Deodália Dias

Identification of sarcosaprophagous Diptera species through DNA barcoding in wildlife forensics

In recent years, forensic entomology has been applied in wildlife crimes, such as neglect cases, animal cruelty and illegal poaching. Likewise in human death investigations, in which insects can help to provide information about postmortem interval (PMI) and corpse transfer, entomology may be an important source of information in animal murder suspicion. The use of insects in forensic context relies primarily on its identification at the species level. To overcome some problems of morphological determination, molecular identification has gained relevance and has been applied frequently in forensic areas. Cytochrome c oxidase I (COI) gene was adopted in DNA barcoding approach. This methodology intends to unify the DNA-based identification using a specific region of mitochondrial DNA. COI sequences have been collected into the BOLD online database, allowing the molecular identification of sequences from unknown specimens. Nonetheless, to achieve a correct identification of an unknown sample, it is necessary that sequences from species under study exist, for comparison, in online databases. Due to the geographic differences, it is of huge importance to have samples from a certain species from its distribution range. In that sense, the aim of this research is to contribute to the potential and accuracy improvement of such databases in identification of species commonly found in wildlife carcasses. A portion of COI was sequenced from 95 specimens of seven species belonging to two families of Diptera (Calliphoridae and Muscidae) found in wildlife carcasses-baited traps in Serra da Estrela (Portugal). All specimens were identified at species level with a high specimen similarity and maximum identity percentage (through BOLD Systems and GenBank online databases, respectively). We also demonstrate the correct discrimination of all species through phylogenic and sequence divergence analyses proposed in DNA barcoding studies, reinforcing the suitability of this marker.

Small Bait Traps as Accurate Predictors of Dipteran Early Colonizers in Forensic Studies

Abstract Insect carrion communities vary among habitats and over time. Concerning the dipteran early colonizers of carrion, the use of small bait traps should be accurate because the odors emitted from meat baits should contain many of the volatile organic compounds emitted from the freshly dead mammals. In addition, this kind of trap is easy to replicate and set in position in a given habitat. In the present study, small bait preferences of early Diptera carrion colonizers were examined in an urban biotope. Specifically, three baits were compared (pork muscle, pork liver, and fish flavored cat food) in respect to the number of specimens and species captured and the presence or absence of oviposition at high and low environmental temperatures. A total of 2371 specimens were trapped, primarily belonging to three insect orders, Diptera, Coleoptera, and Hymenoptera. Diptera was the predominant order, with blowflies (Calliphoridae) being the most representative family, followed by filth flies (Muscidae). The pork muscle bait was responsible for the highest number of captures and the highest diversity. The community of Diptera collected with the most efficient bait, pork muscle, was compared with the carrion communities reported in the literature from the Iberian Peninsula. Similar taxonomic species composition was found regarding Calliphoridae species. A specimen from all species morphologically identified were also identified at a molecular level using the cytochrome c oxidase I (COI) barcode region, and the sequences were submitted to online databases.

Population data of the GlobalFiler® Express loci in South Portuguese population

Allele frequencies and other relevant forensic parameters for 21 loci studied with GlobalFiler(®) Express amplification kit (Life Technologies) were calculated in a population of individuals residing in the south of Portugal. Blood stain samples were obtained from a total of 502 unrelated individuals involved in paternity testing casework and directly PCR amplified with GlobalFiler(®) Express following manufacturers instructions. This kit comprises all the loci included in the extended European Standard Set (ESS) and in the Combined DNA Index System (CODIS), besides the very polymorphic D2S441, D19S433, and SE33. In our laboratory this is used as a screening tool to solve complex cases, as fatherless paternity tests or to help in paternity investigations where there is the need to study additional genetic markers. These studies are necessary to calculate statistical forensic parameters, such as power of discrimination or as power of exclusion. Statistical parameters including heterozigosity, homozigosity and combined power of exclusion were estimated.

Mitochondrial DNA analysis of Rhipicephalus sanguineus s.l. from the western Iberian peninsula

Rhipicephalus sanguineus Latreille (1806) (Ixodida: Ixodidae) is considered to be the most widely distributed tick and to have a vast range of habitats and hosts, including livestock, pets and wildlife. In addition to morphological differences, recent investigations using approaches based on molecular genetic markers have revealed the existence of different R. sanguineus lineages in different geographic regions. In this study, 475 ticks collected from dogs in the western Iberian peninsula were studied both morphologically and genetically, using 12S and 16S rDNA and COI gene markers in order to clarify the controversy over the systematic status of R. sanguineus sensu lato in Western Europe, and to compare the present data with those sourced from studies conducted in other regions of the world. Despite the high morphometric variability, particularly on spiracles in both genders and in female genitalia, data obtained with different genetic molecular markers show very low variability, suggesting the existence of a unique species. In addition, the phylogenetic analysis showed genetic uniformity, supporting the existence of a well‐defined clade consisting of R. sanguineus s.l. specimens from Western Europe that are distinct from R. sanguineus s.l. from Africa. Furthermore, these data corroborate the existence of a polymorphic species in Western Europe, which requires to be consensually redescribed in view of its medical and veterinary importance in pathogen transmission.

Genome-wide SNP data unveils the globalization of domesticated pigs

AbstractBackgroundPigs were domesticated independently in Eastern and Western Eurasia early during the agricultural revolution, and have since been transported and traded across the globe. Here, we present a worldwide survey on 60K genome-wide single nucleotide polymorphism (SNP) data for 2093 pigs, including 1839 domestic pigs representing 122 local and commercial breeds, 215 wild boars, and 39 out-group suids, from Asia, Europe, America, Oceania and Africa. The aim of this study was to infer global patterns in pig domestication and diversity related to demography, migration, and selection.ResultsA deep phylogeographic division reflects the dichotomy between early domestication centers. In the core Eastern and Western domestication regions, Chinese pigs show differentiation between breeds due to geographic isolation, whereas this is less pronounced in European pigs. The inferred European origin of pigs in the Americas, Africa, and Australia reflects European expansion during the sixteenth to nineteenth centuries. Human-mediated introgression, which is due, in particular, to importing Chinese pigs into the UK during the eighteenth and nineteenth centuries, played an important role in the formation of modern pig breeds. Inbreeding levels vary markedly between populations, from almost no runs of homozygosity (ROH) in a number of Asian wild boar populations, to up to 20% of the genome covered by ROH in a number of Southern European breeds. Commercial populations show moderate ROH statistics. For domesticated pigs and wild boars in Asia and Europe, we identified highly differentiated loci that include candidate genes related to muscle and body development, central nervous system, reproduction, and energy balance, which are putatively under artificial selection.ConclusionsKey events related to domestication, dispersal, and mixing of pigs from different regions are reflected in the 60K SNP data, including the globalization that has recently become full circle since Chinese pig breeders in the past decades started selecting Western breeds to improve local Chinese pigs. Furthermore, signatures of ongoing and past selection, acting at different times and on different genetic backgrounds, enhance our insight in the mechanism of domestication and selection. The global diversity statistics presented here highlight concerns for maintaining agrodiversity, but also provide a necessary framework for directing genetic conservation.

Phylogenetic origin of the endemic pigeons from Madeira (Columba trocaz) and Azores Islands (Columba palumbus azorica)

AbstractThis study addresses for the first time the issue of pigeon (genus Columba) phylogeny within the archipelagos of Madeira (Columba trocaz) and Azores (C. palumbus azorica), located in the singular biogeographic area of Macaronesia. The phylogeny of these endemic pigeons was inferred based on mitochondrial (cytochrome b and cytochrome c oxidase I) and nuclear (β-fibrinogen intron 7) genetic markers through multiple approaches. Despite the non-monophyletic pattern for the insular endemic species recovered in the phylogenies, topology tests presented somewhat different results. C. trocaz, the Madeiran endemic species, clustered strongly with the Canarian endemic C. bollii, and these two are thus more closely related to each other than C. bollii to C. junoniae, the other endemic species of Canary Islands, which seems to have diverged independently. Moreover, C. trocaz was found to be phylogenetically closer to C. bollii than to C. palumbus from mainland Europe and Azores Islands. No genetic differentiation was found between the continental C. p. palumbus and the endemic C. p. azorica, which suggests a relatively recent colonisation event of the Azores Islands.ZusammenfassungPhylogenetische Ursprünge endemischer Taubenarten auf Madeira (Columba trocaz) und den Azoren (Columba palumbus azorica) Diese Studie untersucht erstmalig phylogenetische Fragen bei Tauben (Gattung Columba) auf den Inselgruppen Madeira (Columba trocaz) und Azoren (C. palumbus azorica) in der biogeografischen Region Makaronesien. Die Phylogenie dieser endemischen Taubenarten wurde mittels multipler Ansätze auf der Grundlage mitochondrialer (Cytochrom b und Cytochrom-c-Oxidase I) und nukleärer (β-Fibrinogen Intron 7) genetischer Marker rekonstruiert. Obwohl sich in den Phylogenien ein nicht-monophyletisches Muster für die endemischen Inselarten zeigte, führten die Topologie-Tests zu etwas abweichenden Ergebnissen. C. trocaz, die für Madeira endemische Art, war eng mit dem kanarischen Endemiten C. bollii gruppiert; somit sind diese beiden näher miteinander verwandt als C. bollii und C. junoniae, der anderen endemischen Art der Kanaren, welche sich unabhängig davon entwickelt zu haben scheint. Außerdem zeigte sich, dass C. trocaz phylogenetisch C. bollii näher steht als C. palumbus des Europäischen Festlandes und der Azoren. Es gab keine genetische Differenzierung zwischen der Festlandform C. p. palumbus und dem endemischen Taxon C. p. azorica, was für eine erst relativ kürzlich erfolgte Besiedlung der Azoren spricht.

Phylogenetic insights on Mediterranean and Afrotropical Rhipicephalus species (Acari: Ixodida) based on mitochondrial DNA

A multigene phylogeny including 24 Rhipicephalus species from the Afrotropical and Mediterranean regions, based on mitochondrial DNA genes (COI, 12S and 16S), was constructed based on Bayesian inference and maximum likelihood estimations. The phylogenetic reconstruction revealed 31 Rhipicephalus clades, which include the first molecular records of Rhipicephalus duttoni (Neumann), and Rhipicephalussenegalensis (Koch). Our results support the R. pulchellus, R. evertsi and R. pravus complexes as more phylogenetically close to Rhipicephalus (Boophilus) than to the remaining Rhipicephalus clades, suggesting two main monophyletic groups within the genus. Additionally, the phenotypic resembling R. sanguineus s.l. and Rhipicephalusturanicus (Pomerantsev) are here represented by nine clades, of which none of the R. turanicus assemblages appeared as distributed in the Iberian Peninsula. These results not only indicate that both species include more cryptic diversity than the already reported, but also suggest that R. turanicus distribution is less extended than previously anticipated. This analysis allowed to improve species identification by exposing cryptic species and reinforced mtDNA markers suitability for intra/inter-species clarification analyses. Incorporating new species molecular records to improve phylogenetic clarification can significantly improve ticks’ identification methods which will have epidemiologic implications on public health.

Geno- and cyto-toxicity in free-living rodent Mus spretus exposed to simulated onshore oil spill.

AbstractThis study investigated geno- and cyto-toxic damage in the free-living rodent, Mus spretus after exposure to a simulated spill of crude oil on soil. The results revealed increased mutagenicity and cytotoxicity in bone marrow cells and increased DNA damage in blood cells. Exposure to crude oil increased sperm abnormalities, with lasso-like folds being the most common. These results point to the value of this rodent in serving as a sentinel species for the monitoring and prediction of environmental hazards.

Autosomal SNPs study of a population sample from Southern Portugal and from a sample of immigrants from Guinea-Bissau residing in Portugal

In recent years, autosomal single nucleotide polymorphisms (SNPs) have been comprehensively investigated in forensic research due to their usefulness in certain circumstances in complementing short tandem repeats (STRs) analysis, or even for use on their own when analysis of STRs fails. However, as with STRs, in order to properly use SNP markers in forensic casuistic we need to understand the population and forensic parameters in question. As a result of Portugals colonial history during the time of empire, and the subsequent process of decolonization, some African individuals migrated to Portugal, giving rise to large African and African-descendent communities. One of these groups is the community originating from Guinea-Bissau, that in 2014, was enumerated to consist of more than 17,700 individuals with official residency status, more than the third major city of Guinea-Bissau. In order to study the population and forensic parameters mentioned above for the two populations important to our casuistic, a total of 142 unrelated individuals from the South of Portugal and 90 immigrants from Guinea-Bissau (equally non related and all residing in Portugal) were typed with SNaPshot™ assay for all 52 loci included in the SNPforID 52plex.

Fine structure comparison of eggshell from Lucilia sericata and Calliphora vicina, Calliphoridae with forensic importance

In forensic investigations, eggs of blowflies are very commonly found and can become essential for a proper identification of sarcosaprophagous Diptera. However, most of the time they cannot be used as entomological evidence since they are morphologically very similar, have not been studied in detail, and there are no references to distinguish among them in some geographical areas as the Iberian Peninsula, with exception of Calliphora vicina [1]. This study compares the ultrastructural morphology of eggs from Lucilia sericata (Meigen, 1826) and Calliphora vicina Robineau-Desvoidy, 1830, two cryptic species of blowflies frequently found in forensic context in Portugal, through SEM (JEOL JSM 5200 LV). Fourty eggs for each species were analyzed. The use of this technique to differentiate calliphorid-fly eggs, although not widely used, has also been employed by others [2-11]. In these studies, the main structures used to discriminate species are the micropyle apparatus, the plastron and the chorion ornamental arrangements. Our results show that it is possible to distinguish L. sericata from C. vicina based on SEM images through the use of qualitative criteria, as the plastron termination near the micropyle apparatus (Figure 1) and the distinctive aspect of the anastomosis in the islands located at the median area of the plastron (Figure 2), among others. The distinctive features of C. vicina are identical to those described in other studies [1,3,7]. Our structural results for L. sericata are the first ones ever recorded. The morphological identification of insect eggs is very laborious and difficult, and sometimes impossible to be achieved. The employment of SEM allowed to distinguishing between the eggs of Lucilia sericata and Calliphora vicina. It is therefore considered its application in further studies on morphological structures of eggs, as this technique could be used as an important tool in forensic entomology by providing useful data for additional comparisons of other cryptic sarcosaprophagous species.