4.1. Experiment 1: Competition between Nosema ceranae and Nosema apis in Mixed Infection
Our results suggest that
N. ceranae has a negative effect on the longevity of infected bees. The effect seems to be more clearly visible than in case of
N. apis, although in the first experiment (when the bees that survived were frozen at 24th day post infection) only the tendency (a clear one nevertheless) was observed. However, during the second experiment the difference was significant, which confirms the findings of a Canadian study [
36]. However, this contradicts with findings from Sweden and Germany [
35,
37], where such a difference was not found.
This may suggest a geographical/climatic component of the virulence level of N. ceranae compared to N. apis. As mentioned before, in the warmer parts of Europe it causes high colony losses, and in the colder ones it seems to be quite a mild infection.
There is also the experimental design component, which may significantly impact the results. We infected five-day old bees and mentioned authors who infected, respectively, two-week old bees, 48 h old bees and three-day old bees. Younger bees may be more susceptible to
N. ceranae infection since their perytrophic membrane is slightly thinner and may be more easily penetrated by the spores and the shorter life cycle of
N. ceranae than
N. apis (three days, and five days respectively) [
5] enabling faster multiplication. It would also explain why we found higher
N. ceranae DNA copy numbers in mixed infection (
N. apis and
N. ceranae), but also singular infections with either
N. apis or
N. ceranae. Young bees may also be more prone to energetic stress [
7], which is more evident during the infection caused by
N. ceranae than
N. apis. It was proven [
38] that five-day old bees (or slightly older) are the most susceptible to infection. However, the two-week old bees may just be too old already, and their peritrophic membrane too thick to accommodate
Nosema as well as in the case of younger bees. On the other hand, the newly emerged bees are known to be more resistant to
Nosema infection (the reasons of this phenomenon are still unclear), therefore newly emerged bees are always free from infection, even after they chew on infected wax or eat infected food [
38]. Our findings confirm that bees infected with
N. ceranae had higher cumulative mortality than those infected with
N. apis [
39]. The results from our experiment also correspond with the research showing higher virulence of
N. ceranae than
N. apis [
16]. Discrepancies between results may be also due to different temperatures in which bees were kept, namely bees in the Swedish experiment [
35], which were kept in 30 °C, when bees in our experiment were kept in 34 °C, quite similarly to a 2009 study [
40] with which we received comparable results.
We think that infecting young bees and keeping them at a temperature of 34 °C more accurately mimics natural conditions, where bees can be infected during the first days after emergence. During the following days, they mainly stay in warmer parts of the hive because of the tasks performed in the colony, allowing the spores time to multiply. This is why, in field conditions (apart from constant ingestion of spores with food overtime), the numbers of spores in older bees are higher.
In our experiment, infection of individual bees at the 5th day post emergence with
N. ceranae resulted in the first deaths after the 14th day post infection, similar to the case of bees infected with
N. apis (in the second experiment it was 12 and 10 days, respectively), and with the mixture of both species. These findings are similar to the results of another study researching the virulence of both
Nosema species in individual bees [
35]. But another study [
31] found that the first deaths were observed at the 6th day post infection, and all infected bees were dead at the 8th day post infection. The difference between results might have been caused by using bees from different times of the season, namely, in the contradicting paper, late spring bees were used, and in our experiment we used August bees, which are practically winter bees already and have different body constitutions and, in general, live much longer than spring bees. We also did not use CO
2 when handling the bees, since it was proven to shorten their life significantly [
41,
42]. In addition, more recent studies clearly show, that CO
2 treatment influence was so considerable, that the influence of
N. ceranae on the longevity of bees could no longer be observed [
43].
It is also highly possible that bees (either used for the experiment or used to extract spores from) in experiments of most of the contradicting studies, might have been infected with BQCV (partly or entirely), which was not investigated. The results might be due to presence of this virus in the group of bees infected with
N. apis and absence in the group infected with
N. ceranae [
38] and its presence in the
N. apis spore solution [
20]. Adding the BQCV antiserum to
Nosema spore solutions eliminated this possibility in our experiments.
Our finding, that
N. ceranae is highly competitive against
N. apis in mixed infections and reaches much higher spore counts contradicts other results [
20], in which
N. apis was the one with higher (or similar to
N. ceranae) spore counts. This might suggest that there may be several
N. ceranae strains exhibiting different virulence rates, but this was proven untrue [
44], so, in this case, a conclusion that different bee subspecies undergo nosemosis differently seems logical, which is supported by the Danish case [
45] and other studies [
39,
46,
47]. In one of the studies [
38],
A. m. ligustica was used, whereas in others it was Buckfast bees [
36] and
A. m. carnica [
37].
4.2. Experiment 2: Influence of BQCV on the Course of Nosema Infection
In the 1980s [
22], it was proven that bees infected with
N. apis and BQCV lived shorter than bees infected with
N. apis alone. In our study, in the case of
N. ceranae, this effect was expressed even better. This can also be correlated to the higher number of virus copies in bees with
N. ceranae, which, because of a very quick multiplication and causing much more damage to epithelial cells than
N. apis, opened more entrance sites for BQCV. What is more, greater (than with
N. apis) energetic stress caused by
N. ceranae can explain why BQCV would multiply better in
N. ceranae infected bees.
The fact that
Nosema and BQCV act synergistically in adult honeybees and induce high and rapid mortality is in agreement with several other findings [
36,
48] and seems to be universally true.
Occurrence and mean peptide count of viral pathogens and Nosema in honeybee colonies sampled from widespread locations in USA CCD colonies showed, instead, a high frequency of iridescent virus and a low frequency of BQCV [
49].
Bees infected with either species of Nosema and BQCV showed lower spore counts than the ones infected with only Nosema. Since dead bees were investigated, this result was probably due to the synergistic effect of the virus and Microsporidium, which caused faster death of bees, hence the spores could not multiply further. As the effect was much stronger in the case of N. ceranae, as expected, this dissonance is much clearer between bees with N. ceranae, together with BQCV, and bees with only N. ceranae, than in respective groups with N. apis.
Since this experiment was conducted in 2013, we based the design on available publications on this topic [
22,
50], which state that there is no virus propagation, or it is very minor, without the presence of
Nosema sp. However, a more recent study [
48] proved that the virus can multiply on its own, so a design with a control group with only BQCV infected bees is needed.
Further experiments are needed since it would also be desirable to compare the above results with spore and virus counts from live bees, as well as through the experiment. Studies on temperature influence on the course of virus/Nosema infection are also needed.
It has also become more apparent that a thorough comparison of Nosema infection course in different bee subspecies and, simultaneously, different climatic conditions, is needed. Those two factors together may have a greater influence on the course of this infection than previously thought.
This study shows the effects of interaction between N. ceranae, N. apis and black queen cell virus, as well as the effect of both Nosema species on the longevity of bees. All organisms (bees, Nosema), as well as the virus, were of Polish origin and show only the interaction of actors of said origin. As shown above, further multi-direction research is needed to say with certainty what influences the pathogenicity/virulence of N. ceranae, which, in time, may open new paths to controlling Nosema infections and preventing related colony losses.