BackgroundSerological surveys for SARS-CoV-2 were used early in the COVID-19 pandemic to assess epidemiological scenarios. In the municipality of Cascais (Portugal), serological testing combined with a comprehensive socio-demographic, clinical and behavioral questionnaire was offered to residents between May 2020 and beginning of 2021. In this study, we analyze the factors associated with adherence to this municipal initiative, as well as the sociodemographic profile and chronic diseases clinical correlates associated to seropositivity. We aim to contribute with relevant information for future pandemic preparedness efforts.MethodsThis was a cross-sectional study with non-probabilistic sampling. Citizens residing in Cascais Municipality went voluntarily to blood collection centers to participate in the serological survey. The proportion of participants, stratified by socio-demographic variables, was compared to the census proportions to identify the groups with lower levels of adherence to the survey. Univariate and multivariate logistic regression were used to identify socio-demographic, clinical and behavioral factors associated with seropositivity.ResultsFrom May 2020 to February 2021, 19,608 participants (9.2% of the residents of Cascais) were included in the study. Based on the comparison to census data, groups with lower adherence to this survey were men, the youngest and the oldest age groups, individuals with lower levels of education and unemployed/inactive. Significant predictors of a reactive (positive) serological test were younger age, being employed or a student, and living in larger households. Individuals with chronic diseases generally showed lower seroprevalence.ConclusionThe groups with low adherence to this voluntary study, as well as the socio-economic contexts identified as more at risk of viral transmission, may be targeted in future pandemic situations. We also found that the individuals with chronic diseases, perceiving higher risk of serious illness, adopted protective behaviors that limited infection rates, revealing that health education on preventive measures was effective for these patients.

Mosquito-borne diseases kill millions of people each year. Therefore, many innovative research and population control strategies are being implemented but, most of them require large-scale production of mosquitoes. Mosquito rearing depends on fresh blood from human donors, experimentation animals or slaughterhouses, which constitutes a strong drawback since high blood quantities are needed, raising ethical and financial constraints. To eliminate blood dependency and the use of experimentation animals, we previously developed BLOODless, a patented diet that represents an important advance towards sustainable mosquito breeding in captivity. BLOODless diet was used to maintain a colony of Anopheles stephensi for 40 generations. Bloodmeal appetite, fitness, Plasmodium berghei infectivity, whole genome sequencing and microbiota were evaluated over time. Here we show that BLOODless can be implemented in Anopheles insectaries since it allows long-term rearing of mosquitoes in captivity, without a detectable effect on their fitness, infectivity, nor on their midgut and salivary microbiota composition.

Background: The global scale-up of antiretroviral treatment (ART) offers significant health benefits by suppressing HIV-1 replication and increasing CD4 cell counts. However, incomplete viral suppression poses a potential threat for the emergence of drug resistance mutations (DRMs), limiting ART options, and increasing HIV transmission. Objective: We investigated the patterns of transmitted drug resistance (TDR) and acquired drug resistance (ADR) among HIV-1 patients in Portugal. Methods: Data were obtained from 1050 HIV-1 patient samples submitted for HIV drug resistance (HIVDR) testing from January 2022 to June 2023. Evaluation of DRM affecting viral susceptibility to nucleoside/tide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and integrase strand transfer inhibitors (INSTIs) was performed using an NGS technology, the Vela Diagnostics Sentosa SQ HIV-1 Genotyping Assay. Results: About 71% of patients were ART naïve and 29% were experienced. Overall, 20% presented with any DRM. The prevalence of TDR and ADR was 12.6% and 41.1%, respectively. M184V, T215S, and M41L mutations for NRTI, K103N for NNRTI, and M46I/L for PIs were frequent in naïve and treated patients. E138K and R263K mutations against INSTIs were more frequent in naïve than treated patients. TDR and ADR to INSTIs were 0.3% and 7%, respectively. Patients aged 50 or over (OR: 1.81, p = 0.015), originating from Portuguese-speaking African countries (PALOPs) (OR: 1.55, p = 0.050), HIV-1 subtype G (OR: 1.78, p = 0.010), and with CD4 < 200 cells/mm3 (OR: 1.70, p = 0.043) were more likely to present with DRMs, while the males (OR: 0.63, p = 0.003) with a viral load between 4.1 to 5.0 Log10 (OR: 0.55, p = 0.003) or greater than 5.0 Log10 (OR: 0.52, p < 0.001), had lower chances of presenting with DRMs. Conclusions: We present the first evidence on TDR and ADR to INSTI regimens in followed up patients presenting for healthcare in Portugal. We observed low levels of TDR to INSTIs among ART-naïve and moderate levels in ART-exposed patients. Regimens containing PIs could be an alternative second line in patients with intermediate or high-level drug resistance, especially against second-generation INSTIs (dolutegravir, bictegravir, and cabotegravir).

Introduction: Sexually transmitted infections (STIs) continue to occur at high levels. According to the WHO, each year there are an estimated 374 million new infections with syphilis, gonorrhea, chlamydia, and trichomoniasis. STIs are associated with an increased risk of acquiring HIV infection. Migrants are reportedly highly affected by STIs. Objectives: This study aims to characterize factors associated with STIs in a population of HIV-positive migrants living in Portugal. Methodology: This is a cross-sectional observational study of 265 newly diagnosed HIV-1 positive migrants, who were defined as individuals born outside Portugal. This group of people were part of the BESTHOPE study that was developed in 17 Portuguese hospitals between September 2014 and December 2019, and included information collected through sociodemographic and behavioral questionnaires filled in by the migrant patients, clinical questionnaires filled in by the clinicians and HIV-1 genomic sequences generated through resistance testing (Sanger sequencing). A multivariable statistical analysis was used to analyze the association between sociodemographic characteristics, sexual behaviors, HIV testing and sexual infections. Results: Most HIV-1 positive individuals included in the study were men (66.8%) and aged between 25 and 44 years old (59.9%). Men had a higher proportion of STIs when compared to women (40.4% vs. 14.0%) and the majority of men reported homosexual contacts (52.0%). Most men reported having had two or more occasional sexual partners in the previous year (88.8%) and 50.9% reported always using condoms with occasional partners, while 13.2% never used it. For regular partners, only 29.5% of the women reported using condoms, compared to 47.3% of men. Other risk behaviors for acquiring HIV, such as tattooing and performing invasive medical procedures, were more prevalent in men (38.0% and 46.2%, respectively), when compared to women (30.4% and 45.1% respectively) and 4.7% of men reported having already shared injectable materials, with no data for comparison in the case for women. Additionally, 23.9% of women reported having had a blood transfusion while only 10.3% of men reported having had this medical procedure. Meanwhile, 30.9% of the individuals reported having been diagnosed with some type of STI in the last 12 months. In addition, 43.3% of individuals that answered a question about hepatitis reported to be infected with hepatitis B, while 13.0% reported having hepatitis C infection. According to the multivariable analysis, the only transmission route was significantly associated with reports of previous STI infection: men who have sex with men (MSM) were 70% more likely to have been diagnosed with an STI in the past 12 months compared to the heterosexual route. Conclusion: HIV-1 infected men were more likely to report previous STIs than women. On the other hand, most migrant women had a regular sexual partner and never or only sometimes used condoms. This somewhat discrepant findings suggest that gender inequalities may make women unable to negotiate safe sexual practices, resulting in increased susceptibility to infection. However, since migrant women report less STIs, we cannot exclude that these STIs may remain undiagnosed. The implementation of safer sex awareness campaigns for condom use and screening for STIs in women is crucial. On the other hand, health education campaigns for STI knowledge need to be implemented for both MSM and women and their partners.

The comparison of closely related taxa is cornerstone in biology, as understanding mechanisms leading up to differentiation in relation to extant shared characters are powerful tools in interpreting the evolutionary process. Hotspots of biodiversity such as the west-Mediterranean, where many lineages meet are ideal grounds to study these processes. We set to explore the interesting example of Sooty Copper butterflies: widespread Eurasian Lycaena tityrus (Poda, 1761) comes into contact in Iberia with closely related and local endemic, L. bleusei (Oberthur, 1884), which hasn't always been considered a distinct species. An integrative analysis was designed, combining the use of extensive molecular data (five genes), geometric morphometrics analyses, verified and up-to-date distribution data, and environmental niche modelling, aimed at deciphering their true relationship, their placement within European Lycaena and trace their evolutionary history. We revealed several levels of differ-entiation: L. bleusei and L. tityrus appear to be reciprocally monophyletic independent gene-pools, distinct in all genes analysed, having mutually diverged 4.8 Ma ago. L. tityrus but not L. bleusei, further displays a genetic structure compatible with several glacial refugia, where populations assignable to infraspecific taxa surface. Conversely, L. bleusei shows a loss in mtDNA diversity in relation to nuDNA. Morphological analyses differentiate both species according to size and shape but also discriminate strong seasonal and sexual traits and a geographical phenotype segregation in L. tityrus. Finally, updated distribution and its modelling for current and glacial timeframes reveal both species respond differently to environmental variables, defining a mostly para-patric distribution and an overlapping belt where sympatry was recovered. During the last glacial maximum, a wider expansion in L. bleusei distribution explains current isolated populations. Our study highlights the importance of gathering several lines of evidence when deciphering the relationships between closely related populations in the fringe of cryptic species realm.

Background: Investigating the role of late presenters (LPs) in HIV-1 transmission is important, as they can contribute to the onward spread of HIV-1 virus before diagnosis, when they are not aware of their HIV status. Objective: To characterize individuals living with HIV-1 followed up in Europe infected with subtypes A, B, and G and to compare transmission clusters (TC) in LP vs. non-late presenter (NLP) populations. Methods: Information from a convenience sample of 2679 individuals living with HIV-1 was collected from the EuResist Integrated Database between 2008 and 2019. Maximum likelihood (ML) phylogenies were constructed using FastTree. Transmission clusters were identified using Cluster Picker. Statistical analyses were performed using R. Results: 2437 (91.0%) sequences were from subtype B, 168 (6.3%) from subtype A, and 74 (2.8%) from subtype G. The median age was 39 y/o (IQR: 31.0–47.0) and 85.2% of individuals were males. The main transmission route was via homosexual (MSM) contact (60.1%) and 85.0% originated from Western Europe. In total, 54.7% of individuals were classified as LPs and 41.7% of individuals were inside TCs. In subtype A, individuals in TCs were more frequently males and natives with a recent infection. For subtype B, individuals in TCs were more frequently individuals with MSM transmission route and with a recent infection. For subtype G, individuals in TCs were those with a recent infection. When analyzing cluster size, we found that LPs more frequently belonged to small clusters (<8 individuals), particularly dual clusters (2 individuals). Conclusion: LP individuals are more present either outside or in small clusters, indicating a limited role of late presentation to HIV-1 transmission.

The Zika virus (ZIKV) disease caused a public health emergency of international concern that started in February 2016. The overall number of ZIKV-related cases increased until November 2016, after which it declined sharply. While the evaluation of the potential risk and impact of future arbovirus epidemics remains challenging, intensified surveillance efforts along with a scale-up of ZIKV whole-genome sequencing provide an opportunity to understand the patterns of genetic diversity, evolution, and spread of ZIKV. However, a classification system that reflects the true extent of ZIKV genetic variation is lacking. Our objective was to characterize ZIKV genetic diversity and phylodynamics, identify genomic footprints of differentiation patterns, and propose a dynamic classification system that reflects its divergence levels. We analysed a curated dataset of 762 publicly available sequences spanning the full-length coding region of ZIKV from across its geographical span and collected between 1947 and 2021. The definition of genetic groups was based on comprehensive evolutionary dynamics analyses, which included recombination and phylogenetic analyses, within- and between-group pairwise genetic distances comparison, detection of selective pressure, and clustering analyses. Evidence for potential recombination events was detected in a few sequences. However, we argue that these events are likely due to sequencing errors as proposed in previous studies. There was evidence of strong purifying selection, widespread across the genome, as also detected for other arboviruses. A total of 50 sites showed evidence of positive selection, and for a few of these sites, there was amino acid (AA) differentiation between genetic clusters. Two main genetic clusters were defined, ZA and ZB, which correspond to the already characterized 'African' and 'Asian' genotypes, respectively. Within ZB, two subgroups, ZB.1 and ZB.2, represent the Asiatic and the American (and Oceania) lineages, respectively. ZB.1 is further subdivided into ZB.1.0 (a basal Malaysia sequence sampled in the 1960s and a recent Indian sequence), ZB.1.1 (South-Eastern Asia, Southern Asia, and Micronesia sequences), and ZB.1.2 (very similar sequences from the outbreak in Singapore). ZB.2 is subdivided into ZB.2.0 (basal American sequences and the sequences from French Polynesia, the putative origin of South America introduction), ZB.2.1 (Central America), and ZB.2.2 (Caribbean and North America). This classification system does not use geographical references and is flexible to accommodate potential future lineages. It will be a helpful tool for studies that involve analyses of ZIKV genomic variation and its association with pathogenicity and serve as a starting point for the public health surveillance and response to on-going and future epidemics and to outbreaks that lead to the emergence of new variants.

Emerging infectious diseases are one of the main threats to public health, with the potential to cause a pandemic when the infectious agent manages to spread globally. The first major pandemic to appear in the 20th century was the influenza pandemic of 1918, caused by the influenza A H1N1 strain that is characterized by a high fatality rate. Another major pandemic was caused by the human immunodeficiency virus (HIV), that started early in the 20th century and remained undetected until 1981. The ongoing HIV pandemic demonstrated a high mortality and morbidity rate, with discrepant impacts in different regions around the globe. The most recent major pandemic event, is the ongoing pandemic of COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has caused over 5.7 million deaths since its emergence, 2 years ago. The aim of this work is to highlight the main determinants of the emergence, epidemic response and available countermeasures of these three pandemics, as we argue that such knowledge is paramount to prepare for the next pandemic. We analyse these pandemics' historical and epidemiological contexts and the determinants of their emergence. Furthermore, we compare pharmaceutical and non-pharmaceutical interventions that have been used to slow down these three pandemics and zoom in on the technological advances that were made in the progress. Finally, we discuss the evolution of epidemiological modelling, that has become an essential tool to support public health policy making and discuss it in the context of these three pandemics. While these pandemics are caused by distinct viruses, that ignited in different time periods and in different regions of the globe, our work shows that many of the determinants of their emergence and countermeasures used to halt transmission were common. Therefore, it is important to further improve and optimize such approaches and adapt it to future threatening emerging infectious diseases.

Hydra is a freshwater solitary polyp, capable of temporary adhesion to underwater surfaces. The reversible attachment is based on an adhesive material that is secreted from its basal disc cells and left behind on the substrate as a footprint. Despite Hydra constituting a standard model system in stem cell biology and tissue regeneration, few studies have addressed its bioadhesion. This project aimed to characterize the glycan composition of the Hydra adhesive, using a set of 23 commercially available lectins to label Hydra cells and footprints. The results indicated the presence of N-acetylglucosamine, N-acetylgalactosamine, fucose, and mannose in the adhesive material. The labeling revealed a meshwork-like substructure in the footprints, implying that the adhesive is mainly formed by fibers. Furthermore, lectins might serve as a marker for Hydra cells and structures, e.g., many labeled as glycan-rich nematocytes. Additionally, some unexpected patterns were uncovered, such as structures associated with radial muscle fibers and endodermal gland cells in the hypostome of developing buds.

The tiger-fly Coenosia attenuata Stein (Diptera: Muscidae: Coenosiini) is a generalist predator that preys on several pests of greenhouse crops and is considered a biological control agent in the Mediterranean region. Previous behavioural observations identified its preferred prey, but a more in-depth evaluation will benefit from using Polymerase Chain Reaction amplification of prey DNA remains in the gut of this predator. To evaluate the rate of decay and suitability of this method for use in the field assessments, we carried out a laboratory feeding calibration experiment on 355 females of C. attenuata, which were killed at different intervals of time after ingestion (10 time points from 0 to 48 h). The prey species tested were: Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae: Trialeurodini), Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae), Diglyphus isaea (Walker) (Hymenoptera: Eulophidae: Cirrospilini), Bradysia impatiens (Johannsen) (Diptera: Sciaridae) and Drosophila mercatorum Patterson & Wheeler (Diptera: Drosophilidae: Drosophilini). Based on a probit model, amplification success of prey DNA declined exponentially with increasing time after ingestion. The half-time molecular detection differed between species, ranging from an average of 5 h for T. vaporariorum and D. isaea, 6 h for B. impatiens, 15 h for L. huidobrensis to more than 40 h for D. mercatorum. This study confirmed the feasibility of using DNA based detection to identify prey species in the gut contents of C. attenuata and provided calibration curves for a better understanding of predation activity in this agroecosystem.

Squamous cell carcinoma is the most common neoplasm in the oral cavity. Treatment with chemotherapy and radiotherapy can result in dry mouth and hyposalivation, which can be treated with low-level laser therapy. We aimed to report a case of xerostomia and hyposalivation improvement in a patient previously irradiated in the head and neck region who was treated with low-level laser therapy. A 48-year-old male patient complained of dry mouth. He had undergone surgical treatment, chemotherapy, and radiotherapy due to a tongue-based squamous cell carcinoma. Ten low-level laser therapy sessions in the infrared mode with the application of 1 J in extra and intraoral points were planned. Artificial saliva was prescribed. Improvements in xerostomia and hyposalivation were observed using a visual analog scale. Further studies are needed to assess the use of low-level laser therapy for these symptoms to improve the quality of life of these individuals. (C) 2021 Sociedade Portuguesa de Estomatologia e Medicina Dentaria. Published by SPEMD.

Objetivo: relatar um caso de aumento da hiperdensidade da cortical óssea línguo-basal pós-radioterapia emregião de cabeça e pescoço por meio de avaliação imaginológica. Relato de caso: paciente do sexo feminino,80 anos, procurou tratamento odontológico com a finalidade de reabilitação oral com implantes dentários.Na anamnese, relatou histórico de carcinoma espinocelular em regiões distintas da cavidade oral. No exameclínico, notou-se que a paciente era edêntula total e foi solicitado um exame de tomografia computadorizadade feixe cônico. Foi observado, na região anterior inferior, aumento da hiperdensidade da cortical óssea línguo-basal, com osso compacto mais espesso, achado incomum na literatura após radioterapia. Considerações finais: os efeitos da radioterapia em mandíbula estão relatados na literatura. No caso clínico em questão, detectou-se uma alteração pouco notada em pacientes submetidos à radioterapia, fazendo surgir novos questionamentos, sendo eles sobre o diagnóstico dessa modificação na estrutura óssea e suas repercussões.

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.

O carcinoma espinocelular é a neoplasia mais frequente na cavidade oral. O tratamento com quimioterapia e radioterapia pode resultar em xerostomia e hipossalivação, sintomas que podem ser tratados com laserterapia de baixa intensidade. Este trabalho tem como objetivo relatar um caso de melhoria da xerostomia e da hipossalivação num paciente irradiado na região da cabeça e do pescoço tratado com laserterapia de baixa intensidade. Paciente do sexo masculino, 48 anos, queixou-se de boca seca. Tinha sido submetido a tratamento cirúrgico, quimioterapia e radioterapia devido a um carcinoma espinocelular da base da língua. Foi feito o planeamento de 10 sessões de laserterapia de baixa intensidade no modo infravermelho com aplicação de 1 J em pontos extra e intraorais e prescrição de saliva artificial. Foi observada melhoria no quadro por meio de escala visual analógica. São necessários novos estudos para avaliação do tratamento com laserterapia de baixa intensidade para esses sintomas, com o intuito de melhorar a qualidade de vida desses indivíduos.

Following publication of the original article [1], it has been brought to the authors' attention that in their paper (Rodrigues et al. 2016) they reported the genome size based on 2C values (diploid genome) when it is more common to present it as 1C value.

Ongoing climate change is expected to cause an increase in temperature and a reduction of precipitation levels in the Mediterranean region, which might cause changes in many species distributions. These effects negatively influence species gene pools, decreasing genetic variability and adaptive potential. Here, we use mitochondrial DNA and RADseq to analyse population genetic structure and genetic diversity of the bumblebee species Bombus terrestris (subspecies Bombus terrestris lusitanicus), in the Iberian Peninsula. Although this subspecies shows a panmictic pattern of population structure across Iberia and beyond, we found differentiation between subspecies B. t. lusitanicus and B. t. africanus, probably caused by the existence of barriers to gene flow between Iberia and North Africa. Furthermore, the results revealed that the Iberian Peninsula harbours a large fraction of B. terrestris intraspecific genetic variation, with the highest number of mitochondrial haplotypes found when compared with any other region in Europe studied so far, suggesting a potential role for the Iberian Peninsula as a glacial refugium. Our findings strengthen the idea that Iberia is a very important source of diversity for the global genetic pool of this species, because rare alleles might play a role in population resilience against human- or climate-mediated changes.

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.

Roberto Biello*, Thomas Mathers, Qun Liu, Ana S. B. Rodrigues, Ana Carina Neto, Maria Teresa Rebelo, Octávio S. Paulo, Sam Mugford, Sofia Seabra, Saskia Hogenhout Department of Crop Genetics, John Innes Centre, Norwich Research Park, Norwich, United Kingdom Centre for Ecology, Evolution and Environmental Changes (cE3c), Departamento de Biologia Animal, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal

The meadow spittlebug, Philaenus spumarius (L.) (Hemiptera, Aphrophoridae) is a widespread insect species in the Holarctic region, exhibiting a dorsal colour balanced polymorphism. In the Azores the species is known from high elevations in Terceira and São Miguel islands. A sample of 235 individuals from Pico da Vara and Graminhais protected areas (São Miguel, Azores) (between 645 and 935 m a.s.l.), collected in 2000 and 2017, showed a remarkable high frequency of the melanic morphs flavicollis (FLA) and quadrimaculatus (QUA). In addition, a high frequency of melanics was observed in males. We explore the hypotheses for the origin of Azorean colonization and for the high proportion of melanism in the Azorean populations.