Glenn A McConkey ^*

• University of Leeds, Leeds, United Kingdom

We are at a time in science with great prospects for significant contributions by rising stars in parasitology. This field is enhanced by major discoveries and developments in molecular and cell biology; particularly the rapid advancement of genomics, other “omics” and high throughput technologies. The application of molecular techniques from PCR to single-cell sequencing has been poignant in advances in detecting, tracking and characterizing protozoan parasites. The knowledge gained has been utilized for investigating the evolution, epidemiology and biology of these microscopic organisms. In the past several years, the field of molecular biology and genetics of parasites has witnessed a remarkable evolution, particularly in the realms of genomics and high-throughput next-generation sequencing (NGS). The advent of NGS technologies has revolutionized our understanding of their genomes’ complexity and functionality. These advances have enabled the comprehensive analysis of many parasite genomes, leading to the identification of numerous genetic variations and their unique characteristics. The integration of NGS with 'omics' approaches, such as transcriptomics, proteomics, and metabolomics, has facilitated a more holistic view of parasite biological systems. This synergy has been pivotal in unravelling the intricate networks of gene expression regulation, protein interactions, and metabolic pathways. The application of NGS in omics studies has also accelerated the discovery of markers for parasite diagnosis, epidemiology, and therapeutic target identification. Molecular parasitology has greatly benefited from these technological strides, allowing for the exploration of genetic landscapes in diverse parasites at an unprecedented resolution. The use of NGS in molecular genetics has provided insights into the mechanisms of parasite biology, life cycle stages and interactions with the host. The field of genomics has expanded our knowledge of numerous genomes with the Sanger Centre listing 25 annotated parasitic protozoan genomes. Efforts to further expand the number of protist genomes known have led to sequencing of 629 species (Genomes Online Database (GOLD), Joint Genome Institute) and the Protist 10,000 Genomes Project (https://ngdc.cncb.ac.cn/p10k/). This project is currently sequencing 1101 new species and has the goal of 10,000 of the 60,000-200,000 protist species. This has had significant implications for understanding parasite evolution, metabolism, and host evasion. The advances in molecular biology, genetics, and genomics have also been instrumental in the response to global health challenges. Indeed, the COVID-19 pandemic showed how these technologies could be applied on a global scale with technologies such as RT-PCR and NGS, that played a crucial role in the rapid sequencing of the SARS-CoV-2 genome. The techniques, applied in hundreds of testing facilities, were essential for the development of diagnostic tests, vaccines and treatments. Applications of these permitted strain detection, monitoring viral evolution, and tracking. In this Special Issue a series of articles written by rising stars in parasitology apply the transformative developments in molecular biology, molecular genetics and genomics driven by the advancements in NGS and omics technologies, to expand our understanding of parasitic infections and parasite biology. These breakthroughs have not only deepened our understanding of parasites at a molecular level but also opened new avenues for scientific research, with far-reaching implications for disease diagnosis and treatment. The articles in this Special Issue study parasitic protozoa in the phyla Euglenozoa, Metamonada and Apicomplexa with early branching kinetoplastid Trypanosoma brucei and Giardia as well as haemosporidia, respectively. Apicomplexa are a large phyum including Plasmodium, the agent responsible for malaria. Plasmodium species are host-specific with five species (from hundreds described) infecting humans. Coccidian Apicomplexa Cryptosporidium, Cystoisospora and Toxoplasma (that escapes the gut to become systemic) are zoonotic and can be transmitted between animals and humans. These coccidia are intestinal parasites as well as Giardia and Blastocystis which are studied in the articles. Trypansoma brucei, the agent responsible for African sleeping sickness, represents the flagellated kinetoplastids. Amongst the collection in Rising Stars in Parasite and Host: 2023 articles apply molecular biology techniques for identification, epidemiology, evolution, and diagnosis of parasitic infections. Three of the articles[1; 2; 3] utilise molecular techniques involving DNA amplification and sequencing for detection and characterisation of parasitic protozoa with application to diagnosis, screening, and epidemiology. Mutant libraries of T. brucei were developed for unbiased scanning of residue contributions in one study [4]. Articles in the collection employ molecular parasitology to investigate differences in life cycle stages and interactions with the host. Post-transcriptional mRNA editing investigations in T. brucei found life cycle stage differences between stages in vertebrate host with stages in the insect vector in one study [4]. T. gondii infection prompts a systemic immune response but parasites that reach immunoprivileged tissues convert to slow-growing tissue cyst forms that evade removal, establishing a chronic infection. The early immune responses of a type III lineage T. gondii strain were delineated in another study. Immune markers with qRT-PCR found that rather than association of the strain type with particular markers, immune response was associated with the mortality rate of the isolate [5]. Highly complex and weakly understood in host-parasite interactions, T. gondii induces host manipulation and behavior changes in animals that are advantageous in transmission of the parasite to the definitive feline host (termed ‘fatal feline attraction). Changes, at the molecular level, in microflora, neurotransmission and immune activation are evaluated in the final paper in the series [6]. The intestinal protozoan Blastocystis, a member of the Stramenopiles, responsible for diarrhea and gastrointestinal problems has traditionally been diagnosed microscopically by fecal smear. Mei et al [3] in this series developed an isothermal polymerase amplification with lateral flow detection as a rapid, sensitive, specific detection by dipstick. The test was consistent with the sensitivity of prior PCR assays of fecal samples and highly specific for this pathogen. The assay is rapid, easy and does not require microscopy expertise. Detection and differentiation of the intestinal parasites Cryptosporidium, Cystoisospora, and Giardia duodenalis and species identification was completed by PCR and sequencing of fecal samples in one of the studies [1]. Molecular phylogenetic analysis identified subspecies of Giardia in assemblages A, B and C. By this approach zoonotic protozoan parasites were identified and characterised in cat populations. Molecular detection and identification were again applied in a third paper in the series. This time to avian infections to differentiate species of haemosporidia. The initial development of genus-specific detection of avian Plasmodium infection by PCR with species identification by sequencing was conducted in 1996 [7] This has grown as a method for species identification. In the study in the series presented here, a nested PCR approach was developed using broad haemosporidia primers for initial PCR with subsequent nested PCR for genus-specific amplification [2] PCR detection is challenging in birds due to the nucleated red blood cells that increase background. Lineages of Plasmodium, Haemoproteus and Leucocytozoa were detected in a large collection of blood samples from passerine birds by the nested PCR protocol and sequencing. To uncover functional characteristics of a protein central to RNA editing in the parasitic protozoa trypanosomes, a high throughput mutational screening of libraries of mutants was developed. As an early diverged group, characterisation is hampered in comparative, bioinformatic analysis by low homology to known proteins; necessitating experimental mutant analysis. RNA editing in kinetoplastid mitochondria involves uridine insertions and deletions to generate open reading frames in mRNAs. In this study, thousands of variants were tested for their effect on parasite growth in bloodstream forms [4]. This permitted detection of amino acid residues involved functionally but not apparent from conservation in sequence alignments for this highly divergent organism. RNA editing variants differed in their phenotype between vertebrate bloodstream and insect host stages of their life cycle. Host interactions were further investigated in the study on early immune responses in type III T. gondii [5]. The genus Toxoplasma contains a single species but this species is composed of several strains with the three archetypes, type I, type II, type III that are distributed globally. The types differ in virulence with type I virulent in mice and types II and III non-virulent. Type II strains dominate human infections and are most associated with AIDS patients. Most prevalent in immunocompetent individuals with severe ocular toxoplasmosis was type I genotype parasites. Type I is also associated with severe congenital toxoplasmosis. Strain specific differences in immune responses has recently been reviewed by Saeij’s group [8]. Type I and III strains induce a much milder pro-inflammatory response than type II strains. Type III strains, that do not express the parasitic rhoptry protein ROP18, are sensitive in mice to cellular immune-related genes that destroy parasitic vacuoles in mice and hence rely on the virulence factor ROP16 to dampen the initial immune response early after infection. In the study in this series, isolates with different virulence were examined for cytokine levels and an immune marker by qRT-PCR found elevated systemic IFNγ in a high mortality strain whilst it was down-regulated in isolates with low mortality rates in mice. Arguably one of the most interesting facets in biology, at the interface of the parasite with the host, is altered host behavior induced by infection. This effect by T. gondii on mammals is presented in the paper by Prandovszky et al [6]. During stages of chronic infection, when the parasite is encysted in neurons in the host brain, changes in behavior of the intermediate host have been observed (reviewed in Chapter here) [9]. Notably, infected rodents are more active with increased exploratory activity, exhibit delayed arousal, and lose their innate fear of cat odors [10; 11; 12; 13]. Behaviour changes have been observed in a range of infected intermediate hosts from rodents, wolves and chimpanzees to humans [14; 15; 16]. Seroprevalence of T. gondii has been associated with schizophrenia in numerous studies [17]. Alteration in neurotransmission have been observed with changes in neurotransmission. The levels of catecholamines and dopaminergic and noradrenergic signalling is altered during chronic infection [10; 18; 19; 20; 21]. Changes have also been seen in the distribution of γ-aminobutyric acid biosynthetic enzyme GAD67, decreased glutamate transporter in astrocytes, and dendritic spine loss. The number and types of contributors to these neurological and behavioral changes during T. gondii infection remains unclear. Immune activation, parasite products, neuronal signalling, and microflora changes have been posited. Intriguingly, even mice infected with an attenuated T. gondii strain maintain their changes in behavior when the parasite is no longer detectable in the brain; implicating a long-term or permanent host change [22] This could support the involvement of an immune dysfunction, epigenetic changes in the host brain, or microflora dysbiosis induced by infection. Indeed, elevated levels of pro-inflammatory cytokines correlated with increased behavior changes [18]. Recently, T. gondii infection was found to induce DNA methylation changes in the vasopressin receptor gene and the key gene in norepinephrine synthesis in the brain and, importantly, paracrine signalling of the epigenetic changes via extracellular vesicles to uninfected bystander neurons was observed [20; 23]. Evidence of gut microflora changes have been observed during chronic infection; with enrichment of Bacteroidetes [24]. Acute infection of the intestine induces a dysbiosis and an imprint is maintained during chronic infection with multiple studies reporting an enrichment in the Verrucomicrobia. In the paper herein, several possible contributors are discussed with a focus on the potential role/s of gut microbial dysbiosis.