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Conclusions
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The 2D approach has been an immediate instrument to give a qualitative analysis of treated/ill samples compared to control samples. This analysis was used to investigate the effects of formic acid treatments in the APEnet project. A small quantity of spots was identified by mass spectrometry but one protein was particularly interesting. This protein was glutathione- S-transferase. Glutathione S-transferases (EC 2.5.1.18) are thought to play a physiological role in initiating the detoxification of potential alkylating agents including pharmacologically active compounds (Habig et al., 1974). These enzymes catalyze the reaction of such compounds with the -SH group of glutathione, neutralizing their electrophilic sites and rendering the products more water-soluble. In the future it would be interesting to continue this line of research investigating the enzymatic activity of glutathione-S-transferase in treated/control samples in order to discover if it increases or decreases following the treatments.
The 2D approach was also used to investigate the proteases present in samples infected with American foulbrood. In this case 2D zymography was used. At least 4 proteases were revealed in healthy prepupa samples and absent (P1, P2, P3) or partially present (P4) in diseased prepupa samples. The protein pattern of diseased prepupa samples was similar to the protein pattern of healthy pupae from infected hives. Using selective inhibitors of proteases, the proteases could be grouped in two families: the serine proteases or the cysteine proteases.
It was interesting to identify the proteases by mass spectrometry, although this technology used with the gel for zymography (using gelatin as a substrate) showed some problems.
Analysis with 2D zymography have also revealed a protease inhibitor (trypsin or α- chymotrypsin inhibitor) that was present in the healthy prepupa samples but was absent in diseased prepupa samples. We can suppose that this inhibitor is connected to the revealed proteases because in diseased prepupa samples the absence of this inhibitor is simultaneous with the absence of the proteases. The α-chymotrypsin inhibitor was investigated because the
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α-chymotrypsin is responsible for the prophenoloxidase activation in phenoloxidase for the production of melanin.
Regarding to the phenoloxidase and glucose oxidase activities, one and two spectrophotometer protocols were identified, respectively for the phenoloxidase and for the glucose oxidase in order to quantify the activity. Furthermore for each enzyme one protocol was identified with the activity staining method in order to quantify the phenoloxidase and glucose oxidase activities using polyacrylamide gels. The honey bee samples and the bee organs targets to use for the analysis have been identified. Regarding the glucose oxidase activity, the whole head showed a stronger activity than every kind of gland (mandibular and hypopharingeal glands) responsible for the royal jelly production. Regarding the phenoloxidase activity we used two types of samples: the head and the thorax (for the winter bees we also used the abdomen). For the analysis the nurse bee samples (6-12 days of age of image) and the forager bee samples (20-30 days of age of the imago) were preferred.
Phenoloxidase and glucose oxidase had in fact an increase correlated with the age of the honeybees, with a peak when the bee is nurse for the glucose oxidase and two peaks (nurse and forager bees) for the phenoloxidase.
Regarding the different analysis with treated/ill samples (adult or brood bees) the study revealed different phenoloxidase and glucose oxidase activities compared to the control samples (healthy or not treated samples).
About the investigation on treated samples (Amrine and apilifevar), 30 days after the treatment by beekeepers, we could see a return of the enzymatic activity (phenoloxidase and glucose oxidase) to the initial values before the treatment. We can suppose that the honeybees have detoxification processes that eliminate the effects of the treatments.
After a double stress represented by the infection with a parasite and a treatment, the honeybees presented a phenoloxidase or glucose oxidase activity frequently opposite or
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enhanced compared to a single stress scenario. According to Sadd B.M. and Schmid-Hempel P (2006) these effects could be a particular “memory” of the immune system. Although honey bees don’t have immune memory, for example they don’t have antibodies, after a stress the honey bees could be “prepared” to respond to a second stress.
A very strong treatment that caused in summer the death of a lot of honeybees in the productive season, did not have the same effect on the winter bees. Unexpectedly the winter bees survived to this treatment and the phenoloxidase and glucose oxidase activities showed significant changes in the enzymatic activity in response to the treatment.
It could be interesting to investigate the immune system of winter bees and the possibility of a stronger resistance of the winter bee immune system compared to the immune system of the honey bees of the productive season.
A small pilot project was made with the aim to understand if the phenoloxidase and the glucose oxidase activities change in function of the season. The goal could be to indentify the hypothetical ranges of the phenoloxidase and glucose oxidase activities in the different ages of the worker honey bees in order to use them as an instrument to analyse the well being of the colonies in an apiary. The limits of this project are represented by the fact that only one apiary (the experimental apiary of the Department of Veterinary Science) was analyzed during a period of time of only one year. It could be interesting to investigate at least three consecutive years to study the effects of the season on the phenoloxidase and glucose oxidase activities.
The changes in the phenoloxidase and glucose oxidase activities were attributed particularly to the apiary condition (weak or strong families), meteorological conditions (rain, temperature, etc.), to the tasks which the honey bees carry out in a particular month (brood cure, honey production, etc.) and the cares of the beekeepers (treatments, actions to avoid bee pathologies, etc.). Knowing that the honeybees can change phenotypically depending on the
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colony conditions, it could be interesting a second pilot project in order to study the reactions of the bee superorganism with regard to the phenoloxidase and glucose oxidase activities terms in strong stress conditions, for instance starvation, absence of the queen, continued rains, or weakening of the family.
The use of the glucose oxidase and its products (gluconic acid and hydrogen peroxide) against Paenibacillus larvae represents a new approach against this terrible pathology. It opens the
possibility to try to “reinforce” the immune system of the honeybees against pathogens.
This research could be further extended investigating the use of honey and the use of Gluconobacter oxidans, which has an enzyme with the same function of the glucose oxidase and which too produces gluconic acid, to reinforce the immune system.
