Plant quality and local adaptation undermine relocation in a bog specialist butterfly

The butterfly Boloria aquilonaris is a specialist of oligotrophic ecosystems. Population viability analysis predicted the species to be stable in Belgium and to collapse in the Netherlands with reduced host plant quality expected to drive species decline in the latter. We tested this hypothesis by rearing B. aquilonaris caterpillars from Belgian and Dutch sites on host plants (the cranberry, Vaccinium oxycoccos). Dutch plant quality was lower than Belgian one conferring lower caterpillar growth rate and survival. Reintroduction and/or supplementation may be necessary to ensure the viability of the species in the Netherlands, but some traits may have been selected solely in Dutch caterpillars to cope with gradual changes in host plant quality. To test this hypothesis, the performance of Belgian and Dutch caterpillars fed with plants from both countries were compared. Dutch caterpillars performed well on both plant qualities, whereas Belgian caterpillars could not switch to lower quality plants. This can be considered as an environmentally induced plastic response of caterpillars and/or a local adaptation to plant quality, which precludes the use of Belgian individuals as a unique solution for strengthening Dutch populations. More generally, these results stress that the relevance of local adaptation in selecting source populations for relocation may be as important as restoring habitat quality.     

Measuring the immune system of the three‐spined stickleback – investigating natural variation by quantifying immune expression in the laboratory and the wild

Current understanding of the immune system comes primarily from laboratory‐based studies. There has been substantial interest in examining how it functions in the wild, but studies have been limited by a lack of appropriate assays and study species. The three‐spined stickleback (Gasterosteus aculeatus L.) provides an ideal system in which to advance the study of wild immunology, but requires the development of suitable immune assays. We demonstrate that meaningful variation in the immune response of stickleback can be measured using real‐time PCR to quantify the expression of eight genes, representing the innate response and Th1‐, Th2‐ and Treg‐type adaptive responses. Assays are validated by comparing the immune expression profiles of wild and laboratory‐raised stickleback, and by examining variation across populations on North Uist, Scotland. We also compare the immune response potential of laboratory‐raised individuals from two Icelandic populations by stimulating cells in culture. Immune profiles of wild fish differed from laboratory‐raised fish from the same parental population, with immune expression patterns in the wild converging relative to those in the laboratory. Innate measures differed between wild populations, whilst the adaptive response was associated with variation in age, relative size of fish, reproductive status and S. solidus infection levels. Laboratory‐raised individuals from different populations showed markedly different innate immune response potential. The ability to combine studies in the laboratory and in the wild underlines the potential of this toolkit to advance our understanding of the ecological and evolutionary relevance of immune system variation in a natural setting.     

Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis

We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment.     

The effect of pH on glucoamylase production, glycosylation and chemostat evolution of Aspergillus niger

The effect of ambient pH on production and glycosylation of glucoamylase (GAM) and on the generation of a morphological mutant produced by Aspergillus niger strain B1 (a transformant containing an additional 20 copies of the homologous GAM glaA gene) was studied. We have shown that a change in the pH from 4 to 5.4 during continuous cultivation of the A. niger B1 strain instigates or accelerates the spontaneous generation of a morphological mutant (LB). This mutant strain produced approx. 50% less extracellular protein and GAM during both chemostat and batch cultivation compared to another strain with parental-type morphology (PS). The intracellular levels of GAM were also lower in the LB strain. In addition, cultivation of the original parent B1 strain in a batch-pulse bioreactor at pH 5.5 resulted in a 9-fold drop in GAM production and a 5-fold drop in extracellular protein compared to that obtained at pH 4. Glycosylation analysis of the glucoamylases purified from shake-flask cultivation showed that both principal forms of GAM secreted by the LB strain possessed enhanced galactosylation (2-fold), compared to those of the PS. Four diagnostic methods (immunostaining, mild methanolysis, mild acid hydrolysis and beta-galactofuranosidase digestion) provided evidence that the majority of this galactose was of the furanoic conformation. The GAMs produced during batch-pulse cultivation at pH 5.5 similarly showed an approx. 2-fold increase in galactofuranosylation compared to pH 4. Interestingly, in both cases the increased galactofuranosylation appears primarily restricted to the O-linked glycan component. Ambient pH therefore regulates both GAM production and influences its glycosylation.     

Cladogenesis and loss of the marine life-history phase in freshwater galaxiid fishes (Osmeriformes: Galaxiidae)

Switches from migratory (diadromous) to nonmigratory (freshwater) life histories are known to have occurred repeatedly in some aquatic taxa. However, the significance of the loss of diadromy as an initiator for speciation remains poorly understood. The rivers of New Zealand's South Island house a species flock of recently derived nonmigratory galaxiid fishes known as the Galaxias vulgaris complex. Members of this complex are morphologically and genetically similar to the diadromous G. brevipinnis found in New Zealand and southeastern Australia. We hypothesised that South Island's G. vulgaris complex (at least 10 nonmigratory lineages) represents a number of independent radiations from a migratory G. brevipinnis stock, with repeated loss of diadromy. Sequence data were obtained for 31 ingroup samples (G. vulgaris complex and G. brevipinnis) plus four outgroup taxa. A well-resolved phylogeny based on 5039 base pairs of the mitochondrial genome suggests that diadromy has been lost on three separate occasions. Thus, speciation in these galaxiid fishes is partly an incidental phenomenon caused by switches from diadromous to nonmigratory strategies. However, much of the subsequent nonmigratory diversity is monophyletic, suggesting that drainage evolution (vicariance) has also played a major role in cladogenesis. Levels of sequence divergence among major ingroup lineages (1.6-12.7%) suggest that the radiation is considerably older relative to Northern Hemisphere (postglacial) complexes of salmonid, osmerid, and gasterosteid fishes. Sympatric taxa are not monophyletic, suggesting that their coexistence reflects secondary contact rather than sympatric speciation. The monophyly of New Zealand G. brevipinnis is well supported, but both mitochondrial DNA and nuclear sequences indicate that G. brevipinnis is paraphyletic on an intercontinental scale. The divergence (maximum 11.5%) between Tasmanian and New Zealand G. brevipinnis, although large, supports marine dispersal rather than vicariance as the principle biogeographic mechanism on an intercontinental scale.     

Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival

Matrix Gla protein (MGP) is a 14-kD extracellular matrix protein of the mineral-binding Gla protein family. Studies of MGP-deficient mice suggest that MGP is an inhibitor of extracellular matrix calcification in arteries and the epiphyseal growth plate. In the mammalian growth plate, MGP is expressed by proliferative and late hypertrophic chondrocytes, but not by the intervening chondrocytes. To investigate the functional significance of this biphasic expression pattern, we used the ATDC5 mouse chondrogenic cell line. We found that after induction of the cell line with insulin, the differentiating chondrocytes express MGP in a stage-specific biphasic manner as in vivo. Treatment of the ATDC5 cultures with MGP antiserum during the proliferative phase leads to their apoptosis before maturation, whereas treatment during the hypertrophic phase has no effect on chondrocyte viability or mineralization. After stable transfection of ATDC5 cells with inducible sense or antisense MGP cDNA constructs, we found that overexpression of MGP in maturing chondrocytes and underexpression of MGP in proliferative and hypertrophic chondrocytes induced apoptosis. However, overexpression of MGP during the hypertrophic phase has no effect on chondrocyte viability, but it does reduce mineralization. This work suggests that coordinated levels of MGP are required for chondrocyte differentiation and matrix mineralization.