Blood Digestion in the Mosquito, Anopheles stephensi Liston (Diptera: Culicidae): Activity and Distribution of Trypsin, Aminopeptidase, and α-Glucosidase in the Midgut

The activities of trypsin, aminopeptidase, and α-glucosidase were studied in the whole midgut, anterior and posterior midgut, and posterior midgut lumen and epithelium of the mosquito Anopheles stephensi Liston. Trypsin activity was restricted entirely to the posterior midgut lumen. No trypsin activity was found before the blood meal, but activity increased continuously up to 30 h after feeding, and subsequently returned to baseline levels by 60 h. Aminopeptidase was active in anterior and posterior midgut regions before and after feeding. In whole midguts, activity rose from a baseline of ≍3 enzyme units (EU) per midgut to a maximum of 12 EU at 30 h after the blood meal, subsequently falling to baseline levels by 60 h. A similar cycle of activity was observed in the posterior midgut and posterior midgut lumen, whereas aminopeptidase in the posterior midgut epithelium decreased in activity during digestion. Aminopeptidase in the anterior midgut was maintained at a constant low level, showing no significant variation with time after feeding, α-glucosidase was active in anterior and posterior midguts before and at all times after feeding. In whole midgut homogenates, α-glucosidase activity increased slowly up to 18 h after the blood meal, then rose rapidly to a maximum at 30 h after the blood meal, whereas the subsequent decline in activity was less predictable. All posterior midgut activity was restricted to the posterior midgut lumen. Depending upon the time after feeding, >25% of the total midgut activity of α-glucosidase was located in the anterior midgut. The enzyme distributions are consistent with described structural models for digestion in mosquitoes. After blood meal ingestion, proteases are active only in the posterior midgut. Trypsin is the major primary hydrolytic protease and is secreted into the posterior midgut lumen without activation in the posterior midgut epithelium. Aminopeptidase activity is also luminal in the posterior midgut, but cellular aminopeptidases are required for peptide processing in both anterior and posterior midguts. α-glucosidase activity is elevated in the posterior midgut after feeding in response to the blood meal, whereas activity in the anterior midgut is consistent with a nectarprocessing role for this midgut regio

Mosquito cell line glycoproteins: an unsuitable model system for the Plasmodium ookinete-mosquito midgut interaction?

<p>Abstract</p> <p>Background</p> <p>Mosquito midgut glycoproteins may act as key recognition sites for the invading malarial ookinete. Effective transmission blocking strategies require the identification of novel target molecules. We have partially characterised the surface glycoproteins of two cell lines from two mosquito species; <it>Anopheles stephensi </it>and <it>Anopheles gambiae</it>, and investigated the binding of <it>Plasmodium berghei </it>ookinetes to carbohydrate ligands on the cells. Cell line extracts were run on SDS-PAGE gels and carbohydrate moieties determined by blotting against a range of biotinylated lectins. In addition, specific glycosidases were used to cleave the oligosaccharides.</p> <p>Results</p> <p><it>An. stephensi </it>43 and <it>An. gambiae </it>55 cell line glycoproteins expressed oligosaccharides containing oligomannose and hybrid oligosaccharides, with and without α1-6 core fucosylation; N-linked oligosaccharides with terminal Galβ1-3GalNAc or GalNAcβ1-3Gal; <it>O</it>-linked α/βGalNAc. <it>An. stephensi </it>43 cell line glycoproteins also expressed <it>N</it>-linked Galβ1-4R and <it>O</it>-linked Galβ1-3GalNAc. Although <it>P. berghei </it>ookinetes bound to both mosquito cell lines, binding could not be inhibited by GlcNAc, GalNAc or Galactose.</p> <p>Conclusions</p> <p>Anopheline cell lines displayed a limited range of oligosaccharides. Differences between the glycosylation patterns of the cell lines and mosquito midgut epithelial cells could be a factor why ookinetes did not bind in a carbohydrate inhibitable manner. Anopheline cell lines are not suitable as a potential model system for carbohydrate-mediated adhesion of <it>Plasmodium </it>ookinetes.</p

Infection and treatment immunizations for successful parasite vaccines

Since the advent of techniques for the expression of recombinant peptide antigens, the availability of human vaccines for parasitic diseases has been ‘imminent’. Yet vaccines based on recombinant proteins are still largely aspirations, not realities. It is now apparent that vaccine development needs additional knowledge about host protective immune response(s), antigen characteristics, and the delivery required to induce those responses. The most successful immune protection against parasites has been generated by infection and treatment, the induction of protective immunity by truncating the course of an infection with drug treatment. Here, we consider the characteristics of an effective, protective anti-parasite vaccine and propose a conceptual framework to aid parasite vaccine development using malaria and schistosomiasis as examples

Feeding-associated gene expression in sheep scab mites (Psoroptes ovis)

The mite Psoroptes ovis is the causative agent of sheep scab. Although not usually fatal, the disease can spread rapidly and is a serious animal welfare concern. Vaccine development against ectoparasites has primarily focussed on two sources of candidate vaccine antigens – “exposed” antigens that are secreted in saliva during feeding on a host and “concealed” antigens that are usually expressed in the parasite gut and may be involved in digestion. Here, we sought to identify genes encoding proteins important for mite feeding and digestion by a subtractive suppressive hybridisation approach comparing mRNA transcript abundance in “fed” and “starved” mites. The study identified a variety of genes which are up-regulated by feeding mites. These included group 1, 5, 7 and 13 allergens including the previously described cysteine protease Pso o 1. In addition, numerous novel genes were identified here including some encoding potential salivary gland proteins and others encoding proteins which may facilitate feeding such as a serum opacity factor. An olfactory receptor-like protein was identified in the starved mite population which may help the mite to identify a host

Robust, reproducible, industrialized, standard membrane feeding assay for assessing the transmission blocking activity of vaccines and drugs against Plasmodium falciparum.

BackgroundA vaccine that interrupts malaria transmission (VIMT) would be a valuable tool for malaria control and elimination. One VIMT approach is to identify sexual erythrocytic and mosquito stage antigens of the malaria parasite that induce immune responses targeted at disrupting parasite development in the mosquito. The standard Plasmodium falciparum membrane-feeding assay (SMFA) is used to assess transmission-blocking activity (TBA) of antibodies against candidate immunogens and of drugs targeting the mosquito stages. To develop its P. falciparum sporozoite (SPZ) products, Sanaria has industrialized the production of P. falciparum-infected Anopheles stephensi mosquitoes, incorporating quantitative analyses of oocyst and P. falciparum SPZ infections as part of the manufacturing process.MethodsThese capabilities were exploited to develop a robust, reliable, consistent SMFA that was used to assess 188 serum samples from animals immunized with the candidate vaccine immunogen, Pfs25, targeting P. falciparum mosquito stages. Seventy-four independent SMFAs were performed. Infection intensity (number of oocysts/mosquito) and infection prevalence (percentage of mosquitoes infected with oocysts) were compared between mosquitoes fed cultured gametocytes plus normal human O(+) serum (negative control), anti-Pfs25 polyclonal antisera (MRA39 or MRA38, at a final dilution in the blood meal of 1:54 as positive control), and test sera from animals immunized with Pfs25 (at a final dilution in the blood meal of 1:9).ResultsSMFA negative controls consistently yielded high infection intensity (mean = 46.1 oocysts/midgut, range of positives 3.7-135.6) and infection prevalence (mean = 94.2%, range 71.4-100.0) and in positive controls, infection intensity was reduced by 81.6% (anti-Pfs25 MRA39) and 97.0% (anti-Pfs25 MRA38), and infection prevalence was reduced by 12.9 and 63.5%, respectively. A range of TBAs was detected among the 188 test samples assayed in duplicate. Consistent administration of infectious gametocytes to mosquitoes within and between assays was achieved, and the TBA of anti-Pfs25 control antibodies was highly reproducible.ConclusionsThese results demonstrate a robust capacity to perform the SMFA in a medium-to-high throughput format, suitable for assessing large numbers of experimental samples of candidate antibodies or drugs

Towards an Intelligent Tutor for Mathematical Proofs

Computer-supported learning is an increasingly important form of study since it allows for independent learning and individualized instruction. In this paper, we discuss a novel approach to developing an intelligent tutoring system for teaching textbook-style mathematical proofs. We characterize the particularities of the domain and discuss common ITS design models. Our approach is motivated by phenomena found in a corpus of tutorial dialogs that were collected in a Wizard-of-Oz experiment. We show how an intelligent tutor for textbook-style mathematical proofs can be built on top of an adapted assertion-level proof assistant by reusing representations and proof search strategies originally developed for automated and interactive theorem proving. The resulting prototype was successfully evaluated on a corpus of tutorial dialogs and yields good results.Comment: In Proceedings THedu'11, arXiv:1202.453

Direct venous inoculation of Plasmodium falciparum sporozoites for controlled human malaria infection: a dose-finding trial in two centres

BACKGROUND: Controlled human malaria infection (CHMI) accelerates development of anti-malarial interventions. So far, CHMI is done by exposure of volunteers to bites of five mosquitoes carrying Plasmodium falciparum sporozoites (PfSPZ), a technique available in only a few centres worldwide. Mosquito-mediated CHMI is logistically complex, exact PfSPZ dosage is impossible and live mosquito-based interventions are not suitable for further clinical development. METHODS: An open-labelled, randomized, dose-finding study in 18-45 year old, healthy, malaria-naive volunteers was performed to assess if intravenous (IV) injection of 50 to 3,200 aseptic, purified, cryopreserved PfSPZ is safe and achieves infection kinetics comparable to published data of mosquito-mediated CHMI. An independent study site verified the fully infectious dose using direct venous inoculation of PfSPZ. Parasite kinetics were assessed by thick blood smear microscopy and quantitative real time PCR. RESULTS: IV inoculation with 50, 200, 800, or 3,200 PfSPZ led to parasitaemia in 1/3, 1/3, 7/9, and 9/9 volunteers, respectively. The geometric mean pre-patent period (GMPPP) was 11.2 days (range 10.5-12.5) in the 3,200 PfSPZ IV group. Subsequently, six volunteers received 3,200 PfSPZ by direct venous inoculation at an independent investigational site. All six developed parasitaemia (GMPPP: 11.4 days, range: 10.4-12.3). Inoculation of PfSPZ was safe. Infection rate and pre-patent period depended on dose, and injection of 3,200 PfSPZ led to a GMPPP similar to CHMI with five PfSPZ-infected mosquitoes. The infectious dose of PfSPZ predicted dosage of radiation-attenuated PfSPZ required for successful vaccination. CONCLUSIONS: IV inoculation of PfSPZ is safe, well tolerated and highly reproducible. It shall further accelerate development of anti-malarial interventions through standardization and facilitation of CHMI. Beyond this, rational dose selection for whole PfSPZ-based immunization and complex study designs are now possible. TRIAL REGISTRATION: ClinicalTrials.gov NCT01624961 and NCT01771848