Two New Species of Trypanosomatid Parasites Isolated from Heteroptera in Costa Rica

ABSTRACT. Two new trypanosomatid species (Euglenozoa, Kinetoplastea) isolated from the intestinal tract of heteropteran insect hosts were described based on molecular phylogenetic analyses of Spliced Leader (SL) RNA gene repeats, glycosomal glyceraldehyde phosphate dehydrogenase, and small subunit ribosomal RNA genes, as well as by morphology. Leptomonas barvae n. sp., from a mirid host Collaria oleosa, was found to represent one of the closest monoxenous (one host) relatives of the dixenous (two hosts) parasitic genus Leishmania. This finding further supports the origin of these dixenous parasites from monoxenous progenitors in the Neotropics. Blastocrithidia largi n. sp., from a largid host Largus cinctus, is among a few members of this genus available in culture. The species is a close relative of Blastocrithidia triatomae and is a member of a new monophyletic phylogenetic group characterized by formation of straphanger cysts.     

The Immune Response of Hemocytes of the Insect Oncopeltus fasciatus against the Flagellate Phytomonas serpens

The genus Phytomonas includes parasites that are etiological agents of important plant diseases, especially in Central and South America. These parasites are transmitted to plants via the bite of an infected phytophagous hemipteran. Despite the economic impact of these parasites, many basic questions regarding the genus Phytomonas remain unanswered, such as the mechanism by which the parasites cope with the immune response of the insect vector. In this report, using a model of systemic infection, we describe the function of Oncopeltus fasciatus hemocytes in the immune response towards the tomato parasite Phytomonas serpens. Hemocytes respond to infection by trapping parasites in nodular structures and phagocytizing the parasites. In electron microscopy of hemocytes, parasites were located inside vacuoles, which appear fused with lysosomes. The parasites reached the O. fasciatus salivary glands at least six hours post-infection. After 72 hours post-infection, many parasites were attached to the salivary gland outer surface. Thus, the cellular responses did not kill all the parasites.     

Probing into the diversity of trypanosomatid flagellates parasitizing insect hosts in South-West China reveals both endemism and global dispersal

Flagellates of the class Kinetoplastea are known to frequently parasitize insects. We have collected 67 isolates from 407 Heteroptera hosts captured in several locations of South-West China. Their splice leader (SL) RNA gene repeats and small subunit (SSU) rRNA genes were PCR amplified from the infected tissue samples. In most cases, parasites were found in the midgut, rarely the infection was confined to the Malpighian tubes. Phylogenetic analysis of the obtained sequences has significantly expanded the known diversity of these monoxenous parasites. Fifteen typing units were found among these isolates including 11 potentially new species. Four typing units matched the previously known typing units from the Neotropics indicating a global distribution of the respective parasite species. At the same time, new clades appeared, testifying for a certain level of endemism. The host record of the parasites found indicated a variable specificity level of the host–parasite association including several cases of a very broad host range. Our results disprove the “one host – one parasite” paradigm and show that although the global diversity of monoxenous parasites is high, it is not as enormous as suggested earlier. Moreover, phylogenetic analysis revealed the presence, among the isolated strains, of a new Phytomonas species, which is the first documentation of this potentially pathogenic dixenous parasite of plants in China.     

A PCR-based survey on Phytomonas (Euglenozoa: Trypanosomatidae) in phytophagous hemipterans of the Amazon region

ABSTRACT. We have surveyed 244 hemipterans from Western Brazilian Amazônia for the presence of trypanosomatids and identification of members of the genus Phytomonas. Examination by phase microscopy of squashes of insect salivary glands (SG) and digestive tubes (DT) revealed that 44% (108/244) of insects from seven families harbored trypanosomatids. Infections were 5 times more frequent in Coreidae than in all other families together. Smears of SG and DT of the dissected insects were fixed on glass slides with methanol and stained with Giemsa for morphological analysis. DNA was recovered from these preparations and submitted to a PCR assay that permitted amplification of all trypanosomatid genera using primers of conserved sequences flanking a segment of the spliced leader (SL) gene. Upon PCR amplification of the recovered DNA, amplicons were hybridized with an oligonucletide probe (SL3′) complementary to a SL intron sequence specific for flagellates of the genus Phytomonas. Among the trypanosomatid‐positive insects, 38.8% harbored Phytomonas spp., corresponding to an overall Phytomonas prevalence of 17.1% among phytophagous bugs, their putative vectors. Since many Phytomonas are pathogenic in plants, this high prevalence in their vectors emphasizes the permanent risk of exposure to disease by native and cultured plants of the Amazon region.     

Microbial Population Analysis of the Salivary Glands of Ticks; A Possible Strategy for the Surveillance of Bacterial Pathogens

Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (Ixodes ovatus, I. persulcatus and Haemaphysalis flava) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Rickettsia-specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in I. persulcatus female samples. The numbers of bacterial genera identified for the tick species were 71 (I. ovatus), 127 (I. persulcatus) and 59 (H. flava). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was Coxiella. Spiroplasma was detected in Ixodes, and not in H. flava. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female I. persulcatus samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.     

The interaction between Trypanosoma rangeli and the nitrophorins in the salivary glands of the triatomine Rhodnius prolixus (Hemiptera; Reduviidae)

The parasite Trypanosoma rangeli develops in the intestinal tract of triatomines and, particularly in species of the genus Rhodnius, invades the hemolymph and salivary glands, where subsequent metacyclogenesis takes place. Many aspects of the interaction between T. rangeli and triatomines are still unclear, especially concerning the development of the parasite in the salivary glands and how the parasite interacts with the saliva. In this work, we describe new findings on the process of T. rangeli infection of the salivary glands and the impact of infection on the saliva composition. To ensure a complete infection (intestinal tract, hemolymph and salivary glands), 3rd instar Rhodnius prolixus nymphs were fed on blood containing T. rangeli epimastigotes using an artificial feeder. After molt to the 4th instar, the nymphs were inoculated with epimastigotes in the hemolymph. The results showed that the flagellates started to invade the salivary glands by the 7th day after the injection. The percentage of trypomastigotes inside the salivary glands continuously increased until the 25th day, at which time the trypomastigotes were more than 95% of the T. rangeli forms present. The salivary contents from T. rangeli-infected insects showed a pH that was significantly more acidic (<6.0) and had a lower total protein and hemeprotein contents compared with non-infected insects. However, the ratio of hemeprotein to total protein was similar in both control and infected insects. qPCR demonstrated that the expression levels of three housekeeping genes (18S rRNA, β-actin and α-tubulin) and nitrophorins 1–4 were not altered in the salivary glands after an infection with T. rangeli. In addition, the four major nitrophorins (NPs 1–4) were knocked down using RNAi and their suppression impacted T. rangeli survival in the salivary glands to the point that the parasite burden inside the R. prolixus salivary glands was reduced by more than 3-fold. These results indicated that these parasites most likely non-specifically incorporated the proteins that were present in R. prolixus saliva as nutrients, without impairing the biosynthesis of the antihemostatic molecules.     

A putative kinase-related protein (PKRP) from Plasmodium berghei mediates infection in the midgut and salivary glands of the mosquito

The completion of the Plasmodium (malaria) life cycle in the mosquito requires the parasite to traverse first the midgut and later the salivary gland epithelium. We have identified a putative kinase-related protein (PKRP) that is predicted to be an atypical protein kinase, which is conserved across many species of Plasmodium. The pkrp gene encodes a RNA of about 5300 nucleotides that is expressed as a 90 kDa protein in sporozoites. Targeted disruption of the pkrp gene in Plasmodium berghei, a rodent model of malaria, compromises the ability of parasites to infect different tissues within the mosquito host. Early infection of mosquito midgut is reduced by 58-71%, midgut oocyst production is reduced by 50-90% and those sporozoites that are produced are defective in their ability to invade mosquito salivary glands. Midgut sporozoites are not morphologically different from wild-type parasites by electron microscopy. Some sporozoites that emerged from oocysts were attached to the salivary glands but most were found circulating in the mosquito hemocoel. Our findings indicate that a signalling pathway involving PbPKRP regulates the level of Plasmodium infection in the mosquito midgut and salivary glands.     

Lineage‐specific activities of a multipotent mitochondrion of trypanosomatid flagellates

Trypanosomatids are a very diverse group composed of monoxenous and dixenous parasites belonging to the excavate class Kinetoplastea. Here we studied the respiration of five monoxenous species (Blechomonas ayalai, Herpetomonas muscarum, H. samuelpessoai, Leptomonas pyrrhocoris and Sergeia podlipaevi) introduced into culture, each representing a novel yet globally distributed and/or species‐rich clade, and compare them with well‐studied flagellates Trypanosoma brucei, Phytomonas serpens, Crithidia fasciculata and Leishmania tarentolae. Differences in structure and activities of respiratory chain complexes, respiration and other biochemical parameters recorded under laboratory conditions reveal their substantial diversity, likely a reflection of different host environments. Phylogenetic relationships of the analysed trypanosomatids do not correlate with their biochemical parameters, with the differences within clades by far exceeding those among clades. As the S. podlipaevi canonical respiratory chain complexes have very low activities, we believe that its mitochondrion is utilised for purposes other than oxidative phosphorylation. Hence, the single reticulated mitochondrion of diverse trypanosomatids seems to retain multipotency, with the capacity to activate its individual components based on the host environment.     

Sex-specific glands in tephritid fruit flies of the genera Anastrepha,Ceratitis, Dacus and Rhagoletis (Diptera : Tephritidae)

The objectives of this work were to systematically examine males and females of tephritid fruit flies from the genera Anastrepha, Ceratitis, Dacus and Rhagoletis for the presence of possible sex pheromone glands, and to determine whether particular glands may be characteristic of certain genera. Flies from the 4 genera were examined from paraffin embedded histological sections. Pleural epidermal glands and sexually dimorphic, enlarged salivary glands were found in males, but not in females, of 7 species of Anastrepha and in 2 species of Ceratitis. Females of these species do not develop the pleural epidermal glands at all, and although they have salivary glands, the glands do not have the large glandular mass of tissue that is found in males. The pleural glands were similar in all the males, and consisted of a single layer of enlarged, columnar epidermal cells in the pleural region of abdominal segments 3, 4 and 5. The salivary glands of males were divided into 4 types based upon structure.Pleural glands and enlarged salivary glands were searched for, but were not found, in 2 species of Rhagoletis and in 3 species of Dacus. Anal glands, opening externally on the last segment, were confirmed in C. capitata, and were found in C. rosa. Anal glands were not found in any of the other species. A diverticulum from the rectum, the rectal pouch or gland, was confirmed in D. dorsalis, D. cucurbitae and D. tryoni as already published, but this gland was not found in species of other genera. Growth of the pleural glands and salivary glands in males of A. ludens, A. fraterculus and C. capitata was described as a function of age of flies.     

Anopheles Gambiae PRS1 Modulates Plasmodium Development at Both Midgut and Salivary Gland Steps

Background

Invasion of the mosquito salivary glands by Plasmodium is a critical step for malaria transmission. From a SAGE analysis, we previously identified several genes whose expression in salivary glands was regulated coincident with sporozoite invasion of salivary glands. To get insights into the consequences of these salivary gland responses, here we have studied one of the genes, PRS1 (Plasmodium responsive salivary 1), whose expression was upregulated in infected glands, using immunolocalization and functional inactivation approaches.

Methodology/Principal Findings

PRS1 belongs to a novel insect superfamily of genes encoding proteins with DM9 repeat motifs of uncharacterized function. We show that PRS1 is induced in response to Plasmodium, not only in the salivary glands but also in the midgut, the other epithelial barrier that Plasmodium has to cross to develop in the mosquito. Furthermore, this induction is observed using either the rodent parasite Plasmodium berghei or the human pathogen Plasmodium falciparum. In the midgut, PRS1 overexpression is associated with a relocalization of the protein at the periphery of invaded cells. We also find that sporozoite invasion of salivary gland cells occurs sequentially and induces intra-cellular modifications that include an increase in PRS1 expression and a relocalization of the corresponding protein into vesicle-like structures. Importantly, PRS1 knockdown during the onset of midgut and salivary gland invasion demonstrates that PRS1 acts as an agonist for the development of both parasite species in the two epithelia, highlighting shared vector/parasite interactions in both tissues.

Conclusions/Significance

While providing insights into potential functions of DM9 proteins, our results reveal that PRS1 likely contributes to fundamental interactions between Plasmodium and mosquito epithelia, which do not depend on the specific Anopheles/P. falciparum coevolutionary history.     

A Cysteine Protease Inhibitor of Plasmodium berghei Is Essential for Exo-erythrocytic Development

Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.     

The Puf-family RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite

Malaria is a vector-borne infectious disease caused by unicellular, obligate intracellular parasites of the genus Plasmodium. During host switch the malaria parasite employs specialized latent stages that colonize the new host environment. Previous work has established that gametocytes, sexually differentiated stages that are taken up by the mosquito vector, control expression of genes required for mosquito colonization by translational repression. Sexual parasite development is controlled by a DEAD-box RNA helicase of the DDX6 family, termed DOZI. Latency of sporozoites, the transmission stage injected during an infectious blood meal, is controlled by the eIF2alpha kinase IK2, a general inhibitor of protein synthesis. Whether RNA-binding proteins participate in translational regulation in sporozoites remains to be studied. Here, we investigated the roles of two RNA-binding proteins of the Puf-family, Plasmodium Puf1 and Puf2, during sporozoite stage conversion. Our data reveal that, in the rodent malaria parasite P. berghei, Puf2 participates in the regulation of IK2 and inhibits premature sporozoite transformation. Inside mosquito salivary glands puf2(-) sporozoites transform over time to round forms resembling early intra-hepatic stages. As a result, mutant parasites display strong defects in initiating a malaria infection. In contrast, Puf1 is dispensable in vivo throughout the entire Plasmodium life cycle. Our findings support the notion of a central role for Puf2 in parasite latency during switch between the insect and mammalian hosts.     

Salivary glands in Cicadidae (Hemiptera: Cicadoidea): comparative morphology, ultrastructure, and their phylogenetic significance

The present investigation provides information on gross morphology and ultrastructure of salivary glands of species in Cicadidae in detail. The structure of the salivary glands of 11 representative species from 10 genera belonging to three subfamilies of Cicadidae was studied using light microscopy and transmission electron microscopy. In the examined species, the salivary glands are paired structures, and each of which is comprised of a principal gland (pg) and an accessory gland (ag). The pg is divided into anterior and posterior lobes, and both of which consist of numerous long digitate lobules. The lobules at the base of the long digitate lobules of posterior lobe are greatly short; here, we named as “short lobules.” All the lobules vary in size, disposition, length, and shape. The anterior lobe and posterior lobes are connected by an anterior–posterior duct (apd). Two efferent salivary ducts (esd), derived from corresponding posterior lobes, fuse to form a short common duct which enters into the saliva syringe. The ag is composed of a greatly tortuous and folded accessory salivary tube, a gular gland (gg) constituting of several acini, and an accessory salivary duct (asd). The asd joins the esd at the place where the latter emergences. Constituents and arrangement of the salivary glands, the number and shape of the long digitate lobules in the anterior and posterior lobes, and the visibility of the apd were promising characters for the taxonomic and phylogenetic analysis of Cicadoidea. The variations of secretory granules in size, shape, and electron density in lobule cells of pg of Platypleura kaempferi probably indicating different materials are synthesized. The absence of the infoldings of basal plasma membrane in the basal area of the cells and the presence of electron-lucent vesicles in the cytoplasm of the gg cells of P. kaempferi might suggest that the secretions of gg are more watery.