Organ transcriptomes of the lucinid clam Loripes orbiculatus (Poli, 1791) provide insights into their specialised roles in the biology of a chemosymbiotic bivalve

- Organ transcriptomes of the lucinid clam Loripes orbiculatus (Poli, 1791) provide insights into their specialised roles in the biology of a chemosymbiotic bivalve.
- The foot transcriptome is dominated by the biosynthesis of glycoproteins for the construction of mucus tubes, and receptors that mediate the detection of chemical cues in the environment..
- In the deep sea, these symbioses underpin primary productivity that supports an unexpectedly large biomass (hydrothermal vent mussels and tubeworms) in a food scarce environment, thus serving as the foundation of entire ecosystems [1, 2].
- This heavy reliance on symbionts for nutrition is reflected in the mor- phological simplification of the lucinid feeding structures and digestive system.
- Previous studies suggest a high degree of control over symbionts in the gills, as lucinids appear to inhibit symbiont cell division and are able to selectively digest symbionts or re- acquire the same strain from the environment [14–16]..
- Examples include filter feeding and digestion in the digestive tract and sulphide and oxygen acquisition by the foot [7].
- Despite the large number of contigs in the final assembly, only 2.5% of the BUSCOs were identified as duplicates while 20,163 transcripts were functionally annotated with gene ontol- ogy terms.
- This is supported by differential gene expression analyses, which showed that and 999 corset unigenes were most highly expressed in the gill, visceral mass, mantle, and foot, respectively (Fig.
- Immunity and cell death are important processes in the symbiont-housing organ.
- The key components of the main immune signalling pathways conserved across most metazoan line- ages were present in the transcriptome of L.
- We also investigated the repertoire of different pattern recognition receptor (PRR) gene families in the L.
- For ex- ample, more TIR domain containing unigenes (including TLRs) are more abundant in the gills than in other or- gans, while SRCRs and C-type lectins are more abundant in the mantle and visceral mass, respectively (Fig.
- An array of unigenes encoding diverse PRRs are more abundant in the gills, including eight TIR domain-containing (PF01582) unigenes, four SRCRs,.
- The remaining TIR domain-containing unigene highly expressed in the gills is unlikely to mediate MAMP recognition.
- b Domain architectures of eight TIR domain-containing unigenes significantly more highly expressed specifically in the symbiont-housing gills.
- The functions of these domains and the co- expression of this unigene with TLRs in the gills leads us to speculate it could be involved in transducing TLR signals..
- Biological Process (BP) GO terms associated with apop- tosis regulation are enriched amongst the unigenes expressed in the gills (Fig.
- The importance of regulating apoptosis in the gills is further emphasised by the expression of unigenes encoding B-cl2-like and Bax-inhibitor 1-like proteins that supress cell death (Fig.
- It is noteworthy that an expanded repertoire of Death domain-containing genes was reported in the gen- ome of Bathymodiolus platifrons, a deep sea mussel that also hosts chemoautotrophic endosymbionts [19].
- Interest- ingly, TUNEL staining of the gill tissues of lucinid and chemosynthetic deep sea mytilids indicates cell death is prevalent in the ciliated zone of both lucinid and symbiotic deep sea mytilids but not in the bacteriocyte zone [37, 38]..
- These lines of evidence suggest that apoptosis regula- tion is a key process in the symbiont housing organs of chemosynthetic bivalves in general.
- Therefore, one possible explanation is that the high prevalence of apoptosis in ciliated cells and enrichment of apoptosis- related GO terms in the lucinid gills are due to a high cell turnover rate caused by greater oxidative stress..
- The expression of these diverse molecules involved in various arms of innate immunity suggests cells in the lucinid gills have the capacity to detect and raise a com- plex response to bacteria.
- We cannot rule out the possi- bility that the PRRs, immune signalling and effector molecules expressed in the gills are used to defend the host against harmful bacteria because the gills are con- stantly exposed to the water from the environment.
- Unigenes expressed in the gill facilitate the.
- chemoautotrophic metabolism of the endosymbiotic bacteria.
- This is consistent with the enrichment of BP GO terms associated with ion transmembrane transport in the gills, because half of the unigenes contributing to this GO term encode ion chan- nels typical of the animal nervous system known as nico- tinic acetylcholine receptors (Fig.
- Nicotinic acetylcholine receptors and dynein com- plex genes are similarly over-represented in the gills of non-chemosymbiotic bivalves [23], which indicates that these genes are unlikely to be directly involved in the unique symbiont housing role of lucinid gills.
- Two globin domain-containing unigenes are expressed in the gills, one of which groups with the sulphide-binding hemoglobin HBI of the lucinid.
- Through the oxidation of sulphur, endosymbi- onts produce energy that is used to fixed inorganic car- bon in the form of CO 2 into sugars [1].
- Two CA unigenes are expressed in the gills (Fig.
- However, the larger of the two has a predicted GPI anchor site (Serine position 432) that could mediate the attachment of the protein to the cell membrane (Additional file 5).
- A similar secreted CA bear- ing a GPI anchor site is expressed in bacteriocytes of the chemosynthetic vesicomyid clam Phreagena okutanii and is probably responsible for catalysing the conversion of HCO 3 − in the hemolymph into CO 2 that can diffuse into the bacteriocytes [44].
- to these other physiological roles, we hypothesise that the CAs expressed in the gills of L.
- Unigenes encoding SLC4 were not more abundant in the gills compared to the other organs analysed, but we identified a unigene from the SLC26 family of transporters more highly expressed in the gills (Fig.
- This was supported by microscopic evidence indicating low rates of symbiont digestion in the gills.
- 4 Genes expressed specifically in the symbiont-housing gills of Loripes orbiculatus and putatively involved in symbiosis.
- Expressed in the gills are 20 unigenes encoding trans- porters belonging the to the Solute Carrier (SLC), and folate (Additional file 5).
- Fifteen different SLC families are expressed in the gills, with specificities for a diversity of substrates including amino acids, glu- cose, phosphate, monocarboxylates, organic cations and anions, zinc, ascorbic acidother nutritional symbioses..
- The diverse transporters expressed in the L.
- We also identified unigenes from other transporter superfamilies in the gills, including monocarboxylate and organic cation transporters from the Major Facilita- tor Superfamily, and a DUR3-like unigene encoding a urea-proton symporter (Fig.
- DUR3 is thought to be involved in nitrogen transfer between invertebrate hosts (Hydra and the giant clam) and their algal symbionts [54, 55], but the expression of a urea transporter in the lucinid gills is somewhat unexpected because Candidatus Thiodiazotropha endoloripes, the endosymbiont of L.
- orbiculatus DUR3 could be used to provide symbionts with a nitrogen source in the form of urea.
- orbiculatus and its sym- bionts is likely important in the coastal sediments of.
- This is consist- ent with the expression of unigenes encoding proteins involved in lysosomal activity in the gills, including two acid phosphatases, two arylsulfatases, and one Cathepsin L (Fig.
- We also identified two uni- genes from the Membrane Attack Complex/Perforin (MACPF) superfamily (PF01823) expressed in the gills..
- Proteins from this superfamily are pore forming toxins involved in immunity that form holes in the membrane of bacteria leading to cell lysis [59].
- A MACPF gene in the oyster Crassostrea gigas is upregulated under bacter- ial exposure, localised to late endosomes, and is highly expressed in the gills, digestive gland and gonads [60]..
- In fact, many more enzymes typically associated with metazoan digestion are highly expressed in the lucinid visceral mass rather than the gills (Additional file 3).
- The importance of digestion in the visceral mass is fur- ther reflected in the enrichment of BP GO terms with chitin and polysaccharide metabolism, as well as the Glycosyl hydrolase family 9 PFAM domain (PF00759) (Fig.
- These expression patterns suggest the lucinid di- gestive system is able to digest complex polysaccharides that are typical constituents of phytoplankton in the marine environment [61].
- corre- sponding to 25 C-type lectin unigenes – in the visceral mass, suggesting that carbohydrate recognition is im- portant in the lucinid gut.
- In the mosquito gut, C-type lectins bind to microbes to enable antimicro- bial peptide evasion, thereby allowing microbial colon- isation and gut homeostasis [63].
- The BP GO terms enriched in the foot include transmis- sion of nerve impulse, protein glycosylation and carbo- hydrate metabolism (Fig.
- These GO terms correlate with the enrichment of Glycosyl transferase (PF17039 and PF00852), sulfotransferase (PF03567), and von wild- ebrand A (PF00092) PFAM domains in the foot (Fig.
- Therefore, the enrichment of GO terms and PFAM domains associated with glycosylation indicates a battery of genes expressed in the lucinid foot are dedicated to mucus biosynthesis and is consistent with the foot’s ecological and physio- logical functions.
- By contrast, gene expression in the non-symbiotic mussel foot is in- stead dominated by byssal cuticle proteins and other proteins that contribute to connective tissue structures [23], which reflects the mussel foot’s primary role in se- creting byssal threads that facilitate attachment to hard substrates..
- Consequently, the lucinid foot has become a specialised probe for locating and acquiring sulphide in the clam’s surrounding environment [5, 7].
- Three iGluRs unigenes are expressed in the foot, one of which belongs to the NMDA2 subfamily while the remaining two are members of the AKDF sub- family (Additional file 3).
- iGluRs similarly mediate syn- aptic transmission throughout the nervous system, and while they do not directly bind H 2 S, it is interesting that NMDA2 receptor signalling is enhanced by physiological levels of H 2 S in the mammalian nervous system [69, 70]..
- Although little is known about the receptors specific for H 2 S, it is interesting to speculate that the iGluRs expressed in the foot could be involved in coordinating the clam’s behavioural response to sulphide in the environment..
- Based on the Interpro annotations of the unigenes, most of the GPCRs expressed in the foot contain a 7tm_1 domain (PF0001) and have short N-termini regions, which indi- cate that they belong to the rhodopsin family of GPCRs [72].
- GPCRs belonging to this family interact with a broad spectrum of ligands and their expression in the lucinid foot suggests they could play a role in enabling the foot to detect cues in the immediate environment..
- Furthermore, the deployment of non-overlapping sub- sets of GPCRs in the mantle and foot also suggests the molecular set-up for sensing environmental cues is specialised to the distinct functions of each organ (Additional file 3).
- In addition to receptors for chemical stimuli, a unigene encoding a piezo-type mechanosensi- tive ion channel component is also expressed in the foot;.
- The global transcriptomic profiles of the L.
- For example, in addition to detecting resources in the environment, does the foot also play a.
- role in replenishing symbiont abundance by secreting mucus that serves as a chemoattractant and first point of contact a potential symbiont might have with the host? Given the importance of heterotrophy, how do seasonal changes to resource availability in the environ- ment (sediment sulphide and phytoplankton abundance) influence the dynamics of nutritional and immune ex- changes in the symbiont-housing gills? The transcrip- tome we have assembled and the accompanying insights it has provided into molecular basis each lucinid organ’s functions, set up the foundation for future studies to build a picture of how lucinids use their molecular tool- kit to orchestrate the tripartite interactions linking host, symbiont and the environment..
- orbiculatus (roughly 1.5 cm in length) were dug up from 10 to 20 cm deep sediment in the Bay of Fetovaia, Elba, Italy (May 2017).
- Predicted errors in the raw reads were corrected using Rcorrector under default settings and the uncor- rectable reads were removed [74].
- SortMeRNA was used to remove rRNA sequences from the libraries by search- ing against the 5S, 5.8S, 16S, 23S, 18S, and 28S rRNA se- quences in the package’s default rRNA database [75]..
- Although small open reading frames and other non- coding transcripts are likely to have important albeit unknown biological functions, this arbitrary threshold was chosen to reduce the number of transcripts in the final assembly to reduce the computational resources required for subsequent analyses.
- Functional annotation of the predicted proteins.
- implemented through DESeq2 version v1.18.1 according to instructions in the DESeq2 manual [91] and visualised using the R package.
- 0.05) of GO terms in the lists of organ-specific unigenes [94]..
- GO term enrichments – Results of the gene ontology term enrichment analyses of genes highly expressed in each organ Additional file 5.
- Trees and tables – Phylogenetic trees, table of the transporter genes annotated in the L.
- orbiculatus transcriptome, and table of the urease metabolism genes in the genome of Loripes orbiculatus endosymbiont candidatus Thiodiazotropha endoloripes.
- The funding agencies played no role in the design of the study, sample collection, analysis or interpretation of the data nor in the writing of the manuscript..
- Raw sequencing reads have been deposited in the NCBI SRA database under the umbrella Bioproject PRJNA555495, linked to Biosamples SAMN .
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- Light exposure enhances urea absorption in the fluted giant clam, Tridacna squamosa, and up-regulates the protein abundance of a light-dependent urea active transporter, DUR3-like, in its ctenidium.
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