Detection of the initial infective stages of the protozoan parasite Marteilia sydneyi in Saccostrea glomerata and their development through to sporogenesis

DNA probes were used in in situ hybridisation on histological sections of oysters exposed for defined intervals to Marteilia sydneyi infection to reveal the early development of the parasite in the oyster host, Saccostrea glomerata. The initial infective stages enter through the palps and gills whereupon extrasporogonic proliferation results in the liberation of cells into surrounding connective tissue and haemolymph spaces. Following systemic dissemination, the parasite infiltrates the digestive gland and becomes established as a nurse cell beneath the epithelial cells ill a digestive tubule. Here, cell-within-cell proliferation results in the eventual liberation of daughter cells from the nurse cell into spaces between adjacent epithelial cells. None of these stages had previously been described. Proliferation is associated with host responses, including haemocytic infiltration of the connective tissue and diapedesis across tubule epithelia. The responses cease as sporogenesis begins. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Molecular phylogenetics of Lentibulariaceae inferred from plastid rps16 Intron and trnL-F DNA sequences: implications for character evolution and biogeography

Phylogenetic relationships among 75 species of Lentibulariaceae, representing the three recognized genera, were assessed by cladistic analysis of DNA sequences from the plastid rps16 intron and the trnL-F region. Sequence data from the two loci were analyzed both separately and in combination. Consensus trees from all analyses are congruent, and parsimony jackknife results demonstrate strong support for relationships both between and within each of the three demonstrably monophyletic genera. The genus Pinguicula is sister to a Genlisea-Utricularia clade, the phylogenetic structure within this clade closely follows Taylor's recent sectional delimitations based on morphology. Three principal clades are shown within Utricularia, with the basal sections Polypoinpholyx and Pleiochasia together forming the sister lineage of the remaining Utricularia species. Of the fundamental morphological specializations, the stoloniferous growth form apparently arose independently within Genlisea and Utricularia three times, and within Utricularia itself, perhaps more than once. The epiphytic habit has evolved independently at least three times, in Pinguicula, in Utricularia section Phyllaria, and within the two sections Orchidioides and Iperua (in the latter as bromeliad tank-epiphytes). The suspended aquatic habit may have evolved independently within sections Utricularia and Vesiculina. Biogeographic optimization on the phylogeny demonstrates patterns commonly associated with the boreotropics hypothesis and limits the spatial origin of Lentibulariaceae to temperate Eurasia or tropical America.

Analysis of genetic diversity in Cassia brewsteri with randomly amplified DNA fingerprints (RAFS)

Genetic diversity in Cassia brewsteri (F. Muell.) F. Muell. ex Benth. was assessed with Randomly Amplified DNA Fingerprints (RAFs). Thirty accessions of C. brewsteri collected from throughout its natural distribution were analysed with three random decamer primers, along with three accessions of C. tomentella (Benth.) Domin and a single accession of each of C. queenslandica C. T. White and C. marksiana (F. M. Bailey) Domin. The three primers yielded a reproducible amplification profile of 265 scorable polymorphic fragments for the 35 accessions. These molecular markers were used to calculate Nei and Li similarity coefficients between each pair of individuals. A matrix of dissimilarity of each pair of individuals was examined by multidimensional scaling (MDS). The analysis supports the division of C. brewsteri into two subspecies and the suggestion that intergradation of C. brewsteri and C. tomentella can occur where the distributions of these species meet.

Evidence from mitochondrial DNA that head lice and body lice of humans (Phthiraptera : Pediculidae) are conspecific

The specific status of the head and body lice of humans has been debated for more than 200 yr. To clarify the specific status of head and body lice, we sequenced 524 base pairs (bp) of the cytochrome oxidase I (COI) gene of 28 head and 28 body lice from nine countries. Ten haplotypes that differed by 1-5 bp at II nucleotide positions were identified. A phylogeny of these sequences indicates that these head and body lice are not from reciprocally monophyletic lineages. Indeed, head and body lice share three of the 10 haplotypes we found. F-ST values and exact tests of haplotype frequencies showed significant differences between head and body lice. However, the same tests also showed significant differences among lice from different countries. Indeed, more of the variation in haplotype frequencies was explained by differences among lice from different countries than by differences between head and body lice. Our results indicate the following: (1) bead and body lice do not represent reciprocally monophyletic lineages and are conspecific; (2) gene flow among populations of lice from different countries is limited; and (3) frequencies of COI haplotypes can be used to study maternal gene flow among populations of head and body lice and thus transmission of lice among their human hosts.

DNA sequence variation in the ITS-1 rDNA subunit and host relationships in sorghum midge, Stenodiplosis sorghicola (Coquillett) (Diptera : Cecidomyiidae), in Australia

Sequence variation in the internal transcribed spacer (ITS-1) ribosomal DNA subunit was examined for sorghum midge obtained from introduced and native hosts in south-eastern and central Queensland. No variation was observed relative to host plant or geographical distance for midges collected from two introduced hosts, grain sorghum (Sorghum bicolor ) and Johnson grass (S. halepense ); however, sequence differences were observed between midges from introduced and native hosts and among midges from a single native host, slender bluegrass (Dichanthium affine ). No evidence was observed of introduced midges on native hosts, or vice versa. These results agree with previously hypothesised host distributions for native and introduced midges in Australia, and expand the sample of introduced hosts to include Johnson grass. They suggest that Stenodiplosis sorghicola , the principal midge infesting grain sorghum, is also the most common species on Johnson grass. This confirms that Johnson grass plays a role in the population dynamics of S. sorghicola and suggests that midges originating from Johnson grass may influence levels of infestation in grain sorghum.

Arsenic inhibits the repair of DNA damage induced by benzo(a)pyrene

In order to study the effect of arsenic on DNA damage, Sprague-Dawley rats were dosed with sodium arsenite (10 mg/kg) with or without 800 mug of benzo(a)pyrene (BP) by intramammilary injection. The animals were sacrificed on day 1, 3, 5, 10 and 27 and the mammary gland tissues were collected for DNA adduct measurement using a P-32 post-labeling assay. Animals dosed with arsenic alone did not show any DNA adducts. DNA adduct levels in rats dosed with BP alone reached a maximum level by day 5, reducing to 13% of this level by day 27. Adduct levels in rats dosed with arsenic and BP also reached a maximum by day 5 but only 80% of the level observed in the BP group. However, 84% of this amount still remained by day 27. The First Nucleotide Change (FNC) technique was used for the screening of 115 samples of various tissues from mice that had been chronically exposed to sodium arsenate for over 2 years revealed that inorganic arsenic did not attack the two putative hotspots (codons 131 and 154) of the hOGG1 gene. These results support the hypothesis that arsenic exerts its biological activity through DNA repair inhibition. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

In vivo DNA electrotransfer

The use of electrotransfer for DNA delivery to prokaryotic cells, and eukaryotic cells in vitro, has been well known and widely used for many years. However, it is only recently that electric fields have been used to enhance DNA transfer to animal cells in vivo, and this is known as DNA electrotransfer or in vivo DNA electroporation. Some of the advantages of this method of somatic cell gene transfer are that it is a simple method that can be used to transfer almost any DNA construct to animal cells and tissues in vivo; multiple constructs can be co-transfected; it is equally applicable to dividing and nondividing cells; the DNA of interest does not need to be subeloned into a specific viral transfer vector and there is no need for the production of high titre viral stocks; and, as no viral genes are expressed there is less chance of an adverse immunologic reaction to vector sequences. The ease with which efficient in vivo gene transfer can be achieved with in vivo DNA electrotransfer is now allowing genetic analysis to be applied to a number of classic animal model systems where transgenic and embryonic stem cell techniques are not well developed, but for which a wealth of detailed descriptive embryological information is available, or surgical manipulation is much more feasible. As well as exciting applications in developmental biology, in vivo DNA electrotransfer is also being used to transfer genes to skeletal muscle and drive expression of therapeutically active proteins, and to examine exogenous gene and protein function in normal adult cells situated within the complex environment of a tissue and organ system in vivo. Thus, in effect providing the in vivo equivalent of the in vitro transient transfection assay. As the widespread use of in vivo electroporation has really only just begun, it is likely that the future will hold many more applications for this technology in basic research, biotechnology and clinical research areas.

Bracken (Pteridium aquilinum)-induced DNA adducts in mouse tissues are different from the adduct induced by the activated form of the bracken carcinogen ptaquiloside

Following treatment with bracken fern (Pteridium aquilinum) extract and bracken spores a number of DNA adducts were detected by P-32-postlabeling. Three of these adducts have been described previously (Povey et al., Br. J. Cancer (1996) 74, 1342-1348) and in this study, using a slightly different protocol, four new adducts, with higher chromatographic mobility, were detected at levels ranging from 50 to 230% of those previously described, When DNA was treated in vitro with activated ptaquiloside (APT) and analysed by butanol extraction or nuclease P1 treatment, only one adduct was detected by P-32-postlabeling, This adduct was not present in the DNA from mice treated with bracken fern or spores, suggesting either that bracken contains genotoxins other than ptaquiloside or that the metabolism of ptaquiloside produces genotoxins not reflected by activated ptaquiloside. However, as the ATP-derived adduct has been detected previously in ileal DNA of bracken-fed calves, species-specific differences in the metabolism of bracken genotoxins may exist, thereby leading to differences in their biological outcomes. (C) 2001 Academic Press.

Speciation and distribution of the members of the Anopheles punctulatus (Diptera : Culicidae) group in Papua New Guinea

Mosquito collections were made throughout the mainland of Papua New Guinea to identify the members of the Anopheles punctulatus group present and to determine their distribution. Identification was made using morphology, DNA hybridization, and polymerase chain reaction (PCR)-RFLP analysis. Nine members of the group were identified: An. farauti s.s. Laveran, An.farauti 2, An. koliensis Owen, and An. punctulatus Donitz, were common and widespread; An. farauti 4 was restricted to the north of the central ranges where it was common; An. farauti 6 was found only in the highlands above 1,000 m; and An. farauti 3, An. sp. near punctulatus and An. clowi Rozeboom & Knight were uncommon and had restricted distributions. Identification of An. koliensis and An. punctulatus using proboscis morphology was found to be unreliable wherever An. farauti 4 occurred. The distribution and dispersal of the members of the An. punctulatus group is discussed in regard to climate, larval habitats, distance from the coast, elevation, and proximity to human habitation.

Colony-stimulating factor-1 suppresses responses to CpG DNA and expression of toll-like receptor 9 but enhances responses to lipopolysaccharide in murine macrophages

During bacterial infections, the balance between resolution of infection and development of sepsis is dependent upon the macrophage response to bacterial products. We show that priming of murine bone marrow-derived macrophages (BMMs) with CSF-1 differentially regulates the response to two such stimuli, LPS and immunostimulatory (CpG) DNA. CSF-1 pretreatment enhanced IL-6, IL-12, and TNF-alpha production in response to LPS but suppressed the same response to CpG DNA. CSF-1 also regulated cytokine gene expression in response to CpG DNA and LPS; CpG DNA-induced IL-12 p40, IL-12 p35, and TNF-alpha mRNAs were all suppressed by CSF-1 pretreatment. CSF-1 pretreatment enhanced LPS-induced IL-12 p40 mRNA but not TNF-alpha and IL-12 p35 mRNAs, suggesting that part of the priming effect is posttranscriptional. CSF-1 pretreatment also suppressed CpG DNA-induced nuclear translocation of NF-kappaB and phosphorylation of the mitogen-activated protein kinases p38 and extracellular signal-related kinases-1/2 in BMMs, indicating that early events in CpG DNA signaling were regulated by CSF-1. Expression of Toll-like receptor (TLR)9, which is necessary for responses to CpG DNA, was markedly suppressed by CSF-1 in both BMMs and thioglycolate-elicited peritoneal macrophages. CSF-1 also down-regulated expression of TLR1, TLR2, and TLR6, but not the LPS receptor, TLR4, or TLR5. Hence, CSF-1 may regulate host responses to pathogens through modulation of TLR expression. Furthermore, these results suggest that CSF-1 and CSF-1R antagonists may enhance the efficacy of CpG DNA in vivo.

Was there a second adaptive radiation of giant tortoises in the Indian Ocean? Using mitochondrial DNA to investigate speciation and biogeography of Aldabrachelys (Reptilia, Testudinidae)

A radiation of five species of giant tortoises (Cylindraspis ) existed in the southwest Indian Ocean, on the Mascarene islands, and another (of Aldabrachelys ) has been postulated on small islands north of Madagascar, from where at least eight nominal species have been named and up to five have been recently recognized. Of 37 specimens of Madagascan and small-island Aldabrachelys investigated by us, 23 yielded significant portions of a 428-base-pair (bp) fragment of mitochondrial (cytochrome b and tRNA-Glu), including type material of seven nominal species (A. arnoldi, A. dussumieri, A. hololissa, A. daudinii, A. sumierei, A. ponderosa and A. gouffei ). These and nearly all the remaining specimens, including 15 additional captive individuals sequenced previously, show little variation. Thirty-three exhibit no differences and the remainder diverge by only 1-4 bp (0.23-0.93%). This contrasts with more widely accepted tortoise species which show much greater inter- and intraspecific differences. The non-Madagascan material examined may therefore only represent a single species and all specimens may come from Aldabra where the common haplotype is known to occur. The present study provides no evidence against the Madagascan origin for Aldabra tortoises suggested by a previous molecular phylogenetic analysis, the direction of marine currents and phylogeography of other reptiles in the area. Ancient mitochondrial DNA from the extinct subfossil A. grandidieri of Madagascar differs at 25 sites (5.8%) from all other Aldabrachelys samples examined here.

Testing the link between the latitudinal gradient in species richness and rates of molecular evolution

Numerous hypotheses have been proposed to explain latitudinal gradients in species richness, but all are subject to ongoing debate. Here we examine Rohde's (1978, 1992) hypothesis, which proposes that climatic conditions at low latitudes lead to elevated rates of speciation. This hypothesis predicts that rates of molecular evolution should increase towards lower latitudes, but this prediction has never been tested. We discuss potential links between rates of molecular evolution and latitudinal diversity gradients, and present the first test of latitudinal variation in rates of molecular evolution. Using 45 phylogenetically independent, latitudinally separated pairs of bird species and higher taxa, we compare rates of evolution of two mitochondrial genes and DNA-DNA hybridization distances. We find no support for an effect of latitude on rate of molecular evolution. This result casts doubt on the generality of a key component of Rohde's hypothesis linking climate and speciation.

Biochemical and molecular studies using human autopsy brain tissue

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post-mortem. However, their abundance and integrity can exhibit marked intra- and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante- and post-mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.