Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 95, No.2, April 1986, pp. 205-214.© Printed in India.

Digestive enzymes level in the midgut of Adoretus lasiopygusBurmeister (Scarabaeidae: Coleoptera)

A C BHARDWAJPG Department of Zoology, Sanatan Dharm College, Muzaffarnagar 251001, India

MS received 14 May 1984; revised 16 March 1985

Abstract. Enzyme system pertaining to the midgut region of Adore/us lasiopygus Burm. isstudied in vitro. Most of the carbohydrases operate optimally in the mesomesenteron whereasproteinases and lipases behave optimally iiI the metamesenteron region. Optimum conditionsof certain carbohydrases in relation to pH, temperature, substrate concentration and enzymeconcentration have been studied. IX-amylase, IX-glucosidase, P-glucosidase, e-galactosidase andP-galactosidase showed optimum activity at pH 5·7,6·2, 5-0, 5·8and 5·6 respectively.e-Amylase,e-glucosidase and IX-galactosidase are physiologically adapted to high temperature rangingbetween 48°-{i2°C. A positive response of starvation on the secretion ofcellulase, inulinase, IX­and p-galactosidase is observed. Based on this parameter the midgut enzymes of the pest isdivided into two groups i.e, enzymes secreted continually and those secreted under theinfluence of food.

Keywords. Enzymes level; midgut; Adoretus lasiopygUS; Scarabaeidae.

1. Introduction

Utilization of complex molecules in their food by animals is not possible unless theformer get cleaved into simpler forms absorbable through the gut epithelium. Themidgut region of several beetles is adapted physiologically to degrade complexmolecules in their diet with the aid of appropriate enzymes secreted from the midgutepithelium. Krishna (1958), Bhatnagar (1962), Rao and Rastogi (1967), Sakurai (1968),Chinnery (1971), Baker (1976), Shukla and Upadhaya (1978) are some of the recentworkers who have described enzyme systems pertaining to the digestion of food invarious coleopteran families. The scarabaeid beetles are of great economic value butremained untouched for such study after Swingle (1930). The present communication

~

describes exo- and endogeneous digestive enzymes of the Chafer beetle, Adoretuslasiopygus which feeds on the leaf lamina of its host plant, Maqnifera indica Linnaeus.

2. Material and methods

The beetles used in the present investigation were collected from its host plant in thenight during rainy season when they congregate in large numbers. Both field collectedand 24 hours starved individuals were vivisected under stereoscopic binocularmicroscope in cold double distilled water. The midgut region freed from all adheringtissues was incised into morphological distinct parts as described earlier (Bhardwaj1985). For enzyme assay extract of each part of midgut was prepared and stored underrefrigerated conditions. The reaction and control mixtures were prepared (Gael andBhardwaj 1981) and incubated at 3rc ± 2°C for about 5-10 hr under a layer of

205

206 A C Bhardwaj

toluene. The incubated mixtures were analysed for the presence of hydrolytic productofcarbohydrates (Plummer 1971),peptone, casein, lipid, ester (Baldwin and Bell 1967)and urea (Feigl 1947) as detailed elsewhere (Goel and Bhardwaj 1981).

To study the optimum pH ofdifferent carbohydrases buffers used were HCI-sodiumcitrate, 0·1 M for pH 3·5; Soresen's phosphate, 0·1 M for pH 5'5-8·0; glycine-NaOH,0·1 M for pH 8·5-9·0. To obtain temperature optima of carbohydrases reactionmixtures were incubated at temperature ranging from lOo-70°C with an interval oflO°C. To study influence of enzyme concentration on the activity of carbohydrasesextract of pro-, meso- and metamesenteron was prepared separately using 1 midgutregion/rnl to 6 midguts region/rnl (w/v). Substrate concentration ranging from1·0-8·0 % was used t~ see its effect on the enzymes. The following colorimetricprocedure was used for different parameters.

Appropriate buffer and substrate in equal proportions wert mixed and incubated at37°C for 10 min. Later 100 Jll midgut extract was added and reincubated exactly for60 min. The carbohydrase activity was terminated by adding 4 ml 3, 5 dinitrosalicylicacid reagent (Noelting and Bernfeld 1948; Plummer 1971). The mixture so obtainedwas heated for about 5 min at lOO°C in water bath and followed by immediate cooling.The extinction was read at 540 nm with the help of colorimeter. A control and blankwas also prepared and treated in the same way. Enzyme activity was expressed asdifference in absorbance between reaction and control mixtures. The process wasrepeated 4 times and mean is taken.

3. Results and discussion

In vitro studies indicate that midgut of Adoretus lasiopygus is physiologically adaptedto utilize starch, maltose, sucrose, raffinose, melibiose, cellobiose and lactose as maincarbohydrates; simple proteins and very little fat occurring in the diet of pest. Therelative strength of various enzymes is shown in table 1.

Starch constitutes major part of the diet in phytophagous beetles. Its quickhydrolysis is observed with pro- and mesomesenteron extract while reaction remainedrelatively slow with the extract of metamesenteron. As a result of starch hydrolysis theturbidity in the reaction mixture disappears. It indicates presence of dextrogenic orliquefying amylase (Fruton and Simmonds 1965).The analysis of the reaction mixtureelucidates oligosaccharides on the chromatoplate thus confirms amylase activity. Theamylase has cleaved glycosidic linkage of starch probably in the interior of the chainhence it is called as ex-amylase in A.lasiopygus and supports Gilmour's (1962)view aboutthe universal occurrence of ex-amylase in insects. Applebaum (1964) classified amylaseinto (X- and f3-amylase while studying action pattern in the midgut of Tenebrio sp. larva.The nonspecific amylase has been reported earlier by several workers in the beetle theystudied (Lal and Ghai 1958; Krishna 1958; Bhatnagar 1962;Krishna and Saxena 1962;Rao and Rastogi 1967; Sakurai 1968; Chinnery 1971; Shukla and Upadhaya 1978).

The digestion of inulin with metamesenteron extract is evident from the presence offructose on the chromatoplate. The metamesenteron is the only region in the wholemidgut of A. lasiopygus adapted to secrete inulinase and utilizes inulin before beingpassed out.

Among structural polysaccharides like cellulose, pectin and chitin; the cellulose couldonly respond to the metamesenteron extract. It suggests presence ofcellulase in the said

Tab

leI.

Asu

mm

aris

edpr

esen

tati

ono

fin

form

atio

nre

latin

gto

qual

itat

ive

assa

yof

dige

stiv

een

zym

esin

the

diff

eren

tre

gion

so

fth

em

idgu

to

fA

dure

tus

lasi

opyq

usB

urm

.

Enz

yme

dist

ribu

tion

~ ~P

rom

esen

tero

nM

esom

esen

tero

nM

etam

esen

tero

ns:: ... l1> ;:s

E.C

.nu

mbe

rE

nzym

eS

ubst

rate

Fed

Star

ved

Fed

Sta

rved

Fed

Star

ved

~ sC

arbo

hydr

ases

a3-

21·1

a-A

myl

ase

2~~

Sta

rch

++

++

++

++

++

+S·

32·1

·4C

ellu

lase

2~;,

;C

arbo

xym

ethy

lcel

lulo

se-

--

-+

-:A

..3·

2·1·

7In

ulin

ase

2/~

Inul

insu

spen

sion

(Aq.

)-

--

-±

-fi

32·1

·14

Chi

tina

se2

%C

hiti

nsu

spen

sion

(Aq.

)-

--

--

-32

·1·1

5P

olyg

alac

turo

nase

Pect

insu

spen

sion

(Aq.

)-

--

--

-.go ~

32·1

·20

a-G

luco

sida

se5

%M

alto

se,

sucr

ose

++

++

++

++

++

+It:

I

3·2·

\·21

p-G

luco

sida

se5

%C

ello

bios

e+

++

++

++

~3·

2·1·

22a-

Gal

acto

sida

se5

%R

affi

nose

,m

elib

iose

--

++

32·1

·23

p-G

alac

tosi

dase

5%

Lac

tose

+-

++

+-

±±

3·2·

1·26

p-F

ruct

osid

ase

5%

Raf

fino

se,

Phe

nyl-

ji-f

ruct

osid

e3·

2·1·

28a,

a-T

reha

lase

5%

Tre

halo

seP

rote

inas

es30

4·21

·3P

rote

ase-

A2

%C

asei

n+

-±

-+

+±

Pol

ypep

tida

se2

%P

epto

ne±

-+

+-

++

+±

Lip

ases

3·1·

1·1

Car

boxy

lest

eras

eE

thyl

buty

rate

emul

sion

--

+-

++

3·1·

13T

riac

ylgl

ycer

olip

ase

Oliv

eoi

lem

ulsi

on-

-±

-±

Am

idas

e35

·1·5

Ure

ase

I%

Ure

a

++

+,

Bri

skac

tivity

;+

-t-,

mod

erat

eac

tivity

;+

.fee

ble

activ

ity;

±.t

race

acti

vity

.v-,

noac

tivity

.IV 0 -.

.J

208 A C Bhardwaj

region of A. lasiopygus and is a point of significance because hydrolysis of cellulose inthe insects that survive on woody or fibrous diet is brought about by flagellates orbacterial flora of their gut (Cleveland et al 1934). Midgut and the caecal origin ofcellulase in Blaberus discoidalis has been demonstrated by Ehrhardt and Voss (1962).Similarly cellulase has also been reported from the crop and midgut of Byrsotriafumiqata (Fisk and Rao 1964) and in the salivary gland of cockroach (Wharton et al1965). The pectin and chitin remained unaltered in the reaction mixture even after anincrease in incubation time is given.

The occurrence of glucose and fructose on the chromatoplate as end products of thesubstrates maltose and sucrose confirmed the activity ofex-glucosidase in A. lasiopyqus.The activity is moderate in the promesenteron, accelerated in the mesomesenteronwhereas it has dropped towards metamesenteron. In the absence of P-fructosidaseactivity in A. lasiopygus it is presumed that sucrose with ex- and p-linkage iscleaved onlyby ex-glucosidase. The presence of non specific glucosidase is an adaptation on thedigestive part ofA. lasiopygus to utilize more carbohydrates of the diet. The availabilityof ex-glucosidase in the midgut of Dermestes maculatus and Dactylosternum hydro­philioides has been reported by Chinnery (1971) and Shukla and Upadhaya (1978)respectively and support present observation. Similar to the midgut of Latheticusoryzae (Bhatnagar 1962) and Tribolium castaneum (Krishna and Saxena 1962) A.lasiopygus also secretes P-glucosidase. In present instance P-glucosidase activity isobserved moderate in the mesomesenteron whereas the activity droped on either side.The ex- galactosidase of A. lasiopygus digests trisaccharides such as melibiose andraffinose specifically in the mesomesenteron and supports observation made byKrishna and Saxena (1962). The p-galactosidase established in L. oryzae (Bhatnagar1962)and T. castaneum (Krishna and Saxena 1962)throughout the length of midgut, isobserved with optimum activity only in the mesomesenteron of A. lasiopygus. UnlikeBhatnagar (1962),Sakurai (1968), and Chinnery (1971) trehalase activity could not betraced in any region of the midgut of present scarabaeid.

The midgut of A. lasiopygus is also able to utilize simple proteins present in the food.Among these peptone seems to be quickly degraded by polypeptidase in themesomesenteron. The digestion of peptone however, becomes vigorous towardsmetamesenteron probably due to the substantial amount of appropriate enzyme andfavourable medium (Bhardwaj 1982). The casein shows feeble digestion with the extractofmetamesenteron perhaps due to the inadequate amount ofprotease-A. The presentfinding thus supports occurrence of slight proteolytic activity in the midgut of Tenebriomolitor and T. castaneum (Dadd 1956; Brik et al 1962). The end products of theseproteins are further assayed either by secretion ofaminopeptidase or carboxypeptidaseas suggested by Wigglesworth (1965). Carboxylesterase shows feeble activity inmesomesenteron while the activity enhances to moderate in the metamesenteronregion. Triacylglycerolipase shows activity in traces in both meso- and metamesenteronregions of A. lasiopygus.

The digestive enzymes level vividly reveal that the present pest has a complexmodified midgut, demarcated physiologically into pro-, meso- and meta mesenteron.Each region shows optimum hydrolysis affinity towards particular types of substrate.Thus promesenteron digests starch, maltose and sucrose. Mesomesenteron is the chiefsite where hydrolysis of several carbohydrates and peptone takes place. The meta­mesenteron is adapted for the digestion of proteins and lipids. Such a specific digestiveadaptation in A. lasiopygus is highly correlated with the nature and arrangement of

Midgut enzymes in A. lasiopygus 209

epithelial cells in different regions as established histocytologically earlier (Bhardwaj1985).

pH values for optimum activity of insect carbohydrases vary widely and range from5·4-7·5 (Shinoda 1930; Rao and Rastogi 1967; Krishna 1958). The IX-amylase activity ofAi lasiopyqus is quite appreciable in a pH zone 3·5-7·0. The optimum amylolytic activityis recorded at pH 5.7 (figure1) which is almost similar to the optimum amylase pH ofEpilachna oiqintioctopunctata (Sahurai 1968). Krishna and Saxena (1962) establishedslight low pH optimum for the activity ofamylase in T. castaneum larva and adult. TheIX-glucosidase behaves appreciably between pH zone 4·7-6·8. Its optimum activity takesplace at pH 6·2 (figure 2). The fJ-glucosidase appears to be capable of operating underpH ranging from 3·8-6·3. Its optimum activity is noticed at pH 5·0 (figure 3). The IX­galactosidase has an optimum pH 5,8, although this enzyme has noticeable catalyticactivity between pH 5·5-6·5 (figure 4). Krishna and Saxena (1962) recorded pHoptimum 5'4 for this enzyme. The fJ-galactosidase activity falls into relatively wider pHzone which ranges from 4·5-6·6. It has optimum activity at pH 5·6 (figure 5) anobservation almost parallel to Krishna and Saxena (1962).

There is progressive response in the activity ofcarbohydrases of A. lasiopyqus withthe rise of temperature. It has been observed that IX-amylase, x-glucosidase and IX­galactosidase behave optimally at 55°C. Higher temperature stimulates amylolyticactivity in T. molitor (Jankovic et aI1976). In the present case further rise in temperaturedrops enzymatic· activity sharply and ceases almost at 68°C. This suggests thatcarbohydrases of A. lasiopyqus are physiologically adapted to operate at moderately

.., 6000x1Il.-c:

~:J

.- Q)

> u 400c.... 0u0 ..0

L.Q) 01Il 1Ilo ..0

- 0~1IlE 0« 200I "U

Q)

~ 1Il1Il

~0..xQ)

03·0 5·0 7·0

pH

Figure 1. pH profile of z-amylase in midgut regions of A. lasiopygus. .-.Prornesenteron, 0-0 Mesomesenteron, 6-6 Metamesenteron.

210 A C Bhardwaj

,...0~

xV)

>- ........ c> :l.... Q)o <.>0 cQ) 0

.0V)~

0 0'"0 V)

V).0

0 0o V)

:l 0

o '"0

IQ)V)

CS V)

~0-XQ)

600

400

200

o3.0 5·0

pH

7·0

Figure 2. pH profile of CL-g1ucosidase in midgut regions of A. /asioPY9US.

,... 6000.....XV).-

>- 'c.... :l'>.... Q)

o <.>4000 c

0Q) .0V) ~

0 0'"0 V)

V) .00 0o V):l 0o '"0 200I Q)

ca. V)V)

Q)~

0-XQ)

o3·0 5·0

pH

7·0

Figure 3. pH profile of P-g1ucosidase in midgut regions of A. /asioPY9US,

Midgut enzymes in A. lasiopyqus 211

M

oc-, x.-> Vl.-.-

CU0 :J

Qj Qj

Vl U

0 C

\J 0.o

Vl L

.2 0u Vl

0 .o0

0 Vlo 0

I \J

~QjVlVl

e0.XQj

600

400

200

o3·0 5·0

pH

7·0

Figure 4. pH profile of iX-galactosidase in the mesomesenteron of A. lasiopygus.

M

0X

>- II)---- ·C> :J-- Qj 400u u0 cQj 0Vl .£I0 L

"0 0II)

Vl .£I0 0--U Vl 2000 0

0 "0

o QjII)

I II)

(:Q QjL0-xQj

03'0 5·0 7·0

pH

Figure 5. pH profile of p-galactosidase in midgut regions of A. lasiopygus.

212 A C Bhardwaj

~

r"l0X 0 ()I. -Amylase

2 • ()I. - GlucosidaseC 500 t. ex - Galactosida se:J

>0- <lIU

> Ca 400U .0L.a 0

<lI(J)

.0E a 300>0-N (J)

C aw "0

<lI 200(J)(J)

<lIL.

~ 100<lI

010 30 50 70

Temperature (Oel

Figure 6. Impact of temperature on the activity of certain midgut carbohydrases of A.lasiopygus.

high temperatures and behave appreciably at normal temperatures i.e. from 30°-37°Cat which this pest flourishes (figure 6). There is a significant variation in the optimumtemperature for amylase activity. It ranges from 28°--60°C (Day and Powning 1949;Applebaum et al 1961; Rao and Rastogi 1967; Sinha 1975; Jankovic et al 1976; Hori1970) in different groups of insects.

Five substrates with increasing concentration ranging from 1-8 %were used to assessthe influence of it on the activity of respective enzyme. The results indicate thatamylolytic activity is quite moderate even at 1%concentration of starch is used. Thereis very little augmentation in enzymatic activity with further increase in concentration.The IX-galactosidase shows feeble rise in activity with rise of raffinose concentration,beyond 4 %the reaction becomes more or less constant. Likewise 4 %concentration oflactose seems to be appropriate for fJ-galactosidase activity. The e-glucosidase activityenhances sharply with the rise ofcellobiose concentration upto 5 ~~. Further addition ofsubstrate shows a decline in enzymatic activity whereas the activity of fJ-glucosidaseshoots up with increasing concentration of maltose.

Amylase activity in A. lasiopygus shows a linear progressive response with the rise ofenzyme concentration up to a certain limit (5 midgutsjml). Further rise in enzymeconcentration shows feeble amylolytic activity thereafter hydrolysis of starch becomesconstant. A much higher enzyme concentration is recorded for the hydrolysis of starch(6-12 midgutsjml) in Sarcophaga ruficornis and Musca domestica (Sinha 1975).

A significant positive effect of starvation on the secretion ofcellulase, inulinase, c-, fJ­galactosidase, proteinases and esterases have been observed. The remaining carbohyd­rases are secreted continually into the midgut lumen but their secretion however,enhance as food comes in the midgut. Probably the presence of some food moleculesstimulate midgut epithelium for secretion. It is also interesting to note that a particularenzyme in the region of promesenteron inhibits under starvation but this parameter

Midgut enzymes in A. /asiopygus 213

fails to arrest the secretion of same enzyme in the succeding regions or vice versa (table1). It is believed here that different enzymes are secreted from different epithelial cells ofthe same region. A particular sector among these cells responded to the starvation whileremainder cells behaved normally. Thus based on this parameter the enzyme system ofA. lasiopygus can bedivided into two groups i.e.enzyme secreted continually and thosesecreted under the influence of food.

Acknowledgements

Grateful thanks are due to Dr S S Krishna, Department of Zoology, GorakhpurUniversity, Gorakhpur for many meaningful suggestions. The continuous interest andconstant encouragement by Dr S C Goel, during the project is also thankfullyacknowledged.

References

Applebaum A W, Jankovic M and Birk Y 1961 Studies on the midgut amylase activity of Tenebrio molitor L.Larvae; J. Insect Physiol 7 100-108

Applebaum S W 1964The action pattern and physiological role of Tenebrio larval amylase; J. Insect Physiol.10897-906

Baldwin E and Bell D J 1967 Cole's Physiological Chemistry, (New Delhi: Sagar Publications)Baker J E 1976 Properties of midgut protease in the larva of Attagenus megatoma; Insect Biochem. 6 14}-148Bhardwaj A C 1982 On the pH in the midgut of Adoretus lasiopygus Burrn.; Geobios 9 69-70Bhardwaj A C 1985 Some histocytological observations on the midgut of Adoretus lasiopygus burm.

(Scarabaeidae: Coleoptera); Uttar Pradesh J. 2001. 5 164-171Bhatnagar P L 1-962 Digestive enzymes and hydrogen ion-concentration in the gut oflarva Latheticus oryzae

Waterh. (Coleoptera: Tenebrionidae); Indian J. Entomol. 24 19-26Brik Y, Harpaz I, Ishaaya I and Bondi A 1962 A study on the proteolytic activity of beetle Tenebrio and

Tribolium; J. Insect Physiol. 8 417-429Chinnery JAB 1971Carbohydrases of the midgut ofleather beetle, Dermestesmaculatus; J. Insect Physiol. 17

47---{)1Cleveland L R, Hall S R, Sandres E P and Collier J 1934 The wood feeding roach, Cryptocercus, its protozoa

and symbiosis between protozoa and roach; Am. Acad. Arts Sci. 17 185-343Dadd R H 1956 Proteolytic activity in relation to feeding beetles Tenebrio molitor and Dytiscus marqinalis

L.; J. Exp. Bioi. 33 311-324Day M F and Powning R F 1949 A study of process of digestion in certain insects; Aust. 1. Sci. Res. 82

175-215Ehrhardt P and Voss Z 1962 Beitrag zum Wirkungspektrum Kohlenhydratspaltender Fermente und ihre

Verteilung im Verdauungstrakt der Schaben Blaberus discoidalis Sv. und Leucophaea maderae F.(Blattoidea); J. Insect Physiol. 8 165-174

Fisk F Wand Rao B R 1964 Digestive carbohydrases of the Cuban burrowing cockroach; Ann. Entomol. Soc.Am. 5740-44

Feigl F 1947 Qualitative analysis by Spot Test (New York: Elsevier Publishing Co.)Fruton J S and Simmonds S 1965 General Biochemistry (Bombay: Asia Publishing House)Gilmour D 1962 The Biochemistry of Insects (New York: Academic Press)Goel S C and Bhardwaj A C 1981 Physiology ofdigestion in the midgut of Tanymecus sciurus Oliv; Indian J.

Entomol. 43 259-265Hori K 1970Carbohydrases in the gut and salivary glands and the nature of amylase in the gut homogenate of

Lygus disponsi Linnavuori (Hemiptera: Miridae); Appl. Entomol 2001. 5 13-22Jankovic H M, Stanic V and Ivanovic J P 1976 The effect of temperature and crowding on the midgut

amylolytic activity in Tenebrio motitor adults; 1. Insect Physiol. 22 851-853Krishna S S 1958 Physiology of digestion in Trogoderma larva; J. Zool. Soc. India 7 170-176

214 A C Bhardwaj

Krishna S S and Saxena K N 1962 Digestion and absorption of food in Tribolium castaneum (Herbst);Physiol. Zool. 3S 66-78

La! Rand Ghai S 1958 Physiology of digestion in the adult red pumpkin beetle, Aulacophora fooeicollis(Lucas); Indian J. Entomol. 20 37-45

Noelting G and Bernfeld P 1948 Sur les enzymes amylolytiques-II1 La p-amylase: Dosage d' activite etcontrole de l'absence d' cz-amylase; Helv. Chim. Acta 31 286-290

Plummer D T 1971 An Introduction to Practical Biochemistry (England: McGraw Hill)Rao G S and Rastogi S C 1967 Digestive enzymes in the midgut oftenebrionid beetle, Arthroides sp.; Proc.

Zool. Soc. Calcutta 20 69--81Sakurai H 1968 Physiological studies on the digestion ofcoccinellid beetles (Coleoptera: Coccinellidae) with

special reference to their food habits; Appl. Entomol. Zool. 3 130-138Shinoda 0 1930 Contribution to the knowledge of intestinal secretion in insects. III. On the digestive

enzymes of Silk worm; J~ Biochem. 11 345-367Shukla G S and Upadhaya V B 1978 Digestion of food in the adult Dactylostemum hydrophilioides (Macly);

Indian J. Entomol. 40 171-176Sinha M 1975 Amylase activity in the midgut of Sarcophaqa ruficornis and Musca domestica; Entomol. Exp.

Appl. 18 290-296Swingle M C 1930Anatomy and physiology ofthe digestive tract ofJapanese beetle: J. Aqric. Res. 41181-195Wharton D R A, Wharton M L and Lola J 1965 Cellulase in the cockroach, with special reference to

Periplaneta americana (L); J. Insect Physiol. 11 391-404Wigglesworth V B 1965 The Principles of Insect Physiology (London: Methuen and Co. Ltd)