The initial aim of this study on Balb/C mice was to investigate the putative effects on feeding and appetite of isopentenyl pyrophosphate (IPP) and E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), also known as phosphoantigens (pAgs). HMBPP was recently shown to increase blood meal appetite in malaria mosquitoes. Both IPP and HMBPP are metabolites produced by the normal gut microbiota and apicomplexan parasites such as Plasmodium. To explore potential effects on appetite, male and female mice were treated by gavage with these metabolites, and body mass and gene expression were monitored in brain, stomach and small intestine at 3 h and 7 weeks. Body mass gain did not clearly differ between pAg-treated and water control mice. However, beginning between 4 and 7 weeks, the salivary glands of IPP-treated males began to swell. With the autoimmune Sjögren disease (SjD) in mind, we subsequently investigated the salivary glands after 1, 4 and 7 weeks of IPP treatment. Fast gene set enrichment analysis (FGSEA) of marginal zone B-cell (MZB) transcripts from salivary glands, together with B-cell infiltration in both sexes at 4 weeks, suggested similarities to SjD pathology. Using ELISA, we measured serum autoantibodies against Ro52, Ro60 and La. Multivariate analysis at 7 weeks showed treatment-associated trends: levels of anti-Ro52 and anti-La tended to increase in IPP-treated males, but not in females. Notably, IL-6 serum levels displayed a sex-dependent pattern, and PCA analyses of transcriptomic data from brain, stomach and small intestine—though with some exceptions—also indicated differential responses to pAgs between males and females.

V gamma 9V delta 2 T cells, the dominant gamma delta T cell subset in human peripheral blood, are stimulated by phosphoantigens, of which (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, is produced in the apicoplast of malaria parasites. Cell-free media from synchronised Plasmodium falciparum asexual ring, trophozoite, and schizont stage-cultures of high purity as well as media from ruptured schizont cultures, all stimulated V gamma 9V delta 2 T cell proliferation, as did media from pure gametocyte cultures, whereas media from uninfected erythrocytes cultures did not. The media from ruptured schizont cultures and all the asexual and gametocyte stage cultures contained only background iron levels, suggesting that all erythrocyte haemoglobin is consumed as the parasites develop and supporting that the phosphoantigens were released from intact parasitized erythrocytes. The V gamma 9V delta 2 T cell-stimulating agent was not affected by freezing, thawing or heating but was sensitive to phosphatase treatment, confirming its phosphoantigen identity. In summary, phosphoantigens are released from parasitised erythrocytes at all developmental blood stages.

Malaria infection renders humans more attractive to Anopheles gambiae sensu lato mosquitoes than uninfected people. The mechanisms remain unknown. We found that an isoprenoid precursor produced by Plasmodium falciparum, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), affects A. gambiae s. l. blood meal seeking and feeding behaviors as well as susceptibility to infection. HMBPP acts indirectly by triggering human red blood cells to increase the release of CO2, aldehydes, and monoterpenes, which together enhance vector attraction and stimulate vector feeding. When offered in a blood meal, HMBPP modulates neural, antimalarial, and oogenic gene transcription without affecting mosquito survival or fecundity; in a P. falciparum-infected blood meal, sporogony is increased.

Ethnopharmacological relevance: Resistance of the parasites to known antimalarial drugs has provided the necessity to find new drugs from natural products against malaria. The aim of the study was to evaluate the in vitro antiplasmodial activity of some plants used by Traditional Medical Practitioners (TMPs) of Prometra and Rukararwe in malaria treatment in Uganda to provide scientific proof of the efficacies claimed by these Herbalists.

Materials and methods: The air dried samples of Clerodendrum rotundifolium (leaves), Microglossa pyrifolia (leaves), Momordica foetida (leaves) and Zanthoxylum chalybeum (stem bark) used for malaria treatment by TMPs were successively extracted with ethyl acetate, methanol and water to yield twelve extracts. The extracts were tested against the chloroquine-sensitive (NF54) and chloroquine-resistant (FCR3) Plasmodium falciparum strains in vitro using the micro Mark III test which is based on assessing the inhibition of schizont maturation. A compound A was extracted and purified from the stem bark of Z. chalybeum and its structure was identified and confirmed by spectroscopic methods.

Results: Most of the extracts tested (92%) showed an antiplasmodial activity with IC50 < 50 mu g/mL. In spite of successive extractions with different solvents, potent anti-plasmodial activity (IC50 < 5 mu g/mL) was observed in the ethyl acetate, methanol and aqueous extracts of M. pyrifolia and C. rotundifolium. Preferential enrichments of activity into water (IC50 < 15 mu g/mL) and Ethyl acetate (IC50 < 5 mu g/mL) were seen in the case of M. foetida and Z chalybeum respectively. The most active extracts were from C rotundifolium and M. pyrifolia with IC50 values less than 2 mu g/mL. Phytochemical analysis of the extracts revealed the presence of saponins, tannins, flavonoids, alkaloids and cardiac glycocides. Fagaramide isolated from Z chalybeum had a higher activity (IC50 2.85 mu g/mL) against the chloroquine-resistant strain than against the chloroquine-senstive (IC50 16.6 mu g/mL) strain used in the study.

Conclusion: The plant extracts analysed in this study presented an average antiplasmodial activity (58%). This study revealed for the first time the antiplasmodial activity of the plant C. rotundofolium. It's the first time the compound fagaramide (N-isobutyl-3-(3,4-methylene dioxyphenyl) - 2E-propenamide) has been isolated from Z. chalybeum as one of the compounds that contribute to the activity of this plant against P. falciparum.

Four decades ago, immunological research was dominated by the field of lymphoid biology. It was commonly accepted that multicellular eukaryotes defend themselves through phagocytosis. The lack of lymphoid cells in insects and other simpler animals, however, led to the common notion that they might simply lack the capacity defend themselves with humoral factors. This view was challenged by microbiologist Hans G. Boman and co-workers in a series of publications that led to the advent of antimicrobial peptides as a universal arm of the immune system. Besides ingenious research, Boman ignited his work by posing the right questions. He started off by asking himself a simple question: 'Antibodies take weeks to produce while many microbes divide hourly; so how come we stay healthy?'. This led to two key findings in the field: the discovery of an inducible and highly potent antimicrobial immune response in Drosophila in 1972, followed by the characterization of cecropin in 1981. Despite broadly being considered an insect-specific response at first, the work of Boman and co-workers eventually created a bandwagon effect that unravelled various aspects of innate immunity. This article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'.

Elizabethkingia anophelis is a dominant bacterial species in the gut ecosystem of the malaria vector mosquito Anopheles gambiae. We recently sequenced the genomes of two strains of E. anophelis, R26(T) and Ag1, isolated from different strains of A. gambiae. The two bacterial strains are identical with a few exceptions. Phylogenetically, Elizabethkingia is closer to Chryseobacterium and Riemerella than to Flavobacterium. In line with other Bacteroidetes known to utilize various polymers in their ecological niches, the E. anophelis genome contains numerous TonB dependent transporters with various substrate specificities. In addition, several genes belonging to the polysaccharide utilization system and the glycoside hydrolase family were identified that could potentially be of benefit for the mosquito carbohydrate metabolism. In agreement with previous reports of broad antibiotic resistance in E. anophelis, a large number of genes encoding efflux pumps and blactamases are present in the genome. The component genes of resistance-nodulation-division type efflux pumps were found to be syntenic and conserved in different taxa of Bacteroidetes. The bacterium also displays hemolytic activity and encodes several hemolysins that may participate in the digestion of erythrocytes in the mosquito gut. At the same time, the OxyR regulon and antioxidant genes could provide defense against the oxidative stress that is associated with blood digestion. The genome annotation and comparative genomic analysis revealed functional characteristics associated with the symbiotic relationship with the mosquito host.

Elizabethkingia anophelis is a species in the family Flavobacteriaceae. It is a dominant resident in the mosquito gut and also a human pathogen. We present the draft genome sequences of two strains of E. anophelis, R26^T and Ag1, which were isolated from the midgut of the malaria mosquito Anopheles gambiae.

Despite efficient vector transmission, Plasmodium parasites suffer great bottlenecks during their developmental stages within Anopheles mosquitoes. The outcome depends on a complex three-way interaction between host, parasite and gut bacteria. Although considerable progress has been made recently in deciphering Anopheles effector responses, little is currently known regarding the underlying microbial immune elicitors. An interesting candidate in this sense is the pathogen-derived prenyl pyrophosphate and designated phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), found in Plasmodium and most eubacteria but not in higher eukaryotes. HMBPP is the most potent stimulant known of human V gamma 9V delta 2 T cells, a unique lymphocyte subset that expands during several infections including malaria. In this study, we show that V(Y)9V delta 2 T cells proliferate when stimulated with supernatants from intraerythrocytic stages of Plasmodium falciparum cultures, suggesting that biologically relevant doses of phosphoantigens are excreted by the parasite. Next, we used Anopheles gambiae to investigate the immune-and redox-stimulating effects of HMBPP. We demonstrate a potent activation in vitro of all but one of the signaling pathways earlier implicated in the human V(Y)9V delta 2 T cell response, as p38, JNK and PI3K/Akt but not ERK were activated in the A. gambiae 4a3B cell line. Additionally, both HMBPP and the downstream endogenous metabolite isopentenyl pyrophosphate displayed antioxidant effects by promoting cellular tolerance to hydrogen peroxide challenge. When provided in the mosquito blood meal, HMBPP induced temporal changes in the expression of several immune genes. In contrast to meso-diaminopimelic acid containing peptidoglycan, HMBPP induced expression of dual oxidase and nitric oxide synthase, two key determinants of Plasmodium infection. Furthermore, temporal fluctuations in midgut bacterial numbers were observed. The multifaceted effects observed in this study indicates that HMBPP is an important elicitor in common for both Plasmodium and gut bacteria in the mosquito.

A new generation of strategies is evolving that aim to block malaria transmission by employing genetically modified vectors or mosquito pathogens or symbionts that express anti-parasite molecules. Whilst transgenic technologies have advanced rapidly, there is still a paucity of effector molecules with potent anti-malaria activity whose expression does not cause detrimental effects on mosquito fitness. Our objective was to examine a wide range of antimicrobial peptides (AMPs) for their toxic effects on Plasmodium and anopheline mosquitoes. Specifically targeting early sporogonic stages, we initially screened AMPs for toxicity against a mosquito cell line and P. berghei ookinetes. Promising candidate AMPs were fed to mosquitoes to monitor adverse fitness effects, and their efficacy in blocking rodent malaria infection in Anopheles stephensi was assessed. This was followed by tests to determine their activity against P. falciparum in An. gambiae, initially using laboratory cultures to infect mosquitoes, then culminating in preliminary assays in the field using gametocytes and mosquitoes collected from the same area in Mali, West Africa. From a range of 33 molecules, six AMPs able to block Plasmodium development were identified: Anoplin, Duramycin, Mastoparan X, Melittin, TP10 and Vida3. With the exception of Anoplin and Mastoparan X, these AMPs were also toxic to an An. gambiae cell line at a concentration of 25 µM. However, when tested in mosquito blood feeds, they did not reduce mosquito longevity or egg production at concentrations of 50 µM. Peptides effective against cultured ookinetes were less effective when tested in vivo and differences in efficacy against P. berghei and P. falciparum were seen. From the range of molecules tested, the majority of effective AMPs were derived from bee/wasp venoms.

In vector mosquitoes, the presence of midgut bacteria may affect the ability to transmit pathogens. We have used a laboratory colony of Aedes aegypti as a model for bacterial interspecies competition and show that after a blood meal, the number of species (culturable on LuriaBertani agar) that coexist in the midgut is low and that about 40% of the females do not harbor any cultivable bacteria. We isolated species belonging to the genera Bacillus, Elizabethkingia, Enterococcus, Klebsiella, Pantoea, Serratia, and Sphingomonas, and we also determined their growth rates, antibiotic resistance, and ex vivo inhibition of each other. To investigate the possible existence of coadaptation between midgut bacteria and their host, we fed Ae.similar to aegypti cohorts with gut bacteria from human, a frog, and two mosquito species and followed the bacterial population growth over time. The dynamics of the different species suggests coadaptation between host and bacteria, and interestingly, we found that Pantoea stewartii isolated from Ae.similar to aegypti survive better in Ae.similar to aegypti as compared to P.similar to stewartii isolated from the malaria mosquito Anopheles gambiae.