Trypanosomatids are obligate parasites of animals, predominantly insects and vertebrates, and flowering plants. Monoxenous species, representing the vast majority of trypanosomatid diversity, develop in a single host, whereas dixenous species cycle between two hosts, of which primarily insect serves as a vector. To explore in-depth the diversity of insect trypanosomatids including their co-infections, sequence profiling of their 18S rRNA gene was used for true bugs (Hemiptera; 18% infection rate) and flies (Diptera; 10%) in Cuba. Out of 48 species (molecular operational taxonomic units) belonging to the genera Vickermania (16 spp.), Blastocrithidia (7), Obscuromonas (4), Phytomonas (5), Leptomonas/Crithidia (5), Herpetomonas (5), Wallacemonas (2), Kentomonas (1), Angomonas (1) and two unnamed genera (1 + 1), 38 species have been encountered for the first time. The detected Wallacemonas and Angomonas species constitute the most basal lineages of their respective genera, while Vickermania emerged as the most diverse group. The finding of Leptomonas seymouri, which is known to rarely infect humans, confirms that Dysdercus bugs are its natural hosts. A clear association of Phytomonas with the heteropteran family Pentatomidae hints at its narrow host association with the insect rather than plant hosts. With a focus on multiple infections of a single fly host, using deep Nanopore sequencing of 18S rRNA, we have identified co-infections with up to 8 trypanosomatid species. The fly midgut was usually occupied by several Vickermania species, while Herpetomonas and/or Kentomonas species prevailed in the hindgut. Metabarcoding was instrumental for analysing extensive co-infections and also allowed the identification of trypanosomatid lineages and genera.

Biological interactions can greatly influence the abundance of species. This is also true for parasitic species that share the same host. Microsporidia and Rickettsia are widespread intracellular parasites in populations of Paracalliope fluviatilis, the most common freshwater amphipods in New Zealand. Although both parasites coexist in many populations, it is unclear whether they interact with each other. Here, we investigated spatial−temporal dynamics and co-occurrence of the two parasites, Microsporidia and Rickettsia in P. fluviatilis hosts, across one annual cycle and in three different locations. Prevalence of both Microsporidia and Rickettsia changed over time. However, while the prevalence of Rickettsia varied significantly between sampling times, that of Microsporidia did not change significantly and remained relatively low. The two parasites therefore followed different temporal patterns. Also, the prevalence of both parasites differed among locations, though the two species reached their highest prevalence in different locations. Lastly, there was no evidence for positive or negative associations between the two parasite species; the presence of one parasite in an individual host does not appear to influence the probability of infection by the other parasite. Their respective prevalence may follow different patterns among populations on a larger spatial scale due to environmental heterogeneity across locations.

We performed a long-term natural experiment investigating the impact of the diphyllobotriidean cestode Schistocephalus solidus on the body condition and clutch size (CS) of threespine stickleback Gasterosteus aculeatus, its second intermediate host, and the growth of larval parasites in host fish. We tested the hypothesis that single S. solidus infections were more virulent than multiple infections. We also asked whether the metrics of mean and total parasite mass (proxies for individual and total volume, respectively) were consistent with predictions of the resource constraints or the life history strategy (LHS) hypothesis for the growth of, hence exploitation by, larval helminths in intermediate hosts. The samples were drawn from Walby Lake, Alaska in eight of 11 years. Host body condition and CS (egg number per spawning bout) decreased significantly with intensity after adjustments for host size and parasite index. Thus, infections have an increasingly negative impact on measures of host fitness with greater intensity, in contrast to the hypothesis that single infections are more harmful than multiple infections. We also found that mean parasite mass decreased with intensity while total parasite mass increased with intensity as predicted by the LHS hypothesis.

Trophically transmitted parasites must trade-off own growth on one hand and energy drain from the intermediate host on the other hand, since killing the host before transmission to the next host is a dead end for both parasites and hosts. This challenge becomes especially intriguing when multiple parasites find themselves within the same individual host. The tapeworm Schistocephalus solidus may gain more than 98% of its final body mass within few months infecting its three-spined stickleback (Gasterosteus aculeatus) intermediate host. During these months the tapeworms may achieve a mass even larger than its host. We studied virulence of single and multiple infections of S. solidus, by comparing body condition of wild stickleback hosts in two perennial stickleback populations located at high latitudes, and each population was studied in two different years. Our results demonstrated multiple compared with single infections to be a highly significant predictor of the condition of stickleback hosts, with multiple-infected hosts having relatively higher body condition. However, this applied only after adjusting for parasite mass, which was another significant predictor for host condition. Thus, our results suggested that, at a given parasite mass, S. solidus was more harmful towards their host's body condition in single compared with multiple infections.

Although co-infection by multiple groups of pathogens is the norm rather than the exception in nature, most research on the effects of pathogens on their hosts has been largely based on a single or few pathogen species. Nevertheless, the health impact of co-occurring infections is evident, and it is important that scientists should consider pathogen communities rather than single relevant pathogen species when assessing the impact of multiple infections. In this work we illustrate the consequences of neglecting different pathogen taxa (viruses, protozoa, helminths, arthropods) in the explanatory power of a set of Partial Least Squares Regression (PLS-R) models used for exploring the impact of co-infections on the body condition of 57 adult feral cats; 71·5% cats were co-infected by ⩾3 groups of pathogens. The best two PLS-R models provided a first component based on the combination of helminths, protozoa and viruses, explaining 29·15% of body-condition variability. Statistical models, partially considering the pathogen community, lost between 24% and 94% of their explanatory power for explaining the cost of multiple infections. We believe that in the future, researchers assessing the impact of diseases on host life-history traits should take into account a broad representation of the pathogen community, especially during early assessment of the impact of diseases on host health.

Microsporidia are intracellular parasites, frequently infecting the planktonic crustacean Daphnia. Questioning the ability to detect and identify microsporidia with conventional microscopic techniques, we applied molecular methods in order to investigate the distribution and co-infection patterns of this parasite among 8 communities of the Daphnia longispina hybrid complex. Eight microsporidian taxa were detected, including 3 that previously had not been characterized genetically. Microsporidian communities from nearby lakes were found to be more similar to each other, apparently due to short distance dispersal via secondary hosts. Moreover, we detected seasonal (but not interannual) changes in microsporidian community structure. With some microsporidia being host-specific, these changes might have resulted from seasonal changes in host taxon and clonal composition. The 2 dominant and closely related parasite species were found mainly in single infections, whereas another pair of related microsporidians was found predominantly in co-infections; suggesting species-level differences in the ability to colonize infected hosts. By applying molecular methods, we were not only able to unambiguously identify parasite taxa but also to reveal multiple infections that otherwise would have remained undetected. Given the increased level of accuracy and sensitivity, we highly recommend molecular approaches in future parasite surveys of Daphnia infections.

Wolbachia bacteria are among the most common endosymbionts in insects. In Wolbachia research, the Wolbachia surface protein (wsp) gene has been used as a phylogenetic tool, but relationships inferred by single-locus analysis can be unreliable because of the extensive genome recombination among Wolbachia strains. Therefore, a multilocus sequence typing (MLST) method for Wolbachia, which relies upon a set of five conserved genes, is recommended. In this study, we examined whether the alnus ambrosia beetle, Xylosandrus germanus (Blandford), is infected with Wolbachia using wsp and MLST genes. Wolbachia was detected from all tested specimens of X. germanus (n=120) by wsp amplification. Five distinct sequences (i.e. five alleles) for wsp were found, and labeled as wXge1–5. MLST analysis and molecular phylogeny of concatenated sequences of MLST genes identified wXge3 and wXge5 as closely-related strains. The detection rate of wXge4 and wXge1 was 100% and 63.3%, respectively; wXge2, wXge3 and wXge5 were detected from less than 15% of specimens. We performed mitochondrial haplotype analyses that identified three genetic types of X. germanus, i.e. Clades A, B and C. Wsp alleles wXge1, wXge2 and wXge4 were detected in all clade A beetles; wXge2 allele was absent from Clades B and C. We concluded that (i) five wsp alleles were found from X. germanus, (ii) use of MLST genes, rather than the wsp gene, are more suited to construct Wolbachia phylogenies and (iii) wsp alleles wXge2 and wXge3/wXge5 would infect clade A and clade B/C of X. germanus, respectively.

Malaria and filaria infection rates were determined for anopheline mosquitoes collected whilst biting and resting in village houses in Papua New Guinea. The number of anophelines infected with both parasites was greater than expected from the infection rates of each parasite and this difference was significant in resting collections. The excess of multiply infected mosquitoes is probably a result of a vector population composed of individuals with differing numbers of opportunities to become infected. Malaria-positive, Anopheles punctulatus from resting catches had a significantly greater number of Stage 3 Wuchereria bancroftilarvae than malaria-negative mosquitoes. However, multiply infected mosquitoes appear to suffer greater mortality than non-infected or singly infected mosquitoes when the filarial worm reaches the third stage. Any potential increase in transmission resulting from multiple infections is thereby offset by a greater mortality rate in these mosquitoes.

Parasitic worm infections are amongst the most widespread of all chronic human infections. It is estimated that there are more than 3 billion infections in the world today. In many low income countries it is often more common to be infected than not to be. Indeed, a child growing up in an endemic community can expect be infected soon after weaning, and to be infected and constantly reinfected for the rest of her or his life. Infection is most common amongst the poorest and most disadvantaged communities, and is typically most intense in children of school going age. As the risk of morbidity is directly related to intensity of infection, it follows that children are the most at risk from the morbid effects of disease. Multiparasite infections are also common in such communities and there is evidence that individuals harbouring such infections may suffer exacerbated morbidity, making children even more vulnerable. Thus, these infections pose a serious threat to the health and development of children in low income countries. For many years, the need to control these infections has lain uncontested, and with the advent of broad-spectrum anthelminthic drugs that are cheap, safe and simple to deliver, control has at last become a viable option for many communities. Furthermore, there is now increased emphasis being placed on a multispecies approach as a cost-effective mechanism to control the morbidity of virtually all the major helminthic infections of humans.

Multiple infections, those by more than one parasite in the same erythrocyte, may be adaptive for the malarial parasite as a means to ensure fertility. Alternatively they may simply be the consequence of a non-adaptive process forcing several parasites to compete for resources in one host cell. Avian hosts infected with Haemoproteus were medicated with primaquine or injected with saline solution and the density of infection and number, maturity and sex of mature multiple infections counted. Multiple infections depend on density of infection, and maturity is attained rarely and usually by gametocytes of the same sex. The role of multiple infections for fertility insurance is not supported by these results.

We report a survey of four viruses (beet western yellows luteovirus (BWYV), cauliflower mosaic caulimovirus (CaMV), turnip mosaic potyvirus (TuMV), turnip yellow mosaic tymovirus (TYMV)) in five natural populations of Brassica oleracea in Dorset (UK). All four viruses were common; 43% of plants were infected with BWYV, 60% with CaMV, 43% with TuMV and 18% with TYMV. For each virus there were significant differences in the proportion of infected plants among populations, which were not completely explained by differences in the age of plants. Multiple virus infections were prevalent, with 54% of plants having two or more virus types. There were statistically significant associations between pairs of viruses. The CaMV was positively associated with the other three viruses, and BWYV was also positively associated with TuMV. There was no detectable association between BWYV and TYMV, whereas TuMV and TYMV were negatively associated. We suggest these associations result from BWYV, CaMV and TuMV having aphid vectors in common, as aphids are attracted to plants that already have a virus infection. Infected plants were distributed randomly or were very weakly aggregated within populations. The implications of widespread multiple virus infections in natural plant populations are discussed with respect to the release of transgenic plants expressing virus-derived genes.