Are there general rules governing parasite diversity? Small mammalian hosts and gamasid mite assemblages

Parasite biodiversity varies on several scales, and in particular among different host species. Previous attempts at finding relationships between host features and the diversity of the parasite assemblages they harbour have yielded inconsistent results, suggesting strongly that any patterns might be taxon-specific. Here, we examined the potential of three host characteristics (host body mass, basal metabolic rate, and area of the geographical range) as determinants of parasite diversity in one group of ectoparasites, gamasid mites (superfamily Dermanyssoidea), using data from 63 species of small mammalian hosts. Our analyses used three measures of parasite diversity (species richness, the Shannon diversity index, and average taxonomic distinctness), and controlled for sampling effort and phylogenetic influences. Although several significant relationships were observed, they depended entirely on which diversity measure was used, or on which host taxon was investigated (insectivores vs. rodents and lagomorphs). In addition, the present results on patterns of mite diversity were not consistent with those of an earlier study involving roughly the same host taxa and the same biogeographical area, but a different group of ectoparasites, i.e. fleas. Thus, there appears to be no universal determinant of parasite diversity, and associations between host features and parasite diversity probably evolve independently in different host–parasite systems.     

Cubic exact solutions for the estimation of pairwise haplotype frequencies: implications for linkage disequilibrium analyses and a web tool 'CubeX'

Background  

The frequency of a haplotype comprising one allele at each of two loci can be expressed as a cubic equation (the 'Hill equation'), the solution of which gives that frequency. Most haplotype and linkage disequilibrium analysis programs use iteration-based algorithms which substitute an estimate of haplotype frequency into the equation, producing a new estimate which is repeatedly fed back into the equation until the values converge to a maximum likelihood estimate (expectation-maximisation).     

Clonal diversity of the marine trematode Maritrema novaezealandensis within intermediate hosts: the molecular ecology of parasite life cycles

We quantified the clonal diversity of the New Zealand marine trematode Maritrema novaezealandensis (n = 1250) within Zeacumantus subcarinatus snail (n = 25) and Macrophthalmus hirtipes crab (n = 25) intermediate hosts using four to six microsatellite loci, and investigated the potential biological and physical factors responsible for the observed genetic patterns. Individual snails harboured one to five trematode genotypes and 48% of snails were infected by multiple parasite genotypes. Overall, the number of parasite genotypes did not increase with snail size, but was highest in intermediate-sized snails. Significantly larger numbers of parasite genotypes were detected in crabs (relative to snails; P < 0.001), with 16-25 genotypes recovered from individual crabs. Although crabs are typically infected by small numbers of cercariae sourced from many snails, they are occasionally infected by large numbers of cercariae sourced from single snails. The latter cases explain the significant genetic differentiation of trematode populations detected among their crab hosts (F(ST) = 0.009, P < 0.001). Our results suggest that the timing of infection and/or intraspecific competition among parasite clones within snails determine(s) the diversity of parasite clones that snails harbour. The presence of a large number of infected snails and tidal mixing of cercariae prior to infection results in crabs potentially harbouring hundreds of parasite genotypes despite the crabs' territorial behaviour.     

Diversity of trematode genetic clones within amphipods and the timing of same-clone infections

The genetic diversity of trematodes within second intermediate hosts has important implications for the evolution of trematode populations as these hosts are utilized after the parasites reproduce asexually within first intermediate hosts and before sexual reproduction within definitive hosts. We characterised the genetic clonal diversity of the marine trematode Maritrema novaezealandensis within amphipod (Paracalliope novizealandiae) second intermediate hosts using four to six microsatellite loci to determine if multiple copies of identical trematode clones existed within naturally infected amphipods. To determine the relative timing of infections by identical clones within hosts, trematode metacercariae were assigned to six developmental stages and the stages of identical clones were compared. The genotypes of 306 trematodes were determined from 44 amphipods each containing more than one trematode. Six pairs of identical trematode clones were recovered in total (representing five amphipods: 11% of amphipods with greater than one trematode) and all pairs of clones belonged to the same developmental stage. This suggests that identical clone infections are effectively synchronous. A general decrease in the number of metacercariae recovered, prevalence, and mean intensity of infection for each subsequent developmental stage coupled with large numbers of metacercariae (>9) only being recovered from recent infections, supports the occurrence of post-infection amphipod mortality and/or within-host trematode mortality. Taken together, our results indicate that natural infections are characterised by high genetic diversity, but that amphipods also periodically encounter "batches" of genetically identical clones, potentially setting the stage for interactions within and between clonal groups inside the host.     

Multiple Conformers in Active Site of Human Dihydrofolate Reductase F31R/Q35E Double Mutant Suggest Structural Basis for Methotrexate Resistance

Methotrexate is a slow, tight-binding, competitive inhibitor of human dihydrofolate reductase (hDHFR), an enzyme that provides key metabolites for nucleotide biosynthesis. In an effort to better characterize ligand binding in drug resistance, we have previously engineered hDHFR variant F31R/Q35E. This variant displays a >650-fold decrease in methotrexate affinity, while maintaining catalytic activity comparable to the native enzyme. To elucidate the molecular basis of decreased methotrexate affinity in the doubly substituted variant, we determined kinetic and inhibitory parameters for the simple variants F31R and Q35E. This demonstrated that the important decrease of methotrexate affinity in variant F31R/Q35E is a result of synergistic effects of the combined substitutions. To better understand the structural cause of this synergy, we obtained the crystal structure of hDHFR variant F31R/Q35E complexed with methotrexate at 1.7-Å resolution. The mutated residue Arg-31 was observed in multiple conformers. In addition, seven native active-site residues were observed in more than one conformation, which is not characteristic of the wild-type enzyme. This suggests that increased residue disorder underlies the observed methotrexate resistance. We observe a considerable loss of van der Waals and polar contacts with the p-aminobenzoic acid and glutamate moieties. The multiple conformers of Arg-31 further suggest that the amino acid substitutions may decrease the isomerization step required for tight binding of methotrexate. Molecular docking with folate corroborates this hypothesis.Human dihydrofolate reductase (hDHFR)⁶ catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate in a NADPH-dependent manner. 5,6,7,8-Tetrahydrofolate is a cofactor in purine and thymidylate biosynthesis, which are essential metabolites in cell division and proliferation. As a consequence of its essential role in nucleoside biosynthesis, hDHFR has been extensively exploited as a drug target. Inhibition with folate antagonists, or antifolates, arrests cell proliferation. The most effective clinical antifolate to date is methotrexate (MTX (Fig. 1)), a slow, tight-binding competitive inhibitor that displays high affinity for hDHFR (K_i^MTX = 3.4 pm). MTX is currently used to treat a variety of diseases, including cancer (1–3), and autoimmune diseases such as juvenile idiopathic arthritis (4). A number of resistance mechanisms to MTX have been observed in cancer patients, including impaired transport of MTX to the cytoplasm (5) and decreased retention of MTX in the cell (6). Numerous ex vivo studies have reported mutations in the hDHFR gene resulting in an enzyme variant with decreased affinity for MTX (7, 8). These have contributed to increase our understanding of the molecular basis for active-site discrimination between the substrate, DHF, and its competitive inhibitor, MTX. Understanding the molecular interactions that affect tight binding of MTX to the active site of DHFR will contribute to our understanding of antifolate binding to DHFR, which can in turn contribute to the design of more efficient inhibitors.Open in a separate windowFIGURE 1.Chemical structures of hDHFR ligands. Atom numbering is shown on DHF.A considerable number of DHFR active-site variants have been identified in MTX-resistant cancer cell lines (although never in patients) (9) or engineered in vitro to elucidate the role of active site residues in the binding of MTX. Amino acid substitutions at residues Ile-7 (10), Leu-22 (11, 12), Phe-31 (13), Phe-34 (14), Arg-70 (15), and Val-115 (16) have yielded MTX-resistant variants. These residues are all present in the folate-binding pocket (17). Because MTX and DHF bind to the active site of hDHFR in a similar manner, all known substitutions causing a decrease in MTX affinity also decrease DHF affinity and overall catalytic efficiency (7, 16, 18). However, the loss of DHF affinity and catalytic efficiency is generally smaller than the loss of MTX affinity. This is often attributed to formation of different contacts with either ligand due to the 180° inversion of the pterin ring of bound DHF relative to MTX (17, 19).Crystal structures of MTX-resistant point mutants have offered insight into the causes of decreased binding of MTX or other antifolates (17, 20–24). To this day, crystal structures of MTX-resistant hDHFR variants L22F, L22R, and L22Y (12), as well as F31G and F31S (25), complexed to various antifolates, have been reported. Only the L22Y variant has been co-crystallized with MTX. Despite its decreased affinity for MTX (L22Y K_i^MTX = 11 nm versus WT K_i^MTX < 31 pm (18)), the inhibitor in the variant structure was bound in the same way as in the native enzyme, making interpretation of decreased affinity difficult to assess. Nonetheless, the low probability conformation of residue Tyr-22 suggested that the presence of a bulky aromatic residue in this area of the folate-binding pocket generated unfavorable hydrophobic interactions with the 2,4-diaminopterin moiety of the inhibitor (12). This is also expected to reduce DHF substrate binding. Structures of MTX-resistant variants F31G and F31S were obtained complexed to N-[4-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl]methylamino]benzoyl]-l-glutamate (MTXO) (25), a MTX analog in which the 2,4–2,4-diaminopterin moiety is replaced by a 2,4-diaminofuropyrimidine moiety. Superposition of MTXO-bound variants with MTX-bound WT hDHFR revealed that the ligands bind to the active site in an analogous manner. It was suggested that decreased MTX binding in the substituted variants resulted from the loss of van der Waals and hydrophobic contacts established between the native Phe-31 and the p-ABA and 2,4-diaminopterin moieties of MTX. F31G and F31S display a 10-fold decrease in affinity for MTX relative to WT hDHFR (K_i^MTX < 31 pm (18)). Further Phe-31 variants (i.e. F31R; K_i^MTX = 7 nm, 200-fold decrease in MTX affinity) (10) display larger decreases in affinity relative to F31G and F31S. This cannot be rationalized by reduction of side-chain contacts with the inhibitor due to the presence of a smaller side chain.These results illustrate the difficulty of gaining insight into the molecular causes for altered MTX binding. This may be partly attributed to the very tight binding of MTX to the native enzyme, such that binding to resistant variants often remains in the sub-nanomolar or low nanomolar range, where the general mode of ligand binding has not changed appreciably relative to the native enzyme. Combining active-site mutations in hDHFR by protein engineering has been shown to generate variants with greatly decreased affinity to MTX (18, 26). Studying the molecular interactions in highly MTX-resistant hDHFR variants offers the possibility of capturing more important changes in enzyme-ligand interactions.Here, we report detailed observations for the mode of MTX resistance in the combinatorial variant F31R/Q35E. Variant F31R/Q35E is a relevant candidate for better understanding the specific interactions that govern ligand recognition in the folate binding site, because it displays a >650-fold decrease in MTX affinity (K_i^MTX = 21 nm) accompanied by a modest, 9-fold decrease of affinity for the substrate DHF relative to WT hDHFR (18). In addition, we have recently shown that this variant is an efficient selectable marker for various mammalian cell types, including murine hematopoietic stem cells (18).⁷ Because mutations giving rise to MTX resistance are not observed in mammals, and because MTX is approved for human treatment, engineered resistant DHFRs offer great potential as human selective markers ex vivo or in vivo (10, 27, 28). To better understand the effect of either amino acid substitution on each ligand, a kinetic double mutant cycle was constructed with the simple variants F31R and Q35E. The crystal structure of the F31R/Q35E variant was obtained with bound MTX at 1.7-Å resolution, to elucidate the structural basis of MTX resistance in this variant. In addition, molecular docking was performed with the F31R/Q35E structure to evaluate the role of the two substitutions toward folate binding. Overall, the results reveal synergistic effects of the combined substitutions toward loss of MTX binding, characterized by increased disorder of specific residues throughout the active site of the highly MTX-resistant F31R/Q35E variant.     

Maedi-Visna virus was detected in association with virally exposed IVF-produced early ewes embryos

The objective of this study was to determine whether MVV can be transmitted by ovine embryos produced in vitro and whether the zona pellucida (ZP) provides any protection against MVV infection.Zona pellucida (ZP)-intact and ZP-free embryos, produced in vitro, at the 8-16 cell stage, were cocultured for 72h in an insert over an ovine oviduct epithelial cell (OOEC)-goat synovial membrane (GSM) cell monolayer that had been previously infected with MVV (K1514 strain). The embryos were then washed and transferred to either direct contact or an insert over a fresh GSM cell monolayer for 6 h. The presence of MVV was detected using RT-PCR on the ten washing fluids and by the observation of typical cytopathic effects (CPE) in the GSM cell monolayer, which was cultured for 6 weeks.This experiment was repeated 4 times with the same results: MVV viral RNA was detected using RT-PCR in the first three washing media, while subsequent baths were always negative. Specific cytopathic effects of MVV infection and MVV-proviral DNA were detected in GSM cells that were used as a viral indicator and cocultured in direct contact or as an insert with MVV-exposed ZP-free embryos. However, no signs of MVV infection were detected in cells that were cocultured with exposed ZP-intact or non-exposed embryos.This study clearly demonstrates that (i) in vitro, ZP-free, early ovine embryos, which had been exposed to 10³ TCID₅₀/m MVV in vitro, are capable of transmitting the virus to susceptible GSM target cells, and that (ii) the IETS recommendations for handling in vivo produced bovine embryos (use of ZP-intact embryos without adherent material and performing ten washes) are effective for the elimination of in vitro MVV infection from in vitro produced ovine embryos. The absence of interaction between ZP-intact embryos and MVV suggests that the in vitro produced embryo zona pellucida provides an effective protective barrier.     

From Sea to Sea: Canada's Three Oceans of Biodiversity

Evaluating and understanding biodiversity in marine ecosystems are both necessary and challenging for conservation. This paper compiles and summarizes current knowledge of the diversity of marine taxa in Canada''s three oceans while recognizing that this compilation is incomplete and will change in the future. That Canada has the longest coastline in the world and incorporates distinctly different biogeographic provinces and ecoregions (e.g., temperate through ice-covered areas) constrains this analysis. The taxonomic groups presented here include microbes, phytoplankton, macroalgae, zooplankton, benthic infauna, fishes, and marine mammals. The minimum number of species or taxa compiled here is 15,988 for the three Canadian oceans. However, this number clearly underestimates in several ways the total number of taxa present. First, there are significant gaps in the published literature. Second, the diversity of many habitats has not been compiled for all taxonomic groups (e.g., intertidal rocky shores, deep sea), and data compilations are based on short-term, directed research programs or longer-term monitoring activities with limited spatial resolution. Third, the biodiversity of large organisms is well known, but this is not true of smaller organisms. Finally, the greatest constraint on this summary is the willingness and capacity of those who collected the data to make it available to those interested in biodiversity meta-analyses. Confirmation of identities and intercomparison of studies are also constrained by the disturbing rate of decline in the number of taxonomists and systematists specializing on marine taxa in Canada. This decline is mostly the result of retirements of current specialists and to a lack of training and employment opportunities for new ones. Considering the difficulties encountered in compiling an overview of biogeographic data and the diversity of species or taxa in Canada''s three oceans, this synthesis is intended to serve as a biodiversity baseline for a new program on marine biodiversity, the Canadian Healthy Ocean Network. A major effort needs to be undertaken to establish a complete baseline of Canadian marine biodiversity of all taxonomic groups, especially if we are to understand and conserve this part of Canada''s natural heritage.     

Increased ROS Production: A Component of the Longevity Equation in the Male Mygalomorph,Brachypelma albopilosa

Background

The diversity of longevities encountered in wildlife is one of the most intriguing problems in biology. Evolutionary biologists have proposed different theories to explain how longevity variability may be driven by bad genes expression in late life or by gene pleiotropic effects. This reflexion has stimulated, in the last ten years, an active research on the proximal mechanisms that can shape lifespan. Reactive oxygen species (ROS), i.e., the by-products of oxidative metabolism, have emerged as the main proximate cause of ageing. Because ROS are mainly produced by the mitochondria, their production is linked to metabolic rate, and this may explain the differences in longevity between large and small species. However, their implication in the sex difference in longevity within a species has never been tested, despite the fact that these differences are widespread in the animal kingdom.

Methodology/Principal Findings

Mitochondrial superoxide production of hemolymph immune cells and antioxidant and oxidative damages plasma levels were measured in adult male and female B. albopilosa at different ages. We found that female spiders are producing less mitochondrial superoxide, are better protected against oxidative attack and are then suffering less oxidative damages than males at adulthood.

Conclusions/Significance

In tarantulas, once reaching sexual maturity, males have a life expectancy reduced to 1 to 2 years, while females can still live for 20 years, in spite of the fact that females continue to grow and moult. This study evidences an increased exposure of males to oxidative stress due to an increase in mitochondrial superoxide production and a decrease in hemolymph antioxidant defences. Such a phenomenon is likely to be part of the explanation for the sharp reduction of longevity accompanying male tarantula maturity. This opens several fundamental research roads in the future to better understand how reproduction and longevity are linked in an original ageing model.     

Distribution of transfer RNA genes in thePisum sativum chloroplast DNA

Purified chloroplast tRNAs were isolated fromPisum sativum leaves and radioactively labeled at their 3′ end using tRNA nucleotidyl transferase and α³²P-labeled CTP. Pea ctDNA was fragmented using a number of restriction endonucleases and hybridized with thein vitro labeled chloroplast tRNAs by DNA transfer method. Genes for tRNAs have been found to be dispersed throughout the chloroplast genome. A closer analysis of the several hybrid regions using recombinant DNA plasmids have shown that tRNA genes are localized in the chloroplast genome in both single and multiple arrangements. Two dimensional gel electrophoresis of total ct tRNA have identified 36 spots. All of them have been found to hybridize withPisum sativum ctDNA. Using recombinant clones, 30 of the tRNA spots have been mapped inPisum sativum ctDNA.     

Subcellular Localization and Characterization of Vasopressin Binding Sites in the Ventral Septal Area, Lateral Septum, and Hippocampus of the Rat Brain

[Arg8]-Vasopressin (AVP) has been shown to exert characteristic central physiological actions in the ventral septal area of the rat brain. This study reports the characterization of receptors for AVP in synaptic plasma membranes prepared from the ventral septal area, the lateral septum, and the hippocampus. Binding of [3H]AVP was temperature and time dependent, linearly related to protein concentration, saturable, and specific. Scatchard plot analysis suggested the presence of a population of binding sites in the three brain areas with dissociation constants and maximal binding capacities, respectively, of 1.06 +/- 0.39 nM and 24.0 +/- 7.01 fmol/mg of protein (mean +/- SEM; n = 3 for the ventral septal area, 0.92 +/- 0.13 nM and 47.0 +/- 4.96 fmol/mg of protein (n = 3) for the lateral septum, and 0.91 +/- 0.14 nM and 25 +/- 5.02 fmol/mg of protein (n = 3) for the hippocampus. In all three brain regions, the rank order of potencies of several vasopressin analogs, unrelated peptides, and other compounds for competitive displacement of ligand indicated a receptor with properties resembling those of the V1-like receptor for AVP. These data document the presence of a high-affinity, V1-like vasopressin receptor in the rat ventral septal area for which the pharmacological properties are similar to those previously reported in physiological studies.