Insect Biochem. Vol. 19, No. I, pp. 103-108, 1989 0020-1790189 $3.00+0.00 Printed in Great Britain. All fights reserved Copyright © 1989 Pergamon Press pie

fl-GLUCOSIDASE IN FOUR PHYTOPHAGOUS LEPIDOPTERA

S. J. Yu Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, U.S.A.

(Received 1 June 1988; revised and accepted 17 October 1988)

Abstract--~-Glucosiduse in larvae of the fall armyworm, Spodopterafrugiperda (J. E. Smith), was studied using heliein as the substrate. Enzyme activity was found in various tissues, with the midgut exhibiting the highest activity. Among the midgut subeellular fractions, the soluble fraction was the most active. The apparent Km value for the enzyme was 0.63 raM. The enzyme was fairly stable for 8 weeks when stored at -10°C. Activity was mainly located in the 60*/0 fraction when the midgut soluble fraction was fractiouated with ammonium sulfate. The system also hydrolyzed a variety of glucosides including numerous toxic plant alleloehemicals. Substrate specificity of fl-glucosidase was different in the fall armyworm, velvetbean caterpillar, cabbage looper and corn earworm, suggesting a qualitative difference in the enzyme among these species. In general, v-amygdalin, helicin, D(+)-cellobiose, p-nitrophenyl fl-D-glucoside and 4-methylumbelliferyl fl-D-glucoside were the preferred substrates, whereas sinigrin, phloridzin, ,,-solanine, tomatine and linamarin were poor substrates for the enzyme in these insects. Toxicity tests reveal that, in most instances, the glucosides were less toxic than their corresponding aglycones to the fall armyworm, indicating that/~-glucosidase is an activation enzyme.

Key Word Index: fl-glucosidase, glucosides, fall armyworm, velvetbean caterpillar, cabbage looper, corn earworm.

INTRODUCTION

Glycosidases are a group of hydrolytic enzymes which catalyze the hydrolysis of glycosidic linkages in glycosides. A glycoside consists of a glycone (sugar moiety) and an aglycone (nonsugar moiety), the most common glycone being glucose in plants. Nearly all naturally occurring plant glycosides are r-linked O-glycosyl compounds and therefore fl-glycosidases are very important in the metabolism of plant gly- cosides. #-Glycosidases are commonly measured with artificial substrates such as p-nitrophenyl fl-o- glucoside and 4-methylumbelliferyl fl-D-glucoside.

fl-Glycosidases have been reported in certain spe- cies of insects including the migratory locust (Locusta migratoria), the American cockroach (Periplaneta americana), a mealworm (Tenebrio sp.), the bean aphid (Aphis fabae) (Robinson, 1956), the peachtree borer (Synanthedon exitosa) and the lesser peachtree borer (Synanthedon pictipes) (Reilly et al., 1987). The enzyme has also been purified from the proeessionary moth ( Thaumetopoea pityocampa ) (Pratviel-Sosa et aL, 1987)and a xylophagons insect, Phoracantha semipunctata (Chararas and Chipoulet, 1982).

Although insect fl-glycosidases are capable of hydrolyzing glycosides to release carbohydrates, allelochemieal aglycones are generally toxic to in- sects. Therefore, fl-glycosidases in insects may work to their disadvantages when they ingest plant gly- cosides. Very little information is currently available regarding the hydrolysis of plant glycosides by fl-glycosidases in phytophagous insects. Recently, Reilly et al. (1987) reported that fl-glycosidase plays an important role in the adaptability of peachtree borers and lesser peachtree borers to the cyanogenic glucoside-containing peach tree. Peachtree borer lar-

vae have the ability to metabolize prunasin through fl-glycosidase and fl-cyanoalanine synthase, thereby allowing them to utilize peach trees, fl-Glucosidase in this insect was also found to be inducible by the cyanogenic glucoside amygdalin.

This report concerns the initial characterization of fl-glycosidase in fall armyworm larvae. The substrate specificity toward a variety of glucosides was also examined in larvae of the velvetbean caterpillar, cabbage looper and corn earworm.

MATERIALS AND METHODS

Rearing of insects

Larvae of the fall armyworm, Spodopterafrugiperda (J. E. Smith), corn earworm, Heliothis zea (Boddie), cabbage looper, Trichoplusia ni (Hfibner) and velvetbean caterpillar, Anticarsia gemmatalis (Hiibner) were reared on an artificial diet (Burton, 1969). They were maintained in environmental chambers at 25°C with a 16:8 light:dark photoperiod as described previously (Yu, 1982).

Chemicals Helicin, tomatine, D(+)-cellobiose, phloridzin, D-

amygdalin, arbutin, Gt-solanine,p-nitrophenyl ~-D-glucoside, 4-methylumbelliferyl ~-D-glucoside, prunasin, methyl fl-D-glucoside, indoxyl p-v-glucoside, castanosphermine and D-gluconic acid lactone were purchased from the Sigma Chemical Company, St Louis, Mo., U.S.A. Salicin and sinigrin monohydrate were obtained from the Aldrich Chemical Company, Milwaukee, Wis., U.S.A. Linamarin was purchased from the Atomergic Chemical Corporation, Plainview, N.Y., U.S.A. All other chemicals were of anal- ytical quality and purchased from commet~eial suppliers.

Treatment of insects When the toxicity of allelochemicals was studied, groups

of 10 first-iustar fall armyworm larvae were individually fed

103

104 S.J. Yu

artificial diets containing the allelochemicals (prepared by direct mixing) and held in l-oz plastic cups. Larval weights were taken 12 days after feeding began. Adult emergence was also recorded at the end of the experiments.

Enzyme preparation

Groups of 25 midguts were dissected from 2-day-old final instar larvae. Their gut contents were removed to eliminate gut microbes which may contain fl-glycosidases. They were then washed in 1.15% KC1 and homogenized in 25 ml of ice-cold 0.1 M sodium phosphate buffer, pH 7.0, in a motor- driven tissue grinder for 30 s. The crude homogenate was filtered through cheesecloth and the filtered homogenate was centrifuged at 10,000g max for 15 rain in a Beckman L5-50E ultracentrifuge. The pellet which contained cell debris, nuclei and mitochondria was discarded. The super- natant was recentrifuged at 105,000g max for 1 h to obtain the soluble fraction. Unless otherwise stated, the soluble fraction was used as the enzyme source. The above pro- cedures were conducted at 0-4°C.

For study of subcellular distribution of fl-glycosidase, the crude homogenate was first centrifuged at 1000g max for 15 rain to obtain a pellet which contained nuclei and cell debris. The supematant was recentrifuged at 10,000 g max to obtain mitochondria (pellet). The resultant supernatant was again centrifuged at 105,000 g max for 1 h to obtain the microsomal pellet and the final supernatant representing the soluble fraction.

Enzyme assays Unless otherwise stated, fl-glucosidase activity was mea-

sured with helicin as a model substrate. The 5-ml incubation mixture contained the soluble fraction (equivalent to 1.5 mg protein); 0.1 M sodium phosphate, pH 7.0; and 17.5 # mol of helicin. A complete incubation mixture containing boiled tissue was used as blank. Duplicate incubations were con- ducted in a water bath with shaking at 30°C in an atmo- sphere of air for I h. The reaction was stopped by immersing the incubation tube in boiling water for 10 min. All samples were then deproteinized by adding one ml each of 0.3 N barium hydroxide and 0.3 N zinc sulfate to the incubation mixture. The mixture was then centrifuged at 10,000g max for 15 rain to obtain a clear supernatant fraction. Glucose released (upon hydrolysis of helicin) was determined from the supernatant fraction by the glucose oxidase-peroxidase reaction (Sigma Diagnostics, 1984). To this end, 0:5 ml of the supernatant was mixed with 5 ml of the combined enzyme-color reaction solution and incubated at room temperature for 30 rain. At the end of the incubation period, absorbance was measured at 450 urn. fl-Glucosidase activity was expressed as nmol glucose formed/min/mg protein.

The above-mentioned method utilizing the crude ho- mogenate as enzyme source was also used to study fl-glucosidase activity toward other glucosides. However, in the cases of arbutin and p-nitrophenyl fl-D-glucoside, the aglycones (hydroquinone and p-nitrophenol) were analyzed due to the interference of glucose determination in the incubation mixture. To this end, the incubation mixture and incubation conditions were the same as described above. After incubation, hydroquinone in the incubate was extrac- ted with 10 ml of ethyl acetate, dried over anhydrous sodium sulfate and analyzed by high pressure liquid chro- matography. Analysis was performed on a Beckman Series 340 high performance liquid chromatograph equipped with an Altex Ultrasphere-Si column (4.6 mm i.d. x 25 cm). The column was eluted with a 19:1 mixture of hexane and isopropyl alcohol at the rate of I ml/min, p-Nitrophenol was analyzed as described by Yu (1982).

RESULTS

fl-Glucosidase was characterized in the fall army- worm using helicin as a model substrate. The pH

optimum was determined with sodium phosphate buffer (0.1 M) over the pH range 5.7-8.0. Figure l(a) shows that the fl-glucosidase activity was highest at pH 6.9. Enzyme activity was linear with tissue level up to 1.6 mg protein/incubate [Fig. 1 (b)]; it increased with incubation time up to 75 rain [Fig. l(c)]. Activity was inhibited by the known fl-glucosidase inhibitors, castanosphermine (Fellows et al., 1986) and D-gluconic acid lactone (Conchie and Levy, 1957), showing I50 values of 3.08 x 10 -6 and 5.62 x 10 -4 M, respectively. Figure l(d) shows that the fl-glucosidase had the apparent K m value of 0.63 mM and Vm~x value of 17.39 nmol/min/mg protein. Data in Table 1 show that of the subcellular fractions tested, the midgut soluble fraction prepared from fall armyworm larvae contained the highest fl-glucosidase activity. From Table 2, it is seen that enzyme activity was found in various tissues and was mainly located in the midgut. No activity was found in the fat body. It was also observed that using the midgut crude homogenates as enzyme source, the fl-glucosidase activity in the fourth (3.88 nmol/min/mg protein) and fifth instars (4.59 nmol/min/mg protein) was only one-half and two-thirds that in the sixth instar, respectively, show- ing a gradual increase in enzyme activity as larvae matured.

The distribution of fl-glucosidase activity toward helicin, amygdalin and p-nitrophenyl fl-D-glucoside was examined in the protein fractions isolated by amonium sulfate fractionation of the soluble fraction. The results from Table 3 show that the distribution patterns of fl-glucosidase activities toward these sub- strates remained the same among the four fractions. In each case, approx, three-quarters of the total activity was found in the 60% fraction, with one-fifth in the 45% fraction. Both the 75 and 90% fractions contained only a slight activity. The results also show that the specific activity of fl-glucosidase toward each substrate in the four protein fractions was consistent with the distribution pattern of the enzyme activity.

The stability of helicin fl-glucosidase was in- vestigated by storing the active soluble fraction at - 10° C for periods up to 56 days. Periodic assays indicated that enzyme activity declined gradually during the first 4 weeks and then remained approxi- mately the same, showing about 80% of the initial activity by the 56th day [Fig. l(e)].

Data in Table 4 show that the fall armyworm /3-glucosidase system also hydrolyzed a variety of glucosides including toxic plant allelochemicals and artificial substrates. The activity toward these glu- cosides in this insect varied considerably ranging from 0.43 to 7.70nmol/min/mg protein. Among those glucosides studied, helicin, D-amygdalin and D(+)-cellobiose were the preferred substrates, whereas phloridzin, sinigrin and ~,-solanine were poor substrates for the enzyme. The results also show that /~-glucosidase from the velvetbean caterpillar, cabbage looper and corn earworm was active toward various glucosides. However, the total activity ap- peared to be higher in the cabbage looper and velvetbean caterpillar than in the fall armyworm and corn earworm. Moreover, the substrate specificities toward these glucosides were different among the four phytophagous species.

Table 5 summarizes the results of the bioassays of

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l /-Olucosidase in four phytophagous Lepidoptera 105

0 0 40 50 60 Days stored at -100 C

Fig. i. Effect of pH(a), enzyme level (b), incubation time (c), substrate concentration (d) and storage (e) on helicin/~-glucosidase activity in fall a rmyworm larvae. V = product formed (nmol glucose/min/mg protein); [heliein] = substrate concentration (raM). Each point represents the mean of at least 2

determinations.

various glucosides and aglycones with fall army- worm. With the exception of tomatine/tomatidine, all the glucosides were less toxic than their correspond- ing aglycones to the fall armyworm when first-instar

l a rvae were fed art if icial d ie t s c o n t a i n i n g t he c o m - p o u n d s . A m o n g t h o s e s tud ied , t he a g l y c o n e allyl i s o t h i o c y a n a t e a p p e a r e d to be m o s t tox ic to t he insects .

Table 1. Subcellular distribution of helicin ,8-glucosidase activity in the midgut of fall armyworm larvae

~-Glucosidase activityt

Table 2. Localization of helicin ~-glucosidas¢ activity in fall armyworm larvae

B -Glucosidase activityt

Tissue* nmol/min/mg protein nmol/min/tissue Subeellar fraction* nmol/min/mg protein nmol/min/midgut Foregut 9.01 + 0.14 2.57 -6 0.71

Cell debris, nuclei 7.70 -6 0.08 3.18 -6 0.04 Midgut 11.11 + 0.12 15.10 + 0.16 Mitoehondria 3.49 + 0.71 0.07 + 0.01 Hindgut 5.79 + 0.34 0.25 + 0.01 Mierosomes 1.21 + 0.04 0.10 .6 0 . 0 1 Malpighian tubules 5.89 -l- 0.59 0.16 + 0.02 Soluble fraction 13.45 + 0.20 9.61 .6 0.03 Fat body 0 0

*All fractions were prepared from midguts of 2-day-old sixth instar larvae.

tMean -6 SE of 2 experiments, each with duplicate determinations.

*All tissues prepared from 2-day-old sixth instar larvae. Crude homogcnates were used as enzyme source.

tMcan + SE of 2 experiments, each with duplicate determinations.

106 S . J . Y u

Table 3. fl-Glucosidase activity in various protein fractions prepared by ammonium sulfate fractionation of the midgut soluble fraction from fall armyworm larvae

#-Glucosidase

Specific activity* % Ammonium (nmol/min/mg protein) % of total activity

sulfate saturation Helicin Amygdalin PNPG Helicin Amygdalin PNPG

45 10.75 + 0.26 14.64 _+ 0.65 13.51 ± 0.75 20.4 27. I 20.3 60 37.30 + 0.13 32.35_+0.65 43.04_+1.16 75.1 69.2 75.1 75 2.33 ± 0.39 1.55 ± 0.01 2.56 ± 0.01 4.4 3.3 4.4 90 0.91 ±0 .06 1.04 ± 0.13 0 .52±0 .02 0.1 0.4 0.2

*Mean + SE of 2 experiments, each with duplicate determinations. PNPG, p-nitrophenyl ~-o-glucoside.

Table 4. fl-Glucosidase activities toward various glucosides in four species of Lepidoptera

fl-Glucosidase (nmol glucose/min/mg protein)*

Substrate Velvetbean Cabbage Fall Corn (3.5 mM) caterpillar looper armyworm earworm

Salicin 3.79 + 0.29 2.52 _ 0.01 1.53 + 0.05 2.68 _ 0.24 Heticin 5.09 ± 0.26 12.93 + 0.32 7.70 + 0.29 4.10 + 0.09 Phloridzin 0.41 + 0.05 1.12 + 0.15 0.43 __. 0.02 5.90 __. 0.08 Arbut int 2.59 __. 0.33 3.73 + 0.13 2.02 + 0.14 1.89 + 0.09 o-Amygdalin 9.62 + 0.12 3.03 + 0.22 5.48 __. 0.16 10.60 _ 0.58 Prunasin 4.54 _ 0.08 4.36 __. 0.07 1.48 + 0.04 2.29 + 0.02 Linamarin 0.65 _ 0.04 1.40 __. 0.03 1.89 + 0.09 1.29 _+ 0.01

-Solanine 0.49 ± 0.00 1.63 + 0.03 0.63 __. 0.04 2.63 __. 0.20 Tomatine 0.61 _ 0.09 1.99 ± 0.01 1.74 __. 0.36 2.09 + 0.50 Sin±grin 1.78 + 0.26 1.54 + 0.04 0.48 + 0.02 2.02 + 0.33 D(+)-cellobiose 6.67 ± 0.60 3.82 + 0.33 6.95 + 0.27 3.43 + 0.49 Indoxyl fl-o-glucoside 4.68 _ 0.09 5.60 _ 0.35 3.87 + 0.15 3.09 + 0.25 p-Nitrophenyl fl-o-glucosidet 11.22 ± 0.18 4.87 + 0.08 3.30 _+ 0.02 3.87 _+ 0.03 4-Methylumbelliferyl fl-o-glucoside 4.64 ± 0.08 8.27 _+ 0.39 4.34 _+ 0.44 5.20 _+ 0.39 Methyl fl-o-glucoside 1.33 _+ 0.26 2.12 + 0.27 1.35 + 0.23 2.08 _+ 0.21

*Midgut crude homogenates prepared from 2-day-old final instar larvae were used as Mean __. SE of 2 experiments, each with duplicate determinations.

tnmol aglycone/min/mg protein.

enzyme source.

Table 5. Effect of allelochemicals in larval diet on development of fall armyworm

Treatment* % of control

Concn in Larval weight Adult Glucoside Aglycone diet (%) after 12 days emergence

Arbutin 0.5 48 100 1.0 42 100

Hydroquinone 0.5 5 60 1.0 0 0

Salicin 0.5 82 100 1.0 75 100

Saligenin 0.5 70 100 1.0 33 50

Rutin 0.5 16 72 1.0 8 50

Quercetin 0.5 2 0 1.0 1 0

Esculin 0.5 85 80 1.0 79 90

Esculetin 0.5 60 100 1.0 6 10

Sin±grin 0.01 66 100 0.05 100 100

Allyl isothiocyanate 0.01 30 70 Amygdalin 0.05 0 0

0.5 90 90 1.0 93 90

Mandelonitrile 0.5 0 0 1.0 0 0

Tomatine 0.05 48 100 0.1 2 0

Tomatidine 0.05 100 100 0.1 74 100

*First-instar larvae were fed artificial diets containing the allelochemicals.

p-Glucosidase in four phytophagous Lepidoptera 107

DISCUSSION

The results of the present investigation clearly demonstrate that various phytophagous insects con- tain an active ~.glucosidase system which is capable of hydrolyzing numerous glucosides including toxic plant allelocbemicals. Among those tested, p- nitrophenyl /~-D-glucoside, 4-methylumbelliferyl ~-D-glucoside, D(+)-cel lobiose, D-amygdalin and he- licin were the preferred substrates, whereas sinigrin, phloridzin, ~,-solanine, tomatine and finamarin were poor substrates for ~-glucosidase in the velvetbean caterpillar, fall armyworm, corn earworm and cab- bage looper. There was no correlation between the degree of herbivore polyphagy and ~-glucosidase activities among these species.

It is not clear, however, whether the different /~-glucosidase activities observed in each species were due to a single enzyme with a broad substrate specificity or to multiple forms of the enzyme. In the case of fall armyworm larvae, a single enzyme is likely because the distribution patterns of ~-glucosidase activities toward helicin, amygdalin and p- nitrophenyl/~-D-glucoside were the same in the four protein fractions isolated by ammonium sulfate frac- tionation of the midgut cytosol. On the other hand, the differential substrate specificities of ~-glucosidase shown in the four lepidopterous species suggest that this enzyme is different biochemically among these species. It would be necessary to purify and charac- terize the enzyme from these insects in order to answer these questions.

The study of ~-glucosidase in phytophagous in- sects is important not only in understanding the biochemistry of the insects but also in developing new pest management strategies. Plants appear to produce a wide variety of allelochemicals as defensive weapons. These include alkaloids, cyanogenic and triterpenoid glycosides, nonprotein amino acids, phenols and flavenoids. Among these allomones, glycosides seem to play a very important role in host plant resistance to insects. For example, tomatine, an alkaloid glycosides and rutin (quercetin 3-rutinoside) have been shown to be involved in the resistance of tomato to the tomato fruitworm (Heliothis zea) by acting as feeding deterrents (Elliger et al., 1981; Sinden et al., 1978). Tomatine was also related to the resistance of tomato to the Colarado potato beetle (Leptinotarsa decemlineata) (Juvik and Stevens, 1982). Another well-known case of host plant resistance is the involvement of the glycoside in the resistance of corn to the Eurooean corn borer (Ostrinia nubilalis). It was found that DIMBOA (2,4- dihydroxy- 7- methoxy- 1,4- benzoxazin- 3 - one) glucoside was a precusor of the active resistance factor, DIMBOA, in young corn tissue (Klun et al., 1967). Maysin, a flavone glycoside isolated from corn silk, was found to possess antibiotic activity toward the corn earworm (H. zea) and has been implicated in the resistance of corn to this insect (Waiss et al., 1979). Recently, Reilly et al. (1987) showed that the cyanogenic glucoside prunasin was involved in the selection of peach tree by the peachtree borer (S. exitiosa) and the lesser peachtree borer (S. pictipes). The ability of these insects to metabolize prunasin and its toxic metabolite HCN plays a major role in the host plant selection.

In addition, numerous plant glycosides have been found to possess antifeedant activity toward phyto- phagous insects. For example, the phenolic glucoside phloridzin was found to be a deterrent to non-apple feeding aphids, Myzus persicae and Amphorophora agathomica (Montgomery and Am, 1974). The alka- loid glucosides solanine and chaconine inhibited feed- ing of Colorado potato beetle (Sturckow and Low, 1961). Sinigrin, a mustard oil glucoside (glu- cosinolate), was found to act as a chemical barrier to the black swallowtail butterfly (Papilio polysenes) by reducing larval survival and the number of fertile eggs laid by surviving adults (Erickson and Feeny, 1974).

The toxic action of these glycosides mentioned above could have been due to their corresponding aglycones liberated by the action of p-glucosidase. As shown in Table 5, with the exception of tomatine/tomatidine, all the aglycones were more toxic than their corresponding glucosides to fall armyworms. Therefore, high ~-glucosidase activity is obviously detrimental to phytophagous insects when a plant glycoside is ingested.

If we know the substrate specificity of ~- glucosidase and the consequences of the hydrolysis in a phytophagous insect, we can utilize this knowl- edge to develop plant varieties with more plant defensive systems. This could be achieved, for exam- ple, through traditional breeding programs to select for plant varieties possessing higher degrees of re- sistance factors (i.e. toxic glycosides that are not easily degraded or glycosides that are rapidly acti- vated). Alternatively, genetic engineering techniques could be used to transfer selectively the plant toxin- producing genes into a plant to make it more insect resistant. In addition, knowledge gained from the present study can be used to explain species vari- ations in response to resistant plants in phytophagous insects and hence the mechanisms of host plant resistance.

Acknowledgements--This work was supported by USDA (Competitive Research Grants Program) Grant No. 85-CRCR-l-1658. I thank Dr F. Slansky for reviewing the manuscript and Glinda Burnett for typing it. The technical assistance of Same Nguyen and Catherine Medling is also appreciated. Florida Agricultural Experiment Station Jour- nal Series No. 9029.

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