Toscana Virus in Wild-Caught Sand Flies in Portugal, Findings from the National Vector Surveillance Network, 2023.

Phlebotomine sand flies play a crucial role in both human and veterinary medicine, acting as vectors for Leishmania parasites and most known phleboviruses. In Portugal, the REVIVE program, a comprehensive national surveillance network under the Ministry of Health, has included sand fly surveys since 2016. REVIVE aims to identify existing sand fly species in the country, determine which pathogens are circulating among them, and provide actionable insights for prevention and control measures when necessary. In this way, annually, from May to October, health technicians collect sand flies across mainland Portugal with CDC light traps. The collected sand flies are sent to the Centre for Vectors and Infectious Diseases Research for species identification and molecular screening of pathogens. On 21 September 2023, Toscana virus (TOSV), a well-known phlebovirus in the Mediterranean region due to its capacity to cause neurological disease, was detected in a pool of 30 sand flies collected in Algarve, the southernmost region of Portugal. A 668 bp partial sequence of the nucleoprotein gene shows similarity with TOSV strains from Spain. To our knowledge, this is the first detection of TOSV in its vector in this country, having previously only been reported in vertebrate hosts. These findings highlight the important role of ongoing surveillance efforts in monitoring and understanding the dynamics of sand fly-borne diseases in Portugal.

Mosquito management strategies in European rice fields: Environmental and public health perspectives.

Rice is a crucial food source and an important economic activity globally. Rice fields provide habitats for birds and other organisms but also serve as ideal breeding grounds for mosquitoes, including potential vectors such as Culex, Aedes, and Anopheles. There is an urgent need to manage mosquitoes associated with rice crops, as they are important pests and vectors of diverse pathogens. Effective management should rely on cost-effective, legislative, and environmentally sustainable approaches. We gathered information from various sources on surveillance, phenology, mosquito nuisance, vector-borne diseases and control measures in the main rice paddies of the five major rice-producing regions in Europe: Italy, Spain, Greece, Portugal, and France. Mosquito problems in rice paddies are prevalent across most analyzed regions, with entomological and virological surveillance efforts varying in intensity and timing. Aedes caspius mosquitoes significantly contribute to nuisance levels, while recent West Nile virus (WNV) circulation poses the most serious threat, as these habitats support high densities of mosquito vectors such as Culex pipiens, Culex modestus, and Culex perexiguus. Different mosquito control strategies are applied, ranging from centralized programs to localized interventions funded by public entities and implemented by public or private companies. Biological larviciding with Bacillus thuringiensis serovar. israelensis is the primary method used, supplemented by adulticiding during epidemic outbreaks in nearby urban areas. These management approaches reflect diverse regional contexts and highlight the importance of adaptive strategies in addressing mosquito-related challenges across rice paddies in Europe.

Mark-Release-Recapture Trial with Aedes albopictus (Diptera, Culicidae) Irradiated Males: Population Parameters and Climatic Factors.

Aedes albopictus is considered one of the major invasive species in the world and can transmit viruses such as dengue, Zika, or chikungunya. The Sterile Insect Technique (SIT) can be used to suppress the native populations of Ae. albopictus. Mark-release-recapture (MRR) studies are crucial to support the development of the release strategy during the SIT application. Meanwhile, weather conditions can affect the MRR trial's results and it is critical to understand the influence of climatic factors on the results. In October 2022, 84,000 irradiated sterile males were released for three consecutive weeks in Faro, Southern Portugal. Mosquitoes were recaptured by human landing collection (HLC) one, two, four, and six days after release. Generalized linear models with a negative binomial family and log function were used to estimate the factors associated with the number of recaptured mosquitoes, prevalence ratios, and the 95% confidence intervals (CIs). A total of 84,000 sterile male mosquitoes were released, with 528 recaptured (0.8%) by HLC. The prevalence of recaptured mosquitoes was 23% lower when the wind intensity was moderate. Marked sterile males had an average median distance travelled of 88.7 m. The median probability of daily survival and the average life expectancy were 61.6% and 2.1 days, respectively. The wild male population estimate was 443.33 males/ha. Despite no statistically significant association being found with humidity, temperature, and precipitation, it is important to consider weather conditions during MRR trial analyses to obtain the best determinant estimation and a more efficient application of the SIT in an integrated vector management program.

The spread of the invasive mosquito Aedes albopictus (Diptera: Culicidae) in Portugal: a first genetic analysis.

BACKGROUND: Aedes albopictus, commonly known as the Asian tiger mosquito, has become one of the most invasive mosquito species. Over the last 5 decades, it has been introduced and established in various tropical and temperate regions worldwide. First reported in Europe in 1979 in Albania and later in Italy in 1990, the species is now established in 13 European Union (EU)/European Economic Area (EEA) countries and 337 regions (2023). In Portugal, Ae. albopictus was first detected in the Algarve and Penafiel regions in 2017, followed by Alentejo in 2022 and Lisbon in 2023. This mosquito species poses a significant public health risk as a vector for numerous pathogenic viruses, including dengue, Zika, and chikungunya. METHODS: Aedes albopictus collected in Lisbon in 2023 were analyzed using cytochrome c oxidase I (COX) gene sequencing to understand their genetic relationships. RESULTS: Our data indicate that the Ae. albopictus mosquito populations detected in three locations in Lisbon in 2023 correspond to recent but distinct introduction events. CONCLUSIONS: Although there has been no local transmission of Aedes-transmitted viruses in mainland Portugal to date, the spread of the mosquito and increased international travel increase the risk of Aedes-borne disease outbreaks. The ongoing spread of Ae. albopictus in the country and the confirmed multiple introductions in new locations raise awareness of the need to monitor mosquito vectors to control and prevent autochthonous Aedes-borne disease outbreaks.

Molecular signatures of Haemonchus contortus infection in sheep: A comparative serum proteomic study on susceptible and resistant sheep breeds.

Due to the negative impact of Haemonchus contortus in the tropics and subtropics, the detection of serum protein profiles that occur in infected sheep is of high relevance for targeted selective treatment strategies (TST). Herein, we integrated proteomics with phenotypic traits to elucidate physiological mechanisms associated to H. contortus infection in susceptible (Dorper - D) and resistant (Santa Inês - S) sheep breeds. Naïve female lambs were infected with H. contortus third-stage larvae on day zero (D0), and samples were collected weekly, for 28 days. Feces were used for individual fecal egg counts (FEC) blood for packed cell volume (PCV) and serum for specific antibody quantification through ELISA. Sera was collected on D0 (-) and D21 (+), and analyzed using a LC-MS/MS based proteomics approach. FEC, PCV, and anti-H. contortus antibody levels confirmed the absence of infection on D0. On D28 there was a significant difference between the two breeds for logFEC means (D = 3774 and S = 3141, p=0.033) and PCV means (D = 16.3 % and S = 24.3 %, p=0.038). From a total of 754 proteins identified, 68 differentially abundant proteins (DAPs) were noted. Phosphopyruvate hydratase (ENO3) was a DAP in all comparisons, while S+ vs D+ and S- vs D- shared the highest number of DAPs (8). Each of the four experimental groups clustered separately in a principal component analysis (PCA) of protein profile. Among the DAPs, proteins associated with the innate and adaptive immune system were detected when comparing S- vs D- and S+ vs D+. In D-, some proteins were linked to stress response to handling, sampling and heat. Focusing on the consequences of infection in each breed, in the D+ vs D- comparison, upregulated proteins were associated with inflammation control and immune response, where downregulated proteins pointed to a negative impact of infection on tissue anabolism, compromising muscle growth and fat deposition. In the S+ vs S- comparison, upregulated proteins were related to immune response, while the downregulated proteins were possibly linked to muscular development and growth, impaired by infection. Collectively, it can be concluded that ENO3 regulation emerges as a potential factor underlying the differential immune response observed between Santa Inês and Dorper sheep infected with H. contortus. In turn, detected acute phase proteins (APPs) reinforce their relation with infection, inflammation and stress conditions, whereas THEMIS-like may contribute to the immune system in Dorper. GSDMD, Guanylate-binding protein and ACAN warrant further investigation as possible biomarkers for TST strategy development.

Ovarian cancer ascites proteomic profile reflects metabolic changes during disease progression.

Ovarian cancer (OC) patients develop ascites, an accumulation of ascitic fluid in the peritoneal cavity anda sign of tumour dissemination within the peritoneal cavity. This body fluid is under-researched, mainly regarding the ascites formed during tumour progression that have no diagnostic value and, therefore, are discarded. We performed a discovery proteomics study to identify new biomarkers in the ascites supernatant of OC patients. In this preliminary study, we analyzed a small amount of OC ascites to highlight the importance of not discarding such biological material during treatment, which could be valuable for OC management. Our findings reveal that OC malignant ascitic fluid (MAF) displays a proliferative environment that promotes the growth of OC cells that shift the metabolic pathway using alternative sources of nutrients, such as the cholesterol pathway. Also, OC ascites drained from patients during treatment showed an immunosuppressive environment, with up-regulation of proteins from the signaling pathways of IL-4 and IL-13 and down-regulation from the MHC-II. This preliminary study pinpointed a new protein (Transmembrane Protein 132A) in the OC context that deserves to be better explored in a more extensive cohort of patients' samples. The proteomic profile of MAF from OC patients provides a unique insight into the metabolic kinetics of cancer cells during disease progression, and this information can be used to develop more effective treatment strategies.

Update on the composition and distribution of the mosquito fauna (Diptera: Culicidae) in Cabo Verde, a country at risk for mosquito-borne diseases.

Mosquitoes play a critical role as vectors of pathogens affecting both humans and animals. Therefore, understanding their biodiversity and distribution is crucial to developing evidence-based vector control strategies. The current study updated the composition and distribution of mosquito species through a comprehensive survey of all municipalities of Cabo Verde. From October 2017 to September 2018, mosquito larvae and pupae were collected from 814 aquatic habitats. Anopheles gambiae (Giles, 1902) and Culex pipiens (Linnaeus, 1758) complexes were subjected to PCR-based techniques for sibling species identification. Ten mosquito species from 5 genera were identified: Aedes aegypti (Linnaeus, 1762), Aedes caspius (Pallas, 1771), Anopheles arabiensis (Patton, 1905), Anopheles pretoriensis (Theobald, 1903), Culex bitaeniorhynchus (Giles, 1901), Cx. pipiens, Culex quinquefasciatus (Say, 1823), Culex tritaeniorhynchus (Giles, 1901), Culiseta longiareolata (Macquart, 1838), and Lutzia tigripes (de Grandpre & de Charmoy, 1901). Santiago Island reported the highest number of species (n = 8). Ae. aegypti and Cx. quinquefasciatus were the most widely distributed species across the country. An. arabiensis was the sole species identified within the An. gambiae complex. The findings from our study will help guide health policy decisions to effectively control mosquito-borne diseases.

Field evaluation of an automated mosquito surveillance system which classifies Aedes and Culex mosquitoes by genus and sex.

BACKGROUND: Mosquito-borne diseases are a major concern for public and veterinary health authorities, highlighting the importance of effective vector surveillance and control programs. Traditional surveillance methods are labor-intensive and do not provide high temporal resolution, which may hinder a full assessment of the risk of mosquito-borne pathogen transmission. Emerging technologies for automated remote mosquito monitoring have the potential to address these limitations; however, few studies have tested the performance of such systems in the field. METHODS: In the present work, an optical sensor coupled to the entrance of a standard mosquito suction trap was used to record 14,067 mosquito flights of Aedes and Culex genera at four temperature regimes in the laboratory, and the resulting dataset was used to train a machine learning (ML) model. The trap, sensor, and ML model, which form the core of an automated mosquito surveillance system, were tested in the field for two classification purposes: to discriminate Aedes and Culex mosquitoes from other insects that enter the trap and to classify the target mosquitoes by genus and sex. The field performance of the system was assessed using balanced accuracy and regression metrics by comparing the classifications made by the system with those made by the manual inspection of the trap. RESULTS: The field system discriminated the target mosquitoes (Aedes and Culex genera) with a balanced accuracy of 95.5% and classified the genus and sex of those mosquitoes with a balanced accuracy of 88.8%. An analysis of the daily and seasonal temporal dynamics of Aedes and Culex mosquito populations was also performed using the time-stamped classifications from the system. CONCLUSIONS: This study reports results for automated mosquito genus and sex classification using an optical sensor coupled to a mosquito trap in the field with highly balanced accuracy. The compatibility of the sensor with commercial mosquito traps enables the sensor to be integrated into conventional mosquito surveillance methods to provide accurate automatic monitoring with high temporal resolution of Aedes and Culex mosquitoes, two of the most concerning genera in terms of arbovirus transmission.

Mosquito (Diptera: Culicidae) Fauna of a Zoological Park in an Urban Setting: Analysis of Culex pipiens s.l. and Their Biotypes.

Mosquito-borne diseases (MBDs) are important emerging diseases that affect humans and animals. Zoological parks can work as early warning systems for the occurrence of MBDs. In this study, we characterized the mosquito fauna captured inside Lisbon Zoo from May 2018 to November 2019. An average of 2.4 mosquitos per trap/night were captured. Five mosquito species potentially causing MBDs, including Culex pipiens biotypes, were found in the zoo. The sympatric occurrence of Culex pipiens biotypes represents a risk factor for the epizootic transmission of West Nile virus and Usutu virus. The mosquito occurrence followed the expected seasonality, with the maximum densities during summer months. However, mosquito activity was detected in winter months in low numbers. The minimum temperature and the relative humidity (RH) on the day of capture showed a positive effect on Culex pipiens abundance. Contrary, the RH the week before capture and the average precipitation the week of capture had a negative effect. No invasive species were identified, nor have flaviviruses been detected in the mosquitoes. The implementation of biosecurity measures regarding the hygiene of the premises and the strict control of all the animals entering the zoo can justify the low prevalence of mosquitoes and the absence of flavivirus-infected mosquitoes.

Nanoparticles targeting Sialyl-Tn for efficient tyrosine kinase inhibitor delivery in gastric cancer.

Gastric cancer (GC) is the fourth leading cause of cancer-related deaths worldwide and, therefore, it is urgent to develop new and more efficient therapeutic approaches. Foretinib (FRT) is an oral multikinase inhibitor targeting MET (hepatocyte growth factor receptor) and RON (recepteur d'origine nantais) receptor tyrosine kinases (RTKs) that has been used in clinical trials for several solid tumors. Targeted uptake of therapeutic polymeric nanoparticles (NPs) represents a powerful approach in cancer cell drug delivery. Previously, a nanodelivery system composed of polymeric NPs functionalized with B72.3 antibody, which targets the tumor-associated antigen Sialyl-Tn (STn), has been developed. Herein, these NPs were loaded with FRT to evaluate its capacity in delivering the drug to multicellular tumors spheroids (MCTS) and mouse models. The data indicated that B72.3 functionalized FRT-loaded PLGA-PEG-COOH NPs (NFB72.3) specifically target gastric MCTS expressing the STn glycan (MKN45 SimpleCell (SC) cells), leading to a decrease in phospho-RTKs activation and reduced cell viability. In vivo evaluation using MKN45 SC xenograft mice revealed that NFB72.3 were able to decrease tumor growth, reduce cell proliferation and tumor necrosis. NFB72.3-treated tumors also showed inactivation of phospho-MET and phospho-RON. This study demonstrates the value of using NPs targeting STn for FRT delivery, highlighting its potential as a therapeutic application in GC. STATEMENT OF SIGNIFICANCE: Despite the advances in gastric cancer therapeutics, it remains one of the diseases with the highest incidence and mortality in the world. Combining targeted therapies with a controlled drug release is an attractive strategy to reduce drug cytotoxic effects and improve specific drug delivery efficiency to the cancer cells. Thus, we developed nanoparticles loaded with a tyrosine kinase inhibitor and targeting a specific tumor glycan exclusive of cancer cells. In in vivo gastric cancer xenograft mice models, these nanoparticles efficiently reduced tumor growth, cell proliferation and tumor necrosis area and inactivated phosphorylation of targeting receptors. This approach represents an innovative therapeutic strategy with high impact in gastric cancer.

Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma.

LRP1B remains one of the most altered genes in cancer, although its relevance in cancer biology is still unclear. Recent advances in gene editing techniques, particularly CRISPR/Cas9 systems, offer new opportunities to evaluate the function of large genes, such as LRP1B. Using a dual sgRNA CRISPR/Cas9 gene editing approach, this study aimed to assess the impact of disrupting LRP1B in glioblastoma cell biology. Four sgRNAs were designed for the dual targeting of two LRP1B exons (1 and 85). The U87 glioblastoma (GB) cell line was transfected with CRISPR/Cas9 PX459 vectors. To assess LRP1B-gene-induced alterations and expression, PCR, Sanger DNA sequencing, and qRT-PCR were carried out. Three clones (clones B9, E6, and H7) were further evaluated. All clones presented altered cellular morphology, increased cellular and nuclear size, and changes in ploidy. Two clones (E6 and H7) showed a significant decrease in cell growth, both in vitro and in the in vivo CAM assay. Proteomic analysis of the clones' secretome identified differentially expressed proteins that had not been previously associated with LRP1B alterations. This study demonstrates that the dual sgRNA CRISPR/Cas9 strategy can effectively edit LRP1B in GB cells, providing new insights into the impact of LRP1B deletions in GBM biology.

3D Chromatin Architecture Re-Wiring at the CDH3/CDH1 Loci Contributes to E-Cadherin to P-Cadherin Expression Switch in Gastric Cancer.

Cadherins are cell-cell adhesion molecules, fundamental for cell architecture and polarity. E-cadherin to P-cadherin switch can rescue adherens junctions in epithelial tumours. Herein, we disclose a mechanism for E-cadherin to P-cadherin switch in gastric cancers. CDH1 and CDH3 mRNA expression was obtained from 42 gastric tumours' RNA-seq data. CRISPR-Cas9 was used to knock out CDH1 and a putative regulatory element. CDH1-depleted and parental cells were submitted to proteomics and enrichment GO terms analysis; ATAC-seq/4C-seq with a CDH1 promoter viewpoint to assess chromatin accessibility and conformation; and RT-PCR/flow cytometry to assess CDH1/E-cadherin and CDH3/P-cadherin expression. In 42% of gastric tumours analysed, CDH1 to CDH3 switch was observed. CDH1 knockout triggered CDH1/E-cadherin complete loss and CDH3/P-cadherin expression increase at plasma membrane. This switch, likely rescuing adherens junctions, increased cell migration/proliferation, commonly observed in aggressive tumours. E- to P-cadherin switch accompanied increased CDH1 promoter interactions with CDH3-eQTL, absent in normal stomach and parental cells. CDH3-eQTL deletion promotes CDH3/CDH1 reduced expression. These data provide evidence that loss of CDH1/E-cadherin expression alters the CDH3 locus chromatin conformation, allowing a CDH1 promoter interaction with a CDH3-eQTL, and promoting CDH3/P-cadherin expression. These data highlight a novel mechanism triggering E- to P-cadherin switch in gastric cancer.

Differential Protein and Glycan Packaging into Extracellular Vesicles in Response to 3D Gastric Cancer Cellular Organization.

Alterations of the glycosylation machinery are common events in cancer, leading to the synthesis of aberrant glycan structures by tumor cells. Extracellular vesicles (EVs) play a modulatory role in cancer communication and progression, and interestingly, several tumor-associated glycans have already been identified in cancer EVs. Nevertheless, the impact of 3D tumor architecture in the selective packaging of cellular glycans into EVs has never been addressed. In this work, the capacity of gastric cancer cell lines with differential glycosylation is evaluated in producing and releasing EVs when cultured under conventional 2D monolayer or in 3D culture conditions. Furthermore, the proteomic content is identified and specific glycans are studied in the EVs produced by these cells, upon differential spatial organization. Here, it is observed that although the proteome of the analyzed EVs is mostly conserved, an EV differential packaging of specific proteins and glycans is found. In addition, protein-protein interaction and pathway analysis reveal individual signatures on the EVs released by 2D- and 3D-cultured cells, suggesting distinct biological functions. These protein signatures also show a correlation with clinical data. Overall, this data highlight the importance of tumor cellular architecture when assessing the cancer-EV cargo and its biological role.

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.

Syndecan-4 is a maestro of gastric cancer cell invasion and communication that underscores poor survival.

Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that SDC4 knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression.

Characterization and Proteomic Analysis of Plasma EVs Recovered from Healthy and Diseased Dogs with Canine Leishmaniosis.

Dogs are highly valued companions and work animals that are susceptible to many life-threatening conditions such as canine leishmaniosis (CanL). Plasma-derived extracellular vesicles (EVs), exploited extensively in biomarker discovery, constitute a mostly untapped resource in veterinary sciences. Thus, the definition of proteins associated with plasma EVs recovered from healthy and diseased dogs with a relevant pathogen would be important for biomarker development. For this, we recovered, using size-exclusion chromatography (SEC), EVs from 19 healthy and 20 CanL dogs' plasma and performed proteomic analysis by LC-MS/MS to define their core proteomic composition and search for CanL-associated alterations. EVs-specific markers were identified in all preparations and also non-EVs proteins. Some EVs markers such as CD82 were specific to the healthy animals, while others, such as the Integrin beta 3 were identified in most samples. The EVs-enriched preparations allowed the identification of 529 canine proteins that were identified in both groups, while 465 and 154 were only identified in healthy or CanL samples, respectively. A GO enrichment analysis revealed few CanL-specific terms. Leishmania spp. protein identifications were also found, although with only one unique peptide. Ultimately, CanL-associated proteins of interest were identified and a core proteome was revealed that will be available for intra- and inter-species comparisons.

Search for new biomarkers of tolerance to Perkinsus olseni parasite infection in Ruditapes decussatus clams.

The grooved carpet shell (Ruditapes decussatus) is a clam species with high economic and social importance in several European and Mediterranean countries. Production of this species suffered a decline caused by biotic (parasite infection) and abiotic factors (environmental factors, stress, poor management methods and intensive culture of the introduced species Ruditapes philippinarum). The protozoan parasite Perkinsus olseni is also responsible for the decline of production, being nowadays one of the major issues for clam culture. Molecular biomarkers that might represent tolerance of R. decussatus to P. olseni have already been uncovered, shedding light in a possible production improvement by selecting those clams with a strongest immune response. In the present study, new tolerance biomarkers to P. olseni infection in R. decussatus were identified. The haemolymph proteomic profiles of naturally non/low-infected (tolerant) and highly-infected (susceptible) clams by the parasite across several heavy affected areas of Europe were characterized through a shotgun proteomics approach. Also, the mechanisms that might be involved in the responses against the disease in chronic infections were explored. Proteins related to energy restoration and balance, metabolic regulation, energy accumulation, ROS production, lysosomal activity, amino acid synthesis, proteolytic activity, iron regulation, iron withholding, and immune response modulation were significantly regulated in susceptible clams. In the tolerant group, proteins related to phagocytosis regulation, control of cell growth and proliferation, gonadal maturation, regulation of apoptosis, growth modulation, response to oxidative stress, iron regulation, shell development and metabolic regulation were significantly expressed. In summary, the protein expression profile of tolerant individuals suggests that an efficient pathogen elimination mechanism coupled to a better metabolic regulation leads to a tolerance to the parasite infection by limiting the spread through the tissues.

Co-Circulation of Leishmania Parasites and Phleboviruses in a Population of Sand Flies Collected in the South of Portugal.

In the Old World, phlebotomine sand flies from the genus Phlebotomus are implicated in the transmission of Leishmania spp. parasites (Kinetoplastida: Trypanosomatidae) and viruses belonging to the genus Phlebovirus (Bunyavirales: Phenuiviridae). Two of the five sand fly species known to occur in Portugal, Phlebotomus perniciosus and Ph. ariasi, the former being the most ubiquitous, are recognized vectors of Leishmania infantum, which causes visceral leishmaniasis, the most prevalent form of leishmaniasis in the country. Phlebotomus perniciosus is also the vector of the neurotropic Toscana virus, which can cause aseptic meningitis. Entomological surveillance is essential to provide fundamental data about the presence of vectors and the pathogens they can carry. As such, and given the lack of data in Portugal, an entomological survey took place in the Algarve, the southernmost region of the country, from May to October 2018. Polymerase chain reaction assays were performed in order to detect the presence of the above-mentioned pathogens in sand fly pools. Not only were both Leishmania parasites and phleboviruses detected during this study, but more importantly, it was the first time their co-circulation was verified in the same sand fly population collected in Portugal.

Effect of the Interaction between Elevated Carbon Dioxide and Iron Limitation on Proteomic Profiling of Soybean.

Elevated atmospheric CO2 (eCO2) and iron (Fe) availability are important factors affecting plant growth that may impact the proteomic profile of crop plants. In this study, soybean plants treated under Fe-limited (0.5 mM) and Fe-sufficient (20 mM) conditions were grown at ambient (400 µmol mol-1) and eCO2 (800 µmol mol-1) in hydroponic solutions. Elevated CO2 increased biomass from 2.14 to 3.14 g plant-1 and from 1.18 to 2.91 g plant-1 under Fe-sufficient and Fe-limited conditions, respectively, but did not affect leaf photosynthesis. Sugar concentration increased from 10.92 to 26.17 µmol g FW-1 in roots of Fe-sufficient plants and from 8.75 to 19.89 µmol g FW-1 of Fe-limited plants after exposure to eCO2. In leaves, sugar concentration increased from 33.62 to 52.22 µmol g FW-1 and from 34.80 to 46.70 µmol g FW-1 in Fe-sufficient and Fe-limited conditions, respectively, under eCO2. However, Fe-limitation decreases photosynthesis and biomass. Pathway enrichment analysis showed that cell wall organization, glutathione metabolism, photosynthesis, stress-related proteins, and biosynthesis of secondary compounds changed in root tissues to cope with Fe-stress. Moreover, under eCO2, at sufficient or limited Fe supply, it was shown an increase in the abundance of proteins involved in glycolysis, starch and sucrose metabolism, biosynthesis of plant hormones gibberellins, and decreased levels of protein biosynthesis. Our results revealed that proteins and metabolic pathways related to Fe-limitation changed the effects of eCO2 and negatively impacted soybean production.