N. apis and N. ceranae microsporidia are highly specialized unicellular eukaryotic parasites (Adl et al., 2005). These pathogens are responsible for nosemosis, one of the most widespread bee diseases in the world. Bees are infected by spores ingested with water or pollen (Webster et al., 2004; Chen et al., 2008). The spread of nosemosis is largely influenced by weather conditions in different seasons of the year. Sulborska et al. (2019) have reported that Nosema spp. are transmitted with the wind. N. ceranae is regarded as the most ubiquitous endoparasite of honey bees, but N. apis is also frequently encountered in Poland and in the world (Fries, 2010; Higes et al., 2013). However, the infection dynamics and the interactions between Nosema spp. and L. passim in honey bees inhabiting tree trunks and log hives have never been investigated. This study is the first attempt in both Poland and the world to determine the severity of infections caused by Nosema spp. and L. passim in tree trunks. In this study we aimed to evaluate the prevalence of Nosema spp. and L. passim in bee inhabiting tree trunks by real-time PCR and determine relationships in the occurrence of these pathogens.
MATERIAL AND METHODS
Bee samples
The study was performed on twenty-six samples of bees (colonies) collected from tree trunks in north-eastern Poland (21°04′0″-21°41′0″E and 53°09′0″-53°29’0″N). Feral honey bees were sampled only from naturally colonized sites. No clinical disease symptoms like diarrhea, inability to fly, paralysis, gathering in small groups, dead bees on the bottom of the hive with swollen abdomen and legs retracted below the chest, or reduced activity were observed in these colonies. The experiment involved colonies with a life span of at least three seasons, during which the insects were monitored. Workers were sampled from every colony with the use of an open 0.9 l jar that was placed under the exit hole in a hollow tree. When bees entered the jar, the jar was capped, transported to the laboratory and stored at a temperature of −20°C.
DNA analysis
The collected twenty-six samples were analyzed with the use of molecular methods to identify and quantify Nosema spp. and L. passim. Each test was performed on a random sample of sixty worker bee abdomens collected from hollow trees. Whole abdomens of sixty bees per sample were homogenized with a homogenizer. The whole homogenate from each sample was taken for DNA extraction. Genomic DNA was isolated with the Genomic MiniKit (A&A Biotechnology, Gdynia, Polska) according to the manufacturer's instructions. Real-time PCR was conducted with the use of FastStart Essentials DNA Green Master (Roche). The 20-μl reaction mixture contained ca. 150 ng of isolated DNA (1 μl), 10 μl of FastStart Essential DNA Green Master, and 0.5 μl of each primer (with a final concentration of 0.5 μM). It was supplemented to 20 μl with FastStart Essential DNA Green Master Water (PCR grade). The qPCR reaction was carried out in a Light Cycler Nano System thermal cycler (Roche). The reactions were run using a thermal profile consisting of a hold at 95°C for 10 min, followed by 40 cycles of a 2-step amplification consisting of 95°C for 10 s, primer annealing at 54°C for 60 s and 72°C for 15 s, melting at 65°C for 60 s and 95°C for 1 s, and a final hold at 45°C for 600 s. Lotmaria passim was identified with primers LpCytb_F2: 5′-AGTaTGAGCaGTaGGtTTTaTTATa-3′ and LpCytb_R: 5′-gcCAaAcACCaATaACtGGtACt-3 (Vejnovic et al., 2018).
Nosema apis and N. ceranae were identified and quantified with FastStart Essentials DNA Green Master (Roche). The 20-μl reaction mixture contained approximately 150 ng of isolated DNA (1 μl), 10 μl of FastStart Essential DNA Green Master, and 0.5 μl of each primer. It was supplemented to 20 μl with FastStart Essential DNA Green Master Water (PCR grade). The following primers were applied: 321APIS FOR (5′-GGGGGCATGTCTTTGACGTACTATGTA-5′) and 321APIS REV (5′-GGGGGGCGTTTAAAATGTGAAACAACTATG-5′) for N. apis, and 218 MITOC REV (5′-CCCGGTCATTCTCAAACAAAAAACCG-5′) for N. ceranae (Martín-Hernandez et al. 2007). The reactions were run using a thermal profile consisting of a hold at 95°C for 600 s, followed by forty-five cycles of a three-step amplification consisting of 95°C for 20 s, 55°C for 20 s, and 72°C for 15 s, melting at 65°C for 60 s and 95°C for 1 s, and a final hold at 45°C for 600 s. In the real-time PCR assay, L. passim, N. apis and N. ceranae parasites were quantified in bees inhabiting tree trunks with the use of DNA standards (University of Belgrade, Faculty of Veterinary Medicine, Department of Biology, Serbia). In each reaction, standard curves were generated by ten-fold serial dilutions of known concentrations of L. passim, N. apis, and N. ceranae DNA for each parasite separately. Each run contained negative and positive control and experimental samples preformed in duplicates. The gene copy number of L. passim and Nosema spp. were calculated using software program LightCycler Nano (Roche).
Statistical analysis
The statistical analysis was performed using SAS software Version 9.5 (Statistical Analysis System Institute, Cary, NC). The correlation between the analyzed pathogens was determined through the calculation of Spearman's rank correlation coefficient r. The number of copies of the genome Nosema spp. and L. passim identified on the bees were compared using the one-way ANOVA, followed by Tukey test. Differences at p ≤ 0.05 were considered significant.
RESULTS
After the examination of twenty-six feral honeybee colonies, we found that N. ceranae was the predominant pathogen. The presence of N. ceranae was identified in twenty-five samples (96.15%). N. apis in twenty samples (76.92%), L. passim in fourteen samples (53.84%); both pathogens were always accompanied by N. ceranae. While Nosema ceranae alone was detected in only two cases. Coinfections with N. ceranae/L. passim were only noted in three samples (11.5%), coinfections with N. apis/N. Ceranae in nine samples (34.61%) and multiple infections with N. ceranae/N. apis/L. passim in eleven samples (42.3%) (Fig. 1). Statistically significant (F=25.17 p=0.000001) variations in the average gene copy number of L. passim and Nosema spp. were observed in the bees (Fig. 2). A significant correlation was observed between infection with the three analyzed pathogens (N. apis, N. ceranae, L. passim) (r = 0.84) compared to infection with two pathogens only (N. apis and N. ceranae) (r = 0.49).
Fig. 1
Severity of infections caused by L. passim and Nosema spp. in bee samples.
Fig. 2
Average number of copies of the genome per bee.
a, b – the different small letters denote statistically significant differences between the number of copies of the genome of Lotmaria passim and Nosema spp. (one-way ANOVA; Tukey test, p ≤ 0.05)
DISCUSSION
Crithidia mellificae (Langridge & McGhee, 1967) were initially regarded as the most prevalent pathogen of family Trypanosomatidae in honeybee colonies (Ravoet et al., 2013). However, Schwarz et al. (2015) demonstrated that honeybees were most frequently infected by L. passim. Many isolates that had earlier been identified as C. mellificae were reclassified as L. passim, and the latter species is presently regarded as the predominant protozoan species in bees worldwide (Schwarz et al., 2015). In this study, we attempted to evaluate the prevalence of L. passim and Nosema spp. in feral honeybees and to determine potential relationships between the occurrence of these parasites.
As many researchers have demonstrated, L. passim can compromise the health of honeybees, and coinfections with other pathogens can exacerbate colony losses. Tritschler et al. (2017) reported a relationship between the occurrence of Nosema spp. and L. passim in honeybees. Contrary results were reported by Vejnovic et al. (2018), who attributed the observed variations in the prevalence of Nosema spp. and L. passim to different methodological approaches. Vejnovic et al. (2018) studied pooled samples, whereas Tritschler et al. (2017) examined individual bees. Research results are also affected by weather conditions and differences in the prevalence of Nosema spp. in various countries (Sulborska et al., 2019). These pathogens have been identified in 46.7% of bee colonies in Switzerland (Retschnig et al., 2017) and in 95.7% of bee colonies in Serbia (Stevanovic et al., 2016).
Bee colonies are also increasingly being infected with N. ceranae in Poland, which was confirmed in our previous study (Michalczyk, Sokół, & Koziatek, 2015). Nosema ceranae was also the predominant pathogen in the feral honeybee samples analyzed in the present study (Fig. 2). Lotmaria passim always co-occurred with Nosema spp., and considerable differences in the abundance of pathogens were noted between tree trunks. In contrast, Vejnovic et al. (2018) reported no correlation between the occurrence of N. ceranae and L. passim. In the current study, nearly all monitored colonies were infected with N. ceranae, and coinfections with L. passim were additionally observed in fourteen samples. Real-time PCR supported accurate quantification of Nosema sp. and L. passim in the evaluated colonies. Our data presented significantly more cases with L. passim infection with both Nosema spp. compared to cases where L. passim infection was accompanied by a single Nosema spp. This may be due to the exhaustion of the bee's organism and complete loss of its defense capabilities. Therefore, it is subjected to the penetration of the third parasite L. passim. A similar relationship was observed by Ptaszyńska, Paleolog, & Borsuk (2016) and Borsuk et al. (2013). The authors found that N. ceranae infection promoted the proliferation of yeasts in honey bee intestines. In our case, the invasion of three pathogens was intensified, perhaps due to the compromised immunity of the honeybees. N. ceranae (Antùnez et al., 2009) suppresses the immune system, which facilitates the penetration of other parasites into the bee's organism and additional deterioration. This in turn leads to the depopulation and extinction of bee colonies. From the etiological point of view, it will be important to establish which pathogen is the first to colonize the host in future studies. The role played by L. passim in honey bees colonies is still being debated, but there are no doubts that this parasite compromises bee health. A steady increase in the mortality of bee colonies and lower levels of bee activity were reported by Chilean beekeepers (Arismendi et al., 2016). These losses were probably sustained due to the presence of N. ceranae in 18% of the studied apiaries, but L. passim was also detected in 90% of the analyzed samples (Rodríguez et al., 2012; Rodríguez et al., 2014). A similar study was conducted in Belgium by Ravoet et al. (2013), who identified C. mellificae as a new pathogen responsible for the collapse of bee colonies.
In view of the general scarcity of epidemiological data about coinfections with Nosema spp. and L. passim in feral honey bees in other countries, further research is needed to confirm the effect of concurrent pathogenic infections on the decline of bee colonies. The role of L. passim and coinfections with other pathogens, including viral, bacterial and parasitic infections, in bee colonies should be investigated.
