Sequences were annotated by BLASTX homology search against GenBank non redundant protein sequences database . Sequences were only annotated if hits met an expect value of 10 5 and a score of 50 against the database entry. GeneBank/UniProt accession num ber and species match were recorded with each annotation . cDNAs with the same NCBI annotation were considered to represent the same gene. We used the mean expression and corresponding standard deviation to characterize the transcrip tion level of gene groups represented by several cDNAs. Some gene groups recorded high variability of signal in representative cDNAs, which could be explained by their distance from the gene 3 _ end, hybridization efficiency, or by cross hybridization from members of large gene families where numerous subunits or homologues carried identical annotation and may be differentially expressed in a tissue specific manner.
Fig. 1. Relative response of cDNAs following D. magna exposure to methomyl and propanil. Each dot refers to a cDNA, where 2 fold change in intensity was observed in at least one treatment compared to the control. M/C and P/C stand for the log2 transformed fold regulation of treated MEK Signaling Pathway samples to untreated control . Grey squares indicate cDNAs, which were up regulated exclusively by methomyl or propanil and black squares depict stressor specific down regulation by methomyl or propanil . pesticide specific responses . Of the 768 differentially expressed cDNAs, only 354 were successfully anno tated and assigned functional groups . Both pesticides elicited considerable differential transcription within protein biosynthe sis, moulting and energy metabolism .
Few genes were toxicant specific and, of these, more were up regulated than were down regulated MEK Signaling Pathway . 3. Results 3. 1. Global mRNA expression responses of genes 11,505 cDNAs were of sufficient quality to be analysed. When D. magna were exposed to the estimated EC1 concentrations of methomyl and propanil 2781 cDNAs differed significantly from controls in at least one treatment . Of these, 768 cDNAs indicated 2 fold up or down regulation of the mRNA of the associated gene. Propanil exposure significantly changed transcription levels in 551 cDNAs whereas methomyl significantly changed transcription levels in 624 cDNAs. Responses to both pesticides were similar in propor tions of cDNAs up and down regulated.
When the 768 cDNAs are plotted to compare responses between chemicals, it can be seen that many responses are simi lar, which may indicate general mechanisms of cellular response to chemical stress . However, there are also clusters of 3. 2. Responses to methomyl exposure After removal of redundant sequences, a final list representing 161 genes was estab lished. Moulting, protein biosynthesis checkpoint kinase and energy metabolism were clearly the biological processes responding most to the exposure . Genes associated with moulting represented 20. 5% of the response, with many cuticular proteins and chitin deacetylases responding, some up regulated by 8 fold compared to the control . Genes involved in protein biosynthesis were up and down regulated by methomyl in identical pro portions whereas there was more up than down regulated genes involved with energy metabolism, with mitochondrial genes and cytochrome oxidase up regulated by 4 5 fold .
Within neuronal pathways, carboxylesterase and a predicted doughnut like protein kinase were found down regulated by ca. 3 and 4 fold, respectively. Table 2 shows chemical specific differential NF-kB signaling pathway gene transcription. There was a chemical specific induction of genes involved in ion homeostasis, namely the chloride bicarbonate anion exchanger and Na, K ATPase . Methomyl specific up regulation includes also genes involved in signalling pathways and proteins metabolism, as well as genes cod ing for structural proteins and for the protein sulfotransferase , which should be directly related to the exposure to the xenobiotic. Chemical specific down regulation includes two shows which proteins were assigned to each functional mechanism/pathway.
genes related to proteins metabolism , a protein of unknown function and a glycoprotein that may be related with sexual maturation . 3. 3. Responses to propanil exposure After removal of redundant sequences, a final list of 126 genes was produced. The main functional groups altered by exposure to the herbicide were moulting, pro tein biosynthesis, energy metabolism and PARP oxygen transport . Energy metabolism accounted for the highest proportion of genes induced and repressed . Genes belonging to the mitochondrial genome contributing the most , but additional differen tial transcription included ATP synthase, cytochrome C oxidase, amylase and enolase. Other important groups included genes associated with moulting and ribosome related proteins .