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Malaria parasites of the genus Plasmodium are diverse in mammal hosts, infecting five mammalian orders in the Old World, but were long considered absent from the diverse deer family (Cervidae) and from New World mammals. There was a description of a Plasmodium parasite infecting a single splenectomized white-tailed deer (WTD; Odocoileus virginianus) in 1967 but none have been reported since, which has proven a challenge to our understanding of malaria parasite biogeography. Using both microscopy and polymerase chain reaction, we screened a large sample of native and captive ungulate species from across the United States for malaria parasites. We found a surprisingly high prevalence (up to 25%) and extremely low parasitemia of Plasmodium parasites in WTD throughout the eastern United States. We did not detect infections in the other ungulate species nor in western WTD. We also isolated the parasites from the mosquito Anopheles punctipennis. Morphologically, the parasites resemble the parasite described in 1967, Plasmodium odocoilei. Our analysis of the cytochrome b gene revealed two divergent Plasmodium clades in WTD representative of species that likely diverged 2.3 to 6 million years ago, concurrent with the arrival of the WTD ancestor into North America across Beringia. Multigene phylogenetic analysis placed these clades within the larger malaria parasite clade. We document Plasmodium parasites to be common in WTD, endemic to the New World, and as the only known malaria parasites from deer (Cervidae). These findings reshape our knowledge of the phylogeography of the malaria parasites and suggest that other mammal taxa may harbor infection by endemic and occult malaria parasites.

Within mixed-genotype infections of malaria parasites (Plasmodium), the number of genetic clones present is associated with variation in important life history traits of the infection, including virulence. Although the number of clones present is important, how the proportion of those clones varies over time is poorly known. Clonal proportions of the lizard malaria parasite, Plasmodium mexicanum, were assessed in naturally infected free-ranging lizards followed in a mark-recapture program over as long as two warm seasons, the typical life span of the lizard. Clonal proportions were determined by amplifying two microsatellite markers, a method previously verified for accuracy. Most blood samples had been stored for over a decade, so a verification test determined that these samples had not degraded. Although the environment experienced by the parasite (its host) varies over the seasons and transmission occurs over the entire warm season, 68% of infections were stable over time, harboring a single clone (37% of infections) or multiple clones changing only 1-12% maximum comparing any two samples (31% of infections). The maximum change seen in any infection (comparing any two sample periods) was only 30%. A new clone entered three infections (only once successfully), and a clone was lost in only three infections. These results mirror those seen for a previous study of experimentally induced infections that showed little change in relative proportions over time. The results of this study, the first look at how clonal proportions vary over time for any malaria parasite of a nonhuman vertebrate host for natural infections, were surprising because experimental studies show clones of P. mexicanum appear to interact, yet relative proportions of clones typically remain constant over time.

Within genetically diverse infections of malaria parasites ( Plasmodium spp.), the relative proportions of genetic clones in the vertebrate host's blood can influence clonal competition, transmission success, gametocyte sex ratio, and virulence. Clonal proportions depend on establishment success of each clone when they enter a new host and on subsequent differences in rates of asexual replication and clearance. Both of these life history traits could be influenced by clone genotype. To assess genetic (clonal) influences on both establishment success and later changes in relative proportion for the lizard malaria parasite Plasmodium mexicanum , 7 naturally infected fence lizards harboring a single clone of P. mexicanum served as donors to initiate replicate experimental infections containing each of the clones and combinations of 2 clones. Measured were relative establishment success of each clone, change in relative proportions over time, and rate of increase of parasite density and total parasitemia. Relative clonal proportions were determined using microsatellite markers. Rates of increase in the parasitemia and degree of change in relative proportions were not correlated, so both rapidly and slowly growing infections could show either little or substantial change in clonal proportions over time. There was a significant clone effect on establishment efficiency but not on later changes in relative proportions. These results argue for a combination of genetic and environmental (host) effects on the success of P. mexicanum clones in genetically complex infections. The maintenance of genetic variation for establishment success, but not subsequent replication rate or shifts in relative proportion, suggests trade-offs between these traits during life history evolution of malaria parasites.

Evolutionary theory predicts that virulence of parasites for mobile vector insects will be low for natural parasite-host associations that have coevolved. I determined virulence of the malaria parasite of lizards, Plasmodium mexicanum, for its vectors, two species of sand fly (Diptera: Psychodidae), Lutzomyia vexator (Coquillett 1907) and Lutzomyia stewarti (Mangabeira Fo & Galindo 1944), by measuring several life history traits. Developmental rate from egg to eclosion differed for the two species when noninfected. For both sand fly species, developmental rate for each stage (egg to larval hatching, larval period, pupal period) and life span were not altered by infection. Infected sand flies, however, produced fewer eggs. This reduction in fecundity may be a result of lower quality of the blood meal taken from infected lizards (lower concentration of hemoglobin). This report is the first measure of virulence of Plasmodium for an insect vector other than a mosquito and concords with both expectations of theory and previous studies on natural parasite-host associations that revealed low virulence.

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.

Quantifying the relative proportion of coexisting genotypes (clones) of a malaria parasite within its vertebrate host's blood would provide insights into critical features of the biology of the parasite, including competition among clones, gametocyte sex ratio, and virulence. However, no technique has been available to extract such data for natural parasite-host systems when the number of clones cycling in the overall parasite population is likely to be large. Recent studies find that data from genetic analyzer instruments for microsatellite markers allow measuring clonal proportions. We conducted a validation study for Plasmodium mexicanum and Plasmodium falciparum by mixing DNA from single-clone infections to simulate mixed infections of each species with known proportions of clones. Results for any mixture of DNA gave highly reproducible results. The relationship between known and measured relative proportions of clones was linear, with high regression r² values. Known and measured clone proportions for simulated infections followed over time (mixtures) were compared with 3 methods: using uncorrected data, with uncorrected data and confidence intervals constructed from observed experimental error, and using a baseline mixture of equal proportions to calibrate all other results. All 3 methods demonstrated value in studies of mixed-genotype infections sampled a single time or followed over time. Thus, the method should open new windows into the biology of malaria parasites.

Very slight sequence differences in the mitochondrial cytochrome b gene, even single nucleotide substitutions, have been proposed as indicative of different species of avian malaria parasites. However, few studies have examined within-species variation in that gene for Plasmodium or related genera. We examined sequences for the entire cytochrome b gene from Plasmodium mexicanum , a parasite of lizards, for sites where microsatellite markers revealed substantial genetic diversity. For sites where the parasite is geographically genetically differentiated, and may have been isolated for thousands of years, there was no sequence variation (1,153 nucleotides) for >160 infections studied. The low degree of variation found in the cytochrome b gene for two human malaria parasites world-wide, as well as the lack of variation for P. mexicanum , contrast with the substantial variation found in surveys of bird malaria parasites, even in restricted geographic regions.

Gene flow, and resulting degree of genetic differentiation among populations, will shape geographic genetic patterns and possibly local adaptation of parasites and their hosts. Some studies of Plasmodium falciparum in humans show substantial differentiation of the parasite in locations separated by only a few kilometers, a paradoxical finding for a parasite in a large, mobile host. We examined genetic differentiation of the malaria parasite Plasmodium mexicanum, and its lizard host, Sceloporus occidentalis, at 8 sites in northern California, with the use of variable microsatellite markers for both species. These lizards are small and highly territorial, so we expected local genetic differentiation of both parasite and lizard. Populations of P. mexicanum were found to be differentiated by analysis of 5 markers (F(st) values >0.05-0.10) over distances as short as 230-400 m, and greatly differentiated (F(st) values >0.25) for sites separated by approximately 10 km. In contrast, the lizard host had no, or very low, levels of differentiation for 3 markers, even for sites >40 km distant. Thus, gene flow for the lizard was great, but despite the mobility of the vertebrate host, the parasite was locally genetically distinct. This discrepancy could result if infected lizards move little, but their noninfected relatives were more mobile. Previous studies on the virulence of P. mexicanum for fence lizards support this hypothesis. However, changing prevalence of the parasite, without changes in density of the lizard, could also result in this pattern.

Microsatellites, short tandem repeats of nucleotides in the genome, are useful markers to detect clonal diversity within Plasmodium infections. However, accuracy in determining number of clones and their relative proportions based on standard genetic analyzer instruments is poorly known. DNA extracted from lizards infected with a malaria parasite, Plasmodium mexicanum, provided template to genotype the parasite based on three microsatellite markers. Replicate genotyping of the same natural infections demonstrated strong repeatability of data from the instrument. Mixing DNA extracted from several infected lizards simulated mixed-clone infections with known clonal diversity and relative proportions of clones (N = 56 simulations). The instrument readily detected at least four alleles (clones), even when DNA concentrations among clones differed up to tenfold, but alleles of similar size can be missed because they fall within the "stutter" artifact, and rarely does an allele fail to be detected. For simulations of infections that changed their relative proportions over time, changes in relative peak heights on the instrument output closely followed the known changes in relative proportions. Such data are useful for a broad range of studies on the ecology of malaria parasites.

Premunition in Plasmodium spp. is the prevention of superinfection by novel genotypes entering an already established infection in a vertebrate host. Evidence for premunition was sought for the lizard malaria parasite, P. mexicanum, in its natural host, the fence lizard, Sceloporus occidentalis. Clonal diversity (= alleles for the haploid parasite) was determined with the use of 3 microsatellite markers. Both naturally infected lizards (N = 25) and previously noninfected lizards (N = 78) were inoculated intraperitoneally (IP) with blood from donor infections and followed over a 3-mo period. Compared to the success of clonal establishment in all the naive lizards (78/78 successful), clones entering preexisting infections had a significant disadvantage (9/25 successful). The number of preexisting clones (1-2 vs. 3-4) within recipient infections had no effect on the success of superinfection. Infections that excluded entering novel clones did not have higher initial asexual parasitemia, but had a higher initial density of gametocytes, suggesting they were older. Infections allowing superinfection experienced a higher final parasitemia.

Two species of sandflies (Lutzomyia) are competent vectors of Plasmodium mexicanum, a malaria parasite of lizards. The very patchy distribution of sites with high P. mexicanum prevalence in the lizards, and often low or even nil sandfly density at such sites, provoked an evaluation of 2 common lizard ectoparasites, the tick Ixodes pacificus and the mite Geckobiella occidentalis, as potential passive vectors. Plasmodium sp.-specific polymerase chain primers were used to amplify a long segment of the mitochondrial cytochrome b gene that is unlikely to survive intact if the parasite cells are killed within a blood-feeding arthropod. The segment was strongly amplified from sandflies (the positive control for the method) from 1 to 96 hr postfeeding on an infected lizard. For ticks, the gene fragment was poorly amplified at 0 hr postfeed, and not amplified after 2 hr. In contrast, strong amplification of the parasite DNA was observed from mites from 0 to 20 hr postfeed, and weak amplification even at 96 hr.

Plasmodium-specific polymerase chain reaction (PCR) primers allowed detection of infections with very low-level parasitemia for 3 species of malaria parasites infecting Anolis lizards at 2 Caribbean sites, Puerto Rico and Saba, Netherlands Antilles. A verification study, using a single-tube nested PCR to eliminate contamination, showed that infections as low as 1 parasite per millions of erythrocytes could be detected by amplifying a 673 bp fragment of the cytochrome b gene. Very low-level parasitemia infections, subpatent under the microscope, were common in A. sabanus on Saba sites, with no significant seasonal difference (31% of infections appearing uninfected by microscopic examination in summer were found infected by PCR, 38% in winter). At the Puerto Rico site, the subpatent infections were also common in A. gundlachi, but were more prevalent in winter (53%) than in summer (17%). A similar high frequency of subpatent infections is known from studies on human and bird malaria, but a previous PCR-based study on a temperate lizard malaria system found few such low-level infections. Differences in the prevalence of subpatent infections by site and season suggest transmission biology may select for distinct life history strategies by the parasite.

Gametocyte sex ratio of the malaria parasite Plasmodium mexicanum is variable in its host, the western fence lizard (Sceloporus occidentalis), both among infections and within infections over time. We sought to determine the effect of host physiological quality on the gametocyte sex ratio in experimentally induced infections of P. mexicanum. Adult male lizards were assigned to 4 treatment groups: castrated, castrated + testosterone implant, sham implant, and unmanipulated control. No significant difference in gametocyte sex ratio was found among the 4 treatment groups. Two other analyses were performed. A surgery stress analysis compared infection sex ratio of castrated, castrated + testosterone implant, and sham implant groups with the unmanipulated control group. A testosterone alteration analysis compared infection sex ratio of the castrated and castrated + testosterone implant groups with the sham implant and unmanipulated control groups. Again, no significant difference was observed for these 2 comparisons. Thus, physiological changes expected for experimentally induced variation in host testosterone and the stress of surgery were not associated with any change in the gametocyte sex ratio. Also, theex-periment suggests testosterone is not a cue for shaping the sex ratio of gametocytes in P. mexicanum. These results are related to the evolutionary theory of sex ratios as applied to malaria parasites.

The gametocyte sex ratio of Plasmodium mexicanum, a malaria parasite of western fence lizards, was studied in a modified garden experiment. Each of 6 naturally infected lizards was used to initiate 20 replicate-infections in naive western fence lizards. A significant donor effect was observed for the sex ratios of recipient infections at their maximal parasitemia, and this effect was associated with the sex ratio of the donor infection. In 20 infections in which sex ratio was followed during the course of the infection, 9 revealed constant sex ratios and 11 showed an increase in proportion of males over time. Recipient sex ratio was correlated with another life-history trait, a composite of rate of asexual replication and peak parasitemia, such that higher Rate-Peak scores were associated with infections with less female-biased sex ratios. These results are placed into the context of sex ratio theory that concludes that the degree of selfing of parasite genotypes (number of parasite clones) within the vector will influence the evolution of gametocyte sex ratio. The theory predicts that the sex ratio should be under some genetic control and thus be heritable as observed in the experiment. Clonal diversity should also influence the life-history trait, Rate-Peak, which was found to be correlated with sex ratio.

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 prevalence of malaria parasites was studied in the lizard Anolis gundlachi over a 9-yr period at a site in the wet evergreen forest of eastern Puerto Rico. Three forms of the parasite infected the lizards; these were Plasmodium floridense, Plasmodium azurophilum in erythrocytes, and P. azurophilum in white blood cells. Overall prevalence of infection for 8 samples during the study period was significantly higher for males than females (32% of 3,296 males and 22% of 1,439 females). During the study, the site experienced substantial climatic and physical disturbance including rising temperature, droughts, and hurricanes that severely damaged the forest. Parasite prevalence in the first sample, 8 mo after the massive hurricane Hugo, was slightly, though significantly, lower than for subsequent samples. However, overall prevalence was stable during the 9-yr period. The results show malaria prevalence is more constant at the site than found for 2 studies in temperate forests, and that the Puerto Rico system may be an example of the stable, endemic malaria described by standard models for human malaria epidemiology.

Plasmodium mexicanum, a malaria parasite of lizards, exhibits substantial variation among infections in the life-history traits which define its blood-dwelling stages. Such variation in life histories among infections is common in Plasmodium and may influence the ecology and evolution of the parasite's transmission success and virulence. Insight into these issues requires identification of independent traits (some traits may be bound by developmental trade-offs) and the importance of genetic versus host effects producing the variation. We studied 11 life-history traits in 120 induced infections of P. mexicanum in its natural lizard host (20 each from six donor infections). The traits varied among infections and fell into three clusters: rate/peak (rate of increase and peak parasitaemia of asexuals and gametocytes), time (duration of pre-patent period and the infection's growth) and maturity (timing of first gametocytes). Thus, few life-history traits define an infection in the lizard's blood. Donor effects were significant for ten traits and two trait clusters (maturity was the exception) suggesting genetic differences among infections may influence the rate of increase and peak parasitaemia, but not the timing of the first production of gametocytes.

The ecology of 2 parasites, Plasmodium floridense and Plasmodium azurophilum, was studied in the endemic lizard, Anolis sabanus, on Saba island in the eastern Caribbean. Prevalence of the two parasite species was similar, but prevalence varied among sites. Lowest prevalence occurred at dry, windy sites or cool, wet mountain peaks (0-20%); the parasites were more common at most other locations on the island (40-80% infected; overall prevalence = 47%). High and low prevalence sites sometimes were only a few hundred meters apart. Prevalence was similar for males and females but increased with body size except for a decline in the largest ( = oldest) lizards. A surplus of mixed infections (P. floridense and P. azurophilum together in the same host) existed compared with chance proportions. Parasitemia generally was low; 70% of P. floridense infections were < or = 100 parasites/10,000 erythrocytes, and 70% of P. azurophilum infections were < or = 50/10,000. Parasitemia in solitary infections averaged more than twice that seen in mixed infections for both species.

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.

In northern California, western fence lizards, Sceloporus occidentalis, are frequently parasitized by Plasmodium mexicanum, which causes malaria. Animals with this naturally occurring malarial infection are anemic: immature erythrocytes in peripheral blood become abundant (1 to 30 percent), and blood hemoglobin concentration decreases 25 percent. Maximal oxygen consumption decreases 15 percent and aerobic scope drops 29 percent in infected lizards; both correlate with blood hemoglobin concentration. Running stamina, but not burst running speed, is reduced in malarious lizards. There is a hierarchical relation between infection with malaria and effects on hematology, physiological function, and behavioral capacity. The results suggest that malarial infection may have significant effects on the ecology of lizard 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.