Molecular genetic analysis of a captive-breeding program: the vulnerable endemic Jamaican yellow boa

The endemic Jamaican boa (or “yellow boa”, Epicrates subflavus) is a vulnerable species of the Caribbean biodiversity hotspot whose natural populations greatly declined mainly due to predation by introduced species, human persecution, and habitat destruction. A captive breeding program was initiated in 1976 and rationalized in 2002 by the establishment of a European Endangered Species Program. During the last 30 years, more than 600 offspring, of which 80 are still alive today, have been produced and distributed among European host institutions and privates. Here, using nine nuclear microsatellite loci and a fragment of the mitochondrial cytochrome b gene, we (i) determine the natural population from which the founders originate, (ii) identify parental allocation errors and ambiguities in the studbook, and (iii) assess the genetic diversity and estimate levels of inbreeding of the current captive population based on loss of alleles, variance in reproductive success, and relatedness among individuals. Combining measures of relatedness derived from multilocus genotypes with practical parameters such as age of animals and localization of host institutions, we propose mating groups that would maximize genetic diversity in the captive population of the Jamaican boa. Our analyses provide guidance for a more efficient breeding program that, in turn, could be used as the starting point of a repatriation program to increase the probability of the species long-term survival. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Antagonistic bacteria of composted agro-industrial residues exhibit antibiosis against soil-borne fungal plant pathogens and protection of tomato plants from Fusarium oxysporum f.sp. radicis-lycopersici

Rhizospheric and root-associated/endophytic (RAE) bacteria were isolated from tomato plants grown in three suppressive compost-based plant growth media derived from the olive mill, winery and Agaricus bisporus production agro-industries. Forty-four (35 rhizospheric and 9 RAE) out of 329 bacterial strains showed in vitro antagonistic activity against at least one of the soil-borne fungal pathogens, Fusarium oxysporum f.sp. radicis-lycopersici (FORL), F. oxysporum f.sp. raphani, Phytophthora cinnamomi, P. nicotianae and Rhizoctonia solani. The high percentage of total isolates showing antagonistic properties (13%) and their common chitinase and β-glucanase activities indicate that the cell wall constituents of yeasts and macrofungi that proliferate in these compost media may have become a substrate that favours the establishment of antagonistic bacteria to soil-borne fungal pathogens. The selected bacterial strains were further evaluated for their suppressiveness to tomato crown and root rot disease caused by FORL. A total of six rhizospheric isolates, related to known members of the genera Bacillus, Lysinibacillus, Enterobacter and Serratia and one RAE associated with Alcaligenes faecalis subsp. were selected, showing statistically significant decrease of plant disease incidence. Inhibitory effects of extracellular products of the most effective rhizospheric biocontrol agent, Enterobacter sp. AR1.22, but not of the RAE Alcaligenes sp. AE1.16 were observed on the growth pattern of FORL. Furthermore, application of cell-free culture extracts, produced by Enterobacter sp. AR1.22, to tomato roots led to plant protection against FORL, indicating a mode of biological control action through antibiosis.     

Dissociated Neurons and Glial Cells Derived from Rat Inferior Colliculi after Digestion with Papain

The formation of gliosis around implant electrodes for deep brain stimulation impairs electrode–tissue interaction. Unspecific growth of glial tissue around the electrodes can be hindered by altering physicochemical material properties. However, in vitro screening of neural tissue–material interaction requires an adequate cell culture system. No adequate model for cells dissociated from the inferior colliculus (IC) has been described and was thus the aim of this study. Therefore, IC were isolated from neonatal rats (P3_5) and a dissociated cell culture was established. In screening experiments using four dissociation methods (Neural Tissue Dissociation Kit [NTDK] T, NTDK P; NTDK PN, and a validated protocol for the dissociation of spiral ganglion neurons [SGN]), the optimal media, and seeding densities were identified. Thereafter, a dissociation protocol containing only the proteolytic enzymes of interest (trypsin or papain) was tested. For analysis, cells were fixed and immunolabeled using glial- and neuron-specific antibodies. Adhesion and survival of dissociated neurons and glial cells isolated from the IC were demonstrated in all experimental settings. Hence, preservation of type-specific cytoarchitecture with sufficient neuronal networks only occurred in cultures dissociated with NTDK P, NTDK PN, and fresh prepared papain solution. However, cultures obtained after dissociation with papain, seeded at a density of 2×10⁴ cells/well and cultivated with Neuro Medium for 6 days reliably revealed the highest neuronal yield with excellent cytoarchitecture of neurons and glial cells. The herein described dissociated culture can be utilized as in vitro model to screen interactions between cells of the IC and surface modifications of the electrode.     

Development of a multiplex PCR assay for fine-scale population genetic analysis of the Komodo monitor Varanus komodoensis based on 18 polymorphic microsatellite loci

Multiplex PCR assays for the coamplification of microsatellite loci allow rapid and cost-effective genetic analyses and the production of efficient screening protocols for international breeding programs. We constructed a partial genomic library enriched for di-nucleotide repeats and characterized 14 new microsatellite loci for the Komodo monitor (or Komodo dragon, Varanus komodoensis). Using these novel microsatellites and four previously described loci, we developed multiplex PCR assays that may be loaded on a genetic analyser in three separate panels. We tested the novel set of microsatellites for polymorphism using 69 individuals from three island populations and evaluated the resolving power of the entire panel of 18 loci by conducting (i) a preliminary assignment test to determine population(s) of origin and (ii) a parentage analysis for 43 captive Komodo monitors. This panel of polymorphic loci proved useful for both purposes and thus can be exploited for fine-scale population genetic analyses and as part of international captive breeding programs directed at maintaining genetically viable ex situ populations and reintroductions.     

The C609T inborn polymorphism in NAD(P)H:quinone oxidoreductase 1 is associated with susceptibility to multiple sclerosis and affects the risk of development of the primary progressive form of the disease

Oxidative stress plays a pivotal role in the pathogenesis of multiple sclerosis (MS). Inactivating polymorphisms of genes encoding detoxification enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1), could influence susceptibility to MS. To test this hypothesis we performed a case-control study in which we compared the distribution of NQO1 genotypes between 231 MS patients and 380 controls, using both PCR-RFLP and real-time PCR assays. Correlations with MS clinical subtype classification and gender were also evaluated. A significantly higher frequency of the homozygous (T/T) and heterozygous (C/T) NQO1 C⁶⁰⁹T variant genotypes was observed among MS patients compared to controls (P = 0.01), with MS patients showing a 1.5-fold increased risk of carrying at least one variant T allele (P = 0.009). Interestingly, patients belonging to the primary progressive subgroup exhibited a significantly higher incidence of the heterozygous C/T variant genotype, compared to the other forms of MS (P = 0.019). There was no correlation of the NQO1 polymorphism with gender. These results provide the first evidence for a pathogenetic role for the NQO1 C⁶⁰⁹T polymorphism in MS susceptibility and suggest a possible role for the NQO1 genetic background in the development of primary progressive MS.     

Cryptic patterning of avian skin confers a developmental facility for loss of neck feathering

Vertebrate skin is characterized by its patterned array of appendages, whether feathers, hairs, or scales. In avian skin the distribution of feathers occurs on two distinct spatial levels. Grouping of feathers within discrete tracts, with bare skin lying between the tracts, is termed the macropattern, while the smaller scale periodic spacing between individual feathers is referred to as the micropattern. The degree of integration between the patterning mechanisms that operate on these two scales during development and the mechanisms underlying the remarkable evolvability of skin macropatterns are unknown. A striking example of macropattern variation is the convergent loss of neck feathering in multiple species, a trait associated with heat tolerance in both wild and domestic birds. In chicken, a mutation called Naked neck is characterized by a reduction of body feathering and completely bare neck. Here we perform genetic fine mapping of the causative region and identify a large insertion associated with the Naked neck trait. A strong candidate gene in the critical interval, BMP12/GDF7, displays markedly elevated expression in Naked neck embryonic skin due to a cis-regulatory effect of the causative mutation. BMP family members inhibit embryonic feather formation by acting in a reaction-diffusion mechanism, and we find that selective production of retinoic acid by neck skin potentiates BMP signaling, making neck skin more sensitive than body skin to suppression of feather development. This selective production of retinoic acid by neck skin constitutes a cryptic pattern as its effects on feathering are not revealed until gross BMP levels are altered. This developmental modularity of neck and body skin allows simple quantitative changes in BMP levels to produce a sparsely feathered or bare neck while maintaining robust feather patterning on the body.     

The eggshell of the almond wasp Eurytoma amygdali (Hymenoptera, Eurytomidae) - 1. Morphogenesis and fine structure of the eggshell layers

The almond wasp Eurytoma amvgdali (Hymenoptera: Eurytomidac) feeds and oviposits exclusively in almonds and therefore is characterized as an insect of economic importance. Its meroistic polytrophic ovaries include follicles with a tri-partitc configuration. The mature follicles exhibit two filaments occupying the two poles of the egg. One is the micropylar filament while the other might serve for respiration since it is likely that its flattened end layers remain outside the almond fruit. The eggshell is formed by aposition and the follicle cells, which surround the follicle until the end of oogenesis, may be responsible for protein synthesis and secretion which finally lead to the assembly of the eggshell. The eggshell comprises the thin vitelline membrane, possibly a 'wax' layer of waterproofing function, a transluscent layer which appears amorphous even at the end of choriogenesis, a granular layer, including large and small electron-dense granules, and finally a columnar layer very similar to layers found in other insect species of the same or different orders. Peroxidase is histochemicalLY found for the first time in an eggshell of the Hymenoptera order: the tranluscent layer in particular is positively stained (electron-dense). Two possible roles of this peroxidatic activity are discussed, first, in comparison to other fruit-infesting insects, we assume that elastic chorion is produced through the function of peroxidase induced bonds (resilin-type bonds), very important for avoiding premature breaking, while being oviposited through a narrow ovipositor. Second, referring to other studies, this layer can play a bactericidal role for additional embryonic itprotection.     

Optimization of novel combi-molecules: identification of balanced and mixed bcr-abl/DNA targeting properties

Steps toward the identification of combi-molecules with strong abl tyrosine kinase (TK) inhibitory property and significant DNA damaging potential are described. The optimized combi-molecule 13a was shown to induce approximately twofold stronger abl TK inhibitory activity than Gleevec and high levels of DNA damage in chronic myelogenous leukemic cells.