Do trichome-covered fecal cases protect Neochlamisus leaf beetle larvae from arthropodpredators? A test of multiple mechanisms using N. platani

Christopher G. BrownDepartment of Biological Sciences

VU Station B, Box 35-1634Nashville, TN 37235-1634

(615) 936-3369christopher.g.brown@vanderbilt.edu

IntroductionPredation is a strong selective force due to its direct impact in fitness (Vermeij, 1982;

Brodie & Brodie, 1999). In order to avoid or reduce predation, organisms have evolved adiversity of responses (e.g, behavioral, physiological, and morphological). One of the mostbizarre might be animals that build structures out of their own waste materials for defensivepurposes (Weiss, 2006). Perhaps the most elaborate fecal architecture of this kind belongs to thecase-bearing group of chrysomelid beetles, Camptosomata. I propose to test whether the fecalcases of the sycamore leaf beetle, Neochlamisus platani Karren (Coleoptera: Chrysomelidae:Cryptocephalinae), serve to protect larvae from arthropod predators. These larvae are slow-moving and feed openly on leaves, and therefore are quite susceptible to attack from predators(Pasteels et al., 1988). Like other putative defensive structures, the cases may provide a physical(Eisner & Eisner, 2000; Brandt & Mahsberg, 2002) or distasteful barrier (Muller & Hilker, 1999;Morton & Vencl, 1998) to predator attacks.

Each N. platani case is actively constructed of layers of feces molded into a round tube(Brown & Funk, 2005). The case completely encompasses the curved abdomen of the larvae,but they can extend their heads and legs through a ventral opening for movement and feeding(Fig. 1a) (LeSage, 1984; Erber, 1988). Fecal cases begin as a coating that females wrap aroundeggs, but which larvae continuously enlarge to accommodate growth during the larval stages(Erber, 1988; Brown & Funk, 2005). Larvae never leave their cases and are incapable ofbuilding a case de novo. Case enlargement in Neochlamisus is an elaborate process that larvaeperform regularly until the case is sealed to the substrate prior to pupation (Fig. 1b). During thisstage of the life cycle, larvae are immobile and are particularly vulnerable to predation.

N. platani is exceptionally appropriate for the study of antipredatory construction becauseof additional structural aspects of its case that are not known for most other casebearing taxa.Specifically, individual cases of Neochlamisus platani are, to varying degrees, furry inappearance due to an abundance of defensive plant hairs, or trichomes, that are attached to theoutside of their cases (Figs. 1 and 2) (Riley, 1874; Brown & Funk, 2005; Chaboo et al., in prep.).Many plants have evolved trichomes as a defense against defoliation by presenting a physical orchemical barrier that deters small arthropods from reaching and feeding on the plant surface(Bernays, 1991; Valverde et al., 2001; Andres & Connor, 2003). However, some insects havemanaged to bypass these defenses and actually use trichomes for their own protection, e.g., via“trash packets” carried on the dorsum (Eisner et al., 2002; Medeiros & Moreira, 2002). In N.platani, trichomes may serve as camouflage or as a physical barrier to predators. Neochlamisusplatani performs another unique building behavior that may also be associated with defense. Across section of the ‘cap’ left after an adult N. platani emerges from its pupal case, reveals anextra compartment, or ‘attic’, filled with trichomes (Fig. 2) (Brown & Funk, 2005). Thiscompartment sits at the top of the pupal case, above the main chamber that houses the pupa, and

is separated by a thin layer of fecal material. Larvae must somehow bring trichomes into thecase, push them into a pile toward the top of the case, and then seal the pile off from the rest ofthe case interior, sometime before pupation. Examination of several caps of another species ofNeochlamisus whose host plants lack trichomes, N. comptoniae, revealed no extra space betweenthe pupal chamber and the outside wall of the case (Fig. 2c). This trichome attic may providefurther protection from predators by deterring them from breaking through the top of the caseand reaching the larva. Field-collected cases of various Neochlamisus taxa show damage to thisparticular region of the case, suggesting that it is indeed where predators typically seek entry.

Very little work has been published that empirically shows an adaptive function for thefecal cases of any of the casebearing beetles (but see Root & Messina, 1983; Wallace, 1970).However, building and maintaining a case is time consuming and is most likely energeticallycostly (Venner et al., 2003; McKie, 2004; Stevens et al., 1999); construction of the attic, inparticular, demands some functional explanation that justifies this investment. Here I propose adirect test of the hypotheses that fecal cases, trichome incorporation, and the extra compartmentof N. platani provide their bearers with protection from potential arthropod predators. Usingcontinuous-observation assays and various experimental modifications of larval cases, I willobserve the reactions, handling time, and success of three potential predators in response to N.platani larvae. This study will evaluate the relative contributions of different aspects ofNeochlamisus cases for defense and their potential functions in terms of observed predatorbehaviors.

Study animalsNeochlamisus platani are active in spring and summer and inhabit much of the eastern

United States (Karren, 1972; Thompson & Solomon, 1985). Eggs will be collected fromsycamore trees in the field during the spring of 2006 and maintained in the laboratory atVanderbilt University, Nashville, TN. Eggs will be kept in Petri dishes lined with filter paperand housed in plastic boxes lined with moistened paper towels in an incubator at 25 C and a14:10 light: dark schedule. Immediately after hatching, larvae will be individually reared onsycamore leaves in 5 cm Petri dishes and maintained in the same manner as the eggs. Predatoryhemipterans (Podisus maculiventris), field crickets (Achetus domesticus), and ladybird beetlelarvae (Hippodamia convergens) will be ordered from commercial dealers and maintained inplastic boxes. These groups represent an array of generalist predators with diverse feeding habitsthat naturally encounter N. platani.

Experimental treatmentsTo determine the contribution of the various aspects of N. platani fecal cases to thwarting

predators, I will expose each predator to the following experimental treatments: (1) larva withouta case, (2) larva with a trichome-free case with attic intact, (3) larva with a trichome-coveredcase with attic intact, (4) larva with a trichome-free case with attic opened, and (5) larva with atrichome-covered case with attic opened. Separate experimental trials will be performed withactive larvae in the last instar and with pupating animals in cases affixed to the substrate toevaluate the effects of larval movement on predator response.

Previous studies show that cases can be easily and nondestructively removed from activelarvae with soft forceps since larvae are not physically attached to their cases. The volume oftrichomes added to the exterior of the case can be manipulated by raising larvae on eithernaturally pubescent leaves or on leaves from which the trichomes have been manually removed.

Remaining trichomes can be removed with a razor or forceps. In this way, I will obtain animalswhose cases contain high levels of trichomes or no trichomes. The trichome attic can also becarefully opened and emptied without exposing the animal inside, because the attic is sealed offfrom the immature beetle (Fig. 2).

Experimental design and protocolsI will expose predators to the experimental treatments above using small, ca. 15cm x 5cm

x 5cm, plastic containers as arenas. Before the tests, an effective starvation period of predatorswill be employed. A single predator will be placed in each arena 1-2 hours prior to testing toallow it to acclimate to the arena.

At the beginning of each trial, one larva or pupa of a given experimental treatment will beplaced in the center of the arena. Predator and potential prey will then be observed for twohours, during which time I will observe all test animals and record the following every fiveminutes: (1) if the predator is ignoring the prey; (2) if the predator is approaching the prey; (3) ifthe predator is ‘investigating’ the prey (i.e. touching it with the legs, antennae, or mouthparts);(4) if the predator is attacking the prey, e.g., biting or piercing it; and (5) if the predator haskilled the prey. This represents a sequence (from 1 to 5) of increasing levels of threat to N.platani. I will also record any reactions of the larvae that may aid defense, such as: runningaway, shaking the case, consistently orienting any part of the case toward attackers, or holdingthe case firm to the substrate and remaining still. At the end of the trials, I will remove all of thepredators from the arenas and examine the prey more closely in order to determine if trichomeswere removed by the predator, whether or not the case was damaged, and if the prey survived.

No predator or experimental prey will be used in more than one trial. I will run 300 trialsin total, using 100 individuals of each predator taxon and 300 N. platani, in order to test 10replicates of both larvae and pupae from each experimental treatment with each predator (Fig. 3).Each day of the study I will run three sets of 10 simultaneous trials. Each set will consist of onlyone predator type. The predator types will be used consecutively across these three sets. Testswill thus be conducted on each of 10 days.

At the end of these ten-day sessions I will run three additional sets of each experimentaltrial, performing each individually. During this period I will observe one predator and prey paircontinuously and again record the behaviors of the predators listed above, the interactionbetween the two participants, and the timing of events. Each trial will last two hours or until theprey is eaten.

AnalysisThere are a variety of ways to quantify the data collected, and a number of insights that

can be learned from them, but for present purposes I will focus on the degree to which casesthwart the progress of predation. I will thus describe the analysis of the farthest step reached inthe sequence given above in each trial. Data will initially be treated as a random block design totest for a significant day effect. If day is found not to be significant, data will then be treated as athree-way factorial ANOVA with experimental treatment, prey stage, and predator type as fixedfactors to test for significant effects of each factor and their interactions on predation threat. Iwill then use one-way ANOVAs and subsequent a posteriori multiple comparisons (Tukey'shonestly significant difference method) to test for differences among factor levels. Of particularinterest is the pattern of three experienced across the five treatment levels.

Preliminary DataDuring the summer of 2005, I performed a subset of these tests using fourth instar larvae

of Neochlamisus platani and N. bimaculatus (another locally common species) and, as predators,the predaceous wheel bug Arilus cristatus and the omnivorous house cricket Acheta domesticus(Fig. 4). Experimental treatments included N. platani and N. bimaculatus larvae without cases,N. platani larvae with trichome-free cases with attics intact, N. platani larvae with trichome-covered cases with attics intact, and N. bimaculatus larvae with cases that naturally lacktrichomes and attic. Predators regularly attacked the beetles, usually quickly killing anddevouring the case-less larvae. N. bimaculatus larvae with cases experienced a significantlysmaller threat when facing A. domesticus than larvae without cases (P < 0.001). The numbers ofN. platani however were too small to determine any trend. Beetles with cases managed tosurvive multiple attacks and immense damage from the crickets, which left scrapes and holes inseveral cases.

Interpretations and general significanceThese trials will provide knowledge about the function of a unique animal construction

and a number of outcomes are possible. I predict that fecal cases and trichome incorporation willseparately aid Neochlamisus platani in defense against arthropod predators by increasing thesurvival of beetles with cases, attics, and trichomes left intact over the larvae with any of thesecomponents manipulated. I predict that survival of the experimental prey will be reduced in astep-wise manner when each of these items is removed. A significant treatment x stageinteraction will indicate that certain case components do not protect larvae and pupae equally.

Observations of predators and predator damage may show that cases present a physicalbarrier to predator attack if predators attempt, but fail, to eat larvae with any of the casecomponents (Muller & Hilker, 1999). Each additional component may increase the predators'handling time or their propensity to give up an attack. Even if the case itself does not aidsurvival, i.e. predators are able to break through, then the addition of external trichomes and theattic may prove to be key factors in larval survival. If predators avoid cases altogether, butattack caseless beetles, then perhaps cases present a deterrent of unpalatable chemicals, possiblysequestered from the beetles’ host plant (Morton & Vencl, 1998). These trials might also reveal asignificant predator effect indicating that the three tested predators react differently to N. plataniand a significant predator x treatment interaction will indicate that predators react differently tocertain case components.

No one has yet to perform a fully detailed study that evaluates the possibility of anadaptive function provided by the fecal cases of the chrysomelid group Camptosomata. N.platani especially presents an opportunity to evaluate compelling aspects of animal architecture.Further studies may include tests of the chemical components of larvae, pupae, fecalcases, and trichomes and their effects on predators or other potential adaptive functions such asaiding thermoregulation (Root & Messina, 1983; Damman & Cappuccino, 1991) and preventingdehydration (Danks, 2004).

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Fig. 1. Photographs and diagrammatic illustrations depicting external and internal features ofNeochlamisus platani larval and pupal cases. The background has been altered on thephotographs to highlight the external trichomes.

Fig. 2. Trichome attic. (A) Location of attic in lateral view of mature N. platani case. (B) Dorsalview of cross section of apex of N. platani case. (C) Dorsal view of cross section of apexof N. bimaculatus case. (Modified from Brown and Funk 2005)

Fig. 3. Representation of experimental trials and the number of predators that will be exposed totreatments. The treatment numbers correspond to the experimental condition of each N.platani: (1) larva/pupa without a case, (2) larva/pupa with a trichome-free case with atticintact, (3) larva/pupa with a trichome-covered case with attic intact, (4) larva/pupa with atrichome-free case with attic opened, and (5) larva/pupa with a trichome-covered casewith attic opened. One set of each predator type (beetle, cricket, true bug) will be runconsecutively each day of the study. This experiment will replicate each combination 10times, one for each of 10 days of trials.

Fig. 4. Preliminary results from 2005 pilot studies, showing average threat to two species ofNeochlamisus larvae when presented to two predators: field crickets (Archetusdomesticus) and wheel bugs (Arilus cristatus). Sample sizes and standard error bars areshown. See text for explanation of treatments. There is a significant difference inaverage threat experienced by N. bimaculatus larvae with and without cases whenattacked by A. domesticus (P < 0.001).