BRANDON & SOURAKOV: Utetheisa vs. Crotalaria arms race TROP. LEPID. RES., 26(2): 85-92, 2016 85 Evaluation of mechanical defense provided by pericarps of three different Crotalaria species to their seeds against a specialist herbivore, Utetheisa ornatrix: a case for a possible host-herbivore evolutionary arms race Clara J. Brandon and Andrei Sourakov* McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA *Corresponding author: [email protected] Abstract: Past studies that explored the evolutionary arms race between toxic Crotalaria plants and their herbivores have mostly focused on chemical co-evolution. In this highly speciose genus of plants, we hypothesize that other defenses, such as mechanical protection of the seeds, which are exploited by specialist herbivores for their nutrients and alkaloids, have also been evolving, together with the herbivores’ ability to overcome them. To test this hypothesis, we assessed the cost of penetration imposed on a specialist herbivore, the larvae of the Ornate Bella Moth Utetheisa ornatrix, by the pericarps of three Crotalaria species. We tested the Florida native C. pumila, which has supposedly been in co-existence with U. ornatrix in the New World for thousands of years, the introduced species C. spectabilis that is native to Asia, and another introduced species, C. pallida, which is native to Africa, where multiple tiger moth species, including ones ancestral to the genus Utetheisa such as the genus Amphicallia, attack Crotalaria. Our evaluation was based on the ability of larvae to penetrate pods, their mortality, the rate of development of 4th through ultimate instar larvae when reared on open vs. closed pods as diet, and the wing span of resultant adult moths as a measurement of fitness. The cost associated with larval penetration of pericarps was greatest when feeding onC. pallida, followed by C. pumila, with the cost of penetration of C. spectabilis barely detectable in our study. We found that there are important structural and mechanical differences in the cellulose fibers forming the inner layer of the pericarps, which could help explain these differences in cost of penetration. Similar research on otherCrotalaria species and associated tiger moths is required to test the evolutionary arms race hypothesis. Key words: arthropod-plant interaction, herbivore-plant interaction, insect-plant co-evolution, New Associations Hypothesis, plant mechanical defense INTRODUCTION Plants use a variety of defense mechanisms to ward World, while some were introduced by humans in part because off herbivores. These defenses are divided into two broad of their ability to fixate nitrogen and improve soil quality categories: chemical and mechanical. Chemical defenses (Austin, 2004). These plants contain pyrrolizidine alkaloids include using toxic and indigestible substances (Wink & (PAs), which are toxic to many animals, including horses (e.g., Mohamed, 2003), and chemicals that simulate insect hormones LSUAgCenter, 2012), sheep (Maia et al., 2014) and humans and affect insect development (Fowler et al., 2001), act as (e.g. Williams & Molyneux, 1987; Diaz, 2014). Most insects glue that impairs feeding (Harvey et al., 2011), or signal to do not feed on Crotalaria because of the harmful PAs. There parasitoids (DeMoraes et al., 1998) and predators (Guimarães are several exceptions, however. The most notable among them et al., 2006). There are also various mechanical defenses, such in the New World is Utetheisa ornatrix (Ornate Bella Moth) as physical barriers that keep predators from consuming the (Erebiidae: Arctiinae). By consuming Crotalaria the larvae plant tissues, such as hairs, spikes, wax layers, and hard walls of this moth ingest alkaloids, which render them poisonous (Becerra et al., 2001). However, insects constantly evolve ways to many predators (e.g., Eisner & Eisner, 1991; Martins et al., to overcome this resistance and these arms races often lead to 2015) and provide them with precursors for sex pheromones reciprocal evolutionary change (e.g., Ehrlich & Raven, 1964). (e.g., Conner et al., 1981; Conner, 2008). Larvae actively seek Recently, Edger et al. (2015) discussed co-evolutionary, genetic the individual plants and tissues of the plants that contain the and molecular mechanisms of this arms race and concluded that highest concentration of PAs (Hoina et al., 2013), and Walsh the evolution of both plant defenses and resulting adaptations in and Iyengar (2015) showed that competition for the seeds, the herbivores are associated with changes in diversification rates. richest source of alkaloids in a plant, favors larger larvae, and Legumes in the genus Crotalaria (rattlebox plants) (Fabaceae) is affected by the relatedness of the larvae, with siblings more and the tiger moths that specialize in feeding on them is one of likely to maintain control of the resource over non-sibling the many putative examples of plant-herbivore co-evolution. competitors. Cogni et al. (2012) found that there is very little There are over 700 species in the genus Crotalaria, cost to U. ornatrix in the sequestration of PAs, and hence which are found primarily in tropical and subtropical regions larvae past third or fourth instar are attracted to seeds where PA of the world. Some Crotalaria species are native to the New concentration is highest, regardless of the Crotalaria species. 86 TROP. LEPID. RES., 26(2): 85-92, 2016 BRANDON & SOURAKOV: Utetheisa vs. Crotalaria arms race Paradigm to have evolved better defenses against them. The geographic Despite not being negatively affected by the toxins, larvae association of C. spectabilis with Crotalaria-feeding Utetheisa must nevertheless find a way to work through the pericarp to in Asia, where some members of Utetheisa feed on non- reach the seeds, and it has been proposed that the plants have Crotalaria hosts, is likely to be the most recent among the three evolved mechanical defenses in their pericarp to prevent larvae tested species, as might be implied from phylogenetic studies from reaching the seeds (Ferro et al., 2006; Sourakov, 2015). (DaCosta, 2010; Le Roux et al., 2013). A former study demonstrated the high cost in the form of The present study therefore compares the ability of larval mortality of penetration of C. pallida pericarps by young pericarps of three different species of Crotalaria that grow U. ornatrix larvae. It has also been proposed that not only naturally in Florida to protect their seeds from U. ornatrix mechanical defenses, but also the arrangement of seeds on a larvae. We tested the efficiency of U. ornatrix larvae from the plant, such as the number of seeds per pod and whether pods are fourth instar onwards in penetrating pericarps of the native C. single or clustered, might affect how efficiently larvae obtain pumila and the exotic C. spectabilis and C. pallida, and discuss nutrients and PAs from different Crotalaria species (Sourakov, reasons for the observed differences. 2015). In the present study, we compare these defenses in three Crotalaria species in Florida: one native and two exotics. MATERIALS AND METHODS Pericarps in two of the more common Florida native Crotalaria, C. pumila and C. rotundifolia, are quite hard, Host plants were available on the University of Florida resembling peanut shells in consistency, and enclose few seeds campus in Gainesville. Experiments were conducted in the fall that are more watery and less nutritious (including lower PAs) of 2014, during which time seeds were in an appropriate stage than those of the exotics (Sourakov, 2015). For example, at the of development. Younger larvae normally feed exclusively on same stage of development, C. pumila seeds are 89% water vs. leaves, hence in our experiments we used larvae of early fourth 79% in African C. pallida and 77% in Asian C. spectabilis; PAs instar and older. vary in different populations of C. pumila from 0.04% to 0.6%, Three separate experiments were conducted: while in C. spectabilis they can be as high as 3.81% (Sourakov, • Experiment 1 tested native Crotalaria pumila and 2015; Williams & Molyneux, 1987; Burkill, 1995; Hartmann exotic C. spectabilis simultaneously et al., 2004). A larva feeding on the native C. pumila and C. • Experiments 2 and 3 tested exotic C. pallida rotundifolia would presumably have to expend a significant For the purpose of reducing genetic variability within each amount of energy breaking into these pods in order to consume of our trials, we used a split-brood design in experiments 1 and the nutrients and PAs required to complete their development 2 (experiment 3 was conducted using a batch of field-collected (see for example Fig. 3 and a supplementary video at https:// larvae). For Experiments 1 and 2, the two parental moths youtu.be/uA0BuWzqxOc). (fem#1 and fem#2) were netted behind the Florida Museum of In nature, the task will be additionally complicated by Natural History, Gainesville, Florida (29°38.0' N, 82°22.2' W) the fact that these pods are scattered about the plant and each on 22 Oct 2014. That population is associated mostly with C. plant produces only a few of them, so seeking the pods out lanceolata, yet C. pumila, C. spectabilis and C. pallida are also would also require energy and be a risky endeavor, as it would available to that population, although in smaller numbers. The involve crawling on the ground. In contrast, seeds of the Asian moths were fed a sugar and water solution and were kept in C. spectabilis are surrounded by softer pericarps akin to green individual 2 oz. clear plastic cups, in which they laid their eggs. peas. This species’ high alkaloid content may be a response to The larvae were raised inside such cups on C. lanceolata leaves different types of herbivores, as the plant is clearly extremely that were changed three times a week. toxic to vertebrates (e.g., Yan & Huxtable, 1995). Finally, C. At the 21st day of their development, when offspring pallida and C.