COMP ARATIVE STUDY OF THE ABDOMIN AL DEFENSIVE

SYSTEMS IN TENEBRIONID BEETLES

Katsuo KAN印刷

Tenebrionid beetles have a pair of abdominal defensive glands at the apex of

abdomen， and secrete benzoquinones with secondary minor substances against attackers such as predators. The secretions exude from orifices at the interseg-

mental membranes between seventh and eighth sternites. The secretions are

odoriferous， some toxicity and fingers of handlers are dyed brown. It has been

studied the chemical compositions， morphologies and defensive behaviors (Roth 1943， Schildknecht et aI. 1963， Eisner and Mainwald 1968， Happ 1968， Kendall 1968， Tshinkel 1969， 1975， Kanehisa 1969， 1972， Tseng et al. 1971). At earlier

times special species， i. e. Tribolium sps. and Eleodes sps. have been used， recently tested spcies have been increased and can make comparisons. Three

quinones， p-quinone， 2・methylquinoneand 2・ethylquinonewere detected with

few minor hydrocarbons， but some beetles lacked p-quinone， and some beetles secreted naphtoquinone or' phenol.

In the previous reports (Kanehisa 1969， 1972)， it was described different

ratios of quinones among species and quinones toxicities to other insects. This

report describes compositions in additional species and morphological variations

of the abdominal defensive systems of tenebrionid beetles collected in the Chu

goku district of Japan.

MATERIALS AND METHODS

Tenebrionid beetles :酪 etleswere collected from granaries， decaying wood and dry river beds over a year in the Chugoku district， mostly Okayama pre-fecture. Living beetles were brought to the laboratory and the secretions were

collected immediately or the beetles were immersed in ether and stored in a

freezer (-20 t ) until assay. Some beetles were reared in small logs or wheat

f1our. Defensive glands :長 etleswere dissected in a small quantity of water， and

the gland system was observed under a dissection microscope for variations in

shape and size. Some specimens were examined under the scanning electron

microscope (J鉛 L，JSM-50A). Extracts for chemical analysis : (1) Secretions were collected by milking live

beetles on filter papers and the collections were extracted with ether， or secre-tions were collected in ether by immersions of the a bdominal a pexes for several

seconds. (2) Ether stored in a freezer， they contained the secretions with ether soluble lipoidal substances. After removal of beetles and evaporation of ether，

Supported in part by a grant from the Ministry of Education of Japan (1973・85印 27，1975・056140，1977-23側 5)

48 K. Kanehisa

dissolved in methanol， the yellowish lipoidal substances were mostly removed by low solubility in methanoI. This methanol solutions were used for the analysis.

(3) Gland systems， after dissected out， were homogenized with a small quantity

of water and extracted in ether.

Hydroxyphenyl-glucosides : They are probable precursors. Water soluble

components of homogenates were separated by thin layer chromatography， and the characteristic colours were developed by spraying anisaldehyde-sulfate in

methanol (Kanehisa 1972). Arbutin was developed into orange-brown and

phenylglucoside was developed into greenish-brown. For hydrolysis of glu-

cωides， the probable portions of glucosides were scraped off and dissolved in water， then treated with emulsin (beta-glucosidase) for a few hours at 25"(;， sometimes shaken. Then ethanol added and centrifuged， the supernatant was concentrated and rechromatographed for detection of hydrolyzed products， namely hydroquinones and glucose.

Thin layer chromatography : Benzoquinones and their reducing compounds

were c1early separated on silica gel G (Merck)， and the characteristic colours were developed by anisladehyde-sulfate in methanol. The developing solvents， Rf values and colours were described previously (Kanehisa 1972)， some exam. ples are shown in Fig. 2. For the water soluble components， 66% isopropanol :

ethylacetate (1 : 2)， or n-butanol : acetic acid : water (4 : 1 : 1) were used.

Gas chromatography : This was performed on a Hitachi 063 equipped with

T配 andstainless column (ゆ 3mmx 1m) with 25 % polyethylene glycol mono-

stearate on Celite 545， 40;60 mesh at 120"(; ， detector temperature 180"(;. and

injection temperature 195"(; . Retention times were as follows : p-quinone (10.0

min)， 2・methylquinone (11.0 min) and 2-ethylquinone (14.0 min). When samples contained large amounts of quinones， peaks continued and rather

unseparable. Thin layer chromatography was more often used for quinones， but gas chromatography was more effective for detection of secondary components， such as hydrocarbons.

UV assays : The amounts of quinones in the reservoirs were assayed spec・

trophotometrically. Yellowish bands of quinones on TLC were scraped off， dissolved in methanol and after centrifugation， the absorption spectra of the supernatant solutions were measured at 247 nm， and the amounts were cal-culated on a standard curve desCribed previously (Kanehisa 1969).

RESULTS

AII tenebrionid beetles tested had a pair of abdominal defense mechanisms

composed of large reservoirs， slender synthetic lobes connected to the base of the reservoirs and efferent ducts with muscles， lobes and ducts located in a short

distance (Plate 1・5and 6). Some differences were observed in shape and size of

reservoirs， and they were roughly divided into four groups (Fig. 1 and Plate 1).

In some beetles such as Toxicum and Cryphaeus， accessory glands covered the reservoirs (Plate 1・1). The surface of reservoirs of several species， such as

Abdominal Defensive Systems in Tenebrionid B田 tles 49

B A C

川

wv

Fig. 1. Schematic figures of reservoirs and glands. divided by reservoir s問 団 .

Plate 1. Morpholog問。fr田町voirsand glands. 1: Toxicum tricornuJum， 2 : Neatus picipes. 3:

Gonocephalum se:ruale. 4: Neat凶 piciμs. 5: Setenis valgi，向.6 and 7: Phelopatrum scaphoi-

des.4・7: scanning electron micrographs. a: ac信回rygland. r : r田町voir.g : synthetic lobes. m :

m田 cle.e : efferent d uct.

Kanehi姐

Misolampidius and Gonocephalum had milky whitish spots， they are probably one of the synthetic tissues. But many other species lacked this spot. Synthetic

lobes， main sythetic tissues， were slender sexible and soating in hemolymph， their

proximal regions were more or less wide swelling (Plate 1・5-7).

All beetles had and secreted benzoquinones with minor components， but considerable quantitative differences were found among species and in the size of

K. 回

TABLE 1 Relative amounts of benzoquinones in abdominal defensive secretions and

reservoirs of tenebrionid beetles. - : undetected in more than 20

beetles，土 ;trace amounts (sometimes undetectable)，

A，s，C and 0 : relative sizes shown in Fig. 1.

割問。freservolf

cccccc cccccB BAABDDBBB

cccccc C

p.quinone 2・E.quinone2・Me.quinone

-tt 十件

-tt桝-tt ttt -tt ttt -tt ttt -tt ttt

川前

川W

H

廿

掛

川前

桝

H廿

州

+ + ttt +件十件

ttt ttt 十件

川市川廿

H廿

H廿

川

前

桝

benzoquinone

-tt + -tt -tt -tt +

+++件朴件特H廿

H廿

-tt

件+++件特

土土土土土土

+++土

土

+++

++++++

Opatinae Phelopatrum scaph句desMARSEUL Gon町 ephaJumrect必vlkMOTSCHULSKY G. sexuaJe MARSEUL G. coenruum KASZAB G. tersimiJe LEWIS G. corim:eum MOTSCHULSKY Bolitophaginae BolilωItha伊 spa棚田凶 LEWISDiaperinae Diaperis lewisi BA TES Platydema nigroaeneum MOTSCHULSKY P. /turama NAKANE A1phitopha炉 sbij副 ciatusSAY

B画 an師 erotyloid，田 LEwrsCerop，切 sp.

Ulominae Triboliumc.副 taneumHERBST Palor凶 ratzeburgiMARSEUL Uloma bo7lzica MARSEUL U. excisa GEBIEN U. marseuliNAKAIIE

A1phito仇・凶 diaterinusPAIlZER

Tenebrioninae Me>町'Thil制 arcおcelisMARSEUL Tenebio obscur出 FABRrcrusT.mol酌rLrllNE N掘削伊ipesHERBST Setenis vaJgipes MA隠 EULE配 yaJ，由 th凶 violaceipennおMARSEULToxicum tricornutum WATERHOUSE T. fungi7lum LEwrs C，ヲIthaeusd雌 UicusLEwrs

51

C

C

B

B

A

A

A

A

A

+十+

#t +

tt

+++++

Abdominal Defensive Systems in Tenebrionid Beetles

H廿H廿

+

+

+++++

土

+

土

土

土

土

土

土

土

Cnodaloninae TdraphyU凶 lunuliJl"MARSEUL

Hm町erazig%aga MARSEUL

Phoedis helo戸ぬ1esMARSEUL

Misolampinae Misolampidi凶 clavicr凶 MARSEUL

Amarygminae PlesUJphthalmω sp回 abilisHAROLD

P. nigrocyaneus MOTSCHUl.SKY

Strongylium japanum MARSEUL

S. ni，知町umLEWIS

Elixota cu同 aMARSEUL

A

A

A

A

A

+++ + +

+++ + +

Lagriidae L uprops cribrず問問 MARSEUL

L. sinensis MA RSEUL

Hderotars凶 ca円inulaMARSEUL

Alleculidae AJlecula fuliginosa Mll KLlN

A. melanaria MJl.KLlN

glands (Table 1). Among quinones， p-quinone was relatively rare (0・5%)，tut 2・methylquinone(20・40%) and 2・ethylquinone(60・80%) were relatively com・

mon. Two species of Tenebrio contained trace amounts of quinones and some. times undetectable in samples prepared from more than 50 beetles. They have

very small reservoirs and often vacant. No p.quinone was detected in Bolito・

pha伊 s，Tribolium， Palorus， Toxicum and Cryphaeus， these species contained

。

。Cコ

3

。

。

。

。

。通'

'

2

33t

'. 、3

H

EMP R

L

G

Fig. 2. Exampl白 ofthin layer chromatography. 1: Gon町 φhalumsexuale， 2: Tribolium c，由 tan側 m，3:em叫sintreatment of T. c由 taneum.(solvent for glu棚 id田).

H : hydrocarhons， L : lipids， E : 2.ethylquinone， M : 2・n時出ylquinone，P : p・quinone，R : reduc. .

ing compounds of quinones， X : immer関 din ether a bout one day， Y : homogenate of問曽rvoirs，

Z: hom句j'enateof abdominal apex， G : gluc偶 e，U : arbutin， A : larva and pupa， B : adult， C : after emulsin t問 atmentof B.

U R A B C z Y x P z YM x z

52 K. Kanehisa

only two quinones， but two quinones were found large amounts. Species of

Gonα~ephalum， Ul併 na，Setenis， Encyalesthus and Hemicera contained relatively large amounts of p.quinone. Species of Amarygminae contained small amounts

and secreted little quantity， and they had small reservoirs. TLC examples are shown in Fig. 2. Gonocephalum sexuale contained three

quinones， their reducing compounds and hydrocarbons. Reducing compounds

were detected in homogenized preparations， but were almost undetected in secre. tions (Fig. 2・1). Tribolium cωtaneum lacked p.puinone and its reducing com・

pound (Fig. 2・2).

Hydrocarbons were observed in the upper parts of the thin layer

chromatography (Fig. 2) and on some peaks of gas chromatography. There

were three to five peaks under the standard condition described in methods.

Their retention times were from large to small amounts as follows， (A) 20.0 min， (B) 30.0 min， (C) 36.0 min， (0) 16.0 min and (E) 18 min. (A)， (0) and (E) were present in many beetles， but (0) and (E) found in trace amounts. (B)

and (C) were rather scattered， and found in Gonocephalum and Tribolium. They were unidentified in details.

Typical whitish young adults just after emergence could not secrete and

reservoirs were vacant. The contents of the reservoirs increased with aging， and the colour of reservoirs changed from white through yellow， brown， brown.red， and finally red. An example of Tribolium castaneum is shown in Fig. 3. Con.

tents reached the maximum about one month after emergence.

80

260 0¥ =司

王40

320 0

5 10 15 20 25 30 Days after emergence

Fig. 3. Increase in quinon白 afterernergence of Tribolium c，ωtaneum， E:2・ethylquino田， M : 2.rnethylquinone.

Hydroxyphenyl.glucoside， arbutin， or relatives were detected only in adults， and undetected in larva and pupa. Glucosides were clearly detected from two

grams of whole加dyof Tribolium c，出 taneum(Fig. 2・3)and 10 abdomens of

Toxicum tricornutum. These glucosides disappeared after emulsin treatment，

Abdominal Defensive Systems in Tenebrionid Beetles 53

hydrolyzed to hydroquinone and glucose. But glucosides were uncertain in

Gonocephalum， Uloma， Neatus， Setenis， Encyalesthus， and Hemicera， spots on TLC in accordance with arbutin or relatives were unclear， very faint while water soluble samples were prepared from 20・100beetles. We can consider another

unknown precursor and pathway.

Some species near tenebrionid beetles were also tested for comparison.

Three species of Lagriidae and two Alleculidae contained even though contents

and reservoirs were small， but Penthe japana (Tetratomidae) and several species of Mycetophagidae did not contain quinones.

DISCUSSION

AII tested tenebrionid beetles had and secreted benzoquinones with hydro-carbons， as reported in other species by Schildknecht (1963)， Eisner and Main-wald (1966)， Happ (1968)， Tseng et al. (1971)， Tshinkel (1969， 1975) and Kanehisa (1972). In this re卯 rt，some characteristic features were observed， namely dfference of quinone ratios and lJ)orphological variations among tenebri-

onid species. Two species of Tenebrio had trace amounts of quinones and small

reservoirs， sometimes vacant. Amarygminae also contained small amounts of

quinones and small reservoirs. Several species， such as Tribolium and Toxicum lacked p-quinone. Gonocephalum， Uloma and several Tenebrioninae contained and secreted large amounts of quinones， including p-quinone， and had large reservoirs. Many other tested beetle species were intermediary. But none of the

unique features showing species spcificities.

A correlation was apparent between the amounts of quinones and reservoir

size. The secretion amounts did not depend on the reservoir size， but rather depended on the strength of stimulation. AIl tested beetles in this experiment

exuded secretions， did not spray as carabid beetles (Kanehisa and Murase 1977) or Eleodes (Tshinkel 1975). It thus took a few seconds to collect the secretion

and note the odour. This was due to morphological differences in the efferent

ducts between tenebrionid beetles and carabid beetles. The latter has longer

efferent ducts with valves and many muscles producing high pressure， and the former has short duct and less muscles producing low pressure， valve-like struc-ture IS uncertam.

The synthetic lobe shapes are similar to the lobes of benzoquinone secreting

carabid beetles， Brachinus (Kanehisa and Murase 1977)， as compared with m-cresol secreting or fatty acid secreting species. Eisner et al. (1964) described

two types of synthetic cells， ty伊 11町 atedon the outer surface of the reservoirs， and type 2 located in the proximal parts of the microvilli， namely in the synthetic lobes in this report. Type 2 predominant in many beetles tested in this experi-

ment. Synthetic lobes of 10 to 20 long flexible lobes with swelling in the pr。ximal region were easily observed， but the milky whitish spots on the surface of reservoirs， type 2， were not often observed.

Secretions were observed only in adults， and the depositions of quinones in

M K. ~~~

the reservoirs were rather slow. The amounts of reserved quinones increased

with aging after emergence， and it took about one month to reach the maximal

level. These features may indicate synthesis of defensive substances taking place

in only adults. The synthetic velocity was rather slow. Resting beetles do not

secrete and are nOil.odorus for a few hours， while cultured madia of Tribolium gradually changed colours by the probable slow secretion of quinones.

Hydroxyphenyl.glucosides were detected in Tribolium castaneum and

Toxicum tricornutum， and hydrolyzed by emulsin， as odserved in Tribolium and Eleodes (Happ 1968). Their occurrence is u悶巾ininGonocep加 lum，Uloma， Neatus， Setenis， Encyalesthus and Hemicera， even though prepared from 20 to 100 beetles. On the contrary， large amounts of hydrdxyphenyl.glucosides were readily detected in Brachinus beetles usually enough only a few beetles. this

may suggest at least two different synthetic modes : one is oxidation of hydro-

quinone after hydrolysis of hydrophenyl-glucosides (Happ 1968)， and the other is the synthesis of alkylated quinone nuclei， utilizing the aromatic ring of phenyla-lanine and tyrosine (Mainwald et al. 1966). Di伍cultproblem may be caused by

the small amouts of precursors and low synthetic property.

Benzoquinones have toxicity and low insecticidal activity (Kanehisa 1969).

Ozawa et al. (1968) reported the respiratory inhibition of intact mitochondria of

rat liver by benzoquinone derivatives. Benzoquinones have unpleasant odour， and this property may function as a defensive substance against attackers. Hydro-

carbons may act as synergist by their lipophilic activity. A close resemblance

seems present between abdominal defensive glands and the integuments， chi-tinous structure of reservoirs and similar components. Their features are also

assumed present in the pygidial defensive systems of carabid beetles (Kanehisa

aod Murase 1977).

Several species of Lagriidae and Alleculidae have gland systems and ben-

zoquinone secretion as observed by Kendall (1968)， but several species of Tet-ratomidae and Mycetophagidae lack them. An abomioal defensive system may

be one of the characters in the evolutional trends.

SUMMARY

Investigations were conducted on the abdominal defensive systems of 37

species of tenebrionid beetles and several related species.

1. AlI tenebrionid beetles contained benzoquinones with hydrocarbons that

was secreted under stimulation. However there were quantitative differences ;

p-quinone was relatively rare (0・5%)，2・methylquinone(20・40%) and 2-ethyl-

quinone (60・80%) were relatively common. 2. Tenebrio， Phaedis and Amarygminae contained trace amounts of ben-

zoquinones， Gonocephalum， Uloma， Setenis and Hemicera contained large

amounts， and other beetles contained intermediary amounts. The contents were

generally correlated with reservoir size， but the secreted amount was dependent 00 the strength of stimulation.

Abdominal Defensive Systems in Tenebrionid Beetles 55

3. The amounts in the reservoirs increased with aging after emergence.

4. Hydroxyphenyl-glucosides were detected as precursors in several species， while questionable in many other species.

5. The shape of the synthetic lobes were rather similar to the lobes of

benzoquinone secreting cara bid beetles.

ACKNOWLEDGMENT

The authour wish田 tothank Dr. Yutaka Ishida， Kobe Commercial ColJege， for his identification and sugg田 tionson tenebrionid beetles. Thanks are also due to Prof. Yasunobu Yasue， Dr. Kazuo Kawada， Messers. 日isaakiTsumuki， Takashi Shiraga， Masanori Murase， Yukio Shimo and Toshi. kazu Yamashita， Institute for Agricultural and Biological Sciences， Okayama Unive四ity，for their

valuable discussion and ins町 tcollections.

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