Bioindication potential of physiological responses of gypsy moth Lymantria dispar L. (Lepidoptera) on the benzo[a]pyren effects

Intenzivan antropogeni uticaj u poslednjih nekoliko decenija doveo je do značajnog porasta stepena zagađenja biosfere, kojem u velikoj meri doprinosi toksični i kancerogeni organski polutant - policiklični aromatični ugljovodonik, benzo[a]piren. Cilj ove doktorske disertacije bio je procena odgovora fizioloških i molekularnih parametara larvi Lymantria dispar kao potencijalnih biomarkera sredinskog zagađenja benzo[a]pirenom. Ispitan je hronični uticaj dve koncentracije ovog ksenobiotika (5 i 50 ng/g suve mase hrane) na larve L. dispar iz legala sakupljenih u nezagađenoj šumi (planina Majdanpek) i sakupljenih u zagađenoj šumi (okolina rudnika Bor D.O.O), sa svrhom procene efekta porekla populacije. Larve iz nezagađene šume pokazuju pad specifične aktivnosti katalaze u srednjem crevu, ali i značajan porast aktivnosti antioksidativnih enzima - superoksid dismutaze i katalaze, na nivou hemolimfe, dok je u populaciji iz zagađene šume zabeleženo povećanje aktivnosti ova dva enzima samo u okviru srednjeg creva. Detoksifikacioni enzimi, karboksilesteraze, pokazuju osetljivost i povećanje aktivnosti u prisustvu benzo[a]pirena, u srednjem crevu larvi iz nezagađene šume, kao i u mozgu larvi iz zagađene šume, koje pored toga, na nivou srednjeg creva odlikuje značajna indukcija aktivnosti glutation S-transferaza. Acetilholinesteraze nisu osetljive na primenjene koncentracije ovog ksenobiotika. Povećanje ekspresije proteina Hsp70 u okviru srednjeg creva uočeno je kod obe populacije larvi, a na nivou mozga jedino u populaciji iz zagađene šume i to pri dejstvu niže koncentracije benzo[a]pirena. Većina digestivnih enzima nije osetljiva na prisustvo benzo[a]pirena sa izuzetkom tripsina, alkalnih fosfataza i lipaza, koje odlikuje povećanje aktivnosti kod larvi iz nezagađene šume, a larve iz zagađene šume karakteriše jedino indukcija lipazne aktivnosti. Za populaciju larvi iz nezagađene šume svojstven je pad koncentracije ukupnih lipida u hemolimfi i nepromenjen sadržaj trehaloze, dok ovi parametri ne pokazuju promene u populaciji iz zagađene šume. Hronično prisustvo benzo[a]pirena i produženo angažovanje energetski zahtevnih, odbrambenih mehanizama, odrazilo se na komponente fitnesa larvi L. dispar pri čemu se populacija iz nezagađene šume pokazala manje podložnom energetskom deficitu, ispoljavajući smanjenje mase i relativne brzine rasta larvi petog larvenog stupnja, dok larve iz zagađene šume, pored iste promene prethodna dva parametra fitnesa, odlikuje i produženo razviće. Uočeno je da su odgovori antioksidativnih enzima - superoksid dismutaze i katalaze, u hemolimfi, kao i karboksilesteraza i digestivnih enzima - tripsina i alkalnih fosfataza, u srednjem crevu larvi, specifični i potencijalno primenjivi biomarkeri zagađenosti životne sredine sa benzo[a]pirenom za populaciju larvi poreklom iz nezagađene šume. Sličnu primenu, kada je u pitanju populacija larvi iz zagađene šume, mogu naći specifične aktivnosti superoksid dismutaze, katalaza i glutation S-transferaza u srednjem crevu larvi, aktivnost karboksilesteraza i ekspresija Hsp70 u mozgu larvi L. dispar.Over the last few decades intense anthropogenic influence has led to a significant increase of the pollution levels in the biosphere, which can greatly be attributed to the toxic and carcinogenic organic pollutants - polycyclic aromatic hydrocarbones, and especially to the benzo[a]pyrene. The aim of this dissertation was to evaluate responses of the physiological and molecular parameters of the Lymantria dispar larvae as a potential biomarkers of environmental pollution by benzo[a]pyrene. For the purpose of assessing the effects of population origin, L. dispar egg-masses were collected from unpolluted forest (Majdanpek mountain) and polluted forest (vicinity of Bor D.O.O mine). The chronic effects of the two benzo[a]pyrene concentrations in the diet (5 and 50 ng/g dry food weight) on these two population of larvae were investigated. Larvae from unpolluted forest showed a decrease of the catalase specific activity in the midgut, and a significant increase in the activity of antioxidative enzymes - superoxide dismutase and catalase, in the hemolymph, while the population from the polluted forest expressed an increase in the activity of these two enzymes only within the midgut. Detoxification enzymes - carboxylesterases, showed sensitivity and increase in activity in the presence of benzo[a]pyrene in the midgut of larvae from unpolluted forest, as well as in the brain of larvae from polluted forest, which in addition, are characterized by significant induction of glutathione S-transferase activity at the midgut level. Acetylcholinesterases are non inducible to the applied concentrations of this xenobiotic. An increase in expression of Hsp70 protein at midgut level was observed in both larval populations, while only the population from contaminated forest showed rise in brain Hsp70 concentration, after treatment with lower dose of benzo[a]pyrene. Most digestive enzymes are insensitive to the presence of benzo[a]pyrene with the exception of trypsin, alkaline phosphatases, and lipases which demonstrated increased activity in larvae from unpolluted forest. Larvae from polluted forest exibited only induction of lipase activity. The population of non-polluted larvae is characterized by a decrease in the concentration of total lipids in the hemolymph and an unchanged trehalose content, while these parameters do not show changes in the population from the contaminated forest. The chronic presence of benzo[a]pyrene and prolonged engagement of energy-demanding defense mechanisms have reflected on the fitness components of L. dispar larvae, with population from unpolluted forest showing less susceptibility to energy deficiency and exhibiting reduced mass and relative growth rates of the fifth instar larvae, while the larvae from the polluted forest, in addition to the same changes in previous two fitness parameters, have also showed prolonged development. It have been noticed that responses of the antioxidative enzymes - superoxide dismutase and catalase in hemolymph, as well as carboxylesterases and digestive enzymes - trypsin and alkaline phosphatases, from the larvae midgut, are specific and potentially applicable biomarkers of environmental contamination with benzo[a]pyrene for larval populations originally from unpolluted forest systems. Similarly, when it comes to larval populations from contaminated forests, specific activities of superoxide dismutase, catalase and glutathione S-transferases in the larvae midgut, as well as carboxylesterase activity and expression of Hsp70 in the brain of L. dispar larvae, could be potentially used in biomonitoring