The effects of temperature stress and population origin on the thermal sensitivity of Lymantria dispar L. (Lepidoptera: Erebidae) larvae
Increased environmental temperature is one of the most frequent stresses effecting metabolic rate in herbivorous insect species. Our goal was to compare the influence of increased environmental temperature and induced thermotolerance on the activity of midgut phosphatases and brain tissue hsp70 concentration in 5th instar Lymantria dispar larvae originating from an unpolluted and polluted forest. Thermal regimes in this experiment predominantly influenced the ALP activity and it was affected by temperature, population origin, and their combined effect. Tot ACP activity was changed only by the joint effect of temperature and population origin. Brain hsp70 concentration was under a significant individual and joint effect of temperature and population. In both populations, brain tissue hsp70 concentration and ALP activity should be taken under consideration as a battery with biomarker potential for thermal stress in L. dispar larvae as a bioindicator species.
Steps to reproduce
In the autumn (November), L. dispar egg masses were collected at two sites: unpolluted and polluted forest. The first was a mixed oak forest at Kosmaj Mountain, 40 km south-east of Belgrade (coordinates 44°27′56″N 20°33′56″E). These woods are regarded as unpolluted because they are far from direct pollution and are part of the system of protected green areas around Belgrade, where the construction of industrial facilities and traffic infrastructure with potential negative effects on the environment is prohibited by legal regulations. The second site was Lipovica Forest (coordinates 44°38′11″N 20°24′12″E), with mixed Quercus frainetto and Quercus cerris trees, considered a polluted forest since it is located along the border of State Road 22, one of the most frequently used IB-class roads in Serbia. Collected egg masses were kept in a refrigerator at 4 °C until spring (March) when 200 eggs for each experimental group were set for hatching. After hatching in transparent Petri dishes (V = 200 mL), 10 first instar larvae were transferred and reared together at 23 °C with a 12:12 h light: dark photoperiod and relative humidity of 60%, until the third larval instar. Then, five 3rd instar larvae were reared together in the same Petri dish. After molting into the 4th instar, each larva was kept individually until the third day of the 5th instar, when they were sacrificed. Larvae were fed on an artificial diet designed for L. dispar (O’Dell et al., 1985), and food was replaced every 48 h. Each experimental group contained between 50 and 60 larvae.Scheme of experimental groups The optimal temperature for L. dispar larval development is 23 °C, and the control group was reared at this temperature. The highest summer temperature (2007–2010) measured in Serbian Quercus forests at a similar elevation was 28.4 °C, and the lowest 19.6 °C, while the average summer temperature was 26.3 °C (Babić, 2014). Thus, we established variable temperature regimens that included brief (24 h) and daily (72 h) exposures to 28 °C. At sacrifice on the third day of the 5th instar, the caterpillar midguts were dissected out on ice (n = 8 – 11 larval midguts per group for each enzyme assay). Midgut from single larvae was weighed and homogenized in insect physiological saline, as insect fluids have buffer values similar to vertebrates (Harrison, 2001). Homogenization was performed in ice-cold 0.15 M NaCl (final tissue concentration was 100 mg/mL in each sample). A modified method by Nemec and Socha (1988) was used to determine the activity of ALP and ACP. Lysosomal ACP were detected indirectly (Amlabu et al., 2009). -Nemec, V. & Socha, R. Phosphatases and pteridines in Malpigian tubules: a possible marker of the mosaic mutant in Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae). Acta Entomol. Bohemosl. 85, 321–326 (1988).