In addition to genetic factors, environmental factors such as temperature, oxygen, food intake, and nutrition also highly affect the life span of metazoans. Among these, the honeybee Apis mellifera has been studied in great detail mostly due to its long affiliation with humans and contemporary ecological and economic importance (Rueppell et al. 2016). It is also known that a deficient or improper nutrition can have negative consequences on health and pathogens susceptibility (Wang et al. 2014). Moreover, the life span of worker honeybees, average daily brood production and individual productivity of worker bees are among the major factors that affect honey production. Nutrigenomics is a new scientific branch that integrates genomic tools with nutrition research due to the relationship between health and nutrition (Alaux et al. 2011). Use of dietary supplements is common in some countries, as natural forage is not available in some seasons and honeybees must be manually fed. Moreover, reduction in floral abundance and diversity as a result of loss of natural habitats has been a global concern, leading to reduced natural nutrition (pollen and nectar) in many regions (Glavinic et al. 2017). Consequently, supplemental diets have been introduced (Brodschneiderand Crailsheim, 2010; Stanimirovic et al. 2017) that can be used to fight colony losses by alleviating protein stress (DeGrandi-Hoffman et al. 2010). Among the most common supplements for honeybees are amino acids and vitamins (Glavinic et al. 2017); however, scientific research about their influence on honeybees has not yet been well investigated. Research showed that proteins from pollen had a beneficial role on physiological processes, brood rearing, adult population growth and production of royal jelly (Mattila and Otis, 2006; DeGrandi-Hoffman et al. 2008). Natural proteins of pollen are essential for maintaining colony healthy including immune responses, parasite tolerance and survival (DeGrandi-Hoffman et al. 2016; Basualdo et al. 2014), reproductive success (Czekońska et al. 2015), and worker longevity (Li et al. 2014). The efficacy of protein supplements on honey bees varied depending on formulation and composition. Nevertheless, the proper amount of protein needed for honeybees has not yet been precisely determined. Herbert et al. (1977) reported that the mortality rate of honeybees living on a high protein diet (50%) was greater than those on a less protein diet (5 and 10%), which was likely due to defecation disabilities caused by the high levels of protein in their diet (Human et al. 2007). Different pollen-free commercial diets have been produced with various levels of success in enhancing honey bees growth and brood rearing (DeGrandi-Hoffman et al. 2008). Epigenetics studies show changes in gene activity that are inheritable but do not alter the DNA sequence. Many studies have proposed that morphological and behavioral differences between honeybee castes are ascribed to caste-specific protein isoforms, rather than changes in levels of gene expression (Vaiserman et al. 2018). Vitellogenin (Vg), an egg yolk protein, is present in growing oocytes and is used by queens for egg production. Moreover, it functions as an antioxidant to reduce oxidative stress and increase longevity in honeybees and hence, it is considered an indicator of longevity (Alaux et al. 2011). It is showed that bees with higher Vg expression have longer lifespan after paraquat injection, and knockdown of Vg using RNAi treatment significantly shortened lifespan. Thus, assessment of Vg expression in bees can be potentially an indicator of longevity (Seehuus et al. 2006). In addition to Vg, the mitochondrial Mn superoxide dismutase (Sod) gene contributes to the degradation of H2O2and superoxide radicals. In addition to vertebrates, researchers have investigated that SOD1 gene express in insects, such as Drosophila, reporting that this enzyme also plays a significant role in eliminating ROS in insects (Gaál et al. 2006). For instance, Phillips et al. (1989) reported that insects with deactivated SOD1 had shortened lifespans because they are unable to remove reactive oxygen species (ROS). Actually, the effects of ROS removal on life expectancy have examined in several insect species which ultimately highlighted the importance of SOD1 (Parkes et al. 1998; Sun and Tower, 1999; Koo et al. 2016). It is also assumed that the two genes are associated with pollen-induced molecular mechanisms in honeybees that increase life expectancy. Therefore, for the first time, the effect of 12 concentrations of essential amino acids on longevity and fecundity of honeybees was investigated in the present study. The results of this study can be used in diet management of honeybees worldwide.
MATERIALS AND METHODS
Honeybees and feeding
Honeybees (A. mellifera) were obtained from Khorasan Razavi Technical and Professional Center (Mashhad city, Honarestan) according to Wang et al. (2014). First, to stop egg production, a naturally mated queen was kept in a queen cage for 5 days; after which it was moved to a queen excluder box for approximately 24 h. An empty frame was also provided so the entire brood laid by the queen would roughly have the same ages. Then, three weeks after the egg laying, the frame was transferred from the colony to an incubator in a frame hive (dark, 32 ˚C and 70% RH). Finally, the emerging honeybees were partitioned into 78 hives. Therefore, we investigated the effect of 13 treatments of amino acid in 5 replications. The treatment of honeybees is presented in Table 1.
Measurement of productive traits
The experiment was carried out on 5 November to 22 December 2018 to provide sufficient space for growth of brood bees and avoid creating additional space and crowding the colony. The amount of honey production was measured weekly. The population of brood bees was determined using a specially wired frame (5×5 cm2). The population of adult worker bees was measured by estimating the surface of the frame covered by mature bees. In this method, frames fully covered by worker bees were considered as a population frame, whereas partially covered frames were accordingly considered as a fraction of the frame. To measure the amount of pollen collected, pollen traps were used during the flowering period of each treatment and the production was estimated using a digital scale. Resistance and stability of colonies in winter is known to determine the durability and resistance of queens and their colonies as a winter survival parameter. In order to determine winter mortality, starting from mid-December, colonies were weighed at the beginning of each day (before bees left). At the end of winter, the secondary weighing was carried out.
Table 1 The composition of diet provided to the honeybees
The difference between the primary weighing and the secondary weighing (overnight=130 days) right after extracting the stored honey was estimated to represent winter survival (Mohebodini et al. 2013).
RNA extraction and cDNA synthesis
According to Wang et al. (2014), we used liquid nitrogen to flash-freeze honeybees in each group. For real time PCR analysis, 12 samples per hive were collected and, for each sample, heads of 4 bees were pooled during RNA extraction. The heads of the four honeybees were put in a sterile 1.5-mL polypropylene microtube. Then, 200 μL of digestion buffer was added and the sample was homogenized using a sterile microtube pestle. Therefore, we had 6 replicates and each treatment had 12 samples, so we had 156 samples for the entire study. Isolation of total RNA from the heads of honeybees was carried out with the RNeasy mini kit plus (QiaGene, USA). The next step was to prepare cDNA using RT Kit (Thermo, USA).
Real time PCR
The Maxima SYBR Green/ROX qPCR Master Mix Kit (Thermo, USA) was used