Comparison of two domoic acid-producing diatoms: a review

In the past five years, awareness of domoic acid has increased from localized problems in Canada to outbreaks along both North American coasts. The phycotoxin domoic acid causes Amnesic Shellfish Poisoning (ASP) in humans and can be fatal. The known species of phytoplankton responsible for production of domoic acid include some pennate diatom species of the genus Nitzschia, sensu latu, which form stepped chains typical of the Pseudonitzschia. These diatoms are widely distributed, but their life histories and population dynamics are poorly understood. This review addresses histories of occurrences, morphology, geographical distributions, seasonal patterns, growth requirements, domoic acid production, and trophic interactions, with emphasis on a comparison of Pseudonitzschia pungens f. multiseries (Hasle) Hasle and Pseudonitzschia australis Frenguelli. Through continued research it will become possible to provide guidelines for regulatory agencies that protect both the consumer and the seafood industry.     

Electro-immunoblotting characterization ofPseudo-nitzschia multiseries andP. pungens antigens recognized by antibodies directed against whole cells

Separate polyclonal antibodies have previously been developed against the domoic-acid-producingPseudonitzschia multiseries (=Pseudo-nitzschia pungens f.multiseries) and the non-toxicP. pungens (=P. pungens f.pungens). These antibodies bind to the surface of the diatoms as shown by immunofluorescence studies. Here we examine the molecular nature of the antigens by Western blotting (electro-immunoblotting) analysis. The major antigens for both polyclonal antibodies migrated as high molecular-weight diffuse bands, mostly remaining in the stacking gel, using an SDS-PAGE system. The antibodies prepared againstP. multiseries strongly labelled the high molecular-weight antigens of allP. multiseries strains tested and showed little reactivity towardsP. pungens extracts on Western blots.P. pungens antibodies strongly labelled high molecular-weightP. pungens antigens and faintly labelled a fewP. multiseries antigens. The selectivity of the antibodies for their respective species correlates with the results of the immunofluorescence experiments, suggesting that the antigens examined in this study are responsible for the selective labelling in immunofluorescence studies. The electrophoretic mobility and the antibody labelling of antigens were not altered by proteolytic digestion of cell pellets. However, disruption of carbohydrates in the pellets by treatment with periodic acid resulted in loss of the antigen. These data suggest that the major antigens of toxicP. multiseries and non-toxicP. pungens are high molecular-weight (°>100kDa) polysaccharides located on the surface of these diatoms.Author for correspondence     

Feeding,egg production,and egg hatching success of the copepods Acartia tonsa and Temora longicornis on diets of the toxic diatom Pseudo-nitzschia multiseries and the non-toxic diatom Pseudo-nitzschia pungens

In 1987, there was an episode of shellfish poisoning in Canada with human fatalities caused by the diatom Pseudo-nitzschia multiseries, which produced the toxin domoic acid. In order to examine whether domoic acid in this diatom serves as a grazing deterrent for copepods, we compared feeding rates, egg production rates, egg hatching success and mortality of the calanoid copepods Acartia tonsa and Temora longicornis feeding on unialgal diets of the toxic diatom P. multiseries and the similarly-sized non-toxic diatom Pseudo-nitzschia pungens. Copepods were collected in summers of 1994, 1995 and 1996 from Shediac Bay, New Brunswick, Canada, near Prince Edward Island, the site of the 1987 episode of domoic acid shellfish poisoning. Rates of ingestion of the toxic versus the non-toxic diatom by A. tonsa and T. longicornis were similar, with only one significantly different pair of values obtained in 1994, for which A. tonsa had a higher mean rate of ingestion of the toxic than the non-toxic diatom. Thus, domoic acid did not appear to retard grazing. Analyses of copepods with high performance liquid chromatography (HPLC) revealed that copepods accumulated domoic acid when feeding on P. multiseries. Egg production rates of copepods when feeding on P. multiseries and P. pungens were very low, ranging from 0 to 2.79 eggs female^–1 d^–1. There did not appear to be differential egg production or egg hatching success on diets of the toxic and non-toxic diatoms. Mortality of females on the toxic diet was low, ranging from 0 to 20%, with a mean of 13%, and there was no apparent difference between mortality of copepods feeding on toxic versus non-toxic diatoms. Egg hatching success on both diets, although based on few eggs, ranged between 22% and 76%, with a mean percentage hatching of 45%. Diets of the non-toxic diatom plus natural seawater assemblages supplemented with dissolved domoic acid, revealed similar rates and percentages when compared to previous experiments. In summary, none of the variables measured indicated adverse effects on copepods feeding on the toxic compared to the non-toxic diatom.     

Interaction between bacteria and the domoic-acid-producing diatom Pseudo-nitzschia multiseries (Hasle) Hasle; can bacteria produce domoic acid autonomously?

The interaction between bacteria and phytoplankton is increasingly becoming recognised as an important factor in the physiology of toxin production and the dynamics of harmful algal blooms (HABs). Bacteria can play a direct or indirect role in the production of biotoxins once solely attributed to microalgae. Evidence implicating bacteria as an autonomous source paralytic shellfish poisoning biotoxins raises the question of autonomous bacterial toxigenesis of the neurotoxin domoic acid (DA), the cause of amnesic shellfish poisoning. Here, we examine whether the previously observed bacterial enhancement of DA production by Pseudo-nitzschia multiseries (Hasle) Hasle may be attributable to independent biotoxin production by the extra-cellular bacteria associated with this diatom. The growth and toxicity of six cultures of xenic P. multiseries clone CLN-1 were followed for 24 days. Up to day 14 (mid-stationary phase), DA production was not statistically different among culture flasks. On day 14, P. multiseries cells were removed by gentle filtration from a set of triplicate flasks, leaving the bacteria in the filtrate. Following the removal of the algal cells, DA in the filtrate ceased to increase. Instead, DA levels continuously declined. A follow-up experiment determined that this was likely caused by photodegradation rather than by bacterial degradation. We conclude that after removing P. multiseries cells, the extra-cellular bacteria remaining in the filtrate were incapable of autonomous DA toxigenesis, even in the presence of P. multiseries exudates. However, scanning electron microscopy revealed that P. multiseries cells harboured epiphytic bacteria, the importance of which can still not be ruled out in DA production.     

Biocatalytic acylation of carbohydrates with fatty acids from palm fatty acid distillates

Palm fatty acid distillates (PFAD) are by-products of the palm oil refining process. Their use as the source of fatty acids, mainly palmitate, for the biocatalytic synthesis of carbohydrate fatty acid esters was investigated. Esters could be prepared in high yields from unmodified acyl donors and non-activated free fatty acids obtained from PFAD with an immobilized Candida antarctica lipase preparation. Acetone was found as a compatible non-toxic solvent, which gave the highest conversion yields in a heterogeneous reaction system without the complete solubilization of the sugars. Glucose, fructose, and other acyl acceptors could be employed for an ester synthesis with PFAD. The synthesis of glucose palmitate was optimized with regard to the water activity of the reaction mixture, the reaction temperature, and the enzyme concentration. The ester was obtained with 76% yield from glucose and PFAD after reaction for 74 h with 150 U ml⁻¹ immobilized lipase at 40°C in acetone.     

Variation of fatty acid profile with solar cycle in outdoor chemostat culture ofIsochrysis galbana ALII-4

Outdoor chemostat cultures of the marine microalgaIsochrysis galbana at constant dilution rate (0.034 h^–1 ) have been carried out under different weather conditions. Steady-state biomass concentrations were 1.61±0.03 kg m^–3 in May and 0.95±0.04 kg m^–3 in July, resulting in biomass output rates of 0.54 kg m^–3 d^–1 and 0.32 kg m^–3 d^–1 in May and July, respectively. Two patterns of daily variation with the solar cycle were observed in the fatty acid content. Saturated and mono-unsaturated fatty acids (16:0 and 16:1n7) show significant variation with the solar cycle, associated with short-term changes in environmental factors. Palmitic and palmitoleic acids are generated during daylight and consumed during the dark period. However, polyunsaturated fatty acids do not show a significant response to the solar cycle and their changes are associated with long-term variation in environmental factors. The maximum EPA productivity was obtained in May, 14.1 g m^–3 d^–1, which is close to that found in the literature for indoor continuous cultures. Nonetheless, the outdoor EPA content (up to 2.61 % d.wt) was lower than the indoor EPA content from a previous study (5% d.wt).     

The utilization of short-chain monocarboxylic acids as carbon sources for the production of gamma-linolenic acid by Mucor strains in fed-batch culture

The Fischer-Tropsch reaction water, which contains C₂ to C₅ monocarboxylic acids, generated as a co-product of the Sasol industrial oil-from-coal process, constitutes a potential cheap carbon substrate for the production of gamma-linolenic acid (GLA) by selced Mucor species. Three strains of Mucor were each grown in an air-lift reactor operated in a fed-batch, pH-stat mode under N-limitation with a mixture of C₂ to C₅ monocarboxylic acids as both pH titrant and carbon source. The production of GLA from this substrate was evaluated. Growth typically resulted in the rapid assimilation of acetic, n-butyric and n-valeric acids. Although propionic, iso-butyric and iso-valeric acids were assimilated to varying degrees, these acids accumulated in the culture. Mucor circinelloides CBS 203.28 gave the best results in that it assimilated 36% to 100% of each acid, had a biomass yield coefficient of 0.3 (calculated on acids utilized), and contained 28% crude oil, 84% of which comprised neutral lipids with a GLA content of 14.4%, giving 33 mg GLA/g biomass. GLA accumulation coincided with a decrease in the stearic-acid content of the neutral-lipid fraction. The results were comparable with previous results obtained with acetic acid and glucose as sole carbon sources, demonstrating the feasibility of producing GLA from the above mixture of organic acids.     

Effect of supplementation with exogenous fatty acid on the biological properties of a fatty acid requiring auxotroph ofSalmonella typhimurium

The effects of changes in fatty acid composition of the cell membrane on different biological functions ofSalmonella typhimurium have been studied with the help of a temperature sensitive fatty acid auxotroph which cannot synthesise unsaturated fatty acids at high temperature. On being shifted to nonpermissive temperature the cells continue growing for another one and half to two generations. The rates of protein and DNA syntheses run parallel to the growth rate but the rate of RNA synthesis is reduced. Further, there is a gradual reduction in the rate of transport of exogenous uridine and thymidine into the soluble pool. The transport process can be restored by supplementing the growth medium with cis-unsaturated fatty acids but not trans-unsaturated ones although the growth of the cells is resumed by supplementation with eithercis or trans-unsaturated fatty acids. However, supplementation withtrans, trans-unsaturated fatty acids leads to only partial recovery of the transport process. The rate of oxygen uptake is also affected in cells grown in the presence of thetrans-unsaturated fatty acids, elaidic acid and palmitelaidic acid. Analysis of cells grown under different fatty acid supplementation indicate that fatty acid composition of the cell membrane, especially the ratio of unsaturated to saturated fatty acids varies with temperature shift and supplementation of the growth media with fatty acids.     

Labeling of adipocyte membranes by sulfo-N-succinimidyl derivatives of long-chain fatty acids: Inhibition of fatty acid transport

Summary Sulfo-N-succinimidyl derivatives of the long-chain fatty acids, oleic and myristic, were synthesized and covalently reacted with isolated rat adipocytes. The plasma membrane proteins labeled by these compounds and the effect of labeling on the transport of long-chain fatty acids were investigated. Sulfo-N-succinimidyl oleate (SSO) and myristate (SSM) inhibited the transport of fatty acids (by about 70%). Inhibition of fatty acid transport was not a result of alterations in cell integrity, as intracellular water volume was not changed. It did not reflect effects on fatty acid metabolism, since it was observed under conditions where greater than 90% of the fatty acid taken up was recovered in the free form. The inhibitory effect was specific to the fatty acid transport system, as the transport of glucose and the permeation of retinoic acid, a substance with structural similarities to long-chain fatty acids, were unaffected. Sulfosuccinimidyl oleate reacted exclusively with a plasma membrane protein with an apparent size of 85 kDa while sulfosuccinimidyl myristate also labeled a 75-kDa while sulfosuccinimidyl myristate also labeled a 75-kDa protein. These proteins were among the ones labeled by diisothiocyanodisulfonic acid (DIDS) which also inhibits fatty acid transport irreversibly. The data suggest that the 85-kDa protein, which is the only one labeled by all three inhibitors is involved in facilitating membrane permeation of long-chain fatty acids.     

Growth and fatty acid composition of Nannochloropsis sp. grown mixotrophically in fed-batch culture

The cell growth and eicosapentaenoic acid (EPA) yields of Nannochloropsis sp. were enhanced in the fed-batch cultures. With feeding glucose solution, the biomass reached 1.1 g dry wt l(-1) after 10 days' culture, which was 40% higher than that obtained in the batch culture (0.8 g dry wt l(-1)). With supplement of nitrate solution, the biomass reached 1 g dry wt l(-1), and reached the stationary phase 2 days earlier than the others. The maximum of biomass (1.2 g dry wt l(-1)) was obtained with the supplement of the mixture of glucose and nitrate solution. The EPA yields of Nannochloropsis sp. after 10 days' growth in the fed-batch cultures were 52 mg l(-1), 43 mg l(-1) and 56 mg l(-1) with, respectively, addition of nitrate, glucose and both together. In batch culture only 35 mg EPA l(-1) was obtained.     

Isoprenoid quinones and fatty acids ofZoogloea

NineZoogloea strains including the type strain ofZ. ramigera (IAM 12136=ATCC 19544=N.C. Dondero 106) and newly isolated strains were investigated for isoprenoid quinone composition and whole-cell fatty acid profiles. Seven of the tested strains, having phenotypic properties typical ofZoogloea, were characterized by their production of both ubiquinone-8 and rhodoquinone-8 as major quinones, whereas the remaining two strains,Z. ramigera IAM 12669 (=K. Crabtree I-16-M) and IAM 12670 (=P.R. Dugan 115), formed ubiquinone-10 and ubiquinone-8, respectively, as the sole quinone. All rhodoquinone-producing strains contained palmitoleic acid and 3-hydroxy-decanoic acid as the major components of nonpolar and hydroxylated fatty acids, respectively. Marked differences were noted in the fatty acid composition between the strains with and without rhodoquinones. The chemotaxonomic data suggested that the rhodoquinonelacking strains should be excluded from the genusZoogloea. Since there have been no reliable taxonomic tools forZoogloea, rhodoquinone analysis may provide a new criterion of great promise for identifyingZoogloea strains.     

The influence of size on domoic acid concentration in king scallop, Pecten maximus (L.)

Concentrations of domoic acid (DA), the biotoxin responsible for amnesic shellfish poisoning (ASP), exceeding the regulatory limit of 20 μg g⁻¹ have caused restricted harvesting and closures of wild king scallop fisheries. Toxin monitoring programmes have reported significant inter-animal variation in DA concentration between scallops from the same area. For the development of reliable sampling and management protocols an understanding of the magnitude and causes of inter-animal variation in toxin concentration are important. Ten samples were collected from an aquaculture site in Clew Bay, Co. Mayo off the west coast of Ireland between February 2003 and February 2004, each sample comprising 12 scallops of each of the following size groups: small (70–85 mm), medium (85–100 mm), large (100–115 mm) and very large (>115 mm). DA concentration in each hepatopancreas and in composite samples of both gonad and adductor muscle from each size group on each sampling occasion were measured. High inter-animal variability in DA concentration in hepatopancreas was recorded; CVs ranging from 12.5% to 82.5%. One negative correlation (R² = 0.7079) between DA concentration in hepatopancreas and scallop shell length, three positive but weak correlations (R² = 0.4536, 0.3459 and 0.4665) and six no correlations were exhibited. Negative correlations were attributed to faster DA uptake by smaller scallops, positive correlations to faster DA depuration by smaller scallops. If only scallops greater than or equal to 100 mm shell length, the minimum commercial size of this species were considered, no correlation occurred on any of the 10 sampling occasions.     

Changes in lipid composition of Blumeria graminis f.sp. tritici conidia produced on wheat leaves treated with heptanoyl salicylic acid

Treatment of wheat leaves with heptanoyl salicylic acid (HS) and trehalose at concentrations of 0.1 and 15 g l(-1), prior to fungal inoculation, resulted in 40% and 60% protection, respectively, against powdery mildew. The total lipid composition of Blumeria graminis f.sp. tritici (Bgt) conidia, the causal agent of wheat powdery mildew, was compared when produced on wheat leaves, respectively, untreated and treated with the two elicitors, HS and trehalose. An obvious effect was observed on lipid composition (sterol and fatty acid (FA)) of Bgt conidia produced on wheat leaves treated with HS. A total of 16 FA (C12-C24 saturated and unsaturated) as well as unusual methoxylated Fatty Acids (mFA) (3-methoxydocosanoic and 3-methoxytetracosanoic acids) were detected in the conidia. Medium chain FA were predominant in HS treated conidia (64.65%) while long chain fatty acids constituted the major compounds in untreated conidia (62%). The long chain/medium chain FA ratio decreased from 1.8 in the conidia produced on untreated leaves to 0.5 in the conidia obtained from HS treated leaves. When comparing the sterol composition of Bgt conidia produced on leaves treated with HS versus conidia obtained from untreated ones, very important changes within the two major classes can be seen. In particular, 24-methylsterols, e.g., 24-methylenecholesterol and 24-methylcholesta-7,24-dien were reduced by about 82% whereas 24-ethylsterols, e.g., 24-ethylcholesterol and 24-ethylcholesta-5,22-dienol were increased by about 85%. The 24-methylsterols/24-ethylsterols ratio was reduced by ninefold in the conidia produced from HS treated leaves.     

Recent domoic acid closures of shellfish harvest areas in Washington State inland waterways

Several species of the toxigenic diatom Pseudo-nitzschia, together with low concentrations of domoic acid (DA) in shellfish have been observed in Puget Sound, Washington State, since 1991. However, for the first time in September 2003, high-density blooms of Pseudo-nitzschia forced the closure of recreational, commercial, and tribal subsistence shellfish harvesting in Puget Sound. Here we report on the environmental conditions associated with shellfish closures in two inland waterways of Washington State during the Fall 2005. In Sequim Bay, shellfish harvest losses occurred on September 12 following the measurement of elevated macronutrient levels on September 2, and a bloom of P. pseudodelicatissima (up to 13 million cells/L) on September 9. Ambient NH₄ concentrations >12 μM (measured on September 2) were likely due to anthropogenic sources, ostensibly from sewage inputs to Sequim Bay. The closure of a Penn Cove commercial shellfish farm on October 16 was caused by a bloom of P. australis that followed a period of sustained precipitation, elevated Skagit River flow, and persistent southeasterly winds. The relative importance of a number of environmental factors, including temperature, stratification caused by rivers, and nutrient inputs, whether natural or anthropogenic, must be carefully studied in order to better understand the recent appearance of massive blooms of toxigenic Pseudo-nitzschia in the inland waterways of Washington State.     

Methoxylated fatty acids in Blumeria graminis conidia

The total fatty acids (FA) composition of Blumeria graminis f.sp. tritici conidia, the causal agent of wheat powdery mildew, was analyzed as a function of their age. A total of 19 FA (C12-C24 saturated and unsaturated) and unusual methoxylated fatty acids (mFA) were detected in young, intermediate and old conidia. Two very long chain methoxylated FA were identified by GC-MS as 3-methoxydocosanoic and 3-methoxytetracosanoic acids. Medium chain FA were predominant in young conidia (75%, including 13% of mFA) while very long chain fatty acids constituted the major compounds in old conidia (74%, including 30% of mFA). We have shown for the first time that the total FA composition is strongly correlated with the age of B. graminis f.sp. tritici (Bgt) conidia.     

Diversity and distribution of epibiotic bacteria on Pseudo-nitzschia multiseries (Bacillariophyceae) in culture, and comparison with those on diatoms in native seawater

Previous studies on the interaction between bacteria and harmful algal bloom species have mostly considered the bacteria in the bulk solution. Here, we document the abundance and mode of attachment of bacteria growing on the cell surface of the domoic acid-producing diatom Pseudo-nitzschia multiseries (Hasle) Hasle in culture, compared with diatoms in field samples. The epiphytic bacteria were examined by scanning electron microscopy to visualize their morphology and mode of attachment. Two P. multiseries cultures were studied: clone CLN-1 and sub-clone CLN-1-NRC; the latter had been maintained in another laboratory for 2 years. Each of these P. multiseries cultures exhibited a clearly different assemblage of epibiotic bacteria, even though both originated from the same parent culture. The bacterial diversity was greater in clone CLN-1 (nine distinct morphotypes seen) than in sub-clone CLN-1-NRC (six morphotypes). The former clone also produced more domoic acid than the latter. There was a succession of bacterial morphotypes as well as an increase in the number of epiphytic bacteria per diatom cell during the progression from exponential to stationary phase. The most diverse and common morphotypes were rod-shaped cells (e.g. a Caulobacter-like bacterium attached by a discoid holdfast). Epibionts showed a preference for attachment at specific regions of the host diatom frustule, e.g. the raphe or cingulum, locations where organic matter may be extruding from the diatom cell. Most diatom cells carried only one to five bacteria, and up to ca. 60% of the intact diatom cells (although intact cells themselves were infrequent) were still free of epibiotic bacteria at the end of the 31-day batch culture experiment. Sequencing of the SSU rRNA gene showed that five of the eight bacterial strains isolated from the P. multiseries cultures were members of the Alphaproteobacteria, three of the Gammaproteobacteria and one of the Bacteroidetes. A morphologically diverse assemblage of epibiotic bacteria was also found on both centric and pennate planktonic diatoms in natural coastal waters. Of the eight morphotypes recorded, all but two were also found in the cultures. Relatively fewer wild diatom cells carried bacteria compared to cells in culture. We hypothesize that the diversity and abundance of epiphytic bacteria may explain some of the variability seen in the production of DA by different P. multiseries clones, and should be considered as another important and controllable variable that influences diatom cell physiology.     

Note on the occurrence of Pseudo-nitzschia australis and domoic acid in squid from Monterey Bay, CA (USA)

Over the past decade diatom blooms of domoic acid (DA)-producing Pseudo-nitzschia spp. have been responsible for numerous marine mammal and bird mortalities in Monterey Bay, CA. One possible toxin vector is the market squid, Loligo opalescens, a small pelagic mollusk that plays an important role in the near-shore food web of the California Current ecosystem as a favored vertebrate prey species. This study examined the trophic link between toxic Pseudo-nitzschia and L. opalescens using toxin and stomach content analyses of animals collected from Monterey Bay, CA in 2000. Receptor binding assay data (confirmed by tandem mass spectrometry), demonstrated the presence of DA in squid during a toxic Pseudo-nitzschia event, with P. australis frustules observed in stomach samples. Though DA levels were low (<0.5 μg DA g⁻¹ tissue) in L. opalescens during the study period, it is now clear that this potent neurotoxin can occur in squid and is likely delivered through its krill prey species, which are known to retain DA after feeding on toxic Pseudo-nitzschia. Our findings suggest that further study of the relationship between Pseudo-nitzschia blooms and DA contamination of squid is warranted to better evaluate the potential health risk to humans and wildlife associated with this major commercial seafood species and important prey item.     

Distribution of alkanes and fatty acids in theCondalia montana (Rhamnaceae) species complex

The alkane and fatty acid composition of the lipid extracts from leaves of 14 populations ofCondalia: C. microphylla f.xanthocarpa, C. microphylla f.erythrocarpa, C. microphylla f.melanocarpa, C. montana, andC. buxifolia, were determined by gas chromatography. Seventeen alkanes and 14 fatty acids were investigated and a predominance of nonacosane, hentriacontane, palmitic and linoleic acids in all species was found.     

Inhibitory effect of medium-chain-length fatty acids on synthesis of polyhydroxyalkanoates from volatile fatty acids by Ralstonia eutropha

Medium-chain-length fatty acids, such as nonanoic (9:0) and octanoic (8:0) acids, are more toxic to Ralstonia eutropha than volatile fatty acids such as acetic, propionic and butyric acids. Nonanoic acid was degraded to acetic and propionic acids via -oxidation by Ralstonia eutropha for cell growth and synthesis of polyhydroxyalkanoates (PHAs). In a mixture of the fatty acids, utilization of nonanoic acid was depressed by acetic and propionic acids, and vice versa. The PHA accumulation from the volatile fatty acids was decreased from 53% (w/w) of dry cell mass to 23% due to the nonanoic acid. Similar phenomena were also observed with octanoic acid and its metabolic intermediates, acetic and butyric acids.     

Inhibition of methanogens by n- and iso-volatile fatty acids

n-Butyrate, n-valerate and n-caproate were more inhibitory towards Methanobacterium byrantii, Methanobacterium formicicum and Methanosarcina barkeri than the corresponding iso-acids. Butyrate caused maximum inhibition irrespective of isomer. Methanobacterium bryantii was more sensitive to inhibition than Methanobacterium formicicum.The authors are with the Division of Microbial Sciences, Agharkar Research Institute, G.G. Agarkar Road, Pune 411 004, India.