The microneme proteins CTRP and SOAP are not essential for Plasmodium berghei ookinete to oocyst transformation in vitro in a cell free system

<p>Abstract</p> <p>Background</p> <p>Two <it>Plasmodium berghei </it>ookinete micronemal proteins, circumsporozoite and TRAP related protein (CTRP) and secreted ookinete adhesive protein (SOAP) both interact with the basal lamina component laminin. Following gene disruption studies it has been proposed that, apart from their role in motility, these proteins may be required for interactions leading to ookinete-to-oocyst transformation.</p> <p>Methods</p> <p>CTRP and SOAP null mutant <it>P. berghei </it>ookinetes were compared to <it>P. berghei </it>ANKA wild-type for their ability to transform and grow <it>in vitro</it>. To confirm <it>in vitro </it>findings for <it>P</it>. <it>berghei </it>CTRP-KO ookinetes were injected into the haemocoel of <it>An</it>opheles <it>gambiae </it>female mosquitoes.</p> <p>Results</p> <p>Transformation, growth, and viability were comparable for the gene disrupted and wild-type parasites. <it>P</it>. <it>berghei </it>CTRP-KO ookinetes were able to transform into oocysts in the haemocoel of <it>An</it>. <it>gambiae </it>mosquitoes.</p> <p>Conclusion</p> <p>Neither CTRP nor SOAP is required for parasite transformation <it>in vitro</it>. By-passing the midgut lumen allows for the transformation of <it>P</it>. <it>berghei </it>CTRP-KO ookinetes suggesting that it is not required for transformation <it>in vivo</it>.</p

Hymenolepis diminuta: the pathophysiology of infection in the intermediate host, Tenebrio molitor

In T. molitor infected with metacestodes of H. diminuta total haemolymph free protein concentration, determined by the Lowry method, ranged from 62 to 81 mg/ml in females and was significantly lower in males (36-54mg/ml). A 477. increase was detected in female beetles 12 days or more post-infection, no such difference being detected at an earlier stage, nor in males at any age examined. Using SDS PAGE, 13 bands were separated from haemolymph, bands 2/3 and 7/8 being present in greater concentrations in female beetles. Molecular weight determinations and histochemical evidence suggested that these proteins were vitellogenins. Densitometric analysis revealed that these bands alone were elevated in haemolymph from females 12 days post-infection, no such elevation being detected at an earlier stage, nor in infected male beetles. Infection did not affect fat body wet-weight nor protein content. However, in vitro culture of fat bodies with ^C-leucine revealed a 617 decrease in protein synthesis to be associated with infection. In vivo sequestration of labelled proteins by ovaries from infected beetles was significantly decreased, the majority of the label being detected in the vitellogenic fraction of ovary homogenates. Various parameters of fecundity were measured over a period of 30 days; total egg-volume and egg protein content were unaffected by infection. However, the second peak of egg laying was delayed, egg viability decreased and total protein content of eggs reduced in infected females. Sixteen haemolymph free amino acids were detected, total concentrations ranging from 34-84 mM. Although overall concentrations were not affected by infection, significant differenes occurred in individual amino acids, these being most marked in female beetles. The literature concerning host parasite interactions has been reviewed and it is suggested that the above pathological effects may be due to an interference by the parasite with the host endocrine system

Interactions between parasites and insects vectors

This review stresses the importance of studies that will provide a basic understanding of the pathology of parasite-infected vector insects. This knowledge should be a vital component of the very focussed initiatives currently being funded in the areas of vector control. Vector fecundity reduction is discussed as an example of such pathology. Underlying mechanisms are being investigated in a model system, Hymenolepis diminuta-infected Tenebrio molitor and in Onchocerca-infected blackflies and Plasmodium-infected Anopheles stephensi. In all cases, host vitellogenesis is disrupted by the parasite and, in the tapeworm/beetle model, interaction between the parasite and the endocrine control of the insect's reproductive physiology has been demonstrated

Localisation of laminin within Plasmodium berghei oocysts and the midgut epithelial cells of Anopheles stephensi

<p>Abstract</p> <p>Background</p> <p>Oocysts of the malaria parasite form and develop in close proximity to the mosquito midgut basal lamina and it has been proposed that components of this structure play a crucial role in the development and maturation of oocysts that produce infective sporozoites. It is further suggested that oocysts incorporate basal lamina proteins into their capsule and that this provides them with a means to evade recognition by the mosquito's immune system. The site of production of basal lamina proteins in insects is controversial and it is still unclear whether haemocytes or midgut epithelial cells are the main source of components of the mosquito midgut basal lamina. Of the multiple molecules that compose the basal lamina, laminin is known to interact with a number of <it>Plasmodium </it>proteins. In this study, the localisation of mosquito laminin within the capsule and cytoplasm of <it>Plasmodium berghei </it>oocysts and in the midgut epithelial cells of <it>Anopheles stephensi </it>was investigated.</p> <p>Results</p> <p>An ultrastructural examination of midgut sections from infected and uninfected <it>An. stephensi </it>was performed. Post-embedded immunogold labelling demonstrated the presence of laminin within the mosquito basal lamina. Laminin was also detected on the outer surface of the oocyst capsule, incorporated within the capsule and associated with sporozoites forming within the oocysts. Laminin was also found within cells of the midgut epithelium, providing support for the hypothesis that these cells contribute towards the formation of the midgut basal lamina.</p> <p>Conclusion</p> <p>We suggest that ookinetes may become coated in laminin as they pass through the midgut epithelium. Thereafter, laminin secreted by midgut epithelial cells and/or haemocytes, binds to the outer surface of the oocyst capsule and that some passes through and is incorporated into the developing oocysts. The localisation of laminin on sporozoites was unexpected and the importance of this observation is less clear.</p

Rodent malaria-resistant strains of the mosquito, Anopheles gambiae, have slower population growth than -susceptible strains

<p>Abstract</p> <p>Background</p> <p>Trade-offs between anti-parasite defence mechanisms and other life history traits limit the evolution of host resistance to parasites and have important implications for understanding diseases such as malaria. Mosquitoes have not evolved complete resistance to malaria parasites and one hypothesis is that anti-malaria defence mechanisms are costly.</p> <p>Results</p> <p>We used matrix population models to compare the population growth rates among lines of <it>Anopheles gambiae </it>that had been selected for resistance or high susceptibility to the rodent malaria parasite, <it>Plasmodium yoelii nigeriensis</it>. The population growth rate of the resistant line was significantly lower than that of the highly susceptible and the unselected control lines, regardless of whether mosquitoes were infected with <it>Plasmodium </it>or not. The lower population growth of malaria-resistant mosquitoes was caused by reduced post blood-feeding survival of females and poor egg hatching.</p> <p>Conclusion</p> <p>With respect to eradicating malaria, the strategy of releasing <it>Plasmodium</it>-resistant <it>Anopheles </it>mosquitoes is unlikely to be successful if the costs of <it>Plasmodium</it>-resistance in the field are as great as the ones measured in this study. High densities of malaria-resistant mosquitoes would have to be maintained by continuous release from captive breeding facilities.</p

Isolation of Plasmodium berghei ookinetes in culture using Nycodenz density gradient columns and magnetic isolation

BACKGROUND: Large scale in vitro production of the mosquito stages of malaria parasites remains elusive, with only limited success for complete sporogonic development and only one report of development through to infective sporozoites. The initial step in this process is the production, in vitro, of ookinetes from gametocytaemic blood. Methods for isolation of these ookinetes from blood cells have been described; however, in addition to yield often being low, processing time and potential for contamination by erythrocytes remain high. METHODS: This study compares two procedures for retaining mature ookinetes from blood stage cultures, whilst removing red blood cells and other contaminants prior to further culture of the parasite. The well established method of isolation on Nycodenz cushions is compared with a novel method utilizing the innate magnetic properties of the haem pigment crystals found in the cytoplasm of ookinetes. RESULTS: Yield and viability of ookinetes were similar with both isolation methods. However, in our hands magnetic isolation produced a cleaner ookinete preparation much more quickly. Moreover, decreasing the flow rate through the magnetic column could further enhance the yield. CONCLUSION: We recommend the enrichment of an ookinete preparation prior to further culture being performed using the magnetic properties of Plasmodium berghei ookinetes as an alternative to their density. The former technique is faster, removes more erythrocytes, but day-to-day costs are greater

New Prospects for Research on Manipulation of Insect Vectors by Pathogens

International audienc

Hydric stress-dependent effects of Plasmodium falciparum infection on the survival of wild-caught Anopheles gambiae female mosquitoes

<p>Abstract</p> <p>Background</p> <p>Whether <it>Plasmodium falciparum</it>, the agent of human malaria responsible for over a million deaths per year, causes fitness costs in its mosquito vectors is a burning question that has not yet been adequately resolved. Understanding the evolutionary forces responsible for the maintenance of susceptibility and refractory alleles in natural mosquito populations is critical for understanding malaria transmission dynamics.</p> <p>Methods</p> <p>In natural mosquito populations, <it>Plasmodium </it>fitness costs may only be expressed in combination with other environmental stress factors hence this hypothesis was tested experimentally. Wild-caught blood-fed <it>Anopheles gambiae </it>s.s. females of the M and S molecular form from an area endemic for malaria in Mali, West Africa, were brought to the laboratory and submitted to a 7-day period of mild hydric stress or kept with water ad-libitum. At the end of this experiment all females were submitted to intense desiccation until death. The survival of all females throughout both stress episodes, as well as their body size and infection status was recorded. The importance of stress, body size and molecular form on infection prevalence and female survival was investigated using Logistic Regression and Proportional-Hazard analysis.</p> <p>Results</p> <p>Females subjected to mild stress exhibited patterns of survival and prevalence of infection compatible with increased parasite-induced mortality compared to non-stressed females. Fitness costs seemed to be linked to ookinetes and early oocyst development but not the presence of sporozoites. In addition, when females were subjected to intense desiccation stress, those carrying oocysts exhibited drastically reduced survival but those carrying sporozoites were unaffected. No significant differences in prevalence of infection and infection-induced mortality were found between the M and S molecular forms of <it>Anopheles gambiae</it>.</p> <p>Conclusions</p> <p>Because these results suggest that infected mosquitoes may incur fitness costs under natural-like conditions, they are particularly relevant to vector control strategies aiming at boosting naturally occurring refractoriness or spreading natural or foreign genes for refractoriness using genetic drive systems in vector populations.</p