Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15A degrees C and 3.9-fold at 4A degrees C compared to the alga at 25A degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.

Molecular biodiversity of microbial communities in polluted soils and their role in soil phytoremediation

Les métaux lourds (ML) s’accumulent de plus en plus dans les sols à l’échelle mondiale, d’une part à cause des engrais minéraux et divers produits chimiques utilisés en agriculture intensive, et d’autre part à cause des activités industrielles. Toutes ces activités génèrent des déchets toxiques qui s’accumulent dans l’environnement. Les ML ne sont pas biodégradables et leur accumulation cause donc des problèmes de toxicité des sols et affecte la biodiversité des microorganismes qui y vivent. La fertilisation en azote (N) est une pratique courante en agriculture à grande échelle qui permet d’augmenter la fertilité des sols et la productivité des cultures. Cependant, son utilisation à long terme cause plusieurs effets néfastes pour l'environnement. Par exemple, elle augmente la quantité des ML dans les sols, les nappes phréatiques et les plantes. En outre, ces effets néfastes réduisent et changent considérablement la biodiversité des écosystèmes terrestres. La structure des communautés des champignons mycorhiziens à arbuscules (CMA) a été étudiée dans des sols contaminés par des ML issus de la fertilisation à long terme en N. Le rôle des différentes espèces de CMA dans l'absorption et la séquestration des ML a été aussi investigué. Dans une première expérience, la structure des communautés de CMA a été analysée à partir d’échantillons de sols de sites contaminés par des ML et de sites témoins non-contaminés. Nous avons constaté que la diversité des CMA indigènes a été plus faible dans les sols et les racines des plantes récoltées à partir de sites contaminés par rapport aux sites noncontaminés. Nous avons également constaté que la structure de la communauté d'AMF a été modifiée par la présence des ML dans les sols. Certains ribotypes des CMA ont été plus souvent associés aux sites contaminés, alors que d’autres ribotypes ont été associés aux sites non-contaminés. Cependant, certains ribotypes ont été observés aussi bien dans les sols pollués que non-pollués. Dans une deuxième expérience, les effets de la fertilisation organique et minérale (N) sur les différentes structures des communautés des CMA ont été étudiés. La variation de la structure de la communauté de CMA colonisant les racines a été analysée en fonction du type de fertilisation. Certains ribotypes de CMA étaient associés à la fertilisation organique et d'autres à la fertilisation minérale. En revanche, la fertilisation minérale a réduit le nombre de ribotypes de CMA alors que la fertilisation organique l’a augmenté. Dans cette expérience, j’ai démontré que le changement de structure des communautés de CMA colonisant des racines a eu un effet significatif sur la productivité des plantes. Dans une troisième expérience, le rôle de deux espèces de CMA (Glomus irregulare et G. mosseae) dans l'absorption du cadmium (Cd) par des plants de tournesol cultivés dans des sols amendés avec trois niveaux différents de Cd a été évalué. J’ai démontré que les deux espèces de CMA affectent différemment l’absorption ou la séquestration de ce ML par les plants de tournesol. Cette expérience a permis de mieux comprendre le rôle potentiel des CMA dans l'absorption des ML selon la concentration de cadmium dans le sol et les espèces de CMA. Mes recherches de doctorat démontrent donc que la fertilisation en N affecte la structure des communautés des CMA dans les racines et le sol. Le changement de structure de la communauté de CMA colonisant les racines affecte de manière significative la productivité des plantes. J’ai aussi démontré que, sous nos conditions expériemntales, l’espèce de CMA G. irregulare a été observée dans tous les sites (pollués et non-pollués), tandis que le G. mosseae n’a été observé en abondance que dans les sites contaminés. Par conséquent, j’ai étudié le rôle de ces deux espèces (G. irregulare et G. mosseae) dans l'absorption du Cd par le tournesol cultivé dans des sols amendés avec trois différents niveaux de Cd en serre. Les résultats indiquent que les espèces de CMA ont un potentiel différent pour atténuer la toxicité des ML dans les plantes hôtes, selon le niveau de concentration en Cd. En conclusion, mes travaux suggèrent que le G. irregulare est une espèce potentiellement importante pour la phytoextration du Cd, alors que le G. mosseae pourrait être une espèce appropriée pour phytostabilisation du Cd et du Zn.

Studies on the Biology of the Clam Marcia opima Gmelin from kayamkulam Lake

Division of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology

STUDIES ON THE BIOLOGY OF THE WEDGE CLAM DONAX INCARNATUS (GMELIN) FROM THE MALIPPURAM BEACH OF KERALA

The present investigations have considerably enhanced the existing knowledge on the biology and distribution/availability pattern of D.incarnatus in the Malippuram region. The species occurs in good concentration during October - March/April, and disappears from the area during late premonsoon and monsoon months. Recolonising the area in September, it grows fast in the subsequent months. The life span of the species is estimated to be about an year. Studies on the reproductive biology of the species have revealed that there are two spawning peaks, the major peak in February - March and minor peak, in December. The salinity regime of the area influences the reproductive activity. These observations form the original contribution in the thesis. The information on variation in water content, protein,glycogen and lipid levels in relation to reproductive cycle has helped to a better understanding of the gametogenic activity and spawning of the species. Similarly, the findings on salinity tolerance and filtration rate have shown that small sized clams exhibit greater tolerance range than larger clams, and grow at a faster rate with active metabolism. It is hoped that these information would considerably add to the present knowledge of the basic facts which are relevant to the improvement and management of the clam fishery of this region.

Investigations on the biology of Indian Mackerel Rastrelliger kanagurta (Cuvier) along the Central Kerala coast with special reference to maturation, feeding and lipid dynamics

Dept. of Marine Biology, Microbiology & Biochemistry, Cochin University of Science and Technology

Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps

The constitutive production of AMPs in shrimps ensures that animals are able to protect themselves from low-level assaults by pathogens present in the environment. As these molecules play important roles in the shrimp immune defense system, the expression level of these AMPs are possible indicators of the immune state of shrimps. The present study also indicates the antiviral property of AMPs, especially ALF, stressing the importance of their up-regulation through the application of immunostimulants/probiotics as a prophylactic strategy in aquaculture. The present study shows that shrimp defense system is equipped enough to evade WSSV infection to a certain extent, when the animals were maintained on marine yeast and probiotic diet, whereas the control diet fed group succumbed to WSSV infection. This study reveals that marine yeast and probiotic supplemented diet can delay the process of WSSV infection and confer greater protection to the animals. Particularly, the protection conferred by marine yeast, C. haemulonii S27 and Bacillus MCCB101 were highly promising imparting greater hope to the aquaculture community to overcome the prevailing disease problems in aquaculture. It may be inferred from the present study that up-regulation of AMP genes could be effected by the application of immunostimulants and probiotics. Also, AMP expression profile could be used as an effective tool for screening immunostimulants and probiotics for application in shrimp culture. Ultimately, it is likely that no single compound or strategy will provide a solution to the problem of disease within aquaculture and that, in reality, a suite of techniques will be required including the manipulation of the rearing environment, addition of probionts as a matter of routine during culture, and the use of immunostimulants and other supplements during vulnerable growth phases. Finally, the development of good management practices, the control of environmental variables, genetic improvement in the penaeid species, understanding of host-virus interaction, modulation of the shrimp immune system, supported by functional genomics and proteomics of this crustacean, as a whole suggests that the control of WSSV is not far.

Biology of Flat Toadfish,Colletteichthys dussumieri(Valenciennes,1837) of Cochin Estuary

Dept.of Marine Biology,Microbiology and Biochemistry,Cochin University of Science and Technology

Biology, population characteristics and fishery of the speckled shrimp Metapenaeus monoceros (Fabricius, 1798) along the Kerala coast

Crustaceans comprising numerous edible species of prawns, lobsters and crabs inhabiting different ecosystem form significant portion of the aquatic food resources of the world. Among the crustaceans, prawns are the most commercially exploited group and hold premier rank by virtue of their importance as an esteemed food of gourmet and on account of their high export value. Met-ape-naeus manoceras (Fabricius, 1798) which is known IS,Speckled shrimp’ (FAD name) and ‘Brown shrimp’ ( common nameused in the industry) is one of the commercially important marine penaeid prawns of India. During 1995, M. monaceros catch constituted 7.5 Z of the all India marine penaeid prawn landings. M. monoceros attains a maximum length of about 200 mm and has high export potential.Thus realising the growing importance of M. monoceros in the capture fisheries, it was felt, that it would be ideal to carry out detailed study on this species for rational exploitation and management of its fishery. Hence, the present work entitled, “Biology, population characteristics and fishery of the speckled shrimp Hetapenaeus monoceros (Fabricius, 1798) along Kerala coast“ was undertaken by the author. The thesis is laid out in seven chapters comprising TAXONOMY, FOOD AND FEEDING HABITS, AGE AND GROWTH, REPRODUCTION,LENGTH-WEIGHT RELATIONSHIP, FISHERY and POPULATION DYNAMICS

Studies on The Eco Biology of The Indian Pearl Oyster, Pinctada Fucata (Gould)

The present work aims to study induced maturation of the pearl oyster for induced spawning experiments. The work on larval development was done with a view to developing techniques for the artificial rearing of commercially important pearl oyster P fucata, and also to elucidate the principles and problems of tropical bivalve larvae in general for detailed investigations in the future. The present study is designed to probe into the details of the basic aspects of the biology related to the hatchery technology of Pinctada fucata and the understanding of the factors which influence induction of maturation, spawning, larval rearing and spat settlement. This would go a long way in the upgradation of hatchery technology of the Indian Pearl oyster Pinctada fucata fora commercial level seed production..

Marine yeast isolates from Arabian Sea and Bay of Bengal :Biochemical, Molecular and Phylogenetic Analysis

the present study was undertaken with the following objectives: 1. Isolation and identification of yeasts from Arabian Sea and Bay of Bengal. 2. Molecular characterization of yeast isolates and phylogenetic analysis 3. Physiological and biochemical characterization of the isolates. 4. Proximate composition of yeast biomass and bioactive compounds. The Thesis is comprised of six chapters. A general introduction to the topic is given in Chapter1. Isolation and identification of marine yeasts are presented in Chapter 2. Chapter 3 deals with molecular identification and physiological characterization of Non- pigmented yeasts. Molecular identification and physiological characterization of pigmented yeast is presented in Chapter 4. Proximate composition of yeast biomass of various genera and their bioactive compounds are illustrated in Chapter 5. A summary of the results of the present study is given in Chapter 6. References and Appendices are followed

Bacterial Community Composition in Brazilian Anthrosols and Adjacent Soils Characterized Using Culturing and Molecular Identification

Microbial community composition was examined in two soil types, Anthrosols and adjacent soils, sampled from three locations in the Brazilian Amazon. The Anthrosols, also known as Amazonian dark earths, are highly fertile soils that are a legacy of pre-Columbian settlement. Both Anthrosols and adjacent soils are derived from the same parent material and subject to the same environmental conditions, including rainfall and temperature; however, the Anthrosols contain high levels of charcoal-like black carbon from which they derive their dark color. The Anthrosols typically have higher cation exchange capacity, higher pH, and higher phosphorus and calcium contents. We used culture media prepared from soil extracts to isolate bacteria unique to the two soil types and then sequenced their 16S rRNA genes to determine their phylogenetic placement. Higher numbers of culturable bacteria, by over two orders of magnitude at the deepest sampling depths, were counted in the Anthrosols. Sequences of bacteria isolated on soil extract media yielded five possible new bacterial families. Also, a higher number of families in the bacteria were represented by isolates from the deeper soil depths in the Anthrosols. Higher bacterial populations and a greater diversity of isolates were found in all of the Anthrosols, to a depth of up to 1 m, compared to adjacent soils located within 50-500 m of their associated Anthrosols. Compared to standard culture media, soil extract media revealed diverse soil microbial populations adapted to the unique biochemistry and physiological ecology of these Anthrosols.

Spiroplasma in Drosophila melanogaster Populations: Prevalence, Male-Killing, Molecular Identification, and No Association with Wolbachia

Spiroplasma endosymbionts are maternally transmitted bacteria that may kill infected sons resulting in the production of female-biased broods. The prevalence of male killers varies considerably both between and within species. Here, we evaluate the spatial and temporal status of male-killing and non-male-killing Spiroplasma infection in three Brazilian populations of Drosophila melanogaster, nearly a decade after the first occurrence report for this species. The incidence of the male-killing Spiroplasma ranged from close to 0 to 17.7 % (so far the highest estimate for a Drosophila species) with a suggestion of temporal decline in a population. We also found non-male-killing Spiroplasma coexisting in one population at lower prevalence (3-5 %), and we did not detect it in the other two. This may be taken as a suggestion of a spreading advantage conferred by the male-killing strategy. Sequencing two loci, we identified the phylogenetic position of Spiroplasma strains from the three localities, showing that all strains group closely in the poulsonii clade. Due to intensive sampling effort, we were able to test the association between Spiroplasma infections and another widespread endosymbiont, Wolbachia, whose prevalence ranged from 81.8 to 100 %. The prevalence of Wolbachia did not differ between Spiroplasma-infected and uninfected strains in our largest sample nor were the prevalences of the two endosymbionts associated across localities.

Molecular evolution of primate immunodeficiency viruses and hepatitis delta virus

Primate immunodeficiency viruses, or lentiviruses (HIV-1, HIV-2, and SIV), and hepatitis delta virus (HDV) are RNA viruses characterized by rapid evolution. Infection by primate immunodeficiency viruses usually results in the development of acquired immunodeficiency syndrome (AIDS) in humans and AIDS-like illnesses in Asian macaques. Similarly, hepatitis delta virus infection causes hepatitis and liver cancer in humans. These viruses are heterogeneous within an infected patient and among individuals. Substitution rates in the virus genomes are high and vary in different lineages and among sites. Methods of phylogenetic analysis were applied to study the evolution of primate lentiviruses and the hepatitis delta virus. The following results have been obtained: (1) The substitution rate varies among sites of primate lentivirus genes according to the two parameter gamma distribution, with the shape parameter $\alpha$ being close to 1. (2) Primate immunodeficiency viruses fall into species-specific lineages. Therefore, viral transmissions across primate species are not as frequent as suggested by previous authors. (3) Primate lentiviruses have acquired or lost their pathogenicity several times in the course of evolution. (4) Evidence was provided for multiple infections of a North American patient by distinct HIV-1 strains of the B subtype. (5) Computer simulations indicate that the probability of committing an error in testing HIV transmission depends on the number of virus sequences and their length, the divergence times among sequences, and the model of nucleotide substitution. (6) For future investigations of HIV-1 transmissions, using longer virus sequences and avoiding the use of distant outgroups is recommended. (7) Hepatitis delta virus strains are usually related according to the geographic region of isolation. (8) Evolution of HDV is characterized by the rate of synonymous substitution being lower than the nonsynonymous substitution rate and the rate of evolution of the noncoding region. (9) There is a strong preference for G and C nucleotides at the third codon positions of the HDV coding region. ^

Molecular mechanism of PUVA-induced apoptosis in mouse epidermal cells

A combination of psoralens and ultraviolet-A radiation referred to as PUVA, is widely used in the treatment of psoriasis. PUVA therapy is highly effective in killing hyperproliferative cells, but its mechanism of action has not been fully elucidated. Psoralen binds to DNA, and upon photoactivation by UVA, it forms monofunctional adducts and interstrand cross-links. PUVA treatment has been shown to be mutagenic and to produce tumors in animals. In addition, epidemiological studies have reported a 10 to 15 percent increased risk of developing squamous cell carcinoma in individuals treated chronically with PUVA. However, it remains a treatment for skin disorders such as psoriasis because its benefits outweigh its risks. The widespread use of PUVA therapy and its associated cancer risk requires us to understand the molecular mechanisms by which PUVA induces cell death. Immortalized JB6 mouse epidermal cells, p53−/− mice, and Fas Ligand−/− (gld) mice were used to investigate the molecular mechanism by which PUVA kills cells. Treatment of JB6 cells with 10 μg/ml 8-methoxypsoralen followed by irradiation with 20 kJ/m2 UVA resulted in cell death. The cells exhibited morphological and biochemical characteristics of apoptosis such as chromatin condensation, DNA ladder formation, and TUNEL-positivity. PUVA treatment stabilized and phosphorylated p53 leading to its activation, as measured by nuclear localization and induction of p21Waf/Cip1, a transcriptional target of p53. Subsequent in vivo studies revealed that there was statistically significantly less apoptosis in p53 −/− mice than in p53+/+ mice at 72 hours after PUVA. In addition, immunohistochemical analysis revealed more Fas and FasL expression in p53+/+ mice than in p53−/− mice, suggesting that p53 is required to transcriptionally activate Fas, which in turn causes the cells to undergo apoptosis. Studies with gld mice confirmed a role for Fas/FasL interactions in PUVA-induced apoptosis. There was statistically significantly less apoptosis in gld mice compared with wild-type mice 24, 48, and 72 hours after PUVA. These results demonstrate that PUVA-induced apoptosis in mouse epidermal cells requires p53 and Fas/FasL interactions. These findings may be important for designing effective treatments for diseases such as psoriasis without increasing the patient's risk for skin cancer. ^