The studies by Schwarzenberger et al.  and Miyakawa et al.  present interesting first insights into the molecular genetic pathways primarily involved in morphological defense formation in Daphnia pulex and Daphnia magna under predation risk by Chaoborus larvae. A strength of the traditional deductive candidate approach employed in both studies is that it is based on a hypothesis about possible gene regulatory networks (for example, [11, 12]). The finding of moderately up-regulated genes supports the hypothesis of the involvement of IS and JH and the expression of body pattern and morphogenic genes in the development of inducible defense structures. It is interesting to note, however, that although there is strong support for an involvement of JH, no JH receptor has been identified in the Daphnia pulex draft genome, despite considerable efforts.
The clear drawback of the candidate gene approach is its limitation to pathways and genes that are known. Novel pathways or genes relevant in this context may be missed completely. Hence, it is important also to scan for novel differentially expressed genes, as has been done by Miyakawa et al. . Despite a large number of false positives or genes that did not amplify (19 of 22; H Miyakawa, personal communication) and despite the lack of clear homology for the remaining three genes (DD1, DD2, DD3), the detection of three intriguing novel genes highlights the potential of such genomic approaches for the discovery of novel transcripts that play an important role in induced defense formation.
One issue that will be important for future studies to address is how best to quantify changes in gene expression. The sensitivity of quantification in expression studies to different biases underlines the importance of validating expression levels of candidate genes using a great number of biological and also technical replicates. Since even classical housekeeping genes have been proven to be significantly differentially regulated in Daphnia [9, 13], careful selection and evaluation of the internal reference genes for normalization of expression levels is important. If GAPDH is differentially regulated between kairomone-exposed Daphnia and the respective control, this may alter the interpretations of gene response profiles . First results from tiling microarray data show that a large number of genes are potentially differentially regulated (JK Colbourne, personal communication). However, it is important to test with a greater number of biological and technical replicates to confirm up/down-regulation and, also for the microarray data, to assign levels of differential regulation, which can be considered as significant. In this context it should be kept in mind that as long as the kairomones have not been identified and a blend of different chemicals has to be used for induction, some genes might be activated as a side effect, that is, by compounds not related to the chemical alarm cues. Furthermore, predation is only one of several possible stressors to the organisms. Future comparative transcriptional profiling studies need to disentangle general stress responses from predation-specific responses.
With the advent of more sophisticated preparation techniques, tissue-specific expression profiles along a time course will greatly help in understanding fine-scale temporal and spatial gene expressions patterns. In this context, whole genome expression analysis by microarrays or RNA next-generation sequencing techniques will greatly facilitate studying these processes on a fine scale. Such studies should provide detailed insights into regulatory gene networks. Then, large-scale quantitative trait loci mapping, functional validation (at the protein level; Figure 1) and the subsequent extension to different Daphnia pulex clones and different Daphnia species should be performed to understand how predation signals are perceived, transmitted and refined into specific gene/protein regulatory pathways and subsequently into form and function. The fascinating array of inducible defenses manifested by Daphnia provides an interesting model for studying the interaction of genes with the environment. The application of new techniques in conjunction with genome sequence data is likely to yield significant new insights into the genetic basis of inducible defenses and phenotypic plasticty in general.