The potential role of scavenging flies as mechanical vectors of Lagovirus europaeus/GI.2.

The European rabbit (Oryctolagus cuniculus) populations of the Iberian Peninsula have been severely affected by the emergence of the rabbit haemorrhagic disease virus (RHDV) Lagovirus europaeus/GI.2 (RHDV2/b). Bushflies and blowflies (Muscidae and Calliphoridae families, respectively) are important RHDV vectors in Oceania, but their epidemiological role is unknown in the native range of the European rabbit. In this study, scavenging flies were collected between June 2018 and February 2019 in baited traps at one site in southern Portugal, alongside a longitudinal capture-mark-recapture study of a wild European rabbit population, aiming to provide evidence of mechanical transmission of GI.2 by flies. Fly abundance, particularly from Calliphoridae and Muscidae families, peaked in October 2018 and in February 2019. By employing molecular tools, we were able to detect the presence of GI.2 in flies belonging to the families Calliphoridae, Muscidae, Fanniidae and Drosophilidae. The positive samples were detected during an RHD outbreak and absent in samples collected when no evidence of viral circulation in the local rabbit population was found. We were able to sequence a short viral genomic fragment, confirming its identity as RHDV GI.2. The results suggest that scavenging flies may act as mechanical vectors of GI.2 in the native range of the southwestern Iberian subspecies O. cuniculus algirus. Future studies should better assess their potential in the epidemiology of RHD and as a tool for monitoring viral circulation in the field.

Small bait traps as accurate predictors of dipteran early colonizers in forensic studies.

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.

Cicadomorpha Community (Hemiptera: Auchenorrhyncha) in Portuguese Vineyards with Notes of Potential Vectors of Xylella fastidiosa.

Cicadomorpha (Hemiptera) insects are currently responsible for a growing negative impact on the agricultural economy due to their ability to directly damage crops or through the capacity to act as vectors for plant pathogens. The phytopathogenic bacterium Xylella fastidiosa, the causal agent of Pierce's disease in vineyards, is exclusively transmitted by insects of this infraorder. Therefore, knowledge of the Cicadomorpha species and understanding their biology and ecology is crucial. In this work, in 2018 and 2019, the canopy and inter-row vegetation of 35 vineyards distributed in mainland Portugal were sampled to investigate species composition, richness, and diversity of the Cicadomorpha community, with a special focus given to vectors and potential vectors of X. fastidiosa. A total of 11,834 individuals were collected, 3003 in 2018 and 8831 in 2019. Of the 81 species/morphospecies identified, only five are considered vectors or potential vectors of this pathogen, namely, Cicadella viridis (Linnaeus, 1758), Philaenus spumarius (Linnaeus, 1758), Neophilaenus campestris (Fallén, 1805), Lepyronia coleoptrata (Linnaeus, 1758), and N. lineatus (Linnaeus, 1758). Cicadella viridis was the most abundant xylem sap feeder, followed by P. spumarius. In addition, Cicadomorpha that cause direct damage to vines and vectors of grapevine yellows' phytoplasmas were also collected and identified in the sampled vineyards. The results suggested that vectors and potential vectors of X. fastidiosa and a large proportion of the population of Cicadomorpha have a positive correction with inter-row vegetation.

Strategies for Monitoring Microbial Life in Beach Sand for Protection of Public Health.

The 2021 revised guidelines of the World Health Organization recommend monitoring the quality of sand in addition to water at recreational beaches. This review provides background information about the types of beaches, the characteristics of sand, and the microbiological parameters that should be measured. Analytical approaches are described for quantifying fungi and fecal indicator bacteria from beach sand. The review addresses strategies to assess beach sand quality, monitoring approaches, sand remediation, and the proposed way forward for beach sand monitoring programs. In the proposed way forward, recommendations are provided for acceptable levels of fungi given their distribution in the environment. Additional recommendations include evaluating FIB distributions at beaches globally to assess acceptable ranges of FIB levels, similar to those proposed for fungi.

Everyday Life after the First Psychiatric Admission: A Portuguese Phenomenological Research.

Returning to daily life after psychiatric admission can be difficult and complex. We aimed to explore, describe and interpret the lived experience of returning to everyday life after the first psychiatric admission. We designed this research as a qualitative study, using van Manen's phenomenology of practice. We collected experiential material through phenomenological interviews with 12 participants, from 5 June 2018 to 18 December 2018. From the thematic and hermeneutic analysis, we captured seven themes: (1) (un)veiling the imprint within the self; (2) the haunting memories within the self; (3) from disconnection to the assimilation of the medicated body in the self; (4) from recognition to overcoming the fragility within the self; (5) the relationship with health professionals: from expectation to response; (6) the relationship with others: reformulating the bonds of alterity; (7) the relationship with the world: reconnecting as a sense of self. The results allow us to establish the phenomenon as a difficult, complex, demanding and lengthily transitional event that calls into question the person's stability and ability for well-being and more-being. Thus, implementing structured transitional interventions by health services seems crucial. Mental health specialist nurses can present a pivotal role in establishing a helping relationship with recovery-oriented goals, coordinating patients' transitional care, and assuring continuity of care sensitive to the person's subjective experiences, volitions, and resources.

Parasite island syndromes in the context of nidicolous ectoparasites: Fleas (Insecta: Siphonaptera) in wild passerine birds from Azores Archipelago.

Island syndrome, previously established for isolation process of insular vertebrates' populations, have been adapted to insular parasites communities, termed parasite island syndromes. In this work, were studied for the first time the insular syndromes for nidicolous ectoparasites of the bird species, Turdus merula, Sylvia atricapilla, Fringilla coelebs and Erithacus rubecula from Azores and the mainland Portugal. Flea species were only recorded on Azorean birds, namely Dasypsyllus gallinulae and Ctenocephalides felis felis, known as not host-specific parasites. In the absence of shared flea species between mainland and islands birds, a comparison among our fleas prevalence to Azores Islands and mainland fleas prevalence, recorded to others European studies, showed that Azorean host populations undergo higher prevalence than the mainland one. This result was consistent with parasite island syndromes predictions recorded to ectoparasites, hippoboscid flies and chewing lice, that fleas have higher prevalence on the Azores Islands compared to mainland Portugal. However, our results provide a new perspective to parasite island syndromes assumptions, namely in the context of nidicolous ectoparasites that spend only brief periods on the hosts' body.

Size does matter: intraspecific geometric morphometric analysis of wings of the blowfly Chrysomya albiceps (Diptera: Calliphoridae).

Blowflies have forensic, sanitary and veterinary importance, as well as being pollinators, parasitoids and ecological bioindicators. There is still little work with real data and from experiments assessing the relationship between blowflies' morphologic features and environmental and demographic factors. The present work tests whether the variation, in the shape and size, of Chrysomya albiceps (Wiedemann, 1819) wings is influenced by the following factors: 1) time; 2) temperature; 3) sex and; 4) different types of carcasses (pig, dog/cat and whale). Male and female wings from four different sites collected in six different years were used to obtain wing size and shape of C. albiceps. Analyses between wing shape and the variables tested had low explanatory power, even though they had statistical support. However, it was possible to identify differences in wing shape between males and females, with good returns in sex identification. The comparison between wing size and the variables tested showed that wing size has a negative relationship with temperature, significant differences between sexes, slight variation over time and no influence by carcass types. Furthermore, wing size influenced wing shape. Understanding population-specific characteristics of C. albiceps provide important insights about how the species reacts under specific conditions.

Geographic distribution of the V1016G knockdown resistance mutation in Aedes albopictus: a warning bell for Europe.

BACKGROUND: Colonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality < 70%) was recently reported in Ae. albopictus populations from Italy and Spain and associated with the V1016G point mutation in the voltage-sensitive sodium channel gene conferring knockdown resistance (kdr). Genotyping pyrethroid resistance-associated kdr mutations in field mosquito samples represents a powerful approach to detect early signs of resistance without the need for carrying out phenotypic bioassays which require availability of live mosquitoes, dedicated facilities and appropriate expertise. METHODS: Here we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries. RESULTS: The mutation was identified in 12 sites from nine countries (with allele frequencies ranging from 1 to 8%), mostly distributed in two geographical clusters. The western cluster includes Mediterranean coastal sites from Italy, France and Malta as well as single sites from both Spain and Switzerland. The eastern cluster includes sites on both sides of the Black Sea in Bulgaria, Turkey and Georgia as well as one site from Romania. These results are consistent with genomic data showing high connectivity and close genetic relationship among West European populations and a major barrier to gene flow between West European and Balkan populations. CONCLUSIONS: The results of this first effort to map kdr mutations in Ae. albopictus on a continental scale show a widespread presence of the V1016G allele in Europe, although at lower frequencies than those previously reported from Italy. This represents a wake-up call for mosquito surveillance programs in Europe to include PCR-genotyping of pyrethroid resistance alleles, as well as phenotypic resistance assessments, in their routine activities.

Louse flies in Azorean and mainland populations of four Passeriformes species: A new perspective to parasite Island syndromes.

Hippoboscid flies, also known as louse flies, are obligate blood-feeders ectoparasites of birds and mammals. By studying louse fly parasites of four Passeriformes species, Eurasian blackbird (Turdus merula), Eurasian blackcap (Sylvia atricapilla), common chaffinch (Fringilla coelebs) and European robin (Erithacus rubecula), with dissimilar time of colonization of Azores islands, we tested whether: (i) island host populations have lower parasite richness than the mainland one; (ii) island host populations undergo higher parasite prevalence, mean intensities and mean abundance than the mainland one; (iii) island parasite diversity are composed exclusively by specific parasites and (iv) parasite richness is positively correlated with the island area and proximity to the continent. For these purposes, 775 birds were sampled for presence of louse flies, by modified fumigation chamber method, from Azores Islands (São Miguel, Terceira and Flores) and Portugal mainland. Insular and mainland parasite assemblages were statistically compared. We record for the first time to Azores, Ornithomya fringillina and Icosta minor from Portugal mainland. Louse flies had highest prevalence and abundance from Azores Islands compared to those observed in mainland birds, especially blackbirds. The insular parasite diversity of Azores blackbirds, blackcaps and chaffinches was richer than the one observed in mainland population. None of the hippoboscid flies observed on the islands and mainland were host-specific. Thus, our findings provide an upgrade of parasite island syndromes knowledge, in the context of the ectoparasites, namely to the hippoboscid flies case.

Population structure, adaptation and divergence of the meadow spittlebug, Philaenus spumarius (Hemiptera, Aphrophoridae), revealed by genomic and morphological data.

Understanding patterns of population differentiation and gene flow in insect vectors of plant diseases is crucial for the implementation of management programs of disease. We investigated morphological and genome-wide variation across the distribution range of the spittlebug Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Auchenorrhyncha, Aphrophoridae), presently the most important vector of the plant pathogenic bacterium Xylella fastidiosa Wells et al., 1987 in Europe. We found genome-wide divergence between P. spumarius and a very closely related species, P. tesselatus Melichar, 1899, at RAD sequencing markers. The two species may be identified by the morphology of male genitalia but are not differentiated at mitochondrial COI, making DNA barcoding with this gene ineffective. This highlights the importance of using integrative approaches in taxonomy. We detected admixture between P. tesselatus from Morocco and P. spumarius from the Iberian Peninsula, suggesting gene-flow between them. Within P. spumarius, we found a pattern of isolation-by-distance in European populations, likely acting alongside other factors restricting gene flow. Varying levels of co-occurrence of different lineages, showing heterogeneous levels of admixture, suggest other isolation mechanisms. The transatlantic populations of North America and Azores were genetically closer to the British population analyzed here, suggesting an origin from North-Western Europe, as already detected with mitochondrial DNA. Nevertheless, these may have been produced through different colonization events. We detected SNPs with signatures of positive selection associated with environmental variables, especially related to extremes and range variation in temperature and precipitation. The population genomics approach provided new insights into the patterns of divergence, gene flow and adaptation in these spittlebugs and led to several hypotheses that require further local investigation.

An Overview of Zoonotic Disease Outbreaks and its Forensic Management Over Time.

Most emerging or re-emerging infections are vector-borne or zoonotic and can be disseminated worldwide by infected humans or animals. They are a major public health problem and cause a great impact on economy. Zoonotic outbreaks began to be characterized in the 90s, after the creation of Europol and the FBI. Such investigations are carried by forensic pathologists and other specialists to determine whether an outbreak is natural or deliberate. This review will discuss ten zoonotic outbreaks nonrelated to wars focusing on forensic management. In conclusion, some points should be highlighted in the management of a zoonotic outbreak: (i) its diagnosis and detection by forensic pathologists and the coordination of efforts between other specialists are key factors; (ii) communication guidelines and an efficient healthcare system are crucial for any emergency response; (iii) biosafety of all specialists involved must be guaranteed.

Genomic signatures of introgression between commercial and native bumblebees, Bombus terrestris, in western Iberian Peninsula-Implications for conservation and trade regulation.

Human-mediated introductions of species may have profound impacts on native ecosystems. One potential impact with largely unforeseen consequences is the potential admixture of introduced with autochthonous species through hybridization. Throughout the world, bumblebees have been deliberately introduced for crop pollination with known negative impacts on native pollinators. Given the likely allochthonous origin of commercial bumblebees used in Portugal (subspecies Bombus terrestris terrestris and B. t. dalmatinus), our aim was to assess their putative introgression with the native Iberian subspecies B. terrestris lusitanicus. We analysed one mitochondrial gene, cytochrome c oxidase subunit I (COX1) and genomic data involving thousands of genome-wide restriction-site-associated DNA markers (RAD-seq). In the mitochondrial COX1 analyses, we detected one relatively common haplotype in commercial bumblebees, also present in wild samples collected nearby the greenhouses where the commercial hives are used. In the RAD-seq analysis, we found a clear genetic differentiation between native and commercial lineages. Furthermore, we detected candidate hybrids in the wild, as well as putatively escaped commercial bumblebees, some of which being potentially fertile males. Although we cannot assess directly the fitness effects of introgressed alleles, there is a risk of maladaptive allele introgression to the local bumblebee subspecies, which can negatively impact autochthon populations. One immediate recommendation to farmers is for the proper disposal of hive boxes, after their use in greenhouses, so as to minimize the risk of escapees contaminating native populations. On the other hand, the feasibility of using local subspecies B. t. lusitanicus, preferably with local production, should be evaluated.

The Asian tiger mosquito, Aedes (Stegomyia) albopictus (Skuse), a vector of dengue, chikungunya and zika viruses, reaches Portugal (Diptera: Culicidae).

The Asian tiger mosquito, Aedes (Stegomyia) albopictus (Skuse), is a tropical species originally from south-eastern Asia (Hawley, 1988). It has experienced a rampant human-mediated range expansion since the 1970s to now occupy almost the whole of the tropical and subtropical areas of the world. As an eclectic haematophagous species, it attacks humans and is able to use a number of man-made and natural structures where stagnant water is present (Estrada-Franco Craig, 1995). Eggs are able to survive for extended periods of time in complete dryness and diapause over unsuitable cold season, making it an especially resilient species (Hawley, 1988).

Occurrence of helminth parasites in the gastrointestinal tract of wild birds from Wildlife Rehabilitation and Investigation Centre of Ria Formosa in southern Portugal.

This study was carried out in southern Portugal to determine the prevalence of parasitic helminths infecting wild birds. Between September and December of 2013, adult parasites were collected from the gastrointestinal tract of 22 bird specimens (belonging to 12 species) that died in Wildlife Rehabilitation and Investigation Centre of the Ria Formosa. Identified gastrointestinal parasites include the nematodes (Contracaecum sp., Cosmocephalus obvelatus, Desportesius invaginatus, Dispharynx nasuta, Porrocaecum angusticolle and Synhimantus laticeps) and cestodes (Diplophallus sp., Neyraia intricata and Tetrabothrius sp.). The overall level of infection was 54.5% (12/22) and the most frequent helminths present were nematodes (40.9% - 9/22), followed by cestodes (13.6% - 3/22). Helminthic richness was similar in all birds (one species or genera per bird) and the helminth species, except Dispharynx nasuta, exhibited a clear relationship with host diet. Five helminth species (Cosmocephalus obvelatus, Desportesius invaginatus, Dispharynx nasuta, Porrocaecum angusticolle and Neyraia intricata) and two genera (Contracaecum sp. and Diplophallus sp.) were reported for the first time in Portugal and the presence of P. angusticolle in Bonelli's eagle was recorded for the first time across all researched literature.

Chewing lice (Phthiraptera) from wild birds in southern Portugal.

This study was carried out to determine chewing louse species of wild birds in the Ria Formosa Natural Park, located in southern Portugal. In addition, the hypothesis that bird age, avian migration and social behaviour have an impact on the louse prevalence was tested. Between September and December of 2013, 122 birds (belonging to 10 orders, 19 families, 31 genera and 35 species) captured in scientific ringing sessions and admitted to the Wildlife Rehabilitation and Investigation Centre of Ria Formosa were examined for lice. Twenty-six (21.3%) birds were found to be infested with at least one chewing louse species. The chewing lice identified include 18 species. Colonial birds (34.9%) and migratory birds (29.5%) had statistically significant higher prevalence than territorial birds (6.8%) and resident birds (13.1%), respectively. This paper records 17 louse species for the first time in southern Portugal: Laemobothrion maximum, Laemobothrion vulturis, Actornithophilus piceus lari, Actornithophilus umbrinus, Austromenopon lutescens, Colpocephalum heterosoma, Colpocephalum turbinatum, Eidmanniella pustulosa, Nosopon casteli, Pectinopygus bassani, Pseudomenopon pilosum, Trinoton femoratum, Trinoton querquedulae, Craspedorrhynchus platystomus, Degeeriella fulva, Falcolipeurus quadripustulatus, Lunaceps schismatus. Also a nymph of the genus Strigiphilus was collected from a Eurasian eagle-owl. These findings contribute to the knowledge of avian chewing lice from important birds areas in Portugal.

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.

Molecular evidence of polyandry in the citrus mealybug, Planococcus citri (Hemiptera: Pseudococcidae).

The occurrence of polyandry in Planococcuscitri, presumed by earlier observations of mating behavior, was confirmed using microsatellite genotyping of pools of over 400 eggs resulting from controlled crosses of one female with two males. The genetic contribution of both mated males was confirmed in 13 out of 43 crosses. In three crosses it was possible to determine that only the first male fertilized the eggs, which may be due to sperm competition or unviable sperm supply. The microsatellite analysis also allowed the confirmation of aspects of the chromosomal inheritance detected previously in cytogenetic studies in Planococcuscitri, namely that only one of the alleles is transmitted by the male, indicating that the males are functionally haploid, supporting the observation of Paternal Genome Elimination (PGE) in these insects.