Molecular detection of Lotmaria passim in honeybees in Japan

Crithidia mellificae (C. mellificae) and Lotmaria passim (L. passim) are trypanosomatids that infect Apis mellifera. We analyzed the prevalence of C. mellificae and L. passim in six regions of Japan from 2018 to 2019. The detection rate of C. mellificae was 0.0% in all regions, whereas L. passim was detected in 16.7%–66.7% of the honeybees. L. passim was detected at a significantly lower rate in the Cyugoku-Shikoku region than in other regions. Furthermore, phylogenetic analysis of the internal transcribed spacer 1 (ITS1) locus of related species was performed. All the samples in this study could be assigned to the L. passim clade. This study reveals that L. passim infection is predominantly prevalent in Japan. Further epidemiological surveys are needed to clarify the prevalence of C. mellificae infection in honeybees in Japan.     

Trypanosomatid parasites infecting managed honeybees and wild solitary bees

The parasite Crithidia mellificae (Kinetoplastea: Trypanosomatidae) infects honeybees, Apis mellifera. No pathogenic effects have been found in individual hosts, despite positive correlations between infections and colony mortalities. The solitary bee Osmia cornuta might constitute a host, but controlled infections are lacking to date. Here, we challenged male and female O. cornuta and honeybee workers in laboratory cages with C. mellificae. No parasite cells were found in any control. Parasite numbers increased 6.6 fold in honeybees between days 6 and 19 p.i. and significantly reduced survival. In O. cornuta, C. mellificae numbers increased 2–3.6 fold within cages and significantly reduced survival of males, but not females. The proportion of infected hosts increased in O. cornuta cages with faeces, but not in honeybee cages without faeces, suggesting faecal – oral transmission. The data show that O. cornuta is a host of C. mellificae and suggest that males are more susceptible. The higher mortality of infected honeybees proposes a mechanism for correlations between C. mellificae infections and colony mortalities.     

Scanning electron microscope observations on the pathological changes of Malpighian tubules in the worker honeybee,Apis mellifera,infected by Malpighamoeba mellificae

A mixed population of cysts, immature cysts, and trophozoites was observed in the lumen of Malpighian tubules of Apis mellifera infected with Malpighamoeba mellificae. The most distinct pathological change in the infected tubules was swelling of the brush border of epithelial cells. In some areas the brush border was broken down into pieces, and in some cases it was completely engulfed by the parasites. The microvilli of the brush border were long and slender in noninfected tubules. Nuclei were not found in the infected tubules, but were frequently observed in the non-infected tubules. Numerous secretion globules were observed in the cytoplasm of epithelial cells of noninfected Malpighian tubules, but not in the cells of infected tubules. Epithelial cells in the infected tubules were destroyed and consumed by the trophozoites of M. mellificae. Trophozoites possessed protrusions and pits on their surfaces; many also had pseudopodia and apparently consumed the host cell by a mechanism of phagocytosis or endocytosis.     

Honey bee and bumblebee trypanosomatids: specificity and potential for transmission

Abstract 1. Experimental studies of multihost parasite dynamics are scarce. Understanding the transmission dynamics of parasites in these systems is a key task in developing better models of parasite evolution and to make more accurate predictions of disease dynamics. 2. Bumblebee species (Bombus spp.) host the trypanosomatid parasite, Crithidia bombi. Its transmission in the field occurs through the shared use of flowers. Flowers are a perfect scenario for inter‐taxa transmission of diseases because they are used by a wide range of animals. 3. Honey bees host a poorly studied trypanosomatid, Crithidia mellificae. In this study, five questions have been experimentally addressed: (a) Can C. bombi infect honey bees? (b) Can C. mellificae infect bumblebees? (c) Can the honey bee act as a vector for C. bombi? (d) Are C. bombi cells present in honey‐bee faeces? (e) Does C. bombi have an effect on the mortality of honey bees after ingestion? 4. While both parasites were found to be specific to their hosts at the genus level, results suggest that honey bees may play a role in the epidemiology of C. bombi transmission.     

Lotmaria passim (Kinetoplastea: Trypanosomatidae) in honey bees from Argentina

Lotmaria passim (Kinetoplastea) is considered the most prevalent as well as the most virulent trypanosomatid associated to the European honey bee Apis mellifera. We used qPCR to screen for the presence of this parasite in 57 samples from ten Argentinian provinces, and were able to detect its presence throughout most of the country with 41% of the samples testing positive. In a retrospective analysis, we detected L. passim in 73% of honey bee samples from 2006 showing that this flagellate has been widely present in Argentina for at least ~15 years. Additionally, three primer sets for L. passim detection were compared, with the pair that produced smallest PCR product having the best detection capability. Finally, we also found L. passim DNA in 100% (n = 6) of samples of the mite Varroa destructor. The role of this ectoparasite in the lifecycle of Lotmaria, if any, remains unrevealed.     

Varroa destructor changes its cuticular hydrocarbons to mimic new hosts

Varroa destructor (Vd) is a honeybee ectoparasite. Its original host is the Asian honeybee, Apis cerana, but it has also become a severe, global threat to the European honeybee, Apis mellifera. Previous studies have shown that Varroa can mimic a host''s cuticular hydrocarbons (HC), enabling the parasite to escape the hygienic behaviour of the host honeybees. By transferring mites between the two honeybee species, we further demonstrate that Vd is able to mimic the cuticular HC of a novel host species when artificially transferred to this new host. Mites originally from A. cerana are more efficient than mites from A. mellifera in mimicking HC of both A. cerana and A. mellifera. This remarkable adaptability may explain their relatively recent host-shift from A. cerana to A. mellifera.     

Unveiling the Intracellular Survival Gene Kit of Trypanosomatid Parasites

Trypanosomatids are unicellular protozoans of medical and economical relevance since they are the etiologic agents of infectious diseases in humans as well as livestock. Whereas Trypanosoma cruzi and different species of Leishmania are obligate intracellular parasites, Trypanosoma brucei and other trypanosomatids develop extracellularly throughout their entire life cycle. After their genomes have been sequenced, various comparative genomic studies aimed at identifying sequences involved with host cell invasion and intracellular survival have been described. However, for only a handful of genes, most of them present exclusively in the T. cruzi or Leishmania genomes, has there been any experimental evidence associating them with intracellular parasitism. With the increasing number of published complete genome sequences of members of the trypanosomatid family, including not only different Trypanosoma and Leishmania strains and subspecies but also trypanosomatids that do not infect humans or other mammals, we may now be able to contemplate a slightly better picture regarding the specific set of parasite factors that defines each organism''s mode of living and the associated disease phenotypes. Here, we review the studies concerning T. cruzi and Leishmania genes that have been implicated with cell invasion and intracellular parasitism and also summarize the wealth of new information regarding the mode of living of intracellular parasites that is resulting from comparative genome studies that are based on increasingly larger trypanosomatid genome datasets.     

The Limits on Trypanosomatid Morphological Diversity

Cell shape is one, often overlooked, way in which protozoan parasites have adapted to a variety of host and vector environments and directional transmissions between these environments. Consequently, different parasite life cycle stages have characteristic morphologies. Trypanosomatid parasites are an excellent example of this in which large morphological variations between species and life cycle stage occur, despite sharing well-conserved cytoskeletal and membranous structures. Here, using previously published reports in the literature of the morphology of 248 isolates of trypanosomatid species from different hosts, we perform a meta-analysis of the occurrence and limits on morphological diversity of different classes of trypanosomatid morphology (trypomastigote, promastigote, etc.) in the vertebrate bloodstream and invertebrate gut environments. We identified several limits on cell body length, cell body width and flagellum length diversity which can be interpreted as biomechanical limits on the capacity of the cell to attain particular dimensions. These limits differed for morphologies with and without a laterally attached flagellum which we suggest represent two morphological superclasses, the ‘juxtaform’ and ‘liberform’ superclasses. Further limits were identified consistent with a selective pressure from the mechanical properties of the vertebrate bloodstream environment; trypanosomatid size showed limits relative to host erythrocyte dimensions. This is the first comprehensive analysis of the limits of morphological diversity in any protozoan parasite, revealing the morphogenetic constraints and extrinsic selection pressures associated with the full diversity of trypanosomatid morphology.     

Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease.     

Phylogenetic analysis of Nosema ceranae isolated from European and Asian honeybees in Northern Thailand

Nosema ceranae was found to infect four different host species including the European honeybee (A. mellifera) and the Asian honeybees (Apis florea, A. cerana and Apis dorsata) collected from apiaries and forests in Northern Thailand. Significant sequence variation in the polar tube protein (PTP1) gene of N. ceranae was observed with N. ceranae isolates from A. mellifera and A. cerana, they clustered into the same phylogenetic lineage. N. ceranae isolates from A. dorsata and A. florea were grouped into two other distinct clades. This study provides the first elucidation of a genetic relationship among N. ceranae strains isolated from different host species and demonstrates that the N. ceranae PTP gene was shown to be a suitable and reliable marker in revealing genetic relationships within species.     

Comprehensive Bee Pathogen Screening in Belgium Reveals Crithidia mellificae as a New Contributory Factor to Winter Mortality

Since the last decade, unusually high honey bee colony losses have been reported mainly in North-America and Europe. Here, we report on a comprehensive bee pathogen screening in Belgium covering 363 bee colonies that were screened for 18 known disease-causing pathogens and correlate their incidence in summer with subsequent winter mortality. Our analyses demonstrate that, in addition to Varroa destructor, the presence of the trypanosomatid parasite Crithidia mellificae and the microsporidian parasite Nosema ceranae in summer are also predictive markers of winter mortality, with a negative synergy being observed between the two in terms of their effects on colony mortality. Furthermore, we document the first occurrence of a parasitizing phorid fly in Europe, identify a new fourth strain of Lake Sinai Virus (LSV), and confirm the presence of other little reported pathogens such as Apicystis bombi, Aphid Lethal Paralysis Virus (ALPV), Spiroplasma apis, Spiroplasma melliferum and Varroa destructor Macula-like Virus (VdMLV). Finally, we provide evidence that ALPV and VdMLV replicate in honey bees and show that viruses of the LSV complex and Black Queen Cell Virus tend to non-randomly co-occur together. We also noticed a significant correlation between the number of pathogen species and colony losses. Overall, our results contribute significantly to our understanding of honey bee diseases and the likely causes of their current decline in Europe.     

Does a facultative precocious life cycle predispose the marine trematode Proctoeces cf. lintoni to inbreeding and genetic differentiation among host species?

Intraspecific variability in parasite life cycle complexity (number of hosts and species of hosts in the life cycle) may have an impact how parasite genetic variation is partitioned among individual parasites, host individuals or host species within a given area. Among digenean trematodes, a three-host life cycle is common. However, a few species are precocious and may reach sexual maturity in what is typically regarded as the second intermediate host. The objective of this study was to determine whether a precocious life cycle predisposes digeneans to possible inbreeding or genetic subdivision among host species. As a study system, we used the digenean Proctoeces cf. lintoni whose metacercariae precociously mature (facultative) without a cyst wall in the gonads of multiple sympatric species of keyhole limpets (Fissurella spp.), typically regarded as the second intermediate hosts. Genotyped parasites were collected from four species of limpets and the clingfish Sicyases sanguineus, the third and final host where sexual maturity occurs. We found very high microsatellite diversity, Hardy–Weinberg equilibrium over all genotyped individuals, and little to no genetic structuring among parasites collected from the different host species. The fact that metacercariae do not encyst in the keyhole limpets, coupled with the high mixing potential of an aquatic environment, likely promote panmixia in local populations of P. cf. lintoni.     

Host Specificity in the Honeybee Parasitic Mite,Varroa spp. in Apis mellifera and Apis cerana

The ectoparasitic mite Varroa destructor is a major global threat to the Western honeybee Apis mellifera. This mite was originally a parasite of A. cerana in Asia but managed to spill over into colonies of A. mellifera which had been introduced to this continent for honey production. To date, only two almost clonal types of V. destructor from Korea and Japan have been detected in A. mellifera colonies. However, since both A. mellifera and A. cerana colonies are kept in close proximity throughout Asia, not only new spill overs but also spill backs of highly virulent types may be possible, with unpredictable consequences for both honeybee species. We studied the dispersal and hybridisation potential of Varroa from sympatric colonies of the two hosts in Northern Vietnam and the Philippines using mitochondrial and microsatellite DNA markers. We found a very distinct mtDNA haplotype equally invading both A. mellifera and A. cerana in the Philippines. In contrast, we observed a complete reproductive isolation of various Vietnamese Varroa populations in A. mellifera and A. cerana colonies even if kept in the same apiaries. In light of this variance in host specificity, the adaptation of the mite to its hosts seems to have generated much more genetic diversity than previously recognised and the Varroa species complex may include substantial cryptic speciation.     

Proboscis behavioral response of four honey bee Apis species towards different concentrations of sucrose,glucose, and fructose

Honey bees forage for pollen and nectar. Sugar is an important stimulus for foraging and a major source of energy for honey bees. Any differential response of bees to different concentrations of sugary nectar can affect their foraging. The sugar responsiveness of Apis species (Apis dorsata, Apis florea, and Apis cerana) was determined in comparison to that of Apis mellifera by evaluating the proboscis extension response (PER) with eight serial concentrations (0.00001, 0.0001, 0.001, 0.01, 0.1, 0.5, 1.0, and 1.5 M) of sucrose, glucose and fructose. Nectar foragers of bee species (A. dorsata, A. florea, A. cerana, and A. mellifera) exhibited an equal response for sucrose, glucose, and fructose, with no significant differences in their PER at all tested concentrations of these sugars within the same species. The inter-species comparison between Apis species revealed the differential responsiveness to the different concentrations of sugars, and the lowest concentration at which a response occurs was considered as the response threshold of these bee species for sugar solutions. A. mellifera presented significantly higher responsiveness than A. dorsata to low concentrations (0.00001, 0.0001, 0.001, 0.01, and 0.1 M) of sucrose, glucose and fructose. A. mellifera displayed a significantly higher response to water than A. dorsata. A. florea and A. mellifera presented no significant difference in their responsiveness to sucrose, glucose, and fructose at all tested concentrations, and their water responsiveness was also significantly at par but relatively higher in A. mellifera than in A. florea. Likewise, the responsiveness of A. cerana and A. mellifera to different concentrations of sucrose, glucose and fructose was significantly at par with no difference in their water responsiveness. This study represents preliminary research comparing the response of different honey bee species to three sugar types at different concentrations. The results imply that the native species are all better adapted than A. mellifera under local climate conditions.     

Studies on the development biology of Tropilaelaps clareae Delfinado and Baker (Acarina: Laelapidae) vis a vis the threshold stage in the life cycle of Apis mellifera Linn. (Hymenoptera: Apidae).

The biology of the parasitic mite Tropilaelaps clareae Delfinado and Baker (Acarina: Laelapidae) was studied with a view to identify the time of invasion of the mite into the honey bee (Apis mellifera) brood and the threshold stage in the life cycle of the host. Honey bee brood was sequentially sampled on day 0, 4, 8, 12, 16 and 20 of development. Adult T. clareae infested the 8-day larva shortly before its cell was capped. The larvac, protonymphs, deutonymphs and adults of T. clareae were all found parasitising bright red eyed pupae during day 16 of brood development. This was identified as the most parasitised stage in the life cycle of the host. The mite developed from egg to adult in about 8 days.     

Biology and engineering of integrative and conjugative elements: Construction and analyses of hybrid ICEs reveal element functions that affect species-specific efficiencies

Integrative and conjugative elements (ICEs) are mobile genetic elements that reside in a bacterial host chromosome and are prominent drivers of bacterial evolution. They are also powerful tools for genetic analyses and engineering. Transfer of an ICE to a new host involves many steps, including excision from the chromosome, DNA processing and replication, transfer across the envelope of the donor and recipient, processing of the DNA, and eventual integration into the chromosome of the new host (now a stable transconjugant). Interactions between an ICE and its host throughout the life cycle likely influence the efficiencies of acquisition by new hosts. Here, we investigated how different functional modules of two ICEs, Tn916 and ICEBs1, affect the transfer efficiencies into different host bacteria. We constructed hybrid elements that utilize the high-efficiency regulatory and excision modules of ICEBs1 and the conjugation genes of Tn916. These elements produced more transconjugants than Tn916, likely due to an increase in the number of cells expressing element genes and a corresponding increase in excision. We also found that several Tn916 and ICEBs1 components can substitute for one another. Using B. subtilis donors and three Enterococcus species as recipients, we found that different hybrid elements were more readily acquired by some species than others, demonstrating species-specific interactions in steps of the ICE life cycle. This work demonstrates that hybrid elements utilizing the efficient regulatory functions of ICEBs1 can be built to enable efficient transfer into and engineering of a variety of other species.