Association of a P2RX7 gene missense variant with brachycephalic dog breeds.

Missense variants are associated with various phenotypic traits and disorders in dogs. The canine P2RX7 gene, coding the ATP-gated P2X7 receptor ion channel, contains four known missense variants. The current study aimed to examine the presence of these variants in a random sample of pedigree and mixed-pedigree dogs. Exons 3, 8, 11 and 13 of the P2RX7 gene, encoding these four respective variants, in 65 dogs were assessed by Sanger sequencing and combined with existing sequencing data from another 69 dogs. The distribution of these variants was then evaluated in all 134 dogs combined and separately within individual breeds including 35 different pure breeds. The rs23314713 (p.Phe103Leu) and rs23315462 (p.Pro452Ser) variants were present in 47 and 40% of all dogs studied respectively, with the rs23314713 variant associated with brachycephalic breeds. Among pedigree dogs, the rs23314713 and rs23315462 variants were associated with brachycephalic and non-brachycephalic breeds respectively. The rs851148233 (p.Arg270Cys) and rs850760787 (p.Arg365Gln) variants were present only in dogs of Cocker Spaniel and Labrador Retriever pedigrees respectively. No other missense variants were found in exons 3, 8, 11 and 13 of the P2RX7 gene within the dogs. In conclusion, the rs23314713 and rs23315462 missense variants of the P2RX7 gene are present in a large proportion of dogs, with the rs23314713 variant associated with a number of brachycephalic breeds. However, the association of this variant with dogs of bulldog ancestry, not brachycephaly per se, cannot be excluded.

Using COI barcodes to identify forensically and medically important blowflies.

The utility of cytochrome oxidase I (COI) DNA barcodes for the identification of nine species of forensically important blowflies of the genus Chrysomya (Diptera: Calliphoridae), from Australia, was tested. A 658-bp fragment of the COI gene was sequenced from 56 specimens, representing all nine Chrysomya species and three calliphorid outgroups. Nucleotide sequence divergences were calculated using the Kimura-two-parameter distance model and a neighbour-joining (NJ) analysis was performed to provide a graphic display of the patterns of divergence among the species. All species were resolved as reciprocally monophyletic on the NJ tree. Mean intraspecific and interspecific sequence divergences were 0.097% (range 0-0.612%, standard error [SE] = 0.119%) and 6.499% (range 0.458-9.254%, SE = 1.864%), respectively. In one case, a specimen that was identified morphologically was recovered with its sister species on the NJ tree. The hybrid status of this specimen was established by sequence analysis of the second ribosomal internal transcribed spacer (ITS2). In another instance, this nuclear region was used to verify four cases of specimen misidentification that had been highlighted by the COI analysis. The COI barcode sequence was found to be suitable for the identification of Chrysomya species from the east coast of Australia.

Systematics, evolution, and biology of scelionid and platygastrid wasps.

The Platygastroidea comprises two families of parasitoids, Scelionidae and Platygastridae, and nearly 4500 described species. They parasitize a diverse array of insects as well as spiders. Idiobiont endoparasitism of eggs is the putative ground plan biology, as reflected by all scelionids, but most Platygastridae are koinobiont endoparasitoids of immature Auchenorrhyncha, Sternorrhyncha, and Cecidomyiidae. The superfamily is demonstrably monophyletic but its phylogenetic position remains uncertain. Relationships within the Platygastroidea are also poorly known and the group is in need of comprehensive phylogenetic study. Significant information is available on host relationships and biology, although much of this is biased to a few genera of Telenominae that are employed as biocontrol agents. Hosts for many genera are unknown, in particular those that inhabit leaf litter or parasitize solitary host eggs. The Trissolcus basalis-Nezara viridula parasitoid-host association has become a favored model system in ecological, behavioral, and physiological research on insects.

Two distinct genotypes of prtF2, encoding a fibronectin binding protein, and evolution of the gene family in Streptococcus pyogenes.

The group A Streptococcus (GAS) is an important pathogen that is responsible for a wide range of human diseases. Fibronectin binding proteins (FBPs) play an important role in promoting GAS adherence and invasion of host cells. The prtF2 gene encodes an FBP and is present in approximately 60% of GAS strains. In the present study we examined 51 prtF2-positive GAS strains isolated from the Northern Territory of Australia, and here we describe two genotypes of prtF2 which are mutually exclusive. The two genotypes have been identified previously as pfbp and fbaB. We show that these genotypes map to the same chromosomal location within the highly recombinatorial fibronectin-collagen-T antigen (FCT) locus, indicating that they arose from a common ancestor, and in this study these genotypes were designated the pfbp type and the fbaB type. Phylogenetic analysis of seven pfbp types, 14 fbaB types, and 11 prtF2-negative GAS strains by pulsed-field gel electrophoresis (PFGE) produced 32 distinct PFGE patterns. Interpretation of evolution based on the PFGE dendrogram by parsimony suggested that the pfbp type had a recent origin compared to the fbaB type. A comparison of multiple DNA sequences of the pfbp and fbaB types revealed a mosaic pattern for the amino-terminal region of the pfbp types. The fbaB type is generally conserved at the amino terminus but varies in the number of fibronectin binding repeats in the carboxy terminus. Our data also suggest that there is a possible association of the pfbp genotype with sof (84.2%), while the fbaB genotype was found in a majority of the GAS strains negative for sof (90.6%), indicating that these two prtF2 subtypes may be under different selective pressures.

Contrasting rates of mitochondrial molecular evolution in parasitic Diptera and Hymenoptera.

We investigated the putative association between the parasitic lifestyle and an accelerated rate of mt genetic divergence, compositional bias, and gene rearrangement, employing a range of parasitic and nonparasitic Diptera and Hymenoptera. Sequences were obtained for the cox1, cox2, 16S, 28S genes, the regions between the cox2 and atp8 genes, and between the nad3 and nad5 genes. Relative rate tests indicated generally that the parasitic lifestyle was not associated with an increased rate of genetic divergence in the Diptera but reaffirmed that it was in the Hymenoptera. Similarly, a departure from compositional stationarity was not associated with parasitic Diptera but was in parasitic Hymenoptera. Finally, mitochondrial (mt) gene rearrangements were not observed in any of the dipteran species examined. The results indicate that these genetic phenomena are not accelerated in parasitic Diptera compared with nonparasitic Diptera. A possible explanation for the differences in the rate of mt molecular evolution in parasitic Diptera and Hymenoptera is the extraordinary level of radiation that has occurred within the parasitic Hymenoptera but not in any of the dipteran parasitic lineages. If speciation events in the parasitic Hymenoptera are associated with founder events, a faster rate of molecular evolution is expected. Alternatively, biological differences between endoparasitic Hymenoptera and endoparasitic Diptera may also account for the differences observed in molecular evolution.

Intramitochondrial recombination - is it why some mitochondrial genes sleep around?

A new paper by Kajander et al. undermines the general view that mitochondria do not recombine. The authors discovered the existence of 'sublimons', rearranged mitochondrial genomes present at very low levels in healthy human patients. Crucially, the different rearranged mitochondrial genomes can theoretically be interconverted through intramitochondrial recombination. The putative operation of intramitochondrial recombination should impact on our ideas of how mitochondrial genes evolve, particularly with respect to how mitochondrial genomes rearrange.

Identification of key components in the irreversibility of salmon calcitonin binding to calcitonin receptors.

This study investigates the poor reversibility of salmon calcitonin (sCT) binding to rat and human calcitonin receptors. Efficacy of CT and analogue peptides in (125)I-sCT binding competition and cAMP assays was compared with the dissociation kinetics of (125)I-labelled peptides. Assessment was performed on cells stably expressing either rat or human calcitonin receptors. Dissociation kinetics of the antagonists, sCT(8-32) and AC512, revealed that binding was rapidly and completely reversible at the receptors, despite high affinity binding, suggesting that poor reversibility required the active conformation of the receptor. G protein coupling was not essential as the dissociation kinetics of (125)I-sCT binding to cell membranes did not significantly alter in the presence of GTP gamma S. Time course experiments established that the transition to irreversibility was slow, while the reversible component of binding appeared to involve a single population of either receptor states or binding sites. Pre-bound (125)I-human CT dissociated rapidly from the receptors, indicating that not all agonists bound irreversibly. To identify structural features of sCT that contribute to its poor reversibility, dissociation kinetics of sCT analogues with various structural modifications were examined. Increasing truncation of N-terminal residues of sCT analogues led to a corresponding increase in the rate of peptide dissociation. Salmon CT peptides which had been substituted at the N-terminus by 13-21 residues of human CT (hCT) were equipotent with sCT in binding competition and cAMP accumulation assays but exhibited a dissociation rate similar to hCT. In contrast, despite lower affinity and efficacy at the receptors, the chimeric analogue sCT(1-16)-hCT(17-32) displayed poorly reversible binding, similar to sCT. Analysis of the dissociation kinetics of sCT analogues with differing alpha-helix forming potential indicated that the ability to form alpha-helical secondary structure was an important factor in the rate of ligand dissociation. We hypothesise that poor reversibility results from a conformational change in the receptor and/or ligand and that this is dependent, at least in part, on interaction with residues constrained within the alpha-helix of the peptide.

Estimating ancestral geographical distributions: a Gondwanan origin for aphid parasitoids?

We tested the published hypothesis of a Gondwanan origin for the overwhelmingly northern hemisphere aphid parasitoids (Aphidiinae) as follows: (i) finding their sister group by a phylogenetic analysis of the entire Braconidae (Insecta: Hymenopterai using sequence data from approximately 500 bp fragments of both the nuclear 28S (D2 region) and mitochondrial 16S rDNA genes, (ii) using this sister-group relationship and the more informative 28S D2 gene to estimate the phylogeny of the Aphidiinae and (iii) estimating the ancestral distribution for the Aphidiinae using maximum-likelihood and maximum-parsimony methods. Both methods indicated a Gondwanan origin.

Relationships among the cyclostome braconid (Hymenoptera: Braconidae) subfamilies inferred from a mitochondrial tRNA gene rearrangement.

The arrangement of mitochondrial tRNA genes for lysine (K) and aspartate (D) from the junction of the cytochrome oxidase II and ATPase 8 genes was determined in a range of hymenopteran taxa. This indicated that the ancestral arrangement for the order is 'KD', as found in the Diptera (represented by Drosophila and Anopheles) and basal Orthoptera. Most Hymenoptera that evolved after the appearance of parasitism also have the 'KD' arrangement, including noncyclostome braconids. However, most cyclostome braconids have either a 'DK' or a 'DHK' arrangement (where 'H' refers to the tRNA gene for Histidine). In both cases, the aspartate tRNA gene is encoded on the mitochondrial N-strand, rather than the J-strand as is usually the case. This rearrangement identified a monophyletic group not previously recognized, consisting of Rogadinae + Braconinae + Gnamptodontinae + Histeromerinae + Rhyssalinae + Betylobraconinae + Opiinae + Alysiinae. Only one cyclostome subfamily (Doryctinae) retained the 'KD' arrangement, suggesting this to be the most basal of the cyclostome subfamilies, consistent with ectoparasitism being plesiomorphic for the cyclostomes. However, the Aphidiinae also retained the 'KD' arrangement, leaving unresolved the issue of whether they should be included within the cyclostomes.

Evolutionary dynamics of a mitochondrial rearrangement "hot spot" in the Hymenoptera.

The arrangement of tRNA genes at the junction of the cytochrome oxidase II and ATPase 8 genes was examined across a broad range of Hymenoptera. Seven distinct arrangements of tRNA genes were identified among a group of wasps that have diverged over the last 180 Myr (suborder Apocrita); many of the rearrangements represent evolutionarily independent events. Approximately equal proportions of local rearrangements, inversions, and translocations were observed, in contrast to vertebrate mitochondria, in which local rearrangements predominate. Surprisingly, homoplasy was evident among certain types of rearrangement; a reversal of the plesiomorphic gene order has arisen on three separate occasions in the Insecta, while the tRNA(H) gene has been translocated to this locus on two separate occasions. Phylogenetic analysis indicates that this gene translocation is real and is not an artifactual translocation resulting from the duplication of a resident tRNA gene followed by mutation of the anticodon. The nature of the intergenic sequences surrounding this region does not indicate that it should be especially prone to rearrangement; it does not generally have the tandem or inverted repeats that might facilitate this plasticity. Intriguingly, these findings are consistent with the view that during the evolution of the Hymenoptera, rearrangements increased at the same time that the rate of point mutations and compositional bias also increased. This association may direct investigations into mitochondrial genome plasticity in other invertebrate lineages.

Evolutionary relationships among the Braconidae (Hymenoptera: Ichneumonoidea) inferred from partial 16S rDNA gene sequences.

Phylogenetic relationships among the Braconidae were examined using homologous 16S rDNA gene sequence data. Analyses recovered the few well-supported relationships evident in this family from morphological analyses, viz the monophyly of the microgastroid complex of subfamilies, the monophyly of the cyclostome complex of subfamilies (= braconoids), a sister-group relationship between the Alysiinae and Opiinae, and a close relationship between the Helconinae and Blacinae. With respect to the braconoid complex of subfamilies, a sister-group relationship was recovered between Aphidiinae and Mesostoinae, and a clade composed of Gnamptodontinae + Histeromerinae + Rhyssalinae + Aphidiinae + Mesostoinae was also recovered. The Doryctinae and Rogadinae sensu lato (s.l.) were generally not resolved as monophyletic. With respect to the helconoid complex of subfamilies, a sister-group relationship was recovered between Sigalphinae and Agathidinae, whereas Neoneurinae fell out among other helconoid subfamilies. Other relationships among the helconoid subfamilies were unclear from these analyses. With respect to the microgastroid complex of subfamilies, our data conform to morphological estimates, recovering ((Microgastrinae + Miracinae) + Cardiochilinae) + Cheloninae. The topology of our trees suggests that the cyclostome subfamilies are a natural derived group, inferring that endoparasitism (not ectoparasitism) is the ancestral state for the Braconidae, unless all of the ectoparasitic ancestors of the helconoid + microgastroid subfamilies are now extinct.

Purification of calcitonin-like peptides from rat brain and pituitary.

Accumulating evidence supports the existence of nonthyroidal calcitonin (CT)-like peptides, more similar to fish CTs, which may act as endogenous regulators of CT receptors in brain and other tissues. In this study, we have carried out large-scale extractions from Sprague-Dawley rat brain diencephalon and pituitary, and purified a novel, biologically active, CT-like peptide from pituitary. Monitoring of the calcitonin-like activity of the peptides from rat brain and pituitary required different detection systems. While the brain CT cross-reacted with C-terminally directed salmon CT-specific antisera, the pituitary CT did not. However, the pituitary CT was biologically active, exhibiting specific interaction with CT receptors to activate adenylate cyclase. Conventional chromatographic techniques were employed to purify the CT-like peptides. Although the brain CT was not purified to homogeneity, size exclusion chromatography revealed the presence of multiple molecular weight forms of immunoreactive CT. Of these, only the lowest molecular weight form was biologically active. Purification from the pituitary resulted in the isolation of a biologically active peptide with a mass of 3267 Da. This mass differs from the mass of both salmon and thyroid-derived rat CT. Initial amino acid sequencing of the pituitary CT indicated that it was N-terminally blocked. Following aminopeptidase digestion, a unique six amino acid sequence, EKSQSP, was identified. Elucidation of the amino acid composition provided supporting evidence that the peptide was novel and was consistent with a full length peptide of approximately 30 amino acids. These data support the existence of novel, nonthyroidal, CTs which are potential regulators of CT receptor-mediated functions.

Phylogenetic relationships among the microgastroid wasps (Hymenoptera: braconidae): combined analysis of 16S and 28S rDNA genes and morphological data.

Relationships among the microgastroid complex of braconid wasps were investigated using sequence data from the 16S mitochondrial rDNA and 28S (D2 expansion region) nuclear rDNA genes, as well as morphological data. Parsimony analysis of these gene fragments, both separately and combined, indicated that Neoneurus (Neoneurinae) and Ichneutes (Ichneutinae) were no more closely related to the microgastroids than were a range of helconoid taxa. Combined parsimony analysis of the microgastroids indicated the relationships ((Cardiochilinae + Microgastrinae) + Miracinae) + Cheloninae, with Adeliinae falling inside the Cheloninae. Bootstrap proportions for each of these nodes were greater than 70%. Character reweighting (sensu Farris), using the rescaled consistency index, also recovered these relationships. Mapping of lifestyle traits onto this relatively well supported phylogeny indicated that solitary endoparasitism is ancestral for the microgastroids, with a single origin for egg-larval endoparasitism in the Cheloninae + Adeliinae. Mapping of the radiation of the microgastroids into lepidopteran hosts was less clear, due to the specialized biology of the most basal microgastroid clade, the Cheloninae + Adeliinae. Our data are consistent with attack of concealed lepidopteran hosts as the plesiomorphic lifestyle, at least for the Miracinae + Cardiochilinae + Microgastrinae, with radiation into more exposed hosts in the Cardiochilinae + Microgastrinae.

Evidence for AT-transversion bias in wasp (Hymenoptera: Symphyta) mitochondrial genes and its implications for the origin of parasitism.

We inferred the incidence of nucleotide conversions in the COI and 16S rRNA mitochondrial genes of members of the Symphyta and basal Apocrita (Hymenoptera). Character-state reconstructions in both genes suggested that conversions between A and T (AT transversions) occurred much more frequently than any other type of change, although we cannot wholly discount an underlying transition bias. Parsimony analysis of COI nucleotide characters did not recover phylogeny; e.g., neither the Tenthredinoidea nor Apocrita were recovered as monophyletic. However, analysis of COI amino acid characters did recover these relationships, as well as others based on fossil and morphological evidence. Analysis of 16S rRNA characters also recovered these relationships providing conversions between A and T were down-weighted. Analysis of the combined data sets gave relatively strong support for various relationships, suggesting that both data sets supported similar topographies. These data sets, both separately and combined, suggested that the phytophagous Siricidae were more closely related to the predominantly parasitic Apocrita than were the ectoparasitic Orussoidea. This suggests that the wasp parasitic lifestyle did not have a single origin, unless the Siricidae have more recently reverted to phytophagy. Alternatively, parasitism evolved twice independently, once in the Orussoidea and again in the Apocrita. The latter scenario is supported by the observation that the evolution of parasitism was accompanied by a tendency for the larvae to develop inside plant tissues. Adaptations that accompanied the movement of wasps into a confined, wood-boring habitat may have preadapted them to becoming ectoparasitic.

Electrophoretic mobility and glycosylation characteristics of heterogeneously expressed calcitonin receptors.

The translated calcitonin receptor (CTR) complementary DNA sequences contain potential N-linked glycosylation sites within the extracellular N-terminus. We investigated the relative molecular mass (M(r)) and degree of N-linked glycosylation of five cloned CTRs (pig, rat C1a, rat C1b, human I1-ve, and human I1+ve), together with the pig hypothalamic CTR, to analyze the potential contribution of carbohydrate moieties to the molecular identity of these receptors. Receptors were cross-linked to 125I-salmon CT with the homobifunctional reagent bis(sulfosuccinimidyl) suberate. Autoradiographic analysis of the cross-linked receptors, following SDS-PAGE, revealed apparent M(r)S, ranging between 70,000 and 80,000 for the rat, human, and pig hypothalamic receptors. However, the cloned, expressed pig CTR was much smaller (approximately 58,000). The lower M(r) of the cloned pig CTR appeared to be due to absence of N-terminal residues, but this did not impact on ligand-receptor specificity when compared with the hypothalamic pig CTR. Cleavage under nondenaturing conditions of N-linked sugars from the CTRs using endoglycosidase F (Endo F), increased the electrophoretic mobility of all receptors, except the pig CTRs, by approximately 10 kDa. Under denaturing conditions, electrophoretic mobilities increased by approximately 30 kDa for the rat C1a, rat C1b, and humanI1-ve (expressed in human embryonic kidney-293 cells) CTRs and by approximately 20 kDa for the cloned pig, pig hypothalamic, and human CTR isoforms (expressed in baby hamster kidney cells). Competition binding studies using glycosylated and partially deglycosylated (nondenaturing conditions) receptor preparations demonstrated no significant differences in binding affinity or specificity. Thus the CTRs are N-linked glycoproteins whose degree of glycosylation is both cell-type and species dependent.

Increased genetic diversity in mitochondrial genes is correlated with the evolution of parasitism in the Hymenoptera.

A higher AT content and rate of mtDNA sequence divergence was found in parasitic wasps (Apocrita) compared with nonparasitic wasps (Symphyta). The compositional bias was reflected in extreme codon bias for a cytochrome oxidase I protein coding gene fragment as well as in the types of amino acid substitutions that have occurred during the evolution of this gene fragment. In some instances, compositional bias influenced the definition of a conservative amino acid change. The increased rate of mtDNA sequence evolution probably arose during the early Jurassic, coincident with the first appearance of parasitic wasps in the fossil record. Our results suggest a causal link between the rate of sequence divergence and the parasitic lifestyle.

Molecular phylogeny of the insect order Hymenoptera: apocritan relationships.

Phylogenetic relationships among the major groups of hymenopteran insects were investigated by using comparative sequence information from the mitochondrial 16S rRNA gene. The placement of the ectoparasitic Stephanidae as the sister group to the remaining Apocrita confirmed ectoparasitism as the ground plan biology for the Apocrita. Endoparasitism evolved at least eight times within the Apocrita, and the consequent association with polydnaviruses and virus-like particles evolved at least three times. The Evaniomorpha were consistently placed as basal to the remaining Apocrita but were not resolved as monophyletic. The Gasteruptiidae were resolved as the sister group to the Evaniidae, but the relationship between the Trigonalyoidea and the Evanioidea was unclear. The Proctotrupomorpha (sensu Rasnitsyn) was resolved by topology-dependent permutation tail probability (T-PTP) testing as monophyletic, with strong evidence for a sister group relationship between the Platygastroidea and the Chalcidoidea. Strong evidence was found for the monophyly of the Ichneumonomorpha (Ichneumonidae + Braconidae) and the sister-group relationship between the Aculeata (Vespomorpha) and the Ichneumonomorpha.