The masked birch caterpillar, Drepana arcuata (Lepidoptera: Drepanidae) is an excellent model for studying vibratory communication and sociality in larval insects. Vibratory communication occurs throughout development, but the functions of signals are reported to change as larvae change from gregarious to solitary lifestyles. To better understand the sensory ecology of these caterpillars, it is important to study their life history. Here, we describe the morphological and behavioral characteristics of larvae by confirming the number of instars, identifying their distinguishing morphological features, and noting changes in feeding and shelter construction. Five instars were confirmed based on the number of head capsules collected for individuals throughout development, and by using Dyar’s rule, which predicts the number of instars based on geometric growth patterns of head capsules. Frequency distributions of head capsule widths showed five separate peaks, indicating that this is a useful parameter for distinguishing between instars. Other morphological features including body length, shape, and banding patterns of head capsules, and morphology of thoracic verrucae are helpful in distinguishing among instars. Feeding behavior changes from leaf skeletonization in first and second instars to leaf cutting in fourth and fifth instars, with third instars transitioning between these feeding styles as they grow. Early instars typically construct communal silken shelters whereas late instars live solitarily in leaf shelters. These results provide essential life history information on the masked birch caterpillar that will enable future investigations on the proximate and ultimate mechanisms associated with social behavior and communication in larval insects. Key words: caterpillar, behavior, leaf shelter, ontogeny, morphology Larvae of the arched hooktip moth, Drepana arcuata Walker D. arcuata is broadly distributed throughout northern and (Lepidoptera: Drepanidae) have been studied as a model for larval east-southeastern North America (Rose and Lindquist 1997). Host vibratory communication, a poorly understood mode of commu- plants of D. arcuata include Betula papyrifera Marshall (Fagales: nication in juvenile insects (Yack 2016). Late instars are territorial Betulaceae), Betula populifolia Marshall (Fagales: Betulaceae), and generate ritualized acoustic signals to defend leaf shelters from Betula glandulosa Michx. (Fagales: Betulaceae), Betula alleghanien- conspecifics (Yack et al. 2001, 2014; Scott et al. 2010; Guedes et al. sis Britton (Fagales: Betulaceae), Alnus rubra Bong. (Fagales: 2012). Early instars, unlike late instars, are reported to live in small Betulaceae), and Alnus rugosa (incana) (L.) Moench (Fagales: groups and a recent study demonstrates that vibrational signaling is Betulaceae) (Handfield 1999). Previous studies have reported on var- associated with recruitment of conspecifics (Yadav et al. 2017). In ious life history, morphological, behavioral, and physiological traits addition, vibration signals are proposed to function in other social (Packard 1890, Dyar 1895, Beutenmuller 1898, Stehr 1987). Adults interactions among early instars living in groups (Matheson 2011). are medium-sized, broad-winged with hooked tips on the forewings The masked birch caterpillar (adults are referred to as arched hook- (Fig. 1). They possess abdominal ears that are ultrasound-sensitive tip moths) offers great potential for studying the roles of vibratory and proposed to function in bat detection (Surlykke et al. 2003). communication and mechanisms mediating social interactions in lar- Previous reports on immature stages provide mostly anecdotal val insects. To proceed with such investigations, it is important to be details on the morphology or behavior of late instars (sometimes able to identify the larval stages, and to understand how life history referred to as ‘mature’ larvae) (e.g., Dyar 1895, Beutenmuller 1898, traits change with each stage. Stehr 1987). A more detailed study by Packard (1890) provides © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/18/4904262 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Journal of Insect Science, 2018, Vol. 18, No. 1 of 4–6 because of their lower survivorship when reared individually (personal observation, J. E. Yack). Jars and petri dishes were exam- ined daily to collect head capsules for subsequent measurements, to take photographs, to monitor feeding and shelter building activities, and to refresh food supplies. This enabled us to keep track of molting and keep track of head capsules for each individual caterpillar. A few individuals of each instar were preserved in 75% ethanol. Morphology Morphological features were assessed from live individuals, ethanol preserved specimens and from shed head capsules. Each live indi- vidual was examined at least once within 24 h of molting, and the final (fifth) instar was examined for an additional time period to document the prepupal stage. A number of morphological features, including color, setae, verrucae, and body length were recorded from Fig. 1. Adult moth D. arcuata in resting position on a birch leaf. Scale bar: live larvae in their natural resting positions on leaves. Shed head 5 mm. capsules were measured across the widest part for each larval stage (Dyar 1890) for instars I–IV. Because head capsules were deformed morphological descriptions of larval stages, but this study was following ecdysis from fifth instar, these measurements were taken limited in that it followed only a few, unspecified numbers of indi- directly from live larvae on days 3–4 of the fifth instar. viduals and did not provide objective or quantifiable measures for Photographs were taken using a stereomicroscope (Leica distinguishing between larval stages. Furthermore, Packard (1890) M205 C, Leica, Wetzlar, Germany) equipped with a camera (Leica provided limited information on instar-specific behaviors, and for DMC4500, Leica, Wetzlar, Germany). Measurements, z-stacked only certain instars. Previous studies focusing on vibratory commu- images, and videos were obtained using Leica application suite V 4.2. nication (Yack et al. 2001, 2014; Scott et al. 2010; Matheson 2011; A Nikon Coolpix camera (4500, Nikon, Japan) was used to obtain Guedes et al. 2012; Yadav et al. 2017) described some characteristics images of eggs, pupae, and adults. For scanning electron micrographs, of leaf shelters, conspecific communication, predator detection, and head capsules were air-dried and mounted on aluminum stubs, sput- morphological features associated with signal production in uniden- ter-coated with gold-palladium, and examined using a Tesca Vega-II tified ‘early’ or ‘late’ instars. Currently, there are no formal studies XMU scanning electron microscope (XMU VPSEM; Brno, Czech documenting instar-specific morphological and behavioral traits. Republic). Identification and naming of various morphological traits The goals of this study are to document the number of instars, iden- followed the nomenclature of Stehr (1987) and Scoble (1992). tify morphological criteria for distinguishing between instars and to note stage-specific behaviors associated with sociality including feed- Behavioral Observations ing, grouping, and shelter building. Behaviors were monitored and documented with photographs and videotapes at various times following 24 h after the molt for each Methods instar. Feeding style was noted as being either by skeletonization (feeding only on the green tissue between the leaf veins) or cutting Insect Collection and Rearing (whereby the mandibles cut through the full leaf). Shelter construc- D. arcuata (Lepidoptera: Drepanidae) were collected as moths from tion behaviors, including the patterns of silk deposition and location ultraviolet lights at the Queen’s University Biology Station (Chaffey’s on the leaf, were recorded. Records were also made on each instar’s lock, ON, Canada, 44.5788° N, 76.3195° W) and a few other loca- tendency to live solitarily or in groups. However, because the nature tions close to Ottawa, Ontario, Canada (45.4215° N, 75.6972° W) of the rearing process (designed to follow individuals to collect head between May and September, 2010–2015. Gravid females were held capsules) may have impeded the caterpillars’ natural tendencies to in glass jars where they oviposited on paper birch (B. papyrifera) cut- group, a separate study on instar-specific grouping behaviors would tings or brown paper bag clippings. Using a fine paint brush, neonates be required. were transferred to fresh birch cuttings held in plastic vials and reared indoors at room temperature (21–23°C and 16 h: 8 h light:dark). Measurements to Distinguish Larval Stages To determine the number of instars, neonate larvae were followed throughout their development. On the day of hatching, four to six We measured head capsule widths and body lengths of larvae in each neonates (first instars, 59 in total and obtained from >10 females) instar in order to confirm the total number of instars and to dis- were transferred to leaves contained in a polystyrene petri dish tinguish between larval stages. To confirm the number of instars, (Falcon, 100 × 15 mm) (number of petri dishes = 14) lined with mois- in addition to counting the number of shed head capsules by fol- tened paper towels. Larvae of the same age (i.e., hatched within 12 h lowing individuals, we also used Dyar’s rule (Dyar 1890, Gaines of each other) were placed in petri dishes in small groups. First instars and Campbell 1935, Cazado et al. 2014). Dyar’s growth ratio was were kept in petri dishes instead of jars to facilitate collection of their calculated by dividing mean head capsule width of one instar by very small head capsules. After molting to second instar, larvae were the mean head capsule width of the preceding instar and then cal- transferred to twigs of paper birch with 5–10 leaves. Birch twigs were culating the average growth ratio for all instars. We plotted the nat- inserted into the lids of water-filled plastic vials and care was taken ural log of the mean head capsule width for each instar against the to seal the bases of the twigs using reusable adhesive putty (Staples) number of instars and conducted linear regression analysis to deter- to prevent wandering larvae from drowning. Each twig, in turn, was mine if larvae follow a regular geometric growth progression (Dyar enclosed in large glass jars (23.5 × 14 cm) lined with moistened paper 1890, Gaines and Campbell 1935). To identify morphological traits towels (number of jars = 10). Early instars were reared in small groups for distinguishing between larval stages, we plotted both normal Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/18/4904262 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 1 3 distribution values of head capsule widths and body lengths (within subdorsal surface running along the segments from T1 to A10. Pairs 24 h of molting from preceding instar) of larvae in each instar and of brown-black dorsal and subdorsal structures called tubercles by conducted analysis of variance (ANOVA) by using one-way ANOVA Packard (1890) and verrucae by Stehr (1987) are present on thoracic tests followed by Tukey’s HSD in R Studio 1.0.136 (R Core Team (T1, T2, T3) and abdominal segments. We follow Stehr’s term “ver- 2016) package Agricolae (de Mendiburu 2016). All the statistical rucae” to provide descriptions of these setae. Verrucae on thoracic analyses in this study were performed at P < 0.05. (T1, T2, T3) and abdominal segments (A9, A10) are slightly more prominent than those on other segments. The subdorsal verrucae on T1 are the most prominent. Dorsal verrucae on thoracic (T2, T3) Results segments bear one seta whereas the subdorsal verrucae bear two setae. Greyish-black, elongated thoracic legs are present on T1–T3; General Comments on Immature Stages and 4 pairs of light brown, thick, rounded abdominal prolegs are present Rearing on A3–A6, and the anal proleg on A10 is absent. Clear, forked setae Our results confirm that D. arcuata has five larval instars. Twenty- arise from the dorsal and lateral surfaces on all thoracic and abdom- two individuals were followed from neonatal to pupal stages. More inal segments. A uranal plate is formed by the last abdominal seg- data were collected for early than late instars for two reasons (1) ment (A10) and has a small, brownish, conical, bifurcated projection higher mortality was observed in early instars (25%) and; (2) 1–3 (called a “knob” or “process” by Stehr, 1987), is covered with very representatives for each instar were preserved in 75% ethanol for short bristles, and two long setae emerging from the bifurcated tip. reference (Instar I, II, III = 3 larvae of each, IV, V = 2 larvae of Developmental time (mean ± SD, n = 42) = 4.36 ± 0.90 d. each). Development time from hatching to pupation took 16–22 d (mean = 19.32 ± 1.73, n = 22). Feeding and Shelter Construction Post hatching, neonate larvae wander individually until they find a Eggs location to feed and build a shelter, eventually forming small groups. Morphology Larvae construct a tent-like silk shelter by first laying a silk mat on Eggs were smooth, polished, flattened and oval (Fig. 2). Diameter: either the upper or lower leaf surface, followed by spinning silk 0.64–0.84 mm (mean = 0.77 ± 0.03 mm, n = 46). Adults laid eggs in threads, slightly folding the leaf edge. Larvae begin constructing the rows of 2–14 on both the upper and lower surfaces of leaves, as well shelter by first spinning two silk strands on either side of the shelter as on the plant stems, paper bag clippings, and the sides of the glass which they then extend into multiple cell-like units by attaching jars. The color of fertilized eggs changed from yellow when laid, smaller silk strands with slight webbing. The location of the shelter to orange-brown, reddish-brown, and then to black as they neared is variable, with most shelters (~85%) formed at the edges of leaf. hatching. It took approximately 9–11 d for eggs to hatch. Neonates The size of the shelter also varies depending on the size of group hatched at different times, with the exit holes about one-third diam- contributing to shelter construction (e.g., 0.7 cm for a group of two eter of the eggs shells, and oriented away from adjacent eggs. individuals to 2 cm with seven individuals). First instars typically make only one shelter during this stage, and molt within the same First Instar shelter. Shelter building activity alternates with resting, walking, and Morphology feeding behaviors within the shelter. Larvae attach frass to the silk Head capsule width: 0.26–0.31 mm (mean = 0.29 ± 0.01 mm, canopy of the shelter (Fig. 4B). Larvae skeletonize the leaf surface n = 42), body length: 1.75–2.93 mm (mean = 2.36 ± 0.33 mm, within the shelter, with feeding spots of variable size and number n = 42) (Figs. 3 and 4; Tables 1 and 2). Head capsule black, shiny, depending on the number of individuals residing within the shelter. granulated, triangular, rounded on top with a slight notch at the When several larvae reside together in shelters they often, but not dorsal end of epicranial suture; head is approximately the same always, work on edges of the same feeding spot. They extend both width or slightly wider than the body. Body mostly dark brown- the feeding spots and shelters as they skeletonize the leaf tissue. black with bright pale colored prothoracic segment (T1) and abdom- Larvae tended to form small groups within 24 h of placing them in inal segments A1, A7; two dark brownish black parallel lines on the petri dishes. Fig. 2. Eggs of D. arcuata laid in rows on birch leaves. (A) One day old yellow eggs (left) and 3 days old light orange colored eggs (right) laid on the upper surface of a birch leaf. Scale bar: 4 mm; (B) Nine days old dark brown-black colored eggs laid on the underside of a birch leaf. Scale bar: 4mm. (C) Hatched eggs showing exit holes. Scale bar: 500 µm. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/18/4904262 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Journal of Insect Science, 2018, Vol. 18, No. 1 Fig. 3. Light micrographs (left) and scanning electron micrographs (right) of D. arcuata head capsules: (A) first instar. Scale bar: 100 µm; (B) second instar. Scale bar: 200 µm; (C) third instar. Scale bar: 300 µm; (D) fourth instar. Scale bar: 500 µm (E) fifth instar. Scale bar: 500 µm. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/18/4904262 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 1 5 Fig. 4. Lateral (left) views of each instar and dorsal views of larvae within their characteristic shelter (right). (A) First instar lateral view. Scale bar: 500 µm; (B) first instars in shelter. Scale bar: 2,000 µm; (C) second instar lateral view. Scale bar: 1,000 µm; (D) second instars in shelter. Scale bar: 2,000 µm; (E) third instar lateral view. Scale bar: 2,000 µm; (F) third instar in shelter. Scale bar: 3,000 µm; (G) fourth instar lateral view. Scale bar: 2,000 µm; (H) fourth instar in shelter. Scale bar: 3,000 µm; (I) fifth instar lateral view. Scale bar 5,000 µm; (J) fifth instar in shelter. Scale bar: 5,000 µm. Second Instar conical projection at the end of the suranal plate is less well pronounced than in first instars, covered with more conspicuous black setae. A pair of Morphology setae, emerging from the bifurcation, is shorter than the projection itself. Head capsule width: 0.46–0.56 mm (mean = 0.52 ± 0.03 mm, n = 32), Developmental time (mean ± SD, n = 32) = 4.21 ± 0.64 d. body length: 3.09–5.58 mm (mean = 3.85 ± 0.67 mm, n = 32) (Figs. 3 and 4; Tables 1 and 2). Head capsule differs from the first instar primar - ily in color and banding pattern, with the color becoming lighter brown Feeding and Shelter Construction and the appearance of two, not so well pronounced, dark brown trans- Following the molt from first instar, the exoskeleton is consumed in verse bands across the head. Overall, the body color is lighter brown but most cases (in >90% of the cases) while the head capsule is often with the same color pattern as first instars. Lateral verrucae on thoracic attached to the overhanging strands of the silk shelter (in >80% of segment T1 similar in size to the dorsal and subdorsal verrucae on tho- the cases), or present on the floor of silk shelter (<20% of the cases). racic segments T2 and T3; dorsal and subdorsal verrucae are present on Second instars either continue extending the same first instar shelter T2, T3, and lateral verrucae on abdominal segment A9 are slightly more or make a new shelter on the same leaf. Feeding and shelter construc- prominent and conspicuous than in the first instar. Bifurcation of the tion behaviors are similar to those observed in the first instar. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/18/4904262 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6 Journal of Insect Science, 2018, Vol. 18, No. 1 for resting and feeding that consist of only a few (2–4) thick silk Third instar strands with no cells and webbing. Some larvae were observed to Morphology remove frass from the shelter by either backing up and flicking over Head capsule width: 0.77–1.02 mm (mean = 0.92 ± 0.06 mm, the edge of the leaf, or by picking it up in their mandibles, walking n = 28), body length: 4.20–7.50 mm (mean = 5.72 ± 0.91 mm, to the leaf edge and dropping it. Like late third instars, fourth instars n = 28) (Figs. 3 and 4; Tables 1 and 2). Head capsule differs from feed by cutting the leaf and consuming the shelter as they feed. Once the second instar in shape, color, and banding pattern; the shape the larva consumes the entire leaf, it wanders in search of a new leaf becomes slightly round, the color is yellowish-green, and there are to construct a shelter. two clear, brown transverse across the head. Overall, the body color changes to yellowish green with a similar pattern as previous instar. Lateral verrucae on T1 are reduced in size compared to dorsal and Fifth Instar subdorsal verrucae on T2 and T3; dorsal and subdorsal verrucae on Morphology thoracic segments T2, T3, as well as other verrucae on abdominal Head capsule width: 1.56–2.21 mm (mean = 2.01 ± 0.18 mm, segments, are much more prominent than in the second instar; ver- n = 22), body length: 10.00–20.50 mm (mean = 13.95 ± 3.01 mm, rucae on abdominal segments are lighter yellow to red. Thoracic legs n = 22) (Figs. 3–5; Tables 1 and 2). Morphology and color patterns are blackish-brown, and abdominal prolegs are yellowish-brown to of head capsules and body do not change significantly from fourth green. The conical projection at the end of suranal plate is rust col- instar; dorsal abdominal body color turns rust-brown. Dorsal and ored with a black tip and more conspicuous black setae. subdorsal verrucae on thoracic segments T2, T3 more prominent Developmental time (mean ± SD, n = 28) = 3.75 ± 0.81 d. than other verrucae on the body and compared to T2, T3 thoracic verrucae on the previous instar; dorsal verrucae almost double the size of subdorsal ones with a yellow base and bright red tip; lateral Feeding and Shelter Construction verrucae on A9 green and inconspicuous. Prepupal larvae become Following the second instar molt, third instars continue to feed enlarged, with the head capsule noticeably more narrow than the within the same shelter by skeletonizing the leaf tissue, but then tran- body; dorsal surface of abdominal segments turns brownish-red sition to cutting the leaf edges, moving inwards and consuming silk with green thoracic segments; prominent verrucae are only present strands as they feed. Within 24 h of molting, third instars move out on thoracic segments T2, T3. of the early instar shelter and make a new solitary shelter on the edge Developmental time (mean ± SD, n = 22) = 4.41 ± 0.80 d. or tip of a leaf. Leaf skeletonization is no longer observed after this point in their development. Shelter construction involves more fold- ing of the leaf compared to early instars with occasional attachment Feeding and Shelter Construction of frass to the overhanging silk strands. Shelters consist of thicker Same as observed in fourth instar. Prior to pupation, larvae may silk strands joining one end of leaf to another, thus folding the leaf wander and eat multiple leaves before settling into a silk shelter that more so than in earlier instars. Prior to molting into fourth instar, they construct specifically for pupation. A fifth instar feeds for 2–3 d third instars construct a “premolting” shelter that can either be a before entering prepupal stage and then continues feeding for 1–2 d new shelter or a modification of the existing shelter. The premolting before folding the leaf. Once the leaf is entirely folded, it takes 2–3 shelter comprises additional layers of silk, making it denser. d for the pupa to form. This shelter consists of several (~5–10) thick silk strands that fold the leaf edge tightly, encasing the pupa. Fourth Instar Morphology Pupa Head capsule width: 1.20–1.75 mm (mean = 1.46 ± 0.12, n = 25), Morphology body length: 6.62–10.60 mm (mean = 8.50 ± 1.08, n = 25) (Figs. 3 Length: 11.19–13.84 mm (mean = 12.49 ± 0.88, n = 10); width: and 4; Tables 1 and 2). Head capsule differs from previous instar 3.67–4.40 mm (mean = 3.98 ± 0.30, n = 10) (Fig. 5). The pupa is mainly with respect to shape, becoming completely round, and medium to dark brown colored with fine hair-like setae present on bilobed with a prominent, well-defined notch at the epicranial abdominal segments. Darker brown to black colored spiracles are suture. Overall body color changes to lighter green, with a green tho- also present on the abdominal segments. rax and abdominal segments green mottled with brown spots; the Duration: the duration of the pupal stage is temperature depend- pattern is the same as the preceding instar. Lateral verrucae on T1 ent. We did not formally measure this, but in general, the duration reduced in size, becoming flat; dorsal and subdorsal verrucae on T2, is about 2 wks at room temperature (~21–23°C). Pupae can also T3 are more conspicuous and prominent than others on abdominal successfully overwinter for over a year at 4–6°C (J. E. Yack, unpub- segments and compared to the verrucae on T2, T3 of the previous lished observations). instar; dorsal verrucae on T2, T3 are bigger than subdorsal ones with a yellowish-green base and black tip; distinctly visible oval, green Comparisons Between Sizes of Instars spiracles with brown outline present on T1, A1–A8. Thoracic legs Five head capsules were collected for each of the individuals followed and abdominal prolegs are green. The conical projection at the end throughout the larval development from hatching to pupation. When of A10 is bright rust-red colored with a small pair of setae emerging we plotted the distribution of head capsule widths using normalized from the significantly reduced bifurcation. values (Fig. 6A), five distinct peaks were observed. Furthermore, head Developmental time (mean ± SD, n = 25) = 3.55 ± 0.80 d. capsule widths of each instar were significantly different from each other at P < 0.05 (one-way ANOVA; F = 1,797, P < 0.0001 and 4,144 Feeding and Shelter Construction Tukey’s HSD test). This indicated that there are five distinct instars Larvae lay a silk mat on the leaf surface as do previous instars but and head capsule width is a good indicator of instars. Furthermore, while constructing the shelter fold the leaf significantly more so than the natural log of head capsule widths plotted against instars showed third instars. Fourth instars also construct feeding and molting shel- geometric larval growth through development (Fig. 7; Dyar’s aver- ters as described for third instars. Larvae may make multiple shelters age growth ratio: 1.63) hence further confirming five instars for Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/18/4904262 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 1 7 Fig. 7. Natural log of mean head capsule widths plotted against the number of instars. The figure shows a linear geometric progression (R = 0.98, P = 0.0005), confirming five instars for D. arcuata. Table 1. Head capsule measurements for instars (I–V) of D. arcuata Instar N Range (mm) Mean ± SD (mm) Coefficient of Dyar’s variation ratio I 42 0.26–0.31 0.29 ± 0.01 0.04 — II 32 0.46–0.56 0.52 ± 0.03 0.05 1.79 II 28 0.77–1.02 0.92 ± 0.06 0.06 1.79 IV 25 1.20–1.75 1.46 ± 0.12 0.08 1.57 V 22 1.56–2.21 2.01 ± 0.18 0.09 1.40 Fig. 5. Light micrographs of prepupa and pupa of D. arcuata. (A) Prepupal phase showing thick strands characteristic of pupal shelters. Scale bar: 500 µm. (B) As silk dries, it contracts forming a tight leaf enclosure for the Dyar’s ratio (e.g., for instar II): mean head capsule width of Instar II/Mean pupa. Pupa is shown in the inset. Scale bar: 500 µm. head capsule width of instar I Table 2. Body length measurements for instars of D. arcuata Instar N Range (mm) Coefficient of Mean ± SD (mm) variation I 42 1.75–2.93 0.14 2.36 ± 0.33 II 32 3.09–5.58 0.17 3.85 ± 0.67 III 28 4.20–7.50 0.16 5.72 ± 0.91 IV 25 6.62–10.60 0.13 8.50 ± 1.08 V 22 10.00–20.50 0.22 13.95 ± 3.01 test) but as expected there was more overlap in lengths across instars with higher coefficients of variation for each instar than for head capsule widths (Tables 1 and 2; Fig. 6B). Discussion Two main goals of this study were to confirm the number of instars and to establish morphological criteria to distinguish between these instars. By following molts and collecting head capsules for multi- ple individuals, we confirmed that D. arcuata larvae undergo five instars. We further confirmed five instars using Dyar’s rule (1890). Dyar showed a more or less constant, geometric progression in lar- val head capsule widths for 28 larval lepidopteran species. Dyar’s Fig. 6. Distribution of head capsule widths and body lengths for D. arcuata rule (1890) has been used to identify the number of larval stages in instars. (A) Normalized distribution of head capsule widths; (B) normalized several insects (e.g., McClellan and Logan 1994, Delbac et al. 2010, distribution of body lengths recorded at 24 h following molt for each instar. Velásquez and Viloria 2010, Barrionuevo and San Blas 2016). Head D. arcuata. Body lengths for larvae in each instar were also meas- capsule width is the most reliable way to identify a larval stage, as it ured within 24 h of molting. Although we observed five peaks cor - does not change within an instar. responding to five instars with each significantly different from each In addition to head capsule width and body length, a number other (one-way ANOVA; F = 328.8, P < 0.0001 and Tukey’s HSD of other morphological characteristics differed between instars. First 4,144 Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/18/4904262 by Ed 'DeepDyve' Gillespie user on 16 March 2018 8 Journal of Insect Science, 2018, Vol. 18, No. 1 hatching, early instars (I, II) use vibrational signaling in the forma- instars differ from all others based on their characteristic black, tri- tion of small groups (Yadav et al. 2017), whereas in late instars (IV, angular head, and dark brown body. Second instars develop faint V) vibrational signaling is used in territorial encounters (Yack et al. brown bands on their heads with comparatively more prominent 2001, 2014; Guedes et al. 2012). It is still unknown how early instar verrucae on thoracic and abdominal segments. Third instars are the groups are maintained, but vibratory signals likely play a role. Third first to develop two clear, distinct brown transverse bands on the instars exhibited both behaviors in that they resided in the commu- head, with the head becoming more rounded than oval. Between nal shelter for a period following their molt, but within 24–48 h third and fourth instar, the head becomes more rounded, thoracic of molting became solitary. Gregariousness is an important life his- legs and abdominal prolegs become green in color, and the overall tory trait observed in a number of lepidopteran larvae with a large body color becomes lighter green mottled with brown on abdom- number of species living gregariously either throughout or through inal segments. Aside from head capsule width and body size, the a part of their development (Costa and Pierce 1997, Costa 2006). most noteworthy change from fourth to early fifth instar is the very However, the mechanisms mediating sociality in larval Lepidoptera prominent bright red-tipped dorsal verrucae on thoracic segments requires further study. The masked birch caterpillar is an excellent T2 and T3. However, as fifth instars reach the prepupal stage their model to investigate the multimodal mechanisms involved, as they dorsal abdominal surface becomes rust-brown, and the overall body clearly transition from being gregarious to solitary during devel- becomes enlarged with the head size almost half as wide as the body. opment, produce vibratory signals throughout their development, While we did find consistency in body color within instars, this mor - while at the same time possibly using chemical cues associated with phological feature should be further studied in larvae reared on dif- silk in their shelters. In particular, the interesting transitional fea- ferent colored leaves, it has been noted that body color of late instars tures of third instars provide opportunities to study physiological may vary depending on the leaf color (personal observation, J. E. and genetic mechanisms underlying social behavior in larval insects. Yack), and therefore this feature should be used cautiously in distin- guishing between instars, and warrants further attention. Our study is the first to document instar-specific behavioral changes in this species, focusing on feeding style and shelter con- Acknowledgments struction. Feeding style changes from exclusively skeletonizing the We thank Jake Miall for helping with insect collection. This research was leaf surface in first and second instars, to exclusively cutting the leaf funded by the Natural Science and Engineering Council of Canada (2014– in fourth and fifth instars. Third instars exhibit both behaviors, and 05947), the Canadian Foundation for Innovation (9555), and an Early transition from one to the other as they mature. Changes in feeding Researcher Award (ERO7-04-1-44) to J.E.Y. style from skeletonizing in early instars to cutting in late instars have been previously noted for a number of Lepidoptera (Hochuli 2001). 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