©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 12 (4): 6931-6938 (2013) Honey bee lines selected for high propolis production also have superior hygienic behavior and increased honey and pollen stores D. Nicodemo1,2, D. De Jong2, R.H.N. Couto3 and E.B. Malheiros4 1Curso de Zootecnia, Universidade Estadual Paulista, Dracena, SP, Brasil 2Departamento de Genética, Faculdade de Medicina, Universidade de São Paulo, Ribeirão Preto, SP, Brasil 3Departamento de Zootecnia, Universidade Estadual Paulista, Jaboticabal, SP, Brasil 4Departamento de Ciências Exatas, Universidade Estadual Paulista, Jaboticabal, SP, Brasil Corresponding author: D. Nicodemo E-mail: nicodemo@dracena.unesp.br Genet. Mol. Res. 12 (4): 6931-6938 (2013) Received November 1, 2013 Accepted December 16, 2013 Published December 19, 2013 DOI http://dx.doi.org/10.4238/2013.December.19.12 ABSTRACT. Honey bees use propolis to defend against invaders and disease organisms. As some colonies produce much more propolis than others, we investigated whether propolis collecting is associated with disease resistance traits, including hygienic behavior and resistance to the parasitic bee mite, Varroa destructor. The three highest (HP) and three lowest propolis-producing (LP) colonies among 36 Africanized honey bee colonies were initially selected. Queens and drones from these colonies were crossed through artificial insemination to produce five colonies of each of the following crosses: HP♀ X HP♂, LP♀ X HP♂, HP♀ X LP♂, and LP♀ X LP♂. Colonies headed by HP♀ X HP♂ queens produced significantly more propolis than those with HP♀ X LP♂ and LP♀ X HP♂ queens and these in turn produced significantly more propolis than those headed by LP♀ X LP♂ queens. The brood cell 6932 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 12 (4): 6931-6938 (2013) D. Nicodemo et al. uncapping rate of the high-propolis-producing colonies in the hygienic behavior test was significantly superior to that of the other groups. The LP X LP group was significantly less hygienic than the two HP X LP crosses, based on the evaluation of the rate of removal of pin-killed pupae. The HP X HP colonies were significantly more hygienic than the other crosses. No significant differences were found in mite infestation rates among the groups of colonies; although overall, colony infestation rates were quite low (1.0 to 3.2 mites per 100 brood cells), which could have masked such effects. Honey and pollen stores were significantly and positively correlated with propolis production. Key words: Controlled mating; Propolis production; Cleaning behavior; Varroa destructor INTRODUCTION Hygienic behavior, first described in honey bee colonies tolerant to American foul- brood disease (Rothenbuhler and Thompson, 1956), has also been indicated as important for the control of other brood diseases, such as chalkbrood and Varroosis (Spivak, 1996). Along with this behavioral response to brood diseases, honey bees also use propolis to control dis- ease organisms (Kamel et al., 2013) and even to seal off enemies such as vertebrate predators and small hive beetles that they encounter in the colony (Couto and Couto, 2006). Hygienic behavior begins with the detection of sick or dead brood, subsequent uncap- ping of the brood cell and removal of the larva or pupa (Rothenbuhler, 1964; Wilson-Rich et al., 2009). The expression of this characteristic in bees has been widely studied and it is estimated that up to 30 pairs of genes are involved in this process (Moritz, 1988; Kefuss et al., 1996; Gramacho et al., 1999). Environmental factors, such as nectar flow (Momot and Rothenbuhler, 1971; Spivak, 1996; Boecking and Spivak, 1999) and relative humidity of the air and age of the combs (Couto and Couto, 2006) also affect the efficiency of hygienic behavior by honey bees. Propolis is a product elaborated by bees from plant resins; it is used to fill holes and cracks, reduce hive entrances, combat bacteria and fungi, and mummify small dead invaders that the bees cannot remove from the hive (Marcucci, 1996; Park et al., 2002). Besides these utilities, propolis promotes social immunity in the colony, by reducing the dissemination of disease organisms in these social insects (Schmid-Hempel, 1998; Farnesi et al., 2009; Simone et al., 2009; Simone-Finstrom and Spivak, 2012; Roode et al., 2013). Considering that individually, honey bees have a less efficient immune system com- pared to non-social insects (Evans et al., 2006), these benefits of propolis could be part of a group of behavioral mechanisms and other characteristics that social bees have developed to collectively resist diseases and pests (Evans and Spivak, 2010; Simone-Finstrom and Spivak, 2010). When we consider the advantages for the colony of propolis in the hive and the possi- bility of economic gain from selling this product, it is clear that selection for propolis produc- tion is desirable and viable, especially given the high degree of heritability (h2 = 0.87) for this characteristic (Garcia et al., 2013). Hygienic behavior also has a high heritability rate, ranging from 0.52 to 0.65 (Rinderer, 1986; Harbo and Harris, 1999; Garcia et al., 2013). However, Garcia et al. (2013) found no significant correlation between propolis production and hygienic 6933 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 12 (4): 6931-6938 (2013) Propolis production and disease resistance traits in honey bees behavior, which would make indirect selection for one of the characteristics by selecting for the other unviable. Considering that propolis is important for colony health due to its antimicrobial prop- erties (Ghisalberti, 1979; Machado et al., 1991; Marcucci, 1995; Bastos et al., 2008), we ex- amined the relationship between propolis production tendencies, hygienic behavior and sus- ceptibility to the parasitic bee mite, Varroa destructor. MATERIAL AND METHODS Thirty-six Africanized honey bee colonies were evaluated for propolis production. These were wild-type Africanized colonies, maintained in Jaboticabal, São Paulo State, 21°15'22'' latitude south and 48°18'68'' longitude west, at 595 meters altitude. The climate in this area is subtropical, with a hot rainy summer and a relatively dry and cooler winter. The three colonies that were most productive (HP) and the three least productive (LP) were select- ed as parental colonies. Five colonies were produced of each of the following crosses: HP♀ X HP♂, LP♀ X HP♂, HP♀ X LP♂, and LP♀ X LP♂. The inseminated queens were marked to facilitate identification. After 60 days, when all of the worker bees had been replaced, data col- lection was initiated. See Nicodemo et al. (2013) for details of the selection process, propolis production apparatus and production data. Hygienic behavior was evaluated in the progeny colonies using the pin-killing method (Gramacho et al., 1999). One hundred sealed worker brood cells of a comb from each colony were perforated with a needle to kill the pupae. Every 12 h, for three days, the number of un- capped cells and the number of pupae that were completely removed were counted. The number of mites (V. destructor) in brood cells containing worker pupae was eval- uated in 50 comb cells on each side of a brood comb in each progeny colony. An otoscope and a fiberlight were used to help locate and identify the mites and progeny. The mites were separated into age categories, including original adult females, young adult female progeny, female deutonymphs, protonymphs (males were hard to distinguish from protonymphs and were lumped together with the protonymph counts), and eggs (Piccirillo and De Jong, 2003). The pupae that were selected had dark eyes and a light-colored body, and were considered to be approximately 17 days old (Nunes-Silva et al., 2006). Mite infestations on adult bees were determined by collecting approximately 200 bees brushed from brood combs of each colony into a 70% ethanol/water solution, agitating the col- lection jars mechanically for 1 h and straining the mites through a metal screen (De Jong et al., 1982). The number of mites was divided by the number of the bees in the sample. The colony conditions were evaluated by mapping the comb areas of worker brood (eggs, larvae and pupae) and food stores (honey and pollen). The combs of each F1 colony were taken to the laboratory and placed in a frame holder with 2 x 2 cm wire grid to measure the area covered by brood and food (Al-Tikrity et al., 1971). Comparisons between the different groups of F1 colonies were made using analy- sis of variance, in a completely randomized experimental design, with five repetitions, for propolis production, brood infestation by V. destructor and brood and food area in the comb. Hygienic behavior was analyzed in a completely randomized experimental design, with each group subdivided in time, with five repetitions. Pearson’s correlation was used to determine if propolis production per hive correlated with the brood, honey and pollen store comb areas. 6934 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 12 (4): 6931-6938 (2013) D. Nicodemo et al. Comparisons of means were made using the Tukey test, and the data were processed with the SAS program (SAS Institute, 1993). RESULTS The HP♀ X HP♂ colonies were more efficient at uncapping killed brood than all the other types of colonies, uncapping 46% of the perforated cells in the first 12 h, and 76% after 24 h (Figure 1). After 72 h, these high level propolis producing colonies had the highest rate of uncapped cells (97%). The LP♀ X LP♂ colonies had a lower rate of cell uncapping than the mixed cross bees, although the differences were not significant (P > 0.20). Figure 1. Percentage of pin killed brood cells that were uncapped, counted for three days at intervals of 12 h, in five colonies of each cross. The high propolis line cross (HP♀ X HP♂) was significantly more efficient than the hybrid crosses (high line X low line); these in turn were significantly more efficient than the low propolis line cross (LP♀ X LP♂). The superior hygienic behavior in colonies headed by HP X HP queens was again demonstrated when an evaluation of the number of pupae removed from the brood cells was made (Figure 2). The HP♀ X HP♂ bees removed 38 and 68% of the pupae 12 and 24 h after perforation, respectively. After 72 h, 96% of the pupae had been removed. In this evaluation, the colonies with bees produced from reciprocal crosses were similar to each other and signifi- cantly superior to colonies with low propolis producing bees. The performance of the HP♀ X HP♂ group was 28% superior to the LP♀ X LP♂ colonies 24 h after the brood was perforated; this was period when the differences between groups were greatest. The infestation rate with Varroa on adult bees was higher in the HP X HP colonies, lower in the LP X LP colonies, and intermediate in the crosses between HP and LP, although the differences were not significant, possibly because overall, the infestation rates were low (Table 1). There were no significant differences in the Varroa progeny frequencies between the different categories of propolis producing colonies. 6935 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 12 (4): 6931-6938 (2013) Propolis production and disease resistance traits in honey bees There were no significant differences in brood area among the different categories of propolis production colonies (Table 2). When brood area was divided into components (eggs, larvae, pupae), the differences were also not significant; there was also no significant correla- tion with tendency to produce propolis. Figure 2. Percentage of pin-killed brood cells from which the pupae were completely removed, counted for three days at intervals of 12 h, in five colonies of each cross. The high propolis line cross (HP♀ X HP♂) was significantly more efficient than the hybrid crosses (high line X low line); these in turn were significantly more efficient than the low propolis line cross (LP♀ X LP♂). Groups Mites in worker brood cells Adult bees Egg Protonymph Deutonymph New ♀ Original ♀ HP♀ X HP♂ 0.0 ± 0.0 0.2 ± 0.5 0.6 ± 0.6 2.0 ± 1.6 2.8 ± 2.5 3.5 ± 2.5 HP♀ X LP♂ 0.8 ± 1.8 1.2 ± 2.7 0.6 ± 0.6 2.8 ± 4.1 3.2 ± 3.8 2.9 ± 1.9 LP♀ X HP♂ 0.2 ± 0.5 0.4 ± 0.6 0.4 ± 0.6 1.6 ± 0.9 2.8 ± 1.3 2.5 ± 1.5 LP♀ X LP♂ 0.0 ± 0.0 0.0 ± 0.0 0.4 ± 0.6 0.2 ± 0.5 1.0 ± 1.2 1.9 ± 1.2 Table 1. Means ± standard deviation of the number of mites of different stages per 100 worker brood cells, in colonies descendants of colonies selected for high (HP) and low production of propolis (LP). Statistics Brood areas (cm2) Eggs Larvae Pupae Total Means ± SD HP♀ X HP♂ 54 ± 29 118 ± 49 207 ± 73.39 379 ± 150 HP♀ X LP♂ 47 ± 27 173 ± 27 292 ± 46.79 512 ± 102 LP♀ X HP♂ 79 ± 37 174 ± 37 291 ± 34.21 544 ± 84 LP♀ X LP♂ 45 ± 34 124 ± 34 275 ± 82.00 444 ± 128 r 0.36 0.12 -0.04 0.09 P value 0.12 0.60 0.86 >0.20 Table 2. Means ± standard deviation of the area (cm2), in each comb, containing worker bee, eggs, larvae, and pupae in colonies that were descendants of colonies selected for high (HP) and low production of propolis (LP), Pearson’s correlation coefficient (r) and P value. 6936 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 12 (4): 6931-6938 (2013) D. Nicodemo et al. Honey production was significantly correlated with propolis production. The HP♀ X HP♂ colonies had 49% more honey in the comb than the LP♀ X LP♂ colonies (Table 3). Pollen in the comb was also significantly correlated with propolis production. The high line propolis production colonies had 22% more pollen in the comb than the low-line propolis producing colonies. DISCUSSION Gonçalves and Kerr (1970) reported that colonies that are resistant to diseases remove at least 50% of the killed brood within 48 h, completing removal within 72 h. In our experi- ment, all of the types of bees removed more than 50% of the pin-killed pupae within 48 h; however, after 72 h, 3.6, 8.8, 11.2, and 13.4% of the dead pupae had not been removed, in HP♀ X HP♂, HP♀ X LP♂, LP♀ X HP♂ and LP♀ X LP♂ colonies, respectively. Directed selection for propolis production promoted increased hygienic behavior in the colonies. This tendency was not found by Garcia et al. (2013), who only tested colonies selected for high propolis production. In our study, we also included and compared colonies selected for low propolis production, which allowed us to differentiate the degree of hygienic behavior as a function of propolis production tendencies. Using Bayesian inference, Padilha et al. (2013) found a positive correlation between propolis production and hygienic behavior (r = 0.15); the high positive residual correlation (r = 0.65) leads them to conclude that environment has a strong effect on these characteristics. The rate of brood cell infestation by V. destructor was similar among the colony groups; the mean infestation rate varied from 1.0 to 2.6%. It is known that infestation by this mite is influenced by the type of bee and by local climate conditions (De Jong et al., 1984; Moretto et al., 1991; De Jong and Soares, 1997). Infestation rates are higher in temperate climates, making it necessary to apply acaricides or other treatments in order to maintain the colonies alive (De Jong et al., 1984). Among the different types of honey bees, Africanized bees are more tolerant to Varroa than are the European races (Moretto and Mello, 1999; Mar- tin and Medina, 2004). The local tropical climate and the fact that these are Africanized bees contributed to the low rates of infestation with V. destructor. Although infestation with the Varroa mite is known to negatively affect colony produc- tion (De Jong et al., 1984), a tendency for a higher rate of infestation with this mite in colonies selected for propolis production was also found by Padilha et al. (2013), who reported a corre- lation of 0.49 between these two characteristics. However, bees selected for hygienic behavior have been found to be less susceptible to diseases and mite pests (Spivak and Reuter, 1998). Type of colony Honey Pollen Propolis production HP♀ X HP♂ 466 ± 85 121 ± 13 22.4 ± 13.0 HP♀ X LP♂ 316 ± 146 111 ± 27 4.3 ± 2.5 LP♀ X HP♂ 377 ± 44 106 ± 19 4.0 ± 5.2 LP♀ X LP♂ 313 ± 30 99 ± 31 0.65 ± 0.92 r 0.77 0.50 P value <0.01 0.03 Table 3. Means ± standard deviation of the area (cm2) in each comb, of honey and pollen, in each group of five propolis-producing hives. Propolis production (g) is given as a mean for each type of colony. Pearson’s correlations (r) were calculated for food stores as a function of individual colony propolis production. 6937 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 12 (4): 6931-6938 (2013) Propolis production and disease resistance traits in honey bees Generally speaking, we found that the colonies selected for HP also produced more honey and pollen than those selected for LP, suggesting a relationship between these charac- teristics. Manrique and Soares (2002) also found that selection for honey production resulted in increased propolis production, and vice-versa. CONCLUSIONS Colonies that were descendants of a cross between superior propolis producers were significantly more hygienic than those from crosses between low propolis-producing colonies. 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