Targeting of host cell lineages by vertically transmitted, feminising microsporidia

Feminising microsporidian parasites are transmitted vertically from generation to generation of their crustacean hosts. Little is known about the mechanisms underpinning vertical transmission, in particular, parasite transmission to the host gonad during host development. Here, we investigate the burden and distribution of two species of vertically transmitted, feminising microsporidia (Dictyocoela duebenum and Nosema granulosis) during early embryogenesis (zygote to eight-cells) of the Gammarus duebeni host. Parasite burden differs between the two parasites with N. granulosis being higher by a factor of 10. Whilst D. duebenum replicates during the first few host cell divisions, there is no increase in N. granulosis burden. Only merogonic parasite stages were observed in the host embryo. Distribution of both parasites was non-random from the two-cell embryo stage, indicating biased parasite segregation at host cell division. Dictyocoela duebenum burden was low in the germline and somatic gonad progenitor cells but was highest in the ectoderm precursors, leading us to propose that the parasite targets these cells and then secondarily infects the gonad later in host development. Targeting by N. granulosis was less specific although there was a persistent bias in parasite distribution throughout host cell divisions. Parasite burden was highest in the ectoderm precursors as well as the germline progenitors leading us to suggest that, in addition to using the ectodermal route, N. granulosis may also target germline directly. Biased segregation will be adaptive for these parasites as it is likely to lead to efficient transmission and feminisation whilst minimising virulence in the host.     

Local adaptation and enhanced virulence of Nosema granulosis artificially introduced into novel populations of its crustacean host, Gammarus duebeni

Local adaptation theory predicts that, on average, most parasite species should be locally adapted to their hosts (more suited to hosts from local than distant populations). Local adaptation has been studied for many horizontally transmitted parasites, however, vertically transmitted parasites have received little attention. Here we present the first study of local adaptation in an animal/parasite system where the parasite is vertically transmitted. We investigate local adaptation and patterns of virulence in a crustacean host infected with the vertically transmitted microsporidian Nosema granulosis. Nosema granulosis is vertically transmitted to successive generations of its crustacean host, Gammarus duebeni and infects up to 46% of adult females in natural populations. We investigate local adaptation using artificial horizontal infection of different host populations in the UK. Parasites were artificially inoculated from a donor population into recipient hosts from the sympatric population and into hosts from three allopatric populations in the UK. The parasite was successfully established in hosts from all populations regardless of location, infecting 45% of the recipients. Nosema granulosis was vertically (transovarially) transmitted to 39% of the offspring of artificially infected females. Parasite burden (intensity of infection) in developing embryos differed significantly between host populations and was an order of magnitude higher in the sympatric population, suggesting some degree of host population specificity with the parasite adapted to its local host population. In contrast with natural infections, artificial infection with the parasite resulted in substantial virulence, with reduced host fecundity (24%) and survival (44%) of infected hosts from all the populations regardless of location. We discuss our findings in relation to theories of local adaptation and parasite-host coevolution.     

Paramyxean–microsporidian co-infection in amphipods: Is the consensus that Microsporidia can feminise their hosts presumptive?

The current consensus is that Microsporidia belong to a select group of parasites capable of causing both intersexuality and complete sex reversal in their hosts. Indeed, species such as Nosema granulosis and Dictyocoela duebenum, which infect amphipod crustaceans, are regularly referred to as 'feminising microsporidians'. This categorisation is based on a combination of findings: that these species are vertically transmitted and occur at a high prevalence of infection in intersex and female amphipods, that infected amphipod populations are female-biased, and that infected females have significantly female-biased broods with no concurrent increase in mortality. In this study, we report on a population of the amphipod Echinogammarus marinus presenting both female bias and high levels of intersexuality, which are infected with D. deubenum. In keeping with its feminising classification, infection is prevalent in animals presenting female and intersex phenotypes. However, a further screen revealed the presence of a previously unknown paramyxean parasite related to organisms of the genus Marteilia, a group known to cause catastrophic sexual dysfunction in bivalves. We found that the paramyxean parasite was also vertically transmitted, with infections being more prevalent in females and intersex animals. Critically, every animal infected with D. deubenum was also co-infected with the paramyxean, with few animals presenting an independent paramyxean infection. In contrast, co-infection of E. marinus with a paramyxean and the non-feminising microsporidian Dictyocoela berillonum rarely occurred. These observations raise a new hypothesis, namely, that D. duebenum and other feminising microsporidians are not actually capable of host feminisation but instead 'hitch-hike' together with a feminising paramyxean parasite.     

Invasion success of Fibrillanosema crangonycis, n.sp., n.g.: a novel vertically transmitted microsporidian parasite from the invasive amphipod host Crangonyx pseudogracilis

Parasitism is known to be an important factor in determining the success of biological invasions. Here we examine Crangonyx pseudogracilis, a North American amphipod invasive in the United Kingdom and describe a novel microsporidium, Fibrillanosema crangonycis n.sp., n.g. The primary site of infection is the female gonad and the parasite is transovarially transmitted to the eggs. PCR screening reveals a female bias in the distribution of parasites (96.6% of females, N=29; 22.2% of males, N=27), which is indicative of host sex ratio distortion. The morphological and molecular characterisations of this new microsporidium place it outside all currently established genera. On the basis of these differences, we erect the new genus Fibrillanosema n.g. While F. crangonycis is morphologically identical to uncharacterised microsporidia from populations of North American amphipods, it is distinct from microsporidia found in European populations of amphipods. These data support the hypothesis that vertically transmitted parasites may be selectively retained during invasion events. Furthermore where vertical transmission is combined with host sex ratio distortion these parasites may directly enhance host invasion success through increased rates of population growth.     

Evolution of early male-killing in horizontally transmitted parasites

Early male-killing (MK) bacteria are vertically transmitted reproductive parasites which kill male offspring that inherit them. Whereas their incidence is well documented, characteristics allowing originally non-MK bacteria to gradually evolve MK ability remain unclear. We show that horizontal transmission is a mechanism enabling vertically transmitted bacteria to evolve fully efficient MK under a wide range of host and parasite characteristics, especially when the efficacy of vertical transmission is high. We also show that an almost 100% vertically transmitted and 100% effective male-killer may evolve from a purely horizontally transmitted non-MK ancestor, and that a 100% efficient male-killer can form a stable coexistence only with a non-MK bacterial strain. Our findings are in line with the empirical evidence on current MK bacteria, explain their high efficacy in killing infected male embryos and their variability within and across insect taxa, and suggest that they may have evolved independently in phylogenetically distinct species.     

Two species of feminizing microsporidian parasite coexist in populations of Gammarus duebeni

The amphipod crustacean Gammarus duebeni hosts two species of vertically transmitted microsporidian parasites, Nosema granulosis and Microsporidium sp. A. Here it is demonstrated that these co-occurring parasite species both cause infected females to produce female-biased broods. A survey of European G. duebeni populations demonstrates that these two parasites co-occur in six of 10 populations. These findings contrast with the theoretical prediction that two vertically transmitted feminizing parasites should not coexist in a panmictic population of susceptible hosts at equilibrium. Possible explanations for the co-occurrence of the two feminizing microsporidia in G. duebeni include the recent invasion of a new parasite, horizontal transmission of one or both parasites and the spread of alleles for resistance to the dominant parasite in host populations.     

Incomplete feminisation by the microsporidian sex ratio distorter,Nosema granulosis,and reduced transmission and feminisation efficiency at low temperatures

We investigated the effects of temperature on transovarial transmission and feminisation by Nosema granulosis, a microsporidian sex ratio distorter of the brackish water amphipod Gammarus duebeni. There was no difference in parasite transmission efficiency to the F(1) eggs of infected females maintained under two temperature conditions, 5 and 10 degrees C (89 and 86%, respectively). When F(1) individuals were screened as adults, the proportion infected was also similar at both temperatures (74 and 75%, respectively). However, transmission to the eggs of the F(2) generation was significantly reduced at low temperatures (61% at 5 degrees C and 91% at 10 degrees C). In addition, feminisation efficiency was reduced substantially at low temperatures; at 10 degrees C, a calculated 85% of infected males were feminised, but at 5 degrees C only 49% were feminised. This is the first evidence for incomplete feminisation and temperature-dependent transmission and feminisation by this sex ratio distorter. We examine the consequences for parasite spread and maintenance in natural populations using a model to predict parasite prevalence in large populations. Reduced feminisation at low temperatures impedes the spread of the parasite so that it attains a substantially lower frequency, or may even be excluded, from host populations.     

Interactions between frequency-dependent and vertical transmission in host-parasite systems.

We investigate host-pathogen dynamics and conditions for coexistence in two models incorporating frequency-dependent horizontal transmission in conjunction with vertical transmission. The first model combines frequency-dependent and uniparental vertical transmission, while the second addresses parasites transmitted vertically via both parents. For the first model, we ask how the addition of vertical transmission changes the coexistence criteria for parasites transmitted by a frequency-dependent horizontal route, and show that vertical transmission significantly broadens the conditions for parasite invasion. Host-parasite coexistence is further affected by the form of density-dependent host regulation. Numerical analyses demonstrate that within a host population, a parasite strain with horizontal frequency-dependent transmission can be driven to extinction by a parasite strain that is additionally transmitted vertically for a wide range of parameters. Although models of asexual host populations predict that vertical transmission alone cannot maintain a parasite over time, analysis of our second model shows that vertical transmission via both male and female parents can maintain a parasite at a stable equilibrium. These results correspond with the frequent co-occurrence of vertical with sexual transmission in nature and suggest that these transmission modes can lead to host-pathogen coexistence for a wide range of systems involving hosts with high reproductive rates.     

Vector-borne parasites decrease host mobility: a field test of freeze or flee behaviour of willow ptarmigan

Transmission mode has been suggested to be a strong predictor of virulence. According to theory, the transmission of vector-borne parasites should be less dependent on host mobility than directly transmitted parasites. This could select for increased exploitation of host resources in parasites transmitted by vectors, which may be manifested as higher virulence. Here, we test the prediction that there is an association between transmission mode and the effect on host mobility by comparing parasite infection levels and mobility in willow ptarmigan (Lagopus lagopus L.). We examined the endoparasite infracommunities of individual hosts to obtain annual, quantitative data on four vector-transmitted species (Leucocytozoon lovati, Trypanosoma avium, Haemoproteus mansoni and microfilaria), two directly transmitted species (Trichostrongylus tenuis and Eimeria sp.) and two species with indirect life cycles (Hymenolepis microps and Parionella urogalli). We then used observed variations in freeze-or-flee responses of individual willow ptarmigan to assess whether parasite intensities were related to scored freezing responses. From a field data set covering a period of 9 years from a single area, we found that stronger freezing responses were associated with higher intensities of vector-borne parasites, especially with higher intensities of the haemosporidian L. lovati. Freezing responses were not associated with parasites transmitted in other ways. Thus, high intensities of vector-borne parasites tended to reduce host movements, while parasites with other transmission modes did not.     

Molecular data suggest that microsporidian parasites in freshwater snails are diverse

Microsporidian parasites infect almost all invertebrate and vertebrate hosts and have significant effects on individual and population fitness. Phylogenetic analysis demonstrates that the phylum is highly divergent and that some lineages show strong associations with host taxa. We here examine the diversity and distribution of parasites in gastropod molluscs to test for host-parasite co-association. 16 populations representing 10 species of freshwater snails were screened using microsporidian specific small subunit rDNA primers. Four novel microsporidian parasite sequences were detected within populations of three host species from the genera Bulinus, Biomphalaria and Planorbis. Prevalence ranged from 5 to 84%. Phylogenetic analysis of these novel sequences reveals that they group together as a paraphyletic assemblage in the microsporidian tree basal to the two lineages containing the genera Encephalitozoon and Nosema. Preliminary observation of one microsporidian infection, show parasites distributed in all tissue systems of Bulinus globosus. However, infection is most prevalent in the digestive gland while also in the egg sacs, suggesting that the microsporidium is using a mixed strategy of horizontal and vertical transmission in this population.     

Effectively vertical transmission of a Drosophila‐parasitic nematode: mechanism and consequences

1. Long‐term control of insects by parasites is possible only if the parasite populations persist. Because parasite transmission rate depends on host density, parasite populations may go extinct during periods of low host density. Vertical transmission of parasites, however, is independent of host density and may therefore provide a demographic bridge through times when their insect hosts are rare. 2. The nematode Howardula aoronymphium, which parasitises mycophagous species of Drosophila, can experience both horizontal and effectively vertical transmission, relative rates of which depend, in theory at least, on the density of hosts at breeding sites. 3. A nine‐generation experiment was carried out in which nematodes were transmitted either exclusively vertically or primarily horizontally. This experiment revealed that these parasites can persist and exhibit positive population growth even when there is only vertical transmission. 4. Assays at the end of the experiment revealed that the vertically transmitted nematodes had suffered no inbreeding depression and that they were similar to the horizontally transmitted nematodes in terms of virulence, infectivity, within‐host growth rate, and fecundity. Thus, vertical transmission of H. aoronymphium did not appear to compromise the ability of these parasites to control Drosophila populations.     

Microsporidian life cycles and diversity: the relationship between virulence and transmission

The microsporidia are obligate intracellular parasites which have diverse life cycles involving both horizontal and vertical transmission and parasitise a wide range of vertebrate and invertebrate hosts. In this paper we consider the life cycles and diversity of the microsporidia. We focus in particular on the relationship between parasite transmission and virulence and its implications for host-parasite coevolution. The use of horizontal and vertical routes of transmission varies between species and there is a strong link between transmission and virulence. Horizontal transmission is characterised by a high parasite burden and associated pathogenicity. In contrast, vertical transmission is characterised by low virulence, which has led to under-reporting of this important transmission route. Vertically transmitted microsporidia may also cause male killing or feminisation of their host, with implications for host population sex ratio and stability. Phylogenetic analysis shows that vertical transmission occurs in diverse branches of the Microspora. We find that there is evidence for vertical transmission in both vertebrate and invertebrate hosts and conclude that it is a common or possibly even ubiquitous transmission route within this phylum.     

Social network structure and parasite infection patterns in a territorial reptile, the tuatara (Sphenodon punctatus)

We investigated whether the parasite load of an individual could be predicted by its position in a social network. Specifically, we derived social networks in a solitary, territorial reptile (the tuatara, Sphenodon punctatus), with links based on the sharing of space, not necessarily synchronously, in overlapping territories. Tuatara are infected by ectoparasitic ticks (Amblyomma sphenodonti), mites (Neotrombicula spp.) and a blood parasite (Hepatozoon tuatarae) which is transmitted by the tick. We recorded the location of individual tuatara in two study plots twice daily during the mating season (March) in 2 years (2006 and 2007) on Stephens Island, New Zealand. We constructed weighted, directed networks to represent pathways for parasite transmission, where nodes represented individual tuatara and edges connecting the nodes represented the extent of territory overlap among each pair of individuals. We considered a network-based hypothesis which predicted that the in-strength of individuals (the sum of edge weights directed towards a node) in the derived network would be positively related to their parasite load. Alternatively, if the derived social network did not reflect actual parasite transmission, we predicted other factors such as host sex, size or territory size may better explain variation in parasite infection patterns. We found clear positive relationships between the in-strength of tuatara and their tick loads, and infection patterns with tick-borne blood parasites. In particular, the extent that individuals were connected to males in the network consistently predicted tick loads of tuatara. However, mite loads of tuatara were significantly related to host sex, body size and territory size, and showed little association with network measures. The results suggest that the pathway of transmission of parasites through a population will depend on the transmission mechanism of the parasite, but that social networks provide a powerful predictive tool for some parasites.     

Long‐term selection experiment produces breakdown of horizontal transmissibility in parasite with mixed transmission mode

Evolutionary transitions from parasitism toward beneficial or mutualistic associations may encompass a change from horizontal transmission to (strict) vertical transmission. Parasites with both vertical and horizontal transmission are amendable to study factors driving such transitions. In a long‐term experiment, microcosm populations of the protozoan Paramecium caudatum and its bacterial parasite Holospora undulata were exposed to three growth treatments, manipulating vertical transmission opportunities over ca. 800 host generations. In inoculation tests, horizontal transmission propagules produced by parasites from a “high‐growth” treatment, with elevated host division rates increasing levels of parasite vertical transmission, showed a near‐complete loss of infectivity. A similar reduction was observed for parasites from a treatment alternating between high growth and low growth (i.e., low levels of population turn‐over). Parasites from a low‐growth treatment had the highest infectivity on all host genotypes tested. Our results complement previous findings of reduced investment in horizontal transmission and increased vertical transmissibility of high‐growth parasites. We explain the loss of horizontal transmissibility by epidemiological feedbacks and resistance evolution, reducing the frequency of susceptible hosts in the population and thereby decreasing the selective advantage of horizontal transmission. This illustrates how environmental conditions may push parasites with a mixed transmission mode toward becoming vertically transmitted nonvirulent symbionts.     

Population dynamics of a vertically transmitted, parasitic sex ratio distorter and its amphipod host

We investigate the population dynamics of a vertically transmitted, parasitic sex ratio distorter ( Nosema granulosis ) and its amphipod host ( Gammarus duebeni ), using field measurements to quantify and test alternative theoretical models of the interaction. We measure parasite, host population and transmission parameters at four locations on the Isle of Cumbrae, Scotland at monthly intervals for two years. We develop a simple infinite population model and test its predictions for parasite prevalence using field measurements of the parameters. Parasite prevalence is maintained at relatively low levels at all four sites (20–42%), consistently below that predicted. The parasite imposes a slight fitness cost on its host by reducing female fecundity, but this cost cannot account for the relatively low prevalences observed. We also investigate the importance of population structure, comparing parasite prevalence across sites and sampling intervals to look for evidence of spatial and temporal asynchrony as predicted by metapopulation models. We find significant temporal and spatial heterogeneity in parasite prevalence although there was also evidence that parasite dynamics were synchronous across sites. These data suggest that the parasite is unlikely to drive local population dynamics through cycles of extinction and recolonization at the scale measured. As the host (adult) population sex ratio was male-biased, local population crashes are unlikely to be induced by the parasite, contrary to theoretical predictions.     

Patterns of intermediate host use and levels of association between two conflicting manipulative parasites.

For many parasites with complex life cycles, manipulation of intermediate host phenotypes is often regarded as an adaptation to increase the probability of successful transmission. This phenomenon creates opportunities for either synergistic or conflicting interests between different parasite species sharing the same intermediate host. When more than one manipulative parasite infect the same intermediate host, but differ in their definitive host, selection should favour the establishment of a negative association between these manipulators. Both Polymorphus minutus and Pomphorhynchus laevis exploit the amphipod Gammarus pulex as intermediate host but differ markedly in their final host, a fish for P. laevis and a bird for P. minutus. The pattern of host use by these two conflicting manipulative parasites was studied. Their incidence and intensity of infection and their distribution among G. pulex were first examined by analysing three large samples of gammarids collected from the river Tille, Eastern France. Both parasites had low prevalence in the host population. However, temporal fluctuation in the level of parasitic infection was observed. Overall, prevalence of both parasite species was higher in male than in female G. pulex. We then assessed the degree of association between the two parasites among their intermediate hosts, using two different methods: a host-centred measure and a parasite-centred measure. Both measures gave similar results; showing random association between the two acanthocephalan species in their intermediate hosts. We discuss our results in relation to the selective forces and ecological constraints that may determine the pattern of association between conflicting manipulative parasites.     

Potential versus actual contribution of vertical transmission to pathogen fitness

Theory predicts that virulent parasites cannot be maintained at high prevalence if they are only vertically transmitted. However, parasites with high rates of vertical transmission that cause severe reduction in host fitness have been reported. Atkinsonella hypoxylon is a fungal pathogen capable of both vertical and horizontal transmission that drastically reduces its host''s fitness. In contrast with theoretical predictions, field and laboratory observations suggested that the primary mechanism of transmission was vertical. Using randomly amplified polymorphic DNA markers, we investigated the effective contribution of vertical and horizontal transmission to the genetic structure of three natural populations of A. hypoxylon. We found high genotypic diversity and low linkage disequilibrium, indicating that most established genotypes are derived from horizontally transmitted, sexual spores. The low contribution of vertical transmission to the parasite''s fitness despite its high potential might be due to lower establishment of cleistogamous seeds (through which vertical transmission occurs) or lower vigour of vertically transmitted fungal genotypes. Low establishment of vertically infected hosts might explain the persistence of virulent parasites with high apparent vertical transmission. Our results suggest that caution must be taken when using the potential for vertical transmission to make predictions about the evolution of parasite virulence.     

An empirical study of the evolution of virulence under both horizontal and vertical transmission

According to current thinking, a parasite's transmission mode will be a major determinant of virulence, defined as the harm induced by parasites to their hosts. With horizontal transmission, virulence will increase as a byproduct of a trade-off between fitness gained through increased among-host transmission (infectivity) and fitness lost through increased virulence. With vertical transmission, virulence will decrease because a parasite's reproductive potential will be maximized only by decreasing harm to the host, allowing parasite transmission to more host offspring. To test both predictions, we transmitted barley stripe mosaic virus (BSMV) horizontally and then vertically in its host, barley (Hordeum vulgare). After four generations of horizontal transmission, we observed a nearly twofold increase in horizontal infectivity and nearly tripled virulence. After three generations of subsequent vertical transmission, we observed a modest (16%) increase in vertical transmissibility and a large (40%) reduction in virulence. Increased horizontal transmission is often due to increased pathogen replication which, in turn, causes increased virulence. However, we found no correlation between within-host virus concentration and virulence, indicating that the observed changes in virulence were not due to changes in viral titer. Finally, horizontally transmitted BSMV had reduced vertical transmission and vertically transmitted BSMV had reduced horizontal infectivity. These two observations suggest that, in nature, in different host populations with varying opportunities for horizontal and vertical transmission, different viral strains may be favored.     

Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis

Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis.     

Impact of a Novel, Feminising Microsporidium on its Crustacean Host

We describe the transmission and pathogenic effects of a novel, feminising microsporidium, probably a Nasema species, on its crustacean host Gammarus duebeni. The parasite prevalence in the field was high (46% of females were infected) and the parasite was transovarially transmitted to 91% of embryos of infected females. The impact of the parasite on the host was assessed by means of a host breeding experiment. The parasite feminised 66% of infected host young and was transovarially transmitted by these individuals to the next host generation. The parasite differed from other feminising microsporidia in G. duebeni in that early embryos had a high parasite burden (288 parasites per embryo) and the infection was pathogenic, causing a reduction in both the growth rate of young hosts and in adult size. This study suggests that feminising microsporidia are a diverse group in which a variety of host/pathogen relationships have evolved.