Unbiased Transcriptional Comparisons of Generalist and Specialist Herbivores Feeding on Progressively Defenseless Nicotiana attenuata Plants

et al. (2010) Unbiased Transcriptional Comparisons of Generalist and Specialist Herbivores Feeding on Progressively Defenseless Nicotiana attenuata Plants. PLoS ONE 5(1): e8735. doi:10.1371/journal.pone.0008735 Unbiased Transcriptional Comparisons of Generalist and Specialist Herbivores Feeding on Progressively Defenseless Nicotiana attenuata Plants Thomas Baldwin 0 1 Geetha Govind 0 1 Omprakash Mittapalli 0 1 Thasso Griebel 0 1 Silke Allmann 0 1 Sebastian Bo cker 0 1 Ian 0 1 Stewart Plaistow, University of Liverpool, United Kingdom 0 Current address: Department of Entomology, Ohio State University , Wooster, Ohio , United States of America 1 1 Max Planck Institute for Chemical Ecology , Jena, Germany , 2 Faculty of Mathematics and Computer Science, Friedrich Schiller University of Jena , Jena , Germany Background: Herbivore feeding elicits dramatic increases in defenses, most of which require jasmonate (JA) signaling, and against which specialist herbivores are thought to be better adapted than generalist herbivores. Unbiased transcriptional analyses of how neonate larvae cope with these induced plant defenses are lacking. Methodology/Principal Findings: We created cDNA microarrays for Manduca sexta and Heliothis virescens separately, by spotting normalized midgut-specific cDNA libraries created from larvae that fed for 24 hours on MeJA-elicited wild-type (WT) Nicotiana attenuata plants. These microarrays were hybridized with labeled probes from neonates that fed for 24 hours on WT and isogenic plants progressively silenced in JA-mediated defenses (N: nicotine; N/PI: N and trypsin protease inhibitors; JA: all JA-mediated defenses). H. virescens neonates regulated 16 times more genes than did M. sexta neonates when they fed on plants silenced in JA-mediated defenses, and for both species, the greater the number of defenses silenced in the host plant (JA . N/PI . N), the greater were the number of transcripts regulated in the larvae. M. sexta larvae tended to down-regulate while H. virescens larvae up- and down-regulated transcripts from the same functional categories of genes. M. sexta larvae regulated transcripts in a diet-specific manner, while H. virescens larvae regulated a similar suite of transcripts across all diet types. Conclusions/Significance: The observations are consistent with the expectation that specialists are better adapted than generalist herbivores to the defense responses elicited in their host plants by their feeding. While M. sexta larvae appear to be better adapted to N. attenuata's defenses, some of the elicited responses remain effective defenses against both herbivore species. The regulated genes provide novel insights into larval adaptations to N. attenuata's induced defenses, and represent potential targets for plant-mediated RNAi to falsify hypotheses about the process of adaptation. - The co-evolution of plants and insects has primarily been driven by their interactions [1,2,3,4]. Plants respond to herbivore attack with highly evolved, elegantly regulated arrays of responses. Attack triggers at least two types of inducible defense responses: those that involve the production of metabolites that directly retard the growth and development of the herbivores (direct defenses) and those that involve the production of metabolites that indirectly protect plants by attracting the herbivores natural enemies, usually parasitoids and predators (indirect defenses) [5,6,7]. These inducible defense mechanisms are tightly regulated by insect elicitors, likely to curtail the costs of production in the absence of herbivory and to prevent insects from adapting to the plants defenses. The elicitors found in the oral secretions and regurgitants (OS) of the caterpillars enable plants to specifically recognize attack from insects; this recognition is mediated by complex signaling pathways in which jasmonic acid (JA) plays a central role [7,8,9]. The small quantities of OS that are transferred to leaves by the larvae during feeding are sufficient to elicit defense responses [10]. The salivary components are complex and differ among different insect species and some of these differences allow plants to tailor their defense responses to attack from different insect species [11,12,13]. The defense responses elicited in plants by these elicitors are also highly complex, frequently involving the production of metabolites from many different biosynthetic pathways that sometimes function synergistically to confer resistance [14,15,16,17,18]. The attacking insects, on the other hand, do not remain passive, but up-regulate detoxification systems and employ various behavioral responses to counter the plants defense responses [18,19,20]. These counter defense responses are particularly well studied in specialist herbivores that have adapted to their host plants defenses. While generalist herbivores are often deterred by the secondary metabolites produced by their host plants, numerous studies reported that many specialists have evolved effective countermeasures [21,22,23]. Specialist herbivores often have well-developed specific enzymatic systems that allow them to metabolize secondary chemicals; for example, the specialist bruchid beetle metabolizes toxic non-protein amino acids and synthesizes its own amino acids [24,25], and the larvae of the specialist Heliconius convert cyanogenic glycosides to thiols, which they use as a source of nitrogen [26]. Larvae of the lepidopteran Papilio and Helicoverpa genera metabolize furanocoumarins with the help of cytochromeP450-dependent mono-oxygenases [27,28,29,30] and Manduca sexta (Ms) larvae have developed a greater tolerance for nicotine that exceeds that of other insect species that do not regularly attack nicotine-containing plants [31,32,33]. Some specialists can sequester toxic secondary metabolites for their own defense, such as the larvae of the lepidopteran Uthesia ornatrix, which sequesters pyrrolizidine alkaloids to defend themselves against predators [34,35,36,37]. The majority of these examples of plant induced defenses and insect counter responses come from studies that examine the responses of adapted insects to single compounds or classes of compounds. Few have examined the responses of insects in an unbiased fashion to the full complement of defenses that are elicited by herbivore attack. This stands in contrast to the many unbiased transcriptional analyses of plant responses to insect attack [38,39,40,41,42,43]. Nicotiana attenuata, a wild tobacco plant native to the North American Great Basin Desert is regularly attacked by the larvae of specialist (Ms), as well as generalist (Heliothis virescens: Hv) lepidopteran herbivores [44]. This plants responses to attack from Ms larvae have been extensively studied and is known to activate a well-characterized mitogen-activated protein kinase (MAPK) signaling system as soon as it perceives the elicitors, fatty acid-amino acid conjugates, in the OS of Ms larva (...truncated)