Parasite Reproduction Research Articles

DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi.

Malaria parasites are unusual, early-diverging protozoans with non-canonical cell cycles. They do not undergo binary fission, but divide primarily by schizogony. This involves the asynchronous production of multiple nuclei within the same cytoplasm, culminating in a single mass cytokinesis event. The rate and efficiency of parasite reproduction is fundamentally important to malarial disease, which tends to be severe in hosts with high parasite loads. Here, we have studied for the first time the dynamics of schizogony in two human malaria parasite species, Plasmodium falciparum and Plasmodium knowlesi. These differ in their cell-cycle length, the number of progeny produced and the genome composition, among other factors. Comparing them could therefore yield new information about the parameters and limitations of schizogony. We report that the dynamics of schizogony differ significantly between these two species, most strikingly in the gap phases between successive nuclear multiplications, which are longer in P. falciparum and shorter, but more heterogenous, in P. knowlesi. In both species, gaps become longer as schizogony progresses, whereas each period of active DNA replication grows shorter. In both species there is also extreme variability between individual cells, with some schizonts producing many more nuclei than others, and some individual nuclei arresting their DNA replication for many hours while adjacent nuclei continue to replicate. The efficiency of schizogony is probably influenced by a complex set of factors in both the parasite and its host cell.

Symbiopectobacterium purcellii, gen. nov., sp. nov., isolated from the leafhopper Empoasca decipiens.

Bacterial endosymbionts are found in multiple arthropod species, where they play crucial roles as nutritional symbionts, defensive symbionts or reproductive parasites. Recent work has highlighted a new clade of heritable microbes within the gammaproteobacteria that enter into both obligate and facultative symbioses, with an obligately required unculturable symbiont recently given the name Candidatus Symbiopectobacterium. In this study, we describe a culturable rod shaped non-flagellated bacterial symbiont from this clade isolated from the leafhopper Empoasca decipiens. The symbiont is related to the transovarially transmitted 'BEV' bacterium that was first isolated from the leafhopper Euscelidius variegatus by Alexander Purcell, and we therefore name the symbiont Symbiopectobacterium purcellii sp. nov., gen. nov. We further report the closed genome sequence for S. purcellii. The genome is atypical for a heritable microbe, being large in size, without profound AT bias and with little evidence of pseudogenization. The genome is predicted to encode Type II, III and VI secretion systems and associated effectors and a non-ribosomal peptide synthase array likely to produce bioactive small molecules. The predicted metabolism is more complete than for other symbionts in the Symbiopectobacterium clade, and the microbe is predicted to synthesize a range of B vitamins. However, Biolog plate results indicate that the metabolism is depauperate compared to the sister clade, represented by Pectobacterium carotovorum. A quorum-sensing pathway related to that of Pectobacterium species (containing an overlapping expI-expR1 pair in opposite directions and a "solo" expR2) is evidenced, and LC-MS/MS analysis reveals the presence of 3-hydroxy-C10-HSL as the sole N-acylhomoserine lactone (AHL) in our strain. This AHL profile is profoundly divergent from that of other Erwinia and Pectobacterium species which produce mostly 3-oxo-C6- and 3-oxo-C8-HSL and could aid group identification. Thus, this microbe denotes one that has lost certain pathways associated with a saprophytic lifestyle but represents an important baseline against which to compare other members of the genus Symbiopectobacterium that show more profound integration into host biology. The type strain of Symbiopectobacterium purcellii gen. nov., sp. nov. is SyEd1T (LMG 32449T=CECT 30436T).

Widespread phages of endosymbionts: Phage WO genomics and the proposed taxonomic classification of Symbioviridae.

Wolbachia are the most common obligate, intracellular bacteria in animals. They exist worldwide in arthropod and nematode hosts in which they commonly act as reproductive parasites or mutualists, respectively. Bacteriophage WO, the largest of Wolbachia’s mobile elements, includes reproductive parasitism genes, serves as a hotspot for genetic divergence and genomic rearrangement of the bacterial chromosome, and uniquely encodes a Eukaryotic Association Module with eukaryotic-like genes and an ensemble of putative host interaction genes. Despite WO’s relevance to genome evolution, selfish genetics, and symbiotic applications, relatively little is known about its origin, host range, diversification, and taxonomic classification. Here we analyze the most comprehensive set of 150 Wolbachia and phage WO assemblies to provide a framework for discretely organizing and naming integrated phage WO genomes. We demonstrate that WO is principally in arthropod Wolbachia with relatives in diverse endosymbionts and metagenomes, organized into four variants related by gene synteny, often oriented opposite the putative origin of replication in the Wolbachia chromosome, and the large serine recombinase is an ideal typing tool to distinguish the four variants. We identify a novel, putative lytic cassette and WO’s association with a conserved eleven gene island, termed Undecim Cluster, that is enriched with virulence-like genes. Finally, we evaluate WO-like Islands in the Wolbachia genome and discuss a new model in which Octomom, a notable WO-like Island, arose from a split with WO. Together, these findings establish the first comprehensive Linnaean taxonomic classification of endosymbiont phages, including non-Wolbachia phages from aquatic environments, that includes a new family and two new genera to capture the collective relatedness of these viruses.

Interacting host modifier systems control Wolbachia-induced cytoplasmic incompatibility in a haplodiploid mite.

Reproductive parasites such as Wolbachia spread within host populations by inducing cytoplasmic incompatibility (CI). CI occurs when parasite‐modified sperm fertilizes uninfected eggs and is typified by great variation in strength across biological systems. In haplodiploid hosts, CI has different phenotypic outcomes depending on whether the fertilized eggs die or develop into males. Genetic conflict theories predict the evolution of host modulation of CI, which in turn influences the stability of reproductive parasitism. However, despite the ubiquity of CI‐inducing parasites in nature, there is scarce evidence for intraspecific host modulation of CI strength and phenotype. Here, we tested for intraspecific host modulation of Wolbachia‐induced CI in haplodiploid Tetranychus urticae mites. Using a single CI‐inducing Wolbachia variant and mitochondrion, a nuclear panel was created that consisted of infected and cured near‐isogenic lines. We performed a highly replicated age‐synchronized full diallel cross composed of incompatible and compatible control crosses. We uncovered host modifier systems that cause striking variation in CI strength when carried by infected T. urticae males. We observed a continuum of CI phenotypes in our crosses and identified strong intraspecific female modulation of the CI phenotype. Crosses established a recessive genetic basis for the maternal effect and were consistent with polygenic Mendelian inheritance. Both male and female modulation interacted with the genotype of the mating partner. Our findings identify spermatogenesis as an important target of selection for host modulation of CI strength and underscore the importance of maternal genetic effects for the CI phenotype. Our findings reveal that intraspecific host modulation of CI is underpinned by complex genetic architectures and confirm that the evolution of reproductive parasitism is contingent on host genetics.

Host-dependent impairment of parasite development and reproduction in the acanthocephalan model

BackgroundA central question in parasitology is why parasites mature and reproduce in some host species but not in others. Yet, a better understanding of the inability of parasites to complete their life cycles in less suitable hosts may hold clues for their control. To shed light on the molecular basis of parasite (non-)maturation, we analyzed transcriptomes of thorny-headed worms (Acanthocephala: Pomphorhynchus laevis), and compared developmentally arrested worms excised from European eel (Anguilla anguilla) to developmentally unrestricted worms from barbel (Barbus barbus).ResultsBased on 20 RNA-Seq datasets, we demonstrate that transcriptomic profiles are more similar between P. laevis males and females from eel than between their counterparts from barbel. Impairment of sexual phenotype development was reflected in gene ontology enrichment analyses of genes having differential transcript abundances. Genes having reproduction- and energy-related annotations were found to be affected by parasitizing either eel or barbel. According to this, the molecular machinery of male and female acanthocephalans from the eel is less tailored to reproduction and more to coping with the less suitable environment provided by this host. The pattern was reversed in their counterparts from the definitive host, barbel.ConclusionsComparative analysis of transcriptomes of developmentally arrested and reproducing parasites elucidates the challenges parasites encounter in hosts which are unsuitable for maturation and reproduction. By studying a gonochoric species, we were also able to highlight sex-specific traits. In fact, transcriptomic evidence for energy shortage in female acanthocephalans associates with their larger body size. Thus, energy metabolism and glycolysis should be promising targets for the treatment of acanthocephaliasis. Although inherently enabling a higher resolution in heterosexuals, the comparison of parasites from definitive hosts and less suitable hosts, in which the parasites merely survive, should be applicable to hermaphroditic helminths. This may open new perspectives in the control of other helminth pathogens of humans and livestock.

Novel High-Throughput Fluorescence-Based Assay for the Identification of Nematocidal Compounds That Target the Blood-Feeding Pathway.

Hookworm infections cause a neglected tropical disease (NTD) affecting ~740 million people worldwide, principally those living in disadvantaged communities. Infections can cause high morbidity due to their impact on nutrient uptake and their need to feed on host blood, resulting in a loss of iron and protein, which can lead to severe anaemia and impaired cognitive development in children. Currently, only one drug, albendazole is efficient to treat hookworm infection and the scientific community fears the rise of resistant strains. As part of on-going efforts to control hookworm infections and its associated morbidities, new drugs are urgently needed. We focused on targeting the blood-feeding pathway, which is essential to the parasite survival and reproduction, using the laboratory hookworm model Nippostrongylus brasiliensis (a nematode of rodents with a similar life cycle to hookworms). We established an in vitro-drug screening assay based on a fluorescent-based measurement of parasite viability during blood-feeding to identify novel therapeutic targets. A first screen of a library of 2654 natural compounds identified four that caused decreased worm viability in a blood-feeding-dependent manner. This new screening assay has significant potential to accelerate the discovery of new drugs against hookworms.

Altered within- and between-host transmission under coinfection underpin parasite co-occurrence patterns in the wild

Interactions among parasite species coinfecting the same host individual can have far reaching consequences for parasite ecology and evolution. How these within-host interactions affect epidemics may depend on two non-exclusive mechanisms: parasite growth and reproduction within hosts, and parasite transmission between hosts. Yet, how these two mechanisms operate under coinfection, and how sensitive they are to the composition of the coinfecting parasite community, remains poorly understood. Here, we test the hypothesis that the relationship between within- and between-host transmission of the fungal pathogen, Phomopsis subordinaria, is affected by co-occurring parasites infecting the host plant, Plantago lanceolata. We conducted a field experiment manipulating the parasite community of transmission source plants, then tracked P. subordinaria within-host transmission, as well as between-host transmission to naïve recipient plants. We find that coinfection with the powdery mildew pathogen, Podosphaera plantaginis, causes increased between-host transmission of P. subordinaria by affecting the number of infected flower stalks in the source plants, resulting from altered auto-infection. In contrast, coinfection with viruses did not have an effect on either within- or between-host transmission. We then analyzed data on the occurrence of P. subordinaria in 2018 and the powdery mildew in a multi-year survey data set from natural host populations to test whether the positive association predicted by our experimental results is evident in field epidemiological data. Consistent with our experimental findings, we observed a positive association in the occurrence of P. subordinaria and historical powdery mildew persistence. Jointly, our experimental and epidemiological results suggest that within- and between-host transmission of P. subordinaria depends on the identity of coinfecting parasites, with potentially far-reaching effects on disease dynamics and parasite co-occurrence patterns in wild populations.

Intersexuality in a natural population of the terrestrial isopod Porcellioscaber.

Intersex phenotypes are rarely observed in natural isopod populations and their expression is typically associated with infection of Wolbachia, a reproductive parasite that manipulates arthropod reproduction. During an intensive sampling effort of a natural population of the isopod Porcellioscaber, an adult individual was isolated that expressed both male and female traits. The intersex individual exhibited clearly developed external male genitalia and carried multiple eggs in its brood pouch. No Wolbachia infection could be identified in this individual, a result that needs to be approached with caution due to suboptimal DNA preservation for diagnostic PCR assays. Wolbachia were, however, detected in two adult females of the same population, and appear closely related to isolates that infect other terrestrial isopod species. This is the first demonstration that intersex phenotypes can arise under natural conditions in P.scaber.

Discovery of Early-Branching Wolbachia Reveals Functional Enrichment on Horizontally Transferred Genes.

Wolbachia is a widespread endosymbiont of insects and filarial nematodes that profoundly influences host biology. Wolbachia has also been reported in rhizosphere hosts, where its diversity and function remain poorly characterized. The discovery that plant-parasitic nematodes (PPNs) host Wolbachia strains with unknown roles is of interest evolutionarily, ecologically, and for agriculture as a potential target for developing new biological controls. The goal of this study was to screen communities for PPN endosymbionts and analyze genes and genomic patterns that might indicate their role. Genome assemblies revealed 1 out of 16 sampled sites had nematode communities hosting a Wolbachia strain, designated wTex, that has highly diverged as one of the early supergroup L strains. Genome features, gene repertoires, and absence of known genes for cytoplasmic incompatibility, riboflavin, biotin, and other biosynthetic functions placed wTex between mutualist C + D strains and reproductive parasite A + B strains. Functional terms enriched in group L included protoporphyrinogen IX, thiamine, lysine, fatty acid, and cellular amino acid biosynthesis, while dN/dS analysis suggested the strongest purifying selection on arginine and lysine metabolism, and vitamin B6, heme, and zinc ion binding, suggesting these as candidate roles in PPN Wolbachia. Higher dN/dS pathways between group L, wPni from aphids, wFol from springtails, and wCfeT from cat fleas suggested distinct functional changes characterizing these early Wolbachia host transitions. PPN Wolbachia had several putative horizontally transferred genes, including a lysine biosynthesis operon like that of the mitochondrial symbiont Midichloria, a spirochete-like thiamine synthesis operon shared only with wCfeT, an ATP/ADP carrier important in Rickettsia, and a eukaryote-like gene that may mediate plant systemic acquired resistance through the lysine-to-pipecolic acid system. The Discovery of group L-like variants from global rhizosphere databases suggests diverse PPN Wolbachia strains remain to be discovered. These findings support the hypothesis of plant-specialization as key to shaping early Wolbachia evolution and present new functional hypotheses, demonstrating promise for future genomics-based rhizosphere screens.

A chromosome-level genome of the human blood fluke Schistosoma japonicum identifies the genomic basis of host-switching.

The evolution and adaptation of S.japonicum, a zoonotic parasite that causes human schistosomiasis, remain unclear because of the lack of whole-genome data. We construct a chromosome-level S.japonicum genome and analyze it together with 72 samples representing six populations of the entire endemic region. We observe a Taiwan zoophilic lineage splitting from zoonotic populations ∼45,000 years ago, consistent with the divergent history of their intermediate hosts. Interestingly, we detect a severe population bottleneck in S.japonicum, largely coinciding with human history in Asia during the last glacial maximum. We identify several genomic regions underlying natural selection, including GATAD2A and Lmln, both showing remarkable differentiation among different areas. RNAi knockdown suggests association of GATAD2A with parasite development and infection in definitive hosts, while Lmln relates to the specificity of the intermediate hosts. Our study provides insights into the evolution of S.japonicum and serves as a resource for further studies.

Ovary activation correlates with the reproductive potential of honeybee (Apis mellifera) workers if they are in a foreign colony

In eusocial Hymenoptera, the laying of male-determined eggs by workers in an unrelated colony can be a powerful strategy for increasing direct fitness benefits. A recent study showed that honeybee rebels, which are workers that develop under queenless conditions and have high reproductive potential, drift to foreign colonies, with a preference for hopelessly queenless colonies, and act as reproductive parasites. In our experiment, by introducing 5-day-old rebels and normal workers into foreign queenright colonies, we discovered that only rebels developed a reproductive phenotype by activating their ovaries. In a similar experiment with 1-day-old workers, neither rebels nor normal workers displayed such a tendency. We suggest that workers’ reproductive potential could thus be a key parameter activating the ovaries in not only drifting workers but also workers acting as reproductive parasites. Our results also support the hypothesis that the colony recognition cue probably has an environmental, rather than a genetic, origin.

Changes in the Prevalence of Natural Paramphistomum Cercariae Infection in Indoplanorbis and Lymnaea Intermediate Hosts Influenced by Meteorological Factors.

Paramphistomosis is a neglected ruminant parasitic disease caused by trematodes known as Paramphistomum, which has a diheteroxenic life cycle involving freshwater snail genera, i.e., Planorbis and Lymnaea as the intermediate host and mammals as the definitive host. Snail vector distribution, infection with Paramphistomum spp. cercariae, preferred habitat, and their relationship with certain meteorological factors were not investigated in the province Khyber Pakhtunkhwa of Pakistan. Therefore, this study is designed to evaluate the effects of meteorological factors on the occurrence and severity of Paramphistomum spp. cercariae in Indoplanorbis and Lymnaea intermediate snail hosts. For this purpose, a cross-sectional survey was conducted from October 2018 to September 2019, and snails were collected and then identified using snail shell morphology; their infection with Paramphistomum spp. cercariae was determined through microscopy; and descriptive statistics were used to estimate the prevalence of infection and evaluate their occurrence relationship with a certain meteorological factors including temperature, humidity, rainfall, and pan evaporation in different districts of the above-mentioned province of Pakistan, i.e., adjacent areas of Bara and Kabul rivers in district Nowshehra, Kabul River (Sardaryab) of district Charsadda, Kalpani River of district Mardan, and Indus River (Hund) of district Swabi. A total of 2,706 Indoplanorbis (1539) and Lymnaea (1167) snails were collected, in which overall 10.30% shed Paramphistomum spp. cercariae. The highest infection rate was found in the river of district Swabi (13.20%), while the lowest in adjacent rivers of district Nowshehra (8.19%). Meteorological factors play an essential role in the causation of Paramphistomum spp. infection, parasitic reproduction, vector growth, and survival. Due to these factors, high significant prevalence was found in the summer season (11.83), followed by autumn (11.25), which might be due to optimum temperature, relative humidity, and rainfall (p < 0.05). It was concluded from the study that meteorological factors contribute to the prevalence of Paramphistomum species in the Indoplanorbis and Lymnaea, which act as vectors for the disease paramphistomosis, which may lead to the increased intensity of infection outbreaks of the parasite population in humans and domestic animals.

Detection of Wolbachia and Leishmania DNA in sand flies (Diptera: Psychodidae, Phlebotominae) from a focus of cutaneous leishmaniasis in Tabasco, Mexico.

Phlebotomine sand flies are the main vectors of Leishmania genus species worldwide; therefore, the detection of some reproductive parasites, such as Wolbachia, has been considered a possible strategy for biological control. In Mexico, leishmaniasis cases have been recorded in 25 states, yet only two sand fly species have been related to Wolbachia spp. Although the state of Tabasco has a high number of leishmaniasis cases, only few studies have been done on sand fly species. The aim of this study was to analyze the diversity of sand fly species and to detect Wolbachia spp. and/or Leishmania spp. in the captured specimens. Sand flies were collected at the locality of Huimango, Tabasco, Mexico, during October 2019, using nine light traps (CDC) and two Shannon traps per night. The specimens were identified and females were analyzed by PCR for the DNA detection for pathogens. A total of 193 sand fly specimens belonging to five species were morphologically identified. Pintomyia ovallesi was the most abundant species (76.84%), followed by Micropygomyia cayennensis (6.40%). Furthermore, first records of four sand fly species were established for the state of Tabasco, thereby increasing the species richness in the state from four to eight. We observed a natural infection rate of 9.7% (10/103) for Leishmania and 0.91% (1/103) for Wolbachia. The importance of conducting entomological surveys in endemic areas of leishmaniasis in Mexico is highlighted, to determine whether other sand fly species may be potential vectors of Leishmania spp., and if some Wolbachia strains could be relevant for the control of leishmaniasis.

Comparison of the Parasitization of Chelonus inanitus L. (Hymenoptera: Braconidae) in Two Spodoptera Pests and Evaluation of the Procedure for Its Production.

Simple SummarySuccessful integrated pest management of horticultural crops requires the evaluation of natural enemies used in biological control. To improve the mass rearing of Chelonus inanitus (L.) in the factitious host Ephestia kuehniella Zeller, we investigated the influence of host age and number of females parasitizing simultaneously on quality parameters evaluated in the commercial production of biological control agents, such as overall rearing success, duration of life cycle and body size. Under semi-field conditions, we investigated how the competition among females impacted the parasitism success on important pests Spodoptera exigua (Hübner) and S. littoralis (Boisduval). For the parasitoid mass rearing optimization, mature eggs seem to be more convenient because they increased the female percentage in the offspring and shortened the parasitoid life cycle. Furthermore, a high number of females parasitizing simultaneously reduced the emergence of non-parasitized hosts. Under more realistic conditions, the parasitoid effectively controlled both pests, but S. exigua may be a more convenient host because the parasitoid offspring was much larger. With this research, we provide a foundation that aims to better manage these cosmopolitan, highly polyphagous pests that exhibit long-distance migratory potential, alongside the potential use of this natural enemy.Chelonus inanitus (L.) is an egg-larval parasitoid of noctuids Spodoptera exigua (Hübner) and S. littoralis (Boisduval), whose mass rearing or real potential has not been targeted yet. To improve the rearing in the factitious host Ephestia kuehniella Zeller, we investigated the influence of host age and number of females parasitizing simultaneously on the overall rearing success, the influence of host age on the life cycle, and the influence of host species on the parasitoid body size. The proportion of emerging C. inanitus was higher from young host eggs, but more females emerged from mature eggs. Under high parasitoid competition, we observed a reduction in non-parasitized hosts without reducing parasitoid emergence. The parasitoid life cycle was longer in females, but the mismatch between sexes was smaller in mature eggs. The parasitoid size was smaller in the factitious host than in the natural hosts. Under semi-field conditions, we investigated the competition among parasitoid females on the overall parasitism success. The reproductive parasitism was more successful in S. exigua than in S. littoralis, and the maximum emergence was reached with three and four females, respectively. The control of S. littoralis may be attributed to the high developmental mortality, a non-reproductive parasitism that is often underestimated.

Characterization of the endogenous DAF-12 ligand and its use as an anthelmintic agent in Strongyloides stercoralis.

A prevalent feature of Strongyloides stercoralis is a life-long and potentially lethal infection that is due to the nematode parasite's ability to autoinfect and, thereby, self-replicate within its host. Here, we investigated the role of the parasite's nuclear receptor, Ss-DAF-12, in governing infection. We identified Δ7-DA as the endogenous Ss-DAF-12 ligand and elucidated the hormone's biosynthetic pathway. Genetic loss of function of the ligand's rate-limiting enzyme demonstrated that Δ7-DA synthesis is necessary for parasite reproduction, whereas its absence is required for the development of infectious larvae. Availability of the ligand permits Ss-DAF-12 to function as an on/off switch governing autoinfection, making it vulnerable to therapeutic intervention. In a preclinical model of hyperinfection, pharmacologic activation of DAF-12 suppressed autoinfection and markedly reduced lethality. Moreover, when Δ7-DA was administered with ivermectin, the current but limited drug of choice for treating strongyloidiasis, the combinatorial effects of the two drugs resulted in a near cure of the disease.

Pb103 Regulates Zygote/Ookinete Development in Plasmodium berghei via Double Zinc Finger Domains.

Sexual reproduction of Plasmodium parasites takes place in anopheline mosquitoes, where male and female gametes fuse to form zygotes and then ookinetes. These processes are orchestrated by stage-specific protein expression, which is mediated in part by translational repression. Accumulating evidence shows that RNA binding proteins (RBPs) play crucial roles in these processes. Here, we report the characterization of P. berghei 103 (Pb103), which encodes a protein possessing double zinc finger domains (ZFs), an RBP. Reporter parasites expressing azami green fluorescent protein (AGFP) under the endogenous Pb103 gene promoter (Pb103-AGFP reporter) showed that the AGFP fluorescent signal was detected from gametes to ookinetes, while AGFP mRNA was translationally repressed in female gametocytes. The Pb103-disrupted parasites (Pb103(−)) grew and produced gametocytes with similar efficiencies to those of wild-type parasites. However, no oocysts were formed in mosquitoes fed Pb103(−). An in vitro fertilization assay showed abortion at the zygote stage in Pb103(−), suggesting that Pb103 plays a critical role in zygote/ookinete development. Cross-fertilization assays with Pb103(−) and male- or female-sterile parasites revealed that Pb103 was essential exclusively for female gametes. To identify the domains critical for zygote/ookinete development, transgenic parasites expressing partially deleted Pb103 were generated and assayed for ookinete maturation. As a result, deleting either of two ZFs but not the C-terminal region abolished zygote/ookinete development, highlighting the indispensable roles of ZFs in parasite sexual development, most likely via translational repression.

The mTOR Pathway in Platelets Contributes to the Pathophysiology of Experimental Cerebral Malaria

Background: Cerebral malaria is a highly prevalent infectious disease in Sub-Saharan Africa caused by the Plasmodium parasite. The pathogenesis of cerebral malaria results from damaged vascular endothelium induced by parasite sequestration, inflammatory cytokine production and vascular leakage, which results in increased brain permeability and death. While maladaptive responses from immune cells are thought to contribute, growing evidence suggests a crucial role of platelets in malaria pathophysiology. The mammalian target of rapamycin (mTOR) pathway is critical in regulating outcomes in malaria. Previous studies have demonstrated an mTOR specific inhibitor, rapamycin, is protective in a mouse model of experimental cerebral malaria (ECM). However, if the mTOR pathway in platelets specifically contributes to the pathogenesis of malaria is unknown.Methods: Platelet-specific mTOR-deficient (mTOR plt-/-) mice and littermate controls were subjected to a well-established model of ECM, using Plasmodium berghei ANKA. In addition, platelets isolated from human malaria patients were examined for differential regulation of the mTOR pathway using RNA-seq.Results: Platelet RNA-seq and Ingenuity Pathway Analysis from patients infected with P. vivax demonstrated enrichment of mTOR-associated pathways in platelets, such as mTOR signaling and p70S6K signaling, indicating mTOR associated genes are upregulated in human platelets during malaria infection. In mice infected with P. berghei ANKA, the mTOR pathway was activated in bone marrow-megakaryocytes and platelets based on phosphorylation of mTOR and its downstream effector, 4E-BP1. As the mTOR pathway regulates protein translation in platelets, we examined de novo protein synthesis and observed increased protein translation in platelets isolated from mice infected with P. berghei ANKA compared to uninfected controls.To study the specific role of platelet mTOR during ECM pathogenesis, mTOR plt-/- mice and wild-type controls (mTOR plt+/+), were infected with P. berghei ANKA. Platelet deficient-mTOR mice had significantly (p=0.0336) prolonged survival compared to wild-type mice. Increased survival was independent of parasitemia, suggesting platelets did not alter parasite reproduction. While thrombocytopenia and anemia were similar in both genotypes, mTOR plt-/- mice had significantly reduced brain (p=0.0067) and lung (p<0.0001) vascular permeability during late-stage ECM. Interestingly, flow cytometric assessment of leukocyte recruitment to the brain demonstrated a 1.7-fold (p=0.0442) reduction in inflammatory monocytes in platelet-deficient mTOR mice. However, mTOR plt-/- mice had significantly (1.4-fold, p=0.007) more inflammatory monocytes in the blood. Interestingly, circulating platelet-monocytes aggregates were significantly less in mTOR plt-/- compared to mTOR plt+/+ (p=0.0433). Taken together, these results suggest that platelets assist in the recruitment of leukocytes to the brain vasculature during ECM, which is impaired when mTOR is ablated.Conclusions: Our data demonstrates that the mTOR pathway in platelets plays a significant role in malaria pathogenesis. Deletion of platelet mTOR reduces vascular permeability and prolongs survival during ECM. We hypothesize that altered platelet-inflammatory monocyte interactions drive this phenotype. DisclosuresRondina: Platelet Transcriptomics: Patents & Royalties; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding.

A single synonymous nucleotide change impacts the male-killing phenotype of prophage WO gene wmk.

Wolbachia are the most widespread bacterial endosymbionts in animals. Within arthropods, these maternally transmitted bacteria can selfishly hijack host reproductive processes to increase the relative fitness of their transmitting females. One such form of reproductive parasitism called male killing, or the selective killing of infected males, is recapitulated to degrees by transgenic expression of the prophage WO-mediated killing (wmk) gene. Here, we characterize the genotype-phenotype landscape of wmk-induced male killing in D. melanogaster using transgenic expression. While phylogenetically distant wmk homologs induce no sex-ratio bias, closely-related homologs exhibit complex phenotypes spanning no death, male death, or death of all hosts. We demonstrate that alternative start codons, synonymous codons, and notably a single synonymous nucleotide in wmk can ablate killing. These findings reveal previously unrecognized features of transgenic wmk-induced killing and establish new hypotheses for the impacts of post-transcriptional processes in male killing variation. We conclude that synonymous sequence changes are not necessarily silent in nested endosymbiotic interactions with life-or-death consequences.

Time-Varying Effects of Meteorological Variables on Malaria Epidemiology in the Context of Interrupted Control Efforts in the Amazon Rainforest, 2000-2017.

Successful malaria control interventions, mostly based on the training of health workers, distribution of insecticide-treated nets, and spraying, decrease malaria incidence; however, when these interventions are interrupted, a resurgence may occur. In the Peruvian Amazon, after discontinuing the control activities implemented by the PAMAFRO project (2006–2010)-a Global Fund-sponsored project for the strengthening of malaria control and surveillance in multiple countries in Latin America– malaria cases re-emerged dramatically. In parallel, meteorological factors determine the conditions suitable for the development, reproduction, and survival of mosquito vectors and parasites. This study hypothesized that interruption of malaria interventions may have modified the meteorological-malaria relationships over time (i.e., temporal changes in the dose-response between meteorological variables and malaria incidence). In this panel data analysis, we assessed the extent that relationships between meteorological variables and malaria changed temporally using data of monthly malaria incidence due to Plasmodium vivax or P. falciparum in Loreto, Peru (2000–2017). Generalized additive models were used to explore how the effects of meteorological variables changed in magnitude before, during, and after the PAMAFRO intervention. We found that once the PAMAFRO intervention had been interrupted, the estimated effects (dose-response) of meteorological variables on incidence rates decreased for both malaria parasite species. However, these fitted effect estimates did not reach their baseline levels (before the PAMAFRO period); variations of time-varying slopes between 0.45 and 2.07 times were observed after the PAMAFRO intervention. We also reported significant heterogeneity in the geographical distributions of malaria, parasite species, and meteorological variables. High malaria transmission occurred consistently in the northwestern provinces of Loreto Department. Since the end of the PAMAFRO period, a higher effect of precipitation and actual evapotranspiration was described on P. falciparum compared to P. vivax. The effect of temperature on malaria was greater over a shorter time (1-month lag or less), compared with precipitation and actual evapotranspiration (12-month lag). These findings demonstrate the importance of sustained malaria control efforts since interruption may enhance the links between meteorological factors and malaria. Our results also emphasize the importance of considering the time-varying effect of meteorological factors on malaria incidence to tailor control interventions, especially to better manage the current and future climate change crisis.

Effects of supplementation on production and age anticipation at first mating of ewe lambs in tropical pastures.

Studies on the rearing of ewe lambs in the Cerrado are scarce, so the objective was to evaluate the effects of protein-energy supplementation with 1.6 and 2.4% BW on the productive and reproductive performance of ewe lambs raised on pastures of Brachiaria brizantha cv. Marandu. Twenty-four Texel ewe lambs, with a mean age of 5months, were distributed in two treatments with provision of energy protein supplementation at levels of 1.6 and 2.4% BW, formulated for average daily gains of 150 and 200g/day for ewe lambs weighing 20kg, respectively. The experimental delineation adopted was the completely randomized design, with fifteen repetitions per treatment. The performance of the ewe lambs was evaluated by weight at the beginning and end of the breeding season (BS), average daily gain (ADG), and total weight gain (TWG). The count of fecal eggs was performed monthly. The evaluation of the ewe lambs reproductive organs took place at 8months of age. The fertility of the ewe lambs was detected by transrectal ultrasonography at the end of the BS. The supplementation level of 2.4% BW was higher for ADG and TWG; however, it did not influence the other performance characteristics. There was no effect of supplementation on the reproductive characteristics, parasite load, and nutrients of the pasture. Pasture nutrients were influenced by the month of use. Supplementation levels were not effective to ensure the reproductive success of 8-month-old ewe lambs.