Gene Disruption of Honey Bee Trypanosomatid Parasite, Lotmaria passim, by CRISPR/Cas9 System.
ABSTRACT: Two trypanosomatid species, Lotmaria passim and Crithidia mellificae, have been shown to parasitize honey bees to date. L. passim appears to be more prevalent than C. mellificae and specifically infects the honey bee hindgut. Although the genomic DNA has been sequenced, the effects of infection on honey bee health and colony are poorly understood. To identify the genes that are important for infecting honey bees and to understand their functions, we applied the CRISPR/Cas9 system to establish a method to manipulate L. passim genes. By electroporation of plasmid DNA and subsequent selection by drug, we first established an L. passim clone expressing tdTomato or Cas9. We also successfully disrupted the endogenous miltefosine transporter and tyrosine aminotransferase genes by replacement with drug (hygromycin) resistant gene using the CRISPR/Cas9-induced homology-directed repair pathway. The L. passim clone expressing fluorescent marker, as well as the simple method for editing specific genes, could become useful approaches to understand the underlying mechanisms of honey bee-trypanosomatid parasite interactions.
Project description:Commercially and traditionally managed bees were compared for oxidative stress (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and malondialdehyde (MDA)), the prevalence of parasites (Lotmaria passim, Crithidia mellificae and Nosema ceranae/apis) and social immunity (glucose oxidase gene expression). The research was conducted on Pester plateau (Serbia-the Balkan Peninsula), on seemingly healthy colonies. Significant differences in CAT, GST and SOD activities (p < 0.01), and MDA concentrations (p < 0.002) were detected between commercial and traditional colonies. In the former, the prevalence of both L. passim and N. ceranae was significantly (p < 0.05 and p < 0.01, respectively) higher. For the first time, L. passim was detected in honey bee brood. In commercial colonies, the prevalence of L. passim was significantly (p < 0.01) lower in brood than in adult bees, whilst in traditionally kept colonies the prevalence in adult bees and brood did not differ significantly. In commercially kept colonies, the GOX gene expression level was significantly (p < 0.01) higher, which probably results from their increased need to strengthen their social immunity. Commercially kept colonies were under higher oxidative stress, had higher parasite burdens and higher GOX gene transcript levels. It may be assumed that anthropogenic influence contributed to these differences, but further investigations are necessary to confirm that.
Project description:It is still not understood how honey bee parasite changes the gene expression to adapt to the host environment and how the host simultaneously responds to the parasite infection by modifying its own gene expression. To address this question, we studied a trypanosomatid, Lotmaria passim, which can be cultured in medium and inhabit the honey bee hindgut. We found that L. passim decreases mRNAs associated with protein translation, glycolysis, detoxification of radical oxygen species, and kinetoplast respiratory chain to adapt to the anaerobic and nutritionally poor honey bee hindgut during the infection. After the long term infection, the host appears to be in poor nutritional status, indicated by the increase and decrease of take-out and vitellogenin mRNAs, respectively. Simultaneous gene expression profiling of L. passim and honey bee during infection by dual RNA-seq provided insight into how both parasite and host modify their gene expressions.
Project description:Recent declines of wild pollinators and infections in honey, bumble and other bee species have raised concerns about pathogen spillover from managed honey and bumble bees to other pollinators. Parasites of honey and bumble bees include trypanosomatids and microsporidia that often exhibit low host specificity, suggesting potential for spillover to co-occurring bees via shared floral resources. However, experimental tests of trypanosomatid and microsporidial cross-infectivity outside of managed honey and bumble bees are scarce. To characterize potential cross-infectivity of honey and bumble bee-associated parasites, we inoculated three trypanosomatids and one microsporidian into five potential hosts - including four managed species - from the apid, halictid and megachilid bee families. We found evidence of cross-infection by the trypanosomatids Crithidia bombi and C. mellificae, with evidence for replication in 3/5 and 3/4 host species, respectively. These include the first reports of experimental C. bombi infection in Megachile rotundata and Osmia lignaria, and C. mellificae infection in O. lignaria and Halictus ligatus. Although inability to control amounts inoculated in O. lignaria and H. ligatus hindered estimates of parasite replication, our findings suggest a broad host range in these trypanosomatids, and underscore the need to quantify disease-mediated threats of managed social bees to sympatric pollinators.
Project description:Lotmaria passim currently appears to be the predominant trypanosome in honey bees worldwide. Although, the specific effects of L. passim by single or mixed with other gut parasites such as Nosema ceranae on honey bees' health is still unclear. We consequently measured bees' survival, parasite loads, the expression of antimicrobial peptides (AMPs) and vitellogenin gene. Thus, (1) bees naturally infected with L. passim, (2) healthy bees inoculated with Nosema ceranae, (3) bees naturally infected with L. passim and inoculated with N. ceranae and (4) healthy bees (control) were maintained under controlled conditions. Honey bees infected with N. ceranae or with mixed infections of L. passim and N. ceranae had significantly lower survival rates than the control group at 20 days post-inoculation (dpi). A competitive suppression was also detected, provided that the L. passim load was significantly affected by the presence of N. ceranae at 15 dpi. Expressions of the AMPs defensin and hymenoptaecin rapidly (two hours post-inoculation) increased in bees infected with N. ceranae and mixed infections. However, this effect was not continuous. In fact, expressions of abaecin, defensin, hymenoptaecin and vitellogenin decreased drastically at 15 dpi in bees with both single and mixed infections. The decrease in the expression of AMPs and vitellogenin throughout this period was consistent with the reduced survivals observed in this study, indicating that mixed infections of L. passim and N. ceranae, and even into a scenario of competition between them, may have a synergic effect on the survival and immune-related gene expressions (biomarkers) of worker bees.
Project description:Honey bees (Apis mellifera) play a critical role in global food production as pollinators of numerous crops. Recently, honey bee populations in the United States, Canada, and Europe have suffered an unexplained increase in annual losses due to a phenomenon known as Colony Collapse Disorder (CCD). Epidemiological analysis of CCD is confounded by a relative dearth of bee pathogen field studies. To identify what constitutes an abnormal pathophysiological condition in a honey bee colony, it is critical to have characterized the spectrum of exogenous infectious agents in healthy hives over time. We conducted a prospective study of a large scale migratory bee keeping operation using high-frequency sampling paired with comprehensive molecular detection methods, including a custom microarray, qPCR, and ultra deep sequencing. We established seasonal incidence and abundance of known viruses, Nosema sp., Crithidia mellificae, and bacteria. Ultra deep sequence analysis further identified four novel RNA viruses, two of which were the most abundant observed components of the honey bee microbiome (?10(11) viruses per honey bee). Our results demonstrate episodic viral incidence and distinct pathogen patterns between summer and winter time-points. Peak infection of common honey bee viruses and Nosema occurred in the summer, whereas levels of the trypanosomatid Crithidia mellificae and Lake Sinai virus 2, a novel virus, peaked in January.
Project description:Environmental DNA (eDNA) has been proposed as a powerful tool to detect and monitor cryptic, elusive, or invasive organisms. We recently demonstrated that honey constitutes an easily accessible source of eDNA. In this study, we extracted DNA from 102 honey samples (74 from Italy and 28 from 17 other countries of all continents) and tested the presence of DNA of nine honey bee pathogens and parasites (Paenibacillus larvae, Melissococcus plutonius, Nosema apis, Nosema ceranae, Ascosphaera apis,Lotmaria passim, Acarapis woodi, Varroa destructor, and Tropilaelaps spp.) using qualitative PCR assays. All honey samples contained DNA from V. destructor, confirming the widespread diffusion of this mite. None of the samples gave positive amplifications for N. apis, A. woodi, and Tropilaelaps spp. M. plutonius was detected in 87% of the samples, whereas the other pathogens were detected in 43% to 57% of all samples. The frequency of Italian samples positive for P. larvae was significantly lower (49%) than in all other countries (79%). The co-occurrence of positive samples for L. passim and A. apis with N. ceranae was significant. This study demonstrated that honey eDNA can be useful to establish monitoring tools to evaluate the sanitary status of honey bee populations.
Project description:China has the largest number of managed honey bee colonies, which produce the highest quantity of honey and royal jelly in the world; however, the presence of honey bee pathogens and parasites has never been rigorously identified in Chinese apiaries. We thus conducted a molecular survey of honey bee RNA viruses, Nosema microsporidia, protozoan parasites, and tracheal mites associated with nonnative Apis mellifera ligustica and native Apis cerana cerana colonies in China. We found the presence of black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), and sacbrood virus (SBV), but not that of acute bee paralysis virus (ABPV) or Kashmir bee virus (KBV). DWV was the most prevalent in the tested samples. Phylogenies of Chinese viral isolates demonstrated that genetically heterogeneous populations of BQCV, CBPV, DWV, and A. cerana-infecting SBV, and relatively homogenous populations of IAPV and A. meliifera-infecting new strain of SBV with single origins, are spread in Chinese apiaries. Similar to previous observations in many countries, Nosema ceranae, but not Nosema apis, was prevalent in the tested samples. Crithidia mellificae, but not Apicystis bombi was found in five samples, including one A. c. cerana colony, demonstrating that C. mellificae is capable of infecting multiple honey bee species. Based on kinetoplast-encoded cytochrome b sequences, the C. mellificae isolate from A. c. cerana represents a novel haplotype with 19 nucleotide differences from the Chinese and Japanese isolates from A. m. ligustica. This suggests that A. c. cerana is the native host for this specific haplotype. The tracheal mite, Acarapis woodi, was detected in one A. m. ligustica colony. Our results demonstrate that honey bee RNA viruses, N. ceranae, C. mellificae, and tracheal mites are present in Chinese apiaries, and some might be originated from native Asian honey bees.
Project description: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.
Project description:The trade of bumble bees started in the early nineties for pollinator-dependent greenhouse plants. Nowadays, its rearing and transport have received public attention, since managed bees can transfer pathogens to wild bee populations. Therefore, guaranteeing pathogen-free bumble bees is fundamental. The major protein source used in rearing facilities is honey bee-collected pollen. This can carry pathogens, however to date, solid data on the risk of this food source to the health of bumble bees is lacking. Here we performed a large pathogen screening of non-irradiated honey bee-collected pollen to discover particles infective to Bombus terrestris. We identified seven parasites (Apicystis bombi, Ascosphaera apis, Crithidia mellificae, Nosema ceranae, Paenibacillus larvae and two parasites resembling Nosema thomsoni and Microsporidium sp. Oise) and four viruses (CBPV, DWV, IAPV and SBV) in 17 pollen batches from two major European pollen source regions (Spain and Romania). Ascosphaera apis was capable of infecting bumble bees; the larvae showed similar symptoms to chalkbrood disease reported in honey bees. Bumble bee breeding facilities need to be cautious about the potential presence of this disease, which was originally reported in honey bees. Thorough diagnostic and control methods are needed, as risk of spillover to wild bee species is possible.
Project description:Since 2006, honey bee colonies in North America and Europe have experienced increased annual mortality. These losses correlate with increased pathogen incidence and abundance, though no single etiologic agent has been identified. Crithidia mellificae is a unicellular eukaryotic honey bee parasite that has been associated with colony losses in the USA and Belgium. C. mellificae is a member of the family Trypanosomatidae, which primarily includes other insect-infecting species (e.g., the bumble bee pathogen Crithidia bombi), as well as species that infect both invertebrate and vertebrate hosts including human pathogens (e.g.,Trypanosoma cruzi, T. brucei, and Leishmania spp.). To better characterize C. mellificae, we sequenced the genome and transcriptome of strain SF, which was isolated and cultured in 2010. The 32 megabase draft genome, presented herein, shares a high degree of conservation with the related species Leishmania major. We estimate that C. mellificae encodes over 8,300 genes, the majority of which are orthologs of genes encoded by L. major and other Leishmania or Trypanosoma species. Genes unique to C. mellificae, including those of possible bacterial origin, were annotated based on function and include genes putatively involved in carbohydrate metabolism. This draft genome will facilitate additional investigations of the impact of C. mellificae infection on honey bee health and provide insight into the evolution of this unique family.