Chemoautotrophy at the Expense of Thiosulphate: A Supplementary Nutritional Source to the Mangrove Clam-Polymesoda Erosa

Chemolithoautotrophy is a primordial process, where chemical energy converts inorganic carbon to organic. The prevalence of chemosynthesis was examined in the mangrove clam, Polymesoda erosa, and the ambient sediment at Chorao Island, Mandovi estuary, Goa. The sediment system is reducing, organically rich, high in electron donors, acceptors, and inorganic carbon. This clam thrives by immersing 75-90% of its body in suldic sediments. Hence, it is hypothesized that it could have an adaptive mechanism like microbially mediated utilization of reduced sulfur compound, S 2 O 32- (model compound) coupled to inorganic carbon uptake. During spawning, maximum carbonate uptake rates of 449 and 594 nmole C g dry wt -1 h -1 were recorded in the gill and foot, respectively. Next generation sequencing revealed that Thiothrix and other sulfur oxidizers gathered from ambient sediment were present in gill, mantle and foot in the ratio 1:3:14 and 1:5:6, respectively. It is inferred that the clam and these associated bacteria could make an important contribution to chemosynthetic carbon xation. The process could serve as an essential supplementary nutritional source especially during the physiologically feeble spawning phase. To the best of our knowledge, this is the rst report of chemolithoautotrophic process in P. erosa of the family Cyrenidae.


Introduction
Mangrove swamps are one of the most productive ecosystems in the world occupying considerable part of the tropical and subtropical coastlines (Robertson 1986). High organic carbon input from the mangrove forests results in milli-molar sulphide enrichment in the sediment, mainly through anaerobic degradation via sulphate reduction (Attri et al. 2011;Fenchel and Riedl 1970;Laurent 2009;Nedwell et al. 1994; Sahoo and Dhal 2009). The mangrove surface sediment is generally aerobic to micro-aerobic followed by deeper anaerobic layers (Sahoo and Dhal 2009). These redox zones lead to the proliferation and functioning of chemoautotrophic bacteria which x inorganic carbon using the energy derived from the oxidation of sulphide, ammonia, methane and metal ions (Oren 2010). Previous work has been restricted to mangrove molluscs such as Anondontia edentula, Lucina pectinata associated with chemoautotrophic bacteria (Christo et al. 2016;Frenkiel et al. 1996;Lebata 2001; Lebata and Primavera 2001). Such studies have been mainly from the mangroves along the Southeast Asia and Carribean Sea Durand et al. 1996;Pimenov et al. 2002).
Polymesoda erosa is widely distributed in the tropical and subtropical mangrove forests, the adults being more abundant in the high tide regions with 9-12 individuals m − 2 (Clemente and Ingole 2006; Clemente and Ingole 2011; Ingole et al. 1994;Meehan 1982). It inhabits the sub-oxic to anoxic sediments in the intertidal zone. However, its potential for sulphide oxidation dependent inorganic carbon uptake (chemoautotrophy) is not yet understood. Moreover, unlike some clams from other reducing environments, P. erosa possesses a complete digestive system. Besides, this species also has rather small gills, which is uncharacteristic of strict chemosymbiotic bivalves (Morton 1976). Though primarily a lter feeder, its occurrence in organically rich, highly sulphidic and anoxic sediment prompted us to investigate the prevalence of chemoautotrophy mediated by the associated bacteria.
This study attempted to address the S 2 O 3 2− (model compound) utilization and HCO 3 − uptake potential of the bacterial associates of P. erosa to indicate the occurrence of chemoautotrophy in this clam. Further, for the rst time the above processes were measured in gill, foot and mantle tissues during different spawning phases under simulated laboratory conditions. This phenomenon has been described previously in other clams belonging to the families Lucinidae, Thyasiridae and Solemyidae. However, to the best of our knowledge this is the rst report of microbial chemolithoautotrophy in P. erosa of family Cyrenidae.
Ambient sediment samples up to 10 cm below sediment surface (bss) were also collected in triplicate and processed as detailed in Fernandes et al., (2012;2013). The sampling was carried out intermittently from February 2013 to June 2015, covering pre-spawning (January-June), spawning (July-September) and post-spawning (October-December) (Rivonker 1995) (Supplementary Table 1). Morphometry of clams in different phases has been studied and given in (Supplementary Table 2).
Geochemical parameters were measured from the ambient sediment (0-2 cm bss) of P. erosa and overlying water. Water content, porosity and speci c gravity of the sediment for each 2 cm depth up to 10 cm bss were measured by weight/volume method (ASTM 1978).
Eh and pH were measured on eld using Sartorius PT-15 portable probe. Salinity was measured using a hand-held refractometer (ATAGO S/MillE) which was pre-calibrated to zero with distilled water.Overlying water was xed for oxygen estimation as per Winkler's titrimetric method (Strckland and Tarson 1965). Approximately 1 g of sediment was xed in 9 ml of 2 % zinc acetate solution for H 2 S estimation.
Subsequently, weight of the sediment was estimated after drying at 60 °C. Pore water for nutrient analyses was extracted in the laboratory as detailed in Fernandes et al. (2012;2013). Sulphur content in the pore-water was measured iodometrically (referred as 'iodometric sulphur') prior to dilution (Hansen 1973). H 2 S content in the xed sediment sample was measured by Pachmayr's method (Truper and Schlegel 1964). TOC was determined by dichromate digestion by calorimetric method (Azam and Sajjad 2005).
Maintenance of clams in the laboratory and tissue sample treatment The clams were maintained at the prevailing ambient temperature of 30±2 ºC in an aerated tank containing a thin bed of mangrove sediments lled with adequate volume of water collected from the sampling site. After a week of acclimatization, their weight, length, width and height were recorded. The shells were subsequently opened and each clam's tissue was washed with pre-ltered autoclaved 50 % sea water. Further, they were dissected under sterile conditions and gill, foot and mantle tissues were separated. Care was also taken to avoid all contamination across the tissues. Subsequently, about 1 g of wet weight of each tissue was homogenized in 1 ml of 50 % pre-ltered and autoclaved sea water and made up to 10 ml and then serially diluted for measuring the S 2 O 3 2uptake rate and bacterial abundance.
Wet weight was later converted to dry weight by drying at 60 °C till constant weight. Each tissue of 0.1 g was used separately for measuring HCO 3 uptake rate.
TBC was estimated as detailed in Hobbie et al. (1977). TVCa and TVCan were estimated as detailed in Kogure et al. (1984) and LokaBharathi et al. (1988;

Results
P. erosa is generally found in the proximity of vegetation. The pre, post and spawning phases of the clam more or less overlap with the pre-monsoon, post-monsoon and monsoon seasons. The body mass reduced by 27% from an average of 134 ± 44 g during pre-spawning to 98 ± 6 g during post-spawning (Supplementary Table 2).
Distribution of P.erosa. The clams were present at an average density of 10 individuals m − 2 in the mid tide and high tide regions of the Chorao mangrove swamp. Adult clams with maximum size were observed during the peak maturation period (June). Mean weight, length, height and width of the mature adult clam during pre-spawning phase were 134 ± 44 g, 7 ± 0.7 cm, 6 ± 0.9 cm and 4 ± 0.5 cm, respectively (Supplementary Table 2).
Geochemical and geotechnical properties of the ambient sediment of P. erosa. Salinity varied from a mean of 2.1 ± 3 during spawning to27.8 ± 4 during post-spawning. The mangrove sediment pH varied from mildly acidic (6.63 ± 0.3) during the post-spawning to alkaline (7.52 ± 0.6) during the spawning period. In contrast, the lowest Eh in sediment was recorded during the spawning period (Supplementary  Table 3).
The concentrations of potential electron acceptors O 2 and NO 3 − during the seasons were in the order spawning > pre-spawning > post-spawning. During the spawning period, O 2 and NO 3 − concentrations reached up to 64 µM and 12 µM, respectively. On the other hand, NO 2 − concentration up to 0.7 µM was recorded during pre-spawning with high standard deviations. It further showed an exponential decrease through spawning and post-spawning periods. In general, all the three electron acceptors were low during the post-spawning period (Supplementary Table 3). The highest sedimentTOC (total organic carbon) of 40.48 ± 0.25 mg C g − 1 dry sediment was during the post-spawning period and the lowest of 6.93 ± 2.16mg C g − 1 dry sediment was during the pre-spawning period (Supplementary Table 3).
Speci c gravity of the sediment measured up to 10 cm bss varied from 1.67 ± 0.9 at 4-6 cm bss to 2.44 ± 0.3 at 6-8 cm bss. The corresponding reduction in porosity to 28.4 ± 21% was recorded at 4-6 cm bss. Water content at this depth was relatively high up to 8.6 ± 0.2 % compared to upper and lower layers (Supplementary Table 4).
Bacterial abundance in tissues. Total bacterial numbers (TBC) were an order higher in pre-spawning phase compared to spawning and post-spawning phases. Maximum colony forming units (CFU) were retrieved from the foot during all the three phases. Spawning phase was generally characterized by low bacterial numbers except in the mantle which possessed relatively high number of TBC. Total aerobic viable counts (TVCa) and Total anaerobic viable counts (TVCan), outnumbered that of gill and foot.
MPN of S 2 O 3 2− utilizing bacteria was estimated in liquid Leiske's medium. The MPN ranged from 10 4 g − 1 dry wt. in gill to nearly 10 6 g − 1 dry wt.in mantle during post spawning. These tubes mostly served to estimate the S 2 O 3 2− utilization in tissues and sediments.
Microbial community structure of gill, foot and mantle of P. erosa and ambient surface sediment.
Proteobacteria was the most abundant in gill and sediment while Bacteroidetes dominated in foot and mantle. Proteobacteria from gill and sediment clustered together depicting close phylogenetic relatedness whereas that from foot and mantle formed another related group (Fig. 3).
Commonality in the reads between sediment and tissues revealed that Thiothrix sp and other sulphur oxidisers could be harvested from the ambient system (Fig. 4). Table 2 shows the comparative distribution of Thiothrix andother chemoautotrophic genera in these tissues. It also shows that Thiothrix tags in tissue were relatively higher than other chemoautotrophic sulphur oxidizers.NGS (next generation sequence)analyses also showed the dominance of Thiothrix sp among bacteria, and their large standard deviations con rm their heterogenous and patchy distribution. They were present in gill, mantle and foot in the ratio 1:3:14 and 1:5:6, respectively. spawning, gill exhibited relatively higher uptake rate of 0.16 mmole g dry wt − 1 h − 1 than the other two tissues (Fig. 5).
During the post spawning phase, gill and foot regained their uptake e ciency. These tissues consumed S 2 O 3 2− at a higher rate of 0.6 mmole g dry wt − 1 h − 1 and 0.4mmole g dry wt − 1 h − 1 , respectively than during the spawning phase. On the other hand, mantle showed a net release of S 2 O 3 2− (Fig. 5).
Experiments to measure HCO 3 − uptake rates at the expense of S 2 O 3 2− showed interesting variations.
HCO 3 − uptake ( 14 C uptake) rate in tissues. In contrast to S 2 O 3 2− uptake, maximum HCO 3 − uptake rate of 594 nmole C g dry wt − 1 h − 1 with a large standard deviation of ± 605 was measured during spawning phase in the foot. This was followed by gill where the average was 441 nmoles C g dry wt − 1 h − 1 with a standard deviation of ± 156 (Fig. 6). On the other hand, mantle showed maximum HCO 3 − uptake of 116 nmole C g dry wt − 1 h − 1 during pre-spawning and the lowest during spawning. Mantle was able to uptake HCO 3 − at a relatively higher rate of 68 nmole C g dry wt − 1 h − 1 than other tissues during post-spawning in spite of the net S 2 O 3 2− release. However, the mean HCO 3 − uptake rate of tissues was low during the postspawning period (Fig. 6).

Discussion
The sediment texture was silty clay which is known to be highly suitable for bacterial multiplication and activity (Chau et al. 2011;Fallon et al. 1983;Sahoo et al. 2011). This habitat could also be suitable for the clams, as they are also dependent on the sediment bacteria for useful association to thrive in the reducing sub-oxic sediment (Dubilier et al. 2008). The geotechnical properties of these sediments are found to be altered in the presence of the clam. The speci c gravity (1.6%) and porosity (28.6%) get reduced, thus increasing the water content (8.6%) especially at 4-6 cm bss (Supplementary Table 4). Chemosynthetic potential of the clam, P. erosa at the expense of reduced sulphur compounds could be attributed to its bacterial associates. The intensity of the process could vary with the three phases of the clam. The phase wise variation can also result from the interaction with ambient environmental parameters. Since the biological rhythm of spawning, overlaps with the monsoon, there could be interesting variations in the pertinent variables promoting dark carbon xation. Table 5). The TBC comprise of living, dormant and even dead forms (Naik et al. 2016). In the tissues they ranged from 10 8 to 10 9 cellsg dry wt − 1 with maximum numbers in the gill during the pre-spawning phase ( Table 1). Since gills are the principle ltration organ, their direct contact with ambient water facilitates the accumulation of highest number of bacteria per gram of tissue (Dando et al. 1985;Duperron et al. 2013). However, the bacterial fractions enumerated as direct viable counts (TVCa and TVCan) are able to participate in the activity either aerobically or anaerobically. These formed 10% of TBC with anaerobes being marginally higher than aerobes. It is probable that a greater portion of them are facultative aerobes or anaerobes as nearly same orders have been encountered under both the conditions. Only about 0.0001 to 0.001% have been able to form colonies.

Four different fractions of bacterial counts showed signi cant variation across phases (Supplementary
Pre-spawning phase was characterized by the occurrence of large adult clams with maximum body mass of 134 ± 44 g (Supplementary Table 2), harboring high number of CFU of thiosulphate utilizing bacteria up to 10 6 CFU g dry wt − 1 (Table 1). This fraction is viable, able to form colonies and perhaps even actively participate in improving the physiology of the clam in preparation for the subsequent spawning phase. Pre-spawning also coincided with the pre-monsoon which is reported to have the highest chl a (Clemente and Ingole 2009) and also perhaps high abundance of bacteria in the Mandovi estuary where the study site is located. Thus, high food supply enhances lter feeding which in turn could lead to a large bacterial population especially in gill tissue during this phase. In sync with the body mass reduction of clam observed during spawning, decrease in TBC to 10 8 cells g dry wt − 1 was also noted (  Table 5). Earlier studies have also mentioned that sulphur oxidizers preferably occupy certain favorable regions in the invertebrate tissues (Dando et al. 1985). The CFU retrieved under microaerobic condition in the mineral media in this study were 1 to 3 times less than those retrieved under aerobic condition as reported earlier by Podgorsek and Imhoff (1999). Most probable numbers are more than CFU by an order.
While CFU facilitate isolation of sulphur oxidizers for future work, MPN help estimate community numbers and their activity in near in situ conditions. Besides, there is more scope for encountering new lineages of these in liquid media.
Proteobacteria was the most abundant in gill and sediment while Bacteroidetes dominated in foot and mantle in metagenomic community analysis. Since Proteobacteria from various chemosynthetic ecosystems like hydrothermal vents and cold seeps are key players in chemoautotrophy (Thomas et al. 2018). Such observation in mangrove ecosystems also emphasizes their important contribution in dark carbon xation.
Branched lamentous facultative anaerobic Thiothrix like cells of 2 different morphotypes with and without sheath were observed in the total bacterial community of gill, foot and mantle tissues (Fig. 2).
Since Thiothrix species were present in the metagenomic community of all the three tissues and in the ambient sediment (Fig. 4, Table 2), it was assumed that they were free-living forms which were laterally acquired from the sediment. Earlier studies on Thiothrix species showed that they can be free living or associates/symbionts (Dattagupta et al. 2009;Distel et al. 1988;Odintsova et al. 1993). Table 2 also shows that Thiothrix tags in tissue were relatively higher than other chemoautotrophic sulphur oxidizers. Apart from being a mixotroph, the high e ciency of Thiotrhix cells to uptake inorganic carbon to perform chemolithoautotrophy can out compete other bacteria in establishing their growth in the host tissue (Nielsen et al. 2000). Presence of common chemoautotrophic OTUs in clam tissues and sediment suggests horizontal transmission. Such horizontal or environmental transmission of chemoautotrophic bacteria were also observed in the lucinid clams Codakia orbicularis, Lucinoma aequizonata and family Thyasiridae Gros et al. 1999).
Thiosulphate uptake measured in the tissues is assumed to be due to the enzymes in the tissue and/or associated bacteria. PERMANOVA (Permutational Analysis of Variance) analysis con rms the signi cant variation in S 2 O 3 2− uptake rate in gill, foot and mantle tissues across different phases (P = 0.001) (Supplementary Table 6). Though the process was observed through the three spawning phases, the maximum rates were noted during pre-spawning in gill (1.1 mmole g dry wt − 1 h − 1 ) and foot (0.8 mmole g dry wt − 1 h − 1 ). The high rates observed in gill and foot could be attributed to their constant and prolonged interaction with water and sediment bacteria, respectively. The rates then reduced by 1 to 2 orders during spawning and later regained during post-spawning (Fig. 5). Mantle displayed net release of S 2 O 3 2− during post-spawning. This release could perhaps be attributed to the stored S 2 O 3 2− in the tissue that gets released before subsequent uptake. Such alternating periods of release and uptake have also been noted by other workers in chemoautotrophic snails and bivalves (Beinart et al. 2015;Ott et al. 1998;Vrijenhoek 2010). Thiosulphate production in these tissues is governed by aerobic oxidation of stored sulphide, sulphite and sulphur in the absence of external sulphide (Arndt et al.2001). Earlier studies on the mangrove clam Anodontia edentula also showed that H 2 S is drawn through the foot and transferred to blood which is later carried and delivered to endosymbiotic bacteria in the gills. Studies have also shown that in the case of chemoautotrophic symbiosis, the foot of the clam penetrates deeper into the sediment to obtain sulphide (Lebata 2001). Though the average thiosulphate utilization across the three phases was higher in the gill, the study also emphasizes the potential of foot and mantle in utilizing the reduced sulphur compound in the mangrove ecosystem. Signi cant variation in S 2 O 3 2− uptake rate was not detected among tissues during pre-spawning and spawning but was evident during post-spawning (Supplementary Table 7). The rate of S 2 O 3 2− uptake has also been examined per gram wet tissue for comparison with the earlier studies. Of the total number of values measured at different time intervals in triplicate with different tissues, the average rate of 0.1 mmole g wet wt − 1 h − 1 obtained with the gill tissue at 10 minutes incubation was used for comparison. Gill of Solemya reidi from a sludge outfall in California was able to uptake 0.52 to 1.45 µmole g wet wt − 1 h − 1 (Anderson et al. 1987). This rate is 2 to 3 orders less than the maximum rate shown by gills of P. erosa. Similarly, the provannid snails Alviniconcha sp. and Ifremeria nautilei and the mussel Bathymodiolus brevior collected from the vents of Eastern Lau Spreading Centre were able to oxidize S 2 O 3 2− within the range of 4.02 to 5.6 µmole g wet wt − 1 h − 1 (Beinart et al. 2015). Again, these rates are two orders less than the maximum rate shown by P. erosa emphasizing its e ciency in decreasing the ambient sulphide.
In contrast to S 2 O 3 2− uptake rate, PERMANOVA analysis did not show signi cant variation in HCO 3 − uptake rate in tissues across phases (Supplementary Table 7). In tissues, HCO 3 − uptake rate varied from 1.6 to 594 nmole C g dry wt − 1 h − 1 and did not follow the same seasonal trend as in S 2 O 3 2− uptake rate. Maximum rates of carbon uptake were measured during spawning period in gill and foot. Though foot showed peak rate of 594 nmole C g dry wt − 1 h − 1 , with higher deviation, gill was relatively more homogenous in its activity with less deviation (Fig. 6).
In spite of the low S 2 O 3 2− uptake rate during spawning, the high HCO 3 − uptake suggests that either it depends on the stored sulphur compounds (Caro et al., 2007)  Thus, mixotrophy in some symbionts is a complementary strategy to maintain the carbon stock when the supply of inorganic carbon is limited. In such cases, they recycle host metabolic products (Seah et al. 2019). So, it is also possible in the case of clam like P. erosa to harbour obligate autotrophic to heterotrophic bacteria which are able to perform varying degree of mixotrophy linked to anaplerotic reactions.
Average rate of carbon xation was the lowest during post-spawning. However, contrary to other seasons, mantle was relatively more e cient than gill and foot (Fig. 6). In this phase, bivalves were characterized by the lowest body mass (Podgorsek and Imhoff 1999) which is also evident in this study (Supplementary Table 2). Though the tissues regained the S 2 O 3 2− uptake potential and associated bacterial abundance in this phase, the HCO 3 − uptake rate was low. As the animals are spent, the use of complementary nutrition would be minimal. High TOC concentration up to 4% during post-spawning in the ambient sediment of P. erosa also decreases the need for inorganic carbon xation by its bacterial associates. Besides, the lower concentration of ambient dissolved oxygen due to higher salinity could slow down HCO 3 − uptake synchronizing with the changing physiology of the clam. At this stage, the existing mechanism of lter feeding could be enough for sustenance.
Since, most of the studies on HCO 3 − uptake by chemoautotrophic fauna have been represented as rate g wet wt − 1 of the tissue. The maximum HCO 3 − uptake rate of 83 nmole C g wet wt − 1 h − 1 in P. erosa was compared to that of hydrothermal vent and seep invertebrates. This rate in P. erosa was lower than the estimated rate of the provannid gastropod Ifremerianautilei of Lau Basin which was 700 nmole C g wet wt − 1 h − 1 (Beinart et al., 2015). The value in this study was also lower than that of the clam Calyptogena This study highlights that, the different tissue of P. erosa along with their bacterial associates, were capable of xing inorganic carbon at the expense of reduced sulphur compound. Though S 2 O 3 2− uptake was high during the pre-spawning phase in preparation for the ensuing spawning phase, HCO 3 Studies could be extended to other electron donors to understand the full chemoautotrophic potential of the clams and sediment associated bacteria. Including other fauna in the study would help in understanding chemosynthesis in the mangrove sediment more holistically. Participation of mixotrophic processes in sediments could be considered for climate models.
Declarations Figure 1 Sampling site Brown stretch represents the high tide region where clams are available Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 2
Please see the Manuscript le for the complete gure caption.

Figure 3
Heat map showing the taxonomic lineage of microbial communities at phylum level in gill, foot and mantle tissues of P. erosa and its ambient sediment (0-2 cm, bss) G -gill; S-sediment; F-foot; M-mantle; bss-below surface sediment; The colour code indicates the increase in range of relative abundance of sequence reads from red to green.

Figure 4
Pictorial representation of dominant chemoautotrophic archaeal and bacterial genera common in clam tissue and ambient sediment. indicates thiosulphate release rate. With mantle, below zero relates to the added level of thiosulphate. Thus thiosulphate uptake below zero is possible because of the "release" by the tissue