Characterization of the Malaria Pigment (Hemozoin) from the Avian Malaria Parasite Plasmodium lophurae*

SYNOPSIS. Plasmodium lophurae hemozoin (malaria pigment) is a heme-containing protein which is distinctly different from hemoglobin and hematin by immunologic, spectrophotometric, fingerprint, heme-iron, gel filtration, and starch gel electrophoretic analyses. The calculated average molecular weight of P. lophurae hemozoin is ca. 40,000. Hemozoin contains at least 3 antigenic components and shows some indication of cross-reaction with hemoglobin.     

Ovarian histology of the placentotrophic Mabuya mabouya (Squamata, Scincidae)

Ovarian structure and folliculogenesis of females at different reproductive stages are described for the viviparous placentotrophic lizard Mabuya mabouya. The small ovaries have a thin wall formed by the ovarian epithelium and a thin tunica albuginea. One to two germinal beds that contain numerous oogonia and developing primordial follicles are derived from the ovarian epithelium and are next to the ovarian hilum. The ovarian cortex contains follicles at different stages of development, corpora lutea, and atretic follicles. The yolk nucleus and Balbiani complex were not evident in the ooplasm of previtellogenic follicles. The follicular epithelium of these follicles is polymorphic, as in other species of Squamata, but the larger cells are spherical and monolayered rather than pyriform. The zona radiata of the preovulatory follicles is less developed than in lecithotrophic species. These features suggest a decrease in metabolic and absorptive processes during follicular growth. In preovulatory follicles (1.5-1.8 mm diameter), primordial yolk vacuoles and small cortical granules are deposited in the ooplasm instead of fatty yolk platelets, so that only one stage of vitellogenesis is observed. Polyovular atretic follicles occur in some females. Follicular atresia is minimal for preovulatory follicles, but is more frequent in follicles with polymorphic epithelia. In the corpus luteum, the luteal tissue is formed from granulosa cells and luteolysis occurs during the late gastrula -- late neurula embryonic stages. Thus, the maintenance of gestation from the pharyngula to preparturition stages seems to be related to secretion of extraluteal progesterone, possibly of placental origin. These observed ovarian features are related to the high degree of placental complexity of this species and show that the evolution of advanced placentotrophy in species of this lineage has been accompanied by concomitant changes in ovarian function.     

A New Method for Intraerythrocytic Cultivation of Malaria Parasites (Plasmodium coatneyi and P. falciparum)

SYNOPSIS. Infected blood (from the rhesus monkey with P. coatneyi or an Aotus trivirgatus monkey with P. falciparum) was placed in a special vial equipped with an overflow tube 2–3 mm above the bottom of the vial. A slow flow (50 ml per day) of culture medium was maintained over the settled layer of red cells. In favorable experiments both species of parasites went thru 2 cycles of development without addition of fresh erythrocytes. so that late schizonts and young rings were present after 4 as well as after 2 days of incubation. There was, however, no increase in total parasite number.     

Morphogenesis of extraembryonic membranes and placentation in Mabuya mabouya (Squamata, Scincidae)

Topological and histological analyses of Mabuya mabouya embryos at different developmental stages showed an extraembryonic membrane sequence as follows: a bilaminar omphalopleure and progressive mesodermal expansion around the whole yolk sac at gastrula stages; mesodermal split and formation of an exocoelom in the entire embryonic chamber at neurula stages; beginning of the expansion of the allantois into the exocoelom to form a chorioallantoic membrane at pharyngula stages; complete extension of the allantois into the exocoelom between limb-bud to preparturition stages. Thus, a placental sequence could be enumerated: bilaminar yolk sac placenta; chorioplacenta; allantoplacenta. All placentas are highly specialized for nutrient absorption from early developmental stages. The bistratified extraembryonic ectoderm possesses an external layer with cuboidal cells and a microvillar surface around the whole yolk sac, which absorbs uterine secretions during development of the bilaminar yolk sac placenta and chorioplacenta. During gastrulation, with mesodermal expansion a dorsal absorptive plaque forms above the embryo and several smaller absorptive plaques develop antimesometrially. Both structures are similar histologically and are active in histotrophic transfer from gastrula stages until the end of development. The dorsal absorptive plaque will constitute the placentome and paraplacentome during allantoplacental development. At late gastrula-early neurula stages some absorptive plaques form chorionic concavities or chorionic bags that are penetrated by a long uterine fold and seem to have a specialized histotrophic and/or metabolic role. The extraembryonic mesoderm does not ingress into the yolk sac and neither an isolated yolk mass nor a yolk cleft are formed. This derived pattern of development may be related to the drastic reduction of the egg size and obligatory placentotrophy from early developmental stages. Our results show new specialized placentotrophic structures and a novel arrangement of extraembryonic membrane morphogenesis for Squamata.     

Identification of Plasmodium malariae,a Human Malaria Parasite,in Imported Chimpanzees

It is widely believed that human malaria parasites infect only man as a natural host. However, earlier morphological observations suggest that great apes are likely to be natural reservoirs as well. To identify malaria parasites in great apes, we screened 60 chimpanzees imported into Japan. Using the sequences of small subunit rRNA and the mitochondrial genome, we identified infection of Plasmodium malariae, a human malaria parasite, in two chimpanzees that were imported about thirty years ago. The chimpanzees have been asymptomatic to the present. In Japan, indigenous malaria disappeared more than fifty years ago; and thus, it is most likely inferred that the chimpanzees were infected in Africa, and P. malariae isolates were brought into Japan from Africa with their hosts, suggesting persistence of parasites at low level for thirty years. Such a long term latent infection is a unique feature of P. malariae infection in humans. To our knowledge, this is the first to report P. malariae infection in chimpanzees and a human malaria parasite from nonhuman primates imported to a nonendemic country.     

Systematics and biogeography of the Neotropical genus Mabuya,with special emphasis on the Amazonian skink Mabuya nigropunctata (Reptilia,Scincidae)

Phylogenetic analyses using up to 1532 base pairs (bp) of mitochondrial DNA from 106 specimens of Neotropical Mabuya, including 18 of the 19 recognized South American and Mesoamerican species, indicate that most species of the genus are monophyletic, including M. nigropunctata that had previously been reported to be paraphyletic. The present results shows that this species includes three highly divergent and largely allopatric lineages restricted to occidental, meridional, and oriental Amazonia. Our dataset demonstrates that previous claims regarding the paraphyletic status of M. nigropunctata and the phylogenetic relationships within this species complex based on the analysis of three mitochondrial and four nuclear genes (approx. 5000 bp) were erroneous and resulted from two contaminated cytochrome b sequences.The phylogenetic results indicate that diversification in the Neotropical genus Mabuya started approximately in the Middle Miocene (15.5–13.4 Ma). The divergence dates estimated for the Mabuya nigropunctata species complex suggest that the major cladogenetic events that produced the three main groups (occidental (oriental + meridional)) occurred during the Late Miocene. These estimations show that diversification within the M. nigropunctata species complex was not triggered by the climatic changes that occurred during the Pleistocene, as has been suggested by several authors. Rather, our data support the hypothesis that the late tertiary (essentially Miocene epoch) was a period that played a very important role in the generation of biological diversity in the Amazonian forests.Speciation between Mabuya carvalhoi, endemic to the coastal mountain range of Venezuela, and M. croizati, restricted to the Guiana Shield, occurred during the Middle Miocene and may have been as the result of a vicariant event produced by the formation of the present day Orinoco river drainage basin and the consequent appearance of the Llanos del Orinoco, which acted as a barrier to dispersal between these two species. The split between M. bistriata and M. altamazonica and between the occidental and (meridional + oriental) clades of M. nigropunctata fits very well with the biogeographic split between the eastern and western Amazon basins reported for several other taxa.     

The biology and biomass of the skinks Mabuya sechellensis and Mabuya wrightii on Cousin Island, Seychelles (Reptilia: Scincidae)

The skinks Mabuya sechellensis and M. wrightii scavenged among the seabird colonies of Cousin Island. particularly under the colony of Lesser noddies Anous tenuirostris which, in 1979, contained about 60.000 pairs. The density of skinks in different areas of the island did not vary greatly through the year. The mean density of M. sechellensis was 1393 individuals/ha equivalent to a biomass of 21.7 kg/ha and of M. wrightii 320 individuals/ha equivalent to 17.7 kg/ha. These biomass figures are the highest recorded for any terrestrial lizard population. Skink and Lesser noddy density were closely correlated. The skinks deposited fat equivalent to up to about 50% of their lean dry weight during the noddy nesting season but were leaner and lighter at other times. Our calculations of the amount of skink food emanating from the noddy colonies, in the form of faeces, dropped fish and eggs suggest that there is sufficient to maintain a skink population of the density observed.
The skinks bred throughout the year, the larger M. wrightii more frequently than the smaller M. sechellensis , and the young/adult ratio was higher in M. wrigh:htii. The possible relevance of these differences to the two species' coexistence is discussed. The young/adult ratio varied according to skink density; in both species few young were found at high density.
Mean home range sizes of M. sechellensis and M. wrightii were 26·6 and 78· m², respectively.     

A Malaria Parasite, Plasmodium aurulentum sp. nov. from the Neotropical Forest Gecko Thecadactylus rapicaudus

SYNOPSIS. Plasmodium aurulentum sp. nov. from the neotropical forest gecko Thecadactylus rapicaudus in Panama is characterized by oval or round to lenticular gametocytes, 6–22 nuclei in crudely fan-shaped schizonts, and light golden pigment masses. A prominent, pinkish red-staining mass, present in older schizonts, disappears by the time schizonts reach maturation.     

Systematics of Mabuya frenata cochabambae Dunn, 1935 (Reptilia,Sauria, Scincidae) from Inner-Andean Dry Valleys of Bolivia

A combined analysis of 523 bp and 402 bp sequences of the mitochondrial 16S and 12S rRNA genes, respectively, suggests Mabuya frenata cochabambae being regarded a full species, M. cochabambae Dunn, 1935. It is phylogenetically closely related to M. dorsivittata rather than to M. frenata. Mabuya cochabambae is only known from inner-Andean dry valleys of central Bolivia, Departments Cochabamba and Santa Cruz, at elevations of 1300–3800m above sea level.     

Asexual and Sexual Stages of a Malaria Parasite in the Thrombocytes of Tropidurus torquatus (Iguanidae) Infected with Plasmodium tropiduri*

SYNOPSIS. Sexual and asexual stages of a parasite found in the thrombocyte-like cells of some Tropidurus torquatus infected with Plasmodium tropiduri are described. The strong ultrastructural similarities between the gametocytes of this parasite and the gametocytes of P. tropiduri, and the finding of this parasite in lizards inoculated with P. tropiduri suggest that this malaria parasite can develop both in erythrocytes and thrombocytes. Evidence in favor of this hypothesis is discussed.     

Phylogenetic relationships of Australian skinks of the Mabuya group (Reptilia: Scincidae) inferred from mitochondrial DNA sequences

Portions of two mitochondrial genes (12S and 16S ribosomal RNAs) were sequenced to analyze the phylogenetic relationships of the Mabuya group from the Australian region (Corucia, Egernia and Tiliqua). Results indicated the monophyly of these genera and their divergence from Asian and African members of this group. This suggests that the diversity of the Mabuya group in the Australian region has increased through an endemic radiation, not through multiple colonizations from outside. Among the genera from this region, Corucia and Tiliqua were closest to each other. This result contradicts with those of the previous hypotheses on the basis of morphological and immunological data that, respectively, suggested closest affinities between Corucia and Egernia, and Egernia and Tiliqua. We suppose that the morphological characters exclusively joining Corucia and Egernia are actually in plesiomorphic state.     

Seasonal spermatogenic cycle and morphology of germ cells in the viviparous lizard Mabuya brachypoda (Squamata,Scincidae)

We describe seasonal variations of the histology of the seminiferous tubules and efferent ducts of the tropical, viviparous skink, Mabuya brachypoda, throughout the year. The specimens were collected monthly, in Nacajuca, Tabasco state, Mexico. The results revealed strong annual variations in testicular volume, stages of the germ cells, and diameter and height of the epithelia of seminiferous tubules and efferent ducts. Recrudescence was detected from November to December, when initial mitotic activity of spermatogonia in the seminiferous tubules were observed, coinciding with the decrease of temperature, photoperiod and rainy season. From January to February, early spermatogenesis continued and early primary and secondary spermatocytes were developing within the seminiferous epithelium. From March through April, numerous spermatids in metamorphosis were observed. Spermiogenesis was completed from May through July, which coincided with an increase in temperature, photoperiod, and rainfall. Regression occurred from August through September when testicular volume and spermatogenic activity decreased. During this time, the seminiferous epithelium decreased in thickness, and germ cell recruitment ceased, only Sertoli cells and spermatogonia were present in the epithelium. Throughout testicular regression spermatocytes and spermatids disappeared and the presence of cellular debris, and scattered spermatozoa were observed in the lumen. The regressed testes presented the total suspension of spermatogenesis. During October, the seminiferous tubules contained only spermatogonia and Sertoli cells, and the size of the lumen was reduced, giving the appearance that it was occluded. In concert with testis development, the efferent ducts were packed with spermatozoa from May through August. The epididymis was devoid of spermatozoa by September. M. brachypoda exhibited a prenuptial pattern, in which spermatogenesis preceded the mating season. The seasonal cycle variations of spermatogenesis in M. brachypoda are the result of a single extended spermiation event, which is characteristic of reptilian species. J. Morphol. © 2012 Wiley Periodicals, Inc.     

Polymorphism at the Apical Membrane Antigen 1 Gene (AMA1) of the Malaria Parasite Plasmodium falciparum in a Vietnamese Population

The patterns of molecular evolution of the most diverse region of the apical membrane antigen 1 (AMA1) gene in Plasmodium falciparum from a Vietnamese subpopulation (Bao Loc) were investigated. Within the Bao Loc population, the sequenced gene region showed relatively high allelic and nucleotide diversity (0.985 and 0.02694, respectively). Further, the level of population recombination was substantial, resulting in a significant decay of linkage disequilibrium along the gene region. The results suggest that AMA1 is a useful genetic marker for studying the relationships between adaptation of parasite populations (to the human host immune system) and malaria epidemiology.     

Investigating the origin of transoceanic distributions: Mtdna shows Mabuya lizards (Reptilia,Scincidae) crossed the Atlantic twice

Phylogenies with even a rough time scale can be used to investigate the history of non‐volant taxa with disjunct distributions in widely separated land areas that were once connected. Basic methods for doing this are discussed. A partial phylogeny of Mabuya based on mtDNA (305 bp cytochrome b, 379 bp 12S rRNA and 388 bp 16S rRNA) is used to show that this genus invaded tropical America from Africa twice in the last 9 Myr, once reaching the American mainland and once the oceanic island of Fernando de Noronha, two journeys each of at least 3000 km. In general, phylogenetic evidence for multiple invasions is less equivocal than that suggesting a single invasion, which is more prone to sampling artefacts. Two alternative hypotheses explaining the presence of Mabuya in both Africa and tropical America are refuted on the basis of molecular clock considerations, namely that the occurrence of Mabuya in these continents pre‐dated their separation over 100 My ago and that it was introduced from one continent to the other by human activities. Like several other lizard groups that have made successful long‐distance transmarine colonizations, Mabuya has done this on many occasions. Phylogenetic results are also compatible with a SE Asian or Australasian origin of Mabuya followed by westward expansion.     

Diet of the lizard Mabuya agilis (Sauria; Scincidae) in an insular habitat (Ilha Grande, RJ, Brazil).

We examined the stomach contents of 21 specimens of Mabuya agilis (Sauria; Scincidae) collected during February 2001 at the restinga habitat of Praia do Sul, in Ilha Grande, RJ, Brazil. Diet was composed of various types of small arthropods, with no plant material being eaten. Spiders were the most important items in the diet, followed by orthopterans. Apart from the absence of isopterans, the diet of Mabuya agilis from this insular area was similar to those of other conspecific mainland populations. This suggests that factors such as insularity and the absence of other small sympatric lizards may not have a significant effect on the feeding habits of the Praia do Sul population.     

Ultrastructure of the ovarian follicles in the placentotrophic Andean lizard of the genus Mabuya (Squamata: Scincidae)

We studied the ultrastructural organization of the ovarian follicles in a placentotrophic Andean lizard of the genus Mabuya. The oocyte of the primary follicle is surrounded by a single layer of follicle cells. During the previtellogenic stages, these cells become stratified and differentiated in three cell types: small, intermediate, and large globoid, non pyriform cells. Fluid‐filled spaces arise among follicular cells in late previtellogenic follicles and provide evidence of cell lysis. In vitellogenic follicles, the follicular cells constitute a monolayered granulosa with large lacunar spaces; the content of their cytoplasm is released to the perivitelline space where the zona pellucida is formed. The oolemma of younger oocytes presents incipient short projections; as the oocyte grows, these projections become organized in a microvillar surface. During vitellogenesis, cannaliculi develop from the base of the microvilli and internalize materials by endocytosis. In the juxtanuclear ooplasm of early previtellogenic follicles, the Balbiani's vitelline body is found as an aggregate of organelles and lipid droplets; this complex of organelles disperses in the ooplasm during oocyte growth. In late previtellogenesis, membranous organelles are especially abundant in the peripheral ooplasm, whereas abundant vesicles and granular material occur in the medullar ooplasm. The ooplasm of vitellogenic follicles shows a peripheral band constituted by abundant membranous organelles and numerous vesicular bodies, some of them with a small lipoprotein core. No organized yolk platelets, like in lecithotrophic reptiles, were observed. Toward the medullary ooplasm, electron‐lucent vesicles become larger in size containing remains of cytoplasmic material in dissolution. The results of this study demonstrate structural similarities between the follicles of this species and other Squamata; however, the ooplasm of the mature oocyte of Mabuya is morphologically similar to the ooplasm of mature oocytes of marsupials, suggesting an interesting evolutionary convergence related to the evolution of placentotrophy and of microlecithal eggs. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

On the Diversity of Malaria Parasites in African Apes and the Origin of Plasmodium falciparum from Bonobos

The origin of Plasmodium falciparum, the etiological agent of the most dangerous forms of human malaria, remains controversial. Although investigations of homologous parasites in African Apes are crucial to resolve this issue, studies have been restricted to a chimpanzee parasite related to P. falciparum, P. reichenowi, for which a single isolate was available until very recently. Using PCR amplification, we detected Plasmodium parasites in blood samples from 18 of 91 individuals of the genus Pan, including six chimpanzees (three Pan troglodytes troglodytes, three Pan t. schweinfurthii) and twelve bonobos (Pan paniscus). We obtained sequences of the parasites'' mitochondrial genomes and/or from two nuclear genes from 14 samples. In addition to P. reichenowi, three other hitherto unknown lineages were found in the chimpanzees. One is related to P. vivax and two to P. falciparum that are likely to belong to distinct species. In the bonobos we found P. falciparum parasites whose mitochondrial genomes indicated that they were distinct from those present in humans, and another parasite lineage related to P. malariae. Phylogenetic analyses based on this diverse set of Plasmodium parasites in African Apes shed new light on the evolutionary history of P. falciparum. The data suggested that P. falciparum did not originate from P. reichenowi of chimpanzees (Pan troglodytes), but rather evolved in bonobos (Pan paniscus), from which it subsequently colonized humans by a host-switch. Finally, our data and that of others indicated that chimpanzees and bonobos maintain malaria parasites, to which humans are susceptible, a factor of some relevance to the renewed efforts to eradicate malaria.     

Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum

Apicoplasts are vestigial plastids in apicomplexan parasites like Plasmodium, the causative agent of malaria. Apicomplexan parasites are dependant on their apicoplasts for synthesis of various molecules that they are unable to scavenge in sufficient quantity from their host, which makes apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs) are embedded in the outer apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer apicoplast membrane of the human malaria parasite P. falciparum. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer apicoplast membrane.