Komparativno ispitivanje prisustva uzročnika i bolesti pčelinjeg legla tradicionalno i savremeno gajenih pčelinjih društava uz analizu uticaja nekih bioloških i antropogenih faktora

Abstract

Doktroska disertacija

Štetoĉine i patogeni predstavljaju najĉešće uzroke gubitaka pĉelinjih društva. Ameriĉka truleţ (AFB) je bolest pĉelinjeg legla, uzrokovana bakterijom Paenibacillus. larvae, White (1906), moţe se smatrati glavnom pretnjom po zdravlje pĉela, s obzirom da je reĉ o panzootskoj bolesti koja se vrlo brzo širi, ne samo iz košnice u košnicu, sa pĉelinjaka na pĉelinjak iz regije u regiju, već i iz drţave u drţavu i šire. Evropska truleţ je bakterijska bolest pĉelinjeg legla uzrokovana sa više bakterisjkih vrsta gde dominira Melissococcus plutonius. Ova bolest je jako raširena i predstavlja veliki problem u pĉelarstvu smanjujući proizvodne rezulte pĉelinjih zajednica. Kreĉno leglo (CHB) je infekcija izazvana gljivicom Ascosphaera apis izazivajući gubitke komercijalno gajenih pĉelinjih zajednica, naroĉito u kombinaciji sa vrstama mikrosporidija roda Nosema i virusom mešinastog legla. Nozemoza je najĉešća bolest meĊu odraslim pĉelama uzrokovana mirkosporidijama Nosema apis i N. ceranae i ĉesto dovodi do ekonomskih gubitaka u pĉelarstvu. Virusne infekcije pĉela, koje su već odavno dostigle razmere panzootije, ugroţavaju zdravlje pĉela i predstavljaju konaĉne egzekutore pĉelinjih zajdnica. Pĉelinji krpelj Varroa destructor je glavni vektor skoro svih, a naroĉito virusnih infekcija pĉela, koje su postale ozbiljan problem ne samo za komercijalno gajene, već i za pĉelinje zajednice u divljini, zahvaljujući upravo krpelju V. destructor kao fiziĉkom i biološkom vektoru Na osnovu svega navedenog jasno je da je zdravstveno stanje društava u komercijalnom pĉelarstvu izloţeno velikim rizicima, a njihovo ispitivanje i tretman predstavljaju veliki izazov kako za istraţivaĉe tako i za pĉelare. Bolesti odraslih pĉela i pĉelinjeg legla u komercijalnom pĉelarstvu najĉešće su posledica energetskog stresa, nastalog zbog neadekvatne prihrane pĉelinjih zajednica, prvenstveno prevelikom upotrebom sećernog sirupa. U osnovi energetskog stresa je oksidativni stres pĉelinjih zajednica koji se moţe definisati kao disbalans izmeĊu proizvodnje reaktivnih oblika kiseonika i antioksidativne odbrane. Reaktivni oblici kiseonika (ROS) negativno utiĉu na ćelijske funkcije i stvaraju se tokom oksidoredukcionih metaboliĉkih procesa u ćeliji. Oni su ukljuĉeni u regulaciju razliĉitih mehanizama, intercelularne signalizacije, a imaju i baktericidno dejstvo. Medonosne pĉele, kao i ostale ţivotinje, razvile su niz enzimskih mehanizama kojima se odupiru oksidativnom stresu, uklanjajući slobodne radikale. U ovim procesima vaţnu ulogu umaju: superoksid dismutaza (SOD), enzim prisutan u citosolu i mitohondrijama; katalaza (CAT) prisutna u peroksizomima glutation S-transferaza (GST), glutation S transferaza (GST), peroksidaza i tireodoksin/tireoreduktazni sistem. Imunitet pĉela, kao i imunitet u opšte, obuhvata kompleksan sistem koji ima cilj da obezbedi oĉuvanje zdravlja kao i opstanak organizma na osnovu mnogih mehanizama odbrane od patogena i drugih štetnih noksi (fiziĉkih i hemijskih). Medonosna pĉela svoj socijalni imunitet ostvaruje uz pomoć bihejviorlanih mehanizama koji pomaţu zajdnici u odbrani od patogena. Ulaganje u individualni imunitet ima visoku materijalnoenergetsku cenu za društvo, pa su udruţivanjem pĉela (razvoj socijlanog ustrojstva) uspostavljeni zajedniĉki socijalni mehanizmi odbrane koji omogućavaju pĉelinjem društvu efikasniju i ekonomiĉniju borbu protiv agenasa spoljašnje sredine. Za socijalni imunitet pĉela od znaĉaja je enzim glukoza oksidazi (GOX). Ovaj enzim je produkt pĉelinjih egzokrinih ţlezda, a uloga mu je u „konzervaciji― pĉelinjuh proizvoda, meda i perge, tako spreĉavajući njihovo kvarenje. GOX uĉestvuje u procesu katalize -D glukoze do glukonske kiseline i vodonik-peroksida. Vodonik-peroksid ima antiseptiĉka svojstva, tako doprinoseći dodatnoj zaštiti i socijalnom imunitetu pĉela. Cilj istraţivanja je bio da se utvrdi: da li izmeĊu tradicionalno i savremeno gajenih pĉelinjih društava postoje razlike u pogledu prisustva patogena i pojave bolesti pĉelinjeg legla, uz analizu bioloških (ekspresija GOX gena i faktora oksidativnog stresa) i antropogenih faktora (menadţment u pĉelarstvu) na pojavu praćenih bolesti pĉelinjeg legla i odraslih pĉela. Terenski deo istraţivanja je obavljen na prostoru Pešterske visoravni (opština Sjenica, Srbija) na 144 asimptomatske pĉelinje zajednice. Uzorci odraslih pĉela i pĉelinjeg legla su uzeti iz savremenih (DB) košnica i tradicionalnih košnica „trmki―, kako bi se utvrdila zastupljenost patogena legla (Paenibacilus larvae, Melissococus plutonius, Ascosphera apis, virus mešinastog legla, tripanozoma Lotmaria passim i Crithidia mellificae) i odraslih pĉela (virus akutne paralize pĉela, virus hroniĉne paralize pĉela, virus deformisanih krila pĉela, tripanozoma Lotmaria passim i Crithidia mellificae,mikrosporidija Nosema apis i Nosema ceranae ) kao i za utvrĊivanje nivoa oksidativnog stresa analizom aktivnosti enzima: SOD, CAT, GST i koncentracije malondialdehida (MDA). TakoĊe, iz istih košnica uzeti su uzorci za procenu ekspresije GOX gena kao parametra socijlanog imuniteta. Detekcija patogena je obavljena izolacijom bakterija kao i upotrebom PCR i real-time PCR, u skladu sa standardima OIE. Rezultati istraţivanja su pokazali da je meĊu komercijalno gajenim društvima P. larvae bio zastupljen u 16,67% uzoraka, A. apis bio prisutan u 15,83% uzorka, dok je SBV detektovan u 96,67% uzoraka. MeĊutim u leglu pĉela gajenih u tradicionalnim trmka košnicama naĊen je samo SBV u 33,33% uzoraka. Dalja istraţivanja su utvrdila da su kod odraslih pĉela u komercijalno gajnim društvima bili signifikantno (p<0,001) više zastupljeni virusi: ABPV, CBPV i DWV (83,33%, 100,00%, 100,00%, istim redom) u odnosu na društva iz trmki gde je procenat ovih virusa iznosio 33,33% za svaki virus. TakoĊe, sva komercijalno gajena društva bila su inficirana makar jednim od praćenih patogena, za razliku od tradicionalno gajenih pĉela u trmkama meĊu kojima je 66,66% bilo bez patogena. Rezultati su otkrili znaĉajne razlike u aktivnosti CAT, GST i SOD (p<0,01) i koncentracije MDA (p<0,002) izmeĊu komercijalnih i tradicionalnih društava što vodi zakljuĉku da u društvima gajenim u trmkama postoji manji oksidativni stres što je rezultiralo i manjom zastupljenošću svih praćenih patogena. U uzorcima komercijalno gajenih pĉela zastupljenost parazita L. passim i N. ceranae bila je znaĉajno veća (p<0,05; p<0,01, respektivno) u odnosu na uzorke iz tradicionalnih košnica, dok C. mellificae i N. apis nisu detektovane ni u jednom uzorku. Treba istaći, da je prvi put detektovana tripanozoma L. passim u leglu iz obe grupe ispitivanih košnica, komercijalnih i tradicionalnih, pri ĉemu je njena zastupljenost u leglu znaĉajno manja (p<0,01) nego u odraslim pĉelama u komercijalnim društvima, dok se kod tradicionalno gajenih pĉela zastupljenost ove tripanozome izmeĊu odraslih pĉela i legla nije znaĉajno razlikovala. Kod komercijalno gajenih pĉela utvrĊen je znaĉajno veći nivo iRNK za gen GOX (p<0,01) u odnosu na tradicionalno gajena društva, što je verovatno posledica pojaĉane potrebe prvopomenutih da ojaĉaju socijalni imunitet. Komercijalna društva su u odnosu na tradicionalno gajena bila pod većim oksidativnim stresom, a imala su i veću opterećenost pĉelinjim patogenima, kao i veći nivo transkirpcije GOX gena, što je verovatno posledica ĉestih pĉelarskih manipulacija i uznemiravanja komercijalnim društvima, kao i njihovo forsirano ekonomsko iskorišćavanje. Sve to vodi zakljuĉku da populacija pĉela koja se uzgaja na tradicionalan ima veći kapacitet samoodrţanja i otpornija na pĉelinje patogene, energetski (i oksidativni) stres, a da antropogeni faktori, odnosno pĉelarski postupci, imaju negativan uticaj na zdravlje komercijalno gajenih pĉela.

Pests and pathogens are the most common causes of honey bee colony losses. American foulbrood (AFB) is a disease of honey bee brood, caused by the bacterium Paenibacillus larvae, White (1906), can be considered a major threat to bee health, as it is a panzootic disease that spreads very quickly, not only from hive to hive, from apiary to apiary, from region to region, but also from the state to the state and beyond. European foulbrood is a bacterial disease of honey bee brood caused by several bacterial species dominated by Melissococcus plutonius. This disease is widespread and poses a major problem in beekeeping by reducing the production results of honey bee colonies. Chalkbrood disease (CHB) is an infection caused by the fungus Ascosphaera apis causing losses of commercially reared bee colonies, especially in combination with microsporidia species of the genus Nosema sp. and a Sacbrood virus (SBV). Nosemosis is the most common disease among adult bees caused by Nosema apis and N. ceranae, and often leads to economic losses in beekeeping. Viral infections of bees, which have long since reached panzootic proportions, endanger the health of bees and present ultimate executors of bee colonies. Varroa destructor mite is the main vector of almost all, especially viral infections, which have become a serious problem not only for commercially reared, but also for honey bees in the wild, thanks to the mite V. destructor as a physical and biological vector. It is clear that the health status of colonies in commercial beekeeping is exposed to great risks, and their examination and treatment represent a great challenge for both researchers and beekeepers. Diseases of adult bees and bee brood in commercial beekeeping are most often a consequence of energetic stress, caused by inadequate feeding of bee colonies, primarily by excessive use of sugar syrup. The basis of energetic stress is oxidative stress of bee colonies, which can be defined as an imbalance between oxidants and antioxidants in favor of the former. Reactive oxygen species (ROS) negatively affect cellular functions and are generated during oxido-reduction metabolic processes in the cell. They are involved in the regulation of various mechanisms, intercellular signaling, and exert bactericidal activity. Honey bees, like other animals, have developed a number of enzymatic mechanisms that resist oxidative stress, removing free radicals. An important role in these processes is played by: superoxide dismutase (SOD), catalase (CAT) in peroxisomes, ascorbate peroxidase, glutathione S transferase (GST) peroxidase, and the thyrodoxin / thyroreductase system. Immunity of bees, as well as immunity in general, includes a complex system that aims to ensure the preservation of health and survival of the organism based on many defense mechanisms against pathogens and other harmful noxa (physical and chemical). The honey bee achieves its social immunity with the help of behavioral mechanisms that help the community defend itself against pathogens. Investing in individual immunity has a high material and energy price for society, so the association of bees (development of social structure) established joint social defense mechanisms that enable bee society to fight more efficiently and economically against environmental agents. The enzyme glucose oxidase (GOX) is important for the social immunity of bees. This enzyme is a product of bee exocrine glands, and its role is in the "conservation" of bee products, honey and bee bread, thus preventing their spoilage. GOX participates in the process of catalysis of -D glucose to gluconic acid and hydrogen peroxide. Hydrogen peroxide has antiseptic properties, thus contributing to additional protection and social immunity of bees. The aim of the research was to determine: whether there are differences between traditional and modern bee colonies in terms of the presence of pathogens and the occurrence of bee brood diseases, with the analysis of biological (expression level of the GOX gene and oxidative stress factors) and anthropogenic factors (beekeeping management) on monitored bee brood and adult bee diseases. The field part of the research was performed in the area of the Peshterska plateau (Sjenica municipality, Serbia) on 144 asymptomatic bee communities. Samples of adult bees and bee brood were taken from modern (DB) hives and traditional "trmka" hives to determine the presence of brood pathogens (Paenibacilus larvae, Melissococus plutonius, Ascosphera apis, Sacbrood virus, trypanosomatids Lotmaria passim and Crithidia mellificae) and adult bees (acute bee paralysis virus, chronic bee paralysis virus, deformed bee wings virus, trypanosomatids Lotmaria passim and Crithidia mellificae, microsporidia Nosema apis and Nosema ceranae) as well as for determination of oxidative stress levels by analysis of activity of enzymes: SOD, CAT, GST and concentration of malondialdehyde (MDA). From the same hives, samples were taken to estimate the expression the GOX gene as a parameter of social immunity. Pathogen detection was performed by bacterial isolation as well as using PCR and real-time PCR, in accordance with the OIE standards. The results showed that among commercially reared colonies, P. larvae, was represented in 16.67% of samples, A. apis in 15.83%, while SBV was detected in 96.67% of samples. However, in the brood from colonies raised in traditional hives, only SBV in the percentage of 33.33% of samples was found. Further research found that in adult bees from commercially reared colonies, viruses ABPV, CBPV and DWV (83.33%, 100.00%, 100.00%, respectively), were significantly (p <0.001) more represented compared to adult bees from „trmka― hives, where the percentage of these viruses was 33.33% for each. Also, all commercially reared colonies were infected with at least one of the monitored pathogens, in contrast to traditionally reared bees, of which 66.66% were pathogen-free. The results revealed significant differences of activities of CAT, GST and SOD (p<0.01) and MDA concentration (p<0.002) between commercial and traditional colonies, which leads to the conclusion that colonies grown in „trmka― hives had less oxidative stress level, which resulted in lower presence of all monitored pathogens. In samples of bees from commercially bred colonies, the prevalence of parasites L. passim and N. ceranae was significantly higher (p<0.05; p<0.01, respectively) compared to samples from traditional hives, while C. mellificae and N. apis were not detected in any sample. It should be noted that L. passim was detected for the first time in a brood from both groups of examined hives, commercial and traditional, with its presence in the brood significantly lower (p<0.01) than in adult bees in commercial colonies, while in traditionally reared bees, the presence of this trypanosome did not differ significantly between adult bees and brood. In commercially bred bees, a significantly higher level of mRNA for the GOX gene was found (p<0.01) compared to traditionally bred colonies, which is probably a consequence of the increased need of the former to strengthen social immunity. Compared to traditional beekeeping, commercial colonies were under greater oxidative stress, and had a higher load of bee pathogens as well as a higher level of transcription for the GOX gene, which is probably due to frequent beekeeping and harassment of commercial colonies, as well as their forced economic exploitation. All this leads to the conclusion that the population of bees raised in the traditional way is more self-sustaining and resistant to bee pathogens, energetic (and oxidative) stress, and that the anthropogenic factors (beekeeping practices) have a negative impact on the health of commercially reared bees.