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Sea turtle nesting on Casey Key is monitored by morning nest surveys and a nighttime tagging program. Turtles encountered on nighttime tagging surveys are inspected for existing identification tags and, if none are found, metal flipper tags and a Passive Integrated Transponder (PIT) tag are applied for future identification. Tagging and standard measurements are taken immediately following egg deposition. After completing the nesting process, green turtles selected for satellite tagging are detained in a corral where the carapace is cleaned and a satellite tag is bonded to the second vertebral scute using construction epoxy (Coyne et al. 2008). Turtles are released when the epoxy is dry.
Figure 1. Green turtle, carapacial dermatitis. At initial presentation (A), the scutes were roughened, exhibited irregular exfoliation, and, in some areas, were absent, exposing an abnormally pale, mottled epidermis. Forty-four days later (B), inflammation is largely resolved and the skin has regained a more normal green color and surface texture. The arrowheads point to the same area of vertebral scute in each image to illustrate partial resolution of the skin lesions.
Figure 2. Green turtle, carapacial dermatitis. Degenerate heterophils (*) associated with bacteria separate the keratin layers of the scute (S). Also visible are the deeper layers of the epidermis (E) and underlying dermis (D). Hematoxylin and eosin stains used.
On 18 June 2019, two nesting green turtles were observed with skin lesions involving the carapace. The lesions were characterized by multiple scute anomalies, including erosions, easy exfoliation, prominent variation in thickness, and formation of inflammatory exudate (Fig. 1A). The carapace condition precluded satellite tag application due to attachment and permit protocols, including concerns related to the health of the skin and potential for exacerbating disease. Studies conducted on curing temperatures of epoxies indicate that the heat of the exothermic curing process can transmit through a turtle carapace (Mazzarella et al. 2009; Evans N. & D. Evans pers. comm. 2016). Although the moderate heat produced by the epoxy (DeWalt Powers Pure 50+TM) is acceptable for use on a healthy carapace, application to potentially ulcerated skin or epidermis lacking its normal keratin risks potential physical harm. In addition, the epoxy would not adhere well to non- keratinized surfaces, thus increasing the probability of premature tag loss. Finally, Florida Fish and Wildlife Conservation Commission (FWC) Marine Turtle Permit conditions require that telemetry devices not be attached to injured or compromised turtles. Thus, a conservative approach was taken and affected individuals were immediately released without satellite tag application.
In 2019, 11 of the 37 green turtles (29.7%) encountered were observed with the described carapacial lesions. It is possible that more turtles had this condition, but went undetected. Similar lesions have been documented in captive animals where temperature and water quality are possible contributory factors (Chuen-Im et al. 2010; Muñoz et al. 2013). To our knowledge, this carapacial condition has not been characterized in the wild nesting population. Therefore, although it was not the original intention of the study, an investigation into the cause of the carapacial lesions in nesting green sea turtles was undertaken.
After the condition was first observed, a thorough visual examination of the carapace was consistently conducted during the egg-laying phase of nesting to identify affected individuals. Carapace assessments were added to the existing tagging protocol to avoid unnecessary corralling of affected individuals. Carapace lesions were photographed, when possible, after shielding the turtle’s head with a dark towel.
A permit modification (FWC Marine Turtle Permit #19-155) was obtained to allow collection of cytology and histology samples and cultures from affected green turtles. When an individual with lesions was identified, the carapace was rinsed with sterile water prior to collecting two superficial 6 mm scute biopsies at the junction of normal and abnormal skin. One biopsy was placed into a cryovial filled with neutral phosphate-buffered formalin; the other was kept on ice and later frozen at 0° C. Histological examinations of scute biopsies were conducted at NOAA/NMFS/Office of Protected Resources Pathology Laboratory. Once authorized, each biopsy site was additionally swabbed with a rayon-tipped BD BBLTM CultureSwabTM Plus. Biopsy sites were subsequently cleaned with betadine and triple antibiotic ointment was applied to prevent further infection. Culture swabs were stored at room temperature and submitted for aerobic culture (Sarasota Memorial Hospital) using Tryptic Soy Agar with 5% Sheep Blood, Chocolate Blood Agar, MacConkey Agar, and Tryptic Soy Broth incubated at 35°C in CO2. Bacteria species were identified using the VITEK® 2 (bioMérieux, Inc.) microbial identification system.
Eleven affected green turtles, including nine neophytes and two remigrants, were observed between 16 July and 1 August 2019. Scute biopsies were collected from nine turtles. The most consistent histopathological finding (n = 6 individuals) was predominantly heterophilic, superficial dermatitis associated with Gram-negative bacteria (Fig. 2). Fungal colonization of the scute surface was observed in one individual with no evidence of bacteria. Two individuals showed no sign of fungal hyphae or bacteria. There was no histomorphological evidence of viral infection in any individuals.
Table 1. Bacteria identified from areas of carapacial dermatitis affecting four nesting green turtles (Chelonia mydas). Bacillus spp. was found on all individuals while other species were unique to each individual.
Table 2. Comparison of nesting parameters of green turtles encountered by tagging personnel between 29 May and 01 Aug 2019. CCLnt = Curved carapace length from notch to tip.
Culture swab results were obtained from biopsy sites of four turtles and pooled by individual for identification of bacteria. A total of 12 different bacteria were identified (Table 1), each unique to individual turtles except for Bacillus spp., which was found on all individuals. The identified bacteria were primarily Gram-negative species that naturally inhabit marine and soil environments, some of which are previously reported opportunistic pathogens of sea turtles and other reptiles, including Pseudomonas, Vibrio, and Bacillus spp. (Pasmans et al. 2020). A fecal bacterium (Enterobacter cloacae) was identified on one turtle.
Of the 11 green turtles observed with carapacial lesions, all were visually examined and ten were photographed. The severity and distribution of lesions ranged from diffuse, superficial involvement of the carapacial scutes (n = 8), to a few discrete, round ulcers (1-2 cm diameter) exposing bone (n = 1). The lesions were most commonly observed on vertebral (V2 and V3) scutes (n = 8) and in scute margins (n = 5). Six individuals were re-evaluated photographically on subsequent encounters and exhibited partial resolution of the carapacial lesions after 12-44 days (Fig. 1B). With the exception of the carapacial lesions, all turtles were in robust body condition and appeared otherwise healthy based on external examination.
Post-hatch nest inventories were conducted for all green turtle nests to determine clutch size, hatch success, and emergence success. A Wilcoxon rank-sum test (R version 3.6.1) was used to determine if these factors differed between affected turtles (n = 11 individuals, 28 nests) and other green turtles (n = 22 individuals, 29 nests). No difference was observed for any parameter (Table 2). Affected turtles also did not differ from other green turtles in size as measured by curved carapace length (notch to tip) or nest incubation duration (Table 2).
Based on our findings, we characterized the carapace condition as bacterial dermatitis ranging from multifocal to diffuse in terms of the extent to which the carapace was affected. The abnormal gross appearance of the carapace resulted from erosion and loss of scutes, associated infiltration by leukocytes (inflammation), and proliferation of bacteria within affected scutes. The lack of histological evidence of bacterial dermatitis in three of the nine individuals with gross lesions is likely due to the superficial nature of the infections and loss of affected scute during sampling.
Culture results yielded multiple isolates, which was unsurprising given that abnormal skin is easily colonized by bacteria (Glazebrook & Campbell 1990; Aguirre et al. 1994; Boylan et al. 2017). Our results are similar to a study by Santoro et al. (2006), which found a wide variety of beneficial and pathogenic bacteria on healthy, wild nesting green turtles in Costa Rica. It is important to note that we did not culture the skin of unaffected individuals for comparison and thus did not characterize the fauna of normal skin during our observations.
Bacterial dermatitis in sea turtles and other reptiles typically results from abrasions or lacerations of the skin, environmental conditions that favor bacterial growth, or an underlying condition that alters the skin and/or its defenses against microbial infection (Boylan et al. 2017). Therefore, we considered potential underlying causes that may have contributed to carapacial infections on green turtles in our study.
Green sea turtles mate immediately prior to nesting season. Mating behavior is characterized by the male mounting the female, plastron to carapace, with the male using claws to grasp the female carapace (Booth & Peters 1972). Evidence of claw marks and bites have been observed on female carapaces during nesting season (Booth & Peters 1972; Mazzarella, K.T., unpubl. data), and may be associated with secondary infection of the skin (Boylan et al. 2017). We did not find the lesions in this study to be concentrated near the shoulders where males typically take hold, but some degree of more generalized damage to the scutes during mating cannot be completely ruled out.
Several species of barnacles have been documented attached to and sometimes embedded in the skin of sea turtles, including that of the carapace (Frick & Pfaller 2013). Attachment or removal of such epibionts can damage the skin to the degree that secondary infection occurs (Stacy et al. 2017). We did not find any epibionts on affected nesting turtles or see any residual marks suggestive of prior epibiont attachment; however, surface organisms may have detached or been removed by grooming prior to our observations.
Warm water temperatures and daily rain events may contribute to conditions favorable for bacterial growth and poor water quality in the Gulf of Mexico during sea turtle nesting season. Heavy summer rains lead to transport of nutrients into the Gulf of Mexico via surface runoff, overflows of sewage treatment plants, and riverine transport. The Florida Healthy Beaches Program conducts routine bacterial monitoring of beaches and coastal waters (<http://www.floridahealth.gov/environmental-health/beach-water-quality/index.html>). Poor water quality indicators were not documented on the beach or in coastal waters adjacent to the study site prior to or during the study. As the turtles exhibited the dermatitis while nesting, it is possible they encountered poor water quality at their foraging ground or during migration. Investigation into the foraging ground of affected individuals will be further pursued via stable isotope analysis.
Another consideration in this region is exposure to brevetoxins, algal toxins produced by the red tide organism Karenia brevis. It is hypothesized that brevetoxins may have sublethal effects on health and immune function (Perrault et al. 2016; Perrault et al. 2017). FWC hosts statewide red tide (Karenia brevis) status reports (<http://myfwc.com/research/redtide/statewide/>). Southwest Florida experienced a long-term major harmful algal bloom in the form of red tide between November 2017 and February 2019 (<http://myfwc.com/media/21885/bloom-historic-database.pdf>) with low to medium levels recorded in the Marquesas Keys, a known adult green turtle foraging ground (Hart et al. 2013; Herren et al. 2018). Karenia brevis was either not detected or was found at very low (>10,000-100,000 cells/liter) concentrations in Florida during March through June 2019 (<http://www.flickr.com/photos/myfwc/sets/72157635398013168/with/25508900517/>), thus there is no environmental evidence to suggest brevetoxin exposure, particularly high-level exposure, in the months immediately prior to nesting.
The underlying cause(s) of the carapacial infections reported here remains unknown. Although the condition resolved during the course of our study and was not believed to jeopardize survival of affected females; skin infections can be life-threatening in sea turtles as evidenced by observations in stranded animals (Boylan et al. 2017). Continued monitoring is recommended to follow the occurrence of carapace disease in subsequent years. Further study may benefit from more comprehensive sampling of the area nesting aggregation from the start of nesting season in order to better document prevalence and follow progression of carapace disease in more individuals. Furthermore, the addition of hematology and blood chemistry would provide more information about the health status of affected turtles beyond that evident from external examination and reproductive metrics.
Abnormalities affecting skin are an important aspect of aquatic wildlife health monitoring because the skin can reflect injurious external factors, such as water quality, as well as a host of systemic factors, such as diet, immune function, other organ function, and disease states. Emergence of conditions as described here merit attention given myriad concerns related to climate, pollution, and other changes in the marine environment that can affect sea turtle health. In addition, although the primary objective of tagging projects is mark/recapture documentation and tracking animal movements, the health of individual turtles should always be considered when conducting field studies. Thorough examination of turtles during tagging encounters may prevent unnecessary detainment of individuals ineligible for studies, avoid unintended negative effects on individuals with health problems, and may help reveal concerns otherwise undetected.
Acknowledgements. Thanks to Taylor Brunson and Kasey Wade for their tagging and sampling efforts and the morning patrol staff, volunteers, and interns that monitored and inventoried nests. Much appreciation to Lynne Byrd, Whitney Greene, Kelly Sloan, Dean Bagley and Whitney Crowder for providing consultation on the condition and Sue Forrest for interpreting cultures. The Mote Sea Turtle Hospital and Strandings Investigations program provided supplies and sample transport. This project was funded in part by a grant awarded from the Sea Turtle Grants Program. The Sea Turtle Grants Program is funded from proceeds from the sale of the Florida Sea Turtle License Plate. Learn more at <http://www.helpingseaturtles.org>. Work was conducted under Florida Marine Turtle Permits 155 and 048 and Mote Marine Laboratory IACUC 19-04-KMz1.
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