The Transition Ratio of Nosema spp . Spores From Colonies to Honey Versus Honey to Colonies

Honey bees (Apis mellifera L.) are insects that have an important role in plant pollination as well as production of benefical products such as honey, propolis, pollen, royal jelly, bee venom and beeswax. There is a growing interest in bee diseases and loss, which is a major threat to the economy and human health. Nosemosis is an adult honey bee disease which effects its digestive system mostly. The cause for the disease is Nosema apis or Nosema ceranae but the two can be seen together, too. This article aims to explore the transmission of Nosemosis and its effect on honey. For this purpose, a field study was conducted in Muğla province, where 51 pieces were collected from bee yards as spring samples and 51 pieces from bee yards as autumn samples during these two seasons, and 51 honey samples from bee yards were examined during the honey harvest. The results revealed that Nosema spp. which was obtained from honey bee samples collected in spring was more effective on honey samples. Nosema spp. was found to have a linear relation with the infection in the hive. It was determined that the percentage of Nosema spores seen in adults was 1.63%. This result contributed to the literature by providing this ratio used in estimating the level of infection in the colonies by means of honey sampling. Furthermore this is the first study where the contamination risk of honey from the infected colonies is calculated. Hopefully, this study can provide background for further research on the protection of bee colonies and risk assessment against Nosema spp. disease.


Introduction
Honeybees have a crucial role to carry 75% of pollination, so honeybee colony losses is a great threat to the agricultural production globally.Of all the factors that threaten bees, Nosemosis is reported to be the most prevalent factor for colony losses all over the world (Huang et al., 2013;Botias et al., 2013).There is a growing interest in the newly detected microsporidian gut parasite Nosema ceranae because an unusually high honey bee colony mortality coincided with its apparent host-switch from Asian honey bees (Apis cerana) to honey bees (Fries, 2010), as well as its subsequent widespread dispersal (Williams et al., 2008).N. ceranae can cause tissue damage (Higes et al., 2008;Dussauabat et al., 2013), nutritional stress (Mayack & Naug, 2009;Alaux et al., 2010) and suppression of host immunity (Chaimanee et al., 2012).In Spain, N. ceranae is typically associated with reduced colony survivorship (Higes et al., 2008), whereas in other parts of Europe (Genersch et al., 2010) and in North America (Guzman et al., 2010;Villa et al., 2013), its virulence is debated.Possible explanations for this variation include parasite or host genetics (Medici et al., 2012), climate (Chen et al., 2012), nutrition (Alaux et al., 2010), or interactions with other stressors such as environmental contaminants or other parasites (Pettis et al., 2013).That is why disappearing of honeybees must be investigated and in cases like Nosemosis infections, risk assessment should be practiced.
Nosema spp. is the pathogen of Nosemosis, a parasitic disease, which effects the adult bees at a global scale.It is a genus of fungi which belongs to Microsporidia (Fries, 1993(Fries, , 2010)).There are two species Nosema apis and Nosema ceranae.Colonies infected by Nosemosis show the following symptoms: reluctance to fly, abdominal swelling, dyspepsia, and leaving the hive.Recently, this is the most important reason of colony losses reported all over the world.(Botias et al., 2013;Fries, 2010;Fenoy et al., 2009;Chen et al., 2008;Martin-Hernández et al., 2007;Hornitzky, 2005;Webster et al., 2004).According to this result, the correlation variant is bigger than 0 and equal to 1; the correlation between the honey samples and adult bee samples is significant.A graph of Nosema spores count, prepared with the average spring and autumn adult bee samples and honey samples can easily show the correlation (Figure 4).The main reason for selecting Muğla and Pine Honey was to ensure the contamination was only from adult bees and to minimize the effects of other sources of infection like water pollens etc. because during Pine Honey production season, there aren't any pollen sources.In the test performed to understand the correlation between Nosema spp. in adult bee samples and the Nosema spp. in honey Multiple R-squared value has been calculated close to 1.This value is expected to be between 0 and 1 and when it is closer to 1 it proves that the nosema ssp in honey is hive sourced.After defining that the Nosema spp in the honey is sourced by colony infection, the second aim was to prove its statistical significance, thus "one-way ANOVA" was used in this study.According to ANOVA results, if Pr > 0.05, Nosema spp.variable in the hive is significant.The relationship between the Nosema spp.counts in the colony and the Nosema spp.counts in the honey were analyzed by Pearson correlation test and correlation value was defined.The value is a positive value (0.9744065) which proves that there is a direct relation in counts of Nosema spp. between colony and honey.This has importance for further studies to calculate the infection rate in both sides.When the same case applied in flower honey at least the minimum rate of infection from the adult bees can be calculated and then additional other sources of infections can be considered (Peukpiboon et al., 2017).
As it is shown in Figure 4. as the counts of Nosema spp.levels increased the level in the honey also increases unless infection level is not over the threshold value (threshold value = 200.000spores/bee).Under this value Nosema spp.spores are not found in honey and this clears that Nosema spp.spores were found in 39 of 51 honey samples.This also coincides with the contamination from colony to honey ratio 1.63%.
The samples collected were also studied by "paired t-test" statistically in order to define the differences according to seasons and was found p-value < 0.05 significant.Statistically, seasons effect the contamination of Nosema spp.Main reason for this can be migratory beekeeping during spring season.In this season colony number increases and they are kept very close to each other, however during pine honey production period 75% of 7 million colonies migrate to Muğla.
When the transition rate of Nosema spp.from adult bees to honey is studied according to seasons (paired-t test p-value < 0.05) the correlation value is higher in Spring season.In other words, the Nosema spp.inflation in Spring increases the Nosema spp.spore counts more than it does in Autumn.This can be due to the fact that spring time is closer to honey production period and spores can be more viable.Viability of the spores can be less in Autumn samples which has passed a cold winter season.This result matches with other reports from different countries (Malone et al., 2001;Mayack &d Naug 2009;Fenoy et al., 2009;Williams et al., 2014;Gisder et al., 2010;Stevanovic et al., 2010).This is the first study that determines the transition of Nosema spp.spore to honey and its risk ratio.By finding the Nosema spp.spore counts in the colony, out of honey samples will be a corner stone for further studies.This study shows that the higher prevalence of Nosema spp.could be kept at a minimum level by means of using these formulas to calculate Nosema spp.transition cycles from colonies to honey and vice versa.And also, it can determine the migratory beekeeping routes, hive capacities of places etc. to prevent colonies from Nosema diseases.

Table 1 .
Th r Spring and A mples, and bo ) N. apis, (b) N

Table 2 .
The comperative results of Nosema spp.analyses in colonies spring and autumn seasons and honey samples Nosema spp.counts out of adult bees and honey samples collected in spring and autumn were both investigated and the data obtained according to the statistical analysis were presented by R programming output.(Accordingto the Regression Analysis R programming ouput in order to define the origin of Nosema spp.spores)Residual standard error: 0.01695 on 48 degrees of freedom Multiple R-squared: 0.9521, Adjusted R-squared: 0.9501 F-statistic: 476.9 on 2 and 48 DF, p-value: < 2.2e-16The multiple R-square value obtained was very close to 1, meaning that Nosema spp.spores in the honey is mostly caused by Nosema spp.infection of the colony.
This study conducted in Muğla tries to answer the following questions; what is the effect of Nosema spore contamination of honey in production of Pine Honey and the infection rate of Nosema spp.spores to contamination rate?