The first barrier that ensures gut homeostasis is made by host components and is comprised of a mucus layer and immunomodulators. In mammals, these components have been and continue to be thoroughly investigated. The composition and organization of the mucus layers along the different compartments of the digestive tract have been described (35–38). In addition, diverse secreted immune molecules have been identified and functionally characterized, e.g., immunoglobulin A (IgA), RegIIIγ; antimicrobial peptides (AMPs) ( Figure 2 ) (39–45).

The microbial communities residing in the gut are generally referred to as the gut microbiota and while they represent exogenous elements, their integral role to animal physiology is now widely recognized. The metabolic output of the gut microbiota is roughly equivalent to an organ, and this extended phenotype has been referred to as a “forgotten organ” (80). While the microbiome includes primarily beneficial or commensal organisms, on occasion some pathogens visit or current inhabitants become pathogenic or harmful after shifts in microbial community structure, i.e., pathobiont or opportunist (81). Therefore, an important consideration in studies of host-microbiome interactions is knowledge of what are the expected or typical compositions of microbial communities residing within the gastrointestinal tract. For example, does the temporal persistence of specific taxa suggest a functional “core” microbiota of physiological importance, required for optimal animal health or survival? These impressions are important starting points in designing studies that focus on the ecology of the system, such as in vitro polymicrobial interactions influenced by biotic or abiotic factors, and further determining if these outcomes influence host physiology. Dissecting the interplay among diverse members of these complex microbial communities is also relevant in other fields of research, e.g., phylosymbiosis, which aims to understand if reciprocal interactions within a holobiont shapes phylogenies (82, 83).

Studies have been focused on both describing, through sequencing, the diversity of the microbial communities present, i.e., bacteria and viruses, in the Ciona gut ( Figure 4A ) (84, 85), and in isolating and culturing a diverse assortment of microbes and viruses, i.e., bacteria, bacteriophages, and fungi (Dishaw et al. unpublished (62);). Amplicon sequencing of 16S rRNA genes (V4 region; Illumina MiSeq) from Ciona whole gut microbiota, using individuals either starved or not starved, revealed the likely presence of a core bacterial community, consisting of 35 operational taxonomic units (OTUs) shared among animals collected in disparate geographic areas, e.g., San Diego (California, U S A) and Naples (Italy) (85). This core microbiota includes (ordered by abundance): Gram-negative bacteria of Proteobacteria, Bacteroidetes, Fusobacteria, and Verrucomicrobia phyla, and Gram-positive bacteria of Firmicutes and Actinobacteria phyla; however, the abundance of individual core OTUs can vary considerably ( Figure 4B ) (85). Interestingly, these are the same phyla that appear to be most important among many other animals, including humans. The preservation of distinct microbial taxa in the gut of a filter-feeder highlights the likely presence of strong selective forces shaping these communities. Recently, based on morphological and developmental characteristics along with analysis of mitochondrial genome features, i.e., gene order, number and size of noncoding regions, compositional features, and sequence divergence (86), it was recognized that the two cryptic species of Ciona intestinalis, previously named types “A” and “B” (87), have been in fact diverging and resulting in two distinct species, now recognized as Ciona robusta and Ciona intestinalis, respectively (88). Also, it is important to mention that the original draft genome and the subsequent analysis of immunity were performed on Ciona robusta, though at the time recognized as C. intestinalis (23, 89). Analysis of the Ciona gut microbiota reveals that the stably associated core microbial community is shared among both species, which includes animals from the North Atlantic population (represented by the Woods Hole, Cape Cod, Massachusetts, USA) (85), suggesting that these microbes are essential to the Ciona genus. However, some unique taxa between the two species may also suggest, consistent with phylosymbiosis theory, that in Ciona some interplay exists between species divergence and microbiomes (82, 83).

The composition of animal-associated bacterial communities can be affected by a variety of exogenous components and includes their viruses, known as bacteriophages or phages. The characterization of the Ciona gut virome, from animals collected in San Diego (California, USA) over two separate years, has demonstrated that i) the viral community of the gastrointestinal tract is distinct from the surrounding seawater, ii) some temporal variability exists, and iii) phages predominate the viral communities. The top 10 viral contigs share sequence identity with phages infecting described members of the Ciona core microbiota, including Flavobacteria, Shewanella, Pseudoalteromonas, and Vibrio. Further, most of the phages appear to be temperate phages, which most often exist as prophages stably integrated within bacterial genomes. Interestingly, animals that had their guts cleared, i.e., the starvation group, revealed significantly more unique viral sequences (84). This suggests that starvation is stressful on the gut microbiome and this process induces prophage release from lysogenized bacteria. Finally, compartmentalization of viruses was noted in the digestive tract, with the midgut possessing the largest number of unique viral sequences ( Figure 4C ) (84). These findings reaffirm the importance of studying the role of phages in shaping the structure of the Ciona gut microbiome likely involving both lytic infections and lysogenization of bacteria.

Beyond a protective role in preventing damage to the intestinal epithelium caused by food and digestive enzymes (93), an additional function of the mucus layer is to provide a niche for microbial colonization (38, 94–96), which can indirectly protect against overgrowth and/or attack from invading pathogens. As first introduced above, in mammals, a set of large glycoproteins, the MUCs, provide mucus with its viscous properties; they can be either secreted to form a gel-like structure or be produced as transmembrane molecules that help to form the epithelial glycocalyx (36, 97). Around 70–80% of the mass of MUCs is O-glycosylated, with N-acetyl-galactosamine, N-acetyl-glucosamine, galactose, and fucose serving as the main oligosaccharides attached to the protein core (94, 95, 98, 99). This differential glycosylation of MUCs is a means for selection, proliferation, and hence colonization by microbes within the diverse regions of the digestive tract. Indeed, the capacity to adhere to, and often consume, the mucus layer within the gastrointestinal tract serves as the first layer of selection in the regulation establishment of the microbiota (95, 96). Region-specific glycosylation of MUCs along the gastrointestinal tract has been observed in humans and rodents (100, 101), supporting a role for the MUC O-glycans in the selection of the gut microbiota. The preferential binding sites of O-glycans act as ligands for bacterial adhesins, and as carbon and energy sources to the surrounding microbial community. To attach and interact with mucus, symbiotic bacteria can either use mucus-binding proteins or extracellular appendages such as flagella, pili, and fimbriae (95, 96). Some intestinal bacteria secrete enzymes such as glucosidases that facilitate the degradation of MUC oligosaccharides, which can be further utilized/metabolized by other resident microorganisms (97, 102, 103); synergism in carbon-utilization strategies is common in polymicrobial communities. Collaborative strategies are beneficial in the utilization of mucus, since a combination of diverse enzymatic activities is required (104, 105). In mammals, for example, mucolytic bacteria include species as diverse as Akkermansia muciniphila, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bactetroides fragilis, and Ruminococcus torques (96, 102, 106). Conversely, pathogens or pathobionts often use antagonistic strategies to compete with commensal microflora for access to nutrients; thus, availability of carbon sources can influence pathogens and commensal colonization success and their niche adaptation (107, 108).

Another function of gut MUCs is to influence the formation of mucosal biofilms, which are bacteria organized into complex surface-attached communities (109, 110); biofilms confer a variety of advantages as opposed to a planktonic existence. Studies of natural biofilms within intestinal mucus are difficult for various reasons, including the rapid turnover of mucus, i.e., collecting shed mucus could be informative, and a lack of proper preservation techniques. However, in vitro assays reveal that MUCs significantly influence biofilm formation among different strains of bacteria (111–113). In Ciona, a gene product related to human gel-forming MUCs, Ci-GFM1, exists but its function and interaction with gut microbes are not yet known; however, future studies of these molecules could help reveal essential processes of colonization. More attention has been devoted instead to another component of Ciona mucus, chitin. The chitin-rich mucus may serve important structural or physical purposes; a well-known example is the peritrophic matrix of the insect midgut, which, among several functions, helps mediate protection against parasitic infections (56, 114).

Mammals, including humans, lack chitin synthase genes and thus cannot synthesize chitin oligosaccharide chains; instead, chitin sources can only be acquired via diet and can include feeding on arthropods and fungi (115). Various studies of chitin particles and its derivatives, such as chitosan (deacetylated form of chitin), report its role as a prebiotic, alleviating metabolic disorders and modulating the composition of gut microbiota (116, 117), and as an immune modulator (118). This effect of chitin particles in regulating gut microbiota and immune responses has also been noted in fish aquaculture (119, 120). In Ciona, chitin-rich mucus is endogenously produced and exuded as part of the gel layering on the epithelial surface; it also acts as part of a mucus-net that entraps and encases food particles, a process that begins in the pharynx (46). Functionally, disruption of these fibers allows direct microbial contact with gut enterocytes, suggesting an important role as a physical barrier (50). For example, treatment with dextran sulfate sodium (DSS), a compound employed to induce experimental colitis-like phenotypes in mammals (121, 122) by mediating biophysical changes to the structure of mucus, contributes to bacterial colonization of the inner mucus layer and results in inflammatory responses (123, 124). This treatment also induces a similar colitis-like phenotype in Ciona by disrupting the chitin-rich mucus layers and inducing inflammatory responses (47). However, exposure to exogenous chitin microparticles enhances the physical barriers of the intestinal epithelium and attenuates the colitis-like features and inflammation, demonstrating a strong protective function of chitin in Ciona ( Figures 5A–C ) (47); a related protective effect of exogenous chitin has also been observed in the mouse model, where chitin is not endogenously produced (125). Moreover, chitin can be utilized as a carbon source and often in the formation of biofilms, as has been observed in Vibrio cholerae, a facultative human pathogen (126, 127). Gut microbes isolated from Ciona, such as Shewanella sp. and Pseudoalteromonas sp., may be influenced by chitin-utilization pathways that also influence biofilm formation, likely shaping settlement outcomes ( Figures 5D, E ) (46).

Secreted effectors of the immune system, such as antimicrobial peptides (AMPs) and IgA in vertebrates, are other important components of mucosal immunity that integrate into the gut mucus layers and shape the ecology of the microbiome while fortifying barrier defenses. The discovery and characterization of the VCBPs, with their peculiar protein structure (IgV domains and CBD) and production by both blood cells and by the gut epithelium, have made them especially attractive for studies of immune interaction with microbes. So far, among the VCBP molecules, VCBP-C has been most characterized, revealing its interplay with distinct components of the gut environment. Although there is not a clear demonstration of the specific function of each VCBP-C domain, it has been observed that this protein co-localizes with bacteria in the Ciona gut mucus (46) and can bind both Gram-positive (Bacillus cereus) and Gram-negative (Escherichia coli) bacteria, via its first Ig domains ( Figures 6A, B ) (48). It has been demonstrated that these domains are also responsible for the opsonic activity of the protein, binding and presenting bacteria to be recognized by granular amoebocytes, thus increasing bacterial clearance via phagocytosis (48). Additionally, the CBD of VCBP-C can bind to chitin present in the mucus, where its colocalization has been observed since the early stages of development, specifically within the developing intestine ( Figures 3E–I ) (46). Recently, it has been demonstrated that the CBD also binds chitin molecules present in the cell wall, sporangia, and spores of fungi isolated from the Ciona gut ( Figures 6F, G ) (62). Hence, VCBP-C, to our knowledge, is the first secreted Ig-containing immune effector with the capacity to directly promote transkingdom interactions by simultaneously binding diverse gut microbial components; this promiscuity may have broad implications in modulating the establishment, succession, and homeostasis of gut microbiomes (62). Future studies for deciphering the significance of these interactions and the implication for host health are necessary.

Whereas the functional consequences of VCBP-C in bridging interactions between bacteria and fungi remain under investigation, in vitro experiments have demonstrated a role for VCBP-C in directly modulating the formation of bacterial biofilms ( Figures 6C–E ). Upregulation of some biofilms has been noted, for example, in Bacillus sp., Shewanella sp., and Pseudoalteromonas sp. isolated from the Ciona gut; downregulation or no effects have been observed with other bacterial species, while some Vibrio species, for example, seem more affected by the presence of hydrolyzed chitin (46, 128). This VCBP-C-specific activity is concentration-dependent and relies not only on the Ig domains but also on the CBD, whose presence appears to serve an important role (46). This differential influence on biofilm formation among distinct bacterial taxa remains to be explored further.

Although speculative, analogies from a functional point of view can be drawn between VCBP-C and mucosal antibodies from other animals, e.g., IgA in mammals or IgT in fish. In mammals, IgA acts as a component of barrier defenses, regulating bacterial adherence to the epithelial surface via immune exclusion processes that include agglutination and/or opsonization of bacteria (129, 130). Moreover, IgA may also influence non-pathogenic bacterial biofilm formation (131–134). In an analogous manner, VCBP-C may modulate adherence and biofilm formation on Ciona gut epithelial surfaces, where the settlement of some transient bacteria is regulated in a process that resembles immune exclusion by IgA while serving other important roles in maintaining gut homeostasis (46). Likewise, in a Ciona DSS-induced colitis-like model, VCBP-C expression in the gut was found to be up-regulated, likely for defensive purposes, further supporting a role analogous to that of IgA in mammals, where it recognizes commensal bacteria that preferentially affect inflammatory bowel disease (IBD) susceptibility (47, 135, 136). A related role for VCBP-C in modulating dysbiosis is predicted also during animal development (metamorphosis stages), when the gut microbiome is being established. Indeed, juveniles experimentally fed Gram-positive or Gram-negative bacteria revealed a distinct up-regulation of VCBPs. Specifically, B. cereus and E. coli increase mRNA level of VCBP-A and VCBP-C, respectively (49).

As briefly mentioned above, the Ciona gut also expresses TLRs, and this likely contributes to not just an innate recognition of microbial products but helps establish and/or maintain a bidirectional regulation of the microbiome. In mammals, distinct TLRs recognize different microbial ligands that often form conserved shapes or patterns, for example, lipopolysaccharide from Gram-negative bacteria. In Ciona, on the contrary, multiple ligands are recognized by the two TLRs (65) and this promiscuity likely evolved to counter the effects of a reduced assortment of available receptors. These ligands include zymosan, heat-killed Legionella pneumophila, double-stranded RNA (PolyI:C), and flagellin. Their binding to CiTLRs induces the transcriptional activation by NF-kB, and additionally, Poly(I:C) and flagellin trigger tumor necrosis factor α (CiTNFα) expression (65). A slight upregulation of CiTLRs was noted in the DSS mediated colitis-like phenotypes (47). However, other functions of CiTLRs within the Ciona gut, and specifically in regulating or mediating host-microbial symbiosis, require further investigation.

In Ciona, the studies of host-microbe interactions within the gut have thus far been focused on the identification and/or isolation of specific strains from the gut environment. Bacterial and viral components were first characterized via 16S rRNA amplicon and viral metagenomic sequencing, respectively, revealing a stably associated microbial community shared by both species, Ciona robusta and Ciona intestinalis (85), and a viral community predominated by bacteriophages (84, 144). Simultaneous investigations using molecular, cellular, biochemical, and microbiological techniques identified a role for the interaction between host immune effectors, i.e., VCBPs, and the microorganisms present in the digestive tract (46, 59, 62, 145). However, to expand the rate at which discovery is made, these studies will benefit from leveraging -omics approaches. For example, metagenomic and metatranscriptomic sequencing of the Ciona gut, either in its entirety or sub-divided into stomach, midgut, and hindgut, may help to identify the dynamic interactions between host and microbiome that help define factors shaping homeostasis among different seasons and distinct geographic locations. Moreover, metaproteomic and metabolomics may help identify active proteins, enzymatic pathways, and metabolites used for mediating interactions between host and microbes within the gut; recent examples focus on the tunic of three ascidian species, including Ciona sp. (146, 147) ( Figure 7 ). Subsequently derived hypotheses can be tested via experimental approaches, if isolation of the identified components is possible, and combined into functional assays. Indeed, these experiments in Ciona robusta are now augmented with important tools, such as the ability to rear short-term germ-free juveniles, essential in studies of host-microbial interactions (148) ( Figure 7 ). Colonization of germ-free animals, for example, could help facilitate studies aimed at understanding if colonization of the gut shapes developmental outcome, since animals like Ciona sp. complete development while exposed to the environment.

Likewise, leveraging simple in vitro assays, such as microbial biofilm assays, facilitates studies of host effectors (and other isolated factors) with individual components of the microbiome (128); data from these experiments can help pose hypotheses to be tested in vivo. Hence, the use of multi-omics approaches in Ciona, as well as in other organisms, may enable a more holistic view of microbiome composition and function at multiple layers (141, 149), facilitating hypothesis-driven experimentation to ascertain mechanisms of homeostasis and help define roles for immune effectors, and their interactions with specific components of these complex microbial communities.

Research on host-microbial interactions are mostly focused on revealing the significance to host physiology, homeostasis, disease, health, and fitness (150), whereas toxicology studies investigate the effects of chemical compounds on organisms, examining the accumulation, biotransformation, elimination, and effect in tissues (151). However, the epithelium and its associated microbiota lie at the interface between host and its environment, serving as a first line of defense against contaminants and environment stressors. In a recent mini-review, Marie B. and coworkers advocate for the development of a “microbiome-aware ecotoxicology,” emphasizing the importance of investigating host-microbiome interactions in light of ecotoxicological implications, while discussing important conceptual and technical pitfalls associated with study design and interpretation (151). To date, most studies have focused on the effects of environmental contaminants, such as pesticides, antibiotics, heavy metals, nanoparticles, microplastics, or compounds with endocrine-disrupting activity on gut microbiota (17, 152–154), mostly describing changes to the composition of bacterial communities via 16s rRNA sequencing (155–157). Whereas, fewer studies have focused on the functional capability of the gut microbiota in metabolizing a wide range of these xenobiotics (152) or in the production of secondary metabolites that may mediate toxicity to the host (158). Additionally, most of these studies have been performed in mammals; however, since most of the contaminants are released in aquatic environments, an important consideration is how aquatic organisms, which face continuous exposures, are impacted (17). A recent ecotoxicological investigation leveraged Ciona robusta as a model, assessing the impact of the combined effect of both industrial pollution (from the industrial area of Bagnoli-Coroglio, Naples, Italy) and two temporal patterns of turbulence events on the gut environment (159). Four broad categories were investigated: oxidative stress, innate immunity, host-microbial interactions, and integrity of gut epithelial barrier, with a focus on expression patterns of oxidative pathway and immune response genes (159). In particular, evidence for the induction of antioxidant defenses was detected after 7 days of exposure to static, polluted, sediment, with higher levels of glutathione S-transferase and metallothionein gene expression observed. Mechanisms of adaptation to chronic exposure to chemical mixtures was also suggested, revealing that turbulence events inducing polluted sediment suspension and a consequent release of heavy metals and polycyclic aromatic hydrocarbons do not correlate with an antioxidant response in Ciona, but instead with a pro-inflammatory response, as suggested by the increase in gene expression of CiTNFα. On the contrary, the absence of effects on gene expression of molecules involved in the establishment and maintenance of barrier defenses, such as MUC, chitin synthases, and also VCBPs, suggested that the mixture of pollutants did not interfere with the host-microorganisms interaction in the gut (159). However, the availability of a more comprehensive multi-omics approach would further help elucidate, utilizing high throughput and untargeted methods, the impact of environmental pollutants on the composition, behavior, or metabolic potential of gut microbial communities as well as to the predictive interpretations of the functional impacts on the animal host (155).

Invasive species, such as filter-feeders like Ciona robusta, despite being constantly exposed to potential toxins in aquatic environments, often can adapt. For example, they possess adaptable “xenobiotic receptors” and antioxidant defense systems that help regulate their physiological responses to repeated exposures of potentially toxic compounds or other environmental stressors, such as temperature fluctuations (160, 161). Also, as a tunicate, Ciona occupies an unusual ecological and evolutionary position, since it is both an invertebrate and part of a group that is a sister clade to the vertebrates (162). As such, it can complement ecotoxicological studies that leverage other filter-feeding organisms, such as bivalves (which are more distantly related to vertebrates), more often used for marine environmental monitoring (160). Thus, Ciona can be an informative system for understanding how animals respond to or tolerate certain chemicals that are foreign to the body, i.e., xenobiotics. This adaptability also relates to the gut microbiome, which may help modify such compounds in ways that protect or promote the survival of the host; indeed, this can serve as a strong selective force favoring bacteria that can tolerate such exposures and even metabolize the compounds. Therefore, detecting changes to the structure of gut bacterial communities may serve as important bioindicators, while also providing opportunities to predict community-level responses to short- and long-term exposures to environmental pollutants. These observations may help us better understand the capacity of the microbiota to both transform specific environmental pollutants and help adapt the host to contaminated environments (17) ( Figure 7 ).

Furthermore, because the gut microbiota is so metabolically active, it has been regarded as an “invisible organ” that may directly or indirectly modulate the function of drugs. The direct effect includes the biotransformation of drugs or their metabolites into products with altered bioactivities, thus changing their efficacy; whereas, the indirect effect involves more complex host-microbial interactions that affect host pathways for xenobiotic metabolism or transport (163, 164). It is now also evident that the microbiota can help mediate the effect of some drugs that target the immune system, and that changes to the structure or function of the microbiome represent an unanticipated limitation of the treatment (163). These data support the use of Ciona robusta as a model organism for investigating the impact of natural products/drugs on innate immune responses, and for studies of the role of the microbiota in metabolizing drugs and other dietary compounds that may explain observed variation in drug pharmacokinetics (165, 166). These data emphasize the importance of also focusing pharmacogenomics studies on genes encoded by the gut microbiota (163). For the reasons described above, Ciona robusta is particularly suited for studies of host-microbial interactions and can be further leveraged for investigating a role for the gut microbiome in drug metabolism, via in vivo and in vitro approaches, and multi-omics technologies, providing a comprehensive overview of the microbiome’s metabolic impact and function ( Figure 7 ). Indeed, future drug discovery efforts depend on harnessing powerful new technologies and model systems, while integrating information from sequenced genomes, functional genomics, protein profiling, metabolomics, and bioinformatics, in a manner that ensures a comprehensive systems-based analysis that furthers our understanding of the complexities of health and disease (167, 168).