Symbiont diversity and distribution
The present study provides the first evidence of symbiosis in R. alisae, a species of nudibranchs. This is the most multiple symbiosis that have ever been recorded for marine invertebrates. While many organisms establish an exclusively one-on-one relationship with a single microbial species or microbes belonging to the same functional group5,12, there are also organisms that harbor multiple microbial species, in which symbiont–symbiont and host–symbiont interactions occur. Six phylotypes of chemoautotrophic bacteria were reported for mussel Idas sp. from a cold seep area11 and five extracellular symbionts for the gutless oligochaete worm Olavius algarvensis34. However, in these cases, symbioses involving bacteria and marine invertebrates are either endosymbiotic microbes co-occurring inside the host bacteriocytes5,11 or ectosymbiotic microbes associated with the external surfaces of the animals3,4,9,15,34, with the exception of scaly-foot snail from hydrothermal vents having partnerships simultaneously with epi- and endosymbiontic microbes35.
Bacterial symbionts in R. alisae have appeared to be more diverse than was previously known for marine invertebrates. It is evident that the detected symbiont phylotypes differ greatly from all other known symbionts found in marine invertebrates. Labrenzia (Rodobacteriales) and Maritalea (Rhizobiales) have not been recorded as forming symbiotic associations with invertebrates or plants so far, although other members of the families Rodobacteriales and Rhizobiales are well known symbionts14. Strains of Bradyrhizobium, Burkholderia, Achromobacter, and Stenotrophomonas are reported as symbionts of plants, interacting with a vast majority of nodulating legume species and efficient in biological nitrogen fixation36. This may be important when considering the nature of these symbionts in the nudibranch. Symbioses between cyanobacteria and marine organisms are commonly found among marine plants, fungi, sponges, ascidians, corals, and protists37,38. Synechococcus, identified as dominant symbiont clones of R. alisae (Table S2), is a unicellular cyanobacterium common in the marine environment, providing a range of beneficial functions including photosynthesis, nitrogen fixation, UV protection, and production of defensive toxins8,9,37. Symbiotic interactions between actinobacteria and their host have been observed in insects, human, animals, and plants, where the bacteria provide the host with protection against pathogens and produce essential nutrients39. However, none of the members of the clade Actinobacteria recorded in R. alisae are known to live symbiotically.
Arrangement of symbiotic association
Despite the high diversity of bacteria, they are well organized in the host. Dense clusters of rod-shaped bacteria, Labrenzia, Maritalea, Bradyrhizobium, Burcholderia, Achromobacter, and Stenotrophomonas, were found within host-derived vacuoles, referred to as bacteriocytes, inside epithelial cells of R. alisae (Fig. 3). Although such arrangement differs from that typical of bacteriocytes, which are usually considered as specialized cells of the hosts for harboring bacteria, it resembles that reported for scaly-food snail from hydrothermal vents, which harbor symbionts in the esophageal gland35. Bacteriocytes in the gastropod Lurifax vitreus found near hydrothermal vents also constitute a portion of the mantle epithelium; they have large vacuoles containing many live and dividing bacteria40. Each bacteriocyte was densely packed with certain symbionts, and the bacteriocytes were randomly distributed within the epithelium cells. A distinctly regular distribution pattern was observed in the gill epithelium of the mussel Bathymodiolus sp.: the thiotrophic symbionts occupy the apical region, and the methanotrophic symbionts are more abundant in the basal region of bacteriocytes4. In the mussel Idas sp., however, there is no spatial pattern of the six distinct bacterial phylotypes, and the symbionts are mixed within bacteriocytes11.
Synechococcus dominated the cytoplasm of intestinal epithelium and, more rarely, epidermis cells, mainly as specialized cell type referred to as nitrogen-fixing heterocysts. They are visually similar to cyanobacteria from corals and sponges8,37.
The phylogenetic diversity and the spatial organization of the symbiotic community in R. alisae were determined by the 16S rRNA analysis, which was consistent with the results of FISH and TEM. Unlike most symbioses of marine invertebrates when bacteria house specialized host cells5,11 or cover epidermis7,15, symbiotic association of R. alisae exhibited spatial partitioning between symbionts, which were unevenly distributed between the tissues (Table S2). It has been established that different members of the microbial community can complement each other in acquisition of various restrictive nutrients, confirming the importance of the functional diversity of symbionts41. Thus, Stenotrophomonas rhizophila and Bradyrhizobium build a beneficial association in the rhizosphere and can act synergistically on promoting growth and nutrient uptake of soybean36. Cyanobacteria can interact synergistically with beneficial members from the endophytic microbiome of rice seedlings42. The location of bacterium in the organism of R. alisae may, in fact, depend on the specific metabolic and ecological roles that the symbionts play, and also on the interaction with bacterium belonging to different physiological groups.
Nature of symbiosis
Symbiotic associations between microbes and invertebrates are acquired mainly in a nutrient-depleted environment where symbionts usually provide their hosts with essential nutrients and high-energy compounds1. In contrast to known symbioses between microbes and gutless invertebrates, which obtain nutrients exclusively from the bacteria, R. alisae, like most nudibranch species, is a sponge-eating predator. However, due to the lack of adipose tissue, sponges are distinguished by a low lipid content (0.4 to 1.5% of wet weight)43 and also by specific proteinaceous spongin fibers and chitin, a polysaccharide similar to cellulose that can be indigestible for some predators, which together indicate their low nutritional value. Furthermore, R. alisae feeds exclusively on the sponge O. pennata; therefore, in habitats with low prey availability, this nudibranch has to survive starvation while searching for sponge assemblages. We suppose that symbiotic bacteria of R. alisae contribute to the utilization of low-quality food, similarly to symbiotic bacteria from the genera Rhodobacter, Burkholderia, and Aeromonas associated with the detritivorous isopod Asellus aquaticus44.
A fatty acid analysis, as a useful approach to clarifying the nature of symbiosis5,20,32, has confirmed the trophic interaction between symbionts and the nudibranch host (Table S2). Among the fatty acids of symbiotic bacteria in R. alisae, OBFA are a major acyl constituent of membranes in Stenotrophomonas45 and also in Actinobacteria, Arthrobacter, Iamia, Ilumatobacter, and Kocuria46. Cis-vaccenic acid is a major component of Maritalea30. Omega-cyclohexyl tridecanoic acid (cyclo19:0) is specific to Bradyrhizobium47, Burkholderia, and Achromobacter48. Linoleic acid is produced by cyanobacteria including marine species of Synecoccocus33; in nudibranch, it obviously serves as a precursor in the synthesis of arachidonic acid (20:4n-6), thus, providing additional evidence for the transfer of fatty acids from symbionts to the host. Mollusks are capable of converting linoleic acid to arachidonic acid, since they have enzymes required for its synthesis21. The presence of these bacteria-specific markers and the abundance of arachidonic acid confirm the metabolic role of symbionts in the nudibranch host.
Among nutrients, biologically available nitrogen can be considered a restrictive nutrient for the sponge-eating R. alisae, which can be acquired with the help of nitrogen-fixing symbionts, also referred to as diazotrophs. R. alisae harbors Bradyrhizobium, Burkholderia, Achromobacter, and Stenotrophomonas that are efficient in biological nitrogen fixation previously found to be associated with nodulating legume species36. Symbiotic nitrogen fixers are known to be associated with a variety of marine invertebrates such as wood-boring bivalves, corals, sponges, sea urchins, tunicates, and polychaetes7,8,37. Moreover, the protection of the enzyme nitrogenase that catalyzes N2 fixation against oxygen is an important physiological requirement in bacteria such as symbiotic Bradyrhizobium, Burkholderia, Achromobacter, and Stenotrophomonas that are located in bacteriocytes and provide this protection. Synechococcus is known as a nitrogen-fixer37,49. It performs N2 fixation in heterocysts where nitrogenase is restricted under oxic conditions. Indeed, heterocysts of Synechococcus are abundant in the intestine cells of R. alisae (Fig. 5B–D).
Nitrate assimilation is one of the major processes of nitrogen acquisition by many heterotrophic bacteria and cyanobacteria50,51. Symbionts of R. alisae can play an important role in the process of nitrate utilization through denitrification, dissimilatory nitrate reduction, and assimilatory nitrate reduction as a nitrogen source and synthesize it into organic nitrogen. The nitrate reducers, Labrenzia52, Stenotrophomonas53, Maritalea30, and Rhodobacteraceae29 are widely represented in R. alisae. Synechococcus also utilizes nitrate, nitrite, or ammonium for growth50. Thus, symbiotic bacteria may play a significant role in the N-budget of the nudibranch mollusk.
The symbiotic bacteria of R. alisae, including Bradyrhizobium, Maritalea, Labrenzia, Burkholderia, Achromobacter, Stenotrophomonas, Arthrobacter, Iamia, Ilumatobacter, and Kocuria, are known as carboxydotrophic or carbon monoxide (CO) oxidizers54,55. Despite the toxicity of CO for multicellular organisms, numerous aerobic and anaerobic microorganisms can use CO as a source of energy and/or carbon for cell growth56. The marine worm Olavius algarvensis establishes symbiosis with chemosynthetic bacteria using CO, a substrate previously not known to play a role in symbiotic associations with marine invertebrates, as an energy source57. We do not rule out that the R. alisae symbionts also might exploit CO as carbon and energy source. Despite this, assumption may seem impossible taking in account the CO toxicity, but, since many invertebrates (mollusks, tube worm, etc.) use toxic sulfate, thiosulfate, and methane as an energy source1,15, this hypothesis is worth to be addressed.
An important component of skeleton in marine sponges of the family Microcionidae, including O. pennata, is the structural polysaccharide chitin58. Some bacteria are capable of hydrolyzing chitin via the activity of chitinolytic enzymes and can utilize chitin as a source of carbon, nitrogen, and/or energy59. Chitinase activity was documented for strains of Labrenzia60, Burkholderia61, Arthrobacter62, Achromobacter63, Stenotrophomonas64, Alcaligenes65, and actinobacteria59 associated with R. alisae. Thus, these bacteria can work synergistically to digest chitin and spongin, contributing to feeding success of the host nudibranch which depends solely on low-quality, nitrogen- and carbon-deficient food available.
Furthermore, direct evidence has confirmed that many bioactive compounds from invertebrates are produced by symbiotic microorganisms66,67. Many biologically active compounds including toxic and deterrent secretions have been identified in nudibranchs of the family Discodorididae68. Symbiotic bacteria may exhibit toxic activity to provide the host nudibranch with chemical defense against predators and environment. Bacteria, especially actinobacteria, living in a symbiotic relationship with R. alisae may help the host in defense, since nudibranch lack a shell, and secondary metabolites of bacteria can provide them with chemical defense against predators and environment, as has been reported for some marine invertebrates2,9,10.
In complex associations, the integration and coexistence of symbionts depend on supplementary partnerships and mutual contribution to the host’s metabolism41. The most intensively studied cases are highly specialized associations, where both partners can only exist in close relationship with one another. The relatively high diversity of microbes in R. alisae complicates understanding the complex pattern of molecular and cellular interactions between the host and its symbionts.
Source: Ecology - nature.com
