Conflict between parasites with different transmission strategies infecting an amphipod host

Competition between parasites within a host can influence the evolution of parasite virulence and host resistance, but few studies examine the effects of unrelated parasites with conflicting transmission strategies infecting the same host. Vertically transmitted (VT) parasites, transmitted from mother to offspring, are in conflict with virulent, horizontally transmitted (HT) parasites, because healthy hosts are necessary to maximize VT parasite fitness. Resolution of the conflict between these parasites should lead to the evolution of one of two strategies: avoidance, or sabotage of HT parasite virulence by the VT parasite. We investigated two co-infecting parasites in the amphipod host, Gammarus roeseli: VT microsporidia have little effect on host fitness, but acanthocephala modify host behaviour, increasing the probability that the amphipod is predated by the acanthocephalan's definitive host. We found evidence for sabotage: the behavioural manipulation induced by the Acanthocephala Polymorphus minutus was weaker in hosts also infected by the microsporidia Dictyocoela sp. (roeselum) compared to hosts infected by P. minutus alone. Such conflicts may explain a significant portion of the variation generally observed in behavioural measures, and since VT parasites are ubiquitous in invertebrates, often passing undetected, conflict via transmission may be of great importance in the study of host-parasite relationships.     

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.     

Virulence is context-dependent in a vertically transmitted aquatic host–microparasite system

Recent work has suggested that the outcomes of host–symbiont interactions can shift between positive, neutral and negative depending on both biotic and abiotic conditions. Even organisms traditionally defined as parasites can have positive effects on hosts under some conditions. For a given host–parasite system, the effects of infection on host fitness can depend on host vigour, route of transmission and environmental conditions. We monitored sublethal microsporidian infections in populations of Gammarus pseudolimnaeus (Amphipoda: Gammaridae) from four cool water streams in southwestern Michigan, USA. Our objectives were to: (i) infer the mechanism of transmission (horizontal, vertical or mixed) from observed effects of infection on host fitness, (ii) determine if the magnitude of the effects on host fitness is a function of parasite load (infection intensity) compared with simple presence or absence of infection, and (iii) determine if there is variation in parasite effects on host fitness in isolated populations. PCR and DNA sequence analyses revealed that there were two microsporidia present among the four host populations: Dictyocoela sp. and Microsporidium sp. PCR screening of a subset of infected hosts showed that Dictyocoela sp. accounted for 90% of infections and was present in all four G. pseudolimnaeus populations, while Microsporidium sp. was found in two populations but was only relatively common in one. We found very low prevalence in males (∼5%), but high prevalence in females (range: 37–85%). Female fitness was positively associated with infection in two streams, resulting from either higher fecundity or more reproductive bouts. Infection had a negative effect on the number of reproductive bouts in a third population, and no effect on fecundity in a fourth population. Infection intensity explained additional variation in fecundity in one population; females with intermediate infection intensity had higher fecundity than females with either light or heavy infection intensity. Given the high prevalence of infection in females compared with males and the generally weak negative fitness effects coupled with some positive fitness effects, it is likely that both Dictyocoela sp. and Microsporidium sp. are primarily vertically transmitted, feminizing microsporidia. Our results suggest that microsporidian effects on G. pseudolimnaeus fitness were context-dependent and varied with host sex and local environment.     

Association with host mitochondrial haplotypes suggests that feminizing microsporidia lack horizontal transmission

The amphipod crustacean Gammarus duebeni hosts two feminizing microsporidian parasites, Nosema granulosis and Microsporidium sp. Samples of G. duebeni were collected from three sites on the Scottish island of Great Cumbrae and screened for microsporidia using polymerase chain reaction. Associations between the prevalence of the two feminizing parasites and haplotypes of the host mitochondrial gene cytochrome oxidase I (COI) were investigated. The prevalence of both parasites varied significantly among the host's COI haplotypes, suggesting that horizontal transmission is rare or absent in the life cycles of the feminizing microsporidia and that all transmission must therefore be vertical. Life cycles in which all transmission is vertical are common among bacterial parasites but have never before been demonstrated in Eukaryotic parasites.     

Accumulation and exchange of parasites during adaptive radiation in an ancient lake

In the ancient Lake Baikal, Russia, amphipod crustaceans have undergone a spectacular adaptive radiation, resulting in a diverse community of species. A survey of microsporidian parasites inhabiting endemic and non-endemic amphipod host species at the margins of Lake Baikal indicates that the endemic amphipods harbour many microsporidian parasite groups associated with amphipods elsewhere in Eurasia. While these parasites may have undergone a degree of adaptive radiation within the lake, there is little evidence of host specificity. Furthermore, a lack of reciprocal monophyly indicates that exchanges of microsporidia between Baikalian and non-Baikalian hosts have occurred frequently in the past and may be ongoing. Conversely, limitations to parasite exchange between Baikalian and non-Baikalian host populations at the margins of the lake are implied by differences in parasite prevalence and lack of shared microsporidian haplotypes between the two host communities. While amphipod hosts have speciated sympatrically within Lake Baikal, the parasites appear instead to have accumulated, moving into the lake from external amphipod populations on multiple occasions to exploit the large and diverse community of endemic amphipods in Lake Baikal.     

Parasite-mediated predation between native and invasive amphipods

Parasites can structure biological communities directly through population regulation and indirectly by processes such as apparent competition. However, the role of parasites in the process of biological invasion is less well understood and mechanisms of parasite mediation of predation among hosts are unclear. Mutual predation between native and invading species is an important factor in determining the outcome of invasions in freshwater amphipod communities. Here, we show that parasites mediate mutual intraguild predation among native and invading species and may thereby facilitate the invasion process. We find that the native amphipod Gammarus duebeni celticus is host to a microsporidian parasite, Pleistophora sp. (new species), with a frequency of infection of 0-90%. However, the parasite does not infect three invading species, G. tigrinus, G. pulex and Crangonyx pseudogracilis. In field and laboratory manipulations, we show that the parasite exhibits cryptic virulence: the parasite does not affect host fitness in single-species populations, but virulence becomes apparent when the native and invading species interact. That is, infection has no direct effect on G. d. celticus survivorship, size or fecundity; however, in mixed-species experiments, parasitized natives show a reduced capacity to prey on the smaller invading species and are more likely to be preyed upon by the largest invading species. Thus, by altering dominance relationships and hierarchies of mutual predation, parasitism strongly influences, and has the potential to change, the outcome of biological invasions.     

Specific Detection and Localization of Microsporidian Parasites in Invertebrate Hosts by Using In Situ Hybridization

We designed fluorescence in situ hybridization probes for two distinct microsporidian clades and demonstrated their application in detecting, respectively, Nosema/Vairimorpha and Dictyoceola species. We used them to study the vertical transmission of two microsporidia infecting the amphipod Gammarus duebeni.     

Resolution of a taxonomic conundrum: an ultrastructural and molecular description of the life cycle of Pleistophora mulleri (Pfeiffer 1895; Georgevitch 1929)

The classification of a microsporidian parasite observed in the abdominal muscles of amphipod hosts has been repeatedly revised but still remains inconclusive. This parasite has variable spore numbers within a sporophorous vesicle and has been assigned to the genera Glugea, Pleistophora, Stempellia, and Thelohania. We used electron microscopy and molecular evidence to resolve the previous taxonomic confusion and confirm its identification as Pleistophora mulleri. The life cycle of P. mulleri is described from the freshwater amphipod host Gammarus duebeni celticus. Infection appeared as white tubular masses within the abdominal muscle of the host. Light and transmission electron microscope examination revealed the presence of an active microsporidian infection that was diffuse within the muscle block with no evidence of xenoma formation. Paucinucleate merogonial plasmodia were surrounded by an amorphous coat immediately external to the plasmalemma. The amorphous coat developed into a merontogenetic sporophorous vesicle that was present throughout sporulation. Sporogony was polysporous resulting in uninucleate spores, with a bipartite polaroplast, an anisofilar polar filament and a large posterior vacuole. SSU rDNA analysis supported the ultrastructural evidence clearly placing this parasite within the genus Pleistophora. This paper indicates that Pleistophora species are not restricted to vertebrate hosts.     

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.     

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.     

Effect of multiple parasitic infections on the tolerance to pollutant contamination

The horizontally-transmitted acanthocephalan parasite Polymorphus minutus and the vertically-transmitted microsporidian parasite Dictyocoela roeselum have both been shown to influence on the antitoxic responses of mono-infected Gammarus roeseli exposed to cadmium. The present study investigates the effect of this co-infection on the antitoxic defence responses of naturally infected females exposed to cadmium stress. Our results revealed that, depending on the cadmium dose, bi-infection induced only slight, significant increased cell damage in G. roeseli as compared to non-infection. In addition, the antitoxic defence pattern of cadmium-exposed bi-infected hosts was similar to the pattern of cadmium-exposed D. roeselum-infected hosts. Reduced glutathione concentrations, carotenoid levels and γ-glutamylcystein ligase activity decreased, while metallothionein concentrations increased. This similar pattern indicates that host physiology can be controlled to some extent by microsporidia under stress conditions. It supports the hypothesis of a disruption of acanthocephalan effects in the presence of microsporidia. However, the global negative effects of bi-infection on host condition should be tested on more biological models, since competition between parasites depends on life history trade-off.     

Host sexual dimorphism affects the outcome of within‐host pathogen competition

Natural infections often consist of multiple pathogens of the same or different species. When coinfections occur, pathogens compete for access to host resources and fitness is determined by how well a pathogen can reproduce compared to its competitors. Yet not all hosts provide the same resource pool. Males and females, in particular, commonly vary in both their acquisition of resources and investment in immunity, but their ability to modify any competition between different pathogens remains unknown. Using the Daphnia magna–Pasteuria ramosa model system, we exposed male and female hosts to either a single genotype infection or coinfections consisting of two pathogen genotypes of varying levels of virulence. We found that coinfections within females favored the transmission of the more virulent pathogen genotype, whereas coinfections within male hosts resulted in equal transmission of competing pathogen genotypes. This contrast became less pronounced when the least virulent pathogen was able to establish an infection first, suggesting that the influence of host sex is shaped by priority effects. We suggest that sex is a form of host heterogeneity that may influence the evolution of virulence within coinfection contexts and that one sex may be a reservoir for pathogen genetic diversity in nature.     

The artificial infection of the cladoceran Moina macrocopa (Crustacea: Phyllopoda) with the microsporidia Gurleya sp. (Microsporidia: Gurleyidae)

Microcrustaceans Moina macrocopa (Straus, 1820) have been collected in a small vernal pond and cultured under laboratory conditions. The infection of this culture with the microsporidia Gurleya sp. was detected and high virulence of this microsporidian species allowed us to carry out the transmission experiments. Horizontal transmission, the high host specificity and maximal spore production in the hypodermal and fat body cells through 5-6 days after per oral experimental infection have been revealed. Histological investigations demonstrated that Gurleya sp. reduces drastically host reproductive success without a considerable influence on the microcrustaceans' mortality.     

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.     

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.     

Contrasting consequences of different defence strategies in a natural multihost–parasite system

Hosts counteract infections using two distinct defence strategies, resistance (reduction in pathogen fitness) and tolerance (limitation of infection damage). These strategies have been minimally investigated in multi-host systems, where they may vary across host species, entailing consequences both for hosts (virulence) and parasites (transmission). Comprehending the interplay among resistance, tolerance, virulence and parasite success is highly relevant for our understanding of the ecology and evolution of infectious and parasitic diseases. Our work investigated the interaction between an insect parasite and its most common bird host species, focusing on two relevant questions: (i) are defence strategies different between main and alternative hosts and, (ii) what are the consequences (virulence and parasite success) of different defence strategies? We conducted a matched field experiment and longitudinal studies at the host and the parasite levels under natural conditions, using a system comprising Philornis torquans flies and three bird hosts – the main host and two of the most frequently used alternative hosts. We found that main and alternative hosts have contrasting defence strategies, which gave rise in turn to contrasting virulence and parasite success. In the main bird host, minor loss of fitness, no detectable immune response, and high parasite success suggest a strategy of high tolerance and negligible resistance. Alternative hosts, on the contrary, resisted by mounting inflammatory responses, although with very different efficiency, which resulted in highly dissimilar parasite success and virulence. These results show clearly distinct defence strategies between main and alternative hosts in a natural multi-host system. They also highlight the importance of defence strategies in determining virulence and infection dynamics, and hint that defence efficiency is a crucial intervening element in these processes.     

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.     

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.     

THE INTERACTION PHENOTYPE IN THE DROSOPHILA WILLISTONI–SPIROPLASMA SYMBIOSIS

Both the population and coevolutionary dynamics of hereditary male-lethal endosymbionts, found in a wide range of insect species, depend on host fitness and endosymbiont transmission rates. This paper reports on fitness effects and transmission rates in three lines of Drosophila willistoni infected with either male-lethal spiroplasmas or a spontaneous nonmale-lethal mutant. Overall fitness measures were reduced or unaffected by the infection; however, some infected females produced more offspring in early broods. Maternal transmission rates were high, but imperfect, and varied with a female's age, host line, and spiroplasma type. No evidence for paternal or horizontal transmission was found. If an altered temporal pattern of reproduction is not a factor in countering the loss of spiroplasma hosts through imperfect maternal transmission, persistence of this endoparasitism remains unexplained. Tolerance of the infection and ability to transmit bacteria varied with both host and spiroplasma line. Analysis of the interaction between the spontaneous nonmale-lethal mutant and its host suggests this symbiosis has undergone coevolution under laboratory culture.