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Background The consequences of the earth’s daily rotation have led to 24-h biological rhythms in most organisms. Even some parasites are known to have daily rhythms, which, when in synchrony with host rhythms, can optimise their fitness. Understanding these rhythms may enable the development of control strategies that take advantage of rhythmic vulnerabilities. Recent work on protozoan parasites has revealed 24-h rhythms in gene expression, drug sensitivity and the presence of an intrinsic circadian clock; however, similar studies on metazoan parasites are lacking. To address this, we investigated if a metazoan parasite has daily molecular oscillations, whether they reveal how these longer-lived organisms can survive host daily cycles over a lifespan of many years and if animal circadian clock genes are present and rhythmic. We addressed these questions using the human blood fluke Schistosoma mansoni that lives in the vasculature for decades and causes the tropical disease schistosomiasis. Results Using round-the-clock transcriptomics of male and female adult worms collected from experimentally infected mice, we discovered that ~ 2% of its genes followed a daily pattern of expression. Rhythmic processes included a stress response during the host’s active phase and a ‘peak in metabolic activity’ during the host’s resting phase. Transcriptional profiles in the female reproductive system were mirrored by daily patterns in egg laying (eggs are the main drivers of the host pathology). Genes cycling with the highest amplitudes include predicted drug targets and a vaccine candidate. These 24-h rhythms may be driven by host rhythms and/or generated by a circadian clock; however, orthologs of core clock genes are missing and secondary clock genes show no 24-h rhythmicity. Conclusions There are daily rhythms in the transcriptomes of adult S. mansoni, but they appear less pronounced than in other organisms. The rhythms reveal temporally compartmentalised internal processes and host interactions relevant to within-host survival and between-host transmission. Our findings suggest that if these daily rhythms are generated by an intrinsic circadian clock then the oscillatory mechanism must be distinct from that in other animals. We have shown which transcripts oscillate at this temporal scale and this will benefit the development and delivery of treatments against schistosomiasis.

Control and elimination of the parasitic disease schistosomiasis relies on mass administration of praziquantel. Whilst these programmes reduce infection prevalence and intensity, their impact on parasite transmission and evolution is poorly understood. Here we examine the genomic impact of repeated mass drug administration on Schistosoma mansoni populations with documented reduced praziquantel efficacy. We sequenced whole-genomes of 198 S. mansoni larvae from 34 Ugandan children from regions with contrasting praziquantel exposure. Parasites infecting children from Lake Victoria, a transmission hotspot, form a diverse panmictic population. A single round of treatment did not reduce this diversity with no apparent population contraction caused by long-term praziquantel use. We find evidence of positive selection acting on members of gene families previously implicated in praziquantel action, but detect no high frequency functionally impactful variants. As efforts to eliminate schistosomiasis intensify, our study provides a foundation for genomic surveillance of this major human parasite., Schistosomiasis control strategies rely on mass drug administration (MDA) using praziquantel. Here, Berger et al. perform whole-genome sequencing of larvae from infected children across Ugandan regions with differing MDA histories. They find extensive gene flow with limited positive selection suggesting minimal change post MDA.

Schistosomules of the human parasite Schistosoma mansoni are vital for research focusing on the fundamental functional/developmental biology of schistosomes and many anti-schistosomal drug discovery programmes. Through the further evaluation and validation of a recently tested media, HybridoMed Diff 1000 (HM), for the cell-free culture of juvenile schistosomules, we show that while Basch medium was superior to HM for the survival/development of schistosomules, HM represents a viable and attractive alternative for somule culture, particularly to the early liver stage. Adoption of HM for schistosomule culture could facilitate more standardised approaches, which for drug screening should enable improved multi-centre target-hit evaluation.

Malaria poses a significant threat to approximately half of the world's population with an annual death toll close to half a million. The emergence of resistance to front-line antimalarials in the most lethal human parasite species, Plasmodium falciparum (Pf), threatens progress made in malaria control. The prospect of losing the efficacy of antimalarial drugs is driving the search for small molecules with new modes of action. Asexual reproduction of the parasite is critically dependent on the recycling of amino acids through catabolism of hemoglobin (Hb), which makes metalloaminopeptidases (MAPs) attractive targets for the development of new drugs. The Pf genome encodes eight MAPs, some of which have been found to be essential for parasite survival. In this article, we discuss the biological structure and function of each MAP within the Pf genome, along with the drug discovery efforts that have been undertaken to identify novel antimalarial candidates of therapeutic value.

In living cells, the genetic information stored in the DNA sequence is always associated with chromosomal and extra-chromosomal epigenetic information. Chromatin is formed by the DNA and associated proteins, in particular histones. Covalent histone modifications are important bearers of epigenetic information and as such have been increasingly studied since about the year 2000. One of the principal techniques to gather information about the association between DNA and modified histones is chromatin immunoprecipitation (ChIP), also combined with massive sequencing (ChIP-Seq). Automated ChIPmentation procedure is a convenient alternative to native chromatin immunoprecipitation (N-ChIP). It is now routinely used for ChIP-Seq in many model species, using in general roughly 106 cells per experiment. Such high cell numbers are sometimes difficult to produce. Using the human parasite Schistosoma mansoni, whose production requires sacrificing animals and should therefore be kept to a minimum, we show here that automated ChIPmentation is suitable for limited biological material. We define the operational limit as ≥20,000 Schistosoma cells. We also present a streamlined protocol for the preparation of ChIP input libraries.

Schistosomes are parasitic flatworms that cause schistosomiasis, a neglected tropical disease affecting over 200 million people. Schistosomes develop multiple body plans while navigating their complex life cycle, which involves two different hosts: a mammalian definitive host and a molluscan intermediate host. Their survival and propagation depend upon proliferation and differentiation of stem cells necessary for parasite homeostasis and reproduction. Infective larvae released from snails carry a handful of stem cells that serve as the likely source of new tissues as the parasite adapts to life inside the mammalian host; however, the role of these stem cells during this critical life cycle stage remains unclear. Here, we characterize stem cell fates during early intramammalian development. Surprisingly, we find that the esophageal gland, an accessory organ of the digestive tract, develops before the rest of the digestive system is formed and blood feeding is initiated, suggesting a role in processes beyond nutrient uptake. To explore such a role, we examine schistosomes that lack the esophageal gland due to knockdown of a forkhead-box transcription factor, Sm-foxA, which blocks development and maintenance of the esophageal gland, without affecting the development of other somatic tissues. Intriguingly, schistosomes lacking the esophageal gland die after transplantation into naive mice, but survive in immunodeficient mice lacking B cells. We show that parasites lacking the esophageal gland are unable to lyse ingested immune cells within the esophagus before passing them into the gut. These results unveil an immune-evasion mechanism mediated by the esophageal gland, which is essential for schistosome survival and pathogenesis.

Experimental research into malaria biology and pathogenesis has historically focused on two model systems, in vitro culture of the human parasite Plasmodium falciparum and in vivo infections of laboratory animals using rodent parasites. While there is clear value in having a manipulatable animal model for studying malaria, there have occasionally been controversies around how representative the rodent model is of the human disease, and therefore significant emphasis has been placed on the similarities between the two biological systems. By focusing on basic nuclear functions, we wish to highlight that identifying key differences in the parasites and their interactions with their mammalian hosts can be equally informative and provide remarkable insights into the biology and evolution of these important infectious organisms.

Emerging drug resistance and high-attrition rates in early and late stage drug development necessitate accelerated development of antimalarial compounds. However, systematic and meaningful translation of drug efficacy and host-parasite dynamics between preclinical testing stages is missing. We developed an ensemble of mathematical within-host parasite growth and antimalarial action models, fitted to extensive data from four antimalarials with different modes of action, to assess host-parasite interactions in two preclinical drug testing systems of murine parasite P. berghei in mice, and human parasite P. falciparum in immune-deficient mice. We find properties of the host-parasite system, namely resource availability, parasite maturation and virulence, drive P. berghei dynamics and drug efficacy, whereas experimental constraints primarily influence P. falciparum infection and drug efficacy. Furthermore, uninvestigated parasite behavior such as dormancy influences parasite recrudescence following non-curative treatment and requires further investigation. Taken together, host-parasite interactions should be considered for meaningful translation of pharmacodynamic properties between murine systems and for predicting human efficacious treatment.

Primer sequences and raw data are available in the supplementary file S1 at: https://doi.org/10.5281/zenodo.6460010. Samples and associated data were deposited in the Small Mammal Collection at the IRD/CBGP (https://doi.org/10.15454/WWNUPO) as well as at URIB/LARBA/EPAC and Kasetsart University (Thailand). They are available upon request.Preprint version 3 of this article has been peer-reviewed and recommended by Peer Community InInfections.; Trypanosoma lewisi belongs to the so-called atypical trypanosomes that occasionally affect humans. It shares the same hosts and flea vector of other medically relevant pathogenic agents as Yersinia pestis, the agent of plague. Increasing knowledge on the population structure (reproductive mode, population size, dispersal) of this parasite thus represents a challenging but important issue. The use of polymorphic genetic markers, together with suitable population genetics tools, is a convenient way to achieve such objectives. To date, the population biology of T. lewisi is poorly known and, to our knowledge, no population genetics studies have ever been conducted. Here, we present the development of nine microsatellite markers of this species. We investigated their polymorphism in different countries from Africa and South-East Asia from DNAs extracted from the spleen of their rodent reservoirs (essentially rat species). Several amplification problems arose, especially with South-East Asian individuals. This led to retain only those individuals with complete genotypes (most of them originating from West Africa, notably Cotonou, Benin) to ensure an optimal estimate of heterozygosity. Our results pointed towards a mainly (at least 95-99%) clonal mode of propagation, a strong subdivision at the smallest scale available (i.e., urban neighborhoods, i.e. 0.250 km²), and a generation time most probably shorter than 4 months. In future studies, more extensive sampling at smaller geographic scales (i.e., households), within a one- or two-months window and with improved amplification conditions, should lead to a more precise picture of the fine population structure of this parasite.

Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium-independent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4− parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4− parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild-type and Plasmodium falciparum cdpk4− late gametocyte stages to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events, such as DNA replication, mRNA translation, and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation and thereby is critical for parasite transmission to the mosquito vector. IMPORTANCE Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.

After inoculation by the bite of an infected mosquito, Plasmodium sporozoites enter the blood stream and infect the liver, where each infected cell produces thousands of merozoites. These in turn, infect red blood cells and cause malaria symptoms. To initiate a productive infection, sporozoites must exit the circulation by traversing the blood lining of the liver vessels after which they infect hepatocytes with unique specificity. We screened a phage display library for peptides that structurally mimic (mimotope) a sporozoite ligand for hepatocyte recognition. We identified HP1 (hepatocyte-binding peptide 1) that mimics a ~50 kDa sporozoite ligand (identified as phospholipid scramblase). Further, we show that HP1 interacts with a ~160 kDa hepatocyte membrane putative receptor (identified as carbamoyl-phosphate synthetase 1). Importantly, immunization of mice with the HP1 peptide partially protects them from infection by the rodent parasite P. berghei. Moreover, an antibody to the HP1 mimotope inhibits human parasite P. falciparum infection of human hepatocytes in culture. The sporozoite ligand for hepatocyte invasion is a potential novel pre-erythrocytic vaccine candidate., After transmission of Plasmodium sporozoites from infected mosquitoes, parasites first infect hepatocytes. Here, Cha et al. identify a sporozoite ligand (phospholipid scramblase) and the hepatocytic receptor (carbamoyl-phosphate synthetase 1) as relevant for hepatocyte invasion and show that an antibody to hepatocyte-binding peptide 1 (HP1), which structurally mimics the sporozoite ligand, partially protects mice from infection.

Background Skin-penetrating nematodes of the genus Strongyloides infect over 600 million people, posing a major global health burden. Their life cycle includes both a parasitic and free-living generation. During the parasitic generation, infective third-stage larvae (iL3s) actively engage in host seeking. During the free-living generation, the nematodes develop and reproduce on host feces. At different points during their life cycle, Strongyloides species encounter a wide variety of host-associated and environmental bacteria. However, the microbiome associated with Strongyloides species, and the behavioral and physiological interactions between Strongyloides species and bacteria, remain unclear. Results We first investigated the microbiome of the human parasite Strongyloides stercoralis using 16S-based amplicon sequencing. We found that S. stercoralis free-living adults have an associated microbiome consisting of specific fecal bacteria. We then investigated the behavioral responses of S. stercoralis and the closely related rat parasite Strongyloides ratti to an ecologically diverse panel of bacteria. We found that S. stercoralis and S. ratti showed similar responses to bacteria. The responses of both nematodes to bacteria varied dramatically across life stages: free-living adults were strongly attracted to most of the bacteria tested, while iL3s were attracted specifically to a narrow range of environmental bacteria. The behavioral responses to bacteria were dynamic, consisting of distinct short- and long-term behaviors. Finally, a comparison of the growth and reproduction of S. stercoralis free-living adults on different bacteria revealed that the bacterium Proteus mirabilis inhibits S. stercoralis egg hatching, and thereby greatly decreases parasite viability. Conclusions Skin-penetrating nematodes encounter bacteria from various ecological niches throughout their life cycle. Our results demonstrate that bacteria function as key chemosensory cues for directing parasite movement in a life-stage-specific manner. Some bacterial genera may form essential associations with the nematodes, while others are detrimental and serve as a potential source of novel nematicides.

Background Current treatment options for onchocerciasis are sub-optimal, prompting research and development of a safe cure (macrofilaricide). Onchocerca ochengi, a parasite of cattle, is used as a close surrogate for the human parasite O. volvulus in a murine model for pre-clinical screening of macrofilaricides. Skin from naturally infected cattle have been used in previous studies as a reliable source of parasite material. However, there is limited knowledge on how source-related factors such as the microfilaridermia status of the cattle, the nodule load and nodular worm viability may affect survival of male O. ochengi worms implanted in the rodent hosts. Such relationships were investigated in this study. Methods Dermal tissue and nodules were obtained from Gudali cattle, dissected and cultured to obtain migrating microfilariae (mf) and male worms. Emerged male worms were implanted into SCID mice and Gerbils (Meriones unguiculatus) and recovery rates were determined upon 42 days post implantation. Finally, nodules were processed for histology and embryogram analyses to assess the nodular worm viability and fertility, respectively. Results Of the 69 cattle sampled, 24 (34.8%) were mf+ and 45 (65.2%) were mf–. The mean nodule loads were 180.5 ± 117.7 (mf+) and 110.6 ± 102.7 (mf-) (p = 0.0186). The mean male worm harvest from nodules were 76.8 ± 120.3 and 47.2 ± 33.4 (p = 0.2488) for mf+ and mf– cattle, respectively. The number of male worms per 100 nodules were 57/100 and 46/100 nodules for mf+ and mf– cows, respectively. Female worms from nodules of mf– cows had higher counts of both normal and abnormal embryos with higher proportions of dead nodular worms evinced by histology compared to those from mf+ cows. A total of 651 worms were implanted into mice and gerbils, out of which 129 (19.81%) were recovered. Logistic regression analysis indicated that the microfilaridermia status of the cattle (presence of mf) (OR = 4.3319; P = 0.001) is the single most important predictor of the success of male worm recovery after implantation into rodents. Conclusion Microfilaridermic cattle provide a promising source of adult O. ochengi. Male worms from this group of cattle have a better success rate of survival in a murine implant model. Nevertheless, in the programmatic point of view, amicrofilaridermic Gudali cattle would still constitute an important source of O. ochengi male worms with relatively good viability after implantation into rodents., Graphical abstract Image 1, Highlights • There is a need of developing safe macrofilaricide for human onchocerciasis using cattle Onchocerca ochengi. • Skin from naturally infected cattle have been used as a reliable parasite source for development of murine pre-clinical screening models. • The role of source-related factors on O. ochengi production and the success of pre-clinical screening models is assessed. • Worms from positive microfilaridermic cattle are successfully recovered after their implantation into rodents.

Lymphatic filariasis (LF) is a chronic debilitating neglected tropical disease (NTD) caused by mosquito-transmitted nematodes that afflicts over 60 million people. Control of LF relies on routine mass drug administration with antiparasitics that clear circulating larval parasites but are ineffective against adults. The development of effective adulticides is hampered by a poor understanding of the processes and tissues driving parasite survival in the host. The adult filariae head region contains essential tissues that control parasite feeding, sensory, secretory, and reproductive behaviors, which express promising molecular substrates for the development of antifilarial drugs, vaccines, and diagnostics. We have adapted spatial transcriptomic approaches to map gene expression patterns across these prioritized but historically intractable head tissues. Spatial and tissue-resolved data reveal distinct biases in the origins of known drug targets and secreted antigens. These data were used to identify potential new drug and vaccine targets, including putative hidden antigens expressed in the alimentary canal, and to spatially associate receptor subunits belonging to druggable families. Spatial transcriptomic approaches provide a powerful resource to aid gene function inference and seed antiparasitic discovery pipelines across helminths of relevance to human and animal health.

We explored the hypothesis that progesterone direct effect on Trichinella spiralis might be mediated indeed by a new steroid-binding parasite protein. Our first results showed that Progesterone decreases the parasite molting rate. We amplify, isolated, cloned and sequenced the PGRMC2 sequence using specific primers from known species. Furthermore, we expressed the protein and developed an antibody to performance immunofluorescent confocal microscopy, where detected that parasite cells showed expression of a P4-binding protein exclusively located at the oocyte and the parasite´s cuticle. Presence of the PGRMC2 protein in these cells was also confirmed by western blot and flow cytometry. Molecular modeling studies accompanied by computer docking using the sequenced protein showed that PGRMC2 is potentially able to bind steroid hormones such as progesterone, estradiol, testosterone, and dihydrodrotestosterone with different affinities. Phylogenetic analysis and sequence alignment clearly demonstrated that Trichinella spiralis PGRMC2 is related to a steroid-binding protein of another platyhelminths. Progesterone may probably act upon Trichinella spiralis oocytes probably by binding to PGRMC2. This research has implications in the field of host-parasite co-evolution as well as the sex-associated susceptibility to this infection. In a more practical matter, present results may contribute to the molecular design of new drugs with anti-parasite actions.

The consequences of parasite infection for individual hosts depend on key features of host–parasite ecology underpinning parasite growth and immune defense, such as age, sex, resource supply, and environmental stressors. Scaling these features and their underlying mechanisms from the individual host is challenging but necessary, as they shape parasite transmission at the population level. Translating individual-level mechanisms across scales could inherently improve the way we think about feedbacks among parasitism, the mechanisms driving transmission, and the consequences of human impact and disease control efforts. Here, we use individual-based models (IBMs) based on general metabolic theory, Dynamic Energy Budget (DEB) theory, to scale explicit life-history features of individual hosts, such as growth, reproduction, parasite production, and death, to parasite transmission at the population level over a range of resource supplies focusing on the major human parasite, Schistosoma mansoni, and its intermediate host snail, Biomphalaria glabrata. At the individual level, infected hosts produce fewer parasites at lower resources as competition increases. At the population level, our DEB–IBM predicts brief, but intense parasite peaks early during the host growth season when resources are abundant and infected hosts are few. The timing of these peaks challenges the status quo that high densities of infected hosts produce the highest parasite densities. As expected, high resource supply boosts parasite output, but parasite output also peaks at modest to high host background mortality rates, which parallels overcompensation in stage-structured models. Our combined results reveal the crucial role of individual-level physiology in identifying how environmental conditions, time of the year, and key feedbacks within host–parasite ecology interact to define periods of elevated risk. The testable forecasts from this physiologically-explicit epidemiological model can inform disease management to reduce human risk of schistosome infection.

Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium dependent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4− parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4− parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild type and Plasmodium falciparum cdpk4− late gametocyte stages, to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events such as DNA replication, mRNA translation and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation, and thereby is critical for parasite transmission to the mosquito vector.IMPORTANCETransmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.

The stalling global progress in the fight against malaria prompts the urgent need to develop new intervention strategies. Whilst engineered symbiotic bacteria have been shown to confer mosquito resistance to parasite infection, a major challenge for field implementation is to address regulatory concerns. Here, we report the identification of a Plasmodium-blocking symbiotic bacterium, Serratia ureilytica Su_YN1, isolated from the midgut of wild Anopheles sinensis in China that inhibits malaria parasites via secretion of an antimalarial lipase. Analysis of Plasmodium vivax epidemic data indicates that local malaria cases in Tengchong (Yunnan province, China) are significantly lower than imported cases and importantly, that the local vector A. sinensis is more resistant to infection by P. vivax than A. sinensis from other regions. Analysis of the gut symbiotic bacteria of mosquitoes from Yunnan province led to the identification of S. ureilytica Su_YN1. This bacterium renders mosquitoes resistant to infection by the human parasite Plasmodium falciparum or the rodent parasite Plasmodium berghei via secretion of a lipase that selectively kills parasites at various stages. Importantly, Su_YN1 rapidly disseminates through mosquito populations by vertical and horizontal transmission, providing a potential tool for blocking malaria transmission in the field.

BackgroundThe consequences of the earth’s daily rotation have led to 24-hour biological rhythms in most organisms. Even parasites have daily rhythms, which, when in synchrony with host rhythms, optimize their fitness. Understanding these rhythms may enable development of novel control strategies that take advantage of rhythmic vulnerabilities. Recent work on blood-dwelling protozoan parasites has revealed daily rhythms in gene expression, physiology, drug sensitivity and the presence of an intrinsic circadian clock. However, similar studies on metazoan parasites are lacking. The aims of this study were to investigate if a metazoan parasite has daily molecular oscillations, whether they give us insight into how these longer-lived organisms can survive host daily cycles over a life-span of many years and to determine whether canonical metazoan circadian clock genes are present and rhythmic. We addressed these questions using the human blood fluke Schistosoma mansoni, that lives in the vasculature for decades and causes the serious neglected tropical disease schistosomiasis.ResultsUsing round-the-clock transcriptomics of male and female adult worms we discovered that ∼2% of its genes followed a daily pattern of expression. Rhythmic processes included a night-time stress response and a day-time metabolic ‘rush hour’. Transcriptional profiles in the female reproductive system were mirrored by daily patterns in egg laying (eggs are the main drivers of the host pathology). Genes cycling with the highest amplitudes include drug targets and a vaccine candidate. These 24hr rhythms may be driven by host rhythms and/or generated by a circadian clock. However, core clock genes are missing and orthologues of secondary clock genes show no 24hr rhythmicity in transcript abundance.ConclusionsThe daily rhythms identified here reveal temporally compartmentalised internal processes and host interactions over the daily cycle, including processes relevant to within-host survival and between-host transmission. Our findings suggest that if these daily rhythms are generated by an intrinsic circadian clock then the oscillatory mechanism must be distinct from that in other Metazoa. Most importantly, knowing which gene transcripts oscillate at this temporal scale is relevant to functional genomic studies that will lead to the development and delivery of therapeutics against schistosomiasis.

Mosquitoes may feed multiple times during their life span in addition to those times needed to acquire and transmit malaria. To determine the impact of subsequent blood feeding on parasite development in Anopheles gambiae, we examined Plasmodium parasite infection with or without an additional noninfected blood meal. We found that an additional blood meal significantly reduced Plasmodium berghei immature oocyst numbers, yet had no effect on the human parasite Plasmodium falciparum. These observations were reproduced when mosquitoes were fed an artificial protein meal, suggesting that parasite losses are independent of blood ingestion. We found that feeding with either a blood or protein meal compromises midgut basal lamina integrity as a result of the physical distention of the midgut, enabling the recognition and lysis of immature P. berghei oocysts by mosquito complement. Moreover, we demonstrate that additional feeding promotes P. falciparum oocyst growth, suggesting that human malaria parasites exploit host resources provided with blood feeding to accelerate their growth. This is in contrast to experiments with P. berghei, where the size of surviving oocysts is independent of an additional blood meal. Together, these data demonstrate distinct differences in Plasmodium species in evading immune detection and utilizing host resources at the oocyst stage, representing an additional, yet unexplored component of vectorial capacity that has important implications for the transmission of malaria. IMPORTANCE Mosquitoes must blood feed multiple times to acquire and transmit malaria. However, the impact of an additional mosquito blood meal following malaria parasite infection has not been closely examined. Here, we demonstrate that additional feeding affects mosquito vector competence; namely, additional feeding significantly limits Plasmodium berghei infection, yet has no effect on infection of the human parasite P. falciparum. Our experiments support that these killing responses are mediated by the physical distension of the midgut and by temporary damage to the midgut basal lamina that exposes immature P. berghei oocysts to mosquito complement, while human malaria parasites are able to evade these killing mechanisms. In addition, we provide evidence that additional feeding promotes P. falciparum oocyst growth. This is in contrast to P. berghei, where oocyst size is independent of an additional blood meal. This suggests that human malaria parasites are able to exploit host resources provided by an additional feeding to accelerate their growth. In summary, our data highlight distinct differences in malaria parasite species in evading immune recognition and adapting to mosquito blood feeding. These observations have important, yet previously unexplored, implications for the impact of multiple blood meals on the transmission of malaria.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais A família Solanaceae é uma das maiores famílias do reino vegetal, incluindo várias plantas de importância agronômica e médica, sobretudo o tomate, por seu alto consumo a nível mundial. Além de sua importância como alimento, o tomate possui características agronômicas interessantes, a saber, fruto carnoso e folhas compostas, que plantas modelo não possuem, ex. Arabidopsis thaliana. Nesse sentido, o tomate comum (Solanum lycopersicum) tem sido utilizado como modelo para espécies que também apresentam tais características. Devido às características específicas e às condições de cultivo, as variedades de tomate são constantemente melhoradas. No entanto, essa cultura é suscetível a diversas pragas e patógenos. Para auxiliar no controle dessas doenças, a maioria dos produtores opta pelo uso de agentes químicos que, muitas vezes, são caros, às vezes ineficazes e têm efeitos deletérios ao meio ambiente. Uma alternativa para esses problemas é cruzar o tomate cultivado com espécies silvestres. São várias as espécies conhecidas de tomate selvagem, constituídas por recursos genéticos pouco explorados e de grande importância para o melhoramento, pesquisa e desenvolvimento da cultura, como o Solanum pennellii, Solanum galapagense, Solanum chilense, Solanum peruvianum e Solanum pimpinellifolium. Algumas espécies de tomate selvagem têm sido amplamente utilizadas para a construção e mapeamento de populações devido a sua tolerância ao estresse ambiental. Nesse sentido, um melhor entendimento da base molecular do tomate é necessário para se atingir a eficiência e o sucesso na seleção de marcadores associados às características de interesse, como a tolerância biótica e abiótica. Da mesma forma que é importante obter um maior conhecimento sobre as bases moleculares de muitas espécies de plantas, alguns animais, parasitas, bactérias e vírus também necessitam de estudos moleculares para auxiliarem no controle de doenças que acometem os serem humanos. Neste sentido, a esquistossomose humana é uma doença parasitária causada por helmintos do gênero Schistosoma que afeta milhões de pessoas em todo o mundo. Cinco espécies de Schistosoma são responsáveis pela maioria das infecções humanas: Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, Schistosoma mekongi e Schistosoma intercalatum. O S. haematobium afeta o sistema reprodutivo e urinário e tem sido considerado um grande problema de saúde pública na África. A disseminação da doença é dependente da interação entre o parasita e o hospedeiro intermediário. Esta interação é complexa e determinada por alguns genes envolvidos na infectividade do parasita e suscetibilidade do hospedeiro. Portanto, compreender os mecanismos de regulação gênica é extremamente importante para compreender as interações parasita-hospedeiro e quais genes podem contribuir para a infecção. O progresso atual no sequenciamento do genoma de plantas e animais tem gerado informações úteis para auxiliar no estudo da diversidade genética de muitas espécies. Nos anos 2000 foram sequenciados os genomas de S. galapagense, S. peruvianum, S. haematobium, S. lycopersicum, S. pennellii, S. pimpinellifolium e S. chilense. A interpretação dos genomas do esquistossomo melhorou nosso entendimento da biologia molecular desses parasitas, permitindo a identificação e caracterização de genes ainda não descobertos. A disponibilidade de um genoma de referência de tomate forneceu um arcabouço para a análise genômica da família Solanaceae, gerando uma fonte de informações importantes para seu melhoramento molecular. Além disso, o sequenciamento de genomas de espécies selvagens de tomate forneceu um recurso valioso para a compreensão de várias características proeminentes, como mudanças na resposta ao déficit hídrico, resistência biótica e abiótica e metabolismo. Entretanto, várias partes desses genomas precisam ser exaustivamente estudadas e anotadas, uma vez que o sequenciamento do genoma e o depósito das sequências em bancos de dados públicos são apenas os passos iniciais do estudo. Estudos recentes têm mostrado a importância da regulação gênica envolvendo várias classes de pequenos RNAs, seu sistema de processamento e desempenho celular em diferentes organismos. Os principais representantes dessa classe de pequenos RNAs são os microRNAs (miRNAs), cujas formas de regulação podem envolver a inibição do processo de tradução, degradação de RNAm ou silenciamento de genes, seja por complementaridade de alvos ou por sinalização de modificações no DNA em regiões específicas do genoma. Diferentes proteínas atuam para gerar miRNAs maduros específicos para a regulação pós-transcricional. Em plantas e animais estas vias de síntese dos miRNAs possuem algumas diferenças. Estratégias computacionais têm sido usadas para identificar miRNAs e proteínas envolvidas em suas vias em vários organismos. Os métodos computacionais são amplamente utilizados na identificação de genes, uma vez que alguns transcritos são expressos apenas sob certas condições ou em células específicas. Assim, as técnicas computacionais auxiliam no processo de descoberta de novos miRNAs, utilizando todas as informações contidas no genoma e/ou no transcriptoma independentemente da amostragem. Além disso, tais métodos são úteis para a previsão de precursores de miRNA e seus alvos. Diante da importância de tais RNAs não codificantes na regulação da expressão gênica, é importante estudarmos miRNAs maduros e precursores, bem como os genes envolvidos na via de miRNA nestas espécies de tomate cultivado e selvagens para auxiliar na elucidação de processos biológicos, bem como seus desempenhos no nível celular. Também em S. haematobium, o conhecimento destas pequenas moléculas e sua via de processamento pode ajudar a entender o ciclo de vida desse parasita, seu mecanismo de infectividade e na busca de novos métodos de controle da esquistossomose. Além da via de produção dos miRNAs, outras vias de defesa são importantes em algumas plantas. Diferentes aleloquímicos presentes em espécies de Solanum têm sido associados à resistência a pragas, como metil-cetonas, sesquiterpenos e acilaçúcares. A seleção para altos teores destes aleloquímicos poderia, desta maneira, constituir-se uma técnica eficiente de seleção indireta para resistência a pragas. Esses fitoquímicos purificados podem agir contra as pragas reduzindo o desenvolvimento larval, prejudicando a alimentação e a ovoposição de muitas pragas do tomate. Tendo em vista a grande importância do tomate e a alta taxa de aplicação de agrotóxicos na cultura, um maior conhecimento sobre as bases moleculares do tomate faz-se necessário na seleção de marcadores associados a características importantes em resposta ao estresse biótico e abiótico. Portanto, compreender os miRNAs, sua via de processamento e os genes relacionados com a produção de metabólitos poderá auxiliar na compreensão do seu envolvimento na resistência a artrópodes, principalmente pela produção de acilaçúcar e a regulação pós transcricional mediada pelos miRNAs. Tendo em vista essas questões, o Capítulo I apresentará a fundamentação teórica sobre os miRNAs, acilaçucares e suas vias, além da importância de estudar estas moléculas em S. lycopersicum, S. pennellii, S. galapagense, S. chilense, S. peruvianum, S. pimpinellifolium e S. haematobium. No Capítulo II serão expostos os artigos publicados e submetidos referente às pesquisas realizadas durante o doutorado. O tomate cultivado, Solanum lycopersicum, é uma das frutas mais comuns na indústria global de alimentos e, junto com o tomate selvagem Solanum pennellii, são espécies amplamente utilizadas no desenvolvimento de melhores cultivares. Os microRNAs atuam na regulação do mRNA, inibindo sua tradução e / ou promovendo sua degradação. ARGONAUTA e DICER são importantes proteínas envolvidas nesses processos. Este estudo teve como objetivo identificar e caracterizar genes envolvidos nas vias de processamento de miRNAs, moléculas de miRNAs e genes-alvo no genoma de ambas as espécies por meio de análise in silico. Também validamos a expressão de genes e miRNAs em diferentes bibliotecas de NGS, além de validar alguns miRNAs usando RT-PCR quantitativa. Identificamos 70 proteínas em S. lycopersicum e 108 em S. pennellii envolvidas no processamento de pequenos RNAs. Destes, 28 e 32 participam de vias de processamento de miRNA, respectivamente. Identificamos 343 miRNAs maduros, 226 pré-miRNAs em 87 famílias, incluindo 192 miRNAs não identificados anteriormente, pertencentes a 38 novas famílias em S. lycopersicum. Em S. pennellii, encontramos 388 miRNAs maduros, 234 pré-miRNAs contidos em 85 famílias. Todos os miRNAs encontrados em S. pennellii não foram publicados, sendo identificados pela primeira vez em nosso estudo. Além disso, identificamos 2.471 e 3.462 miRNA diferentes alvos em S. lycopersicum e S. pennellii, respectivamente. Genes-chave controlam a infectividade do Schistosoma haematobium causando esquistossomose. Um método para entender a regulação desses genes pode auxiliar no desenvolvimento de novas estratégias de controle da esquistossomose, como o silenciamento mediado por microRNAs (miRNAs). Os miRNAs têm sido estudados em espécies de esquistossomos e desempenham papéis importantes na regulação pós-transcricional de genes e nas interações parasita-hospedeiro. No entanto, a identificação e caracterização em todo o genoma de novos miRNAs e seus genes de vias e sua expressão gênica não foram explorados profundamente no genoma e no transcriptoma de S. haematobium. Neste sentido, este estudo teve como objetivo identificar e caracterizar miRNAs maduros, precursores e seus genes de vias no genoma de S. haematobium. Predição computacional e caracterização de miRNAs e genes envolvidos na via de miRNA do genoma de S. haematobium no SchistoDB. A análise de domínio conservado foi realizada usando os bancos de dados PFAM e CDD. Um algoritmo robusto foi aplicado para identificar miRNAs maduros e seus precursores. A caracterização dos miRNAs precursores foi realizada utilizando scripts RNAfold, RNAalifold e Perl. Identificamos e caracterizamos 14 proteínas envolvidas na via de miRNA, incluindo ARGONAUTE e DICER em S. haematobium. Além disso, 149 miRNAs maduros e 131 miRNAs precursores foram identificados no genoma, incluindo novos miRNAs. A via de miRNA ocorre em S. haematobium, incluindo miRNAs endógenos e componentes da via de miRNA, sugerindo um papel deste tipo de RNAs não codificantes na regulação gênica do parasita. Os resultados encontrados neste trabalho abrirão um novo caminho para o estudo de miRNAs na biologia de S. haematobium, auxiliando no entendimento do mecanismo de silenciamento gênico do parasita humano Esquistossomo. Os acilaçucares são aleloquímicos secretados por tricomas do tipo IV nas folhas e têm sido associados à resistência a artrópodes-praga em espécies de Solanaceae. Neste estudo, os genes putativos envolvidos no metabolismo de acilaçucar foram identificados, caracterizados e validados em tomate cultivado, Solanum lycopersicum, e em tomate selvagem, Solanum pennellii. Oitenta e sete genes foram identificados em S. lycopersicum e 77 em S. pennellii envolvidos na via de acilaçucar, incluindo as proteínas-chave desidrogenase cetoácido de cadeia ramificada E2 (BCKD E2), 2-isopropilmalato sintase A (IPMSA) e treonina desaminase / desidratase (TD). As proteínas putativas do tomate exibiram conservação usando distribuição de domínio conservado e sítio ativo em comparação com proteínas ortólogas da via de acilaçucar. A análise filogenética das proteínas putativas BCKD E2, IPMSA e TD mostrou que todas as prováveis proteínas do tomate se agruparam com seus ortólogos das espécies de Solanaceae, corroborando com a distribuição da árvore da vida. Os transcritos BCKD E2 e IPMSA de S. pennellii apresentaram maior expressão gênica em relação aos transcritos de S. lycopersicum, sugerindo que a via do acilaçúcar é mais ativa em tomate selvagem do que em cultivado, corroborando com a literatura. Dado o importante papel dos aleloquímicos produzidos em Solanaceae, os resultados contribuem para uma melhor compreensão dos acilaçucares e suas vias de processamento e abrem oportunidades para estudar sua relação com a resistência biótica nessas importantes espécies e outras espécies de Solanaceae. The Solanaceae family is one of the largest families in the plant kingdom, including several plants of agronomic and medical importance, especially tomatoes, due to their high consumption worldwide. In addition to its importance as a food, tomatoes have interesting agronomic characteristics, namely, fleshy fruit and compound leaves, which model plants do not have, eg. Arabidopsis thaliana. In this sense, the common tomato (Solanum lycopersicum) has been used as a model for species that also have such characteristics. Due to their specific characteristics and growing conditions, tomato varieties are constantly being improved. However, this crop is susceptible to several pests and pathogens. To help control these diseases, most producers choose to use chemical agents, which are often expensive, sometimes ineffective and have harmful effects on the environment. An alternative to these problems is to cross the cultivated tomato with wild species. There are several known species of wild tomatoes, constituted by little explored genetic resources and of great importance for the improvement, research and development of the culture, such as Solanum pennellii, Solanum galapagense, Solanum chilense, Solanum peruvianum and Solanum pimpinellifolium. Some species of wild tomatoes have been widely used for the construction and mapping of populations due to their tolerance to environmental stress. In this sense, a better understanding of the molecular basis of tomatoes is necessary to achieve efficiency and success in the selection of markers associated with the characteristics of interest, such as biotic and abiotic tolerance. In the same way that it is important to obtain a greater knowledge about the molecular bases of many species of plants, some animals, parasites, bacteria and viruses also need molecular studies to assist in the control of diseases that affect humans. In this sense, human schistosomiasis is a parasitic disease caused by helminths of the genus Schistosoma that affects millions of people worldwide. Five species of Schistosoma are responsible for the majority of human infections: Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, Schistosoma mekongi and Schistosoma intercalatum. S. haematobium affects the reproductive and urinary systems and has been considered a major public health problem in Africa. The spread of the disease is dependent on the interaction between the parasite and the intermediate host. This interaction is complex and determined by some genes involved in the parasite's infectivity and host susceptibility. Therefore, understanding the mechanisms of gene regulation is extremely important to understand the parasite-host interactions and which genes can contribute to the infection. Current progress in sequencing the genome of plants and animals has generated useful information to assist in the study of the genetic diversity of many species. In the 2000s, the genomes of S. galapagense, S. peruvianum, S. haematobium, S. lycopersicum, S. pennellii, S. pimpinellifolium and S. chilense were sequenced. The interpretation of schistosome genomes has improved our understanding of the molecular biology of these parasites, allowing the identification and characterization of genes not yet discovered. The availability of a tomato reference genome provided a framework for the genomic analysis of the Solanaceae family, generating a source of important information for its molecular improvement. In addition, the sequencing of genomes of wild tomato species has provided a valuable resource for understanding several prominent characteristics, such as changes in response to water deficit, biotic and abiotic resistance and metabolism. However, several parts of these genomes need to be thoroughly studied and annotated, since sequencing the genome and depositing the sequences in public databases are only the initial steps of the study. Recent studies have shown the importance of gene regulation involving several classes of small RNAs, their processing system and cell performance in different organisms. The main representatives of this class of small RNAs are microRNAs (miRNAs), whose forms of regulation may involve inhibiting the translation process, mRNA degradation or gene silencing, either by target complementarity or by signaling changes in DNA in regions specific to the genome. Different proteins act to generate mature miRNAs specific for post-transcriptional regulation. In plants and animals, these miRNA synthesis pathways have some differences. Computational strategies have been used to identify miRNAs and proteins involved in their pathways in various organisms. Computational methods are widely used to identify genes, since some transcripts are expressed only under certain conditions or in specific cells. Thus, computational techniques assist in the process of discovering new miRNAs, using all the information contained in the genome and / or the transcriptome regardless of the sample. In addition, such methods are useful for predicting miRNA precursors and their targets. Given the importance of such non-coding RNAs in the regulation of gene expression, it is important to study mature miRNAs and precursors, as well as the genes involved in the miRNA pathway in these species of cultivated and wild tomatoes to assist in the elucidation of biological processes, as well as their performances at the cellular level. Also in S. haematobium, the knowledge of these small molecules and their processing pathway can help to understand the life cycle of this parasite, its mechanism of infectivity and in the search for new methods to control schistosomiasis. In addition to the miRNA production pathway, other defense pathways are important in some plants. Different allelochemicals present in Solanum species have been associated with resistance to pests, such as methyl ketones, sesquiterpenes and acyl sugars. The selection for high levels of these allelochemicals could, in this way, constitute an efficient technique of indirect selection for resistance to pests. These purified phytochemicals can act against pests by reducing larval development, impairing the nutrition and oviposition of many tomato pests. In view of the great importance of tomatoes and the high rate of application of pesticides in the crop, greater knowledge about the molecular basis of tomatoes is necessary in the selection of markers associated with important characteristics in response to biotic and abiotic stress. Therefore, understanding the miRNAs, their processing pathway and the genes related to the production of metabolites may help to understand their involvement in resistance to arthropods, mainly due to the production of acyl sugar and post-transcriptional regulation mediated by miRNAs. In view of these issues, Chapter I will present the theoretical background on miRNAs, acylacars and their pathways, in addition to the importance of studying these molecules in S. lycopersicum, S. pennellii, S. galapagense, S. chilense, S. peruvianum, S. pimpinellifolium and S. haematobium. In Chapter II, articles published and submitted regarding research carried out during the doctorate will be exposed. Cultivated tomato, Solanum lycopersicum, is one of the most common fruits in the global food industry and together with the wild tomato Solanum pennellii, are both species widely used in developing better cultivars. microRNAs act on mRNA regulation, inhibiting their translation and/or promoting its degradation. Important proteins involved in these processes are ARGONAUTA and DICER. This study aimed to identify and characterize genes involved in miRNA processing pathways, miRNAs molecules and target-genes in the genome of both species using in silico analysis. We also validated the genes and miRNAs expression in different NGS libraries, in addition to validating some miRNAs using quantitative RT-PCR. We identified 70 putative proteins in S. lycopersicum and 108 in S. pennellii involved in small RNAs processing. Of these, 28 and 32 participate in miRNA processing pathways, respectively. We identified 343 mature miRNAs, 226 pre-miRNAs in 87 families, including 192 miRNAs not previously identified, belonging to 38 new families in S. lycopersicum. In S. pennellii, we found 388 mature miRNAs, 234 pre-miRNAs contained in 85 families. All miRNAs found in S. pennellii were unpublished, being first identified in our study. Furthermore, we identified 2471 and 3462 miRNA different target in S. lycopersicum and S. pennellii, respectively. Key genes control the infectivity of the Schistosoma haematobium causing schistosomiasis. A method for understanding the regulation of these genes might help in developing new strategies to control schistosomiasis, such as the silencing mediated by microRNAs (miRNAs). The miRNAs have been studied in schistosome species and they play important roles in the post-transcriptional regulation of genes, and in parasite-host interactions. However, genome-wide identification and characterization of novel miRNAs and their pathway genes and their gene expression have not been explored deeply in the genome and transcriptome of S. haematobium. Identify and characterize mature and precursor miRNAs and their pathway genes in the S. haematobium genome. Computational prediction and characterization of miRNAs and genes involved in miRNA pathway from S. haematobium genome on SchistoDB. Conserved domain analysis was performed using PFAM and CDD databases. A robust algorithm was applied to identify mature miRNAs and their precursors. The characterization of the precursor miRNAs was performed using RNAfold, RNAalifold and Perl scripts. We identified and characterized 14 putative proteins involved in miRNA pathway including ARGONAUTE and DICER in S. haematobium. Besides that, 149 mature miRNAs and 131 precursor miRNAs were identified in the genome including novel miRNAs. miRNA pathway occurs in the S. haematobium, including endogenous miRNAs and miRNA pathway components, suggesting a role of this type of non-coding RNAs in gene regulation in the parasite. The results found in this work will open up a new avenue for studying miRNAs in the S. haematobium biology in helping to understand the mechanism of gene silencing in the human parasite Schistosome. Acylsugars are allelochemicals secreted by type IV trichomes in leaves and have been associated with resistance to arthropod pests in Solanaceae species. In this study, the putative genes involved in the acylsugar metabolism were identified, characterized, and validated in cultivated tomato, Solanum lycopersicum, and wild tomato, Solanum pennellii. Eighty-seven putative genes were identified in S. lycopersicum and 77 in S. pennellii involved in the acylsugar pathway, including the key proteins branched-chain keto acid dehydrogenase E2 (BCKD E2), 2-isopropylmalate synthase A (IPMSA), and threonine deaminase/dehydratase (TD). The putative tomato proteins displayed conservation using conserved domain distribution and active site comparing to ortholog proteins from the acylsugar pathway. Phylogenetic analysis of the putative proteins BCKD E2, IPMSA, and TD showed that all putative tomato proteins grouped with their orthologs from the Solanaceae species, corroborating with the distribution of the tree of life. The BCKD E2 and the IPMSA transcripts from S. pennellii showed higher gene expression compared to S. lycopersicum transcripts, suggesting that the acylsugar pathway is more active in wild than in cultivated tomato, corroborating with the literature. Given the important role of the allelochemicals produced in Solanaceae, the results contribute to a better understanding of acylsugars and their processing pathways and open up opportunities to study their relationship with biotic resistance in these important species and other Solanaceae species. Tese (Doutorado) 2023-02-24

All insects rely on an innate immune system to eliminate potentially lethal parasites and pathogens. In kissing bugs, this immune system comprises a plethora of elements that first recognize parasites and pathogens as non-self, followed by a multi-faceted response to eliminate them. There is an intriguing molecular interplay between kissing bugs and pathogens; the insects wish to eliminate pathogens while not eliminating the obligate intestinal microbial symbionts on which they depend for survival. Beneficial bacteria and the human parasite Trypanosoma cruzi survive in the lumen of the intestinal tract but are eliminated if they enter the hemocoel. The closely related parasite, Trypanosoma rangeli, however, survives in the hemocoel and later establishes in the salivary glands. Our understanding of triatomine-pathogen interactions has expanded dramatically due to the availability of new molecular and genetic tools that allow us to understand the expression and function of immune molecules, and the mechanisms by which they function. The immune system in triatomines has three main components: physical barriers, cellular responses (phagocytosis, nodulation, and encapsulation), and humoral factors (antimicrobial peptides, lectins, reactive oxygen and nitrogen species, and the phenoloxidase cascade). Here, we describe the current knowledge and adaptations of the innate immune system of triatomines, how it interacts with pathogens and parasites, and the challenges we face in studying these interactions.

Over 250 million people suffer from schistosomiasis, a tropical disease caused by parasitic flatworms known as schistosomes. Humans become infected by free-swimming, water-borne larvae, which penetrate the skin. The earliest intra-mammalian stage, called the schistosomulum, undergoes a series of developmental transitions. These changes are critical for the parasite to adapt to its new environment as it navigates through host tissues to reach its niche, where it will grow to reproductive maturity. Unravelling the mechanisms that drive intra-mammalian development requires knowledge of the spatial organisation and transcriptional dynamics of different cell types that comprise the schistomulum body. To fill these important knowledge gaps, we perform single-cell RNA sequencing on two-day old schistosomula of Schistosoma mansoni. We identify likely gene expression profiles for muscle, nervous system, tegument, oesophageal gland, parenchymal/primordial gut cells, and stem cells. In addition, we validate cell markers for all these clusters by in situ hybridisation in schistosomula and adult parasites. Taken together, this study provides a comprehensive cell-type atlas for the early intra-mammalian stage of this devastating metazoan parasite., Schistosomes undergo several develepmental stages during infection of humans. Here, the authors perform single-cell RNA sequencing on the earliest intra-mammalian stage of Schistosoma mansoni and generate a comprehensive cell-type atlas for this human parasite.

Malaria is infamous for the massive toll it exacts on human life and health. In the face of this intense selection, many human populations have evolved mechanisms that confer some resistance to the disease, such as sickle-cell hemoglobin or the Duffy null blood group. Less understood are adaptations in other vertebrate hosts, many of which have a longer history of co-evolution with malaria parasites. By comparing malaria resistance adaptations across host species, we can gain fundamental insight into host-parasite co-evolution. In particular, understanding the mechanisms by which non-human primate immune systems combat malaria may be fruitful in uncovering transferable therapeutic targets for humans. However, most research on primate response to malaria has focused on a single or few loci, typically in experimentally-infected captive primates. Here, we report the first transcriptomic study of a wild primate response to a malaria-like parasite, investigating gene expression response of red colobus monkeys (Piliocolobus tephrosceles) to natural infection with the malaria-like parasite, Hepatocystis. We identified colobus genes with expression strongly correlated with parasitemia, including many implicated in human malaria and suggestive of common genetic architecture of disease response. For instance, the expression of ACKR1 (alias DARC) gene, previously linked to resistance in humans, was found to be positively correlated with parasitemia. Other similarities to human parasite response include induction of changes in immune cell type composition and, potentially, increased extramedullary hematopoiesis and altered biosynthesis of neutral lipids. Our results illustrate the utility of comparative immunogenetic investigation of malaria response in primates. Such inter-specific comparisons of transcriptional response to pathogens afford a unique opportunity to compare and contrast the adaptive genetic architecture of disease resistance, which may lead to the identification of novel intervention targets to improve human health.Author SummaryThe co-evolutionary arms race between humans and malaria parasites has been ongoing for millennia. Fully understanding the evolved human response to malaria is impossible without comparative study of parasites in our non-human primate relatives. Though laboratory primates are fruitful models, the complexity of wild primates infected in a natural transmission system may be a more suitable comparison for contextualizing malaria infections in human patients. Here, we investigate the genetic mechanisms underlying the immune response to Hepatocystis, a close relative of human-infective malaria, in a population of wild Ugandan red colobus monkeys. We find that the genes involved have considerable overlap with those active in human malaria patients. Like Plasmodium, Hepatocystis induces changes in blood cell type and may cause the host to produce blood components outside of the bone marrow or alter metabolism related to the production of lipids. Our work helps to identify the genetic mechanisms underlying the arms race between primates and malaria parasites, providing fundamental evolutionary insight. Such comparative work on the interaction between wild non-human primates and malaria parasites can identify ways in which primates have evolved resistance to malaria parasites, and further investigation of such implicated genes may lead to novel potential therapeutic and vaccine targets.

Onchocerciasis also known as river blindness is a neglected tropical disease and the world's second-leading infectious cause of blindness in humans; it is caused by Onchocerca volvulus. Current treatment with ivermectin targets microfilariae and transmission and does not kill the adult parasites, which reside within subcutaneous nodules. To support the development of macrofilaricidal drugs that target the adult worm to further support the elimination of onchocerciasis, an in-depth understanding of O. volvulus biology especially the factors that support the longevity of these worms in the human host (>10 years) is required. However, research is hampered by a lack of access to adult worms. O. volvulus is an obligatory human parasite and no small animal models that can propagate this parasite were successfully developed. The current optimized 2-dimensional (2-D) in vitro culturing method starting with O. volvulus infective larvae does not yet support the development of mature adult worms. To overcome these limitations, we have developed and applied 3-dimensional (3-D) culture systems with O. volvulus larvae that simulate the human in vivo niche using in vitro engineered skin and adipose tissue. Our proof of concept studies have shown that an optimized indirect co-culture of in vitro skin tissue supported a significant increase in growth of the fourth-stage larvae to the pre-adult stage with a median length of 816–831 μm as compared to 767 μm of 2-D cultured larvae. Notably, when larvae were co-cultured directly with adipose tissue models, a significant improvement for larval motility and thus fitness was observed; 95% compared to 26% in the 2-D system. These promising co-culture concepts are a first step to further optimize the culturing conditions and improve the long-term development of adult worms in vitro. Ultimately, it could provide the filarial research community with a valuable source of O. volvulus worms at various developmental stages, which may accelerate innovative unsolved biomedical inquiries into the parasite’s biology., Author summary The filarial nematode Onchocerca volvulus is an obligatory human parasite and the causative agent of onchocerciasis, better known as river blindness. In 2017, more than 20 million infections with O. volvulus were estimated worldwide, 99% of the patients live in Africa. Current international control programs focus on the reduction of microfilaridermia by mass drug administration of ivermectin. However, to meet the elimination goals, additional treatment strategies are needed that also target the adult worms. As this parasite is obliged to humans, there are no small animal models that sustain the full life cycle of the parasite, thus greatly impeding the research on this filarial nematode. To overcome these drawbacks, we have developed co-culture systems based on engineered human skin and adipose tissue that represent the in vivo niche of O. volvulus adult worms that improved the culturing conditions and the development to the pre-adult stages of the parasite. Furthermore, our new culture approach could significantly reduce the use of surrogate animal models currently used for macrofilaricidal drug testing.

Neglected parasitic diseases remain a major public health issue worldwide, especially in tropical and subtropical areas. Human parasite diversity is very large, ranging from protozoa to worms. In most cases, more effective and new drugs are urgently needed. Previous studies indicated that the gold(I) drug auranofin (Ridaura&reg, ) is effective against several parasites. Among new gold(I) complexes, the phosphole-containing gold(I) complex {1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl (abbreviated as GoPI) is an irreversible inhibitor of both purified human glutathione and thioredoxin reductases. GoPI-sugar is a novel 1-thio-&beta, d-glucopyranose 2,3,4,6-tetraacetato-S-derivative that is a chimera of the structures of GoPI and auranofin, designed to improve stability and bioavailability of GoPI. These metal-ligand complexes are of particular interest because of their combined abilities to irreversibly target the essential dithiol/selenol catalytic pair of selenium-dependent thioredoxin reductase activity, and to kill cells from breast and brain tumors. In this work, screening of various parasites&mdash, protozoans, trematodes, and nematodes&mdash, was undertaken to determine the in vitro killing activity of GoPI-sugar compared to auranofin. GoPI-sugar was found to efficiently kill intramacrophagic Leishmania donovani amastigotes and adult filarial and trematode worms.

We investigated the prevalence, morphological characters and molecular classifications of trematode cercariae in freshwater snails randomly collected from 59 sampling localities in Bangkok from May 2018 to March 2019. We used a crushing technique to observe the cercarial stage inside each snail body and amplified the internal transcribed spacer 2 regions of cercarial DNA using polymerase chain reaction methodology. The associated phylogenetic tree was reconstructed using Bayesian inference analyses. A total of 517 of 15 621 examined snails were infected with trematode cercariae, and the infected snails were classified into 11 species of seven families with a 3.31% overall prevalence of the infection. The Bithynia siamensis siamensis snail displayed the highest prevalence of infection (16.16%), whereas the Physella acuta snail exhibited the lowest prevalence (0.08%) of infection. Eight morphological types of cercariae were observed. The highest prevalence of infection was observed in mutabile cercaria (1.86%). Based on molecular investigations, the phylogram revealed eight cercaria types assigned to at least nine digenean trematode families, of which five belong to groups of human intestinal flukes. Although, with the exception of schistosome cercaria, trematode cercariae are not known to directly damage humans, understanding the general biology of trematode cercariae (including diversity, distribution, infection rates and host range) is important and necessary for the prevention and control of parasitic transmission that impacts aquatic cultivations, livestock farming and human health.

The phylum Apicomplexa encompasses 6000 ubiquitous animal parasites, including Plasmodium, the most deadly human parasite on Earth. Anciently parasitic lineages, like apicomplexans, lose core metabolic pathways over time, as they evolve less costly scavenging mechanisms. The recent description of a mutualistic apicomplexan, Nephromyces, from deep within this parasitic group, opened the possibility of an evolutionary innovation that allowed an escape from a parasitic lifestyle. Nuclear genome data from Nephromyces, as well as the three bacterial symbionts that live within this species complex, demonstrate that the bacteria within Nephromyces contribute essential cofactors and amino acids that have enabled Nephromyces to abandon a parasitic lifestyle. Among these, bacterial lipoic acid appears to be a key cofactor for the reduction of virulence in Nephromyces. However, whereas we use FISH microscopy to reveal that each individual Nephromyces harbors no more than one endosymbiont type, no single bacterial endosymbiont can account for all missing metabolites. Based on the unique habitat of Nephromyces, as well as genomic, culturing, and wild population data, we conclude that Nephromyces has evolved as an extraordinary clade of codependent species, unlike any previously described.

Onchocerciasis also known as river blindness is a neglected tropical disease and the world’s second-leading infectious cause of blindness in humans; it is caused by Onchocerca volvulus. Current therapies kill microfilariae but fail to kill the adult parasites, which reside within subcutaneous nodules. To support a more target-driven drug development that can also cure and thus support the elimination of this disease, an in-depth understanding of O. volvulus biology especially the factors that support the longevity of these worms in the human host (>10 years) is required. However, research is hampered by a lack of access to adult worms. O. volvulus is an obligatory human parasite and no small animal models that can propagate this parasite were successfully developed. The current optimized 2-dimensional (2-D) in vitro culturing method starting with O. volvulus infective larvae does not yet support the development of mature adult worms. To overcome these limitations, we have developed 3-dimensional (3-D) culture systems that simulated the human in vivo niche using in vitro engineered skin and adipose tissue. We show that an optimized indirect co-culture of in vitro skin tissue with fourth-stage larvae supported a significant increase in growth until the pre-adult stage with a median length of 816 – 831 μm as compared to 767 μm of 2-D cultured larvae. Notably, when larvae were co-cultured directly with adipose tissue models, a significant improvement for larval motility and thus fitness was observed; 95 % compared to 26 % in the 2-D system. These promising co-culture concepts are a first step to further improve the long-term development of adult worms in vitro, and thus provide the filarial research community with a valuable source of O. volvulus worms at various developmental stages, which may accelerate innovative unsolved biomedical inquiries into the parasite’s biology.Author summaryThe filarial nematode Onchocerca volvulus is an obligatory human parasite and the causative agent of onchocerciasis, better known as river blindness. In 2017, more than 20 million infections with O. volvulus were estimated worldwide, 99 % of the patients live in Africa. Current international control programs focus on the reduction of microfilaridermia by mass drug administration of ivermectin. However, to meet the elimination goals, additional therapy strategies are needed that also target the adult worms. As this parasite is obliged to humans, there are no small animal models that sustain the full life cycle of the parasite, thus greatly impeding the research on this filarial nematode. To overcome these drawbacks, we developed co-culture systems based on engineered human skin and adipose tissue that represent the in vivo niche of O. volvulus in which we were able to establish improved conditions of culturing to the pre-adult stages of the parasite. Furthermore, our new culture approach could significantly reduce the use of animal models currently used for drug testing of surrogate larvae.

Trypanosomatid parasites are causative agents of important human and animal diseases such as sleeping sickness and leishmaniasis. Most trypanosomatids are transmitted to their mammalian hosts by insects, often belonging to Diptera (or true flies). These are called dixenous trypanosomatids since they infect two different hosts, in contrast to those that infect just insects (monoxenous). However, it is still unclear whether dixenous and monoxenous trypanosomatids interact similarly with their insect host, as fly-monoxenous trypanosomatid interaction systems are rarely reported and under-studied–despite being common in nature. Here we present the genome of monoxenous trypanosomatid Herpetomonas muscarum and discuss its transcriptome during in vitro culture and during infection of its natural insect host Drosophila melanogaster. The H. muscarum genome is broadly syntenic with that of human parasite Leishmania major. We also found strong similarities between the H. muscarum transcriptome during fruit fly infection, and those of Leishmania during sand fly infections. Overall this suggests Drosophila-Herpetomonas is a suitable model for less accessible insect-trypanosomatid host-parasite systems such as sand fly-Leishmania., Author summary Trypanosomes and Leishmania are parasites that cause serious Neglected Tropical Diseases (NTDs) in the world’s poorest people. Both of these are dixenous trypanosomatids, transmitted to humans and other mammals by biting flies. They are called dixenous as they can establish infections in two different types of hosts– insect vectors and mammals. In contrast, monoxenous trypanosomatids usually only infect insects. Despite establishment in the insect’s midgut being key to transmission of NTDs, events during early establishment inside the insect are still unclear in both dixenous and monoxenous parasites. Here, we study the interaction between a model insect–the fruit fly Drosophila melanogaster–and its natural monoxenous trypanosomatid parasite Herpetomonas muscarum. We show that both the genome of this parasite, and gene regulation at early stages of infection have strong parallels with Leishmania. This work has begun to identify evolutionarily conserved aspects of the process by which trypanosomatids establish in insects, thus potentially highlighting key checkpoints necessary for transmission of dixenous parasites. In turn, this might inform new strategies to control trypanosomatid NTDs.

Background The Plasmodium genus of malaria parasites encodes several families of antigen-encoding genes. These genes tend to be hyper-variable, highly recombinogenic and variantly expressed. The best-characterized family is the var genes, exclusively found in the Laveranian subgenus of malaria parasites infecting humans and great apes. Var genes encode major virulence factors involved in immune evasion and the maintenance of chronic infections. In the human parasite P. falciparum, var gene recombination and diversification appear to be promoted by G-quadruplex (G4) DNA motifs, which are strongly associated with var genes in P. falciparum. Here, we investigated how this association might have evolved across Plasmodium species – both Laverania and also more distantly related species which lack vars but encode other, more ancient variant gene families. Results The association between var genes and G4-forming motifs was conserved across Laverania, spanning ~ 1 million years of evolutionary time, with suggestive evidence for evolution of the association occurring within this subgenus. In rodent malaria species, G4-forming motifs were somewhat associated with pir genes, but this was not conserved in the Laverania, nor did we find a strong association of these motifs with any gene family in a second outgroup of avian malaria parasites. Secondly, we compared two different G4 prediction algorithms in their performance on extremely A/T-rich Plasmodium genomes, and also compared these predictions with experimental data from G4-seq, a DNA sequencing method for identifying G4-forming motifs. We found a surprising lack of concordance between the two algorithms and also between the algorithms and G4-seq data. Conclusions G4-forming motifs are uniquely strongly associated with Plasmodium var genes, suggesting a particular role for G4s in recombination and diversification of these genes. Secondly, in the A/T-rich genomes of Plasmodium species, the choice of prediction algorithm may be particularly influential when studying G4s in these important protozoan pathogens.

Schistosomes kill 250,000 people every year and are responsible for serious morbidity in 240 million of the world's poorest people. Despite their profound global impact, only a single drug (praziquantel) is available to treat schistosomiasis, highlighting the need to better understand schistosome biology to drive the development of a new generation of therapeutics. A major barrier to this goal is the paucity of large-scale datasets exploring schistosome gene function. Here, we describe the first large-scale RNA interference screen in adult Schistosoma mansoni examining the function of over 2000 genes representing approximately 20 percent of the protein coding genome. More than 250 genes were found to have phenotypes affecting neuromuscular function, tissue integrity, stem cell maintenance, and parasite survival. Leveraging these data, we bioinformatically prioritized several compounds with in vitro activity against parasites and validated p97, a component of the ubiquitin proteasome system, as a drug target in the worm. We further reveal a potentially druggable protein kinase-signaling module involving the TAO and STK25 kinases that are essential for maintaining the transcription of muscle-specific mRNAs. Importantly, loss of either of these kinases results in paralysis and death of schistosomes following surgical transplantation into a mammalian host. We anticipate this work will invigorate studies into the biology of these poorly studied organisms and expedite the development of new therapeutics to treat an important neglected tropical disease.

Correction to: Chapter 9 in: David J. Timson (ed.), Schistosoma mansoni: Methods and Protocols, Methods in Molecular Biology, vol. 2151.

Contains fulltext : 220061.pdf (Publisher’s version ) (Closed access) Parasites of the genus Plasmodium infect a wide range of mammalian hosts including humans, primates, bats and arboreal rodents. A hallmark of Plasmodium spp. is the very narrow host range, indicative of matching parasite-host coevolution. Accordingly, their respective genomes harbour many unique genes and gene families that typically encode proteins involved in host cell recognition and remodelling. Whether and to what extent conserved proteins that are shared across Plasmodium spp. also exert distinct species-specific roles remains largely untested. Here, we present detailed functional profiling of the female gametocyte-specific ATP-binding cassette transporter gABCG2 in the murine parasite Plasmodium berghei and compare our findings with data from the orthologous gene in the human parasite Plasmodium falciparum. We show that P. berghei gABCG2 is female-specific and continues to be expressed in zygotes and ookinetes. In contrast to a distinct localization to Iipid-rich gametocyte-specific spots as observed in P. falciparum, the murine malaria parasite homolog is found at the parasite plasma membrane. Plasmodium berghei lacking gABCG2 displays fast asexual blood-stage replication and increased proportions of female gametocytes, consistent with the corresponding P. falciparum knock-out phenotype. Strikingly, cross-species replacement of gABCG2 in either the murine or the human parasite did not restore normal growth rates. The lack of successful complementation despite high conservation across Plasmodium spp. is an indicator of distinct adaptations and tight parasite-host coevolution. Hence, incompatibility of conserved genes in closely related Plasmodium spp. might be more common than previously anticipated.

International audience; DNA-binding proteins play critical roles in many major processes such as development and sexual biology of Schistosoma mansoni, and are important for the pathogenesis of schistosomiasis. Chromatin immunoprecipitation (ChIP) experiments followed by sequencing (ChIP-seq) are useful to characterize the association of genomic regions with posttranslational chemical modifications of histone proteins. Challenges in the standard ChIP protocol have motivated recent enhancements in this approach, such as reducing the number of cells required and increasing the resolution. In this chapter, we describe the latest advances made by our group in the ChIP methods to improve the standard ChIP protocol to reduce the number of input cells required and to increase the resolution and robustness of ChIP in S. mansoni.

Intraspecific competition between co-infecting parasites can influence the amount of virulence, or damage, they do to their host. Kin selection theory dictates that infections with related parasite individuals should have lower virulence than infections with unrelated individuals, because they benefit from inclusive fitness and increased host longevity. These predictions have been tested in a variety of microparasite systems, and in larval stage macroparasites within intermediate hosts, but the influence of adult macroparasite relatedness on virulence has not been investigated in definitive hosts. This study used the human parasite Schistosoma mansoni to determine whether definitive hosts infected with related parasites experience lower virulence than hosts infected with unrelated parasites, and to compare the results from intermediate host studies in this system. The presence of unrelated parasites in an infection decreased parasite infectivity, the ability of a parasite to infect a definitive host, and total worm establishment in hosts, impacting the less virulent parasite strain more severely. Unrelated parasite co-infections had similar virulence to the more virulent of the two parasite strains. We combine these findings with complementary studies of the intermediate snail host and describe trade-offs in virulence and selection within the life cycle. Damage to the host by the dominant strain was muted by the presence of a competitor in the intermediate host, but was largely unaffected in the definitive host. Our results in this host-parasite system suggest that unrelated infections may select for higher virulence in definitive hosts while selecting for lower virulence in intermediate hosts.

Entamoeba histolytica is a human parasite that causes amoebiasis, a disease that affects the colon and liver and is prevalent worldwide. This protozoan requires a high concentration of iron to survive and reproduce. Iron modulates the expression of parasite virulence factors, including hemoglobinases, hemoglobin-binding proteins and cysteine proteases, as well as proteins related to the amoebic cytoskeleton. This review summarizes the virulence factors that are affected by iron, resulting in upregulation or downregulation of E. histolytica genes. This review also discusses the functionality of iron in the mechanisms of pathogenesis.

Objective:Demodex is the most common external human parasite. It has been suggested that the Demodex infection may be associated with malnutrition, nutritional deficiencies, reduced immunity, and poor hygiene. The patients who benefitted from humanitarian aid in Indonesia, often demonstrated the abovementioned risk factors. The aim of the present report was to assess the prevalence of Demodex infestation in ophthalmic patients in Indonesian.Patients and methods:A total of 217 individuals (132 women) with ocular discomfort, who presented to the ophthalmologist in mission points in Indonesia (Bali, Java, and Papua, the province of Indonesia in the New Guinea Island) were checked for the presence of Demodex on their eyelashes and underwent an anthropometric examination.Results:The prevalence of ocular demodicosis varies from 71.2% in Bali to 89.2% in Papua. There were no statistically significant differences between the islands, urban or rural areas, or between sexes. A statistically significant difference was found between nutritional status and Demodex infestation (P = 0.0007). Malnourished and overweight patients had odds of being infested with Demodex that were approximately 8 and 3 times higher, respectively, compared with subjects with a normal body mass index.Conclusions:Ocular demodicosis is very common in medically underserved areas of Bali, Java, and Papua, where it exceeds the frequency observed in countries of the northern hemisphere. An abnormal nutritional status comprising of both malnutrition and being overweight appears to be a risk factor for Demodex infection. This issue requires further research.

Background. Fascioliasis is an important parasitic disease. In the northern region of Brazil, a human parasite infection has been reported through a coprological survey. Eggs of Fasciola hepatica were found in fecal samples of 11 individuals. Knowledge of the infection in animals or the presence of snails is necessary to address the possibility of the parasite cycle occurrence in that region. The aim of this study was to describe the transmission of human fascioliasis in Canutama, Amazonas, in Western Amazonia, Brazil. Methods. Serological (ELISA and Western Blot, WB) and parasitological analyses were carried out in humans. In addition, the presence of the intermediate snail host within the community was examined. Results. A total of 434 human samples were included in the study, of which 36 (8.3%) were reactive by ELISA and 8 (1.8%) were reactive by WB. Fasciola hepatica eggs were found in one human sample. The occurrence of the intermediated host was recorded and 31/43 specimens were identified as Lymnaea columella. Conclusion. Canutama constitutes a focus of transmission of human fascioliasis. This study describes the first serological survey for human fascioliasis, as well as its simultaneous occurrence in human hosts and possible intermediates performed in northern Brazil.