Symbiosis with intracellular bacteria is essential for the nutrition of ticks, particularly through the biosynthesis of B vitamins. Yet, ticks of the genus Ixodes, which include major vectors of human pathogens, lack the nutritional symbionts usually found in other tick genera. This paradox raises questions about the mechanisms that Ixodes ticks use to prevent nutritional deficiencies. Nonetheless, Ixodes ticks commonly harbor other symbionts belonging to the order Rickettsiales. Although these obligate intracellular bacteria are primarily known as human pathogens, Rickettsiales symbionts often dominate the Ixodes microbial community without causing diseases. They also significantly influence Ixodes physiology, synthesize key B vitamins, and are crucial for immatures. These findings underscore unique associations between Rickettsiales and Ixodes ticks distinct from other tick genera.

Rhipicephalus sanguineus, a repulsive obligate blood feeder, is a three-host tick inflicting tremendous damage. Blood-sucking initiates tick-pathogen-host interactions along with alterations in the expression levels of numerous bioactive ingredients. Key molecules regulating blood meals were identified using the transcriptomic approach. A total number of 744 transcripts showed statistically significantly differential expression including 309 significantly upregulated transcripts and 435 significantly downregulated transcripts in semiengorged female ticks compared to unfed ticks, all collected in 2021. The top 10 differentially upregulated transcripts with explicit functional annotations included turripeptide OL55-like protein, valine tRNA ligase-like protein and ice-structuring glycoprotein-like protein. The top 10 differentially down-regulated transcripts were uncharacterized proteins. Gene Ontology (GO) enrichment analysis revealed four associated terms in the cellular component category and 16 in the molecular function category among the top 20 terms. Differentially expressed genes (DEGs) were enriched in GO terms ID 0000323 (lytic vacuole) and ID 0005773 (vacuole). The top 20 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included metabolism, cellular processes, organismal systems and human diseases. The DEGs were enriched in the KEGG term ID: ko-04142 (lysosome pathway) associated with intracellular digestion in the tick midgut epithelium. Molecular markers annotated via comparative transcriptomic profiling were expected to be candidate markers for the purpose of tick control.

Chagas disease is transmitted to humans by obligatory hematophagous insects of Triatominae subfamily, which feeds on various hosts to acquire their nutritional sustenance derived from blood proteins. Hemoglobin (Hb) digestion is a pivotal metabolic feature of triatomines, representing a key juncture in their competence toward Trypanosoma cruzi; however, it remains poorly understood. To explore the Hb digestion pathway in Rhodnius prolixus, a major Chagas disease vector, we employed an array of approaches for activity profiling of various midgut-associated peptidases using specific substrates and inhibitors. Dissecting the individual contribution of each peptidase family in Hb digestion has unveiled a predominant role played by aspartic proteases and cathepsin B-like peptidases. Determination of peptidase-specific cleavage sites of these key hemoglobinases, in conjunction with mass spectrometry-based identification of in vivo Hb-derived fragments, has revealed the intricate network of peptidases involved in the Hb digestion pathway. This network is initiated by aspartic proteases and subsequently sustained by cysteine proteases belonging to the C1 family. The process is continued simultaneously by amino and carboxypeptidases. The comprehensive profiling of midgut-associated aspartic proteases by quantitative proteomics has enabled the accurate revision of gene annotations within the A1 family of the R. prolixus genome. Significantly, this study also serves to illuminate a potentially important role of the anterior midgut in blood digestion. The expanded repertoire of midgut-associated proteases presented in this study holds promise for the identification of novel targets aimed at controlling the transmission of Chagas disease.

Global changes have increased the risk of emerging infectious diseases, which can be prevented or mitigated by studying host-parasite interactions, among other measures. Bats and their ectoparasitic flies of the families Streblidae and Nycteribiidae are an excellent study model but, so far, our knowledge has been restricted to fragmented records at a local scale. To help boost research, we assembled a data set of bat-fly interactions from 174 studies published between 1904 and 2022 plus three original data sets. Altogether, these studies were carried out at 650 sites in the Neotropics, mainly distributed in Mexico, Brazil, Argentina, southern USA, and Colombia, among other countries. In total, our data set contains 3984 interaction records between 237 bat species and 255 fly species. The bat species with the largest number of recorded interactions were Carollia perspicillata (357), Artibeus jamaicensis (263), and Artibeus lituratus (228). The fly species with the largest number of recorded interactions were Trichobius joblingi (256), Megistopoda aranea (235), and Megistopoda proxima (215). The interaction data were extracted, filtered, taxonomically harmonized, and made available in a tidy format together with linked data on bat population, fly population, study reference, sampling methods and geographic information from the study sites. This interconnected structure enables the expansion of information for each interaction record, encompassing where and how each interaction occurred, as well as the number of bats and flies involved. We expect BatFly to open new avenues for research focused on different levels of ecological organization and spatial scales. It will help consolidate knowledge about ecological specialization, resource distribution, pathogen transmission, and the drivers of parasite prevalence over a broad spatial range. It may also help to answer key questions such as: Are there differences in fly prevalence or mean infestation across Neotropical ecoregions? What ecological drivers explain those differences? How do specialization patterns vary among fly species in the Neotropics? Furthermore, we expect BatFly to inspire research aimed at understanding how climate and land-use changes may impact host-parasite interactions and disease outbreaks. This kind of research may help us reach Sustainable Development Goal 3, Good Health and Wellbeing, outlined by the United Nations. The data are released under a Creative Commons Attribution 4.0 International License.

In arthropods, hematophagy has arisen several times throughout evolution. This specialized feeding behavior offered a highly nutritious diet obtained during blood feeds. On the other hand, blood-sucking arthropods must overcome problems brought on by blood intake and digestion. Host blood complement acts on the bite site and is still active after ingestion, so complement activation is a potential threat to the host\'s skin feeding environment and to the arthropod gut enterocytes. During evolution, blood-sucking arthropods have selected, either in their saliva or gut, anticomplement molecules that inactivate host blood complement. This review presents an overview of the complement system and discusses the arthropod\'s salivary and gut anticomplement molecules studied to date, exploring their mechanism of action and other aspects related to the arthropod-host-pathogen interface. The possible therapeutic applications of arthropod\'s anticomplement molecules are also discussed.

Clogmia albipunctata (Williston, 1893) is a non-hematophagous insect belonging to the order Diptera, suborder Nematocera (Lower Diptera) and family Psychodidae. In the present work, we investigated how C. albipunctata control their midgut pH under different physiological conditions, comparing their midgut physiology with some nematoceran hematophagous species. The C. albipunctata midgut pH was measured after ingestion of sugar, protein and under the effect of the alkalinizing hormone released in the hemolymph of the hematophagous sand fly Lutzomyia longipalpis obtained just after a blood meal. The midgut pH of unfed or sugar-fed C. albipunctata is 5.5-6, and its midgut underwent alkalinization after protein ingestion or under treatment with hemolymph collected from blood fed L. longipalpis. These results suggested that in nematocerans, mechanisms for pH control seem shared between hematophagous and non-hematophagous species. This kind of pH control is convenient for successful blood digestion. The independent evolution of many hematophagous groups from the Lower Diptera suggests that characteristics involved in midgut pH control were already present in non-hematophagous species and represent a readiness for adaptation to this feeding mode.

Blood feeding is a secondary adaptation in hematophagous bugs. Many proteins are secreted in the saliva that are devoted to coping with the host\'s defense and to process the blood meal. Digestive enzymes that are no longer required for a blood meal would be expected to be eventually lost. Yet, in many strictly hematophagous arthropods, α-amylase genes, which encode the enzymes that digest starch from plants, are still present and transcribed, including in the kissing bug Rhodnius prolixus (Hemiptera, Reduviidae) and its related species, which transmit the Chagas disease. We hypothesized that retaining α-amylase could be advantageous if the bugs occasionally consume plant tissues. We first checked that the α-amylase protein of Rhodnius robustus retains normal amylolytic activity. Then we surveyed hundreds of gut DNA extracts from the sylvatic R. robustus to detect traces of plants. We found plant DNA in 8% of the samples, mainly identified as Attalea palm trees, where R. robustus are usually found. We suggest that although of secondary importance in the blood-sucking bugs, α-amylase may be needed during occasional plant feeding and thus has been retained.

The common bed bug Cimex lectularius (Linnaeus 1758) is an ectoparasite that feeds preferably on human blood, being considered an important public health issue. Blood-feeding is a challenging process for hematophagous organisms, and one of the inherent risks with this kind of diet is the liberation of high doses of free heme after the digestion of hemoglobin. In order to deal with this potent cytotoxic agent, such organisms have acquired different defense mechanisms. Here, we use UV-visible spectrophotometry and infrared spectroscopy to show that C. lectularius crystalizes free heme to form the much less dangerous compound, hemozoin. According to our results, the peak of formation of hemozoin in the intestinal contents occurred 4-5 days after the blood meal, primarily in the posterior midgut. The quantification of the rate of conversion of heme to hemozoin revealed that at the end of digestion all the heme was in the form of hemozoin. Inhibition of the synthesis of hemozoin using the anti-malarial drug quinine led to an increase in both catalase activity in the intestinal epithelium and the mortality of the bed bugs, indicating that the insects were unable to cope with the oxidative stress generated by the overload of free heme. The data presented here show for the first time how C. lectularius deals with free heme, and how the process of formation of hemozoin is essential for the survival of these insects. Since resistance to insecticides is a common feature among field populations of bed bugs, there is an urgent need to develop alternative control methods. Thus, targeting the synthesis of hemozoin emerges as a possible novel strategy to fight bed bugs.

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.

OBJECTIVE: Insects that live exclusively on vertebrate blood utilize symbiotic bacteria as a source of essential compounds, e.g., B vitamins. In louse flies, the most frequent symbiont originated in genus Arsenophonus, known from a wide range of insects. Here, we analyze genomic traits, phylogenetic origins, and metabolic capacities of 11 Arsenophonus strains associated with louse flies. We show that in louse flies, Arsenophonus established symbiosis in at least four independent events, reaching different stages of symbiogenesis. This allowed for comparative genomic analysis, including convergence of metabolic capacities. The significance of the results is twofold. First, based on a comparison of independently originated Arsenophonus symbioses, it determines the importance of individual B vitamins for the insect host. This expands our theoretical insight into insect-bacteria symbiosis. The second outcome is of methodological significance. We show that the comparative approach reveals artifacts that would be difficult to identify based on a single-genome analysis.