vertebrate embryos are highly variable within and between species and depend on the incubation temperature [27]. The incubation temperature influences its duration and the degree of development [28], at low temperatures they increase the time and decrease the rate of development, while at high temperature they decrease the duration and increase the rate of development [4, 29, 30, 31]. In this way, these temperatures need to be taken into account when comparing, since eggs incubated at different temperatures can present divergent results. In K. scorpioides the eggs were incubated with a controlled average temperature of 28 ° C. The observations, even similar to other reported species, had some important differences and they may be linked to the morphological characters of the interspecific variations, while the incubation temperature influences the differences related to the embryonic stage.
The morphological characteristics of the embryonic development of the species K. scorpioides showed similar states, with few differences, to other turtles. Some characteristics were found delayed or accelerated in comparison to the findings of [10], for the turtle Chelydra serpentina. In addition, strong evidence from this study, supports the hypothesis that the morphological characteristics of the embryonic development of this species, resemble the other chelonians, presenting some variations in the appearance and disappearance of structures.
The information generated by the analysis of the embryonic development of K. scorpioides provides a basis for research with species of the same genus and also with other species of freshwater tortoises, since the temperature and humidity conditions that the eggs are submitted to during the incubation act in the variation of this period, in the speed of metabolic reactions and consequently in the development of the embryo. Another aspect in the difference between the chronology of development in turtles, can be attributed to the necessary morphological characteristics to each species according to their habitat as a result of the evolution process itself. Thus, the present study contributes to the understanding of the various stages of embryonic evolution and reproductive management of this controlled species in captivity, taking into account the incubation temperature, and it is worth emphasizing the importance of expanding the studies to understand the intraspecific variations.