Unexpected occurrence of Aeromonas species in the cutaneous mucus of Barbour's seahorses (Hippocampus barbouri)


 Because the skin microbiota is valuable for elucidating health status and common disorders in the host, this study aimed to describe the bacterial community composition of the skin of Barbour’s seahorse ( Hippocampus barbouri ) using high-throughput sequencing of 16S ribosomal RNA genes. Water and sediment samples from the surrounding environment were also analyzed for comparative purposes. The results revealed the presence of operational taxonomic units (OTUs) affiliated to the Aeromonas genus in the skin, whose abundance was slightly similar to the sediment samples. Given that some Aeromonas species are known to be important pathogens in humans and fish, these results may provide information to show the dependency of the skin microbial composition on the environment, as well as determine whether the presence of Aeromonas species has implications on seahorse health.


Introduction
Although seahorses (Hippocampus) play important roles in the ecosystem, they are constantly at risk because of trade for medicinal and ornamental purposes (IUCN 2019). Moreover, seahorses are not exempt from diseases, which could make the situation even more critical. In fact, some studies have reported skin diseases of diverse etiology in wild and farmed seahorses (Balcázar et al. 2010a(Balcázar et al. , 2011LePage et al. 2014). In a recent study carried out by our research team, we observed the presence of Aeromonas in the cutaneous mucus of healthy Hippocampus barbouri (Ortega et al. 2021). It should be noted that Aeromonas species are ubiquitous bacteria primarily recovered from aquatic ecosystems (Janda and Abbot 2010), as they are commonly found in freshwater bodies, estuaries (Chaix et al. 2017), and even seawater (Chenia and Duma 2017). Despite that some Aeromonas species are potential pathogens of sh (Vivekanandhan et al. 2005), there is limited information on the prevalence of these microorganisms in seahorses. Thanks to the implementation of cutting-edge sequencing technologies, a full picture of bacterial community composition can be obtained, thereby increasing our knowledge on the role of skin microbiota in health and disease. Here we used the Illumina sequencing technology to explore the bacterial community structure and composition within the skin ecosystem of Barbour's seahorses, with particular emphasis on members belonging to the Aeromonadaceae family.

Materials And Methods
Eleven healthy Barbour's seahorses and samples from the surrounding environment (water and sediment) were collected from coral reefs off the coast of were de ned at 99% sequence similarity of 16S rRNA genes. This value was used to de ne a core set of representative sequences, which were used for phylogenetic analyses. The weighted UniFrac test was applied to determine whether two or more communities have the same structure (Lozupone et al. 2007). A heatmap was also generated showing the relative abundance of OTUs assigned to the Aeromonadaceae family across the samples, which were classi ed using the EzBioCloud database (Yoon et al. 2017). Phylogenetic analyses were performed by using MEGA version 6.0 (Tamura et al. 2013). Distances (distance options according to the Kimura 2-parameter model) and clustering with the neighbor-joining method were determined by using bootstrap values for 1,000 replications.

Results And Discussion
After normalizing to avoid any bias due to the difference in the total number of sequences, bacterial community structure was analyzed using the weighted UniFrac test (sensitive to abundances of taxa), whose results demonstrated that the relative abundance of OTUs (de ned at 99% similarity) was signi cantly different (p<0.001) among groups.
Overall taxonomic characterization of the bacterial community was conducted at the family level ( Figure 1A). Sequences a liated with the Shewanellaceae family were dominant in the skin of female Barbour's seahorses and sediment samples (41.6 and 37.8%, respectively), whereas sequences a liated with the Bacillaceae family were dominant in the skin of male Barbour's seahorses (59.1%). Moreover, water samples (WS) were dominated by sequences a liated with Flavobacteriaceae and Vibrionaceae families (7.2 and 6.7%, respectively). Although sequences a liated with the Aeromonadaceae family were found in all samples, its abundance was very low in water samples (0.4%). Speci cally, sequences a liated with the Aeromonadaceae family had relative abundances of 7.6 and 5.5% in the skin of female and male Barbour's seahorses, respectively. Interestingly, a relatively high abundance of sequences a liated with the Aeromonadaceae family was found in sediment samples (21.4%). Because the sequences belonging to this family were abundant, a phylogenetic analysis was carried out to establish its taxonomic a liation. A phylogenetic dendrogram of selected OTUs was then constructed using the neighbor-joining method (Fig. 1B). The phylogenetic analysis revealed that these OTUs grouped with known Aeromonas species. The closest described relative of OTU 6 and OTU 588 was A. taiwanensis LMG 24683, whereas OTU 91 was closely related to A. tecta CECT 7082, OTU 104 to A. sanarellii LMG 24682, OTU 462 to A. bivalvium CECT 7113, OTU 582 to A. jandaei CECT 4228, and OTU 708 to A. rivipollensis LMG 26313. Although Aeromonas species share high levels of similarity based on 16S rRNA gene analysis (Carnahan and Joseph 2005), our analyses provide valuable information on the diversity of Aeromonas species, thereby validating the reliability of our ndings. The above mentioned Aeromonas species have been also observed in shes intended for human consumption such as tilapia and salmonids (Figueras and Beaz-Hidalgo 2014), bivalve mollusks (Galbis et al. 2007), whereas other Aeromonas species have been reported in clinical and environmental samples (Carnahan et al. 1991;Demarta et al. 2008;Alperi et al. 2010;Marti et al. 2015).
The relative abundance of each OTUs classi ed as Aeromonas species in the skin of H. barbouri was compared to sediment and water samples through a heatmap (Fig. 2). The map showed that the abundance of Aeromonas species from the skin of H. barbouri was more related to the sediments compared to water. This is consistent with the overall OTU representations. Aeromonas species detected in the skin of H. barbouri samples were greatly in uenced by the environment where they thrive. Based on Larsen et al. (2013), the structure and composition of the skin microbiota are likely to be impacted by several variables including abiotic factors linked to geographic locality and season, as well as biotic factors related to the nutrient potential or antimicrobial components of sh mucus. Given the interface in uencing this skin microbiota, water and sediments were considered (Chiarello et al. 2015), and in this case, the sediments had a higher in uence on skin microbiota. Our results were also supported by recent ndings, where the skin-microbiome assemblage of marine organisms is strongly associated with the surrounding sediments (Kawamoto et al. 2021).
The genus Aeromonas is a member of the Aeromonadaceae family, which consists of Gram-negative bacteria commonly found in aquatic environments, some of which are capable of causing disease in humans, sh, and other aquatic animals (Abu-Elala et al. 2015; Bravo and Figueras 2020). As inhabitants of marine environments, sh and other seafood are the most common sources for isolating these microorganisms (Pessoa et al. 2019), supporting their occurrence in seahorses. Moreover, Aeromonas spp. are known to be opportunistic pathogens for sh, and under stressful conditions such as an increase in water temperature, poor water quality, excessive handling, etc., they can cause epidemic outbreaks (Beaz-Hidalgo et al. 2010). However, the severity of disease cases depends on the concentration of these microorganisms (Hu et al. 2012). Some changes of the skin microbiota based on its phylogenetic composition may affect its functions, thereby upsetting its homeostatic interactions with the host and eventually favoring disease development (Chiarello et al. 2015).
Considering that H. barbouri specimens in this study were directly collected from the wild with no trace of skin disease, they can be considered apparently healthy. As a result, further research is needed to understand the differences in the composition of a healthy and diseased H. barbouri.

Conclusions
High-throughput sequencing technology revealed the presence of sequences a liated to Aeromonas species in the skin of Hippocampus barbouri. Interestingly, Aeromonas species were also observed in the sediments, which seem to be the most probable source of these species. Although some Aeromonas species are known to be important pathogens, the presence of these species in this study may not have been affecting the health status of Hippocampus barbouri yet. However, further studies are required to explore the implications of these species on seahorse health.