Experimental test for premunition in a lizard malaria parasite (Plasmodium mexicanum)

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.     

Use of PCR for detection of subpatent infections of lizard malaria: implications for epizootiology

The estimated prevalence of a malaria parasite, Plasmodium mexicanum , of western fence lizards, Sceloporus occidentalis , was compared using two techniques: microscopic examination of blood smears, and nested PCR amplification of the 18S small subunit rRNA gene. Two sites in northern California, USA were investigated, one with known long-term high prevalence of the parasite (30% by blood smear scanning), and one with low prevalence (6%). The nested PCR readily detected very low-level infections (< 1 parasite per 10 000 erythrocytes); such infections are often subpatent by normal microscopic examination. False negatives (scored as not infected after scanning the blood smear, but found infected via PCR) were rare at both sites (4% at the high-prevalence site, 6% at the low-prevalence site). However, a greater proportion of infections was detected only by PCR at the low-prevalence site (50% vs. 9%). If 50% of the infections sustain very weak parasitaemia where lizards are rarely infected, this would accord with hypotheses that predict that parasites should reduce infection growth when transmission is uncommon. The study demonstrates that PCR is a powerful tool to detect very low-level malarial infections in vertebrate hosts, including those with nucleated erythrocytes.     

Clonal diversity within infections and the virulence of a malaria parasite, Plasmodium mexicanum

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.     

Clonal diversity of a malaria parasite, Plasmodium mexicanum, and its transmission success from its vertebrate-to-insect host

Infections of the lizard malaria parasite Plasmodium mexicanum are often genetically complex within their fence lizard host (Sceloporus occidentalis) harbouring two or more clones of parasite. The role of clonal diversity in transmission success was studied for P. mexicanum by feeding its sandfly vectors (Lutzomyia vexator and Lutzomyia stewarti) on experimentally infected lizards. Experimental infections consisted of one, two, three or more clones, assessed using three microsatellite markers. After 5 days, vectors were dissected to assess infection status, oocyst burden and genetic composition of the oocysts. A high proportion (92%) of sandflies became infected and carried high oocyst burdens (mean of 56 oocysts) with no influence of clonal diversity on these two measures of transmission success. Gametocytemia was positively correlated with transmission success and the more common vector (L. vexator) developed more oocysts on midguts. A high proportion (74%) of all alleles detected in the lizard blood was found in infected vectors. The relative proportion of clones within mixed infections, determined by peak heights on pherograms produced by the genetic analyser instrument, was very similar for the lizard’s blood and infections in the vectors. These results demonstrate that P. mexicanum achieves high transmission success, with most clones making the transition from vertebrate-to-insect host, and thus explains in part the high genetic diversity of the parasite among all hosts at the study site.     

Life history of a malaria parasite (Plasmodium mexicanum): independent traits and basis for variation

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.     

Clonal diversity of a lizard malaria parasite, Plasmodium mexicanum, in its vertebrate host, the western fence lizard: role of variation in transmission intensity over time and space

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.     

Saurian malaria in Kenya: epidemiological features of malarial infections in lizard populations of the West Pokot District

During investigations into the prevalence of malarial parasites among lizards in the West Pokot District in Kenya, 179 lizards comprising eight species were caught. Examination of the Giemsa-stained smears made from their blood showed that 34 lizards were infected with Plasmodium species. Fifteen lizards were infected with a single species of Plasmodium and 19 carried multiple infections, the maximum, in four lizards, was four species. There were 19 combinations of parasite infections. Seventeen Plasmodium species were identified, the commonest being P. icipeensis. Only two of the eight lizard species were infected: the skink Mabuya striata and the agamid Agama agama. Eight of the Plasmodium species infected both; another eight species infected M. striata only but three of these have been described from different lizard families elsewhere in Africa. P. robinsoni infected A. agama only, although it was first described from another lizard family in another part of Africa. The epidemiological significance of these results is discussed.     

Manipulation of the vertebrate host's testosterone does not affect gametocyte sex ratio of a malaria 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.     

Two New Trypanosomes from California Toads and Lizards

SYNOPSIS Trypanosoma bufophlebotomi n. sp. and T. scelopori n. sp. are described from the California toad Bufo boreas halophilus and the western fence lizard Sceloporus occidentalis , respectively. T. bufophlebotomi occurred in 15% of 39 toads examined and is characterized by a juxtanuclear, bipolar-staining kinetoplast. The parasite may share a common ancestry with another sandfly-transmitted trypanosome of toads from China. T. scelopori occurred in 0.4% of 758 lizards examined for malarial parasites. It may also develop in sandflies.     

Life history of a malaria parasite (Plasmodium mexicanum) in its host, the western fence lizard (Sceloporus occidentalis): host testosterone as a source of seasonal and among-host variation?

The course of infection of a malaria parasite (Plasmodium mexicanum) is highly variable in its host, the fence lizard (Sceloporus occidentalis). However, a seasonal trend is superimposed on this variation such that gametocyte production is intensified during mid- to late summer. Host testosterone levels follow a similar seasonal fluctuation and are variable among individual lizards. We sought to determine if testosterone levels affect seasonal and among-host variation in 11 P. mexicanum life history traits: rate of increase in level of infection (3 measures), peak parasitemia (3 measures), duration of increase (3 measures), time to detectable infection, and timing of production of gametocytes. We followed the course of infection in 125 male S. occidentalis, each randomly assigned to 1 of 4 treatment groups: castrated, castrated and implanted with exogenous testosterone, sham implanted, and unmanipulated controls. Median values for the 11 life history traits did not differ among treatment groups, and variances were homogeneous among the treatment groups for 10/11 traits. However, elevated testosterone significantly reduced the variation in timing of the onset of gametocyte production. Therefore, testosterone does not appear to be a primary regulator of P. mexicanum life history, yet testosterone may have some effect on when gametocytes first become detectable.     

The Occurrence and Development of Plasmodium mexicanum in the Western Fence Lizard Sceloporus occidentalis

SYNOPSIS. From various localities in California, 81 (21%) of 381 Sceloporus occidentalis were found infected with Plasmodium mexicanum. Variations in asexual reproduction and host response during the acute period of development are discussed.
Appearance of initial infections in early March and the subsequent increase in natural incidence of the parasite, the absence of infections in hatchlings, and the persistence of numerous gametocytes in hibernating lizards suggest a period of spring transmission.     

Male gametocyte fecundity and sex ratio of a malaria parasite, Plasmodium mexicanum

Evolutionary theory predicts that the sex ratio of Plasmodium gametocytes will be determined by the number of gametes produced per male gametocyte (male fecundity), parasite clonal diversity and any factor that reduces male gametes' ability to find and combine with female gametes. Despite the importance of male gametocyte fecundity for sex ratio theory as applied to malaria parasites, few data are available on gamete production by male gametocytes. In this study, exflagellating gametes, a measure of male fecundity, were counted for 866 gametocytes from 26 natural infections of the lizard malaria parasite, Plasmodium mexicanum. The maximum male fecundity observed was 8, but most gametocytes produced 2-3 gametes, a value consistent with the typical sex ratio observed for P. mexicanum. Male gametocytes in infections with higher gametocytaemia had lower fecundity. Male fecundity was not correlated with gametocyte size, but differed among infections, suggesting genetic variation for fecundity. Fecundity and sex ratio were correlated (more female gametocytes with higher fecundity) as predicted by theory. Results agree with evolutionary theory, but also suggest a possible tradeoff between production time and fecundity, which could explain the low fecundity of this species, the variation among infections, and the correlation with gametocytaemia.     

Vectors of the filaria Thamugadia ivaschkini (Splendidofilariidae) in the southern Turkmen SSR

1903 specimens of sandflies and mosquitoes were examined for their spontaneous infection with larvae of Thamugadia ivaschkini. The experimental infection of sandflies and mosquitoes was conducted on infected lizards. It has been established that the vectors of this filaria in the south of Turkmenia are sandflies of the genus Phlebotomus and Sergentomyia arpaclensis. The development of the helminth carries out in thoracic muscles of sandflies, the first infective larvae appear in 7 to 17 days after the infection of vectors.     

Gametocyte sex ratio of a malaria parasite: experimental test of heritability

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.     

Malarial parasitism and male competition for mates in the western fence lizard,Sceloporus occidentalis

Summary The effect of malarial parasitism on the ability of male western fence lizards, Sceloporus occidentalis, to compete for access to females was assessed experimentally. Pairs of male lizards, one infected with the malarial parasite, Plasmodium mexicanum, and the other not infected, were matched by size and color and placed in large seminatural outdoor enclosures along with an adult female lizard. Infected males displayed to females and to other males less often than did noninfected male lizards. Noninfected lizards were dominant in social interactions more often than malarious animals, based on duration and intensity of agonistic encounters toward the other male, and time spent with the female. Thus, malarial infection hinders the ability of male fence lizards to compete for mates.     

The use of faecal counts for estimating populations of wall lizards (Podarcis muralis)

Measurements of the rates at which droppings of wall lizards ( Podarcis muralis ) on Jersey appeared in, and disappeared from, the environment made it possible to formulate a simple mathematical model for the dynamics of faecal pellet populations. The model shows how, for a given lizard population, the number of droppings changes with time according to weather conditions. It was used to estimate lizard densities at a number of sites from daily pellet counts. There were significant differences in the densities of lizards between sites; possible reasons for these are discussed.     

Absence of measurable malaria-induced mortality in western fence lizards (Sceloporus occidentalis) in nature: a 4-year study of annual and over-winter mortality

Theoretical models of parasite virulence often quantify virulence by mortality. However, there is a lack of empirical studies of parasite-induced host mortality because it is often difficult to quantify in natural populations. I have estimated annual and over-winter mortality in a population of fence lizards (Sceloporus occidentalis) infected with a malaria parasite, Plasmodium mexicanum, in northern California. The duration of time a lizard was observed (an estimate of life-span) throughout the 4-year observation period, or following winter, was not related to either infection status or maximum parasitemia. In contrast to previous laboratory studies of this parasite-host system, I found no evidence of parasite-induced host mortality in nature.     

Amplification of a New Region of DNA in an Unselected Laboratory Stock of L. tarentolae: The T Region

A new DNA amplification is described from an isolate of the lizard parasite Leishmania tarentolae . This DNA is present in up to 50 copies in the Trager line of this species and present but not amplified in all other lines tested. This amplification has been named the T amplification (for Tarentolae/Trager). Restriction enzyme digestion and electrophoresis of total DNA reveal amplified fragments totalling 19 kb following staining with ethidium bromide, a finding confirmed by the use of specific hybridization probes. Much of the amplified T DNA occurs as extra-chromosomal circular molecules. No cross-hybridization was observed between the T region and other amplified DNA of Leishmania , or the maxicircle of L. tarentolae , nor was resistance to methotrexate, chloroquine or primaquine detected in the T-amplified line. Combined with our previous results showing H region amplification in 2 other unselected lab stocks, these data demonstrate the prevalence of apparently spontaneous gene amplifications in L. tarentolae .     

Amplification of a new region of DNA in an unselected laboratory stock of L. tarentolae: the T region

A new DNA amplification is described from an isolate of the lizard parasite Leishmania tarentolae. This DNA is present in up to 50 copies in the Trager line of this species and present but not amplified in all other lines tested. This amplification has been named the T amplification (for Tarentolae/Trager). Restriction enzyme digestion and electrophoresis of total DNA reveal amplified fragments totalling 19 kb following staining with ethidium bromide, a finding confirmed by the use of specific hybridization probes. Much of the amplified T DNA occurs as extra-chromosomal circular molecules. No cross-hybridization was observed between the T region and other amplified DNA of Leishmania, or the maxicircle of L. tarentolae, nor was resistance to methotrexate, chloroquine or primaquine detected in the T-amplified line. Combined with our previous results showing H region amplification in 2 other unselected lab stocks, these data demonstrate the prevalence of apparently spontaneous gene amplifications in L. tarentolae.     

Prevalence of malaria parasites (Plasmodium floridense and Plasmodium azurophilum) infecting a Puerto Rican lizard (Anolis gundlachi): a nine-year study

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.