Abstract

   In hermatypic scleractinian corals, photosynthetic fixation of CO[2]
   and the production of CaCO[3] are intimately linked due to their
   symbiotic relationship with dinoflagellates of the Symbiodiniaceae
   family. This makes it difficult to study ion transport mechanisms
   involved in the different pathways. In contrast, most ahermatypic
   scleractinian corals do not share this symbiotic relationship and thus
   offer an advantage when studying the ion transport mechanisms involved
   in the calcification process. Despite this advantage, non-symbiotic
   scleractinian corals have been systematically neglected in
   calcification studies, resulting in a lack of data especially at the
   molecular level. Here, we combined a tissue micro-dissection technique
   and RNA-sequencing to identify calcification-related ion transporters,
   and other candidates, in the ahermatypic non-symbiotic scleractinian
   coral Tubastraea spp. Our results show that Tubastraea spp. possesses
   several calcification-related candidates previously identified in
   symbiotic scleractinian corals (such as SLC4-γ, AMT-1like, CARP, etc.).
   Furthermore, we identify and describe a role in scleractinian
   calcification for several ion transporter candidates (such as SLC13,
   -16, -23, etc.) identified for the first time in this study. Taken
   together, our results provide not only insights about the molecular
   mechanisms underlying non-symbiotic scleractinian calcification, but
   also valuable tools for the development of biotechnological solutions
   to better control the extreme invasiveness of corals belonging to this
   particular genus.

   Subject terms: Physiology, Transcriptomics, Marine biology

Introduction

   In scleractinian corals (Cnidaria, Anthozoa), also known as stony
   corals, calcification leads to the formation of a biomineral composed
   of two fractions, one made of calcium carbonate (CaCO[3]) in the
   mineral form of aragonite^[34]1–[35]3, and the other made of organic
   molecules^[36]4–[37]6. Based on the ability of scleractinian corals to
   build reef structures, they are functionally divided into two main
   groups, namely, hermatypic (i.e. reef-building) and ahermatypic (i.e.
   non-reef-building). The majority of hermatypic corals hosts symbiotic
   dinoflagellates of the Symbiodinacae family^[38]7 in their tissues,
   commonly known as zooxanthellae^[39]8. This symbiotic association,
   which is lacking in most ahermatypic corals, provides the nutritional
   foundation for the host metabolism and boosts calcification in
   nutrient-poor tropical waters^[40]9.

   Given the ability of hermatypic corals to build reefs, and given the
   economic and ecological importance associated with reef
   structures^[41]10, symbiotic scleractinian corals have been a major
   focus of calcification research over the years^[42]2,[43]11. Whereas,
   ahermatypic non-symbiotic scleractinian corals have not been
   extensively studied and to date they remain under-represented
   especially in terms of molecular data^[44]12. These corals, however,
   should not be neglected as they represent important resources for
   scleractinian calcification research. This is because, in symbiotic
   scleractinian corals, calcification is linked to the photosynthetic
   fixation of CO[2]—both at the spatial as well as the temporal
   scales—which makes it difficult to disentangle these processes.
   Whereas, non-symbiotic scleractinian corals allow studying the
   transport mechanisms involved in calcification without the confounding
   factor of symbiosis^[45]13. In addition, studying calcification in
   non-symbiotic scleractinian corals further allows obtaining comparative
   information on the different scleractinian calcification strategies,
   therefore aiding in a better understanding of how calcification evolved
   within this order.

   One of the main questions surrounding scleractinian calcification is
   how (i.e. via which molecular tools) corals promote a favorable
   environment for calcification^[46]14. As in other biological groups,
   coral calcification is a biologically controlled process, meaning that
   the precipitated mineral is not a byproduct of metabolic processes
   (also known as biologically induced biomineralization), but rather
   under strict biological and physiological control^[47]15,[48]16. This
   control is exerted by a specialized tissue called the calicoblastic
   epithelium, that comprises the calcifying calicoblastic cells^[49]17.
   These cells control and promote calcification by modifying the chemical
   composition at the sites of calcification, which comprise intracellular
   vesicles and the extracellular calcifying medium (ECM)^[50]14. As
   recently suggested, calcification begins with the formation of
   amorphous calcium carbonate (ACC) nanoparticles, within intracellular
   vesicles, in the calicoblastic cells. ACC nanoparticles are then
   released via exocytosis into the ECM^[51]18. Here, ACC nanoparticles
   attach (i.e. nanoparticle attachment) and crystallize, while ions fill
   the interstitial spaces between them (i.e. ion-by-ion filling). Both,
   nanoparticle attachment and ion-by-ion filling processes require the
   calicoblastic cells to regulate ion transport and their concentration
   at the sites of calcification^[52]2,[53]19. Furthermore, the
   calicoblastic cells also secrete an organic matrix which may stabilize
   ACC in the intracellular vesicles and play other roles, such as aiding
   and promoting ACC crystallization^[54]20–[55]24.

   Ion (i.e. calcium, carbonate, protons, and others) transport, to and
   from the sites of calcification, is of particular
   interest^[56]2,[57]19. For instance, calcium and carbonate ions, the
   building blocks of the coral skeleton, have to be constantly supplied
   to the sites of calcification to sustain its growth^[58]25. Whereas,
   protons must be removed from the sites of calcification to increase the
   aragonite saturation state, prevent dissolution of calcium carbonate
   nanoparticles, and promote ion-by-ion filling mechanisms^[59]14.

   Over the years, the ion transport model underlying scleractinian
   calcification has been well characterized for the calicoblastic cells
   through physiological and molecular studies^[60]26–[61]28. However,
   such understanding is only partial and many calcification-related ion
   transporters still need to be identified. When searching for
   calcification-related candidates, different approaches are possible.
   One is the so called “targeted” approach and is based on the analysis
   of genes and/or proteins that have been chosen a priori- generally
   based on known biological functions in other model systems. This
   approach is extremely powerful for studying the genetic architecture of
   complex traits, such as calcification, in addition to being an
   effective approach for direct gene discovery^[62]29. Nevertheless,
   although the targeted approach has led to the identification of some of
   the most relevant calcification-related candidates in scleractinian
   calcification^[63]13,[64]30–[65]32, it is largely limited by the
   requirement of existing knowledge about the gene(s) under
   investigation. To overcome this limitation, other approaches, the
   so-called “broad” approaches, have been developed throughout the years.
   Broad approaches have the potential to discover novel candidates and
   pathways that have not been previously considered in the context of
   calcification, thus allowing a more holistic understanding of the
   process. These approaches have been performed at different levels,
   including the transcriptomic one, which relies on the use of
   RNA-sequencing (RNA-seq) technology^[66]33–[67]35. To date, however,
   the use of RNA-seq to identify calcification-related candidates has
   been limited to analyzing coral molecular responses to environmental
   parameters known to influence calcification (such as light^[68]33 and
   CO[2]^[69]36), and only one study, performed in the symbiotic
   scleractinian coral Stylophora pistillata, has analyzed genes being
   more highly expressed in the coral calcifying tissue^[70]37.

   Therefore, given the high potential of broad approaches in discovering
   novel candidates, and given the scarce amount of data available for
   non-symbiotic scleractinian corals^[71]12, we have performed, in this
   study, RNA-seq on coral species belonging to the ahermatypic
   non-symbiotic scleractinian genus Tubastraea (Lesson, 1829)^[72]38.
   Tubastraea corals include invasive saltwater species^[73]39–[74]42 that
   were introduced into the southwestern Atlantic on oil platforms^[75]42.
   Since the late 1980s, these corals have been colonizing the rocky
   shores of the southeastern Brazilian coast^[76]40. Their rapid spread
   and growth provides them a competitive advantage and, therefore,
   represent a serious risk for endemic biodiversity loss^[77]43. In the
   absence of innovation in control methods, the dispersal of Tubastraea
   is expected to continue. In this context, calcification studies are
   fundamental to a better understanding of the life histories and
   population ecology of this genus. Of particular interest is the rapid
   linear skeletal growth of Tubastraea that could increase the
   competitiveness of these species^[78]44. In this study, we searched for
   calcification-related candidates, by sequencing the whole transcriptome
   from total colonies and oral fractions (i.e. fractions devoid of the
   aboral tissues that contain the calicoblastic cells) of Tubastraea
   spp., obtained through a tissue micro-dissection technique. After
   assembling and annotating a highly complete transcriptome for
   Tubastraea spp., we identified and analyzed genes enriched in the total
   colony transcriptomes compared to the oral fraction transcriptomes. The
   analysis included both a comparison with calcification-related
   candidates previously characterized in symbiotic scleractinian corals,
   as well as a search for novel calcification-related ion transporter
   candidates.

   This study provides insights into the molecular mechanisms underlying
   non-symbiotic scleractinian calcification and identifies valuable tools
   for the development of biotechnological solutions to better control the
   extreme invasiveness of corals belonging to this genus.

Results

Sequence read data and raw data pre-processing

   RNA sequencing was performed for two sample groups, total colony and
   oral fraction (i.e. fraction devoid of the aboral tissues containing
   the calcifying calicoblastic cells), of three independent biological
   replicates (n = 3) of Tubastraea spp. Both groups, obtained through a
   previously developed micro-dissection protocol^[79]45, produced a total
   of 539,331,300 raw reads with an average of 44.9 ± 8.7 (mean ± SD)
   million read pairs per sample. Raw reads were subjected to quality
   trimming, which included adaptor removal, yielding a total of
   369,357,576 trimmed reads.

De novo transcriptome assembly and quality assessment

   Trimmed reads were subjected to de novo whole transcriptome assembly
   using Trinity, after being further reduced to 73,014,522 by in silico
   normalization. Trinity assembler produced 691,300 transcripts, which
   were then clustered into 48,638 unigenes (i.e. uniquely assembled
   transcripts) with N[50] 1206 bp. Based on BUSCO, the assembled
   transcriptome was highly complete with 94.9% ortholog genes from
   Eukaryota database being present with low fragmentation (2.7%), missing
   (2.4%), and duplication (15.7%). In addition, ExN[50] statistics showed
   that the maximum N[50] value was on E97% with 1644 bp N[50] length
   (Fig. [80]1).

Figure 1.

   [81]Figure 1
   [82]Open in a new tab

   Expression-dependent N[50] (ExN[50]), as calculated against a fraction
   of the total expressed data (Ex). ExN[50] at the point of assembly
   saturation (97%) and traditional N[50] are highlighted.

Functional annotation

   To evaluate the completeness of the transcriptome library, functional
   annotation—including GO terms, EggNOG and KEGG pathway enrichment
   analysis—of the whole transcriptome of Tubastraea spp. was performed
   using Blastx results and OmicsBox. A summary of the whole transcriptome
   assembly and annotation results is listed in Table [83]1.

Table 1.

   Summary of the transcriptome assembly, annotation, and differential
   abundance analysis.
   Total number of contigs                                 75,006
   Total number of unigenes                                48,638
   Blast annotated                                         59,505
   InterProScan annotated                                  75,005
   GO annotated                                            18,246
   EggNOG annotated                                        44,319
   KEGG annotated                                          4,203
   Differentially expressed genes FDR < 0.05, LogFC <  ± 1 4,483
   Enriched genes in the total vs oral                     3,174
   Enriched genes in the oral vs total                     1,309
   [84]Open in a new tab

Differential expression analysis

   To identify differentially expressed genes between the total colony and
   the oral fraction, we first selected genes that had count per millions
   (CPM) more than 1 in at least two samples. Differential expression
   analysis was then performed using OmicsBox, followed by
   Benjamini–Hochberg multiple test correction. A total of 4,483 genes
   were reported to be differentially expressed (FDR < 0.05, LogFC <  ± 1)
   between the total colony and the oral fraction (Table [85]1). Of these,
   3,174 genes were significantly enriched in the total colony compared to
   the oral fraction, and 1,309 genes were significantly enriched in the
   oral fraction compared to the total colony. Differentially Expressed
   Genes (DEGs) have been clustered using Pearson’s correlation and
   displayed in a heatmap (Fig. [86]2). In this heatmap, biological
   replicates (1, 2 and 3) show strong clustering within the same group
   (Total and Oral), which are also clearly separated. In addition, a
   Multi-Dimensional Scaling (MDS) plot was performed to examine the
   homogeneity across biological replicates (Fig. [87]3). According to the
   MDS results, biological replicates showed strong clustering within each
   group and each group formed a distinct cluster.

Figure 2.

   Figure 2
   [88]Open in a new tab

   Heatmap and hierarchical cluster of 4,483 differentially expressed
   transcripts. The heatmap was generated using trimmed mean of M-values
   (TMM). Sample clustering was done using Pearson’s correlation. The
   Z-score scale is shown in the top-right corner ranging from − 1 (blue)
   to + 1 (violet).

Figure 3.

   [89]Figure 3
   [90]Open in a new tab

   MDS plot of the data set. Samples (n = 6) are separated by the
   biological replicate in the first dimension and by the coral fraction
   in the second dimension.

Comparison of calcification-related candidates between non-symbiotic and
symbiotic scleractinian corals

   To assess whether calcification-related candidates, previously
   identified in symbiotic scleractinian corals, were also present in
   Tubastraea spp., we performed a search for known homologs in the
   Tubastraea spp. transcriptome. Based on their role and their
   cellular/extracellular localization, these candidates can be divided
   into two groups: ion membrane transporters/enzymes and skeletal organic
   matrix proteins. Ion membrane transporters/enzymes comprise: Ammonium
   Transporters (AMTs)^[91]46, voltage gated proton channels
   (H[v]CNs)^[92]47, Na^+/H^+ exchanger (SLC9A)^[93]47, the SoLute Carrier
   4-γ (SLC4-γ)^[94]31, Plasma Membrane Ca^2+-ATPases (PMCAs)^[95]30 and
   the Voltage-Gated Ca^2+-Channel (VGCC)^[96]32. Whereas, skeletal
   organic matrix proteins comprise: Coral Acid-Rich Proteins
   (CARPs)^[97]35, neurexins^[98]48, galaxins^[99]49 and Small
   Cysteine-Rich Proteins (SCRiPs)^[100]50. Carbonic Anhydrases
   (CAs)^[101]34,[102]51 fall in between these two groups as they have
   been also identified in the organic matrix of the coral
   skeleton^[103]13. Our results show that, among the 75,006 contigs
   (Table [104]1), Tubastraea spp. possesses 55 protein sequences
   homologous to calcification-related candidates of symbiotic
   scleractinian corals (Table [105]S1). Of these, only 21 (38%) are
   differentially expressed between the total colony and the oral
   fraction: 10/21 are enriched in the oral fraction compared to the total
   colony (CARP-1, -2 and -3, CA-4/5, 10, 11, 12/13/14 and 15, AMT3 and
   one galaxin-like) and 11/21 are enriched in the total colony compared
   to the oral fraction (CARP-4 and -5, SLC4-γ, CA-6, -2/16 and 3, two
   galaxin-like proteins and one galaxin, AMT1-like and SCRiPs)
   (Fig. [106]4 and Table [107]S2). It is important to note that
   Tubastraea spp. CARPs and CAs have been named according to the
   phylogenetic trees provided in Figures S1 and S2.

Figure 4.

   [108]Figure 4
   [109]Open in a new tab

   Heatmap of calcification-related genes differentially expressed between
   the total colony and the oral fraction.

   Our results also show that 34 (62%) of the 55 protein sequences are not
   differentially expressed between the total colony and the oral
   fraction, and thus are not found in the heatmap (Fig. [110]4). These
   included: H[v]CNs, SLC9s, PMCAs, and VGCC.

Functional annotation and identification of unigenes putatively involved in
coral calcification

   DEGs were annotated using the same databases used for the whole
   transcriptome annotation. First, GO-term enrichment analysis using
   Fisher’s exact test was performed to infer which biological processes
   are associated with the enriched genes in the total colony compared to
   the oral fraction. Our results show 13 enriched GO-terms, including
   biological processes associated with “carbohydrate metabolic process”,
   “extracellular space”, “cell adhesion”, “extracellular matrix” and
   “extracellular matrix organization” (Fig. [111]5).

Figure 5.

   [112]Figure 5
   [113]Open in a new tab

   Gene Ontology Enrichment Analysis GO-terms associated with enriched
   genes in the total colony compared to the oral fraction.

   EggNOG functional annotation of the enriched genes in the total colony
   compared to the oral fraction was then performed. A total of 2,841 out
   of 3,174 enriched genes (88.6%) are functionally annotated into 23 COG
   functional categories, including inorganic ion transport and metabolism
   (P), and intracellular trafficking, secretion and vesicular transport
   (U) (Fig. [114]6).

Figure 6.

   [115]Figure 6
   [116]Open in a new tab

   EggNOG classifications of enriched genes in the total colony compared
   to the oral fraction EggNOG categories are shown on the horizontal axis
   as alphabets with corresponding category names on the right.

   Finally, using the KO term, provided by the EggNOG mapper, of each
   annotated gene, we performed KEGG annotation. KEGG annotation further
   divided genes into multiple families. Among these, 39 KO terms are
   associated with ion transporters (Table [117]2).

Table 2.

   List of enriched transcripts, involved in ion transport, in the total
   colony compared to the oral fraction.
   KO term Ion transporters unigenes Functional annotation FC
   K14611 TRINITY_DN1292_c0_g1_i21.p1 Solute carrier family 23 (nucleobase
   transporter), member 1 916.1
   K15381 TRINITY_DN15243_c0_g1_i12.p1 MFS transporter, FLVCR family,
   disrupted in renal carcinoma protein 2 500.7
   K05655 TRINITY_DN13741_c0_g1_i13.p1 ATP-binding cassette, subfamily B
   (MDR/TAP), member 8 333.1
   K06580 TRINITY_DN11254_c0_g1_i6.p1 SLC42A; ammonium transporter Rh
   317.7
   K22190 TRINITY_DN43565_c0_g1_i3.p1 Membrane-spanning 4-domains
   subfamily A member 3/4/8/12/15/18 206.8
   K21893 TRINITY_DN88858_c0_g1_i5.p1 Neuroglobin 182.3
   K21396 TRINITY_DN29747_c0_g1_i6.p1 ATP-binding cassette, subfamily G
   (WHITE), eye pigment precursor transporter 100.7
   K15104 TRINITY_DN18817_c0_g1_i1.p1 Solute carrier family 25
   (mitochondrial oxoglutarate transporter), member 11 95.7
   K21396 TRINITY_DN10851_c0_g1_i5.p1 ATP-binding cassette, subfamily G
   (WHITE), eye pigment precursor transporter 85.5
   K08187 TRINITY_DN104999_c0_g1_i1.p1 Solute carrier family 16
   (monocarboxylic acid transporters), member 10 62.1
   K17769 TRINITY_DN45389_c0_g1_i4.p1 Mitochondrial import receptor
   subunit TOM22 61.4
   K13868 TRINITY_DN7767_c0_g1_i15.p1 Solute carrier family 7 (L-type
   amino acid transporter), member 9/15 53.2
   K14387 TRINITY_DN6094_c0_g1_i13.p1 Solute carrier family 5 (high
   affinity choline transporter), member 7 52.7
   K14613 TRINITY_DN38892_c0_g1_i1.p1 Solute carrier family 46 (folate
   transporter), member 1 49.4
   K09866 TRINITY_DN9220_c0_g1_i2.p1 Aquaporin-4 43.4
   K08204 TRINITY_DN84629_c0_g1_i1.p1 Solute carrier family 22 (organic
   anion transporter), member 7 42.5
   K05673 TRINITY_DN43332_c0_g1_i2.p1 ATP-binding cassette, subfamily C
   (CFTR/MRP), member 4 39.9
   K08187 TRINITY_DN57711_c0_g1_i2.p1 Solute carrier family 16
   (monocarboxylic acid transporters), member 10 39.2
   K15109 TRINITY_DN17474_c0_g1_i7.p1 Solute carrier family 25
   (mitochondrial carnitine/acylcarnitine transporter), member 20/29 35.1
   K15108 TRINITY_DN12359_c10_g1_i3.p1 Solute carrier family 25
   (mitochondrial thiamine pyrophosphate transporter), member 19 32.2
   K05643 TRINITY_DN170738_c0_g1_i2.p1 ATP-binding cassette, subfamily A
   (ABC1), member 3 23.7
   K14445 TRINITY_DN38284_c0_g1_i1.p1 Solute carrier family 13
   (sodium-dependent dicarboxylate transporter), member 2/3/5 16.4
   K05673 TRINITY_DN29216_c2_g1_i3.p1 ATP-binding cassette, subfamily C
   (CFTR/MRP), member 4 13.6
   K21396 TRINITY_DN29747_c0_g1_i3.p1 ATP-binding cassette, subfamily G
   (WHITE), eye pigment precursor transporter 12.4
   K05673 TRINITY_DN73454_c0_g1_i2.p1 ATP-binding cassette, subfamily C
   (CFTR/MRP), member 4 8.9
   K08204 TRINITY_DN23500_c0_g1_i4.p1 Solute carrier family 22 (organic
   anion transporter), member 7 8.8
   K14686 TRINITY_DN90716_c0_g1_i1.p1 Solute carrier family 31 (copper
   transporter), member 1 8.6
   K21862 TRINITY_DN29586_c0_g1_i7.p1 Voltage-gated cation channel 8.5
   K21988 TRINITY_DN5910_c4_g1_i1.p1 Transmembrane channel-like protein
   7.4
   K14611 TRINITY_DN1292_c0_g1_i23.p1 Solute carrier family 23 (nucleobase
   transporter), member 1 6.8
   K08204 TRINITY_DN23500_c0_g1_i3.p1 Solute carrier family 22 (organic
   anion transporter), member 7 6.5
   K14445 TRINITY_DN20897_c0_g1_i1.p1 Solute carrier family 13
   (sodium-dependent dicarboxylate transporter), member 2/3/5 6.4
   K15109 TRINITY_DN22386_c0_g1_i6.p1 Solute carrier family 25
   (mitochondrial carnitine/acylcarnitine transporter), member 20/29 6.4
   K14994 TRINITY_DN9280_c0_g1_i8.p1 Solute carrier family 38
   (sodium-coupled neutral amino acid transporter), member 7/8 6.3
   K05039 TRINITY_DN7450_c0_g1_i13.p1 Solute carrier family 6, member 6
   5.7
   K15015 TRINITY_DN68697_c0_g1_i1.p1 Solute carrier family 32 (vesicular
   inhibitory amino acid transporter) 5.7
   K08187 TRINITY_DN26257_c3_g1_i4.p1 Solute carrier family 16
   (monocarboxylic acid transporters), member 10 5.3
   K11536 TRINITY_DN3340_c0_g2_i6.p1 Pyrimidine nucleoside transport
   protein 5.3
   K05038 TRINITY_DN12243_c1_g1_i1.p1 Solute carrier family 6, member 5/9
   5.1
   [118]K00799 TRINITY_DN12289_c0_g1_i14.p1 Glutathione S-transferase 4.8
   K21862 TRINITY_DN9832_c0_g1_i2.p1 Voltage-gated cation channel 4.6
   K08204 TRINITY_DN19075_c0_g1_i1.p1 Solute carrier family 22 (organic
   anion transporter), member 7 4.3
   K08187 TRINITY_DN16679_c0_g2_i1.p1 Solute carrier family 16
   (monocarboxylic acid transporters), member 10 4.2
   K14453 TRINITY_DN16687_c0_g1_i7.p1 Solute carrier family 26 4.2
   K14683 TRINITY_DN5423_c0_g1_i6.p1 Solute carrier family 34
   (sodium-dependent phosphate cotransporter) 4.2
   [119]K03320 TRINITY_DN16401_c0_g1_i7.p1 Ammonium transporter, Amt
   family 4.1
   K05048 TRINITY_DN609_c1_g1_i13.p1 Solute carrier family 6, member
   15/16/17/18/20 3.9
   K21396 TRINITY_DN10851_c0_g1_i4.p1 ATP-binding cassette, subfamily G
   (WHITE), eye pigment precursor transporter 3.8
   K05048 TRINITY_DN609_c1_g1_i14.p1 Solute carrier family 6, member
   15/16/17/18/20 3.6
   K05678 TRINITY_DN8486_c0_g1_i4.p1 ATP-binding cassette, subfamily D
   (ALD), member 4 3.5
   K05048 TRINITY_DN2910_c0_g1_i4.p1 Solute carrier family 6, member
   15/16/17/18/20 3.3
   [120]K00799 TRINITY_DN1669_c0_g1_i3.p1 Glutathione S-transferase 3.2
   K15281 TRINITY_DN9775_c0_g1_i5.p1 Solute carrier family 35 3.2
   K14995 TRINITY_DN19648_c0_g1_i1.p1 Solute carrier family 38
   (sodium-coupled neutral amino acid transporter), member 9 3.2
   K06580 TRINITY_DN1639_c0_g1_i6.p1 SLC42A; ammonium transporter Rh 3.1
   K12385 TRINITY_DN8972_c0_g1_i9.p1 Niemann-Pick C1 protein 3.1
   K05038 TRINITY_DN8948_c0_g1_i15.p1 Solute carrier family 6, member 5/9
   2.9
   K15015 TRINITY_DN14356_c0_g1_i5.p1 Solute carrier family 32 (vesicular
   inhibitory amino acid transporter) 2.9
   K15381 TRINITY_DN1196_c2_g2_i3.p1 MFS transporter, FLVCR family,
   disrupted in renal carcinoma protein 2 2.9
   K11518 TRINITY_DN2445_c7_g1_i4.p1 Mitochondrial import receptor subunit
   TOM40 2.8
   K14613 TRINITY_DN9535_c0_g1_i2.p1 Solute carrier family 46 (folate
   transporter), member 1 2.7
   K12385 TRINITY_DN2483_c0_g1_i6.p1 Niemann-Pick C1 protein 2.5
   K05643 TRINITY_DN2705_c1_g1_i6.p1 ATP-binding cassette, subfamily A
   (ABC1), member 3 2.3
   K05399 TRINITY_DN12573_c1_g1_i4.p1 Lipopolysaccharide-binding protein
   2.1
   [121]Open in a new tab

Discussion

   The “calcification toolkit” is the collective term documented and/or
   hypothesized to be involved in biomineral formation at various stages
   of an organism’s life history^[122]52. Out of all the toolkit
   components, proteins have been the most intensively
   characterized^[123]26,[124]53,[125]54. As a result, proteomic studies
   have suggested that, although proteins from distant organisms share
   common properties^[126]53, each taxon-specific suite appears to have
   evolved independently through convergent and co-option evolution. This
   has led to variable contributions, from new lineage- and
   species-specific proteins, to the “calcification toolkit”, which show
   contrasting rates of conservation between and within lineages^[127]55.
   Several tools of the “calcification toolkit” have also been identified
   in scleractinian corals^[128]48,[129]56, yet to date only few
   experiments have been conducted and solely for symbiotic
   species^[130]54. Other than being particularly attractive for
   calcification studies because of the lack of symbiotic dinoflagellates
   in their tissues, corals belonging to the Tubastraea genus have been
   the focus of numerous biological^[131]57–[132]59 and ecological
   research studies^[133]60,[134]61 aiming at identifying key parameters
   underlying their invasiveness. Nevertheless, their “calcification
   toolkit”, which may include specific components providing these corals
   with an advantage in terms of calcification strategies, has never been
   investigated at the molecular level. In this study, we aimed to fill
   this knowledge gap by searching for candidates of the “calcification
   toolkit” in the non-symbiotic scleractinian coral Tubastraea spp. using
   a tissue micro-dissection technique to remove the oral fraction (easily
   accessible and free of the calicoblastic cells) from the total colony
   of Tubastraea spp. This previously developed technique has already been
   used in the past and has contributed to the identification of some of
   the most frequently searched and studied candidates in a wide range of
   calcifying metazoans^[135]26,[136]62,[137]63, other than
   corals^[138]31,[139]45,[140]47. By coupling this technique with
   RNA-seq, we have then identified and analyzed differentially expressed
   genes with a focus on those enriched in the total colony compared to
   the oral fraction. Indeed, these genes are specific of the aboral
   tissues and include calicoblastic cell-specific genes, that could play
   a role in calcification. This is supported by our results showing that,
   although many genes are ubiquitously expressed in the total colony—and
   thus in both oral and aboral tissues -, others are differentially
   expressed, with clearly distinct expression profiles between the total
   colony and the oral fraction (Figs. [141]2 and [142]3). It follows that
   the different expression profiles reflect specific gene functions
   related to the oral and aboral tissues. Amongst the 3,174
   aboral-specific genes (Table [143]1), we identified most
   calcification-related candidates previously described as part of the
   “calcification toolkit” of symbiotic scleractinian corals
   (Fig. [144]4). These include the bicarbonate transporter
   SLC4-γ^[145]64. SLC4-γ has been proposed to play a role both in the
   regulation of intracellular HCO[3]^- homeostasis—which is critical to
   buffer excess of H^+ generated during CaCO[3] precipitation—and the
   supply of HCO[3]^- to the calcifying cells in several organisms,
   including sea urchins^[146]63,[147]65, mussel^[148]62,
   coccolithophores^[149]66 and corals^[150]31,[151]67. We also identified
   an ammonium transporter belonging to the AMT1 sub-clade (Fig. [152]4).
   AMT transporters have been suggested to play a role in calcification in
   multiple metazoans, including mollusks^[153]68–[154]70 and symbiotic
   scleractinian corals^[155]71–[156]73. Although their role in coral
   calcification still needs to be investigated in detail, it has been
   suggested that AMT1 transporters mediate pH regulation in the ECM by
   transporting NH[3] into the ECM which could buffer excess of protons.
   Organic matrix proteins, including 2 CARPs and 1 SCRiP, were also
   identified (Fig. [157]4). CARPs are proteins with dominant Low
   Complexity Domains (LCDs) that have been described in the secreted
   organic matrix of biominerals in different metazoan
   taxa^[158]74–[159]77. CARPs have been identified also in previous
   proteomic studies on coral skeletons^[160]35, where they have been
   suggested to play a role in CaCO[3] formation given their high affinity
   to positively charged ions (i.e. Ca^2+)^[161]78–[162]80. SCRiPs,
   instead, are a family of putatively coral-specific genes for which
   different roles have been suggested based on their molecular features
   (i.e. presence of signal peptide, high amino acidic residues content
   and cysteine-rich)^[163]50. Moreover, three galaxins-like proteins and
   three CAs were also identified (Fig. [164]4). Galaxin was first
   identified in the exoskeleton of the scleractinian coral Galaxea
   fascicularis and was described as a tandem repeat structure with a
   di-cysteine motif fixed at nine positions^[165]49. Since this
   discovery, galaxin homologs have been observed in the exoskeleton of
   other scleractinian species^[166]81–[167]83, as well as in
   mollusks^[168]84 and squid^[169]85. It has also been shown that galaxin
   is associated with the developmental onset of calcification after
   larval stage in Acropora millepora^[170]86. Whereas CAs are
   metallo-enzymes that catalyze the reversible hydration of CO[2] into
   HCO[3]^-, the source of inorganic carbon for CaCO[3] precipitation. In
   metazoans, CAs belong to a multigenic family and are widely known to be
   involved in calcification in diverse metazoans such as sponge
   spicules^[171]87,[172]88, mollusk shells^[173]89,[174]90, sea urchin
   skeleton^[175]91, and bird eggshells^[176]92, as well as scleractinian
   corals^[177]34,[178]51. In Tubastraea aurea, CAs were previously
   identified both in the coral tissues and the skeletal organic matrix,
   where they have been suggested to play a direct role in
   calcification^[179]13. Here, we have identified 3 CAs with higher
   expression in the total colony compared to the oral fraction, which
   suggests a potential role in calcification.

   The presence of these candidates amongst the aboral-specific genes of a
   non-symbiotic scleractinian coral strongly suggests a
   calcification-related function and, in a wider context, further
   supports the hypothesis of a “common calcification toolbox” in
   scleractinian corals, as previously suggested^[180]93.

   However, several components of the toolbox have not been identified
   amongst the aboral-specific genes of Tubastraea spp. (Fig. [181]4).
   These include: (1) voltage-gated H^+ channels (H[v]CN), that have been
   suggested to participate in the pH[i] homeostasis of calcifying
   coccolithophore cells^[182]94, in the larval development and shell
   formation of the blue mussel^[183]62 and in the calicoblastic cells of
   several symbiotic scleractinian coral species^[184]47,[185]95; (2)
   SLC9s, that have been suggested to play a role in H^+ removal during
   trochophore development in mussels^[186]62 and coral
   calcification^[187]47, (3) Plasma Membrane Ca^2+-ATPase (PMCA), which
   have been suggested to take part in Ca^2+ supply to the sites of
   calcification in mussels^[188]62, as well as in the pH[ECM] regulation
   of the ECM in corals^[189]30, neurexins, that connect the calicoblastic
   cells to the extracellular matrix in corals^[190]48 and (4)
   Voltage-Gated Ca^2+-channels (VGCC), which have been suggested to
   facilitate Ca^2+ transport in the calcifying epithelium of
   oysters^[191]96 and corals^[192]30. One possible explanation of these
   results is that gene gain/loss or even change of protein function has
   occurred during scleractinian evolutionary history, resulting in a
   different “calcification toolkit”. This is further supported by the
   hypothesis that, although calcification-related proteins from distant
   organisms share common properties^[193]53, they have evolved
   independently, through convergent evolution and co-option, in each
   taxon, thus resulting in contrasting rates of conservation between and
   within lineages^[194]55.

   In addition to comparing these calcification-related candidates between
   non-symbiotic and symbiotic scleractinian corals, we have also searched
   for novel ion transporter candidates of the “calcification toolkit” in
   Tubastraea spp. by focusing on the rest of the aboral-specific genes.
   To explore their involvement in biological processes, we performed a GO
   enrichment analysis, and showed that, among several processes, these
   aboral-specific genes are enriched in “extracellular space”, “cell
   adhesion”, “extracellular matrix” and “extracellular matrix
   organization” (Fig. [195]5). These results highlight the importance of
   extracellular matrices in the aboral tissues, in which they play a
   pivotal role in the spatial organization of the cells, as they organize
   them according to their function. Some examples include both the
   organic extracellular matrix (ECM), which facilitates cell–cell and
   cell-substrate adhesion with the help of desmocytes^[196]97, as well as
   the skeletal organic matrix (SOM), which facilitates the controlled
   deposition of the CaCO[3] skeleton. Also, the expression of genes in
   the aboral tissues linked to “carbohydrate metabolic processes”
   suggests an enrichment of biochemical processes involved in
   carbohydrate metabolism, which may ensure a constant supply of energy
   needed to support the energy-demanding process of
   calcification^[197]98,[198]99.

   Moreover, EggNOG annotation (Fig. [199]6) shows that some of the
   aboral-specific genes belong to the following categories: “inorganic
   ion transport and metabolism” and “intracellular trafficking,
   secretion, and vesicular transport”, thus underling the importance of
   membrane and vesicular transport linked to calcification in the aboral
   tissues^[200]2,[201]14,[202]18. These results are supported by recent
   observations of intracellular vesicles moving towards the calcification
   site both in corals^[203]14,[204]18 and sea urchins^[205]100.
   Calcification-related ions have been suggested to be highly
   concentrated in these vesicles in order to promote the formation of ACC
   nanoparticles, which are successively deposited into the calcification
   compartment where crystallization occurs^[206]14. The regulation of
   endocytosis and vesicular transport between membrane-bound cellular
   compartments is therefore strictly necessary in coral calcification,
   and the identification of genes related to these pathways, among the
   Tubastraea spp. aboral-specific genes, further underlines their
   importance also in non-symbiotic scleractinian species.

   KEGG analysis further allowed us to identify a list of candidates that
   could play a role in calcification related ion transport (Table
   [207]2). The list includes several genes belonging to the ammonium
   transporter family (AMT/Rh/MEP), notably, AMT and Rh homologs. As well
   as for AMT transporters, also Rh transporters have been suggested to be
   involved in coral calcification. Rh homologs have been identified in
   the calicoblastic epithelium of the symbiotic scleractinian coral
   Acropora yongei, where they have been suggested to mediate a possible
   pathway for CO[2]—a critical substrate for CaCO[3] formation—in the
   ECM^[208]101. The identification of these genes also among the
   Tubastraea spp. aboral-specific ones strongly suggests a direct role of
   these transporters in non-symbiotic scleractinian calcification.

   We also identified a large number of transporters belonging to the
   SoLute Carrier (SLC) families that, in vertebrates, constitute a major
   fraction of transport-related genes ^[209]102 (Table [210]2). Some of
   these members (SLC7, SLC25 and SLC35) have been previously reported to
   be involved in coral thermal stress, while others (SLC26) have been
   proposed to participate in coral larval development^[211]103, as well
   as cellular pH and bicarbonate metabolism^[212]31.

   Two plasma-membrane homologs belonging to the SLC13 family have also
   been identified (Table [213]2). These transporters function as
   Na^+-coupled transporters for a wide range of tricarboxylic acid (TCA)
   cycle intermediates^[214]104, and have been widely described in
   vertebrates for their role in calcification^[215]105–[216]108. The
   tricarboxylic acid citrate has also been found to be strongly bound to
   the bone nanocrystals in fish, avian, and mammalian bone^[217]109,
   whereas in corals no study has shown the presence of citrate in the
   skeleton. In invertebrates, SLC13 members have been mainly described
   for their role in nutrient absorption^[218]110,[219]111, as they
   provide TCA cycle metabolites, that are used for the biosynthesis of
   macromolecules, such as lipids and proteins^[220]2,[221]20. These
   macromolecules are among the principal components of the skeletal
   organic matrix and SLC13 members might contribute to their transport
   into the coral aboral tissues.

   SLC16 family members, and precisely the monocarboxylic acid
   transporters (MCTs), are also enriched in the total colony compared to
   the oral fraction (Table [222]2). Members of the SLC16 family comprise
   several subfamilies that differ in their substrate
   selectivity^[223]112. In corals and sea anemones, SLC16 subfamilies
   transporting aromatic amino acids have been mostly characterized for
   their role in nutrient exchange between the coral host and its
   symbionts^[224]36,[225]113, while no information is available for those
   transporting monocarboxylic acids. In human, MCTs function as pH[i]
   regulatory transporters by mediating the efflux of monocarboxylic acid
   (predominantly lactate) and H^+, in tissues undergoing elevated
   anaerobic metabolic rates^[226]114,[227]115, and in Tubastraea spp.
   they might be involved in H^+ extrusion at the sites of calcification
   perhaps functioning as pH[i] regulators.

   Interestingly, members of the SLC23 family (FC = 916.1), which comprise
   ascorbic acid transporters, are the most enriched in the total colony
   compared to the oral fraction, in Tubastraea spp. (Table [228]2). This
   result is in agreement with another RNA-seq study performed on swimming
   and settled larvae of the coral Porites astreoides, also showing that
   an SLC23 transporter is among the most highly expressed ion
   transporters in larvae initiating calcification^[229]116. Ascorbic acid
   is an essential enzyme cofactor that participates in a variety of
   biochemical processes, most notably collagen
   synthesis^[230]117,[231]118. Collagen is a fibrillar protein, that
   forms one of the main components of extracellular matrices^[232]119.
   Previous studies have shown that the addition of ascorbic acid
   stimulates collagen production in many metazoans^[233]120–[234]122,
   including corals^[235]119. It is thus possible that SLC23 transporters
   might provide ascorbic acid to the aboral tissues, and potentially the
   calcifying cells, which use it to promote collagen that, together with
   other ECM proteins, builds a structural framework for the recruitment
   of calcium binding proteins, as previously
   suggested^[236]48,[237]123–[238]125.

   Last but not least, our study also identified so-called “dark genes”,
   i.e., genes that lack annotation^[239]126, within the list of
   aboral-specific genes. These genes are potentially equally important,
   as they are expressed in the aboral tissues along with other genes with
   known functions in calcification. It is therefore possible that “dark
   genes” and calcification-related genes may be linked, as they can be
   involved in the same pathway e. g. enzymes and/or regulatory factors.
   Heterologous expression of “dark genes” in model systems, easy to
   manipulate and with available molecular tools for visualizing gene
   expression and protein localization (e. g. Nematostella vectensis)
   could be taken in consideration to investigate their role and
   contribute to functional annotation^[240]127.

Conclusion

   The Tubastraea spp. transcriptome here provided is a fundamental tool
   which promises to provide insights not only about the genetic basis for
   the extreme invasiveness of this particular coral genus, but also to
   understand the differences between calcification strategies adopted by
   symbiotic and non-symbiotic scleractinian corals at the molecular
   level. The analysis of the aboral-specific genes of Tubastraea spp.
   revealed numerous candidates for a potential role in scleractinian
   calcification, including both previously described candidates (SLC4-γ,
   AMT-1like) and novel ion transporters (SLC13, −16, −23, and others)
   (Fig. [241]7). Future studies will then be required to better dissect
   the precise mechanisms behind these candidates and may offer further
   knowledge which could lead to the development of novel biotechnological
   strategies for prevention, management, and control of this and other
   invasive species.

Figure 7.

   Figure 7
   [242]Open in a new tab

   Model of a calicoblastic cell from a non-symbiotic scleractinian coral
   showing aboral specific ion transporter candidates identified in this
   study. Cellular localization of ion transporters on the apical and
   basolateral membrane is hypothetical and has been assigned based on
   previous immunolocalization studies and/or existing literature in other
   model systems. Dark green arrows indicate putative biological processes
   in which ion transporters could be involved. Abbreviation: ECM:
   Extracellular Calcifying Medium.

Methods

Biological material and experimental design

   Experiments were conducted on non-symbiotic corals belonging to the
   Tubastraea genus. Corals belonging to this genus possess poorly defined
   taxonomic features and several unidentified morphotypes that severely
   challenge species identification^[243]128. Three independent Tubastraea
   spp. colonies, of unknown genotype, were collected in Australia in
   February 2018. The colonies were purchased from De JONG Marine Life,
   Netherland and imported with the CITES N°2018MC39519. These were grown
   in the long-term culture facilities at the Centre Scientifique de
   Monaco in aquaria supplied with seawater from the Mediterranean Sea
   (exchange rate 2% h^−1 and flow rate of 20 L/h), under the following
   controlled conditions: semi-open circuit, temperature of 26 °C and no
   light. Corals were fed daily with frozen rotifers and twice a week with
   live Artemia salina nauplii.

Micro-dissection, RNA isolation and sequencing from Tubastraea spp.

   Three biological replicates of Tubastraea spp. were micro-dissected by
   separating the oral fraction from the total colony. Then, RNA was
   extracted from each fraction, as previously described^[244]47.
   Preparation of mRNAs, fragmentation, cDNA synthesis, library
   preparation, and sequencing using Illumina HiSeq™ 2000 were performed
   at the King Abdullah University of Science and Technology
   (KAUST)^[245]93.

Data analysis pipeline

   Data analysis pipeline contained three major sections including: raw
   data pre-processing, de novo transcriptome assembly and post-processing
   of the transcriptome. First, raw reads of six individual libraries were
   subjected to quality trimming, using the software Trimmomatic (version
   0.36)^[246]129. This step consisted in trimming low quality bases,
   removing N nucleotides, and discarding reads below 36 bases long. Then,
   contaminant sequences were removed, using the software BBDuk^[247]130,
   by blasting raw reads against a previously created contaminant_DB of
   the most common contaminant species—including Symbiodiniaceae. Clean
   and trimmed reads from all samples were then pooled together and
   further assembled using Trinity software (version 2.8.0) with default
   parameters^[248]131. The in-silico normalization was performed within
   Trinity prior to de novo assembly. To obtain sets of non-redundant
   transcripts, we applied the following filtering steps: (1) transcripts
   with more than 95% of identity were clustered together using CD-HIT
   software^[249]132 and (2) all likely coding regions were filtered by
   selecting the single best open reading frame (ORF) per transcript,
   using TransDecoder (version 3.0.0)^[250]133. Also, in the latter step,
   transcripts with ORFs < 100 base pairs (bp) in length were removed
   before performing further analyses. The final transcriptome (referred
   to as transcriptome_all) was subjected to quality assessment via
   generation of ExN50 statistics, using “contig_ExN50_statistic.pl”, and
   examination of orthologs completeness, using BUSCO (version 3) against
   eukaryota_odb10 database^[251]134. Transcriptome_all was then aligned
   against NCBI’s non-redundant metazoan databases using Blastx^[252]135,
   with a cutoff E-value of < 10^–15, and the alignment results were used
   to annotate all the unigenes (= uniquely assembled transcripts). For
   their further annotation and classification, OmicsBox software (version
   2.0.36)^[253]136 was used to assign Gene Ontology (GO) terms^[254]137,
   Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups
   (EggNOG)^[255]138 and Kyoto Encyclopedia of Genes and Genomes (KEGG)
   pathway enrichment analysis^[256]139. Additionally, differential
   abundance analysis to identify differentially expressed genes (DEGs)
   was performed using OmicsBox^[257]136. To convert the RNA-Seq data into
   quantitative measure of gene expression, we calculated the number of
   RNA-Seq reads mapping to transcriptome_all. Transcripts that had at
   least a log fold change (LogFC) of ± 1 with a false discovery rate (FDR
   or adjusted p-value) less than 0.05 were considered as differentially
   expressed.

Supplementary Information

   [258]Supplementary Information 1.^ (3.9MB, tiff)
   [259]Supplementary Information 2.^ (3.9MB, tiff)
   [260]Supplementary Information 3.^ (14KB, docx)
   [261]Supplementary Information 4.^ (3.9MB, tiff)
   [262]Supplementary Information 5.^ (3.9MB, tiff)

Acknowledgements