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 specific 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). This could be attributed to the bio-turbating activity of the clam and perhaps other thiobiotic fauna (Sahoo et al. 2011). All these physical changes brought about by the clam could affect the geotechnical properties of the sediment which in turn affect the responses by both free and associated bacteria and physiology of the clam. Such effect of fauna on the physical properties of the sediment had been mentioned earlier by Rhoads and Boyer (Rhoads and Boyer 1982).
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 fixation.
Four different fractions of bacterial counts showed significant variation across phases (Supplementary Table 5). The TBC comprise of living, dormant and even dead forms (Naik et al. 2016). In the tissues they ranged from 108 to 109 cellsg dry wt− 1 with maximum numbers in the gill during the pre-spawning phase (Table 1). Since gills are the principle filtration 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.
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 106CFU 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 filter 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 108cells g dry wt− 1 was also noted (Table 1) (Rivonker and Parulekar 1995). High heterogeneity in the distribution was observed in the TBC of gill, TVCa of gill and foot, TVCan and CFU of all the three tissues, suggesting patchy distribution. However, significant variations of bacterial parameters across tissues was absent, except for CFU (P < 0.01) (Supplementary 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 fixation.
Branched filamentous 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 efficiency 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. 1996; 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 confirms the significant variation in S2O32−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 S2O32− during post-spawning. This release could perhaps be attributed to the stored S2O32− 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 H2S 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. Significant variation in S2O32− uptake rate was not detected among tissues during pre-spawning and spawning but was evident during post-spawning (Supplementary Table 7). The rate of S2O32− 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− 1obtained 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 S2O32− 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 efficiency in decreasing the ambient sulphide.
In contrast to S2O32− uptake rate, PERMANOVA analysis did not show significant variation in HCO3−uptake rate in tissues across phases (Supplementary Table 7). In tissues, HCO3−uptake rate varied from 1.6 to 594 nmole C g dry wt− 1h− 1 and did not follow the same seasonal trend as in S2O32− 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− 1h− 1, with higher deviation, gill was relatively more homogenous in its activity with less deviation (Fig. 6).
In spite of the low S2O32− uptake rate during spawning, the high HCO3−uptake suggests that either it depends on the stored sulphur compounds (Caro et al., 2007) or other electron donors like CH4 and NH4+. This phase was also characterized by highest concentrations of electron donors (H2S: 385 ± 394 µM, Iodimetric sulphur: 161 ± 244 µM and NH4+: 143 ± 217 µM) and acceptors (O2: 64 ± 25 µM and NO3−: 12 ± 4 µM) in the Chorao mangrove sediment. Both the physiology of the clam and the geochemistry of the ambient water and sediment facilitate the HCO3−uptake in tissue during the spawning phase. The physiology of the clam helps in converting the stored sulphur compounds to S2O32− to help in CO2 fixation when external supply of H2S dissipates. Another aspect relevant to spawning phase is the high rate of dissolution of CO2 and O2 due to freshening of waters during monsoon which coincides with spawning. Continuous supply of potential electron acceptor O2 and the carbon source CO2 facilitates carbon fixation by bacterial associates. Since spawning retards the feeding rate in bivalves, the symbiont/associate based chemoautotrophy could emerge as an additional complementary nutritional source to the host.
Such complementary nutritional sources can also be accomplished by anaplerotic reactions. Anaplerotic reactions generate some of the key intermediates of the citric acid cycle which are also used as precursors in other biosynthetic pathways to produce glucose, fatty acids and essential amino acids (Owen et al. 2002). Some bacteria like the green sulphur bacteria Chlorobaculum tepidum can utilize CO2 and organic molecules like acetate and pyruvate during mixotrophic growth. This is accomplished through a unique carbon and energy metabolism (Feng et al. 2010). The bacteria produce acetyl CoA from acetate. Further, Pyruvate:ferredoxin oxidoreductase convert acetyl-CoA and CO2 to pyruvate. This reaction is promoted by reduced ferredoxin generated during phototrophic growth. Here, anaplerotic pathway maintains the interaction of autotrophic and heterotrophic pathways and drives mixotrophy more efficiently. Relevance of anaplerotic pathways in carbon uptake was also noted in the Roseobacter clade of marine bacteria (Tang et al. 2009).
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 fixation was the lowest during post-spawning. However, contrary to other seasons, mantle was relatively more efficient 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 S2O32− uptake potential and associated bacterial abundance in this phase, the HCO3−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 fixation by its bacterial associates. Besides, the lower concentration of ambient dissolved oxygen due to higher salinity could slow down HCO3−uptake synchronizing with the changing physiology of the clam. At this stage, the existing mechanism of filter feeding could be enough for sustenance.
Since, most of the studies on HCO3−uptake by chemoautotrophic fauna have been represented as rate g wet wt− 1of the tissue. The maximum HCO3−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 magnifica from Galápagos Rift which showed 900 nmole C g wet wt− 1 h− 1 Childress (Childress et al. 1991; Henry et al. 2008). The tubeworm Ridgeia piscesae from JFR vents and Lamellibrachia cf. luymesi from cold seep were able to take up 3.5 and 2.4 µmole C g wet wt− 1 h− 1 at the expense of H2S (Freytag et al. 2001; Nyholm et al. 2008). The vent mussel Bathymodiolus brevior and the clam Solemya reidi from the sludge outfall in Santa Monica Bay also exhibited closer rates of 1.4 and 2.4 µmole C g wet wt− 1 h− 1, respectively. The maximum reported rate of 26.8 µmole C g wet wt− 1 h− 1 in the vent tube worm Riftiapachyptila and 24.7 µmole C g wet wt− 1 h− 1 in the snail Alviniconchahessler (Girguis and Childress 2006; Girguis and Childress 2011) were 3 orders higher than the rate obtained for P. erosa. Muddy clam Myrteaspinifera from Norway exhibited chemosynthetic rate at the expense of H2S ranging from 72 to 96 nmole C g wet wt− 1 h− 1. Rate showed by this deep lying clam found at > 30-meter depth (Dando et al. 1985) is close to the rate of P. erosa. Also, the rate reported for cold seep mytilid from the Gulf of Mexico53falls within the range shown by P. erosa. Estimated value of average carbon uptake by 10 individuals m− 2 of P. erosa (standing stock of 1 kg) over the year constitutes 24 mg C m− 2. It is also interesting to note that S2O32− uptake rate of P. erosa was nearly 100 times more than invertebrates from other reducing habitats like vents and seeps, but CO2 fixation is on the lower end of the range suggesting a lower efficiency of the chemoautotrophic bacterial associates. This could be due to the prevalence of the organically rich ecosystem which has lower rates of chemoautotrophy as opposed to oligotrophic region as in the Central Indian Basin (Das et al. 2011) which could promote higher rates.
Besides, the availability of high concentration of electron donors and acceptors, scarcity of organic carbon as substrate and prevailing higher temperatures facilitate much higher rates of chemoautotrophy at most hydrothermal vents. However, dark carbon fixation in mangrove ecosystem is mainly governed by the concentration of electron donors. Though there are no previous records on the role of foot and mantle tissues to chemoautotrophy, our studies clearly showed that the mantle could be the main contributor during post-spawning. However, large standard deviations encountered in the measurement suggest high spatial heterogeneity within the tissue (Duperron et al. 2013).
This study highlights that, the different tissue of P. erosa along with their bacterial associates, were capable of fixing inorganic carbon at the expense of reduced sulphur compound. Though S2O32− uptake was high during the pre-spawning phase in preparation for the ensuing spawning phase, HCO3−uptake was maximum during spawning period coinciding with the monsoon. Ambient geochemistry of the sediment showing high concentrations of electron donors and acceptors in non-limiting concentrations during spawning period, suggests their role in enhancing chemosynthetic production. This chemosynthetic conversion of CO2 by these clams especially during spawning could serve as a supplementary source of nutrition constituting about 24 mg C m− 2 when the clams are physiologically feeble.
Most of the studies on chemosynthesis till date have revolved around hydrothermal vents and cold seep systems or wetlands like salt marshes and sea grass meadows. Such studies have not yet been covered in the Indian mangrove ecosystem. It throws light on the extent of chemoautotrophy prevailing in mixotrophic representative of family Cyrenidae and prompts future attempts on other sub surface dwelling sediment fauna. Participation of mixotrophs in chemoautotrophy could have wide ranging implications in climate modelling and geoengineering.
Future Scope
Down core profiling of both S2O32− oxidizing potential and HCO3− uptake would help compare activity in sediments with the clam and help appreciate their contribution to carbon fixation on a wider spatial scale. 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.