Morphology and morphometry of morphotypes in the population of Artemia franciscana (Kellogg, 1906) from salterns of the southeastern coast of India.

We document the morphology, morphometric variations among the morphotypes of the brine shrimp, Artemia franciscana. From the samples collected at four different locations in South India, Tamil Nadu viz. Kelambakkam, Vedaranyam, Tuticorin and Nagarcoil we identified six morphotypes: M1, M3, M4 in males and F1, F2, F3 in females. The Scanning electron micrographs of male morphotypes show distinct variation in the basal width, shape and number of cuticular cones on the second antennae. Similarly, the female morphotypes show various shape and sizes of the ovisac with or without spines. However, the cyst surface topography is smooth without any specific variation/ornamentation in all three female morphotypes. Multivariate analysis of eighteen morphological traits measured in males and fifteen in females to elucidate the intraspecific variations among morphotypes indicate significant dissimilarity between males and females. Furthermore, relative length measurements showed distinct morphometric variation of traits between the morphotypes encountered at different sampling sites.

The pre- and postembryonic development of Artemia franciscana (Anostraca: Artemiidae).

Artemia franciscana is a universal live feed in aquaculture, and it has been reported as an invasive species in many Asian hypersaline ecosystems. The present observations illustrated the pre- and postembryonic development stages of the A. franciscana population confined to the Indian saltern of Kelambakkam. We observed their growth patterns during various hydration periods with specific time intervals. Results showed differences in the development stages with respect to unique identity. Interestingly, a period of hydration showed notable cellular movement toward clockwise positions in the hydrating cysts. After 10 h of hydration, blastocoel appeared, accelerating the dynamic route of nuclei movement. At the end of the invagination, the embryo burst out of the cyst, and a sequence of emerging stages was noted. With reference to light microscopic observations, a series of developmental stages were observed, and each instar was documented by developing limb buds of nauplii. Excitingly, the 10th and 11th instar stages reveal sexual differentiation between male and female individuals. Thus, the laboratory culture study clearly documented the different developmental stages with their specific characteristic features. However, further molecular study would provide a cellular basis for understanding the early development of A. franciscana.

Bioaccumulation of TBT and Its Cellular Toxic Effects on the Freshwater Prawn Macrobrachium rosenbergii.

To test the toxic effects of tributyltin (TBT), Macrobrachium rosenbergii were exposed to three concentrations of TBT viz. 10 ng/L, 100 ng/L and 1000 ng/L for 90 days. The bioaccumulation of TBT level varied in hepatopancreas based upon dose dependent manner. Histopathological results revealed the reduction in basement membrane thickness, disruption of the hepatopancreatic tubules and abnormal lumen in hepatopancreas of TBT treated prawns. The ultrastructure of the control prawn showed normal architecture of cellular organelles with prominent nuclei in hepatocytes. On the other hand, many vacuoles, irregular arrangements of microvilli, swollen mitochondria, distorted rough endoplasmic reticulum cisternaes and abnormal nucleus were seen in the TBT treated group. Further, the biochemical and vitellogenin content were altered remarkably due to TBT exposure. It directly indicated that TBT had conspicuously inhibited the vitellogenesis. Therefore, it was inferred that the administration of TBT has considerably affected the hepatopancreatic functions in M. rosenbergii.

Ultrastructure of spermatozoa in cobia, Rachycentron canadum (Linnaeus, 1766).

Ultrastructure and development of spermatozoa in cobia, Rachycentron canadum are described. Sections through the testis show different developmental stages viz, Spermatocytes, spermatids and sperm. Spermatozoa of R. canadum exhibit the configuration of uniflagellated, anacrosomal Type I aquasperm, typical for externally fertilizing fish. Mature spermatozoon is seen with a prominent head and long cylindrical flagellum. Ultrastructure of sperm shows invaginated 'U' shaped nucleus and other organelles. The mitochondrial matrix is electron-dense with irregular arrangement of the cristae. The nucleus reveals a deep invagination (nuclear fossa) in which the centriolar complex is located. The centriolar complex lies inside the nuclear fossa and is composed of a proximal and a distal centriole. The two centrioles are placed perpendicular to each other. The flagellum has a typical eukaryotic organization (microtubule doublets 9 + 2 pattern) and measures around 36.21 ±â€¯0.42 µm in length. This study for the first time provides a comprehensive detail on the ultrastructure and developmental process of sperm in cobia, R. canadum.

Cryopreservation of sperm in Grey mullet Mugil cephalus (Linnaeus, 1758).

The aim of this study was to document the effects of cryopreservation on sperm motility and viability in Grey mullet Mugil cephalus. Cryopreservation of sperm was attempted by using two extenders ringer solution for marine fish (RSMF) and V2 extender (V2E) and cryoprotectants dimethylacetamide (DMA), dimethylsulfoxide (DMSO), ethylene glycol (EG), glycerol (GLY), propylene glycol (PG) and methanol (MeOH). Cryoprotectants were assessed at different concentrations individually as well as in combination with varying equilibration times (10 and 30min). For optimization of freezing rate, four freezing protocols (-5, -10, -20 and -30°C/min) were evaluated. After achieving final temperature, samples were plunged in liquid nitrogen (-196°C) and stored for a week. Samples were subsequently thawed in a water bath at 30°C for assessment of sperm motility and viability. Results indicated that cryomedium constituting of V2E extender+10% glycerol with a dilution ratio of 1:1 (sperm: cryomedium) at an equilibration time of 5 to- 10min and freezing rate of -20°C/min was more desirable compared with other factors that were assessed. Use of this protocol resulted in retaining the greatest sperm motility grade 3.0±0.0 (50%-80% sperm movement, fast swimming) and 48.19±3.12% of sperm viability. The results of the present study, therefore, provide base-line data for establishing a protocol for sperm cryopreservation in M.cephalus. Further studies are, however, required for optimization of most suitable sperm cryopreservation protocol.

Detection of metal induced cytopathological alterations and DNA damage in the gills and hepatopancreas of green mussel Perna viridis from Ennore Estuary, Chennai, India.

This study report the impact of heavy metals on cytopathology and DNA damage in the gills and hepatopancreas of Perna viridis collected from Ennore estuary and the Kovalam coastal waters. Principal Component Analysis (PCA) showed significant differences among all variables at the scale of plots. The ultrastructural alterations such as lack of microvilli, distorted mitochondria, electron dense particles and the presence of large mucous droplets were common in the gill and hepatopancreatic cells of mussels from Ennore estuary. However, the gill and hepatopancreatic cells of P. viridis from Kovalam revealed normal compartmentalization of cells. The percentage of tail DNA in the mussels from Ennore estuary was recorded as 12.44 and 10.14% in the gills and hepatopancreas respectively. Overall, it has been demonstrated that the Comet and cytopathological assays are useful biomarkers to assess the level of pollution and it provide reliable information on ecotoxicology and genotoxicology of coastal waters.

Spatio-temporal distribution, source, and genotoxic potential of polycyclic aromatic hydrocarbons in estuarine and riverine sediments from southern India.

In order to categorize the distribution, source, and effects of polycyclic aromatic hydrocarbons (PAHs) in aquatic systems of southern India, chemical and toxicological analyses were performed on surface and core sediments, collected from Adyar river, Cooum river, Ennore estuary, and Pulicat lake near Chennai city. The total PAH concentration in surface sediment ranged from 13 to 31,425ng/g with a mean value of 4320ng/g; the concentration was markedly higher in Cooum river compared to that at other sites. The historical PAH dissemination in core samples in the Cooum river, Ennore estuary, and Pulicat lake ranged from 30 to 31,425ng/g, from 8.6 to 910ng/g, and from 62 to 546ng/g, respectively. Surface sediments were predominantly contaminated with low molecular weight (LMW) PAHs. Historical profiles suggest that PAH contamination in the area is now greater than it had been in the past. PAH accumulation in Pulicat lake was distinct from that at other locations where high molecular weight (HMW) PAHs were predominant. DNA damage in HepG2 cells treated with sediment extracts from different locations showed a good correlation with their respective total PAH levels. Statistical analysis revealed that 3-ring and 4-ring PAHs may synergistically contribute to the genotoxic potency compared to others in sediments. The study also showed that a majority of PAHs in the study area indicated a petrogenic origin. Based on the enrichment and toxicological assessment of PAHs in sediments, Cooum river was shown to suffer the highest biological impairment among the studied water bodies.

Distribution and Relationships of Antimicrobial Resistance Determinants among Extended-Spectrum-Cephalosporin-Resistant or Carbapenem-Resistant Escherichia coli Isolates from Rivers and Sewage Treatment Plants in India.

To determine the distribution and relationship of antimicrobial resistance determinants among extended-spectrum-cephalosporin (ESC)-resistant or carbapenem-resistant Escherichia coli isolates from the aquatic environment in India, water samples were collected from rivers or sewage treatment plants in five Indian states. A total of 446 E. coli isolates were randomly obtained. Resistance to ESC and/or carbapenem was observed in 169 (37.9%) E. coli isolates, which were further analyzed. These isolates showed resistance to numerous antimicrobials; more than half of the isolates exhibited resistance to eight or more antimicrobials. The blaNDM gene was detected in 14/21 carbapenem-resistant E. coli isolates: blaNDM-1 in 2 isolates, blaNDM-5 in 7 isolates, and blaNDM-7 in 5 isolates. The blaCTX-M gene was detected in 112 isolates (66.3%): blaCTX-M-15 in 108 isolates and blaCTX-M-55 in 4 isolates. We extracted 49 plasmids from selected isolates, and their whole-genome sequences were determined. Fifty resistance genes were detected, and 11 different combinations of replicon types were observed among the 49 plasmids. The network analysis results suggested that the plasmids sharing replicon types tended to form a community, which is based on the predicted gene similarity among the plasmids. Four communities each containing from 4 to 17 plasmids were observed. Three of the four communities contained plasmids detected in different Indian states, suggesting that the interstate dissemination of ancestor plasmids has already occurred. Comparison of the DNA sequences of the blaNDM-positive plasmids detected in this study with known sequences of related plasmids suggested that various mutation events facilitated the evolution of the plasmids and that plasmids with similar genetic backgrounds have widely disseminated in India.

Bisphenol A and other bisphenol analogues including BPS and BPF in surface water samples from Japan, China, Korea and India.

Concentrations of eight bisphenol analogues (BPs) including BPA, BPS, and BPF were determined in surface waters collected from select rivers in Japan, Korea, China, and India. BPA was found at a concentration in the range of several tens to several hundreds of nanograms per liter in most of the rivers surveyed and some of the highest concentrations (54-1950 ng/L) were found in rivers in Chennai, India. Concentrations of BPF were one to two orders of magnitude higher than those of BPA in river and sea waters collected from Japan, Korea and China, which suggested that BPF is a major contaminant in surface waters in several Southeast Asian countries. BPF concentrations as high as 2850 ng/L were found in the Tamagawa River in Japan. The flux of BPs through riverine discharges into Tokyo Bay was calculated to be approximately 5.5 t per year. Based on the flux estimates and the mass of BPF found in water column and sediment in Tokyo Bay, it was found that BPF degrades faster than BPA in the environment. Elevated concentrations of BPF found in surface waters suggest the need for further studies to determine the fate and toxicity of this compound.

Monitoring of genotoxicity in marine zooplankton induced by toxic metals in Ennore estuary, Southeast coast of India.

The present study provides preliminary in-situ data on genetic integrity of marine zooplankton. Paracalanus parvus, Oithona rigida and Euterpina acutifrons were collected during four different seasons (summer, pre-monsoon, monsoon and post-monsoon) from 2011 to 2012 in Ennore and Kovalum estuaries. DNA damage levels in different zooplankton were analyzed by comet assay and were correlated with different environmental stressors. Spatial and temporal variations in DNA damage was observed in all the species. Zooplankton from Ennore estuary showed significantly lower genetic integrity. Particulate, sediment, and zooplankton fractions of Pb, Ni, Cu, Cr and Co were associated with high DNA damage during the period of lowest pH, salinity and dissolved oxygen. Zn and Cd showed lower genotoxic impact than the other metals. Feeding modes strongly influenced the genetic integrity in the zooplankton species studied. These results support the use of comet assay as a tool in effectively monitoring genotoxicity in marine plankton communities.

An integrated use of multiple biomarkers to investigate the individual and combined effect of copper and cadmium on the marine green mussel (Perna viridis).

The present study documents individual and combined sub-lethal effect of one redox active (copper) and one non-redox active (cadmium) metal on green mussel (Perna viridis). The mussels were exposed to 60 µg L(-1) of Cu and 150 µg L(-1) of Cd (individually and in combination) for 21 days. Histopathological and ultrastructural studies revealed significant metal induced alterations such as vacuolization, fusion of gill lamellae, enhance mucous deposition, hyperplasia and necrosis in gills. Antioxidant enzyme assays revealed significant increase in superoxide dismutase (SOD), glutathione S-transferase (GST) and glutathione peroxidase (GPx) activity. Similarly, single exposure to Cd and Cu caused significant induction in Malate dehydrogenase (MDH) activity. However, combined Cu+Cd exposure modulated suppression in MDH activity. Unlike MDH, Cu and Cd individual exposure resulted in a decrease in esterase (EST) activity, but their combined exposure caused an induction. Non-enzymatic biomarkers such as lipid peroxidation (LPO) and metallothionein (MT) levels showed no significant change in response to Cu exposure, whereas, individual Cd exposure or Cd exposure in combination with Cu caused significant changes in their levels. Comet assay revealed a significant increase in DNA damage upon metal exposure. These results indicate that Cu (redox active) and Cd (non-redox active) can induce measurable physiological, biochemical as well as genotoxic perturbations in mussels even at sub-lethal concentrations. A monitoring programme based on the biomarkers discussed here would be useful to study the effect of metal pollutants reaching the coastal waters.

Influence of short term exposure of TBT on the male reproductive activity in freshwater prawn Macrobrachium rosenbergii (De Man).

In the present study, the effect of tributyltin (TBT) on the histopathological and hormonal changes during spermatogenesis in freshwater prawn Macrobrachium rosenbergii was documented. Three experimental concentrations such as 10, 100 and 1,000 ng/L were selected and exposed to prawns for 45 days. After TBT exposure, the reproductive activities like sperm count and sperm length were decreased when compared with control. Further, abnormal structure of the seminiferous tubule, decrease in spermatozoa concentration, diminution of the seminiferous tubule membrane and the abundance of spermatocytes in the testis were noticed in treated prawns. Interestingly, radioimmunoassay clearly revealed the reduction of testosterone level in TBT exposed groups. Thus, TBT has considerably reduced the level of testosterone and caused the impairment of spermatogenesis in the freshwater male prawn M. rosenbergii.

The application of histo-cytopathological biomarkers in the mud crab Scylla serrata (Forskal) to assess heavy metal toxicity in Pulicat Lake, Chennai.

The concentrations of heavy metals and their associated structural deformities in the gills, muscles and hepatopancreas of Scylla serrata from Pulicat Lake were determined and compared with crabs live along Kovalam coast. The concentrations of metals were high in the hepatopancreas and gills of crab from Pulicat Lake, whereas, low in crab from the Kovalam coast. Data were visualized using a principal component analysis (PCA). Significant differences were found for all variables at the plot scale; however, the overall variation was relatively low for muscle tissues in both stations. The structural deformities observed in the gills, hepatopancreas and muscle was due to metal toxicity, and the degree of damage was correlated with the elevated metal concentration. The results showed significant metal accumulation and histo-cytological lesions in the crabs from Pulicat Lake. The results suggest that these biomarkers are useful for assessing the impact of metal pollution in the coastal environments.

Ultrastructural changes during spermatogenesis, biochemical and hormonal evidences of testicular toxicity caused by TBT in freshwater prawn Macrobrachium rosenbergii (De Man, 1879).

The present investigation documents the impact of tributyltin (TBT) on the ultrastructural variation of spermatogenesis in freshwater prawn Macrobrachium rosenbergii. The environmentally realistic concentration of TBT can cause damages to the endocrine and reproductive physiology of crustaceans. In this context, three concentrations viz. 10, 100, and 1000 ng/L were selected and exposed to prawns for 90 days. The TBT exposed prawn exhibited decrease the reproductive activity as evidenced by sperm count and sperm length compared to control. Histopathological results revealed the retarded testicular development, abnormal structure of seminiferous tubule, decrease in the concentration of spermatozoa, diminution of seminiferous tubule membrane, abundance of spermatocytes and vacuolation in testis of treated prawns. Ultrastructural study also confirmed the impairment of spermatogenesis in treated prawns. Furthermore, radioimmunoassay (RIA) clearly documented the reduction of testosterone level in TBT exposed groups. Thus, TBT substantially reduced the level of male sex hormone as well as biochemical constituents which ultimately led to impairment of spermatogenesis in the freshwater male prawn M. rosenbergii.

Effects of heavy metals on antioxidants and expression of HSP70 in different tissues of Milk fish (Chanos chanos) of Kaattuppalli Island, Chennai, India.

Distribution of heavy metals and its associated oxidative stress, ultrastructure and expression of HSP 70 were studied in varies tissues of Chanos chanos collected from polluted sites compared with the fish collected from less polluted sites of Kaattuppalli Island. The concentrations of copper, lead, zinc, cadmium, manganese and iron were quantified in gills and liver. The results showed marked differences between the two sites as well as significant variations within the tissues. The decreasing trend of metals in the tissues of fish sampled from both polluted and less polluted sites was in the order of Fe>Mn>Zn>Cu>Pb>Cd. Overall, the highest metal concentrations were found in the fish collected from polluted sites. Similarly increase of antioxidant enzymes biomarkers due to heavy metals was also evident in gills and liver of the fish collected from polluted sites. These tissues were further investigated by scanning and electron microscopy and the results were compared with the reference less polluted sites. The presence of large lipid droplets in liver and increase of mucous cells in gills were some of the most noticeable alterations observed and were related to heavy metal contaminants. It is concluded that scanning, ultrastructural and useful of HSP70 biomarkers for heavy metal induced oxidative stress, and demonstrate that precautions need to be taken in polluted sites of Kaattuppalli Island in order to prevent heavy metal pollution that can occur in the future.

Impact of TBT on the vitellogenesis and sex hormones in freshwater prawn Macrobrachium rosenbergii (De Man, 1879).

BACKGROUND: Tributyltin (TBT) is a ubiquitous persistent xenobiotic that can be found in freshwater, estuarine and marine ecosystem. TBT is a strong endocrine disrupting compound (EDC) that can cause toxic threat to aquatic organisms. Imposex, sexual deformities and endocrine dysfunctions are the causes of TBT to most of the aquatic organisms. Effect of TBT on the vitellogenesis and sex hormonal changes in Macrobrachium rosenbergii has never been reported. Hence, the present investigation was undertaken to find out the impact of TBT on histological changes in the different reproductive tissues, sex hormonal alterations and level of biomarkers like vitellogenin and vitellin in M. rosenbergii. RESULTS: The present investigation documents the possible impact of tributyltin (TBT) on the vitellogenesis in freshwater female prawn M. rosenbergii. TBT at 10 ng/l, 100 ng/l and 1000 ng/l concentrations were exposed individually to prawns for a period of three months. At higher concentration of 1000 ng/l, the ovarian development was arrested and ovary remained at spent stage. At lower concentration of TBT (10 ng/l), the development proceeded up to early vitellogenic stage. At intermediate concentration of 100 ng/l TBT, the ovary remained at pre vitellogenic stage and thereafter no development was noticed. Histological results indicated the normal ovarian development with vitellogenic oocytes, filled with yolk globules in control prawn. On the other hand, the TBT treated groups showed reduction in yolk globules, fusion of developing oocytes and abundance of immature oocytes. Immunofluorescence staining denoted the remarkable reduction in vitellin content in ovary of TBT treated prawn. Hence, TBT had conspicuously inhibited the vitellogenesis by causing hormonal imbalance in M. rosenbergii. CONCLUSION: TBT had notably inhibited the vitellogenesis due to hormonal imbalance. This endocrine dysfunction ultimately impaired the oogenesis in the freshwater female prawn M. rosenbergii.

Integrated use of histological and ultrastructural biomarkers in Mugil cephalus for assessing heavy metal pollution in Ennore estuary, Chennai.

Bioaccumulation of heavy metals and its associated histological perturbations were studied in various tissues of Mugil cephalus collected from Ennore estuary and compared with the fish collected from off-shore region. The concentration of copper, lead, zinc, cadmium, manganese and iron were quantified in gills, liver and muscle. The results showed marked differences between the two sites as well as significant variations within the tissues. The decreasing trend of metals in the tissues of fish sampled from both Ennore estuary and off-shore was in the order of Fe>Mn>Zn>Cu>Pb>Cd. Overall, the highest metal concentrations were found in the fish collected from Ennore estuary. The accumulation in the gills and liver of M. cephalus was found to be quite high in comparison with the muscle. These tissues were further investigated by light and electron microscopy and the results were compared with the reference site (off-shore region). The presence of large lipid droplets in liver and increase of mucous cells in gills were some of the most noticeable alterations observed and were related to heavy metal contaminants. It is concluded that histopathological and ultrastructural biomarkers provide reliable and discriminatory data to augment heavy metal pollution in Ennore estuary. Therefore, long-term monitoring is necessary to assess the eco-health of the Ennore estuarine environment by choosing bio-indicator species like M. cephalus, which provide accurate, reliable measurements of environmental quality.

Perchlorate contamination of groundwater from fireworks manufacturing area in South India.

Perchlorate contamination was investigated in groundwater and surface water from Sivakasi and Madurai in the Tamil Nadu State of South India. Sensitive determination of perchlorate (LOQ = 0.005 µg/L) was achieved by large-volume (500 µL) injection ion chromatography coupled with tandem mass spectrometry. Concentrations of perchlorate were <0.005-7,690 µg/L in groundwater (n = 60), <0.005-30.2 µg/L in surface water (n = 11), and 0.063-0.393 µg/L in tap water (n = 3). Levels in groundwater were significantly higher in the fireworks factory area than in the other locations, indicating that the fireworks and safety match industries are principal sources of perchlorate pollution. This is the first study that reports the contamination status of perchlorate in this area and reveals firework manufacture to be the pollution source. Since perchlorate levels in 17 out of 57 groundwater samples from Sivakasi, and none from Madurai, exceeded the drinking water guideline level proposed by USEPA (15 µg/L), further investigation on human health is warranted.

Biomarkers of metal toxicity and histology of Perna viridis from Ennore estuary, Chennai, south east coast of India.

Distribution of heavy metals and its associated histological perturbations were studied in the soft tissues of Perna viridis collected from Ennore estuary and compared with the less polluted Kovalam coast. The concentration of copper, lead, zinc, cadmium, manganese and iron were quantified in gills, digestive gland and adductor muscle. The results showed marked differences between the two sites as well as significant variations within the tissues. Among the heavy metals analyzed, lead and cadmium recorded very low in the soft tissues of mussel. Heavy metal levels in tissues of mussel collected from Ennore estuary were in the order of gills>digestive gland>adductor muscle, while it was digestive gland>gills>adductor muscle in the mussel sampled from Kovalam coast. The decreasing trend of metals in the tissues of mussels sampled from both Ennore estuary and Kovalam coast was in the order of Fe>Mn>Zn>Cu>Pb>Cd. Overall, the highest metal concentrations were found in the mussel collected from Ennore estuary. The metal accumulation in the gills and digestive gland of Perna viridis was found to be quite high in comparison with the adductor muscle. These soft tissues were further investigated by light microscopy and the results were compared with the reference site (Kovalam coast). These results suggest that thickening of the digestive epithelium, hemocytic infiltration in the gills and myodegeneration in the muscle tissue are useful histological biomarkers for heavy metal induced stress, and demonstrate that precautions need to be taken in Ennore estuary in order to prevent heavy metal pollution that can occur in the future.

Preservation, development, and hatching of resting eggs in the freshwater rotifer Brachionus calyciflorus pallas.

The resting eggs of Brachionus calyciflorus produced under laboratory by manipulating various environmental conditions were subjected to preservation for long-term storage. Preservation strategies included as storing freshly collected resting eggs, dry resting eggs, and storing at 4°C. Results showed that the resting eggs preserved by drying as they were able to maintain the viability without much variation in the hatchability. The scanning electron micrographs clearly depicted the surface damage as well as change in the shape and surface topography due to the storage. The resting eggs thus stored were traced for their normal embryonic development, during which as many as 11 stages were traced and time taken for each stage of development was observed. Microscopic observations revealed that the first four stages represented the development of embryo and thereafter around 16-18 h extrution of neonate from egg occurred. At 18-19 h of development the characteristic of anterior and posterior spines appeared in the neonate. Just released neonate, after 4-5 h of development reached adult stage. Overall the hatching and embryonic development of the resting eggs preserved by "drying" were normal and hence this method could be adopted for long-term storage.