Hepatozoon cf. terzii (Sambon & Seligman, 1907) infection in the snake boa constrictor constrictor from north Brazil: transmission to the mosquito Culex quinquefasciatus and the lizard Tropidurus torquatus

Specimens of Hepatozoon-infected Boa constrictor constrictor were obtained from localities in Pará State, north Brazil. Gametocytes in erythrocytes of the peripheral blood measured 10 x 2.5-16.2 x 3.7 microns. They were similar to those described as Haemogregarina terzii by Sambon & Seligmann (1907) in B. c. constrictor, in that they did not distort the infected erythrocyte, and their dimensions approximated those given by Carini (1947). Lungs and liver of infected snakes contained actively dividing meronts of a single type, and cysts containing two to six cystozoites were also present in the liver. Our initial feeding of Culex quinquefasciatus on infected snakes consistently resulted in a heavy death-rate of the engorged mosquitoes, with only a few surviving till the 9th day post feeding. These contained numerous oocysts which were undivided or in early stages of division. A fifth and final experiment, however, provided a few mosquitoes surviving up to 21 days post infection (dpi), and these contained fully sporulated oocysts measuring 190-200 microns in diameter and containing over 60 sporocysts of 19-30 microns in diameter. The number of sporozoites in each sporocyst was estimated as approximately 50. The nature of the parasite's sporogonic cycle in the mosquito thus justifies inclusion of this haemogregarine in the genus Hepatozoon. Two wild-caught specimens of the lizard Tropidurus torquatus were fed with mosquitoes containing fully developed oocysts (21 dpi). When sacrificed, three months later, large numbers of dizoic, tetrazoic and hexazoic cysts were demonstrated in their livers. Cystozoites released from these cysts were shown to possess a conspicuous refractile body.     

Ultrastructural Features of Cystic and Merogonic Stages of Hepatozoon sipedon (Apicomplexa: Adeleorina) in Northern Leopard Frogs (Rana pipiens) and Northern Water Snakes (Nerodia sipedon) from Ontario, Canada

The cystic and merogonic stages of the haemogregarine Hepatozoon sipedon , infecting Northern water snakes ( Nerodia sipedon sipedon ) and Northern leopard frogs ( Rana pipiens ), respectively, in Ontario, Canada, were investigated by transmission electron microscopy. Cysts, which were observed in the liver of Northern leopard frogs ( Rana pipiens ) after these anurans ingested mosquitoes ( Culex pipiens ) containing oocysts of the parasite, harboured two cystozoites, each of which contained a large crystalloid inclusion anterior to the nucleus. Two types of meronts were observed in snakes that were fed the liver of infected frogs. Macromeronts, which matured in endothelial cells of the liver approximately 16 d after snakes ingested infected frogs, contained about 50 large macromerozoites. Macromerozoites emerged from macromeronts, entered the bloodstream of the snake, and reinfected endothelial cells. Micromeronts, which matured about 34 d post-inoculation, contained about 150 micromerozoites that infected erythrocytes and transformed into gamonts. The ultrastructural features of micromeronts and macromeronts differed only slightly: immature macromeronts and macromerozoites contained numerous amylopectin and lipid inclusions, whereas immature micromeronts and micromerozoites did not contain amylopectin inclusions and featured fewer, smaller lipid inclusions. A comparison of cystic stages among Hepatozoon species in different groups of vertebrates is presented with respect to their structure and evolutionary significance.     

Malaria-induced reduction of fecundity during the first gonotrophic cycle of Anopheles Stephensi mosquitoes

Abstract. Anopheles stephensi mosquitoes which had fed upon mice infected with Plasmodium yoelii nigeriensis malaria parasites produced significantly fewer eggs than mosquitoes fed on an uninfected mouse. Fecundity reduction was more pronounced when the bloodmeal contained malaria gametocytes and the mosquitoes developed oocysts. Egg production and haematin excretion were correlated for uninfected bloodfed mosquitoes; the presence of P.y. nigeriensis in the blood affected this relationship. Reduced fecundity was associated with a significant reduction of bloodmeal size (measured by haematin excretion) in mosquitoes which ingested gametocytaemic blood. The bloodmeal size in mosquitoes fed on parasitaemic blood without gametocytes was not significantly reduced. The use of haematin assays for determination of bloodmeal size in mosquitoes is discussed.     

Plasmodium gallinaceum: Erythrocyte factor essential for zygote infection of Aedes aegypti

Zygotes of Plasmodium gallinaceum, fertilized in vitro and fed to Aedes aegypti mosquitoes through a membrane, formed oocysts only when a substance in the cytoplasm of uninfected erythrocytes was present. The relation between erythrocyte volume and infectivity was linear (1:1.2) up to a 50% hematocrit. The intraerythrocytic substance was both nondialyzable and poorly soluble in plasma. By carboxymethyl cellulose chromatography, cytoplasmic constituents eluted at pH 8.6 supported the same infection as control blood did; but higher and lower pH eluates supported none. Dialyzable factors present in the plasma, but absent from M199, enhanced infection but were not essential. Zygotes developed normally to ookinetes in the gut of plasma-fed mosquitoes, or when cultured in plasma or M199. Ookinetes from culture formed normal oocysts when fed to mosquitoes in blood or when injected with M199 into the hemocoels of unfed females. Mosquitoes fed infected blood containing lima bean or soybean trypsin inhibitor were unable to digest the erythrocytes and, although normal ookinetes developed, no oocysts formed. It appears from this and histological evidence that an erythrocyte substance, released by mosquito digestion, is needed for ookinete invasion of the gut epithelium.     

Plasmodium cynomolgi: the comparative infectiousness of individual rhesus monkeys

Five rhesus monkeys were infected with the malaria parasite Plasmodium cynomolgi Mayer. Anopheles stephensi Liston mosquitoes were fed on each monkey over the period of prepremunitive gametocytemia. Individual monkeys did not differ significantly in either mean daily gametocyte count (median = 1300 gametocytes per mosquito blood meal volume per day) or mean daily oocyst production (median = 34 oocysts per mosquito per day). Significant differences among monkeys in daily oocyst/gametocyte conversion ratio were attributable to essentially random correlation effects. The observed range in duration of the period of prepremunitive gametocytemia was 14–43 days. Total oocyst production over this period, as calculated for a unit mosquito biting rate of one per day, ranged from 130 to 2800 oocysts. The overall efficiency of conversion of gametocytes to oocysts in A. stephensi was estimated at 0.02 oocysts per gametocyte.     

Gametogenesis and Sporogony of Hepatozoon Mocassini (Apicomplexa: Adeleina: Hepatozoidae) In an Experimental Mosquito Host, Aedes Aegypti

ABSTRACT. The sexual life cycle of the hemogregarine Hepatozoon mocassini was studied in Aedes aegypti , an experimental mosquito host, using transmission electron microscopy. Gamonts were observed leaving the host snake erythrocyte as early as 30 min after mosquitoes ingested infected blood, and some gamonts had penetrated the gut epithelial cells by this time. Six hours post-feeding, gamonts were identified within cells of the abdominal fat body. Twenty-four hours post-feeding, gamonts were often entrapped within the peritrophic membrane, but were no longer observed within the gut wall. Parasites pairing up in syzygy and undergoing sexual differentiatioe were observed within fat cells at this time, and by 48 hours post-feeding, well-developed macro- and microgametocytes as well as microgametes were discernible. Developing zygotes observed 3 days post-feeding were enclosed within a panoitophorous vacuole. By day 6, multinucleate oocysts with crystalloid bodies in the cytoplasm were seen. Sporazoites developing within sporocysts appeared by day 12. Seventeen days post-feeding, mature oocysts with sporocysts containing approximately 16 sporozoites were observed upon dissection of mosquitoes. Large crystalloid bodies no longer bound by rough endopbsmic reticulum were located anterior and posterior to the sporozoite nucleus. Free sporozoites were not observed.     

Resistance of early midgut stages of natural Plasmodium falciparum parasites to high temperatures in experimentally infected Anopheles gambiae (Diptera: Culicidae)

We studied the effects of high temperature, 30 and 32 versus 27 C on early Plasmodium falciparum development in Anopheles gambiae experimentally infected with gametocytes from 30 volunteers with mean density of 264.1 gametocytes/microl blood (range: 16-1,536/microl). From several batches of mosquitoes, fed by membrane feeding, midguts of individual mosquitoes were dissected at 24 hr for ookinete enumeration and at 7 days to quantify oocysts. There were temperature-related differences in mean ookinete intensity per mosquito midgut, with 9.71 +/- 1.6 at 27 C, 9.85 +/- 2.32 at 30 C, and 3.89 +/- 0.81 at 32 C. The prevalence of oocyst infection decreased with an increase in temperatures from 15.9 to 8.5 to 6.4% at 27, 30, and 32 C, respectively. The average oocyst intensities for the infected mosquitoes increased with temperatures from 2.9 at 27 C to 3.5 at 30 C, and to 3.3 at 32 C. However, the success of infections was reduced at 30 and 32 C, and resulted in greater losses during consecutive inter-stage parasite development. The most significant impact of high temperatures occurred at the transition between macrogametocytes and ookinetes, whereas the transition between ookinetes and oocysts apparently was not affected. In contrast to other reports, exposure of mosquitoes infected with natural parasites to high temperatures did not eliminate preoocyst stages, as has been observed from laboratory studies using the NF-54 strain of P. falciparum. This observation of parasite resistance to high temperatures is consistent with the natural situation in tropical environments where perennial malaria transmission occurs during hot dry seasons.     

Comparative studies on the infectivity of Plasmodium berghei gametocytes and ookinetes for gnotobiotic and xenobiotic Anopheles stephensi

Previous studies indicated that gnotobiotic Anopheles stephensi mosquitoes were less susceptible to infection with Plasmodium berghei than xenobiotic ones (Munderloh and Kurtti, 1985). Groups of 100 to 200 mosquitoes were fed on infected hamsters, heparinized gametocytemic blood (via a membrane feeder), and in vitro-formed ookinetes suspended in blood (membrane feeder). Xenobiotic A. stephensi were readily infected by all 3 routes. Gnotobiotic mosquitoes consistently acquired infection after engorging on hamsters (average level of infected females in 8 experiments: 54.1%), but the parasite yield was low (average number of oocysts per infected female: 21.6). In 7 experiments where gnotobiotic A. stephensi were membrane-fed infected hamster blood, an average of only 8.8% of the females became infected, harboring a mean of 2.4 oocysts, and in 7 additional cases no infection was achieved. This pattern was reversed when gnotobiotic A. stephensi were fed ookinetes. A larger proportion of them became infected (mean level of infection in 8 experiments: 76.2%) and they acquired a higher mean number of oocysts per female (94.4) than did xenobiotic mosquitoes. Thus, gnotobiotic A. stephensi are as able as xenobiotic ones to support the sporogonic development of P. berghei, but are less able to support ookinete development.     

Defined medium supporting development of cleansed Plasmodium berghei ookinetes in Anopheles stephensi.

Hamsters blood infected with Plasmodium berghei was cultured in vitro for the development of ookinetes. The ookinetes were separated from blood components, suspended in various defined media and fed to Anopheles stephensi through a membrane. The development of the oocysts and infective sporozoites was recorded. Mosquitoes infected with ookinetes suspended in L15 formulated into L15-B, L15-D (a medium specially modified for this purpose), IPL-41 or 199 media with no proteins added, developed at least as many oocysts as the control mosquitoes fed ookinetes suspended in blood. Ookinetes suspended in the L15-B medium yielded more oocysts than after feeding ookinetes suspended in L15-B with 5% casein. Sporozoites from mosquitoes maintained on blood, L15-B, L15-D, or L15-B with 5% casein were shown to be infective to hamsters. Mosquitoes fed ookinetes suspended in sucrose solutions showed very few oocysts, but the yield was increased when a blood meal was given 2-4 days after the infective meal. Some of the oocysts which had developed from the ookinetes suspended in artificial media were found to have degenerated. The described system could be potentially useful for a study of the interaction between the vector physiology and the parasite. The possible use of the system to learn which media should be developed in the future for in vitro cultivation of oocysts is discussed.     

An ecological study of Caiman crocodilus crocodilus inhabiting savanna lagoons in the Venezuelan Guayana

Summary Mark and recapture studies were carried out for three and a half years on a population of Caiman c. crocodilus inhabiting a savanna lagoon system in the Venezuelan Guayana. Sub-adult and adult caimans migrated from permanent lagoon refuges to temporary lagoons during the wet season. A distinct homing response by artificially displaced caimans was observed.The wet season was the most significant time of the year for both feeding and growth. It was estimated that caimans take 6 years to reach a size of 97 cm. Thereafter the growth rate was variable. During a dry year there was little growth, but during a wet year a large caiman could increase in length by up to 10 cm.During the first 18 months of life, young caimans remained near the nest site. Older caimans dispersed and competed for territories which resulted in a high incidence of damage, particularly to the tails, as a result of intraspecific fighting.     

The fate of Hepatozoon species naturally infecting Florida black racers and watersnakes in potential mosquito and soft tick vectors, and histological evidence of pathogenicity in unnatural host species.

Haemogregarine parasites, derived from the Florida snakes Coluber constrictor and Nerodia fasciata and ingested by Aedes aegypti, completed sporogony within the hemocoeles of nearly all fed mosquitoes in 14-18 days, and produced oocysts typical of Hepatozoon. However, mortalities and morbidity were high in the Culex which had fed on the Coluber. Oocysts were not found in any Ornithodoros turicata (Argasidae) which fed upon either snake host, but many sections of fed ticks had gametocyte-like cells within the gut lumen. Most lizards, Anolis carolinensis and Anolis sagrei, infected per os with oocysts derived from both snake species developed infections. Infections in the lizards were largely confined to hepatic schizonts with few parasites found in erythrocytes. Unlike naturally infected snake hosts, Hepatozoon schizonts in livers of lizards were often either surrounded by an unidentified dark pigment or heavily infiltrated with mononuclear inflammatory cells.     

The peritrophic membrane as a barrier: its penetration by Plasmodium gallinaceum and the effect of a monoclonal antibody to ookinetes

We studied the point at which a monoclonal antibody (mAb C5) to a surface protein (Pgs25) on Plasmodium gallinaceum ookinetes blocked the infection of Aedes aegypti mosquitoes. The antibody did not block the development of zygotes to ookinetes in vitro. Development of ookinetes to oocysts in the mosquito was blocked to the same extent whether zygotes grew to ookinetes in the presence of mAb C5 or the antibody was added after the ookinetes had reached full development. When ookinetes developed in vitro in the presence of mAb C5, antibody remained on the surface of the parasite for the next 50 hr and did not block attachment to the peritrophic membrane. When ookinetes were fed to mosquitoes, two subpopulations of mosquitoes were observed (high numbers of oocysts per midgut and low numbers of oocysts per midgut). mAb C5 reduced the number of oocysts per midgut in the subpopulation that had low numbers of oocysts. The subpopulation that had high numbers of oocysts was unaffected by antibody, indicating that the antibody did not block invasion of the midgut epithelium. When mAb C5 was fed with gametocytes, the parasites invaded the epithelium at the same time (between 30 and 35 hr after the blood meal) as in controls, although at a markedly reduced rate. The ultrastructural observations were consistent with a block of parasites within the peritrophic membrane and not with a block at the epithelium, as parasites were not seen to accumulate within the space between the peritrophic membrane and the epithelium. The mechanism by which mAb C5 to Pgs25 of P. gallinaceum blocks the penetration of the peritrophic membrane remains undefined. We present evidence that the parasite modifies the peritrophic membrane during penetration, an observation first made for Babesia microti during penetration of the peritrophic membrane in Ixodes ticks. Ookinetes in the absence of antibodies appeared to disrupt the layers of the peritrophic membrane, suggesting an enzymatic mechanism for penetration.     

Sporogonic development of Hepatozoon americanum (Apicomplexa) in its definitive host, Amblyomma maculatum (Acarina)

Light microscopic observations of the sporogonic development of Hepatozoon americanum are described in its acarine host, Amblyomma maculatum. Laboratory-reared nymphal ticks were fed on 2 dogs infected with H. americanum. Nymphal ticks were sampled daily, starting 3 days after being placed on a parasitemic dog, until 18 days after infestation (PI), and then every 3 or 4 days until replete nymphs molted. Ticks were examined as unstained wet mounts and hematoxylin-eosin-stained paraffin sections. Gametes were found within the gut cells of nymphs 4 and 6 days PI. Although differentiation of gamonts into gametes was not detected, syngamy and sporogony were observed. Sporogony appears to occur wholly within tick gut cells, followed by release of mature oocysts into the hemocoel. The earliest evidence of sporoblast formation was observed 23 days PI and of sporozoite formation, 10 days later. Mature oocysts were first found 42 days PI in newly molted adult ticks. Most adult ticks (>98%) that were dissected contained mature oocysts. Oocysts were multisporocystic, and sporocysts contained a variable number of sporozoites. Oocysts in various stages of development were often seen within the same tick, and the number of mature oocysts ranged from 4 to 573.     

Differential infectivity of Plasmodium for mosquitoes

The four human malarias - Plasmodium falciparum, P. vivax, P. ovale and P. malariaecan - canonly be transmitted by mosquitoes of the genus Anopheles, although not all species (nor all strains) of these mosquitoes are equally susceptible. Moreover, there are many other plasmodial parasites of other mammals and birds, that can infect other genera of mosquito. What determines this level of vector-parasite specificity? Malarial gametocytes, ingested by a feeding mosquito, must transform to gametes, fuse to form zygotes, and then, as ookinetes, migrate to the mosquito's midgut epithelium to develop as oocysts that release sporozoites to infect the mosquito's salivary glands. During this process, the blood- fed mosquito is developing its peritrophic membrane lining the gut. In this article, the Guthors examine these parallel processes in three sets of mosquito-parasite models, suggesting that parasite-vector specificity may depend on a balance between speed of parasite development versus speed of formation of the peritrophic membrane which can act as a barrier to ookinete migration and establishment in the midgut epithelium.     

Complement effects on the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. I. Resistance of zygotes to the alternative pathway of complement

Gametocytes are the intraerythrocytic stages of malaria parasites that infect mosquitoes. When gametocytes of the chicken malaria parasite Plasmodium gallinaceum are ingested by a mosquito they become extracellular in the mosquito midgut, form gametes, and fertilize within 10 to 15 min after the insect has taken a blood meal. Gametocytes of P. gallinaceum were infectious when fed to Aedes aegypti mosquitoes in blood meals containing native serum from chickens or from the non-host species, man or sheep. Gametocytes stimulated to undergo gametogenesis and to fertilize in vitro were also infectious when fed to mosquitoes in native chicken serum. However, native serum from most non-host species, including sheep and man, suppressed the infectivity of newly fertilized zygotes to mosquitoes and lysed the zygotes in vitro. These effects were shown to be due to the activity of the alternative pathway of complement (APC) in the serum of the non-host species. After mild trypsin treatment, the zygotes of P. gallinaceum no longer infected mosquitoes in the presence of native chicken serum, although in heat-inactivated chicken serum their infectivity was normal. We conclude that trypsin-sensitive components on the zygotes surface protect them from destruction by the APC of their native host. The ability of gametocytes of P. gallinaceum to infect mosquitoes in the presence of native human serum is probably due to proteases that inactivate the APC of human serum before the gametes and zygotes emerge as extracellular parasites in the blood meal.     

The transmission-blocking effects of antimalarial drugs revisited: fitness costs and sporontocidal effects of artesunate and sulfadoxine-pyrimethamine

Assays used to evaluate the transmission-blocking activity of antimalarial drugs are largely focused on their potential to inhibit or reduce the infectivity of gametocytes, the blood stages of the parasite that are responsible for the onward transmission to the mosquito vector. For this purpose, the drug is administered concomitantly with gametocyte-infected blood, and the results are evaluated as the percentage of reduction in the number of oocysts in the mosquito midgut. We report the results of a series of experiments that explore the transmission-blocking potential of two key antimalarial drugs, artesunate and sulfadoxine-pyrimethamine, when administered to mosquitoes already infected from a previous blood meal. For this purpose, uninfected mosquitoes and mosquitoes carrying a 6 day old Plasmodium relictum infection (early oocyst stages) were allowed to feed either on a drug-treated or an untreated host in a fully factorial experiment. This protocol allowed us to bypass the gametocyte stages and establish whether the drugs have a sporontocidal effect, i.e. whether they are able to arrest the ongoing development of oocysts and sporozoites, as would be the case when a mosquito takes a post-infection treated blood meal. In a separate experiment, we also explored whether a drug-treated blood meal impacted key life history traits of the mosquito relevant for transmission, and if this depended on their infection status. Our results showed that feeding on an artesunate- or sulfadoxine-pyrimethamine-treated hosts has no epidemiologically relevant effects on the fitness of infected or uninfected mosquitoes. In contrast, when infected mosquitoes fed on an sulfadoxine-pyrimethamine-treated host, we observed both a significant increase in the number of oocysts in the midgut, and a drastic decrease in both sporozoite prevalence (?30%) and burden (?80%) compared with the untreated controls. We discuss the potential mechanisms underlying these seemingly contradictory results and contend that, provided the results are translatable to human malaria, the potential epidemiological and evolutionary consequences of the current preventive use of sulfadoxine-pyrimethamine in malaria-endemic countries could be substantial.     

High gametocyte complexity and mosquito infectivity of Plasmodium falciparum in the Gambia

The purpose of this work was to determine the infectivity to mosquitoes of genetically diverse Plasmodium falciparum clones seen in natural infections in the Gambia. Two principal questions were addressed: (i) how infectious are gametocytes of sub-patent infections, particularly at the end of the dry season; and (ii) are all clones in multiclonal infections equally capable of infecting mosquitoes? The work was carried out with two cohorts of infected individuals. Firstly, a group of 31 P. falciparum-infected people were recruited in the middle of the dry season (May, 2003), then examined for P. falciparum at the beginning (August 2003) and middle (October, 2003) of the transmission season. On each occasion, we examined the genotypes of asexual forms and gametocytes by PCR and RT-PCR, as well as their infectivity to Anopheles gambiae using membrane feeds. One individual gave rise to infected mosquitoes in May, and two in August. Different gametocyte genotypes co-existed in the same infection and fluctuated over time. The mean multiplicity of infection was 1.4, 1.7 and 1.5 clones in May, August and October, respectively. Second, a group of patients undergoing drug-treatment during August 2003 was tested for asexual and gametocyte genotypes and their infectivity to mosquitoes. Forty-three out of 100 feeds produced infections. The genetic complexity of the parasites in mosquitoes was sometimes greater than that detectable in the blood on which the mosquitoes had fed. This suggested that gametocytes of clones existing in the blood below PCR detection limits at the time of the feed were at least as infectious to the mosquitoes as the more abundant clones. These findings emphasise the crucial role of gametocyte complexity and infectivity in generating the remarkable diversity of P. falciparum genotypes seen in infected people, even in an area of seasonal transmission.     

Antibodies to Anopheles midgut reduce vector competence for Plasmodium vivax malaria

Anopheles tessellatus mosquitoes ingested Plasmodium vivax gametocytes in human erythrocytes suspended in rabbit sera with and without anti-mosquito midgut antibodies. When the mosquito bloodmeal contained anti-midgut antibodies, fewer oocysts of P. vivax developed on the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. Complement inactivated serum also reduced the infection rate and load. A second bloodmeal containing anti-midgut antibodies, given 48 or 72 h later, did not enhance the transmission-blocking effect. IgG purified from antimidgut sera was shown to mediate the transmission-blocking effect.