吃軟不吃硬：警戒色昆蟲的神祕...

國立臺灣師範大學生命科學系碩士論文

吃軟不吃硬：警戒色昆蟲的神祕

次級防禦機制

Too hard to swallow: A secret secondary

defense of an aposematic insect

研究生：王露翊Lu-Yi Wang

指導教授：林仲平博士 Dr. Chung-Ping Lin

黃文山博士 Dr. Wen-San Huang

中華民國 106 年 11 月 01 日

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致謝

長長兩年多的碩士生涯，短短的精采的過完了。回首過程中的一切辛苦與

挑戰，我何其幸運的能夠沉浸其中，享受著與這個有趣的題目相伴的甜蜜時光。

感謝我的指導老師林仲平博士，他總是能夠適時的為我打氣，每次與他談話後

總覺得實驗又充滿希望！也謝謝共同指導我的黃文山博士，和口試委員顏聖紘博

士，點出論文的問題與建議。

實驗室與師大共同努力的夥伴們（惠芸、若凡、祐薰學姊、Wataru、小 A、

緊張哥、一休哥、小哈、震邑、Pincess、Leo、Ian、劉班、俊佑、阿薇、中中），

感謝他們的陪伴與時間，陪我聊天放鬆；謝謝馥慈師母，替我處理出國與出差的

各種繁瑣行政工作。謝謝溫泉扮演著野外工作與生活中不可或缺的要角；也謝謝

昆蟲館唐欣潔館長與春捲龍學長，協助我飼養實驗所需的象鼻蟲與提供食草；還

有為實驗犧牲的象鼻蟲們，辛苦你們了。感謝阿磐學長在統計與 R上提供許多

幫助；謝謝Dr.HamedRajabi 在昆蟲結構與物理名詞上給了許多建議。

最後感謝我的家人，義無反顧的支持我想做的事情。我很幸運能擁有你們！

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中文摘要

1. 抵禦掠食者攻擊是獵物適應上重要的一環。獵物的次級防禦機制（例如毒

性）常伴隨著警戒色的出現，並可藉由避免被捕食讓掠食者與獵物彼此獲

利。因此警戒色獵物被預期具有和警戒色彩一起演化的有效次級防禦機制。

2. 早在將近一百五十年前，華萊士就已經提出球背象鼻蟲 (Pachyrhynchus) 以

鮮豔的花紋來警告掠食者，並以堅硬的外骨骼作為防禦的假說；然而這項假

說至今依然無實驗證據驗證。本研究檢驗球背象鼻蟲的次級防禦機制假說，

並探討堅硬外骨骼在球背象鼻蟲身上所扮演的生態功能。

3. 我們以斯文豪氏攀木蜥蜴（Japalura swinhonis）作為掠食者，實驗設計四組

象鼻蟲處理（軟/硬；有足鉤/無足鉤）進行操控性行為實驗測試其存活率。

並以比較破壞球背象鼻蟲的外骨骼，和掠食者共域昆蟲獵物所需的力量 (牛

頓)，與掠食者的咬合力大小，來了解球背象鼻蟲的硬度在生態上的重要性。

最後用氣相色譜質譜法 (GC-MS) 分析球背象鼻蟲體內是否有毒性或驅避

性的化合物。

4. 所有「硬」球背象鼻蟲被蜥蜴攻擊後都立即被吐出且存活。相較之下「軟」

球背象鼻蟲則在被多次咀嚼後吞下。此外不論足鉤存在與否，蜥蜴對球背象

鼻蟲的捕食結果都與上述相同。

5. 「硬」球背象鼻蟲的硬度顯著高於共域昆蟲的平均硬度和當地蜥蜴族群的平

均咬合力。

6. GC-MS分析出球背象鼻蟲體內四種可能的化合物（ethyl propyl ether、

diethylene glycol、butylated hydroxy toluene以及 cadalene）均無毒性或對脊

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椎動物有驅避的功能。

7. 本研究指出具警戒色物種的硬度是一項有效抵禦捕食者的次級防禦機制，亦

提供ㄧ個探討脊椎動物掠食者與具有警戒色獵物之間時空互動的研究模

式。

關鍵字：防禦機制、警戒色、硬度、斯文豪氏攀木蜥蜴、球背象鼻蟲、捕食－

被捕食交互作用、次級防禦、台灣

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Abstract

1. Anti-predator strategies are significant components of adaptation for prey species.

The advance of the secondary defenses like toxins have led to the evolution of

warning colours that benefit both prey and predators by mutual avoidance.

Therefore aposematic prey are expected to possess effective secondary defenses

evolved together with their warning colours.

2. This study tested the hypothesis of secondary defensive function and ecological

significance of the hard body in aposematic Pachyrhynchus weevils, which was

pioneered by Alfred Russel Wallace nearly 150 years.

3. We used predation trials of Japalura tree lizards to assess the survivorship of

‘hard’ (mature) vs. ‘soft’ (teneral) and ‘claw’ (intact) vs. ‘clawless’ (surgically

removed) weevils. The ecological significance of the weevil’s hard body was

evaluated by assessing the hardness of the weevils, local insect prey, and the bite

forces of lizard populations. The existence of toxins or deterrents of the weevil

was examined by Gas Chromatography-Mass Spectrometry (GC-MS).

4. All ‘hard’ weevils were spat out and survived the attack by the lizards. By contrast,

the ‘soft’ weevils were chewed and subsequently swallowed. The results were the

same regardless of presence or absence of the weevil’s tarsal claws.

5. The hardness of ‘hard’ Pachyrhynchus weevil was significantly higher than the

average hardness of insect prey in the same habitat and mean bite forces of local

lizards.

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6. The four candidate compounds of the weevil identified by GC-MS (ethyl propyl

ether, diethylene glycol, butylated hydroxy toluene and cadalene) had no known

toxic or repellent functions against the vertebrates.

7. These results reveal that the hardness of aposematic prey functions as effective

secondary defense, and provide a framework for understanding spatio-temporal

interactions between vertebrate predators and aposematic insect prey.

Key-words: Anti-predator strategy, aposematic colour, hardness, Japalura lizard,

Pachyrhynchus weevil, predator-prey dynamics, secondary defense, Taiwan

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Contents

致謝 ................................................................................................................................. i

中文摘要 ....................................................................................................................... ii

Abstract ......................................................................................................................... iv

Introduction .................................................................................................................... 1

Materials and methods ................................................................................................... 5 Ethics statement ..................................................................................................................... 5

Weevil and lizard ................................................................................................................... 5

Colour and mobility of ‘hard’ and ‘soft’ weevils .................................................................. 7

Behavioral trials .................................................................................................................... 9

Insect prey and hardness measurement ............................................................................... 10

Chemical analysis ................................................................................................................ 12

Statistical analyses .............................................................................................................. 13

Results .......................................................................................................................... 13 Effects of the hardness and claws ........................................................................................ 13

Hardness of prey and lizard’s bite force ............................................................................. 15

Biochemical compounds of P. sarcitis kotoensis ................................................................. 16

Discussion .................................................................................................................... 17 Effective secondary defense ................................................................................................. 17

Multiple secondary defenses ............................................................................................... 19

Mechanical mechanism of hardness .................................................................................... 20

Adaptive defense against lizard’s predation ....................................................................... 21

References .................................................................................................................... 24

Figures………………………………………………………………………………..31

Supplementary tables………………………………………………………………...39

Supplementary movies……………………………………………………………….56

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“… tropical weevils which have the elytra and the whole covering of the body so hard

as to be a great annoyance to the entomologist, …, they cannot be pinned without first

drilling a hole to receive the pin, and it is probable that all such find a protection in

this excessive hardness.”

“The insects which others imitate always have a special protection, which leads them

to be avoided as dangerous or uneatable by small insectivorous animals; some have a

disgusting taste …; others have such a hard and stony covering that they cannot be

crushed or digested;…”

Alfred Russel Wallace, 1867

Introduction

Predation is one of the most visible selective forces driving the ecology and evolution

of organisms in nature (Abrams 2000). Therefore, evolving effective anti-predator

strategies is a significant component of adaptation for many prey species. Diverse

defensive strategies have evolved at a range of specific stages of the encounters

between predators and preys (Stevens 2013). These anti-predator strategies can be

classified as the ‘primary’ and ‘secondary’ defenses depending on the timing when

they are performed (Ruxton, Sherratt & Speed 2004). The primary defenses serve to

avoid detection (crypsis) by operating before predators initiate prey-catching

behaviour, or to prevent pursuit by advertising themselves unprofitable to predators

(mimicry, warning signals) (Robinson 1969; Edmunds 1974). The secondary defenses

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are post-detection defenses that function to increase the chances of surviving

prey-capturing process (deflection or startle), or to make an encounter unprofitable to

predators (e.g. spines, stings and toxins) (Robinson 1969; Edmunds 1974). The

development of the secondary defenses using chemical or physical deterrents have led

to the evolution of warning signals in prey that benefit both well-defended prey and

their potential predators by mutual avoidance (Summers & Clough 2001; Sherratt &

Beatty 2003; Ruxton et al. 2004). Well-known examples of co-evolution between

warning signals and prey defenses include the generation of aposematic colouration

and toxicity in the poison frogs (Summers & Clough 2001; Maan & Cummings 2011)

and ladybird beetles (Blount et al. 2012).

Pachyrhynchus weevils (Germar, 1824) (Coleoptera: Curculionidae: Entiminae:

Pachyrhynchini) are a group of brilliant metallic-coloured weevils distributing in the

Old World tropics (Fig. 1a) (Wallace 1895; Schultze 1923; Tseng et al. 2017). The

observations of diverse colours of more abundant Pachyrhynchus weevils (as models)

being mimicked by several relatively rare species of longhorn beetles (Cerambycidae:

Doliops) suggested the aposematic function of their colouration (Wallace 1867;

Dickerson et al. 1928; Barševskis 2013). A recent manipulative experiment showed

the first empirical evidences that the conspicuous colouration of Pachyrhynchus

weevils could function as effective warning signals (the primary defense) to prevent

predacious pursuit by Japalura lizards (Fig. 1c) (Tseng et al. 2014). In those

behavioural trials, the lizards attacked weevils without conspicuous colours at higher

rates than weevils with intact colours. During those trials, the lizards were observed to

display irritating behavioral response (spitting out & throwing off) after biting the

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weevils, indicating the unpalatability of the prey. Moreover, the weevils frequently

survived the lizard’s attack without visible harm to their bodies, suggesting the

existence of an effective secondary defense, which is expected to have evolved in

concert with their warning colouration.

Alfred Russel Wallace first hypothesized that the excessive hardness of

aposematic Pachyrhynchus weevils serves as a defense mechanism against small

insectivorous animals (morphological defense hypothesis) (Wallace 1867). Despite

that the defensive function appears to be a charming and conceivable explanation for

these weevils with completely fused and rigid elytra, approximately 150 years after

Wallace’s proposal we still do not understand the ecological and adaptive significance

of the hard body in these weevils, and have no solid empirical evidences to evaluate

this hypothesis. Nevertheless, our earlier observations of only extremely large lizards

(thus higher biting force; ~ 3% of the trials) can consume the weevils pointed to the

feasibility of Wallace’s hypothesis (Tseng et al. 2014). Alternatively, other external

rigid morphologies such as sharp claws and spines of insect’s legs can often be used

as effective weapons upon being swallowed by small vertebrate predators (Eisner,

Eisner & Siegler 2005; Ruxton et al. 2004). Pachyrhynchus weevils have three pairs

of elongated legs with strong grip on the objects (Fig. 1e) (Schultze 1923; Starr &

Wang 1992). The sharp tarsal claws at the tips of these legs combined with strong grip

could potentially cause the irritating behavioral response of the lizards, which have

soft tongues and mouth cavities.

In addition to morphological (mechanical) defense, the secondary defense of

animals can often take the form of toxic or distasteful chemicals (chemical defense

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hypothesis) (Ruxton et al. 2004). Although chemical defense is the most common

secondary defensive strategies in aposematic prey, so far no specialized secretory

glands or storage for toxic chemicals have been found in Pachyrhynchus weevils

(Pasteels, Grégoire & Rowell-Rahier 1983). Therefore, these weevils are less likely to

be chemically defended. However, a few Pachyrhynchus weevils are known to feed

on poisonous plants (e.g., sea poison tree, Barringtonia asiatica, Lecythidaceae in P.

sonani) (Chen et al. 2017), thus their bodies may potentially contain distasteful

chemicals or plant-derived toxins.

This study investigated the secondary defensive function and ecological

significance of the hard body in Pachyrhynchus weevils. First, the morphological

defense hypothesis was tested using manipulative experiments of lizard predation, by

comparing the survivorship between the ‘hard’ (mature) and ‘soft’ (teneral) weevils

(Figs 1a & b), and between the ‘claw’ (intact) and ‘clawless’ (surgically removed)

weevils (Figs 1e & f). If the weevils used the hard bodies or tarsal claws as the

secondary defenses against the lizards, the prediction was that after the encounters

with lizards, either the ‘hard’ weevils would have a higher survival rate than the ‘soft’

ones, or that the ‘claw’ weevils would survive better than the ‘clawless’ counterparts.

Next, the ecological relevance and adaptive significance of the weevil’s hard body

was assessed by measuring and comparing the hardness of the weevils and other

insect prey species found in the lizard’s natural habitat. Because the bite capacity is an

important functional trait and ecological indicator for the width of diet niche in the

lizards (Herrel et al. 2001; Meyers & Irschick 2015), the hardness (measured as force

at failure) was compared to the bite forces of the local lizard populations. If the hard

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body of the weevils represented an important adaptive defense mechanism within the

potential prey spectrum of the lizards, the prediction was that the hardness of the

weevils would be higher than that of the other potential preys in lizard’s habitat. The

hardness of the weevils was also expected to be higher than the average bite force of

the local lizard populations. Finally, the chemical defense hypothesis was excluded by

analyzing biochemical content of the weevils for the existence of toxins or deterrents

of the vertebrates. The prediction was to find no toxic or distasteful chemicals in the

weevils.

Materials and methods

Ethics statement

The permission of using the protected Pachyrhynchus weevils was granted by the

Forestry Bureau, Council of Agriculture, Taiwan (permit no. 1031700770 &

1041700842). The Institutional Animal Care and Use Committee of the National

Taiwan Normal University approved the behavioural experiments using Japalura

lizards (No. 105012). The animal ethics protocols of the Wildlife Conservation Act of

Taiwan were followed throughout the experiments and no harmful effect to the lizards

was observed. All lizards were released to their capturing locations after the

experiments and the weevils were fed and kept in the laboratory until they died.

Weevil and lizard

Six adults (3 males and 3 females) of endemic Taiwanese Pachyrhynchus sarcitis

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kotoensis Kôno, 1930 (Kôno 1930) (Fig. 1a) were collected by hands from Orchid

Island in August of 2015. This Pachyrhynchus species was chosen for the study

because it is relatively larger and easier to rear in the laboratory. The collected

weevils were reared individually in cylinder plastic containers (8cm diameter, 6cm

height) in the laboratory at 25°C and under 12-day/12-night cycles. Once a week, they

were supplied with fresh leaves of the known host plant, Leea guineensis (Leeaceae).

These weevils were later placed together in the containers and allowed to mate freely.

The eggs and the first instar larvae produced by these individuals were reared inside

the stems of their host plants. The plant stems were individually kept in transparent

plastic boxes (5.4cm x 4.3cm x 4.0cm) at 25°C with 12-day/12-night cycles. Once

eclosed, the teneral adults stayed in their pupal chambers for approximately five days.

The cuticles of the teneral adults remained soft (unsclerotized) for at least seven days

after they emerged from the chambers. For the following behavioral trials, the ‘soft’

weevils were defined as the individuals being within the first five days of emergence

from the pupal chambers (Fig. 1b); the ‘hard’ weevils were defined as those having

emerged for more than two months (Fig. 1a). The ‘clawless’ weevils (Fig. 1f) were

made by removing all of their tarsal claws with scissors.

The diurnal tree lizards, Japalura swinhonis (Agamidae) (Fig. 1c) are common

arboreal predators in lowland forests of Taiwan, Orchid and Green Islands. They are

sit-and-wait predators (Huang 2007) and agamid lizards use mainly visual cues for

prey detection (Cooper Jr 1989), and feed on the arthropods including weevils (Huang

2007). Therefore, J. swinhonis was selected as the testing predator for Pachyrhynchus

weevils in this study. 78 adult lizards (male: n = 60, snout-vent length (SVL) = 73.16

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± 4.79 mm; female: n = 18, SVL = 66.57 ± 4.13 mm) were collected using a noose

between May and October of 2016 in the forests of Taipei Zoo (24°59'36.4" N,

121°34'51.9" E). The allopatrically distributed (thus likely naïve) Taipei lizard

population was used to increase the attacking rate of weevils in the behavioural trials

because the sympatric lizards attack weevils at lower frequencies (Tseng et al. 2014).

However, the male body size of Taipei’s lizards were smaller than that of Green

Island (SVL, male: 76.594 ± 4.440 mm, n = 92; female: 63.403 ± 5.094 mm, n = 80)

and Orchid Island populations (SVL, male: 75.927 ± 4.865, n = 80; female: 69.596 ±

4.061 mm, n = 89), which were used for measuring bite forces (Wang et al.

unpublished data). The captured lizards were kept individually in plastic containers

(34cm x 17cm x 24cm) at 25-30°C with water ad libitum in the laboratory for three

days before the experiments. The three-day deprivation of food prior to the

behavioural trials was used to increase the level of hunger in lizards. Each lizard was

used only once in the behavioural trials. The fifth toe of right hind legs of the lizards

was clipped and used as a marker before releasing to prevent recapture.

Colour and mobility of ‘hard’ and ‘soft’ weevils

Before conducting the behavioural trials, we measured the colouration and daily

rhythm of ‘hard’ and ‘soft’ weevils to examine the level of their resemblance in

appearance and mobility. Reflectance spectra of the colouration were measured with a

spectrometer (detection range: 250-800 nm, Jaz spectrometer, Ocean Optics, Inc.,

Dunedin, Florida) connected to a reflection probe (ZFQ-13101) illuminated by a

deuterium-tungsten halogen light source (DH-2000-BAL). The spectrometer was

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calibrated with a reflectance standard (WS-1-SL). For each individual, we measured

the three blue spots (Fig. 1d, red circles) and three black background areas (Fig. 1d,

white circles). The light source was positioned 1mm perpendicularly above the focal

area of the weevil body surface (diameter of the illuminated area: ~ 2mm). The three

measurements of the blue spots and black areas from each individual were averaged.

The hue (wavelength of the apex of the spectrum), brightness (reflectance ratio of the

apex of the spectrum), and saturation (area under the spectral curve between the

visible wavelengths of diurnal lizards, 440 - 625nm; Yewers et al. 2015) of the

measured spectrum were calculated. The ultraviolet-sensitive (UVS) (364 - 383 nm)

spectrum was excluded from the calculation because of the low UV reflectance (<

40%) of the colouration (Fig. 2a). No significant difference between the colour of

‘hard’ and ‘soft’ weevils was detected (Fig. 2a) (two-sample t test, n = 4; hue, ‘hard’:

555.36 ± 19.76 nm, ‘soft’: 547.15 ± 8.73 nm, t = 0.761, p = 0.476; brightness, ‘hard’:

128.97 ± 4.79%, ‘soft’: 125.00 ± 3.99%, t = 1.274, p = 0.250; saturation, ‘hard’: 66.40

± 2.99%, ‘soft’: 67.23 ± 1.90%, t = -0.454, p = 0.666).

The daily rhythm of the weevils (n: ‘hard’ = 10, ‘soft’ = 10) were recorded at

25°C for six consecutive days using a video monitor (DS-VR7160H, Der Shuenn,

Taipei, Taiwan). The weevils were fed once on the third day. Each weevil was placed

in a petri dish (diameter: 90mm, height: 15mm) divided into four equal quadrants. We

recorded the number of weevil’s movements between the quadrants in the first 10

minutes of every two hours. The total numbers of movements for each individual

during the six days were summed up as a proxy of its mobility. The daily rhythm of

the two weevil groups was similar (Fig. 2b) (number of movements per day, ‘hard’:

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42.5 ± 15.9, ‘soft’: 44.6 ± 15.9; two-sample t test of daytime scores, n = 10, t = -0.376,

p = 0.7113). These results suggested that the ‘hard’ and ‘soft’ weevils represent prey

items with equal appearance and mobility for the visually oriented lizard predator.

Behavioral trials

To test the effectiveness of weevil’s hard exoskeleton and tarsal claws against

predation by lizards, the weevils were randomly divided into four treatments (n = 78):

(1) ‘hard’ weevils with claws, (2) ‘hard’ weevils without claws, (3) ‘soft’ weevils

with claws, (4) ‘soft’ weevils without claws. The trial arena was a transparent plastic

container (58cm x 30cm x 40cm) with the bottom padded with soil (Fig. 3a). The

three vertical planes of the container were covered with light brown cardboard paper

while one side was left transparent for recording by a camcorder (HDR-XR200,

SONY, Tokyo, Japan). The operator and the camcorder were located behind a

cardboard (75cm x 100cm) with an opening (diameters, 10cm) to minimizing the

disturbance. The trials were carried out between 10 am and 6 pm and at 26-30°C

when the lizards were most active. The lizards were placed into the arena three

minutes prior to the trials for accommodation. The lizards appeared to settle down

within one minute of introducing by not running around or climbing out of the

container. The weevils were tied with a black cotton thread, and the opposite side of

the thread was tied to the center of a transparent plastic bar (58cm x 3cm x 0.3cm).

The length of the thread was ~ 43cm to confine the weevil’s movement to the center

of the arena for video recording. Three minutes after placing the lizard, the weevil

was introduced into the arena by hanging the plastic bar above the arena. The weevil

10

was placed at the center of the arena (about 30cm diameter) and approximately

15-30cm away from the lizard that often positioned itself at the periphery. The trial

started when the lizard was aware of the weevil by turning its eyes or head toward the

prey and ended after the lizard have stopped response to the prey. The survival rate,

number of bites, and prey handling time (when the lizard had the first physical contact

with the weevil until the lizard stopped responding to it) were calculated from the

videos.

Insect prey and hardness measurement

To assess the level and distribution of hardness in the lizard’s prey community,

the insects occurring within the habitats of lizards were sampled using hands,

sweeping nets, light traps, and pitfalls in Orchid and Green Islands in September and

October of 2015. The insects were collected along the forest trails where most lizards

could be found (eight sites in Orchid Island for five days and four sites in Green

Island for four days). The light traps (high-intensity discharge lamps, 35W, 6000K,

Cnight, Guangdong, China) were set up in Orchid Island for two hours/nights. The

lepidopterans and spiders were not sampled because they are considered to be ‘soft’

preys (Verwaijen, Van Damme & Herrel 2002). One to two individuals of each insect

species were obtained and later stored at a -20°C freezer in the laboratory until the

measurement. All voucher insect specimens of this study were archived in the insect

collection of the laboratory (Table S1).

The hardness of the insects (field-collected insect prey and the ‘hard’ and ‘soft’

weevils) was measured following Herrel et al. 2001. The ‘force at failure’ of the elytra

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(abdomen) or the thorax of the insects was measured to represent the ‘hardness’ of the

subjects. The ‘failure’ was defined as the completely loss of load-carrying capacity of

the structure (Collins 1993). The definition of ‘hardness’ in biological studies are

debatable and this term often refers to the phenomena of combining effects of

material and structural properties (e.g., for alternative ‘intractability’ in Evans &

Sanson 2005). Here we use ‘hardness’ to indicate the maximum force at failure for

crushing an insect’s exoskeleton. The upper and lower jaws of the lizard were

dissected, cleaned and then embedded in resin (model 40200029, Struers, Ballerup,

Denmark; diameter: 25mm) with the teeth rows exposed (H in Fig. 3b) to build the

biting platform (F & G in Fig. 3b). The upper part of the platform (F in Fig. 3b) was

connected to a force transducer (model 9203, Kistler Inc., Wintherthur, Swizerland)

(D in Fig. 3b), which was attached to the lower end of a camera mount on a copy

stand (E in Fig. 3b) and connected to the charge amplifier (model 5995). The lower

part of the platform was placed below the upper part at a position where the two jaws

can match completely. These insect specimens was measured at 25°C after they were

fully thawed in the laboratory. The specimen was positioned perpendicularly with

respect to the tooth rows. The upper platform was slowly moved down until the

exoskeleton of the insect was broken or crashed, and the amount of forces exerted on

the transducer was recorded simultaneously. The breakdown of the exoskeleton of the

specimen was recognized by a structural failure on the exoskeleton. The maximum

force needed to break the specimen’s exoskeleton was recorded as a proxy for the

hardness of the insect. These measurements were compared with the bite forces of the

J. swinhonis populations from Orchid and Green Islands (Wang et al. unpublished

12

data).

Chemical analysis

The existence of potential deterrent chemicals within the body of P. sarcitis

kotoensis was examined using the Gas Chromatograph-Mass Spectrometer (GC-MS).

The thorax and abdomen of three reared P. sarcitis kotoensis adults were separated

and immersed in 3ml of ether for 10 minutes of chemical extraction. 0.5µl of the

extraction was used for the GC-MS analysis. The GC-MS system consisted a gas

chromatograph (model: TRACE™ 1300), a mass selective detector (model: ISQ™,

Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) and the GC column

(inside diameter: 0.25mm, length: 30m, film thickness: 0.25um) (model: Rxi-5MS,

13423, Restek Corporation, Bellefonte, Pennsylvania, USA). The inlet temperature

was 280°C and helium was the carrier gas with the constant flow at 1 ml/min. The

initial temperature was 50°C and rose with a rate of 10°C/min to 300°C. This

temperature was held for 10 minutes without delay of the solvent. The peaks with

higher relative abundance were identified and compared with the spectrum libraries

(NIST, Gaithersburg, Maryland, USA, and WILEY 10R, Hoboken, New Jersey,

USA). If the SI (similarity index) and RSI (reverse similarity index) value of the

candidate were both higher than 800, it was considered as a possible chemical

compound extracted from the weevils (Tian et al. 2014). Then this compound was

identified and determined if it represents a potential repellent for vertebrate predators

in the online Pherobase (El-Sayed 2014).

13

Statistical analyses

The two-sample t tests were used to compare the reflectance spectra and daily

rhythm (daytime) between ‘hard’ and ‘soft’ weevils, and Mann-Whitney U test was

used to compare the daily rhythm of day and nighttime. Fisher’s exact test, negative

binomial regression, and survival analysis were employed to compare the survival rate,

number of bites, and prey handling duration in behavioral trials, respectively. In

negative binomial regression and survival analysis, we used the elytra length of the

weevils and the snout-vent length of the lizards as covariates. These statistics were

followed by Tukey post hoc tests to examine the differences between treatment

groups. Welch two sample t-test was used to compare the hardness between ‘hard’

and ‘soft’ weevils, and linear regression analyses were conducted to assess the

relationship between prey hardness and body size. All statistical analyses were carried

out in R 3.3.1 (R Development Core Team 2016).

Results

Effects of the hardness and claws

Immediately after introducing the weevils into the trial arena, the lizards

perceived the presence of the prey by turning their eyes and heads toward them

(movie 1 & 2). Then the lizards approached and bit the weevils (often the abdomen)

using their lateral teeth rows. The weevils were attacked by the lizards within an

average of 18.8 seconds (18.8 ± 22.9 sec, n = 59) after the introduction of the prey.

Approximately one half of the lizards (51%, n = 76) showed no predatory response

14

toward the weevils after more than 3 minutes, suggesting that they either were not

interested, neophobia, or avoided attacking aposematic preys. After the lizard’s first

attack, the weevils were either spat out (movie 1, 00:50) or consumed (movie 2, 00:40)

by the lizards. All ‘hard’ weevils were spat out immediately by the lizards and

survived the first attack. All rejected weevils have no visible physical damage to the

body and lived for more than two months after the trials (survival rate = 100%, n = 40)

(Fig. 4a), except that two individuals were injured and suffered a small depression on

the surface of lateral abdomen. The lizards made no more predatory attempt after

spitting out the weevils (movie 1). By contrast, all ‘soft’ weevils were chewed

continuously and subsequently swallowed by the lizards (movie 2) (Fig. 4a) (survival

rate = 0%; Fisher’s exact test: n = 38; ‘hard’ with claws vs. ‘soft’ with claws: p <

0.001; ‘hard’ without claws vs. ‘soft’ with claws: p < 0.001; ‘hard’ with claws vs.

‘soft’ without claws: p < 0.001; ‘hard’ without claws vs. ‘soft’ without claws: p <

0.001). The results were the same regardless of tarsal claws (Fig. 4a) (‘hard’ with

claws vs. ‘hard’ without claws: p = 1; ‘soft’ with claws vs. ‘soft’ without claws: p =

1).

The lizards bit ‘soft’ weevils significantly more times than ‘hard’ weevils (Fig.

4b) (mean number of bites = 1.5 ± 1.0 ‘hard’, 80.3 ± 34.0 ‘soft’; negative binomial

regression: χ2 = 227.8264, p < 0.001) (‘hard’ with claws vs. ‘soft’ with claws: Z =

18.991, p < 0.001; ‘hard’ without claws vs. ‘soft’ with claws: Z = -19.014, p < 0.001;

‘hard’ with claws vs. ‘soft’ without claws: Z = 19.146, p < 0.001; ‘hard’ without

claws vs. ‘soft’ without claws: Z = 19.044, p < 0.001), with no effect of tarsal claws

(‘hard’ with claws vs. ‘hard’ without claws: Z = -0.476, p = 1; ‘soft’ with claws vs.

15

‘soft’ without claws: Z = -0.060, p = 1). The lizards spent significantly more time

handling prey in ‘soft’ than in ‘hard’ weevils (Fig. 4c) (mean handling time = 0.7 ±

0.5 sec ‘hard’, 106.5 ± 102.4 sec ‘soft’; survival analysis: n = 78, χ2 = 202.98, p <

0.001) (‘hard’ with claws vs. ‘soft’ with claws: Z = 22.992, p < 0.001; ‘hard’ without

claws vs. ‘soft’ with claws: Z = -22.937, p < 0.001; ‘hard’ with claws vs. ‘soft’

without claws: Z = 21.637, p < 0.001; ‘hard’ without claws vs. ‘soft’ without claws: Z

= 22.049, p < 0.001). Again, there was no significant difference between treatments

with and without tarsal claws (Fig. 4c) (‘hard’ with claws vs. ‘hard’ without claws: Z

= -0.109, p = 0.914; ‘soft’ with claws vs. ‘soft’ without claws: Z = -1.954, p = 0.102).

Hardness of prey and lizard’s bite force

The prey hardness increased allometrically with size for most prey species

measured in the habitats (Fig. 5) [log10(Force at failure) = -0.9389 + 1.5885

log10(Body length), adjusted R2 = 0.5262, p < 0.001, AIC = 69.94692; Force at failure

= -9.2195 + 1.5678 Body length, adjusted R2 = 0.6494, p < 0.001, AIC = 503.2208],

with the exception that grasshoppers (Acr, Acrididae), owlflies (Asc, Ascalaphidae),

dragonflies (Lib, Libellulidae) and katydids (Tet, Tettigoniidae) were larger but softer

insect prey. The ‘hard’ and ‘soft’ weevils deviated from the regression of hardness

versus body length by being harder and softer than the prey of similar sizes,

respectively (Fig. 5). The ‘hard’ P. sarcitis kotoensis (32.6 ± 9.1 N, n = 17) were

significantly harder than their ‘soft’ counterparts (0.6 ± 0.6 N, n = 10) (Welch two

sample t-test, t = 14.46, p < 0.001) and the average hardness of insect prey in the same

habitat (Fig. 5) (9.95 ± 11.98 N, n = 72), except for the two rhinoceros beetles,

16

Xylotrupes philippinensis, 93.5 N and 60.8 N, black arrow 1 & 2, Fig. 5). The ‘hard’

P. sarcitis kotoensis were also harder than most other weevil species (Cur) in the

habitat, except for Aclees hirayamai (black arrow 3, Fig. 5). The average hardness of

‘hard’ P. sarcitis kotoensis was higher than the mean bite force of J. swinhonis from

Green Island (males: 27.97 ± 9.55 N, n = 91; females: 8.14 ± 2.42 N, n = 80) and

Orchid Island (males: 29.66 ± 9.62 N, n = 80; females: 12.03 ± 2.80 N, n = 89) (Fig.

5), indicating that most lizards from these two islands lack sufficient bite force

capable of penetrating the sclerotized exoskeletons of P. sarcitis kotoensis, especially

for females and smaller males. As what we would expect from insects mimicking

Pachyrhynchus, the hardness of a longhorn beetle, Doliops similis from Orchid Island

was merely 11.5 N (Table S1), which was much lower than that of ‘hard’ P. sarcitis

kotoensis.

Biochemical compounds of P. sarcitis kotoensis

We selected twenty peaks with the highest relative abundance from the master

chromatogram of the extraction of P. sarcitis kotoensis to compare with the spectrum

library (Fig. 6). Five of them, including the solvent (ethane; RT: 1.72, ST: 935, RST:

936, CAS number: 60-29-7), had both SI and RSI values higher than 800. The four

candidate compounds in P. sarcitis kotoensis were identified as ethyl propyl ether,

diethylene glycol, butylated hydroxy toluene and cadalene. These chemicals can be

found in other animals or plants but have no toxic or repellent functions, and do not

fall into the 13 categories of known insect toxins and repellents against vertebrate

predators (Blum 2012).

17

Discussion

Effective secondary defense

The study provides the first empirical evidences to support the morphological

defense hypothesis pioneered by A. R. Wallace almost 150 years ago. Our findings

clearly indicate that the hardness of aposematic Pachyrhynchus functions as a

secondary defense against the lizards, whereas the tarsal claw serves no defense, and

no toxin or repellent is stored in the weevil. This secondary defense of hardness is

highly effective in at least three intriguing features. First, the hardness of exoskeleton

significantly increased the survival of the weevils under lizard’s attack. In fact, the

survival rate was 100% for the ‘hard’ weevils. Almost all rejected weevils by the

lizard were undamaged and resumed walking immediately after the attack. By

contrast, the ‘soft’ weevils were all consumed by the lizards during the staged

encounters. These results suggest that the hardness of exoskeletion is an important

fitness component of morphological adaptation for aposematism in Pachyrhynchus.

Given that physical attacks by the predators are often costly to the prey in terms of

injury, escaping time or survival (Ruxton et al. 2004), it was very surprising to

observe that Pachyrhynchus with such highly effective defense as hardness that they

practically suffered no cost in terms of injury and survival. The high survival of

Pachyrhynchus under lizard’s attack can provide an empirical evidence for

evolutionary origin of aposematic colours. Because one of the conundrum of evolving

aposematic colours is that a novel conspicuous individual will be detected easily in

18

the population, and its rarity will make it challenging for predators to learn to avoid

the colour signal in future encounter (Fisher 1958). However, at the beginning of

evolving warning signals, if a rare novel conspicuous prey such as the case in

Pachyrhynchus can survive the initial attack, then the survived aposematic individual

will make it easier for predators to recognize the colour signal, thus the warming

colour can increase its frequency in the prey population (Ruxton et al. 2004).

Secondly, the hardness of weevil’s exoskeleton immediately triggered strong

aversive responses of the lizards after biting the ‘hard’ weevils (such as instantly

shaking heads and spitting out the prey). Similar to some of colourful Eupholus and

Trigonopterus species (Seago et al. 2009; Riedel et al. 2013), the Pachyrhynchus are

unique among those highly sclerotized flightless weevils to possess the most diverse

aposematic colours (Wallace 1895; Schultze 1923). Our findings indicate that this

anti-predation strategy is very effective in strengthening the aposematism in colourful

Pachyrhynchus weevils, by developing a robust association between warning displays

and prey unprofitability. Our earlier study found that after only a single encounter the

naïve lizard predators rapidly learned to avoid weevils for at least more than 23 days

(Tseng et al. 2014). The lizards can instantly learn from the weevil’s unprofitability

and reduce future attacks when they encounter prey items with similar warning

signals. Therefore, the inclusive fitness of aposematic weevils that confer such

defense may subsequently be raised.

Finally, the secondary defense using the hardness is a ‘secret weapon’ (sensu

lato Eisner, Eisner & Siegler 2005) in the sense that, similar to internally stored

chemical defenses in toxic prey, it is invisible and unexpected by the naïve predators.

19

Aposematic prey species that use internally stored toxic chemicals as the source of

defenses are well known in the nature. However, other invisible defense strategies

such as heavy armature of a harvestman (da Silva Souza & Willemart 2011) and hard

body used by aposematic weevils in this study are poorly studied (Caro 2017). Our

results suggest that, in additional to surviving the attack, the ‘secret weapon’ of

weevils simultanously strengthens lizard’s ‘unexpectancy’ via their outstanding

hardness compared to most insect prey with similar and even larger sizes in the

habitat. This high level of hardness largely explain the instant and strong aversive

responses displayed by the lizards after biting weevils with their teeth.

Multiple secondary defenses

This study showed that the hardness of Pachyrhynchus weevils is the main

secondary defenses against lizard predators. However, like other flightless weevils

such as Trigonopterus (Van De Kamp et al. 2014), Pachyrhynchus employs a series

of secondary defensive strategies when encounters human predators, by firstly turning

itself around the tree branches or leaves to hide, then performing a sudden drop-off

from the trees, and finally a death-feigning on the ground (Wang et al. personal

observations). This series of secondary defensive strategies may be essential in

evading predators at initial stage of prey recognition and pursuing, especially when

weevils are soft (i.e., newly emerged without a ‘hard’ defense) or encountering

predators with greater bite forces. Nevertheless, it is still unknown whether ‘soft’

weevils could perform drop-off and death-feigning equally well as that of ‘hard’

individuals.

20

Mechanical mechanism of hardness

The mechanical mechanism of achieving the outstanding hardness for a small

sized insect such as Pachyrhynchus remains elusive. The mechanical properties of

insect’s cuticle like strength and stiffness depend on many physical and chemical

factors, including arrangement of cuticle microfibers, protein and water content, and

process of pigmentation and sclerotization (Andersen 2010; Klocke & Schimitz 2011).

A recent study indicated that an endosymbiont Nardonella specialized in cuticle

formation and hardening of Pachyrhynchus host by tyrosine provisioning, suggesting

the important role of the bacterial symbiosis for the weevil’s hardness (Anbutsu et al.

2017). The microstructure of elytral cuticle in weevils is phylogenetically conserved

to have a ‘weevil-specific’ combination of interlocking of exo- and endocuticle, an

endocuticle with distinct ovoid macrofibers embedded in a matrix, and small angles

between successive endocuticular layers (Van de Kamp, Riedel & Greven 2016).

These microstructural attributes or their modifications may have contributed to the

hardness of Pachyrhynchus. However, the cuticular microstructures in other hard

flightless weevils such as Trigonopterus do not deviate from the weevil’s uniform

ground plan but instead possess thicker elytra (Van de Kamp et al. 2016). Besides

cuticular microstructures and thickness, sclerotization (Hopkins & Kramer 1992),

heavy metals and halogens (Schofield, Nesson & Richardson 2002) can enhance

mechanic strength of insect cuticles. The mechanical behaviour of an insect body is

the result of combining material properties and geometric characteristics. Thus the

geometry of an organism can modify the stress distribution and reduce the stress

21

concentration to prevent mechanical failure (e.g., coiling mollusk shells in Rajabi et al.

2014). Because the Pachyrhynchus has a peculiar dome-shaped thorax, abdomen and

densely interlocked elytra, these oval architectures may build these weevils more

robust bodies as a defense strategy against predators.

Adaptive defense against lizard’s predation

This study provided convincing evidences to indicate that the hardness of

aposematic Pachyrhynchus weevils is ecologically important and represents an

adaptive defense mechanism against the predation of tree lizards. The results

suggested that the hardness of the weevils was among the highest in the prey spectrum

of the tree lizards and higher than the average bite force of the lizards from Green and

Orchid Islands. Therefore, the predation of small vertebrate predators like tree lizards

may have acted as primary driving/maintaining forces for the evolution of hardness in

Pachyrhynchus weevils. At present, Japalura swinhonis is the only known natural

predator of Pachyrhynchus weevils in these two islands (Huang 2007). The other

eight lizard species of the islands are less likely to be major predators of the weevils

due to their habitat and prey preference (Chen et al. 2008; Li et al. 2010) (Table S2).

Pachyrhynchus’s hard body likely has been originated in the common ancestors of

relates like Eupyrgops, Metapocyrtus and Polycatus in the Philippine Archipelago and

neighboring islands (Schultze 1923), therefore the arboreal lizards of Southeast Asia

such as Gekko spp. might also be important in promoting the origin of weevil’s hard

exoskeletons.

Multiple selective forces of diverse predator community may each play an

22

important role in driving and maintaining the evolution of aposematic colours and

associated defense strategies (Willink et al. 2014). In addition to lizard’s (reptilian)

predation, avian and mammalian predations constitute the other two major selective

forces for tropical insects with aposematic or cryptic colouration (Mappes et al. 2014;

Roslin et al. 2017). Avian predation is likely to be essential for the evolution of

aposematism and defense strategies in Pachyrhynchus weevils because birds in

general possess a wide range of colour spectrum (Bennett & Théry 2007), and they

are primary predators for aposematic insects (Iniesta, Ratton & Guerra 2017). In

Green and Orchid Islands, there are about 62 bird species that eat insects as parts of

their diet (Chen et al. 2008; Li et al. 2010; Severinghaus et al. 2012) (Table S3).

Among them, 12 species [emerald dove (Chalcophaps indica), large hawk-cuckoo

(Cuculus sparverioides), Himalayan cuckoo (Cuculus saturatus), lesser coucal

(Centropus bengalensis), common kingfisher (Alcedo atthis), brown shrike (Lanius

cristatus), Japanese paradise-flycatcher (Terpsiphone atrocaudata), brown-eared

bulbul (Microscelis amaurotis), lowland white-eye (Zosterops meyeni),

white-shouldered starling (Sturnus sinensis), eyebrowed thrush (Turdus obscurus),

and blue rock thrush (Monticola solitarius)] inhibit the same habitat and are probable

avian predators of Pachyrhynchus (Table S3). At present no bite forces of avian fauna

from Green and Orchid islands is available for comparison. However, the bite forces

of the vertebrates are often strongly correlated with head sizes and shapes (Anderson,

McBrayer & Herrel 2008). The average bite force of small to median size passerines

ranges was 9.3 ± 10.5 N (2.9-38.4 N, n = 18, Estrildidae & Fringillidae, Van der Meij

& Bout 2006), suggesting that the hardness of Pachyrhynchus weevils (32.6 ± 9.1 N)

23

may be effective against predation from most passerine birds of similar sizes.

Nevertheless, the shape beaks and pecking behavior of most birds alone may be

proven effective to penetrate Pachyrhynchus’s exoskeleton. Our field observations

suggested that Pachyrhynchus weevils were defenseless and could be easily pecked

and swallowed by adult domestic chickens. However, smaller domestic chicks were

observed to spend considerable amount of time pecking the weevils, but could not

successfully swallow them. These preliminary observations and the bite force

measurement of birds together suggest that the hardness of Pachyrhynchus weevils

may only be effective against smaller avian predators in their habitats.

There are 14 mammal species found in Green and Orchid Islands that may

consume insects (Chen et al. 2008; Li et al. 2010), in which 10 species [masked palm

civet (Paguma larvata), Chinese white-toothed shrew (Crocidura rapax tadae & C. r.

lutaoensis), Pallas's squirrel (Callosciurus erythraeus thaiwanensis), house mouse

(Mus musculus), lesser rice field rat (R. losea), Tanezumi rat (R. tanezumi), greater

bandicoot rat (Bandicota indica), brown rat (R. norvegicus) and black rat (R. rattus)]

occur in Pachyrhynchus’s habitats. Tanezumi rats were observed to forage on tree

trunks of weevil’s habitat during the day and nighttime, and are likely to be one of

their mammal predators, with capable bite force of penetrating their exoskeleton

(24.7-76.8 N, brown rat, R. norvegicus, Cox et al. 2012). Future studies on identifying

the major predators and assessing spatio-temporal variation of predation in

Pachyrhynchus weevils will help us to better understand the ecology and evolution of

the hardness in aposematic prey.

24

References

Abrams, P.A. (2000) The evolution of predator-prey interactions: theory and evidence.

Annual Review of Ecology and Systematics, 31, 79-105.

Anbutsu, H., Moriyama, M., Nikoh, N., Hosokawa, T., Futahashi, R., Tanahashi, M.,

Meng, X.Y., Kuriwada, T., Mori., N., Oshima., K., Hattori, M., Fujie, M., Satoh,

N., Maeda, T., Shigenobu, S., Koga, R. & Fukatsu, T. (2017) Small genome

symbiont underlies cuticle hardness in beetles. Proceedings of the National

Academy of Sciences of the United States of America, 201712857.

Andersen, S.O. (2010) Insect cuticular sclerotization: a review. Insect Biochemistry

and Molecular Biology, 40, 166-178.

Anderson, R.A., McBrayer, L.D., & Herrel, A. (2008) Bite force in vertebrates:

opportunities and caveats for use of a nonpareil whole‐animal performance

measure. Biological Journal of the Linnean Society, 93, 709-720.

Barševskis, A. (2013) Contribution to the knowledge of the genus Doliops

Waterhouse, 1841 (Coleoptera: Cerambycidae). Baltic Journal of Coleopterology,

13, 73-89.

Bennett, A.T. & Théry, M. (2007) Avian color vision and coloration:

multidisciplinary evolutionary biology. The American Naturalist, 169, S1-S6.

Blount, J.D., Rowland, H.M., Drijfhout, F.P., Endler, J.A., Inger, R., Sloggett, J.J.,

Hurst, G.D.D., Hodgson, D.J. & Speed, M.P. (2012) How the ladybird got its

spots: Effects of resource limitation on the honesty of aposematic signals.

Functional Ecology, 26, 334-342.

25

Blum, M. (2012) Chemical Defenses of Arthropods. Elsevier.

Caro, T. (2017) Wallace on coloration: contemporary perspective and unresolved

insights. Trends in Ecology & Evolution, 32, 23-30.

Chen, P.C., Chang, M.H., Chen, S.F., Li, Z.L., Chen, S.L., Lin, H.C. & Chu, C.W.

(2008) The Research of Terrestrial Vertebrate Fauna on the Green Island.

Marine National Park Headquarters.

Chen, Y.T., Tseng, H.Y., Jeng, M.L., Su, Y.C., Huang, W.S. & Lin, C.P. (2017)

Integrated species delimitation and conservation implications of an endangered

weevil Pachyrhynchus sonani (Coleoptera: Curculionidae) in Green and Orchid

Islands of Taiwan. Systematic Entomology, 42, 796-813.

Collins, J.A. (1993) Failure of materials in mechanical design: analysis, prediction,

prevention. John Wiley & Sons.

Cooper Jr, W.E. (1989) Absence of prey odor discrimination by iguanid and agamid

lizards in applicator tests. Copeia, 472-478.

Cox, P.G., Rayfield, E.J., Fagan, M.J., Herrel, A., Pataky, T.C. & Jeffery, N. (2012)

Functional evolution of the feeding system in rodents. PLoS ONE, 7, e36299.

Dickerson, R.E., Merrill, E.D., McGregor, R.C., Schultze, W., Taylor, E.H. & Herre,

A.W. (1928) Distribution of Life in the Philippines. Bureau of Printing.

Edmunds, M. (1974) Defence in Animals: A Survey of Anti-Predator Defences.

Longman Publishing Group.

Eisner, T., Eisner, M., & Siegler, M. (2005) Secret weapons: defenses of insects,

spiders, scorpions, and other many-legged creatures. Harvard University Press.

El-Sayed, A.M. (2014) The Pherobase: Database of pheromones and semiochemicals.

26

The Pherobase.

Evans, A.R., & Sanson, G.D. (2005) Biomechanical properties of insects in relation to

insectivory: cuticle thickness as an indicator of insect ‘hardness’ and

‘intractability’. Australian Journal of Zoology, 53, 9-19.

Fisher, R.A. (1958) The genetic theory of natural selection. Dover.

Herrel, A., Damme, R.V., Vanhooydonck, B., & Vree, F.D. (2001) The implications

of bite performance for diet in two species of lacertid lizards. Canadian Journal

of Zoology, 79, 662-670.

Hopkins, T.L. & Kramer, K.J. (1992) Insect cuticle sclerotization. Annual Review of

Entomology, 37, 273-302.

Huang, W.S. (2007) Ecology and reproductive patterns of the agamid lizard Japalura

swinhonis on an east Asian island, with comments on the small clutch sizes of

island lizards. Zoological Science, 24, 181-188.

Iniesta, L.F.M., Ratton, P. & Guerra, T.J. (2017) Avian predators avoid attacking

artificial aposematic millipedes in Brazilian Atlantic Forest. Journal of Tropical

Ecology, 33, 89-93.

Klocke, D., & Schmitz, H. (2011) Water as a major modulator of the mechanical

properties of insect cuticle. Acta Biomaterialia, 7, 2935-2942.

Li, Z.D., Su, Y., Li, Z.M., Guo, S.J., Wu, T.Y., Lin, Y.J., Yang, D.Q., Liang, Y.Z., Yu,

H.Y., Qiu, B.H., Hsu, S.T., Lin, Y.Z., Liu, Z.H., Weng, R.H., Ye, R.K., Ye, C.B.,

Wu, Y.X., Tseng, Y.H., Pan, T.Z., Tsai, Z.H. Su, X.Q., Chen, Y.J. & Tsai, J.H.

(2010) The Inventory of Natural Systems in Orchid Island. Marine National Park

Headquarters.

27

Maan, M.E. & Cummings, M.E. (2011) Poison frog colors are honest signals of

toxicity, particularly for bird predators. The American Naturalist, 179, E1-E14.

Mappes, J., Kokko, H., Ojala, K., & Lindström, L. (2014) Seasonal changes in

predator community switch the direction of selection for prey defences. Nature

Communications, 5, 5016.

Meyers, J.J. & Irschick, D.J. (2015) Does whole-organism performance constrain

resource use? A community test with desert lizards. Biological Journal of the

Linnean Society, 115, 859-868.

Pasteels, J.M., Grégoire, J.C. & Rowell-Rahier, M. (1983) The chemical ecology of

defense in arthropods. Annual Review of Entomology, 28, 263-289.

R Development Core Team. (2016) R: A Language and Environment for Statistical

Computing. Vienna, Austria. ISBN 3-900051-07-0. URL

http://www.R-project.org.

Rajabi, H., Darvizeh, A., Shafiei, A., Eshghi, S. & Khaheshi, A. (2014) Experimental

and numerical investigations of Otala lactea’s shell–I. Quasi-static analysis.

Journal of the Mechanical Behavior of Biomedical Materials, 32, 8-16.

Riedel, A., Sagata, K., Surbakti, S., Tänzler, R., & Balke, M. (2013) One hundred and

one new species of Trigonopterus weevils from New Guinea. ZooKeys, 280, 1.

Robinson, M.H. (1969) Defenses against visually hunting predators.Evolutionary

Biology, 3, 5-59.

Roslin, T., Hardwick, B., Novotny, V., Petry, W.K., Andrew, N.R., Asmus, A., ... &

Cameron, E.K. (2017) Higher predation risk for insect prey at low latitudes and

elevations. Science, 356, 742-744.

28

Ruxton, G.D., Sherratt, T.N. & Speed, M.P. (2004) Avoiding Attack: The Evolutionary

Ecology of Crypsis, Warning Signals and Mimicry. Oxford University Press.

Schofield, R.M.S., Nesson, M.H. & Richardson, K.A. (2002) Tooth hardness

increases with zinc-content in mandibles of young adult leaf-cutter ants.

Naturwissenschaften, 89, 579-583.

Schultze, W. (1923) A monograph of the pachyrrhynchid group of the Brachyderinae,

Curculionidae: Part I. The genus Pachyrhynchus Germar. The Philippine Journal

of Science, 23, 609-673.

Seago, A.E., Brady, P., Vigneron, J.P., & Schultz, T.D. (2009) Gold bugs and beyond:

a review of iridescence and structural colour mechanisms in beetles (Coleoptera).

Journal of the Royal Society Interface, 6, S165-S184.

Severinghaus, L.L., Ding, T.S., Fang, W.H., Lin, W.H., Tsai, M.C., & Yen, C.W.

(2012) The avifauna of Taiwan 2nd edition. Taipei, Taiwan: Forestry Bureau of

Council of Agriculture of Executive Yuan.

Sherratt, T.N. & Beatty, C.D. (2003) The evolution of warning signals as reliable

indicators of prey defense. The American Naturalist, 162, 377-389.

da Silva Souza, E. & Willemart, R.H. (2011) Harvest-ironman: heavy armature, and

not its defensive secretions, protects a harvestman against a spider. Animal

Behaviour, 81, 127-133.

Starr, C.K. & Wang, H.Y. (1992) Pachyrhynchine weevils (Coleoptera: Curculionidae)

of the islands fringing Taiwan. Journal of Taiwan Museum, 45, 5-14.

Stevens, M. (2013) Sensory Ecology, Behaviour, and Evolution. Oxford University

Press.

29

Summers, K. & Clough, M.E. (2001) The evolution of coloration and toxicity in the

poison frog family (Dendrobatidae). Proceedings of the National Academy of

Sciences of the USA, 98, 6227-6232.

Tian, H., Zhan, P., Deng, Z., Yan, H. & Zhu, X. (2014) Development of a flavour

fingerprint by GC‐MS and GC‐O combined with chemometric methods for the

quality control of Korla pear (Pyrus serotina Reld). International Journal of

Food Science & Technology, 49, 2546-2552.

Tseng, H.Y., Lin, C.P., Hsu, J.Y., Pike, D.A. & Huang, W.S. (2014) The functional

significance of aposematic signals: geographic variation in the responses of

widespread lizard predators to colourful invertebrate prey. PLoS ONE, 9,

e91777.

Tseng, H.Y., W.S. Huang, M.L. Jeng, R.J.T. Villanueva, O.M. Nuñeza & Lin, C.P.

(2017) Complex inter-island colonization and peripatric founder speciation

promote diversification of flightless Pachyrhynchus weevils in the

Taiwan-Luzon volcanic belt. Journal of Biogeography, in press.

Van De Kamp, T., dos Santos Rolo, T., Vagovič, P., Baumbach, T., & Riedel, A.

(2014) Three-dimensional reconstructions come to life-interactive 3D PDF

animations in functional morphology. PLoS ONE, 9, e102355.

Van de Kamp, T., Riedel, A. & Greven, H. (2016) Micromorphology of the elytral

cuticle of beetles, with an emphasis on weevils (Coleoptera: Curculionoidea).

Arthropod Structure and Development, 45, 14-22.

Van der Meij, M.A.A., & Bout, R.G. (2006) Seed husking time and maximal bite

force in finches. Journal of Experimental Biology, 209, 3329-3335.

30

Verwaijen, D., Van Damme, R., & Herrel, A. (2002) Relationships between head size,

bite force, prey handling efficiency and diet in two sympatric lacertid lizards.

Functional Ecology, 16, 842-850.

Wallace, A.R. (1867) Mimicry and other protective resemblance among animals.

Westminster and Foreign Quarterly Review, 32, 1-43.

Wallace, A.R. (1895) Natural Selection and Tropical Nature: Essays on Descriptive

and Theoretical Biology. Macmillan, London, UK.

Willink, B., Garcia-Rodriguez, A., Bolaños, F., & Pröhl, H. (2014). The interplay

between multiple predators and prey colour divergence. Biological Journal of the

Linnean Society, 113, 580-589.

Yewers, M.S., McLean, C.A., Moussalli, A., Stuart-Fox, D., Bennett, A.T.D. & Knott,

B. (2015) Spectral sensitivity of cone photoreceptors and opsin expression in two

colour-divergent lineages of the lizard Ctenophorus decresii. Journal of

Experimental Biology, 218, 1556-1563.

31

Figure 1. Pachyrhynchus sarcitis kotoensis and experimental manipulation. (a) ‘Hard’

and (b) ‘soft’ P. sarcitis kotoensis. (c) The predator, Japalura swinhonis lizard. (d)

The locality for measuring the colour spectrum of the blue spots (red circles) and the

black background (white circles) of P. sarcitis kotoensis. (e) ‘Claw’ (intact) and (f)

‘clawless’ (surgically removed) tarsi of P. sarcitis kotoensis. Images by L.Y. Wang (a

& b) and F.C. Hsu (c).

32

Figure 2. The reflectance spectra and daily rhythm of ‘hard’ and ‘soft’ Pachyrhynchus

sarcitis kotoensis in the behavioural experiments. (a) The reflectance spectra of blue

spot and dark background colours on the cuticles of the weevils (‘hard’ n = 4, ‘soft’ n

= 4) were measured using a spectrometer (Jaz spectrometer, Ocean Optics, Inc.,

Dunedin, Florida). The colour and grey shaded regions are the 95% confidence

intervals of the measurements. (b) The daily rhythm of the weevils (‘hard’ n = 10,

‘soft’ n = 10) were recorded for six consecutive days using a video monitor

(DS-VR7160H, Der Shuenn, Taipei, Taiwan). The weevils were placed in petri dishes

divided into four equal quadrants. The number of the weevil’s movements between

the quadrants in the first 10 minutes of every two hours was calculated. The shaded

regions are the standard deviation of the number of movements.

0

50

100

150

300 400 500 600 700

Reflc

etan

ce ra

tio (%

)

Wavelength (nm)

Hard weevil / blue spot

Hard weevil /black background

Soft weevil / blue spot

Soft weevil /black background 0

25

50

75

Num

ber o

f mov

emen

ts

SoftHard

7:00 A

M

7:00 P

M

Time

7:00 A

M

7:00 P

M

7:00 A

M

7:00 P

M

7:00 A

M

7:00 P

M

7:00 A

M

7:00 P

M

7:00 A

M

7:00 P

M

7:00 A

M

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

(a) (b)

33

Figure 3. The arena and recording set up for behavioral trials of Japalura swinhonis’s

predation on Pachyrhynchus sarcitis kotoensis (a). A: The bottom of the plastic

container was padded with soil. The three inner vertical planes of the plastic container

were covered with cardboard paper while left one side transparent for video recording.

B: The cardboard was used to hide the operator and video camera from disturbing the

lizards. C: The video camera (HDR-XR200, SONY, Tokyo, Japan). The weevil was

introduced to the arena using a black cotton thread tied between its thorax and

abdomen, and attached to the center of a transparent plastic bar. Experimental set-up

for measuring the hardness of insect prey (b). The force transducer (D) was attached

to the lower end of the camera mount on the copy stand (E). The upper and lower

jaws of the lizard were embedded in resin platforms (F) and (G), respectively. The

upper jaw was attached to the force transducer and the lower jaw was placed directly

below the upper jaw to match the upper one. The prey was oriented perpendicularly

with respect to the lower tooth rows (H).

D EF

G

(a) (b)

AB

C 5cm

15cm

10cm

58cm

3cm

43cm

30cm

40cm

H

GH

25mm

34

0

25

50

75

100

Surv

ival r

ate

(%)

0

25

50

75

Num

ber o

f bite

s

0

50

100

150

200

Prey

han

dling

dur

ation

(s)

HardSoft

HardSoft

HardSoft

Hard with claws

Softwith claws

Hard without claws

Softwithout claws

a a

b b

bb

a a

a a

b

b

(a)

(b)

(c)

n=20 n=20n=19 n=19

35

Figure 4. The survivorship of the weevil, Pachyrhynchus sarcitis kotoensis and

predatory response of the tree lizard, Japalura swinhonis in the behavioral trials. The

‘hard’ treatments were significantly different from ‘soft’ ones in (a) survival rate, (b)

number of bites, and (c) prey handling duration of the lizards. a and b indicate groups

with significant differences (Tukey post hoc tests, p < 0.001).

36

Figure 5. The hardness of Pachyrhynchus sarcitis kotoensis and insect prey from

Green and Orchid Islands. The regression line [log10(Force at failure) =

-0.9389+1.5885log10(Body length), adjusted R2 = 0.5262, p < 0.001, AIC = 69.94692]

is the correlation between log transformed hardness and body length by excluding

four insect families (Arc, Asc, Lib & Tet). The horizontal lines are the mean bite

forces of male and female tree lizards, Japalura swinhonis from Green and Orchid

Islands, with shaded regions indicating the 95% confidence intervals of the

measurements. Acr, Acrididae (Horned grasshoppers); Ant, Anthribidae (Fungus

weevils); Aph, Aphrophoridae (Spittlebugs); Asc, Ascalaphidae (Owlflies); Bla,

Acr

Acr

AphAsc

Bla

Bla

Bup

CarCar

CeraCera

Cera

Cera

Cera

Cerc

Cerc

Chr

Chr

ChrChr

Chr

Cur

Cur

CurCur

Cur

Ant

Bre

CurCur

Ela

Ela

For

ForFla

Tro

Tr oTro

Tro

Gry

GryGry

Hyb

Hyb

Lib

LucLuc

Lyc

Lyg

NitNit

Pen

PenPen

Pen

Pen Pyr

Sca

Sca

Sca

Sca

Sca

Sca

Sca

Sca

Sca

Sca

Sca

Sca

Ten

Ten

Tet

Tet

Tet

25

50

75

20 40 60Body length (mm)

Forc

e at

failu

re (N

)

Insect families

WeevilHard P. sarcitis

Soft P. sarcitis

Lizard

Orchid Island

Green Island

Orchid Island

Green Island

♀

♂

♂

♀

1

2

3

AcrAntAphAscBlaBreBupCarCeraCercChrCurElaFla

ForGryHybLibLucLycLygNitPenPyrScaTenTetTro

00

37

Blaberidae (Cockroaches); Bre, Brentidae (Straight-snouted weevils); Bup,

Buprestidae (Jewel beetles); Car, Carabidae (Ground beetles); Cera, Cerambycidae

(Long-horned beetles); Cerc, Cercopidae (Froghoppers); Chr, Chrysomelidae (Leaf

Beetles); Cur, Curculionoidea (Weevils); Ela, Elateridae (Click beetles); Fla, Flatidae

(Flatid planthoppers); For, Formicidae (Ants); Gry, Gryllidae (Crickets); Hyb,

Hybosoridae (Scavenger scarab beetles); Lib, Libellulidae (Dragonflies); Luc,

Lucanidae (Stag beetles); Lyc, Lycidae (Net-winged beetles); Lyg, Lygaeoidea

(Chinch bugs); Nit, Nitidulidae (Sap beetles); Pen, Pentatomoidea (Stink bugs); Pyr,

Pyrgomorphidae (Gaudy grasshoppers); Sca, Scarabaeidae (Scarab beetles), Ten,

Tenebrionidae (Darkling beetles); Tet, Tettigoniidae (Katydids); Tro, Tropiduchidae

(Tropiduchid planthoppers).

38

Figure 6. The master chromatogram and identified candidate compounds from the

extraction of Pachyrhynchus sarcitis kotoensis. The compounds that have SI

(similarity index) and RSI (relative similarity index) higher than 800 are shown in the

figure with retention time (RT). The chemical profile of these compounds are: Ethane

(solvent), RT: 1.72, SI: 935, RSI: 936, Chemical Abstract Service (CAS) registry

number: 60-29-7; Ethyl propyl ether, RT: 1.85, SI: 867, RSI: 890, CAS#: 628-32-0;

Diethylene glycol, RT: 5.30, SI: 967, RSI: 967, CAS#: 111-46-6; Butylated hydroxy

toluene, RT: 13.17, SI: 941, RSI: 945, CAS#: 128-37-0; Cadalene, RT: 15.65, SI: 947,

RSI: 956, CAS#: 483-78-3

5 10 15 20 25 30 35Time (min)

10

20

30

40

50

60

70

80

90

100R

elat

ive

abun

danc

e (%

)

00

RT: 1.72Ethane

RT: 1.85Ethyl propyl ether

RT: 5.30Diethylene glycol

RT: 13.17Butylated hydroxy toluene

RT: 15.65Cadalene

39

Table S1. List of insect prey species and hardness measurements from Green and Orchid Islands.

No. Order Family Species Common name Chinese name ID Abb. Body

part a

Hardn

ess (N)

Body

weight

(g)

Body

lengt

h

(mm)

Body

width

(mm)

Site b

36 Odonata Libellulidae/

蜻蜓科 Pantala flavescen Dragonfly 薄翅蜻蜓 Lu-Yi Wang Lib AN 2.8 0.302 48.62 8.40 OI

32 Orthoptera Acrididae/蝗科

Horned

Grasshopper Lu-Yi Wang Acr TX 5.3 0.279 53.49 5.12 OI

64 Orthoptera Acrididae

Horned

Grasshopper Lu-Yi Wang Acr TX 3.3 1.458 61.87 7.83 GI

7 Orthoptera Gryllidae/蟋蟀科

Cricket

Lu-Yi Wang Gry AN 0.7 0.052 5.55 1.91 OI

39 Orthoptera Gryllidae

Cricket

Lu-Yi Wang Gry AN 4.3 1.100 25.24 8.62 OI

57 Orthoptera Gryllidae

Cricket

Lu-Yi Wang Gry AN 3.7 0.068 12.01 3.44 GI

21 Orthoptera Pyrgomorphidae/

錐頭蝗科

Gaudy

grasshopper Lu-Yi Wang Pyr TX 2.6 0.103 20.65 3.48 OI

38 Orthoptera Tettigoniidae/

螽斯科

Katydid

Lu-Yi Wang Tet TX 1.8 4.472 52.70 9.40 OI

65 Orthoptera Tettigoniidae

Katydid

Lu-Yi Wang Tet AN 2.7 0.243 36.92 4.82 GI

30 Orthoptera Tettigoniidae Mecopoda elongata Katydid 臺灣騷斯 Feng-Chuan Hsu Tet AN 15.2 2.337 16.66 8.48 OI

40

49 Blattodea Blaberidae/

匍蜚蠊科

Cockroach

Lu-Yi Wang Bla TX 0.5 0.070 14.54 6.52 OI

62 Blattodea Blaberidae

Cockroach

Lu-Yi Wang Bla TX 6.4 0.283 27.33 11.55 GI

80 Hemiptera Aphrophoridae/

尖胸沫蟬科 Ariptyelus sp. Spittlebug

Shun-Chern

Tsaur Aph AN 1.1 0.011 6.72 2.81 GI

9 Hemiptera Cercopidae/

沫蟬科 Eoscarta botelensis Froghopper 蘭嶼曙沫蟬

Hsien-Tzung

Shih Cerc AN 0.7 0.009 7.08 3.48 OI

17 Hemiptera Cercopidae Eoscarta botelensis Froghopper 蘭嶼曙沫蟬 Hsien-Tzung

Shih Cerc AN 0.9 0.008 7.04 3.07 OI

68 Hemiptera Flatidae/

青翅飛蝨科 Mimophantia maritima

Flatid

planthopper 禾草褐蛾蠟禪

Shun-Chern

Tsaur Fla AN 1.4 0.016 6.89 3.09 GI

23 Hemiptera Lygaeoidea/

長蝽科 Neoletheus sp. Chinch bug

Jing-Fu Tsai Lyg AN 0.5 0.007 6.27 2.25 OI

26 Hemiptera Pentatomoidea/

蝽科 Lampromicra myakona Stink bug

Jing-Fu Tsai Pen AN 4.3 0.078 10.96 5.44 OI

52 Hemiptera Pentatomoidea Canto ocellatus Stink bug 角盾蝽 Jing-Fu Tsai Pen AN 4.8 0.206 21.12 10.00 OI

34 Hemiptera Pentatomoidea Glaucia sp. Stink bug


61 Hemiptera Pentatomoidea Leptocorisa acuta Stink bug 禾蛛緣椿象 Jing-Fu Tsai Pen AN 2.7 0.156 16.65 2.99 GI

45 Hemiptera Pentatomoidea Plautia sp. Stink bug


78 Hemiptera Tropiduchidae/ Kallitaxila sinica Tropiduchid Shun-Chern Tro AN 0.8 0.010 6.16 2.74 GI

41

軍配飛蝨科 planthopper Tsaur

79 Hemiptera Tropiduchidae Kallitaxila sinica Tropiduchid

planthopper

Shun-Chern

Tsaur Tro AN 0.8 0.018 6.97 3.09 GI

76 Hemiptera Tropiduchidae Sogana hopponis Tropiduchid

planthopper 軍配飛蝨

Shun-Chern

Tsaur Tro AN 2.0 0.017 6.05 2.52 GI

81 Hemiptera Tropiduchidae Sogana hopponis Tropiduchid

planthopper

Shun-Chern

Tsaur Tro AN 2.9 0.023 11.58 3.96 GI

53(2) Coleoptera Anthribidae/

長角象鼻蟲科 Anthribidae sp. Fungus weevil

Chen-Fu Hsu Ant AN 2.5 0.023 8.80 2.95 OI

53 Coleoptera Brentidae/

三錐象鼻蟲科 Baryrrhynchus sp.

Straight-snoute

d weevil Chen-Fu Hsu Bre AN 11.0 0.025 13.94 3.28 OI

83 Coleoptera Buprestidae/

吉丁蟲科 Chrysodema dalmanni Jewel beetle 粉彩吉丁蟲 Feng-Chuan Hsu Bup AN 33.9 0.636 26.23 9.00 GI

67 Coleoptera Carabidae/

步行蟲科 Calleida sp. Ground beetle

Lan-Wei Yeh Car AN 1.8 0.022 9.83 3.02 GI

44 Coleoptera Carabidae Cosmodela batesi Ground beetle 臺灣八星虎甲蟲 Lu-Yi Wang Car AN 2.5 0.128 16.71 5.43 OI

22 Coleoptera Cerambycidae/

天牛科 Ceresium longicorne

Long-horned

beetle 長角姬天牛 Sin-Yan Shih Cera AN 1.6 0.009 8.04 1.85 OI

10 Coleoptera Cerambycidae Doliops similis Long-horned

beetle 擬硬象天牛 Sin-Yan Shih Cera AN 11.5 0.243 14.61 5.84 OI

42

31 Coleoptera Cerambycidae Epepeotes ambigenus Long-horned

beetle 蘭嶼縱紋長角天牛 Sin-Yan Shih Cera AN 39.9 0.623 24.69 8.00 OI

3 Coleoptera Cerambycidae Olenecamptus bilobus Long-horned

beetle 五星白天牛 Sin-Yan Shih Cera AN 15.3 0.118 17.25 4.22 OI

48 Coleoptera Cerambycidae Sybra sp. Long-horned

beetle Sin-Yan Shih Cera AN 6.8 0.042 11.28 2.78 OI

77 Coleoptera Chrysomelidae/

金花蟲科 Cleorina janthina Leaf beetle 小溝腳猿金花蟲 Chi-Feng Lee Chr AN 1.3 0.006 3.67 2.28 GI

18 Coleoptera Chrysomelidae Pyrrhalta sp. Leaf beetle

Chi-Feng Lee Chr AN 3.6 0.010 4.68 2.43 OI

28 Coleoptera Chrysomelidae Monolepta ongi Leaf beetle 蘭嶼長腳螢金花蟲 Chi-Feng Lee Chr AN 1.5 <0.001 3.67 2.25 OI

47 Coleoptera Chrysomelidae Rhyparida sakisimensis Leaf beetle 巨島嶼猿金花蟲 Chi-Feng Lee Chr AN 2.4 0.010 4.79 2.91 OI

75 Coleoptera Chrysomelidae Rhyparida sakisimensis Leaf beetle 巨島嶼猿金花蟲 Chi-Feng Lee Chr AN 3.0 0.022 5.15 3.12 GI

6 Coleoptera Curculionidae/

象鼻蟲科 Aclees hirayamai Weevil 白波粉象鼻蟲 Chen-Fu Hsu Cur AN 29.9 0.149 16.65 5.86 OI

37 Coleoptera Curculionidae Mechistocerus sp. Weevil

Chen-Fu Hsu Cur AN 19.7 0.067 9.75 4.23 OI

13 Coleoptera Curculionidae Metapocyrtus immeritus Weevil 紅足鏽象鼻 Chen-Fu Hsu Cur AN 10.5 0.040 8.23 3.36 OI

14 Coleoptera Curculionidae Metapocyrtus immeritus Weevil 紅足鏽象鼻 Chen-Fu Hsu Cur AN 19.0 0.061 10.04 3.83 OI

73 Coleoptera Curculionidae Metapocyrtus sp. Weevil

Chen-Fu Hsu Cur AN 13.7 0.048 9.13 3.87 GI

74 Coleoptera Curculionidae Metapocyrtus sp. Weevil

Chen-Fu Hsu Cur AN 17.9 0.095 10.99 4.36 GI

p1 Coleoptera Curculionidae Pachyrhynchus sarcitis Weevil 大圓斑球背象鼻蟲 Lu-Yi Wang Cur AN 29.5 0.250 13.94 3.28 OI

43

(hard)

160514 Coleoptera Curculionidae Pachyrhynchus sarcitis

(hard) Weevil 大圓斑球背象鼻蟲 Lu-Yi Wang Cur AN 49.0 0.306 17.12 6.43 OI

160505

D Coleoptera Curculionidae

Pachyrhynchus sarcitis




p5 Coleoptera Curculionidae Pachyrhynchus sarcitis












p11 Coleoptera Curculionidae Pachyrhynchus sarcitis Weevil 大圓斑球背象鼻蟲 Lu-Yi Wang Cur AN 37.1 0.255 17.22 6.45 OI

44

(hard)














(soft) Weevil 大圓斑球背象鼻蟲 Lu-Yi Wang Cur AN 2.1 0.132 16.24 6.07 OI

170604

A Coleoptera Curculionidae



170604B Coleoptera Curculionidae Pachyrhynchus sarcitis


170604C Coleoptera Curculionidae Pachyrhynchus sarcitis Weevil 大圓斑球背象鼻蟲 Lu-Yi Wang Cur AN 0.6 0.194 16.30 6.05 OI

45

(soft)

170531

A Coleoptera Curculionidae



170531B Coleoptera Curculionidae Pachyrhynchus sarcitis


ZY1 Coleoptera Curculionidae Pachyrhynchus sarcitis








43 Coleoptera Curculionidae Platypus sp. Weevil

Hao Su Cur AN 1.8 0.025 6.92 2.91 OI

42 Coleoptera Elateridae/

叩頭蟲科 Agrypnus formosanus Click beetle

Jui-Fan Hsieh Ela AN 8.7 0.182 17.11 8.71 OI

40 Coleoptera Elateridae Cryptalaus larvatus Click beetle 雙紋褐叩頭蟲 Jui-Fan Hsieh Ela AN 31.9 0.556 27.07 8.66 OI

15 Coleoptera Hybosoridae/

駝金龜科 Phaeochrous emarginatus

Scavenger

scarab beetle 緣邊駝金龜 Chun-Lin Li Hyb AN 7.9 0.067 10.79 5.58 OI

16 Coleoptera Hybosoridae Phaeochrous emarginatus Scavenger 緣邊駝金龜 Chun-Lin Li Hyb AN 13.3 0.044 10.09 4.99 OI

46

scarab beetle

8 Coleoptera Lucanidae/

鍬形蟲科 Metallactulus parvulus Stag beetle 姬扁鍬形蟲 Chun-Lin Li Luc AN 12.7 0.132 13.87 6.34 OI

20 Coleoptera Lucanidae Metallactulus parvulus Stag beetle 姬扁鍬形蟲 Chun-Lin Li Luc AN 15.5 0.219 16.39 7.84 OI

82 Coleoptera Lycidae/紅螢科

Net-winged

beetle Lu-Yi Wang Lyc AN 2.8 0.050 13.80 5.28 GI

19 Coleoptera Nitidulidae/

出尾蟲科

Sap beetle

Wenbe Hwang Nit AN 3.1 0.016 6.44 3.59 OI

46 Coleoptera Nitidulidae

Sap beetle

Wenbe Hwang Nit AN 2.9 0.020 6.85 3.60 OI

56 Coleoptera Scarabaeidae/

金龜子科

Anomala expansa

lutaoensis Scarab beetle 綠島青銅金龜 Chun-Lin Li Sca AN 37.3 1.111 23.09 14.16 GI

63 Coleoptera Scarabaeidae Anomala expansa

lutaoensis Scarab beetle 綠島青銅金龜 Chun-Lin Li Sca AN 49.4 1.213 25.18 14.18 GI

4 Coleoptera Scarabaeidae Anomala siniopyga Scarab beetle 櫻花青銅金龜 Chun-Lin Li Sca AN 10.5 0.157 12.72 6.59 OI

5 Coleoptera Scarabaeidae Anomala siniopyga Scarab beetle 櫻花青銅金龜 Chun-Lin Li Sca AN 14.5 0.205 12.57 7.09 OI

66 Coleoptera Scarabaeidae Anomala siniopyga Scarab beetle 櫻花青銅金龜 Chun-Lin Li Sca AN 15.4 0.421 15.41 9.34 GI

33 Coleoptera Scarabaeidae Maladera hongkongica Scarab beetle 香港絨毛金龜 Chun-Lin Li Sca AN 3.7 0.122 11.49 5.89 OI

58 Coleoptera Scarabaeidae Maladera annamensis Scarab beetle 安南絨毛金龜 Chun-Lin Li Sca AN 1.4 0.015 8.15 4.71 GI

50 Coleoptera Scarabaeidae Papuana philippinica Scarab beetle 菲律賓圓金龜 Chun-Lin Li Sca AN 41.3 14.393 23.13 11.26 OI

1 Coleoptera Scarabaeidae Xylotrupes philippinensis Scarab beetle 蘭嶼姬兜蟲 Chun-Lin Li Sca AN 60.8 18.590 30.80 16.68 OI

47

peregrinus

2 Coleoptera Scarabaeidae Xylotrupes philippinensis

peregrinus Scarab beetle 蘭嶼姬兜蟲 Chun-Lin Li Sca AN 41.2 21.370 34.03 19.11 OI


peregrinus Scarab beetle 蘭嶼姬兜蟲 Chun-Lin Li Sca AN 32.8 1.810 31.74 17.03 OI


peregrinus Scarab beetle 蘭嶼姬兜蟲 Chun-Lin Li Sca AN 93.5 3.582 45.98 21.55 GI

59 Coleoptera Tenebrionidae/

擬步行蟲科 Gonocephalum sp. Darkling beetle

Lu-Yi Wang Ten AN 3.3 0.008 4.65 2.65 GI

41 Coleoptera Tenebrionidae Promethis sulcigera Darkling beetle 南洋長擬步行蟲 Chi-Feng Lee Ten AN 22.7 1.002 27.65 11.43 OI

60 Neuroptera Ascalaphidae/

長角蛉科 Owlfly

Zhen-Yi Chen Asc AN 1.2 0.129 31.52 3.92 GI

12 Hymenoptera Formicidae/蟻科 Polyrhachis dives Ant 黑棘山蟻 Feng-Chuan Hsu For AN 1.9 0.010 4.97 2.03 OI

11 Hymenoptera Formicidae Polyrhachis moesta Ant 哀愁棘山蟻 Feng-Chuan Hsu For AN 2.4 0.006 4.48 1.60 OI a We measured the force at failure of an insect individual on either abdomen (AN) or thorax (TX). b The insects were collected either from Green Island (GI) or Orchid Island (OI).

48

Table S2. List of candidate mammal and reptile predators of Pachyrhynchus weevils from Green and Orchid Islands (Chen et al. 2008; Li et al.

2010). We selected the candidate predators (*) based on their diet and distribution. Class Family Species Common name Chinese name Location

Mammalia Soricidae Crocidura rapax tadae Chinese white-toothed shrew 蘭嶼長尾麝鼩 Orchid Island

Soricidae Crocidura rapax lutaoensis Chinese white-toothed shrew 綠島長尾麝鼩 Green Island

Rhinolophidae Hipposideros armiger terasensis Formosan leaf-nosed bat 台灣葉鼻蝠 Green Island

Rhinolophidae Rhinolophus monoceros Formosan lesser horseshoe bat 台灣小蹄鼻蝠 Orchid Island

Molossidae Tadarida insignis European free-tailed bat 游離尾蝠 Orchid Island

Vespertilionidae Pipistrellus abramus Japanese house bat 東亞家蝠 Green Island

Viverridae *Paguma larvata Masked palm civet 白鼻心 Green Island; Orchid Island

Sciuridae *Callosciurus erythraeus thaiwanensis Pallas's squirrel 赤腹松鼠 Green Island

Muridae *Mus musculus House mouse 家鼷鼠 Green Island

Muridae *Rattus losea Lesser ricefield rat 小黃腹鼠 Green Island; Orchid Island

Muridae *Rattus tanezumi Tanezumi rat 亞洲家鼠 Green Island; Orchid Island

Muridae *Bandicota indica Greater bandicoot rat 鬼鼠 Orchid Island

Muridae *Rattus norvegicus Brown rat 溝鼠 Green Island; Orchid Island

Muridae *Rattus rattus Black rat 玄鼠 Orchid Island

Reptilia Agamidae *Japalura swinhonis Swinhoe's tree lizard 斯文豪氏攀蜥 Green Island; Orchid Island

Lacertidae Takydromus luyeanus

台灣草蜥 Green Island

Lacertidae Takydromus sauteri Koshun grass lizard 梭德氏草蜥 Green Island; Orchid Island

49

Scincidae Emoia atrocostata Mangrove skink 岩岸島蜥 Green Island; Orchid Island

Scincidae *Eutropis longicaudata Long-tailed sun skink 長尾南蜥 Green Island; Orchid Island

Scincidae *Eutropis multifasciata East Indian brown mabuya 多線南蜥 Green Island; Orchid Island

Scincidae *Plestiodon leucostictus

白斑石龍子 Green Island

Scincidae *Sphenomorphus incognitus Brown forest skink 股鱗蜓蜥 Green Island; Orchid Island

Scincidae *Eutropis cumingi Cuming's mabuya 庫氏南蜥 Orchid Island

50

Table S3. List of candidate bird predators (*) of Pachyrhynchus weevils from Green (GI) and Orchid Islands (OI).

Family Species Common name

Chinese

name Site

Wing

length

(mm)

Body

weight

(g) Diet Habitat Status

Seasonalit

y Rarity

Anatidae Anas poecilorhyncha Spot-billed duck 斑嘴鴨 OI 273

omnivore wetland migratory winter

Ardeidae Bubulcus ibis Cattle egret 牛背鷺 GI; OI 240

invertebrate & small

vertebrate farm, mangrove migratory

summer

& winter

Ardeidae Butorides striatus Striated heron 綠簑鷺 OI 200

fish, frog & insect riverbank & pond resident

Ardeidae Egretta garzetta Little egret 小白鷺 GI 246

carnivore (fish, aquatic

insect, shrimp & frog)

farm, swamp &

pond resident

Ardeidae Egretta sacra Pacific reef egret 岩鷺 GI; OI

fish, crab & insect rocky shores resident

Ardeidae

Ixobrychus

cinnamomeus Cinnamon bittern 栗葦鷺 OI 150

aquatic invertebrate, fish,

frog swamp & pond resident

Ardeidae Ixobrychus sinensis Yellow bittern 黃葦鷺 GI; OI 145


frog

swamp &

mangrove migratory winter

Ardeidae

Gorsachius

melanolopahus

Malaysian night

heron 黑冠麻鷺 OI 275

earthworm, insect, lizard,

frog forest resident

Ardeidae Nycticorax nycticorax

Black-crowned

night heron 夜鷺 OI 284


frog

swamp, mangrove,

& riverbank resident

Falconidae Falco amurensis Amur falcon 紅腳隼 OI

insect & rat grassland, swamp straggler

51

& forest

Falconidae Falco tinnunculus Common kestrel 紅隼 GI; OI 244

carnivore (rat, reptile &

insect) grassland migratory winter

Accipitridae Circus spilonotus

Eastern marsh

harrier 東方澤鷂 GI; OI 435

rat & bird grassland migratory winter Yes

Accipitridae Milvus migrans Black kite 黑鳶 GI; OI 455

bird, rat & frog

port, fish farm,

river ＆reservoir resident

Accipitridae Pandion haliaetus Osprey 魚鷹 GI; OI 445

fish rivebank & pond migratory winter

Rallidae Porzana pusilla Baillon's crake 小田雞 OI 87.5 45 omnivore

farm, riverbank &

swamp migratory winter Yes

Rallidae Rallus aquaticus Water rail 秧雞 OI

omnivore riverbank & pond resident

Yes

Rallidae Rallina eurizonoides Slaty-legged crake 灰腳斑秧雞 OI 131

omnivore

forest, farm &

mangrove resident

Yes

Rallidae Rallina fasciata Red-legged crake 紅腳斑秧雞 OI 121.7

omnivore farm & forest

straggler Yes

Scolopacidae Calidris ruficollis Rufous-necked stint 紅頸濱鷸 OI 97.5

clamworm, crustacean,

clam & insect coastline migratory winter

Scolopacidae Gallinago gallinago Common snipe 田鷸 GI; OI 124

omnivore

farm, riverbank &

swamp migratory winter

Scolopacidae Numenius phaeopus Whimbrel 中杓鷸 OI 222

crustacean, clam,

clamworm, insect & coastline migratory transit

52

earthworm

Laridae Chlidonias leucopterus White-winged tern 白翅黑浮鷗 OI 201

aquatic insect, small fish &

tadpole

coastline, estuary,

wetland & farm migratory

spring &

fall

Laridae Stercorarius pomarinus Pomarine skua 中賊鷗 OI

small mammal, fish,

crustacean & insect coastline & estuary migratory transit Yes

Laridae Sterna albifrons Little tern 小燕鷗 GI; OI 184

fish, shrimp & insect

coastline &

wetland migratory summer

Columbidae *Chalcophaps indica Emerald dove 翠翼鳩 GI; OI 144.5

omnivore forest resident

Cuculidae *Centropus bengalensis Lesser coucal 小鴉鵑 OI 156

omnivore open area & bush resident

Cuculidae *Cuculus sparverioides Large hawk-cuckoo 鷹鵑 OI 203

omnivore forest migratory summer

Cuculidae *Cuculus saturatus Himalayan cuckoo 中杜鵑 GI; OI 185

omnivore forest migratory summer

Strigidae Otus elegans Elegant scops owl 優雅角鴞 OI 169 115 insectivore forest; nocturnal resident

Strigidae Otus sunia Oriental scops owl 東方角鴞 OI 149

carnivore

savanna, forest in

the wetland & city

park; nocturnal migratory transit

Strigidae Asio otus Long-eared owl 長耳鴞 OI 284

carnivore forest migratory winter Yes

Strigidae Ninox scutulata Brown hawk-owl 褐鷹鴞 GI; OI 238

insectivore, also small

mammal forest; nocturnal resident

Caprimulgidae Caprimulgus affinis Savanna nightjar 南亞夜鷹 OI 187

insectivore river bed resident

Apodidae Apus nipalensis House swift 家雨燕 GI; OI 134

insectivore (only flying farm & city resident

53

insect)

Apodidae Apus pacificus Fork-tailed swift 叉尾雨燕 GI; OI 175

insectivore (only flying

insect) near the sea resident

Alcedinidae *Alcedo atthis Common kingfisher 翠鳥 GI; OI 72

fish, insect & aquatic

crustacean

riverbank &

mangrove resident

Alcedinidae Halcyon coromanda Ruddy kingfisher 赤翡翠 OI 122.8

omnivore forest near the sea migratory transit

Alcedinidae Todiramphus chloris Collared kingfisher 白領翡翠 OI

fish & lizard

forest near the sea

& farm straggler Yes

Meropidae Merops ornatus Rainbow bee eater 彩虹蜂虎 GI


insect) forest

straggler Yes

Campephagidae Pericrocotus ethologus Long-tailed minivet 長尾山椒 OI

insectivore forest

straggler

Laniidae *Lanius cristatus Brown shrike 紅尾伯勞 GI; OI 82.6

omnivore forest edge migratory winter

Dicruridae Dicrurus macrocercus Black drongo 大卷尾 GI; OI 138

insectivore farm & city resident

Rostratulidae Bombycilla japonica Japanese waxwing 朱連雀 OI

omnivore forest

straggler

Hirundinidae Hirundo rustica Barn swallow 家燕 OI 111.12 14.5 insectivore open area & farm migratory

summer&

winter

Hirundinidae Hirundo tahitica Pacific swallow 洋燕 GI; OI 104.6 19


insect) farm resident

Monarchidae

*Terpsiphone

atrocaudata

Japanese

paradise-flycatcher 紫壽帶 OI 88 19 insectivore forest migratory summer

54

Corvidae Corvus dauuricus Daurian jackdaw 東方寒鴉 OI

omnivore forest & farm

straggler

Corvidae Corvus splendens House crow 家烏鴉 OI

omnivore human community

straggler

Alaudidae Alauda gulgula Oriental skylark 小雲雀 OI 98 21.7 omnivore open area & farm resident

Pycnonotidae *Microscelis amaurotis Brown-eared bulbul 棕耳鵯 GI; OI 128 73 omnivore forest resident

Zosteropidae *Zosterops meyeni Lowland white-eye 低地繡眼 GI; OI 41.1

omnivore forest & bush resident

Sturnidae Sturnus cineraceus

White-cheeked

starling 灰椋鳥 GI; OI

omnivore farm migratory winter

Sturnidae Sturnus philippensis

Chestnut-cheeked

starling 紫背椋鳥 OI

omnivore farm migratory transit Yes

Sturnidae Sturnus sericeus Red-billed starling 絲光椋鳥 OI 118

omnivore farm migratory winter

Sturnidae *Sturnus sinensis

White-shouldered

starling 灰背椋鳥 GI; OI

omnivore savanna & park migratory winter

Sturnidae Sturnus vulgaris European starling 歐洲椋鳥 OI

omnivore open area & farm migratory winter Yes

Turdidae *Turdus obscurus Eyebrowed thrush 白眉鶇 GI 127.7 63 omnivore forest migratory winter

Muscicapidae *Monticola solitarius Blue rock thrush 藍磯鶇 GI; OI 112.7 53 omnivore open area migratory winter

Fringillidae Carpodacus erythrinus Common rosefinch 普通朱雀 OI

omnivore bush & forest

straggler Yes

Fringillidae Carduelis flammea Common redpoll 白腰朱頂雀 OI

omnivore

forest along the

coastline &

grassland straggler Yes

Emberizidae Emberiza Pine bunting 白頭鵐 OI

omnivore open area & farm

straggler

55

leucocephalos

Emberizidae Emberiza pallasi Pallas's bunting 葦鵐 OI

omnivore swamp & bush

straggler

56

Movie 1. ‘Hard’ weevil. A Pachyrhynchus sarcitis kotoensis weevil was introduced in

the arena (00:12), and a Japalura swinhonis lizard noticed the weevil by shifting its

body (00:20) and turning its head toward the weevil (00:27). The lizard approached

the weevil (00:48) when it resumed moving, and then spitted it out immediately

(00:51) after giving it a bite (00:50) without any visible damage to the weevil.

57

Movie 2: ‘Soft’ weevil. A Pachyrhynchus sarcitis kotoensis weevil was introduced in

the arena (00:12), and a Japalura swinhonis lizard noticed the weevil by turning its

head (00:30) and quickly approached the weevil (00:35). The lizard jumped onto the

weevil (00:39), bit on its abdomen (00:40) and started chewing it continuously

(00:41). After ~ 40 seconds, the weevil was completely consumed (01:19).

吃軟不吃硬：警戒色昆蟲的神祕...

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