Search

Understanding the consequences of environmental fluctuations for parasite dynamics requires a long-term view stretching over many transmission cycles. Here we studied the dynamics of three malaria parasites (Plasmodium azurophilum, P. leucocytica and P. floridense) infecting the lizard Anolis gundlachi, in the rainforest of Puerto Rico. In this malaria-anole system we evaluated temporal fluctuations in individual probability of infection, the environmental drivers of observed variation and consequences for host body condition and Plasmodium parasites assemblage. We conducted a total of 15 surveys including 10 from 1990 to 2002 and five from 2015 to 2017. During the early years, a lizard's probability of infection by all Plasmodium species appeared stable despite disturbances ranging from two hurricanes to short droughts. Over a longer timescale, probability of infection and overall prevalence varied significantly, following non-linear relationships with temperature and rainfall such that highest prevalence is expected at intermediate climate measures. A perplexing result was that host body condition was maximized at intermediate levels of rainfall and/or temperature (when risk of infection was highest), yet we found no significant decreases in body condition due to infection. Plasmodium parasite species composition varied through time with a reduction and near local extinction of P. floridense. Our results emphasize the need for long-term studies to reveal host-parasite dynamics, their drivers and consequences.

Changes in population allele frequencies may be driven by several forces, including selection and drift, and are revealed only by sampling over many generations. Such studies, however, are rare for protist parasites. Microsatellite allele frequencies for 4 loci were followed in a population of Plasmodium mexicanum, a malaria parasite of lizards in California USA at 1 site from 1978 to 2010. Rapid turnover of the lizards indicates the parasite was studied for a minimum of 33 transmission cycles and possibly twice that number. Sample sizes ranged from 841 to 956 scored parasite clones per locus. DNA was extracted from frozen dried blood and blood removed from stained blood smears from the earliest years, and a verification study demonstrated DNA from the blood smears provided valid genetic data. Parasite prevalence and effective population size (Ne) dropped after 2000, remaining lower for the next decade. For 2 loci, allele frequencies appeared stable for the first 2 decades of the study, but changed more rapidly after the decline in prevalence. Allele frequencies changed more gradually for the other 2 loci. Genetic drift could account for changes in allele frequencies, especially after the drop in prevalence and Ne, but the force of selection could also have driven the observed patterns.

Sex ratio theory predicts that malaria parasites should bias gametocyte production toward female cells in single-clone infections because they will experience complete inbreeding of parasite gametes within the vector. A higher proportion of male gametocytes is favoured under conditions that reduce success of male gametes at reaching females such as low gametocyte density or attack of the immune system later in the infection. Recent experimental studies reveal genetic variation for gametocyte sex ratio in single-clone infections. We examined these issues with a study of experimental single-clone infections for the lizard malaria parasite Plasmodium mexicanum in its natural host. Gametocyte sex ratios of replicate single-clone infections were determined over a period of 3-4 months. Sex ratios were generally female biased, but not as strongly as expected under simple sex ratio theory. Gametocyte density was not related to sex ratio, and male gametocytes did not become more common later in infections. The apparent surplus of male gametocytes could be explained if male fecundity is low in this parasite, or if rapid clotting of the lizard blood reduces male gamete mobility. There was also a significant clone effect on sex ratio, suggesting genetic variation for some life-history trait, possibly male fecundity.

Both verbal and mathematical models of parasite virulence predict that genetic diversity of microparasite infections will influence the level of costs suffered by the host. We tested this idea by manipulating the number of co-existing clones of Plasmodium mexicanum in its natural vertebrate host, the fence lizard Sceloporus occidentalis. We established replicate infections of P. mexicanum made up of 1, 2, 3, or >3 clones (scored using 3 microsatellite loci) to observe the influence of clone number on several measures of parasite virulence. Clonal diversity did not affect body growth or production of immature erythrocytes. Blood haemoglobin concentration was highest for the most genetically complex infections (equal to that of non-infected lizards), and blood glucose levels and rate of blood clotting was highest for the most diverse infections (with greater glucose and more rapid clotting than non-infected animals). Neither specific clones nor parasitaemia were associated with virulence. In this first experiment that manipulated the clonal diversity of a natural Plasmodium-host system, the cost of infection with 1 or 2 clones of P. mexicanum was similar to that previously reported for infected lizards, but the most complex infections had either no cost or could be beneficial for the host.

Malaria parasites in the genus Plasmodium are now placed within 11 subgenera based on morphology under the light microscope, life-history traits, and host taxon. The phylogenetic significance of these characters, however, is problematic because the observed variation could be homoplasious. Using Plasmodium infections found in 2632 birds of many avian families collected in the USA, and several samples from other locations, we compared identifications to subgenus based on morphology in blood smears with a 2-gene molecular phylogeny (the first for avian Plasmodium) to determine if the 5 avian Plasmodium subgenera represent monophyletic groups. Phylogenetic trees recovered by parsimony, likelihood, and Bayesian methods presented nearly identical topologies. The analysis allowed testing the hypothesis of monophyly for the subgenera. Monophyly of the subgenera Haemamoeba, Huffia, and Bennettinia was supported by the analysis. The distinctive morphology of Haemamoeba species appears to have evolved once. Most samples identified to Novyella also fell within a monophyletic clade with the exception of 2 samples that fell basal to all other avian Plasmodium. Samples of the subgenus Giovannolaia did not form a monophyletic group. Thus, the characters used by parasitologists for over a century to define subgenera of Plasmodium vary in their phylogenetic significance.

More than 200 species of avian Haemosporidia (genera Plasmodium, Haemoproteus, and Leucocytozoon) have been described based primarily on morphological characters seen in blood smears. Recent molecular studies, however, suggest that such methods may mask a substantial cryptic diversity of avian haemosporidians. We surveyed the haemosporidians of birds sampled at 1 site in Israel. Parasites were identified to species based on morphology, and a segment of the parasite's cytochrome b gene was sequenced. We compared 3 species concepts: morphological, genetic, and phylogenetic. Fifteen morphological species were present. Morphological species that occurred once within our dataset were associated with a unique gene sequence, displayed large genetic divergence from other morphological species, and were not contained within clades of morphological species that occurred more than once. With only 1 exception, morphological species that were identified from multiple bird hosts presented identical sequences for all infections, or differed by few synonymous substitutions, and were monophyletic for all phylogenetic analyses. Only the morphological species Haemoproteus belopolskyi did not follow this trend, falling instead into at least 2 genetically distant clades. Thus, except for H. belopolskyi, parasites identified to species by morphology were supported by both the genetic and phylogenetic species concepts.

Sex ratio theory posits that the adaptive proportion of male to female gametocytes of a malaria parasite within the vertebrate host depends on the degree of inbreeding within the vector. Gametocyte sex ratio could be phenotypically flexible, being altered based on the infection's clonal diversity, and thus likely inbreeding. This idea was tested by manipulating the clonal diversity of infections of Plasmodium mexicanum in its lizard host, Sceloporus occidentalis. Naive lizards were inoculated with infected blood from a single donor or 3 pooled donors. Donors varied in their gametocyte sex ratios (17-46%, male), and sex ratio theory allowed estimation of the clonal diversity within donor and recipient infections. Phenotypic plasticity would produce a correlation between donor and recipient infections for infections initiated from a single donor, and a less female-biased gametocyte sex ratio in recipients that received a mixed blood inoculum (with predicted higher clonal diversity) than recipients receiving blood from a single donor. Neither pattern was observed. Gametocyte sex ratio of most infections ranged from 35 to 42% male, expected if clonal diversity was high for all infections. Alternative explanations are suggested for the observed variation of gametocyte sex ratio among P. mexicanum infections.

The life-cycle of Plasmodium depends on transmission of the parasite from the vertebrate host into its vector when the insect takes a bloodmeal. Transmission success may depend in part on the parasite's gametocyte density and sex ratio in the blood. P. mexicanum, a parasite of fence lizards in California, USA, exploits the sandfly Lutzomyia vexator as its vector. In experimental transmissions using naturally infected lizards as donors of blood, transmission success (measured as percentage of vectors infected and number of parasite oocysts on the insect's midgut) was positively related to gametocyte density, although density above 20/1000 erythrocytes did not improve transmission. Sex ratio (proportion of microgametocytes in the infection) was positively correlated with gametocyte density. Transmission improved with higher proportion of microgametocytes, but partial correlations revealed that this was a result only of higher gametocyte densities. These results agree with the theory of virulence and sex ratios because single clone infections should produce a more female-biased sex ratio and grow to the minimum parasitaemia that would maximize clonal transmission, whereas multiple clone infections will be closer to a 1:1 sex ratio and yield a higher parasitaemia when each clone competes for transmission to the vector.

The negative consequences of parasitic infection (virulence) were examined for two lizard malaria parasite-host associations: Plasmodium agamae and P. giganteum, parasites of the rainbow lizard, Agama agama, in Sierra Leone, West Africa; and P. mexicanum in the western fence lizard, Sceloporus occidentalis, in northern California. These malaria species vary greatly in their reproductive characteristics: P. agamae produces only 8 merozoites per schizont, P. giganteum yields over 100, and P. mexicanum an intermediate number. All three parasites appear to have had an ancient association with their host. In fence lizards, infection with malaria is associated with increased numbers of immature erythrocytes, decreased haemoglobin levels, decreased maximal oxygen consumption, and decreased running stamina. Not affected were numbers of erythrocytes, resting metabolic rate, and sprint running speed which is supported by anaerobic means in lizards. Infected male fence lizards had smaller testes, stored less fat in preparation for winter dormancy, were more often socially submissive and, unexpectedly, were more extravagantly coloured on the ventral surface (a sexually dimorphic trait) than non-infected males. Females also stored less fat and produced smaller clutches of eggs, a directly observed reduction in fitness. Infected fence lizards do not develop behavioural fevers. P. mexicanum appears to have broad thermal buffering abilities and thermal tolerance; the parasite's population growth was unaffected by experimental alterations in the lizard's body temperature. The data are less complete for A. agama, but infected lizards suffered similar haematological and physiological effects. Infected animals may be socially submissive because they appear to gather less insect prey, possibly a result of being forced into inferior territories. Infection does not reduce clutch size in rainbow lizards, but may lengthen the time between clutches. These results are compared with predictions emerging from several models of the evolution of parasite virulence. The lack of behavioural fevers in fence lizards may represent a physiological constraint by the lizards in evolving a thermal tolerance large enough to allow elimination of the parasite via fever. Such constraints may be important in determining the outcome of parasite-host coevolution. Some theory predicts low virulence in old parasite-host systems and higher virulence in parasites with greater reproductive output. However, in conflict with this argument, all three malarial species exhibited similar high costs to their hosts.

Sex ratio theory usually predicts an equilibrium sex ratio and equal proportions of males and females in a population, including the progenitors of the reproductive cells of protozoans. This proposal was tested with three species of malarial parasites of lizards, Plasmodium mexicanum of the western fence lizard, and P. agamae and P. giganteum of the African rainbow lizard, using single samples from naturally infected lizards, repeated samples from free-ranging lizards (P. mexicanum only), and repeated samples from laboratory maintained animals. Macrogametocytes were usually more abundant than microgametocytes, and were slightly larger, revealing a typically greater investment of resources by the progenitors of female reproductive cells. However, the proportion of microgametocytes varied among the three species and among infections within each species of Plasmodium. The sex ratio of gametocytes often remained constant within infections followed over time even if the absolute number of gametocytes was changing. However, the equilibrium sex ratio of gametocytes varied among those infections that had an unchanging microgametocyte proportion. Thus, although an equilibrium sex ratio apparently occurs for most infections, there appears to be no characteristic proportion of microgametocytes for any of the species. Potential explanations for this conflict with theory are presented.