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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., (© 2024 Institute of Zoology, Chinese Academy of Sciences.)

Amblyomma sculptum is widely distributed in Brazil and is the main vector of Rickettsia rickettsii, the causative agent of the Brazilian spotted fever (BSF). Tick gut proteins play an essential role in blood feeding, digestion, and protection of gut epithelium. Therefore, many of these were investigated as potential vaccine targets for tick-control strategies. The present study aimed to select transcripts corresponding to putative immunogenic proteins in the A. sculptum gut epithelial membrane, produce recombinant proteins and evaluate them as antigens against A. sculptum infestations. Three gut proteins - AsMucin, AsAPP, and AsLAMP - and a chimeric protein (rAsChimera) based on 22 peptides containing putative B cell epitopes from seven different gut proteins were evaluated as anti-A. sculptum antigens. Mice immunizations revealed that all recombinant targets elicited humoral response with significantly increased IgG levels compared to controls. For rAsChimera, IgG levels remained significantly higher than controls up to 75 days after the end of the immunization. Challenge trials revealed that vaccination with the chimeric protein was the most effective against A. sculptum, inducing 100 % nymph mortality and reaching 80.8 % efficacy against females. The other three proteins did not induce relevant protection, as AsAPP had only 26.6 % efficacy, whereas AsMucin and AsLAMP induced no protection. These data indicate that targeting gut protein immunogenic regions may be an effective strategy for a vaccine formulation againstA. sculptum., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ricardo Nascimento Araujo has patent #BR 10 2019 018609 7 pending to INPI- Instituto Nacional de Propriedade Intelectual. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Malaria is a severe public health problem in several developing tropical and subtropical countries. Anopheles aquasalis is the primary coastal malaria vector in Central and South America and the Caribbean Islands, and it has the peculiar feature of living in water with large changes in salinity. Recent research has recognised An. aquasalis as an important model for studying the interactions of murine and human Plasmodium parasites. This study presents the complete genome of An. aquasalis and offers insights into its evolution and physiology. The genome is similar in size and gene content to other Neotropical anophelines, with 162 Mb and 12,446 protein-coding genes. There are 1387 single-copy orthologs at the Diptera level (eg. An. gambiae, An. darlingi and Drosophila melanogaster). An. aquasalis diverged from An. darlingi, the primary malaria vector in inland South America, nearly 20 million years ago. Proteins related to ion transport and metabolism belong to the most abundant gene families with 660 genes. We identified gene families relevant to osmosis control (e.g., aquaporins, vacuolar-ATPases, Na+/K+-ATPases, and carbonic anhydrases). Evolutionary analysis suggests that all osmotic regulation genes are under strong purifying selection. We also observed low copy number variation in insecticide resistance and immunity-related genes for all known classical pathways. The data provided by this study offers candidate genes for further studies of parasite-vector interactions and for studies on how anophelines of brackish water deal with the high fluctuation in water salinity. We also established data and insights supporting An. aquasalis as an emerging Neotropical malaria vector model for genetic and molecular studies., (© 2023. The Author(s).)

Anophelines are vectors of malaria, the deadliest disease worldwide transmitted by mosquitoes. The availability of genomic data from various Anopheles species allowed evolutionary comparisons of the immune response genes in search of alternative vector control of the malarial parasites. Now, with the Anopheles aquasalis genome, it was possible to obtain more information about the evolution of the immune response genes. Anopheles aquasalis has 278 immune genes in 24 families or groups. Comparatively, the American anophelines possess fewer genes than Anopheles gambiae s . s ., the most dangerous African vector. The most remarkable differences were found in the pathogen recognition and modulation families like FREPs, CLIP and C-type lectins. Even so, genes related to the modulation of the expression of effectors in response to pathogens and gene families that control the production of reactive oxygen species were more conserved. Overall, the results show a variable pattern of evolution in the immune response genes in the anopheline species. Environmental factors, such as exposure to different pathogens and differences in the microbiota composition, could shape the expression of this group of genes. The results presented here will contribute to a better knowledge of the Neotropical vector and open opportunities for malaria control in the endemic-affected areas of the New World.

The complement system is a primary component of the vertebrate innate immune system, and its activity is harmful to microorganisms and parasites. To evade complement attack, some pathogens, such as viruses, bacteria, and protozoa, can interact with complement regulatory proteins from their hosts. Our research group has described the ability of Leishmania species to bind Factor H from human serum and use it as a tool to evade the complement system. However, there is no description of the interaction of Leishmania with other complement regulatory proteins, such as the C4b-binding protein (C4bBP), a negative regulator of classical and lectins complement system pathways. The results presented in this manuscript suggest that Leishmania infantum, L. amazonensis, and L. braziliensis recruit C4bBP from human serum. The uptake of C4bBP by L. infantum was studied in detail to improve our understanding of this inhibitory mechanism. When exposed to this complement regulator, parasites with inactivated GP63 bind to C4bBP and inactivate C4b deposited on their surface after serum exposure. This inactivation occurs by the action of Factor I, a complement system protease. In addition to the C4bBP-Factor I inactivation mechanism, the surface parasite protease GP63 can also inactivate soluble C4b molecules and probably that C4b molecules deposited on the parasites surface. This manuscript shows that Leishmania has two independent strategies to inactivate C4b molecules, preventing the progress of classical and lectins pathways. The identification of the C4bBP receptor on the Leishmania membrane may provide a new vaccine target to fight leishmaniasis., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The sand fly Lutzomyia longipalpis is the main vector of Leishmania infantum in the Americas. Female sand flies ingest sugar-rich solutions and blood, which are digested in the midgut. Digestion of nutrients is an essential function performed by digestive enzymes, which require appropriate physiological conditions. One of the main aspects that influence enzymatic activity is the gut pH, which must be tightly controlled. Considering second messengers are frequently involved in the coordination of tightly regulated physiological events, we investigated if the second messenger cAMP would participate in the process of alkalinization in the abdominal midgut of female L. longipalpis. In midguts containing the indicator dye bromothymol-blue, cAMP stimulated the alkalinization of the midgut lumen. Through another technique based on the use of fluorescein as a pH indicator, we propose that cAMP is involved in the alkalinization of the midgut by activating HCO3 - transport from the enterocyte's cytoplasm to the lumen. The results strongly suggested that the carrier responsible for this process would be a HCO3 - /Cl - antiporter located in the enterocytes' apical membrane. Hematophagy promotes the release of alkalinizing hormones in the hemolymph; however, when the enzyme adenylyl cyclase, responsible for cAMP production, was inhibited, we observed that the hemolymph from blood-fed L. longipalpis' females did not stimulate midgut alkalinization. This result indicated that hormone-stimulated alkalinization is mediated by cAMP. In the present study, we provide evidences that cAMP has a key role in the control of intestinal pH., (© 2021 Institute of Zoology, Chinese Academy of Sciences.)

The haematophagy process by arthropods has been one of the main targets of studies in the parasite-host interaction, and the kissing-bug Rhodnius prolixus, vector of the protozoan Trypanosoma cruzi, has been one of the main models for such studies. Still in the 1980s, it was identified that among the salivary proteins for disrupting vertebrate host homeostasis, lipocalins were among the most relevant proteins for this process. Since then, 30 lipocalins have been identified in the salivary glands of R. prolixus, that promotes vasodilatation, prevents inflammation, act as anticoagulants and inhibits platelet aggregation. The present work aims to identify new lipocalins from R. prolixus, combining transcriptome and genome data. Identified new genes were mapped and had their structure annotated. To infer an evolutionary relationship between lipocalins, and to support the predicted functions for each lipocalin, all amino acid sequences were used to construct phylogenetic trees. We identified a total of 29 new lipocalins, 3 new bioaminogenic-biding proteins (which act to inhibit platelet aggregation and vasodilation), 9 new inhibitors of platelet aggregation, 7 new apolipoproteins and 10 lipocalins with no putative function. In addition, we observed that several of the lipocalins are also expressed in different R. prolxius tissues, including gut, central nervous system, antennae, and reproductive organs. In addition to newly identified lipocalins and a mapping the new and old lipocalins in the genome of R. prolixus, our study also carried out a review on functional status and nomenclature of some of the already identified lipocalins. Our study reinforces that we are far from understanding the role of lipocalins in the physiology of R. prolixus, and that studies of this family are still of great relevance., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Control of Chagas disease in endemic countries is primarily accomplished through insecticide spraying for triatomine vectors. In this context, pyrethroids are the first-choice insecticide, and the evolution of insect resistance to these insecticides may represent an important barrier to triatomine control. In insects, cytochrome P450s are enzymes involved in the metabolism of xenobiotics and endogenous chemicals that are encoded by genes divided into different families. In this work, we evaluated the role of three Rhodnius prolixus CYP4EM subfamily genes during blood meal and after deltamethrin exposure. CYP4 gene members were expressed in different insect organs (integument, salivary glands (SGs), midgut, fat body and malpighian tubules) at distinct transcriptional levels. CYP4EM1 gene was highly expressed in the SG and was clearly modulated after insect blood meal. Injection of CYP4EM1dsRNA promoted significant reduction in mRNA levels of both CYP4EM1 and CYP4EM2 genes and induced deleterious effects in R. prolixus nymphs subsequently exposed to sublethal doses of deltamethrin (3.4 or 3.8 ng/nymph treated). The higher dose reduced the survival over time and increased susceptibility of R. prolixus nymphs to deltamethrin. A better understanding of this mechanism can help in developing of more efficient strategies to reduce Trypanosoma cruzi vector transmission in Americas., (© 2021 Royal Entomological Society.)

Escaping the complement system is an important step in the establishment of infections. Some pathogens have acquired the ability to inactivate the complement system to ensure successful infection. This has been observed in parasites from the genus Leishmania, which inactivate C3b molecules deposited on their surface through the membrane protease GP63. In the present study, we describe a new mechanism that also acts through C3b inactivation. This mechanism involves the binding of the complement regulatory molecule factor H from serum. Factor H signals a plasma protease (factor I) to inactivate C3b molecules deposited on the surface of the parasites. According to our results, Leishmania infantum, L. amazonensis, and L. braziliensis recruit factor H from human serum. The absorption of factor H by L. infantum was studied in detail to better understand how it works. L. infantum binds factor H from human serum and factor H-like proteins from dog serum. When exposed to purified factor H, promastigotes bind this regulatory molecule and inactivate C3b in the presence of factor I. This indicates the existence of an as yet unidentified factor H-binding outer surface molecule functioning as a receptor. The two mechanisms (GP63 and factor H binding) work independently, as Leishmania promastigotes with inhibited GP63 can easily inactivate C3b molecules on the surface of the parasite. The identification of the factor H receptor could lead to the development of a vaccine target for leishmaniasis control, as blocking antibodies to factor H binding could impair the mechanism of C3b inactivation, making the parasite more susceptible to the complement system., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Culex quinquefasciatus is a mosquito species with an anthropophilic habit, often associated with areas with poor sanitation in tropical and urban regions. Adult males and females feed on sugars but only females feed on blood in natural conditions for egg maturation. During haematophagy, female C. quinquefasciatus transmit pathogens such as the West Nile virus, Oropouche virus, various encephalitis viruses, and Wuchereria bancrofti to human hosts. It has been observed in laboratory conditions that male C. quinquefasciatus may feed on blood during an artificial feed. Experiments were carried out to understand how males and females of this species deal with human complement activation. Our results showed that female C. quinquefasciatus, but not males, withstand the stress caused by the ingestion of normal human serum. It was observed that the salivary gland extracts from female mosquitoes were able to inhibit the classical and lectin pathways, whereas male salivary gland extracts only inhibited the lectin pathway. The male and female intestinal contents inhibited the classical and lectin pathways. Neither the salivary glands nor the intestinal contents from males and females showed inhibitory activity towards the alternative pathway. However, the guts of male and female C. quinquefasciatus captured factor H from the human serum, permitting C3b inactivation to its inactive form iC3b, and preventing the formation of the C3 convertase. The activity of the antioxidant enzyme catalase is similar in C. quinquefasciatus females and males. This article shows for the first time that males from a haematophagous arthropod species present human anti-complement activity in their salivary gland extracts and gut contents. The finding of an activity that helps to protect the damage caused by blood ingestion in sugar-feeding male mosquitoes suggests that this may be a pre-adaptation to blood-feeding., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Panstrongylus megistus is the most important vector of Chagas disease in Brazil. Studies show that the principal factor hindering the control of triatomines is reinfestation of houses previously treated with insecticides. Studies at the microgeographic level are therefore necessary to better understand these events. However, an efficient molecular marker is not yet available for carrying out such analyses in this species. The aim of the present study was to identify and characterize microsatellite loci for future population genetic studies of P. megistus., Methods: This study work consisted of five stages: (i) sequencing of genomic DNA; (ii) assembly and selection of contigs containing microsatellites; (iii) validation of amplification and evaluation of polymorphic loci; (iv) standardization of the polymorphic loci; and (v) verification of cross-amplification with other triatomine species., Results: Sequencing of males and females generated 7,908,463 contigs with a total length of 2,043,422,613 bp. A total of 2,043,690 regions with microsatellites in 1,441,091 contigs were obtained, with mononucleotide repeats being the most abundant class. From a panel of 96 loci it was possible to visualize polymorphisms in 64.55% of the loci. Of the 20 loci genotyped, the number of alleles varied from two to nine with an average of 4.9. Cross-amplification with other species of triatomines was observed in 13 of the loci., Conclusions: Due to the high number of alleles encountered, polymorphism and the capacity to amplify from geographically distant populations, the microsatellites described here show promise for utilization in population genetic studies of P. megistus.

Amblyomma sculptum is the main tick associated with human bites in Brazil and the main vector of Rickettsia rickettsii , the causative agent of the most severe form of Brazilian spotted fever. Molecules produced in the salivary glands are directly related to feeding success and vector competence. In the present study, we identified sequences of A. sculptum salivary proteins that may be involved in hematophagy and selected three proteins that underwent functional characterization and evaluation as vaccine antigens. Among the three proteins selected, one contained a Kunitz_bovine pancreatic trypsin inhibitor domain (named AsKunitz) and the other two belonged to the 8.9 kDa and basic tail families of tick salivary proteins (named As8.9kDa and AsBasicTail). Expression of the messenger RNA (mRNA) encoding all three proteins was detected in the larvae, nymphs, and females at basal levels in unfed ticks and the expression levels increased after the start of feeding. Recombinant proteins rAs8.9kDa and rAsBasicTail inhibited the enzymatic activity of factor Xa, thrombin, and trypsin, whereas rAsKunitz inhibited only thrombin activity. All three recombinant proteins inhibited the hemolysis of both the classical and alternative pathways; this is the first description of tick members of the Kunitz and 8.9kDa families being inhibitors of the classical complement pathway. Mice immunization with recombinant proteins caused efficacies against A. sculptum females from 59.4% with rAsBasicTail immunization to more than 85% by immunization with rAsKunitz and rAs8.9kDa. The mortality of nymphs fed on immunized mice reached 70-100%. Therefore, all three proteins are potential antigens with the possibility of becoming a new tool in the control of A. sculptum ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Costa, Ribeiro, Melo-Junior, Gontijo, Sant’Anna, Pereira, Pessoa, Koerich, Oliveira, Valenzuela, Giunchetti, Fujiwara, Bartholomeu and Araujo.)

The active locomotion of ticks is directly associated with the epidemiology of tick-borne diseases, as it has important implications for the interaction of ticks with their hosts and their dispersion in the environment. In an attempt to elucidate the factors involved in the dispersion of Amblyomma sculptum, the present work aimed to characterize different aspects of the active locomotion of A. sculptum nymphs under laboratory conditions. To this end, nymphs were placed on a string at a 70° inclination and their walking activity was recorded daily along with their survival period. During their lifetime, ticks walked an average of 110 m. Their locomotion was not in a straight line and nymphs changed direction 142 times throughout their lifetimes. The mean distance walked per experimental day was 1.8 m, while the average walking distance before changing direction was 52 cm. The distance walked per experimental day reduced over time. The survival of ticks was affected by walking; resting nymphs survived for over 6 months, while the survival of those that walked daily was reduced to approximately 62 days. The results showed that A. sculptum nymphs were able to cover distances of over 100 m throughout their lifetimes, but they walked short distances at a time and constantly changed direction. This behavior indicates that ticks are not able to disperse over long distances by means of active locomotion., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

In sandflies, males and females feed on carbohydrates but females must get a blood meal for egg maturation. Using artificial blood meals, this study aimed to understand how galactosamine interferes with sandfly digestive physiology. We also used galactosamine to manipulate the digestive physiology of Lutzomyia longipalpis to investigate its influence on sandfly digestion and Leishmania development within their insect vectors. Galactosamine was capable to reduce Lu. longipalpis trypsinolytic activity in a dose-dependent manner. This effect was specific to galactosamine as other similar sugars were not able to affect sandfly trypsin production. An excess of amino acids supplemented with the blood meal and 15 mM galactosamine was able to abrogate the reduction of the trypsinolytic activity caused by galactosamine, suggesting this phenomenon may be related to an impairment of amino acid detection by sandfly enterocytes. The TOR inhibitor rapamycin reduces trypsin activity in the L. longipalpis midgut. Galactosamine reduces the phosphorylation of the TOR pathway repressor 4EBP, downregulating TOR activity in the gut of L. longipalpis. Galactosamine reduces sandfly oviposition, causes an impact on sandfly longevity and specifically reduces sandfly gut proteases whereas increasing α-glycosidase activity. The administration of 15 and 30 mM galactosamine increased the number of promastigote forms of Le. mexicana and Le. infantum in galactosamine-treated L. longipalpis. Our results showed that galactosamine influences amino acid sensing, reduces sandfly gut protease activity through TOR downregulation, and benefits Leishmania growth within the Lu. longipalpis gut., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aedes aegypti is the main urban vector of dengue virus, chikungunya virus and Zika virus due to its great dispersal capacity and virus susceptibility. A. aegypti feed on plant-derived sugars but females need a blood meal for egg maturation. Haematophagous arthropods need to overcome host haemostasis and local immune reactions in order to take a blood meal. In this context, molecules present in the saliva and/or intestinal contents of these arthropods must contain inhibitors of the complement system (CS). CS salivary and/or intestinal inhibitors are crucial to protect gut cells of haematophagous arthropods against complement attack. The present work aimed to investigate the anti-complement activity of A. aegypti intestinal contents on the alternative, classical and lectin pathways of the human complement system. Here we show that A. aegypti gut contents inhibited the human classical and the lectin pathways but not the alternative pathway. The A. aegypti gut content has a serine protease able to specifically cleave and inactivate human C4, which is a novel mechanism for human complement inactivation in haematophagous arthropods. The gut of female A. aegypti was capable of capturing human serum factor H (a negative complement modulator), unlike males. C3 molecules in recently blood-fed female A. aegypti remain in their original state, being inactivated to iC3b soon after a blood feed. A transmission-blocking vaccine using these complement inhibitory proteins as antigens has the potential to interfere with the insect's survival, reproductive fitness and block their infection by the arboviruses they transmit to humans., Competing Interests: Declaration of competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In Lutzomyia longipalpis females, which are the main vectors of Leishmania infantum in the Americas, hematophagy is crucial for ovary development. The control of pH in the midgut during blood digestion is important to the functioning of the digestive enzymes, which release amino acids in the luminal compartment that are then transported through the enterocytes to the hemolymph for delivery to the ovary and other organs. In the present work, we investigated transport systems known as LuloPATs that are present in the midgut of L. longipalpis but not in other organs. These transporters achieve symport of amino acids with H + ions, and one of them (LuloPAT1) is orthologous to a transporter described in Aedes aegypti. According to our results, the transcription levels of LuloPAT1 increased significantly immediately after a blood meal. Based on the variation of the fluorescence of fluorescein with the pH of the medium, we developed a technique that shows the acidification of the cytoplasm of gut cells when amino acids are cotransported with H + from the lumen into the enterocytes. In our experiments, the midguts of the sandflies were dissected and opened longitudinally so that added amino acids could enter the enterocytes via the lumen (PAT carriers are apical). LuloPAT1 transporters are part of a complex of mechanisms that act synergistically to promote gut alkalinization as soon as blood intake by the vector occurs. In dissected but not longitudinally opened midguts, added amino acids could only enter through the basolateral region of enterocytes. However, alkalinization of the lumen was observed because the entry of some amino acids into the cytoplasm of enterocytes triggers a luminal alkalinization mechanism independent of LuloPATs. These findings provide new perspectives that will enable the characterization of the set of signaling pathways involved in pH regulation within the L. longipalpis midgut., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Complement inhibitors are present in all hematophagous arthropods. Lutzomyia longipalpis is an important vector of Leishmania infantum, the etiologic agent of visceral leishmaniasis in the Americas. Studies with this vector identified complement inhibitors and respective inhibitory mechanisms. Despite the studies conducted with L. longipalpis, there is a gap in the knowledge about what happens in vivo with the complement present in the blood ingested. The experiments reported here show that the soluble inhibitor present in the intestinal lumen can act on the classical pathway of the human complement system by inhibiting the cascade soon after the activation of the C4 component. This means that this inhibitor can inhibit both the classical and lectin pathways. In the absence of salivary or gut inhibitors, the intestinal epithelium can activate the alternative pathway. At the same time, it can activate the lectin and the classical pathways by binding of MBL as well as by an antibody-independent C1 deposition mechanism. Without the salivary and intestinal inhibitors, the sand fly midgut epithelium may be more susceptible to complement attack as indicated by the C9/C3 deposition ratio when compared with intestines after a blood feed on a human host. In L. longipalpis, most of the C3 molecules present inside the midgut after a blood meal are found in their native form (not activated C3) or are present as iC3b (its inactivated form). C3b inactivation to iC3b, on the intestinal surface, is probably performed by a mechanism involving the uptake of factor H by the intestinal epithelium. Factor H is a negative complement regulator present in the plasma. Collectively, these results indicate how the complement inhibitors are necessary for a successful hematophagy in a sand fly model., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Whole mitogenome sequences (mtDNA) have been exploited for insect ecology studies, using them as molecular markers to reconstruct phylogenies, or to infer phylogeographic relationships and gene flow. Recent Anopheles phylogenomic studies have provided information regarding the time of deep lineage divergences within the genus. Here we report the complete 15,393 bp mtDNA sequences of Anopheles aquasalis, a Neotropical human malaria vector. When comparing its structure and base composition with other relevant and available anopheline mitogenomes, high similarity and conserved genomic features were observed. Furthermore, 22 mtDNA sequences comprising anopheline and Dipteran sibling species were analyzed to reconstruct phylogenies and estimate dates of divergence between taxa. Phylogenetic analysis using complete mtDNA sequences suggests that A. aquasalis diverged from the Anopheles albitarsis complex ~28 million years ago (MYA), and ~38 MYA from Anopheles darlingi. Bayesian analysis suggests that the most recent ancestor of Nyssorhynchus and Anopheles + Cellia was extant ~83 MYA, corroborating current estimates of ~79-100 MYA. Additional sampling and publication of African, Asian, and North American anopheline mitogenomes would improve the resolution of the Anopheles phylogeny and clarify early continental dispersal routes., Competing Interests: The authors have declared that no competing interests exist.

Ornithodoros rostratus is a South American argasid tick which importance relies on its itchy bite and potential as disease vector. They feed on a wide variety of hosts and secrete different molecules in their saliva and intestinal content that counteract host defences and help to accommodate and metabolize the relatively large quantity of blood upon feeding. The present work describes the transcriptome profile of salivary gland (SG) and midgut (MG) of O. rostratus using Illumina sequencing. A total of 8,031 contigs were assembled and assigned to different functional classes. Secreted proteins were the most abundant in the SG and accounted for ~67% of all expressed transcripts with contigs with identity to lipocalins and acid tail proteins being the most representative. On the other hand, immunity genes were upregulated in MG with a predominance of defensins and lysozymes. Only 10 transcripts in SG and 8 in MG represented ~30% of all RNA expressed in each tissue and one single contig (the acid tail protein ORN-9707) represented ~7% of all expressed contigs in SG. Results highlight the functional difference of each organ and identified the most expressed classes and contigs of O. rostratus SG and MG.

Philornis is a neotropical genus of muscid fly that interacts with birds and may affect the development and survival of the birds' offspring. Although Philornis is a relatively common parasite, there is a lack of information about Philornis hosts in several parts of the Americas. In this study, two nests of the Rufousfronted Thornbird ( Phacellodomus rufifrons) were collected in Pedro Leopoldo, southeast Brazil. The first contained four nestlings of advanced age (about 20 d old) and a recently emerged Philornis torquans female adult fly. The second nest contained three nestlings (less than 7 d old) and several Philornis torquans subcutaneous larvae. One of the nestlings was infested by 53 larvae, which had attacked several parts of its body and caused individual wounds containing 1 to more than 15 larvae. The length of the larvae ranged from 3 to 18 mm and only one was a second instar; the remaining 69 were third instars. The pupal period lasted 9-13 d. In total, 71 larvae were collected from the nest, with nestling parasitism varying from 7 to 53 larvae (mean- 23.7±25.5 larvae/nestling).

Innate immunity is an ancient and conserved defense system that provides an early effective response against invaders. Many immune genes of Anopheles mosquitoes have been implicated in defense against a variety of pathogens, including plasmodia. Nevertheless, only recent work identified some immune genes of Anopheles aquasalis mosquitoes upon P. vivax infection. Among these was a GATA transcription factor gene, which is described here. This is an ortholog of GATA factor Serpent genes described in Drosophila melanogaster and Anopheles gambiae. Gene expression analyses showed an increase of GATA-Serpent mRNA in P. vivax-infected A. aquasalis and functional RNAi experiments identified this transcription factor as an important immune gene of A. aquasalis against both bacteria and P. vivax. Besides, we were able to identify an effect of GATA-Serpent knockdown on A. aquasalis hemocyte proliferation and differentiation. These findings expand our understanding of the poorly studied A. aquasalis-P. vivax interactions and uncover GATA-Serpent as a key player of the mosquito innate immune response., Competing Interests: The authors have declared that no competing interests exist.

Anopheline mosquitoes are vectors of malaria parasites. Their saliva contains anti-hemostatic and immune-modulator molecules that favor blood feeding and parasite transmission. In this study, we describe the inhibition of the alternative pathway of the complement system (AP) by Anopheles aquasalis salivary gland extracts (SGE). According to our results, the inhibitor present in SGE acts on the initial step of the AP blocking deposition of C3b on the activation surfaces. Properdin, which is a positive regulatory molecule of the AP, binds to SGE. When SGE was treated with an excess of properdin, it was unable to inhibit the AP. Through SDS-PAGE analysis, A. aquasalis presented a salivary protein with the same molecular weight as recombinant complement inhibitors belonging to the SG7 family described in the saliva of other anopheline species. At least some SG7 proteins bind to properdin and are AP inhibitors. Searching for SG7 proteins in the A. aquasalis genome, we retrieved a salivary protein that shared an 85% identity with albicin, which is the salivary alternative pathway inhibitor from A. albimanus. This A. aquasalis sequence was also very similar (81% ID) to the SG7 protein from A. darlingi, which is also an AP inhibitor. Our results suggest that the salivary complement inhibitor from A. aquasalis is an SG7 protein that can inhibit the AP by binding to properdin and abrogating its stabilizing activity. Albicin, which is the SG7 from A. albimanus, can directly inhibit AP convertase. Given the high similarity of SG7 proteins, the SG7 from A. aquasalis may also directly inhibit AP convertase in the absence of properdin., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Due to an abundance of repetitive DNA, the annotation of heterochromatic regions of the genome such as the Y chromosome is problematic. The Y chromosome is involved in key biological functions such as male-fertility and sex-determination and hence, accurate identification of its sequences is vital. The hemipteran insect Rhodnius prolixus is an important vector of Chagas disease, a trypanosomiasis affecting 6-7 million people worldwide. Here we report the identification of the first Y-linked genes of this species., Results: The R. prolixus genome was recently sequenced using separate libraries for each sex and the sequences assembled only with male reads are candidates for Y linkage. We found 766 such candidates and PCR tests with the ten largest ones, confirmed Y-linkage for all of them, suggesting that "separate libraries" is a reliable method for the identification of Y-linked sequences. BLAST analyses of the 766 candidate scaffolds revealed that 568 scaffolds contained genes or part of putative genes. We tested Y-linkage for 36 candidates and found that nine of them are Y-linked (the PCR results for the other 25 genes were inconclusive or revealed autosomal/X-linkage). Hence, we describe in this study, for the first time, Y-linked genes in the R. prolixus genome: two zinc finger proteins (Znf-Y1 and Znf-Y2), one metalloproteinase (Met-Y), one aconitase/iron regulatory protein (Aco-Y) and five genes devoid of matches in any database (Rpr-Y1 to Rpr-Y5). Expression profile studies revealed that eight genes are expressed mainly in adult testis (some of which presented a weak expression in the initial developmental stages), while Aco-Y has a gut-restricted expression., Conclusions: In this study we showed that the approach used for the R. prolixus genome project (separate sequencing of male and female DNA) is key to easy and fast identification of sex-specific (e.g. Y chromosome sequences). The nine new R. prolixus Y-linked genes reported here provide unique markers for population and phylogenetic analysis and further functional studies of these genes may answer some questions about sex determination, male fertility and Y chromosome evolution in this important species.

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.

Y chromosomes, with their reduced effective population size, lack of recombination, and male-limited transmission, present a unique collection of constraints for the operation of natural selection. Male-limited transmission may greatly increase the efficacy of selection for male-beneficial mutations, but the reduced effective size also inflates the role of random genetic drift. Together, these defining features of the Y chromosome are expected to influence rates and patterns of molecular evolution on the Y as compared with X-linked or autosomal loci. Here, we use sequence data from 11 genes in 9 Drosophila species to gain insight into the efficacy of natural selection on the Drosophila Y relative to the rest of the genome. Drosophila is an ideal system for assessing the consequences of Y-linkage for molecular evolution in part because the gene content of Drosophila Y chromosomes is highly dynamic, with orthologous genes being Y-linked in some species whereas autosomal in others. Our results confirm the expectation that the efficacy of natural selection at weakly selected sites is reduced on the Y chromosome. In contrast, purifying selection on the Y chromosome for strongly deleterious mutations does not appear to be compromised. Finally, we find evidence of recurrent positive selection for 4 of the 11 genes studied here. Our results thus highlight the variable nature of the mode and impact of natural selection on the Drosophila Y chromosome., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The heme molecule is the prosthetic group of many hemeproteins involved in essential physiological processes, such as electron transfer, transport of gases, signal transduction, and gene expression modulation. However, heme is a pro-oxidant molecule capable of propagating reactions leading to the generation of reactive oxygen species. The blood-feeding insect Rhodnius prolixus releases enormous amounts of heme during host blood digestion in the midgut lumen when it is exposed to a physiological oxidative challenge. Additionally, this organism produces a hemolymphatic heme-binding protein (RHBP) that transports heme to pericardial cells for detoxification and to growing oocytes for yolk granules and as a source of heme for embryo development. Here, we show that silencing of RHBP expression in female fat bodies reduced total RHBP circulating in the hemolymph, promoting oxidative damage to hemolymphatic proteins. Moreover, RHBP knockdown did not cause reduction in oviposition but led to the production of heme-depleted eggs (white eggs). A lack of RHBP did not alter oocyte fecundation. However, produced white eggs were nonviable. Embryo development cellularization and vitellin yolk protein degradation, processes that normally occur in early stages of embryogenesis, were compromised in white eggs. Total cytochrome c content, cytochrome c oxidase activity, citrate synthase activity, and oxygen consumption, parameters that indicate mitochondrial function, were significantly reduced in white eggs compared with normal dark red eggs. Our results showed that reduction of heme transport from females to growing oocytes by RHBP leads to embryonic mitochondrial dysfunction and impaired embryogenesis.

Two species of the genus Trypanosoma infective to humans have been extensively studied at a cell and molecular level, but study of the third, Trypanosoma rangeli, remains in relative infancy. T. rangeli is non-pathogenic, but is frequently mistaken for the related Chagas disease agent Trypanosoma cruzi with which it shares vectors, hosts, significant antigenicity and a sympatric distribution over a wide geographical area. In this study, we present the T. rangeli gene expression profile as determined by the generation of ESTs (Expressed Sequence Tags) and ORESTES (Open Reading Frame ESTs). A total of 4208 unique high quality sequences were analyzed, composed from epimastigote and trypomastigote forms of SC-58 and Choachí strains, representing the two major phylogenetic lineages of this species. Comparative analyses with T. cruzi and other parasitic kinetoplastid species allowed the assignment of putative biological functions to most of the sequences generated and the establishment of an annotated T. rangeli gene expression database. Even though T. rangeli is apathogenic to mammals, genes associated with virulence in other pathogenic kinetoplastids were found. Transposable elements and genes associated mitochondrial gene expression, specifically RNA editing components, are also described for the first time. Our studies confirm the close phylogenetic relationship between T. cruzi and T. rangeli and enable us to make an estimate for the size of the T. rangeli genome repertoire ( approximately 8500 genes)., (Copyright 2010 Elsevier B.V. All rights reserved.)

Classically, Y chromosomes are thought to originate from X chromosomes through a process of degeneration and gene loss. Now, the availability of 12 Drosophila genomes provides an opportunity to study the origin and evolution of Y chromosomes in an informative phylogenetic context. Surprisingly, the majority of Drosophila Y-linked genes are recent acquisitions from autosomes and Y chromosome gene gains are more frequent than gene losses. Moreover, the Drosophila pseudoobscura Y chromosome lacks homology with the Y of most Drosophila species. Thus, the Drosophila Y has a different evolutionary history from canonical Y chromosomes (such as the mammalian Y) and it also might have a different origin.

Chromosomal organization is sufficiently evolutionarily stable that large syntenic blocks of genes can be recognized even between species as distantly related as mammals and puffer fish (450 million years (Myr) of divergence). In Diptera, the gene content of the X chromosome and the autosomes is well conserved: in Drosophila more than 95% of the genes have remained on the same chromosome arm in the 12 sequenced species (63 Myr of divergence, traversing 400 Myr of evolution), and the same linkage groups are clearly recognizable in mosquito genomes (260 Myr of divergence). Here we investigate the conservation of Y-linked gene content among the 12 sequenced Drosophila species. We found that only a quarter of the Drosophila melanogaster Y-linked genes (3 out of 12) are Y-linked in all sequenced species, and that most of them (7 out of 12) were acquired less than 63 Myr ago. Hence, whereas the organization of other Drosophila chromosomes traces back to the common ancestor with mosquitoes, the gene content of the D. melanogaster Y chromosome is much younger. Gene losses are known to have an important role in the evolution of Y chromosomes, and we indeed found two such cases. However, the rate of gene gain in the Drosophila Y chromosomes investigated is 10.9 times higher than the rate of gene loss (95% confidence interval: 2.3-52.5), indicating a clear tendency of the Y chromosomes to increase in gene content. In contrast with the mammalian Y chromosome, gene gains have a prominent role in the evolution of the Drosophila Y chromosome.

The Y chromosome and other heterochromatic regions present special challenges for genome sequencing and for the annotation of genes. Here we describe two new genes (ARY and WDY) on the Drosophila melanogaster Y, bringing its number of known single-copy genes to 12. WDY may correspond to the fertility factor kl-1.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

Like in other trypanosomatids D-glucose is a crucial source of energy to Trypanosoma rangeli, a non-pathogenic parasite that in Central and South America infects triatomine vectors and different mammalian species, including humans. In several trypanosome species, D-glucose transporters were already described and cloned. In this study, we characterized the D-glucose transport activity present in 2 life-stage forms of T. rangeli (epimastigotes and trypomastigotes) using D-[U-14C]glucose as substrate. Our results indicate that T. rangeli transports D-glucose with high affinity in both epimastigote (Km 30 microM) and trypomastigotes (Km 80 microM) life-forms. Both transport activities were inhibited by Cytochalasin B and Phloretin, indicating that probably D-glucose uptake in T. rangeli is mediated by facilitated diffusion of the sugar. Significant differences were observed between epimastigotes and trypomastigotes in relation to their affinity for D-glucose analogues, and the predicted amino acid sequence of a putative D-glucose transporter from T. rangeli (TrHT1) showed a larger identity with the T. cruzi D-glucose transporter encoded by the TcrHT1 gene than with other transporters already characterized in trypanosomatids.

The life cycle of Triatoma klugi Carcavallo, Jurberg, Lent & Galvão 2001 was compared under laboratory conditions using two groups of the F1 generation obtained from field-collected bugs. Among the 100 nymphs weekly fed on mice (Group A) or chicken (Group B), 77% of Group A and 67% of Group B reached the adult stage, and the mean time from the first nymphal stage to adult was 190.08 +/- 28.31 days and 221.23 +/- 40.50, respectively. The average span in days for each stage per group and the number of blood meals required for each stage were also evaluated. The overall mortality rate was 23% and 33% for Groups A and B, respectively. The mean number of eggs laid per month in a three-month period was of 56.20, 51.70 and 73.20 for Group A, and 64.50, 53.50 and 38.71 for Group B. Despite the blood source, comparative analysis revealed no statistically significant differences in the life cycle of T. klugi under laboratory conditions. Infection rates over 60% were observed for both Trypanosoma cruzi strains tested. Even revealing high infection rates of the hemolymph by T. rangeli strains, T. klugi revealed no salivary gland infections and was not able to transmit the parasite.

In the present study, we report a simple method to induce high Trypanosoma rangeli differentiation in vitro, producing a large number of infective trypomastigote forms. Parasites from SC-58 (Brazil) and Choachi (Colombia) strains were cultivated at 27 degrees C in TC-100, Grace and DMEM media, each supplemented with 5% fetal bovine serum and prepared at three distinct pHs (6.0, 7.0, 8.0). Differentiation was microscopically evaluated at 0, 3 and 6 days after cultivation in each medium by determining the percentage of trypomastigotes in Giemsa-stained smears. Our data revealed similar results for both T. rangeli strains, showing (after 6 days of cultivation in DMEM medium, pH 8.0) the presence of about 80% of trypomastigotes. These culture-derived trypomastigotes proved to be infective to both Balb-C mice and Rhodnius spp, reaching the triatomine's salivary glands. Our results describe a new and easy method to induce high T. rangeli differentiation in vitro, allowing further studies on the antigenic constitution of trypomastigotes.

The susceptibility of four Rhodnius species to different Trypanosoma rangeli strains was evaluated using both intracoelomic inoculation and oral infection. Rhodnius prolixus, Rhodnius domesticus, Rhodnius neglectus and Rhodnius nasutus were infected with Trypanosoma rangeli Macias (Venezuela), Choachi (Colombia) and SC-58 (Brazil) strains, revealing distinct haemolymph and salivary glands infection rates. The obtained infection rates were revealed to be dependent on the method of infection and the triatomine species. Our results suggest the existence of a high adaptation between the strain and the local vector.