Progression of Plasmodium berghei through Anopheles stephensi Is Density-Dependent

It is well documented that the density of Plasmodium in its vertebrate host modulates the physiological response induced; this in turn regulates parasite survival and transmission. It is less clear that parasite density in the mosquito regulates survival and transmission of this important pathogen. Numerous studies have described conversion rates of Plasmodium from one life stage to the next within the mosquito, yet few have considered that these rates might vary with parasite density. Here we establish infections with defined numbers of the rodent malaria parasite Plasmodium berghei to examine how parasite density at each stage of development (gametocytes; ookinetes; oocysts and sporozoites) influences development to the ensuing stage in Anopheles stephensi, and thus the delivery of infectious sporozoites to the vertebrate host. We show that every developmental transition exhibits strong density dependence, with numbers of the ensuing stages saturating at high density. We further show that when fed ookinetes at very low densities, oocyst development is facilitated by increasing ookinete number (i.e., the efficiency of ookinete–oocyst transformation follows a sigmoid relationship). We discuss how observations on this model system generate important hypotheses for the understanding of malaria biology, and how these might guide the rational analysis of interventions against the transmission of the malaria parasites of humans by their diverse vector species

Global urban environmental change drives adaptation in white clover

Urbanization transforms environments in ways that alter biological evolution. We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale

Female inheritance of malarial lap genes is essential for mosquito transmission.

Members of the LCCL/lectin adhesive-like protein (LAP) family, a family of six putative secreted proteins with predicted adhesive extracellular domains, have all been detected in the sexual and sporogonic stages of Plasmodium and have previously been predicted to play a role in parasite-mosquito interactions and/or immunomodulation. In this study we have investigated the function of PbLAP1, 2, 4, and 6. Through phenotypic analysis of Plasmodium berghei loss-of-function mutants, we have demonstrated that PbLAP2, 4, and 6, as previously shown for PbLAP1, are critical for oocyst maturation and sporozoite formation, and essential for transmission from mosquitoes to mice. Sporozoite formation was rescued by a genetic cross with wild-type parasites, which results in the production of heterokaryotic polyploid ookinetes and oocysts, and ultimately infective Deltapblap sporozoites, but not if the individual Deltapblap parasite lines were crossed amongst each other. Genetic crosses with female-deficient (Deltapbs47) and male-deficient (Deltapbs48/45) parasites show that the lethal phenotype is only rescued when the wild-type pblap gene is inherited from a female gametocyte, thus explaining the failure to rescue in the crosses between different Deltapblap parasite lines. We conclude that the functions of PbLAPs1, 2, 4, and 6 are critical prior to the expression of the male-derived gene after microgametogenesis, fertilization, and meiosis, possibly in the gametocyte-to-ookinete period of differentiation. The phenotypes detectable by cytological methods in the oocyst some 10 d after the critical period of activity suggests key roles of the LAPs or LAP-dependent processes in the regulation of the cell cycle, possibly in the regulation of cytoplasm-to-nuclear ratio, and, importantly, in the events of cytokinesis at sporozoite formation. This phenotype is not seen in the other dividing forms of the mutant parasite lines in the liver and blood stages

RT-PCR Analysis of <i>pblap</i> Expression in Day 10 Oocysts

<p>RT-PCR on total RNA isolated from midguts of <i>An. stephensi</i> infected with <i>Δpblap1</i> × <i>Δpbs47</i> (lanes 1 and 2), <i>Δpblap2</i> × <i>Δpbs47</i> (lanes 3 and 4), <i>Δpblap4</i> × <i>Δpbs47</i> (lanes 5 and 6), <i>Δpblap6</i> × <i>Δpbs47</i> (lanes 7 and 8), <i>Δpblap1</i> × <i>Δpbs48/45</i> (lanes 9 and 10), <i>Δpblap2</i> × <i>Δpbs48/45</i> (lanes 11 and 12), <i>Δpblap4</i> × <i>Δpbs48/45</i> (lanes 13 and 14), and <i>Δpblap6</i> × <i>Δpbs48/45</i> (lane 15 and 16). Top panel, PCR for the respective <i>pblap</i> gene as indicated above the panel; bottom panel, control PCR for <i>a-tubulin;</i> RT, reverse transcriptase. Fragments of the expected size were amplified for <i>pblap2</i> (399 bp), <i>pblap4</i> (482 bp), <i>pblap6</i> (381 bp), and <i>α-tubulin</i> (432 bp). A weak signal for <i>pblap1</i> (540 bp) was observed on longer exposures in both crosses. Some genomic DNA contamination was detected for <i>Δpblap6</i> × <i>Δpbs47</i> (lane 8, bottom panel), but the <i>pblap6</i> PCR product from cDNA (lane 7, top panel) can be distinguished from amplification of genomic DNA by the absence of a 310-bp intron.</p

Transmission Electron Micrographs of <i>wt</i> and <i>Δpblap</i> Oocysts

<div><p>All images taken on day 13 p.i. unless otherwise indicated. Scale bar = 1 μm (A, B, F) or 5 μm (C–E). ep, midgut epithelium.</p><p>(A) <i>wt</i> oocyst showing normal morphology of the endoplasmic reticulum (er).</p><p>(B) <i>Δpblap2</i> oocyst showing extensive expansion of the endoplasmic reticulum (er).</p><p>(C) <i>Δpblap1</i> oocyst (day 27 p.i.) showing extensive expansion of the endoplasmic reticulum (er) and some budding sporozoites (s).</p><p>(D) <i>wt</i> oocyst showing normal morphology following cytokinesis to produce hundreds of daughter sporozoites (s).</p><p>(E) <i>Δpblap2</i> oocyst showing extensive degeneration and few nuclei (some of which are labelled n).</p><p>(F) Degenerate <i>Δpblap4</i> oocyst showing prominent melanization (m) of the extracellular oocyst wall (cw) which appears to spread into the mosquito basal lamina (bl).</p></div

Oocyst Morphology of <i>wt</i> and <i>Δpblap</i> Parasites

<p>Differential interference contrast images of <i>wt</i> day 21 p.i. (A), <i>Δpblap2</i> day 13 p.i. (B), <i>Δpblap4</i> day 18 p.i. (C), and <i>Δpblap6</i> day 21 p.i. oocysts (D) in <i>An. stephensi</i>. Most <i>wt</i> oocysts have undergone sporulation (open arrow). No sporozoite formation is observed in <i>Δpblap</i> infections, and oocysts appear either immature/enlarged (open arrowhead) or degenerate/vacuolated (closed arrowhead). Some <i>Δpblap4</i> oocysts are melanized (closed arrow). Scale bar = 20 μm.</p

Functional characterization of an LCCL-lectin domain containing protein family in Plasmodium berghei.

Using bioinformatic, proteomic, immunofluorescence, and genetic cross methods, we have functionally characterized a family of putative parasite ligands as potential mediators of cell-cell interactions. We name these proteins the Limulus clotting factor C, Coch-5b2, and Lgl1 (LCCL)-lectin adhesive-like protein (LAP) family. We demonstrate that this family is conserved amongst Plasmodium spp. It possesses a unique arrangement of adhesive protein domains normally associated with extracellular proteins. The proteins are expressed predominantly, though not exclusively, in the mosquito stages of the life cycle. We test the hypothesis that these proteins are surface proteins with 1 member of this gene family, lap1, and provide evidence that it is expressed on the surface of Plasmodium berghei sporozoites. Finally, through genetic crosses of wild-type Pblap1+ and transgenic Pblap1- parasites, we show that the null phenotype previously reported for sporozoite development in a Pblap1- mutant can be rescued within a heterokaryotic oocyst and that infectious Pblap1 sporozoites can be formed. The mutant is not rescued by coparasitization of mosquitoes with a mixture Pblap1+ and Pblap1- homokaryotic oocysts

P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions

The ookinete surface proteins (P25 and P28) are proven antimalarial transmission-blocking vaccine targets, yet their biological functions are unknown. By using single (Sko) and double gene knock-out (Dko) Plasmodium berghei parasites, we show that P25 and P28 share multiple functions during ookinete/oocyst development. In the midgut of mosquitoes, the formation of ookinetes lacking both proteins (Dko parasites) is significantly inhibited due to decreased protection against lethal factors, including protease attack. In addition, Dko ookinetes have a much reduced capacity to traverse the midgut epithelium and to transform into the oocyst stage. P25 and P28 are partially redundant in these functions, since the efficiency of ookinete/oocyst development is only mildly compromised in parasites lacking either P25 or P28 (Sko parasites) compared with that of Dko parasites. The fact that Sko parasites are efficiently transmitted by the mosquito is a compelling reason for including both target antigens in transmission-blocking vaccines

The South African 24-hour movement guidelines for birth to 5 years: An integration of physical activity, sitting behavior, screen time, and sleep

Background: In December 2018, the South African 24-hour movement guidelines for birth to 5 years were released. This article describes the process used to develop these guidelines. Methods: The Grading of Recommendations Assessment, Development, and Evaluation-ADOLOPMENT approach was followed, with some pragmatic adaptions, using the Australian guidelines for the early years as a starting point. A consensus panel, including stakeholders in early childhood development and academics, was formed to assist with the development process. Results: At a face-to-face meeting of the panel, global and local literatures were considered. Following this meeting, a first draft of the guidelines (including a preamble) was formulated. Further reviews of these drafts by the panel were done via e-mail, and a working draft was sent out for stakeholder consultation. The guidelines and preamble were amended based on stakeholder input, and an infographic was designed. Practical tips documents were also developed for caregivers of birth to 5-year-olds and early childhood development practitioners. The guidelines (and accompanying documents) were released at a launch event and disseminated through various media channels. Conclusions: These are the first movement guidelines for South African and the first such guidelines for this age group from a low- and middle-income country