Ceranae Infection Research Articles

Nosema ceranae Infection Promotes Proliferation of Yeasts in Honey Bee Intestines.

BackgroundNosema ceranae infection not only damages honey bee (Apis melifera) intestines, but we believe it may also affect intestinal yeast development and its seasonal pattern. In order to check our hypothesis, infection intensity versus intestinal yeast colony forming units (CFU) both in field and cage experiments were studied.Methods/FindingsField tests were carried out from March to October in 2014 and 2015. N. ceranae infection intensity decreased more than 100 times from 7.6 x 108 in March to 5.8 x 106 in October 2014. A similar tendency was observed in 2015. Therefore, in the European eastern limit of its range, N. ceranae infection intensity showed seasonality (spring peak and subsequent decline in the summer and fall), however, with an additional mid-summer peak that had not been recorded in other studies. Due to seasonal changes in the N. ceranae infection intensity observed in honey bee colonies, we recommend performing studies on new therapeutics during two consecutive years, including colony overwintering. A natural decrease in N. ceranae spore numbers observed from March to October might be misinterpreted as an effect of Nosema spp. treatment with new compounds. A similar seasonal pattern was observed for intestinal yeast population size in field experiments. Furthermore, cage experiments confirmed the size of intestinal yeast population to increase markedly together with the increase in the N. ceranae infection intensity. Yeast CFUs amounted to respectively 2,025 (CV = 13.04) and 11,150 (CV = 14.06) in uninfected and N. ceranae-infected workers at the end of cage experiments. Therefore, honey bee infection with N. ceranae supported additional opportunistic yeast infections, which may have resulted in faster colony depopulations.

Colony Level Prevalence and Intensity of Nosema ceranae in Honey Bees (Apis mellifera L.).

Nosema ceranae is a widely prevalent microsporidian parasite in the western honey bee. There is considerable uncertainty regarding infection dynamics of this important pathogen in honey bee colonies. Understanding the infection dynamics at the colony level may aid in development of a reliable sampling protocol for N. ceranae diagnosis, and provide insights into efficient treatment strategies. The primary objective of this study was to characterize the prevalence (proportion of the sampled bees found infected) and intensity (number of spores per bee) of N. ceranae infection in bees from various age cohorts in a colony. We examined N. ceranae infection in both overwintered colonies that were naturally infected with N. ceranae and in quadruple cohort nucleus colonies that were established and artificially inoculated with N. ceranae. We also examined and quantified effects of N. ceranae infection on hypopharyngeal gland protein content and gut pH. There was no correlation between the prevalence and intensity of N. ceranae infection in composite samples (pooled bee samples used for analysis). Our results indicated that the prevalence and intensity of N. ceranae infection is significantly influenced by honey bee age. The N. ceranae infection prevalence values from composite samples of background bees (unmarked bees collected from four different locations in a colony) were not significantly different from those pertaining to marked-bee age cohorts specific to each sampling date. The foraging-aged bees had a higher prevalence of N. ceranae infection when compared to nurse-aged bees. N. ceranae did not have a significant effect on hypopharyngeal gland protein content. Further, there was no significant difference in mean gut pH of N. ceranae infected bees and non-infected bees. This study provides comprehensive insights into N. ceranae infection dynamics at the colony level, and also demonstrates the effects of N. ceranae infection on hypopharyngeal gland protein content and midgut pH.

Bombus brasiliensis Lepeletier (Hymenoptera, Apidae) infected with Nosema ceranae (Microsporidia)

Heavy infections caused by a microsporidium were detected in midgut epithelium cells of two adult workers of the bumble bee Bombus brasiliensis Lepeletier collected near Puerto Iguazú, Misiones province, Argentina. Microsporidium rRNA (16S small subunit) was amplified by 218MITOC primers and produced amplicons indicating presence of Nosema ceranae Fries et al., a virulent pathogen of more than 20 bee species, possibly involved in Apis mellifera L. Colony Collapse Disorder. Campaigns in search of B. brasiliensis between 2008 and 2015 have revealed a possible narrower range in the southeastern area of its known distribution. Effects of N. ceranae infections could be modulating their populations and should not be overlooked. In addition, the wide host range of this microsporidium makes it a potential threat to several endemic bees such as stingless (Meliponini) and orchid bees (Euglossini).

First detection of Nosema ceranae in New Zealand honey bees

Nosema is a genus of 81 microsporidian parasites, two of which affect honey bees. The advent of molecular methods has facilitated the differentiation between Nosema apis and Nosema ceranae. N. ceranae infection was detected by polymerase chain reaction (PCR) in an apiary on the Coromandel Peninsula of New Zealand during investigation into unusual colony losses in early spring 2010, and was subsequently found in other areas of the country. While no association could be confirmed between apiary health status and presence of N. ceranae, a significantly higher proportion of positive apiaries were detected in spring compared with autumn. PCR for N. ceranae conducted in three different laboratories showed fair agreement. Minor assay differences and sample level variation in Nosema are the most likely explanation for differences in results.

Nosema spp. infections cause no energetic stress in tolerant honeybees.

Host-pathogen coevolution leads to reciprocal adaptations, allowing pathogens to increase host exploitation or hosts to minimise costs of infection. As pathogen resistance is often associated with considerable costs, tolerance may be an evolutionary alternative. Here, we examined the effect of two closely related and highly host dependent intracellular gut pathogens, Nosema apis and Nosema ceranae, on the energetic state in Nosema tolerant and sensitive honeybees facing the infection. We quantified the three major haemolymph carbohydrates fructose, glucose, and trehalose using high-performance liquid chromatography (HPLC) as a measure for host energetic state. Trehalose levels in the haemolymph were negatively associated with N. apis infection intensity and with N. ceranae infection regardless of the infection intensity in sensitive honeybees. Nevertheless, there was no such association in Nosema spp. infected tolerant honeybees. These findings suggest that energy availability in tolerant honeybees was not compromised by the infection. This result obtained at the individual level may also have implications at the colony level where workers in spite of a Nosema infection can still perform as well as healthy bees, maintaining colony efficiency and productivity.

Parasite infection accelerates age polyethism in young honey bees.

Honey bees (Apis mellifera) are important pollinators and their health is threatened worldwide by persistent exposure to a wide range of factors including pesticides, poor nutrition, and pathogens. Nosema ceranae is a ubiquitous microsporidian associated with high colony mortality. We used lab micro-colonies of honey bees and video analyses to track the effects of N. ceranae infection and exposure on a range of individual and social behaviours in young adult bees. We provide detailed data showing that N. ceranae infection significantly accelerated the age polyethism of young bees, causing them to exhibit behaviours typical of older bees. Bees with high N. ceranae spore counts had significantly increased walking rates and decreased attraction to queen mandibular pheromone. Infected bees also exhibited higher rates of trophallaxis (food exchange), potentially reflecting parasite manipulation to increase colony infection. However, reduction in queen contacts could help bees limit the spread of infection. Such accelerated age polyethism may provide a form of behavioural immunity, particularly if it is elicited by a wide variety of pathogens.

Honey bee microRNAs respond to infection by the microsporidian parasite Nosema ceranae

In order to study the effects of Nosema ceranae infection on honey bee microRNA (miRNA) expression, we deep-sequenced honey bee miRNAs daily across a full 6-day parasite reproduction cycle. Seventeen miRNAs were differentially expressed in honey bees infected by N. ceranae that potentially target over 400 genes predicted to primarily involve ion binding, signaling, the nucleus, transmembrane transport, and DNA binding. Based on Enzyme Code analysis, nine biological pathways were identified by screening target genes against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, seven of which involved metabolism. Our results suggest that differentially expressed miRNAs regulate metabolism related genes of host honey bees in response to N. ceranae infection.

Effect of dietary supplementation of Bifidobacterium and Lactobacillus strains in Apis mellifera L. against Nosema ceranae.

Nosema ceranae is a widespread microsporidium of European honeybee Apis mellifera L. affecting bee health. The ban of Fumagillin-B (dicyclohexylammonium salt) in the European Union has driven the search for sustainable strategies to prevent and control the infection. The gut microbial symbionts, associated to the intestinal system of vertebrates and invertebrates and its impact on host health, are receiving increasing attention. In particular, bifidobacteria and lactobacilli, which are normal inhabitants of the digestive system of bees, are known to protect their hosts via antimicrobial metabolites, immunomodulation and competition. In this work, the dietary supplementation of gut bacteria was evaluated under laboratory conditions in bees artificially infected with the parasite and bees not artificially infected but evidencing a low natural infection. Supplemented bacteria were selected among bifidobacteria, previously isolated, and lactobacilli, isolated in this work from healthy honeybee gut. Four treatments were compared: bees fed with sugar syrup (CTR); bees fed with sugar syrup containing bifidobacteria and lactobacilli (PRO); bees infected with N. ceranae spores and fed with sugar syrup (NOS); bees infected with N. ceranae and fed with sugar syrup containing bifidobacteria and lactobacilli (NP). The sugar syrup, with or without microorganisms, was administered to bees from the first day of life for 13 days. N. ceranae infection was carried out individually on anesthetised 5-day-old bees. Eight days after infection, a significant (P<0.05) lower level of N. ceranae was detected by real-time PCR in both NP and PRO group, showing a positive effect of supplemented microorganisms in controlling the infection. These results represent a first attempt of application of bifidobacteria and lactobacilli against N. ceranae in honeybees.

Are commercial probiotics and prebiotics effective in the treatment and prevention of honeybee nosemosis C?

The study was conducted to investigate the effect of Lactobacillus rhamnosus (a commercial probiotic) and inulin (a prebiotic) on the survival rates of honeybees infected and uninfected with Nosema ceranae, the level of phenoloxidase (PO) activity, the course of nosemosis, and the effect on the prevention of nosemosis development in bees. The cells of L. rhamnosus exhibited a high rate of survival in 56.56 % sugar syrup, which was used to feed the honeybees. Surprisingly, honeybees fed with sugar syrup supplemented with a commercial probiotic and a probiotic + prebiotic were more susceptible to N. ceranae infection, and their lifespan was much shorter. The number of microsporidian spores in the honeybees fed for 9 days prior to N. ceranae infection with a sugar syrup supplemented with a commercial probiotic was 25 times higher (970 million spores per one honeybee) than in a control group fed with pure sucrose syrup (38 million spores per one honeybee). PO activity reached its highest level in the hemolymph of this honeybee control group uninfected with N. ceranae. The addition of probiotics or both probiotics and prebiotics to the food of uninfected bees led to the ~2-fold decrease in the PO activity. The infection of honeybees with N. ceranae accompanied an almost 20-fold decrease in the PO level. The inulin supplemented solely at a concentration of 2 μg/mL was the only administrated factor which did not significantly affect honeybees’ survival, the PO activity, or the nosemosis infection level. In conclusion, the supplementation of honeybees’ diet with improperly selected probiotics or both probiotics and prebiotics does not prevent nosemosis development, can de-regulate insect immune systems, and may significantly increase bee mortality.

Nosema ceranae alters a highly conserved hormonal stress pathway in honeybees.

Nosema ceranae, an emerging pathogen of the western honeybee (Apis mellifera), is implicated in recent pollinator losses and causes severe energetic stress. However, whether precocious foraging and accelerated behavioural maturation in infected bees are caused by the infection itself or via indirect energetic stress remains unknown. Using a combination of nutritional and infection treatments, we investigated how starvation and infection alters the regulation of adipokinetic hormone (AKH) and octopamine, two highly conserved physiological pathways that respond to energetic stress by mobilizing fat stores and increasing search activity for food. Although there was no response from AKH when bees were experimentally infected with N. ceranae or starved, supporting the notion that honeybees have lost this pathway, there were significant regulatory changes in the octopamine pathway. Significantly, we found no evidence of acute energetic stress being the only cause of symptoms associated with N. ceranae infection. Therefore, the parasite itself appears to alter regulatory components along a highly conserved physiological pathway in an infection-specific manner. This indicates that pathogen-induced behavioural alteration of chronically infected bees should not just be viewed as a coincidental short-term by-product of pathogenesis (acute energetic stress) and may be a result of a generalist manipulation strategy to obtain energy for reproduction.

Permanent prevalence of Nosema ceranae in honey bees (Apis mellifera) in Hungary

Nosema ceranae is present in honey bee (Apis mellifera L.) colonies worldwide. Studies on the comparative virulence of N. ceranae and N. apis showed significant differences in individual mortality, and the prevalence of N. ceranae seems to be predominant in both the continental and the Mediterranean climate regions. This study attempted to monitor the geographical and seasonal distribution of these two Nosema species in Hungary, using a simple laboratory method. The distribution of N. ceranae and N. apis infection rates along all seasons was homogeneous (P = 0.57). In co-infected samples, the intensity of N. ceranae infection was always significantly higher than that of N. apis infection (P < 0.001). The infection rate of infected bees in exterior samples was higher than in interior samples in each season; however, the differences were not statistically significant. The species N. ceranae had been present in Hungary already in 2004. Statistical analysis of data shows that the infection level is best represented by sampling exterior bees to establish the proportion of infected bees rather than by determining the mean spore count.

Antimicrosporidian activity of sulphated polysaccharides from algae and their potential to control honeybee nosemosis

Nosemosis is one of the most common and widespread diseases of adult honeybees. The causative agents, Nosema apis and Nosema ceranae, belong to microsporidia some obligate intracellular eukaryotic parasites. In this study, 10 sulphated polysaccharides from algae were evaluated for their antimicrosporidian activity. They were first shown to inhibit the in vitro growth of the mammal microsporidian model, Encephalitozoon cuniculi. The most efficient polysaccharides were then tested for their ability to inhibit the growth of Nosema ceranae in experimentally-infected adult honeybees. Two polysaccharides extracted from Porphyridium spp. did not show any toxicity in honeybees and one of them allowed a decrease of both parasite load and mortality rate due to N. ceranae infection. A decrease in parasite abundance but not in mortality rate was also observed with an iota carrageenan. Our results are promising and suggest that algal sulphated polysaccharides could be used to prevent and/or control bee nosemosis.

Identification of candidate agents active against N. ceranae infection in honey bees: establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.

Molecular Identification of Nosema ceranae and Nosema apis in Native Bulgarian Honey Bee (Apis mellifera rodopica)

Molecular Identification of Nosema ceranae and Nosema apis in Native Bulgarian Honey Bee (Apis mellifera rodopica) The aim of current study is to investigate the presence of two main microsporidian parasites N. apis and N. ceranae in Bulgarian local honey bee. The discrimination among two Nosema species was carried out on 18 individual samples. The 16S (SSU) rDNA gene region was analyzed and duplex PCR assay was performed. The results showed (1) the dominance of N. ceranae infection in Bulgaria (2) N. ceranae most likely switched its host to A. mellifera, probably within the last decade (3) N. ceranae is now a parasite on A. mellifera across worldwide. The rapid, long-distance dissemination of N. ceranae is likely due to transport of infected honey bees and/or by increased mobility of people, goods and livestock.

Spore Loads May Not be Used Alone as a Direct Indicator of the Severity of Nosema ceranae Infection in Honey Bees Apis mellifera (Hymenoptera:Apidae).

Nosema ceranae Fries et al., 1996, a microsporidian parasite recently transferred from Asian honey bees Apis cerana F., 1793, to European honey bees Apis mellifera L., 1758, has been suspected as one of the major culprits of the worldwide honey bee colony losses. Spore load is a commonly used criterion to describe the intensity of Nosema infection. In this study, by providing Nosema-infected bees with sterilized pollen, we confirmed that pollen feeding increased the spore loads of honey bees by several times either in the presence or absence of a queen. By changing the amount of pollen consumed by bees in cages, we showed that spore loads increased with an increase in pollen consumption. Nosema infections decrease honey bee longevity and transcription of vitellogenin, either with or without pollen feeding. However, the reduction of pollen consumption had a greater impact on honey bee longevity and vitellogenin level than the increase of spore counts caused by pollen feeding. These results indicate that spore loads may not be used alone as a direct indicator of the severity of N. ceranae infection in honey bees.

Retrospective study of the Nosema ceranae infection of honey bee colonies in Iran (2004-2013).

Nosemosis is the most common disease in adult bees. Nosema apis and Nosema ceranae species are agents of important economic losses to beekeepers around the world. The severity of disease at various area is different. Previously, N. apis was observed in areas with a long winter, especially in late winter and early spring. But in recent years, disease has been reported in the warm seasons. The studies indicated that a new species as N. ceranae is involvement in loss and mortality in adult bees. Therefore, diagnosis and differentiation of Nosema species is importance at colony collapse disorders (CCD). The aim of this Research was a retrospective study on Nosema samples isolated from apiaries. Forty- one Nosema Sp. Positive samples were collected from five provinces during 2004 to 2013. The samples were tested by multiplex PCR method using both primers of N. ceranea and N. apis were simultaneously. All of samples were positive for N. ceranea. The products were sent for sequencing. The results show that N. ceranea has spread in Iran, from previous years almost simultaneously with other parts of the world.

Infectivity and virulence of Nosema ceranae and Nosema apis in commercially available North American honey bees

Nosema ceranae infection is ubiquitous in western honey bees, Apis mellifera, in the United States and the pathogen has apparently replaced Nosema apis in colonies nationwide. Displacement of N. apis suggests that N. ceranae has competitive advantages but N. ceranae was significantly less infective and less virulent than N. apis in commercially available lineages of honey bees in studies conducted in Illinois and Texas. At 5days post eclosion, the most susceptible age of adult bees tested, the mean ID50 for N. apis was 359 spores compared to 3217 N. ceranae spores, a nearly 9-fold difference. Infectivity of N. ceranae was also lower than N. apis for 24-h and 14-day worker bees. N. ceranae was less infective than reported in studies using European strains of honey bees, while N. apis infectivity, tested in the same cohort of honey bees, corresponded to results reported globally from 1972 to 2010. Mortality of worker bees was similar for both pathogens at a dosage of 50 spores and was not different from the uninfected controls, but was significantly higher for N. apis than N. ceranae at dosages ⩾500 spores. Our results provide comparisons for evaluating research using different ages of bees and pathogen dosages and clarify some controversies. In addition, comparisons among studies suggest that the mixed lineages of US honey bees may be less susceptible to N. ceranae infections than are European bees or that the US isolates of the pathogen are less infective and less virulent than European isolates.

Sex-Specific Differences in Pathogen Susceptibility in Honey Bees (Apis mellifera)

Sex-related differences in susceptibility to pathogens are a common phenomenon in animals. In the eusocial Hymenoptera the two female castes, workers and queens, are diploid and males are haploid. The haploid susceptibility hypothesis predicts that haploid males are more susceptible to pathogen infections compared to females. Here we test this hypothesis using adult male (drone) and female (worker) honey bees (Apis mellifera), inoculated with the gut endoparasite Nosema ceranae and/or black queen cell virus (BQCV). These pathogens were chosen due to previously reported synergistic interactions between Nosema apis and BQCV. Our data do not support synergistic interactions between N. ceranae and BQCV and also suggest that BQCV has limited effect on both drone and worker health, regardless of the infection level. However, the data clearly show that, despite lower levels of N. ceranae spores in drones than in workers, Nosema-infected drones had both a higher mortality and a lower body mass than non-infected drones, across all treatment groups, while the mortality and body mass of worker bees were largely unaffected by N. ceranae infection, suggesting that drones are more susceptible to this pathogen than workers. In conclusion, the data reveal considerable sex-specific differences in pathogen susceptibility in honey bees and highlight the importance of ultimate measures for determining susceptibility, such as mortality and body quality, rather than mere infection levels.

Nosema ceranae and queen age influence the reproduction and productivity of the honey bee colony

SummaryThe strength of the honey bee colony and the quality concerning brood production, number of worker bees and honey yield depend substantially upon the characteristics of the queen. Reproductive and productive capacities of the colony, as two important physiological qualities can depend a great deal on the age of the queen. Over the years, the laying ratio of queens could decrease depending on the age; therefore colonies headed by one-year-old queens have proven to be more efficient considering colony population and honey production than ones with older queens. Nosema ceranae infection is a possible reason for the decrease in reproductive capacities and the reduction in the honey production of infected colonies. With the help of numerous pheromones, the queen can preserve colony homeostasis, however exhausted by N. ceranae infection she might compromise the renewal of worker population. The aim of this research is to investigate the influence of N. ceranae on reproduction and productivity of honey bee colonies headed by queens of different age. Experiment was conducted on 27 honey bee colonies equalized with regard to brood, food source, number of worker bees and Nosema infection. In the three year investigation period (2009–2012), Nosema detection, identification and spore load per each colony, as well as parameters of colony reproduction (such as uncapped and capped brood area, queen egg-laying rate and colony strength) and productivity (the total extracted honey and the winter honey stores) were recorded. Concerning the influence of the age of the queens on followed parameters we compared one, two and three-years old queens and found significantly higher reproductive and productive values in favour of younger ones. Significant differences regarding the effects of N. ceranae infection on productive and reproductive characteristics could be explained with a higher capacity of one year old queens to compensate the effects of Nosema infection; a capacity which gradually declines in the second and the third year.

Four quantitative trait loci associated with low Nosema ceranae (Microsporidia) spore load in the honeybee Apis mellifera

Nosema ceranae has been recently introduced into the honeybee Apis mellifera as a novel microsporidian gut parasite. To locate the genetic region involved in N. ceranae infection tolerance, we fed N. ceranae spores to haploid drones of a F1 hybrid queen produced from a cross between a queen of a Nosema-resistant bred strain and drones of susceptible colonies. The spore loads of the infected F1 drones were used as the phenotype to identify quantitative trait loci (QTLs) associated with N. ceranae spore load. One hundred forty-eight infected drones were individually genotyped with microsatellite markers at an average marker distance of 20 cM along the genome. Four QTLs were significantly associated with low spore load, explaining 20.4 % of total spore load variance. Moreover, a candidate gene Aubergine (Aub) within the major QTL region was significantly overexpressed in drones with low spore loads than in those with high spore loads. Our results confirm the genetic basis of Nosema tolerance in the selected strain and show that both additive effects and epistatic interactions among the QTLs interfere with the tested phenotype.