Green sea turtles (Chelonia mydas) are found in tropical and subtropical oceans, migrating to and from their nesting beaches. After the pelagic phase, during which they are omnivorous, the green sea turtles move to the coastal areas and become herbivorous, spending the majority of their life in areas where their diet consists largely of seagrass and/or macroalgae. Juveniles often become residents of their feeding grounds for several years until they mature (Mortimer 1982; Bjorndal 1997; Spotila 2004). This species is susceptible to fibropapillomatosis (FP), a debilitating disease characterized by the development of skin tumors (Adnyana et al. 1997). The first report of FP was in 1936, in a female from Key West (Smith and Coates 1938). FP was first reported in Brazil in 1986, in the state of Espírito Santo. According to data collected from 2000 to 2005, the Brazilian prevalence of FP (in areas monitored by the TAMAR/ICMBio Project) is approximately 15% (Baptistotte 2007). FP is most frequently reported in sea turtles at coastal feeding grounds (Ene et al. 2005; Chaloupka et al. 2009), and it has been well established that green sea turtles use the coastal areas more than other marine turtle species (Hirth 1997). This coastal behavior may partially explain why this species is apparently more susceptible than others.

The Chelonid herpesvirus 5 (ChHV5) has been proposed as the primary etiological agent of FP, and green sea turtles with many tumors can act as superspreaders in the ocean (Herbst and Klein 1995; Lackovich et al. 1999; Work et al. 2014). The presence of this alphaherpesvirus was detected in 95% of natural infections and researchers reported 100% of association between this virus and tumors induced experimentally (Quackenbusch et al. 2001; Ene et al. 2005). However, studies revealed that this potential hypothesis could not explain several aspects of the pathogeneses of FP in sea turtles, leading to the conclusion that its etiology is likely multifactorial, in which several genetic, environmental, and biological cofactors (including host immune response) may also play a significant role (Herbst and Klein 1995; Work et al. 2009; Van Houtan et al. 2010). In particular, immunosuppression is also frequently present in sea turtles with FP (Aguirre et al. 1995).

The investigation of physiological processes through blood sample analyzes constitutes an important conservation tool (Bolten and Bjorndal 1992; Chansue et al. 2011). Flow cytometry is one of the best available methods to evaluate vertebrate immune function modulation. In green sea turtles, this technique has been used to identify leukocyte populations, leukocyte nuclear damage caused by environmental pollutants, and to assess phagocytosis and oxidative burst activities (Matson et al. 2005; Hays and McBee 2007; Muñoz et al. 2009, 2014; Rossi et al. 2009). Phagocytosis and oxidative burst are particularly fundamental aspects of the innate immunity and antimicrobial defenses, as shown by in vitro assays broadly used to assess the health and immune status of vertebrates (Hasui et al. 1989; Lehmann et al. 2000; Kumar and Rai 2011; Muñoz et al. 2014). Furthermore, the evaluation of reactive oxygen species (ROS) and phagocytosis can be used as an indicator of the immune status of these animals (Labrada-Martagón et al. 2011).

Because immunosuppression has been associated with FP, we hypothesized whether it could also influence the innate response in green sea turtles. To test this hypothesis we used flow cytometry to evaluate leukocyte activity in blood samples of green sea turtles with and without FP.

METHODS

RESULTS

Flow cytometry analysis identified three cell populations: lymphocytes (Lym; R1), monocytes (Mon; R2), and granulocytes (Gran; R3) (Fig. 1), confirmed by optical microscopy. Phagocytosis and oxidative burst results between green sea turtles with and without FP did not differ. However, we observed significant difference between stimulus and control group for phagocytosis results: green sea turtles without FP, Lym (p < 0.01), Mon (p < 0.001), and Gran (p < 0.05); and green sea turtles affected by FP, Lym (p < 0.001), Mon (p < 0.001), and Gran (p < 0.001) (Fig. 2).

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There was no difference among Δ_MFI-values of cell populations (R1, R2, and R3) from green sea turtles without FP. However, cells types from specimens with FP produced different activity levels. Lym and Mon had significantly higher phagocytosis than Gran (p < 0.05 and p < 0.001, respectively), whereas Lym showed lower oxidative burst activity than Gran and Mon (p < 0.01 and p < 0.001, respectively) (Fig. 3).

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DISCUSSION

Flow cytometry identified 3 well-defined regions corresponding to lymphocytes, monocytes, and granulocytes, contrasting with a study by Muñoz et al. (2014), in which the authors reported 2 regions after evaluating blood samples from green sea turtles using FACSCalibur one presenting high granularity and variable size (heterophils) and another had low granularity and less variable size (lymphocytes). However, this study evaluated cell populations on forward scatter (FSC) in linear scale, whereas our study used the logarithmic scale. We attempted to confirm leukocyte subpopulations using the BD FACSAria flow cytometer (based on relative size and complexity), obtaining conclusive results only for cells placed at R1 (88.33% of lymphocytes, 10% of heterophils, and 1% of monocytes). Slides prepared in cytocentrifuge (Cytospin FANEM 248) containing cells found at R2 and R3 demonstrated the presence of monocytes and granulocytes, respectively, but quantification of these cells was not possible because of their poor post-sorting morphology.

We observed an increase of MFI in stimulated samples when PMA was applied for oxidative burst analyzes but without significant differences between control and stimulated groups. Studies on blood samples of Centropomus parallelus fish indicated a significant increase in monocytes and thrombocytes phagocytosis following naphthalene exposure and a decrease of oxidative burst activity after PMA stimulation (Affonso 2006). We observed that monocytes exhibited a higher oxidative burst than lymphocytes, also described by Rousselet et al. (2013) in loggerhead sea turtles (Caretta caretta). PMA is considered a good indicator of cellular metabolism and bactericidal activity. Studies with fish reported the effects on oxidative burst in PMA stimulated cells (Ortuño et al. 1999; Stosik et al. 2002; Serada et al. 2005).

Heterophils were the most frequently counted granular cells on blood extensions and on slides prepared following Percoll isolation. Based on the small number of eosinophils, we conclude that our results indicate further heterophil phagocytic activity and ROS production varying according with the applied stimuli, aside from the phagocytic capacity of mononuclear cells. Eosinophil phagocytic activity has been previously reported for common snapping turtle (Chelydra serpentina) and C. caretta (Mead and Borysenko 1984; Rousselet et al. 2013). Studies focused on quantifying the phagocytic capacity of leucocytes in blood samples from juvenile C. mydas, reported heterophil and mononuclear cells phagocytic percentages of Ovalbumin–Alexa 647 of approximately 61%–75% and 35%–46%, respectively (Muñoz et al. 2014). We found mean phagocytosis values of 41.54%/37.24% (turtles without FP/with FP) for monocytes and 28.21%/32.81% (turtles without FP/with FP) for heterophils. Our values were not as high as those reported by Muñoz et al. (2014), probably because of different stimuli and the separation of mononuclear cells analysis between monocytes and lymphocytes. We observed that lymphocytes presented higher Δ_MFI during phagocytosis than oxidative burst analysis; nevertheless, we did not use biomarkers to identify B and T lymphocytes. Moreover, lymphocytes from green sea turtles with FP exhibited higher phagocytosis (17.25%) than lymphocytes from green sea turtles without FP (6.95%), an important result considering the viral component of this disease. The phagocytic activity of B lymphocytes in reptiles has already been reported by Zimmerman et al. (2010) and was not observed in mammalian B lymphocytes (Vidard et al. 1996; Li et al. 2006).

In the present study, monocytes exhibited similar phagocytic response as the ones previously reported for C. mydas, C. parallelus, and C. caretta (Affonso 2006; Rossi et al. 2009; Rousselet 2013).

Finally, although there was no significant difference in leukocyte activity between green sea turtles with and without FP, we only found significant differences among leukocyte subpopulations of green sea turtles with FP. Flow cytometry may be considered a valid tool in the evaluation of cell activity, able to elucidate some aspects of the immunity of green sea turtles with and without FP and greatly contribute to future studies in the field of sea turtle conservation.