The distribution and population dynamics of the honey bee pathogens Crithidia mellificae and Lotmaria passim in New Zealand

<p>The honey bee Apis mellifera is experiencing colony losses across the world, this is not the first time in history colony losses have been reported. New molecular detection methods such as real-time PCR allow the detection and analysis of pathogens present in colonies, quickly and reliably. Of the pathogens that the honey bee is host to, trypanosomes are one of the least understood and trypanosome interactions within the honey bee host remain largely unknown. Using the bumble bee as a model for this host-parasite relationship. The trypanosome C. bombi is known to cause a reduced ability to gain nutrients from food and an overall decrease in efficiency of queens in founding colonies in spring. These negative correlations are significant enough in the bumble bee to warrant investigation into trypanosomes in the honey bee. The trypanosome C. mellificae was first described in the honey bee in 1967. A screening study in 2009 included a test for and detected the trypanosome in modern honey bee samples. In 2013 C. mellificae was identified as a contributory factor to overwintering colony losses when co-infected with N. ceranae. Following studies detected trypanosomes and led to the characterisation of a new species, L. passim in 2013. Lotmaria passim was first detected in New Zealand in 2014 however no subsequent studies had been undertaken to identify the distribution and dynamics of trypanosomes in New Zealand honey bee colonies. My goal in this study was to identify the presence of trypanosomes in New Zealand. In an overview study of 47 honey bee colonies from across New Zealand, 46 were positive for the L. passim species. Identified by sequencing of the GAPDH gene. A yearlong study of 15 colonies revealed that the infection rate of L. passim was consistent throughout the year and very low genetic variation was detected. Lotmaria passim was detected in all parts of New Zealand sampled in this study and often in high levels. A positive correlation was detected when L. passim was present in addition to N. apis. There was no detection of C. mellificae in my study. The lack of detection of C. mellificae may suggest that the species is not present, or that it is in such low levels it cannot yet be detected. In parallel to this trypanosome study two Nosema spp. and DWV were also examined. Nosema apis was found to be more prevalent than N. ceranae, which was not present in any South Island samples. A strong positive correlation was detected between the two Nosema spp. DWV showed a high level of variation likely a reflection of differing Varroa management practices in apiaries in this study. This study of trypanosomes is the first of its kind in New Zealand identifying the presence and population dynamics of L. passim. This in conjunction with data on Nosema spp. and DWV will be of value to the New Zealand apiculture industry and contribute to global honey bee health studies.</p>

The distribution and population dynamics of the honey bee pathogens Crithidia mellificae and Lotmaria passim in New Zealand

<p>The honey bee Apis mellifera is experiencing colony losses across the world, this is not the first time in history colony losses have been reported. New molecular detection methods such as real-time PCR allow the detection and analysis of pathogens present in colonies, quickly and reliably. Of the pathogens that the honey bee is host to, trypanosomes are one of the least understood and trypanosome interactions within the honey bee host remain largely unknown. Using the bumble bee as a model for this host-parasite relationship. The trypanosome C. bombi is known to cause a reduced ability to gain nutrients from food and an overall decrease in efficiency of queens in founding colonies in spring. These negative correlations are significant enough in the bumble bee to warrant investigation into trypanosomes in the honey bee. The trypanosome C. mellificae was first described in the honey bee in 1967. A screening study in 2009 included a test for and detected the trypanosome in modern honey bee samples. In 2013 C. mellificae was identified as a contributory factor to overwintering colony losses when co-infected with N. ceranae. Following studies detected trypanosomes and led to the characterisation of a new species, L. passim in 2013. Lotmaria passim was first detected in New Zealand in 2014 however no subsequent studies had been undertaken to identify the distribution and dynamics of trypanosomes in New Zealand honey bee colonies. My goal in this study was to identify the presence of trypanosomes in New Zealand. In an overview study of 47 honey bee colonies from across New Zealand, 46 were positive for the L. passim species. Identified by sequencing of the GAPDH gene. A yearlong study of 15 colonies revealed that the infection rate of L. passim was consistent throughout the year and very low genetic variation was detected. Lotmaria passim was detected in all parts of New Zealand sampled in this study and often in high levels. A positive correlation was detected when L. passim was present in addition to N. apis. There was no detection of C. mellificae in my study. The lack of detection of C. mellificae may suggest that the species is not present, or that it is in such low levels it cannot yet be detected. In parallel to this trypanosome study two Nosema spp. and DWV were also examined. Nosema apis was found to be more prevalent than N. ceranae, which was not present in any South Island samples. A strong positive correlation was detected between the two Nosema spp. DWV showed a high level of variation likely a reflection of differing Varroa management practices in apiaries in this study. This study of trypanosomes is the first of its kind in New Zealand identifying the presence and population dynamics of L. passim. This in conjunction with data on Nosema spp. and DWV will be of value to the New Zealand apiculture industry and contribute to global honey bee health studies.</p>

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.

First description ofLotmaria passimandCrithidia mellificaehaptomonad stage in the honeybee hindgut

The remodelling of flagella into attachment structures is a common and important event in the insect stages of the trypanosomatid life cycle. Among their hymenopteran hosts,Lotmaria passimandCrithidia mellificaecan parasitizeApis mellifera, and as a result they might have a significant impact on honeybee health. However, there are details of their life cycle and the mechanisms underlying their pathogenicity in this host that remain unclear. Here we show that bothL. passimpromastigotes andC. mellificaechoanomastigotes differentiate into haptomonad stage covering the ileum and rectum of honeybees. These haptomonad cells remain attached to the host surface via zonular hemidesmosome-like structures, as revealed by Transmission Electron Microscopy. Hence, for the first time this work describes the haptomonad morphotype of these species and their hemidesmosome-like attachment inApis mellifera, a key trait exploited by other trypanosomatid species to proliferate in the insect host hindgut.Author summaryIn recent years, the mortality of European Honeybees (Apis mellifera) has risen worldwide due to a variety of factors, including their infection by parasites. Former studies have linked the presence of several trypanosomatids species, beingLotmaria passimandCrithidia mellificaethe most prevalent ones, with this increase in mortality. Although previous studies have shown that trypanosomatid infection reduces the lifespan of bees, there is little information regarding their development in the gut when honeybees become infected. Here, for the first time we describe the haptomonad morphotype of these two trypanosomatid species inA. mellifera. The most characteristic feature of haptomonads is the extensive remodelling of the flagellum and the formation of junctional complexes at the host gut wall. The presence of this morphotype in the honeybee hindgut increases our understanding of the life cycle of these species and their possible pathogenic mechanisms. We found that they can multiply while attached and that their disposition, covering the hindgut walls, could hinder host nutrient uptake and consequently, represent a pathogenic mechanism itself. This attachment could also be a key stage in the life-cycle to prevent the trypanosomatids leaving the host prematurely, ensuring transmission through infective morphotypes.

Detection of Lotmaria passim, Crithidia mellificae and Replicative Forms of Deformed Wing Virus and Kashmir Bee Virus in the Small Hive Beetle (Aethina tumida)

Knowledge regarding the honey bee pathogens borne by invasive bee pests remains scarce. This investigation aimed to assess the presence in Aethina tumida (small hive beetle, SHB) adults of honey bee pathogens belonging to the following groups: (i) bacteria (Paenibacillus larvae and Melissococcus plutonius), (ii) trypanosomatids (Lotmaria passim and Crithidia mellificae), and (iii) viruses (black queen cell virus, Kashmir bee virus, deformed wing virus, slow paralysis virus, sacbrood virus, Israeli acute paralysis virus, acute bee paralysis virus, chronic bee paralysis virus). Specimens were collected from free-flying colonies in Gainesville (Florida, U.S.A.) in summer 2017. The results of the molecular analysis show the presence of L. passim, C. mellificae, and replicative forms of deformed wing virus (DWV) and Kashmir bee virus (KBV). Replicative forms of KBV have not previously been reported. These results support the hypothesis of pathogen spillover between managed honey bees and the SHB, and these dynamics require further investigation.