Characterization of Respiratory, Skin, and Cloaca Microbiota in Mediterranean Loggerhead and Green Sea Turtle Populations

doi:10.21203/rs.3.rs-5108643/v1

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Characterization of Respiratory, Skin, and Cloaca Microbiota in Mediterranean Loggerhead and Green Sea Turtle Populations

https://doi.org/10.21203/rs.3.rs-5108643/v1

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Animals are considered biological units with their microbiota, which is composed of commensal, pathogenic, and symbiont bacteria. This microbiota is shaped by the environment and through changes in the animal life history, and it plays a key role in the physiology and fitness of its host. Sea turtles are known to be good indicators of marine ecosystem health, and it is known that their populations are declining in the Mediterranean. Herein, we characterize the upper respiratory, cloaca, and skin microbiota of loggerhead sea turtles under rehabilitation at the Israeli Sea Turtle Rescue Center located in Mikhmoret, Israel, and the Stazione Zoologica Anton Dohrn located in Napoli, Italy to compare sea turtles’ microbiota in the Eastern Mediterranean Sea (EMS) and the Western Mediterranean Sea. Furthermore, we characterize the breath, skin, and cloaca microbiota of green sea turtles kept in captivity for conservation purposes at the Israeli Sea Turtle Rescue Center. Our results showed significant composition differences between the three groups of sea turtles, their surrounding water, and between the organs assessed within and between species. Additionally, we identified core bacterial taxa for the organs sampled in each sea turtle group. The dominant bacteria in all turtle groups belonged to the Gammaproteobacteria, Bacteroidia and Alphaproteobacteriaclasses. Our study is the first one to enable the comparison of the microbial composition of two loggerhead populations in the Eastern and Western Mediterranean. The results aligned with previous findings regarding the dominant bacterial groups and constituted baseline data for the core microbial communities’ composition on wild chelonians as sentinel species in the Tyrrhenian and Levantine basins. The differences in the bacterial profile enable the identification of the turtles' species, sex, and environment. Changes in the microbiota can also indicate their health status. Additionally, the microbiome characterization of the Israeli green sea turtle population allows the understanding of the microbial community composition of a breeding stock aimed to repopulate the EMS to improve its conservation status practically extinct in the area.

loggerhead sea turtles

green sea turtles

respiratory

gut

skin

microbiome

Western

Eastern Mediterranean

Animals are increasingly viewed not as autonomous entities but as biological units or “holobionts”, consisting of the host and its symbiotic organisms (Marchesi and Ravel 2015). Therefore, microbes associated with their hosts have a key impact on their fitness (Bordenstein and Theis 2015). Microbes colonize diverse organs within the host body starting at birth, displaying different assemblages in skin, eyes, ears, mouth, nostrils, lungs, small/large intestine, and urogenital cavities (Hoffman et al., 2016, West et al., 2019). Disruption of the microbiota (change in composition or relative abundance) is defined as dysbiosis, leading to a potential reduction in microbiome function, which can, in turn, result in the capability loss of effective resistance to infectious diseases by the host (Holmes et al., 2011).

Bacterial communities can influence their host's health, physiology, behavior, and ecology (Apprill 2014, Apprill 2017a), and play a key role in the normal function of healthy multicellular organisms. This includes the development of the neurological and immune systems, as well as nutrient absorption in the gut (Marchesi & Ravel 2015, Colston and Jackson 2016, Simon et al., 2019). The host-microbiome association may be symbiotic or pathogenic (Lizé & Lewis 2020), and alteration in microbiota composition can promote shifts towards pathogenic organisms (Bleich and Fox 2015). In wild animals, microbiome differences have been documented between those inhabiting degraded habitats and those in less disturbed areas, with the former showing less diverse microbial communities (Bahrndorff et al., 2016). Although symbiotic bacteria can protect their host by stimulating the immune system or via competitive exclusion (Wiles et al., 2016), they may turn into opportunistic pathogens under adverse environmental conditions (Stevens et al., 2021).

Sea turtles are excellent indicators of ecosystem health due to their long lifespan and juveniles of almost all species spend several years maturing in oceanic waters and then returning to neritic waters that are used as foraging and reproduction areas (Aguirre & Tabor 2004). Coastal waters are particularly susceptible to human-induced environmental degradation. Therefore, sea turtles may be considered an early warning system for the ecological status, enabling action to control threats (Aguirre and Lutz 2004). The loggerhead sea turtle (Caretta caretta) is globally classified as vulnerable to extinction by the International Union for Conservation of Nature (IUCN). However, although the Mediterranean population is listed as Least Concern (Casale and Tucker 2017). Conversely, the Mediterranean green sea turtles (Chelonia mydas) are listed as endangered (Seminoff 2023). Both species are threatened and declining in the Mediterranean Sea due to habitat degradation (e.g., loss of nest beaches), bycatch in marine fisheries or entanglement, boat strikes, marine debris, and exposure to chemical contaminants (Casale and Tucker 2017, Seminoff, 2023). Numerous studies have been conducted on the microbiome of sea turtles, providing valuable insights into various aspects of their microbial communities. However, most of these studies have focused on specific regions or conditions. Studies that have explored the sea turtles’ respiratory microbiome have focused mainly on diseases. For instance, McNally et al. (2021) have recently described the microbiota of cold-stunned Kemp’s Ridley sea turtles recently admitted to the rehabilitation center on the Northwest Atlantic exhibiting a marked dominance of the Vibrionaceae family. Likewise, respiratory and oral bacteria isolated from healthy and sick loggerhead sea turtles of the Western Mediterranean Sea showed a higher abundance of gram-negative bacteria in bronchioalveolar lavage samples (Ciccarelli et al., 2020; Trotta et al., 2021a). Additional microbiome studies have described carapace and skin microbial communities of loggerhead sea turtles in Croatia and Italy and identified several species of gram-negative bacteria (Kanjer et al., 2022; Trotta et al., 2021b).

Greater efforts have been made to explore the gastrointestinal microbiome of loggerhead, Kemp Ridley, and green sea turtles in the North Pacific (McDermid et al., 2020), Northwest Atlantic (McNally 2021a,b), Gulf of Mexico (Price et al., 2017), Western Mediterranean (Filek et al., 2021, Biagi et al., 2019, Arizza et al., 2019, Abdelrhman et al., 2016) as well as Australian population (Ahasan et al., 2017, 2018). According to these studies, bacterial communities may vary by age class, turtle species, and origin (captured in the wild, sampled during rehabilitation, or stranded on the coast). Differences were shown based on their diets (green turtles are known to be generalists as juveniles and herbivores in adulthood, and loggerheads are known to be carnivores). Despite these studies, there remains a significant gap in knowledge regarding the microbiome of sea turtle species in the Eastern Mediterranean Sea.

Hence, our study focused on the microbiome of the two sea turtle species (C. caretta and C. mydas) in the Mediterranean Sea, located in rehabilitation centers in Israel and Italy. We examined the influence of geographical location, anatomical niche (nostrils, breath, skin, and cloaca), and sex on microbiota composition and diversity. Our results provide baseline data that could be used to indicate the health status of these endangered sea turtle species, potentially guiding future decisions about the rehabilitation process and thus ensuring their conservation.

Study Site and Sea Turtle Sample Collection

On the Eastern Mediterranean, microbiota samples were taken from 13 loggerhead sea turtles Caretta caretta recently admitted to the Sea Turtle Rescue Center - Nature and Parks Authority of Israel in Mikhmoret, Israel (32.402052°N, 34.857307°E) (https://en.parks.org.il). Additionally, 23 green sea turtles Chelonia mydas, maintained in captivity as part of the breeding stock for conservation purposes were sampled, as well as three turtles from the Jerusalem Aquarium. On the Western Mediterranean, samples were taken from 11 loggerhead sea turtles recently admitted to the Stazione Zoologica Anton Dohrn in Napoli, Italy (40.832648°N, 14.235457°E) (https://www.szn.it/index.php/it/centro-tartarughe-marine/centro-ricerche-tartarughe-marine). Water samples (1.5 L) from each tank (loggerheads) and each group tank (green turtles) were collected and filtrated upon arrival to the laboratory using single-use Nalgene Rapid Flow Filters (Thermo Scientific, USA). All samples were stored at -20°C until DNA extraction. Mean (± SD) curve carapace length (CCL) was measured. Data of each sampled individual is detailed in Table S1. Samples were obtained from different anatomical locations (nostrils, skin, and cloaca) using a sterile swab (Copan Diagnostics, United States). Breath samples were taken in green sea turtles, where an empty Petri dish was placed on the nares while breathing was stimulated, and subsequently, after a swab was taken of the Petri dish content. The animals were handled avoiding unnecessary stress with the permit N. 3233543 from the Nature and Parks Authority of Israel from 2019 to 2021 and the Registro Ufficiale U.0000992 of the Italian Ministry of Ecological Transition of Italy from 2020.

During the rehabilitation process, loggerhead sea turtles were housed separately in individual tanks of filtered seawater, and they were offered food items of local fish and squid once a day. Every two days, tanks were cleaned using regular bleach, and the water was replaced (Levy et al., 2019). Green sea turtles were kept in two large tanks, one for males and one for females.

DNA extraction, PCR amplification, and amplicon sequencing

DNA was extracted using DNeasy powerSoil kit (Qiagen, CA, United States) following the manufacturer’s protocol. DNA quantity and purity have been estimated using NanoDrop One (NanoDrop Ins., Thermo Scientific). PCR amplification of the microbial subunit ribosomal rRNA gene fragments from isolated DNA was performed using universal primers (515F: GTGYCAGCMGCCGCGGTAA and 806R: GGACTACNVGGGTWTCTAAT) targeting V4 regions of microbial SSU RNA genes. Amplicons were generated using a two-stage PCR amplification protocol as described previously in Naquib et al. (2018). Cycling conditions for the first stage PCR included the denaturation step of 98°C for 15s, annealing at 50°C for 20s, and extension at 72°C for 30s for ten cycles with an additional 25 cycles with annealing at 62°C for 20s (35 cycles in total). The second PCR (95°C for five min, followed by eight cycles of 95°C for 30s, 60°C for 30s and 72°C for 30s), library preparation (including sample barcoding and addition of PhiX DNA spike-in control), pooling and sequencing (Illumina MiniSeq, Fluidigm, South San Francisco, CA; Item# 100–4876) were performed at the Research Resources Center (GRC) within the Research Resources Center (RRC) at the University of Illinois at Chicago (UIC). Raw sequence data is available in the NCBI SRA database under BioProject ID PRJNA1159730 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA1159730?reviewer=886hsem9i113qilcg67aq5v43r).

Sequence data processing

The Dada2 pipeline (dada2 package, R version 1.18.0) was used for sequence data processing (Callahan et al., 2016). Sequences were filtered and trimmed for quality using the ‘filterAndTrim’ command with the parameters maxN set to zero, maxEE set to ‘2’, and trimLeft set to 20 bp. The truncLen was set to 153 bp for both forward and reverse reads. The sequence error estimation model was calculated using default parameters using the ‘learnErrors’ option. The dada2 algorithm for error correction was applied with the ‘dada’ command using default parameters. Sequences were merged using the ‘mergePairs’ command with minimum overlap set at 8 bp. Following, sequencing runs/batches were merged using the Dada2 command ‘mergeSequenceTables’. Following, the suspected chimera was detected and removed using the command ‘removeBimeraDenovo’. A count table including each ASV (Amplicon Sequence Variant) in each sample was produced. To obtain a taxonomic assignment for each ASV, each ASV sequence was aligned to the ARB-Silva small subunit rRNA database (version Silva_nr_138.1) using the command ‘assignTaxonomy’ with default parameters, but minBoot set at 80%. A count table, adjoined with taxonomic assignment for each ASV was produced. All sequences with length < 250 bases or length > 254 bases were removed. In addition, ASVs of non-bacterial origin were filtered out, including chloroplast, mitochondria, Archaea, and unclassified origin. In total, 2,853,650 reads and 8,169 unique ASVs were obtained.

Data Analysis

Community structure, composition, and similarity

The community structure of the organs sampled was assessed by calculating the relative abundance at the taxon levels. A permutational analysis of variance (PERMANOVA) was conducted using the "adonis" function in the R package vegan to assess and compare the influence of species or organs on variations in microbiota composition. This analysis was based on Bray-Curtis dissimilarities calculated from CSS normalized count data. The permutation parameter was set to 999. Differences were considered significant for p < 0.05. Post hoc pairwise PERMANOVA tests were conducted using the R package “pairwiseAdonis”. Non-metric multidimensional scaling (NMDS) analysis was performed based on Bray-Curtis dissimilarities using CSS-normalized counts with R package vegan. Stress values were considered at < 0.2. The P values obtained were corrected using Bonferroni post-hoc for multiple tests. The Kruskal-Wallis test was used to compare the Shannon index between loggerhead and green turtle samples and between the anatomic locations within each turtle species. When required, post-hoc tests were conducted using the Dunn multiple comparison test. P-values were adjusted with the Bonferroni method.

Sea turtles were sampled throughout 2019 and 2021 in two Sea Turtle Rescue Centers, located in Mikhmoret, Israel (IL), and Napoli, Italy (IT). Microbial samples were collected from 24 loggerheads (IL=13, IT=11), including three organs (nostrils, skin, and cloaca), as well as 26 green sea turtles kept in captivity (IL), including three sample sites (breath, skin, and cloaca). In total 137 samples from sea turtles and 17 samples from their surrounding water were analyzed. The IL loggerhead sea turtles were composed of adult individuals (CCL= 66.78±3.69), whereas the IT sea turtles were composed mainly of juveniles (CCL= 57.03±16.30). The green turtles were primarily composed of adults (CCL= 73.67±15.63) (Table S1).

Sea turtle microbiota and its drivers

The similarity of the microbial samples was assessed and compared using NMDS analysis. Significant differences were identified between the microbiome of the three groups of sampled turtles and between each turtle group and its surrounding water (Figure 1A and Table S2 ). The factors of site and species explained about 26% of the variance (Table S2). When comparing the bacterial community composition, we found two dominant Class groups (Gammaproteobacteria and Bacteroidia) shared by all sea turtle groups. The Gammaproteobacteria class was represented by the Orders, Pseudomonadales, Enterobacteriales, and Burkholderiales (the latter was significantly higher in green turtles). Flavobacteria represented the Bacteroidea class in all turtle groups, while Chitinophagales was significantly dominant only in IT loggerhead. In addition, Cyanobacteria was found only in green turtles, and Campylobacteria was mainly registered IL turtles (Figure 1B).

To examine the drivers contributing to the variation in the sea turtles' microbial profile, we focused on the loggerhead species and compared the samples from IL and IT. Figure 2 demonstrates a significant division of the bacterial samples according to (mainly) the site (IL and IT) and a second division according to the organs. The site, organ, and the interaction between them explain ~17% of those differences. Significant differences were also found in the comparison between the organs in each group of turtles (cloaca, skin, and nostrils) and between all the pairs of turtle organs (IL vs IT) (Table 1).

Bacterial Profiles by Sex and Organ in Green Turtles

The breeding stock of green turtles, maintained in Michmoret, includes separate pools of males and females exposed to equal conditions, which enables the isolation of the sex parameter and examination of its effect. The bacterial profile was mainly clustered according to the organs (explained ~11.8% of the variance), where the parameter of the sex was secondary but significant as well and in total (included their interaction) explained ~15.3% of the variance (Figure 3A, Table S3). A comparison of the organs within each sex showed significant variation. However, when comparing male versus female organ pairs, significant differences were only seen in the cloaca and skin (Table S3). The dominant groups of bacteria at a family level were similar between the sex in each organ and differed mainly in their relative abundance. In the cloaca, Nisaeaceae, Marinilabiliaceae, Helicobacteraceae and Bacilii were the most common in both sexes, while Candidatus Jidaibacter was enriched in females and Limnotrichaeceae and Ardenticatenaceae in males. The skin was characterized by Fokiniaceae, Rhodocyclaceae and unclassified Bacteroidia when the latter had higher relative abundance in female. Flavobacteriaceae, Rhodobacteraceae and Nesseriaceae were dominant in the breath samples (Figure 3B). It should be emphasized that a few individuals (n=3, labelled as ‘Unknown’ in Figure 3A) were juveniles, and therefore their sex could not be determined. In addition, the nostril samples were not included in this analysis since they were only sampled from these juveniles.

Organs-Specific Bacterial Composition

We grouped the samples according to the organs to focus on the bacteria-organ relationship. The main bacterial groups (above 10% relative abundance) were characterized for each organ and later for each group of turtles (Figure 4A, B). Gammaproteobacteria and Bacteroidia were the most dominant classes in all organs, constituting 60-80% of the bacterial community. In addition, Cyanobacteria was found only in the skin, while Campylobacter and Fusobacteria were found only in the cloaca (according to the 10% cutoff) (Figure 4A). Figure 4B characterized all the families (above 2.5% relative abundance) of the main classes when Gammaproteobacteria was found as the most diverse class and included 16 different families. Gammaproteobacteria was represented by Moraxellaceae and Cardiobacteriacea in the nostril samples of IL loggerheads, and by Methylophagaceae and Moraxellaceae in IT loggerheads. The dominant families of Bacteroidia were Flavobacteriaceae and Saprospiraceae in nostril and skin samples whereas Crocinitomicaceae were observed only in the nostrils. Exemplar dominant families were Rhodobacteraceae, which appeared in all samples, mainly in nostril and skin, whereas Cyanobcteria was observed only in the skin of green turtles, and Oligoflexaceae was significantly dominant in IT loggerhead turtles. The bacterial families in the cloaca were unique compared to the other organs. Regarding Gammaproteobacteria, Neisseriaceae and Halomonadaceae were the predominant families of green sea turtles, whereas Vibrionaceae, Pseudomanadaceae, and Shewaellacea dominated the cloacal samples of IL loggerheads; and Cardiobacteriaceae of IT loggerheads. In the Bacteroidia class, the families Weeksellaceae, Marinifilaceae, and Bacteroidaceae were dominante and appeared only in cloaca samples, whereas Paludibacteraceae was found only in the Green turtles. Other dominant cloaca families were Campylobacteraceae, and Fusobacteriaceae in green and Loggerhead (IL and IT) sea turtles, respectively.

Sea Turtle Bacterial Diversity

The diversity of the three sea turtle groups and their organs was calculated using the Shannon index to evaluate differences between them. The Kruskal-Wallis test revealed significant differences in bacterial diversity between the IL sea turtles (loggerhead and green turtles) and IT sea turtles (loggerheads), with the lowest diversity value observed in the IT group (Figure 5A, Table S4). When comparing the diversity index between the anatomical sample sites, significant differences were identified only in the loggerhead from IL (skin vs cloaca) and green sea turtles (skin and breath vs. the cloaca). However, the diversity indices consistently indicated higher values in skin samples compared to cloacal samples across all turtle groups (Figure 5B-D).

We characterized the upper respiratory tract, cloaca, and skin microbiome of loggerhead and green sea turtles of the Eastern (EMS) and Western Mediterranean Sea (WMS). To our knowledge this is the first study that compares the microbiota of the two sea turtle populations from the Tyrrhenian and the Levantine Sea. We observed differences in microbiome composition between the sea turtle groups assessed and within organs. In addition, we found differences in the microbial community composition between sexes in green sea turtles. Our results show the different core bacterial communities among two locations in the Mediterranean Sea, between turtle species, location, and organs. It is well known that mucosal surfaces (such as nostrils and cloacal mucosa) are critical habitats for microbial communities, serving as the primary pathogen defense system preventing opportunistic and pathogen colonization (Neish 2014). Moreover the skin is the primary barrier and defense against the exterior (Manire et al., 2017). Therefore, the anatomic sites selected in our study are in permanent contact with water and influenced by their environment, as evidenced by Bregman et al. (2023). Additionally, intrinsic ontogeny changes sea turtles' experiments during their lifetime, modeling their organ microbiome (Price et al., 2017).

Sea turtle microbiota and its drivers

It is known that the microbiota is influenced by geography (IT vs IL), diet, captivity, long or short-term rehabilitation process, age, sex, stress events, physiological changes, and dietary factors that could shape the microbial composition of the sea turtles’ organs (Colston and Jackson 2016, McNally et al., 2021a, Yuan et al., 2015). For instance, captive animals have a different microbial composition compared to the same species in the wild (Keenan et al., 2013), and geographic locations also shape those differences (marine vs terrestrial iguanas) (Lankau et al., 2012, Ahasan et al,. 2018). In this regard, it is important to emphasize that the Mediterranean Sea is known to be oligotrophic with a productivity gradient from West to East (Brosset et al., 2017) with higher productivity localized in the WMS (Gulf of Lion) (Bănaru et al., 2013). In contrast, the EMS has low biological production, high temperature, and salinity and is ultra-oligotrophic (Brosset et al., 2017). Thus, these regional differences are important drivers of the results obtained in IT and IL loggerhead populations. Dietary preferences (carnivores, herbivores, and omnivores) lead to shifts in the gut microbiome of mammals (Ley et al., 2008), with a similar pattern observed in reptiles such as the cottonmouth snake (Colston et al., 2015). The different anatomical locations where the sample was taken also influence the predominance of certain bacterial groups (Colston et al., 2015). For example, different portions of the intestine tract show bacterial dissimilarities in the same reptile species (Burmese python) (Costello et al., 2010, Khol et al., 2017).

Age class is also responsible for microbiota differences (Colston and Jackson 2016). Indeed, the gut microbiome of juvenile reptiles develops from environmental exposure, as was reported in hatchling iguanas that consume soil as they exit the nest and acquire it through coprophagy (Troyer 1984); the latter behavior was also reported in several species of turtles and tortoise (Yuan et al., 2015). In our study, the IT loggerhead population sampled was mainly composed of juveniles and sub-adults, whereas the IL population was mainly composed of adults; hence, the microbiome differences between both populations might be associated intrinsically with the ontogeny changes sea turtles’ experiment during their life cycle. Recently, Filek et al. (2024) identified differences in the bacterial community composition, structure, and richness of cloacal samples of juvenile and adult loggerheads from IT and Croatia. These differences are attributed to the loggerhead’s important ontogenetic shift from an oceanic inhabiting period while they are juveniles with an omnivore opportunistic diet to neritic zones as they subsequently recruit to grow and complete sexual maturation with a carnivore diet in several ocean basins (Bolten 2003, Marshall et al. 2012). Conversely, in the Mediterranean Sea, this species had a different strategy in which juveniles, subadults, and adults share their feeding grounds (Filek et al., 2024 and reference therein). Nevertheless, their prey preference is intrinsically associated with their age based on their jaw size, bite force, and diving ability (from soft oceanic prey when juveniles to hard benthic prey as they mature) (Marshall et al., 2012). Those prey preferences shape their gut microbiota.

Sheelings et al. (2020) show evidence that gut microbial composition and sea turtle phylogeny are intrinsically linked and that a process of coevolution likely exists between host and microbial community composition (Sheelings et al., 2020). These authors identified that the core gut microbiota was composed of Proteobacteria, Bacteroidota, and Actinobacteria in the seven species of sea turtles. Interestingly, physiological changes in females of all species of sea turtles during the nesting season show a predominance of Proteobacteria in cloaca samples (Sheelings et al., 2020).

Organs-Specific Bacterial Composition

Nostrils

Since the oral cavity and nostrils share several bacterial communities due to their connectivity and the lack of information about nostril microbial community composition, we analyzed our results considering the oral microbiota described in different sea turtle species. Filek et al. (2021) have identified Proteobacteria, Bacteroidia and Firmicutes as the dominant phyla in oral samples of stranded and rehabilitated sea turtles from Italy and Croatia, similar to our results. The reason behind the differences related to Firmicutes might be related to the fact that the oral microbial assemblages are less stable than cloacal regarding the turtles’ changing environment (Filek et al., 2021) and thus, the wildlife composition changes during the rehabilitation process.

Moraxellaceae, Flavobacteraceae, and Rhodobacteraceae families reported in nostril samples of IL and IT loggerheads herein have also been reported in oral samples of green and Kemp’s Ridley sea turtles of the northwestern Atlantic (McNally 2021b). The Kemp’s Ridley sea turtle is a carnivore species with similar diet preferences as loggerheads (McNally 2021b), therefore, the oral bacteria could be shaped by those preferences. The Methylophagaceae family reported herein in IT loggerheads has also been identified as part of the microbiome of estuarine seagrass leaves (Trevathan et al., 2020). Conversely, McKnight et al. (2020) reported Bacteroidetes as Australia's predominant phylum of freshwater turtles. It is important to highlight that sea turtles open their mouth to inhale air when they come to the water’s surface, thus it might be possible that bacteria present in the water tanks, which could be contaminated with fecal content, might enter the oral or nasal cavity.

Skin

Few studies have focused their attention on sea turtle skin microbial composition. Kanjer et al. (2022) identified Gammaproteobacteria, Alphaproteobacteria, Bacteroidia, and Cyanobacteria as the dominant bacteria classes in Loggerheads skin of the Adriatic, Ionian, and Tyrrhenian Seas resembling the results we obtained in the IL loggerhead population, whereas the IT loggerheads of our study share the Bacteroidia and Alphaproteobacterial classes with Kanjer et al. (2022) research as the most abundant bacteria group. Those bacteria classes are part of the loggerhead prey microbiota and could attach to the sea turtle’s skin while foraging. It is important to mention that we identified the Oligoflexia class as a key component of the IT loggerhead sea turtles as was previously reported in the Western Mediterranean Sea (Kanjer et al. 2022). Similar to our findings in green turtles, Loghmannia et al. (2023) identified that Proteobacteria was the dominant bacteria phylum, followed by Bacteroidia and Cyanobacteria on the carapace of green sea turtles of the Persian Gulf. These authors found that Gammaproteobacteria (Pseudomonadaceae family) and Alphaproteobacterial (Rhodobacteraceae family) were the dominant classes of bacteria, with certain similarities with our findings on the Eastern Mediterranean population of green sea turtles. Likewise, McKnight et al. (2020) identified Bacteroidia, Gammaproteobacteria, and Alphaproteobacteria as key bacteria class components of the skin (head) and shell of freshwater turtles of Australia (Krefft’s river turtles). It is important to highlight that the skin of IL loggerhead and IL green sea turtles showed Moraxellaceae as the dominant family of bacteria as it was registered on skin samples of dusky sharks on the Israeli coast (Bregman et al., 2023).

Cloaca

Our study identified Gammaproteobacteria as the predominant bacterium class in cloaca samples of the two species of sea turtles assessed, followed by Bacteroidia, Campylobacteria (Green Sea turtles), and Fusobacteria (Loggerhead Sea turtles). Vibrionaceae, Bacteriodaceae, and Fusobacteriaceae were the predominant families of bacteria in loggerhead sea turtles of IL, while Weeksellaceae, Marinifilaceae, and Cardiobacteriaceae were the dominant bacteria families in Loggerheads of IT. Our results were aligned with Biagi et al. (2019); these authors emphasized that loggerhead sea turtles of the Western Mediterranean share the gut microbial community with carnivore marine mammals rather than herbivorous sea turtles. It is important to highlight that Fusobacteria was also described as the core gut microbiome of the American alligator, and in other animals that feed on carrion (Keenan et al., 2013). Arizza et al. (2019) and Abdelrhman et al. (2016) described a high abundance of Firmicutes, Bacteroidota, and Proteobacteria phyla in the fecal microbiome composition of loggerheads of the Western Mediterranean Sea. Results that differ from our study (Firmicutes vs Fusobacteriota) and could be explained by the differences between the gastrointestinal portion sampled (fecal samples from large intestine vs. cloaca samples), and the origin of the animals (stranded vs, bycaught turtles). The differences could be caused by the intrinsic function of the cloaca that harbors the hindgut, reproductive, and urinary tract bacteria. Likewise, this organ is in constant contact with the marine environment (Price et al. 2017) and could harbor marine bacteria. A microbial community mainly represented by Gammaproteobacteria and Alphaproteobacteria has been previously described in several sea turtles’ prey items such as seaweed (Saha et al., 2019), sponges (Karimi et al., 2019), and bivalves (Timmins-Schiffman et al., 2021). In loggerheads, polysaccharides are metabolized by Bacteroidota and contribute to the digestion of items that have carbohydrates (Arizza et al., 2019).

Herein, Proteobacteria was the dominant phylum in captive green sea turtles. Firmicutes were less in proportion, which was described following the pattern of stranded turtles determined by Ahassan et al. (2017). In contrast, Price et al. (2017) described Bacteroidetes as the predominant phyla in wild-capture green sea turtles of the Gulf of Mexico. This could be explained by the type of diet that does not resemble exactly free-ranging green turtles based on algae and seagrass. In captivity, the animals ate lettuce and, occasionally, animal protein. In our study, a high abundance of Snodgrassella sp. (family Neisseriaceae) was found in green turtles; this bacterium species was reported as a component of mucosal surfaces in several animals such as dolphins, iguanas, dogs, and cats (Liu et al., 2015). Moreover, sex differences found herein in cloaca samples of green sea turtles could be explained by the action of the reproductive hormones; for instance, the human gut microbiota is associated with the metabolism of estrogen and androgen by the excretion and circulation of these hormones (Yoon and Kim 2021). Also, there have been significant differences between alpha and beta diversity in fish gut microbiota associated with sex (Bates et al., 2022).

Sea Turtle Bacterial Diversity

The higher diversity of IL loggerheads compared to IT could be explained by the age class. Adult sea turtles have been exposed to a greater number of microorganisms during their lifespan compared to juveniles. It is important to emphasize that coastal waters are impacted by sewage of human origin that pours a significant load of bacteria into the sea environment. Moreover, the ontogeny developmental history could drive this exposure, with juveniles growing offshore and subadults and adults using migratory corridors to reach neritic waters with completely different bacterial ensembles. Those microbial ontogenetic differences have been described in juvenile green turtles from pelagic and neritic habitats (Price et al., 2017). However, they were highlighted during the beta diversity comparison and were attributed to habitat differences, as well as different dietary resources. Skin samples had higher diversity than breath, nostrils, and cloaca, which could be explained by their extensive surface, which might allow a wider area for bacterial adherence. Likewise, this could be explained by the external condition of the skin, which does not provide a filtering barrier of microorganisms as the internal organs offer. Indeed, the skin microbiome has intimate contact with the environment and could reflect its diversity (Lavrinienko et al. 2018). Identifying core bacterial communities in several organs of different species of sea turtles could help us to understand those factors that shape the microbial composition differences, and thus guide us in the proper husbandry practices that might minimize the dysbiosis process and enhance the correct treatment of their diseases during their rehabilitation process to guarantee a successful recovery and reintroduction to their natural habitats. Moreover, this bacteria ensemble could shift during migration, food availability, physiological key changes, and winter (associated with metabolic suppression) following the Ana Karenin principle proposed by Zaneveld et al. (2017).

Our study characterizes and compares, for the first time, the loggerhead microbiota of the Mediterranean Sea turtle population from the Eastern and Western basins to understand how these communities compose their organs and systems. We identified significant differences based on their age class and geographic distribution (which determine their food preferences), information that can contribute to their recovery treatment during the rehabilitation process. Furthermore, we characterized the microbiota of adult green sea turtles used in Israel as the breeding stock to reintroduce hatchlings in their natural habitats to repopulate the EMS and improve the conservation status of this species nearly extinct in the area. We observed considerable differences between the microbial composition of the two loggerhead populations, between the two species assessed, as well as between intra and interspecies ’organs. Significant sex differences were also identified in the cloacal composition of green sea turtles. Microbial higher diversity has been observed in loggerheads and green sea turtles of the EMS. Our results align with previous findings regarding the dominant bacterial groups and give an insight into the possible bacteria taxa that constitute the core bacteria of each sea turtle species in the Mediterranean Basin.

Ethics approval and consent to participate

This animal study was authorized with permits issued by Israel Nature and Parks Authority (n. 3233543) and the Registro Ufficiale U.0000992 of the Italian Ministry of Ecological Transition of Italy.

Consent for publication

All authors have read and approved the final version of the manuscript, and they consent to its publication in Environmental Microbiome. All authors take full responsibility for the integrity and accuracy of the data presented.

Availability of data and materials

The data presented in this study have been deposited in the NCBI repository, accession number: PRJNA1159730, with the following URL: http://www.ncbi.nlm.nih. gov/bioproject/PRJNA1159730. Access to the data will be granted upon acceptance of the article for publication

Competing interests

We declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.

Funding

The research leading to these results received partial funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 730984, ASSEMBLE Plus project.

Authors' contributions

VB: Sampling and processing, Investigation, Formal analysis, Writing the original draft, Writing review and editing, SH: Data curation, sampling, animal husbandry, Writing review and editing. AA: Data curation, sampling, animal husbandry, writing review and editing. AP: Data curation, sampling, animal husbandry, writing review and editing. ML: Formal analysis, review, and editing. YL: Data curation, conceptualization, animal restraint, writing review, and editing. GK: Data curation, sampling, animal restraint, and husbandry, review, and editing, OR: Data curation, sampling, animal restraint, and husbandry, review, and editing, DT: Conceptualization, writing review and editing. DMorick & DMeron: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, writing original draft, writing review and editing.

Acknowledgements

We would like to thank the personnel and volunteers of the Israel Sea Turtle Rescue Center (ISTRC) Nature and Parks Authority and the Stazione Zoologica Anton Dohrn Napoli for their great effort in this project and their routine work. Additionally, we thank Liz Kaufman from the Tisch Family Zoological Gardens in Jerusalem: The Biblical Zoo and Israel Aquarium for her kind assistance.

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Table 1 is available in the Supplementary Files section

No competing interests reported.

Table1.xlsx
Table 1. Effect of site and organ factors on the bacterial composition of loggerhead turtles (Israel vs Italy). The factors were examined by a pairwise Dunn test. R2 values describe the relative contribution of each factor to the variation in microbiota composition. Asterisks represent significant p-values.
TableS1.xlsx
TableS2.xlsx
TableS3.xlsx
TableS4.xlsx

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Characterization of Respiratory, Skin, and Cloaca Microbiota in Mediterranean Loggerhead and Green Sea Turtle Populations

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Abstract

Figures

Background

Methods

Study Site and Sea Turtle Sample Collection

DNA extraction, PCR amplification, and amplicon sequencing

Sequence data processing

Data Analysis

Community structure, composition, and similarity

Results

Discussion

Sea turtle microbiota and its drivers

Organs-Specific Bacterial Composition

Nostrils

Skin

Cloaca

Sea Turtle Bacterial Diversity

Conclusions

Declarations

References

Table

Additional Declarations

Supplementary Files

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