Effects of temperature and size class on the gut digesta microbiota of the sea urchin Tripneustes ventricosus

Introduction

Only approximately 1% of the global prokaryotic biodiversity has been successfully cultured in laboratory conditions using conventional methods (Schleifer, 2004; López-García & Moreira, 2008). The challenge of understanding the culturability of many bacterial taxa has been addressed by employing alternative technological approaches. The advancement of molecular techniques has significantly enhanced our comprehension of the prokaryotic biodiversity (Vartoukian, Palmer & Wade, 2010). Currently, one of the most widely used methods for characterizing prokaryotic communities in marine invertebrates like echinoderms, involves culture-independent identification through 16S ribosomal RNA gene sequencing (Hakim et al., 2015; Pagán-Jiménez et al., 2019; Hastuti, Fatma & Tridesianti, 2023). The number of studies on naturally occurring wild sea urchins has also increased in the last decades (Hakim et al., 2019; Faddetta et al., 2020; Ketchum et al., 2021; Rodríguez-Barreras, Tosado-Rodríguez & Godoy-Vitorino, 2021).

Over the last decade, sequencing the 16S rRNA gene through amplicon sequencing has emerged as a quick and affordable method for analyzing microbiota composition and diversity including the host gut microbiota associated with the digestive system, across animal phyla ranging from invertebrates to vertebrates (Lee & Hase, 2014; Grinevich et al., 2024). Recently, there has been a significant increase in knowledge about how environmental elements can impact the makeup and behavior of prokaryotic communities (Ward et al., 2017; Fontaine, Novarro & Kohl, 2018; Sepulveda & Moeller, 2020; Traving et al., 2021).

Sea urchins have been widely used in host-microbiota studies among marine invertebrates (Hakim et al., 2019; Schwob et al., 2020; Miller et al., 2021). The biological fitness of echinoderms, including sea urchins, heavily depends on the symbiotic relationship with their microbiota, which performs essential functions to the organism’s resilience (Ho et al., 2016; Carrier & Reitzel, 2018; Schuh et al., 2020). Echinoderms rely on diverse microorganisms within their bodies to carry out vital processes such as nutrient metabolism, immune system modulation, and protection against pathogens (Schuh et al., 2020). The intricate interdependence between echinoderms and their microbiota underscores the critical role of symbiotic interactions in their host survival and performance (Carrier & Reitzel, 2019, 2020; Carrier et al., 2021). For example, a recent study stated that symbiosis in the sea urchin Brisaster townsendi plays different roles in the host nutrition (Ziegler et al., 2020), while another study found the occurrence and importance of a photosynthetic bacteria as a nutrition supporter in the seastar Mithrodia clavigera (Galac, Bosch & Janies, 2016).

Changes in microbial communities are often associated with changes in environmental conditions (Dang et al., 2023; Zeng et al., 2023). Temperature has emerged in the literature as a prominent abiotic factor and a reliable predictor, driving significant shifts in prokaryotic taxa within thermally variable habitats (Ketchum et al., 2021). The gut microbiota plays a critical role in host phenotypic plasticity (Kolodny & Schulenburg, 2020) in many ways through morphological changes, physiological adaptations, behavioral responses, and life history strategies (Gotthard & Nylin, 1995). Modifications in environmental temperature can result in significant changes to the gut microbiota diversity in echinoderms (Gao et al., 2014; Brothers et al., 2018). Conversely, the microbiota, through their metabolites, can serve as a feedback mechanism, enhancing host plasticity in thermoregulatory mechanisms (Khakisahneh et al., 2020).

Another driving factor explored is the relationship between aging and host microbiota (Kawamoto & Hara, 2024). Traditionally, studies have primarily focused on understanding the consequences of changes in microbiota on nutrition and immune-related processes in both invertebrates and vertebrates (Clark & Walker, 2018; Maynard & Weinkove, 2018; Derrien, Alvarez & de Vos, 2019; Miró et al., 2020). However, understanding how prokaryotic taxonomic composition changes with age in marine invertebrates, particularly echinoderms, remains limited. In addition, the dynamic nature of the gut microbiota in response to seasonal changes underscores the adaptability of marine invertebrates to diverse environmental conditions. The dynamic nature of the gut microbiota in response to seasonal changes underscores the adaptability of marine invertebrates to diverse environmental conditions seasonal dynamics of the gut microbiota in marine invertebrates have important implications for marine ecosystems’ overall health and resilience (Ketchum et al., 2021). Seasonal shifts in microbial communities can influence nutrient cycling, disease resistance, and the overall fitness of the host organisms (Lee, Wong & Qian, 2009).

Sea urchins are considered suitable models for microbiota studies due to their anatomical simplicity, ecological importance, ease of collection and maintenance. The White Sea urchin, Tripneustes ventricosus (Lamark, 1816), plays a pivotal role in shaping coastal ecosystems, influencing benthic communities through algae grazing, and contributing to nutrient cycling (Lawrence & Agatsuma, 2007). The species is considered one of the largest regular echinoids in the western Atlantic and the Caribbean (Hendler et al., 1995; Rodríguez-Barreras, Sabat & Calzada-Marrero, 2013). T. ventricosus is characterized by a rapid growth, sexual maturity, and short longevity (McPherson, 1965). It usually inhabits back-reef areas dominated by marine flowering plants like Thalassia testudinum and Syringodium filiforme (Tertschnig, 1989; Hendler et al., 1995). This sea urchin T. ventricosus is primarily herbivorous and plays an important role in the dynamic of seagrass meadows. A dietary characterization of the sea urchin, using gut content analysis by stable isotopes and DNA-metabarcoding, revealed the eukaryotic composition of the ingested material (Maciá & Robinson, 2008; Rodríguez-Barreras et al., 2016, 2020).

While the gut and epibiotic microbiota in large urchins have been studied (Rodríguez-Barreras, Tosado-Rodríguez & Godoy-Vitorino, 2021; Rodríguez-Barreras et al., 2023), there is still a gap in our understanding about the dynamic of the gut prokaryotic community between size classes. Additionally, considering the rise of ocean temperatures across different seasons (Williams, Williams & Logan, 2023), it becomes critical to understand how the host microbiota responds to seasonal changes in temperature. Therefore, the objectives of this study were (1) comparisons of the gut microbiota in T. ventricosus during February (low temperature) and August (high temperature), and (2) comparing the gut microbiota between small and large size classes. Our hypothesis states that the gut microbiota will likely change between the two collection periods and between individual size class.

Materials and Methods

Results

Temperature changes in the gut microbiota among large animals are more significant than among collection sites

Most differences at the phylum level include a higher relative abundance of Bacteroidota in August (average 39.8% in August vs. 28.8% in February), while Firmicutes were higher in February (31.9% vs. 6% in August (Fig. 1A, Table S3). At the genus level, a higher relative abundance of Desulfatolea (~7.4%), Propionigenium (23.7%) and Roseimarinus (15.9%) e in high-temperature period samples, while Candidatus Hepatoplasma (~16.5%), Fusibacter (11.9%), and Roseimarinus (15.9%) had greater relative abundance in February (Fig. 1B, Table S3). Across seasons, a permutational statistical test based in ASVs confirmed significant dissimilarities in bacterial community composition between seasons (Permanova, p = 0.001) (Table S4). Inter-community features were highlighted using DEICODE compositional biplots. The analysis revealed that the genera Fusibacter were dominant in February samples; while in August, in addition to Propionigenium, the order Bacteroidales, and the genera Roseimarinus, Fusibacter, Desulfotalea, and Photobacterium also dominated. Propionigenium were dominant in the two time periods (Fig. 2A). Additionally, alpha diversity analyses revealed significant differences between seasons. There was an increase Shannon diversity in February (p = 0.0022) (Fig. 2B), and in observed features (p = 0.0022), while faith_pd remained similar between seasons (p = 0.390) (Fig. 2D). The seasonal dynamics of the gut microbiota, analyzed using MaAsLin2, identified a total discriminating 176 ASVs at the genus level (Fig. 3), out of which only 12 showed significant differentiation (FC = 1.5; FDR p = 0.05). Specifically, seven taxa exhibited a significant decrease of at least 1.5-fold in February compared to August, including Desulfotalea, Sediminispirochaeta, SCGC_AAA286_E23, Marinimicrobia SAR406 clade, SG8_4, Ferrimonas, and Woesearchaeales. On the other hand, five other taxa displayed a significant increase in relative abundances of at least 1.5-fold in February compared to August, namely MSBL3, Haloferula, Cutibacterium, RF39, and Candidatus Hepatoplasma (Fig. 3).

Changes in gut microbiota linked with size class

The sample size distribution consisted of six small individuals from ICB and 17 large individuals from ICB, CGD, and PBD, collected exclusively during August. Size classes were significantly different in horizontal test diameter (p = 0.004) (Table S2). Statistical tests (PERMANOVA strata) confirmed that the bacterial composition did not differ based on size in T. ventricosus even when correcting for sample collection site (p = 0.789) (Table S3). Small urchin samples displayed a lower relative abundance of Fusobacteria and higher relative abundance of Bacteroidota and Proteobacteria than large individuals at phylum level (Fig. 4A). At the genus level, Propionigenium had greater relative abundance in large urchins, while Photobacterium and Roseimarinus had greater relative abundance in small urchins (Fig. 4B).

The LEfSe analysis were visualized using box plots, showing that Ferrimonas (p-value = 5.15E−05) (Fig. 5A) and Propionigenium (p = 4.85E−05) (Fig. 5B) were more dominant in the large sea urchins. A DEICODE resulting biplot revealed that the genera Persicobacter and Spirochaetota (g__V2072-189E03) had greater relative abundance in small urchins. In contrast, Roseimarinus, Photobacterium, Desulfotea, and Propionigenium had greater relative abundance in large urchins (Fig. 6A). The alpha diversity did not reach statistical significance, but small urchins exhibited apparently more diversity than large urchins (p = 0.141) (Fig. 6B). Additionally, the observed features (p = 0.52861), and faith_pd (p = 0.44121) remained similar between size classes (Figs. 6C and 6D).

Discussion

This is the first study characterizing the gut microbiota of T. ventricosus, exploring the effect of water temperature and size class. This approach offers an in-depth understanding of the species’ gut microbiota dynamics. The novelty of this manuscript lies in understanding how the fecal microbiota of the white sea urchin changes between size classes and in response to temperature changes in the sea urchin Tripneustes ventricosus. An initial characterization of the fecal pellet microbiota conducted during February, when water temperature is usually lower, was publicly available (Rodríguez-Barreras, Tosado-Rodríguez & Godoy-Vitorino, 2021); however, the fecal microbiota during August, when water temperature is higher, remained unknown. Therefore, we characterized for the first time the gut digesta microbiota for August and compared it with the February samples. Additionally, the fecal pellet microbiota of small individuals was characterized for the first time and compared with that of large sea urchins during the same time period, allowing us to analyze how it changes between small and large sea urchins. The gut microbiota displayed changes in response to environmental fluctuation of abiotic parameters, like temperature more than by spatial variations, however, we consider this a big limitation in our study, as temperature and size evaluations are restricted to one sampling site and there are no variations in the animal sizes. Future studies should include a broader characterization of size classes and seasons to support any correlations. The composition and relative abundance of the gut microbiota in T. ventricosus during August and February were likely linked to annual changes in water temperature rather than pH or salinity. Water temperature changes were significant between February and August, while the other two measured abiotic parameters remained similar between both periods. The slight increase in alpha diversity found in this study during February could be related to bacteria genera being more evenly distributed in February than August. Nonetheless, we assume this is an important limitation of the study as other abiotic parameters such as pollutants (heavy metals, plastics, or chemicals that induce stress to the gut microbiota), water flow and currents (removing specific communities), differences in oxygen levels, or nutrient variations due to changes in diet (availability of macroalgae) (Masasa et al., 2021), which may all account for changes in the gut microbiota of these invertebrates, and these factors remain to be evaluated.

Recent studies have reported differences in microbial communities across seasons, illustrating the effect of environmental factors (Logue, Findlay & Comte, 2015; Karl et al., 2018; Ketchum et al., 2021). Microorganisms from the Phyla Proteobacteria, Bacteroidetes, and Fusobacteria have been found to colonize the gut system in sea urchins (Pagán-Jiménez et al., 2019; Faddetta et al., 2020; Feng et al., 2021). In this study, we reported a higher relative abundance of the phyla Bacteroidota and Fusobacteriota and a reduction in relative abundance of the phylum Firmicutes, probably related to an increase in water temperature in August. Firmicutes have been previously found in high relative abundance associated with water algae and gut digest microbiota samples in the temperate sea urchin Strongilocentrotus purtpuratus at 13.1 °C (Hakim et al., 2019). This temperature is lower than those experienced by sea urchins in the Caribbean (Table S1), and probably water temperature could be the fact behind the reduction of Firmicutes reduction during February and indicates a less tolerance or lower performance of the groups at higher water temperatures. Indeed, a reduction in the relative abundance of Firmicutes was also detected with the increase in temperature in the gut microbiota of the bivalve Mytilus coruscus (Li et al., 2018). Another study reported a significant increase in the relative abundance of the Phylum Firmicutes from autumn to spring in the sea cucumber Stichopus japonicus inhabiting temperate waters (Feng et al., 2021). However, the lack of abundant studies in wild sea urchins makes it difficult to discuss the influence of water temperature. On the other hand, while the previously mentioned phyla experienced seasonal fluctuations in relative abundance, the phylum Proteobacteria remained relatively stable between February and August. However, this behavior was not observed in the intestinal microbiota of the sea cucumber Holothuria scabra, where Proteobacteria, especially the genus Vibrio, was found to have a higher relative abundance during the rainy season (Plotieau et al., 2013).

Seven genera experienced a reduction in relative abundance in samples collected in February. One of them was Desulfotalea, a sulfate-reducing bacteria known for forming symbiotic associations with invertebrates in anaerobic or sulfate-rich conditions (Rabus et al., 2004). They can contribute to the host’s energy needs by metabolizing chemical compounds such as hydrogen sulfide. This genus is more abundant under lower water temperature, contrasting with our results (Grim et al., 2023). A potential explanation could be related with the fact that free-living species of Desulfotalea react in a different way to host Desulfotalea species, but also free-living species tend to be more affected by additional environmental factors such as the light incidence (Grim et al., 2023). A second genus that experienced a remarkable decrease from August to February in relative abundance was Ferrimonas. This genus is commonly found in aquatic environments and uses iron as an energy source through dissimilatory iron reduction (Rosselló-Mora et al., 1995; Fan et al., 2013). Warmer water temperatures led to an increase in the abundance of Propionigenium. Interestingly, another study found a similar pattern in Tripneustes gratilla, which was associated with the consumption of Ulva (Masasa et al., 2021).

On the contrary, at least five taxa increased in relative abundances in February. Cutibacterium, formerly known as Propionibacterium, is a Gram-positive bacterium commonly found in sebaceous areas of the skin (Lee, Byun & Kim, 2019). However, its presence in the samples may be due to human contamination and not because it constitutes a regular component of the microbiota of T. ventricosus. A second genus, Haloferula, was recently identified in samples from Sea Cucumber Apostichopus japonicus and its relative abundance associated with water temperature fluctuations (Kang et al., 2023). The candidate genus of small, cell wall-less bacteria in the Class Mollicutes, Candidatus Hepatoplasma, has been found in insects, specifically beetles, in their hepatopancreatic tissues (Leclercq et al., 2014). Candidatus Hepatoplasma are believed to be vertically transmitted and potentially mutualistic (Leclercq et al., 2014). While research has focused on the interactions with insects, a study in sea urchins has already established the detection of this taxa in sea urchins (Hakim et al., 2016). Limited information is available for the RF39 and MSBL3 strains. One potential seasonal factor associated with host microbiota shift could be nutrient availability. For example, a recent study demonstrated a high dynamisms level of the gut microbiota (Bengtsson et al., 2024), while other study found the ability of invertebrates to respond under seasonal changes in food supply spectrum (Kivistik et al., 2023).

Studying the gut digesta microbiota dynamic between small and large stages of wild caught sea urchins and other echinoderms offers valuable insights into the dynamic nature of these microbial communities during different life stages (Clark & Walker, 2018; Carrier et al., 2021). Previous studies have demonstrated significant changes in the gut microbiota during the transition from small to large urchins. However, few studies have been conducted addressing this issue in invertebrates (Onitsuka et al., 2015; Miró et al., 2020; Popkes & Valenzano, 2020), with limited research focusing on echinoderms (Zhao et al., 2019; Carrier et al., 2021; Marangon et al., 2023). Our findings revealed a size-related effect, providing insights into the progression of microbiota associated with different life stages. These studies concluded that small sea urchins exhibit higher microbiota diversity compared to adults, what agrees with our findings that also revealed that small T. ventricosus exhibited a slightly alpha diversity in their gut digesta microbiota compared to large urchins. This trend could be also related to significantly lower counts of Ferrimonas and Propionigenium. This difference in counts could be associated with the occurrence of more groups in small sea urchins. Particularly, Propionigenium tends to be a dominant group in adult sea urchins (Yao et al., 2019). Additionally, a higher relative abundance of Ferrimonas in adults could be related to the aestivation process and the reproductive cycle (Kang et al., 2023). A higher relative abundance of Ferrimonas has been found during this complex physiological process, which takes place during the summer season, coinciding with the period when our samples were taken. While large individuals invest more in reproduction, small individuals usually have undeveloped or absent gonads and therefore do not engage in reproductive activities (Hendler et al., 1995). Consequently, these two genera could serve as biomarkers for adult T. ventricosus.

The changes in the gut microbial community with size could be linked to feeding preferences. Literature has reported that sea urchins experience a dietary shift when transitioning from small to large size classes (Zann et al., 1987; Grosso et al., 2022). Small T. ventricosus possess smaller mouths in contrast to large individuals, potentially explaining the differences in microbiota composition due to their ability to ingest different kind of particles. Furthermore, the aging process may lead to a gradual decline in biodiversity, favoring genera associated with immune responses and dysbiosis through evolutionary symbiosis (Carrier et al., 2021). A recent study conducted with the tropical sea urchin of the genus Echinometra sp., found differences among life stages, where small urchins exhibited a higher relative abundance of the Class Oxyphotobacteria (within the Phylum Cyanobacteria) compared to large urchins (Marangon et al., 2023). This finding agrees with our results, and other studies conducted in marine invertebrates where the microbial community display important changes across the animal life cycle (Bernasconi et al., 2019; Quigley et al., 2020). On the other hand, despite of lack of significant differences in alpha diversity found between small and large urchins, the slightly higher diversity found in small urchins is a pattern observed in sea urchins, related with natural transitions that occurs alongside life history (Carrier et al., 2021). Overall, taxa reported here such as Psychromonas, Fusibacter, Propionigenium or Photobacterium, can be considered keystones species in sea urchins as they were not only found in our study, but also in other sea urchin species (Rodríguez-Barreras, Tosado-Rodríguez & Godoy-Vitorino, 2021; Ruiz-Barrionuevo et al., 2024) as well as Tripneustes gratilla (Masasa et al., 2021) or Lytechinus variegatus (Hakim et al., 2016).

Conclusions

Our study unravels the gut digest microbiota of T. ventricosus, focusing on the understudied aspects of seasonal and age-related dynamics, and underscores the importance of the gut microbiota of wild sea urchins and their potential associations with environmental variables. Comprehending the factors that influence gut microbial shifts is of utmost importance due to the significance of the microbiota in the overall function of the holobiont (Pita et al., 2018), being particularly critical due to rapid climate change (Konopka, 2009). Understanding the effect of temperature in gut bacteria will lead to valuable insights into these organisms’ ecological and physiological adaptations to changing environmental conditions. Our findings suggest the existence of specific microbial profiles associated with different life stages in T. ventricosus, emphasizing the importance of life-stage-related factors in shaping the gut digesta microbiota. By demonstrating slight size-class changes in the gut digesta microbiota between small and large urchins, we highlight the dynamic nature of the host bacterial community throughout the animal’s life cycle. By exploring the seasonal dynamics of the sea urchin gut microbiota influenced by fluctuating ocean conditions, and studying how microbial communities evolve from small to large urchins, we contribute unique insights guiding broader strategies for the conservation and sustainable management of coastal environments. Further studies should include a greater number of samples and collection sites to strengthen our capacity for drawing conclusions about T. ventricosus and to generalize to other similar sea urchin species in the Caribbean basin.

Supplemental Information