Plasmodiumoocysts respond with dormancy to crowding and nutritional stress

Abstract

11Malaria parasites develop and grow as oocysts in the mosquito for several days before being able to 12 infect another human. During this time, mosquitoes take regular bloodmeals to replenish their 13 nutrient and energy reserves needed for flight and reproduction. We hypothesized that supplemental 14 bloodmeals are critical for oocyst growth and that experimental infection protocols, typically 15 involving a single bloodmeal, cause nutritional stress to developing oocysts. Therefore, 16 enumerating oocysts independently of their growth and differentiation state may lead to erroneous 17 conclusions regarding the efficacy of malaria transmission blocking interventions. We tested this 18 hypothesis in Anopheles coluzzii mosquitoes infected with human and rodent parasites Plasmodium 19 falciparum and Plasmodium berghei, respectively. We find that oocyst growth rates decrease at late 20 developmental stages as infection intensities increase; an effect exacerbated at very high infection 21intensities. Oocyst growth and differentiation can be restored by supplemental bloodmeals even at 22 high infection intensities. We show that high infection intensities as well as starvation conditions 23 reduce RNA Polymerase III activity in oocysts unless supplemental bloodmeals are provided. Our 24 data suggest that oocysts respond to crowding and nutritional stress by employing a dormancy-like 25 strategy and urge development of alternative methods to assess the efficacy of transmission 26 blocking interventions. 27 28 Main text 29 2The lifecycle of the malaria parasite Plasmodium inside a mosquito vector begins with gametocytes 30 ingested by a female mosquito during a bloodmeal. In the mosquito midgut lumen, gametes fuse to 31 form zygotes that develop into motile ookinetes. Ookinetes traverse the midgut-epithelium and 32 transform into oocysts. Multiple rounds of endomitotic replications inside an oocyst result in 33 production of thousands of sporozoites, a process known as sporogony. Upon oocyst rupture, 34 sporozoites migrate to the salivary gland for transmission to humans via a mosquito bite. 35 Current malaria control efforts focus on development of transmission blocking interventions 36including transmission blocking vaccines and drugs as well as genetically engineered mosquitoes 37 expressing antimalarial effectors 1,2 . Experimental mosquito infections with Plasmodium parasites, 38 and microscopic enumeration of oocysts is the gold standard for evaluation of transmission 39 reduction, whereby mosquitoes are offered an infectious bloodmeal and maintained on sugar 40 solution until oocysts are counted 3 . Infections often lead to high oocyst loads and consequently 41 massive proliferation of sporozoites, causing depletion of nutrients in the vector 4-7 . For example, 42 ultra-high intensity infections of Anopheles stephensi mosquitoes with the rodent parasite P. 43 berghei leads to reduced number of sporozoites reaching the salivary glands and high mosquito 44 mortality compared with lower infection intensities 8 . Hig...

Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential

“…Here, we demonstrate that multiple blood feedings significantly accelerate parasite growth, shortening the time required for sporozoites to appear in the salivary glands. These findings are consistent with infection studies in closely related mosquito species that showed that an additional blood meal during development either boosts P. falciparum sporozoite intensities(21,22,31) or increases oocyst size (20-22), although none analyzed parasite growth rates after the second meal. Our study conclusively links the effects of an additional blood meal to increased parasite developmental rates and a shortened EIP.…”

“…Moreover, multiple feeds may be required even within a single reproductive cycle due to interrupted feeding or to nutrient deprivation during the larval stage (pre-gravid behavior) (19). Additional blood meals may therefore potentially influence oocyst growth and the EIP, as suggested by reports showing an additional feed can increase P. falciparum oocyst size (20, 21) and salivary gland sporozoite numbers at a given time point (21,22).…”

Untitled

“…Here, we demonstrate that multiple blood feedings significantly accelerate parasite growth, shortening the time required for sporozoites to appear in the salivary glands. These findings are consistent with infection studies in closely related mosquito species that showed that an additional blood meal during development either boosts P. falciparum sporozoite intensities or yields [22][23][24][25][26]34] or increases oocyst size [21][22][23], although none analyzed parasite growth rates after the second meal. Our study conclusively links the effects of an additional blood meal to increased parasite developmental rates and a shortened EIP.…”

Estimating the extrinsic incubation period of malaria using a mechanistic model of sporogony

“…Current estimates indicate climate change has the potential to change the number of people at risk of malaria (56,60,61), though there is uncertainty due to the complexity of how the parasite and given that our model assumes the EIP is determined on a per-oocyst basis and that developmental times are independent within an individual mosquito which needs to be verified experimentally. What's more, other processes may be operating: emerging experimental evidence hints that a decrease in resource availability per parasite (at high parasite loads), as determined by the parasite load and the number of times the mosquito blood-feeds, may decrease the parasite development rate (48,67). Since (a) transmission is highly sensitive to changes in EIP, (b) transmission blocking interventions cause a decline in oocyst intensity (38), and (c) parasite load in the field may be higher than previously thought (37), the influence of parasite load on EIP merits further investigation.…”