Long-term blood-free rearing of Anopheles mosquitoes with no effect on fitness, Plasmodium infectivity nor microbiota composition

Funding Information: We would like to thank Lidia M. D. Gon\u00E7alves (Principal Investigator at the Faculdade de Farm\u00E1cia from Universidade de Lisboa) for technical support. This work was financed by Funda\u00E7\u00E3o para a Ci\u00EAncia e Tecnologia Aga-Khan De-velopment Network (FCT-AGAKHAN/541725581/2019) and has received resources funded by the European Union\u2019s Horizon 2020 research and innovation programme under grant agree-ments No 731060 (Infravec2). JM, SGS, ACA and HS are supported by the Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia (FCT Portugal) for funds to GHTM/IHMT-NOVA (GHTM-UID/04413/2020). JM is recipient of a contract by FCT (DOI: https://doi.org/10.54499/CEECIND/00450/2017/CP1415/CT0001). SGS was funded by FCT, Portugal, through contrato-programa 1567 (https://doi.org/ https://doi.org/10.54499/CEECINST/00 102/2018/CP1567/CT0040). Publisher Copyright: © The Author(s) 2024.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.publishersversionpublishe

Are There Differences between Sexes?

Funding Information: This study was supported by European Funds through grant \u2018Bio-Molecular and Epidemiological Surveillance of HIV Transmitted Drug Resistance, Hepatitis Co-Infections and Ongoing Transmission Patterns in Europe\u2014BEST HOPE\u2014(project funded through HIVERA: Harmonizing Integrating Vitalizing European Research on HIV/Aids, grant 249697); by FCT for funds to GHTM-UID/Multi/04413/2013 and GHTM-UID/04413/2020; by the MigrantHIV project (financed by FCT: PTDC/DTP-EPI/7066/2014), by Characterization of drug-resistance TB and HIV, and associated socio-behavioral factors among migrants in Lisbon, Portugal project financed by Funda\u00E7\u00E3o para a Ci\u00EAncia e Tecnologia to GHTM-UID/04413/2020 and LA-REAL-LA/P/0117/2020.GHTM-UID/Multi/04413/2013 and by Integriv project (financed by FCT: PTDC/SAU-INF/31990/2017). Publisher Copyright: © 2024 by the authors.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.publishersversionpublishe

Predictable phenotypic, but not karyotypic, evolution of populations with contrasting initial history

This study was financed by Portuguese National Funds through FCT - ‘Fundação para a Ciência e Tecnologia’ within the projects PTDC/BIA-BEC/098213/2008, PTDC/BIA-BIC/2165/2012 and cE3c Unit FCT funding UID/BIA/00329/2013. I.F. had a PhD grant (SFRH/BD/60734/2009), P.S. has a Post Doc grant (SFRH/BPD/86186/2012) and S.G.S. has a Post Doc grant (SFRH/BPD/108413/2015) from FCT. M.S. is funded by grant CGL2013-42432-P from the Ministerio de Economía y Competitividad (Spain) and grant 2014 SGR 1346 from Generalitat de Catalunya. The datasets generated during and/or analysed during the current study are available in the figshare repository, at https://doi.org/10.6084/m9.figshare.4797550.The relative impact of selection, chance and history will determine the predictability of evolution. There is a lack of empirical research on this subject, particularly in sexual organisms. Here we use experimental evolution to test the predictability of evolution. We analyse the real-time evolution of Drosophila subobscura populations derived from contrasting European latitudes placed in a novel laboratory environment. Each natural population was sampled twice within a three-year interval. We study evolutionary responses at both phenotypic (life-history, morphological and physiological traits) and karyotypic levels for around 30 generations of laboratory culture. Our results show (1) repeatable historical effects between years in the initial state, at both phenotypic and karyotypic levels; (2) predictable phenotypic evolution with general convergence except for body size; and (3) unpredictable karyotypic evolution. We conclude that the predictability of evolution is contingent on the trait and level of organization, highlighting the importance of studying multiple biological levels with respect to evolutionary patterns.Publisher PDFPeer reviewe

Different genomic changes underlie adaptive evolution in populations of contrasting history

Funding Information: This work was supported by Portuguese National Funds through “Fundac¸ão para a Ciência e a Tecnologia” (projects PTDC/ BIA-BEC/098213/2008, PTDC/BIA-BIC/2165/2012 and cE3c Unit FCT funding UID/BIA/00329/2013, grants SFRH/BD/ 60734/2009 to I.F. and SFRH/BPD/86186/2012 to P.S.). We thank Miguel Lopes-Cunha for help in the laboratory, Francisco Pina-Martins for help with computing, Josiane Santos and Ana Sofia Quina for discussions, and Mauro Santos and Anthony Long for advice on the study and comments on the manuscript. We also thank the three anonymous reviewers for their constructive suggestions.Experimental evolution is a powerful tool to understand the adaptive potential of populations under environmental change. Here, we study the importance of the historical genetic background in the outcome of evolution at the genomewide level. Using the natural clinal variation of Drosophila subobscura, we sampled populations from two contrasting latitudes (Adraga, Portugal and Groningen, Netherlands) and introduced them in a new common environment in the laboratory. We characterized the genome-wide temporal changes underlying the evolutionary dynamics of these populations, which had previously shown fast convergence at the phenotypic level, but not at chromosomal inversion frequencies. We found that initially differentiated populations did not converge either at genome-wide level or at candidate SNPs with signs of selection. In contrast, populations from Portugal showed convergence to the control population that derived from the same geographical origin and had been long-established in the laboratory. Candidate SNPs showed a variety of different allele frequency change patterns across generations, indicative of an underlying polygenic basis. We did not detect strong linkage around candidate SNPs, but rather a small but long-ranging effect. In conclusion, we found that history played a major role in genomic variation and evolution, with initially differentiated populations reaching the same adaptive outcome through different genetic routes.publishersversionpublishe

Integrative analysis reveals the divergence and speciation between sister Sooty Copper butterflies Lycaena bleusei and L. tityrus

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 (Oberthür, 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 differentiation: 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 parapatric 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.info:eu-repo/semantics/publishedVersio

Population genomics of Bombus terrestris reveals high but unstructured genetic diversity in a potential glacial refugium

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.info:eu-repo/semantics/publishedVersio

History, chance and selection during phenotypic and genomic experimental evolution : replaying the tape of life at different levels

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Applying Next-Generation Sequencing to Track HIV-1 Drug Resistance Mutations Circulating in Portugal

Funding Information: This research was funded by FCT in the Scopus of INTEGRIV project (PTDC/SAU-INF/31990/2017), FCT MARVEL (PTDC/SAU-PUB/4018/2021), Calouste Gulbenkian Foundation (FCG), under the ENVOLVE Ci\u00EAncia PALOP program that funded the HITOLA project (N\u00BA.250466), Africa Research Excellence Fund (AREF) (AREF-312-CRUZ-F-C0931), Science and Technology Development Project (PDCT) within the scope of the MUTHIVAO project (N\u00BA.36 MESCTI/PDCT/2022), and by funds from FCT to GHTM-UID/04413/2020 and LA-REAL-LA/P/0117/2020. SGS was funded by FCT, Portugal, through a program contract (CEECINST/00102/2018/CP1567/CT0040). CiiEM has provided support through Project 10.54499/UIDB/04585/2020, funded by FCT. Publisher Copyright: © 2024 by the authors.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).publishersversionpublishe

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.Peer reviewe

4Pipe4-A 454 data analysis pipeline for SNP detection in datasets with no reference sequence or strain information

This work was fully supported by projects SOBREIRO/0036/2009 (under the framework of the Cork Oak ESTs Consortium), PTDC/BIA-BEC/098783/2008 and PTDC/AGR-GPL/119943/2010 from Fundação para a Ciência e Tecnologia (FCT) – Portugal. F. Pina-Martins was funded by FCT grant SFRH/BD/51411/2011, under the PhD program “Biology and Ecology of Global Changes”, Univ. Aveiro & Univ. Lisbon, Portugal. D. Batista was funded by FCT grant SFRH/BPD/104629/2014