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Author: Donna Huber

On the origin and evolution of the mosquito male-determining factor Nix

Background and workflow.

The mosquito family Culicidae is divided into two subfamilies named the Culicinae and Anophelinae. Nix, the dominant male-determining factor, has only been found in the culicines Aedes aegypti and Ae. albopictus, two important arboviral vectors that belong to the subgenus Stegomyia. Here we performed sex-specific whole-genome sequencing and RNAseq of divergent mosquito species and explored additional male-inclusive datasets to investigate the distribution of Nix. Except for the Culex genus, Nix homologs were found in all species surveyed from the Culicinae subfamily, including 12 additional species from three highly divergent tribes comprising 4 genera, suggesting Nix originated at least 133-165 MYA. Heterologous expression of one of three divergent Nix ORFs in Ae. aegypti resulted in partial masculinization of genetic females as evidenced by morphology and doublesex splicing. Phylogenetic analysis suggests Nix is related to femaleless (fle), a recently described intermediate sex-determining factor found exclusively in anopheline mosquitoes. Nix from all species has a conserved structure, including three RNA-recognition motifs (RRMs), as does fle. However, Nix has evolved at a much faster rate than fle. The RRM3 of both Nix and fle are distantly related to the single RRM of a widely distributed and conserved splicing factor transformer-2 (tra2). RRM3-based phylogenetic analysis suggests this domain in Nix and fle may have evolved from tra2 or a tra2-related gene in a common ancestor of mosquitoes. Our results provide insights into the evolution of sex-determination in mosquitoes and will inform broad applications of mosquito-control strategies based on manipulating sex ratios towards the non-biting males.

James K Biedler, Azadeh Aryan, Yumin Qi, Aihua Wang, Ellen O Martinson, Daniel A Hartman, Fan Yang, Atashi Sharma, Katherine S Morton, Mark Potters, Chujia Chen, Stephen L Dobson, Gregory D Ebel, Rebekah C Kading, Sally Paulson, Rui-De Xue, Michael R Strand, Zhijian Tu. Mol Biol Evol. 2023 Dec 21:msad276. doi: 10.1093/molbev/msad276. Online ahead of print.

Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy

Ultrastructural expansion microscopy (U-ExM) workflow and summary of parasite structures imaged in this study.
Ultrastructural expansion microscopy (U-ExM) workflow and summary of parasite structures imaged in this study.

Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample by ~4.5×. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have cataloged 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.

Benjamin Liffner, Ana Karla Cepeda Diaz, James Blauwkamp, David Anaguano, Sonja Frolich, Vasant Muralidharan, Danny W Wilson, Jeffrey D Dvorin, Sabrina Absalon. Elife. 2023 Dec 18:12:RP88088. doi: 10.7554/eLife.88088.

Advances in the cellular biology, biochemistry, and molecular biology of acidocalcisomes

Fig 1 Ultrastructure of acidocalcisomes
Fig 1 Ultrastructure of acidocalcisomes.

Acidocalcisomes are organelles conserved during evolution and closely related to the so-called volutin granules of bacteria and archaea, to the acidocalcisome-like vacuoles of yeasts, and to the lysosome-related organelles of animal species. All these organelles have in common their acidity and high content of polyphosphate and calcium. They are characterized by a variety of functions from storage of phosphorus and calcium to roles in Ca2+ signaling, osmoregulation, blood coagulation, and inflammation. They interact with other organelles through membrane contact sites or by fusion, and have several enzymes, pumps, transporters, and channels.

Roberto Docampo. Microbiol Mol Biol Rev. 2023 Dec 15:e0004223. doi: 10.1128/mmbr.00042-23.

Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors

Fig 1. Workflow for the selection of the optimal qPCR assay.
Fig 1. Workflow for the selection of the optimal qPCR assay.

 

Background: Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease.

Methodology/principal findings: Current World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay.

Conclusions/significance: Given the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays.

 

Mary Doherty, Jessica R Grant, Nils Pilotte, Sasisekhar Bennuru, Kerstin Fischer, Peter U Fischer, Sara Lustigman, Thomas B Nutman, Kenneth Pfarr, Achim Hoerauf, Thomas R Unnasch, Hassan K Hassan, Samuel Wanji, Patrick J Lammie, Eric Ottesen, Charles Mackenzie, Steven A Williams. PLoS Negl Trop Dis. 2023 Dec 14;17(12):e0011815. doi: 10.1371/journal.pntd.0011815.

Trypanosoma cruzi heme responsive gene (TcHRG) plays a central role in orchestrating heme uptake in epimastigotes

Trypanosoma cruzi, a heme auxotrophic parasite, can control intracellular heme content by modulating heme responsive gene (TcHRG) expression when a free heme source is added to an axenic culture. Herein, we explored the role of TcHRG protein in regulating the uptake of heme derived from hemoglobin in epimastigotes. We demonstrate that the endogenous TcHRG (protein and mRNA) responded similarly to bound (hemoglobin) and free (hemin) heme. Endogenous TcHRG was found in the flagellar pocket boundaries and partially overlapping with the mitochondrion. On the other hand, endocytic null parasites were able to develop and exhibited a similar heme content compared to wild type when fed with hemoglobin, indicating that endocytosis is not the main entrance pathway for hemoglobin-derived heme in this parasite. Moreover, the overexpression of TcHRG led to an increase in heme content when hemoglobin was used as the heme source. Taken together, these results suggest that the uptake of hemoglobin-derived heme likely occurs through extracellular proteolysis of hemoglobin via the flagellar pocket, and this process is governed by TcHRG. In sum, T. cruzi epimastigotes control heme homeostasis by modulating TcHRG expression independently of the available source of heme.

Evelyn Tevere, Cecilia Beatriz Di Capua, Nathan Michael Chasen, Ronald Drew Etheridge, Julia Alejandra Cricco. FEBS J. 2023 Dec 13. doi: 10.1111/febs.17030.

Increased environmental microbial diversity reduces the disease risk of a mosquitocidal pathogen

Fig 6 Ch_R13E2-SpR systemically infects A. aegypti larvae.
Fig 6 Ch_R13E2-SpR systemically infects A. aegypti larvae.

The host-specific microbiotas of animals can both reduce and increase disease risks from pathogens. In contrast, how environmental microbial communities affect pathogens is largely unexplored. Aquatic habitats are of interest because water enables environmental microbes to readily interact with animal pathogens. Here, we focused on mosquitoes, which are important disease vectors as terrestrial adults but are strictly aquatic as larvae. We identified a pathogen of mosquito larvae from the field as a strain of Chromobacterium haemolyticum. Comparative genomic analyses and functional assays indicate this strain and other Chromobacterium are mosquitocidal but are also opportunistic pathogens of other animals. We also identify a critical role for diversity of the environmental microbiota in disease risk. Our study characterizes both the virulence mechanisms of a pathogen and the role of the environmental microbiota in disease risk to an aquatic animal of significant importance to human health.

Zhiwei Kang, Vincent G Martinson, Yin Wang, Kerri L Coon, Luca Valzania, Michael R Strand. mBio. 2023 Dec 6:e0272623. doi: 10.1128/mbio.02726-23.

Blood meals from ‘dead-end’ vertebrate hosts enhance transmission potential of malaria-infected mosquitoes

graphical abstract

Ingestion of an additional blood meal(s) by a hematophagic insect can accelerate development of several vector-borne parasites and pathogens. Most studies, however, offer blood from the same vertebrate host species as the original challenge (for e.g., human for primary and additional blood meals). Here, we show a second blood meal from bovine and canine hosts can also enhance sporozoite migration in Anopheles stephensi mosquitoes infected with the human- and rodent-restricted Plasmodium falciparum and P. berghei, respectively. The extrinsic incubation period (time to sporozoite appearance in salivary glands) showed more consistent reductions with blood from human and bovine donors than canine blood, although the latter’s effect may be confounded by the toxicity, albeit non-specific, associated with the anticoagulant used to collect whole blood from donors. The complex patterns of enhancement highlight the limitations of a laboratory system but are nonetheless reminiscent of parasite host-specificity and mosquito adaptations, and the genetic predisposition of An. stephensi for bovine blood. We suggest that in natural settings, a blood meal from any vertebrate host could accentuate the risk of human infections by P. falciparum: targeting vectors that also feed on animals, via endectocides for instance, may reduce the number of malaria-infected mosquitoes and thus directly lower residual transmission. Since endectocides also benefit animal health, our results underscore the utility of the One Health framework, which postulates that human health and well-being is interconnected with that of animals. We posit this framework will be further validated if our observations also apply to other vector-borne diseases which together are responsible for some of the highest rates of morbidity and mortality in socio-economically disadvantaged populations.

Ashutosh K Pathak, Justine C Shiau, Rafael C S Freitas, Dennis E Kyle. One Health. 2023 Jun 9:17:100582. doi: 10.1016/j.onehlt.2023.100582. eCollection 2023 Dec.

Undergraduate Research Experience Sparked Interest in Parasitology for Graduate Student

doctoral student Victoria Mendiola

My name is Victoria Mendiola and I am a PhD candidate in Dennis Kyle’s lab studying drug-induced dormancy in Plasmodium falciparum, the parasite responsible for malaria. I have been at UGA for four years but originally received my BSc in Biology and MSc of Integrative Biology from Kennesaw State University in Kennesaw, GA.

My interest in infectious diseases stems from an NSF REU research internship where I was first introduced to the complexities of parasite-host interactions on an organismal level by studying hookworm infections in South American fur seals (SAFS) in the Gottdenker Lab at UGA’s College of Veterinary Medicine.

During my REU, I fell in love with Athens and the scientific community in the area but the large number of tropical disease parasitologists solidified my reason for choosing UGA to continue my studies.

My doctoral research focuses on developing novel high-content imaging assays to incorporate Artemisinin-induced dormant Plasmodium falciparum recovery into the current understanding of drug treatment, therapeutics, and prevention. Of the species of Plasmodium that infect humans, P. falciparum is the deadliest and, unfortunately, is becoming resistant to current treatment options.

In August 2023, I received the CTEGD Training in Tropical and Emerging Global Diseases fellowship. In addition to providing up to two years of funding, there is also the opportunity for a capstone experience. I plan to use the capstone project opportunity to gain essential in-field, on-site training to complement my current wet lab skillset.

My long-term career goal is to utilize my diverse training in physiology, developmental biology, cellular biology, and infectious diseases to design, optimize, and implement phenotypic and behavioral assays in the context of drug discovery and parasite homeostasis.

For students who are interested in joining the Center for Tropical and Emerging Global Diseases, I suggest they take every opportunity to talk to other researchers in and out of their field and organism of study. The sense of community within the CTEGD is unparalleled and should be utilized at every given opportunity. The friends I have made in and outside of the lab is one of my favorite things about being here at UGA (but the local festivals are really fun too).

Support trainees like Victoria by giving today to the Center for Tropical & Emerging Global Diseases.

The mosquito Aedes aegypti requires a gut microbiota for normal fecundity, longevity and vector competence

Mosquitoes shift from detritus-feeding larvae to blood-feeding adults that can vector pathogens to humans and other vertebrates. The sugar and blood meals adults consume are rich in carbohydrates and protein but are deficient in other nutrients including B vitamins. Facultatively hematophagous insects like mosquitoes have been hypothesized to avoid B vitamin deficiencies by carryover of resources from the larval stage. However, prior experimental studies have also used adults with a gut microbiota that could provision B vitamins. Here, we used Aedes aegypti, which is the primary vector of dengue virus (DENV), to ask if carryover effects enable normal function in adults with no microbiota. We show that adults with no gut microbiota produce fewer eggs, live longer with lower metabolic rates, and exhibit reduced DENV vector competence but are rescued by provisioning B vitamins or recolonizing the gut with B vitamin autotrophs. We conclude carryover effects do not enable normal function.

Ruby E Harrison, Xiushuai Yang, Jai Hoon Eum, Vincent G Martinson, Xiaoyi Dou, Luca Valzania, Yin Wang, Bret M Boyd, Mark R Brown, Michael R Strand. Commun Biol. 2023 Nov 13;6(1):1154. doi: 10.1038/s42003-023-05545-z.