Do trichome-covered fecal cases protect Neochlamisus leaf beetle larvae from arthropod

predators? A test of multiple mechanisms using N. platani

Christopher G. Brown
Department of Biological Sciences
VU Station B, Box 35-1634
Nashville, TN 37235-1634
(615) 936-3369
christopher.g.brown@vanderbilt.edu
Introduction
Predation 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 a
diversity of responses (e.g, behavioral, physiological, and morphological). One of the most
bizarre might be animals that build structures out of their own waste materials for defensive
purposes (Weiss, 2006). Perhaps the most elaborate fecal architecture of this kind belongs to the
case-bearing group of chrysomelid beetles, Camptosomata. I propose to test whether the fecal
cases 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 around
eggs, 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 of
building a case de novo. Case enlargement in Neochlamisus is an elaborate process that larvae
perform regularly until the case is sealed to the substrate prior to pupation (Fig. 1b). During this
stage of the life cycle, larvae are immobile and are particularly vulnerable to predation.
N. platani is exceptionally appropriate for the study of antipredatory construction because
of 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 in
appearance due to an abundance of defensive plant hairs, or trichomes, that are attached to the
outside 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 or
chemical 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 have
managed 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. Neochlamisus
platani performs another unique building behavior that may also be associated with defense. A
cross section of the cap left after an adult N. platani emerges from its pupal case, reveals an
extra compartment, or attic, filled with trichomes (Fig. 2) (Brown & Funk, 2005). This
compartment 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 the
case, push them into a pile toward the top of the case, and then seal the pile off from the rest of
the case interior, sometime before pupation. Examination of several caps of another species of
Neochlamisus whose host plants lack trichomes, N. comptoniae, revealed no extra space between
the pupal chamber and the outside wall of the case (Fig. 2c). This trichome attic may provide
further protection from predators by deterring them from breaking through the top of the case
and reaching the larva. Field-collected cases of various Neochlamisus taxa show damage to this
particular 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 the
fecal 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 energetically
costly (Venner et al., 2003; McKie, 2004; Stevens et al., 1999); construction of the attic, in
particular, demands some functional explanation that justifies this investment. Here I propose a
direct test of the hypotheses that fecal cases, trichome incorporation, and the extra compartment
of N. platani provide their bearers with protection from potential arthropod predators. Using
continuous-observation assays and various experimental modifications of larval cases, I will
observe 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 of
Neochlamisus cases for defense and their potential functions in terms of observed predator
behaviors.
Study animals
Neochlamisus platani are active in spring and summer and inhabit much of the eastern
United States (Karren, 1972; Thompson & Solomon, 1985). Eggs will be collected from
sycamore trees in the field during the spring of 2006 and maintained in the laboratory at
Vanderbilt University, Nashville, TN. Eggs will be kept in Petri dishes lined with filter paper
and housed in plastic boxes lined with moistened paper towels in an incubator at 25 C and a
14:10 light: dark schedule. Immediately after hatching, larvae will be individually reared on
sycamore leaves in 5 cm Petri dishes and maintained in the same manner as the eggs. Predatory
hemipterans (Podisus maculiventris), field crickets (Achetus domesticus), and ladybird beetle
larvae (Hippodamia convergens) will be ordered from commercial dealers and maintained in
plastic boxes. These groups represent an array of generalist predators with diverse feeding habits
that naturally encounter N. platani.
Experimental treatments
To 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 without
a case, (2) larva with a trichome-free case with attic intact, (3) larva with a trichome-covered
case with attic intact, (4) larva with a trichome-free case with attic opened, and (5) larva with a
trichome-covered case with attic opened. Separate experimental trials will be performed with
active larvae in the last instar and with pupating animals in cases affixed to the substrate to
evaluate the effects of larval movement on predator response.
Previous studies show that cases can be easily and nondestructively removed from active
larvae with soft forceps since larvae are not physically attached to their cases. The volume of
trichomes added to the exterior of the case can be manipulated by raising larvae on either
naturally 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 animals
whose cases contain high levels of trichomes or no trichomes. The trichome attic can also be
carefully opened and emptied without exposing the animal inside, because the attic is sealed off
from the immature beetle (Fig. 2).
Experimental design and protocols
I 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 predators
will be employed. A single predator will be placed in each arena 1-2 hours prior to testing to
allow it to acclimate to the arena.
At the beginning of each trial, one larva or pupa of a given experimental treatment will be
placed in the center of the arena. Predator and potential prey will then be observed for two
hours, during which time I will observe all test animals and record the following every five
minutes: (1) if the predator is ignoring the prey; (2) if the predator is approaching the prey; (3) if
the 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 has
killed 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: running
away, shaking the case, consistently orienting any part of the case toward attackers, or holding
the case firm to the substrate and remaining still. At the end of the trials, I will remove all of the
predators from the arenas and examine the prey more closely in order to determine if trichomes
were 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 trials
in total, using 100 individuals of each predator taxon and 300 N. platani, in order to test 10
replicates 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 only
one predator type. The predator types will be used consecutively across these three sets. Tests
will thus be conducted on each of 10 days.
At the end of these ten-day sessions I will run three additional sets of each experimental
trial, performing each individually. During this period I will observe one predator and prey pair
continuously and again record the behaviors of the predators listed above, the interaction
between the two participants, and the timing of events. Each trial will last two hours or until the
prey is eaten.
Analysis
There 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 cases
thwart the progress of predation. I will thus describe the analysis of the farthest step reached in
the sequence given above in each trial. Data will initially be treated as a random block design to
test for a significant day effect. If day is found not to be significant, data will then be treated as a
three-way factorial ANOVA with experimental treatment, prey stage, and predator type as fixed
factors to test for significant effects of each factor and their interactions on predation threat. I
will then use one-way ANOVAs and subsequent a posteriori multiple comparisons (Tukey's
honestly significant difference method) to test for differences among factor levels. Of particular
interest is the pattern of three experienced across the five treatment levels.
Preliminary Data
During 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 lack
trichomes and attic. Predators regularly attacked the beetles, usually quickly killing and
devouring the case-less larvae. N. bimaculatus larvae with cases experienced a significantly
smaller threat when facing A. domesticus than larvae without cases (P < 0.001). The numbers of
N. platani however were too small to determine any trend. Beetles with cases managed to
survive multiple attacks and immense damage from the crickets, which left scrapes and holes in
several cases.
Interpretations and general significance
These 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 will
separately aid Neochlamisus platani in defense against arthropod predators by increasing the
survival of beetles with cases, attics, and trichomes left intact over the larvae with any of these
components manipulated. I predict that survival of the experimental prey will be reduced in a
step-wise manner when each of these items is removed. A significant treatment x stage
interaction 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 physical
barrier to predator attack if predators attempt, but fail, to eat larvae with any of the case
components (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 aid
survival, i.e. predators are able to break through, then the addition of external trichomes and the
attic may prove to be key factors in larval survival. If predators avoid cases altogether, but
attack caseless beetles, then perhaps cases present a deterrent of unpalatable chemicals, possibly
sequestered from the beetles host plant (Morton & Vencl, 1998). These trials might also reveal a
significant predator effect indicating that the three tested predators react differently to N. platani
and a significant predator x treatment interaction will indicate that predators react differently to
certain case components.
No one has yet to perform a fully detailed study that evaluates the possibility of an
adaptive 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, fecal
cases, and trichomes and their effects on predators or other potential adaptive functions such as
aiding thermoregulation (Root & Messina, 1983; Damman & Cappuccino, 1991) and preventing
dehydration (Danks, 2004).
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Fig. 1. Photographs and diagrammatic illustrations depicting external and internal features of
Neochlamisus platani larval and pupal cases. The background has been altered on the
photographs to highlight the external trichomes.
Fig. 2. Trichome attic. (A) Location of attic in lateral view of mature N. platani case. (B) Dorsal
view of cross section of apex of N. platani case. (C) Dorsal view of cross section of apex
of N. bimaculatus case. (Modified from Brown and Funk 2005)
Fig. 3. Representation of experimental trials and the number of predators that will be exposed to
treatments. 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 attic
intact, (3) larva/pupa with a trichome-covered case with attic intact, (4) larva/pupa with a
trichome-free case with attic opened, and (5) larva/pupa with a trichome-covered case
with attic opened. One set of each predator type (beetle, cricket, true bug) will be run
consecutively each day of the study. This experiment will replicate each combination 10
times, one for each of 10 days of trials.
Fig. 4. Preliminary results from 2005 pilot studies, showing average threat to two species of
Neochlamisus larvae when presented to two predators: field crickets (Archetus
domesticus) and wheel bugs (Arilus cristatus). Sample sizes and standard error bars are
shown. See text for explanation of treatments. There is a significant difference in
average threat experienced by N. bimaculatus larvae with and without cases when
attacked by A. domesticus (P < 0.001).