DOI: 10.1111/eea.12633
Seasonal variation in the activity of selected antioxidant enzymes and malondialdehyde level in worker honey bees 2 , Dusko P. Snezana Orcic1 , Tatjana Nikolic1, Jelena Purac1*, Branko Sikoparija 3 1 4 5 Blagojevic , Elvira Vukasinovic , Nada Plavsa , Jevrosima Stevanovic & Danijela Kojic1 1
Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi Sad, Republic of Serbia, 2BioSense Institute – Research Institute for Information Technologies in Biosystems, University of Novi Sad, Trg Dositeja Obradovica 6, 21000 Novi Sad, Republic of Serbia, 3Institute for Biological Research ‘Sinisa Stankovic’, University of Belgrade, Bulevar Despota Stefana, 11000 Belgrade, Republic of Serbia, 4Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovica 8, 21000 Novi Sad, Republic of Serbia and 5Faculty of Veterinary Medicine, University of Belgrade, Bulevar Oslobodjenja 18, 11000 Belgrade, Republic of Serbia Accepted: 20 July 2017
Key words: oxidative stress, superoxide dismutase, catalase, glutathione S-transferase, floral composition of honey, melyssopalinology, Apis mellifera, Hymenoptera, Apidae
Abstract
The recent decline in managed honey bee populations, Apis mellifera L. (Hymenoptera: Apidae), has caused scientific, ecological, and economic concern. Research into the formation of reactive oxygen species (ROS), antioxidative defense mechanisms, and oxidative stress can contribute to our understanding of bee survival and conservation of this species. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) enzymes together with levels of malondialdehyde (MDA) were measured in summer and winter honey bees sampled from three colonies. One colony was stationary (C1), entering the winter period having accumulated Robinia pseudoacacia L. (Fabaceae) honey, and two were migratory (C2 and C3), entering the winter period with mainly Tilia (Malvaceae) and Brassica (Brassicaceae) honey, respectively. Compared to summer workers, winter worker bees had decreased SOD and GST activity, and MDA level, whereas CAT activity increased in all three colonies. We also demonstrated that seasonality is the main factor responsible for changes in antioxidant enzymes and MDA levels in worker honey bees. Overall, our results indicate a difference between summer and winter worker bees, pointing at a reduced level of antioxidant enzyme defenses during overwintering which may be due to a decrease in production of ROS. The decreased levels of MDA measured in winter honey bees confirm this. As ROS are actively used by insects as a defense mechanism to fight pathogens, we suggest that reduced production of ROS contributes to higher susceptibility of winter honey bees to infections and reduced overwinter survival.
Introduction Due to the highly complex and significant role bees play in the ecosystem and the progressive decline in their number (Neumann & Carreck, 2010), honey bees, Apis mellifera L. (Hymenoptera: Apidae), have been the subject of many studies in recent years (Potts et al., 2010). It is thought that the impact of multiple pathogens and environmental
*Correspondence: Jelena Purac, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi Sad, Republic of Serbia. E-mail:
[email protected]
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stressors weaken the immunity of bees (vanEngelsdorp & Meixner, 2010), and affect their physiology and development. Environmental factors associated with different seasons of the year, such as temperature and availability of food, influence the activity of honey bees throughout the year. This is particularly pronounced in temperate climates of the northern hemisphere, where specific strategies have evolved to survive low winter temperatures (G€atschenberger et al., 2013). During the warmer months (April– August), the colony consists mainly of summer worker bees, which are highly active both in foraging and nest maintenance. From August to October the number of
© 2017 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 165: 120–128, 2017
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summer bees decreases while the population of winter bees increases (Fluri et al., 1982). The activity of winter bees is low during cold periods when there is no foraging for food and bees feed on stored honey, which provides the energy needed for thermoregulation inside the hive (Stabentheiner et al., 2003). The lifespan of a bee in winter is considerably longer than that of summer bees. Worker bees live 6 months or longer in winter compared to only 1 month in summer. Variations in environmental factors are known to affect oxidative stress levels in insects (Lalouette et al., 2011). Honey bees are susceptible to oxidative stress because of their lifestyle (Korayem et al., 2012). During flight, requirements for oxygen are extremely high (Krogh & Weis-Fogh, 1951) and due to their tracheal respiratory system, oxygen comes into direct contact with tissue cells. Honey bee food (i.e., nectar and pollen) contains allelochemicals and phenolics whose metabolic oxidation produces reactive oxygen species (ROS) (Mittapalli et al., 2007; Pecci, 2011). An imbalance between ROS formation and antioxidative defense mechanisms leads to oxidative stress (Sharma et al., 2012). For example, an overproduction of ROS has the potential to cause disruption of cell lipids, proteins, and nucleic acids, resulting in oxidative stress (Birben et al., 2012). Polyunsaturated fatty acids in membrane phospholipids are major targets of lipid peroxidation, which affects the physiological function of cell membranes. The end product of these reactions is malondialdehyde (MDA), a marker of lipid peroxidation and consequently oxidative stress (Gaweł et al., 2003). With the exception of the respiratory chain in mitochondria, ROS are formed as byproducts of various metabolic pathways (Sharma et al., 2012) and have numerous positive roles in the cell such as defense against infectious agents and a regulatory role in signaling. The cellular concentration of ROS is maintained by the antioxidant system (AOS), which includes both enzymatic and non-enzymatic mechanisms. Although the main components of the AOS have been preserved during evolution, there are unique adaptations among the various animal groups (Felton & Summers, 1995; Corona & Robinson, 2006). The AOS of the honey bee is capable of dealing with high levels of oxidative stress (Nikolenko et al., 2012). Its main enzymatic components, 38 antioxidant genes, have been annotated manually and their comparative analysis with dipteran species indicates differences in honey bee lifestyle and the quantity of pro-oxidant molecules ingested with food (Corona & Robinson, 2006). Antioxidative systems in honey bees are also involved in the neutralization of xenobiotics, including insecticides and heavy metal ions (Chakrabarti et al., 2015; Nikolic et al., 2015). Furthermore, honey bee antioxidant enzymes are
important for queen activity and longevity (Corona et al., 2005), viability of germinal cells (Weirich et al., 2002; Collins et al., 2004), and life span plasticity (M€ unch et al., 2008). The most important antioxidant enzymes are superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) (Birben et al., 2012). It is particularly important to determine the activity of antioxidative enzymes in relation to population dynamics (i.e., summer and winter bees) because more bees die in managed hives over winter (Genersch et al., 2010) suggesting that winter bees may have a compromised immune function and higher susceptibility to diseases (Steinmann et al., 2015). Korayem et al. (2012) analyzed the antioxidative enzymes (SOD, CAT, and ascorbate peroxidase) and total peroxide concentrations during the active (summer) and moderately active (autumn) season in honey bees. They reported that increased levels of honey bee antioxidative enzymes and total peroxide concentrations correlated positively with the intense honey bee activities during the warmer seasons. However, they did not study variations in oxidative stress and antioxidative enzymes during winter or the role of MDA as a reliable indicator of oxidative stress. Although they are used by insects as defense mechanisms to fight pathogens (Nikolenko et al., 2012), ROS also constitute a major driving force in aging by introducing deleterious macromolecular damage (Harman, 1956, 1981). Therefore, it could be hypothesized that higher ROS production during periods of increased activity in the summer shortens the life span of bees (M€ unch et al., 2008), but decreases their susceptibility to common pathogens. It is particularly important that levels be maintained during the winter, when bees are most susceptible to infections. In this study, we tested whether there are differences in the level of oxidative stress between summer and winter worker bees, to further our understanding of honey bee colony losses in temperate regions. The focus of this research was to study the activity of some antioxidative enzymes (SOD, CAT, and GST) and the level of MDA in summer (August) and winter (March) worker bees fed with honeys of various floral compositions. The aim was to investigate possible differences in the effectiveness of antioxidant defenses and the level of oxidative stress in summer and winter worker bees.
Materials and methods Collection of worker bees and honey samples
Honey bee workers (A. mellifera sspec. carnica) and honey stored by them in the combs were sampled from apiaries in Vojvodina Province, Republic of Serbia (46°050 35.0″– 44°550 35.2″N, 19°100 40.7″–20°390 34.5″E). Sampling was
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carried out in August 2013 (summer worker bees and honey stored for overwintering) and in March 2014 before significant foraging activity from the hive could be observed (winter worker bees). The samples were taken from one stationary (C1) and two migratory colonies (C2, C3). The colonies were strong and healthy (ca. 20 000 bees in each colony), with no clinical signs of infectious diseases. To ensure that we sampled summer workers in August and winter workers in March, older bees were taken from marginal frames of the honey super (Medrzycki et al., 2013) from each colony, placed in plastic vials, immediately frozen in dry ice, and stored at 20 °C for analysis within the following 2 months. To assess the character of the colony food stored for overwintering, one fully capped frame without brood was removed from the honey super of each colony (C1–3) and the honey was extracted for further melissopalynological analysis. All the colonies used for the experiments were managed by the same beekeeper, suggesting that their genetic backgrounds were similar. Melyssopalinology analysis
Qualitative pollen analysis was performed according to the ‘harmonized methods of melissopalynology’ (von der Ohe et al., 2004). A minimum of 500 pollen grains per sample were counted and identified using reference slides and identification atlases. Along with the pollen from nectiferous plants, honey dew elements (HDE) were counted, i.e., algae, fungal spores and hyphae, anemophilous pollen, and pollen of nectar free plants (Ricciardelli d’Albore, 1998). The relative frequency of pollen and honeydew (Louveaux et al., 1978) were used to characterize the floral origin of honey consumed by overwintering bees. Enzymatic assays and MDA level
Activities of SOD, CAT, and GST, and MDA levels were determined in homogenates made from entire bodies of honey bee workers and expressed as units (U) of enzyme activity per mg of protein or nmol per mg of protein, respectively. For each colony, analysis of enzyme activities and MDA level in summer and winter worker bees included five biological replicates (i.e., five pools of eight worker bees taken at the same time) measured in technical triplicates. Whole bees were frozen in liquid nitrogen, ground to a powder with a pestle and mortar, and subsequently homogenized in ice-cold Tris-HCl buffer, pH 7.4 (10% wt/vol). Crude homogenates were centrifuged at 10 000 g (4 °C) for 10 min and stored at 20 °C until biochemical assays were performed. Protein concentration was determined using the Bradford (1976) method, with bovine serum albumin (BSA) as a protein standard. GST activity was determined spectrophotometrically at 340 nm
using 1-chloro-2,4-dinitrobenzene (DTNB) as the substrate (Habig et al., 1974). CAT activity was measured at 240 nm with H2O2 as the substrate (Claiborne, 1985), and SOD activity at 550 nm according to the method of McCord & Fridovich (1968) in cytochrome c (Fe3+)/xanthine/xanthine oxidase system. MDA levels were measured according to the method described by Slater (1984) which is based on the principle that MDA, i.e., the specific product of lipid peroxidation, reacts with thiobarbituric acid (TBA) to form a colored complex with maximum absorption at 532 nm. Statistical analysis
Averages of the measurements on multiple aliquots of each pooled sample for each parameter were used as the data set for the analysis. Data were analyzed with STATISTICA v.13 software (Dell license no. 135-213-166). Statistical significance of differences among activities of SOD, CAT, and GST, and MDA level was calculated with two-way ANOVA with season (S) and colony (E) as factors. Subsequently, means were separated by Tukey’s HSD test (a = 0.05). Significant difference among the six groups of bees — i.e., three colonies (C1–3) and two seasons (summer, winter): SC1–3 and WC1–3 — was estimated with a 95% confidence interval. Principal component analysis (PCA) was used to examine and visualize the interrelations among the variables analyzed. Canonical discriminant analysis was applied to examine group differences in the coordination of individual antioxidant enzymes between particular seasons and colonies (Jovanovic-Galovic et al., 2007). This method of analysis calculates differences between groups by the composition of individual enzymes.
Results Pollen profile
The honey samples contained only minor amounts of honeydew (ratio HDE/nectiferous plants 0.05
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