20.500.12556/DKUM-60792 Spremembe v srednjem črevesu in goltnih žlezah po okužbi medonosne čebele (Apis mellifera carnica) s sporami Nosema spp. Changes to the ventriculus and hypopharyngeal glands of honeybee (Apis mellifera carnica) caused by Nosema spp. infection Medonosna čebela (A. mellifera) spada med najpomembnejše, vsekakor pa tudi najštevilčnejše opraševalce kmetijskih pridelkov. Živi v specifični socialni združbi z izrazitim kastnim sistemom. Troti in matica predstavljajo reproduktivne osebke v družini za razliko od delavk, ki so sociološko sterilne. V družini opravljajo naloge oskrbovanja zalege, matice in mladih čebel z matičnim mlečkom ali drugo hrano, gradijo satje, varujejo panj pred vsiljivci in nabirajo mano, nektar, cvetni prah in vodo. Delavke lahko zaradi prezgodnje smrtnosti ali subletalnih učinkov, zaradi katerih niso sposobne opravljati katere od naštetih nalog, družini ne koristijo več. Kadar je število takšnih delavk veliko, ta izguba prizadene celotno čebeljo družino. Zato smo v pričujoči nalogi preverili, kakšni so subletalni vplivi okužbe s pogostima parazitoma iz družine Nosema spp. na srednje črevo in goltne žleze čebel delavk. S pomočjo tehnike TUNEL za zaznavanje celične smrti smo ugotavljali pojavnost odmiranja celic na tkivnih rezinah srednjega črevesa in goltnih žlez, neokuženih ali z N. apis oziroma N. ceranae okuženih čebel. Poleg tega smo ocenili vpliv okužbe na morfologijo obeh organov. Premer goltnih žlez smo uporabili kot indikator razvitosti žlez in analizirali njegovo povezanost s težo glave. Ugotovili smo, da okužba z N. apis povzroča povečano smrtnost celic epitela srednjega črevesa. Delež TUNEL pozitivnih jeder pri neokuženih delavkah se je ves čas gibal okoli desetih odstotkov, kar je približno petina pozitivnih jeder, ugotovljenih pri z N. apis okuženih delavkah. Po 15. dnevu je število upadlo na raven prvih. V črevesju z N. ceranae okuženih delavk je bilo število apoptotičnih jeder znatno višje od kontrol tretji. dan, po 12. dnevu pa je povsem upadlo na z neokuženimi delavkami primerljivo raven. Pri čebelah, okuženih z Nosema spp., so bile spore 15. dan prisotne v večini epitelnih celic, zgradba srednjega črevesa je bila močno spremenjena in je kazala znake degeneracije. Okužba z Nosema spp. je negativno delovala na razvoj goltnih žlez in njihovo sekretorno aktivnost. Pri okužbi z N. ceranae smo zabeležili signifikanten (P < 0,05) negativen vpliv okužbe na razvitost goltnih žlez na vse dni testiranja in na 12. ter 21. dan za okužbo z N. apis. Na histološki ravni smo pri okuženih delavkah v hipofaringealnih žlezah ugotovili zmanjšanje količine citoplazme in sekretornih veziklov, ki so znak aktivnosti žlez. Povišan je bil tudi delež celične smrti v sekretornih celicah goltnih žlez okuženih čebel. Ker v nobenem izmed preiskovanih vzorcev goltnih žlez nismo ugotovili spor Nosema spp. sklepamo, da žleze niso primarno mesto okužbe. Atrofija žlez pri okuženih delavkah je tako verjetno posledica slabšega izkoriščanja hrane zaradi poškodb, ki jih povzroči razmnoževanje parazitskih gliv v črevesnem epiteliju. Premeri goltnih žlez so bili v neposredni korelaciji s težo glave pri delavkah iz vseh treh skupin. Okužba z N. ceranae se je širila z okuženih na neokužene delavke, ne pa tudi na matice v poskusnih plemenilnikih. Razvoj okužbe pri neokuženih delavkah je z zamikom sledil poteku pri sočasno naseljenih delavkah, ki so prejele 80.000 spor N. ceranae. Honey bees (A. mellifera) are among most important and widely distributed crop pollinators. They form honey bee colonies - specific social structures with caste system. The drones and the honey bee queen represent reproductive individuals of the colony, unlike the sterile honey bee workers. Workers perform tasks such as feeding the larvae, the queen and young bees with royal jelly or other types of food, building comb, protecting the nest from intruders and gathering nectar, pollen and water. Their premature death or sublethal effects that render them incompetent of performing the tasks makes the workers no longer of use to the society. When the number of affected individuals is high it can cause colony collapse. Therefore we analyzed the sublethal effects of two common honey bee parasites belonging to the genus Nosema, focusing on the worker bees midgut and hypopharyngeal glands. We evaluated cell death in midgut and hypopharyngeal gland tissue of uninfected compared to N. apis and N. ceranae infected bees using the TUNEL techique. Simultaneously, we monitored the effect of infection on morphological characteristics of both organs. We used acinar diameter as an indicator of glandular development and analyzed its connection to fresh head weight. N. apis infection caused significantly elevated level of death in midgut epithelial cells. Proportion of TUNEL positive nuclei in uninfected workers remained around 10 % throughout the experiment, being at about one fifth of the positive nuclei established in N. apis infected workers. Significantly higher level of apoptotic nuclei was detected in N. ceranae infected versus uninfected midgut on the third day. Cell death gradually decreased to the common level after the 15th day in N. apis infected bees and after the 12th day in N. ceranae infected bees. The spores were present in most midgut epithelial cells and the morfology of the organ was markedly altered showing signs of degeneration by day 15 after infection. Nosema spp. infection had a negative influence on the development of hypopharyngeal glands and their secretory activity. The effect was significant (P < 0,05) in N. ceranae infected bees on all tested days and only on the 12th and 21st day in N. apis infected bees. Hypopharyngeal glands of infected honeybees had a scarcer cytoplasm and contained less secretory vacuoles. The presence of the former is a sign of glandular activity.The level of cell death in hypopharyngeal glands was elevated in Nosema infected bees. None of the investigated samples of glandular tissue sections contained spores of Nosema spp., therefore we conclude that the hypopharyngeal glands are not the primary site of infection. Glandular atrophy in infected individuals is most likely the consequence of diminished intestinal absorption of the nutritive compounds due to the damages in midgut epithelium caused by the propagation of parasites. Hypopharyngeal acini diameter was in direct correlation with fresh head weight. N. ceranae infection spread readily from infected to uninfected workers in mating nuclei, but not to the queens. Spread of spores to healthy bees was recorded from workers receiving 80.000 spores of N. ceranae in mating nuclei. Apis mellifera srednje črevo goltne žleze Nosema spp. celična smrt Apis mellifera midgut hypopharyngeal glands Nosema spp. cell death true true false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2016-07-04 19:47:26 2016-08-05 13:19:21 2022-06-24 03:15:27 2019-07-11 11:58:22 2016 0 Maribor 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 URN:SI:UM:DK:LNIB3NBD DOK_Soklic_Mateja_2016.pdf DOK_Soklic_Mateja_2016.pdf 1 51A6ABF825EF51E8436E84FB7BC6D301 040fe8bbae1d3fab97be1ec70fbc15587fac4f5ddd8c05698ef80670d168b8df f7e73ba4-7c0c-11eb-bb7a-00155d0001ca https://dk.um.si/Dokument.php?lang=slv&id=97800 Fakulteta za kmetijstvo in biosistemske vede 0 0 0