Cold hardiness in relation to trace metal stress in the freeze-avoiding beetle Tenebrio molitor
Introduction
Metals from natural or anthropogenic sources represent an important part of the chemical environment. Trace metals like Cu and Zn are essential for normal growth and metabolism of organisms, but they may also produce toxic effects at high concentrations (Kruk, 1998). Cadmium is a non-essential heavy metal that can be taken up by cells through voltage-gated calcium channels located in the plasma membrane. Intracellularly cadmium accumulates by binding to cytosolic and nuclear material, causing a variety of damaging effects like inhibition of zinc-containing enzymes, lipid peroxidation and disturbance of protein synthesis (Beyersmann and Hechtenberg, 1997). Even though soft bodied animals like springtails and earthworms can take up metal directly, metals mostly enter the food chain via plants (Kabata-Pendias and Pendias, 1992). The metals may cause injury by direct toxic actions, but they have also been shown to cause damage more indirectly by interfering with cold hardening processes important for winter survival in insects (Zachariassen and Lundheim, 1995) and earthworms (Holmstrup et al., 1998; Bindesbøl et al., 2005).
Insects that adaptively allow their body fluids to supercool when they become exposed to low temperatures (freeze avoidance) often produce antifreeze proteins (AFPs) (Zachariassen, 1985; Duman, 2001). This family of proteins is defined by their ability to depress the non-equilibrium freezing point of water while not affecting the melting point. This separation of melting and freezing point is called thermal hysteresis (DeVries, 1971, DeVries, 1986). The phenomenon is caused by adsorption of antifreeze proteins to the ice crystals (Raymond and DeVries, 1977; Knight et al., 1991; Kristiansen and Zachariassen, 2005). Their function appears to be to promote supercooling by preventing inoculative freezing across the body wall (Gehrken, 1992; Olsen et al., 1998) and masking of ice nucleating structures within the body fluids (Olsen and Duman, 1997a, Olsen and Duman, 1997b).
The phenomenon of thermal hysteresis was first described by Ramsay (1964), during his study of the cryptonephridial rectal complex of Tenebrio molitor. It was later discovered that this phenomenon was caused by proteins (Grimestone et al., 1968). These proteins have been identified as a family of AFP isoforms which vary both in length and extent of glycosylation (Liou et al., 1999). The thermal hysteresis activity in the hemolymph increases with larval development in T. molitor (Graham et al., 2000), but exposure to conditions normally associated with the winter season (e.g. low temperature, desiccation, starvation, short photoperiod) has been shown to further increase the thermal hysteresis activity (Patterson and Duman, 1978; Horwath et al., 1996; Graham et al., 2000).
The purpose of this investigation was to study the effects of exposure to Cu, Zn and Cd on the production of AFPs in the larvae of the freeze avoiding beetle T. molitor.
Section snippets
Insect rearing
A laboratory culture of T. molitor larvae (Blades Biological, Edenbridge, Kent, UK) was established and maintained on wheat bran at 25 °C and long day (18:6 h, L:D). The animals were sprayed with distilled water once a week to provide adequate moisture.
Stress treatment
Animals were exposed to three different concentrations of Cd (0.005, 0.05 or 0.5 mg/g), Cu (0.1, 1 or 10 mg/g) or Zn (0.5, 5 or 50 mg/g) in the food. The metal was administered via the food since ingestion is the most likely route for metal uptake in
Distribution of body mass for the different treatments
Eggs developed into medium sized larvae in all the groups except those that were exposed to the highest levels of Zn (5 and 50 mg/g) and the highest level of Cu (10 mg/g). In these groups none of the larvae developed beyond the 1st instar (see Table 1). Only a small number of larvae (>10) developed in the winter-acclimated groups exposed to 0.1 mg/g Cu and 0.5 mg/g Cd. There was no significant difference in the body mass between the different groups of summer-acclimated animals (F=1.22 with 6 and
Distribution of body mass for the different treatments
The occurrence of only small 1st instar larvae in the groups exposed to the two highest levels of Cu (1 and 10 mg/g) and in the groups exposed to the highest level of Zn (5 mg/g) clearly indicates that the level of chronic metal stress in these groups was too high to sustain normal larval development. This is not surprising since these levels were far above the normal range for copper (0.005–0.025 mg Cu/g dry weight) and zinc (0.02–0.4 mg/g dry weight) in plants (Reeves and Baker, 2000). The small
Conclusions
The main finding of this study was that metal stress causes a reduction in the hemolymph hysteresis activity in T. molitor larvae acclimated to summer conditions. The reduction in hysteresis activity was accompanied by reduced levels of AFP YL-3 transcription, suggesting reduced levels of AFPs. Winter-acclimation of metal stressed larvae restored both the level of AFP YL-3 mRNA and hemolymph hysteresis activity to normal levels. The reduced hysteresis activity may have fitness consequences in
Acknowledgments
We thank Anne Skjetne Mortensen for technical assistance and helpful suggestions. This work was financed by the Norwegian research Council (NFR) Project no. 147710/432.
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