Jpn. J. Environ. Entomol. Zool. 27（4）：133－139（2017） 環動昆 第 27 巻 第 4 号：133－139（2017） Original Article

Relationships between environmental factors and cocoon color morphs of a slug , flavescens in the field

Mariko Furukawa1), Kosuke Nakanishi1)2) and Takayoshi Nishida1)

1) Graduate School of Environmental Science, The University of Shiga Prefecture, Hassaka 2500, Hikone, Shiga 522-8533, Japan 2) Lake Biwa Museum, Oroshimo 1091, Kusatsu, Shiga 525-0001, Japan

（Received: January 6 , 2017；Accepted: January 27 , 2017）

Abstract The slug caterpillar moth, Walker forms a hard cocoon on twigs, branches and trunks of host trees. Color morphs of the cocoon are very variable, largely falling into two types: bold striped and non-bold striped. However, there is little information on environmental factors associated with the occurrence of the two color morphs. We conducted field censuses of cocoon color morphs, recorded factors potentially correlated with morph type (host-tree species, height and circumference of the cocoon construction site), and examined their relationships with morph type. We collected 94 cocoons (33 bold-striped and 61 non-bold-striped cocoons) from 17 host-tree species among 47 tree species examined. There was a significant negative effect of the circumference of the cocoon construction site on the occurrence of the bold-striped type, indicating that cocoons constructed on thinner twigs or branches are more likely to be of the bold-striped type. There were no significant effects of tree species or of the height of the cocoon construction site. Comparison with previous studies on the same host-tree species suggested that the cocoon color morphs differ locally.

Key words: Cocoon color morphs, host species, , polymorphism, slug caterpillar

Introduction because intraspecific communication and thermoregulation are unlikely functions of the color morphs of this life stage. Thus, Color polymorphism is widely distributed among . color polymorphism in cocoons probably serves exclusively This phenomenon, which may influence the performance and for predator avoidance. Despite the putative importance of fitness of individuals (Forsman et al., 2008), is considered to cocoon color polymorphism in predator avoidance, little is serve three main functions in animals: thermoregulation, known about environmental factors associated with the intraspecific communication, and predator avoidance (Endler, occurrence of color polymorphism in cocoons. We therefore 1978). Color polymorphism is well represented among ; examined relationships between environmental conditions and e.g., in pygmy grasshoppers (Tetrix), color and body-marking the color morphs of the cocoons of Monema flavescens Walker, polymorphisms serve all three functions: thermoregulation 1855 (: Limacodidae), as previously reported by (Ahnesjö and Forsman, 2006), mate recognition (Tsurui, 2011), Ishii (1984). and predator avoidance (Ahnesjö and Forsman, 2006; Tsurui et Monema flavescens forms a hard spheroidal cocoon on the al., 2010). surfaces of twigs, branches and trunks of host trees. Because of the multifunctional nature of color Overwintering occurs at the prepupal stage within the cocoon polymorphism, it is often difficult to identify its adaptive (Okajima and Takeda, 1932). It is well documented that the significance. We focused on color polymorphism of cocoons patterning of cocoon color morphs of M. flavescens

Corresponding author: [email protected]

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Materials and Methods

The Monema flavescens is a polyphagous slug moth and is distributed across Japan, China, Taiwan, Korea and southeastern Siberia (Esaki, 1957; Umeya, 2003). Okajima and Takeda (1932) and Mutuura et al (1969) described the life history and general ecology of M. flavescens. In Japan, one or Fig. 1 The cocoon color morphs of M. flavescens; two generations occur per year. The coloration is aposematic in non-bold-striped type (left) and bold-striped type (right). larvae, which are armed with sharp spines. They often cause Scale bar: 1 cm. severe pain to humans and the species is regarded as a noxious varies widely, largely falling into two types: ‘bold striped’ pest (Mutuura et al., 1969). In bivoltine populations, adults of (cocoons entirely covered with black and white stripes) and the overwintering generation emerge from cocoons in early ‘non-bold striped’ (cocoons entirely or partly covered with summer, mate and lay eggs individually on leaf surfaces. The non-bold stripes, or entirely brownish) (Fig. 1). Previous larvae feed on the leaves of various trees, construct cocoons on studies suggested that bold-striped cocoons were common on the tree surface, and emerge as first-generation adults in late twigs (Okajima and Takeda, 1932; Shinozaki, 1953; Shimbo summer. Larvae of the overwintering generation form hard and Ishida, 1954; Ishii, 1984), and that larger cocoons spheroidal cocoons on the surfaces of twigs, branches and (measured across the shorter diameter) included greater trunks and overwinter at the prepupal stage within the cocoons proportions of the non-bold-striped types (Shimbo and Ishida, (Okajima and Takeda, 1932). 1954). The color morphs of M. flavescens cocoons are highly However, these observations were fragmentary and did not variable. A few studies have documented behavioral processes quantitatively analyze relationships between environmental that potentially may be involved in determination of this factors and the frequency of different cocoon color morphs in variability (Ishii et al., 1984; Shimbo and Inaba, 2003). The the field. Among many potentially important environmental fully grown larva of M. flavescens spins silk over the body factors, we focused on three; tree species, height above the surface and regurgitates a proteinaceous fluid, which is spread ground (cocoon-site height), and the circumference of the over the silken film. Calcium oxalate crystals from the cocoon construction site. Tree species was included because Malpighian tubules are then expelled through the anus, which host plants are known to affect the color polymorphisms of is moved in a variable manner, resulting in a range of color larvae (Fink, 1995) and in some of the previous studies, the morph patterns; protein-rich parts become dark brown and the censuses included different tree species from those at the remaining parts are whitish. current study site (Okajima and Takeda, 1932; Shinozaki, 1953; Shimbo and Ishida, 1954). Tree height and the Field census circumference of the cocoon construction site were considered We examined host-tree utilization by M. flavescens cocoons as separate factors because previous reports suggested that the and recorded cocoon color morphs for 364 trees of 47 species greater abundance of bold-striped cocoons on thinner branches that grew in the campus of the University of Shiga Prefecture (Okajima and Takeda, 1932; Shinozaki, 1953; Shimbo and (USP, 35°17′N, 136°15′E), Hikone, Shiga Prefecture (Table 1). Ishida, 1954; Ishii, 1984) may simply be a consequence of the Twenty censuses of overwintering cocoons formed in the 2011 greater heights of cocoon construction sites above the ground. autumn were conducted between July 6, 2012 and November 1, Therefore, we conducted field censuses on the cocoon color 2012 (July 6, 19, 25, 27; August 4, 10, 16, 24, 30; September 6, morphs of M. flavescens with respect to three factors: host 27; October 3, 5, 7, 11, 12, 18, 19, 25; November 1). plant species, and the heights and circumferences of cocoon Before the census we removed all cocoons that had been construction sites, to elucidate environmental factors that may formed in previous generations. We examined all cocoons be quantitatively correlated with the different cocoon color formed lower than 2 m above the ground, ignoring those above morphs. 2 m, using census methods based on those used in a previous study (Sawada et al., 2008). A visual census was conducted for

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Table 1 List of tree species investigated, and the number of overwintering cocoons formed on each tree

species in 2011. The scientific names of trees were based on Yonekura and Kajita (2003).

No. of trees No. of cocoons Cocoon Family Species With Non-bold Bold † Total density cocoon striped striped Ginkgoaceae Ginkgo biloba L., 1771 10 0 0 0 0 Magnoliaceae Magnolia grandiflora L., 1759 50000 M. kobus DC., 1817 10 0 0 0 0 Lauraceae Cinnamomum camphora (L.) J.Presl, 1825 10 0 0 0 0

Hamamelidaceae Hamamelis japonica Siebold et Zucc., 1845 3 1 1 0 0.3 Liquidambar formosana Hance, 1866 10 0 0 0 0 Ulmaceae Ulmus parvifolia Jacq., 1798 10 3 8 3 1.1 Zelkova serrata (Thunb.) Makino, 1903 10 1 1 0 0.1 Myricaceae Morella rubra Lour., 1790 7 0 0 0 0 Fagaceae Lithocarpus edulis (Makino) Nakai, 1916 10 0 0 0 0 Quercus acutissima Carruth., 1862 10 4 3 1 0.4

Q. glauca Thunb., 1784 9 0 0 0 0 Q. myrsinifolia Blume, 1850 14 0 0 0 0 Q. serrata Murray, 1784 9 1 1 0 0.1 Betulaceae Alnus hirsuta (Spach) Turcz. ex Rupr. 11 11 2.0 var. sibirica (Spach) C.K.Schneid., 1916 Betula platyphylla Sukaczev 30 00 0 var. japonica (Miq.) H.Hara, 1865

Theaceae Camellia japonica L., 1753 80 00 0 C. sasanqua Thunb., 1784 10 0 0 0 0 Ternstroemia gymnanthera (Wight et Arn.) Bedd., 1871 3 0 0 0 0 Salicaceae Salix babylonica L., 1753 10 0 0 0 0 Ebenaceae kaki Thunb., 1780 10 3 4 6 1.0 Styracaceae Styrax japonica Siebold et Zucc., 1837 6 1 1 0 0.2 Rosaceae Cerasus × yedoensis (Matsum.) A.V.Vassil., 1957 10 3 3 6 0.9 Cerasus. jamasakura (Siebold ex Koidz.) H.Ohba, 1992 10 0 0 0 0 C. spachiana Lavalée ex H.Otto var. spachiana , 1879 10 1 1 2 0.3 C. subhirtella (Miq.) Sokolov 'Autumnalis', 2007 1 0 0 0 0 Pseudocydonia sinensis (Thouin) C.K.Schneid., 1906 5 0 0 0 0

Eriobotrya japonica (Thunb.) Lindl., 1821 8 1 1 0 0.1 Photinia glabra (Thunb.) Maxim., 1873 10 1 1 0 0.1 Mimosaceae Acacia dealbata Link, 1822 3 0 0 0 0 Albizia julibrissin Durazz., 1772 9 0 0 0 0

Caesalpiniaceae Cercis chinensis Bunge, 1833 1 1 11 3 14.0 Lythraceae Lagerstroemia indica L., 1759 10 1 0 1 0.1 Cornaceae Cornus kousa Buerger ex Hance subsp. Kousa, 1865 2 1 1 0 0.5 C. florida L., 1753 20 0 0 0 0 Aquifoliaceae Ilex rotunda Thunb., 1784 6 0 0 0 0 Euphorbiaceae Triadica sebifera (L.) Small, 1933 13 0 0 0 0 Hippocastanaceae Aesculus turbinata Blume, 1847 8 0 0 0 0

Aceraceae Acer buergerianum Miq., 1865 10 2 3 0 0.3 A. amoenum Carrière 100000 var. matsumurae (Koidz.) K.Ogata, 1965 A. palmatum Thunb., 1784 10 4 20 10 3.0 A. pycnanthum K.Koch, 1864 8 0 0 0 0 Rutaceae Citrus tachibana (Makino) Tanaka, 1924 5 0 0 0 0 Oleaceae Ligustrum lucidum Aiton, 1810 8 0 0 0 0

Osmanthus fragrans Lour. var. aurantiacus Makino, 1902 3 0 0 0 0 Scrophulariaceae Paulownia tomentosa (Thunb.) Steud., 1841 1 0 0 0 0 Caprifoliaceae Viburnum odoratissimum Ker Gawl. 50 00 0 var. awabuki (K.Koch) Zabel, 1902 Total 364 30 61 33 0.3 † Cocoon density = Number of cocoons / tree investigated.

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10 min for each tree by two or three investigators. For each cocoon, we recorded the tree species, cocoon-site height (height above ground of the cocoon formation point), branch circumference (circumference of the twig, branch or trunk onto which the cocoon adhered), and the cocoon color morph type (bold striped or non-striped). We measured cocoon-site height and branch circumference using measuring tapes (for cocoon-site height: model S16-35NBP, Muratec-KDS Corp, Kyoto, Japan; for branch circumference: model KA-15, Promart, Kanagawa, Japan). After the measurement, we removed the cocoons from the trees. We analyzed factors affecting cocoon color morphs using a generalized linear model with cocoon color morph as the response variable (1, bold-striped type; 0, non-bold-striped type), and tree species, cocoon-site height and branch Fig. 2 The heights (cm) above the ground of twigs, branches circumference as explanatory variables. Cocoon-site height and and trunks where M. flavescens constructed cocoons. The thick horizontal line within the box represents the median. The branch circumference data were standardized (normalized) bottom and top of the box show the 1st and 3rd quartiles, before the analysis. We assumed a binomial distribution of the respectively. Whiskers show ranges from the smallest and response variable and applied logit as a link function. The best largest values within 3× the inter-quartile ranges. combination of explanatory variables was selected using the Akaike information criterion (AIC) with stepwise selection. We used the Mann–Whitney U test to analyze cocoon-site height and branch circumference between two cocoon color morphs. Fisher's exact tests for count data were conducted to compare the utilization of tree species in the field between the two color morphs. All analyses were conducted using R ver.3.2.1 (R core team, 2015).

Results

A total of 94 cocoons of M. flavescens were found on 30 individual trees of 17 tree species, among 364 trees of 47 species examined (Table 1). Cercis chinensis harbored the greatest density of cocoons per plant, followed by Acer palmatum and Alnus hirsute (Table 1). Among the 94 cocoons 33 (35.1%) and 61 (64.9%) cocoons Fig. 3 The circumferences (cm) of twigs, branches and trunks where M. flavescens constructed cocoons. The thick horizontal were of the bold-striped and non-bold-striped types, line within the box represents the median. See caption of Fig. 2 respectively (Table 1). The bold-striped cocoons were found for detailed explanation of boxes and whiskers. Circles show on 9 tree species, with the greatest density per plant on C. outliers. chinensis，followed by A. palmatum and A. hirsute (Table 1). The ratio of host tree species in the field was not significantly for the bold-striped and the non-bold-striped cocoons, different between the two color morphs (Fisher’s exact test, p = respectively (highly significant difference between the two 0.36). The median cocoon-site heights were 90.0 cm and 106.0 types; Mann–Whitney U test, p < 0.001) (Fig. 3). cm for the bold-striped and non-bold-striped cocoons, The best combination of explanatory variables selected by respectively, without significant difference between the two AIC was cocoon-site height and branch circumference, types (Mann–Whitney U test, p = 0.48) (Fig. 2). followed by branch circumference alone (Table 2). In the The median branch circumferences were 3.1 cm and 18.9 cm model with the lowest AIC, a significant negative effect on the

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Table 2 Results of the generalized linear models for the proportion of the bold stripe morph of M. flavescens cocoons.

Coefficient of explanatory variables

Model AIC ΔAIC (Intercept) Cocoon-site height Branch circumference Plant species

1 86.122 0 -1.277** -0.472 -2.811** -

2 86.594 0.472 -1.096** - -2.354** -

3 104.503 18.381 -0.486* -0.180 - - Null 122.970 36.848 -0.598** * p <0.01, ** p < 0.001 (Wald test) - Excluded variables from the model by stepwise selection using AIC. occurrence of bold-striped cocoons was detected only for Previous studies have indicated that the proportion of branch circumference (estimated coefficient = -0.089, Z = bold-striped cocoons on Japanese persimmon, Diospyros kaki, -3.078, p < 0.001, Wald test). at Hikone, Shiga Prefecture was 54.9% (503 among 917 cocoons examined) (Shimbo and Ishida, 1954) and 20–30% at Discussion Matsumotodaira, Nagano Prefecture (Okajima and Takeda, 1932). Okajima and Takeda (1932) reported that approximately The composition of host-tree species was not significantly 70% of cocoons were bold striped on maple in Kagoshima. Our different between the two color morphs in the field. The larvae observation of 35.1% bold-striped cocoons, in comparison with of M. flavescens construct cocoons on the branches and trunks previous results, suggested that the frequency of cocoon color of the food plants (Ishii et al., 1984), suggesting that materials morphs varies among localities or years. derived from host plants do not affect the cocoon color morphs Differences in census methods (e.g. differences in census of this species. periods or branch congestion) between our study and previous Cocoon-site height was not significantly different between studies, might have affected the results. We doubt the the two color morphs. In contrast, branch circumference was importance of these factors because the exploratory highly significantly different between the two color morphs. environments of M. flavescens (farm lands and city gardens) Furthermore, the best combination of factors using AIC are similar among the studies. We propose two hypotheses that identified cocoon-site height and branch circumference as could account for the different cocoon color morph ratios explanatory variables, followed by the branch circumference among the studies. First, the larvae are able to change the alone. The coefficient of the explanatory variables in the model cocoon color morphs facultatively depending on the properties of the best combination of factors using AIC indicated that of the cocoon construction points. Second, both the cocoon branch circumference strongly affected, and cocoon-site height color morphs and cocoon construction sites are genetically slightly affected, the cocoon color morph, independently of determined, and the corresponding gene frequencies differ host-tree species (Table 2). That is, bold-striped cocoons were among the study regions. The two hypotheses are not mutually commoner on thin branches or twigs, almost irrespective of exclusive. To determine which hypothesis is more adequate, tree species or cocoon-site height. we are planning further experiments incorporating both We postulated that the adaptive significance of cocoon color environmental and genetic factors. morphs is related to predator avoidance. This could explain the greater abundance of bold-striped cocoons on thin branches. Acknowledgements First, bold stripes might enhance cryptic effects because they blend into a background of thinner twigs. Second, the bold We wish to express our appreciation to Dr. H. Sawada for stripe might be an aposematic signal to visually hunting his valuable advice and kind support. We also thank Ms. K. predators. Since the bold-striped cocoons are situated on thin Matsuyama and Mr. N. Hidaka for help with the field survey. branches or twigs, it may be difficult for small birds to attack them while perching. To elucidate which explanation is more plausible, further study is needed.

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ecological studies of Cnidocampa flavesens Walker. Bull. References Kagoshima Imp. Coll. Agr. Forest. 10: 219–299 (in Japanese). Ahnesjö, J. and A. Forsman (2006) Differential habitat R Core Team (2015) R: A language and environment for selection by pygmy grasshopper color morphs; interactive statistical computing. http://www.R-project.org/ (accessed effects of temperature and predator avoidance. Evol. Ecol. June 18, 2015) 20: 235–257. Sawada, H., Y. Hori, S. Nishida, T. Matsumoto and T. Nishida Endler, J. A. (1978) A predator’s view of color patterns. (2008) Population dynamics of an invasive grub moth, Evol. Ecol. 11: 319–364. lepida (Cramer) that damages ornamental trees: Esaki, T. (1957) Monema flavescens. In “Icones heterocerorum the seasonal and annual fluctuations of the cocoon density. japonicorum in coloribus naturalibus (first volume)”, p. Jpn. J. Environ. Entomol. Zool. 19: 115–124. 159, Hoikusha, Osaka (in Japanese). Shimbo, T. and M. Inaba (2003) The constitution of cross Fink, L. S. (1995) Foodplant effects on colour morphs of section over the stripy cocoon of Cnidocampa flavescens. Eumorpha fasciata caterpillars (Lepidoptera: Sphingidae). Bull. Shiga Soc. Nat. 5: 21–23 (in Japanese). Biol. J. Linnean. Soc. 56: 423–437. Shimbo, T. and H. Ishida (1954) Preliminary note on the Forsman, A., J. Ahnesjö, S. Caesar and M. Karlsson (2008) A ecological studies of the pattern on the cocoon of slug model of ecological and evolutionary consequences of moth. Scientific reports of Shiga Agricultural College 5: color polymorphism. Ecology 89: 34–40. 8–9 (in Japanese). Ishii, S. (1984) Studies on the cocoon of the oriental moth, Shinozaki, J. (1953) On the cocoon of slug moth. Low Temp. Monema (Cnidocampa) flavescens, (Lepidoptera: Sci. 10: 127–136 (in Japanese). Limacodidae). II. Construction of the cocoon and Tsurui, K. (2011) Ecological study on adaptive significance of appearance of dark brown stripes. Jpn. J. Appl. Entomol. color-marking polymorphism in a pygmy grasshopper, Zool. 28: 167–173 (in Japanese). Tetrix japonica. Doctoral Thesis, Kyoto University, Ishii, S., T. Inokuchi, J. Kanazawa and C. Tomizawa (1984) Kyoto. Studies on the cocoon of the oriental moth, Monema Tsurui, K., A. Honma and T. Nishida (2010) Camouflage (Cnidocampa) flavescens, (Lepidoptera: Limacodidae). III. effects of various colour-marking morphs against different Structure and composition of the cocoon in relation to backgrounds of microhabitats in a polymorphic pygmy hardness. Jpn. J. Appl. Entomol. Zool. 28: 269–273 (in grasshopper Tetrix japonica. PLoS ONE 5: e11446. Japanese). Umeya, K. (2003) Monema flavescens. In “Agricultural insect Mutuura, A., Y. Yamamoto, I. Hattori, H, Kuroko, T. Kodama, pests in Japan” (Umeya, K. and T. Okada, eds), pp. T. Yasuda, S. Moriuchi and T. Saitou (1969) Monema 902–903, Zennokyo, Tokyo (in Japanese). flavescens. In “Early stages of Japanese in colour”, Yonekura, K. and T. Kajita (2003) BG Plants. Japanese name - p. 56, Hoikusha, Osaka (in Japanese). Scientific name Index (YList). http://ylist.info (accessed Okajima, G. and N. Takeda (1932) Preliminary notes on the Jan. 5, 2017)

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野外におけるイラガ繭の色斑二型の出現要因

古川真莉子 1)・中西康介 1)2)・西田隆義 1)

1) 滋賀県立大学大学院環境科学研究科

2) 滋賀県立琵琶湖博物館

イラガは固い繭を樹木の幹や枝に形成する．この繭の色斑には大きな変異があり，縞模様型（全面が鮮明な白黒

模様）と不鮮明型（不鮮明な縞模様，または全体的に不鮮明な茶褐色）に大別される．しかし，繭の色班型の違い

に関わる環境条件について知見は乏しかった．そこで本研究では，イラガ繭の色班型の出現頻度と環境条件との関

係を，野外調査により明らかにすることを目的とした．調査対象とした 47 種の樹種のうち，17 種でイラガの繭が

見つかり，94 個の繭を採集した．縞模様型の繭は，そのうち 9 種の樹種から 33 個採集した．縞模様の有無に対す

る，繭形成樹種，繭を形成した場所の地面からの高さおよび枝・幹の太さとの関係を解析したところ，繭が形成さ

れた部分の枝の太さと負の関係があり，細い枝ほど縞模様型の割合が高いことがわかった．また，同じ樹種で行な

われた先行研究の結果と比較すると，縞模様型の割合は地域によって異なることが示された．

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