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Changes in several physiological and biochemical parameters have been reported to be associated with FP in green turtles. They include anemia, immunosuppression, hypoproteinemia, hypoalbuminemia, hypoglycemia, uremia, electrolyte imbalance, increased activity of liver enzymes, low levels of cholesterol and triglycerides, propensity to acquire systemic bacterial infections, and alterations in the number of white blood cells (Foley et al. 2005; Work & Balazs 1999; Aguirre & Balazs 2000; Santos et al. 2015). Furthermore, previous studies have reported a relationship between the hematological status of turtles and the severity of tumors (Work & Balazs 1999; Santos et al. 2015). According to Balazs (1991), tumor scores reflect the spectrum of severity of FP in green turtles. In advanced stages of the disease, clinical tests usually indicate acidosis, imbalance in the ratio between calcium and phosphorus concentrations, anaemia, hypoproteinemia paralleled by hypoglobulinemia and hypoalbuminemia, hypoglycemia, uremia, and increased activity of liver enzymes (Aguirre et al. 1995; Work & Balazs 1999; Aguirre & Balazs 2000; Santos et al. 2015). Immunosuppression may also occur paralleled by bacteremia (Work et al. 2001, 2003).

However, these reported alterations are generalized responses to stressors and clear evidence of a primary response that could be used as a reliable biomarker of FP in green turtles is still lacking. This is likely because blood parameters in sea turtles can be affected by several intrinsic and extrinsic factors (Aguirre et al. 1995). For example, the wide reference ranges for many biochemistry markers reported in green turtles with FP could be associated with factors such as gender or body size of the specimens, both of which are known to affect biochemistry. Previous studies have been performed on individuals of both sexes and of a wide range of body sizes (Work & Balazs 1999; Aguirre & Balazs 2000;), which could hamper the identification of a potential and reliable biomarker of the disease.

The coastal zone of the southern Atlantic Ocean is an important feeding area and habitat for the development of juvenile green turtles. In Brazil, the first case of FP in green turtles was reported in 1986 by the Marine Turtle (TAMAR) Project (Baptistotte et al. 2005). Since then, an increase in the prevalence of the disease has been reported in several studies across the TAMAR project region. For example, Mehnert et al. (2001) reported an increase along the Brazilian coast between 1990 and 1999 and the prevalence of FP in juvenile green turtles from the Ubatuba coastal region of Brazil rose from 0 to 24% in the 12 years from 1986 to 1998 (Rossi et al. 2009).

The aim of the present study is to identify a primary biochemical response that could be used as a potential biomarker of FP for use in future evaluation and monitoring of health status of immature and juvenile green turtles found in coastal waters of the southern Atlantic Ocean.

Green turtles were captured using purse seine and scuba diving activities from January 2011 to March 2012 in the TAMAR Project area (23°26’S, 45°05’W, Ubatuba, São Paulo State, southeastern Brazil). The turtles that ended up trapped in the purse seines were used in the study; in addition, turtles were captured swimming freely to complete the sampling. Turtles were transferred to the TAMAR Project facilities for blood sample collection and physical examination, as described below. Blood samples (5 ‒ 10 ml) of 36 green turtles (C. mydas) were collected by puncture of the dorsal cervical sinus using disposable 10-ml syringes with 25 x 7-gauge needles. This procedure is considered a minimally invasive technique (Owens & Ruiz 1980). Blood samples were immediately transferred to harvesting tubes without anticoagulant. All procedures were performed under a permit of the Brazilian Ministry of Environment (permit # 25829-2 SisBio/ICMBio/ MMA).

After blood sampling, each turtle was subjected to a visual examination, including evaluation of general physical condition and the presence of external FP tumors. Therefore, the presence of internal tumors in green turtles assessed as clinically normal cannot be ruled out. Considering that levels of hematological and serum biochemical parameters may differ according to the severity of the tumors (Santos et al. 2015; Hirama et al. 2014), green turtles were grouped and analyzed according to this condition (score 0: non-afflicted with FP; score 1: lightly afflicted with FP; score 2: moderately afflicted with FP; and score 3: heavily afflicted with FP), as described by Work & Balazs (1999).

After visual examination, curved carapace length (CCL; to the nearest 0.1 cm) was measured. Green turtles were then tagged on their front flippers, using metal Inconel style flipper tags provided by the TAMAR Project, placed in the center of the first or second scale proximal to the body of the turtle; turtle’s with FP score 0 were immediately released close to the site of capture. Individuals afflicted with FP had their tumors removed surgically and were maintained for some time for observation and recovery. Once deemed sufficiently recovered, they were released near their site of capture.

Immediately after collection, sampled blood was divided into 2 tubes, one with heparin and one without anticoagulant. Whole blood was transferred to duplicate microcapillary tubes and centrifuged for 5 min using a microhematocrit centrifuge (Spin 1000, Microspin, Brazil). Hematocrit value was expressed as the average percentage value observed between the two hematocrit capillary tubes. Heparinized blood was used for the total leukocyte (WBC) and red blood cells (RBC) counts, which were performed using a Neubauer chamber. For each sample, two blood smears were also prepared, one fixed in methanol, and one stained with Wright-Giemsa stain (Campbell 2014). Differential leukocyte counting (heterophil, lymphocyte, eosinophil and monocyte) was performed manually; cells were identified using data reported in the literature regarding the morphology of sea turtle cells (Casal & Orós 2007; Zhang et al. 2011; Acevedo et al. 2012).

Immediately after the hematocrit analysis, the remainder of the whole blood was centrifuged at 1,800 x g for 5 min (Centribio 80- 2B, Centribio, China). There was no visual evidence of hemolysis. Serum obtained was transferred into cryogenic vials kept on dry ice, transferred to the laboratory, and stored in an ultrafreezer (-80 °C) until analysis.

Serum biochemical parameters analyzed included cortisol, glucose, cholesterol, triglycerides, uric acid, urea, creatinine, total protein, albumin, globulin, bilirubin (total, direct and indirect), sodium, potassium, magnesium, chloride, calcium, and phosphorus concentration, as well as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), creatine kinase (CK), lactate dehydrogenase (LDH), and 3-hydroxy-3-methylglutaryl- CoA reductase (HMGR) activity. Additionally, serum testosterone concentration was measured for sex identification (Bolten & Bjorndal 1992; Owens 1997).

Commercial reagent kits used to perform the serum biochemistry analyses were purchased from Labtest Diagnóstica (Lagoa Santa, MG, Brazil) and Sigma-Aldrich (St. Louis, MO, USA). Analyses were performed using a flame photometer (Micronal, Campo Grande, MS, Brazil), an automated Roche Cobas Mira Classic Chemistry Analyzer (Roche Molecular Systems, Branchburg, NJ, USA) and a microplate reader (Victor, PerkinElmer, Waltham, MA, USA).

For all parameters, data were expressed as X ±SE. For each parameter, data normality was checked by the normal probability plot of raw residuals while homogeneity of variances was verified using the Cochran C test. Data on CCL, hematocrit, eosinophils, basophils, glucose, uric acid, potassium, magnesium, phosphorus, ALT, ALP, GGT, CK, LDH, and HMGR were mathematically transformed (decimal logarithmic transformation) to meet the analysis of variance (ANOVA) assumptions (data normality and homogeneity of variances). Mean values for all parameters were compared using one-way ANOVA followed by the Fisher LSD test. In all cases, the significance level adopted was 95% (α = 0.05). Mean values of HMGR activity and serum cholesterol concentration were significantly different among the groups of green turtles. Therefore, they were subjected to the Product- Moment correlation analysis. In all cases, the significance level adopted was 5% (α = 0.05) (Sokal & Rohlf 1995).

Among the 36 green turtles sampled, 31 individuals (CCL = 40.3 ±1.6 cm; BM = 8.8 ±1.3 kg) showed serum testosterone concentration <10 pg/ml and were considered as being females (Bolten & Bjorndal 1992; Owens 1997). The CCL of these individuals ranged from 29.3 to 62.0 cm, which is in the range of sizes for immature and juvenile green turtles. Among the 31 green turtles analyzed in the present study, 14 individuals were non-afflicted with FP (score 0; CCL = 36.8 ±1.2 cm) while 17 green turtles had FP (external tumors). Among those with FP, 5 green turtles were lightly afflicted with FP (score1; CCL = 47.1 ±5.7 cm), 5 green turtles were moderately afflicted with FP (score 2; CCL = 37.6 ±2.1 cm), and 7 green turtles were heavily afflicted with FP (score 3; CCL= 44.4 ±4.2 cm). There were no significant differences in CCL among these groups of green turtles.

No significant difference was observed in hematological parameters among the four groups of green turtles analyzed (Table 1). However, green turtles moderately or heavily afflicted with FP showed significantly lower serum cholesterol concentration than those non-afflicted or lightly afflicted with FP (Table 2). Also, green turtles with FP (lightly, moderately and heavily afflicted with FP) had significantly reduced serum HMGR activity respect with those non-afflicted with FP (Table 3). Indeed, a significant and positive correlation was observed between serum HMGR activity and cholesterol concentration (r = 0.48; p = 0.01).

Table 1. Hematological parameters in juvenile female green sea turtles with and without fibropapillomatosis (FP). Individuals were collected in coastal waters of the southern Atlantic Ocean (Ubatuba, southeastern Brazil) from January 2011 to March 2012. They were grouped according to the severity of tumors. Data are expressed as X±SE, with sample size in parentheses. Same letters indicate mean values are not significantly different (p<0.05).

Table 2. Serum biochemical parameters in juvenile female green sea turtles with and without fibropapillomatosis (FP). Individuals were collected in coastal waters of the southern Atlantic Ocean (Ubatuba, southeastern Brazil) from January 2011 to March 2012. They were grouped according to the severity of tumors. Data are expressed as X±SE, with sample size in parentheses. Same letters indicate mean values are not significantly different (p<0.05).

Table 3. Serum enzyme activity in juvenile female green sea turtles with and without fibropapillomatosis (FP). Individuals were collected in coastal waters of the southern Atlantic Ocean (Ubatuba, southeastern Brazil) from January 2011 to March 2012. They were grouped according to the severity of tumors. Data are expressed as X±SE, with sample size in parentheses. Same letters indicate mean values are not significantly different (p<0.05). AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; GGT: gamma glutamyl transferase; CK: creatine kinase; LDH: lactate dehydrogenase; HMGR: 3-hydroxy-3-methylglutaryl-CoA reductase.

Analysis of blood parameters is a useful tool to evaluate the health condition of turtles, as they can provide information for the diagnosis and prognosis of diseases. Furthermore, they have been used as indicators of physiological changes due to illness, stress or exposure to environmental contaminants (Omonona et al. 2011). Therefore, hematological and blood chemistry analyses can be considered important steps in determining the physiological and pathological conditions in turtles (Gelli et al. 2009).

Among all the reported effects, it is worth noting that only 2 out of the 34 parameters analyzed in the present study significantly varied among the groups of green turtles with different tumor scores. In this case, reduced serum HGMR activity and blood serum cholesterol concentration were observed in the green turtles heavily (score 3) or moderately (score 2) afflicted with FP compared with those lightly (score 1) or non-afflicted (score 0) with FP. Furthermore, serum HMGR activity was also lower in green turtles lightly afflicted with FP than in those non-afflicted with FP.

According to Aguirre & Balazs (2000), turtles less than 35 cm are immature and those with CCL within the range of 35 to 65cm are juveniles. This scheme for grouping sea turtles was also adopted by Labrada-Martagón (2010). Therefore, according to this scheme, immature and juvenile female green turtles were evaluated in the present study. Also, based on CCL range, green turtles analyzed in the present study could be considered as post- pelagic juveniles (Santos et al. 2015). In this context, it is worth noting that no significant difference in CCL was observed among the four groups of green turtles analyzed in the present study. Therefore, conditions described above may have minimized the potential high variability in the response of biochemical and physiological parameters which would be associated with intrinsic factors, such as sex and body size (Aguirre et al. 1995; Camacho et al. 2013). Indeed, they could help to explain the discrepancy among our findings and those from previous studies with green turtles with and without FP (Foley et al. 2005; Aguirre et al. 1995; Work & Balazs 1999; Aguirre & Balazs 2000; Santos et al. 2015).

It is important to note that reduced serum cholesterol concentration, as observed in the present study, was also reported for green turtles with FP from other regions (Aguirre et al. 1995; Aguirre & Balazs 2000; Work et al. 2001, 2003). This finding suggests that the observed drop in serum cholesterol concentration may be a primary response of green turtles to FP. In turn, the reduced serum cholesterol concentration may be related to the reduced activity of serum HMGR observed in green turtles afflicted with FP. Indeed, the level of reduction in serum cholesterol concentration (53.6%) was paralleled by a quite similar reduction (48.5%) in serum HMGR activity. Furthermore, a significant and positive correlation was observed between these two parameters in this study. It is worth noting that HGMR catalyzes the four- electron reduction of 3-hydroxy-3-methylglutaryl-CoA (HMG- CoA) to coenzyme A (CoA) and mevalonate, which is the rate- limiting step in sterol biosynthesis (Kritchevsky & Kritchevsky 1992; Holdgate et al. 2003). Therefore, an inhibition of serum HMRG activity would induce a lower rate of cholesterol synthesis, thus leading to a reduced level of serum cholesterol, as observed in green turtles with FP evaluated in the present study.

In addition to the influence of the reduced HMGR activity, as discussed above, a higher rate of cholesterol oxidation could also help to explain the lower concentration of serum cholesterol observed in green turtles moderately and heavily afflicted with FP compared with those non-afflicted or lightly afflicted with FP. Some possible causes of a higher rate of cholesterol oxidation would be an excessive exposure to ultraviolet radiation (Morin et al. 1991) and/or the oxidative stress induced by exposure to environmental contaminants (Monserrat et al. 2007). Although the current thinking is that FP is associated with a herpesvirus infection (Rodenbusch et al. 2012, 2014), these environmental factors may trigger processes that influence FP expression and lesions (Aguirre et al. 1994; Santos et al. 2010; Van Houtan et al. 2014). Therefore, future studies should address the influence of UV and aquatic contaminants on the rate of cholesterol oxidation in green turtles for a better understanding of FP etiology. In fact, abnormally low concentration of serum cholesterol is reported as being a reliable biomarker of malignancy in humans (Ahn et al. 2009).

In summary, data reported in the present study indicate that reduced serum HMGR activity and cholesterol concentration are adequate and reliable biomarkers of FP in immature and juvenile female green turtles. Further studies are needed to determine whether these biomarkers may also be applied to juvenile male green turtles.

Acknowledgments. The authors thank Roberta Vargas (Laboratório Dr. Vargas, Rio Grande, RS, Brazil) and Paloma Calábria Carvalho for laboratory assistance and José Henrique Becker, Max Rondon Werneck, Renato Velloso da Silveira, Antônio Mauro Correa (TAMAR Project, Ubatuba, SP, Brazil) and Lucas Feijó Bianchini (Universidade Federal do Rio Grande-FURG, Rio Grande, RS, Brazil) for field assistance. Financial support was provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, DF, Brazil; Instituto Nacional de Ciência e Tecnologia de Toxicologia Aquática, grant #573949/2008-5), Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES, Brasília, DF, Brazil; Programa Ciências do Mar), and the International Development Research Center (IDCR, Ottawa, ON, Canada; Project #104519-003). CCS was a PhD fellow from the Brazilian CNPq. AB is a fellow from the Brazilian CNPq (grant #304430/2009-9) and is supported by the International Canada Research Chair Program from IDRC.

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