Promastigote EPS secretion and haptomonad biofilm formation as evolutionary adaptations of trypanosomatid parasites for colonizing honeybee hosts.

Bees are major pollinators involved in the maintenance of all terrestrial ecosystems. Biotic and abiotic factors placing these insects at risk is a research priority for ecological and agricultural sustainability. Parasites are one of the key players of this global decline and the study of their mechanisms of action is essential to control honeybee colony losses. Trypanosomatid parasites and particularly the Lotmaria passim are widely spread in honeybees, however their lifestyle is poorly understood. In this work, we show how these parasites are able to differentiate into a new parasitic lifestyle: the trypanosomatid biofilms. Using different microscopic techniques, we demonstrated that the secretion of Extracellular Polymeric Substances by free-swimming unicellular promastigote forms is a prerequisite for the generation and adherence of multicellular biofilms to solid surfaces in vitro and in vivo. Moreover, compared to human-infective trypanosomatid parasites our study shows how trypanosomatid parasites of honeybees increases their resistance and thus resilience to drastic changes in environmental conditions such as ultralow temperatures and hypoosmotic shock, which would explain their success thriving within or outside their hosts. These results set up the basis for the understanding of the success of this group of parasites in nature and to unveil the impact of such pathogens in honeybees, a keystones species in most terrestrial ecosystems.

Trypanosomatids in honey bee colonies in Spain: A new specific qPCR method for specific quantification of Lotmaria passim, Crithidia mellificae and Crithidia bombi.

Bee trypanosomatids have not been widely studied due to the original belief that these organisms were not pathogenic to honey bees. However, trypanosomatids have been linked to increased winter mortality in honey bee colonies in recent years and it has been shown that these pathogens can shorten a honey bee worker's lifespan in laboratory conditions. These studies found that this mortality corresponded to dose-dependent infection. Although Lotmaria passim is the most prevalent species worldwide, the natural load in colonies remains poorly investigated. Here we describe a new highly specific and sensitive qPCR method that allows the differentiation and quantification of the parasitic load of each of the three most common trypanosomatid species described to date in honey bee colonies: L. passim, Crithidia mellificae, and Crithidia bombi. We have used this new method to analyze honey bee colonies in central Spain and confirm that L. passim is the most common species and the one with higher parasitic loads in the colonies, which increased over the years, being higher in spring than in autumn. Crithidia mellificae was present along the study, with the highest prevalence in autumn 2019 and lately it was only found in non-quantifiable loads. Crithidia bombi was not detected in any of the colonies analyzed.

Imidacloprid increases the prevalence of the intestinal parasite Lotmaria passim in honey bee workers.

A challenge in bee protection is to assess the risks of pesticide-pathogen interactions. Lotmaria passim, a ubiquitous unicellular parasite in honey bees, is considered harmful under specific conditions. Imidacloprid causes unpredictable side effects. Research indicates that both L. passim and imidacloprid may affect the physiology, behavior, immunity, microbiome and lifespan of honey bees. We designed cage experiments to test whether the infection of L. passim is affected by a sublethal dose of imidacloprid. Workers collected at the time of emergence were exposed to L. passim and 2.5 µg/L imidacloprid in the coexposure treatment group. First, samples of bees were taken from cages since they were 5 days old and 3 days postinfection, i.e., after finishing an artificial 24 h L. passim infection. Additional bees were collected every two additional days. In addition, bees frozen at the time of emergence and collected from the unexposed group were analyzed. Abdomens were analyzed using qPCR to determine parasite load, while corresponding selected heads were subjected to a label-free proteomic analysis. Our results show that bees are free of L. passim at the time of emergence. Furthermore, imidacloprid considerably increased the prevalence as well as parasite loads in individual bees. This means that imidacloprid facilitates infection, enabling faster parasite spread in a colony and potentially to surrounding colonies. The proteomic analysis of bee heads showed that imidacloprid neutralized the increased transferrin 1 expression by L. passim. Importantly, this promising marker has been previously observed to be upregulated by infections, including gut parasites. This study contributes to understanding the side effects of imidacloprid and demonstrates that a single xenobiotic/pesticide compound can interact with the gut parasite. Our methodology can be used to assess the effects of different compounds on L. passim.

Frequent Parasitism of Apis mellifera by Trypanosomatids in Geographically Isolated Areas with Restricted Beekeeping Movements.

Trypanosomatids form a group of high prevalence protozoa that parasitise honey bees, with Lotmaria passim as the predominant species worldwide. However, the knowledge about the ecology of trypanosomatids in isolated areas is limited. The Portuguese archipelagos of Madeira and Azores provide an interesting setting to investigate these parasites because of their geographic isolation, and because they harbour honey bee populations devoid of two major enemies: Varroa destructor and Nosema ceranae. Hence, a total of 661 honey bee colonies from Madeira and the Azores were analysed using different molecular techniques, through which we found a high prevalence of trypanosomatids despite the isolation of these islands. L. passim was the predominant species and, in most colonies, was the only one found, even on islands free of V. destructor and/or N. ceranae with severe restrictions on colony movements to prevent the spread of them. However, islands with V. destructor had a significantly higher prevalence of L. passim and, conversely, islands with N. ceranae did not shown any significant correlation with the trypanosomatid. Crithidia bombi was detected in Madeira and on three islands of the Azores, almost always coincident with L. passim. By contrast, Crithidia mellificae was not detected in any sample. A high-throughput sequencing analysis distinguished two main haplotypes of L. passim, which accounted for 98% of the total sequence reads. This work suggests that L. passim and C. bombi are parasites that have been associated with honey bees predating the spread of V. destructor and N. ceranae.

Bee Trypanosomatids: First Steps in the Analysis of the Genetic Variation and Population Structure of Lotmaria passim, Crithidia bombi and Crithidia mellificae.

Trypanosomatids are among the most prevalent parasites in bees but, despite the fact that their impact on the colonies can be quite important and that their infectivity may potentially depend on their genotypes, little is known about the population diversity of these pathogens. Here we cloned and sequenced three non-repetitive single copy loci (DNA topoisomerase II, glyceraldehyde-3-phosphate dehydrogenase and RNA polymerase II large subunit, RPB1) to produce new genetic data from Crithidia bombi, C. mellificae and Lotmaria passim isolated from honeybees and bumblebees. These were analysed by applying population genetic tools in order to quantify and compare their variability within and between species, and to obtain information on their demography and population structure. The general pattern for the three species was that (1) they were subject to the action of purifying selection on nonsynonymous variants, (2) the levels of within species diversity were similar irrespective of the host, (3) there was evidence of recombination among haplotypes and (4) they showed no haplotype structuring according to the host. C. bombi exhibited the lowest levels of synonymous variation (πS= 0.06 ± 0.04 %) - and a mutation frequency distribution compatible with a population expansion after a bottleneck - that contrasted with the extensive polymorphism displayed by C. mellificae (πS= 2.24 ± 1.00 %), which likely has a more ancient origin. L. passim showed intermediate values (πS= 0.40 ± 0.28 %) and an excess of variants a low frequencies probably linked to the spread of this species to new geographical areas.

Hot and sour: parasite adaptations to honeybee body temperature and pH.

Host temperature and gut chemistry can shape resistance to parasite infection. Heat and acidity can limit trypanosomatid infection in warm-blooded hosts and could shape infection resistance in insects as well. The colony-level endothermy and acidic guts of social bees provide unique opportunities to study how temperature and acidity shape insect-parasite associations. We compared temperature and pH tolerance between three trypanosomatid parasites from social bees and a related trypanosomatid from poikilothermic mosquitoes, which have alkaline guts. Relative to the mosquito parasites, all three bee parasites had higher heat tolerance that reflected body temperatures of hosts. Heat tolerance of the honeybee parasite Crithidia mellificae was exceptional for its genus, implicating honeybee endothermy as a plausible filter of parasite establishment. The lesser heat tolerance of the emerging Lotmaria passim suggests possible spillover from a less endothermic host. Whereas both honeybee parasites tolerated the acidic pH found in bee intestines, mosquito parasites tolerated the alkaline conditions found in mosquito midguts, suggesting that both gut pH and temperature could structure host-parasite specificity. Elucidating how host temperature and gut pH affect infection-and corresponding parasite adaptations to these factors-could help explain trypanosomatids' distribution among insects and invasion of mammals.

Detection of Lotmaria passim in Africanized and European honey bees from Uruguay, Argentina and Chile.

Trypanosomatids affecting honey bees, Crithidia mellificae and Lotmaria passim, have been poorly studied in South America. We therefore analyzed their presence in Africanized and European honeybees from Uruguay, Argentina and Chile collected between 1990 and 2011 and assessed their association with other bee parasites and pathogens. Crithidia mellificae was not detected while L. passim was wide-spread. This report shows that L. passim has been present in this region at least since 2007 and it infects both Africanized and European honey bees. L. passim infected colonies showed high V. destructor parasitization levels, suggesting an association between them.

Differential diagnosis of the honey bee trypanosomatids Crithidia mellificae and Lotmaria passim.

Trypanosomatids infecting honey bees have been poorly studied with molecular methods until recently. After the description of Crithidia mellificae (Langridge and McGhee, 1967) it took about forty years until molecular data for honey bee trypanosomatids became available and were used to identify and describe a new trypanosomatid species from honey bees, Lotmaria passim (Evans and Schwarz, 2014). However, an easy method to distinguish them without sequencing is not yet available. Research on the related bumble bee parasites Crithidia bombi and Crithidia expoeki revealed a fragment length polymorphism in the internal transcribed spacer 1 (ITS1), which enabled species discrimination. In search of fragment length polymorphisms for differential diagnostics in honey bee trypanosomatids, we studied honey bee trypanosomatid cell cultures of C. mellificae and L. passim. This research resulted in the identification of fragment length polymorphisms in ITS1 and ITS1-2 markers, which enabled us to develop a diagnostic method to differentiate both honey bee trypanosomatid species without the need for sequencing. However, the amplification success of the ITS1 marker depends probably on the trypanosomatid infection level. Further investigation confirmed that L. passim is the dominant species in Belgium, Japan and Switzerland. We found C. mellificae only rarely in Belgian honey bee samples, but not in honey bee samples from other countries. C. mellificae was also detected in mason bees (Osmia bicornis and Osmia cornuta) besides in honey bees. Further, the characterization and comparison of additional markers from L. passim strain SF (published as C. mellificae strain SF) and a Belgian honey bee sample revealed very low divergence in the 18S rRNA, ITS1-2, 28S rRNA and cytochrome b sequences. Nevertheless, a variable stretch was observed in the gp63 virulence factor.

Effect of topoisomerase inhibitors and DNA-binding drugs on the cell proliferation and ultrastructure of Trypanosoma cruzi.

Trypanosomatids present unusual organelles, such as the kinetoplast that contains the mitochondrial DNA arranged in catenated circles. The nucleus of these protozoa presents distinct domains during interphase as well as a closed mitosis. DNA topoisomerases modulate the topological state of DNA by regulating supercoiling of the double-stranded DNA during replication, transcription, recombination and repair. Because topoisomerases play essential roles in cellular processes, they constitute a potential target for antitumour and antimicrobial drugs. In this study, the effects of various topoisomerase inhibitors and DNA-binding drugs were tested on the cellular proliferation and ultrastructure of the Trypanosoma cruzi epimastigote form Blastocrithidia culicis was used as a comparative model, which has a more relaxed kinetoplast DNA (kDNA) organization. The results showed that the eukaryotic topoisomerase I inhibitors camptothecin and rebeccamycin were the most effective compounds in the arrest of T. cruzi proliferation. Of the eukaryotic topoisomerase II inhibitors, mitoxantrone, but not merbarone, was effective against cell proliferation. The prokaryotic topoisomerase II inhibitors norfloxacin and enoxacin targeted the kinetoplast specifically, thus promoting ultrastructural kDNA rearrangement in B. culicis. Of the DNA-binding drugs, berenil caused remarkable kDNA disorganization. With the exception of camptothecin, there have been no previous evaluations of the compounds tested here on trypanosomatid ultrastructure. In conclusion, inhibitors of the same class may have different effects on trypanosomatid proliferation and ultrastructure. The results obtained in this work may help to reveal the mechanism of action of different topoisomerase inhibitors in trypanosomatids.

Trypanosomatids in dogs belonging to individuals with chronic chagas'disease living in Botucatu town and surrounding region, São Paulo State, Brazil

Among domestic animals, dogs are considered to be the major reservoirs of trypanosomatids and, due to their proximity to man, the presence of these parasites in dogs is an alert to actions aiming at triatomine control. Fifty dogs (26 males and 24 females), aged from 2 months to 15 years, belonging to 30 chronic Chagas’ disease individuals from 15 different municipalities in the western region of São Paulo State, Brazil, were subjected to blood collection for the following tests: artificial xenodiagnosis, blood culture, and Polymerase Chain Reaction (PCR). Forty-three (86%) out of 50 dogs were positive to at least one of the tests performed; 34 (68%) were positive to xenodiagnosis, 30 (60%) to blood culture, and 25 (50%) to PCR for T. cruzi and/or T. rangeli. Although triatomines were not detected during the intra and peridomiciliary inspections in the dog owners’ residences, the results obtained demonstrate that there is a transmission cycle whereby triatomine vector may be participating in the infection epidemiological chain

Parasite genotypically related to a monoxenous trypanosomatid of dog's flea causing opportunistic infection in an HIV positive patient

An HIV positive patient presenting a clinical picture of visceral leishmaniasis co-infection was submitted to a bone marrow aspiration after admission to hospital. Amastigotes forms were seen in the bone marrow aspirate and the parasite grew in culture as promastigotes. Molecular analyses showed that the flagellates isolated did not belong to the genera Leishmania, Trypanosoma or Sauroleishmania. It was not possible to establish infection in laboratory animals. In vitro culture of mouse peritoneal macrophages revealed the invasion of the host cells by the flagellates and their killing 48 hr after infection. Opportunistic infection with an insect trypanosomatid was suspected. Further hybridization analyses against a pannel of different monoxenous and heteroxenous trypanosomatids showed kDNA cross-homology with Leptomonas pulexsimulants a trypanosomatid found in the dog's flea.

Morphological evidence by scanning electron microscopy of excretion of metacyclic forms of Trypanosoma cruzi in vector's urine

Observando-se, em microscopia eletrônica de varedura, formas flageladas do Trypanosoma cruzi presas a cutícula da glândula retal de ninfas infectadas de Dipetalogaster maxima verificaram-se nítidas diferenças antes e depois da alimentaçäo. Antes, viam-se numerosos tripomastigotas metacíclicos entre os abundantes epimastigotas que formavam o tapete de flagelados, ao passo que nos insetos que urinavam dentro das 24 horas após o repasto os metacíclicos eram raros, indicando que haviam sido desprendidos pelo fluxo urinário. Foi notado, as vezes, um tipo assimétrico de divisäo celular, originando um epi e um tripomastigota. Nos flagelados dos epimastigotas a presença de dilataçöes a diferentes níveis sugere que lugares secundários de aderência podem ser comuns

Morphological evidence by scanning electron microscopy of excretion of metacyclic forms of Trypanosoma cruzi in vector's urine.

Comparison by scanning electron microscopy (SEM) of Trypanosoma cruzi flagellates attached to the cuticle of the rectal gland of infected Dipetalogaster maxima nymphs, showed marked differences before and after feeding. Before feeding numerous metacyclic trypomastigotes were observed among the abundant epimastigotes that formed the carpet of flagellates. On the other hand, in insects that were allowed to urinate for 24 hours after a meal, the metacyclics were scarce, indicating that they had been detached by the urine flow. An asymmetric type of cell division, probably originating both an epi- and a trypomastigote, was occasionally observed. The occurrence of swellings at different levels of the flagella of epimastigotes suggests that secondary sites of attachment may be common.