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1. Plasmodium falciparum Calcium-Dependent Protein Kinase 4 is Critical for Male Gametogenesis and Transmission to the Mosquito Vector

   https://doi.org/10.1128/mBio.02575-21  

   Kumar, Sudhir; Haile, Meseret T.; Hoopmann, Michael R.; Tran, Linh T.; Michaels, Samantha A.; Morrone, Seamus R.; Ojo, Kayode K.; Reynolds, Laura M.; Kusebauch, Ulrike; Vaughan, Ashley M.; et al (December 2021, mBio)

   Weiss, Louis M. (Ed.)

   ABSTRACT Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium-independent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4 − parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4 − parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild-type and Plasmodium falciparum cdpk4 − late gametocyte stages to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events, such as DNA replication, mRNA translation, and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation and thereby is critical for parasite transmission to the mosquito vector. IMPORTANCE Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies. 

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2. The Genome of Plasmodium gonderi : Insights into the Evolution of Human Malaria Parasites

   https://doi.org/10.1093/gbe/evae027  

   Cepeda, Axl S.; Mello, Beatriz; Pacheco, M. Andreína; Luo, Zunping; Sullivan, Steven A.; Carlton, Jane M.; Escalante, Ananias A.; dos Reis, ed., Mario (February 2024, Genome Biology and Evolution)

   Abstract Plasmodium species causing malaria in humans are not monophyletic, sharing common ancestors with nonhuman primate parasites. Plasmodium gonderi is one of the few known Plasmodium species infecting African old-world monkeys that are not found in apes. This study reports a de novo assembled P. gonderi genome with complete chromosomes. The P. gonderi genome shares codon usage, syntenic blocks, and other characteristics with the human parasites Plasmodium ovale s.l. and Plasmodium malariae, also of African origin, and the human parasite Plasmodium vivax and species found in nonhuman primates from Southeast Asia. Using phylogenetically aware methods, newly identified syntenic blocks were found enriched with conserved metabolic genes. Regions outside those blocks harbored genes encoding proteins involved in the vertebrate host-Plasmodium relationship undergoing faster evolution. Such genome architecture may have facilitated colonizing vertebrate hosts. Phylogenomic analyses estimated the common ancestor between P. vivax and an African ape parasite P. vivax-like, within the Asian nonhuman primates parasites clade. Time estimates incorporating P. gonderi placed the P. vivax and P. vivax-like common ancestor in the late Pleistocene, a time of active migration of hominids between Africa and Asia. Thus, phylogenomic and time-tree analyses are consistent with an Asian origin for P. vivax and an introduction of P. vivax-like into Africa. Unlike other studies, time estimates for the clade with Plasmodium falciparum, the most lethal human malaria parasite, coincide with their host species radiation, African hominids. Overall, the newly assembled genome presented here has the quality to support comparative genomic investigations in Plasmodium. 

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3. The seminal odorant binding protein Obp56g is required for mating plug formation and male fertility in Drosophila melanogaster

   https://doi.org/10.7554/eLife.86409  

   Brown, Nora C; Gordon, Benjamin; McDonough-Goldstein, Caitlin E; Misra, Snigdha; Findlay, Geoffrey D; Clark, Andrew G; Wolfner, Mariana Federica (December 2023, eLife)

   In Drosophila melanogaster and other insects, the seminal fluid proteins (SFPs) and male sex pheromones that enter the female with sperm during mating are essential for fertility and induce profound post-mating effects on female physiology. The SFPs in D. melanogaster and other taxa include several members of the large gene family known as odorant binding proteins (Obps). Work in Drosophila has shown that some Obp genes are highly expressed in the antennae and can mediate behavioral responses to odorants, potentially by binding and carrying these molecules to odorant receptors. These observations have led to the hypothesis that the seminal Obps might act as molecular carriers for pheromones or other compounds important for male fertility, though functional evidence in any species is lacking. Here, we used functional genetics to test the role of the seven seminal Obps in D. melanogaster fertility and the post-mating response (PMR). We found that Obp56g is required for male fertility and the induction of the PMR, whereas the other six genes are dispensable. We found males lacking Obp56g fail to form a mating plug in the mated female’s reproductive tract, leading to ejaculate loss and reduced sperm storage, likely due to its expression in the male ejaculatory bulb. We also examined the evolutionary history of these seminal Obp genes, as several studies have documented rapid evolution and turnover of SFP genes across taxa. We found extensive lability in gene copy number and evidence of positive selection acting on two genes, Obp22a and Obp51a. Comparative RNAseq data from the male reproductive tract of multipleDrosophilaspecies revealed that Obp56g shows high male reproductive tract expression in a subset of taxa, though conserved head expression across the phylogeny. Together, these functional and expression data suggest that Obp56g may have been co-opted for a reproductive function over evolutionary time. 

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4. Reproductive Proteins Evolve Faster Than Non-reproductive Proteins Among Solanum Species

   https://doi.org/10.3389/fpls.2021.635990  

   Moyle, Leonie C.; Wu, Meng; Gibson, Matthew J. (April 2021, Frontiers in Plant Science)

   null (Ed.)

   Elevated rates of evolution in reproductive proteins are commonly observed in animal species, and are thought to be driven by the action of sexual selection and sexual conflict acting specifically on reproductive traits. Whether similar patterns are broadly observed in other biological groups is equivocal. Here, we examine patterns of protein divergence among wild tomato species ( Solanum section Lycopersicon ), to understand forces shaping the evolution of reproductive genes in this diverse, rapidly evolving plant clade. By comparing rates of molecular evolution among loci expressed in reproductive and non-reproductive tissues, our aims were to test if: (a) reproductive-specific loci evolve more rapidly, on average, than non-reproductive loci; (b) ‘male’-specific loci evolve at different rates than ‘female’-specific loci; (c) genes expressed exclusively in gametophytic (haploid) tissue evolve differently from genes expressed in sporophytic (diploid) tissue or in both tissue types; and (d) mating system variation (a potential proxy for the expected strength of sexual selection and/or sexual conflict) affects patterns of protein evolution. We observed elevated evolutionary rates in reproductive proteins. However, this pattern was most evident for female- rather than male-specific loci, both broadly and for individual loci inferred to be positively selected. These elevated rates might be facilitated by greater tissue-specificity of reproductive proteins, as faster rates were also associated with more narrow expression domains. In contrast, we found little evidence that evolutionary rates are consistently different in loci experiencing haploid selection (gametophytic-exclusive loci), or in lineages with quantitatively different mating systems. Overall while reproductive protein evolution is generally elevated in this diverse plant group, some specific patterns of evolution are more complex than those reported in other (largely animal) systems, and include a more prominent role for female-specific loci among adaptively evolving genes. 

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5. Needle in a Haystack: A Droplet Digital Polymerase Chain Reaction Assay to Detect Rare Helminth Parasites Infecting Natural Host Populations

   https://doi.org/10.1111/1755-0998.14131  

   Fouilloux, Chloe_A; Neeno‐Eckwall, Eric; Srinivas, Ipsita; Compton, Jonathan_S; Sampson, Josh; Weber, Jesse; Wolf, Cole; Hund, Amanda; Berini, III, John; Alexander, Heather; et al (June 2025, Molecular Ecology Resources)

   ABSTRACT Helminths infect humans, livestock, and wildlife, yet remain understudied despite their significant impact on public health and agriculture. Because many of the most prevalent helminth‐borne diseases are zoonotic, understanding helminth transmission among wildlife could improve predictions and management of infection risks across species. A key challenge to understanding helminth transmission dynamics in wildlife is accurately and quantitatively tracking parasite load across hosts and environments. Traditional methods, such as visual parasite identification from environmental samples or infected hosts, are time‐consuming, while standard molecular techniques (e.g., PCR and qPCR) often lack the sensitivity to reliably detect lower parasite burdens. These limitations can underestimate the prevalence and severity of infection, hindering efforts to manage infectious diseases. Here, we developed a multiplexed droplet digital PCR (ddPCR) assay to quantify helminth loads in aquatic habitats using 18S rRNA target genes. UsingSchistocephalus solidusand their copepod hosts as a case study, we demonstrate ddPCR's sensitivity and precision. The assay is highly reproducible, reliably detecting target genes at concentrations as low as 1 pg of DNA in lab standards and field samples (multi‐species and eDNA). Thus, we provide a toolkit for quantifying parasite load in intermediate hosts and monitoring infection dynamics across spatio‐temporal scales in multiple helminth systems of concern for public health, agriculture, and conservation biology. 

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