Morphometric measurements of Africanized honeybee queens kept in an incubator or in queen banking

This study aimed to estimate live weight, the length and the width of wing and abdomen and the length, the width and the height of thorax in newly emerged Africanized honeybee queens up to 192 hours and their influence on final quality regarding appropriate storage techniques in queen banking or incubator and to verify the possibility of using these parameters as selection criteria in honeybee breeding programs. Queen honeybees were produced in summer, autumn and winter. The queen production method used throughout the experiments was adapted from Doolittle, and 68 the queens were weighed at emergence. Data were analyzed in R software by regression analysis of queen weight at emergence according to life time after emergence, in two types of storage. The weight of queens emerged in queen banking and incubator was significantly different (p < 0.05), with mean values of 165.61 and 157.04 mg, respectively. Queens kept in an incubator and in queen banking showed no significant differences (p > 0.05) in wing length, abdomen length and thorax height. The weight of queen at emergence can be used as a selection criterion in honeybee breeding programs.


Introduction
Some characteristics are little influenced by the environment but highly correlated with honey production, and can assist in selecting colonies with greater production capacity for more efficient analysis.Kahya et al. (2008) suggested that queen weight might be one of these characteristics and the queen can transmit this characteristic via drone.
The haplo-diploid sex determination system in Apis mellifera enables the transference of 100% of the genetic material of the drone from its mother to its offspring, because it is originated from an egg without fertilization (Cobey, 2007).Tarpy et al. (2000) reported a potential selection for newly emerged queens, as there is variation in the reproductive potential.
Queen reproductive potential is correlated with weight at emergence (Gilley et al., 2003;Tarpy et al., 2000).On the other hand, Kahya et al. (2008) found no significant correlation between queen weight at emergence and number of ovarioles.Harano et al. (2007) reported that even when the ovaries are poorly developed, the abdomen of queens with potential for high production of eggs is heavy, which can adversely affect long mating flights, so that the temporary weight loss becomes a benefit to the flight.
The thorax is the locomotor center of insects, grouping the legs and wings containing the muscles that move them.Furthermore, natural fertilization occurs in flight and thus depends on wings to perform this movement (Moritz & Southwick, 1992).Faquinello et al. (2011) associated the morphometric characteristics of newly emerged queens with royal jelly production in mini-hives and found potential selection for production, based on the abdomen width.Costa-Maia et al. (2011) estimated genetic and phenotypic parameters, via Bayesian inference, for weight and morphometric measurements of wing and abdomen in newly emerged Africanized honeybee queens and registered potential selection in all characteristics separately.Akyol et al. (2008) reported that queen weight is the most important among morphometric characteristics assessed given the positive correlation between queen weight and the brood area of the colony, the number of sperm and the diameter of spermatheca.In addition to this correlation, Akyol et al. (2006) observed a variation between morphological and physiological characteristics between subspecies and ecotypes.
Therefore, this study aimed to estimate queen weight at emergence and morphometric characteristics of the wing, abdomen and thorax of Africanized honeybee queens regarding storage techniques after emergence in queen banking or incubator.

Material and methods
Matrixes colonies that supply larvae located at the apiary in the farm at Universidade Estadual de Maringá were used to produce queens in summer, autumn and winter.Queens were produced by the method adapted from Doolittle (1889) throughout the experiments.On the day of larvae grafting, the unsealed brood comb was taken to the Laboratory of Queen Production at appropriate temperature (34 ± 2ºC) and humidity (60 ± 10%) to maintain the quality of the larvae until the time of grafting.
The grafted larvae were subsequently placed in mini-hives consisting of two overlapping nuclei separated by a queen excluder screen.The lower nuclei was composed of five combs and the upper, of four combs and a frame with cell cups spaced on cell bars (Mouro & Toledo, 2004).This cup bar frame consisted of 28 acrylic cups, 14 at the upper bar and 14 at the lower bar.During the experimental period, every 12 days, mini-hives were managed following method proposed by Faquinello et al. (2011), and received sugar syrup at a ratio of 2: 1 sugar and water.
On the tenth day after the grafting, bars with accepted cell cups were removed from the minihives and taken to the Laboratory of Queen Production.To house them, we prepared 20 mLglass vials containing a strip of paper, so that when a new queen emerged it could move and reduce the risk of injury.Each glass vial was identified as by bar and mini-hive.Later, the vials with cell cups were taken to the incubator at an average temperature of 34ºC and 60% humidity.The technique of providing queen food (made of powdered sugar and honey) inside the vial for maintenance was not used because it could interfere with the queen weight at emergence.
Newly emerged queens were anesthetized with CO 2 for determining measures of body weight (mg) on a precision balance AL500 MARTE from 0.001 to 500 g, the length and width of the right forewing and abdomen (cm), the length, width and height of the thorax (cm) by using a caliper accurate to 0.01 mm.The length and width measurements were taken from the right forewing, and the abdomen length was measured at a relaxed position in width to the third segment.
Queens were then placed in an introduction cage and the variation in weight was monitored every hour for six hours per day after emergence to check its variation.Morphometric measurements were taken again at the last weight assessment for possible variations within this period due to dehydration after emergence.
Queens were stored in the bacteriological incubator 002CB or in the queen banking, 28 in the incubator and 40 in the queen banking.In the incubator, each queen was housed in a JZsBZs TM cage and received four nurse worker honeybees and queen candy.The queen banking consisted of a populous colony with a queen excluder screen in mini-hive system with young worker honeybees ensuring the care and maintenance of virgin queens.Weight and morphometric measurements of the queens stored in both systems were monitored once a day over an eight day-period, i.e. until the reproductive viability of the queen.
We used the Bayesian inference, implemented in R software Development Core Team (2011).Data were tested for the effect of life time of the queen on the cha For thi the eff effects, fixed ef  2011) reported that queens with larger thorax are prone to mate with a higher number of drones and also found a positive correlation between the thorax width with mating frequency and number of sperm stored.Africanized honeybee queen weight at emergence in relation to life time for the two types of storage is presented in Figure 4.The weight of the queens housed in queen banking was higher (p < 0.05) than those stored in incubator; the mean values were 165.61 and 157.04 mg respectively.The live weight of virgin queens can be influenced by the time of the grafting, the type of the grafting and the mini-hive used for rearing larvae, indicating that there is potential for development (Padilha et al., 2013).The type of storage for the honeybee queens after emergence is part of the care for the efficient reproduction, since there is a waiting time required for their introduction in their respective colony.Gilley et al. (2003) and Kahya et al. (2008) verified an association between reproductive quality of the queen with weight at emergence and Akyol et al. (2008) concluded that heavier queens had higher mating rates and started laying eggs earlier than lighter queens.As a result, queen weight is studied as a selection criterion in honeybee breeding programs (Souza et al., 2013) due to high heritability.Rangel et al. (2013) observed that a honeybee colony can be evaluated as the extended phenotype of its queen, and thus the selection working mostly at the colony level may be equivalent at the individual level.Tarpy et al. (2012) did not notice a correlation between queen body size and mating success.

The
Regarding weight, the average weight of honeybee queens at emergence was lower than that obtained by Tarpy et al. (2000) in European honeybee queens, in which the body weight was 188.00 mg.However, with Africanized honeybee queens, Souza et al. (2013) found values of 180 to 200 mg, while Tarpy et al. (2011;2012) reported values of 206.6 and 218.7 mg for European honeybee queens.Even with lower weight of European honeybee queens, they showed a more hygienic behavior, once their infestation index by Varroa destructor is lower compared with the European queen (Guerra Júnior et al., 2010).
Furthermore, it is noteworthy that the highest weight loss occurred in the first hours after the emergence indicating that the queen must be weighed immediately after emergence to obtain accurate data.Nevertheless et al. (2004) reported that the body weight of queens should be determined two days after emergence, because previous measurements can cause an error due to weight variation.The variation in the queen weight in relation to the life time was efficiently explained by the fitted model (r 2 = 0.64).
Africanized honeybee colonies are characterized by producing cell cups all year round, regardless of the age of the queen, both in times of food scarcity and abundance, with a weekly average of four cell cups (Toledo et al., 2012).This can be explained by the low number of drones to fertilize this queen.This fact is very important because if the beekeeper allow their colonies to replace their queens alone, they usually produce new queens from older larvae, resulting in the production of low-quality queens (Tarpy et al., 2011), which are produced with a higher frequency during the year, and cause the production of these colonies to remain low.Wang et al. (2014) concluded that nurse honeybees controlled the larval growth and the number of ovarioles, by temporarily manipulating the delivery of food and that the response of larvae to food differs according to the larval stage and genotype.The body mass of the larvae was more sensitive to nutrition during the first (L1) and fourth instar (L4), whereas the number of ovarioles was more sensitive to the food in the fifth instar (L5).Therefore, during all larval stages, the conditions of temperature and humidity must be carefully controlled to obtain higher quality queens.
Besides that, Andi & Ahmadi (2014) recommended supplemental vitamin C at 2,000 mg L -1 of syrup in the spring food (1:1, sugar: water) to the colonies, because it increases the brood area, the population of the colony and can help to increase body protein content and the body weight.The intention pointed out here is that not only genetic origin but also management techniques and rearing conditions of the rearing nurse honeybees and/or future queens are critical when seeking higher quality, because these factors are closely related (Oldroyd et al., 2014).

Conclusion
The storage of honeybee queens, either in an incubator or in queen banking, has no influence on the forewing length, abdomen length and thorax height.However, Africanized honeybee queens kept in queen banking emerged with greater weight than those stored in an incubator.Thereby, there is potential selection of Africanized honeybee queens based on their weight, since it is very important to weigh queen soon after its emergence to be efficiently used as a tool in honeybee breeding programs.

Figure 3 .
Figure 3. Thorax height (mm) of Africanized honeybee queens in relation to life time (h) after emergence, stored in queen banking or incubator.

Figure 4 .
Figure 4. Weight (mg) of Africanized honeybee queens in relation to life time (h) after emergence, stored in queen banking or incubator.
of reproductive viability, once after the mating flight queens remain in the hive.However, during swarming periods queens are essential because they fly with the workers in search of a new place to nest.Delaney et al. ( gá, v. 37, n. 1, p. 2 Acta Scientiarum.Animal Sciences Maringá, v. 37, n. 1, p. 91-96, Jan.-Mar., 2015 proximity