Structural and functional comparisons of salivary α-glucosidases from the mosquito vectors Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus.

Mosquito vectors of medical importance both blood and sugar feed, and their saliva contains bioactive molecules that aid in both processes. Although it has been shown that the salivary glands of several mosquito species exhibit α-glucosidase activities, the specific enzymes responsible for sugar digestion remain understudied. We therefore expressed and purified three recombinant salivary α-glucosidases from the mosquito vectors Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus and compared their functions and structures. We found that all three enzymes were expressed in the salivary glands of their respective vectors and were secreted into the saliva. The proteins, as well as mosquito salivary gland extracts, exhibited α-glucosidase activity, and the recombinant enzymes displayed preference for sucrose compared to p-nitrophenyl-α-D-glucopyranoside. Finally, we solved the crystal structure of the Ae. aegypti α-glucosidase bound to two calcium ions at a 2.3 Ångstrom resolution. Molecular docking suggested that the Ae. aegypti α-glucosidase preferred di- or polysaccharides compared to monosaccharides, consistent with enzymatic activity assays. Comparing structural models between the three species revealed a high degree of similarity, suggesting similar functional properties. We conclude that the α-glucosidases studied herein are important enzymes for sugar digestion in three mosquito species.

Typing of Leishmania isolates from vectors and leporids of the Madrid (Spain) outbreak.

In 2009, a large outbreak of leishmaniasis, associated with environmental changes, was declared near Madrid (Spain), in which Phlebotomus perniciosus was the vector, whereas the main reservoirs were hares and rabbits. Analysis of isolates from humans, vectors and leporids from the focus identified the Leishmania infantum ITS-Lombardi genotype. However, multilocus enzyme electrophoresis (MLEE), the reference technique for Leishmania typing, and sequencing of the hsp70 gene, a commonly used marker, were not performed. In the present study, 19 isolates from P. perniciosus (n = 11), hares (n = 5) and rabbits (n = 3) from the outbreak area, all characterized as ITS-Lombardi in previous studies, were analysed by MLEE and hsp70 sequencing. The hsp70 results confirmed that all the analysed strains are L. infantum. However, by MLEE, 4 different zymodemes of L. infantum were identified based on variable mobilities of the NP1 enzyme: MON-34 (NP1100, n = 11), MON-80 (NP1130, n = 6), MON-24 (NP1140, n = 1) and MON-331 (NP1150, n = 1). The relative frequency of these zymodemes does not correspond to their usual occurrence in Spain. Moreover, MON-34 and MON-80 were found in P. perniciosus, hares and rabbits for the first time. These findings continue to provide insights into the outbreak and call for further studies with a higher number of strains.

Mapping the risk of exposure to Crimean-Congo haemorrhagic fever virus in the Iberian Peninsula using Eurasian wild boar (Sus scrofa) as a model.

Crimean-Congo haemorrhagic fever (CCHF) virus (CCHFV) is a tick-borne zoonotic pathogen that can cause a lethal haemorrhagic disease in humans. Although the virus appears to be endemically established in the Iberian Peninsula, CCHF is an emerging disease in Spain. Clinical signs of CCHFV infection are mainly manifested in humans, but the virus replicates in several animal species. Understanding the determinants of CCHFV exposure risk from animal models is essential to predicting high-risk exposure hotspots for public health action. With this objective in mind, we designed a cross-sectional study of Eurasian wild boar (Sus scrofa) in Spain and Portugal. The study analysed 5,291 sera collected between 2006 and 2022 from 90 wild boar populations with a specific double-antigen ELISA to estimate CCHFV serum prevalence and identify the main determinants of exposure probability. To do so, we statistically modelled exposure risk with host- and environment-related predictors and spatially projected it at a 10 × 10 km square resolution at the scale of the Iberian Peninsula to map foci of infection risk. Fifty-seven (63.3 %) of the 90 populations had at least one seropositive animal, with seroprevalence ranging from 0.0 to 88.2 %. Anti-CCHFV antibodies were found in 1,026 of 5,291 wild boar (19.4 %; 95 % confidence interval: 18.3-20.5 %), with highest exposure rates in southwestern Iberia. The most relevant predictors of virus exposure risk were wild boar abundance, local rainfall regime, shrub cover, winter air temperature and soil temperature variation. The spatial projection of the best-fit model identified high-risk foci as occurring in most of western and southwestern Iberia and identified recently confirmed risk foci in eastern Spain. The results of the study demonstrate that serological surveys of CCHFV vector hosts are a powerful, robust and highly informative tool for public health authorities to take action to prevent human cases of CCHF in enzootic and emergency settings.

Titin domains with reduced core hydrophobicity cause dilated cardiomyopathy.

The underlying genetic defect in most cases of dilated cardiomyopathy (DCM), a common inherited heart disease, remains unknown. Intriguingly, many patients carry single missense variants of uncertain pathogenicity targeting the giant protein titin, a fundamental sarcomere component. To explore the deleterious potential of these variants, we first solved the wild-type and mutant crystal structures of I21, the titin domain targeted by pathogenic variant p.C3575S. Although both structures are remarkably similar, the reduced hydrophobicity of deeply buried position 3575 strongly destabilizes the mutant domain, a scenario supported by molecular dynamics simulations and by biochemical assays that show no disulfide involving C3575. Prompted by these observations, we have found that thousands of similar hydrophobicity-reducing variants associate specifically with DCM. Hence, our results imply that titin domain destabilization causes DCM, a conceptual framework that not only informs pathogenicity assessment of gene variants but also points to therapeutic strategies counterbalancing protein destabilization.

Just a matter of size? Evaluating allometry and intersexual heterometry in Pagurus bernhardus using ratios and indices (Decapoda, Anomura).

Heterochely denotes the presence of dissimilarly sized chelipeds on opposite sides of the body, a prevalent occurrence in diverse crustaceans. Conversely, heterometry pertains to the quantifiable disparities in size between these chelipeds. Both chelipeds hold pivotal roles in activities such as foraging, mating, and defense. Consequently, individuals of both genders in heterochelic species exhibit this morphological pattern. Previous studies have identified sexual dimorphism in cheliped size, with males displaying larger major chelipeds compared to females, albeit solely relying on propodus length as a size proxy and focusing solely on the major cheliped. In our study, we meticulously examined 190 specimens of the common European hermit crab Pagurus bernhardus from two collections. We sought to elucidate allometric relationships and assess whether heterometry exhibited sex-based differences when adjusting for body size by using ratios. Our findings revealed that male chelipeds displayed hyperallometric growth relative to females, and all three calculated heterometry indices exhibited significant disparities between the sexes. Consequently, male specimens exhibited larger major and minor chelipeds, even when theoretically matched for body size with females. This phenomenon may be attributed, among other factors, to male-male contests. Should indirect mate selection favor males with larger chelipeds in proportion to their body size, this dynamic could potentiate sexual selection in their favor.

Aedes aegypti D7 long salivary proteins modulate blood feeding and parasite infection.

Mosquito saliva facilitates blood meal acquisition through pharmacologically active compounds that prevent host hemostasis and immune responses. Here, we generated two knockout (KO) mosquito lines by CRISPR/Cas9 to functionally characterize D7L1 and D7L2, two abundantly expressed salivary proteins from the yellow fever mosquito vector Aedes aegypti. The D7s bind and scavenge biogenic amines and eicosanoids involved in hemostasis at the bite site. The absence of D7 proteins in the salivary glands of KO mosquitoes was confirmed by mass spectrometry, enzyme-linked immunosorbent assay, and fluorescence microscopy of the salivary glands with specific antibodies. D7-KO mosquitoes had longer probing times than parental wildtypes. The differences in probing time were abolished when mutant mice resistant to inflammatory insults were used. These results confirmed the role of D7 proteins as leukotriene scavengers in vivo. We also investigated the role of D7 salivary proteins in Plasmodium gallinaceum infection and transmission. Both KO lines had significantly fewer oocysts per midgut. We hypothesize that the absence of D7 proteins in the midgut of KO mosquitoes might be responsible for creating a harsh environment for the parasite. The information generated by this work highlights the biological functionality of salivary gene products in blood feeding and pathogen infection. IMPORTANCE During blood feeding, mosquitoes inject saliva into the host skin, preventing hemostasis and inflammatory responses. D7 proteins are among the most abundant components of the saliva of blood-feeding arthropods. Aedes aegypti, the vector of yellow fever and dengue, expresses two D7 long-form salivary proteins: D7L1 and D7L2. These proteins bind and counteract hemostatic agonists such as biogenic amines and leukotrienes. D7L1 and D7L2 knockout mosquitoes showed prolonged probing times and carried significantly less Plasmodium gallinaceum oocysts per midgut than wild-type mosquitoes. We hypothesize that reingested D7s play a vital role in the midgut microenvironment with important consequences for pathogen infection and transmission.

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes.

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life parameter data from genetically modified mosquitoes and comparing them to mosquitoes lacking transgenes from the same genetic background. This manuscript illustrates how to measure common life history traits in the mosquito Aedes aegypti, including fecundity, wing size and shape, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These parameters were chosen because they reflect reproductive success, are simple to measure, and are commonly reported in the literature. The representative results quantify fitness costs associated with either a gene knock-out or a single insertion of a gene drive element. Standardizing how life parameter data are collected is important because such data may be used to compare the health of transgenic mosquitoes generated across studies or to model the transgene fixation rate in a simulated wild-type mosquito population. Although this protocol is specific for transgenic Aedes aegypti, the protocol may also be used for other mosquito species or other experimental treatment conditions, with the caveat that certain biological contexts may require special adaptations.

Guianensin, a Simulium guianense salivary protein, has broad anti-hemostatic and anti-inflammatory properties.

Background: Salivary glands from blood-feeding arthropods secrete several molecules that inhibit mammalian hemostasis and facilitate blood feeding and pathogen transmission. The salivary functions from Simulium guianense, the main vector of Onchocerciasis in South America, remain largely understudied. Here, we have characterized a salivary protease inhibitor (Guianensin) from the blackfly Simulium guianense. Materials and methods: A combination of bioinformatic and biophysical analyses, recombinant protein production, in vitro and in vivo experiments were utilized to characterize the molecula mechanism of action of Guianensin. Kinetics of Guianensin interaction with proteases involved in vertebrate inflammation and coagulation were carried out by surface plasmon resonance and isothermal titration calorimetry. Plasma recalcification and coagulometry and tail bleeding assays were performed to understand the role of Guianensin in coagulation. Results: Guianensin was identified in the sialotranscriptome of adult S. guianense flies and belongs to the Kunitz domain of protease inhibitors. It targets various serine proteases involved in hemostasis and inflammation. Binding to these enzymes is highly specific to the catalytic site and is not detectable for their zymogens, the catalytic site-blocked human coagulation factor Xa (FXa), or thrombin. Accordingly, Guianensin significantly increased both PT (Prothrombin time) and aPTT (Activated partial thromboplastin time) in human plasma and consequently increased blood clotting time ex vivo. Guianensin also inhibited prothrombinase activity on endothelial cells. We show that Guianensin acts as a potent anti-inflammatory molecule on FXa-induced paw edema formation in mice. Conclusion: The information generated by this work highlights the biological functionality of Guianensin as an antithrombotic and anti-inflammatory protein that may play significant roles in blood feeding and pathogen transmission.

Same parasite, different outcomes: unraveling the epidemiology of Leishmania infantum infection in Brazil and Spain.

Human leishmaniosis caused by Leishmania infantum is an important health problem worldwide. One of the main aspects arousing interest is the epidemiological scenario surrounding Le. infantum infection in the New World (NW) and Old World (OW). This parasite was introduced to the Americas during European colonization leading to different epidemiology outcomes, even more enigmatic in the face of global changes. Thus, this review aims to highlight the differences and similarities between Le. infantum epidemiology between Brazil (NW) and Spain (OW), as both countries are leading the total number of leishmaniosis cases in their respective continents. Grounded on a systemic view, this article also draws attention to possible common innovative strategies to rethink ways of controlling infections caused by Le. infantum.

Anopheles salivary apyrase regulates blood meal hemostasis and drives malaria parasite transmission.

Mosquito salivary proteins play a crucial role in regulating hemostatic responses at the bite site during blood feeding. In this study, we investigate the function of Anopheles gambiae salivary apyrase (AgApyrase) in Plasmodium transmission. Our results demonstrate that salivary apyrase interacts with and activates tissue plasminogen activator, facilitating the conversion of plasminogen to plasmin, a human protein previously shown to be required for Plasmodium transmission. Microscopy imaging shows that mosquitoes ingest a substantial amount of apyrase during blood feeding which reduces coagulation in the blood meal by enhancing fibrin degradation and inhibiting platelet aggregation. Supplementation of Plasmodium infected blood with apyrase significantly enhanced Plasmodium infection in the mosquito midgut. In contrast, AgApyrase immunization inhibited Plasmodium mosquito infection and sporozoite transmission. This study highlights a pivotal role for mosquito salivary apyrase for regulation of hemostasis in the mosquito blood meal and for Plasmodium transmission to mosquitoes and to the mammal host, underscoring the potential for new strategies to prevent malaria transmission.

A deeper insight into the sialome of male and female Culex quinquefasciatus mosquitoes.

INTRODUCTION: During evolution, blood-feeding arthropods developed a complex salivary mixture that can interfere with host haemostatic and immune response, favoring blood acquisition and pathogen transmission. Therefore, a survey of the salivary gland contents can lead to the identification of molecules with potent pharmacological activity in addition to increase our understanding of the molecular mechanisms underlying the hematophagic behaviour of arthropods. The southern house mosquito, Culex quinquefasciatus, is a vector of several pathogenic agents, including viruses and filarial parasites that can affect humans and wild animals. RESULTS: Previously, a Sanger-based transcriptome of the salivary glands (sialome) of adult C. quinquefasciatus females was published based on the sequencing of 503 clones organized into 281 clusters. Here, we revisited the southern mosquito sialome using an Illumina-based RNA-sequencing approach of both male and female salivary glands. Our analysis resulted in the identification of 7,539 coding DNA sequences (CDS) that were functionally annotated into 25 classes, in addition to 159 long non-coding RNA (LncRNA). Additionally, comparison of male and female libraries allowed the identification of female-enriched transcripts that are potentially related to blood acquisition and/or pathogen transmission. CONCLUSION: Together, these findings represent an extended reference for the identification and characterization of the proteins containing relevant pharmacological activity in the salivary glands of C. quinquefasciatus mosquitoes.

An insight into the female and male Sabethes cyaneus mosquito salivary glands transcriptome.

Mosquitoes are responsible for the death and debilitation of millions of people every year due to the pathogens they can transmit while blood feeding. While a handful of mosquitoes, namely those in the Aedes, Anopheles, and Culex genus, are the dominant vectors, many other species belonging to different genus are also involved in various pathogen cycles. Sabethes cyaneus is one of the many poorly understood mosquito species involved in the sylvatic cycle of Yellow Fever Virus. Here, we report the expression profile differences between male and female of Sa.cyaneus salivary glands (SGs). We find that female Sa.cyaneus SGs have 165 up-regulated and 18 down-regulated genes compared to male SGs. Most of the up-regulated genes have unknown functions, however, odorant binding proteins, such as those in the D7 protein family, and mucins were among the top 30 genes. We also performed various in vitro activity assays of female SGs. In the activity analysis we found that female SG extracts inhibit coagulation by blocking factor Xa and has endonuclease activity. Knowledge about mosquitoes and their physiology are important for understanding how different species differ in their ability to feed on and transmits pathogens to humans. These results provide us with an insight into the Sabethes SG activity and gene expression that expands our understanding of mosquito salivary glands.

Evaluation of Aedes aegypti Feeding Status.

Female mosquitoes need vertebrate blood for egg development. Evaluating mosquito behavior is essential for determining the ability of a mosquito to blood feed. Blood feeding experiments are often performed using artificial membrane feeders; however, such experiments do not represent realistic scenarios in which a mosquito injects saliva into the host to prevent host hemostatic responses. Vertebrate animal models are therefore more representative of a natural blood feeding event. Here, we describe a methodology to evaluate mosquito blood feeding success that can be used to compare blood feeding between mosquito groups-for instance, wild-type versus transgenic mosquitoes lacking salivary proteins or field-collected versus laboratory-reared mosquitoes. We also include a simple procedure to measure blood meal size, allowing for a more quantitative assessment of feeding status. The volume of ingested blood directly affects mosquito fecundity and fertility, important markers of fitness. The methods described herein can be used to evaluate transmission-blocking vaccines, insecticides, or fitness costs associated with transgenic mosquitoes.

Evaluation of Aedes aegypti Penetration, Probing, and Feeding Times on Mice.

In mosquitoes, the intradermal search for vertebrate blood (probing time) corresponds to the time taken from initial insertion of the mouthparts in the skin until visualization of the initial engorgement of blood in the midgut. Probing time evaluation provides useful information on the ability of a mosquito to initiate successful blood feeding. In this protocol, we describe how to determine feeding parameters in Aedes aegypti, a widely distributed mosquito that transmits several deadly pathogens, including yellow fever, dengue, Zika, and Chikungunya viruses. We focus on the different steps of a blood feeding event, including penetration, probing, interprobing, and feeding time. Penetration time corresponds to the insertion of the stylets into the host skin and usually lasts <10 sec. Probing time or intradermal search for blood involves saliva secretion into the skin. Some researchers group penetration and probing time as the exploratory phase for blood. Feeding time is an active phase of blood ingestion and engorgement. Feeding parameters depend on mosquito behaviors and these measurements are visually taken by the investigator. We include a video that provides a close look at a mosquito feeding event in which penetration, probing, and feeding times can be observed. To record these experimental times, one must closely watch the mosquito feeding behavior including stylet penetration in the host skin, visualization of the first traces of blood in the midgut, engorgement of the midgut, and removal of stylets from the skin.

Determination of Mosquito Probing and Feeding Time to Evaluate Mosquito Blood Feeding.

Hematophagous arthropods are animals that feed on vertebrate blood for egg production. Mosquitoes must pierce the host skin, locate blood vessels, and extract blood without being noticed. Mosquito stylets lacerate host tissues, triggering the activation of the three branches of hemostasis, or stopping of blood flow: vasoconstriction, platelet aggregation, and coagulation. Mosquitoes inject saliva into the host skin during their intradermal search for blood (also called probing), and salivary proteins counteract hemostasis. Blood feeding dynamics have been traditionally described by observational studies, in which researchers using magnifying glasses watched mosquitoes in the act of blood feeding. These studies provided the foundation for protocols to evaluate mosquito blood feeding in a more quantitative manner. Here, we introduce mosquito blood feeding biology with a focus on the feeding steps, which include penetration, probing, and feeding. Understanding blood feeding dynamics is crucial for evaluating probing time and other relevant parameters derived from blood feeding, such as blood meal size, fecundity, and fertility. Other considerations, including the relationship between probing and pathogen transmission and novel technologies to address blood feeding, are also discussed.

The relationship between cognition and functioning in Bipolar Disorder: An investigation using functional imaging during working memory performance.

The psychosocial functioning of individuals suffering from bipolar disorder (BD) has a significant impact on prognosis and quality of life. The aim of this study was to assess brain functional correlates of psychosocial functioning in BD individuals during the performance of a working memory task. Sixty-two subjects (31 euthymic BD individuals and 31 matched healthy controls) underwent structural and functional magnetic resonance imaging scanning while performing the 1- and 2-back versions of the n-back task (1-back and 2-back). The Functional Assessment Short Test (FAST) and its subdomains were used to assess functioning. Whole brain analysis revealed only overall activation differences between BD patients and healthy controls, but the patients showed failure of de-activation in the medial frontal cortex. Six clusters of significant inverse correlation with the FAST scores were found in the dorsolateral prefrontal cortex, the superior parietal cortex, and temporo-occipital regions bilaterally, and in the left inferior frontal cortex. Cognitive and occupational functioning were the subdomains most significantly associated with brain activation in these clusters. The results suggest that poor psychosocial functioning in BD individuals is associated with hypoactivation in a range of cortical regions, including the fronto-parietal working memory network and inferior temporo-occipital regions.

Regio- and Stereoselectivity of the Norrish-Yang Photocyclization of Dialkyl 1,2-Diketones: Solution versus Solid State Photochemistry of Two Polymorphs.

As shown by X-ray crystallography, crystals of 3ß-acetoxy-16,17-seco-17,20-dioxopregn-5-ene-16-nitrile are dimorphic. The regioselectivity of the Norrish-Yang type II photocyclization under visible light of this steroidal 1,2-diketone, which bears primary, secondary, and tertiary nonequivalent abstractable γ-hydrogens, dramatically increases in the crystalline state of both polymorphs. X-ray crystallography and molecular mechanics calculations reveal crystal structure-solid state photochemistry relationships.

Performing Immunohistochemistry in Mosquito Salivary Glands.

Studying protein localization in mosquito salivary glands provides novel insights on the function and physiological relevance of salivary proteins and also provides an avenue to study interactions between mosquitoes and pathogens. Salivary proteins display compartmentalization. For example, proteins involved in blood feeding are stored in the medial and distal lateral lobes, whereas proteins related to sugar metabolism localize to the proximal portion of the lateral lobes. Immunohistochemistry assays use antibodies raised against recombinant salivary proteins to reveal the protein localization and interactions within the tissue. In this assay, permeabilization of the salivary glands allows the antibodies to enter the cells and bind their target proteins. The primary antibody-antigen complexes are later marked with fluorescently labeled secondary antibodies. Antibodies that recognize pathogen-specific proteins can also be incorporated in these assays, providing information about pathogen localization within the salivary glands or pathogen interactions with mosquito salivary proteins. Here, we introduce immunohistochemistry assays for use in mosquito salivary glands.

A Simple Method for Immunohistochemistry and Imaging of Mosquito Salivary Glands.

Immunohistochemistry is a valuable technique that provides information on protein localization and interactions in tissues. Mosquito salivary gland immunohistochemistry requires the meticulous dissection of a delicate tissue. The integrity of the salivary glands must be closely monitored throughout the entire process to prevent structural damage and loss of saliva. This protocol describes a series of simple steps to perform salivary gland immunohistochemistry including tissue dissection, permeabilization, immunostaining, mounting, and imaging by confocal microscopy.