Journal of Insect Science, (2018) 18(1): 12; 1–6 doi: 10.1093/jisesa/iey001 Short Communication Developmental Irregularity and Abnormal Elytra Formation in the Oriental Wood Borer Induced by Physical Disturbance Lee-Jin Bong,1,2,4,* Kok-Boon Neoh,3,* and Tsuyoshi Yoshimura1 1Laboratory of Innovative Humano-habitability, Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan, 2Current address: National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan, 3Department of Entomology, National Chung Hsing University, 250 Guoguang Road, Taichung 40227, Taiwan, and 4Corresponding author, e-mail: [email protected] *These authors contributed equally to this article. Subject Editor: John Ewer Received 21 September 2017; Editorial decision 19 December 2017 ABSTRACT Teratological deformity in arthropods may arise spontaneously or be induced by chemicals, mutagens, diseases, and physical disturbance. Coleopterans are known to be sensitive to physical disturbance, but detailed studies of how physical disturbance affects the juvenile development and causes deformities are limited. In this study, larval oriental wood borers Heterobostrychus aequalis (Waterhouse) in three weight categories (2 to <10 mg, 10 to <26 mg, and 26 to 36 mg) were used to examine the development of immature beetles when exposed to daily physical disturbance (i.e., handling). Morphological changes induced in the immature and adult stages were observed for disturbed individuals. Handled larvae were highly susceptible to physical disturbance, as none of them survived to the end of the experiment, regardless of their initial weight group. Unhandled larvae molted twice before turning into pupae, whereas handled larvae took longer to develop and underwent irregular molting. Handled larvae also exhibited various levels of morphological abnormality in the pupal stage; in particular, the elytra were deformed, the abdomen and head capsule were broader than normal. It is speculated that the irregularity and deformity observed in the handled larvae might be due to hormone alteration resulting from physical disturbance. In addition, the extensively exposed abdominal parts of deformed beetles might have increased the risk of dehydration and disease attack, which would have resulted in premature mortality. Key words: Bostrichidae, deformity, ecdysteroid, juvenile hormone, octopamine Morphological deformity is widely reported in many arthropods. It Coleoptera: Tenebrionidae) from wheat flour prolonged the larval can arise spontaneously (idiopathic) due to cell division failure, and incubation period and caused high pre-adult mortality. Based on it also can be induced by chemicals (Martinez et al. 2001, Watson these published records, two common phenomena occur during dis- et al. 2014, Barbosa et al. 2015), mutagens (Hiyama et al. 2012, turbance: a prolonged pre-adult developmental period and premature Akimoto 2014), diseases (Neoh and Lee 2011, Rocío et al. 2011), mortality. However, to date no study has focused on how physical and physical disturbance (Spence et al. 1996). Excessive physical dis- disturbance affects the development of immature beetles and causes turbance due to handling (e.g., pouring and rotation) of wheat has deformities, which eventually lead to low adult emergence. decreased adult emergence of several stored product pests (Bailey Bostrichidae are among the most destructive wood-attack- 1969, Loschiavo 1978). For example, mortality of the immature (i.e., ing beetles in forestry and lumber industries (Peters et al. 2002). prepupae and pupae) grain weevil Sitophilus granaries (L.) increased Bong (2015) reported that 65% of the Bostrichidae infestations in with increasing pouring height and speed. Bailey (1969) suggested Japan were caused by species of Lyctus. Although few cases involv- that this premature mortality may be attributable to cuticle rupture ing Heterobostrychus (i.e., Heterobostrychus hamatipennis Lesne that eventually caused water loss in the larvae, excessive secretion and Heterobostrychus aequalis [Waterhouse]) were reported, the of neurosecretory materials that paralyzed the larvae, and lack of prevalence of the attacks are gradually increasing in Japan (Bong oxygen consumption by the disturbed larvae. Mertz and Robertson 2015). Heterobostrychus species are not host specific and attack a (1970) reported that daily sifting of Tribolium castaneum (Herbst; wide range of timber types. Due to their cryptic nature of residing © 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/ 1 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 [email protected] 2 Journal of Insect Science, 2018, Vol. 18, No. 1 inside wood, direct assessment of the beetle life stage development 2.5 mm diameter × 1.0 mm depth was drilled in the center of the is impossible. artificial diet and the larva was placed in the hole. The block was In this study, we aimed to examine the development of H. aequa- stacked with another piece of diet block and tied with a rubber band. lis exposed to physical disturbance and the relationship between To induce physical disturbance, the larva was picked up using fine frequency of physical disturbance and pupation rate and morpho- forceps and then returned to the diet block (referred to as handled logical deformity. We also provide a detailed biometric descriptor of hereafter). The block was checked for any exuviae left by the larva deformed pupae. during molting. The handling was repeated on weekly basis. The experiment was terminated soon after the handled larvae died or emerged into adults. The dead pupae were preserved in 80% alcohol Materials and Methods for further biometric analysis. Control larvae were left untouched Adult H. aequalis were reared in a walk-in environmental chamber until the adult emerged (referred as unhandled hereafter). We deter- at 26 ± 2°C and 65 ± 10% relative humidity located at the Research mined the number of moltings that occurred based on the exuviae Institute for Sustainable Humanosphere, Kyoto University, Japan. found at the end of the experiment (until adult emergence). The beetles were provided with an artificial diet block (8.0 cm length A total of 21 pupae, representing all the pupae derived from han- × 4.0 cm width × 2.0 cm depth) consisting of a mixture of 24% dled larvae (across all weight groups), and five pupae from unhan- dried yeast (Asahi Food and Health Care, Tokyo, Japan), 50% starch dled larvae were subjected to biometric analysis. The following parts (Nacalai Tesque, Kyoto, Japan), and 26% lauan wood sawdust of each specimen were observed using a BX51 stereomicroscope (Shorea spp.) (Kartika and Yoshimura 2013). Three weight groups (Olympus, Tokyo, Japan) and measured using ImageJ Software: of larval H. aequalis (based on weight: 2 to <10 mg, 10 to <26 mg, 1) length of head to tip of abdomen, 2) maximum width of ab- and 26 to 36 mg) were used in this study. Twelve to seventeen larvae domen, 3) length of head, 4) width of head, 5) length of abdomen, of each weight group were used. Each test larva was kept individ- 6) length of elytra, and 7) width of elytra. Each specimen also was ually with an artificial diet block (2.5 cm length × 2.5 cm width scored from 0 (absent) to 3 (severe) based on the degree of elytra × 0.5 cm depth) as harborage and food source. A hole measuring deformities and the presence of black spots (Fig. 1). Fig. 1. Types of pupal teratology. (A–B) Ventral and dorsal views of a normal pupa, which was scored as 0. (C–D) Deformed pupa with only wrinkled elytra (we) (score 1) and a minimal spot on the dorsal abdomen, indicated with an arrow (score 1). (E–F) Deformed pupa with divergent elytra (di) and short left elytra (score 2). Minimal spot on the ventral abdomen (score 1). (G–H) Pupa with multiple deformities of the elytra. The left elytra was short and wrinkled, whereas the right elytra was diverted and wrinkled (score 3). Spots were present on the ventral abdomen and left elytra (score 2). (I–J) Pupa with multiple deformities, including wrinkled elytra and distorted left elytra (dt) that curled inside (score 3). High density of spots was present on the ventral and dorsal abdomen and elytra (score 3). Scale bar, 1 mm. Journal of Insect Science, 2018, Vol. 18, No. 1 3 We used the head width, length of elytra, and degree of elytra de- of H. aequalis. We assumed that younger larvae were subjected to formity, which combined explained 91.6% of the total variance (see longer duration of physical disturbance throughout their develop- discriminant function analysis), in subsequent multivariate analyses. ment. We also used the weight of larvae as a surrogate for larval A morphological variation index (MVI) based on the differences instar stage and included this parameter as a predictor. We used gen- of the head width and length of elytra between each handled and eralized linear models (GLMs) with a binomial error distribution unhandled larva-derived pupa was generated (Warwick 1985). The (logit link), for which the response variable was larval pupation; values of MVI were summed for each individual (Supplementary a Gaussian error distribution, for which the response variable was Appendix S1). MVI; and a Poisson error distribution (log link), for which the re- The molting numbers between handled and unhandled individual sponse variable was the score values for the degree of elytra deform- was compared using Mann-Whitney U. The morphological var- ities. All analyses were performed using SPSS version 11.0 (SPSS Inc., iations between handled and unhandled larva-derived pupae were Chicago, IL) at α = 0.05. compared using Student’s t-test. We also performed discriminant function analysis to identify the morphological parameters that dis- criminate between deformed and non-deformed pupae (Neoh and Results and Discussion Lee 2011).