Fine- and local- scale genetic structure of <Emphasis Type="Italic">Dysoxylum malabaricum,</Emphasis> a late-successional canopy tree species in disturbed forest patches in the Western Ghats,India

Dysoxylum malabaricum (white cedar) is an economically important tree species, endemic to the Western Ghats, India, which is the world’s most densely populated biodiversity hotspot. In this study, we used variation at ten nuclear simple sequence repeat loci to investigate genetic diversity and fine scale spatial genetic structure (FSGS) in seedlings and adults of D. malabaricum from four forest patches in the northern part of the Western Ghats. When genetic variation was compared between seedlings and adults across locations, significant differences were detected in allelic richness, observed heterozygosity, fixation index (F _IS), and relatedness (P < 0.05). Reduced genetic diversity and increased relatedness at the seedling stage might be due to fragmentation and disturbance. There was no FSGS at the adult stage and FSGS was limited to shorter distance classes at the seedling stage. However, there was clear spatial genetic structure at the landscape level (<50 km), regardless of age class, due to limited gene flow between forest patches. A comparison of the distributions of size classes in the four locations with published data from a more southern area, showed that large trees (diameter at breast height, DBH, >130 cm) are present in the southern sacred forests but not in the northern forest reserves. This pattern is likely due to stronger harvesting pressure in the north compared to the south, because in the north there are no cultural taboos regulating the extraction of natural resources. The implications for forest conservation in this biodiversity hotspot are discussed.     

Genetic Structure,Diversity and Long Term Viability of a Medicinal Plant,Nothapodytes nimmoniana Graham. (Icacinaceae), in Protected and Non-Protected Areas in the Western Ghats Biodiversity Hotspot

Background and Question

The harvesting of medicinal plants from wild sources is escalating in many parts of the world, compromising the long-term survival of natural populations of medicinally important plants and sustainability of sources of raw material to meet pharmaceutical industry needs. Although protected areas are considered to play a central role in conservation of plant genetic resources, the effectiveness of protected areas for maintaining medicinal plant populations subject to intense harvesting pressure remain largely unknown. We conducted genetic and demographic studies of Nothapodytes nimmoniana Graham, one of the extensively harvested medicinal plant species in the Western Ghats biodiversity hotspot, India to assess the effectiveness of protected areas in long-term maintenance of economically important plant species.

Methodology/Principal Findings

The analysis of adults and seedlings of N. nimmoniana in four protected and four non-protected areas using 7 nuclear microsatellite loci revealed that populations that are distributed within protected areas are subject to lower levels of harvesting and maintain higher genetic diversity (H_e = 0.816, H_o = 0.607, A = 18.857) than populations in adjoining non-protected areas (H_e = 0.781, H_o = 0.511, A = 15.571). Furthermore, seedlings in protected areas had significantly higher observed heterozygosity (H_o = 0.630) and private alleles as compared to seedlings in adjoining non-protected areas (H_o = 0.426). Most populations revealed signatures of recent genetic bottleneck. The prediction of long-term maintenance of genetic diversity using BOTTLESIM indicated that current population sizes of the species are not sufficient to maintain 90% of present genetic diversity for next 100 years.

Conclusions/Significance

Overall, these results highlight the need for establishing more protected areas encompassing a large number of adult plants in the Western Ghats to conserve genetic diversity of economically and medicinally important plant species.     

Matrix-Assisted Refolding,Purification and Activity Assessment Using a ‘Form Invariant’ Assay for Matrix Metalloproteinase 2 (MMP2)

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed ‘form invariant’ assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.     

Do Ecological Niche Model Predictions Reflect the Adaptive Landscape of Species?: A Test Using Myristica malabarica Lam., an Endemic Tree in the Western Ghats,India

Ecological niche models (ENM) have become a popular tool to define and predict the “ecological niche” of a species. An implicit assumption of the ENMs is that the predicted ecological niche of a species actually reflects the adaptive landscape of the species. Thus in sites predicted to be highly suitable, species would have maximum fitness compared to in sites predicted to be poorly suitable. As yet there are very few attempts to address this assumption. Here we evaluate this assumption. We used Bioclim (DIVA GIS version 7.3) and Maxent (version 3.3.2) to predict the habitat suitability of Myristica malabarica Lam., an economically important tree occurring in the Western Ghats, India. We located populations of the trees naturally occurring in different habitat suitability regimes (from highly suitable to poorly suitable) and evaluated them for their regeneration ability and genetic diversity. We also evaluated them for two plant functional traits, fluctuating asymmetry – an index of genetic homeostasis, and specific leaf weight – an index of primary productivity, often assumed to be good surrogates of fitness. We show a significant positive correlation between the predicted habitat quality and plant functional traits, regeneration index and genetic diversity of populations. Populations at sites predicted to be highly suitable had a higher regeneration and gene diversity compared to populations in sites predicted to be poor or unsuitable. Further, individuals in the highly suitable sites exhibited significantly less fluctuating asymmetry and significantly higher specific leaf weight compared to individuals in the poorly suitable habitats. These results for the first time provide an explicit test of the ENM with respect to the plant functional traits, regeneration ability and genetic diversity of populations along a habitat suitability gradient. We discuss the implication of these resultsfor designing viable species conservation and restoration programs.     

A model for the diffusion of fluorescent probes in the septate giant axon of earthworm. Axoplasmic diffusion and junctional membrane permeability.

The diffusion of the three fluorescent probes dichlorofluorescein, carboxyfluorescein, and Lucifer Yellow within the septate median giant axon of the earthworm was monitored using fluorometric methods. A diffusion model was derived that allowed computation of the apparent axoplasmic diffusion coefficient, junctional membrane permeability (septal membranes), and plasma membrane permeability for each probe. Dichlorofluorescein and carboxyfluorescein have similar apparent axoplasmic diffusion coefficients, which were reduced by a factor of eight relative to that predicted from the Einstein-Stokes equation. Nonspecific reversible binding appears to be the major cause of the retarded diffusion coefficients. Junctional membrane permeability for dichlorofluorescein was 4.7 to 73-fold greater than that for carboxyfluorescein. This difference could not be explained on the basis of molecular size but can be explained by the difference in charge between the two molecules. Diffusion coefficients and junctional membrane permeabilities remained constant with time for both dyes. The diffusion of Lucifer Yellow within the axoplasm and permeability through the junctional membranes did not remain constant with time but declined. From this it was inferred that Lucifer Yellow experienced a slow, irreversible binding to axoplasmic elements. All three probes had finite plasma membrane permeabilities.     

Multichannel recordings from membranes which contain gap junctions.

We have studied multichannel patch-clamp recordings in earthworm axon septal membranes that contain gap junctions. Though all channels have the same conductance and selectivity, the probabilities of the conductance levels in the majority of the recordings could not be fit by assuming independent and identical channels; in these cases, we found that at least two different open probabilities were required to explain the data. The data thus suggest that, within one junctional membrane complex, there exists a heterogenous channel population of similar but not identical channel types. The analysis also revealed cases where cooperativity between individual channels was the only explanation for the amplitude histograms of the observed multichannel activity. The conclusions drawn are based on a theoretical analysis of multichannel current-amplitude histograms. We derive two tests for independent and identical channels. We analyze the effects of mode shifting. These results are based on the ratio of peaks in the histograms; they are independent of the number of channels in the patch and the model of channel gating. In some cases failure to fulfill the criteria of these tests implied an interdependence or cooperativity between channels. Lastly, we have devised statistical tests for stability of the recording in the presence of variance due to finite sample size.     

Cost-Effectiveness of “Golden Mustard” for Treating Vitamin A Deficiency in India

Background

Vitamin A deficiency (VAD) is an important nutritional problem in India, resulting in an increased risk of severe morbidity and mortality. Periodic, high-dose vitamin A supplementation is the WHO-recommended method to prevent VAD, since a single dose can compensate for reduced dietary intake or increased need over a period of several months. However, in India only 34 percent of targeted children currently receive the two doses per year, and new strategies are urgently needed.

Methodology

Recent advancements in biotechnology permit alternative strategies for increasing the vitamin A content of common foods. Mustard (Brassica juncea), which is consumed widely in the form of oil by VAD populations, can be genetically modified to express high levels of beta-carotene, a precursor to vitamin A. Using estimates for consumption, we compare predicted costs and benefits of genetically modified (GM) fortification of mustard seed with high-dose vitamin A supplementation and industrial fortification of mustard oil during processing to alleviate VAD by calculating the avertable health burden in terms of disability-adjusted life years (DALY).

Principal Findings

We found that all three interventions potentially avert significant numbers of DALYs and deaths. Expanding vitamin A supplementation to all areas was the least costly intervention, at $23–$50 per DALY averted and $1,000–$6,100 per death averted, though cost-effectiveness varied with prevailing health subcenter coverage. GM fortification could avert 5 million–6 million more DALYs and 8,000–46,000 more deaths, mainly because it would benefit the entire population and not just children. However, the costs associated with GM fortification were nearly five times those of supplementation. Industrial fortification was dominated by both GM fortification and supplementation. The cost-effectiveness ratio of each intervention decreased with the prevalence of VAD and was sensitive to the efficacy rate of averted mortality.

Conclusions

Although supplementation is the least costly intervention, our findings also indicate that GM fortification could reduce the VAD disease burden to a substantially greater degree because of its wider reach. Given the difficulties in expanding supplementation to areas without health subcenters, GM fortification of mustard seed is an attractive alternative, and further exploration of this technology is warranted.