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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.

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.

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.

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.

The ratio of male to female gametocytes seen in infections of Plasmodium and related haemosporidian parasites varies substantially, both within and among parasite species. Sex ratio theory, a mainstay of evolutionary biology, accounts for this variation. The theory provides an algebraic solution for the optimal sex ratio that will maximize parasite fitness. A crucial term in this solution is the probability of selfing by clone-mates within the vector (based on the clone number and their relative abundance). Definitive tests of the theory have proven elusive because of technical challenges in measuring clonal diversity within infections. Newly developed molecular methods now provide opportunities to test the theory with an exquisite precision.

Ecological and evolutionary theory predicts that genetic diversity of microparasites within infected hosts will influence the parasite replication rate, parasitemia, transmission strategy, and virulence. We manipulated clonal diversity (number of genotypes) of the malaria parasite, Plasmodium mexicanum, in its natural lizard host and measured important features of the infection dynamics, the first such study for any natural Plasmodium-host association. Hosts harboring either a single P. mexicanum clone or various combinations of clones (scored via three microsatellite markers) were established. Production of asexually replicating stages (meronts) and maximal meront parasitemia did not differ by clonal diversity, nor did timing of first production of transmission stages (gametocytes). However, mean rate of gametocyte increase and maximal gametocyte parasitemia were greater for hosts with mixed-clone infections. Characteristics of infections were more variable in hosts with mixed-clone infections than with single-clone infections except for first production of gametocytes. One or more of the parasite reproductive traits were extreme in 20 of 52 hosts with mixed-clone infections. This was not associated with specific clones, but diversity itself. The overall pattern from studies of clonal diversity for human, rodent, and now reptile malaria parasites confirms that the genetic diversity of infections in the vertebrate host is of central importance for the ecology of Plasmodium.

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.

Within the vertebrate host, infections of a malaria parasite (Plasmodium) could include a single genotype of cells (single-clone infections) or two to several genotypes (multiclone infections). Clonal diversity of infection plays an important role in the biology of the parasite, including its life history, virulence, and transmission. We determined the clonal diversity of Plasmodium mexicanum, a lizard malaria parasite at a study region in northern California, using variable microsatellite markers, the first such study for any malaria parasite of lizards or birds (the most common hosts for Plasmodium species). Multiclonal infections are common (50-88% of infections among samples), and measures of genetic diversity for the metapopulation (expected heterozygosity, number of alleles per locus, allele length variation, and effective population size) all indicated a substantial overall genetic diversity. Comparing years with high prevalence (1996-1998 = 25-32% lizards infected), and years with low prevalence (2001-2005 = 6-12%) found fewer alleles in samples taken from the low-prevalence years, but no reduction in overall diversity (H = 0.64-0.90 among loci). In most cases, rare alleles appeared to be lost as prevalence declined. For sites chronically experiencing low transmission intensity (prevalence approximately 1%), overall diversity was also high (H = 0.79-0.91), but there were fewer multiclonal infections. Theory predicts an apparent excess in expected heterozygosity follows a genetic bottleneck. Evidence for such a distortion in genetic diversity was observed after the drop in parasite prevalence under the infinite alleles mutation model but not for the stepwise mutation model. The results are similar to those reported for the human malaria parasite, Plasmodium falciparum, worldwide, and support the conclusion that malaria parasites maintain high genetic diversity in host populations despite the potential for loss in alleles during the transmission cycle or during periods/locations when transmission intensity is low.

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.