Abstract: Background and Objective: Chlorophyllin derivatives, mammal-safe pesticides, are using recently to control many agricultural and medical pests such as lepidopteran and mosquitoes population. The objective of this work was to assess the possible environmental risks for the chlorophyllin derivatives field application, particularly related to pollinating insect, Apis mellifera. Materials and Methods: In this study, the foraging worker honey bees fed on tri-sodium copper and magnesium chlorophyllin LC50 (10–5 and 3×10–3 M L–1, respectively) were used to measure the total antioxidant capacity, superoxide dismutase and glutathione S-transferase. Also, midgut cells were papered for the comet assay. Results: The tri-sodium Cu-chlorophyllin increased the TAC, SOD and GST activities in the body homogenate of honey bees in response to the oxidative stress of both chlorophyll and Cu elements. Conclusion: Furthermore, the results of the genotoxicity experiment revealed that Cu-chlorophyllin caused DNA damage in the gut cells more than Mg-chlorophyllin and its effect might be due to copper elements.
INTRODUCTION
Honey bees are the most important species that pollinate flowering plants and among a few species that are domesticated by humans for agricultural and industrial economic purposes1. Honey bee, Apis mellifera has a major role in sustaining food security and maintaining biodiversity for the natural ecosystems 2. This important insect faces many complex stressors: Biological include pathogens, parasite and microorganisms3-5, environmental include: Temperature and humidity6, chemicals include: Metal and metalloid contaminants7,8, different types of plant protection products such as insecticides, acaricides, herbicides and fungicides have applied to control agricultural pests9. In addition, the anthropogenic activities associated with urbanization and industrialization has created localized and regional pollution problems10. A lot of publications have studied the sub-lethal and accumulation effects of many natural and synthetic pesticides groups on honey bee life such as fitness, foraging activity, flight activity, sensorial ability, neurotoxicity, detoxification, metabolism and oxidative stress11. Less study focused on enzymatic and molecular responses of honey bees using genomic, metabolomic and transcriptomic techniques and biomarkers12.
The detoxification systems of insects, especially honey bees, respond quickly to chemical and biological stresses13,14. In honey bees, oxidative stress due to different biotic and biotic factors was examined in many kinds of literature and the activity of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione s-transferase (GST), glutathione peroxidase (GPx), peroxidase (POD)15-17. Chlorophyllin derivatives have a new mode of action on insects and consider environmentally safe compounds.
This photoactive compound is accumulated inside the insect body and upon exposure to sunlight, the reactive oxygen species (ROS) is produced that cause cellular and molecular destruction18-20. Copper chlorophyllin has been added safely as animal nutrition21. The reduction of mosquitos’ population in endemic areas using chlorophyllin derivatives have been recorded22,23. Afify et al.24 showed that Cu and Mg-chlorophyllin caused death to Culex pipiens larvae due to physical damage of tissues and direct effect on their biochemical and physiological parameters. Recently, Nassar et al.25 have studied the toxicity effect of both copper and magnesium tri-sodium chlorophyllin on forage honey bee’s A. mellifera L., in laboratory and semi-field conditions. The LC50 of Cu and Mg-chlorophyllin measured in the laboratory were 10–5 and 10–3 M L–1, respectively. Few studies on the cytotoxicity and genotoxicity impact of pesticides (insecticides, fungicides and acaricides) on honey bees have been done12,26,27. In the current work, the biochemical effects, total antioxidant capacity TAC, SOD and GST enzyme activities, were measured in the total body homogenates of forage bees exposed to LC50 of Cu and Mg chlorophyllin for 3 days in light and dark conditions. Furthermore, the genotoxic impact was estimated as DNA damage using Comet assay. This work presented the biochemical responses of honey bees to human safe pesticides, chlorophyllin derivatives which are crucial for their implementation to control pests without affecting the beneficial pollinators in fields worldwide.
MATERIALS AND METHODS
Honey bee sampling: Laboratory experiments were carried out with honey bee foragers of Apis mellifera L. (Hymenoptera: Apidae) from December, 2015 to October, 2018. The adult workers were obtained from the Apiculture station, Faculty of Agriculture, Cairo University, Giza, Egypt, where honey bee colonies were maintained according to the standard commercial technique in the field. For this study, foraging bees were used when they start performing external tasks28,29. Based on farming records, no obvious diseases were observed on units or colonies and pesticides free. According to Nassar et al.25 the foraging workers were collected from the front of the hives in a plastic bag then transferred into experimental foam containers (10×7×12 cm) and left overnight before treatment. The bees were fed a 50% (w/v) sucrose solution with or without the chlorophyllin derivatives ad libitum at 25±2°C with 65±5% RH, a photoperiod of 8:16 (light: dark) for control light (CL) and treated light group (TL). The third group was fed on sucrose solution with chlorophyllin derivatives and kept in dark conditions (TD). The used LC50 of Cu-chlorophyllin and Mg-chlorophyllin were 10–5 and 3×10–3 M L–1, respectively. The fourth group of bees were collected from the field (FC). Bees were collected at the end of the first, second and third day of the feeding. The bodies of five honey bees were homogenized in 500 μL ice-cold phosphate buffer saline (pH 7.4). The homogenates were centrifuged at 10,000 g for 15 min at 4°C and the supernatant was stored at -20°C in the freezer till use for further analyses. Total antioxidant capacity (TAC), superoxide dismutase (SOD) and glutathione S-transferase (GST), as well as protein content, were assayed in bee body homogenate.
Antioxidant enzyme assay: Total antioxidant capacity (TAC) was assayed according to Koracevic et al.30. TAC was determined using a commercial antioxidant colourimetric assay kit (Biodiagnostic and research reagent, Egypt). The absorbance read against blank at 505 nm and total antioxidant concentration results were expressed in mM L–1.
Superoxide dismutase (SOD): Superoxide dismutase (SOD) has been assayed according to Nishikimi et al.31. The total SOD from cytosol, mitochondria and extracellular spaces inhibit the phenazine methosulphate-mediated (PMS) reduction of nitroblue tetrazolium dye (NBT). The SOD was measured as the increase in absorbance at 560 nm. The SOD activity can be expressed as a function of the protein (U mg–1 protein).
Glutathione S-transferase (GST): Glutathione S-transferase (GST) activity was determined using Habig et al.32 methods. Total GST activity (cytosolic and microsomal) kit (Biodiagnostic and research reagent, Egypt) has been used. The conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione is increased by an increase in absorbance at 340 nm and is directly proportional to the GST activity in the sample. Enzyme activity was converted into the number of units (U) per 1 mg of protein.
The proteins were assayed using Biuret reagent (Biodiagnostic and research reagent, Egypt) at a wavelength of 550 nm33. Simply, protein produces a violet colour with Biuret reagent, alkaline cupric sulfate and the intensity of which is proportional to their concentrations.
Detection of DNA damage: The comet assay was used to analyze the percent of DNA damage in the gut cells of worker honey bees after feeding on Cu-chlorophyllin (LC50 = 10–5 M L–1) or Mg-chlorophyllin (LC50 = 3×10–3 M L–1) and exposure to sunlight for 3 days (8 hrs per day). Honey bees fed on 50% sucrose solution and exposure to sunlight was used as control. Briefly, the guts of three honey bees for each treatment were maintained with 200 μL of PBS and centrifuged at 1000 rpm for 10 min. Then, 90 μL of low melting agarose was added to 10 μL of isolated cells and loaded on microscope slides, pre-coated with 1.5% normal melting point agarose (NMA). Next, covered the slide and then slides were coaled on ice. After agarose solidified, we removed the cover slips and slides were immersed in a lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 0.25 M NaOH, 1% TritonX-100 and 10% dimethylsulfoxide (DMSO), pH 10.0) for 24 hrs at 4°C. After lysis, slides were placed in a horizontal gel electrophoresis tank and DNA was allowed to unwind for 20 min in electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH 13). Electrophoresis was carried out at 24 V and 270 mA, at 4°C, for 20 min. Then neutralize the samples in 0.4 M Tris–HCl (pH 7.4), fixed with methanol and allowed to dry overnight at room temperature before staining with ethidium bromide (2 μg mL–1). Comets were analyzed with an Axio fluorescence microscope (Carl Zeiss, Germany) with an excitation filter of 524 nm and a barrier filter of 605 nm. Three replicates were prepared and each of them consisted of a pool of 3 guts. The most common parameters analyzed were the percentage DNA in the tail (DNA%), tail moment and tail length34.
Statistical analysis: Averages of the measurements on multiple aliquots of each sample for each treatment were used as the data set for the analysis. Statistical significance of differences among activities of TAC, SOD and GST level was calculated with two-way ANOVA. Subsequently, means were separated by Tukey’s HSD test (a = 0.05).
RESULTS
Antioxidant protection (TAC, SOD and GST activities): The TAC, SOD and GST activities showed a significant increase in the body homogenate of bees fed on Tri-sodium Cu-chlorophyllin and exposed to light compared to that fed on tri-sodium Mg-chlorophyllin under the same experimental conditions.
Total antioxidant capacity (TAC): The bees fed on Cu-chlorophyllin showed a significant increase of the measured TAC in the first, second and third day of feeding under light (TL: 3.59±0.01, 3.36±0.02 and 2.82±0.2 mM L–1, respectively) and dark condition (TD: 2.37±0.03, 2.55±0.09 and 2.47±0.00 mM L–1, respectively) Table 1 and Fig. 1a. Furthermore, FC bees little increase of TAC compared to the control light group, significant difference, p<0.01 (Table 1 and Fig. 1b). On the other hand, TAC measured in body homogenates of Mg-chlorophyllin fed bees have no significant increase compared to the CL group and were nearly the same TAC measured in the FC group. (Supplemented Table 1).
SOD activity: The highest SOD activities have been measured in the bees fed on Cu-chlorophyllin and exposed to light (0.43, 0.32 and 0.41 U μg–1 protein) Table 1 and Fig. 2. Then it was followed by SOD activities of the dark group and finally, the field-collected group (Table 1 and Fig. 2a). The SOD activity measured in the body homogenate of the Mg-chlorophyllin fed bees was almost the same in TL (0.28, 0.27 and 0.28 U μg–1 protein) and TD (0.25, 0.26 and 0.27 U μg-1 protein) Table 1 and Fig. 2b and Supplemented Table 2.
GST activity: Cu- chlorophyllin fed group exposed to light, TL, showed the highest GST activities (2.4, 3.07 and 2.56 U μg–1 protein) Table 1 and Fig. 3a. The GST activities measured from FC bees were observed to be higher than that measured from the TD group (Table 1, Fig. 3a and Supplemented Table 3).
| Fig. 1(a-b): | Concentrations of TAC in adult Apis mellifera body homogenate after being treated with (a) 10–5 M L–1 copper chlorophyllin and (b) 3×10–3 magnesium chlorophyllin for different times CL: Control light, TL: Treated light, TD: Treated dark and FC: Field collected groups, *,**Represent significant differences as compared to the CL group at p<0.01 and p<0.000, respectively |
| Table 1: | Biochemical parameters of Apis mellifera homogenate after treated with 10–5 M L–1 copper and 3×10–3 M L–1 magnesium photosensitizer chlorophyllin for different times | ||||
Photosensitizer |
CL |
TL |
TD |
FC |
|
| TP (μg μL–1) | Cu |
0.99±0.2 |
1.8±0.04 |
1.29±0.18 |
1.08±0.2 |
0.88±0.03 |
2.1±0.09 |
1.18±0.05 |
1.08±0.2 |
||
1.22±0.01 |
1.88±0.1 |
1.46±0.2 |
1.11±0.2 |
||
Mg |
0.98±0.2 |
1.56±0.14 |
1.19±0.06 |
1.08±0.2 |
|
1.01±0.4 |
1.76±0.03 |
1.22±0.03 |
1.08±0.2 |
||
0.99±0.11 |
1.84±0.08 |
1.35±0.1 |
1.08±0.2 |
||
| TAC (mM L–1) | Cu |
2.01±0.12 |
3.59±0.01 |
2.37±0.03 |
2.22±0.05 |
1.53±0.12 |
3.36±0.02 |
2.55±0.09 |
2.22±0.05 |
||
1.60±0.3 |
2.82±0.2 |
2.47±0.00 |
2.22±0.05 |
||
Mg |
2.01±0.06 |
2.37±0.00 |
2.28±0.01 |
2.22±0.05 |
|
1.85±0.00 |
2.42±0.01 |
2.39±0.00 |
2.22±0.05 |
||
1.80±0.01 |
2.42±0.02 |
2.40±0.03 |
2.22±0.05 |
||
| SOD (U μg–1 protein) | Cu |
0.18±0.0218 |
0.43±0.0595 |
0.35±0.0409 |
0.21±0.064 |
0.18±0.0174 |
0.32±0.0466 |
0.27±0.0599 |
0.23±0.064 |
||
0.168±0.042 |
0.41±0.058 |
0.20±0.0460 |
0.21±0.064 |
||
Mg |
0.19±0.0485 |
0.28±0.057 |
0.25±0.0429 |
0.21±0.064 |
|
0.18±0.050 |
0.27±0.057 |
0.26±0.0474 |
0.23±0.064 |
||
0.19±0.0388 |
0.28±0.030 |
0.27±0.040 |
0.21±0.064 |
||
| GST (U μg–1 protein) | Cu |
1.51±0.07 |
2.40±0.25 |
2.01±0.14 |
2.00±0.25 |
1.28±0.04 |
3.07±0.17 |
2.26±0.17 |
2.41±0.25 |
||
1.31±0.14 |
2.56±0.06 |
1.98±0.27 |
2.45±0.25 |
||
Mg |
1.51±0.09 |
1.92±0.34 |
1.26±0.43 |
2.17±0.25 |
|
1.47±0.68 |
2.32±0.01 |
1.65±0.20 |
2.22±0.25 |
||
1.53±0.05 |
2.67±0.05 |
1.97±0.30 |
2.01±0.25 |
||
Honey bees treatment condition: CL: Control in light condition, TL: Treated in light condition, TD: Treated in dark condition and FC: Field collected bees, Measured biochemical parameters: TP: Total protein concentration in μg μ–L1, TAC: Total antioxidant capacity in mM L–1, SOD: Superoxide dismutase activity in U μg–1 protein and GST: Glutathione S-transferase activity in U μg–1 protein, (each measure represented as Mean±Standard error) |
|||||
On the other hand, Mg-chlorophyllin fed bees exposed to light and dark conditions showed a gradual increase in GST activities with the highest value in the third day of feeding (TL 2.67±0.05 and TD 1.97±0.3 U μg–1 protein, respectively) Table 1 and Fig. 3b. Moreover, the GST activities measured from FC bees were higher than the TD group Table 1, Fig. 3b and Supplemented Table 3).
| Fig. 2(a-b): | Activities of SOD in adult Apis mellifera body homogenate after being treated with (a) 10–5 M L–1 copper chlorophyllin and (b) 3×10–3 magnesium chlorophyllin for different times CL: Control light, TL: Treated light, TD: Treated dark and FC: Field-collected groups, *,**Represent significant differences as compared to the CL group at p<0.01 and p<0.000, respectively |
| Fig. 3(a-b): | Activities of GST in adult Apis mellifera body homogenate after being treated with (a) 10–5 M L–1 copper chlorophyllin and (b) 3×10–3 magnesium chlorophyllin for different times CL: Control light, TL: Treated light, TD: Treated dark and FC: Field-collected groups, *,**Represent significant differences as compared to the CL group at p<0.01 and p<0.000, respectively |
| Table 2: | Comet parameters in the adult Apis mellifera gut cells treated with 10–5 copper or 3×10–3 M L–1 magnesium chlorophyllin for three days | |||
Photosensitizer |
CL |
TL |
TD |
|
| Tail length (μm) | Cu |
1.24±0.251 |
3.26±0.424 |
2.56±0.424 |
Mg |
1.40±0.532 |
2.06±0.44 |
1.60±0.265 |
|
| DNA damage in tail (%) | Cu |
12.88±1.43 |
23.73±1.65 |
17.92±1.89 |
Mg |
12.86±2.44 |
16.34±1.43 |
13.76±1.49 |
|
| Each measure represented as Mean±Standard error, Honey bees treatment condition: CL: Control exposed to light, TL: Treated exposed to light and TD: Treated kept in dark | ||||
Genotoxic effect of chlorophyllin derivatives on honey bee: Gut cells of honey bees fed on Cu or Mg-chlorophyllin for three days were used for quantitative analysis of DNA damage by comet assay and expressed as tail length (TL) (μm), DNA tail% and the tail moment. The midgut cell nuclei with tail-like extension was an indication for DNA damage while the intact ones were almost rounded (Fig. 4a). The obtained TL (μm) in Cu-chlorophyllin fed bees in light (TL: 3.26±0.424 μm) and dark condition (TD: 2.56±0.424 μm) showed a significant increase compared with the control group (Fig. 4b).
Fig. 4(a-c): |
Different cell damage stages in the comet assay in Apis mellifera worker after treatment with (a) Chlorophyllin derivatives and exposed to sunlight, (b and c) Comet parameters in the adult Apis mellifera gut cells after 3 days fed with 10–5 M L–1 copper or 3×10–3 magnesium chlorophyllin CL: Control light, TL: Treated light and TD: Treated dark groups, *,**Represent significant differences as compared to the CL group at p<0.01 and p<0.000, respectively |
Also, the DNA damage measured as (DNA% in Tail) presented a clear and significant indication of genotoxicity of chlorophyllin derivatives. The percentage of DNA damage was 23.73±1.65 and 16.34±1.43 in the bees’ gut cells fed on Cu-chlorophyllin and Mg-chlorophyllin which were exposed to sunlight (TL), Table 2 and Fig. 4c. The minimum percent of DNA in the tail was measured in sugar-fed bees exposed to light, CL (12.86±2.44). Under the dark condition, the Cu-chlorophyllin fed bees showed DNA damage (17.92±1.89) more than the Mg-chlorophyllin fed bee (13.67±1.49) under Table 2 and Fig. 4c.
DISCUSSION
All aerobic organisms possess antioxidant systems, which function to prevent oxidative damage of ROS resulting from internal metabolism or due to exposure to insecticides including organophosphates, carbamates and pyrethroids7,11. The present study was aimed to assess the oxidative stress and genotoxicity effects in A. mellifera under the influence of Cu or Mg-chlorophyllin. The TAC of bee body homogenate was significantly increased upon feeding on 10–5 M L–1 Cu-chlorophyllin for three days under light and dark conditions compared to the control and field-collected groups (Table 1 and Fig. 1a). In the contrast, the Mg-chlorophyllin fed bees showed a little increase in TAC compared to the control group and it was almost the same TAC measured in the TD and FC groups (Table1 and Fig. 1b). Similarly, Nicewicz et al.35 observed a higher TAC (enzymatic and non-enzymatic antioxidant) in the fat body of rural bees. The increased TAC met the elevation of oxidative stress upon feeding on chlorophyllin and thus the antioxidant capabilities36. The SOD enzyme is one of the antioxidant protection systems against Reactive Oxygen Species (ROS)37,38, which increased due to exposure to many agrochemicals (such as pesticides, herbicides, extra) and heavy metals39-41. SOD activity was significantly increased in Cu-chlorophyllin fed bees and its highest activities were on the first day of feeding both in TL (0.32±0.04663 U μg–1 protein) and TD (0.35±0.0409 U μg–1 protein) groups (Table 1 and Fig. 2a). These results were inconsistent with Bernardes et al.8, who have observed a significant increase in CAT and SOD activities of the stingless bee Partamona helleri exposed to CuSO4 (LC50 = 142.95 μg mL–1). Also, CAT and SOD increased in the foragers of P. helleri that were exposed to the insecticide fipronil41, indicating that different agrochemicals can induce oxidative stress on these bees.
GST is a multifunctional enzyme that works to neutralize toxic compounds by conjugating them to glutathione for removal from cells42-45. So, GST activity in Cu-chlorophyllin fed bees referred to the metabolic actions against both the chlorophyllin and copper elements parts. These results agreed with Nikolić et al.46, who recorded that an increase in Cu and Cd concentration added to sucrose fed to bees caused an increase of three GST activities and expression level Furthermore, the measured GST activity in the field-collected bees was higher than that in the Mg-chlorophyllin fed bees which might reflect the harmful substances such as pesticides and xenobiotics the bees were exposed in the field. These results agreed with Orčić et al.47, who indicated that SOD and GST activity increased in summer workers compared to winter worker bees.
Animal cells exposed to ROS that are released due photopesticide activity have shown a different level of cellular compartment damage48,49. The single-cell electrophoresis, comet assay is used for studying environmental pollution risk at the level of genetic materials50,51. There is no information on the effects of chronic exposure to photo-pesticides on honey bees. This is, to our knowledge, the first study that utilized comet assay to reveal genotoxicity of photo-pesticides on worker forage bees. The DNA damage measured as percent of Tail DNA was significantly higher in gut cells of forage bee fed on Cu-chlorophyllin compared to Mg-chlorophyllin. This damage was also highly and significantly correlated with the accumulation of chlorophyllin derivatives in the bee body and can be reduced through the active antioxidant system (high efficiency of release dynamic) of honey bees which decreased the photooxidation stress of Cu-chlorophyllin and Mg-chlorophyllin by reducing their accumulated concentration in two days maximum. This result may be due to Cu metal which produces OH that interacts with cellular redox leading to ROS formation and oxidative DNA damage52,53. Also, the DNA repair system is reduced by the free metal51-55. This study presented an assessment of domesticated honey bee response to photo-pesticide exposures which could be critical to determine non-target pesticide impacts on such economically important insects.
CONCLUSION
The finding of this research recommended chlorophyllin derivatives field application to control pest insects such as mosquitoes and lepidopteran larvae without affecting the beneficial insect such as a honey bee. We are preparing to study the effect of the same chlorophyllin derivatives on other carnivores and parasitoids insects. The overall aim is to recover the natural balance of organisms destroyed by human activities.
SIGNIFICANCE STATEMENT
The applied objective of this study is to use a safe and quick method to assess the expected toxicity of chlorophyll derivatives, photosynthetically, before recommending its use in the open field on large scale. This study will help researchers to use chlorophyllin derivatives in the field control of pests within the safe concentration for honey bees, as there is not a sufficient number of studies on the environmental toxicity of these compounds on beneficial organisms. Thus a new theory about biosafety can be followed.
| Supplemented Table 1: | Effect of experimental conditions (EC) on TP and TAC in adult Apis mellifera body homogenate after treated with 10–5 M L–1 copper and 3×10–3 M L–1 magnesium chlorophyllin for different times | |||||||
Photosensitizer |
Time (days) |
Factor |
Sum of squares |
df |
Mean square |
Fcalculated |
p-value |
|
| TP | Cu |
First |
EC |
1.309 |
3 |
0.436 |
15.305 |
0.001 |
Error |
0.228 |
8 |
0.028 |
|||||
Total |
1.537 |
11 |
||||||
Second |
EC |
2.636 |
3 |
0.879 |
68.256 |
0.000 |
||
Error |
0.103 |
8 |
0.013 |
|||||
Total |
2.739 |
11 |
||||||
Third |
EC |
1.048 |
3 |
0.349 |
18.456 |
0.001 |
||
Error |
0.151 |
8 |
0.019 |
|||||
Total |
1.199 |
11 |
||||||
Mg |
First |
EC |
0.577 |
3 |
0.192 |
5.886 |
0.02 |
|
Error |
0.262 |
8 |
0.033 |
|||||
Total |
0.839 |
11 |
||||||
Second |
EC |
1.039 |
3 |
0.346 |
31.915 |
0.000 |
||
Error |
0.087 |
8 |
0.011 |
|||||
Total |
1.126 |
11 |
||||||
Third |
EC |
1.313 |
3 |
0.438 |
25.559 |
0.000 |
||
Error |
0.137 |
8 |
0.017 |
|||||
Total |
1.45 |
11 |
||||||
| TAC | Cu |
First |
EC |
4.543 |
3 |
1.514 |
338.43 |
0.000 |
Error |
0.036 |
8 |
0.004 |
|||||
Total |
4.579 |
11 |
||||||
Second |
EC |
5.198 |
3 |
1.733 |
272.835 |
0.000 |
||
Error |
0.051 |
8 |
0.006 |
|||||
Total |
5.248 |
11 |
||||||
Third |
EC |
2.359 |
3 |
0.786 |
23.665 |
0.000 |
||
Error |
0.266 |
8 |
0.033 |
|||||
Total |
2.625 |
11 |
||||||
Mg |
First |
EC |
0.211 |
3 |
0.07 |
42.545 |
0.000 |
|
Error |
0.013 |
8 |
0.002 |
|||||
Total |
0.224 |
11 |
||||||
Second |
EC |
0.599 |
3 |
0.2 |
201.465 |
0.000 |
||
Error |
0.008 |
8 |
0.001 |
|||||
Total |
0.607 |
11 |
||||||
Third |
EC |
0.745 |
3 |
0.248 |
254.769 |
0.000 |
||
Error |
0.008 |
8 |
0.001 |
|||||
Total |
0.753 |
11 |
||||||
Measured biochemical parameters: TP: Total protein concentration in μg μL–1 and TAC: Total antioxidant capacity in mM L–1. p>0.05: Insignificant effect, p<0.000, p<0.01 and p<0.05: Significant effect |
||||||||
| Supplemented Table 2: | Activities of GST and SOD in adult Apis mellifera body homogenate after treated with 10–5 M L–1 copper and 3×10–3 magnesium photosensitizer chlorophyllin for different times | |||||||
Photosensitizer |
Time (days) |
Factors |
Sum of squares |
df |
Mean square |
Fcalculated |
p-value |
|
| GST | Cu |
First |
First |
1.196 |
3 |
0.399 |
10.665 |
0.004 |
Error |
0.299 |
8 |
0.037 |
|||||
Total |
1.495 |
11 |
||||||
Second |
EC |
4.917 |
3 |
1.639 |
53.779 |
0.000 |
||
Error |
0.244 |
8 |
0.030 |
|||||
Total |
5.161 |
11 |
||||||
Third |
EC |
2.910 |
3 |
0.970 |
24.467 |
0.000 |
||
Error |
0.317 |
8 |
0.040 |
|||||
Total |
3.228 |
11 |
||||||
Mg |
First |
EC |
1.494 |
3 |
0.498 |
5.369 |
0.026 |
|
Error |
0.742 |
8 |
0.093 |
|||||
Total |
2.236 |
11 |
||||||
Second |
EC |
1.576 |
3 |
0.525 |
3.719 |
0.061 |
||
Error |
1.130 |
8 |
0.141 |
|||||
Total |
2.706 |
11 |
||||||
Third |
EC |
1.988 |
3 |
0.663 |
24.659 |
0.000 |
||
Error |
0.215 |
8 |
0.027 |
|||||
Total |
2.203 |
11 |
||||||
| SOD | Cu |
First |
EC |
0.125 |
3 |
0.042 |
18.12 |
0.001 |
Error |
0.018 |
8 |
0.002 |
|||||
Total |
0.143 |
11 |
||||||
Second |
EC |
0.032 |
3 |
0.011 |
4.198 |
0.046 |
||
Error |
0.02 |
8 |
0.003 |
|||||
Total |
0.052 |
11 |
||||||
Third |
EC |
0.108 |
3 |
0.036 |
12.77 |
0.002 |
||
Error |
0.023 |
8 |
0.003 |
|||||
Total |
0.131 |
11 |
||||||
Mg |
First |
EC |
0.015 |
3 |
0.005 |
1.912 |
0.206 |
|
Error |
0.02 |
8 |
0.003 |
|||||
Total |
0.035 |
11 |
||||||
Second |
EC |
0.015 |
3 |
0.005 |
1.922 |
0.205 |
||
Error |
0.02 |
8 |
0.003 |
|||||
Total |
0.035 |
11 |
||||||
Third |
EC |
0.015 |
3 |
0.005 |
2.532 |
0.130 |
||
Error |
0.015 |
8 |
0.002 |
|||||
Total |
0.03 |
11 |
||||||
Measured biochemical parameters: SOD: Superoxide dismutase activity in U μg–1 protein and GST: Glutathione S-transferase activity in U μg–1 protein. p>0.05: Insignificant effect, p<0.000, p<0.01 and p<0.05: Significant effect |
||||||||
| Supplemented Table 3: | Comet parameter in the adult gut cell of Apis mellifera after fed with 10–5 M L–1 copper and 3×10–3 M L–1 magnesium chlorophyllin for three days | ||||||
Photosensitizer |
Factor |
Sum of squares |
df |
Mean square |
Fcalculated |
p-value |
|
| TL | Cu |
EC |
6.313 |
2 |
3.156 |
22.801 |
0.002 |
Error |
0.831 |
6 |
0.138 |
||||
Total |
7.143 |
8 |
|||||
Mg |
EC |
0.685 |
2 |
0.343 |
1.888 |
0.231 |
|
Error |
1.089 |
6 |
0.182 |
||||
Total |
1.775 |
8 |
|||||
| TM | Cu |
EC |
0.072 |
2 |
0.036 |
1.395 |
0.318 |
Error |
0.155 |
6 |
0.026 |
||||
Total |
0.228 |
8 |
|||||
Mg |
EC |
0.764 |
2 |
0.382 |
53.042 |
0.000 |
|
Error |
0.043 |
6 |
0.007 |
||||
Total |
0.807 |
8 |
|||||
| DNA (%) | Cu |
EC |
176.88 |
2 |
88.44 |
31.815 |
0.001 |
Error |
16.679 |
6 |
2.78 |
||||
Total |
193.559 |
8 |
|||||
Mg |
EC |
19.725 |
2 |
9.862 |
2.895 |
0.132 |
|
Error |
20.437 |
6 |
3.406 |
||||
Total |
40.162 |
8 |
|||||
CL: Control light, TL: Treated light and TD: Treated, p>0.05: Insignificant effect, p<0.000, p<0.01 and p<0.05: Significant effect |
|||||||