Factor Affecting Sex Ratios of a Mermithid Parasite of Mosquitoes

The ratio of male to female Reesimermis nielseni Tsai and Grundmann, a nematode parasite of mosquito larvae, increased as the number of parasites per host increased. Hosts with a single nematode produced 9% males compared with essentially 100% males in hosts with more than 7 parasites; hosts with 3 nematodes produced about equal numbers of males and females. Males of R. nielseni generally emerged before females because of the earlier death of multiple-infected mosquitoes. The species of the host mosquito influenced the sex ratio, but the size of a specific host at the time of invasion did not. Host diet also had a noticeable influence on the sex ratio of the nematode: singly infected hosts from a starved population produced 92% males compared with 13% in the normally fed group. The importance of these factors in the mass rearing of R. nielseni is discussed.     

Stability of within-host–parasite communities in a wild mammal system

Simultaneous infection by multiple parasite species is ubiquitous in nature. Interactions among co-infecting parasites may have important consequences for disease severity, transmission and community-level responses to perturbations. However, our current view of parasite interactions in nature comes primarily from observational studies, which may be unreliable at detecting interactions. We performed a perturbation experiment in wild mice, by using an anthelminthic to suppress nematodes, and monitored the consequences for other parasite species. Overall, these parasite communities were remarkably stable to perturbation. Only one non-target parasite species responded to deworming, and this response was temporary: we found strong, but short-lived, increases in the abundance of Eimeria protozoa, which share an infection site with the dominant nematode species, suggesting local, dynamic competition. These results, providing a rare and clear experimental demonstration of interactions between helminths and co-infecting parasites in wild vertebrates, constitute an important step towards understanding the wider consequences of similar drug treatments in humans and animals.     

Generation and functional characterisation of Plasmodium yoelii csp deletion mutants using a microhomology-based CRISPR/Cas9 method

CRISPR/Cas9 is a powerful genome editing method that has greatly facilitated functional studies in many eukaryotic organisms including malaria parasites. Due to the lack of genes encoding enzymes necessary for the non-homologous end joining DNA repair pathway, genetic manipulation of malaria parasite genomes is generally accomplished through homologous recombination requiring the presence of DNA templates. Recently, an alternative double-strand break repair pathway, microhomology-mediated end joining, was found in the Plasmodium falciparum parasite. Taking advantage of the MMEJ pathway, we developed a MMEJ-based CRISPR/Cas9 (mCRISPR) strategy to efficiently generate multiple mutant parasites simultaneously in genes with repetitive sequences. As a proof of principle, we successfully produced various size mutants in the central repeat region of the Plasmodium yoelii circumsporozoite surface protein without the use of template DNA. Monitoring mixed parasite populations and individual parasites with different sizes of CSP-CRR showed that the CSP-CRR plays a role in the development of mosquito stages, with severe developmental defects in parasites with large deletions in the repeat region. However, the majority of the csp mutant parasite clones grew similarly to the wild type P. yoelii 17XL parasite in mice. This study develops a useful technique to efficiently generate mutant parasites with deletions or insertions, and shows that the CSP-CRR plays a role in parasite development in mosquito.     

Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles stephensi Mosquitoes

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity.     

Complex interactions among a nematode parasite (Daubaylia potomaca), a commensalistic annelid (Chaetogaster limnaei limnaei), and trematode parasites in a snail host (Helisoma anceps)

Many biotic interactions can affect the prevalence and intensity of parasite infections in aquatic snails. Historically, these studies have centered on interactions between trematode parasites or between trematodes and other organisms. The present investigation focuses on the nematode parasite Daubaylia potomaca and its interactions with a commensal, Chaetogaster limnaei limnaei , and a variety of trematode species. It was found that the presence of C. l. limnaei indirectly increased the mean intensity of D. potomaca infections, apparently by acting as a restraint for various trematode parasites, particularly the rediae of Echinostoma sp. In turn, Echinostoma sp. rediae adversely affected the mean intensity of D. potomaca by their consumption of both juvenile and adult nematodes present in tissues of the snail. These organisms not only belong to 3 different phyla but occupy distinct trophic levels as well. The complex interactions among these 3 organisms in the snail host provide an excellent example of biotic interactions influencing the infection dynamics of parasites in aquatic snails.     

Cystatins from filarial parasites: evolution, adaptation and function in the host-parasite relationship

Cystatins, together with stefins and kininogens, are members of the cystatin superfamily of cysteine protease inhibitors (CPI) present across the animal and plant kingdoms. Their role in parasitic organisms may encompass both essential developmental processes and specific interactions with the parasite's vector and/or final host. We summarise information gathered on three cystatins from the human filarial nematode Brugia malayi (Bm-CPI-1, -2 and -3), and contrast them those expressed by other parasites and by the free-living nematode Caenorhabditis elegans. Bm-CPI-2 differs from C. elegans cystatin, having acquired the additional function of inhibiting asparaginyl endopeptidase (AEP), in a manner similar to some human cystatins. Thus, we propose that Bm-CPI-2 and orthologues from related filarial parasites represent a new subset of nematode cystatins. Bm-CPI-1 and CPI-3 share only 25% amino acid identity with Bm-CPI-2, and lack an evolutionarily conserved glycine residue in the N-terminal region. These sequences group distantly from the other nematode cystatins, and represent a second novel subset of filarial cystatin-like genes. Expression analyses also show important differences between the CPI-2 and CPI-1/-3 groups. Bm-cpi-2 is expressed at all time points of the parasite life cycle, while Bm-cpi-1 and -3 expression is confined to the late stages of development in the mosquito vector, terminating within 48h of infection of the mammalian host. Hence, we hypothesise that CPI-2 has evolved to block mammalian proteases (including the antigen-processing enzyme AEP) while CPI-1 and -3 function in the milieu of the mosquito vector necessary for transmission of the parasite.     

Genetic basis of encapsulation response in Anopheles gambiae

The phenomenon of encapsulation of invading organisms is widespread in insects. Co-evolution has produced an intricate balance between the immune responses of the host and immune-suppressive (or immune-evading) properties of the parasite. Genome-wide genetic mapping revealed different loci in Anopheline mosquitoes were involved in melanotic encapsulation of different malaria parasites. Certain isolates of human malaria parasites can still suppress or avoid the immune response from refractory mosquitoes. Similar interactions with parasitoids were observed in Drosophila melanogaster. Species-specific encapsulation locus was identified for two parasitoids, respectively, and virulent strain of parasitoid can suppress the immune system of an otherwise resistant fruitfly. It is believed that the encapsulation loci in both mosquitoes and fruitfly may encode gene products that function at the early stages of parasite/parasitoid recognition or immediate signaling events. Future research on membrane receptor molecules and their roles in insect immunity will yield interesting insights into mosquito-parasite interactions.     

Rhoptry neck protein 11 has crucial roles during malaria parasite sporozoite invasion of salivary glands and hepatocytes

The malaria parasite sporozoite sequentially invades mosquito salivary glands and mammalian hepatocytes; and is the Plasmodium lifecycle infective form mediating parasite transmission by the mosquito vector. The identification of several sporozoite-specific secretory proteins involved in invasion has revealed that sporozoite motility and specific recognition of target cells are crucial for transmission. It has also been demonstrated that some components of the invasion machinery are conserved between erythrocytic asexual and transmission stage parasites. The application of a sporozoite stage-specific gene knockdown system in the rodent malaria parasite, Plasmodium berghei, enables us to investigate the roles of such proteins previously intractable to study due to their essentiality for asexual intraerythrocytic stage development, the stage at which transgenic parasites are derived. Here, we focused on the rhoptry neck protein 11 (RON11) that contains multiple transmembrane domains and putative calcium-binding EF-hand domains. PbRON11 is localised to rhoptry organelles in both merozoites and sporozoites. To repress PbRON11 expression exclusively in sporozoites, we produced transgenic parasites using a promoter-swapping strategy. PbRON11-repressed sporozoites showed significant reduction in attachment and motility in vitro, and consequently failed to efficiently invade salivary glands. PbRON11 was also determined to be essential for sporozoite infection of the liver, the first step during transmission to the vertebrate host. RON11 is demonstrated to be crucial for sporozoite invasion of both target host cells – mosquito salivary glands and mammalian hepatocytes – via involvement in sporozoite motility.     

Intracellular melanization of the larvae of Dirofilaria immitis in the malpighian tubules of the mosquito, Aedes sollicitans

Frequent melanization of larvae of the nematode Dirofilaria immitis parasitizing the Malpighian tubules of the mosquito, Aedes sollicitans, has been observed. Melanized and nonmelanized larvae in the Malpighian tubules were examined using light and electron microscopy. The results indicate that the pattern of melanin deposition and the ultrastructural characteristics of the pigment around the worms are identical to that observed on nematodes which have undergone humoral melanization in other dipteran insects. In the Malpighian tubules, no contact between the intracellular melanized nematodes and the hemolymph or hemocytes was observed. The results suggest that the Malpighian tubules of this species of mosquito are capable of inducing a melanotic response to invading nematode parasites. It is proposed that this is an example of “humoral” melanization at an intracellular site.     

Inter- and intraspecific conflicts between parasites over host manipulation

Host manipulation is a common strategy by which parasites alter the behaviour of their host to enhance their own fitness. In nature, hosts are usually infected by multiple parasites. This can result in a conflict over host manipulation. Studies of such a conflict in experimentally infected hosts are rare. The cestode Schistocephalus solidus (S) and the nematode Camallanus lacustris (C) use copepods as their first intermediate host. They need to grow for some time inside this host before they are infective and ready to be trophically transmitted to their subsequent fish host. Accordingly, not yet infective parasites manipulate to suppress predation. Infective ones manipulate to enhance predation. We experimentally infected laboratory-bred copepods in a manner that resulted in copepods harbouring (i) an infective C plus a not yet infective C or S, or (ii) an infective S plus a not yet infective C. An infective C completely sabotaged host manipulation by any not yet infective parasite. An infective S partially reduced host manipulation by a not yet infective C. We hence show experimentally that a parasite can reduce or even sabotage host manipulation exerted by a parasite from a different species.     

Diversity and specificity in the interaction between <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> and the pathogen <Emphasis Type="Italic">Serratia marcescens</Emphasis>

Background  

Co-evolutionary arms races between parasites and hosts are considered to be of immense importance in the evolution of living organisms, potentially leading to highly dynamic life-history changes. The outcome of such arms races is in many cases thought to be determined by frequency dependent selection, which relies on genetic variation in host susceptibility and parasite virulence, and also genotype-specific interactions between host and parasite. Empirical evidence for these two prerequisites is scarce, however, especially for invertebrate hosts. We addressed this topic by analysing the interaction between natural isolates of the soil nematode Caenorhabditis elegans and the pathogenic soil bacterium Serratia marcescens.     

Effects of Host Size and Parasite Burden on Sex Ratio in the Mosquito Parasite Octomyomermis muspratti

The ratio of Octomyomermis muspratti to the host mosquito at the time of exposure had little effect on the ratio of male to female parasites that resulted. However, the ratio of males to females increased as the number of parasites/host increased. Hosts with a single nematode produced fewer than 1% males in comparison with hosts with 8 parasites which produced about 40% males; hosts with 10 or more nematodes generally produced more male than female nematodes. Males of O. muspratti usually emerged before females because of the earlier death of multiply-infected mosquitoes. The size of the host at the time of invasion bad no significant influence on nematode sex ratios. Since mating is apparently necessary for reproduction in O. muspratti, the low male to female ratios that occur will be important in developing successful mass production techniques.     

A New Role of the Mosquito Complement-like Cascade in Male Fertility in Anopheles gambiae

Thioester-containing protein 1 (TEP1) is a key immune factor that determines mosquito resistance to a wide range of pathogens, including malaria parasites. Here we report a new allele-specific function of TEP1 in male fertility. We demonstrate that during spermatogenesis TEP1 binds to and removes damaged cells through the same complement-like cascade that kills malaria parasites in the mosquito midgut. Further, higher fertility rates are mediated by an allele that renders the mosquito susceptible to Plasmodium. By elucidating the molecular and genetic mechanisms underlying TEP1 function in spermatogenesis, our study suggests that pleiotropic antagonism between reproduction and immunity may shape resistance of mosquito populations to malaria parasites.     

Anopheles gambiae genome: perspectives for malaria control

Malaria, a disease that infects 300 million people throughout the world and kills more than a million people, mostly children in sub-Saharan Africa, involves three organisms. The human host where the disease is seen, the protozoan Plasmodium parasite and the mosquito. The parasite is transmitted to humans only by the mosquito vector, which in sub-Saharan regions is generally Anopheles gambiae. Malaria along with AIDS and tuberculosis are killing large numbers of people and crippling the economies of the affected African countries. Though an enormous effort has been made during the past twenty years to develop vaccines to block malaria in humans, the incidence of the disease is increasing in Africa. The reasons for this development include a breakdown in mosquito control related to increased insecticide resistance, as well as increased parasite resistance to antimalarial drugs. It is clear that new methods of Anopheles mosquito control are needed to ameliorate the medical and economic situation in sub-Saharan Africa. As a step toward new malaria control methods, the international Plasmodium falciparum and Anopheles gambiae consortia have carried out the full genome sequencing of the most deadly malaria parasite and the most efficient vector. These, combined with the human genome sequence, provide the genomic infrastructure for a better understanding of the complex interactions within the malaria triad. This essay discusses possible strategies as to how the Anopheles genome can contribute to malaria control.     

Selective Defecation and Selective Foraging: Antiparasite Behavior in Wild Ungulates?

Selective defecation and selective foraging are two potential antiparasite behaviors used by grazing ungulates to reduce infection by fecal–oral transmitted parasites. While there is some evidence that domestic species use these strategies, less is known about the occurrence and efficacy of these behaviors in wild ungulates. In this study, I examined whether wild antelope use selective defecation and selective foraging strategies to reduce exposure to gastrointestinal nematode parasites. By quantifying parasite levels in the environment in relation to the defecation patterns of three species, dik‐dik (Madoqua kirkii), Grant's gazelle (Gazella granti), and impala (Aepyceros melampus), I found that nematode larval concentrations in pasture were higher in the vicinity of clusters of feces (dung middens) compared to single fecal pellet groups or dung‐free areas. In addition, experimental feeding trials in free‐ranging dik‐dik showed that individuals selectively avoided feeding near concentrations of feces. Given that increased parasite contamination was found in the immediate vicinity of fecal clusters, fecal avoidance could help reduce host consumption of parasites and may therefore be an effective antiparasite behavior for certain species. On the other hand, while the concentration of parasite larvae in the vicinity of middens coupled with host avoidance of these areas during grazing could reduce host contact with parasites, results showing a positive correlation between the number of middens in a habitat and larval abundance at control sites suggest that dung middens might increase and not decrease overall host exposure to parasites. If this is the case, dung midden formation may not be a viable antiparasite strategy.     

A very large C-loop in EGF domain IV is characteristic of the P28 family of ookinete surface proteins

The P28 family of proteins are 28 kDa proteins expressed on the surface of sexual stages—zygote, ookinete and young oocyst stages—of Plasmodium species when the parasite resides inside the mosquito midgut. Together with P25 proteins, P28 proteins protect the parasite from the harsh proteolytic environment prevailing inside the mosquito midgut. Vaccines against these proteins induce antibodies in vertebrate hosts that are capable of inhibiting parasite development in the mosquito midgut, thus preventing transmission of the parasite from the mosquito to another human host. These transmission-blocking vaccines are helpful in reducing the burden caused by malaria, which affects 300–600 million, and kills 1–3 million, people annually. The purpose of this study was to structurally characterise six members of the P28 family of ookinete surface proteins with the help of homology modelling, to compare these proteins in terms of transmission blocking and host parasite interactions, and to analyse phylogenetic relationships within the P28 family and with the P25 family. Our results indicate that all the members of the P28 family studied have four EGF domains arranged in triangular fashion with a very big C loop present in EGF domain IV, which could serve as a diagnostic feature of the P28 family as this loop is absent in the P25 family of ookinete surface proteins. The models of the P28 family of ookinete surface proteins obtained may help in understanding the biology of the parasite inside the mosquito midgut, and in designing transmission-blocking vaccines against malaria in the absence of experimentally determined structures of these important surface proteins. An erratum to this article can be found at     

Dirofilaria immitis: effect on the longevity of Aedes trivittatus

Laboratory studies on vector mortality as related to parasite burden revealed that the mosquito Aedes trivittatus showed considerable tolerance to infection with the filarial nematode Dirofilaria immitis. Although mosquitoes exposed to a dog with a high microfilaremia (347 microfilariae/20 mm³) had a significant increase in mortality during the first 16 days postexposure, 66% of the mosquitoes lived long enough for complete parasite development to occur. Those mosquitoes exposed to a dog with a low microfilaremia (62 microfilariae/20 mm³) had no significant increase in mortality. There was a strong negative correlation between parasite burden and mosquito survival, but only mosquitoes harboring more than 15 juveniles had an increased chance of dying before D. immitis could develop to the infective stage. The retention of microfilariae within the blood clot and peritrophic membrane of A. trivittatus seems beneficial to this vector-parasite system by reducing the parasite burden and increasing mosquito longevity.     

Malaria Parasite-Synthesized Heme Is Essential in the Mosquito and Liver Stages and Complements Host Heme in the Blood Stages of Infection

Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-¹⁴C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.     

Why do fish have so few roundworm (nematode) parasites?

Synopsis Although they are the oldest and most diverse members of the subphylum, the fishes have relatively few nematode parasites in comparison with other vertebrate classes. It is hypothesized that this paucity of parasite species has occurred because nematode parasites first evolved in terrestrial hosts and only a few lines of these parasites were able to transfer to fish after the appearance of heteroxeny (use of intermediate hosts) and paratenesis (use of transport hosts). The inability of nematodes to initiate parasitism in aquatic ecosystems restricted fish parasites mainly to forms first adapted to terrestrial vertebrates and at the same time deprived large groups of aquatic invertebrates such as the crustaceans, annelids and molluscs of a nematode parasite fauna.Invited editorial