Journal of Apicultural Research

ISSN: 0021-8839 (Print) 2078-6913 (Online) Journal homepage: http://www.tandfonline.com/loi/tjar20

Supplementation of the honey bee diet with

vitamin C: The effect on the antioxidative system
of Apis mellifera carnica brood at different stages

Marek Farjan, Magorzata Dmitryjuk, Zbigniew Lipiski, Elbieta

Biernat-opieska & Krystyna towska

To cite this article: Marek Farjan, Magorzata Dmitryjuk, Zbigniew Lipiski, Elbieta
Biernat-opieska & Krystyna towska (2012) Supplementation of the honey bee diet with vitamin
C: The effect on the antioxidative system of Apis mellifera carnica brood at different stages, Journal
of Apicultural Research, 51:3, 263-270, DOI: 10.3896/IBRA.1.51.3.07

To link to this article: https://doi.org/10.3896/IBRA.1.51.3.07

Published online: 02 Apr 2015.

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 Journal of Apicultural Research 51(3): 263-270 (2012) IBRA 2012
DOI 10.3896/IBRA.1.51.3.07

ORIGINAL RESEARCH ARTICLE

Supplementation of the honey bee diet with vitamin C:

The effect on the antioxidative system of Apis mellifera
carnica brood at different stages
Marek Farjan1, Magorzata Dmitryjuk1, Zbigniew Lipiski2, Elbieta Biernat-opieska1 and
Krystyna towska1*
1
Biochemistry Department, Faculty of Biology, University of Warmia and Mazury, Oczapowskiego 1A Str., 10-719 Olsztyn, Poland.
2
Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima Str.10, 10-748 Olsztyn, Poland.

Received 28 May 2011, accepted subject to revision 12 April 2012, accepted for publication 27 May 2012.

*Corresponding author: Email: k.zoltowska@uwm.edu.pl

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Summary
Diet in the winter has a vital effect on the survival and condition of a honey bee (Apis mellifera) colony in the spring. The effect of
supplementation of the diet with vitamin C (ascorbic acid) on the total antioxidant status (TAS), glutathione content, and activity of 4
antioxidative enzymes: superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), and glutathione transferase (GST) of honey bee brood
developing in the spring was studied. Twelve stages, from newly hatched larvae to emerging adult worker bees were studied, allowing
changes in the antioxidant profile during brood development to be determined for the first time. It was shown that bees are more exposed to
oxidative stress after emergence. In workers emerging in colonies after supplementation with vitamin C, higher contents of protein and
glutathione, and higher activities of peroxidase, catalase, and glutathione transferase were observed. Vitamin C did not alter brood weight
increase, and the level of protein in emerged workers was higher than in the control group. The mean of bee losses over winter were about
33% lower in colonies receiving vitamin C.

Suplementacin de la dieta de la abeja de la miel con vitamina

C: El efecto sobre el sistema antioxidante de la cra de Apis
mellifera carnica en diferentes etapas
Resumen
La dieta durante el invierno tiene un efecto importante en la supervivencia y el estado de las colmenas de la abeja de la miel (Apis mellifera)
durante la primavera. Se ha estudiado el efecto de la suplementacin de la dieta con vitamina C (cido ascrbico) sobre el estado antioxidante
total (TAS), el contenido de glutatin y la actividad de cuatro enzimas antioxidantes: superxido dismutasa (SOD), peroxidasa (POX), catalasa
(CAT), y glutatin transferasa (GST) en la cra de abejas en desarrollo durante la primavera. Se han estudiado doce etapas, desde larvas
recin nacidas hasta la emersin de las abejas obreras adultas, lo que permite determinar por primera vez los cambios en el perfil
antioxidante durante el desarrollo de la cra. Se ha demostrado que las abejas estn ms expuestas a estrs oxidativo despus de la
emergencia. Los mayores contenidos de protena y glutatin, y las mayores actividades de peroxidasa, catalasa y glutatin transferasa se
observaron en las obreras que emergen en las colmenas tras la suplementacin con vitamina C. La vitamina C no alter el aumento del peso
de la cra, y el nivel de protena en las obreras emergentes fue mayor que en el grupo control. La media de las prdidas de abejas durante el
invierno fue un 33% menor en las colmenas que recibieron la vitamina C.

Keywords: honey bee brood, Apis mellifera carnica, vitamin C, ascorbic acid, oxidative stress, antioxidants, oxidative enzymes
 264
Introduction
Reactive oxygen species (ROS) are continuously generated in
metabolic processes of aerobes. An organism tries to keep a balance
between the amount of ROS and antioxidant processes. The
overproduction of ROS (so called oxidative stress) may lead to
damage of basic molecules: peroxidation of lipids, nucleic acid
disruption, modification of amino acids in proteins, altering their
biological activity. An efficient antioxidative system is of particular
importance for insects, which have high rate of metabolism, which
naturally generates huge amounts of free radicals (Candy et al., 1997).
To prevent the undesired ROS effects, organisms developed defensive
mechanisms. They include both enzymatic and non-enzymatic
components (Seehuus et al., 2006). The measure of ability to
counteract oxidation is the Total Antioxidant Status (TAS).
For the honey bee (Apis mellifera) the assessment of antioxidative (3:1) syrup supplemented with vitamin C (Biofactor; Poland) at a rate
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capability became easier after the sequencing of the honey bee
genome. Based on this, Corona and Robinson (2006) showed at least
38 genes coding for antioxidative proteins. The most important
antioxidative enzymes in the insect are superoxide dismutase (SOD),
catalase (CAT), peroxidases (POX). Insects have lost genes coding for
two important antioxidative enzymes, glutathione peroxidase and
glutathione reductase. Their functions are covered by very active
thioredoxin peroxidase and thioredoxin reductase. In insects,
glutathione S-transferases (GST) also function with peroxidase activity eyes (P1); with pale-pink eyes (P2); with pink eyes (P3); with brown
(Corona and Robinson, 2006).
The second group are low-molecular antioxidants, which react
with ROS in a less specific manner than the enzymes, so are
universal, and can interchangeably play protective functions (Colven
and Pinnell, 1996). In insects, a glutathione-ascorbic acid redox cycle
is active, which is similar to plants. This cycle allows efficient
scavenging of hydrogen peroxide formed in enzymatic reactions
(Summers and Felton, 1993; Krishnan et al., 2009). Vitamin C
(ascorbic acid) participation in proline and tyrosine hydroxylation was
confirmed in insects (Briggs, 1962). Vitamin C is not thought to be
essential for bees, but this assumption still needs more studies to be
completely resolved. It is, however, sometimes recommended for
administration in large doses, especially in artificial diets (Black, 2006) layer was collected for biochemical analyses.
based on the lack of estimation of vitamin C demand in bees.
Honey bee colonies are particularly endangered in winter.
Prolonged winter can lead to exhaustion of food reserve, and a high
density of often weakened individuals facilitates disease spread
(Brodschneider and Crailsheim, 2010). As a result, extended losses in
bee colonies are noted after winter in many countries (Brodschneider
et al., 2010; Van Engelsdorp et al., 2010). Knowledge of the helpful
effect of vitamin C on immunity promoted us to apply it as a dietary
supplement for wintering bees. In humans, vitamin C is commonly
used for prevention of viral and bacterial infections, and as a
cancerogenesis inhibitor (Goodwin and Brodwick, 1995; Hemila, 1997). in katals (kat). Peroxidase activity was measured according to Chance
Farjan, Dmitryjuk, Lipiski, Biernat-opieska, towska
Herbert et al. (1985) demonstrated enhancement of honey bee brood
rearing when an artificial diet was supplemented with a high dose
(2 g / kg) of vitamin C. He concluded that, at least partially, the
increase the brood production results from the antioxidative
properties of the vitamin C. The aim of this study was therefore to
observe the effects of vitamin C administration to honey bees on the
activity of antioxidant systems of developing brood. This system has
not been studied before, so this study fills a gap in our knowledge of
this important stage in honey bee development.
Materials and methods
Eight A. m. carnica colonies headed by sister queens were kept at a
site 15 km from Olsztyn, Poland. Four were fed with sugar: water
of 1.8 mg per 1 kg syrup. The remaining four colonies received pure
syrup as a control. Feeding began in September 2007 to enable bees
to hoard the winter stores (13 l per colony) and a second took place
on 1 March 2008. Sections of brood comb were sampled from all
colonies on 10 May 2008. Brood was divided into the following
developmental stages (Jay, 1962; 1963): 1-2-day-old larvae (L1/2);
3-day-old larvae (L3); 4-day-old larvae (L4); 6-day-old larvae (L6);
cocoon-spinning larvae (L7); prepupae (PP); and pupae with white
eyes and yellow thorax (P4); with black eyes and dark thorax (P5).
The samples were then rinsed in 0.9% NaCl, carefully dried on filter
paper, and weighed. From fifteen to twenty five brood samples (three
individuals in each) were created within each of the brood stages.
With L1/2, L3, and L4 the sample sizes were larger, of 30, 20, and 10
individuals, respectively. One-day-old adult workers (A) were also
examined. The material was stored at -70C for further analyses.
For vitamin C content measurements, brood samples were
homogenized in 0.9% NaCl at 0.1 g of fresh weight per 1 ml of the
solution. The homogenization lasted for 2 minutes, at 5000 rpm, and
was performed over an ice bath. The homogenate was centrifuged for
10 minutes at 15000g, at 4C. The supernatant from below the fatty
The protein content was analysed using the Bradford (1976)
method. Before the assay, the extract was diluted 5 times with 0.9%
NaCl. Glutathione concentration was measured according to Ellman
(1959). Total Antioxidant Status (TAS) was assayed using a Randox
Laboratories Ltd. kit according to the manufacturers instructions.
Results were calculated as Trolox equivalents per 100 mg of fresh
weight. The activity of superoxide dismutase (SOD) was measured
according to Podczasy and Wei (1988). Catalase was determined
according to Aebi (1984). Samples were diluted 20 times prior to
assaying with 0.2 M phosphate buffer, pH 7.0. Results were expressed
 Honey bee brood antioxidative system and vitamin C supplementation 265

Table 1. The body weights and protein contents of honey bee worker brood. * one or two- (L1/2), three- (L3), four- (L4) and six- day old
larvae (L6), spinning larvae (L7), prepupae (PP), pupae with white- (P1), pale pink- (P2), pink- (P3), brown eyes and yellow thorax (P4),
pupae with dark eyes and dark thorax (P5), freshly emerged imago (A). **The following developmental stages are marked by different small
letters. A small letter placed by a given mean means that this mean are significantly different from the mean marked just by that small letter.
*** The capital letters A - indicate significant differences between means of groups within the same stage of development (p<0.05),
**** n - number of analyzed samples.

Body weights (mg) Protein concentration (mg/100mg)

Stage*
Control group Group with ascorbate Control group Group with ascorbate

n**** Mean SD Mean SD Mean SD Mean SD

b-l** b-l k.l, A*** b-f,i-l

L1/2 10 a 31.45 9.68 33.98 8.70 22.8 3.9 27.0 1.2

a,c-l a,c-l c,k,l a,e,i-l

L3 12 b 63.76 10.10 57.99 9.68 21.6 4.3 18.0 2.0
a,b,d-l a,b,d-l a,b,d-f,k,l a,b,f-l
L4 12 c 90.34 8.70 87.06 9.68 15.7 1.0 14.7 0.9
a-c,e-l a-c,e,g-l c,k,l, A a,g-l,
L6 13 d 167.01 9.68 156.23 10.10 19.9 1.7 15.6 1.0
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a-d,f-l a,c,d,f-l c,k,l, A a,b,f-h,k,l

L7 12 e 188.66 11.20 179.25 6.50 19.2 0.9 13.8 0.5
a-e,i-l a-c,e,l c,k,l a,c,i-l
PP 15 f 149.32 6.50 139.58 10.45 19.7 1.4 18.2 2.1
a-e,i,l a-e,l c,k,l c-e,i-l
P1 25 g 147.09 10.1 137.03 8.90 16.6 2.3 21.9 4,1
a-e a-e,l c,k,l c-e,i-l
P2 18 h 138.00 8.90 135.98 11.2 18.6 2.0 21.3 2.3
a-e a-e c,k,l, A a,b,d,f--l
P3 20 i 132.08 6.50 129.34 11.2 17.5 1.5 13.7 0,6
a-g a-e c,k,l, A a-d,f-i,l
P4 15 j 128.66 13.50 128.87 8.90 15.8 2.1 12.3 0.2
a-f a-e
P5 18 k 128.89 9.68 126.37 9.68 10.1a-j, A
0.9 12.1 a-i,l
0.7
a-h a-h a-j, A a-k
A 23 l 116.730 10.99 112.11 11.40 8.0 0.4 9.9 0.6

and Maehly (1955), and was expressed in international units (U). All
the enzymatic activities were calculated per 1 mg of protein.
Vitamin C content was measured with modified Rutkowski et al.
(2002) method. The brood samples were homogenized on and ice
bath with 8% acetic acid at 10 ml of acid per 2 g of fresh weight. The
solution of 113.6 M L-ascorbic acid (Sigma) was used as the standard.
The vitamin C content was calculated per 100 mg of fresh weight.
This method requires large amounts of biological material for the
analysis, so the level of vitamin C was assayed only once each in the
pupal and adult stages.
Statistical analysis
All results underwent statistical analysis with use of Statistica 8
software (StatSoft Inc.). In order to determine statistically significant
differences between means of groups at p < 0.05, one-way ANOVA
analysis with Tukey test for different n was performed (see Tables).
Results
The winter of 2007-8 was relatively mild in our study region. This was
probably one reason why winter losses in the experimental apiary
were not large (Mean of 12.54% and 8.36% in the control and
experimental groups respectively. Analysis of the results was
performed: i. between developmental stages and; ii. between diets. In
Tables 1- 4, mean values and standard deviations are presented, as
well as the results of statistical analysis.
Enriching diet with vitamin C did not have a significant effect on
the brood weight. Changes in body weight during the development of
both brood groups were similar (Table 1). The highest protein content
was found in the youngest developmental stages L1/2. In capped
brood, it slightly fluctuated, and a clear decrease was seen in the end
of the development. In the vitamin C supplemented group, the protein
content was significantly higher in the youngest larvae (L1/2) and in
the final phase of development (P5 pupa and adult) in comparison
with analogous stages of the control group (Table 1).
During the development of both groups, the TAS was almost
constant. The enrichment of bee diet with vitamin C had a weak
impact on the value of TAS (Table 2). The glutathione level increased
during larval development of both groups, and was the highest in the
spinning larva stage. It was significantly lower in the prepupae (PP)
(Table 2). Generally, supplementation of diet with vitamin C had a
slight effect on glutathione content, but it is worth mentioning that
adults in the experimental group had significantly higher
 266 Farjan, Dmitryjuk, Lipiski, Biernat-opieska, towska

Table 2. Total antioxidant status (TAS) and glutathione content in worker development.

TAS Glutathione
* (Trolox equivalents/100mg) (mg/100 mg tissue)
Stage
Group with
Control group Control group Group with ascorbate (A)
ascorbate (A)

n**** mean SD mean SD mean SD mean SD

a,c-l** c-h
L1/2 10 a 96.08 0.95 97.31 0.65 42.29 11.20 43.12 13.5

a,d,e,i-l c-g,j-l
L3 12 b 96,38 0,35 97.44 0.98 71.15 6.50 55.24 10.10
a,d,e,i-l a,d,e,i-l
L4 12 c 96.40 0.52 97.50 1.54 67.88 10.10 69.56 8.70
a-c,f,h-l a-c,.f,i-l
L6 13 d 96.39 0.62 97.54 0.56 87.62 8.90 89.52 9.68
a-c,f,h-l a-c,.f,h-l
L7 12 e 96.36 0.65 97.64 0.62 98.27 9.68 99.52 10.10
a,d,e,i-l a,b,d,e,i-l
PP 15 f 96.46 0.65 97.76 0.62 68.38 10.11 72.60 6.50
a,i-l a-c,f,i-l
P1 25 g 96.47 0.62 97.78 0.65 84.75 8.70 89.21 11.20
a,d,e,i-l a,b,d,e,i-l
P2 18 h 96.48 0.73 97.59 0.42 67.82 9.68 72.15 8,90
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a,c-h,l c-h,j
P3 20 i 96.37 0.42 97.44 0.65 37.48 11.20 40.62 9.68
a-h,l b-h
P4 15 j 96.30 0.65 97.43 0.57 21.59 9.68 22.66 9.68
a-h,l b-h
P5 18 k 95.98 0.57 97.44 0.72 19.12 11.22 28.14 10.10
a-l, A*** b-h
A 23 l 95.50 0.87 96.65 0.57 4.86 6.51 24.20 8.70

Table 3. The activity of superoxide dismutase (SOD) and peroxidase (POX) during development of honey bee brood.

SOD POX
(mU/mg protein) (mU/mg protein)
Stage*
Group with ascorbate
Control group Control group Group with ascorbate (A)
(A)

n**** SD SD SD SD
k,l** k,l b,c,f,g c, f-i,k
L1/2 10 a 1.28 0.09 1.14 0.08 27.6 1.7 27.8 2.7
k,l k,l a,h,l
L3 12 b 1.29 0.10 1.13 0.09 37.0 2.5 35.9 6.3
k,l i-l a,e a,e
L4 12 c 1.34 0.08 1.24 0.08 39.7 4.7 40.0 5.1
k,l i-l l
L6 13 d 1.36 0.09 1.22 0.10 32.9 6.5 32.1 6.3
k,l i-l c,f-h,k
L7 12 e 1.37 0.11 1.23 0.06 29.1 6.7 29.9 4.1
k,l, A*** i-l h,l, A
PP 15 f 1.4 0.06 1.17 0.21 34.5 4.7 49.3a,b,d,e,h-j,l 5.0
k,l i-l a,h,l a,d,e,l
P1 25 g 1.35 0.10 1.25 0.08 41.2 5.8 42.0 2.8
k,l i-l b,c, A a,d,e,l
P2 18 h 1.33 0.08 1.23 0.32 27.0 3.7 40.0 1.5
f d,e,g,h a,e
P3 20 i 1.14 0.13 1.04 0.08 27.9 9.5 37.7 4.1
c-h d,e,g,h a,l
P4 15 j 1.11 0.09 1.01 0.13 34.9 5.8 35.0 5.2
a-h d,e,g,h l a,d,e,l
P5 18 k 1.08 0.08 0.95 0.05 33.2 6.6 42.8 3.2
a-h d-h
A 23 l 1.05 0.10 0.95 0.06 23.0b,d,f,g,j,k, A 2.1 33.0 f,k
4.0

concentration of GSH than in the control group (Table 2). During vitamin C from 0.43 0.043 mol/g to 0.68 0.038 mol/g,
pupation in both groups, an increase in the vitamin C content was respectively. As a result, the differences in mean values for P1 pupae
observed, in the control group from 0.41 0.022 mol/g in P1 stage and adults within the group were statistically significant (Fig. 1).
to 0.66 0.032 mol/g in adults, and in the group receiving the
 Honey bee brood antioxidative system and vitamin C supplementation 267

Table 4. The activity of catalase (CAT) and glutathione S-transferase (GST) during development of honey bee brood.

CAT Glutathion S-transferase

(kat/mg protein) (U/mg protein)

Stage*
Group with Group with
Control group Control group
ascorbate (A) ascorbate (A)

n**** SD SD SD SD

L1/2 10 a 12.0a,c,d,f-l, A*** 0.7 14.9 g-l** 0.8 10.36 f,h,k,l, A

0.96 12.19 b,e,f
0.79
a,e-l g-l f,h-l, A d-f
L3 12 b 14.2 1.0 14.1 0.9 11.84 1.32 14.68 1.3

L4 12 c 14.8a,e-l 0.9 14.4g-l 0.9 10.54 2.68 14.30 e,f

2.55
a, e-l g-l g-j
L6 13 d 14.1 1.0 14.2 1.0 10.28 2.07 10.34 1.74

L7 12 e 10.5b-d,h,j-l, A 1.1 13.1g-l 0.6 9.60 2.21 9.94 a-c,h-l

0.77
a-d,j-l, A h-l a,b a-c,g-k
PP 15 f 9.3 0.7 12.5 1.0 8.18 0.40 9.88 1.68

P1 25 g 8.9a-d,k,l, A 1.0 11.1a-e, i-l 0.8 9.78 f,h,j-l, A

1.15 13.51 d-f
1.37
a-d,k,l, A a-f,j-l a,b, A d-f
P2 18 h 8.3 0.9 9.8 1.1 8.91 0.86 13.34 0.92
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P3 20 i 8.2a-d,k,l 1.3 9.1 a-g,j-l

0.8 8.88 b, A
1.83 13.00 a,d-f
0.51
a-d,k,l a-i b,g, A a,d-f
P4 15 j 7.4 0.6 7.3 0.8 8.70 1.16 13.18 0.84
a-j, A a-i a,b,g, A d-f
P5 18 k 4.7 0.9 6.8 0.8 7.86 1.12 12.86 1.12
a-j, A a-i a,b,g, A e
A 23 l 4.5 0.1 6.6 0.7 8.20 1.42 12.32 1.32

Fig. 1. The concentration of vitamin C in the extract from worker pupae stages (P) and newly emerged adult (A) bees. Different letters at the
top of bars indicate significant differences between means (p < 0.05).
 268
Superoxide dismutase (SOD) activity in brood was not high (Table 3). It
fluctuated slightly in both the groups to P2 pupa stage. In the older
pupae it decreased, and was the lowest in one-day old worker bees.
In the vitamin C receiving group, the enzyme activity was lower than
in the control, but the means differences, beside the prepupa stage,
were not statistically significant (Table 3). Unlike SOD, the activity of
peroxidases changed clearly during brood development in both the
groups. The lowest activity during the development was observed in
the emerged worker bees of the control group. From the prepupa
stage the activity of peroxidase was higher in the experimental group
than in the control. The differences between means for PP, P2 and A
stages were statistically significant (Table 3). Brood catalase was an
enzyme of very high activity (Table 4), at least 7 orders higher than in Saltykova et al. (2007) it seems that, at least in middle- and east-
case of peroxidase (Table 3). GST activity during the whole brood
development was high (Table 4). Catalase and glutathione transferase decomposing hydrogen peroxide. Glutathione transferase also has
activities developmental change profile were similar in both the
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groups. Newly emerged workers of the experimental group had
significantly higher concentration of both enzymes than bees of the
control group (Table 4).
Discussion
One of the basic indices determining the physiological state of honey
bees is body weight. Changes in brood weight during the
development of the control group were similar to those described
previously (Hrassingg and Crailsheim, 2005). According to Winston
(1987), the normal weight of young worker bees varies between 81-
151 mg. Mean weight of newly emerged individuals in our experiment
(116.73 10.10 mg) thus did not differ from that given by Winston
(1987). Vitamin C administered to the bees did not significantly
change the brood body weight. Similarly, Herbert et al. (1985) and
Anderson (2004) did not observe that supplementation of diet with
vitamin C significantly influenced bee weight.
The developmental profile of protein content change suggests
that probably during the last stages of worker bee metamorphosis,
after the exhaustion of other storage material, proteins become a
source of energy. In the experimental group in capped larvae and in
P3 and P4 pupa stages, the protein content was significantly lower
than in the control group (Table 1). Decrease in protein content is
thought to be an indicator of deterioration of physiological state of an
organism (Duay et al., 2003), but this suggestion seems unjustifiable
in relation to our results. Significantly higher protein content in freshly control group, especially at the initial period of metamorphosis (stages
emerged worker bees of the group receiving Vitamin C in comparison
to the control and results of other biochemical markers argue for this
fact.
The ability of the honey bee to defend itself against ROS seems to vitamin C will counteract the effects of oxidative stress under
be strongly limited compared to other insects, as indicated by a lower
number of genes coding for antioxidative proteins in the genome
Farjan, Dmitryjuk, Lipiski, Biernat-opieska, towska
(Corona and Robinson, 2006; Claudinos et al., 2006; Oakeshott et al.,
2010). Our results imply that emerging workers are less well
protected against ROS than pupae because all antioxidants have the
lowest values in this stage (Tables 2, 3 and 4). This situation may be
unfavourable for them because they encounter new factors, which
previously had limited influence on pupae because of wax cappings,
which act as a barrier with antioxidative potential (Sammataro et al.,
2010).
Discussion of our results on the activity of antioxidative enzymes
is difficult because insects are very diverse, and there are only a few
data on the Apidae (Weirich et al., 2002; Papdopoulos et al., 2004;
Lipiski and towska, 2005). Based on our results and those of
European bee races, catalase is the most important enzyme
high activity during development of A. m. carnica. GST beside high
antioxidant activity, also removes toxic metabolites and xenobiotics
from organism (Claudinos et al., 2006). There is thus a value of a diet
enriched with vitamin C, in that it increases activity of this important
enzyme.
We assumed that supplementation of diet with vitamin C will
cause considerable increase of the TAS, but in fact this increase was
very small (Table 2). Furthermore, a rise in TAS value resulted rather
from the elevation of glutathione than the ascorbic acid content.
According to Linster and Van Schaftingen (2007), glutathione is a
regulator for ascorbic acid production, and vitamin C synthesis is
negatively correlated with glutathione content. It seems that in honey
bee pupae, the vitamin C content is monitored, and as in insects in
general, bees are able to increase its production or accelerate its
degradation when its supply is excessive in the food (Colven and
Pinnell, 1996; Benzie, 2003). There were no significant differences in
vitamin C content between pupae from the group receiving or not
receiving vitamin C supplementation. The second important
observation in this work was that in pupae of both the groups, vitamin
C content gradually increased, so differences between the first pupal
stage and emerging worker bees were significant. This observation
can be considered as a confirmation of the hypothesis of Herbert et al.
(1985) that Vitamin C synthesis occurs in bees because pupation is a
period of starvation in insect development, so vitamin C increase had
to have an endogenous source.
A beneficial effect of vitamin C supplementation was elevation of
catalase and glutathione transferase activity in comparison to the
L7 P1), when the peroxidase level was higher. In pupae and adult
bees, significantly higher glutathione content was found compared to
the control, suggesting that worker bees from the group receiving
unfavourable environmental conditions more efficiently. We observed
in parallel conducted research similar positive effects of vitamin C on
 Honey bee brood antioxidative system and vitamin C supplementation
carbohydrate metabolism during brood development (unpublished
data).
The diet supplementation with vitamin C positively influenced
some of the studied physiological and biochemical indicators
including: lower winter mortality of bees, and protein content, TAS,
glutathione, and antioxidative enzyme (peroxidase, catalase, and
glutathione transferase) activity in emerging worker bees. These
results suggest that vitamin C can be recommended as a natural,
safe, and relatively cheap diet supplement, elevating resistance to
stress factors (including diseases, in which oxidative stress plays a key
role) of wintering bees and spring generation of worker bees.
Acknowledgements
This study was supported by grant number N N308 282933 from the
Downloaded by [37.76.6.175] at 03:11 10 December 2017
Ministry of Science and Higher Education, Poland.
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