Journal of Apicultural Research 51(3): 252-262 (2012)
© IBRA 2012
DOI 10.3896/IBRA.1.51.3.06
ORIGINAL RESEARCH ARTICLE
The influence of formic acid on the body surface proteolytic system at different developmental stages in Apis mellifera L. workers Aneta Joanna Strachecka1*, Jerzy Paleolog1, Grzegorz Borsuk1 and Krzysztof Olszewski1 1
Department of Biological Basis of Animal Production, Faculty of Animal Biology and Breeding, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland. Received 27 December 2011, accepted subject to revision 22 March 2012, accepted for publication 17 May 2012. *Corresponding author: Email:
[email protected]
Summary To verify the hypothesis that formic acid (FA) has a suppressive effect on the proteolytic system of Apis mellifera cuticle depending on the developmental stage of the insects, 1 to 4-day-old larvae, 8-day-old larvae, pupae, 1-day-old workers, and foragers were sampled from FAtreated and untreated colonies for seven weeks. Hydrophilic (H+) and hydrophobic protein (H-) solutions were washed out from the sampled individual body surfaces. Subsequently, protein concentration, protease activities and protease inhibitor activities were determined. Antifungal and antibacterial activities were also determined.Two-week exposure to FA increased H+ and H- protein concentration but when it was prolonged to three to seven weeks, the concentration fell below the level of untreated colonies. FA treatment decreased H+ protease activities in workers and larvae, but increased them in pupae. H- protease activities oscillated (destabilisation) up and down relative to the control level which was steady. Asparagine and serine proteases were present on apian cuticles independently of the developmental stage, while FA application additionally activated thiolic proteases. FA treatment considerably decreased both H+ and H- natural protease inhibitor activities in larvae and pupae but mostly increased them in workers. Antifungal and antibacterial activities of the body surface washings (in vivo microbiological tests) were suppressed in workers and larvae treated with FA. FA treatment may suppress proteolytic resistance of the bee cuticle but the responses of larvae and workers are different.
El efecto de ácido fórmico sobre el sistema proteolítico cuticular en varios estadios en el desarollo de las abejas obreras Apis mellifera L. Resumen Para investigar la hipótesis según la que ácido fórmico (FA) tiene efecto supresor sobre el sistema proteolítico cuticular de Apis mellifera en función del estadio en el desarrollo de los insectos, de las colonias tratadas con ácido fórmico y de las que no lo fueron se recogió durante siete semanas larvas de desde 1 hasta 4 días de edad, así como las de 8 días de edad, pupas, abejas obreras de un día de edad y abejas recolectoras. Soluciones proteínicas hidrofílicas (H+) e hidrofóbicas (H-) fueron deslavadas de las superficies de los cuerpos abejunos individuales. Subsiguientemente, se determinó: la concentración de proteínas, las actividades de proteasas y las actividades de inhibidores de proteasas. Se determinó también las actividades antifúngicas y antibacterianas.La exposición de 2 semanas de duración al ácido fórmico aumentó la concentración de proteínas H+ y H- pero, cuando la exposición fue prolongada hasta entre 3 y 7 semanas, la concentración cayó debajo del nivel de las colonias no tratadas con ácido fórmico. El tratamiento con ácido fórmico redujo la actividad de las proteasas H+ en las abejas obreras y larvas pero la incrementó en las pupas. Las actividades de las proteasas H- oscilaban (desestabilización) respecto al nivel testigo que permanecía estable. Las proteasas aspárticas y serinas proteinasas fueron presentes en las cutículas de las abejas independientemente del estadio en su desarrollo, mientras que la aplicación de ácido fórmico activó por añadidura las tiol-proteasas. El
Formic acid and the honey bee body surface proteolytic system
253
tratamiento con ácido fórmico considerablemente redujo las actividades tanto de los inhibidores naturales de proteasas H+ como de los de proteasas H- en las larvas y pupas pero en la mayoría de los casos las incrementó en las abejas obreras. Las actividades antifúngicas y antibacterianas de las lavaduras (los ensayos microbiológicos in vivo) fueron inhibidas en las abejas obreras y larvas tratadas con ácido fórmico. Tratamiento con ácido fórmico puede inhibir la resistencia proteolítica cuticular de las abejas pero las reacciones de las larvas y abejas obreras son diferentes. Keywords: proteases, protease inhibitors, Apis mellifera, formic acid, developmental phases
Introduction
defence systemand that this effect may depend on the bee
Varroa destructor is a global apicultural problem (Medina Medina et
surface proteolytic system activity of bees is associated with
al., 2002) and implicated in Colony Collapse Disorder (CCD) (Buczek,
antifungal and antibacterial activities.
developmental stage or worker age, in particular whether the body-
2009) as pyrethroid- and amitraz-resistant mite populationshave been identified in Europe and the USA (Milani, 1995; Elzen et al., 2000; Lipiński and Szubstarski, 2007). Acaricide treatment can also result in contamination of bee products (Colin, 1997; Buczek, 2009), so the
Materials and methods
use of formic acid has been promoted (Imdorf et al., 2003). Treated
Our experiments were conducted in Lublin, Poland in August 2008
workerbees may, however, abscond (Kasprzak and Hartwig, 2005),
and 2009. The Lublin region had hot weather at the time, with
the apian midgut might be damaged (Howis et al., 2010) or bee
occasional summer storms. Five experimental plus two control
immunity may decrease (Lipiński and Szubstarski, 2007).
colonies in the first year, and six experimental plus three control
Proteases and protease inhibitors are active in extra- and
colonies (of an equal strength and structure) in the second year were
intracellular proteolysis and participate in zymogene activation, the
established. The field study lasted 7 weeks in each year. No formic
release of hormones and active proteins from their precursors,
acid treatment was performed until the second week of the study.
transport through the cell membranes, protein compound ordering
From that moment until the seventh week, the experimental colonies
and receptor activation (Barrett, 1999; Bode et al., 1999; Otlewski et
were treated with formic acid vapours (concentration 60%) coming
al., 2001; Deraison, 2004; Walter and Clélia, 1994; Strachecka et al.,
from 200ml P.P.H. SOLO plastic evaporators placed within the hives,
2008). They are present in the bee alimentary duct, haemolymph,
whilst the control colonies were left untreated. The daily evaporation
moult liquid and venom (Bode et al., 1999; Lima et al., 2000; Malone
volume was 30-34 g. From each colony,three pooled samples of ten 1
et al., 2004; Evans et al., 2006; Strachecka et al., 2008). They also
-day- to 4-day-old larvae, ten 8-day-old larvae, ten pupae, seven 1-
play an important role in the body surface defence, as confirmed in
day-old workers, and seven foragers, respectively, were collected
humans (Bogaczewicz et al., 2004), frogs (Milani, 1995; Zhao et al.,
twice each week. The material was sampled every Monday and
2005), silkworms, Drosophila melanogaster (Zou et al., 2006), and
Thursday. The adults were stored in germ-free bags (10 cm x 15 cm)
even in potatoes (Solanum tuberosum) (Hermosa et al., 2006). Recent studies have been the first to show that the active layer of
and the larvae and pupae in sterile Eppendorf tubes (2 ml) at -8⁰C for 1-2 months.In total, 1050 samples (3 samples x 2 samplings per week
proteases (serine-, cysteine-, asparagine-, and metalloproteases) and
x 7 weeks x 5 developmental stages x 5 colonies; 9240 items) were
protease inhibitors is of particular importance to the bee cuticle
collected from the control hives and 2310 samples (20328 items) from
defence (Grzywnowicz et al., 2009; Strachecka et al., 2008; 2010).
the experimental ones.
This anti-pathogen barrier could be suppressed by diverse external
The samples were then successively thawed and rinsed in 10 ml of
factors (Strachecka et al., 2010). On the other hand, knowledge of
distilled water for 20 seconds in order to remove impurities. The
the side effects of common drugs is crucial for a long-lasting therapy,
rinsings were discarded because proteins were not found in them
for example against V. destructor. It has been reported that formic
using the Lowry method, as modified by Schacterle and Pollack
acid treatment is harmful to bee larvae (Gregorc et al., 2004) and
(1973). Subsequently, the samples were shaken/rinsed for 4 min. at
may promote development of some diseases, especially mycosis
3400 rpm with the addition of 10 ml distilled water, and finally, after
(Howis et al., 2010; bee farmers, personal communications). The
filtering through Miracloth, a solution was obtained that mostly
knowledge of mutual relations between pathogens and theirs hosts is
contained hydrophilic proteins. The solution from each sample was
also important. Frączek et al. (2010) studied body-surface proteolytic
then divided into 3 portions (2 ml each), poured into three Eppendorf
system in V. destructor, which opened a new area for the study of the tubes, and frozen again at -40⁰C. This procedure produced: portion a cuticle proteolytic system, in the context of parasite-host relations. The purpose of our research was therefore to test the hypothesis that formic acid has a negative effect on the bee cuticle proteolytic
- for determining protease and protease inhibitor activities; portion b for determining antifungal and antibacterial activities in vivo; and portion c - reserve. Afterwards, the solid sample components that
254
Strachecka, Paleolog, Borsuk, Olszewski
remained on the Miracloth were again shaken/rinsed (4 min at 3400rpm) in a 1% Triton X-100 (detergent) solution in distilled water
Results
(10 ml). As with the first rinsing, 3 portions (2 ml) were created, but
Protein concentration
now with mostly hydrophobic proteins. The entire procedure resulted
Body surface hydrophobic protein concentration was higher than
in a total of 20,160 portions.
hydrophilic (Fig. 1) but it was consistently lower in young adult
From each portion a, 62 µl of solution were taken to create 70
workers and foragers than in larvae and pupae. Concentrations of
pooled samples. Each sample was created by mixing the solutions
each protein type in the control colonies remained at the same steady
originating from one developmental stage (5 categories) within one
level throughout the seven consecutive weeks. Shorter than a week’s
week (7 categories) and one group (2 categories; control/
exposure to formic acid did not affect the concentration at any
experimental). The optimal pH values, at which the protein activities
developmental stage. Levels of both types of proteins markedly
were high, were determined separately for each of the pooled
increased after two weeks of treatment, however. Levels proceeded
samples and amounted to 2.2 - 3.4, 6.4 - 7.6, and 8.2 - 11.6.
to decrease in bees of all ages, but most dramatically in larvae and
Therefore, we decided to determine the activities of acidic, neutral
pupae.
and alkaline proteases/protease inhibitors at pH 2.4, 7.0 and 11.2,
Proteolytic activity
respectively. Next, the remainder (1.938 ml) of portion a was analysed as
Formic acid treatment decreased hydrophilic surface protease
follows: 1. The general protein content by the Lowry method, as
activities (Table 1) in foragers and 1-day-old workers (pH 2.4, 7.0,
modified by Schacterle and Pollack (1973); 2. The proteolytic activity
and 11.2). In 1-4-day-old-larvae, a decrease was observed at pH 2.4
in relation to different substrates (gelatine, haemoglobin, ovoalbumin, and 11.2 but in 8-day-old-larvae only at pH 2.4. In pupae, however, albumin, cytochrome C, casein), according to the modified Anson
the activities were mostly increased. In the other cases (pH), the
(1938) method. In consequence, albumin was considered as the
activities were destabilised and oscillated up and down in comparison
optimal substrate for the future analysis; 3. The proteolytic activity in
with the control colonies that remained at a stable constant level. The
relation to the diagnostic inhibitors of proteolytic enzymes (pepstatin
treatment destabilised hydrophobic protease activities (Table 2),
A, PMSF, iodoacetamide, o-phenanthroline), by the Lee and Lin
causing them to oscillate up and down compared to the control level,
(1995) method; 4. The activity of acidic, neutral and alkaline
which remained stable. In week seven the activities significantly
proteases according to the modified Anson (1938) method;and 5. The increased in most treated bees, regardless of developmental stage. levels of natural inhibitors of acidic, neutral and alkaline proteases,
Metallo-proteases were not identified in any of the samples tested
based on the Lee and Lin (1995) method.
(Table 3). Formic acid application activated thiolic-proteases in all
Portion b acquired from 8-day-old larvae/foragers within rd
th
experimental/control colonies and at the 3 /7 week were pooled
cases, regardless of the developmental stage. Asparagine- and serineproteases were present on the bee cuticle in all samples tested.
separately (2 x 2 x 2 = 8 pooled samples) and then lyophilized to determine antifungal and antibacterial activities. To this end, the
Natural protease inhibitor activity
lyophilizates were combined with 200 µl distilled water. Subsequently,
Formic acid treatment considerably decreased both hydrophilic and
10 µl of the mixtures were plated on the following culture media,
hydrophobic natural surface protease inhibitor activities in larvae and
using double plates: 1. SABG (Sabouraud, 1892)to determine activity
pupae (Tables 4 and 5), whilst in foragers the activities were
in relation to Aspergillus niger; 2. YPD (Murthy et al., 1975) to
increased, except for hydrophilic proteases at pH 2.4. In one-day-old
determine activity in relation to Candida albicans; and 3. LB (Bertani,
workers, hydrophilic protease activities mostly rose but in the case of
1952) to determine activity in relation to Staphylococcus aureus
hydrophobic proteases, they decreased. In the control colonies, the
(ATCC 25923), Bacillus subtilis (ATCC 6633), Micrococcus luteus
activities remained at a stable/constant level.
(ATCC 7468), Salmonella typhimurium (ATCC 13311), Pseudomonas
aeruginosa (ATCC 17853), Escherichia coli (ATCC 10536). In an additional survival test, the bacteria were transferred from the surface on which the growth of B. subtilis had been inhibited onto
Antifungal and antibacterial activity Only foragers and 8-day-old larvae were assayed (Table 6). In none of the samples were activities towards M. luteus and E. coli observed.
a new base and observed for renewed growth or its absence.In all the The control samples suppressed the growth of A. niger and C. albicans microbiological tests, each of the dishes was photographed (SONY
(antifungal), as well as B. subtilis, S. aureus,S. typhimurium,and
α100) to determine the area in which there was no microorganism
P. aeruginosa (antibacterial). The samples collected from formic acid-
growth, using the MultiScan Base software (Version 14.02).In order to treated colonies were active only towards C. albicans, B. subtilis and verify the influence of formic acid on protease- and protease inhibitor
S. aureus (hydrophilic). The same pattern, with few exceptions
activity, one-way ANOVA and the Duncan multiple range test (SAS
(bacteria), was observed in the hydrophobic samples. In the case of
Institute Version 9.13., license 86636) were used.
C. albicans the treatment increased the fungal growth area. The
Formic acid and the honey bee body surface proteolytic system
255
Fig. 1. Protein concentration (C) on A. mellifera body surface at different developmental stages and worker age-castes during the seven consecutive weeks of the field study.
Explanations: The values for the 1-4-day-old larvae, 8-day-old larvae and pupae in the control colonies were almost the same. Therefore, they were plotted together (on average; dotted line). The values for the 1-day-old workers and foragers were also almost the same and were plotted together (on average; dashed line). Formic acid treatment was performed from the 2nd to the 7th week. Arrow – the week in which the colonies started to be treated with formic acid.
microorganism survival test additionally showed that the forager
temporarily impeded their growth. This was reflected in a higher
samples were able to kill B. subtilis, whereas the larval samples only
proteolytic activity in worker cuticles (Tables 1, 2, 4, and 5).
256
Strachecka, Paleolog, Borsuk, Olszewski
Table 1. The activity of body surface hydrophilic proteases (U/mg) at different developmental stages and worker ages of A. mellifera treated with formic acid in comparison to the untreated control.*The protease activities in the treated bees were significantly different (P ≤ 0.05) from those observed in the controls. The results for the control colonies were very similar to each other, so only the particular ranges for the seven consecutive weeks were presented. se: standard error. Shadowed cells: protease activities were considerably decreased by the treatment. Underlined: protease activities were considerably increased by the treatment. In the first and second weeks of the experiment, the activities in the treated bees were not different from those observed in the controls.
pH
factor
control
2.4
formic acid
control
7.0
formic acid
control
11.2
formic acid
week
1-4-day-old larvae
8-day-old larvae
pupae
1-day-old workers
foragers
Average
± se
Average
± se
Average
± se
Average
± se
Average
± se
1-7
1.09-1.86
0.19
3.68-3.89
0.19
3.48-4.59
0.14
33.71-34.31
0.34
12.00-12.66
0.38
1
1.32
0.03
3.68
0.04
3.48
0.03
33.99
0.07
12.20
0.06
2
1.45
0.04
3.72
0.05
3.51
0.04
34.01
0.11
12.23
0.05
3
0.99
0.03
3.64
0.03
3.49
0.04
34.03
0.45
12.30
0.03
4
2.56*
0.23
0.59*
0.07
22.69*
12.77
7.01*
0.90
5.96*
1.51
5
0.35*
0.02
2.56*
0.12
15.64*
0.33
0.46*
0.01
0.26*
0.01
6
0.04*
0.01
0.27*
0.01
0.14*
0.01
0.01*
0.01
0.37*
0.31
7
0.21*
0.01
2.89*
0.16
44.44*
0.57
3.53*
1.79
2.03*
0.07
mean
0.83
0.77
1.99
0.56
17.281
7.76
9.009
8.99
4.99
4.90
1-7
1.28-1.44
0.19
4.35-4.66
0.31
8.21-8.62
0.29
32.17-37.11
0.87
11.79-13.97
2.01
1
1.32
0.04
4.37
0.05
8.34
0.04
36.12
0.06
11.99
0.07
2
1.35
0.03
4.41
0.04
8.40
0.03
36.30
0.04
12.03
0.05
3
1.39
0.03
4.46
0.11
8.66
0.05
37.09
0.09
12.15
0.02
4
1.67
0.31
4.41
0.55
28.25*
2.76
23.08*
1.32
7.24*
0.39
5
0.31*
0.02
4.92
0.07
15.84*
0.18
0.80*
0.16
2.80*
0.05
6
0.00*
0.00
0.15*
0.01
0.29*
0.02
0.44*
0.41
0.02*
0.01
7
13.57*
0.32
18.57*
0.12
22.48*
0.09
138.09*
0.45
4.53*
0.09
mean
3.38
0.98
6.50
0.89
15.11
2.11
39.91
24.67
5.53
1.16
1-7
3.31-3.77
0.29
1.16-1.72
0.52
2.68-2.82
0.28
35.88-37.28
1.32
15.04-15.80
0.73
1
3.45
0.06
1.20
0.07
2.72
0.04
36.01
0.10
15.68
0.02
2
3.46
0.10
1.23
0.10
2.74
0.05
36.15
0.11
15.65
0.04
3
3.48
0.25
1.11
0.04
2.48
0.04
38.46
0.17
11.28*
0.06
4
6.86*
0.56
9.15*
1.57
21.69*
5.05
25.80*
4.29
9.70*
1.37
5
0.38*
0.06
6.39*
0.26
13.61*
0.09
21.23*
0.61
4.68*
0.15
6
0.01*
0
0.09*
0.01
0.04*
0.01
0.01*
0.00
0.01*
0.00
7
0.92*
0.03
7.65*
0.11
22.24*
0.16
7.62*
0.17
4.74*
0.06
mean
2.33
2.43
4.88
2.33
12.01
8.56
18.50
6.54
5.67
0.51
Formic acid and the honey bee body surface proteolytic system
257
Table 2. The body surface hydrophobic proteases activity (U/mg) at different developmental stages and worker ages of A. mellifera treated with formic acid in comparison to the untreated control.*The protease activities in the treated bees were significantly different (P ≤ 0.05) from those observed in the control ones The results for the control colonies were very similar to each other, so only the particular ranges for the seven consecutive weeks were presented. se: standard error. Shadowed cells: protease activities were considerably decreased by the treatment. Underlined: protease activities were considerably increased by the treatment. In the first and second weeks of the experimental period, the activities in the treated bees were not different from those observed in the controls. 1-4-day-old larvae pH
2.4
factor
week
control
formic acid
control
7.0
formic acid
control
11.2
formic acid
8-day-old larvae
pupae
1-day-old workers
foragers
Average
± se
Average
± se
Average
± se
Average
± se
Average
± se
1-7
14.06-14.87
0.14
8.78-9.85
0.34
3.74-3.94
0.21
34.39-39.74
1.09
12.15-15.00
2.01
1
14.12
0.07
8.82
0.09
3.84
0.10
39.35
0.12
13.52
0.07
2
14.20
0.09
8.88
0.08
3.82
0.04
39.42
0.11
13.56
0.09
3
15.38
0.19
9.64
0.08
1.21*
0.03
39.59
0.04
15.85
0.06
4
7.03*
0.26
2.49*
0.21
2.66*
0.07
1.66*
0.05
0.67*
0.02
5
3.86*
0.12
2.78*
0.11
4.39*
0.16
18.19*
0.54
4.74*
0.05
6
11.61*
0.24
7.39*
0.12
9.74*
0.09
31.22*
0.15
17.88*
0.56
7
34.03*
0.18
17.96*
0.09
24.45*
0.12
65.86*
0.17
56.88*
0.43
mean
14.38
8.78
8.06
5.56
8.49
7.65
31.31
10.87
18.88
9.76
1-7
5.15-5.61
0.36
1.15-1.95
0.43
1.36-1.56
0.23
7.16-8.16
0.95
11.78-14.23
1.7
1
5.35
0.09
1.24
0.11
1.52
0.01
7.98
0.06
11.99
0.09
2
5.32
0.11
1.27
0.10
1.51
0.02
7.94
0.05
11.98
0.08
3
5.29
0.03
1.20
0.01
0.63*
0.01
8.41
0.12
11.96
0.01
4
3.21*
0.11
1.85
0.02
7.89*
0.15
5.72*
0.05
5.36*
0.08
5
4.29*
0.12
10.38*
0.29
4.45*
0.07
38.10*
0.65
5.69*
0.19
6
0.19*
0.02
3.68*
0.09
15.23*
0.31
21.75*
1.00
4.76*
0.05
7
6.55*
0.11
10.44*
0.06
11.91*
0.26
5.97*
0.18
18.69*
0.13
mean
3.91
0.98
5.51
3.76
8.02
4.12
15.99
9.98
9.28
8.76
1-7
13.32-15.73
1.35
7.36-8.24
1.01
9.65-9.89
0.21
21.96-22.94
1.53
10.77-11.33
1.02
1
15.44
0.08
7.92
0.06
9.82
0.07
22.12
0.09
10.81
0.05
2
15.45
0.04
7.94
0.05
9.85
0.04
22.05
0.07
10.82
0.01
3
15.46
0.09
7.94
0.06
9.13
0.38
21.99
0.03
11.88
0.13
4
0.76*
0.02
4.34*
0.11
15.43*
0.31
14.42*
0.14
4.53*
0.05
5
3.77*
0.22
8.51
0.10
3.62*
0.05
18.61*
0.16
6.12*
0.28
6
4.27*
0.31
4.96*
0.17
3.59*
0.08
7.11*
0.39
1.21*
0.05
7
51.14*
2.16
7.55
0.09
24.31*
0.13
63.59*
0.89
55.36*
1.45
mean
15.07
17.67
6.66
2.76
11.21
5.98
25.13
10.18
15.68
9.54
258
Strachecka, Paleolog, Borsuk, Olszewski
Table 3. The presence of body surface protease activity (yes/no) in relation to proper substrates in bees treated with formic acid, in comparison to the untreated control (pooled samples). Substrate o- phenanthroline (metallo-proteases)
iodoacetamide (thiolic-proteases)
pepstatin A (asparagine-proteases)
PMSF (serine-proteases)
pH
Control
Formic acid
2.4
no
no
7.0
no
no
11.2
no
no
2.4
no
yes
7.0
no
yes
11.2
no
yes
2.4
yes
yes
7.0
yes
yes
11.2
yes
yes
2.4
yes
yes
7.0
yes
yes
11.2
yes
yes
Table 4. The activity of body surface natural hydrophilic protease inhibitors (U/mg) at different developmental stages and worker ages of A. mellifera treated with formic acid in comparison to the untreated control.*The inhibitor activities in the treated bees are significantly different (P
