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.

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.

Antiparasitic effects of three floral volatiles on trypanosomatid infection in honey bees.

Trypanosomatid gut parasites are common in pollinators and costly for social bees. The recently described honey bee trypanosomatid Lotmaria passim is widespread, abundant, and correlated with colony losses in some studies. The potential for amelioration of infection by antimicrobial plant compounds has been thoroughly studied for closely related trypanosomatids of humans and is an area of active research in bumble bees, but remains relatively unexplored in honey bees. We recently identified several floral volatiles that inhibited growth of L. passim in vitro. Here, we tested the dose-dependent effects of four such compounds on infection, mortality, and food consumption in parasite-inoculated honey bees. We found that diets containing the monoterpenoid carvacrol and the phenylpropanoids cinnamaldehyde and eugenol at > 10-fold the inhibitory concentrations for cell cultures reduced infection, with parasite numbers decreased by > 90 % for carvacrol and cinnamaldehyde and > 99 % for eugenol; effects of the carvacrol isomer thymol were non-significant. However, both carvacrol and eugenol also reduced bee survival, whereas parasite inoculation did not, indicating costs of phytochemical exposure that could exceed those of infection itself. To our knowledge, this is the first controlled screening of phytochemicals for effects on honey bee trypanosomatid infection, identifying potential treatments for managed bees afflicted with a newly characterized, cosmopolitan intestinal parasite.

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.

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.

Influence of the endosymbiont on the interaction of Crithidia deanei with macrophages

Crithidia deanei é um tripanosomatídeo näo patogênico que possui no seu citoplasma um endosimbionte semelhante a uma bactéria. Parasitas sem a presença do endosimbionte podem ser obtidos após tratamento com cloranfenicol. Macrófagos peritoniais de camundongo cultivados in vitro endocitam mais parasitas que possuem endosimbionte do que parasitas sem endosimbionte. Os parasitas foram observados dentro de vacúolos citoplasmáticos. Observamos que os parasitos sem endosimbionte endocitiados por macrófagos eram rapidamente digeridos, ao contrário do que ocorria con os parasitos com endosimbionte, que foram observados dentro dos macrófagos 48 horas após o início da interaçäo. Estes resultados indicam que a presença do endocimbionte parece interferir na interaçäo parasito-célula

Influence of the endosymbiont on the interaction of Crithidia deanei with macrophages

Crithidia deanei é um tripanosomatídeo nõo patogÛnico que possui no seu citoplasma um endosimbionte semelhante a uma bactéria. Parasitas sem a presenþa do endosimbionte podem ser obtidos após tratamento com cloranfenicol. Macrófagos peritoniais de camundongo cultivados in vitro endocitam mais parasitas que possuem endosimbionte do que parasitas sem endosimbionte. Os parasitas foram observados dentro de vacúolos citoplasmáticos. Observamos que os parasitos sem endosimbionte endocitiados por macrófagos eram rapidamente digeridos, ao contrário do que ocorria con os parasitos com endosimbionte, que foram observados dentro dos macrófagos 48 horas após o início da interaþõo. Estes resultados indicam que a presenþa do endocimbionte parece interferir na interaþõo parasito-célula (AU)

Ultrastructural differences between species of trypanosomatids with and without endosymbionts.

Species of trypanosomatids without endosymbionts (Leptomonas seymouri, L. collosoma, L. samueli, crithidia fasciculata, C. luciliae, C. acanthocephali, Herpetomonas megaseliae, H. mariadeanei, H. samuelpessoai, H. muscarum muscarum, Trypanosoma cruzi) and species of trypanosomatids with endosymbionts (Crithidia deanei, C. oncopelti, Blastocrithidia culicis) were comparatively studied by means of electron microscopy. Artificially aposymbiotic strains derived from species with symbiont were also included in the survey. Species with symbiont were found to differ in some ultrastructural aspects from the group of species without symbiont. Paraxial rods of flagella or intraflagellar structure were found exclusively in species without symbiont. Peripheral branching of mitochondria, accompanied by absence of subpellicular microtubules in sites where the mitochondrial branches are appressed to the cell membrane, were found exclusively in species with symbiont. Networks of kinetoplast DNA fibrils were found to be larger and looser in species with symbiont. Symbiont-free strains of species with symbiont retained the same morphological characteristics of their parental species.