Influence of Diet and Density on Laboratory Cannibalism Behaviors in Gypsy Moth Larvae (Lymantria dispar L.)

Influence of Diet and Density on Laboratory Cannibalism Behaviors in Gypsy Moth Larvae (Lymantria dispar L.) Charles J. Mason, Zachary Cannizzo & Kenneth F. Raffa Journal of Insect Behavior ISSN 0892-7553 Volume 27 Number 6 J Insect Behav (2014) 27:693-700 DOI 10.1007/s10905-014-9458-0 1 23

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J Insect Behav (2014) 27:693 700 DOI 10.1007/s10905-014-9458-0 Influence of Diet and Density on Laboratory Cannibalism Behaviors in Gypsy Moth Larvae (Lymantria dispar L.) Charles J. Mason & Zachary Cannizzo & Kenneth F. Raffa Revised: 13 June 2014 /Accepted: 25 June 2014 / Published online: 3 July 2014 # Springer Science+Business Media New York 2014 Abstract The gypsy moth (Lymantria dispar) is an insect folivore that feeds on a broad range of hosts, and undergoes intermittent outbreaks that cause extensive tree mortality. Like many other herbivorous insects, gypsy moth larvae consume a substrate that is low in nitrogen. Gypsy moth larvae have been known to cannibalize under crowded conditions in the laboratory. In this study, we assessed the influence of nitrogen and density on cannibalism behavior in gypsy moth larvae. Cannibalism rates increased with decreased nitrogen and increased density. There was no interaction between these two parameters. Developmental experiments confirmed that low dietary nitrogen is detrimental, in agreement with previous studies. In a second experiment, we assessed the influence of previous cannibalism experiences on subsequent cannibalism behavior. Gypsy moth larvae that had previously cannibalized other larvae subsequently exhibited higher cannibalism rates than those larvae that had not cannibalized. In conclusion, low nitrogen, high larval density, and previous cannibalism experience are important factors contributing to gypsy moth larval cannibalism. Future studies are needed to estimate benefits to larvae, and to more closely approximate field conditions. Keywords Gypsy moth. cannibalism. nitrogen. density. plant-insect interactions Introduction Plants properties that adversely affect insect herbivores include both chemical and physical defenses (Carmona et al. 2011; Mithöfer and Boland 2012), and low concentrations of nutrients that are required for insect growth and reproduction. Nitrogen, among the most critical nutrients for insect development, typically occurs in lower quantities in most plant species and tissues than herbivores require (Mattson 1980). C. J. Mason: Z. Cannizzo : K. F. Raffa (*) Department of Entomology, University of Wisconsin-Madison, 1630 Linden Dr., Madison, WI 53706, USA e-mail: raffa@entomology.wisc.edu

694 J Insect Behav (2014) 27:693 700 Insect herbivores employ several physiological and behavioral mechanisms to cope with limited nitrogen contents, including nutritional symbioses (Douglas 2009), omnivory (Coll and Guershon 2002), compensatory feeding (for ex. Lindroth et al. 1997), and differential substrate feeding (Moreau et al. 2003). While many of these mechanisms are under strong genetic control, there are also more variable behaviors, such as cannibalism, that may supplement nitrogen and caloric intake. Cannibalism occurs among many animal species, including a diversity of arthropods. Cannibalism is not simply confined to stressed populations, but rather is a common response to changing environmental conditions (Fox 1975). Cannibalism in non-carnivorous insects is taxonomically diverse, occurring within many orders, and is frequently observed in Lepidoptera larvae (Richardson et al. 2010). Cannibalism occurs in many lepidopteran families, and can be among the largest sources of mortality in natural populations (Stinner et al. 1977). In addition to density, food quality is considered an important driver of cannibalism events (Kakimoto et al. 2003). The influence of cannibalism on Lepidoptera larval performance appears to be beneficial, as several studies have reported gains in development (Joyner and Gould 1985), diet processing (Raffa 1987), and survival (Chilcutt 2006) when compared to suboptimal diets. In addition to the direct benefits of cannibalism on insect fitness, cannibalism of natural populations may have indirect effects. For example, Chapman et al. (2000) found that cannibalism in Spodoptera frugiperda (Smith) can reduce the risk of predators responding to herbivory. Collectively, cannibalism potentially has important, yet poorly understood, consequences on larval fitness and population dynamics in a diversity of insect species. The gypsy moth (Lymantria dispar L.) is a foliage-feeding polyphagous herbivore that undergoes intermittent population outbreaks, in which they cause severe landscape-scale defoliation (Elkinton and Liebhold 1990; Davidsonetal. 1999). The gypsy moth has an extensive host range (Liebhold et al. 1995), encompassing a breadth of physical and chemical defenses, and nutritional qualities. Nitrogen availability is a major limitation in the performance of gypsy moth larvae (Hough and Pimentel 1978; Barbosa and Greenblatt 1979; Lindroth et al. 1997). The incorporation of nitrogen into eggs is a integral component of gypsy moth reproduction (Montgomery 1982), where larger eggs have higher quantities of vitellogenin proteins (Capinera et al. 1977). Egg size has been suggested to influence individual interactions, that may scale to population level-processes (Leonard 1970; CapineraandBarbosa1976). Like other herbivorous Lepidoptera, gypsy moth larvae partially contend with nitrogen limitations through compensatory feeding (Lindroth et al. 1997). Gypsy moth larvae cannibalize siblings in the laboratory (eg., Leonard and Doane 1966), although the factors influencing this behavior are not well understood. We assessed three factors that potentially contribute to gypsy moth cannibalism under laboratory conditions. First, we tested the effects of experimentally controlled larval densities and nitrogen concentrations on cannibalism rates. We then evaluated how previous cannibalism experiences influenced subsequent larval behaviors and performance. We used artificial diets modified to emulate nitrogen concentrations found in foliage, and densities mimicking field populations.

J Insect Behav (2014) 27:693 700 695 Methods Artificial Diet Preparation and Insect Rearing Wheat germ-based artificial diets were made following the methods described by Stockhoff (1993). We modified the casein and α-cellulose content to correspond to 1.25, 2.25, 3.0, and 4.75 % nitrogen weight to volume. These nitrogen concentrations represent contents found in foliar tissues in tree species among gypsy moths hosts over the growing season (Hough and Pimentel 1978). All other components of the artificial diet were identical among the various nitrogen treatments. Gypsy moth eggs were obtained from the USDA APHIS rearing facility in Otis, MA. The egg masses were hatched in 8.5 cm-diameter plastic petri dishes in a growth chamber under a 16 h light : 8 h dark photoperiod at 25 C. Preliminary experiments indicated newly hatched larvae suffered extensive mortality under low N diet concentrations, so all newly hatched larvae were first reared with 3.0 % N diet until molting into the 2nd stadium when the treatments were administered. Diet was replaced as needed, every two to three days. Effects of Diet and Larval Density on Cannibalism and Development Upon molting into the second star, larvae were randomly assigned a diet and density treatment. Larvae were randomly selected, and reared in 8.5 cm-diameter plastic petri dish arenas at densities of 5, 10, or 20 larvae per dish. Each arena was assigned one of four nitrogen treatments. Each group of insects was treated as an individual replicate, and each diet by density combination had four replicate arenas. Total cannibalism and the development of larvae were assessed three times per week. After four weeks, four, or all of the remaining larvae, were randomly selected and reared individually until pupation. The total cannibalism, days until pupation, and pupal masses were recorded for each experiment. Effect of Previous Cannibalism Experience on Subsequent Behaviors We assessed the effects of previous cannibalism experiences on subsequent cannibalism rates using two cohorts of gypsy moth larvae. Gypsy moths were hatched from two different egg masses 48 h apart and reared on 3.0 % nitrogen diet. When larvae from the first cohort (C1) molted into the 2nd stadium, they were reared in 24 well plates paired with a single larvae from the younger cohort (C2). In addition to the treatments to the C1 larvae, 80 C2 larvae were reared individually. All larvae were reared on 1.25 % nitrogen diet upon separation. Cannibalism by the C1 larvae was recorded every 12 h. Upon molting into 3rd stadium, C1 larvae that either had or had not engaged in cannibalism were placed into a separate 8.5 cm diameter arenas containing four C2 larvae. Larvae were fed 1.25 % nitrogen diet for two weeks, and diet was replaced every two to three days. The amount of cannibalism per arena was determined after two weeks.

696 J Insect Behav (2014) 27:693 700 Statistical Analyses All data were analyzed in the statistical package R v 3.0.1 (R Core Team 2013). The influences of dietary nitrogen content and larval density on gypsy moth cannibalism behavior were analyzed using a two-way analysis of variance in which both variables were treated as continuous. The influences of diet, density, and cannibalism rate (Note: for any treatment, this overall rate does not distinguish between which larvae did or did not cannibalize, so potential benefits from this behavior cannot be quantified) on the time to pupation and pupal mass were analyzed by fitting linear models to the data. We then analyzed the effects of density and cannibalism under each individual nitrogen treatment. The influence of previous cannibalism events on the proportion of subsequent larval cannibalism was analyzed using a two-sample t-test. Results General Observations of Cannibalism Behavior among Gypsy Moth Larvae Gypsy moth larvae cannibalized sibling larvae, but not eggs containing unhatched siblings. Cannibalism did not occur among first instar larvae, but rather began in the second instar. Oftentimes, the cannibalized individual was either initiating ecdysis or immediately following the molt. Older instars more readily cannibalized larvae that were not preparing to molt than did younger instars. Diet and Density Influence Cannibalism Rates Cannibalism rates by gypsy moth larvae increased under both low nitrogen (F 1,44 = 52.12, P<0.001) and high density treatments (F 1,44 =19.18, P<0.001; Fig. 1). There was no interaction effect (F 1,44 =0.31, P=0.58). Larval cannibalism was observed in at least one sample under all nitrogen and density combinations. At low larval densities, the level of cannibalism was similar among all but the lowest nitrogen treatment. At Fig. 1 Mean proportion of cannibalism (±1 standard error) by gypsy moth larvae under different nitrogen concentrations and larval densities. Nitrogen and density both had significant effect on larval cannibalism (P<0.001). The interaction had no effect (P=0.58)

J Insect Behav (2014) 27:693 700 697 higher densities, a distinction in cannibalism amounts between the other nitrogen treatments was observed, where the highest (4.75 %) had the least amount of cannibalism and increased as a factor of nitrogen concentration in the diet. The developmental studies confirmed that low dietary nitrogen decreases the size of resulting pupae (Table 1). As expected, increasing nitrogen content increased pupal mass and trended toward decreased time to pupation, but the latter effect was not statistically significant. Density showed an unexpected inverse relationship with development time, but this appears to be anomalous because this effect was always lost when analyzed within nitrogen categories. Similarly, cannibalism rate within a treatment showed a putative direct relationship with development time, but again cautious interpretation is needed, both because we cannot distinguish which larvae had cannibalized and this correlation was always lost within nitrogen categories. Previous Cannibalism Experience Increases Subsequent Cannibalism Behaviors Among larvae reared in unequally aged pairs, approximately half of the older, 2nd instar larvae exhibited cannibalism by the time they molted into the 3rd instar. Gypsy moth larvae that had cannibalized previously consumed more larvae than those larvae that had not engaged in a previous cannibalism event (Fig. 2). This amounted to an 85.7 % increase in cannibalism compared to the control insects over the same time (t df=6 =4.24, P=0.005). Discussion The increase in cannibalism rates with decreasing nitrogen content and increasing density may relate to the natural conditions under which this polyphagous, eruptive folivore feeds. Individual gypsy moth larvae commonly feed on many leaves, often of multiple species, having a broad range of nutritional qualities (Stoyenoff et al. 1994). Also, larvae from different population phases consume foliage of variable qualities, Table 1 Results of linear model fitting effects of diet, density, sex, and cannibalism on gypsy moth development time and pupal mass Time to pupation (days) Pupal Mass (g) Estimate t-value P-value Estimate t-value P-value All nitrogen treatments intercept 48.46 21.25 < 0.001 0.53 7.79 < 0.001 nitrogen 0.70 1.08 0.282 0.06 3.22 0.002 density 0.39 2.30 0.023 <0.01 0.50 0.617 nitrogen:density 0.03 0.60 0.548 <0.01 0.03 0.973 sex (male) 8.35 7.85 < 0.001 0.42 13.13 < 0.001 cannibalism a 7.94 3.52 < 0.001 0.05 0.80 0.425 a Cannibalism corresponds to the amount of cannibalism in the treatments rather than whether the insect, itself, had cannibalized

698 J Insect Behav (2014) 27:693 700 Fig. 2 Influence of previous cannibalism behavior on subsequent cannibalism rates. Larvae either had no cannibalism experience (black) or a single, previous cannibalism experience (gray). Asterisk indicates significant difference between treatments at P=0.005 various forest community structures provide larvae with different host species and age compositions, and seasonal fluctuations yield different degrees of phenological synchrony and foliar suitability (Elkinton and Liebhold 1990; Rossiter 1991; Hunter and Lechowicz 1992; Kleinerl and Montgomery 1994). This variability, specifically in nitrogen content and density, can influence the rate of cannibalism, at least under controlled laboratory conditions (Fig. 1). Increased cannibalism in response to low dietary nitrogen may be common in Lepidoptera (Al-Zubaidi and Capinera 1983). Additional factors that contributed to gypsy moth cannibalism were larval age and experience. We did not observe cannibalism in early first instar larvae. This is not unique to gypsy moth (Goodbrod and Goff 1990), but contrasts with some other insects (Pienkowskp 1964; Agarwala and Dixon 1992). Size differential is probably an important factor (Hopper et al. 1996), and the large quantities of urticating hairs covering gypsy moth larvae may pose a physical barrier against early but not later stadia. We observed that cannibalism began when insects were undergoing ecdysis early in the second instar, and continued throughout development. We found that larvae that had previously cannibalized had a significantly higher rate of cannibalism (Fig. 2). Our observations noted that treatments in which larval cannibalism was high tended to include more individuals that were much larger than others within comparable nitrogen availabilities and densities. However, our experimental design did not allow for comparative measurements between larvae that did or did not cannibalize, so future experiments are needed to test if this behavior represents a form of compensatory feeding. It may be that cannibalism provides an immediate benefit, enabling a larva to obtain enough nitrogen and calories to successfully molt into the next instar. In our study we attempted to emulate natural conditions by providing diet with realistic nitrogen concentrations and population along a continuum. However, two factors in nature for which we could not account were variation in foliar quality over the insect s lifetime and the opportunity for prey to escape cannibals. Also, higher-scale and tritrophic effects of cannibalism can include population regulation (Stinner et al. 1977), through reduced risk of predation (Chapman et al. 2000), culling of sick

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