Artemisinin-resistant Leishmania parasite modulates host cell defense mechanism and exhibits altered expression of unfolded protein response genes.

Artemisinin, extracted from a medicinal herb Artemisia annua, is widely used to treat malaria and has shown potent anticancer activity. Artemisinin has been found to be effective against experimental visceral and cutaneous leishmaniasis. Despite extensive research to understand the complex mechanism of resistance to artemisinin, several questions remain unanswered. The artesunate (ART)-resistant line of Leishmania donovani was selected and cellular mechanisms associated with resistance to artemisinin were investigated. ART-resistant (AS-R) parasites showed reduced susceptibility towards ART both at promastigote and amastigote stage compared with ART sensitive (WT) parasites. WT and AS-R parasites were both more susceptible to ART at the early log phase of growth compared with late log phase. AS-R parasites were more infective to the host macrophages (p < 0.05). Evaluation of parasites' tolerance towards host microbicidal mechanisms revealed that AS-R parasites were more tolerant to complement-mediated lysis and nitrosative stress. ROS levels were modulated in presence of ART in AS-R parasites infected macrophages. Interestingly, infection of macrophages by AS-R parasites led to modulated levels of host interleukins, IL-2 and IL-10, in addition to nitric oxide. Additionally, AS-R parasites showed upregulated expression of genes of unfolded protein response pathway including methyltransferase domain-containing protein (HSP40) and flagellar attachment zone protein (prefoldin), that are reported to be associated with ART resistance in Plasmodium falciparum malaria. This study presents in vitro model of artemisinin-resistant Leishmania parasite and cellular mechanisms associated with ART resistance in Leishmania.

Plant genotype and induced defenses affect the productivity of an insect-killing obligate viral pathogen.

Plant-mediated variations in the outcomes of host-pathogen interactions can strongly affect epizootics and the population dynamics of numerous species, including devastating agricultural pests such as the fall armyworm. Most studies of plant-mediated effects on insect pathogens focus on host mortality, but few have measured pathogen yield, which can affect whether or not an epizootic outbreak occurs. Insects challenged with baculoviruses on different plant species and parts can vary in levels of mortality and yield of infectious stages (occlusion bodies; OBs). We previously demonstrated that soybean genotypes and induced anti-herbivore defenses influence baculovirus infectivity. Here, we used a soybean genotype that strongly reduced baculovirus infectivity when virus was ingested on induced plants (Braxton) and another that did not reduce infectivity (Gasoy), to determine how soybean genotype and induced defenses influence OB yield and speed of kill. These are key fitness measures because baculoviruses are obligate-killing pathogens. We challenged fall armyworm, Spodoptera frugiperda, with the baculovirus S. frugiperda multi-nucleocapsid nucleopolyhedrovirus (SfMNPV) during short or long-term exposure to plant treatments (i.e., induced or non-induced genotypes). Caterpillars were either fed plant treatments only during virus ingestion (short-term exposure to foliage) or from the point of virus ingestion until death (long-term exposure). We found trade-offs of increasing OB yield with slower speed of kill and decreasing virus dose. OB yield increased more with longer time to death and decreased more with increasing virus dose after short-term feeding on Braxton compared with Gasoy. OB yield increased significantly more with time to death in larvae that fed until death on non-induced foliage than induced foliage. Moreover, fewer OBs per unit of host tissue were produced when larvae were fed induced foliage than non-induced foliage. These findings highlight the potential importance of plant effects, even at the individual plant level, on entomopathogen fitness, which may impact epizootic transmission events and host population dynamics.

Effects of parasite pressure on parasite mortality and reproductive output in a rodent-flea system: inferring host defense trade-offs.

Evaluating host resistance via parasite fitness helps place host-parasite relationships within evolutionary and ecological contexts; however, few studies consider both these processes simultaneously. We investigated how different levels of parasite pressure affect parasite mortality and reproductive success in relationship to host defense efforts, using the rodent Gerbillus nanus and the flea Xenopsylla conformis as a host-parasite system. Fifteen immune-naïve male rodents were infested with 20, 50, or 100 fleas for four weeks. During this time number of new imagoes produced per adult flea (our flea reproductive output metric), flea mortality, and change in circulating anti-flea immunoglobulin G (our measure of adaptive immune defense) were monitored. Three hypotheses guided this work: (1) increasing parasite pressure would heighten host defenses; (2) parasite mortality would increase and parasite reproductive output would decrease with increasing investment in host defense; and (3) hosts under high parasite pressure could invest in behavioral and/or immune responses. We predicted that at high infestation levels (a) parasite mortality would increase; (b) flea reproductive output per individual would decrease; and (c) host circulating anti-flea antibody levels would increase. The hypotheses were partially supported. Flea mortality significantly increased and flea reproductive output significantly decreased as flea pressure increased. Host adaptive immune defense did not significantly change with increasing flea pressure. Therefore, we inferred that investment in host behavioral defense, either alone or in combination with density-dependent effects, may be more efficient at increasing flea mortality and decreasing flea reproductive output than antibody production during initial infestation in this system.

Comparative biological traits of perkinsozoan parasitoids infecting marine dinoflagellates.

The number of perkinsozoan parasitoid species known to infect dinoflagellates has increased to 11 over the last two decades. However, most of the current knowledge about the autecology of perkinsozoan parasitoids of dinoflagellates has derived from studies of one or two species, thereby making it difficult to directly compare their biological traits at the same time and even their potentials as biological control agents if they are to be exploited to mitigate harmful dinoflagellate blooms in the field. This study investigated total generation time, the number of zoospores produced per sporangium, zoospore size, swimming speed, parasite prevalence, zoospore survival and success rate, and host range and susceptibility for five perkinsozoan parasitoids. Four of the species (Dinovorax pyriformis, Tuberlatum coatsi, Parvilucifera infectans, and P. multicavata) were from the family Parviluciferaceae and one (Pararosarium dinoexitiosum) was from the family Pararosariidae, with dinoflagellate Alexandrium pacificum employed as a common host. Distinct differences in the biological traits of the five perkinsozoan parasitoid species were found, suggesting that the fitness of these parasitoids for the common host species differs. These results thus offer useful background information for the understanding of the impacts of parasitoids on the natural host population and for the design of numerical modeling including the host-parasitoid systems and biocontrol experiments in the field.

Sociality and parasite transmission.

Parasites and their social hosts form many different relationships. But what kind of selection regimes are important? A look at the parameters that determine fitness of the two parties suggests that social hosts differ from solitary ones primarily in the structure of transmission pathways. Because transmission is, both, the physical encounter of a new host and infecting it, several different elements determine parasite transmission success. These include spatial distance, genetic distance, or the temporal and ecological niche overlaps. Combing these elements into a 'generalized transmission distance' that determines parasite fitness aids in the identification of the critical steps. For example, short-distance transmission to genetically similar hosts within the social group is the most frequent process under sociality. Therefore, spatio-genetical distances are the main driver of parasite fitness. Vice versa, the generalized distance identifies the critical host defences. In this case, host defences should be primarily selected to defend against the within-group spread of an infection, especially among closely related group members.

Paraphysoderma sedebokerense Infection in Three Economically Valuable Microalgae: Host Preference Correlates with Parasite Fitness.

The blastocladialean fungus Paraphysoderma sedebokerense parasitizes three microalgae species of economic interest: Haematococcus pluvialis, Chromochloris zofingiensis and Scenedesmus dimorphus. For the first time, we characterized the developmental stages of isolated fungal propagules in H. pluvialis co-culture, finding a generation time of 16 h. We established a patho-system to compare the infection in the three different host species for 48 h, with two different setups to quantify parameters of the infection and parameters of the parasite fitness. The prevalence of the parasite in H. pluvialis and C. zofingiensis cultures was 100%, but only 20% in S. dimorphus culture. The infection of S. dimorphus not only reached lower prevalence but was also qualitatively different; the infection developed preferentially on senescent cells and more resting cysts were produced, being consistent with a reservoir host. In addition, we carried out cross infection experiments and the inoculation of a mixed algal culture containing the three microalgae, to determine the susceptibility of the host species and to investigate the preference of P. sedebokerense for these microalgae. The three tested microalgae showed different susceptibility to P. sedebokerense, which correlates with blastoclad's preference to the host in the following order: H. pluvialis > C. zofingiensis > S. dimorphus.

Common host variation drives malaria parasite fitness in healthy human red cells.

The replication of Plasmodium falciparum parasites within red blood cells (RBCs) causes severe disease in humans, especially in Africa. Deleterious alleles like hemoglobin S are well-known to confer strong resistance to malaria, but the effects of common RBC variation are largely undetermined. Here, we collected fresh blood samples from 121 healthy donors, most with African ancestry, and performed exome sequencing, detailed RBC phenotyping, and parasite fitness assays. Over one-third of healthy donors unknowingly carried alleles for G6PD deficiency or hemoglobinopathies, which were associated with characteristic RBC phenotypes. Among non-carriers alone, variation in RBC hydration, membrane deformability, and volume was strongly associated with P. falciparum growth rate. Common genetic variants in PIEZO1, SPTA1/SPTB, and several P. falciparum invasion receptors were also associated with parasite growth rate. Interestingly, we observed little or negative evidence for divergent selection on non-pathogenic RBC variation between Africans and Europeans. These findings suggest a model in which globally widespread variation in a moderate number of genes and phenotypes modulates P. falciparum fitness in RBCs.

Decomposing parasite fitness reveals the basis of specialization in a two-host, two-parasite system.

The ecological specialization of parasites-whether they can obtain high fitness on very few or very many different host species-is a determining feature of their ecology. In order to properly assess specialization, it is imperative to measure parasite fitness across host species; to understand its origins, fitness must be decomposed into the underlying traits. Despite the omnipresence of parasites with multiple hosts, very few studies assess and decompose their specialization in this way. To bridge this gap, we quantified the infectivity, virulence, and transmission rate of two parasites, the horizontally transmitted microsporidians Anostracospora rigaudi and Enterocytospora artemiae, in their natural hosts, the brine shrimp Artemia parthenogenetica and Artemia franciscana. Our results demonstrate that each parasite performs well on one of the two host species (A. rigaudi on A. parthenogenetica, and E. artemiae on A. franciscana), and poorly on the other. This partial specialization is driven by high infectivity and transmission rates in the preferred host, and is associated with maladaptive virulence and large costs of resistance in the other. Our study represents a rare empirical contribution to the study of parasite evolution in multihost systems, highlighting the negative effects of under- and overexploitation when adapting to multiple hosts.

Antimonial susceptibility and in vivo behaviour of Leishmania major isolates collected in Algeria before and after treatment.

The repercussions of cutaneous leishmaniasis therapy on the behaviour and drug susceptibility of Leishmania major parasites is poorly documented. This study explored the link between antimonial susceptibility and in vivo behaviour in Leishmania major isolates collected before and after treatment in Algeria. This study was performed on 3 isolates collected from patients prior to treatment and paired with 3 isolates collected from the same patient after treatment failure. Their in vitro susceptibility towards trivalent (SbIII) and pentavalent (SbV) antimony were ascertained, and their in vivo behaviour was evaluated by determining their capacity to disseminate, proliferate and induce lesions in mice. No relationship was observed between in vitro antimony resistance and parasite fitness in the murine model.

Host heterogeneity affects both parasite transmission to and fitness on subsequent hosts.

Infectious disease dynamics depend on the speed, number and fitness of parasites transmitting from infected hosts ('donors') to parasite-naive 'recipients'. Donor heterogeneity likely affects these three parameters, and may arise from variation between donors in traits including: (i) infection load, (ii) resistance, (iii) stage of infection, and (iv) previous experience of transmission. We used the Trinidadian guppy, Poecilia reticulata, and a directly transmitted monogenean ectoparasite, Gyrodactylus turnbulli, to experimentally explore how these sources of donor heterogeneity affect the three transmission parameters. We exposed parasite-naive recipients to donors (infected with a single parasite strain) differing in their infection traits, and found that donor infection traits had diverse and sometimes interactive effects on transmission. First, although transmission speed increased with donor infection load, the relationship was nonlinear. Second, while the number of parasites transmitted generally increased with donor infection load, more resistant donors transmitted more parasites, as did those with previous transmission experience. Finally, parasites transmitting from experienced donors exhibited lower population growth rates on recipients than those from inexperienced donors. Stage of infection had little effect on transmission parameters. These results suggest that a more holistic consideration of within-host processes will improve our understanding of between-host transmission and hence disease dynamics.This article is part of the themed issue 'Opening the black box: re-examining the ecology and evolution of parasite transmission'.

The Allee effect and elimination of neglected tropical diseases: a mathematical modelling study.

Elimination and control programmes for neglected tropical diseases (NTDs) are underway around the world, yet they are generally informed by epidemiological modelling only to a rudimentary degree. Chief among the modelling-derived predictors of disease emergence or controllability is the basic reproduction number R0. The ecological systems of several of the NTDs include density-dependent processes--which alter the rate of e.g. parasite establishment or fecundity--that complicate the calculation of R0. Here we show how the forms of the density-dependent functions for a model of the NTD lymphatic filariasis affect the effective reproduction number Reff. We construct infection transmission models containing various density-dependent functions and show how they alter the shape of the Reff profile, affecting two important epidemiological outcome variables that relate to elimination and control programmes: the parasite transmission breakpoint (or extinction threshold) and the reproduction fitness, as measured by Reff. The current drive to control, eliminate or eradicate several parasitic infections would be substantially aided by the existence of ecological Allee effects. For these control programmes, the findings of this paper are encouraging, since a single positive density dependency (DD) can introduce a reasonable chance of achieving elimination; however, there are diminishing returns to additional positive DDs.

RNA granules present only in extracellular toxoplasma gondii increase parasite viability.

Toxoplasma gondii is an obligate intracellular parasite. When searching for a new cell to invade, the parasites have to confront the stress of being exposed to the extracellular environment. The mechanisms by which T. gondii survives outside the host cells are poorly understood. In this work we show that extracellular parasites form mRNA aggregates with characteristics of stress granules. Intracellular tachyzoites or bradyzoites do not form mRNA granules. We tested different stimuli that trigger granule formation in vitro and discovered that a buffer that mimics the host cell cytosol ionic composition (high potassium) strongly induces granule formation, suggesting that the granules arise when the parasites come in contact with the host cell cytosol during egress. We examined the importance of granule formation for parasite viability and show that the parasite populations that are able to form granules have a growth advantage, increased invasion, and decreased apoptosis in the extracellular environment. Overall, granule formation improves the fitness of extracellular parasites and increases the efficiency of the lytic cycle.

DYNAMIC AND GENETIC CONSEQUENCES OF VARIATION IN HORIZONTAL TRANSMISSION FOR A MICROPARASITIC INFECTION.

Transmission to a new host is a critical step in the life cycle of a parasite. Variation in the characteristics of the transmission process, for example, due to host demography, is assumed to select for different variants of the parasite. We have experimentally tested how variation in the time to transmission (early or late after infection) and exposure to adverse conditions outside the host (immediate or delayed contact with new host) interact to determine the success of the infection in the next host, using the trypanosome Crithidia bombi infecting its bumblebee host, Bombus terrestris. These two experimentally manageable steps mimic the processes of within- and among-host selection for the parasite. We found that early transmission led to higher infection success in the next host as did immediate contact with the new host. However, there was no interaction between the two parameters as would be expected if early-transmitted variants, resulting from rapid multiplication within the host, would be less adapted to the conditions encountered during the between-host transfer or infection of the next host. Furthermore, typing the genetic variability of the parasites with microsatellites showed that the four different transmission routes of our experiment selected for different degrees of allelic diversity of the infecting parasite populations. The results support the idea that variation in the transmission process selects for different genotypic variants of the parasite. At the same time, the relationship of allelic diversity with infection intensity suggested that the coinfection model of May and Nowak (1995) may be appropriate, where each parasite is able to infect and multiply independent of others within the same host.