Atsushi Mochizuki1,2, Naoto Haruyama3, Hideshi Naka1

______

Proceedings of the Tenth International Symposium on Neuropterology. Piran, Slovenia, 2008. Devetak, D., Lipovšek, S. & Arnett, A.E. (eds). Maribor, Slovenia, 2010. Pp. 209–214.

______

Larval head capsule markings of Chrysoperla nipponensis (Neuroptera: Chrysopidae) vary according to rearing temperature

Atsushi Mochizuki1,2, Naoto Haruyama3, Hideshi Naka1 & Masashi Nomura3 1National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaraki 305-8504, Japan; 2E-mail: [email protected] 3Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan

Abstract. There are two larval types, differentiated by head markings, within the Japanese green lacewing, C. nipponensis, which are called type A and type B according to their different adult courtship songs. The two types are thought to be cryptic species that are reproductively isolated. The head of the type A larva has one pair of longitudinal dark brown or blackish-brown stripes on the dorsal side of the cranium, whereas that of type B has heavy blackish-brown markings, distinctly separated by a pale Y-shaped line. The larval head markings of C. nipponensis type A become black and broad when reared at low temperatures and those of type B show narrower markings when reared at high temperature. Since the larval head markings of C. carnea s. str. change little with temperature, this temperature-mediated phenotypic plasticity may be a characteristic unique to C. nipponensis.

Key words: Japanese green lacewing, Chrysoperla nipponensis, larval head markings, larval types, cryptic species, Chrysoperla carnea sensu stricto

Introduction

The Japanese green lacewing has until recently been regarded as conspecific to the Eurasian Chrysoperla carnea (Stephens). However, Brooks (1994) revised it to C. nipponensis (Okamoto) based on adult external morphological differences such as the color of the gradate crossveins of the wings, which are black in C. nipponensis and green in C. carnea. Tsukaguchi (1995) found that there are two larval types, differentiated by head markings, within the Japanese green lacewing, C. nipponensis, which in his paper he calls C. carnea type A and type B. He described the head of the type A larva as having one pair of longitudinal dark brown or blackish- brown stripes on the dorsal side of the cranium, and that of type B having heavy blackish-brown markings, distinctly separated by a pale Y-shaped line. Taki et al. (2005) reported that the larval types correspond to different song phenotypes. Little cross-hybridization was observed in the laboratory, suggesting them to be reproductively isolated cryptic species. Differences of the larval head markings have been thought to be thus an easy way of differentiating type A from type B.

In early spring, we often find larvae with wide black stripes and darkly pigmented markings on the center of the frons, which resemble the head markings of type B larva, in areas where type B has not yet been collected. The courtship song of the emerged adult is identical to that of type A. Their larval offspring, reared at 25˚C, revealed the same head markings as type A. We hypothesized that the larval head markings of C. nipponensis type A turn black and broad at low temperatures and examined changes in larval head markings of C. nipponensis type A at different rearing temperatures, comparing them with those of C. nipponensis type B and C. carnea sensu stricto.

The offspring of two field-collected populations of C. nipponensis were used. One population was collected in Tsukuba, Ibaraki, Japan, and their offspring were confirmed by adult song type to be type A. The other was collected in Sasagamine, Niigata, Japan, and the offspring were confirmed to be type B. Chrysoperla carnea s. str. larvae (Kagetaro, Sautter & Stepper, Ammerbuch, Germany) were purchased from Arysta LifeScience Corporation (Tokyo, Japan) and their species confirmed by courtship song (Taki et al., 2005; C. S. Henry, personal communication). Females were reared on a honey-yeast extract-water diet at 25˚C, 16L8D (Hagen & Tassan, 1966; Henry, 1979). Eggs were collected within one day of oviposition and the larvae were reared separately on frozen eggs of Ephestia kuehniella (Biotop, France) at 15, 20, 25 or 27.5˚C, at 16L8D as the same method by Mochizuki & Mitsunaga (2004). Twenty larvae each in the 2nd and 3rd instars, two days after molting at each temperature, were frozen at –80˚C before taking digital photos. The dorsal sides of the larval heads were photographed under a binocular microscope.

Fig. 1. Method of measuring the proportion of head markings.

The larval head images were processed using Photoshop 7.0J (Adobe Systems, San Jose, CA); and Image J (Japanese edition) 1.37v, build 4 (Bioarts Co. Ltd., Fukuoka, Japan) was used to measure the areas of the head or head markings. Images were rotated, since the heads are bilaterally symmetrical, and transformed to an 8-bit grayscale. The background (except the head of the larva), compound eyes, antennae and mandibles were erased manually after transformation to grayscale. Two binary (black/white) copies were created from the image: H (whole black head images) and M (only the black head markings on the vertex). Each black area (H and M) was measured, and the M/H was calculated as the proportion of head markings (Fig. 1). Twenty replicates were done for each instar at each temperature and the proportion was compared by Tukey-HSD test.

210 Results and discussion

Fig. 2 shows typical photos of head markings of 3rd instar larvae reared at different temperatures. The proportion of head markings (M/H) is compared for each instar of each species in Table 1. We did not compare the head markings of the first instar larvae, since the markings were unclear. In both 2nd and 3rd instar larvae of C. nipponensis type A, black markings on the larval head increased with decreased rearing temperature. At 25 and 27.5˚C, there was only one pair of longitudinal blackish-brown stripes on the head, and the percentage of the black stripe area did not significantly change; however, when reared at 15 and 20˚C, the stripes became wider, and larger back markings appeared on the center of the frons between the antennae. The percentage of areas carrying black markings was significantly higher than in those reared at 25 and 27.5˚C.

Fig. 2. Example of larval head markings when reared at different temperatures.

In C. nipponensis type B, markings covered almost all the dorsal head, and the percentage of areas of black markings did not change until 25˚C; however, when reared at 27.5˚C, the area of black markings decreased significantly.

211 Table 1. Changes in larval head markings with rearing temperature.

Species/Type Instars Temperature Area % ± SD *

C. nipponensis A 2nd 15℃ 65,0 ± 0,11 a 20℃ 59,5 ± 0,09 a 25℃ 33,7 ± 0,08 b 27.5℃ 37,6 ± 0,08 b

C. nipponensis A 3rd 15℃ 50,5 ± 0,07 a 20℃ 46,2 ± 0,08 a 25℃ 32,4 ± 0,07 b 27.5℃ 30,7 ± 0,05 b

C. nipponensis B 2nd 15℃ 78,5 ± 0,08 a 20℃ 77,5 ± 0,08 a 25℃ 72,8 ± 0,09 a 27.5℃ 65,7 ± 0,08 b

C. nipponensis B 3rd 15℃ 75,6 ± 0,05 a 20℃ 72,5 ± 0,06 ab 25℃ 67,0 ± 0,09 b 27.5℃ 52,8 ± 0,11 c

C. carnea s.str. 2nd 15℃ 47,8 ± 0,12 a 20℃ 46,4 ± 0,08 a 25℃ 43,0 ± 0,09 a 27.5℃ 35,0 ± 0,05 b

C. carnea s.str. 3rd 15℃ 32,4 ± 0,08 ns 20℃ 31,1 ± 0,04 ns 25℃ 30,0 ± 0,06 ns 27.5℃ 28,7 ± 0,05 ns

*Different letters indicate significant differences at 0.05 (Tukey's HSD test).

On the other hand, the larval head of C. carnea s. str. tended to darken at lower temperatures, but no significant change was observed at the 3rd instar at any temperature; and the area of black dorsolateral stripes became narrower only in the 2nd instar at 27.5˚C. These marked changes in larval head markings with rearing temperature may therefore be characteristic unique to C. nipponensis. The similarity of the character of types A and type B may demonstrate the very small genetic distance between the two types, as supported by molecular phylogenetics (Haruyama et al., 2008).

At the same temperature, the percentage of area of head markings was significantly different among C. nipponensis types A, type B and C. carnea s. str., for both the 2nd and 3rd instar at 15 and 20˚C and for the 2nd instar at 25˚C was statistically the same in between C. nipponensis type A and C. carnea s. str. for 3rd instar at 25˚C and for both 2nd and 3rd instar at 27.5˚C (Table 2). At any temperature, the proportion of larval head markings of C. nipponensis type B was the highest. We have reared more than 100 individuals of type A and type B larvae from various localities throughout Japan and have observed little variation in the character of larval head markings at 25˚C. Species diagnosis to distinguish between C. nipponensis type A or type B is very easy, provided one is comparing the head markings of larvae reared at 25˚C. Type B abounds in mountainous places and northern areas, and type A predominates in the lowlands, but their habitat sometimes overlaps. Field- collected larvae from different locations therefore need to be more carefully differentiated. Polymorphism or polyphenism in response to environmental factors, such as temperature and day length, is known in many insects (Nijhout, 1999). For example, adult butterflies with darker wings often emerge during cool seasons. Darker

212 Table 2. Differences in larval head markings at various rearing temperature.

Species/Type Instars Temperature Area % ± SD *

C. nipponensis A 2nd 15℃ 65,0 ± 0,11 b C. nipponensis B 78,5 ± 0,08 a C. carnea s.str. 47,8 ± 0,12 c

C. nipponensis A 3rd 15℃ 50,5 ± 0,07 b C. nipponensis B 75,6 ± 0,05 a C. carnea s.str. 32,4 ± 0,08 c

C. nipponensis A 2nd 20℃ 59,5 ± 0,09 b C. nipponensis B 77,5 ± 0,08 a C. carnea s.str. 46,4 ± 0,08 c

C. nipponensis A 3rd 20℃ 46,2 ± 0,08 b C. nipponensis B 72,5 ± 0,06 a C. carnea s.str. 31,1 ± 0,04 c

C. nipponensis A 2nd 25℃ 33,7 ± 0,08 c C. nipponensis B 72,8 ± 0,09 a C. carnea s.str. 43,0 ± 0,09 b

C. nipponensis A 3rd 25℃ 32,4 ± 0,07 b C. nipponensis B 67,0 ± 0,09 a C. carnea s.str. 30,0 ± 0,06 b

C. nipponensis A 2nd 27.5℃ 37,6 ± 0,08 b C. nipponensis B 65,7 ± 0,08 a C. carnea s.str. 35,0 ± 0,05 b

C. nipponensis A 3rd 27.5℃ 30,7 ± 0,05 b C. nipponensis B 52,8 ± 0,11 a C. carnea s.str. 28,7 ± 0,05 b

*Different letters indicate significant differences at 0.05 (Tukey's HSD test). colors are thought to be advantageous in a cool temperature, because the insect’s body temperature rises faster in sunlight (e.g., Kingsolver & Wiernasz, 1991; Kingsolver & Huey, 1998; Nijhout, 1999). Seasonal polyphenism of body color in some lepidopteran larvae is assumed to be related to their thermoregulation (Fields & McNeil, 1988; Goulson, 1994; Hazel, 2002).

In C. nipponensis larvae, a change to darker head markings at cooler temperatures may be involved in thermoregulation. However, no phenotypic plasticity of larval head markings in response to temperature was found in C. carnea s. str., native to northern Europe, which is cooler than Japan. Since we used commercially reared C. carnea s. str. for this study, the larvae might not be genetically variable and their head markings might not be affected by temperature. Further studies will be necessary on the phenotypic plasticity of larval head markings with temperature in other species of the C. carnea group.

References

Brooks, S. J. 1994. A taxonomic review of the common green lacewing genus Chrysoperla (Neuroptera: Chrysopidae). Bull. Br. Nat. Hist. (Ent.), 63: 137–210.

Fields, P. G. & McNeil, J. M. 1988. The importance of seasonal variation in hair coloration for thermoregulation of Ctenucha virginica larvae (Lepidoptera: Arctiidae). Physiol. Entomol., 13: 165–175.

213

Goulson, D. 1994. Determination of larval melanization in the moth, Mamestra brassicae, and the role of melanin in thermoregulation. Heredity, 73:471–479.

Hagen, K. S. & Tassan, R. L. 1966. The influence of protein hydrolysates of yeast and chemically defined diet upon fecundity of Chrysopa carnea Stephens. Vest. Csl. Spot. Zool., 30: 219–227.

Haruyama, N., Mochizuki, A., Duelli, P., Naka, H. & Nomura, M. 2008. Green lacewing phylogeny, based on three nuclear genes (Chrysopidae, Neuroptera). Syst, Entomol., 33: 275–288.

Hazel, W. N. 2002. The environmental and genetic control of seasonal polyphenism in larval color and its adaptive significance in a swallowtail butterfly. Evolution, 56: 342–348.

Henry, C. S. 1979. Acoustical communication during courtship and mating in the green lacewing Chrysopa carnea (Neuroptera: Chrysopidae). Ann. Entomol. Soc. Am., 72: 68–79.

Kingsolver, J. G. & Huey, R. B. 1998. Evolutionary analyses of morphological and physiological plasticity in thermally variable environments. Am. Zool., 38: 545–560.

Kingsolver, J. G. & Wiernasz, D. C. 1991. Seasonal polyphenism in wing-melanin pattern and thermoregulatory adaptation in Pieris butterflies. Am. Nat., 137: 816–830.

Mochizuki, A. & Mitsunaga, T. 2004. Non-target impact assessment of the introduced green lacewing, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) on the indigenous sibling species, C. nipponensis (Okamoto) through interspeciﬁc predation. Appl. Entomol. Zool., 39: 217–219.

Nijhout, F. 1999. Control mechanisms of polyphenic development in insects. Bioscience, 49: 181–192.

Taki, H., Kuroki, S. & Nomura, M. 2005. Taxonomic diversity within the Japanese green lacewing, Chrysoperla carnea (Neuroptera: Chrysopidae), identified by courtship song analyses and crossing tests. J. Ethol., 23: 57–61.

Tsukaguchi, S. 1995. Chrysopidae of Japan (Insecta, Neuroptera). Personal publication, Osaka.

214