INTRODUCTION

The green turtle (Chelonia mydas) is distributed worldwide and has a very abundant and diversified parasitic fauna (^{Santoro et al. 2006}, ^{Greiner 2013}). In Brazil, studies on the parasitic fauna of C. mydas revealed a considerable occurrence of helminths of the class Digenea (^{Werneck and Silva 2015}, ^{Binoti et al. 2016}, ^{Gomes et al. 2017}). Between 2004 and 2011, ^{Werneck and Silva (2015)} analyzed the helminth composition in juvenile green turtles for the first time in Brazil, and ^{Binoti et al. (2016)} and ^{Gomes et al. (2017)} characterized these helminths and the ecological aspects of the gastrointestinal helminth community in juveniles of C. mydas.

Diagnosis of helminthiasis in sea turtles is usually made during necropsy through the collection and study of adult parasites (^{Greiner and Mader 2006}, ^{Chapman et al. 2019}); it is not common to perform coproparasitological exams, although they are possible and justified to use as a noninvasive method (^{Greiner and Mader 2006}). ^{Marangi et al. (2020)} conducted the first multicenter coprological survey of free-living sea turtle (Caretta caretta) helminth communities, considering that the main method for diagnosing endoparasites in these animals is through fecal examination for egg detection.

The identification of eggs is based on their morphology and morphometry, with consideration of the structures developing within the egg, egg shape, length, width of thorns or filaments in the poles, number of filaments, and filament length. However, some eggs observed in fecal samples are not identifiable to species or genera. In some cases, the dynamics of egg elimination in feces have been little explored. Furthermore, it is unclear how the digenea adults found in places outside the digestive system eliminate their eggs in the host feces. For the spirorchiid life cycle, 3 main possible shedding pathways have been hypothesized, including through feces, expectoration, and postmortem decomposition or scavenging of a carcass with egg dispersion (^{Chapman et al. 2019}). ^{Santoro et al. (2020)} obtained an enormous quantity of eggs containing live miracidium from carcasses of sea turtles positive for spirorchiid infection, which suggests that viable eggs might be shed through the feces or dispersed after postmortem decomposition.

Due to the lack of articles on the morphology and morphometry of eggs in turtle helminths, the aim of this study was to morphologically and morphometrically characterize helminth eggs recovered in sedimentation coproparasitological exams and to analyze their relationship with helminth species recovered during necropsy of juvenile specimens of green turtles (C. mydas).

MATERIALS AND METHODS

Thirty-six juvenile specimens of green turtle (C. mydas, Linnaeus 1758) (Testudines, Chelonidae) found dead on the southern coast of the state of Espírito Santo, Brazil, from March to August 2015 were necropsied, and the contents of the gastrointestinal tract were inspected for parasites. The animals were rescued by the Beach Monitoring Program of the Campos and Espírito Santo basins, in a stretch of approximately 200 km (from the southern to the middle of Espírito Santo coast), between the beaches of Nova Almeida/Serra (20°02ʹ20ʺ S, 40°10ʹ50ʺ W) and Marobá/Presidente Kennedy (21°11ʹ45ʺ S, 40°55ʹ54ʺ W).

The collected parasite specimens were fixed in acetic formaldehyde alcohol (AFA) for 24 h between 2 glass slides and then packed in plastic tubes containing 70% alcohol. The slide assembly was performed according to the laboratory protocol following the steps of clarification, staining, dehydration, and diaphanization. The identification of adult helminths was based on morphological evaluation according to ^{Werneck (2007)} and ^{Fernandes and Kohn (2014)}, and the eggs were evaluated morphologically and morphometrically.

To perform the coproparasitological examination, stool samples were collected directly from the rectal ampoule during the necropsy. The feces were processed according to the sedimentation technique described by ^{Foreyt (2005)}, and the observed eggs were collected and placed on slides, classified under an optical microscope according to morphology (^{Greiner 2013}), and quantified according to morphotype. A comparison was made between eggs from coprological analyses and eggs observed in the collected adult flukes.

RESULTS

Of the 36 necropsied turtles, it was not possible to collect samples from 2 of them because of absence of fecal content. Of the 34 coproparasitological exams performed, 31 gave positive results for the presence of helminth eggs. Four different types of eggs were found and classified according to their morphology (Fig. 1) and morphometry (Table 1).

Figure 1 Photomicrograph of the types of eggs recovered by coproparasitological exams. (a) egg type 1, (b) egg type 2, (c) egg type 3, and (d) egg type 4. 

Adult helminths were collected in 34 of the 36 turtles evaluated, and 18 species of trematodes belonging to 4 families were found (Table 2). Eggs measured directly inside the adult helminths fixed on lamina showed morphological diversity in relation to size, with lengths ranging from 19.07 to 99.28 μm and widths ranging from 11.35 to 45.66 μm, with an average length of 45.52 ± 28.23 μm and an average width of 24.81 ± 16.06 μm. This size variation in eggs measured inside adult parasites may be related to the maturation cycles of eggs before their oviposition. The egg measurements of each helminth species are specified in Table 1. One parasite species (Learedius learedi) did not present eggs inside the specimen, making morphometric analysis impossible.

DISCUSSION

Although eggs are generally more abundant than adult worms, microscopic identification of eggs is difficult due to similarities in the morphology of eggs from congeners or even members of different genera given the frequency of mixed infections in the same individual (^{Chapman et al. 2017}, ^{Stacy et al. 2017}). The eggs found in this study are morphologically similar to the few descriptions existing in the researched literature. In this study, the main limitation was comparing the relation of the egg morphometry observed in the adult helminths collected during necropsy and correlating with the presence of the eggs recovered in coproparasitological examinations. The adult helminths found in this work (Table 2) were previously described by ^{Gomes et al. (2017)} in the same region and were observed to have a major prevalence for Cricocephalus albus, Metacetabulum invaginatum, and Neoctangium travassosi (^{Gomes et al. 2017}).

^{Greiner (2013)} used a sedimentation technique as a diagnostic principle, similar to this study. The ^{Foreyt (2005)} technique was also used to describe egg measurements for the species C. albus, Charaxicephalus robustus, Pronochephalus obliquus, and Schizamphistomum scleroporum. ^{Marangi et al. (2020)} also performed coprological exams using morphological and morphometric characteristics to identify eggs, as reported by ^{Greiner (2013)}.

This size variation in eggs measured inside adult parasites may be related to the maturation cycles of eggs before their oviposition. In relation to egg morphometry in adult helminths, ^{Werneck (2007)} obtained results similar to those of the present study for the egg measurements of C. albus, Cricocephalus megastomun, Deuteribaris proteus, M. invaginatum, N. travassosi, Pleurogonius longiusculus, Polyangium linguatula, and P. obliquus. ^{Santoro et al. (2007)}, when examining adult specimens of Pleurogonius tortugueroi isolated from C. mydas, observed eggs measuring on average 27.00 μm in length by 14.00 μm in width, values close to those found in this work for parasites of other species of the same genus: Pleurogonius linearis (33.17 × 15.98 μm), Pleurogonius lobatos (25.87 × 12.41 μm), and P. longiusculus (24.67 × 12.29 μm).

In this study, helminths Deuterobaris intestinalis, D. proteus, N. travassosi, and S. scleroporum presented egg sizes close to each other and morphometrically similar to egg type 1. ^{Greiner (2013)} identified eggs similar to these, 98.00 × 60.00 μm, as S. scleroporum and D. proteus. This same author also reported eggs similar to types 2 and 3, belonging to helminths of the genera Carettacola (88.00 × 38.00 μm) and Hapalotrema (414.00 × 36.00 μm), respectively, belonging to the family Spirorchiidae. ^{Work (2005)} found eggs similar to types 2, 3, and 4 with measurements of 135.00 × 67.00 μm, 276.00 × 37.00 μm, and 45.00 × 30.00 μm, respectively, being identified as helminths belonging to the family Spirorchiidae.

Some studies that microscopically analyzed tissue samples (spleen, lungs, intestine, kidneys, and heart) (^{Santoro et al. 2007b}) and fecal samples of C. mydas (^{Santoro et al. 2020}) identified eggs similar to types 3 and 4 of this study as corresponding to eggs of spirochiid helminths (Digenea: Spirorchiidae) of the genera Hapalotrema and Neospirorchis, respectively. ^{Jerdy et al. (2019)} also recorded the observation of spiral-shaped eggs with a circular appearance, similar to species of the genus Neospirorchis, in ocular tissue of C. mydas. Finding helminth eggs of the family Spirorchiidae in the coproparasitological examination reveals important data for the study of the ecology of these parasites, since their life cycle is not yet well elucidated (^{Chapman et al. 2019}).

Four different types of eggs were found and identified according to morphology and morphometry in the coproparasitological examination. Type 1 eggs have the characteristics of helminth eggs of the families Microscaphidiidae and Cladorchiidae, and the eggs of types 2, 3, and 4 have characteristics of helminth eggs of the family Spirorchiidae.