Primary myiasis in dog caused by Phaenicia eximia (Diptera: Calliphoridae) and preliminary mitochondrial DNA analysis of the species in Brazil

A case of primary myiasis in a dog caused by Phaenicia eximia (Robineau-Desvoidy) in Brazil is presented. A young and healthy female dog, Canis familiaris, approximate to 10 d old and still under maternal care, was found to have several eggs and Is: instars larvae in its abdomen and urogenital regions. Samples were collected in Campinas, São Paulo, and transferred to the laboratory for rearing and identification. A comparative analysis of the mitochondrial DNA (mtDNA) with 12 restriction enzymes in 2 sampled populations of P. eximia collected in different hosts (live dog and bovine carcass) and in the same locality revealed that 4, EcoRI, EcoRV, HaeIII, and MspI were suitable for detecting mtDNA markers in the 2 populations.

Analysis of the mitochondrial DNA of Schizophrenia patients by next generation sequencing

Mitochondrial DNA (mtDNA), a maternally inherited 16.6-Kb molecule crucial for energy production, is implicated in numerous human traits and disorders. It has been hypothesized that the presence of mutations in the mtDNA may contribute to the complex genetic basis of schizophreniadisease, due to the evidence of maternal inheritance and the presence of schizophrenia symptoms in patients affected of a mitochondrial disorder related to a mtDNA mutation. The present project aims to study the association of variants of mitochondrial DNA (mtDNA), and an increased risk of schizophrenia in a cohort of patients and controls from the same population. The entire mtDNA of 55 schizophrenia patients with an apparent maternal transmission of the disease and 38 controls was sequenced by Next Generation Sequencing (Ion Torrent PGM, Life Technologies) and compared to the reference sequence. The current method for establishing mtDNA haplotypes is Sanger sequencing, which is laborious, timeconsuming, and expensive. With the emergence of Next Generation Sequencing technologies, this sequencing process can be much more quickly and cost-efficiently. We have identified 14 variants that have not been previously reported. Two of them were missense variants: MTATP6 p.V113M and MTND5 p.F334L ,and also three variants encoding rRNA and one variant encoding tRNA. Not significant differences have been found in the number of variants between the two groups. We found that the sequence alignment algorithm employed to align NGS reads played a significant role in the analysis of the data and the resulting mtDNA haplotypes. Further development of the bioinformatics analysis and annotation step would be desirable to facilitate the application of NGS in mtDNA analysis.

Phylogenetic analysis of 16S mitochondrial DNA data in sloths and anteaters

We sequenced part of the 16S rRNA mitochondrial gene in 17 extant taxa of Pilosa (sloths and anteaters) and used these sequences along with GenBank sequences of both extant and extinct sloths to perform phylogenetic analysis based on parsimony, maximum-likelihood and Bayesian methods. By increasing the taxa density for anteaters and sloths we were able to clarify some points of the Pilosa phylogenetic tree. Our mitochondrial 16S results show Bradypodidae as a monophyletic and robustly supported clade in all the analysis. However, the Pleistocene fossil Mylodon darwinii does not group significantly to either Bradypodidae or Megalonychidae which indicates that trichotomy best represents the relationship between the families Mylodontidae, Bradypodidae and Megalonychidae. Divergence times also allowed us to discuss the taxonomic status of Cyclopes and the three species of three-toed sloths, Bradypus tridactylus, Bradypus variegatus and Bradypus torquatus. In the Bradypodidae the split between Bradypus torquatus and the proto-Bradypus tridactylus / B. variegatus was estimated as about 7.7 million years ago (MYA), while in the Myrmecophagidae the first offshoot was Cyclopes at about 31.8 MYA followed by the split between Myrmecophaga and Tamandua at 12.9 MYA. We estimate the split between sloths and anteaters to have occurred at about 37 MYA.

Genetic variation and demographic history of the Haplochromis laparogramma group of Lake Victoria-An analysis based on SINEs and mitochondrial DNA

More than 500 endemic haplochromine cichlid species inhabit Lake Victoria. This striking species diversity is a classical example of recent explosive adaptive radiation thought to have happened within the last similar to 15,000 years. In this study, we examined the population structure and historical demography of 3 pelagic haplochromine cichlid species that resemble in morphology and have similar niche, Haplochromis (Yssichromis) laparogramma, Haplochromis (Y.) pyrrhocephalus, and Haplochromis (Y.) sp. "glaucocephalus". We investigated the sequences of the mitochondrial DNA control region and the insertion patterns of short interspersed elements (SINEs) of 759 individuals. We show that sympatric forms are genetically differentiated in 4 of 6 cases, but we also found apparent weakening of the genetic differentiation in areas with turbid water. We estimated the timings of population expansion and species divergence to coincide with the refilling of the lake at the Pleistocene/Holocene boundary. We also found that estimates can be altered significantly by the choice of the shape of the molecular clock. If we employ the nonlinear clock model of evolutionary rates in which the rates are higher towards the recent, the population expansion was dated at around the event of desiccation of the lake ca. 17,000 YBP. Thus, we succeeded in clarifying the species and population structure of closely related Lake Victoria cichlids and in showing the importance of applying appropriate clock calibrations in elucidating recent evolutionary events. (C) 2009 Elsevier B.V. All rights reserved.

Simultaneous analysis of nuclear and mitochondrial DNA, mRNA and miRNA from backspatter from inside parts of firearms generated by shots at "triple contrast" doped ballistic models

When a firearm projectile hits a biological target a spray of biological material (e.g., blood and tissue fragments) can be propelled from the entrance wound back towards the firearm. This phenomenon has become known as "backspatter" and if caused by contact shots or shots from short distances traces of backspatter may reach, consolidate on, and be recovered from, the inside surfaces of the firearm. Thus, a comprehensive investigation of firearm-related crimes must not only comprise of wound ballistic assessment but also backspatter analysis, and may even take into account potential correlations between these emergences. The aim of the present study was to evaluate and expand the applicability of the "triple contrast" method by probing its compatibility with forensic analysis of nuclear and mitochondrial DNA and the simultaneous investigation of co-extracted mRNA and miRNA from backspatter collected from internal components of different types of firearms after experimental shootings. We demonstrate that "triple contrast" stained biological samples collected from the inside surfaces of firearms are amenable to forensic co-analysis of DNA and RNA and permit sequence analysis of the entire mtDNA displacement-loop, even for "low template" DNA amounts that preclude standard short tandem repeat DNA analysis. Our findings underscore the "triple contrast" method's usefulness as a research tool in experimental forensic ballistics.

Analysis of microsatellite mutations in the mitochondrial DNA of Saccharomyces cerevisiae

In the nuclear genome of Saccharomyces cerevisiae, simple, repetitive DNA sequences (microsatellites) mutate at rates much higher than nonrepetitive sequences. Most of these mutations are deletions or additions of repeat units. The yeast mitochondrial genome also contains many microsatellites. To examine the stability of these sequences, we constructed a reporter gene (arg8m) containing out-of-frame insertions of either poly(AT) or poly(GT) tracts within the coding sequence. Yeast strains with this reporter gene inserted within the mitochondrial genome were constructed. Using these strains, we showed that poly(GT) tracts were considerably less stable than poly(AT) tracts and that alterations usually involved deletions rather than additions of repeat units. In contrast, in the nuclear genome, poly(GT) and poly(AT) tracts had similar stabilities, and alterations usually involved additions rather than deletions. Poly(GT) tracts were more stable in the mitochondria of diploid cells than in haploids. In addition, an msh1 mutation destabilized poly(GT) tracts in the mitochondrial genome.

PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.

Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit, we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays.

Conservation genetics of North Atlantic loggerhead sea turtles: analysis of nuclear and mitochondrial DNA

[EN] Complex population structure has been described for the loggerhead sea turtle (Caretta caretta), revealing lower levels of population genetic structure in nuclear compared to mitochondrial DNA assays. This may result from mating during spatially overlapping breeding migrations, or male-biased dispersal as previously found for the green turtle (Chelonia mydas). To further investigate these multiple possibilities, we carried out a comparative analysis from twelve newly developed microsatellite loci and the mitochondrial DNA control region (~804 bp) in adult females of the Cape Verde Islands (n=158), and Georgia, USA (n=17).

Phylogeography and historical demography of the neotropical stingless bee Melipona quadrifasciata (Hymenoptera, Apidae): incongruence between morphology and mitochondrial DNA

The stingless bees are among the most abundant and ecologically important social invertebrates in tropical communities. The Neotropical stingless bee Melipona quadrifasciata has two subspecies: M. quadrifasciata quadrifasciata and M. quadrifasciata anthidioides. The main difference between subspecies are the yellow metassomal stripes, which are continuous in M. q. quadrifasciata and discontinuous in M. q. anthidioides. Recently, two populations were described with continuous stripes and inhabiting clearly disjunct areas in relation to M. q. quadrifasciata. We sequenced 852 bp of the mtDNA COI gene from 145 colonies from 56 localities, and for the first time performed a detailed phylogeographic study of a neotropical stingless bee. Phylogenetic analyses revealed the existence of two clades exhibiting a south to north distribution: southern populations comprise the subspecies M. q. quadrifasciata, and northern populations are composed of M. q. anthidioides and two disjunct populations with continuous stripes. The divergence time of these two phylogroups was estimated between 0.233 and 0.840 million years ago in the Pleistocene, a period of climatic changes and geomorphological alterations in the Neotropical region. No evidence of genetic structure in relation to the tergal stripes was found, indicating that the morphological trait regarding the pattern of stripes on tergites is not an accurate diagnostic for the subspecies of M. quadrifasciata.

No relationship found between point heteroplasmy in mitochondrial DNA control region and age range, sex and haplogroup in human hairs

The analysis of heteroplasmy (presence of more than one type of mitochondrial DNA in an individual) is used as a tool in human identification studies, anthropology, and most currently in studies that relate heteroplasmy with longevity. The frequency of heteroplasmy and its correlation with age has been analyzed using different tissues such as blood, muscle, heart, bone and brain and in different regions of mitochondrial DNA, but this analysis had never been performed using hair samples. In this study, samples of hair were sequenced in order to ascertain whether the presence or not of heteroplasmy varied according to age, sex and origin of haplogroup individuals. The samples were grouped by age (3 groups), gender (male and female) and haplogroup of origin (European, African and Native American), and analyzed using the chi-square statistical test (chi(2)). Based in statistical results obtained, we conclude that there is no relationship between heteroplasmy and sex, age and haplogroup origin using hair samples.

Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples

Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C-stretch and CA repeat. To observe which CA ""alleles"" were present in each tissue, PCR products were cloned and re-sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.

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.