Observation of the in vitro production of male gametes of the human malaria parasite Plasmodium falciparum through the process of exflagellation

Exflagellation is the term given to the production of male gametes by sporozoans, in particular the Plasmodium parasite that causes malaria in a wide range of vertebrates including humans. In the malaria life cycle this key event occurs naturally within the midgut of a female Anopheles mosquito a few minutes after it takes a blood meal infected with Plasmodium gametocytes [1]. This phenomenon also happens spontaneously in vitro and thus may be observed in cultures of fresh parasitized blood under the light microscope (1000 x magnification under oil immersion) [2].


Case report
Exflagellation is the term given to the production of male gametes by sporozoans, in particular the Plasmodium parasite that causes malaria in a wide range of vertebrates including humans.In the malaria life cycle this key event occurs naturally within the midgut of a female Anopheles mosquito a few minutes after it takes a blood meal infected with Plasmodium gametocytes [1].This phenomenon also happens spontaneously in vitro and thus may be observed in cultures of fresh parasitized blood under the light microscope (1000 x magnification under oil immersion) [2].
We report the rare observation in vitro of male (micro)gametogenesis by the major human malaria parasite P. falciparum (Figure 1).While this process may be recorded by video, by which its explosively energetic nature can readily be appreciated, it is not straightforward to capture by still photography because the parasite oscillates vigorously and thus moves continually in and out of the microscope's two-dimensional plane of focus.This photograph is an unusually clear and detailed image of this metabolically dynamic and visually striking event.The nucleus of the parent male gametocyte divides rapidly three times to form eight daughter nuclei, each of which has an associated axoneme.This nuclear material moves to the surface of the gametocyte to be extruded into peripheral cytoplasmic processes as the spindle-shaped microgametes with visibly identifiable flagella.Gametogenesis may be viewed typically between 10 to 25 minutes after removal of cultures of parasitized blood from an incubator maintained at 37 °C.The gas mixture of choice is 1% oxygen, 3% carbon dioxide and 96% nitrogen.
Under in vitro conditions exflagellation is controlled solely by the drop in air temperature from 37 °C at which P. falciparumcontaining human blood cultures are routinely maintained to the ambient laboratory temperature (of around 22-24 °C), the pH rise this brings being mediated by a fall in carbon dioxide tension as the blood equilibrates with the atmosphere [2].This cascade mimics the environmental changes that occur to the gametocytes in the gut of the mosquito which exhibits a pH of approximately 7.8 as compared to a pH of 7.4 in human blood.
The visual recording of microgametogenesis in vitro is a reliable indicator that oocysts and sporozoites will most likely be produced in vivo following membrane-feeding of a batch of the same blood culture to adult female mosquitoes.Typically, in a successfully fed mosquito oocysts may be observed on the outer midgut wall 10 days post infection, while sporozoites may be dissected from the salivary glands a further 7 days later [3].These sexual stages of the parasite, which occur only within the mosquito host, are required in order to research vector infectivity.
The artificial triggering of male gametogenesis is therefore crucial for investigating the sexual stages of P. falciparum.This is a prerequisite to the successful infectivity of P. falciparum for the insectary standard species of mosquito, A. stephensi, which may be routinely achieved using the protocol described by ourselves previously [4].This provides a powerful tool to enable the conduct of a wide range of molecular and cellular studies of the sexual stages of P. falciparum in vivo.

Discussion
Despite a recent reduction in the annual toll of morbidity and mortality the mosquito-borne infectious disease malaria remains a major global public health concern, with an incidence of several hundred million clinical cases and up to 3 million deaths each year [5].
While it has rendered ineffectual many strategies for infection control, increasing resistance to drugs of the aetiological agent, protozoan Plasmodium parasites, and to insecticides of the vector, Anopheles mosquitoes, is demanding novel perspectives to counter both the transmission of infection and burden of the disease [6].Of the six species of malaria that are known to infect humans, P. falciparum is by far the deadliest and therefore the one towards which most control measures are focused.
A principal reason for the modest success of one strategy, antimalaria vaccine design, is our inadequate understanding of the developmental processes that operate, and antigens expressed, during the differentiation of parasite stages [7], including those within the Anopheles vector.Immunity to sexual stages of malaria, termed transmission-blocking immunity, may operate against gametocytes in the vertebrate host and against gametes in the midgut and ookinetes, the motile form of the Plasmodium parasite that traverses the mosquito midgut epithelium to initiate sporogony [8].
In order to facilitate detailed molecular, biochemical and immunological studies of Plasmodium parasite progression within the Anopheles mosquito, it is necessary to produce sufficient numbers of fully mature oocysts and sporozoites [9].This requires the in vitro cultivation of gametocytes and the laboratory-manipulated induction of gametogenesis prior to a mosquito blood feed.The exflagellation event shown in Figure 1 is a strong predictor of a successful outcome to this sexual stage parasite harvesting process.
It should be noted that P. falciparum is a highly dangerous human pathogen that is classified internationally as requiring biosafety level 3 containment [10].Hence, when maintaining this parasite in human blood in vitro all appropriate health and safety measures must be performed in adherence to local regulations and institutional guidelines.Any isolate of P. falciparum that is used routinely for laboratory-based research purposes should be susceptible to standard antimalarial drugs.
Copyright: ©2018 Looker M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Figure 1 .
Figure 1.Male (micro)gametocyte of the human malaria parasite Plasmodium falciparum, cloned line 3D7A, cultivated in vitro in group O, rhesus group-positive human erythrocytes.Exflagellation (arrowed) -extrusion of eight motile, flagella-like microgametes with vigorous movement.Blood film, wet mount, 1000 x magnification under oil immersion