Clogmia albipunctata (Williston, 1893) midgut physiology: pH control and functional relationship with Lower Diptera (nematoceran) especially with hematophagous species.

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.

Evasion of the complement system by Leishmania through the uptake of C4bBP, a complement regulatory protein, and probably by the action of GP63 on C4b molecules deposited on parasite surface.

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.

cAMP: A second messenger involved in the mechanism of midgut alkalinization in Lutzomyia longipalpis.

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.

Evasion of the complement system by Leishmania through the uptake of factor H, a complement regulatory protein.

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.

Historical Perspective and Biotechnological Trends to Block Arboviruses Transmission by Controlling Aedes aegypti Mosquitos Using Different Approaches.

Continuous climate changes associated with the disorderly occupation of urban areas have exposed Latin American populations to the emergence and reemergence of arboviruses transmitted by Aedes aegypti. The magnitude of the financial and political problems these epidemics may bring to the future of developing countries is still ignored. Due to the lack of effective antiviral drugs and vaccines against arboviruses, the primary measure for preventing or reducing the transmission of diseases depends entirely on the control of vectors or the interruption of human-vector contact. In Brazil the first attempt to control A. aegypti took place in 1902 by eliminating artificial sites of eproduction. Other strategies, such as the use of oviposition traps and chemical control with dichlorodiphenyltrichlorethane and pyrethroids, were successful, but only for a limited time. More recently, biotechnical approaches, such as the release of transgenics or sterile mosquitoes and the, development of transmission blocking vaccines, are being applied to try to control the A. aegypti population and/or arbovirus transmission. Endemic countries spend about twice as much to treat patients as they do on the prevention of mosquito-transmitted diseases. The result of this strategy is an explosive outbreak of arboviruses cases. This review summarizes the social impacts caused by A. aegypti-transmitted diseases, mainly from a biotechnological perspective in vector control aimed at protecting Latin American populations against arboviruses.

The role of LuloPAT amino acid/proton symporters in midgut alkalinization in the sandfly Lutzomyia longipalpis (Diptera - Psychodidae).

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.

How Lutzomyia longipalpis deals with the complement system present in the ingested blood: The role of soluble inhibitors and the adsorption of factor H by midgut.

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.

Wolbachia introduction into Lutzomyia longipalpis (Diptera: Psychodidae) cell lines and its effects on immune-related gene expression and interaction with Leishmania infantum.

BACKGROUND: The leishmaniases are important neglected diseases caused by Leishmania spp. which are transmitted by sand flies, Lutzomyia longipalpis being the main vector of visceral leishmaniasis in the Americas. The methodologies for leishmaniasis control are not efficient, causing 1.5 million reported cases annually worldwide, therefore showing the need for development of novel strategies and interventions to control transmission of the disease. The bacterium Wolbachia pipientis is being used to control viruses transmitted by mosquitoes, such as dengue and Zika, and its introduction in disease vectors has been effective against parasites such as Plasmodium. Here we show the first successful establishment of Wolbachia into two different embryonic cell lines from L. longipalpis, LL-5 and Lulo, and analysed its effects on the sand fly innate immune system, followed by in vitro Leishmania infantum interaction. RESULTS: Our results show that LL-5 cells respond to wMel and wMelPop-CLA strains within the first 72 h post-infection, through the expression of antimicrobial peptides and inducible nitric oxide synthase resulting in a decrease of Wolbachia detection in the early stages of infection. In subsequent passages, the wMel strain was not able to infect any of the sand fly cell lines while the wMelPop-CLA strain was able to stably infect Lulo cells and LL-5 at lower levels. In Wolbachia stably infected cells, the expression of immune-related genes involved with downregulation of the IMD, Toll and Jak-Stat innate immune pathways was significantly decreased, in comparison with the uninfected control, suggesting immune activation upon Wolbachia transinfection. Furthermore, Wolbachia transinfection did not promote a negative effect on parasite load in those cells. CONCLUSIONS: Initial strong immune responses of LL5 cells might explain the inefficiency of stable infections in these cells while we found that Lulo cells are more permissive to infection with Wolbachia causing an effect on the cell immune system, but not against in vitro L. infantum interaction. This establishes Lulo cells as a good system for the adaptation of Wolbachia in L. longipalpis.

The small non-coding RNA response to virus infection in the Leishmania vector Lutzomyia longipalpis.

Sandflies are well known vectors for Leishmania but also transmit a number of arthropod-borne viruses (arboviruses). Few studies have addressed the interaction between sandflies and arboviruses. RNA interference (RNAi) mechanisms utilize small non-coding RNAs to regulate different aspects of host-pathogen interactions. The small interfering RNA (siRNA) pathway is a broad antiviral mechanism in insects. In addition, at least in mosquitoes, another RNAi mechanism mediated by PIWI interacting RNAs (piRNAs) is activated by viral infection. Finally, endogenous microRNAs (miRNA) may also regulate host immune responses. Here, we analyzed the small non-coding RNA response to Vesicular stomatitis virus (VSV) infection in the sandfly Lutzoymia longipalpis. We detected abundant production of virus-derived siRNAs after VSV infection in adult sandflies. However, there was no production of virus-derived piRNAs and only mild changes in the expression of vector miRNAs in response to infection. We also observed abundant production of virus-derived siRNAs against two other viruses in Lutzomyia Lulo cells. Together, our results suggest that the siRNA but not the piRNA pathway mediates an antiviral response in sandflies. In agreement with this hypothesis, pre-treatment of cells with dsRNA against VSV was able to inhibit viral replication while knock-down of the central siRNA component, Argonaute-2, led to increased virus levels. Our work begins to elucidate the role of RNAi mechanisms in the interaction between L. longipalpis and viruses and should also open the way for studies with other sandfly-borne pathogens.

Triatomines (Hemiptera, Reduviidae) blood intake: Physical constraints and biological adaptations.

In order to efficiently obtain blood from their vertebrate hosts, bloodsucking arthropods have undergone an evolutionary selection process leading to specialist adaptations in their feeding apparatus (mouthparts and suction pumps) and salivary molecules. These adaptations act to counteract haemostasis, inflammation, and immune responses in their vertebrate hosts. The association of haematophagous arthropods with vertebrate hosts during a blood feed allows the transmission of pathogens between their hosts and vectors in a tripartite interaction. Feeding mechanisms in haematophagous arthropod species have been the subject of studies over at least eight decades worldwide, as a consequence of the importance of vector-borne diseases and their impact on human health. Here we review studies of the feeding mechanisms of triatomine bugs, with a particular focus on factors that influence their feeding performance when obtaining a blood meal from different vertebrate hosts.

Physiological characterization of the hematophagy of Ornithodoros rostratus (Acari: Argasidae) on live hosts.

Ornithodoros rostratus is an argasid tick and its importance is based on its hematophagy and the resulting transmission of pathogens such as Rickettsia rickettsii and Coxiella burnetii to its vertebrate hosts. In the face of a lack of physiological studies related to hematophagy in argasid ticks, this paper aims to identify and characterize the events that occur throughout the feeding by O. rostratus on live hosts. Electrical signals and alterations on the feeding site were monitored using intravital microscopy and electromyography. The analyses allowed for the characterization of four distinct events: suction, salivation, chelicerae movements and inactivity. Feeding was divided into two distinct phases: (1) penetration of mouthparts (when only salivation and chelicerae movements occurred) and the formation of the feeding pool (salivation and chelicerae movements with the first signs of suction) and (2) engorgement, during which chelicerae movements ceased and blood intake took place in feeding complexes (salivation followed by suction). Variations in patterns of the electrical signals, suction frequency and salivation showed four distinct sub-phases: (2a) suction with electrical signals of irregular shape, increased suction frequency and decreased salivation frequency throughout blood feeding; (2b) suction with electrical signals of symmetrical shape, high suction rates (3.8 Hz on average) and feeding complexes lasting for 7.7 s; (2c) suction with electrical signals of irregular shape, high suction frequency and feeding complex lasting 11.5 s; and (2d) electrical signals with no profile and the longest feeding complexes (14.5 s). Blood feeding ended with the withdrawal of the mouthparts from the host's skin.

Life cycle of Ornithodoros rostratus (Acari: Argasidae) ticks feeding on mice under laboratory conditions.

Ornithodoros rostratus Aragão is an argasid tick found in Bolivia, Paraguay, Argentina and Brazil. Only limited studies about O. rostratus have been conducted and several aspects of their life cycle differ among studies or remain unexplored. In order to better elucidate the biology of O. rostratus, the present work describes its life cycle when feeding on mice under laboratory conditions. To complete their life cycle on mice, O. rostratus goes through a larval stage, 3-6 nymphal instars (nymph 1-6) and adult male and female. Adults can be originated from nymph 3-6. Nymphs 4 with higher weight after feeding tend to originate adults. Adults originated from early instars tended to be lighter. Females tended to be heavier than males. Larvae needed on average 2.7 days to complete their blood meal whereas other instars ranged from 17.3 to 78.3 min. The capacity to ingest blood was higher in larvae and females in comparison to males. The preecdysis period ranged from 5 to 12.5 days. After one blood meal, females remain on average 15.2 ± 5.8 days laying 276.8 ± 137.2.9 eggs. Females originated from nymph 4 had similar oviposition time, egg incubation and conversion ingested blood/number of eggs produced, but presented lower initial weigh and weigh gain, generating fewer eggs. Our results added novel information on O. rostratus biology and was discussed considering the variability of argasid populations and in context with the differences about their life cycle described in previous works.