Jos. J. Schall

A MOLECULAR PHYLOGENY OF MALARIAL PARASITES RECOVERED FROM CYTOCHROME b GENE SEQUENCES

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.

Parasite-mediated competition in Anolis lizards

SummaryOn 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.

Morphological versus molecular identification of avian Haemosporidia: an exploration of three species concepts

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 parasites 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.

Prevalence of a malarial parasite over time and space: Plasmodium mexicanum in its vertebrate host, the western fence lizard Sceloporus occidentalis

1. We studied patterns of abundance of the malarial parasite Plasmodium mexicanum in its vertebrate host, the western fence lizard Sceloporus occidentalis, over a 13-year period at 51 study sites in northern California, USA. Abundance of the vectors, sandflies in the genus Lutzomyia, was also studied among sites during a single warm season, and among nights at one site during two seasons. 2. The parasite differed in prevalence (per cent of lizards infected) in males and females; males were more often infected. Prevalence increased with body size (= older lizards were more often infected). 3. Malaria prevalence varied among sites: 0-50% of lizards were infected. Topography explains part of this variation because malaria was rare or absent at sites. > 500 m elevation. However, sites at lower elevations, even when within short distances of one another, varied in malaria prevalence. 4. Abundance of vectors was not related to prevalence of malaria at a site; sandflies were found at some sites where malaria was rare or absent, including those at high elevations. Wind speed, relative humidity and sky brightness did not affect numbers of sandflies active, but vectors were active only when air temperature was > 16°C. 5. Parasite prevalence varied among years. Environmental conditions (temperature, rainfall and plant biomass produced at the study region) were not correlated with parasite prevalence. The changes in prevalence over time resembled a cycle of long duration (10 years). 6. Classical models developed for study of malaria in humans suggest explanations for variation in prevalence of P. mexicanum among sites, and a more recent theory suggests an explanation for the possible long-duration cycle observed in this study

Testing Models of Optimal Diet Assembly by the Generalist Herbivorous Lizard Cnemidophorus Murinus

The diet of the predominantly herbivorous Bonaire Island whiptail lizard (Teiidae: Cnemidophorus murinus) was examined to assess three models of diet selection by generalist herbivores. These models were: single-nutrient maximizing, toxin avoiding, and nutritional wisdom. At three sites we gathered data on the diet of the lizard over a full year (both wet and dry seasons), on the relative abundance of all plant food types available to the lizards during that period, and on the nutritional composition of each plant type. Thirteen nutritional variables were measured, including content of energy, protein, minerals, water, and potentially toxic plant secondary compounds, and digestibility of protein. The lizards were generalist feeders, consuming a wide variety of flowers, fruits, leaves, nectar, and some animal material. Most stomachs contained more than one food type, suggesting C. murinus typically samples several kinds of plant materials each day. The lizards were selective with regard to foods eaten; most foods were not taken in proportion to their availability in the environment. Potential plant foods varied in nutritional quality, but no one nutrient was correlated with dietary preferences. Multivariate analysis revealed that preference or avoidance of a potential food type could be predicted by a combination of nutritional properties, but these differed among sites. Despite differing plant assemblages at each site, annual intake of nutrients by the lizards was similar among sites. The results best support the nutritional wisdom hypothesis; the Bonaire whiptail lizard may assemble its diet to obtain the proper balance of required nutrients, while avoiding dangerous levels of plant secondary compounds.

Parasites and showy males: malarial infection and color variation in fence lizards

SummaryHamilton 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.

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.

The sex ratio of Plasmodium gametocytes

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.

Lizard malaria: cost to vertebrate host's reproductive success

Plasmodium mexicanum is a common malarial parasite of the western fence lizard, Sceloporus occidentalis , in northern California, USA. Infected female lizards store substantially less fat during the summer activity season and produce smaller clutches of eggs than do non-infected animals. Stored fat is utilized in the production of eggs; the energy content of the decrement in stored fat is approximately equal to the energy content of the average reduction in number of eggs. Thus, there is ongoing strong selective pressure on the host to evolve appropriate anti-parasite measures.

Virulence of lizard malaria: the evolutionary ecology of an ancient parasite-host association.

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 parasites population growth was unaffected by experimental alterations in the lizards 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.