Freshwater trematodes differ from marine trematodes in patterns connected with division of labor.

Background: Prior research suggests that trematode rediae, a developmental stage of trematode parasites that reproduce clonally within a snail host, show evidence of division of labor (DOL). Single-species infections often have two morphologically distinct groups: small rediae, the 'soldiers', are active, aggressive, and do not appear to reproduce; large rediae, the 'reproductives', are larger, sluggish, and full of offspring. Most data supporting DOL come from trematodes infecting marine snails, while data from freshwater trematodes are more limited and generally do not supported DOL. The shorter lifespan typical of freshwater snails may partially explain this difference: defending a short-lived host at the expense of reproduction likely provides few advantages. Here, we present data from sixty-one colonies spanning twenty species of freshwater trematode exploring morphological and behavioral patterns commonly reported from marine trematodes believed to have DOL. Methods: Trematode rediae were obtained from sixty-one infected snails collected in central Vermont, USA. A portion of the COI gene was sequenced to make tentative species identifications ('COI species'). Samples of rediae were photographed, observed, and measured to look for DOL-associated patterns including a bimodal size distribution, absence of embryos in small rediae, and pronounced appendages and enlarged pharynges (mouthparts) in small rediae. Additional rediae were used to compare activity levels and likelihood to attack heterospecific trematodes in large vs. small rediae. Results: Many of the tests for DOL-associated patterns showed mixed results, even among colonies of the same COI species. However, we note a few consistent patterns. First, small rediae of most colonies appeared capable of reproduction, and we saw no indication (admittedly based on a small sample size and possibly insufficient attack trial methodology) that small rediae were more active or aggressive. This differs from patterns reported from most marine trematodes. Second, the small rediae of most colonies had larger pharynges relative to their body size than large rediae, consistent with marine trematodes. We also observed that colonies of three sampled COI species appear to produce a group of large rediae that have distinctly large pharynges. Conclusions: We conclude that these freshwater species likely do not have a group of specialized non-reproductive soldiers because small rediae of at least some colonies in almost every species do appear to produce embryos. We cannot rule out the possibility that small rediae act as a temporary soldier caste. We are intrigued by the presence of rediae with enlarged pharynges in some species and propose that they may serve an adaptive role, possibly similar to the defensive role of small 'soldier' rediae of marine trematodes. Large-pharynx rediae have been documented in other species previously, and we encourage future efforts to study these large-pharynx rediae.

Distribution of clones among hosts for the lizard malaria parasite Plasmodium mexicanum.

BACKGROUND: Malaria parasites reproduce asexually, leading to the production of large numbers of genetically identical parasites, here termed a clonal line or clone. Infected hosts may harbor one or more clones, and the number of clones in a host is termed multiplicity of infection (MOI). Understanding the distribution of parasite clones among hosts can shed light on the processes shaping this distribution and is important for modeling MOI. Here, I determine whether the distribution of clones of the lizard malaria parasite Plasmodium mexicanum differ significantly from statistical distributions commonly used to model MOI and logical extensions of these models. METHODS: The number of clones per infection was assessed using four microsatellite loci with the maximum number of alleles at any one locus used as a simple estimate of MOI for each infection. I fit statistical models (Poisson, negative binomial, zero-inflated models) to data from four individual sites to determine a best fit model. I also simulated the number of alleles per locus using an unbiased estimate of MOI to determine whether the simple (but potentially biased) method I used to estimate MOI influenced model fit. RESULTS: The distribution of clones among hosts at individual sites differed significantly from traditional Poisson and negative binomial distributions, but not from zero-inflated modifications of these distributions. A consistent excess of two-clone infections and shortage of one-clone infections relative to all fit distributions was also observed. Any bias introduced by the simple method for estimating of MOI did not appear to qualitatively alter the results. CONCLUSIONS: The statistical distributions used to model MOI are typically zero-truncated; truncating the Poisson or zero-inflated Poisson yield the same distribution, so the reasonable fit of the zero-inflated Poisson to the data suggests that the use of the zero-truncated Poisson in modeling is adequate. The improved fit of zero-inflated distributions relative to standard distributions may suggest that only a portion of the host population is located in areas suitable for transmission even at small sites (<1 ha). Collective transmission of clones and premunition may also contribute to deviations from standard distributions.

Genetic differentiation over a small spatial scale of the sand fly Lutzomyia vexator (Diptera: Psychodidae).

BACKGROUND: The geographic scale and degree of genetic differentiation for arthropod vectors that transmit parasites play an important role in the distribution, prevalence and coevolution of pathogens of human and wildlife significance. We determined the genetic diversity and population structure of the sand fly Lutzomyia vexator over spatial scales from 0.56 to 3.79 km at a study region in northern California. The study was provoked by observations of differentiation at fine spatial scales of a lizard malaria parasite vectored by Lu. vexator. METHODS: A microsatellite enrichment/next-generation sequencing protocol was used to identify variable microsatellite loci within the genome of Lu. vexator. Alleles present at these loci were examined in four populations of Lu. vexator in Hopland, CA. Population differentiation was assessed using Fst and D (of Cavalli-Sforza and Edwards), and the program Structure was used to determine the degree of subdivision present. The effective population size for the sand fly populations was also calculated. RESULTS: Eight microsatellite markers were characterized and revealed high genetic diversity (uHe = 0.79-0.92, Na = 12-24) and slight but significant differentiation across the fine spatial scale examined (average pairwise D = 0.327; F ST = 0.0185 (95 % bootstrapped CI: 0.0102-0.0264). Even though the insects are difficult to capture using standard methods, the estimated population size was thousands per local site. CONCLUSIONS: The results argue that Lu. vexator at the study sites are abundant and not highly mobile, which may influence the overall transmission dynamics of the lizard malaria parasite, Plasmodium mexicanum, and other parasites transmitted by this species.

Local mate competition and transmission bottlenecks: a new model for understanding malaria parasite and other sex ratios.

The local mate competition model from sex ratio theory predicts female-biased sex ratios in populations that are highly subdivided during mating, and is thought to accord well with the population structure of malaria parasites. However, the selective advantage of female-biased sex ratios comes from the resulting increase in total reproductive output, an advantage the transmission biology of malaria parasite likely reduces. We develop a mathematical model to determine how bottlenecks in transmission that cause diminishing fitness returns from female production affect sex ratio evolution. We develop four variations of this model that incorporate whether or not parasite clones have the ability to detect others that occupy the same host and whether or not the number of clones affects the total mating population size. Our model indicates that transmission bottlenecks favor less female-biased sex ratios than those predicted under LMC. This effect is particularly pronounced if clones have no information about the presence of coexisting clones and the number of mating individuals per patch is fixed. The model could extend our understanding of malaria parasite sex ratios in three main ways. First, it identifies inconsistencies between the theoretical predictions and the data presented in a previous study, and proposes revised predictions that are more consistent with underlying biology of the parasite. Second, it may account for the positive association between parasite density and sex ratio observed within and between some species. Third, it predicts a relationship between mortality rates in the vector and sex ratios, which appears to be supported by the little existing data we have. While the inspiration for this model came from malaria parasites, it should apply to any system in which per capita dispersal success diminishes with increasing numbers of females in a patch.

Testing sex ratio theory with the malaria parasite Plasmodium mexicanum in natural and experimental infections.

The malaria parasite (Plasmodium) life history accords well with the assumptions of local mate competition (LMC) of sex ratio theory. Within a single meal of the blood-feeding vector, sexually dimorphic gametocyte cells produce gametes (females produce one, males several) that mate and undergo sexual recombination. The theory posits several factors drive the Plasmodium sex ratio: male fecundity (gametes/male gametocyte), number and relative abundance of parasite clones, and gametocyte density. We measured these traits for the lizard malaria parasite, Plasmodium mexicanum, with a large sample of natural infections and infections from experiments that manipulated clonal diversity. Sex ratio in single-clone infections was slightly female-biased, but matched predictions of theory for this low-fecundity species. Sex ratio was less female-biased in clonally diverse infections as predicted by LMC for the experimental, but not natural infections. Gametocyte density was not positively related to sex ratio. These results are explained by the P. mexicanum life history of naturally low clonal diversity and high gametocyte production. This is the first study of a natural malaria system that examines all traits relevant to LMC in individual vertebrate hosts and suggests a striking example of sex ratio theory having significance for human public health.

Substratum preference of Philophthalmus sp. cercariae for cyst formation under natural and experimental conditions.

Selection on parasites should favor adaptations that maximize the probability of transmission to the definitive host, such as the preference for and use of intermediate hosts or encystment substrata that are likely to be consumed by the definitive host. Eye flukes in the genus Philophthalmus are passed to their definitive avian host through the ingestion of metacercariae encysted on hard substrata. The life cycle of these parasites is generally well understood; however, there is almost no information on substratum use or preference of the cercariae of these parasites. In this study, we combine a survey of naturally occurring substrata with experimental, laboratory-based choice tests to determine the preferred substratum of Philophthalmus sp. and whether this preference is affected by the presence and density of pre-existing cysts. A concordance between natural and experimental data show a preference for the shells of multiple species of snail over other hard substrata that are common at the field site, including seaweed, other molluscs, and crustaceans. In addition, we found that cercariae preferred substrata with pre-existing cysts and that this preference seemed to increase with increasing cyst density. Such a preference should lead to an aggregated distribution of cysts among snail shells that may benefit the parasite by increasing the number of potential mates that become established in the definitive host. The identification of a preferred substratum also may help to identify potential definitive hosts that were previously unknown.