The project was funded by the National Science Centre (Poland) based on the decision number 2015/17/B/NZ9/03607.

GENERAL DESCRIPTION

As pollinator insects, bees play an important role in the ecosystem and economy. As many as 75% of crop plant species worldwide are pollinated by bees and other insects. Since 2003, there has been a rapid decrease in bee abundance in Europe and economic impact on crop productivity. Bee colony collapse results in shortages of basic commodities such as oil products or fruits and vegetables and in huge economic losses in the production of honey and bee products. This dangerous phenomenon is thought to be associated with e.g. bee diseases, with nosemosis as the most prevalent problem. Nosemosis is a contagious disease of adult bees, well known in beekeeping and veterinary medicine as one of the invasive diseases of the Apis mellifera honeybee. In our climate, nosemosis is caused by intracellular parasites representing microsporidia, i.e. Nosema apis and Nosema ceranae. In 2008, intense infestation with Nosema spp. was reported from over half of apiaries in Poland. The disease causes anatomical and physiological changes in bees, affecting the fitness of the entire colony. Bees die within a very short time, i.e. 8-10 days after infection. Death is a result of the development of Nosema spores (microsporidia) in the midgut epithelium and the resulting high level of hunger leading to energy stress. Microsporidia are extremely resistant to adverse environmental conditions and can survive for several years retaining germinability and ability to infect healthy hosts. Hence, nosemosis control is difficult and the search for new drugs against the disease proves extremely important. Until now, no effective drugs against nosemosis, which would also be harmless to bees, has been developed. The antibiotic fumagillin that was used from the 50s of the last century has been withdrawn in the European Union. Fumagillin does not exhibit 100% effectiveness; moreover, many reports indicate development of resistance to the antibiotic in Nosema spp.

As shown by preliminary investigations conducted by our team, porphyrins can become the basis for development of future formulations for nosemosis control. Porphyrins, i.e. the so-called “pigments of life” constitute a group of extremely important compounds that play a key role in essential life processes. They are involved in many biochemical processes, e.g. oxygen transport and storage [hemoglobin/myoglobin], biocatalytic oxidation, and detoxification of organic compounds (peroxidase and cytochrome P-450), electron transfer (cytochromes b and c), and photosynthesis (chlorophyll). These compounds appear to have high application potential that can be used in many areas, e.g. chemistry, biotechnology, and medicine. In particular, the interactions of porphyrin with light arouse great interest due to the possibility of using photochemical properties of porphyrins in photodynamic therapy targeted at destruction of tumour tissues and control of pathogenic microorganisms.

Scientific literature provides little information about the light-independent antimicrobial properties of porphyrins. Our previous laboratory research showed that these compounds contribute to substantial reduction of the number of spores detected in nosemosis-infected bees. The use of these unique, newly discovered properties requires elucidation of the mechanisms of the antimicrosporidial activity of porphyrinoids and investigations of the effect of these compounds on living organisms. Therefore, the aim of the project is to analyse potential mechanisms involved in inhibition of nosemosis development by porphyrinoid compounds and to characterise their novel biological properties.

In the implementation of the proposed research objective, we are planning to carry out synthesis of amide derivatives of protoporphyrin IX (characterised by increased bioavailability) and to use commercial water-soluble porphyrins with various functional side groups. The tested compounds will be administered the form of sugar syrup to healthy and Nosema microsporidia-infected honeybees, in which the level of infestation will be monitored in the so-called cage tests. We intend to determine the level of porphyrinoid bioactivity towards nosemosis based on the ability of the compounds to reduce the number of spores and bee survivability. We want to analyse the effects of the selected compounds on the parameters of the bees’ immune system as well as their direct impact on Nosema microsporidia and secretory and reproductive functions in bees. An important task of the project is to analyse correlations between the ability of porphyrinoids to destroy spores, their photochemical stability, and doses and bee survival and fitness (in apiary tests).

The proposed investigations will provide guidelines for rational synthesis of derivatives with biological properties relevant for Nosema spp. control. In turn, elucidation of the mechanisms of the action of porphyrinoids will be important in future studies on the optimisation of the conditions of using these compounds in combating these parasites in honeybees and other animals infected by microsporidia. Since acquisition of one-third of all food products is dependent on pollination by bees and other pollinators, the prospective results of the project may have great importance to economy. In this context, it is worth recalling the well-known thought attributed to Albert Einstein: “If the bee disappeared off the face of the Earth, man would only have four years left to live. No more bees, no more pollination, no more plants, no more animals, no more man”.

Research project objectives/hypothesis

The aim of the project is to elucidate the mechanism of the bioactivity of porphyrin compounds, observed by our team (patent application no. P.408774), towards nosemosis, i.e. a dangerous disease of bees infected by intracellular microsporidia from the genus Nosema spp. We intend to analyse potential mechanisms involved in inhibition of nosemosis development by these compounds: their direct impact on Nosema microsporidia and stimulation of immune response in bees. Furthermore, we are planning to characterise the biological properties of porphyrinoids and determine the correlations between their chemical structure and “anti-microsporidial” activity. We will take into account the impact of external factors and biochemical and physical parameters on their biocidal efficiency and biological function of bees.

Research project methodology

To implement the presented research aim, we are planning to use amide derivatives of protoporphyrin IX (PPIX) as well as commercial water-soluble porphyrins with diverse side functional groups. Synthesis of PPIX derivatives (characterised by increased bioavailability) will be carried out in in the 15TH IChO PAN and based on binding natural endo- and exogenous carboxylic amino acids or short peptides to its groups. The analysed compounds and fumagillin will be administered as a control antifungal agent in the form of sugar syrups to healthy honeybees and individuals artificially infected with Nosema spp. microsporidia; the latter bees will be monitored for 14 days post-infection to assess the infection rate. The level of porphyrinoid bioactivity in inhibiting nosemosis will be determined on the basis of their ability to reduce the number of spores in bees infected by microsporidia and bee survivability. The investigations will be conducted in laboratory conditions with the use of cage tests. The aim of the task is to select the most effective compounds and check to what extent the presence of a complexed metal ion or the type of functional groups and their position in the porphyrin ring influence their bioactivity. Simultaneously, we intend to determine the physico-chemical stability of porphyrinoids in sugar solutions based on the analysis of absorption, excitation, and emission spectra. This will allow verification of the biological activity of the tested compounds in terms of their stability and determination of the impact on bee survivability and biological functions. Moreover, we are going to analyse the effects of the action of these compounds on the immune system in bees. We will determine antimicrobial properties of hemolymph, expression of selected immune genes, and the level of activity of proteins important for the immune system in bees (phenol oxidase, lysozyme). In order to assess the direct effect of porphyrinoids on microsporidia, spores isolated from infected bees will be incubated in vitro with selected compounds. Next, we will evaluate the ability of spores to infect and develop in bee guts and assess their morphology using scanning electron microscopy. Additionally, in microsporidia, we will analyse the mechanism of methionine aminopeptidase type-2 inhibition, which is responsible for the antifungal activity of fumagillin, an antibiotic used recently in microsporidiasis treatment in bees and humans. Another important task in the project is to analyse interrelations between the ability of porphyrinoids to destroy spores, their photochemical stability, doses, and bee survivability and fitness. We are planning to examine (in apiary tests) the effect of selected biochemical-physical parameters on the secretory and reproductive functions in bees by assessment of the ability of worker bees to produce royal jelly and measurements of the brood surface in nucleus colonies. Moreover, we will examine the presence of porphyrinoids in dissected throat and salivary glands as well as in the fat body and hemolymph. All results and observed relationships observed in the tasks will be used for determination of the most probable mechanism of the anti-microsporidial activity of the tested porphyrinoids.

We plan to carry out the following research tasks in a period of three years:

  1. Synthesize the amide derivatives of protoporphyrin IX
  2. Screening of porphyrinoid compounds of different chemical structure for their bioactivity in inhibiting the progress of nosemosis in infected bees. Study the effects of porphyrinoids on healthy bees;
  3. Analyze the results of task 2 and determine the relationship between the chemical structure of porphyrinoids and theirantimicrosporidial activity and physico-chemical stability in sugar solutions;
  4. Analysis of the effect of porphyrinoids on the parameters of the immune system of bees and on the morphology and degree of infectivity of Nosema sp. spores;
  5. Carry out experiments to determine the impact of the biochemical and physical parameters of the selected porphyrinoids on their therapeutic effectiveness and the biological functions of the bees using apiary tests.

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