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Sublethal effects of toxicants may cumulate over time and become apparent only when test organisms are exposed for multiple generations. In this study we determined phenotypic effects and transcriptional responses in the parthenogenetic soil invertebrate Folsomia candidaover four generations, followed by two generations of recovery. Animals were exposed to two metal-based nanomaterials (NMs): copper oxide (CuO) and tungsten carbide–cobalt (WCCo), both homogenously mixed in with the soil. Survival and reproduction were not affected in any of four consecutive generations of F. candidaexposed to CuO-NM at concentrations as high as 6400 mg Cu per kg dry LUFA 2.2 soil. WCCo-NM affected reproduction and survival from the third generation onwards, with EC50values between 2400 and 5600 mg NM per kg dry soil, but recovery was seen in recovery generations 1 and 2 when kept in clean soil. Histological investigations showed that WCCo-NM (3200 mg kg−1) induced tissue damage and loss of villi from the gut epithelial cells. Expression of four target genes known to be responsive to stress were investigated by quantitative PCR at different exposure levels and in different generations. Expression of all genes was significantly affected by NMs even though exposures were below toxic threshold concentrations. In addition, gene expression did not always return to control levels during consecutive recovery over two generations in clean soil. This shows that gene expression assays can detect physiological alterations cumulating from one generation to the next initially without visible effects on phenotypic variables such as reproduction. The possibility of multi-generation carry-over of sublethal toxicity needs more attention in environmental risk assessment.

Although nearly all organisms are subject to fluctuating temperature regimes in their natural habitat, little is known about the genetics underlying the response to thermal conditions, and even less about the genetic differences that cause individual variation in thermal response. Here, we aim to elucidate possible pathways involved in temperatureinduced phenotypic plasticity of growth rate. Our model organism is the collembolan Orchesella cinctathat occurs in a wide variety of habitats and is known to be adapted to local thermal conditions. Because sequence information is lacking in O. cincta, we constructed cDNA libraries enriched for temperatureresponsive genes using suppression subtractive hybridization. We compared gene expression of O. cinctawith steep thermal reaction norms high plasticity to those with flat thermal reaction norms low plasticity for juvenile growth after exposure to a temperature switch composed of a cooling or a warming treatment. Using suppression subtractive hybridization, we found differential expression of ten nuclear genes, including several genes involved in energy metabolism, such as pantothenate kinase and carbonic anhydrase. In addition, seven mitochondrial genes were found in the cloned subtracted library, but further analysis showed this was caused by allelic variation in mitochondrial genes in our founder population, and that a specific haplotype was associated with high thermal responsiveness. Future work will focus on candidate genes from pathways such as the oxidative phosphorylation and biosynthesis of coenzyme A which are possibly involved in thermal responsiveness of juvenile growth rate. J. Exp. Zool. Mol. Dev. Evol. 310B:137–147, 2008. © 2007 WileyLiss, Inc.

Since 1986 perizin has been used in many European countries to combat the mite Varroa jacobsoni, a parasite of the honeybee. We have studied the long-term presence of coumaphos, the active ingredient of perizin, by analyzing honey and wax for residues. We distinguished between the direct transfer of coumaphos into wax by contamination during treatment and the indirect transfer through wax production by bees. To study the indirect transfer, we treated colonies with perizin and removed the combs, thus forcing the bees to produce new wax. The newly produced wax and honey were analyzed for the presence of coumaphos. Wax from colonies that had not been treated with perizin for 6 mo and up to 18 mo still contained coumaphos (7 and 1 µg/g wax, respectively). Moreover, newly produced wax from the colonies that had not been treated with perizin for 6 mo also contained coumaphos (˜17% of the amount found in old wax). No residues were found in honey. Only small amounts of coumaphos (±l‰ of the amount administered) were found in newly produced wax when colonies were fed a perizin-sugar solution. The largest amounts of contamination were found in the first samples secreted; samples taken 3-4 d after application contained only 1-5% of the amount secreted on the 1st d. Coumaphos is transferred mainly into the wax directly as a consequence of the application of perizin to combs, and the acaricide is persistent in the wax.