Received: 10 March 2022 Accepted for publication: 27 July 2022 Slov Vet Res 2022; 59 (3): 159–67 DOI 10.26873/SVR-1498-2022 UDC 638.12:638.144:638.124.8 Original Research Article Introduction Honeybees are one of the most important pollinators for the agriculture and food production. The density of honeybee colonies can be very versatile in different parts of the world, depending mostly upon availability of nectar flow. Facing the overpopulated area with honeybee colonies can lead to decrease of health status, frequent infections with pathogens and exploitative competition with wild bees, like bumblebees (1). A success and production in beekeeping sector always depends on the weather conditions, climate change, health condition of the colonies and changes in the environment due to human PERFORMANCE AND Nosema spp. SPORE LEVEL IN YOUNG HONEYBEE (Apis mellifera carnica, Pollmann 1879) COLONIES SUPPLEMENTED WITH CANDIES Maja Ivana Smodiš Škerl1*, Ivana Tlak Gajger2 1Agricultural Institute of Slovenia, Hacquetova ulica 17, SI-1000 Ljubljana, Slovenia, 2Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia *Corresponding author, E-mail: maja.smodis.skerl@kis.si Abstract: We evaluated the efficacy of supplementation with protein, yeast, or sugar candies of young honeybee colonies origi- nated from artificial swarms, by measuring strength and determining Nosema spp. spore level in adult bees in summer period. At the same time, we aimed to assess longevity of adult worker bees after feeding the same type of candies in controlled laboratory condition. The highest survival was found in Yeast and Protein candy group. On the contrary, the field study showed that artificial swarms produced significantly more pupae (2510.4 cm2, p=0.0001) in the 1st period of measurement, and more larvae (964.8 cm2, p=0.003) and frames with bees (5.6, p=0.008) in the 2nd period by feeding non-protein candy. In the 3rd period of evaluation of young colonies, the Sugar candy group had the highest number of frames covered by adult bees and honey stores, respectively (5.6, p=0.0009; 3432.0 cm2, p=0.015). Sugar candy group produced the largest area of wax cells, however, the differences were not statistically significant. Nosema spp. spore level was checked quantitatively in adult bees. The lowest infection was statistically significant in Yeast candy group in June (4.35 million spores per bee, p=0.02), but insignificant in September. Supplementing ar- tificial swarms Sugar candy offers the most promising potential for development of productive young colony. The findings of our study could help beekeepers to choose the effective candy supplement for optimal development of artificial swarms. Key words: Apis mellifera; artificial swarms; young colonies; supplements; candies; development; Nosema spp.; longevity interventions. The essential honey bee colony needs must cover a proper quantity of quality food for productive development, reproduction, and honey yield (2), to overcome the stress caused by pesticides and pathogens. A honey bee colony needs pollen to meet the needs for protein, lipids, and vitamins (2, 3, 4). The lack of pollen diversity and diminished quantity affects the colony in a healthy brood production, increases infections due to deterioration of immune system defence mechanisms and shortens the life span of bees (4-11). Carbohydrates are the source of energy and structural storage polysaccharides (in plants there are as starch and inulin; in animals they are stored as glycogen and chitin). In pollen there are up to 41 % of sugars, but 50 % of them are starch and cell wall constituents and very hard to digest for the honey bees (4). Therefore, floral M. I. Smodiš Škerl, I. Tlak Gajger160 nectar and pollination drops are the richest source of carbohydrates for adult bees containing glucose, fructose, and sucrose, depending on the flowering periods and types of plants available. Honeydew is another source of carbohydrates for honey bees containing glucose, fructose, and sucrose like nectar, but also more complex sugars (maltose, melezitose etc.). It is usually available during nectar dearth and occurs on leaves of fir, larch, pine, oak, spruce etc. Nectar and honeydew are collected by bee foragers, taken to the bee hive, processed, and stored in wax cells as ripe honey. Bees need round 4 kg of honey to produce 500 g of wax or young bees at age 12 to 18 days need on average 20 kg of sugars (fructose, glucose, sucrose) and a significant amount of proteins to produce 1 kg of wax (4, 12). Most insects can regulate protein and carbohydrates from natural food sources for optimal growth and survival (13). Different ratios of carbohydrate to protein affect social insect physiology and ability to survive. The optimal balance of nutrients can be determined by the geometric framework (13, 14). Adult honey bees lived longer on a pure carbohydrate or low protein diet (15). Paoli et al. (16) showed that honey bees of different age and behavioural have different nutritional requirements. The mortality of adult bees fed high amino acid diet was quite high. Also confirmed in ants, where Dussutour and Simpson (2012) (17) showed a reduction in ant worker lifespan when feeding a high protein diet. On contrary, Archer et al. (2013) (18) found that honey bees exposed to environmental stressors (e.g. low temperatures, nicotine) and fed with high protein diet (1:3=P:C) had lower mortality. However, they found that workers in this experiment did not adjust their intake to improve their survival after being exposed to the stressed condition. Feeding protein also extended the survival ability of adult bees after infection with Nosema sp. (19). In beekeeping management, the establishment of young colonies is a frequently used practice in early summer to use the natural reproduction of the colony and to prevent unwanted swarming. New colonies established in May and June need round 1.5 kg of mostly young honeybees and a young, mated queen (20). As such they represent a swarm as a new young colony that must be placed by the beekeeper on another location than its primary colony. When placing the adult bees in a hive with frames and a wax comb foundation only, a newly established young colony requires constant food supplementation (20, 21) to support comb building and foragers start to bring nectar and pollen in the hive and the queen starts egg-laying. The choice of a food supplement is therefore a key factor to bring young colonies in the best shape and increase their potential of productiveness. In our study we fed the bees with different candies in laboratory conditions to compare longevity and consumption rates. Another important aim of our study was to evaluate the performance and level of Nosema spp. spores in young honey bee colonies established from artificial swarms, supplemented with different candies in summer, in the apiary conditions. Material and methods The cage trial was established according to the standard methods for maintaining adult bees in controlled conditions (22). Combs with emerging worker bees were obtained from two colonies, placed in an incubator (34.5° C) and left overnight. On the next day, round 1.000 newly-emerged adult bees were collected and randomly put into plastic cages (8 x 12 cm) of air hoarding cages with around 80 openings of ~ 2 mm to provide circulation, and two larger holes for plastic feeders to deliver water and food. 50 bees were introduced in each cage, having 5 replicates per group. Bees were fed Yeast, Protein or Sugar candy, or sugar syrup (1:1, w:v) as a control. The cages with adult bees were kept in a darkened incubator at 28 (±1) °C. Mortality and food consumption were recorded on daily basis. The feeders were weighted on daily basis and the food replaced every 2 or 3 days. The artificial swarms were established in spring (May) from honeybee colonies (Apis mellifera carnica, Pollmann 1879), weighing 1.5 kg of young, mixed-aged bees (weight of worker bees) each and transferred into boxes for swarms (Multibox®, Croatia) (Fig. 1) and left in a dark place overnight. The next day they were transferred into new LR (Langstroth hive) hives (30.5 x 50.5 x 24.3 cm) with 7 frames of AŽ (Alberti-Žnideršič, 410 x 260 mm) with a new wax foundations inserted. In total 18 hives were installed to Jable location (Middle-Slovenian region, 46°08›26.0»N 14°33›22.5»E). The young, mated queen bees were introduced, originating from the same queen breeding operation. Performance and Nosema spp. spore level in young honeybee (Apis mellifera carnica, Pollmann 1879) colonies … 161 Artificial swarms were divided into three groups: (1) Yeast candy, (2) Protein candy and (3) Sugar candy. Colonies were continuously fed with one home-made and two commercial candies: (1) Yeast – grounded cane sugar, 5% baker’s yeast and water, (2) Protein candy – Medopip plus®, Pip d.o.o., Pisarovina, Croatia, (sugar and vitamins) and (3) Sugar candy – Apifonda®, Südzucker, Germany, (sugar), Figure 1. Young colonies were evaluated three times in summer according to Liebefeld method (23). Adult bees from the side frames were collected for Nosema spp. spores quantification twice, at the beginning of feeding in June and later in September. The abdomens of bees were used to estimate the Nosema spp. spores’ prevalence and intensity as determined by light microscopy techniques, described by Cantwell (24). Briefly, bees’ abdomens (20 bees, 3 replicates per colony) were macerated using a mortar and pestle in 1 ml of distilled water/bee. Further, a drop of the solution was placed on a hemocytometer and Nosema spp. spores were counted under a microscope, at 400 x magnification. The experimental colonies were checked for overwintering ability by being inspected in March of the following year. Figure 1: A – Artificial swarms in multiboxes and LR hives with 7-framed wax-foundations. B – Swarms in the hives were established and fed with candy A B All data were analyzed using the RStudio (2021.09.0, PBC, Boston, USA). The data were expressed as mean ± standard error (SE). The lifespan was calculated using Kaplan-Meier curves of honey bee survival, and a log-rank test was performed for significant differences between curves. Measurements of colonies and Nosema spp. were analyzed using one-way ANOVA with Bonferroni corrected t-test. Results In the controlled laboratory conditions, worker bees from Yeast and Protein candy group lived longer compared to Sugar candy group (Fig. 2) having a significant difference between groups. The highest consumption was determined in group fed sugar syrup followed by Yeast candy group (Fig. 3). In young colonies, adult bees were building wax cells most intensively in Sugar candy group in all measured periods, but the differences were not significant (Table 1). Brood area was statistically significant in Sugar and Yeast candy group (1st Period) and later on in Yeast and Protein candy group. Comparing the amount of pollen stores there were no differences, and the highest honey storage was in Sugar group (Table 1). However, we noticed that in the same group the content in the wax cells was white assuming that workers stored candy (personal observations). Number of frames with adult bees was the highest in Sugar candy group showing statistically significant differences from M. I. Smodiš Škerl, I. Tlak Gajger162 Yeast candy group (2nd period), and later the differences were significant in all groups in the last observation period (Table 1). In young colonies, adult bees were building wax cells most intensively in Sugar candy group in all measured periods, but the differences were not significant (Table 1). Brood area was statistically significant in Sugar and Yeast candy group (1st Period) and later on in Yeast and Protein candy group. Comparing the amount of pollen stores there were no differences, and Figure 2: Survival of worker bees in controlled conditions. Kaplan-Mayer survival analysis Figure 3: Cumulative consumption of candies and sugar syrup by worker bees under controlled conditions the highest honey storage was in Sugar group (Table 1). However, we noticed that in the same group the content in the wax cells was white assuming that workers stored candy (personal observations). Number of frames with adult bees was the highest in Sugar candy group showing statistically significant differences from Yeast candy group (2nd period), and later the differences were significant in all groups in the last observation period (Table 1). Artificial swarms Group 1st period 2nd period 3rd period Mean ±SE p-values Mean ±SE p-values Mean ±SE p-values Wax production (cm2) Y 6162 735.79 0.18 6666 909.96 0.26 8947.2 376.53 0.08 S 7780.8 440.8 8308.8 850.61 10896 864 P 7073.14 453.22 8081.14 378.27 10628.57 473.42 Eggs (cm2) Y 384 143.67 0.019 240 150.52 0.08 518.4 120.14 0.18S 840 71.6 648 109.46 624 112.83 P 648 54.17 480 82.48 785.14 77.45 Larvae (cm2) Y 198 128.64 0.04 276* 173.48 0.003 676.8 142.47 0.85S 652.8 138.79 964.8 161.78 782.4 140.27 P 336 72.76 843.43* 50.62 754.29 105.9 Pupae (cm2) Y 492* 202.35 0.0001 522* 318.45 0.001 2078.4* 531.46 0.015S 2510.4* 311.87 1987.2 369.19 3115.2 406.33 P 1690.29 138.65 2057.14* 59.88 3960* 296.72 Bees (no. of frames) Y 3.5 0.29 0.02 4* 0 0.008 3.8* 0.2 0.0009S 4.6 0.24 5.6* 0.24 5.6* 0.24 P 4.29 0.18 4.86 0.26 4.86* 0.26 Pollen stores (cm2) Y / 348 79.3 0.5 177.6 70.63 0.8S / 235.2 98.78 249.6 84.72 P / 219.43 55.78 240 76.08 Honey stores (cm2) Y / 1698 420.33 0.51 1272* 197.47 0.015S / 2188.8 507.16 3432* 664.8 P / 1614.86 230.42 1827.43 229.45 Times [days] Su riv al [% ] 1.00 0.75 0.50 0.25 0.00 Sugar candy Protein candy Sugar syrup Yeast candy p < 0.0001 0 10 20 30 40 Day Cu m ul at iv e co ns um pt io n (g ) 200 150 100 50 0 Sugar candy Protein candy Sugar syrup Yeast candy 0 10 20 30 40 Performance and Nosema spp. spore level in young honeybee (Apis mellifera carnica, Pollmann 1879) colonies … 163 Table 1: Performance of young colonies established from artificial swarms in summer (1st period – 27.6., 2nd peri- od – 2.7., 3rd period – 27.7.). N = 4-7, Y – Yeast candy, S – Sugar candy, P – Protein candy. SE – standard error, * significant differences (ANOVA, Bonferroni corrected t-test) Artificial swarms Group 1st period 2nd period 3rd period Mean ±SE p-values Mean ±SE p-values Mean ±SE p-values Wax production (cm2) Y 6162 735.79 0.18 6666 909.96 0.26 8947.2 376.53 0.08 S 7780.8 440.8 8308.8 850.61 10896 864 P 7073.14 453.22 8081.14 378.27 10628.57 473.42 Eggs (cm2) Y 384 143.67 0.019 240 150.52 0.08 518.4 120.14 0.18S 840 71.6 648 109.46 624 112.83 P 648 54.17 480 82.48 785.14 77.45 Larvae (cm2) Y 198 128.64 0.04 276* 173.48 0.003 676.8 142.47 0.85S 652.8 138.79 964.8 161.78 782.4 140.27 P 336 72.76 843.43* 50.62 754.29 105.9 Pupae (cm2) Y 492* 202.35 0.0001 522* 318.45 0.001 2078.4* 531.46 0.015S 2510.4* 311.87 1987.2 369.19 3115.2 406.33 P 1690.29 138.65 2057.14* 59.88 3960* 296.72 Bees (no. of frames) Y 3.5 0.29 0.02 4* 0 0.008 3.8* 0.2 0.0009S 4.6 0.24 5.6* 0.24 5.6* 0.24 P 4.29 0.18 4.86 0.26 4.86* 0.26 Pollen stores (cm2) Y / 348 79.3 0.5 177.6 70.63 0.8S / 235.2 98.78 249.6 84.72 P / 219.43 55.78 240 76.08 Honey stores (cm2) Y / 1698 420.33 0.51 1272* 197.47 0.015S / 2188.8 507.16 3432* 664.8 P / 1614.86 230.42 1827.43 229.45 In June, there were significant differences in Nosema spp. spore level, with the highest level in Sugar candy and the lowest in Yeast candy group (p<0,05). Comparing the level of Nosema spp. spores in September of the same year, the infection was low and non-significant (Table 2). Table 2: Nosema spp. spores in worker bees in young colonies expressed in million per bee. N = 4-7, Y – yeast candy, S – Sugar candy, P – Protein candy. SE – standard error, * p>0.05. (ANOVA, Bonferroni corrected t-test). Group June September Mean ±SE p- value Mean ±SE p-value Yeast 4.35* 1.97 0.02 1.68 0.59 0.48Sugar 14.96* 2.27 4.29 1.85 Protein 12.55 2.74 4.03 2.14 The colonies from Yeast candy group survived the winter successfully, however the other two groups lost one colony each by the time of the first inspection in early spring. Discussion An artificial swarm of honey bees is a very vulnerable young colony, as it possesses a small number of adult workers in comparison to the colony with brood and food stores. Young colony needs continuous food intake to allow worker bees to remain in the hive, building wax cells and later nursing young brood. Our study provides insights on how long worker bees live when fed protein or non-protein candies having no access to honey and pollen at the same time, and how M. I. Smodiš Škerl, I. Tlak Gajger164 small, nucleus colonies perform during and after being fed the same type of candies. The results of the field study show that the young colonies produced more brood feeding non- protein candy having at the same time the highest number of Nosema spp. spores in the same experimental group. On the contrary, adult bees in cages lived longer being fed protein or yeast candy, indicating that cage trials by itself do not provide adequate information on performance of bees. The bees in artificial swarms were of mixed age, physiologically in the stage of building wax, and therefore the nutritional needs differed from the bees in cages. However, the overwintering was less successful in groups fed commercial sugar and protein candy. This result can also be connected to some other factors that affect survival ability of honey bee colonies (Varroa, viruses etc.) (24), indicating that trials with bees in controlled conditions, among others, provide the insight in longevity and nutritional requirements (26). For the last few decades, the apicultural sector is expected to reach a high production of honey and a strong resilience to honey bee diseases and at the same time to overwinter colonies successfully. Unfortunately, there are several factors that hinder the development of colonies and challenge the beekeepers. Changing climate affects colony development and redistribution of honey plants (27, 28, 29, 30), and adaptation cycle of plants and bees to these sudden changes is very slow. The abundance and quality of pollen and nectar is changing and is therefore very unstable natural food source for bees (27, 31). Malnourished colonies are very sensitive and susceptible to infections of pathogens and stress due to pesticide exposure (32), and even in some cases being capable to adapt, eventually those colonies will die (33). Nevertheless, beekeepers need to supply all types of colonies with food supplements and substitutes for bees to fill the gap in food shortage and according to season, health status and needs (artificial swarms, queen production etc.). There are many food supplements available on the market and beekeepers mostly prepare syrups containing white sugar (saccharose) and water (34), and some mix sugar patties with or without additives (i.e., pollen, yeast, vitamins, and minerals etc.) (34, 35, 36) to feed their colonies. In our study, we used a home-made and two commercial candies, that are commonly used by the beekeepers. We found that sugar candy (Apifonda) showed the best results in a short term to establish a young colony. Concerning Nosema spp. infection, several recent papers report that different additives are potentially effective to prevent or eliminate Nosema spp. spores and/or support development of honey bee colonies: pre/ probiotics (37), EM probiotic (38), anti-nosema products (39), medicinal mushroom (40), plant extract (41, 42, 43), microalga (44), Chlorella (45), Cyanobacteria (46), pentadecapeptide BPC 157 (47), and other artificial diet (36). Sammataro and Weiss (2013) (48) compared productivity of colonies supplemented with sucrose or high fructose corn syrup (HFCS) and reported that group fed sugar syrup produced more wax, brood, and adult bee population that HFCS group. However, studies on effects of food supplements and additives on productive colonies are abundant, but there is a lack of research for artificial swarms or young colonies. Health status of worker bees in swarms and quality queen is therefore essential for optimal development and productivity of young colonies and the disease in colonies requires control (49). Regarding the health status, young colonies originated from artificial swarms were analysed for Nosema spp. in our study. The spores were detected in adult bees due to possible previous infection of honey bee colonies that were used for the experiment, and the spore load in June was the lowest in Yeast candy group and decreased in all groups in September. Moreover, all the colonies fed Yeast candy were able to survive the winter. Microsporidia Nosema apis and N. ceranae (cause of Nosemosis type C) infect the midgut of bees and reproduce in epithelium cells of the gut (50, 51). Research of Nosema infection (52, 53, 54) shows that Nosemosis influences the strength of the colony and the honey yield (55). It should be pointed out, that through the stages of swarm manipulation (or queen production etc.) beekeepers must consider the negative impact of Nosema infection and prevent transfer of pathogen by adopting good beekeeping practice (56). There are some differences in feeding various type of candies that might affect development and performance of young colonies. In our case we noticed differences in brood production, number of adult bees and honey stores in the hive, and longevity of workers in cages. At this point more studies of feeding supplements and effects on physiology and productivity should be done at the individual bee and colony (swarm) level to prevent colony failure. Performance and Nosema spp. spore level in young honeybee (Apis mellifera carnica, Pollmann 1879) colonies … 165 Acknowledgments We are grateful to Mitja Nakrst and David Kozamernik for their contribution in the field and laboratory data collection. 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Ocenjevali smo razvoj družin in določali število spor Nosema spp. pri odraslih čebelah v poletnem obdobju. V laboratori- jskih pogojih smo krmili čebele delavke z isto vrsto pogač. Najboljše preživetje smo ugotovili v skupinah, ki so prejele pogačo s kvasom oziroma beljakovinami. Nasprotno pa je poskus v družinah pokazal, da je bilo v 1. obdobju merjenja bistveno več bub (2510,4 cm2, p = 0,0001), v 2. obdobju pa več ličink (964,8 cm2, p = 0,003) in okvirjev s čebelami (5,6, p = 0,008) pri krmljenju s sladkorno pogačo. V 3. obdobju ocenjevanja mladih družin je imela skupina s sladkorno pogačo največ pokritih okvirjev z odraslimi čebelami in zalog medu (5,6, p = 0,0009; 3432,0 cm2, p = 0,015). Skupina s sladkorno pogačo je zgradila največjo površino satja, vendar razlike niso bile statistično značilne. Število spor Nosema spp. je bilo kvantitativno preverjeno pri odras- lih čebelah. Najnižja okužba je bila statistično značilna v skupini s pogačo s kvasom v juniju (4,35 milijona spor na čebelo, p = 0,02), septembra pa spremembe niso bile signifikantne. Dodajanje sladkorne pogače umetnim rojem se je pokazalo kot najbolj obetavno za razvoj produktivnih mladih čebeljih družin. Ugotovitve naše študije bi lahko pomagale čebelarjem pri izbiri učinkovitega dodatka pogač za optimalen razvoj umetnih rojev. Ključne besede: Apis mellifera; umetni roji; mlade družine; dodatki; pogače; razvoj; Nosema spp.; dolgoživost