ABSTRACT: Microarray Construction The D. magna microarray was constructed using DNA fragments from three sources: i) Stress-specific cDNAs were generated using Selective Subtractive Hybridisation (SSH) on organisms exposed to the stressors shown in Table 1. SSH was performed with forward and reverse subtraction using a PCR-Select cDNA Subtraction Kit (Clontech Laboratories Inc, USA). cDNA was prepared from mRNA extracted from batches of 100 D. magna, 48 h old, using a Straight AM-CM-"M-BM-,s mRNA Isolation Kit System (Novagen Inc., UK) (2,029 clones). ii) ESTs obtained from a cDNA library from unexposed mixed aged organisms obtained from a 3-4 week old culture (Watanabe et al. 2005) (10,272 clones). iii) cDNAs isolated following an SSH, as above, between 15 adults carrying eggs and 75 juveniles (Soetaert et al. 2006) (1,143 clones). Gene fragment annotation Sequencing of the 3,172 SSH ESTs was carried out with M13-Long primers. Blast searches were performed on specific fragments that responded significantly to the exposure treatment. Sequences were annotated according to BlastX homology search against Uniprot Swissprot (http://www.ebi.ac.uk/swissprot) and Uniprot Trembl (invertebrate section, http://www.ebi.ac.uk/trembl) March 2006. Sequences were only annotated if they were found to have a Blast hit with the expect value smaller than 0.00001 and a score above 50. GeneBank/UniProt Accession number and speciesM-CM-"M-BM-, match were recorded with each annotation. We printed PCR products generated from the above, onto glass slides. All spots have been sequenced except for (iii), only a few available and some sequencing failed for various reasons. Therefore we have the sequences for around 90% of the spots. BLAST searches revealed a large proportion (nr 60%) had no significant match with existing entries in GenBank/Uniprot/Trembl. Because of low confidence we've not considered them, otherwise this would lead to huge and erroneous assumptions. All (i and ii) sequences are available either on GenBank or on our website. All the spots that came out as significant on our experiments were reviewed and only those that have been annotated, with significant BLAST match, are being published, with respective Accession numbers, species match and E-values. Fragments were therefore described as M-CM-"M-BM-,SunnanotatedM-CM-"M-BM-,M-CM-= where no significant difference was measured or M-CM-"M-BM-,Sno significant matchM-CM-"M-BM-,M-CM-= for those that were significant but no matches were found. Significant genes were annotated respectively as described above. Dose Reponse Tests were carried out using the developed microarray on cadmium, ph, lufenuron, calcium limitation and ibuprofen; details below: Cadmium: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to sublethal concentrations of cadmium chloride from 0, 10, 33 to 60 M-CM-^BM-BM-5g L-1 (corresponding to 0, 6.13, 20.24 and 36.79 M-CM-^BM-BM-5g L-1 Cd2+), in quadruplicates. pH: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to pH ranging from 8.0 (controls), 5.5, 5.3 and 5.2, in quadruplicates. pH was maintained by adding HCl on a daily basis. Lufenuron: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to sublethal concentrations of C14 labelled, lufenuron ranging from 0, 0.35, 0.59 to 1.00 ug L-1, in quadruplicates. Calcium: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to calcium chloride limitation ranging from 195.87 (culture condition), 20, 10 and 5 mg L-1 (CaCo3), in quadruplicates. Ibuprofen: RNA was obtained from populations of 300 D. magna, 24-48 h old, exposed for 24 h to ibuprofen (IB) sodium; 0 (control), 20, 40 and 80 mg L-1, in *quadruplicates. *One replicate at 80 mg L-1 was lost, thus this treatment was done in triplicate. IB was measured as the pharmacological active ingredient. Juvenile D. magna were used in order to restrict the response to genes associated with stress responses and growth, rather than genes associated with different stages of reproduction. Following exposure, daphnids were stored in RNAlater (Ambion, UK) at -80M-CM-^BM-BM-0C.
