- Email: [email protected]
Digestive carbohydrases in the salivary gland and midgut of several phytophagous bugs
Comp. Biochem. Physiol., 1975, Vol. 5013,pp. 145 to 151. PergamonPress. Printed in Great Britain
DIGESTIVE CARBOHYDRASES IN THE SALIVARY GLAND AND MIDGUT OF SEVERAL PHYTOPHAGOUS BUGS* K. HoR1 Division of Entomology and Plant Physiology, Obihiro Zootechnical University, Obihiro, Hokkaido, 080 Japan (Received 19 December 1973)
Abstract--1. Carbohydrases in the salivary gland and midgut were compared among several phytophagous bugs with different feeding habits, Lygus disponsi, L. saundersi, Adelphoeoris suturalis and Orthoeephalus funestus (Miridae), Palomena angulosa and Eurydema rugosum (Pentatomidae) and Coreus marginatus (Coreidae). 2. All the bugs had amylase, phleinase, ~- and fl-glucosidase and ~- and fl-galactosidase in the midgut. Carbohydrase variety in the salivary gland was poor. 3. Salivary pectinase was detected in all the mirid bugs. 4. In the salivary gland, C. marginatus had fl-glucosidase, c~- and fl-galactosidase, P. angulosa c~galactosidas¢ and O. funestus ~-galactosidase. 5. A distinct cellulolytic activity was detected in the midgut of P. angulosa, E. rugosum and C. marginatus. 6. The relationship between the taxonomic position or the feeding habit of the bug and the presence of carbohydrase in the alimentary system is discussed. INTRODUCTION THE FOOD and feeding habits of insects vary very greatly and the digestive enzymes are adapted for their respective foods (House, 1965; Dadd, 1970). Therefore, study of the digestive enzymes of insects forms an important basis for study of the injury caused by them and of their digestive physiology. There have been many studies on the presence of digestive enzymes in phytophagous bugs (Baptist, 1941; Goodchild, 1952; Nuorteva, 1954; Saxena, 1954a, b, 1955, 1958; Nuorteva & Laurema, 1961; Ford, 1962; Rastogi, 1962; Rastogl & Datta Gupta, 1962a, b; Khan & Ford, 1967; Mathur & Thakar, 1969; Hori, 1970a, b; Kumar, 1970; Takanona & Hori, 1974), but relatively few comparative studies among species and among bugs with different feeding habits. Takanona & Hori (1974) stated that there was no specificity of species or family with respect to the digestive enzymes of phytophagous bugs except salivary pectinase. In order to prove this statement, however, many more species must be compared with one another. The present paper deals with the carbohydrases contained in the salivary gland and midgut of several phytophagous bugs with different feeding habits. MATERIALS AND METHODS
(Miridae), a polyphagous bug though it has a special food preference for cruciferous plants; Lygus saundersi (Miridae), an oligophagous bug whose feeding is limited to some grasses, Adelphocoris suturalis (Miridae), an oligophagous bug having a food preference for legumes; Orthocephalus funestus (Miridae), presumably a monophagous bug feeding only on mugworts; Palomena angulosa (Pentatomidae), a polyphagous bug feeding on various herbs and trees; Eurydema rugosum (Pentatomidae), an oligophagous bug having a food preference for cruciferous plants; and Coreus marginatus (Coreidae), presumably a polyphagous bug. Thirty adults (not starving), with the exception of P. angulosa (twentythree adults) and C. marginatus (eighteen adults), were collected from the field for the assay. Preparation of salivary and midgut enzyme solutions
The enzyme solutions were prepared by homogenizing the whole salivary gland or the whole midgut according to the procedure employed previously by Hori (1970c). Reaction mixture and incubation
The reaction mixture consisted of 1.0 ml substrate solution adjusted to the proper pH with McIlvaine buffer (Tables 1 and 2) and 1.0 ml enzyme solution, to which 30 mg chloramphenicol was added as antibiotic. Incubations were carried out for 24 hr at 37°C.
Insects used Carbohydrases were compared among the following seven species with different feeding habits: Lygus disponsi
Assay for carbohydrase activity
* Contribution No. 56 from the Laboratory of Entomology, Obihiro Zootechnical University.
Activities were determined by the colorimetric method of Somogyi-Nelson (Hori, 1970c) and/or the paper chromatography method (Hori, 1970a). 145
146
K. Ho~a RESULTS
Quantitative assay for carbohydrase F o r t h e quantitative assay, soluble starch, inulin, phlein, carboxymethyl cellulose, trehalose, sucrose and melezitose were used as the substrates. The results are shown in Table 1. The salivary enzyme of L. disponsi hydrolyzed moderately starch, and weakly phlein and trehalose, while the midgut enzyme hydrolyzed strongly sucrose, moderately starch and melezitose, and weakly trehalose and phlein. L. saundersi hydrolyzed moderately starch and very weakly sucrose by the salivary enzyme, and strongly starch and sucrose, moderately melezitose and weakly phlein and trehalose by the midgut enzyme. ,4. suturalis hydrolyzed weakly starch and only a little phlein, trehalose and sucrose by the salivary enzyme, while hydrolyzing strongly starch and sucrose, moderately melezitose and weakly phlein by the midgut enzyme. The salivary enzyme of O. funestus hydrolyzed very weakly phlein, trehalose and sucrose while the midgut enzyme hydrolyzed strongly starch and sucrose, moderately melezitose and weakly inulin, phlein and trehalose. P. angulosa hydrolyzed strongly trehalose, moderately starch and weakly sucrose by the salivary enzyme, while hydrolyzing strongly starch, trehalose, sucrose and melezitose and weakly inulin, phiein and cellulose by the midgut enzyme. The salivary
enzyme of E. rugosum only hydrolyzed very weakly trehalose, whereas the midgut enzyme hydrolyzed strongly starch, trehalose, sucrose and melezitose and weakly phlein and cellulose. C. marginatus hydrolyzed strongly sucrose, moderately trehalose, relatively weakly starch and inulin and weakly melezitose by the salivary enzyme, while hydrolyzing strongly sucrose and melezitose, moderately starch and trehalose and weakly cellulose, inulin and phlein by the midgut enzyme.
Qualitative assay for carbohydrase F o r the qualitative assay, maltose, sucrose, trehalose, melezitose, cellobiose, melibiose, lactose, isomaltose, ralfmose, c~-methylglucoside, salicin, gentiobiose and pectic acid were used as the substrates. The results are shown in Table 2. L. disponsi hydrolyzed maltose, trehalose, isomaltose and pectic acid by the salivary enzyme and all the substrates used by the midgut enzyme. The salivary enzyme of L. saundersi hydrolyzed weakly only maltose and pectic acid, whereas the midgut enzyme hydrolyzed all the substrates. A. suturalis hydrolyzed maltose, sucrose, trehalose and pectic acid by the salivary enzyme, while hydrolyzing all the substrates by the midgut enzyme. Isomaltose, salicin and gentiobiose were not used for the test. O. funestus hydrolyzed maltose, sucrose, trehalose, lactose and
Table 1. Digestive carbohydrase activities in the salivary gland and midgut of several bugs determined by Somogyi-Nelson colorimetric method
Starch
Inulin
Melezitose
1 '0
1.0
1 "0
l '0
1.0
1"0
6.0
6.0 7-0 6"0 Activity (mg glucose per bug)
5.5
6.0
0"784 0"834
0.002 0
0"029 0-062
0"010 0.005
0-078 0'136
0 2'540
0"012 0.780
0-538 1"723
0.004 0-011
0-012 0.073
0.016 0.011
0 0.072
0.027 1'956
0.011 0'411
0"137 2-717
0.004 0.016
0.044 0.107
0 0-015
0"024 0.831
0.038 3-080
0.007 0-760
0.008 2.445
0 0"062
0.027 0"107
0-008 0-020
0.031 0.098
0.025 2.209
0 0'368
0.396 3'294
0.020 0"120
0.003 0.078
0-018 0.071
2.935 3'059
0.171 2"941
0.015 2'471
0.008 1'896
0 0.005
0.010 0"027
0.010 0"031
0.020 2.440
0 2"004
0 1"370
0"259 0.422
0.152 0-074
0-007 0-044
0.007 0-133
0.834 0-678
3.494 3.685
0-069 2-977
* Salivary gland,
tMidgut.
1.0
Sucrose
6.0 Species and organ
L. disponsi SG* MGt L. saundersi SG MG A. suturalis SG MG O. funestus SG MG P. angulosa SG MG E. rugosum SG MG C. marginatus SG MG
Substrate (concentration % : pH) C.M. Phlein Cellulose Trehalose
(1.0,
6-0)
Gentiobiose
Glucose G a l a c t o u r o n i c acidll
Glucose
Glucose Glucose Fructose Glucose Maltose Glucose Fructose Glucose Glucose and galactose Glucose and galactose Glucose Glucose and galactose Fructose Melibiose Glucose
Degradation product
+
+ +
-
+
+
+
+ + + + + + . + + + + + + + + + + + + + -
MGt
+
-
-
+ :~ + . --+ -
SG*
L. disponsi
.
+
-
-
-
. --
+ -
SG
. .
+
+
+
+
+ + + + + -+
+
+ + + + + +
MG
L. saundersi
. .
+ +
--
-
--
-
+ _+ +
.
.
+
+
+ + + +
+ + + + _+ + . + . + + + + + +
MG
A. suturalis SG
* Salivary gland. 1" M i d g u t . ~. + + , l a r g e a m o u n t ; + , s m a l l a m o u n t ; + , t r a c e ; - , a b s e n c e . § G r o w t h of fungi in the reaction mixture. II P r e s o ~ c e o f s e v e r a l d e g r a d a t i o n p r o d u c t s o t h e r t h a n g a l a c t o u r o n i c a c i d .
(1.0, 6.0) P e c t i c a c i d (1-0, 6-2)
Salicin
G l u c o s i d e (1.0, 6.0)
C ¢ l l o b i o s e (1.0, 5.5) M e l i b i o s e (1.0, 5.5) L a c t o s e (1.0, 6.0) I s o m a l t o s e (1-0, 5-5) R a f l i n o s e (1.0, 6.0)
T r c h a l o s e (1-0, 6"0) M e l e z i t o s e (1.0, 6.0)
M a l t o s e (1.0, 5.5) S u c r o s e (1.0, 5.5)
Substrate (concentration, pH)
.
+
-
-
-
.
_+
_+ + + . -
SG
.
+
+
+
+ +
+
.
+ +
+ + + + +
+ + + + + + . +
MG
O. funestus
Species and organs
.
.
+ + §
-
-
-
+ + -+ + + -
+ + + + . -
SG
.
+
+ §
+
+
+
+ _+ + + + + + + + + + .
+ + + + + + +
MG
P. angulosa
.
.
-
-
-
-
--
-
SG
.
+
+
+
+
+
+ + + + + + + + + + +
+ + + + + + +
MG
E. rugosum
T a b l e 2. D i g e s t i v e c a r b o h y d r a s e s in t h e s a l i v a r y g l a n d a n d m i d g u t o f s e v e r a l b u g s d e t e r m i n e d b y p a p e r c h r o m a t o g r a p h y m e t h o d
-
-
-
+ + + + + -+ -+ -
SG
++
++
++
_
+
+
+
+
++ +
MG
C. marginatus
7.
~
o
0
"~
N"
o
o~"
148
K. Horn
pectic acid by the salivary enzyme, while hydrolyzing all the substrates except isomaltose, which was not tested, by the midgut enzyme. P. angulosa hydrolyzed maltose, sucrose, trehalose, melibiose, isomaltose, raffmose and pectic acid by the salivary enzyme and all the substrates by the midgut enzyme. The salivary enzyme of E. rugosum hydrolyzed nothing, whereas the midgut enzyme hydrolyzed everything. C. marginatus hydrolyzed maltose, sucrose, trehalose, melezitose, melibiose and raffinose by the salivary enzyme and all the substrates except pectic acid by the midgut enzyme. Gentiobiose was not used for the test. DISCUSSION Current classifications of sugar-splitting enzymes are based on the view that specificity depends largely on the nature of the monosaccharide contributing its reducing group to the glycosidic bond, and on the ~- or 8-form of this linkage. This concept implies that a single enzyme may split many substrates. Besides specific trehalase, four oligosaccharidases are postulated on the basis of substrates hydrolyzed: general c~-glucosidase (hydrolyzing maltose, sucrose, trehalose in part, melezitose, isomaltose, a-methylglucoside); 8-glucosidase (cellobiose, gentiobiose, salicin); c~-galactosidase (melibiose, raffinose); 8galactosidase (lactose, o-nitrophenolgalactoside). Hydrolysis of sucrose and raffinose by a 8-fructofuranosidase is refuted by several arguments: a single p H optimum for sucrose hydrolysis is the same as that of ~-glucosidase action on c~-methylglucoside and melezitose; no action on inulin, which would probably be split by 8-fructosidase; competitive inhibition of sucrose hydrolysis by added glucose or ct-glucosides but not fructose or raffinose; and hydrolysis of raffinose by extracts deficient in ~-galactosidase (Dadd, 1970). F r o m the above viewpoint and the results obtained in the present study, carbohydrases present in the alimentary system of the several bugs are summarized in Table 3. L. disponsi: the salivary gland has amylase, pectinase and weakly active phleinase. Moreover, it seems to contain specific maltase and trehalase, since the salivary enzyme hydrolyzes maltose and trehalose but not sucrose and melezitose. The midgut possesses amylase, phleinase, c~- and 8glucosidase, c~- and fl-galactosidase and pectinase. Hori (1970a) reported the presence of fl-fructosidase in the midgut of the same bug, but the present more detailed study indicates that it should be changed to c~-glucosidase, because the enzyme does not hydrolyze inulin. L. saundersi: the salivary gland includes amylase and pectinase. F o r the same reason as mentioned in the case of L. disponsi, it seems to contain specific maltase, although the activity is very weak. The midgut possesses amylase, phleinase, c~- and fl-glucosidase, a- and fl-galactosidase and pectinase. A. suturalis: the salivary gland has
amylase, pectinase, weakly active phleinase and a specific c~-glucosidase, which acts only weakly on melezitose. It appears to contain also a specific maltase, since the enzyme acts more strongly on maltose than on sucrose. The midgut contains amylase, phleinase, ~- and fl-glucosidase, a- and flgalactosidase and pectinase. In addition to these enzymes, it appears to have trehalase, because the enzyme hydrolyzes trehalose as strongly as melezitose, and also fl-fructosidase, because the enzyme acts on inulin and degrades raffinose to glucose or galactose, fructose and melibiose. O. funestus: the salivary gland includes pectinase, and weakly active phleinase, a-glucosidase and fl-galactosidase. The midgut contains amylase, inulase, phleinase, c~- and fl-glucosidase, c~- and 8-galactosidase, pectinase and weakly active cellulase. P. angulosa: the salivary gland has amylase, c~-glucosidase and~-galactosidase. Also it seems to include specific trehalase, since trehalose is hydrolyzed much more strongly than sucrose by the enzyme. Whether or not the pectinase detected is of salivary origin is not clear, because fungi have grown in the reaction mixture. The midgut includes amylase, inulase, phleinase, cellulase, 8-glucosidase, c~- and 8-galactosidase and ~glucosidase, which acts strongly on melezitose. It appears to have a specific trehalase, since the enzyme acts on trehalose as strongly as on sucrose. F o r the same reason as mentioned above, the presence of pectinase in the midgut is open to question. E. rugosum: the salivary gland contains only a trace of trehalase. However, amylase seems to exist occasionally, as the previous study of Hori (1968) demonstrated the presence of amylase. Hori (1968) also reported the presence of pectinase in the salivary gland, but the present more detailed study did not demonstrate any trace of this substance. The midgut carries amylase, cellulase, fl-glucosidase, c~- and flgalactosidase and weakly active phleinase and pectinase. It has also a specific ~x-glucosidase, which acts strongly on melezitose, and seems to have a specific trehalase, which acts on trehalose as strongly as on sucrose. C. marginatus: the salivary gland holds amylase, inulase and weakly active /3glucosidase and 8-galactosidase. It has also a specific a-glucosidase, which acts only a little on melezitose. The midgut contains amylase, cellulase, o~- and 8-glucosidase, c~-galactosidase and weakly active inulase, phleinase and 8-galactosidase. In general, it may be concluded that the carbohydrase variety is poor in the salivary gland but rich in the midgut. This is in accordance with the results hitherto obtained by a number of investigators. It is known that amylase is contained in the salivary gland of many phytophagous bugs (Baptist, 1941; Goodchild, 1952; Nuorteva, 1954; Saxena, 1954a, b; Ford, 1962; Rastogi, 1962; Rastogi & Datta Gupta, 1962a, b; Khan & Ford, 1967; Mathur & Thakar, 1969; Hori, 1970c; Takanona & Hori, 1974). All the bugs used in the present study
+ + :~ . + . + + +
SG*
. + . + + + + + + + + +
+ +
MGt
L. disponsi
. . + +
-
+ .
SG
.
+
+
+ § -+ +
.
+
SG
. + + + +
+ + + + + + +
+ + + +
MG
,4. suturalis
no activity.
+ + . + . + + + + +
MG
L. saundersi
_+ + +
+
SG + + + + _ + + + + + + + + +
MG
O. funestus
Species a n d o r g a n
+ + + + + + + II + + + + + ¶ + +
+ +
MG
+ -+ + + + + ¶
SG
P. angulosa
C a r b o h y d r a s e s d e t e c t e d in t h e s a l i v a r y g l a n d a n d m i d g u t o f s e v e r a l b u g s
* Salivary gland. 1 Midgut. + + , strong activity; + , w e a k activity; + , slight activity; - , § c~-Glucosidase v e r y w e a k l y a c t i n g o n m e l e z i t o s e . JI ~ - G l u c o s i d a s e s t r o n g l y a c t i n g o n m e l e z i t o s e . ¶ G r o w t h o f f u n g i in t h e r e a c t i o n m i x t u r e .
Amylase Inulase Phleinase Ccllulase a-Glucosidase /3-Glucosidase ~-Galactosidase t/-Galactosidase Pectinase Maltase Trehalase /3-Fructosidase
Enzyme
T a b l e 3.
+
-
SG
+ +
+ + + + + + II + + + + + +
MG
E. rugosum
+ + -+ + § + + -
SG
+ + + + + + + + + + --
MG
C. marginatus
=
t~r
,~ o x~ :r
~.
o ~. o ~" ~"
150
K. Horn
had amylase in their salivary glands. It appears that amylase is contained quite commonly in this organ in almost all phytophagous bugs with few exceptions. No one has demonstrated the existence of inulase and phleinase in the salivary gland of heteropterous insects. It is of interest that C. marginatus includes inulin hydrolytic enzyme in the salivary gland. The relationship between the feeding habits and the presence of the enzyme should be investigated in the future. Phleinase or phlein hydrolytic enzyme was contained in the salivary gland of the three species of the family Miridae, although activities were very weak. Invertase and maltase have been found in the salivary gland of several bugs (Baptist, 1941; Goodchild, 1952; Rastogi, 1962), but it remains unascertainable whether they are generally a-glucosidase or fl-fructosidase and specifically maltase. Khan & Ford (1967) found that Dysdercusfasciatus had c~-glucosidase in the salivary gland. The present study demonstrated this enzyme in the glands of A. suturalis, O. funestus, P. angulosa and C. marginatus. This enzyme appears to exist sporadically in the suborder Heteroptera, irrespective of taxonomic position. fl-Glucosidase has been detected only in the salivary gland of D. fasciatus (Khan & Ford, 1967). C. marginatus also had fl-glucosidase in this organ. As the enzyme is included universally in plants, the presence of the enzyme in this organ is of great interest, and therefore it may be important to investigate the significance of the enzyme on the digestive physiology of the phytophagous bugs. aand fl-Galactosidase have never been detected in the salivary gland of any heteropterous insect. In the present study, it was found that the salivary enzymes of P. angulosa and C. marginatus showed, respectively, strong and weak activity of c~-galactosidase, and that of O. funestus showed very weak activity of /3-galactosidase. Further comparative studies on carbohydrases among species would clarify the physiological function of these enzymes. In Heteroptera, salivary pectinase has been detected only in bugs of the family Miridae (Adams & McAllan, 1958; Laurema & Nuorteva, 1961; Strong & Kruitwagen, 1968; Takanona & Hori, 1974). Also in the present study, salivary pectinase was found only in mirid bugs, except for P. angulosa whose pectinase origin was not obvious, because of fungi growing in the reaction mixture. As mentioned by Takanona & Hori (1974), salivary pectinase seems to be a special though not an absolute feature of the family Miridae, since the bugs not belonging to Miridae rarely have this enzyme (Hori, unpublished data). Takanona & Hori (1974) found a specific maltase in the salivary gland of Stenotus binotatus (Miridae). Likewise, the salivary enzyme of the three species of mirid bugs used showed an activity of maltase. Amylase is contained in the midgut of all the phytophagous bugs which have been investigated so far (Goodchild, 1952; Saxena, 1964a, b, 1955;
Rastogi & Datta Gupta, 1962a, b; Mathur & Thakar, 1969; Hori, 1970a; Takanona & Hori, 1974). All the bugs studied in the present work also had amylase in their midguts. Inulase and phleinase have never been investigated with respect to heteropterous insects. The present author found inulase or inulin-hydrolytic enzyme in the midgut of A. suturalis, O. funestus, P. angulosa and C. marginatus, though it was not obvious whether the enzyme originated from the bugs or their food plants. All the bugs investigated contained phleinase or phlein-hydrolytic enzyme in the midgut, which might be of insect origin, because it was detected also in the salivary gland of some bugs. Phlein is a specific polysaccharide contained in roots, stems and pales or glumes of various grasses. Thus the presence of phlein-hydrolytic enzyme in the alimentary system of the bugs is of much interest in relation to their nutrition and feeding habits. Many bugs have invertase and maltase in the midgut (Goodchild, 1952; Saxena, 1954a, b, 1955; Rastogi & Datta Gupta, 1962a, b; Mathur & Thakar, 1969), but it remains obscure whether these are general aglucosidase or/3-fructosidase and specific maltase. Khan & Ford (1967) and Takanona & Hori (1974) suggested that c~-glucosidase was contained in the midgut of D.fasciatus and Stenotus binotatus. All the bugs investigated here manifested a distinct activity of this enzyme in the midgut. It is probable that almost all heteropterous insects have c~-glucosidase in their midguts, fl-Glucosidase is included in the midgut of Leptocorisa varicornis (Saxena, 1954a, b), D. fasciatus (Khan & Ford, 1967) and S. binotatus (Takanona & Hori, 1974). The present author also found this enzyme in the midgut of all the bugs investigated, though its origin was still obscure. It is known that c~-galactosidase is present in the midgut of D. fasciatus and S. binotatus (Khan & Ford, 1967; Takanona & Hori, 1974). All the bugs used in the present study possessed c~-galactosidase in their midguts. Further investigations are needed to determine whether c~-galactosidase is commonly contained in the midgut of heteropterous insects or not. Mathur & Thakar (1969) confirmed the presence of lactase in the midgut of Clavigrella gibbosa. This enzyme may possibly be what corresponds to fl-galactosidase. All the bugs investigated here had fl-galactosidase in their midguts. Pectinase in the midgut of mirid bugs might have come from the salivary glands. The feeding habits differed greatly in the bugs used in the present study, whereas the variety of carbohydrase in the alimentary system varied little from bug to bug, with a few exceptions. This fact may indicate that the nutritional components of the food plants of the bugs are qualitatively very little different among themselves, though appreciably different quantitatively. It is probable that salivary pectinase of mirid bugs is closely related to their feeding habits, lacerate- and flush-feeding type.
Digestive carbohydrases in phytophagous bugs Acknowledgement--The author wishes to express his warmest thanks to Professor Y. Nishijima of the Obihiro Zootechnical University for valuable discussions and criticisms and for reading the manuscript. REFERENCES
ADAMS J. B. & MCALLAN J. W. (1958) Pectinase in certain insects. Can. J. ZooL 36, 305-308. BAPTISTB. A. (1941) The morphology and physiology of the salivary glands of Hemiptera-Heteroptera. Q. di miscrosc. Sci. 83, 91-139. DADD R. H. (1970) Digestion in insect. In Chemical Zoology (Edited by FLORK1NM. & SCHEERB. T.), Vol. 5, pp. 117-145. Academic Press, London. FORD J. B. (1962) Studies on the digestive processes of Dysdercus fasciatus Sign. Ann. appl. Biol. 50, 355. GOODCHILD A. J. P. (1952) A study of the digestive system of the West African cacao capsid bugs (Hemiptera: Miridae). Proc. zool. Soc. Lond. 122, 543-572. HORI K. (1968) Some properties and developmental changes in occurrence of the salivary amylase of the cabbage bug, Eurydema rugosum Motschulsky (Hemiptera: Pentatomidae). Appl. Ent. ZooL 3, 198-202. HORI K. (1970a) Carbohydrases in the gut and the salivary gland, and the nature of the amylase in the gut homogenate of Lygus disponsi Linnavuori (Hemiptera: Miridae). Appl. Ent. Zool. 5, 13-22. HORI K. (197013) Some properties of protease in the gut and in the salivary gland of Lygus disponsi Linnavuori (Hemiptera, Miridae). Res. Bull. Obihiro Univ. 6, 318-324. HORI K. (1970c) Some properties of amylase in the salivary gland of Lygus disponsi (Hemiptera). J. Insect Physiol. 16, 373-386. House H. L. (1965) Digestion. In The Physiology of lnsecta (Edited by ROCKSTEIN M.), pp. 815-862. Academic Press, New York. KHAN M. R. • FORD J. B. (1967) The distribution and localization of digestive enzymes in the alimentary canal and salivaryglands of the cotton stainer, Dysdercus fasciatus. J. Insect Physiol. 13, 1619-1628. KUMARR. (1970) Occurrence of proteases in the salivary glands of cacao-capsids (Heteroptera: Miridae). J. New York Entomol. 78, 198-200. LAUREMAS. & NUORTEVAP. (1961) On the occurrence of pectin polygalacturonase in the salivary glands of Heteroptera and Homoptera Auchenorrhyncha. Ann. ent.fenn. 27, 89-93.
151
MATHUR L. M. L. & T ~ A. V. (1969) Digestive physiology of tur pod bug, Clavigrella gibbosa Spin. (Coreidae: Heteroptera). IndianJ. Ent. 31, 251-257. NUOnTEVA P. (1954) Studies on the salivary enzymes of some bugs injuring wheat kernels. Ann. ent. fenn. 20, 102-104. NUOnTEVA P. & LAtmZMAS. (1961) Observations on the activity of salivary proteases and amylases in Dolycoris baccarum (L.) (Her., Pentatomidae). Ann. ent. fenn. 27, 93-97. RASTOGXS. C. (1962) On the salivary enzymes of some phytophagous and predaceous Heteroptera. Sci. Cult. 28, 478-480. R~TOGXS. C. & DATTAGUPTAA. K. (1962a) The pH and the digestive enzymes of the red pumpkin bug, Coridius janus (Fabr.) (Heteroptera: Dinidoridae). Proc. zooL Soc. 15, 57-64. P~,STOGXS. C. & DATrA GUPTAA. K. (1962b) Studies on the physiology of digestion in the alimentary canal and salivary glands of Odontopus nigricornis Stal. (Heteroptera: Pyrrhocordiae). Indian J. Ent. 23, 106-115. SAXENAK. N. (1954a) Physiology of digestion in Lepto. corisa varicornis Fabr. (Heteroptera: Coreidae). Current Sci. 23, 132. SAXENAK. N. (1954b) Physiology of the alimentary canal of Leptocorisa varicornis Fabr. (Hemiptera: Coreidae). J. zool. Soc. India 6, 111-122. SAXENA K. N. (1955) Studies on the passage of food, hydrogen-ion concentration and enzymes in the gut and salivary glands of Dysdercus koenigii Fabr. (Pyrrhocoridae: Heteroptera). J. zool. Soc. India 7, 145-154. SAXENAK. N. (1958) Digestion and absorption of carbohydrates in the alimentary canal of the red cotton bug, Dysdercus koenigii Fabr. (Heteroptera: Pyrrhocoridae). Physiol. Zorl. 31, 129-138. STRONG F. E. & KRtqTWAGEN E. C. (1968) Polygalacturonase in the salivary apparatus of Lygus hesperus (Hemiptera). J. Insect Physiol. 14, 1113-1119. TAKANONAT. & Horn K. (1974) Digestive enzymes in the salivary gland and midgut of the bug Stenotus binotatus. Comp. Biochem. Physiol. 47A, 521-528. Key Word Index--Carbohydrases; salivary gland; phytophagus insects; phleinase; pectinase.
DIGESTIVE CARBOHYDRASES IN THE SALIVARY GLAND AND MIDGUT OF SEVERAL PHYTOPHAGOUS BUGS* K. HoR1 Division of Entomology and Plant Physiology, Obihiro Zootechnical University, Obihiro, Hokkaido, 080 Japan (Received 19 December 1973)
Abstract--1. Carbohydrases in the salivary gland and midgut were compared among several phytophagous bugs with different feeding habits, Lygus disponsi, L. saundersi, Adelphoeoris suturalis and Orthoeephalus funestus (Miridae), Palomena angulosa and Eurydema rugosum (Pentatomidae) and Coreus marginatus (Coreidae). 2. All the bugs had amylase, phleinase, ~- and fl-glucosidase and ~- and fl-galactosidase in the midgut. Carbohydrase variety in the salivary gland was poor. 3. Salivary pectinase was detected in all the mirid bugs. 4. In the salivary gland, C. marginatus had fl-glucosidase, c~- and fl-galactosidase, P. angulosa c~galactosidas¢ and O. funestus ~-galactosidase. 5. A distinct cellulolytic activity was detected in the midgut of P. angulosa, E. rugosum and C. marginatus. 6. The relationship between the taxonomic position or the feeding habit of the bug and the presence of carbohydrase in the alimentary system is discussed. INTRODUCTION THE FOOD and feeding habits of insects vary very greatly and the digestive enzymes are adapted for their respective foods (House, 1965; Dadd, 1970). Therefore, study of the digestive enzymes of insects forms an important basis for study of the injury caused by them and of their digestive physiology. There have been many studies on the presence of digestive enzymes in phytophagous bugs (Baptist, 1941; Goodchild, 1952; Nuorteva, 1954; Saxena, 1954a, b, 1955, 1958; Nuorteva & Laurema, 1961; Ford, 1962; Rastogi, 1962; Rastogl & Datta Gupta, 1962a, b; Khan & Ford, 1967; Mathur & Thakar, 1969; Hori, 1970a, b; Kumar, 1970; Takanona & Hori, 1974), but relatively few comparative studies among species and among bugs with different feeding habits. Takanona & Hori (1974) stated that there was no specificity of species or family with respect to the digestive enzymes of phytophagous bugs except salivary pectinase. In order to prove this statement, however, many more species must be compared with one another. The present paper deals with the carbohydrases contained in the salivary gland and midgut of several phytophagous bugs with different feeding habits. MATERIALS AND METHODS
(Miridae), a polyphagous bug though it has a special food preference for cruciferous plants; Lygus saundersi (Miridae), an oligophagous bug whose feeding is limited to some grasses, Adelphocoris suturalis (Miridae), an oligophagous bug having a food preference for legumes; Orthocephalus funestus (Miridae), presumably a monophagous bug feeding only on mugworts; Palomena angulosa (Pentatomidae), a polyphagous bug feeding on various herbs and trees; Eurydema rugosum (Pentatomidae), an oligophagous bug having a food preference for cruciferous plants; and Coreus marginatus (Coreidae), presumably a polyphagous bug. Thirty adults (not starving), with the exception of P. angulosa (twentythree adults) and C. marginatus (eighteen adults), were collected from the field for the assay. Preparation of salivary and midgut enzyme solutions
The enzyme solutions were prepared by homogenizing the whole salivary gland or the whole midgut according to the procedure employed previously by Hori (1970c). Reaction mixture and incubation
The reaction mixture consisted of 1.0 ml substrate solution adjusted to the proper pH with McIlvaine buffer (Tables 1 and 2) and 1.0 ml enzyme solution, to which 30 mg chloramphenicol was added as antibiotic. Incubations were carried out for 24 hr at 37°C.
Insects used Carbohydrases were compared among the following seven species with different feeding habits: Lygus disponsi
Assay for carbohydrase activity
* Contribution No. 56 from the Laboratory of Entomology, Obihiro Zootechnical University.
Activities were determined by the colorimetric method of Somogyi-Nelson (Hori, 1970c) and/or the paper chromatography method (Hori, 1970a). 145
146
K. Ho~a RESULTS
Quantitative assay for carbohydrase F o r t h e quantitative assay, soluble starch, inulin, phlein, carboxymethyl cellulose, trehalose, sucrose and melezitose were used as the substrates. The results are shown in Table 1. The salivary enzyme of L. disponsi hydrolyzed moderately starch, and weakly phlein and trehalose, while the midgut enzyme hydrolyzed strongly sucrose, moderately starch and melezitose, and weakly trehalose and phlein. L. saundersi hydrolyzed moderately starch and very weakly sucrose by the salivary enzyme, and strongly starch and sucrose, moderately melezitose and weakly phlein and trehalose by the midgut enzyme. ,4. suturalis hydrolyzed weakly starch and only a little phlein, trehalose and sucrose by the salivary enzyme, while hydrolyzing strongly starch and sucrose, moderately melezitose and weakly phlein by the midgut enzyme. The salivary enzyme of O. funestus hydrolyzed very weakly phlein, trehalose and sucrose while the midgut enzyme hydrolyzed strongly starch and sucrose, moderately melezitose and weakly inulin, phlein and trehalose. P. angulosa hydrolyzed strongly trehalose, moderately starch and weakly sucrose by the salivary enzyme, while hydrolyzing strongly starch, trehalose, sucrose and melezitose and weakly inulin, phiein and cellulose by the midgut enzyme. The salivary
enzyme of E. rugosum only hydrolyzed very weakly trehalose, whereas the midgut enzyme hydrolyzed strongly starch, trehalose, sucrose and melezitose and weakly phlein and cellulose. C. marginatus hydrolyzed strongly sucrose, moderately trehalose, relatively weakly starch and inulin and weakly melezitose by the salivary enzyme, while hydrolyzing strongly sucrose and melezitose, moderately starch and trehalose and weakly cellulose, inulin and phlein by the midgut enzyme.
Qualitative assay for carbohydrase F o r the qualitative assay, maltose, sucrose, trehalose, melezitose, cellobiose, melibiose, lactose, isomaltose, ralfmose, c~-methylglucoside, salicin, gentiobiose and pectic acid were used as the substrates. The results are shown in Table 2. L. disponsi hydrolyzed maltose, trehalose, isomaltose and pectic acid by the salivary enzyme and all the substrates used by the midgut enzyme. The salivary enzyme of L. saundersi hydrolyzed weakly only maltose and pectic acid, whereas the midgut enzyme hydrolyzed all the substrates. A. suturalis hydrolyzed maltose, sucrose, trehalose and pectic acid by the salivary enzyme, while hydrolyzing all the substrates by the midgut enzyme. Isomaltose, salicin and gentiobiose were not used for the test. O. funestus hydrolyzed maltose, sucrose, trehalose, lactose and
Table 1. Digestive carbohydrase activities in the salivary gland and midgut of several bugs determined by Somogyi-Nelson colorimetric method
Starch
Inulin
Melezitose
1 '0
1.0
1 "0
l '0
1.0
1"0
6.0
6.0 7-0 6"0 Activity (mg glucose per bug)
5.5
6.0
0"784 0"834
0.002 0
0"029 0-062
0"010 0.005
0-078 0'136
0 2'540
0"012 0.780
0-538 1"723
0.004 0-011
0-012 0.073
0.016 0.011
0 0.072
0.027 1'956
0.011 0'411
0"137 2-717
0.004 0.016
0.044 0.107
0 0-015
0"024 0.831
0.038 3-080
0.007 0-760
0.008 2.445
0 0"062
0.027 0"107
0-008 0-020
0.031 0.098
0.025 2.209
0 0'368
0.396 3'294
0.020 0"120
0.003 0.078
0-018 0.071
2.935 3'059
0.171 2"941
0.015 2'471
0.008 1'896
0 0.005
0.010 0"027
0.010 0"031
0.020 2.440
0 2"004
0 1"370
0"259 0.422
0.152 0-074
0-007 0-044
0.007 0-133
0.834 0-678
3.494 3.685
0-069 2-977
* Salivary gland,
tMidgut.
1.0
Sucrose
6.0 Species and organ
L. disponsi SG* MGt L. saundersi SG MG A. suturalis SG MG O. funestus SG MG P. angulosa SG MG E. rugosum SG MG C. marginatus SG MG
Substrate (concentration % : pH) C.M. Phlein Cellulose Trehalose
(1.0,
6-0)
Gentiobiose
Glucose G a l a c t o u r o n i c acidll
Glucose
Glucose Glucose Fructose Glucose Maltose Glucose Fructose Glucose Glucose and galactose Glucose and galactose Glucose Glucose and galactose Fructose Melibiose Glucose
Degradation product
+
+ +
-
+
+
+
+ + + + + + . + + + + + + + + + + + + + -
MGt
+
-
-
+ :~ + . --+ -
SG*
L. disponsi
.
+
-
-
-
. --
+ -
SG
. .
+
+
+
+
+ + + + + -+
+
+ + + + + +
MG
L. saundersi
. .
+ +
--
-
--
-
+ _+ +
.
.
+
+
+ + + +
+ + + + _+ + . + . + + + + + +
MG
A. suturalis SG
* Salivary gland. 1" M i d g u t . ~. + + , l a r g e a m o u n t ; + , s m a l l a m o u n t ; + , t r a c e ; - , a b s e n c e . § G r o w t h of fungi in the reaction mixture. II P r e s o ~ c e o f s e v e r a l d e g r a d a t i o n p r o d u c t s o t h e r t h a n g a l a c t o u r o n i c a c i d .
(1.0, 6.0) P e c t i c a c i d (1-0, 6-2)
Salicin
G l u c o s i d e (1.0, 6.0)
C ¢ l l o b i o s e (1.0, 5.5) M e l i b i o s e (1.0, 5.5) L a c t o s e (1.0, 6.0) I s o m a l t o s e (1-0, 5-5) R a f l i n o s e (1.0, 6.0)
T r c h a l o s e (1-0, 6"0) M e l e z i t o s e (1.0, 6.0)
M a l t o s e (1.0, 5.5) S u c r o s e (1.0, 5.5)
Substrate (concentration, pH)
.
+
-
-
-
.
_+
_+ + + . -
SG
.
+
+
+
+ +
+
.
+ +
+ + + + +
+ + + + + + . +
MG
O. funestus
Species and organs
.
.
+ + §
-
-
-
+ + -+ + + -
+ + + + . -
SG
.
+
+ §
+
+
+
+ _+ + + + + + + + + + .
+ + + + + + +
MG
P. angulosa
.
.
-
-
-
-
--
-
SG
.
+
+
+
+
+
+ + + + + + + + + + +
+ + + + + + +
MG
E. rugosum
T a b l e 2. D i g e s t i v e c a r b o h y d r a s e s in t h e s a l i v a r y g l a n d a n d m i d g u t o f s e v e r a l b u g s d e t e r m i n e d b y p a p e r c h r o m a t o g r a p h y m e t h o d
-
-
-
+ + + + + -+ -+ -
SG
++
++
++
_
+
+
+
+
++ +
MG
C. marginatus
7.
~
o
0
"~
N"
o
o~"
148
K. Horn
pectic acid by the salivary enzyme, while hydrolyzing all the substrates except isomaltose, which was not tested, by the midgut enzyme. P. angulosa hydrolyzed maltose, sucrose, trehalose, melibiose, isomaltose, raffmose and pectic acid by the salivary enzyme and all the substrates by the midgut enzyme. The salivary enzyme of E. rugosum hydrolyzed nothing, whereas the midgut enzyme hydrolyzed everything. C. marginatus hydrolyzed maltose, sucrose, trehalose, melezitose, melibiose and raffinose by the salivary enzyme and all the substrates except pectic acid by the midgut enzyme. Gentiobiose was not used for the test. DISCUSSION Current classifications of sugar-splitting enzymes are based on the view that specificity depends largely on the nature of the monosaccharide contributing its reducing group to the glycosidic bond, and on the ~- or 8-form of this linkage. This concept implies that a single enzyme may split many substrates. Besides specific trehalase, four oligosaccharidases are postulated on the basis of substrates hydrolyzed: general c~-glucosidase (hydrolyzing maltose, sucrose, trehalose in part, melezitose, isomaltose, a-methylglucoside); 8-glucosidase (cellobiose, gentiobiose, salicin); c~-galactosidase (melibiose, raffinose); 8galactosidase (lactose, o-nitrophenolgalactoside). Hydrolysis of sucrose and raffinose by a 8-fructofuranosidase is refuted by several arguments: a single p H optimum for sucrose hydrolysis is the same as that of ~-glucosidase action on c~-methylglucoside and melezitose; no action on inulin, which would probably be split by 8-fructosidase; competitive inhibition of sucrose hydrolysis by added glucose or ct-glucosides but not fructose or raffinose; and hydrolysis of raffinose by extracts deficient in ~-galactosidase (Dadd, 1970). F r o m the above viewpoint and the results obtained in the present study, carbohydrases present in the alimentary system of the several bugs are summarized in Table 3. L. disponsi: the salivary gland has amylase, pectinase and weakly active phleinase. Moreover, it seems to contain specific maltase and trehalase, since the salivary enzyme hydrolyzes maltose and trehalose but not sucrose and melezitose. The midgut possesses amylase, phleinase, c~- and 8glucosidase, c~- and fl-galactosidase and pectinase. Hori (1970a) reported the presence of fl-fructosidase in the midgut of the same bug, but the present more detailed study indicates that it should be changed to c~-glucosidase, because the enzyme does not hydrolyze inulin. L. saundersi: the salivary gland includes amylase and pectinase. F o r the same reason as mentioned in the case of L. disponsi, it seems to contain specific maltase, although the activity is very weak. The midgut possesses amylase, phleinase, c~- and fl-glucosidase, a- and fl-galactosidase and pectinase. A. suturalis: the salivary gland has
amylase, pectinase, weakly active phleinase and a specific c~-glucosidase, which acts only weakly on melezitose. It appears to contain also a specific maltase, since the enzyme acts more strongly on maltose than on sucrose. The midgut contains amylase, phleinase, ~- and fl-glucosidase, a- and flgalactosidase and pectinase. In addition to these enzymes, it appears to have trehalase, because the enzyme hydrolyzes trehalose as strongly as melezitose, and also fl-fructosidase, because the enzyme acts on inulin and degrades raffinose to glucose or galactose, fructose and melibiose. O. funestus: the salivary gland includes pectinase, and weakly active phleinase, a-glucosidase and fl-galactosidase. The midgut contains amylase, inulase, phleinase, c~- and fl-glucosidase, c~- and 8-galactosidase, pectinase and weakly active cellulase. P. angulosa: the salivary gland has amylase, c~-glucosidase and~-galactosidase. Also it seems to include specific trehalase, since trehalose is hydrolyzed much more strongly than sucrose by the enzyme. Whether or not the pectinase detected is of salivary origin is not clear, because fungi have grown in the reaction mixture. The midgut includes amylase, inulase, phleinase, cellulase, 8-glucosidase, c~- and 8-galactosidase and ~glucosidase, which acts strongly on melezitose. It appears to have a specific trehalase, since the enzyme acts on trehalose as strongly as on sucrose. F o r the same reason as mentioned above, the presence of pectinase in the midgut is open to question. E. rugosum: the salivary gland contains only a trace of trehalase. However, amylase seems to exist occasionally, as the previous study of Hori (1968) demonstrated the presence of amylase. Hori (1968) also reported the presence of pectinase in the salivary gland, but the present more detailed study did not demonstrate any trace of this substance. The midgut carries amylase, cellulase, fl-glucosidase, c~- and flgalactosidase and weakly active phleinase and pectinase. It has also a specific ~x-glucosidase, which acts strongly on melezitose, and seems to have a specific trehalase, which acts on trehalose as strongly as on sucrose. C. marginatus: the salivary gland holds amylase, inulase and weakly active /3glucosidase and 8-galactosidase. It has also a specific a-glucosidase, which acts only a little on melezitose. The midgut contains amylase, cellulase, o~- and 8-glucosidase, c~-galactosidase and weakly active inulase, phleinase and 8-galactosidase. In general, it may be concluded that the carbohydrase variety is poor in the salivary gland but rich in the midgut. This is in accordance with the results hitherto obtained by a number of investigators. It is known that amylase is contained in the salivary gland of many phytophagous bugs (Baptist, 1941; Goodchild, 1952; Nuorteva, 1954; Saxena, 1954a, b; Ford, 1962; Rastogi, 1962; Rastogi & Datta Gupta, 1962a, b; Khan & Ford, 1967; Mathur & Thakar, 1969; Hori, 1970c; Takanona & Hori, 1974). All the bugs used in the present study
+ + :~ . + . + + +
SG*
. + . + + + + + + + + +
+ +
MGt
L. disponsi
. . + +
-
+ .
SG
.
+
+
+ § -+ +
.
+
SG
. + + + +
+ + + + + + +
+ + + +
MG
,4. suturalis
no activity.
+ + . + . + + + + +
MG
L. saundersi
_+ + +
+
SG + + + + _ + + + + + + + + +
MG
O. funestus
Species a n d o r g a n
+ + + + + + + II + + + + + ¶ + +
+ +
MG
+ -+ + + + + ¶
SG
P. angulosa
C a r b o h y d r a s e s d e t e c t e d in t h e s a l i v a r y g l a n d a n d m i d g u t o f s e v e r a l b u g s
* Salivary gland. 1 Midgut. + + , strong activity; + , w e a k activity; + , slight activity; - , § c~-Glucosidase v e r y w e a k l y a c t i n g o n m e l e z i t o s e . JI ~ - G l u c o s i d a s e s t r o n g l y a c t i n g o n m e l e z i t o s e . ¶ G r o w t h o f f u n g i in t h e r e a c t i o n m i x t u r e .
Amylase Inulase Phleinase Ccllulase a-Glucosidase /3-Glucosidase ~-Galactosidase t/-Galactosidase Pectinase Maltase Trehalase /3-Fructosidase
Enzyme
T a b l e 3.
+
-
SG
+ +
+ + + + + + II + + + + + +
MG
E. rugosum
+ + -+ + § + + -
SG
+ + + + + + + + + + --
MG
C. marginatus
=
t~r
,~ o x~ :r
~.
o ~. o ~" ~"
150
K. Horn
had amylase in their salivary glands. It appears that amylase is contained quite commonly in this organ in almost all phytophagous bugs with few exceptions. No one has demonstrated the existence of inulase and phleinase in the salivary gland of heteropterous insects. It is of interest that C. marginatus includes inulin hydrolytic enzyme in the salivary gland. The relationship between the feeding habits and the presence of the enzyme should be investigated in the future. Phleinase or phlein hydrolytic enzyme was contained in the salivary gland of the three species of the family Miridae, although activities were very weak. Invertase and maltase have been found in the salivary gland of several bugs (Baptist, 1941; Goodchild, 1952; Rastogi, 1962), but it remains unascertainable whether they are generally a-glucosidase or fl-fructosidase and specifically maltase. Khan & Ford (1967) found that Dysdercusfasciatus had c~-glucosidase in the salivary gland. The present study demonstrated this enzyme in the glands of A. suturalis, O. funestus, P. angulosa and C. marginatus. This enzyme appears to exist sporadically in the suborder Heteroptera, irrespective of taxonomic position. fl-Glucosidase has been detected only in the salivary gland of D. fasciatus (Khan & Ford, 1967). C. marginatus also had fl-glucosidase in this organ. As the enzyme is included universally in plants, the presence of the enzyme in this organ is of great interest, and therefore it may be important to investigate the significance of the enzyme on the digestive physiology of the phytophagous bugs. aand fl-Galactosidase have never been detected in the salivary gland of any heteropterous insect. In the present study, it was found that the salivary enzymes of P. angulosa and C. marginatus showed, respectively, strong and weak activity of c~-galactosidase, and that of O. funestus showed very weak activity of /3-galactosidase. Further comparative studies on carbohydrases among species would clarify the physiological function of these enzymes. In Heteroptera, salivary pectinase has been detected only in bugs of the family Miridae (Adams & McAllan, 1958; Laurema & Nuorteva, 1961; Strong & Kruitwagen, 1968; Takanona & Hori, 1974). Also in the present study, salivary pectinase was found only in mirid bugs, except for P. angulosa whose pectinase origin was not obvious, because of fungi growing in the reaction mixture. As mentioned by Takanona & Hori (1974), salivary pectinase seems to be a special though not an absolute feature of the family Miridae, since the bugs not belonging to Miridae rarely have this enzyme (Hori, unpublished data). Takanona & Hori (1974) found a specific maltase in the salivary gland of Stenotus binotatus (Miridae). Likewise, the salivary enzyme of the three species of mirid bugs used showed an activity of maltase. Amylase is contained in the midgut of all the phytophagous bugs which have been investigated so far (Goodchild, 1952; Saxena, 1964a, b, 1955;
Rastogi & Datta Gupta, 1962a, b; Mathur & Thakar, 1969; Hori, 1970a; Takanona & Hori, 1974). All the bugs studied in the present work also had amylase in their midguts. Inulase and phleinase have never been investigated with respect to heteropterous insects. The present author found inulase or inulin-hydrolytic enzyme in the midgut of A. suturalis, O. funestus, P. angulosa and C. marginatus, though it was not obvious whether the enzyme originated from the bugs or their food plants. All the bugs investigated contained phleinase or phlein-hydrolytic enzyme in the midgut, which might be of insect origin, because it was detected also in the salivary gland of some bugs. Phlein is a specific polysaccharide contained in roots, stems and pales or glumes of various grasses. Thus the presence of phlein-hydrolytic enzyme in the alimentary system of the bugs is of much interest in relation to their nutrition and feeding habits. Many bugs have invertase and maltase in the midgut (Goodchild, 1952; Saxena, 1954a, b, 1955; Rastogi & Datta Gupta, 1962a, b; Mathur & Thakar, 1969), but it remains obscure whether these are general aglucosidase or/3-fructosidase and specific maltase. Khan & Ford (1967) and Takanona & Hori (1974) suggested that c~-glucosidase was contained in the midgut of D.fasciatus and Stenotus binotatus. All the bugs investigated here manifested a distinct activity of this enzyme in the midgut. It is probable that almost all heteropterous insects have c~-glucosidase in their midguts, fl-Glucosidase is included in the midgut of Leptocorisa varicornis (Saxena, 1954a, b), D. fasciatus (Khan & Ford, 1967) and S. binotatus (Takanona & Hori, 1974). The present author also found this enzyme in the midgut of all the bugs investigated, though its origin was still obscure. It is known that c~-galactosidase is present in the midgut of D. fasciatus and S. binotatus (Khan & Ford, 1967; Takanona & Hori, 1974). All the bugs used in the present study possessed c~-galactosidase in their midguts. Further investigations are needed to determine whether c~-galactosidase is commonly contained in the midgut of heteropterous insects or not. Mathur & Thakar (1969) confirmed the presence of lactase in the midgut of Clavigrella gibbosa. This enzyme may possibly be what corresponds to fl-galactosidase. All the bugs investigated here had fl-galactosidase in their midguts. Pectinase in the midgut of mirid bugs might have come from the salivary glands. The feeding habits differed greatly in the bugs used in the present study, whereas the variety of carbohydrase in the alimentary system varied little from bug to bug, with a few exceptions. This fact may indicate that the nutritional components of the food plants of the bugs are qualitatively very little different among themselves, though appreciably different quantitatively. It is probable that salivary pectinase of mirid bugs is closely related to their feeding habits, lacerate- and flush-feeding type.
Digestive carbohydrases in phytophagous bugs Acknowledgement--The author wishes to express his warmest thanks to Professor Y. Nishijima of the Obihiro Zootechnical University for valuable discussions and criticisms and for reading the manuscript. REFERENCES
ADAMS J. B. & MCALLAN J. W. (1958) Pectinase in certain insects. Can. J. ZooL 36, 305-308. BAPTISTB. A. (1941) The morphology and physiology of the salivary glands of Hemiptera-Heteroptera. Q. di miscrosc. Sci. 83, 91-139. DADD R. H. (1970) Digestion in insect. In Chemical Zoology (Edited by FLORK1NM. & SCHEERB. T.), Vol. 5, pp. 117-145. Academic Press, London. FORD J. B. (1962) Studies on the digestive processes of Dysdercus fasciatus Sign. Ann. appl. Biol. 50, 355. GOODCHILD A. J. P. (1952) A study of the digestive system of the West African cacao capsid bugs (Hemiptera: Miridae). Proc. zool. Soc. Lond. 122, 543-572. HORI K. (1968) Some properties and developmental changes in occurrence of the salivary amylase of the cabbage bug, Eurydema rugosum Motschulsky (Hemiptera: Pentatomidae). Appl. Ent. ZooL 3, 198-202. HORI K. (1970a) Carbohydrases in the gut and the salivary gland, and the nature of the amylase in the gut homogenate of Lygus disponsi Linnavuori (Hemiptera: Miridae). Appl. Ent. Zool. 5, 13-22. HORI K. (197013) Some properties of protease in the gut and in the salivary gland of Lygus disponsi Linnavuori (Hemiptera, Miridae). Res. Bull. Obihiro Univ. 6, 318-324. HORI K. (1970c) Some properties of amylase in the salivary gland of Lygus disponsi (Hemiptera). J. Insect Physiol. 16, 373-386. House H. L. (1965) Digestion. In The Physiology of lnsecta (Edited by ROCKSTEIN M.), pp. 815-862. Academic Press, New York. KHAN M. R. • FORD J. B. (1967) The distribution and localization of digestive enzymes in the alimentary canal and salivaryglands of the cotton stainer, Dysdercus fasciatus. J. Insect Physiol. 13, 1619-1628. KUMARR. (1970) Occurrence of proteases in the salivary glands of cacao-capsids (Heteroptera: Miridae). J. New York Entomol. 78, 198-200. LAUREMAS. & NUORTEVAP. (1961) On the occurrence of pectin polygalacturonase in the salivary glands of Heteroptera and Homoptera Auchenorrhyncha. Ann. ent.fenn. 27, 89-93.
151
MATHUR L. M. L. & T ~ A. V. (1969) Digestive physiology of tur pod bug, Clavigrella gibbosa Spin. (Coreidae: Heteroptera). IndianJ. Ent. 31, 251-257. NUOnTEVA P. (1954) Studies on the salivary enzymes of some bugs injuring wheat kernels. Ann. ent. fenn. 20, 102-104. NUOnTEVA P. & LAtmZMAS. (1961) Observations on the activity of salivary proteases and amylases in Dolycoris baccarum (L.) (Her., Pentatomidae). Ann. ent. fenn. 27, 93-97. RASTOGXS. C. (1962) On the salivary enzymes of some phytophagous and predaceous Heteroptera. Sci. Cult. 28, 478-480. R~TOGXS. C. & DATTAGUPTAA. K. (1962a) The pH and the digestive enzymes of the red pumpkin bug, Coridius janus (Fabr.) (Heteroptera: Dinidoridae). Proc. zooL Soc. 15, 57-64. P~,STOGXS. C. & DATrA GUPTAA. K. (1962b) Studies on the physiology of digestion in the alimentary canal and salivary glands of Odontopus nigricornis Stal. (Heteroptera: Pyrrhocordiae). Indian J. Ent. 23, 106-115. SAXENAK. N. (1954a) Physiology of digestion in Lepto. corisa varicornis Fabr. (Heteroptera: Coreidae). Current Sci. 23, 132. SAXENAK. N. (1954b) Physiology of the alimentary canal of Leptocorisa varicornis Fabr. (Hemiptera: Coreidae). J. zool. Soc. India 6, 111-122. SAXENA K. N. (1955) Studies on the passage of food, hydrogen-ion concentration and enzymes in the gut and salivary glands of Dysdercus koenigii Fabr. (Pyrrhocoridae: Heteroptera). J. zool. Soc. India 7, 145-154. SAXENAK. N. (1958) Digestion and absorption of carbohydrates in the alimentary canal of the red cotton bug, Dysdercus koenigii Fabr. (Heteroptera: Pyrrhocoridae). Physiol. Zorl. 31, 129-138. STRONG F. E. & KRtqTWAGEN E. C. (1968) Polygalacturonase in the salivary apparatus of Lygus hesperus (Hemiptera). J. Insect Physiol. 14, 1113-1119. TAKANONAT. & Horn K. (1974) Digestive enzymes in the salivary gland and midgut of the bug Stenotus binotatus. Comp. Biochem. Physiol. 47A, 521-528. Key Word Index--Carbohydrases; salivary gland; phytophagus insects; phleinase; pectinase.