Ana Rita Oliveira

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.

Planning Vascular Access in Peritoneal Dialysis—Defining High-Risk Patients

Background: Peritoneal dialysis (PD) is an effective renal replacement technique. However, every year a considerable number of patients are transferred to hemodialysis (HD). Our aim was to identify those at risk, in order to place an arteriovenous fistula (AVF). Methods: Case-control study enrolling all prevalent patients in 2014 and 2015 in our clinic. Groups: 72 case patients who were transferred definitively to HD, 111 control patients (remaining on PD, transplanted, recovered renal function, or deceased). Results: A total of 183 patients were eligible, with a mean age of 55.2 ± 14.8 years, 56.3% male, 31.1% diabetic, and 49.7% on continuous ambulatory PD. The mean follow-up time was 42.1 ± 25.6 months. Eighty-five patients had an AVF. The groups differed in diabetic nephropathy etiology, and in some PD-related characteristics (Kt/V, creatinine clearance, residual renal function, mean ultrafiltration, natriuretic peptide, peritonitis, hospitalizations, and hypervolemia). In multivariate analysis, Kt/V < 1.7 (odds ratio [OR] 3.00, 95% confidence interval [CI]: 1.20 – 7.50], albumin < 35 g/L (OR 4.03, 95% CI: 1.26 – 12.92), number of hospitalizations 1 to 3 (OR 2.74, 95% CI: 1.15 – 6.53) and 4 or more (OR 10.48, 95% CI: 3.62 – 30.36), and 2 or more peritonitis episodes (OR 2.50, 95% CI: 1.03 – 6.07) were predictors of PD transfer to HD. In those patients who were transferred to HD, 34 initiated HD by AVF, 2 needed a catheter due to a non-functioning AVF, and 36 did not have an AVF needing catheter placement. Conclusions: Low Kt/V, low albumin, higher number of hospitalizations, and peritonitis were factors associated with PD transfer to HD, probably indicative of a high-risk PD population where arteriovenous access should be weighed.

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.

Development of a Musculotendon Model Within the Framework of Multibody Systems Dynamics

Human movement is the result of a complex and synergistic interaction between the musculoskeletal and the central nervous system. As result, muscles contract coordinately to produce forces that are transmitted by tendons to the skeletal system, causing its movement or keeping its pose. Often neglected in current muscle models, the elastic properties of tendons play a significant role in the dynamic interaction between the muscular and skeletal systems, influencing the force transmission, energy storage and transfer, and joint control. The aim of this work is to present in detail the necessary steps to incorporate a musculotendon model in the framework of a multibody systems dynamics formulation. A methodology to compute the musculotendon forces and activations is presented based on the use of a Hill-type muscle model assembled in series with a spring-like element defined according to the elastic properties of the tendon. The proposed methodology can be applied, without significant changes, to both inverse and forward dynamic analyses of biomechanical systems. Three daily activities with different levels of musculotendon recruitment are analyzed from an inverse dynamics perspective. The selected activities are walking, running and jumping. The movement data characterizing these activities were acquired experimentally in a movement laboratory. A 3D biomechanical model of the human body, described with natural coordinates and encompassing 43 musculotendon actuators per leg, is proposed to assess the performance of the presented musculotendon model and of its incorporation on the referred multibody dynamics framework. The influence of the introduction of a compliant tendon model on the produced muscle forces and activation patterns is analyzed in face of those same results produced by the same biomechanical model defined with infinitely stiff (or rigid) tendons. Results revealed that the introduction of the tendon model allows muscles to work, predominantly, on their optimal configuration as the dynamic equilibrium generated between muscle and tendon prevents the muscle from support all musculotendon deformation. This not only reduces the activations needed to perform the required contractile forces but it also considerably prevents the development of non-physiological passive forces.

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.