Molecular basis of honey bee (Apis mellifera, L.) response to heavy metal stress

Abstract

<p>Istraživanja&nbsp; u&nbsp; ovoj&nbsp; doktorskoj&nbsp; disertaciji&nbsp; su&nbsp; bila&nbsp; usmerena&nbsp; ka&nbsp; razumevanju<br />molekularnih&nbsp; mehanizama&nbsp; koji&nbsp; se&nbsp; aktiviraju&nbsp; kod medonosne pčele (<em>Apis mellifera, L</em>.)&nbsp; kao&nbsp; odgovor&nbsp; na&nbsp; stres&nbsp; izazvan&nbsp; jonima&nbsp; te&scaron;kih&nbsp; metala,&nbsp; &scaron;to&nbsp; zbog važnosti medonosne pčele kao opra&scaron;ivača&nbsp; ima&nbsp; poseban&nbsp; značaj&nbsp; i&nbsp; očekuje&nbsp; se da će doprineti očuvanju ove vrste. Istraživanja su bila podeljena u tri faze. U prvoj fazi za analize&nbsp; su&nbsp; kori&scaron;ćene populacije pčela sa lokaliteta sa različitim&nbsp; antropolo&scaron;kim uticajem&nbsp; i izmerena je koncentracija metala u pčelama, pergi i medu, kao i relativna genska ekspresija i aktivnost antioksidativnih enzima. U drugoj fazi&nbsp; pčele&nbsp; su&nbsp; u kontrolisanim laboratorijskim uslovima bile&nbsp; izložene subletalnim dozama jona te&scaron;kih&nbsp;metala&nbsp; (bakra,&nbsp; kadmijuma i olova), nakon čega&nbsp; su&nbsp; izmereni parametri koji ukazuju na redoks status i nivo oksidativnog stresa. Rezultati prve dve faze su pokazali&nbsp; da se ekspresija gena i aktivnost antioksidativnih enzima (superoksid dismutaze, katalaze i glutation&nbsp;<em> S</em>-transferaze)&nbsp; razlikuje u zavisnosti od stepena urbanizacije i industrijalizacije, dok je izlaganje bakru i kadmijumu u kontrolisanim uslovima u trajanju od 48 h dovelo samo do promene&nbsp; u ekspresiji gena&nbsp; i u većini slučajeva ekspresija je bila dozno zavisna od koncentracije metala.&nbsp; Olovo je uzrokovalo promene u koncentraciji glutationa i sulfhidrilnih grupa proteina, &scaron;to govori o tome da helacija olova može da bude prvi mehanizam odbrane od toksičnih efekata ovog metala.&nbsp; U trećoj fazi bioinformatičkom analizom je&nbsp; pronađen metalotionein medonosne pčele i ispitana je njegova funkcija u za&scaron;titi od toksičnih efekata jona te&scaron;kih metala.&nbsp; Utvrđeno je da pčele poseduju jedan&nbsp;gen za metalotionein, koji kodira mali protein sa regionima bogatim cisteinom za koje mogu da se vežu joni metala. Indukcija genske ekspresije metalotioneina medonosne pčele nakon izlaganja metalima i povećana tolerancija bakterija koje ekspresuju rekombinantni metalotionein na metale je potvrdila da metalotionein medonosne pčele ima ulogu u homeostazi bioelemenata i detoksikaciji potencijalno toksičnih metala.&nbsp; Dobijeni rezultati predstavljaju osnovu za buduća istraživanja uticaja&nbsp; jona te&scaron;kih metala na medonosnu pčelu i predstavljaju važan korak u sveobuhvatnoj proceni uticaja stresogenih faktora iz životne sredine na pčele.</p>

<p>Research in this doctoral thesis&nbsp; focuses&nbsp; on&nbsp; understanding the molecular mechanisms activated in the honey bee (Apis mellifera L.) as a response to stress caused by exposure to heavy metal ions. Because of the importance of honeybees as pollinators, this has special significance and is expected to contribute to the&nbsp; conservation of this species. Studies have been divided into three phases. In the first phase, bee populations from&nbsp; three localities under&nbsp; different anthropological influence were used and the concentrations of metals in the bees, honey and bee bread&nbsp; (perga), as well as relative gene expression and activity of antioxidant enzymes were measured. In the second phase, bees&nbsp; were exposed to sublethal doses of heavy metal ions (copper, cadmium and lead) under controlled laboratory conditions, after which parameters that indicate redox status and oxidative stress were determined. The results of the first two phases showed that&nbsp; gene expression and activity of antioxidant enzymes (superoxide dismutase, catalase, and glutathione&nbsp; S-transferase) varies depending on the degree of urbanization and industrialization, while exposure to copper and cadmium in controlled conditions for 48 h&nbsp; resulted only in a change in gene expression in the majority of cases, and the expression was dose-dependent on the concentration of the metal. Lead has caused changes in the concentration of glutathione and sulfhydryl groups of proteins, which indicates&nbsp; that chelation may be the first defense mechanism against&nbsp; the&nbsp; toxic effects of this metal. In the third stage, honeybee metallothionein was identified by bioinformatic analysis and its function in&nbsp; protection against the toxic effects of heavy metal ions was examined. It has been found that&nbsp; honeybees have one metallothionein gene, which encodes a small protein with cysteine-rich regions that may bind metal ions. The induction of metallothionein gene expression after exposure of honeybees to metals and increased tolerance of bacteria that express recombinant metallothionein confirmed that this protein plays a role in the homeostasis of bioelements and detoxification of potentially toxic metals. These results form the basis for future research on the impact of&nbsp; heavy metal pollution on the honey bee and represent an important step in the comprehensive assessment of the impact of stress factors from the environment on honey bees.</p>