Karyotype instability in the ponerine ant genus Diacamma

The queenless ponerine ant Diacamma ceylonense and a population of Diacamma from the Nilgiri hills which we refer to as `nilgiri', exhibit interesting similarities as well as dissimilarities. Molecular phylogenetic study of these morphologically almost similar taxa has shown that D ceylonense is closely related to `nilgiri' and indicates that `nilgiri' is a recent diversion in the Diacamma phylogenetic tree. However, there is a striking behavioural difference in the way reproductive monopoly is maintained by the respective gamergates (mated egg laying workers), and there is evidence that they are genetically differentiated, suggesting a lack of gene flow To develop a better understanding of the mechanism involved in speciation of Diacamma, we have analysed karyotypes of D. ceylonense and `nilgiri' In both, we found surprising inter-individual and intra-individual karyotypic mosaicism. The observed numerical variability, both at intra-individual and inter-individual levels, does not appear to have hampered the sustainability of the chromosomal diversity in each population under study Since the related D. indicum, displays no such intra-individual or inter-Individual variability whatsoever under identical experimental conditions, these results are unlikely to he artifacts. Although no known mechanisms can account for the observed karyotypic variability of this nature, we believe that the present findings on the ants under study would provide opportunities for exciting new discoveries concerning the origin, maintenance and significance of intra-individual and inter-individual karyotypic mosaicism.

Population genetic structure and conservation of Asian elephants (Elephas maximus) across India

This study examines the population genetic structure of Asian elephants (Elephas maximus) across India, which harbours over half the world's population of this endangered species. Mitochondrial DNA control region sequences and allele frequencies at six nuclear DNA microsatellite markers obtained from the dung of free-ranging elephants reveal low mtDNA and typical microsatellite diversity. Both known divergent clades of mtDNA haplotypes in the Asian elephant are present in India, with southern and central India exhibiting exclusively the β clade of Fernando et al. (2000), northern India exhibiting exclusively the α clade and northeastern India exhibiting both, but predominantly the α clade. A nested clade analysis revealed isolation by distance as the principal mechanism responsible for the observed haplotype distributions within the α and β clades. Analyses of molecular variance and pairwise population FST tests based on both mitochondrial and microsatellite DNA suggest that northern-northeastern India, central India, Nilgiris (in southern India) and Anamalai-Periyar (in southern India) are four demographically autonomous population units and should be managed separately. In addition, evidence for female philopatry, male-mediated gene flow and two possible historical biogeographical barriers is described.

Flying between Sky Islands: The Effect of Naturally Fragmented Habitat on Butterfly Population Structure

High elevation montane areas are called ``sky islands'' when they occur as a series of high mountains separated by lowland valleys. Different climatic conditions at high elevations makes sky islands a specialized type of habitat, rendering them naturally fragmented compared to more continuous habitat at lower elevations. Species in sky islands face unsuitable climate in the intervening valleys when moving from one montane area to another. The high elevation shola-grassland mosaic in the Western Ghats of southern India form one such sky island complex. The fragmented patches make this area ideal to study the effect of the spatial orientation of suitable habitat patches on population genetic structure of species found in these areas. Past studies have suggested that sky islands tend to have genetically structured populations, possibly due to reduced gene flow between montane areas. To test this hypothesis, we adopted the comparative approach. Using Amplified Fragment Length Polymorphisms, we compared population genetic structures of two closely related, similar sized butterfly species: Heteropsis oculus, a high elevation shola-grassland specialist restricted to the southern Western Ghats, and Mycalesis patnia, found more continuously distributed in lower elevations. In all analyses, as per expectation the sky island specialist H. oculus exhibited a greater degree of population genetic structure than M. patnia, implying a difference in geneflow. This difference in geneflow in turn appears to be due to the natural fragmentation of the sky island complexes. Detailed analysis of a subset of H. oculus samples from one sky island complex (the Anamalais) showed a surprising genetic break. A possible reason for this break could be unsuitable conditions of higher temperature and lower rainfall in the intervening valley region. Thus, sky island species are not only restricted by lack of habitat continuity between montane areas, but also by the nature of the intervening habitat.

Rapid divergence of ecotypes of an invasive plant

Invasive species demonstrate rapid evolution within a very short period of time allowing one to understand the underlying mechanism(s). Lantana camara, a highly invasive plant of the tropics and subtropics, has expanded its range and successfully established itself almost throughout India. In order to uncover the processes governing the invasion dynamics, 218 individuals from various locations across India were characterized with six microsatellites. By integrating genetic data with niche modelling, we examined the effect of drift and environmental selection on genetic divergence. We found multiple genetic clusters that were non-randomly distributed across space. Spatial autocorrelation revealed a strong fine-scale structure, i.e. isolation by distance. In addition, we obtained evidence of inhibitory effects of selection on gene flow, i.e. isolation by environmental distance. Perhaps, local adaptation in response to selection is offsetting gene flow and causing the populations to diverge. Niche models suggested that temperature and precipitation play a major role in the observed spatial distribution of this plant. Based on a non-random distribution of clusters, unequal gene flow among them and different bioclimatic niche requirements, we concluded that the emergence of ecotypes represented by two genetic clusters is underway. They may be locally adapted to specific climatic conditions, and perhaps at the very early stages of ecological divergence.

Differences in host species relationships and biogeographic influences produce contrasting patterns of prevalence, community composition and genetic structure in two genera of avian malaria parasites in southern Melanesia

1. Host-parasite interactions have the potential to influence broadscale ecological and evolutionary processes, levels of endemism, divergence patterns and distributions in host populations. Understanding the mechanisms involved requires identification of the factors that shape parasite distribution and prevalence. 2. A lack of comparative information on community-level host-parasite associations limits our understanding of the role of parasites in host population divergence processes. Avian malaria (haemosporidian) parasites in bird communities offer a tractable model system to examine the potential for pathogens to influence evolutionary processes in natural host populations. 3. Using cytochrome b variation, we characterized phylogenetic diversity and prevalence of two genera of avian haemosporidian parasites, Plasmodium and Haemoproteus, and analysed biogeographic patterns of lineages across islands and avian hosts, in southern Melanesian bird communities to identify factors that explain patterns of infection. 4. Plasmodium spp. displayed isolation-by-distance effects, a significant amount of genetic variation distributed among islands but insignificant amounts among host species and families, and strong local island effects with respect to prevalence. Haemoproteus spp. did not display isolation-by-distance patterns, showed marked structuring of genetic variation among avian host species and families, and significant host species prevalence patterns. 5. These differences suggest that Plasmodium spp. infection patterns were shaped by geography and the abiotic environment, whereas Haemoproteus spp. infection patterns were shaped predominantly by host associations. Heterogeneity in the complement and prevalence of parasite lineages infecting local bird communities likely exposes host species to a mosaic of spatially divergent disease selection pressures across their naturally fragmented distributions in southern Melanesia. Host associations for Haemoproteus spp. indicate a capacity for the formation of locally co-adapted host-parasite relationships, a feature that may limit intraspecific gene flow or range expansions of closely related host species.

Efficacious Gene Silencing in Serum and Significant Apoptotic Activity Induction by Survivin Downregulation Mediated by New Cationic Gemini Tocopheryl Lipids

Nonviral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We present here six new tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient coliposomal formulations derived from each of these geminis and a helper lipid, dioleoylphosphatidylethanolamine (DOPE), were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized coliposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz., HEK 293T, HeLa, and Caco-2, significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and in the presence of serum (FBS). Notably, the knockdown activity of coliposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR, and Western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin, significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines.

alpha-Tocopherol derived lipid dimers as efficient gene transfection agents. Mechanistic insights into lipoplex internalization and therapeutic induction of apoptotic activity

In this report, we present cationic dimeric (gemini) lipids for significant plasmid DNA (pDNA) delivery to different cell lines without any marked toxicity in the presence of serum. Six gemini lipids based on alpha-tocopherol were synthesized, which differed in their spacer chain lengths. Each of these gemini lipids mixed with a helper lipid, 1,2-dioleoyl phosphatidyl ethanolamine (DOPE), was capable of forming stable aqueous suspensions. These co-liposomal systems were examined for their potential to transfect pEGFP-C3 plasmid DNA into nine cell lines of different origins. The transfection efficacies noticed in terms of EGFP expression levels using flow cytometry were well corroborated using independent fluorescence microscopy studies. Significant EGFP expression levels were reported using the gemini co-liposomes, which counted significantly better than one well known commercial formulation, Lipofectamine 2000 (L2 K). Transfection efficacies were also analyzed in terms of the degree of intracellular delivery of labeled plasmid DNA (pDNA) using confocal microscopy, which revealed an efficient internalization in the presence of serum. The cell viability assays performed using optimized formulations demonstrated no significant toxicity towards any of the cell lines used in the study. We also had a look at the lipoplex internalization pathway to profile the uptake characteristics. A caveolae/lipid raft route was attributed to their excellent gene transfection capabilities. The study was further advanced by using a therapeutic p53-EGFP-C3 plasmid and the apoptotic activity was observed using FACS and growth inhibition assay.

Co-liposomes of redox-active alkyl-ferrocene modified low MW branched PEI and DOPE for efficacious gene delivery in serum

Herein, we present six new lipopolymers based on low molecular weight, branched polyethylenimine (BPEI 800 Da) which are hydrophobically modified using ferrocene terminated alkyl tails of variable lengths. The effects of degree of grafting, spacer length and the redox state of ferrocene in the lipopolymers on the self assembly properties were investigated in detail by TEM, AFM, DLS and zeta potential measurements. The assemblies displayed an oxidation induced increase in the size of the aggregates. The co-liposomes comprising the lipopolymer and a helper lipid, 1,2-dioleoyl phosphatidyl ethanolamine (DOPE), showed excellent gene (pDNA) delivery capability in a serum containing environment in two cancer cell lines (HeLa and U251 cells). Optimized formulations showed remarkably higher transfection activity than BPEI (25 kDa) and were also significantly better than a commercial transfection reagent, Lipofectamine 2000 as evidenced from both the luciferase activity and GFP expression analysis. Oxidation of ferrocene in the lipopolymers led to drastically reduced levels of gene transfection which was substantiated by reduced cellular internalization of fluorescently labelled pDNA as detected using confocal microscopy and flow cytometry. Moreover, the transfection inactive oxidized lipopolyplexes could be turned transfection active by exposure to ascorbic acid (AA) in cell culture medium during transfection. Endocytosis inhibition experiments showed that gene expression mediated by reduced formulations involved both clathrin and caveolae mediated pathways while the oxidized formulations were routed via the caveolae. Cytotoxicity assays revealed no obvious toxicity for the lipopolyplexes in the range of optimized transfection levels in both the cell lines studied. Overall, we have exploited the redox activity of ferrocene in branched PEI-based efficient polymeric gene carriers whose differential transfection activities could be harnessed for spatial or temporal cellular transfections.

Efficacious redox-responsive gene delivery in serum by ferrocenylated monomeric and dimeric cationic cholesterols

Herein, we present the design and synthesis of new redox-active monomeric and dimeric (gemini) cationic lipids based on ferrocenylated cholesterol derivatives for gene delivery. The cationic cholesterols are shown to be transfection efficient after being formulated with the neutral helper lipid DOPE in the presence of serum (FBS). The redox activity of the resulting co-liposomes and their lipoplexes could be regulated using the alkanyl ferrocene moiety attached to the ammonium head groups of the cationic cholesterols. Atomic force microscopy (AFM), dynamic light scattering (DLS) and zeta potential measurements were performed to characterize the co-liposomal aggregates and their complexes with pDNA. The transfection efficiency of lipoplexes could be tuned by changing the oxidation state of the ferrocene moiety. The gene transfection capability was assayed in terms of green fluorescence protein (GFP) expression using pEGFP-C3 plasmid DNA in three cell lines of different origins, namely Caco-2, HEK293T and HeLa, in the presence of serum. The vesicles possessing ferrocene in the reduced state induced an efficient transfection, even better than a commercial reagent Lipofectamine 2000 (Lipo 2000) as evidenced by flow cytometry and fluorescence microscopy. All the co-liposomes containing the oxidized ferrocene displayed diminished levels of gene expression. Gene transfection events from the oxidized co-liposomes were further potentiated by introducing ascorbic acid (AA) as a reducing agent during lipoplex incubation with cells, leading to the resumption of transfection activity. Assessment of transfection capability of both reduced and oxidized co-liposomes was also undertaken following cellular internalization of labelled pDNA using confocal microscopy and flow cytometry. Overall, we demonstrate here controlled gene transfection activities using redox-driven, transfection efficient cationic monomeric and dimeric cholesterol lipids. Such systems could be used in gene delivery applications where transfection needs to be performed spatially or temporally.