Molecular identification of crocodile species using novel primers for forensic analysis

All crocodilians are under varying degrees of threat due to over exploitation and these species have been listed in Appendix I or II of CITES. The lack of molecular techniques for the identification of confiscated samples makes it difficult to enforce the law. Conclusive forensic identification of species requires a complete gene sequence which is difficult in case of degraded samples. We have developed two novel sets of primers to amplify two partial cytochrome b gene sequences of six crocodile species i.e. Crocodylus palustris, Crocodylus porosus, Crocodylus siamensis, Crocodylus niloticus, Gavialis gangeticus and Caiman crocodilus. These partial sequences were edited to give a complete cyt b gene sequence, which can be used as an effective tool for forensic authentication of crocodile species. A phylogeny of crocodile species was reconstructed using these sequences. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these ancient species.     

Evaluation of genus <Emphasis Type="Italic">Cenchrus</Emphasis> based on malondialdehyde,proline content,specific leaf area and carbon isotope discrimination for drought tolerance and divergence of species at DNA level

Cenchrus (family Poaceae) is an important component of major grass covers of the world. Largely it is apomictic and both annual and perennial species exist in nature. Variations in contents of malondialdehyde, proline, specific leaf area and carbon isotope discrimination for drought tolerance were estimated among eight prominent species of Cenchrus. Simultaneously, genetic variations were also estimated by employing 187 RAPD primers. Of these, 23 primers did not react, 2 performed poorly and 7 produced many non-scorable bands and one primer yielded a single monomorphic band. Rest of the 154 primers generated one or more unambiguously scorable fragments. Twelve hundred and four of the 1,296 putative loci were polymorphic (93%) between at least one pair-wise comparisons among eight species. Dice coefficient and neighbor-joining algorithm analyses showed clustering patterns that fit with the known habitat of the species except perennial, C. myosuroides which formed a node between two annuals species. When these species were subjected to water stress tolerance test, a correlation (r = 0.612) between specific leaf area (SLA) and carbon isotope discrimination (CID) and difference in levels of drought tolerance based parameters among eight species were observed. Of the eight species investigated two annuals viz., C. biflorus and C. echinatus showed highest level of genetic similarity which was also evident from the similar levels of SLA, MDA, proline contents and carbon isotope discrimination values observed in these two species.     

Metabolic burden as reflected by maintenance coefficient of recombinant Escherichia coli overexpressing target gene

Metabolic burden as a consequence of overexpression of target gene in a recombinant strain of E. coli 1727 has been analyzed with respect to maintenance energy coefficient (m). The values of m for the host, uninduced recombinant and IPTG induced recombinant were determined to be 0.12, 0.17 and 0.32 g.g^-1.h^-1 respectively. Transient plasmid instability and nearly 33% fall in maximum specific growth rate were observed under conditions of enhanced requirements for maintenance energy.     

Carbohydrate metabolism in growing rice seedlings under arsenic toxicity

We studied in the seedlings of two rice cultivars (Malviya-36 and Pant-12) the effect of increasing levels of arsenic in situ on the content of sugars and the activity of several enzymes of starch and sucrose metabolism: alpha-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2), starch phosphorylase (EC 2.4.1.1), acid invertase (EC 3.2.1.26), sucrose synthase (EC 2.4.1.13) and sucrose phosphate synthase (EC 2.4.1.14). During a growth period of 10-20 d As2O3 at 25 and 50 microM in the growth medium caused an increase in reducing, non-reducing and total soluble sugars. An increased conversion of non-reducing to reducing sugars was observed concomitant with As toxicity. The activities of alpha-amylase, beta-amylase and sucrose phosphate synthase declined, whereas starch phosphorylase, acid invertase and sucrose synthase were found to be elevated. Results indicate that in rice seedlings arsenic toxicity causes perturbations in carbohydrate metabolism leading to the accumulation of soluble sugars by altering enzyme activity. Sucrose synthase possibly plays a positive role in synthesis of sucrose under As-toxicity.     

Development and characterization of microsatellite markers from tropical forage Stylosanthes species and analysis of genetic variability and cross-species transferability

A limited number of functional molecular markers has slowed the desired genetic improvement of Stylosanthes species. Hence, in an attempt to develop simple sequence repeat (SSR) markers, genomic libraries from Stylosanthes seabrana B.L. Maass & 't Mannetje (2n=2x=20) using 5' anchored degenerate microsatellite primers were constructed. Of the 76 new microsatellites, 21 functional primer pairs were designed. Because of the small number of primer pairs designed, 428 expressed sequence tag (EST) sequences from seven Stylosanthes species were also examined for SSR detection. Approximately 10% of sequences delivered functional primer pairs, and after redundancy elimination, 57 microsatellite repeats were selected. Tetranucleotides followed by trinucleotides were the major repeated sequences in Stylosanthes ESTs. In total, a robust set of 21 genomic-SSR (gSSR) and 20 EST-SSR (eSSR) markers were developed. These markers were analyzed for intraspecific diversity within 20 S. seabrana accessions and for their cross-species transferability. Mean expected (He) and observed (Ho) heterozygosity values with gSSR markers were 0.64 and 0.372, respectively, whereas with eSSR markers these were 0.297 and 0.214, respectively. Dendrograms having moderate bootstrap value (23%-94%) were able to distinguish all accessions of S. seabrana with gSSR markers, whereas eSSR markers showed 100% similarities between few accessions. The set of 21 gSSRs, from S. seabrana, and 20 eSSRs, from selected Stylosanthes species, with their high cross-species transferability (45% with gSSRs, 86% with eSSRs) will facilitate genetic improvement of Stylosanthes species globally.     

Genetic Connectivity among Populations of an Endangered Snake Species from Southeastern Australia (Hoplocephalus bungaroides, Elapidae)

For endangered species that persist as apparently isolated populations within a previously more extensive range, the degree of genetic exchange between those populations is critical to conservation and management. A lack of gene flow can exacerbate impacts of threatening processes and delay or prevent colonization of sites after local extirpation. The broad-headed snake, Hoplocephalus bungaroides, is a small venomous species restricted to a handful of disjunct reserves near Sydney, Australia. Mark-recapture studies have indicated low vagility for this ambush predator, suggesting that gene flow also may be low. However, our analyses of 11 microsatellite loci from 163 snakes collected in Morton National Park, from six sites within a 10-km diameter, suggest relatively high rates of gene flow among sites. Most populations exchange genes with each other, with one large population serving as a source area and smaller populations apparently acting as sinks. About half of the juvenile snakes, for which we could reliably infer parentage, were collected from populations other than those in which we collected their putative parents. As expected from the snakes' reliance on rocky outcrops during cooler months of the year, most gene flow appears to be along sandstone plateaux rather than across the densely forested valleys that separate plateaux. The unexpectedly high rates of gene flow on a landscape scale are encouraging for future conservation of this endangered taxon. For example, wildlife managers could conserve broad-headed snakes by restoring habitats near extant source populations in areas predicted to be least affected by future climate change.     

The ossification centers in the lower end of tibiotarsus in domestic fowl.

The morphogenesis of lower end of tibia in chick is studied commonly but the process of ossification of the same has received very little attention so far. The present study is directed to throw some light on the appearance of ossification centers in the lower end of tibiotarsus of chick. The histology of lower end of tibiotarsus was studied by procuring developing tibiotarsi from chick embryos (20) of 6th day incubation till hatching and 3 post hatched chicks. The transparancies of chick embryos at different incubation periods and post hatched chicks were prepared by Dawson's Alizarin staining method. Three cartilage center (tibial, fibulare and intermedium) appeared in 6...9 days of incubation period in the tarsal region. These gradually fused with the lower end of tibia. Three ossification centres developed in the lower end of tibiotarsus. One for intermedium appeared on 16th day and two fotibial and fibulare on 20th day. All these three centres could be located in the transparancies of the chick embryos in tarsal region. The present study proves that the three cartilages centres maintain their individuality during the ossification process even though those fuse completely with the lower end of tibia in chick. The centers for tibial and fibulare are similar to epiphyseal centres of mammals in histological details.     

First isolation of Neospora caninum from an aborted bovine fetus in Spain

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum-specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.     

Evaluation of Eupatorium cannabinum Linn. oil in enhancement of shelf life of mango fruits from fungal rotting

Essential oils extracted from 17 higher plants belonging to different families were screened against Botryodiplodia theobromae and Colletotrichum gloeosporioides causing stem end rot disease and anthracnose disease in mango respectively. The essential oil of Eupatorium cannabinum was found to be fungitoxic in nature against both the mango-rotting fungi. Eupatorium oil was standardized through physico-chemical and fungitoxic properties. Gas Liquid Chromatography (GLC) analysis of the oil led to the identification of 16 components, which represented 77.97% of the oil. Germacrene D (16.11%) was found to be the major component. The oil showed a broad fungitoxic spectrum and was recorded to be more efficient than some synthetic fungicides. The oil also showed an inhibitory effect on pectinase and cellulase enzymes. The oil enhanced the shelf life of mango fruits by protecting from fungal rotting when tested as a fumigant. The LD₅₀ of Eupatorium oil was found to be 22.01 ml/kg body weight on mammalian mice.