Efficacy of amorphous TiOx-coated surfaces against micro- and macrofouling through laboratory microcosms and field studies.

In this study, Soda Lime Glass (SLG) and Stainless Steel (SS316L) substrata coated with Titanium oxide (TiOx) were tested for their efficacy in the laboratory microcosms and in field against micro- and macrofouling. Laboratory microcosm studies were conducted for five days using natural biofilms, single-species diatom (Navicula sp.), and bacterial biofilms, whereas field observations were conducted for 30 days. The TiOx-coating induced change in the mean contact angle of the substratum and rendered SS316L more hydrophilic and SLG hydrophobic, which influenced the Navicula sp. biofilm, and bacterial community structure of the biofilm. Overall, the TiOx-coated SS316L showed minimal microfouling, whereas non-coated SLG exhibited greater efficacy in deterring/preventing macrofouling organisms. Moreover, the reduction in macrofouling could be attributed to high abundance of Actinobacteria. Unraveling the mechanism of action needs future studies emphasizing biochemical processes and pathways.

Ecological Impacts of Aged Freshwater Biofilms on Estuarine Microbial Communities Elucidated Through Microcosm Experiments: A Microbial Invasion Perspective.

Inadvertent introductions of alien species via biofilms as a vector released through ballast water are of environmental importance, yet their consequences are not much known. In the present study, biofilm communities developed in an inland freshwater port under in situ and dark conditions were subjected to long-term dark incubations. Subsequently, the impact of these aged biofilms as vectors on estuarine water column communities were evaluated using microcosm experiments in the laboratory. Variations in biofilm and planktonic microbial communities were quantified using quantitative PCR.Upon prolonged dark incubation, a shift in bacterial diversity with an increase in tolerant bacterial communities better adapted to stress was observed. Actinobacteria were the dominant taxa in both aged biofilms upon dark incubations. The laboratory studies indicated that on exposure of these biofilms to estuarine water, resuscitation of Vibrio alginolyticus, V. parahaemolyticus, and V. cholerae from a dormant state existing in these biofilms to culturable form was observed. Moreover, the results revealed that both the biofilm types can pose a threat to the environment, but the degree of risk can be attributed to the imbalance caused by significant changes in the surrounding estuarine microbial communities. Consequently, this may result in either proliferation or decline of some genera with different metabolic potential and resuscitation of pathogenic forms not present earlier, thereby influencing the ecology of the environment. Quantifying these effects in the field using biofilm metagenomes with an emphasis on virulent species and understanding traits that enable them to adapt to changing environments is a way forward.

Response of the bacterial metagenome in port environments to changing environmental conditions.

Port environments are highly dynamic and hotspots for marine bioinvasion. This study investigated the bacterial diversity at two geographically distant ports (Mangalore-marine port; and Haldia-riverine port) using next-generation sequencing during southwest monsoon and non-monsoon (Pre-monsoon) seasons. During southwest monsoon, at both marine and riverine ports, operational taxonomic units (OTUs) affiliated to bacteria reported to have hydrocarbon degrading ability were observed. Whereas during pre-monsoon, a significant increase in benthic bacterial OTUs was evident at the marine port, and the riverine port was characterized by oceanic species OTUs. Results suggest that the dynamics of prevalent environmental conditions, driven by seasons, led to emergence of ecologically relevant bacteria, many of which have been observed for the first time in Indian coastal waters. Their presence could be used as indicators of prevailing environmental conditions and nature of anthropogenic influence in port ecosystems. Unravelling functional roles of such ecologically relevant species is a way forward.

Sedimentary organic matter composition from tropical ports with distinct geographic and morpho-hydrodynamic characteristics: Evaluation through multiple biochemical markers.

Increasing coastal urbanization and shipping activity-related environmental pollution advocate the importance of assessment of port ecosystems. Fatty acid biomarkers, elemental components, and biopolymers were used to evaluate the composition of sedimentary organic matter and benthic trophic status of Kolkata (freshwater, enclosed docks) and Kandla (seawater, macrotidal) ports of India. The sediment fatty acid composition indicated relatively fresh and energy-rich organic matter of phytoplankton and bacterial origin inside Kolkata port than the outside riverine station and Kandla port. Biopolymeric carbon (BPC), used as an indicator of trophic status, revealed eutrophic condition in Kolkata port with high accumulation of organic matter of autochthonous origin, attributed to poor water flushing and input of anthropogenic wastes. In contrast, Kandla port was meso-oligotrophic, rich in bacteria, and terrestrial plant-derived materials. Such an assessment of ports' trophic status helps to evaluate the health of the ecosystem and in management practices.

Changes in the ballast water tank bacterial community during a trans-sea voyage: Elucidation through next generation DNA sequencing.

Ballast water (BW) mediated bioinvasion is one of the greatest threats to the health of aquatic ecosystems. Bacteria, unlike higher organisms, are transferred in large numbers through BW. Owing to their abundance and potential pathogenicity, they pose a direct threat to the prevailing microbiome in the recipient waters and also to human health. This study investigated the changes in the BW tank bacterial community during a trans-sea voyage from Visakhapatnam port, located along the east coast of India (Bay of Bengal) to Mumbai port, located along the west coast of India (Arabian Sea). Next generation sequencing was used to explore the unculturable segment of bacteria. The BW tank conditions led to a decrease in photoautotrophs and non-spore forming bacteria. On the other hand, biofilm forming and antibiotic producing bacteria, nutrient limiting condition sustaining bacteria, and those capable of synthesizing enzymes prerequisite for active metabolism under stress, increased over time. The shifts in the bacterial community were dependent on mechanisms adopted by the clades to cope with the BW tank conditions. Functional prediction of the bacterial community revealed a significant increase in the core metabolic functions, which enabled the survival of such bacteria. As the voyage progressed, an increase in the total viable bacteria in BW tanks could be attributed to the decrease in the abundance of phytoplankton and zooplankton. At the end of the voyage, the bacterial community in the BW tanks was significantly different, and the species diversity and richness were higher than that of the natural seawater (source water). Pathogenic species were more abundant during mid-voyage than at the end of the voyage, suggesting that voyage duration influences the pathogenic bacterial community. Investigating the fate of the discharged bacterial population at the deballasting point is a way forward in the assessment of marine bioinvasion.

Influence of salinity stress on bacterial community composition and ß-glucosidase activity in a tropical estuary: Elucidation through microcosm experiments.

The influence of changing salinity on community composition and functional activity (Bacterial Production (BP) and ectoenzyme activity) of major bacterial taxa was evaluated using microcosm experiments in a tropical monsoon influenced estuary. Natural bacterial inocula at different salinities, representing marine, brackish, and freshwater, were inter-transferred and elucidated their response with an emphasis on community composition and ß-Glucosidase (BGase) activity. The results revealed a significant decrease in the total bacterial count (TBC) and BP on the translocation of bacterial inocula to different salinity conditions in the case of freshwater bacteria. However, a significant increase in BGase activity coupled with shifts in the studied bacterial groups was evident in the case of marine as well as freshwater bacteria. Quantitative PCR (qPCR) revealed a shift in major bacterial taxa upon translocation to different waters, which was dependent on salinity and the source of inocula. Redundancy and qPCR analyses showed that members belonging to Gammaproteobacteria and Betaproteobacteria were higher, and possibly influenced BGase activity in marine and freshwater, respectively. Translocation of marine inocula to brackish and freshwater resulted in an emergence of Bacteroidetes, Actinobacteria, and Betaproteobacteria, respectively. Whereas, when freshwater inocula were translocated to marine or brackish water, Alphaproteobacteria and Gammaproteobacteria taxa emerged, and this was coupled with increased BGase activity. In contrast, brackish water bacteria showed a strong persistence in bacterial community composition when translocated to different salinities within this estuary. Such phylogenetic persistence or changes suggests species level shifts in specific bacterial taxa, and unravelling the same using different functional gene markers would ascertain their role in organic matter processing and is way forward.

Habitat characteristics mediated partitioning of economically important bivalves in a tropical monsoon-influenced estuary.

Bivalves are benthic organisms inhabiting coastal marine habitats, especially estuarine tidal and mudflats. Due to their high stocking density and rich protein content, they form a major part of the artisanal fishery resource around the world. A clear partitioning in the population of bivalves, Paphia malabarica (Chemnitz, 1782), and Meretrix casta (Gmelin, 1791) in southern (Chicalim) and northern (Siridao) bank of a tropical Zuari estuary influenced by the monsoon along the Indian west coast, is evidenced. This study unravels the reasons for their partitioning in this estuary. Paphia malabarica is an exclusive inhabitant of Chicalim which has silty-sandy sediment, whereas M. casta is exclusive to Siridao, a sandy habitat. Observations showed that this segregation is facilitated by the semi-enclosed nature of habitat at Chicalim with the high amount of degraded and aged sediment organic carbon, high chlorophyll a, elemental, and biochemical components, whereas Siridao experiences the high impact of tidal currents, low sediment organic carbon, and high water column chlorophyll a. The habitat in Siridao gets exposed to UV radiation during low tide, reducing the photosynthetic oxygen production, turning the habitat to periodic anoxia indicated by differences in the TOC:TS ratio. However, such conditions may not influence M. casta, which can derive oxygen from the water column. The fatty acids specific to diatoms, dinoflagellates, higher plants, and partially degraded organic matter in the tissues of P. malabarica indicate their ability to source the food from the sediment and water column, whereas in tissues of M. casta, higher dinoflagellate-specific fatty acids followed by diatom and bacteria indicate water column-derived food. Chicalim can be considered an actively coupled benthic-pelagic habitat, and Siridao as an uncoupled habitat. Thus, the diverse flux of food particles, species-specific feeding ecology, and local hydrodynamics operating at these habitats could be the determining factors in the partitioning of the bivalves. Graphical abstract .

Seasonal variation in ß-glucosidase-producing culturable bacterial diversity in a monsoon-influenced tropical estuary.

The episodic monsoon condition in a tropical estuarine environment inputs high allochthonous materials that are rich in carbohydrates and are mainly broken down by enzymatic hydrolysis thus alter both organic matter and microbial community composition of an estuary. ß-Glucosidases are one of the enzymes mediating the degradation of carbohydrates and are majorly produced by heterotrophic bacteria. The present study elucidated ß-glucosidase-producing culturable bacterial diversity and measured their activity during non-monsoon and monsoon seasons in Zuari estuary. The results revealed that both bacterial abundance and ß-glucosidase activity decreased significantly from non-monsoon to monsoon, whereas phylogenetic diversity increased. Majority of ß-glucosidase producers during non-monsoon belonged to the members of Bacillales (53%), Pseudomonadales (26%), and Vibrionales (11%) which shifted to the members of Enterobacteriales (51%), Bacillales (14%), Alteromonadales (12%), Aeromonadales (9%), Xanthomonadales (7%), Pseudomonadales (5%), and Flavobacteriales (2%) during the monsoon. The shift in bacterial community structure points out the occurrence of different allochthonous forms with carbohydrate-metabolizing ability during the monsoon, and their relevance in ecology and health of this estuary can be elucidated by studying their functional diversity and is a step ahead.

Immunoecology of the short neck clam Paphia malabarica (Chemnitz, 1782) in a tropical monsoon-influenced estuary.

Understanding the variability in organism's immunological response is crucial for predicting changes at population or community level. The present study investigated the immunoecology of a commercially valuable clam Paphia malabarica in a tropical monsoon-influenced estuary. Clams were collected monthly during a year cycle, which coincided with pre-monsoon (February-May), monsoon (June-September) and post-monsoon seasons (October-January). For assessment of immune functioning, selected hemocyte parameters (total hemocyte concentration, hemocyte mortality, lysosomal content, esterase activity, reactive oxygen species production, and phagocytic activity) were analyzed using flow cytometry. Simultaneously, clam's condition index, nutrients, chlorophyll a, dissolved oxygen, pH, temperature and bacterial density were also measured at the sampling site. Our results exhibited seasonal patterns in hemocyte functioning with the highest activity during the pre-monsoon season (suggestive of a suitable harvesting period) and lowest during monsoon (suggestive of a critical biological period). The critical biological period for P. malabarica was marked with compromised immune parameters inflicted by low salinity, food availability, and possibly high bacterial abundance. Also, the involvement of reproductive stress altering the hematological functioning in P. malabarica cannot be ruled out. Nutrients, dissolved oxygen, pH and temperature could not explain much of the hemocyte variability. The present study has further validated the usefulness of hemocyte as a suitable marker for understanding immunoecology of P. malabarica which is of prime importance, especially in a monsoon-influenced tropical estuarine environment. The findings of our research will be constructive in monitoring natural as well as cultivated bivalve populations of economic and ecological relevance.

Involvement of inducible nitric oxide synthase (iNOS) in immune-functioning of Paphia malabarica (Chemnitz, 1782).

In recent years, the role of inducible nitric oxide synthase (iNOS) isoform has been widely studied because of its immunological relevance in higher organisms as well as invertebrates including bivalves. However, little is known about the immunological role of iNOS in Paphia malabarica defense mechanism. In this study, we immunodetected the presence of iNOS in P. malabarica hemocytes using antibody N9657 monoclonal anti-nitric oxide synthase. In addition, increased iNOS activity was evident in response to a higher bacterial dosage (Vibrio parahaemolyticus and V. cholerae), highlighting the dose-dependent iNOS activity induction. Also, higher bacterial survivability was observed in the presence of iNOS inhibitor, i.e., S-methylisothiourea hemisulphate (SMIS) thus, validating the bactericidal role of iNOS. These findings implicate the involvement of iNOS in immune-functioning of P. malabarica. Future work should focus on elucidating the expression and regulation of pathogenesis in P. malabarica, involving iNOS.

Influence of Darkness and Aging on Marine and Freshwater Biofilm Microbial Communities Using Microcosm Experiments.

Ballast tank biofilms pose an additional risk of microbial invasion if sloughed off during ballasting operations, yet their significance and invasion biology is poorly understood. In this study, biofilms developed in marine and freshwater locations were exposed to prolonged darkness and aging by mimicking ballast water conditions in the laboratory. Upon prolonged darkness, the decay of phytoplankton, as indicated by the decrease in chlorophyll a in marine biofilms, led to remineralization and enhanced bacterial and protist populations. However, the same trend was not observed in the case of freshwater biofilms wherein the microbial parameters (i.e., bacteria, protists) and chlorophyll a decreased drastically. The bacterial community structure in such conditions was evaluated by real-time quantitative PCR (qPCR), and results showed that the biofilm bacterial communities changed significantly over a period of time. α-Proteobacteria was the most stable taxonomic group in the marine biofilms under dark conditions. However, ß-proteobacteria dominated the freshwater biofilms and seemed to play an important role in organic matter remineralization. γ-Proteobacteria, which includes most of the pathogenic genera, were affected significantly and decreased in both the types of biofilms. This study revealed that marine biofilm communities were able to adapt better to the dark conditions while freshwater biofilm communities collapsed. Adaptation of tolerant bacterial communities, regeneration of nutrients via cell lysis, and presence of grazers appeared to be key factors for survival upon prolonged darkness. However, the fate of biofilm communities upon discharge in the new environment and their invasion potential is an important topic for future investigation.

Effect of pea crab Pinnotheres vicajii (Chhapgar, 1957) on immunocompetence of bivalve Paphia malabarica (Chemnitz, 1782).

The endosymbiont-host relationship between a pea crab and its bivalve host has been studied previously using bivalve's multi-physiological parameters. The present study is first of its kind that uses hemocyte's immune functionality to evaluate the symbiotic relationship between bivalve Paphia malabarica and its symbiont crab, Pinnotheres vicajii. The sampling was carried out at two stations (differed in nutrient concentration, productivity, and bacterial abundance) located along the southwest coast of India. The results showed lower immunocompetence in the case of bivalves infested with pea crabs. The lowering of hemocyte population was the most prominent and perhaps the initial reaction in response to focal inflammation or wound inflicted by the pea crabs. A significant lowering of phagocytosis, lysozyme, and esterase activity was also observed, indicating the detrimental impact of crab infestation. Interestingly, no significant correlation was found between parasitism and condition index despite the lower immunocompetence, validating the effectiveness of using hematological parameters in comparison to other life-history traits. This indicates that the immune parameters being directly related to the organism's metabolic state can elucidate the early effect of stressors, therefore, proving to be a better proxy for understanding a host-pathogen relationship. Disparity found in bivalve's immunocompetence between the sampling sites could be linked to either variation in environmental parameters, the size of the symbiont or the infection level. Our preliminary results provide a direction towards examining immunodepression in parasitized bivalves by considering ex-situ controlled parasitism induction along with varying environmental conditions. In view of this, efforts must be directed towards minimizing pea crab's outbreaks, especially in cultivated shellfish farms. Future studies should elucidate molecular mechanisms involved in the immunocompetence of P. malabarica, hosting the parasitic pea crabs.

Immune response of the short neck clam Paphia malabarica to salinity stress using flow cytometry.

Paphia malabarica is a predominant and commercially important bivalve in India, persistently challenged by wavering salinity in a monsoon-influenced estuary. To examine the organism's immunological response under such a condition we challenged P. malabarica with different salinities (0, 5, 15, 25 and 35) for varied periods using a two-way experimental approach (in vitro and in vivo). This is the first study to report the response of P. malabarica hemocytes to salinity stress from a monsoon-influenced estuary on the southwest coast of India. Evaluation of total hemocytes count, mortality, lysosomal content, reactive oxygen species production, phagocytic and esterase activity was carried out using flow cytometric analysis. In both the experimental conditions, hemocyte parameters were significantly compromised at lower salinities (0 and 5) with an evident immuno-salinity tolerance range of 15-35. The damaging impact of 0 and 5 salinities on hemocyte function intensified with a longer exposure period, indicating that prolonged exposure to low salinity could be detrimental to bivalve wellness if they are pushed beyond their tolerance range which is usually observed during the monsoon. Further studies should focus on the interactive effect of salinity tagged with different stressors influencing biology of P. malabarica.

Elucidation of the tidal influence on bacterial populations in a monsoon influenced estuary through simultaneous observations.

The influence of tides on bacterial populations in a monsoon influenced tropical estuary was assessed through fine resolution sampling (1 to 3 h) during spring and neap tides from mouth to the freshwater end at four stations during pre-monsoon, monsoon and post-monsoon seasons. Higher abundance of total bacterial count (TBC) in surface water near the river mouth, compared to the upstream, during pre-monsoon was followed by an opposite scenario during the monsoon When seasonally compared, it was during the post-monsoon season when TBC in surface water was highest, with simultaneous decrease in their count in the river sediment. The total viable bacterial count (TVC) was influenced by the depth-wise stratification of salinity, which varied with tidal fluctuation, usually high and low during the neap and spring tides respectively. The abundance of both the autochthonous Vibrio spp. and allochthonous coliform bacteria was influenced by the concentrations of dissolved nutrients and suspended particulate matter (SPM). It is concluded that depending on the interplay of riverine discharge and tidal amplitude, sediment re-suspension mediated increase in SPM significantly regulates bacteria populations in the estuarine water, urging the need of systematic regular monitoring for better prediction of related hazards, including those associated with the rise in pathogenic Vibrio spp. in the changing climatic scenarios.

Daily variations in pathogenic bacterial populations in a monsoon influenced tropical environment.

Changing climatic conditions have influenced the monsoon pattern in recent years. Variations in bacterial population in one such tropical environment were observed everyday over two years and point out intra and inter annual changes driven by the intensity of rainfall. Vibrio spp. were abundant during the monsoon and so were faecal coliforms. Vibrio alginolyticus were negatively influenced by nitrate, whereas, silicate and rainfall positively influenced Vibrio parahaemolyticus numbers. It is also known that pathogenic bacteria are associated with the plankton. Changes in the abundance of plankton, which are governed mainly by environmental changes, could be responsible for variation in pathogenic bacterial abundance during monsoon, other than the land runoff due to precipitation and influx of fresh water.

Association of bacteria with marine invertebrates: implications for ballast water management.

Bacteria associated with plankton are of importance in marine bioinvasions and the implementation of ship's ballast water treatment technologies. In this study, epibiotic and endobiotic bacteria associated with zooplankton, including barnacle nauplii, veliger larvae, and adults of the copepod Oithona sp., were characterized and quantified. Barnacle nauplius and veliger larva harbored ~4.4 × 10(5)cells ind(-1) whereas Oithona sp. had 8.8 × 10(5)cells ind(-1). Computation of bacterial contribution based on biovolume indicated that despite being the smallest zooplankton tested, veliger larvae harbored the highest number of bacteria, while barnacle nauplii, the largest of the zooplankton, tested in terms of volume contributed the least. Pulverization of zooplankton led to an increase in bacterial numbers; for example, Vibrio cholerae, which was initially 3.5 × 10(3), increased to 5.4 × 10(5)CFU g(-1); Escherichia coli increased from 5.0 × 10(2) to 1.3 × 10(4)CFU g(-1); and Streptococcus faecalis increased from 2.1 × 10(2) to 2.5 × 10(5)CFU g(-1), respectively. Pulverized zooplankton was aged in the dark to assess the contribution of bacteria from decaying debris. Aging of pulverized zooplankton led to emergence of Chromobacterium violaceum, which is an opportunistic pathogen in animals and humans.

Characterization of bacteria in ballast water using MALDI-TOF mass spectrometry.

To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time.

Relevance of biofilm bacteria in modulating the larval metamorphosis of Balanus amphitrite.

Natural microbial communities found on different substrata exposed to the marine environment, including barnacle shell surfaces, are reported to have varying influences on the settlement and metamorphosis of competent cypris larvae. Experiments were carried out to compare the influence of settlement-inducing compounds from the bacteria isolated from the shell surface of Balanus amphitrite on its larval metamorphosis. The effect of multispecies bacterial film was also assessed. The production of different molecules by the bacteria was influenced by the nutrient media under which they were grown. It was observed that the promotory multispecies bacterial film turned to inhibition mode in the presence of the adult extract of the barnacle, indicating that bacteria-adult extract interactions alter the synthesis of different compounds produced by bacteria. The studies also show that the waterborne and the surface-associated cues from the bacteria function differentially in mediating larval metamorphosis. Understanding the complexities involved in such interactions and identification of the factors governing them would be a step forward.

Larval development and post-settlement metamorphosis of the barnacle Balanus albicostatus Pilsbry and the serpulid polychaete Pomatoleios kraussii Baird: Impact of a commonly used antifouling biocide, Irgarol 1051.

AbstractThe impact of a commonly-used antifouling algicide, Irgarol 1051, on the larval development and post-settlement metamorphosis of the barnacle, Balanus albicostatus Pilsbry (Crustacea: Cirripedia), and the larval metamorphosis of a serpulid polycheate, Pomatoleios kraussii Baird, was evaluated. In the case of B. albicostatus, larval mortality increased with an increase in the concentration of Irgarol 1051, and there was a shift in the larval stage targeted from advanced instars to early instars. Nauplii that survived to the cyprid instar stage when reared in the presence of Irgarol 1051 showed prolonged instar and total naupliar duration when compared to the controls. The post-settlement metamorphosis of cyprids significantly varied with Irgarol concentration and also with biofilm age. One and 2-d-old untreated biofilms showed higher metamorphosis when compared to 5-d-old biofilms. However, when the biofilms that promoted cyprid metamorphosis were treated with Irgarol 1051 at low concentrations, metamorphosis rates decreased. Cyprids were prevented from metamorphosing completely by biofilms treated at the highest concentration of Irgarol 1051. Inhibition of metamorphosis was also observed in the case of competent polychaete larvae when exposed to Irgarol 1051 compared to those exposed to metamorphosis inducers such as 3-iso-butyl-1-methylxanthine (IBMX) and natural biofilms. Identification of the pathway(s) that caused the promotory biofilms to become toxic when exposed to Irgarol 1051 is discussed.