Single-cell RNA sequencing of Trypanosoma brucei from tsetse salivary glands unveils metacyclogenesis and identifies potential transmission blocking antigens.

Tsetse-transmitted African trypanosomes must develop into mammalian-infectious metacyclic cells in the fly's salivary glands (SGs) before transmission to a new host. The molecular mechanisms that underlie this developmental process, known as metacyclogenesis, are poorly understood. Blocking the few metacyclic parasites deposited in saliva from further development in the mammal could prevent disease. To obtain an in-depth perspective of metacyclogenesis, we performed single-cell RNA sequencing (scRNA-seq) from a pool of 2,045 parasites collected from infected tsetse SGs. Our data revealed three major cell clusters that represent the epimastigote, and pre- and mature metacyclic trypanosome developmental stages. Individual cell level data also confirm that the metacyclic pool is diverse, and that each parasite expresses only one of the unique metacyclic variant surface glycoprotein (mVSG) coat protein transcripts identified. Further clustering of cells revealed a dynamic transcriptomic and metabolic landscape reflective of a developmental program leading to infectious metacyclic forms preadapted to survive in the mammalian host environment. We describe the expression profile of proteins that regulate gene expression and that potentially play a role in metacyclogenesis. We also report on a family of nonvariant surface proteins (Fam10) and demonstrate surface localization of one member (named SGM1.7) on mature metacyclic parasites. Vaccination of mice with recombinant SGM1.7 reduced parasitemia early in the infection. Future studies are warranted to investigate Fam10 family proteins as potential trypanosome transmission blocking vaccine antigens. Our experimental approach is translationally relevant for developing strategies to prevent other insect saliva-transmitted parasites from infecting and causing disease in mammalian hosts.

Mutualist-Provisioned Resources Impact Vector Competency.

Many symbionts supplement their host's diet with essential nutrients. However, whether these nutrients also enhance parasitism is unknown. In this study, we investigated whether folate (vitamin B9) production by the tsetse fly (Glossina spp.) essential mutualist, Wigglesworthia, aids auxotrophic African trypanosomes in completing their life cycle within this obligate vector. We show that the expression of Wigglesworthia folate biosynthesis genes changes with the progression of trypanosome infection within tsetse. The disruption of Wigglesworthia folate production caused a reduction in the percentage of flies that housed midgut (MG) trypanosome infections. However, decreased folate did not prevent MG trypanosomes from migrating to and establishing an infection in the fly's salivary glands, thus suggesting that nutrient requirements vary throughout the trypanosome life cycle. We further substantiated that trypanosomes rely on symbiont-generated folate by feeding this vitamin to Glossina brevipalpis, which exhibits low trypanosome vector competency and houses Wigglesworthia incapable of producing folate. Folate-supplemented G. brevipalpis flies were significantly more susceptible to trypanosome infection, further demonstrating that this vitamin facilitates parasite infection establishment. Our cumulative results provide evidence that Wigglesworthia provides a key metabolite (folate) that is "hijacked" by trypanosomes to enhance their infectivity, thus indirectly impacting tsetse species vector competency. Parasite dependence on symbiont-derived micronutrients, which likely also occurs in other arthropod vectors, represents a relationship that may be exploited to reduce disease transmission.IMPORTANCE Parasites elicit several physiological changes in their host to enhance transmission. Little is known about the functional association between parasitism and microbiota-provisioned resources typically dedicated to animal hosts and how these goods may be rerouted to optimize parasite development. This study is the first to identify a specific symbiont-generated metabolite that impacts insect vector competence by facilitating parasite establishment and, thus, eventual transmission. Specifically, we demonstrate that the tsetse fly obligate mutualist Wigglesworthia provisions folate (vitamin B9) that pathogenic African trypanosomes exploit in an effort to successfully establish an infection in the vector's MG. This process is essential for the parasite to complete its life cycle and be transmitted to a new vertebrate host. Disrupting metabolic contributions provided by the microbiota of arthropod disease vectors may fuel future innovative control strategies while also offering minimal nontarget effects.

Transcriptome Profiling of Trypanosoma brucei Development in the Tsetse Fly Vector Glossina morsitans.

African trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals, have a complex digenetic life cycle between a mammalian host and an insect vector, the blood-feeding tsetse fly. Although the importance of the insect vector to transmit the disease was first realized over a century ago, many aspects of trypanosome development in tsetse have not progressed beyond a morphological analysis, mainly due to considerable challenges to obtain sufficient material for molecular studies. Here, we used high-throughput RNA-Sequencing (RNA-Seq) to profile Trypanosoma brucei transcript levels in three distinct tissues of the tsetse fly, namely the midgut, proventriculus and salivary glands. Consistent with current knowledge and providing a proof of principle, transcripts coding for procyclin isoforms and several components of the cytochrome oxidase complex were highly up-regulated in the midgut transcriptome, whereas transcripts encoding metacyclic VSGs (mVSGs) and the surface coat protein brucei alanine rich protein or BARP were extremely up-regulated in the salivary gland transcriptome. Gene ontology analysis also supported the up-regulation of biological processes such as DNA metabolism and DNA replication in the proventriculus transcriptome and major changes in signal transduction and cyclic nucleotide metabolism in the salivary gland transcriptome. Our data highlight a small repertoire of expressed mVSGs and potential signaling pathways involving receptor-type adenylate cyclases and members of a surface carboxylate transporter family, called PADs (Proteins Associated with Differentiation), to cope with the changing environment, as well as RNA-binding proteins as a possible global regulators of gene expression.

Transcript Abundance of Putative Lipid Phosphate Phosphatases During Development of Trypanosoma brucei in the Tsetse Fly.

African trypanosomes (Trypanosoma brucei spp.) cause devastating diseases in sub-Saharan Africa. Trypanosomes differentiate repeatedly during development in tsetse flies before gaining mammalian infectivity in fly salivary glands. Lipid phosphate phosphatases (LPPs) are involved in diverse biological processes, such as cell differentiation and cell migration. Gene sequences encoding two putative T. brucei LPP proteins were used to search the T. brucei genome, revealing two additional putative family members. Putative structural features and transcript abundance during parasite development in tsetse fly were characterized. Three of the four LPP proteins are predicted to have six transmembrane domains, while the fourth shows only one. Semiquantitative gene expression revealed differential regulation of LPPs during parasite development. Transcript abundance for three of the four putative LPP genes was elevated in parasites infecting salivary glands, but not mammalian-infective metacyclic cells in fly saliva, indicating a potential role of this family in parasite establishment in tsetse salivary glands.

Immunogenicity and Serological Cross-Reactivity of Saliva Proteins among Different Tsetse Species.

Tsetse are vectors of pathogenic trypanosomes, agents of human and animal trypanosomiasis in Africa. Components of tsetse saliva (sialome) are introduced into the mammalian host bite site during the blood feeding process and are important for tsetse's ability to feed efficiently, but can also influence disease transmission and serve as biomarkers for host exposure. We compared the sialome components from four tsetse species in two subgenera: subgenus Morsitans: Glossina morsitans morsitans (Gmm) and Glossina pallidipes (Gpd), and subgenus Palpalis: Glossina palpalis gambiensis (Gpg) and Glossina fuscipes fuscipes (Gff), and evaluated their immunogenicity and serological cross reactivity by an immunoblot approach utilizing antibodies from experimental mice challenged with uninfected flies. The protein and immune profiles of sialome components varied with fly species in the same subgenus displaying greater similarity and cross reactivity. Sera obtained from cattle from disease endemic areas of Africa displayed an immunogenicity profile reflective of tsetse species distribution. We analyzed the sialome fractions of Gmm by LC-MS/MS, and identified TAg5, Tsal1/Tsal2, and Sgp3 as major immunogenic proteins, and the 5'-nucleotidase family as well as four members of the Adenosine Deaminase Growth Factor (ADGF) family as the major non-immunogenic proteins. Within the ADGF family, we identified four closely related proteins (TSGF-1, TSGF-2, ADGF-3 and ADGF-4), all of which are expressed in tsetse salivary glands. We describe the tsetse species-specific expression profiles and genomic localization of these proteins. Using a passive-immunity approach, we evaluated the effects of rec-TSGF (TSGF-1 and TSGF-2) polyclonal antibodies on tsetse fitness parameters. Limited exposure of tsetse to mice with circulating anti-TSGF antibodies resulted in a slight detriment to their blood feeding ability as reflected by compromised digestion, lower weight gain and less total lipid reserves although these results were not statistically significant. Long-term exposure studies of tsetse flies to antibodies corresponding to the ADGF family of proteins are warranted to evaluate the role of this conserved family in fly biology.

The peritrophic matrix mediates differential infection outcomes in the tsetse fly gut following challenge with commensal, pathogenic, and parasitic microbes.

The insect gut is lined by a protective, chitinous peritrophic matrix (PM) that separates immunoreactive epithelial cells from microbes present within the luminal contents. Tsetse flies (Glossina spp.) imbibe vertebrate blood exclusively and can be exposed to foreign microorganisms during the feeding process. We used RNA interference-based reverse genetics to inhibit the production of a structurally robust PM and then observed how this procedure impacted infection outcomes after per os challenge with exogenous bacteria (Enterobacter sp. and Serratia marcescens strain Db11) and parasitic African trypanosomes. Enterobacter and Serratia proliferation was impeded in tsetse that lacked an intact PM because these flies expressed the antimicrobial peptide gene, attacin, earlier in the infection process than did their counterparts that housed a fully developed PM. After challenge with trypanosomes, attacin expression was latent in tsetse that lacked an intact PM, and these flies were thus highly susceptible to parasite infection. Our results suggest that immunodeficiency signaling pathway effectors, as opposed to reactive oxygen intermediates, serve as the first line of defense in tsetse's gut after the ingestion of exogenous microorganisms. Furthermore, tsetse's PM is not a physical impediment to infection establishment, but instead serves as a barrier that regulates the fly's ability to immunologically detect and respond to the presence of these microbes. Collectively, our findings indicate that effective insect antimicrobial responses depend largely upon the coordination of multiple host and microbe-specific developmental factors.

Starting small: using microbiology to foster scientific literacy.

In order to achieve scientific literacy for all students, Bard College recently implemented Citizen Science, a common January course for all first-year students. Structured around the question 'how do we reduce the global burden of disease?', this course uses microbiological tools to develop an understanding of potential answers.

Insights into the trypanosome-host interactions revealed through transcriptomic analysis of parasitized tsetse fly salivary glands.

The agents of sleeping sickness disease, Trypanosoma brucei complex parasites, are transmitted to mammalian hosts through the bite of an infected tsetse. Information on tsetse-trypanosome interactions in the salivary gland (SG) tissue, and on mammalian infective metacyclic (MC) parasites present in the SG, is sparse. We performed RNA-seq analyses from uninfected and T. b. brucei infected SGs of Glossina morsitans morsitans. Comparison of the SG transcriptomes to a whole body fly transcriptome revealed that only 2.7% of the contigs are differentially expressed during SG infection, and that only 263 contigs (0.6%) are preferentially expressed in the SGs (SG-enriched). The expression of only 37 contigs (0.08%) and 27 SG-enriched contigs (10%) were suppressed in infected SG. These suppressed contigs accounted for over 55% of the SG transcriptome, and included the most abundant putative secreted proteins with anti-hemostatic functions present in saliva. In contrast, expression of putative host proteins associated with immunity, stress, cell division and tissue remodeling were enriched in infected SG suggesting that parasite infections induce host immune and stress response(s) that likely results in tissue renewal. We also performed RNA-seq analysis from mouse blood infected with the same parasite strain, and compared the transcriptome of bloodstream form (BSF) cells with that of parasites obtained from the infected SG. Over 30% of parasite transcripts are differentially regulated between the two stages, and reflect parasite adaptations to varying host nutritional and immune ecology. These differences are associated with the switch from an amino acid based metabolism in the SG to one based on glucose utilization in the blood, and with surface coat modifications that enable parasite survival in the different hosts. This study provides a foundation on the molecular aspects of the trypanosome dialogue with its tsetse and mammalian hosts, necessary for future functional investigations.

Transcript expression analysis of putative Trypanosoma brucei GPI-anchored surface proteins during development in the tsetse and mammalian hosts.

Human African Trypanosomiasis is a devastating disease caused by the parasite Trypanosoma brucei. Trypanosomes live extracellularly in both the tsetse fly and the mammal. Trypanosome surface proteins can directly interact with the host environment, allowing parasites to effectively establish and maintain infections. Glycosylphosphatidylinositol (GPI) anchoring is a common posttranslational modification associated with eukaryotic surface proteins. In T. brucei, three GPI-anchored major surface proteins have been identified: variant surface glycoproteins (VSGs), procyclic acidic repetitive protein (PARP or procyclins), and brucei alanine rich proteins (BARP). The objective of this study was to select genes encoding predicted GPI-anchored proteins with unknown function(s) from the T. brucei genome and characterize the expression profile of a subset during cyclical development in the tsetse and mammalian hosts. An initial in silico screen of putative T. brucei proteins by Big PI algorithm identified 163 predicted GPI-anchored proteins, 106 of which had no known functions. Application of a second GPI-anchor prediction algorithm (FragAnchor), signal peptide and trans-membrane domain prediction software resulted in the identification of 25 putative hypothetical proteins. Eighty-one gene products with hypothetical functions were analyzed for stage-regulated expression using semi-quantitative RT-PCR. The expression of most of these genes were found to be upregulated in trypanosomes infecting tsetse salivary gland and proventriculus tissues, and 38% were specifically expressed only by parasites infecting salivary gland tissues. Transcripts for all of the genes specifically expressed in salivary glands were also detected in mammalian infective metacyclic trypomastigotes, suggesting a possible role for these putative proteins in invasion and/or establishment processes in the mammalian host. These results represent the first large-scale report of the differential expression of unknown genes encoding predicted T. brucei surface proteins during the complete developmental cycle. This knowledge may form the foundation for the development of future novel transmission blocking strategies against metacyclic parasites.

Blood parasites in birds from Madagascar.

Madagascar has long been recognized for its unique and diverse biota. In particular, significant effort has been made to establish baseline population data to better conserve the endemic avifauna. During field expeditions between 1993 and 2004, birds were mist-netted at 11 different sites, at elevations from 60 m to 2,050 m above sea level. Data on endemic status, forest type, and habitat preference were recorded. Thin blood films from 947 birds, belonging to 26 families and 64 species, were examined by light microscopy to determine the prevalence of blood parasites. Of these 947 birds, 30.7% were infected by at least one species of blood parasite, 26.8% of which were infected by more than one species. Species of Haemoproteus were the most prevalent (17.4%), followed by microfilariae (11.0%), Leucocytozoon spp. (9.4%), Plasmodium spp. (1.9%), Trypanosoma spp. (0.9%), and Babesia spp. (0.2%). Species level identifications confirmed the presence of 47 species of hemosporidians and trypanosomes, which is notably high and mirrors the diversity of their avian hosts. Eleven (23.4%) of these parasite species were new to science and thought to be endemic to the island. Significant differences in prevalence were observed by sample site, forest type (humid vs. dry), and habitat preference. Birds from all elevational zones sampled were infected, although not all parasite genera were present in each zone. Four of the six endemic avian families or subfamilies (Bernieridae, Brachypteraciidae, Philepittinae [Eurylaimidae], and Vangidae) were sampled and found to be parasitized. Of the families with the largest sample sizes, the Zosteropidae and Ploceidae had the highest prevalence of infection (65.6% and 49.3%, respectively). The vectors of hematozoan parasites in Madagascar are currently unknown. These results add to the current knowledge of avian parasitism in Madagascar and are of particular interest for the conservation of endemic species, as well as threatened or endangered populations.

Infections with immunogenic trypanosomes reduce tsetse reproductive fitness: potential impact of different parasite strains on vector population structure.

The parasite Trypanosoma brucei rhodesiense and its insect vector Glossina morsitans morsitans were used to evaluate the effect of parasite clearance (resistance) as well as the cost of midgut infections on tsetse host fitness. Tsetse flies are viviparous and have a low reproductive capacity, giving birth to only 6-8 progeny during their lifetime. Thus, small perturbations to their reproductive fitness can have a major impact on population densities. We measured the fecundity (number of larval progeny deposited) and mortality in parasite-resistant tsetse females and untreated controls and found no differences. There was, however, a typanosome-specific impact on midgut infections. Infections with an immunogenic parasite line that resulted in prolonged activation of the tsetse immune system delayed intrauterine larval development resulting in the production of fewer progeny over the fly's lifetime. In contrast, parasitism with a second line that failed to activate the immune system did not impose a fecundity cost. Coinfections favored the establishment of the immunogenic parasites in the midgut. We show that a decrease in the synthesis of Glossina Milk gland protein (GmmMgp), a major female accessory gland protein associated with larvagenesis, likely contributed to the reproductive lag observed in infected flies. Mathematical analysis of our empirical results indicated that infection with the immunogenic trypanosomes reduced tsetse fecundity by 30% relative to infections with the non-immunogenic strain. We estimate that a moderate infection prevalence of about 26% with immunogenic parasites has the potential to reduce tsetse populations. Potential repercussions for vector population growth, parasite-host coevolution, and disease prevalence are discussed.

Prevalence and distribution of pox-like lesions, avian malaria, and mosquito vectors in Kipahulu Valley, Haleakala National Park, Hawai'i, USA.

We determined prevalence and altitudinal distribution of introduced avian malarial infections (Plasmodium relictum) and pox-like lesions (Avipoxvirus) in forest birds from Kipahulu Valley, Haleakala National Park, on the island of Maui, and we identified primary larval habitat for the mosquito vector of this disease. This intensively managed wilderness area and scientific reserve is one of the most pristine areas of native forest remaining in the state of Hawai'i, and it will become increasingly important as a site for restoration and recovery of endangered forest birds. Overall prevalence of malarial infections in the valley was 8% (11/133) in native species and 4% (4/101) in nonnative passerines; prevalence was lower than reported for comparable elevations and habitats elsewhere in the state. Infections occurred primarily in 'Apapane (Himatione sanguinea) and Hawai'i 'Amakihi (Hemignathus virens) at elevations below 1,400 m. Pox-like lesions were detected in only two Hawai'i 'Amakihi (2%; 2/94) at elevations below 950 m. We did not detect malaria or pox in birds caught at 1,400 m in upper reaches of the valley. Adult mosquitoes (Culex quinquefasciatus) were captured at four sites at elevations of 640, 760, 915, and 975 m, respectively. Culex quinquefasciatus larvae were found only in rock holes along intermittent tributaries of the two largest streams in the valley, but not in standing surface water, pig wallows, ground pools, tree cavities, and tree fern cavities. Mosquito populations in the valley are low, and they are probably influenced by periods of high rainfall that flush stream systems.

Leucocytozoon atkinsoni n. sp. (Apicomplexa: Leucocytozoidae) from the avian family Timaliidae.

Investigators of haematozoa of the Timaliidae have reported the presence of two species of Leucocytozoon Berestneff, 1904, i.e. L. liothricis Laveran & Marullaz, 1914 and L. timaliae Bennett, Earlé & Pierce, 1993. Blood films collected from 42 wild-caught babblers in Madagascar were stained and examined for the presence of haematozoa using a compound microscope. To date, no species of avian haematozoa have been reported from babblers in Madagascar, although haematozoa have been observed. In the present study, we report a new species of Leucocytozoon, L. atkinsoni n. sp., whose morphometrics fall between those reported for the two previously described species from timaliids. The parasite is capped by the host cell nucleus covering 38% of its perimeter. L. atkinsoni n. sp. was found to have a marked, intensely staining, nucleolus as well as vacuoles in the parasite cytoplasm, in contrast to both L. liothricis and L. timaliae. Remnants of the host cell cytoplasm are commonly observed in cells infected with L. atkinsoni, a characteristic not reported in association with either of the previously described species from these hosts.

Haemoproteids of the avian family Dicruridae (the drongos).

Dicrurids are a widespread avian family in Africa and Asia. Earlier surveys of this family in these areas have reported the presence of hematozoa and 1 species of Haemoproteus, i.e., Haemoproteus dicruri (De Mello, 1935). One species of drongo occurs in Madagascar and has not been examined previously. Blood smears collected from wild-caught crested drongos, Dicrurus forficatus, in Madagascar were examined using a compound microscope for the presence of hematozoa. A new species, Haemoproteus khani, is described in this study. This new species has circumnuclear gametocytes, in contrast to the halteridial H. dicruri. In addition, H. dicruri is reported for the first time from the crested drongo and is redescribed. This is the first report of hematozoa in drongos of Madagascar.

A new species of Plasmodium from Malagasy vangas.

During a recent examination of blood smears from Malagasy birds, a species of avian Plasmodium unlike those currently known was observed. All infected birds were members of the Vangidae, which is endemic to Madagascar and the Comoro Islands. Plasmodium parvulum n. sp. is described, and classified as a member of the subgenus Haemamoeba because of gametocyte and schizont shape, displacement of the host cell nucleus, as well as distortion of the host cell. Round, rosettelike schizonts with 6-8 merozoites, clumped refractile granules, and little cytoplasm were observed. Both schizonts and mature, round gametocytes rotated and displaced the erythrocyte nucleus. A brief comparison to P. relictum is included.

Hematozoa of the avian family Brachypteraciidae (the ground-rollers).

The Brachypteraciidae is an avian family endemic to Madagascar. Members of this family were mist-netted in Madagascar, and blood smears were made to screen for the presence of hematozoa. Smears were stained with Giemsa and examined at x100, x160, and x1000 for hematozoa. Three new species of avian hematozoa from wild-caught ground-rollers in Madagascar are described. Haemoproteus goodmani n. sp. is found in the pitta-like ground-roller (Atelornis pittoides), whereas Haemoproteus forresteri n. sp. and Leucocytozoon frascai n. sp. are from the rufous-headed ground-roller (Atelornis crossleyi). These represent the first hematozoa described from this family.

Hematozoa of the avian family Philepittidae (the asities and sunbird asities).

Blood smears from birds in the Philepittidae, endemic to Madagascar, were examined for the presence of hematozoa. All slides were read on a compound microscope at x100, x160, and x1000. To date, no species of avian hematozoa has been reported from asities in Madagascar, although parasites have been observed. Leucocytozoon greineri n. sp. is described from the common sunbird asity (Neodrepanis coruscans) and is also reported from the velvet asity (Philepitta castanea). It is a relatively small species of Leucocytozoon, less than 2 times the size of uninfected erythrocytes. The parasite is capped by the host cell nucleus, covering 38% of the parasite perimeter. This represents the first description of hematozoa from this family.

Hematozoa of the avian family Vangidae (the vangas).

To date, limited surveys have been conducted on the endemic avifauna of Madagascar with regard to hematozoa. Wild-caught birds from the Vangidae, endemic to Madagascar and the Comoros Islands, were mist-netted, and blood smears were made. Slides were examined for the presence of hematozoa at x100, x160, and x1000 using a light microscope. Parasites were measured using established techniques, and morphometrics were compared. On the basis of their distinctive morphologies and morphometrics, 4 new species of avian hematozoa are described in this study. Haemoproteus vangii n. sp. and Leucocytozoon lairdi n. sp. occur in the blue vanga (Cyanolanius madagascarinus), whereas Haemoproteus madagascariensis n. sp. and Leucocytozoon bennetti n. sp. are described from the hook-billed vanga (Vanga curvirostris). These represent the first hematozoa described from this family.

Behavioral observations of adolescent Holstein heifers cloned from adult somatic cells.

Cloning using somatic cells offers many potential applications in biomedicine and basic research. The objective of this study was to test whether clones from the same genotype can be used as models to study the genetic influences of behavior. Specifically, several aspects of the behavior of four prepubertal heifers cloned from somatic cells of a 13-year-old Holstein cow along with age-matched control heifers were compared to determine whether juvenile clones from an aged adult behave similarly to their age-matched controls, and whether clones with identical genetic makeup exhibit any behavioral trends. Behavioral observations or behavior challenge tests were conducted to compare the following traits: vocalization, play behavior, movement frequencies, grooming, curiosity, and companion preference, as well as dominance and aggressiveness. From play behavior, movements and vocalization, we observed that these four juvenile clones of an aged genetic donor did not show behavioral indications of aging and were similar to their counterparts of comparable chronological age except that they tended to play less than controls. Behavioral trends were also observed in the clones that indicated that they exhibited higher levels of curiosity, more grooming activities and were more aggressive and dominant than controls. Furthermore, these four clones preferred each other or the donor as companions, which may indicate genetic kin recognition.