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The life history of a parasite describes its partitioning of assimilated resources into growth, reproduction, and transmission effort, and its precise timing of developmental events. The life cycle, in contrast, charts the sequence of morphological stages from feeding to the transmission forms. Phenotypic plasticity in life history traits can reveal how parasites confront variable environments within hosts. Within the protist phylum Apicomplexa major clades include the malaria parasites, coccidians, and most diverse, the gregarines (with likely millions of species). Studies on life history variation of gregarines are rare. Therefore, life history traits were examined for the gregarine Monocystis perplexa in its host, the invasive earthworm Amynthas agrestis at three sites in northern Vermont, United States of America. An important value of this system is the short life-span of the hosts, with only seven months from hatching to mass mortality; we were thus able to examine life history variation during the entire life cycle of both host and parasite. Earthworms were collected ( N  = 968 over 33 sample periods during one host season), then parasites of all life stages were counted, and sexual and transmission stages measured, for each earthworm. All traits varied substantially among individual earthworm hosts and across the sites. Across sites, timing of first appearance of infected earthworms, date when transmission stage (oocysts packed within gametocysts) appeared, date when number of both feeding (trophic) cells and gametocysts were at maximum, and date when 100% of earthworms were infected differed from 2-8 weeks, surprising variation for a short season available for parasite development. The maximal size of mating cells varied among hosts and across sites and this is reflected in the number of oocysts produced by the gametocyst. A negative trade-off was observed for the number of oocysts and their size. Several patterns were striking: (1) Prevalence reached 100% at all sites by mid season, only one to three weeks after parasites first appeared in the earthworms. (2) The number of parasites per host was large, reaching 300 × 10 3 cells in some hosts, and such high numbers were present even when parasites first appeared in the host. (3) At one site, few infected earthworms produced any oocysts. (4) The transmission rate to reach such high density of parasites in hosts needed to be very high for a microbe, from >0.33% to >34.3% across the three sites. Monocystis was one of the first protist parasites to have its life cycle described (early 19 th century), but these results suggest the long-accepted life cycle of Monocystis could be incomplete, such that the parasites may be transmitted vertically (within the earthworm's eggs) as well as horizontally (leading to 100% prevalence) and merogony (asexual replication) could be present, not recognized for Monocystis , leading to high parasitemia even very early in the host's season., Competing Interests: The authors declare there are no competing interests., (©2024 Keller and Schall.)

Apolocystis bosanqueti n. sp., a parasite of an important invasive earthworm in North America, Amynthas agrestis, is described from a site in northern Vermont. The earthworm host follows an annual life cycle in Vermont, so the entire life cycle of the parasite can be observed in 7 mo. In spring, the parasites were first seen in juvenile worms as paired gamonts (suggesting precocious association). These paired gamonts mature into gametocytes that form an opaque structure, with a thick gelatinous envelope (epicyst), that becomes full of zygotes. The resulting gametocyst becomes packed with ∼105 fusiform oocysts. The mature orbicular gametocysts are large (∼1 mm in diameter) and visible to the naked eye through the body wall of the host's anterior segments. The new species most resembles Apolocystis herculea described from many lumbricid earthworm species in Europe but differs from that parasite because Ap. herculea infects the intestinal wall in the posterior of the host rather than the anterior segments. A survey of 9 other earthworm species sympatric with Am. agrestis revealed that only Amynthas tokioensis, also an invasive species, was infected with Ap. bosanqueti, albeit much less commonly. Diagnosis for the family Monocystidae is problematic because cardinal characters are lacking, and the commonly cited character, a trophozoite with no anterior differentiation, is violated in most genera placed in the family. For the first time, a molecular phylogeny is presented that includes 3 genera of monocystids with diverse cell morphology (including the new species) and supports the monophyly of the family. The only morphological character that may be used to diagnose the Monocystidae is the morphology of oocysts, which are fusiform with extended terminal tips. A comparison of oocysts from 7 parasites recovered from local earthworms, including from 3 monocystid species in the phylogeny, confirms the utility of this diagnostic trait. The 2 hosts of the new species were most likely introduced from Japan, so the range of Apolocystis likely extends into East Asia., (© American Society of Parasitologists 2023.)

The invasive Asian earthworms, Amynthas tokioensis and A. agrestis , have been successful in entering North American forests in recent decades, with significant damage to both soils and above-ground environments. This success could be driven in part by a polyploid genetic system and parthenogenetic reproduction, often suggested as benefits for invasive species. Therefore, we assessed the genetic population structure, genetic diversity, and reproductive system of both species using morphological traits and panels of microsatellite markers. A total of 216 A. tokioensis and 196 A. agrestis from six sites in Vermont USA were analyzed. Although all worms were morphologically hermaphroditic, all the A. agrestis lacked the male pore (the structure allowing pass of sperm between individuals), and only 19% of the A. tokioensis possessed the male pore. All A. tokioensis earthworms were triploid (scored for three alleles for at least 1 locus, and usually several), and A. agrestis was a mix of triploid and diploid individuals. Notable was the high proportion (80%) of A. agrestis earthworms that were diploid at one site. There was clearly clonal reproduction, with identical seven- locus genotypes observed for earthworms from each site, with as many as 45 individuals with the identical genotype at one site. However, the earthworms were also genetically diverse, with 14 genotypes observed for A. tokioensis and 54 for A. agrestis , and with many singleton genotypes (a single individual). Most genotypes (71% for A. tokioensis and 92% for A. agrestis ) were found at a single site. The greatest number of genotypes was found at a commercial nursery where fully 23/26 A. agrestis earthworms were singleton genotypes. As expected for the pattern of private clone alleles at sites, several measures of geographic genetic differentiation were positive, and as expected for triploid systems, an AMOVA analysis showed high within-individual genetic diversity. The paradox of clear clonal reproduction, but with a great number of genotypes for each species, and the mix of triploid and diploid individuals could be explained if the worms have been sexually reproductive, with the switch to the uniparental system only recently (or even if sexual reproduction is episodic). Last, a large number of microsatellite loci were recovered for each species and there sequence and suggested PCR primers are provided for free use by other researchers., Competing Interests: The authors declare there are no competing interests., (©2022 Nouri-Aiin et al.)

Stomatocystis goerresi sp. n., a gregarine (phylum Apicomplexa, Monocystidae) parasite of an important invasive earthworm in North America, Amynthas tokioensis (Beddard), is described. This is the second species placed into the genus, and details of its morphology and life cycle support Stomatocystis Bandyopadhyay, Mitra et Göçmen, 2006 as a valid taxon. The new species is described using standard nomenclature, measurements, shape descriptors, and photographs of living cells. The parasite was found only in A. tokioensis, and absent in sympatric earthworm species, suggesting it arrived when the earthworms were introduced from their origin from Japan. The species is distinctive from the type species in the genus, S. indica Bandyopadhyay, Mitra et Göçmen, 2006, in being substantially larger in all stages, found in only the host's seminal vesicles, and found in a different host species from East Asia. The distinctive trophozoites/gamonts develop a large funnel structure ringed with a collar of pronounced ridges, and the funnel appears even in the smallest cells. This funnel varies greatly in relative size (to the cell body) and shape, sometimes forming a large fan. The life cycle of S. goerresi is described including distinctive syzygy in which the funnels fuse and then produce a large cell with local centres of isogamete production (thus sex without gender). Gametes are large ( ~5 μm) spheres with complex tips. Oocyst production is large, > 1,000 per mature gametocyst. The genus Stomatocystis is placed into the Monocystidae, but the life cycle of the new species differs from those of other monocystid taxa, which may mean the Monocystidae are not monophyletic or life cycles are variable within the family. Prevalence of S. goerresi at the type locality was high (~ 90%). The parasites destroy the earthworm's organ of sperm self-storage thus eliminating the male function in the hermaphroditic host which may influence the ability of the earthworm to invade and be successful at new sites.

Monocystis perplexa n. sp., a parasite of an important invasive Japanese earthworm in North America, Amynthas agrestis, is described from a site in Vermont. An improved standard for Monocystis species descriptions is proposed including a standard nomenclature to reduce synonymies, a standard set of biometrics and shape descriptions for living cells, and a DNA genomic sequence for the 18S rRNA (∼1,700 base pairs). Comparing morphologies of Monocystis parasites in sympatric earthworm species indicates that M. perplexa is specific to A. agrestis in the study region. Also, polymerase chain reaction primers specific to M. perplexa amplified samples of A. agrestis earthworms taken from several sites in Japan. This suggests the parasite entered North America from Japan, the origin of the invasive Amynthas earthworm, and thus M. perplexa would be the first Monocystis described from the diverse Japanese Amynthas earthworms and the first from East Asia. Monocystis perplexa was found in every population of A. agrestis surveyed in Vermont, always reaching 100% prevalence by late summer (the host has an annual life cycle in Vermont). The 18S gene sequence differed from that of Monocystis agilis from the sympatric earthworm Lumbricus terrestris (the only other sequence available for Monocystis), and a genetic similarity tree places them closest among other gregarines. Many of the 95 described species of Monocystis are very similar in morphology (based on species descriptions), so the 18S gene can act as a barcode for Monocystis species and thus will help to eliminate both synonymies and reveal cryptic species., (© American Society of Parasitologists 2020.)

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.

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.

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.

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., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

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.

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.

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.

Vertebrate hosts of malaria parasites (Plasmodium) often harbour two or more genetically distinct clones of a single species, and interaction among these co-existing clones can play an important role in Plasmodium biology. However, how relative clonal proportions vary over time in a host is still poorly known. Experimental mixed-clone infections of the lizard malaria parasite, Plasmodium mexicanum, were followed in its natural host, the western fence lizard using microsatellite markers to determine the relative proportions of two to five co-existing clones over time (2-3 months). Results for two markers, and two PCR primer pairs for one of those, matched very closely, supporting the efficacy of the method. Of the 54 infections, 67% displayed stable relative clonal proportions, with the others showing a shift in proportions, usually with one clone outpacing the others. Infections with rapidly increasing or slowly increasing parasitemia were stable, showing that all clones within these infections reproduced at the same rapid or slow rate. Replicate infections containing the same clones did not always reveal the same growth rate, final parasitemia or dominant clone; thus there was no clone effect for these life history measures. The rate of increase in parasitemia was not associated with stable versus unstable relative proportions, but infections with four to five clones were more likely to be unstable than those with two to three clones. This rare look into events in genetically complex Plasmodium infections suggests that parasite clones may be interacting in complex and unexpected ways., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

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.

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.

During spring-summer 2003-2004, the avian community was surveyed for hemosporidian parasites in an oak (Quercus spp.) and madrone (Arbutus spp.) woodland bordering grassland and chaparral habitats at a site in northern California, a geographic location and in habitat types not previously sampled for these parasites. Of 324 birds from 46 species (21 families) sampled (including four species not previously examined for hemosporidians), 126 (39%) were infected with parasites identified as species of one or more of the genera Plasmodium (3% of birds sampled), Haemoproteus (30%), and Leucocytozoon (11%). Species of parasite were identified by morphology in stained blood smears and were consistent with one species of Plasmodium, 11 species of Haemoproteus, and four species of Leucocytozoon. We document the presence of one of the parasite genera in seven new host species and discovered 12 new parasite species-host species associations. Hatching-year birds were found infected with parasites of all three genera. Prevalence of parasites for each genus differed significantly for the entire sample, and prevalence of parasites for the most common genus, Haemoproteus, differed significantly among bird families. Among families with substantial sample sizes, the Vireonidae (63%) and Emberizidae (70%) were most often infected with Haemoproteus spp. No evidence for parasite between-genus interaction, either positive or negative, was found. Overall prevalence of hemosporidians at the northern California sites and predominance of Haemoproteus spp. was similar to that reported in most other surveys for the USA, Canada, and the Caribbean islands.

Phylogenetic analysis of genomic data allows insights into the evolutionary history of pathogens, especially the events leading to host switching and diversification, as well as alterations of the life cycle (life-history traits). Hundreds, perhaps thousands, of malaria parasite species exploit squamate reptiles, birds, and mammals as vertebrate hosts as well as many genera of dipteran vectors, but the evolutionary and ecological events that led to this diversification and success remain unresolved. For a century, systematic parasitologists classified malaria parasites into genera based on morphology, life cycle, and vertebrate and insect host taxa. Molecular systematic studies based on single genes challenged the phylogenetic significance of these characters, but several significant nodes were not well supported. We recovered the first well resolved large phylogeny of Plasmodium and related haemosporidian parasites using sequence data for four genes from the parasites' three genomes by combining all data, correcting for variable rates of substitution by gene and site, and using both Bayesian and maximum parsimony analyses. Major clades are associated with vector shifts into different dipteran families, with other characters used in traditional parasitological studies, such as morphology and life-history traits, having variable phylogenetic significance. The common parasites of birds now placed into the genus Haemoproteus are found in two divergent clades, and the genus Plasmodium is paraphyletic with respect to Hepatocystis, a group of species with very different life history and morphology. The Plasmodium of mammal hosts form a well supported clade (including Plasmodium falciparum, the most important human malaria parasite), and this clade is associated with specialization to Anopheles mosquito vectors. The Plasmodium of birds and squamate reptiles all fall within a single clade, with evidence for repeated switching between birds and squamate hosts.

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.

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.

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.

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.

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.

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.

A phylogeny of haemosporidian parasites (phylum Apicomplexa, family Plasmodiidae) was recovered using mitochondrial cytochrome b gene sequences from 52 species in 4 genera (Plasmodium, Hepatocystis, Haemoproteus, and Leucocytozoon), including parasite species infecting mammals, birds, and reptiles from over a wide geographic range. Leucocytozoon species emerged as an appropriate out-group for the other malarial parasites. Both parsimony and maximum-likelihood analyses produced similar phylogenetic trees. Life-history traits and parasite morphology, traditionally used as taxonomic characters, are largely phylogenetically uninformative. The Plasmodium and Hepatocystis species of mammalian hosts form 1 well-supported clade, and the Plasmodium and Haemoproteus species of birds and lizards form a second. Within this second clade, the relationships between taxa are more complex. Although jackknife support is weak, the Plasmodium of birds may form 1 clade and the Haemoproteus of birds another clade, but the parasites of lizards fall into several clusters, suggesting a more ancient and complex evolutionary history. The parasites currently placed within the genus Haemoproteus may not be monophyletic. Plasmodium falciparum of humans was not derived from an avian malarial ancestor and, except for its close sister species, P. reichenowi, is only distantly related to haemospordian parasites of all other mammals. Plasmodium is paraphyletic with respect to 2 other genera of malarial parasites, Haemoproteus and Hepatocystis. Explicit hypothesis testing supported these conclusions.

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.

Anolis lizards from Puerto Rico (five species from one site), Curaçao and Aruba in the southern Caribbean (2 populations), and 22 populations from 14 islands in the eastern Caribbean were surveyed for blood parasites (two species of Plasmodium and haemogregarines). Literature records for gut helminths from nine of these populations were added to the data set. Dorsal body color and dewlap color of males were also observed and classified into objective classes with no subjective view of showiness. These data were used to test the among-species prediction of the Hamilton-Zuk hypothesis which states that species harboring more harmful parasites over their evolutionary history will be more likely to evolve extravagant sexually dimorphic traits. Critics have noted important shortcomings in previous tests of the prediction; here we corrected for these errors. Parasite loads (prevalence and number of species) and dorsal and dewlap color varied substantially among the populations sampled. However, there was no association of parasite load with color either in a broad analysis or when correcting for phylogenetic relationships among the lizard species.

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.

Plasmodium giganteum and P. agamae, parasites of the rainbow lizard, Agama agama, in West Africa were studied to determine the nature of any interspecific interactions between the two malaria species. The plasmodia are distributed in A. agama throughout the mesic zone of Africa; P. agamae is sometimes found as a solitary malaria species in populations of the lizard, but P. gigateum has not been found alone. In 3170 lizards from Sierra Leone the prevalence of lizard malaria at 22 sites varied considerably (8-90% of lizards were infected), but the ratio of the two species was similar among sites (52-91% P. agamae). Larger lizards were more often infected. Mixed infections occurred 2-5 times more often than expected by chance. Parasite density within individual hosts, or parasitemia, was similar for each species when alone or in mixed infection. Natural infections followed in laboratory lizards stayed at constant levels for as long as 211 days. The two species use different classes of host cells (P. giganteum in immature cells and P. agamae in mature erythrocytes) and may have different periods of peak transmission. Analysis of the data does not support a neutral relationship between P. giganteum and P. agamae, nor ongoing competition for resources or heterologous immunity. The data best support facilitation in which P. agamae alters the host in a way that allows more successful establishment of P. giganteum.

On many small Caribbean islands, two species of Anolis lizard coexist, but the two are typically very different in body size. The two Anolis of St. Maarten, however, are exceptional because they are similar in size and are known to be strongly competitive. One species, A. gingivinus, appears the stronger competitor and occurs throughout the island; the other, A. wattsi, is found only in the central hills. The malarial parasite Plasmodium azurophilum very rarely infects A. wattsi, but in some locations is very common in A. gingivinus. Wherever malaria infects A. gingivinus, A. wattsi is present, but wherever malaria is absent, only A. gingivinus occurs. This pattern of coincidence of malaria and coexistence of both Anolis is observed over distances of only a few hundred meters. The parasite infects both red and white blood cells of A. gingivinus and causes important pathology: immature erthrocytes increase in abundance, blood hemoglobin decreases, monocytes and neutrophils increase, and infected white cells are less likely to produce acid phosphatase. These results argue that malaria mediates competition between the two species and determines the present distribution of the lizards on St. Maarten. This kind of parasite-mediated competition could be common if susceptibility to parasitic infection varies among competitors. The distribution of malaria in the Anolis of Caribbean islands suggests this parasite can play an important role in Anolis community ecology.

The lizard malarias are a taxonomically and ecologically diverse group of parasites that offer excellent models for research on the ecology of malaria in free-ranging non-human vertebrate hosts. Studies over the past decade show that plasmodia of lizards can play an important role in the ecology and behavior of their hosts. The behavior of malarial infections in lizards also reveals unsuspected variation in the life history of Plasmodium.

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.

Geographic variation in the number of coexisting plant and animal species (species density) often follows repeated patterns; best known is the general increase in species richness from temperate to tropical latitudes. Here we undertake a quantitative analysis of geographic trends in species density for the terrestrial vertebrate faunas of the United States and Australia. Trends in numbers of species of amphibians, reptiles, birds, and mammals are described and are correlated with geographic variation in abiotic environmental measures. Intercontinental comparisons reveal general patterns as well as intriguing and profound differences in vertebrate distributions.

Hamilton and Zuk (1982) proposed that the quality of male showy traits reflects genetically-based resistance to parasites and can be used by females to select mates that are less prone to parasitic attack. The hypothesis requires that a particular state of a variable showy trait should be associated with parasite infection. We tested this idea with a population of western fence lizards, Sceloporus occidentalis, infected with the malarial parasite, Plasmodium mexicanum. Ventral color pattern is strongly dimorphic in fence lizards and varies greatly among males in this population. Malaria-infected males exhibited significantly more black and less pale on their ventral surface than did noninfected males of similar body size. This difference was not a function of differing ages of infected and noninfected animals of the same body size. However, logistic regression demonstrated that females using male ventral color as a gauge of infection status would only marginally improve their chance of choosing a noninfected lizard over random selection of mates.

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.