QUALITY OF HONEY BEE QUEENS THROUGH PHYSICAL CHARACTERS AND COLONY PERFORMANCE F. Hatjina, M. Bieńkowska, L. Charistos, R. Chlebo, C. Costa, M. M. Dražić, J. Filipi, A. Gregorc, E. Nehova Ivanova, N. Kezic, J. Kopernicky, P. Kryger, M. Lodesani, V. Lokar, M. Mladenovic, B. Panasiuk, P. Pavlov Petrov, S. Rašić, M. I. Smodis Skerl, F. Vejsnæs, Jerzy Wilde* * University of Warmia and Mazury in Olsztyn, Poland. Summary The term “quality” in relation to queens and drones refers to certain quanti- tative physical and / or behavioural characters. It is generally believed that a high quality queen should have the following physical characteristics: high live weight; high number of ovarioles; large size of spermatheca; high number of spermatozoa in spermatheca; and be free from diseases and pests. It is, however, also known that the performance of a honey bee colony is the result of its queen’s function as well as of that of the drones that mated with her. These two approaches are often considered together and give a general picture of the queen production technique and selection. Here we describe the most common and well known anatomical, physiological, behavioural and performance characters related to the queens, as measured in different European countries: the live weight of the virgin queen (Bulgaria); the live weight of the laying queen (Bulgaria, Italy); the diameter and volume of spermatheca (Bulgaria, Greece, Slovenia); the number of ovarioles (Greece, Italy, Slovenia); the weight of ovaries (Slovenia); the num- ber of spermatozoa in spermatheca (Italy, Poland, Slovenia); the brood pat- tern (Bulgaria, Greece); the egg laying ability / fecundity (Bulgaria); the brood production (Croatia, Serbia); the colony population development (Croatia, Serbia, Slovakia); the honey production (Croatia, Denmark, Serbia, Slovakia); the hygienic behaviour (Croatia, Denmark, Serbia, Slovakia); the defence behaviour (Croatia); the calmness / sitting on the comb (Croatia, Denmark); and swarming (Croatia, Denmark). Especially for the weight of the queen, the number of ovarioles, the volume of the spermatheca and the number of spermatozoa, data per country proved its own accuracy by repe- tition through the years. We also report that when instrumentally insemi- nated queens are kept under mass production conditions (in small cages in queen banks and with low number of attendants) they can transfer the se- men to their spermatheca and clear their oviducts more efficiently when they have been inseminated with small amounts of semen in two or three sequences (but not four), compared to those inseminated with the same amount of semen at once (Poland). Furthermore, we had an inside view of the sanitary conditions of the colony: a. through the health status of the queen (nosema plus virus analysis) (Slovenia); and b. evaluating the nosema load of worker bees (Denmark) and of the queens (Greece). This is a collaborative work initiated by members of the WG 4 of CO- LOSS, the ‘breeding group’, which have now founded a constitutional network named ‘Research Network for Sustainable Bee Breeding’. Ref. Article: Hatjina et al. (2014) A review of methods used in some European countries for assessing the quality of honey bee queens through their physical charac- ters and the performance of their colonies. Journal of Apicultural Research, 53(3): 337-363. DOI 10.3896/IBRA.1.53.3.02 Conclusions and Perspectives The use of standard, high-quality queens is a prerequisite for any re- search on colony development and behaviour as well as for economically successful beekeeping. In this work we reviewed several methods for as- sessing the queen’s quality accompanied by examples from different countries (they do not represent the only methods used in each country). The presented data also represents a validation of each methodology, as it includes original data records of two or three years. Different coun- tries, influenced by beekeeping traditions and socio-economic systems, apply different protocols and standards for assessing the physical and performance characters which could determine the ‘quality’ of a mated queen. Breeding programs exists in many countries, but each one is dealing with the quality or the characters the queen is transferring to her progeny in a different way. The same evaluation methods are applied in a similar or a modified way to meet the requirements of each coun- try’s needs. Insemination procedure is commonly used in some coun- tries while almost completely absent in others. It has been made obvious that the quality of a queen is neither a single attribute nor even a group of attributes. It is rather the collective result of several groups of attributes such as: a) the physiological and biologi- cal ones that have been influenced by the reproduction process (body size and wing length, weight, number of ovarioles, diameter of sper- matheca); b) the physiological and biological ones that have been influ- enced by the fertilisation process (empty oviducts, number of sperm in spermatheca, time of onset of oviposition, genetic variability of sperm); c) the behavioural/performance attributes of the queen which reflect the inherited traits by both, the queen and the drones with which she has mated, but have also been influenced by the environmental conditions (honey production, colony development, aggressiveness, swarming, hygi- enic behaviour, disease prevalence). Obviously common beekeeping practices can also affect the outcome. Nevertheless, the genetic origin of the queen as well as the genetic origin of the drones is the base line of the whole reproductive process of the reproductive female offspring and the means of expressing the performance characters. TABLE 1. Standard methods used For physical characters For colony development or per- formance Density of worker brood Amount of brood and population Fecundity of the queen Calmness Weight of the queen Gentleness Weight of the ovaries Swarming tendency No of ovarioles Honey production Diameter of spermatheca Hygienic behaviour No of spermatozoa in spermatheca Infestation of Nosema spp. in the colony Presence of Nosema spp. spores in queens Varroa tolerance Virus analysis Table 2. Seasonal variability in live weight (mg) of laying queens of Bulgar- ian honey bees: Range (minimum and maximum); Cv% - coefficient of variability; % - punctuality index. x S Croatia (Fig. 1) Fig. 1. Average strength of test colonies in Continental and Mediterranean region. Denmark (Fig. 2) Fig. 2. The percentage of Nosema spp. spore negative samples in Denmark has increased since the start of the breeding programme from 20-40 % negative samples to over 90 % in the last few years. Bulgaria (Table 2) Greece (Fig. 3) Fig. 3. Average number of ovarioles for each population tested. Italy (Fig. 4) Fig. 4. Relation between queen weight and ovariole number. Poland (Table 3) Table 3. The percentages of single and multiple inseminated queens with empty oviducts 48 hours after last insemination and the number of spermatozoa in spermatheca of theses queens (group B). a,b,c,d – Sig- nificant differences at P≤0.05, (after ArcSin transformation). Month n Range x S X Cv% % x S April 55 198-284 250.5±6.4 10.56 2.56 May 55 232-320 272.7±5.1 7.75 1.88 June 55 230-294 261.0±5.2 6.95 2.01 July 55 232-292 264.2±6.5 7.83 2.46 Serbia (Table 4) Table 4. Quantitative data (average surface covered by bees, the average surface of brood and the average Acacia nectar crop) collected during a complete evaluation cycle (2008-2009) from two Serbian selection centres. Slovakia (Table 5) Table 5. Honey harvest between the years 2007 – 2009 – queens deliv- ered in 2006. Queen code year 2007 (kg) year 2008 (kg) year 2009 (kg) Total (kg) % TEST060021 93.00 32.00 0.00 125.00 85.15 TEST060022 72.00 19.00 0.00 9100 61.99 TEST060023 71.00 0.00 0.00 71.00 48.36 TEST060024 77.00 16.00 0.00 93.00 63.35 TEST060026 75.00 23.00 23.00 121.00 82.42 TEST060028 85.00 23.00 67.00 175.00 119.21 TEST060029 55.00 28.00 70.00 153.00 104.22 TEST060030 59.00 29.00 66.00 154.00 104.90 TEST060033 66.00 24.00 24.00 114.00 77.65 Average tested queens colonies 72.56 24.25 50.00 146.81 100.00 Average con- trol group 82.00 23.00 52.00 157.00 106.94 Slovenia (Table 6) Table 6. Average weights of queens and ovaries; number of spermatozoa and volume of spermatheca. mean ± SD Min Max mean ± SD Min Max Year 2006 (n = 324) Year 2008 (n = 288) Queen weights (mg) 208.40 15.31 184.70 233.30 209.50 11.96 153.70 262.20 Ovary weights (mg) 69.83 11.08 53.30 86.89 73.05 9.83 38.70 126.20 Year 2006 (n = 239) Year 2008 (n = 171) No. of spermato- zoa (x 10 6 ) 3.30 1.68 0.80 6.01 4.96 1.14 3.34 6.69 Volume of sper- matheca (mm 3 ) - - - - 0.890 0.106 0.744 1.070 Line 2008 2009 First spring in- spection Second spring inspection Crop (kg) Autumn First spring in- spection Second spring inspection Crop (kg) Bees Brood Bees Brood Bees Brood Bees Brood Bees Brood Kraljevo selection center 1 N° of colonies 13 13 13 13 13 13 13 13 13 12 12 11 Average 2.98 4.88 8.63 6.15 3.46 2.98 2.75 2.5 3.85 5.37 5.68 22.82 Standard de- viation 0.95 1.26 3.26 0.97 2.54 0.74 0.39 0.9 1.01 1.26 1.2 6.63 2 N° of colonies 13 13 13 13 13 11 11 10 10 8 8 11 Average 2.15 4.26 6.78 5.82 3.77 2.65 2.55 2.18 2.77 5.1 4.95 21.64 Standard de- viation 0.7 1.05 2.97 0.93 1.92 0.62 0.45 0.56 1 2.2 1.64 6.79 3 N° of colonies 11 11 11 11 11 9 9 13 13 10 10 10 Average 2.23 4.1 7.73 5.59 3.09 2.56 2.29 2.47 3.4 4.98 5.96 24.05 Standard de- viation 0.98 1.3 3.64 1.33 1.87 0.45 0.48 0.74 0.8 1.07 1.05 7.13 4 N° of colonies 12 12 12 12 12 10 10 10 10 10 10 9 Average 3.03 5.05 7.03 6.48 4.33 2.68 2.93 2.81 3.41 5.28 5.58 26.67 Standard de- viation 1.02 0.94 1.54 0.58 1.78 0.57 0.62 0.4 0.81 1.46 1.6 3.28 Vranje selection center 1 N° of colonies 10 10 10 10 10 10 11 7 7 7 7 7 Average 2.79 2.56 4.3 4.22 2.9 2.06 0.23 1.03 2.06 4.11 5.79 22.86 Standard de- viation 1.26 1.24 2.35 1.84 1.43 0.72 0.19 0.34 0.52 1.05 0.83 2.97 2 N° of colonies 7 7 7 7 7 7 7 6 5 5 5 5 Average 3.21 2.93 4.14 5.09 2.64 1.36 0.2 1.02 1.56 3.54 3.98 18.8 Standard de- viation 1.28 1.31 1.76 1.61 1.25 0.58 0.08 0.33 0.47 1.85 1.91 5.81 3 N° of colonies 8 8 8 8 8 8 8 7 7 7 7 7 Average 2.5 2.18 3.75 4.8 3.06 1.5 0.18 0.87 1.93 3.74 4.94 20 Standard de- viation 0.35 0.05 0.58 0.73 1.8 0.35 0.05 0.28 0.77 1.45 1.99 3.87 4 N° of colonies 8 8 8 8 8 8 8 6 6 6 6 6 Average 1.89 2.23 4.64 4.98 3.19 1.01 0.23 1.03 2.02 3.97 4.83 20.83 Standard de- viation 0.41 0.1 0.45 0.39 1.46 0.2 0.1 0.35 0.39 0.8 0.49 1.94 Dose of semen No of queens Queens with empty oviducts Queens with semen filled ovi- ducts Dead queens N % Number of sper- matozoa (millions) Sd N % N % 1 x 6 µl 2 x 3 µl 3 x 2 µl 30 61 38 25 57 36 83.3 ab 93.4 c 94.7 c 3.494 a 3.953 ab 4.007 b 1.08 1.29 1.07 4 2 - 13.3ab 3.3a - 1 2 2 1.3a 3.3a 5.3b 1 x 8 µl 2 x 4 µl 4 x 2 µl 25 100 29 21 90 18 84.0 ab 90.0 c 62.1 a 3.902 ab 4.009 b 5.269 c 0.78 1.40 1.56 4 5 10 16.0ab 5.0a 34.5b - 5 1 - 5.0b 3.4a Total Average 283 247 87.3 3.319 1.38 25 8.8 13 4.6