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Effects of protease inhibitor concentration and combinations on the survival, growth and gut enzyme activities of the black field cricket, Teleogryllus commodus
Pergamon
0022-1910(94)00034-4
J. Insrtr Ph.wioI. Vol. 40. No. 9, pp. 803-R I I, 1994 Copyright C 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910/94 $7.00 + 0.00
Effects of Protease Inhibitor Concentration and Combinations on the Survival, Growth and Gut Enzyme Activities of the Black Field Cricket, Teleogryllus commodus E. P. J. BURGESS,* W. A. LAINGT
C. A. MAIN,*
P. S. STEVENS,*
J. T. CHRISTELLER,?
A. M. R. GATEHOUSE,f
Received 22 October 1993; revised 18 February 1994
Growth and survival responses were determined for the black field cricket Teleogryllus commodus fed 6 different protease inhibitors (SBTI, WGI-1, POT-l, POT-2, BPTI and CpTI), either singly or in combination, at a range of concentrations in diets containing 3 different levels of casein. Several inhibitors caused high levels of mortality at concentrations as low as 0.1% (w/v) on diets with low or moderate casein. In all cases the efficacy of the protease inhibitors in reducing cricket growth was strongly influenced by the level of dietary casein as well as by the concentration of the inhibitor. POT-2 and SBTI were particularly effective. Two-way combinations of protease inhibitors caused significant mortality and reduced growth as effectively as the best single inhibitors at all dietary casein levels. A synergistic effect was obtained from the combination of POT-1 (binds strongly to SAAPLpNA hydrolysing enzyme) and WGI-1 (binds strongly to trypsin). A time-series study using POT-2 showed that this inhibitor caused continual suppression in the activity of the digestive endopeptidases trypsin and SAAPLpNA hydrolysing enzyme, but no reduction in the activity of the exopeptidase leucine aminopeptidase. Increased levels of enzyme inhibition at the higher POT-2 concentration were directly reflected in the feeding trials where greater growth suppression was also observed. Protease inhibitors
Orthoptera
Teleogrylh
commodus
INTRODUCTION
Insect resistance
Digestive protease activity
inhibitors under the control of the constitutive CaMV 35s promoter at levels of approx. l-2% of the total soluble protein, have displayed significantly enhanced levels of resistance to several insect pests (Gatehouse et al., 1992). The black field cricket, Teleogryllus commodus (Walker) (Orthoptera: Gryllidae), is a pest of pasture in New Zealand and Australia (Blank, 1982; Williams et al., 1982). Procedures have been developed for genetically transforming white clover (Trifolium repens) (White and Greenwood, 1987; Voisey et al., 1993, 1994; White and Voisey, 1994), the major pasture legume in New Zealand, and we are currently evaluating protease inhibitors as possible insect resistance factors for this plant. We initially identified and characterized the midgut digestive proteases of crickets, then surveyed a range of protease inhibitors in vitro for their ability to bind with these enzymes (Christeller et al., 1990). These data allowed us to select a smaller range of inhibitors for subsequent testing in vivo. In a further study, we showed that protease inhibitors reduced cricket growth rate when ingested at 0.33% w/v of
Protease inhibitors are proteins or polypeptides which bind with proteolytic enzymes, and, in plants which produce them, are thought to provide a form of natural defense against herbivorous insects (Green and Ryan, 1972; Ryan, 1979; Broadway et al., 1986). Direct evidence for a role of protease inhibitors in providing protection in the field was first reported by Gatehouse et al. (1979) and this trait was initially exploited by conventional plant breeding (Redden et al., 1983). Protease inhibitors constitute a major class of proteins which at present are being used in the development of insect-resistant transgenic crops (Hilder et al., 1987; Johnson et al., 1989; White et al., 1990; Gatehouse et al., 1992; McManus et al., 1994). Plants expressing protease *The Horticulture and Food Research Institute of New Zealand, Private Bag 92169, Auckland, New Zealand. tThe Horticulture and Food Research Institute of New Zealand, Private Bag 11030, Palmerston North, New Zealand. SDepartment of Biological Sciences, University of Durham, DHl 3LE, England. 803
804
E. P. J. BURGESS
artificial diets containing casein as the source of nutritive protein (Burgess et al., 1991). To gain a better understanding of the mode of action of protease inhibitors on crickets, the current study determined the effects of altering the dietary concentration of a range of inhibitors on cricket growth rate, survival and digestive enzyme activity when fed in artificial diets containing 0.5, 1 and 3% casein. In addition, the progressive effects of protease inhibitor ingestion on gut protease activity were determined by analysing the digestive enzyme activities of crickets fed POT-2 in a time-series.
MATERIALS Source
AND METHODS
of crickets
Neonate crickets were obtained within 24 h of hatching from eggs in a laboratory colony which had been reared using a method adapted from that of Singh and Charles (1975) as follows: the light-dark regime used in the present colony was 16: 8 h, eggs and first to thirdinstar nymphs were stored at 30°C while later-instar nymphs and adults were kept at 25 f 1°C all nymphs and adults were fed on a diet of whole wheat grain, lucerne meal, rolled oats and dog biscuits (Pal Meaty Bites@‘,Uncle Ben’s of Australia). Experimental
conditions
Experiments were conducted in a controlled temperature room run at 25 f 1°C with a light-dark regime of 16 : 8 h. Crickets were stored individually in clear plastic tissue culture dishes, 23 mm deep and 86 mm in diameter, with vented lids. Each dish was lined with a 70 mm diameter filter paper disc which was moistened daily with water. Diet was provided ad libitum in plugs cut with a cork-borer (8 mm diameter). Random assignment of crickets to treatments, combined with the individual rearing of each cricket in a separate dish, and random positioning of each dish within the experimental room resulted in each cricket being a separate replicate. Base diets
Agar-based artificial diets were made to a recipe based on that of Malone and Wigley (1990) modified to improve cricket feeding, growth and survival as described in Burgess et al. (1991). Base diets with different casein levels (specified below, all with 3% wheatgerm w/v) were made with 80% of total water (by volume) and autoclaved. Ingredients which were not autoclaved were added once the diet had cooled to 70°C. Following this, the remaining 20% of water, containing either protease inhibitor, or additional casein (as controls to match the additions of protease inhibitor) or no additive (plain base diets, termed blank controls), was mixed with aliquots of base diet. To minimize variation between treatments, each of the base diets (at the different casein levels) was made in a single batch so that all treatments
et al.
at a given casein level were identical in all respects except for the additive. Growth rate calculation
Cricket growth during the period from neonate to 6 weeks is exponential, thus the relationship between log weight and time is linear for any given cricket during this time (Burgess et al., 1991). In the present study, relative growth rate was determined for each individual as the slope of this linear relationship, providing a measure of the rate of change of weight relative to initial weight in units of g/g/week. The average relative growth rate for each treatment was calculated as the mean of the rates for all individuals within that treatment. Determination of gut enzyme activities
On termination of each treatment, crickets were coldanaesthetized, dissected and their whole intact guts, which normally contained food, were frozen in liquid nitrogen. Guts were individually homogenized in 1 ml of 0.1 M Tris-HCl buffer, pH 8.0, centrifuged at lO,OOOg, 4°C for 5 min, and the supernatant assayed for enzyme activity as described in Christeller et al. (1990). The gut activity levels of the endopeptidases, SAAPLpNA hydrolysing enzyme and trypsin, and the exopeptidase leucine aminopeptidase (LAP) (expressed on a weight of insect basis) were determined. Efects of protease inhibitor concentration and combinations on cricket survival, growth and digestive enzyme activity Bioassay conditions and design. Protease inhibitor and control treatments were prepared on three base diets with 0.5, 1 and 3% casein w/v, respectively. Each treatment diet was fed to 24 neonate crickets, with the exception of the blank controls, which were fed to 48 crickets on both the 0.5 and 3% casein diets, and to 96 crickets on the 1% casein diet (reflecting a higher number of protease inhibitor treatments on this diet). Crickets were weighed as neonates at the start of the experiment and then at 7 day intervals to an accuracy of f 10,ug. Each treatment was terminated when most crickets in that treatment had grown sufficiently to allow dissection for gut enzyme analysis, or had all died. Protease inhibitors. The protease inhibitors tested were Kunitz soybean trypsin inhibitor (SBTI, purchased from Sigma Chemical Company), potato proteinase inhibitors 1 and 2 (POT-l, Bryant et al., 1976 and POT-2, Melville and Ryan, 1972), wheatgerm trypsin inhibitor 1 (WGI-1, Odani et al., 1986), cowpea trypsin inhibitor (CpTI, Gatehouse et al., 1980) and bovine pancreatic trypsin inhibitor (BPTI, extracted by Dr Lance Smith, personal communication). POT-l, POT-2, WGI-1 and CpTI were purified by the authors according to the published methods referred to above. Protease inhibitor concentration and dietary casein level. Cricket growth is strongly inhibited by selected
protease inhibitors in a diet with a limiting level of protein (Burgess et al., 1991) and the relationship
EFFECTS OF PROTEASE
INHIBITORS
between casein level and growth suggests that approximately half the maximum growth rate would be expected on a diet containing 1% casein. White clover leaves are known to contain 1 to 2% soluble protein (M. McManus, personal communication). For these reasons, a base diet with 1% casein was used in the present experiment to test protease inhibitors at the widest range of concentrations. Inhibitors, or further casein as a control, were added to this base diet at 0.03, 0.1, 0.3, 0.6 and 1% (w/v). On the 0.5 and 3% casein diets fewer protease inhibitors were tested, and only at 3 levels (0. 1, 0.3 and 0.6%). Due to the limited availability of BPTI, CpTI and SBTI, these protease inhibitors were offered at a reduced range of concentrations. BPTI was fed to crickets at only 0.3% in the 0.5% casein diet, and at 0.03, 0.1 and 0.3% on the 1% casein diet, but was not offered on the 3% diet. CpTI and SBTI were offered at up to only 0.3% on the 0.5 and 3% casein diets, but at the full concentration range (up to 1“A) on the 1% casein diet. Protease inhibitor combinations. Protease inhibitors which bind to both trypsin and SAAPLpNA (Nsuccinyl-r_-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide) hydrolysing enzyme extracted from cricket guts (POT-l and POT-2) reduce cricket growth more than those which bind exclusively with either trypsin (WGI-1) or SAAPLpNA hydrolysing enzyme (SBTI) (Burgess et al., 1991). In the present study, to determine the effect of combining protease inhibitors with complementary binding properties, POT-l, a powerful SAAPLpNA hydrolysing enzyme inhibitor with moderate trypsin binding, POT-2, a trypsin inhibitor with some affinity for SAAPLpNA hydrolysing enzyme, and WGI-1, a strong trypsin inhibitor, were fed to crickets in three two-way combinations. Each inhibitor was added at 0.3%, giving a total concentration of 0.6%. EfSects of POT-2
on digestive
enzyme
activity
with time
Pre-conditioning of crickets. To obtain developmentally synchronized crickets large enough to allow dissection of a sample of individuals at the start of the experiment, a cohort of crickets which hatched in the laboratory colony over a 24-h period was collected, stored en masse, and fed standard colony food until 2 weeks old. For 1 week following this, they were pre-conditioned by being fed plain base diet containing 1% casein. Bioassay conditions and design. Five experimental treatments were prepared on a 1% casein base diet as follows: POT-2 or additional casein, added at 0.1 and 0.6% (w/v). and a water only addition (plain base diet as a blank control). Three hundred and ten 3-week-old crickets which had been pre-conditioned as above were randomly selected, individually weighed, and 60 were placed on each of the 5 treatment diets in individual tissue culture dishes (as above). The remaining 10 crickets were weighed, dissected, and their guts stored for enzyme analysis. On days 1, 2, 4, 7, 14 and 21 following this, 10 crickets from
ON T. COMMODUS
805
each diet were randomly chosen, weighed, and gutted. When all crickets had been dissected, guts were individually assayed for enzyme activities. This procedure was repeated with a further replicate of 310 crickets treated identically but collected from the same colony 1 week after the first replicate.
RESULTS Effects of protease inhibitor concentration and combinations on cricket survival, growth and digestive enzyme activity Effect of single protease inhibitors on survival and growth. Over a 6-week period, SBTI, POT-l, POT-2 and BPTI caused dramatic reductions in cricket survival on the low casein (0.5%) diet [Fig. l(a)]. On the 1% casein level diet, SBTI and POT-2 killed nearly all crickets [Fig. l(b)], while on the 3% casein diet, the inhibitors tested had comparatively little effect on survival [Fig. 1(c)l. Treatments were terminated after differing lengths of time ranging from 6 to 12 weeks depending on when crickets in any given treatment had grown large enough to allow dissection. Overall, only 1.5% of crickets fed protease inhibitors in a 0.5% casein diet survived this 12-week period, compared with 39% of blank controls. This figure increased to 15% survival for protease inhibitor treatments on the 1% casein diet compared to 69% of blank controls, and 40% on the high casein diet, on which 71% of controls survived. Abbott’s Formula (Abbott, 1925) was used to calculate corrected mortalities due to protease inhibitors of 96,78 and 44% on low, medium and high casein diets, respectively. On each of the 3 base diets, relative growth rates at each inhibitor concentration were compared using analysis of variance. The standard error of the difference (SED) from each analysis was used to make individual comparisons between growth rates on inhibitor diets and growth rate on the plain base diet (blank control). Bartlett’s Test for Homogeneity of Variances (Miller, 1986) was used to check that the use of a single SED for all comparisons at a given inhibitor concentration was acceptable. On the 0.5% casein diet, in addition to causing mortality, all 5 protease inhibitors which were added at 0.1% (SBTI, WGI-1, POT-l, POT-2 and CpTI) effectively reduced cricket growth when compared to the blank control (P < 0.05) [Fig. 2(a)]. On this diet, increasing inhibitor concentration up to 0.6% (using WGI-1, POT-l and POT-2) did not alter the level of growth reduction achieved. BPTI, which was added at only 0.3%, had a similar effect to the other protease inhibitors. On the 1% casein diet none of the inhibitors significantly reduced growth at 0.03%, but all 6 were effective at 0.1% (P < 0.05) [Fig. 2(b)]. Increasing the concentration from 0.1 to 0.3% caused a slight decrease in growth for most protease inhibitors, but further
806
E. I? J. BURGESS
et al.
increases in inhibitor concentration to 0.6 and 1% (tested for all but CpTI) had no further effect. The growth rate on the 0.6% WGI-1 treatment is, however, not significantly different from that on the blank control diet, proving an exception to this trend. Despite this result, Fig. l(b) shows that all crickets consuming 0.6% WGI-1 had died within 6 weeks. On the 3% casein diet, higher concentrations of
0.6 I C’ --* - ..
0.5
POT-l POT-2
0.4 0.3
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-
- Casein
0.2 0.1. “.”
0 . 0 l
x f * 0 A 0 ..‘.
__-----
@----+ij..
0.0
SBTI &Q-J-j POT-l POT-2 BPTI CpTl Casein Pl +P2 Pl +w1 pz+w1
:
. _.
0.2
0.4
0.6
0.8
1.0
0.2
0.4
0.2
0.4
0.6
0.8
1.0
-0 . . .
rJB u 0.6
0.8
1.0
a)
>
0.0
0.0
. . .
0.0
---.. --.. -. .- -
0.2
0.4
0.6
0.8
0.0
’
0.0
1.0
0.1
I
0.0 0.0
Percent
0.8
0.6
.
t 0.4
,
*
,’
i
0.2
FIGURE 1. Effect of protease inhibitors at a range of concentrations on cricket survival after 6 weeks. (a) 0.5% dietary casein; (b) 1% dietary casein; and (c) 3% dietary casein. Proportion surviving on each of the plain base diets (blank controls) appears in a-c on the y axis, connected to the line representing the casein addition controls. In a, where several treatments resulted in complete mortality (POT-2 and BPTI at 0.3%, POT-2, Pl + P2, PI + Wl and P2 + Wl at 0.6%), symbols have been slightly offset below zero for clarity. N = 24 in all treatments except blank controls which comprised 48 in a and c, and 96 in b. In the key, PI refers to POT-I, P2 to POT-2 and Wl to WGI-1.
0.2
0.4
protease
0.6
0.8 _
inhibitor
(g/l
1.0
_
OOml)
FIGURE 2. Effect of.protease inhibitors at a range of concentrations on cricket growth rate. (a) 0.5% dietary casein; (b) 1% dietary casein; and (c) 3% dietary casein. Growth rate on each of the plain base diets (blank controls) appears in sac on the y axis, connected to the line representing the casein addition controls. Standard errors (SE) were similar for all protease inhibitors at each specified concentration, hence a single pooled estimate of SE is plotted at each level of inhibitor (offset above or below for clarity). In c, it was appropriate to pool SEs for single inhibitors and combinations of inhibitors separately. The pooled SE referring to the combinations is plotted below the SE for single inhibitors at 0.6%. N = 24 in all treatments except blank controls which comprised 48 in a and c, and 96 in b. In the key, Pl refers to POT-l. P2 to POT-2 and WI to WGI-1.
protease inhibitors were needed to reduce cricket growth, and growth reductions were not as great as those observed on diets with less protein. SBTI and WGI-1 were the only inhibitors of the 5 tested found to be effective at 0.1% (although WGI-1 did not reduce
EFFECTS
OF PROTEASE
INHIBITORS
growth at 1%) while CpTI and POT-2 became effective at 0.3%, and POT-l at 0.6% [Fig. 2(c)]. Efect of protease inhibitor combinations on survival and growth. On the 0.5% casein diet, all crickets ingesting protease inhibitor combinations died within 6 weeks [Fig. l(a)], while on the 1% casein diet there were no survivors of the POT-l + WGI-1 treatment, and only 1 and 2 survivors on the POT-l + POT-2 and POT2 + WGI-1 treatments, respectively [Fig. l(b)]. Such high levels of mortality were not observed on the 3% casein diet, where survival was lowest on the POT-2 and POT-l + WGT-I treatments [Fig. l(c)]. At all 3 casein concentrations, the 3 combinations of protease inhibitors, offered at a total concentration of 0.6%, reduced growth as effectively as POT-2, the most powerful single inhibitor, at the same concentration (Fig. 2). Cornell (198 1, p. 24) has provided a mathematical definition of synergy in response to two components of a mixture. The joint effect of the 2 components that are in the proportions x : 1 - x is additive if it can be written 8, .Y+ pz (1 - x) where 8, and /I* are the respective responses for x = 1 (all component 1) and x = 0 (all component 2). Where this relationship does not apply, Cornell defines the joint effect as synergistic. In the present study we define the joint action of inhibitors to be synergistic if the response to a combination of inhibitors is not intermediate between the responses for the two inhibitors separately, where the total inhibitor concentration is held constant. This is a more stringent requirement for synergy than that of Cornell. At all casein levels, the combination of POT-l, which has
TABLE
I. Gut enzyme
activity
Enzymr
Base diet (% casein)
Trypsin
0.5
levels for blank
1.0
controls
Additive, concentration
ON T. COMMODUS
a high binding affinity with cricket SAAPLpNA hydrolysing enzyme, and WGI-1 which strongly inhibits cricket trypsin (Christeller et al., 1990), reduced growth significantly more than either single protease inhibitor at the same total concentration. The growth rate on 0.6% POT-l was always lower than that on 0.6% WGI-1 (P CO.01 on 0.5% casein, P
(plain base diets) and treatments Enzyme activity (nmol/g/min) R (SE)
(%)
807
Blank control WGI-I (0.6)
186 (31) 33 (28)
Blank control WGI-I (0.6)
97 (16) 4.6 (3.8)
which demonstrated
a response*
level Proportion of control activity
N
P value
0.18
13 3
0.05
40 4
0.01
34 23 17 19
0.01 0.01 0.01
0.05 3.0
SAApLpNA hydrolysing enzyme
1.0
3.0
Leucine aminopeptidase
1.0 3.0
*Only treatments
in which
more than
Blank control WGI-I (0.1) WGI-I (0.3) WGI-I (0.6)
112(17) 16.6 (9.1) 28.6 (9.8) 10.5 (3.1)
Blank control POT-l (0.03) POT-I (0.6) Blank control POT-l (0.1) POT-l (0.3)
188 64 9.8 159 11.5 19.0
Blank control CpTI (0.1) Blank control POT-2 (0.1) POT-2 (0.6) Casein (0.6)
857 (123) 1746(192) 692 (88) 1341(147) 1520 (208) 308 (5 1)
2 crickets
survived
are included
(24) (16) (7.5) (20) (3.7) (8.0)
in the table.
0.15 0.26 0.09
0.34 0.05 0.07 0.12
2.0 1.9 2.2 0.45
40 13 4 34 21 19 40 7 34 19 13 16
0.05 0.01 0.01 0.01
0.05 0.05 0.05 0.05
808
E. P. J. BURGESS
treatments (plain base diets) with those on all other treatments (Table 1). Crickets from treatments on the same base diet (with the same level of casein) were compared, but only where more than 2 insects survived to be gutted. This showed that WGI-1, which binds strongly with trypsin in vitro (Christeller et al., 1990) when ingested at concentrations ranging from 0.1 to 1%, reduced gut trypsin activity on all three base diets to values ranging from 0.05 to 0.25 of the activities measured for control insects. POT- 1, which binds to the SAAPLpNA hydrolysing enzyme in vitro, when fed to crickets in medium and high casein diets at concentrations as low as 0.03% and up to l%, decreased SAAPLpNA hydrolysing activity to levels ranging from 0.05 to 0.34 of those of crickets on control diets. The response to POT-I on the low casein diet could not be measured as all crickets died on that treatment. LAP activity was doubled by CpTI at 0.1 on the medium casein diet. POT-2 at 0.1 and 0.6% on the high casein diet increased LAP activity to 1.9 and 2.2 times control levels. LAP activity was decreased by the highest level of casein offered in the experiment, when 0.6% casein was added to a base diet containing 3% casein, reducing activity to 0.45 that of controls. Efects
et al.
PO”/
ca)
of POT-2 on digestive enzyme activity with time
In all treatments, there was a notable decrease in the activity of LAP between the initial sample of crickets taken on day 0, and all following samples, including those taken 24 h later [Fig. 3(c)]. In contrast, over the first 2 days of this experiment, activity of trypsin and SAAPLpNA hydrolysing enzyme increased rapidly on the control and casein addition treatments [Fig. 3(a and b)]. Trypsin levels remained elevated throughout the experiment on the diet with the highest casein level, while on the blank control and 0.1% casein addition diets, trypsin activity decreased after an early peak but remained higher than the day 0 reading. SAAPLpNA hydrolysing activity also gradually diminished after this initial peak. There was a tendency for increased SAAPLpNA hydrolysing enzyme and trypsin activities to be associated with higher casein levels. Ingestion of POT-2 caused an immediate and ongoing depression in the activity of SAAPLpNA hydrolysing enzyme [Fig. 3(b)] and a continuing suppression of trypsin [Fig. 3(a)]. POT-2 has been shown to bind with both of these enzymes in vitro (Christeller et al., 1990). No such effect was demonstrated with LAP [Fig. 3(c)] with which POT-2 has no binding affinity. The depression caused by 0.6% POT-2 was greater than that by 0.1% POT-2 for both enzymes, and this was reflected in a greater growth suppression achieved at the higher concentration, as observed in the previous experiment [Fig. 2(b)]. DISCUSSION
Neonate crickets suffered high levels of mortality and significant growth reductions as a result of ingesting
600
0
21
7
Time
(l&s)
FIGURE 3. Progressive effect of potato protease inhibitor 2 in a 1% casein diet on protease activities within the cricket gut. (a) trypsin activity; (b) SAAPLpNA hydrolysing activity; (c) Ieucine aminopeptidase activity. Within each treatment standard errors (SE) were similar at all sampling occasions, hence a single pooled SE was calculated for each treatment. These are plotted to the right of the corresponding protease level measured on day 21, the final sampling day, with the appropriate symbol to identify which treatment is represented, and are offset for clarity. N = 20 for each data point.
protease inhibitors at low concentrations in low and medium casein level diets. Protease inhibitor toxicity has been shown to increase with diminishing protein quality (Broadway and Duffey, 1988). Cricket growth on white clover is considerably less than that on an artificial diet containing protein at equivalent levels (Burgess et al., 1990, 1991). It is thus likely that protease inhibitors expressed in transgenic white clover will be effective against crickets.
EFFECTS
OF PROTEASE
INHIBITORS
Broadway and Duffey (1986) found that ingestion of SBTI (an inhibitor of lepidopteran trypsin) and POT-2 (which inhibits lepidopteran trypsin and chymotrypsin) at 0.18% dietary wet weight (a dose thought comparable to that found in tomato, Gustafson and Ryan, 1976) caused hyperproduction of trypsin, and inhibition of growth and development in the larval noctuids Spodopteru exigua and Helicoverpa zea (as Heliothis zea). They proposed that the elevated tryptic activity resulting from protease inhibitor ingestion led to a depletion of sulphur-containing amino acids, resulting in a reduction in insect growth. Evidence for this was provided when the anti-nutritional effect of the protease inhibitors was alleviated by supplementation of the diet with methionine. Gatehouse and Boulter (1983) had previously shown that survival of the cowpea bruchid Callosobruchus maculatus was increased by addition of methionine to resistant cowpea meals containing CpTI. Broadway and Duffey (1986) also noted that SBTI and POT-2 caused different patterns of growth inhibition in S. exigua and H. zea, in diets with a range of casein levels. While S. exigua growth was inhibited over the entire range of casein levels (from 0 to 2.4%) that of H. zea was inhibited at low and high protein, but not at the mid-point. They concluded that insect growth was inhibited more at high protein levels because the additional dietary casein induced high levels of tryptic activity, resulting in further depletion of sulphur-containing amino acids. In the study reported here, cricket growth was significantly reduced by protease inhibitor levels as low as 0.1% (w/v) on diets with low and moderate levels of protein which were limiting to growth. In a high protein diet, however, higher protease inhibitor concentrations were required to reduce growth, and protease inhibitors had less effect. Although the addition of casein was shown in the time-series study to somewhat increase the gut activity of both trypsin and SAAPLpNA hydrolysing enzyme in crickets, in contrast to the results of Broadway and Duffey, this was not reflected in increased effectiveness of protease inhibitors at high protein levels. Work with other insects has shown that at higher levels of dietary protein, greater proteolytic activity is induced (Chen, 1978) requiring a higher concentration of inhibitor for deactivation. However, we only observed small changes in proteolytic activity with increased casein in the diet. We suggest that the requirement for higher protease inhibitor levels to saturate inhibition at higher casein concentrations may be due to casein out-competing the protease inhibitors for some of the proteases. Christeller et al. (1990) showed that protease inhibitors could reduce casein hydrolytic activity by a maximum of about 85% in crickets. In the case of POT-l, this reduction could reflect strong inhibition of SAAPLpNA hydrolysing enzyme, weaker inhibition of trypsin and possibly other proteases, and no inhibition of the remaining 15% of proteolytic activity. Where a protease inhibitor has a high binding affinity with a protease, it is unlikely that casein would out-compete the inhibitor
ON 7’. COMMODUS
809
for the enzyme. Casein could, however, successfully compete for binding to weakly inhibited enzymes, and under these conditions, protein digestion by the insect would be dependent on the remaining proteolytic activity of such enzymes. The reduction in effectiveness of protease inhibitors observed at higher dietary casein concentrations could thus be attributable to restoration of digestion by weakly inhibited proteases. In the present study, ingestion of protease inhibitors in some cases resulted in decreased gut activity of endopeptidases. In the first experiment described, the high levels of mortality caused by the ingestion of many of the protease inhibitors prevented determination of enzyme responses to several treatments, but we found that trypsin activity was reduced by WGI-1, and SAAPLpNA hydrolysing activity was reduced by POT1. These results reflect the high levels of in vitro inhibition of trypsin by WGI-1 and of SAAPLpNA hydrolysing enzyme by POT-l (Christeller et al., 1990). In the time-series bioassay, which was conducted over 3 weeks, POT-2 ingestion induced ongoing reductions in both SAAPLpNA hydrolysing enzyme and trypsin activity, also corresponding with in vitro binding. However, the reduced activities contrast with the results of an earlier study, in which neonate crickets ingested 0.33% (w/v) POT-2, POT-l, WGI-1 or SBTI over a 6 week period (Burgess et al., 1991). In the previous study, POT-2 caused increased gut activity levels of SAAPLpNA hydrolysing enzyme, trypsin and LAP, SBTI caused an increase in SAAPLpNA hydrolysing activity, and the other two inhibitors had no significant effect on protease activities. The observed enzyme hyperproduction suggested the operation of a complex system involving a feedback mechanism determining and adjusting enzyme levels within the gut, and was consistent with the model proposed by Broadway and Duffey (1986). The current results were obtained from crickets which were initially 3 weeks old, and were fed POT-2 at 0.1% and 0.6% (w v) for up to 3 weeks. The reduced enzyme activities suggest that the growth reduction in these crickets could have resulted from a simple mechanism whereby POT-2 bound with digestive enzymes, and thus deactivated them within the gut, diminishing the digestive capacity. The differences observed in gut protease activity levels between the earlier trial and this one could be attributed either to the difference in ages of crickets when they first consumed protease inhibitors or, in the case of POT-2, to the difference in duration of the period over which crickets were fed protease inhibitors. The potency of protease inhibitors has been shown to be age-related to some extent in rats, with young and old animals being very much more susceptible (Grant et al., 1993). It is possible that after a further 3 weeks the crickets in the present time-series experiment may have responded to prolonged POT-2 ingestion by demonstrating increased protease activity levels. Further experiments are planned to elucidate the mechanisms involved.
810
E. P. J. BURGESS
In the time-series bioassay, the dramatic decrease in LAP activity in all treatments over the initial 24 h of the experiment may have resulted from the change from mass rearing of crickets to individual rearing, which provided each cricket with greater access to food. The availability of an unlimited supply of diet could then have stimulated the increased production of the endopeptidases SAAPLpNA hydrolysing enzyme and trypsin observed on all the control treatments, and a corresponding reduction in the exopeptidase, LAP. In all experiments, growth was never reduced to below a minimum relative growth rate of about 0.05 to 0.08 g/g/week. The gut contains a mixture of proteases, some of which would not be bound by the protease inhibitor. Consequently, even when susceptible proteases were completely inactivated by the presence of an inhibitor, other proteases would be able to degrade casein to provide amino acids for growth, albeit at a much slower rate. Perhaps the protease inhibitor itself might act as a source of amino acids, being degraded by proteases to which it has no binding affinity. This is supported by the synergistic effect of the POT-l + WGI-1 combination, which exhibits a broader spectrum of inhibition than either single inhibitor. POT-2 was consistently more effective in reducing cricket growth and survival than either POT-l or WGI1. However, when the latter two were combined, they acted synergistically to become as powerful as POT-2. The synergy resulting from this combination on base diets with three different levels of casein would suggest that the transformation of plants with multiple protease inhibitor genes could afford enhanced protection against insect pests, but that a single protease inhibitor such as POT-2, which binds with both trypsin and SAAPLpNA hydrolysing enzyme, may be as potent as this particular synergistic combination with respect to crickets. Johnson et al. (1989) found the trypsin/chymotrypsin inhibitory activity of inhibitor II from both potato and tomato to be more potent in reducing the growth of Manduca sexta than inhibitor I from tomato, which was a powerful inhibitor of chymotrypsin alone. Previous plant transformations with combinations of single resistance genes has shown that transformation of tobacco with the genes for both CpTI and the pea lectin (P-Let) had an additive effect on Heliothis uirescens (Boulter et al., 1990). Transformation of tobacco with a combination of a protease inhibitor from squash seed (CMTI) and a Bacillus thuringiensis var, kurstaki protein (Btk) in the form of a CMTI-Btk HD-1 fusion protein, resulted in enhanced levels of insecticidal activity (Macintosh et al., 1990), compared to the activity of the protease inhibitor or the Bt endotoxin alone. However this result has not been reproduced by other workers using different insecticidal protease inhibitors (Tabashnik et al., 1992; A. M. R. Gatehouse, personal communication). Protease inhibitor expression levels of approx. 1% of the total soluble leaf protein have been detected in tobacco plants transformed with a Bowman-Birk type trypsin inhibitor gene from cowpea, and this signifi-
et al.
cantly reduced survival and feeding damage by the tobacco budworm (Heliothis virescens) (Hilder et al., 1987). The presence of foreign tomato or potato proteinase inhibitor II in transgenic tobacco leaves at > 100 ,ug/g of tissue severely retarded the growth of larval tobacco hornworm (Manduca sexta) which fed on the leaves (Johnson et al., 1989). The results of the present study suggest that if similar expression levels are achieved within white clover, the target plant for pest resistance, cricket growth and survival are likely to be severely affected. REFERENCES Abbott W. S. (1925) A method of computing the effectiveness of an insecticide. J. Econ. Em. 18, 265-267. Blank R. H. (1982) The pest status of Teleogryllus commodus in New Zealand. In Proceedings of the 3rd Australasia Conference on Grassland Invertebrate Ecology (Ed. Lee K. E.), pp. 3340. South Australian Government Printer, Adelaide. Boulter D., Edwards G. A. Gatehouse A. M. R., Gatehouse J. A. and Hilder V. A. (1990) Additive protective effects of different plantderived insect resistance genes in transgenic tobacco plants. Crop Prot. 9, 351-354. Broadway R. M. and Duffey S. S. (1986) Plant proteinase inhibitors: mechanism of action and effect on the growth and digestive physiology of larval Heliothis sea and Spodoptera exigua. J. Insect Physiol. 32, 827-823. Broadway R. M. and Duffey S. S. (1988) The effect of plant protein quality on insect digestive physiology and the toxicity of plant proteinase inhibitors. J. Insect Physiol. 34, 1111&l 117. Broadway R. M., Duffey S. S., Pearce G. and Ryan C. A. (1986) Plant proteinase inhibitors: A defense against herbivorous insects? Entomologia exp. appl. 41, 33338. Bryant J., Green T. R., Gurusaddaiah T. and Ryan C. D. (1976) Proteinase inhibitor II from potatoes; isolation and characterisation of its promoter components. Biochemistry 15, 3418-3424. Burgess E. P. J.. Stevens P. S., Beuning L. L., Christeller J. T. and Laing W. A. (1990) Feeding, survival and growth of the black field cricket, Teleogryllus commodus, on potential model plants for genetic transformation. Proc. 43rd NZ Weed and Pest Contr. Conf... 352-355. Burgess E. P. J., Stevens P. S., Keen G. K., Laing W. A. and Christeller J. T. (1991) Effects of protease inhibitors and dietary protein level on the black field cricket, Teleogryllus commodus. Entomologia exp. appl. 61, 123-130. Chen P. S. (1978) Protein synthesis in relation to cellular activation and deactivation. In Biochemistry of Insects (Ed. Rockstein M.), pp. 145-203. Academic Press, New York. Christeller J. T., Laing W. A., Shaw B. D. and Burgess E. P. J. (1990) Characterisation and partial purification of the digestive proteases of the black field cricket, Teleogryllus commodus (Walker): Elastase is a major component. Insect Biochem. 20, 1577164. Cornell J. A. (1981) Experiments with mixtures: designs, models, and the analysis of mixture data. In Wile,> Series in Probability and Mathematical Statistics. John Wiley & Sons, Inc., U.S.A. Dunnet C. W. (1955) A multiple comparisons procedure for comparing several treatments with a control. J. Am. Slat. Ass. 50, 1096-l 121. Gatehouse A. M. R. and Boulter D. (1983) Assessment of the antimetabolic effects of trypsin inhibitors from cowpea (I’igna ungulatu) and other legumes on development of the Bruchid beetle Callosobruchus maculatus. J. Sci. Fd Agric. 34, 345-350. Gatehouse A. M. R., Gatehouse J. A., Dobie P., Kilminster A. M. and Boulter D. (1979) Biochemical basis of insect resistance in Vigna unguiculata J. Sci. Fd Agric. 30, 9488958. Gatehouse A. M. R., Gatehouse J. A. and Boulter D. (1980) Isolation and characterisation of trypsin inhibitors from cowpea (Vigna uneuiculata). n ~~ , Phvtochemistry , i 19., 751-756.
EFFECTS
OF PROTEASE
INHIBITORS
Gatehouse A. M. R., Boulter D. and V. A. Hilder (1992) Potential of plant-derived genes in the genetic manipulation of crops for insect resistance. In Plant Genetic Manipulation for Crop Protection (Eds Gatehouse A. M. R.. Hilder V. A. and Boulter D.), pp. 155-181. Biotechnology in Agriculture No. 7. Wallingford. U.K. C.A.B. International. Grant G., Dorward P. M. and Pusztai A. (1993) Pancreatic enlargement is evident in rats fed diets containing raw soybeans (Glycine ma\-) or cowpeas (Vigna unguiculata) for 800 days but not in those fed kidneybeans (Phaseolus oulgaris) or lupinseed (Lupinus angusti.folius). J. Nun. 123, 2207-2215. Green T. R. and Ryan C. A. (1972) Wound-induced proteinase inhibitors in plant leaves: a possible defense mechanism against insects. Science 175, 776777. Gustafson G. and Ryan C. A. (1976) Specificity of protein turnover in tomato leaves. Accumulation of proteinase inhibitors, induced with the wound hormone, protease inhibitor IF. J. mol. Chem. 251, 70047010. Hilder V. A.. Gatehouse A. M. R., Sherman S. E., Barker R. F and Boulter D. (1987) A novel mechanism for insect resistance engineered into tobacco. Nature 330, 160-163. Johnson R., Narvaez J.. An G. and Ryan C. (1989) Expression of proteinase inhibitors I and II in transgenic tobacco plants: Effects on natural defense against Manduca sexta larvae. Proc. natn. Acad. SC;. U.S.A. 86, 9871-9875. Macintosh S-C.. Kishore G. M., Perlak F. J., Marrone P. G., Stone T. B.. Sims S. R. and Fuchs R. L. (1990) Potentiation of BaciNus hringiensis insecticidal activity by serine protease inhibitors. J. agric. Fd Chem. 38, 114551152. Malone L. A. and Wigley P. J. (1990) A practical method for rearing Argentine Stem Weevil Listronotus bonariensis (Coleoptera: Curculionidae) in the laboratory. N.Z. Bzt. 13, 87789. McManus M. T., White D. W. R. and McGregor P. G. (1994) Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests. Transgenic Res. 3, 50-58. Melville J. C. and Ryan C. A. (1972) Chymotryptic inhibitor I from potatoes. Large scale preparation and characterisation of its subunit components. J. hiol. Chem. 247, 3445-3453. Miller R. G. Jr. (1986) Beyond ANOVA. Basics of Applied Staristics. John Wiley & Sons, New York. Odani S.. Koido T. and Ono T. (1986) Wheatgerm trypsin inhibitors. Isolation and structural characterisation of the Bowman-Birk type. J. Biochem. 100, 975 983. Redden R. J.. Dobie P. and Gatehouse A. M. R. (1983) The inheritance of seed resistance to Callosobruchus maculatus F. in
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cowpea (Vigna unguiculata L. Walp.) I. Analysis of parental, F I, F2, F3 and backcross seed generations. Aust J. agric. Res. 34, 681695. Ryan C. A. (1979) Proteinase inhibitors. In Herbiuores: Their Interaction nith Secondary Plant Merabolites (Eds Rosenthal G. A. and Janzen D. H.), pp. 599618. Academic Press, New York. Singh P. and Charles J. G. (1975) A list of laboratory cultures and rearing methods of terrestrial arthropods in New Zealand. Bulletin 3, Entomological Society of New Zealand. Tabashnik B. E., Finson, N. and Johnson M. W. (1992) Two protease inhibitors fail to synergize Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). J. econ. Em. 85. 2082-2087. Voisey C. R., White D. W. R.. Dudas B., Appleby R. D.. Ealing P. M. and Scott A. G. (1994) Agrobacterium-mediated transformation of white clover using direct shoot organogenesis. PI. C’eII Rep. 13, 309--3 14. Voisey C. R., White D. W. R., McGregor P. G. and Barker B. L. (1993) Transformation of white clover with a gene encoding a lepidopteran-active fi -endotoxin from Bacillus thuringiensis var. berliner 1715. Proc. XVII Int. Grass/. Congr.. New Zealand and Queensland, 1993. 1175-l 176. White D. W. R. and Greenwood D. (1987) Transformation of the forage legume Tr{fo/ium repens L. using binary Agrobacterium rectors. PI. Molec. Biol. 8, 461469. White D. W. R. and Voisey C. R. (1994) Prolific direct plant regeneration from cotyledons of white clover. PI. Ceil Rep. 13, 303-30s. White D. W. R.. McManus M. T.. Ealing P. M. and McGregor P. G. (1990) Expression of modified potato proteinase inhibitor II genes in transgenic plants. J. cell. Biochem. (Suppl.) 14E, 312. Williams P.. Stahle P. P., Gagen S. J. and Murphy G. D. (1982) Strategies for control of the black field cricket, Teleogr)Nus commodus (Walker). In Proceedings of the 3rd Australasia Conference on Grassland Invertebrate Ecology (Ed. Lee K. E.). pp. 33-40. South Australian Government Printer, Adelaide.
Acknou$edgements-Tony Cooper DSIR Physical Sciences, and John Maindonald, Horticulture and Food Research Institute of New Zealand, provided statistical advice and analysis for this study. BPTI was extracted by Dr Lance Smith. Meat Research Institute of New Zealand, Box 617, Hamilton. New Zealand. Funding for this work was provided by the Foundation for Research Science and Technology contract Cl0 248.
0022-1910(94)00034-4
J. Insrtr Ph.wioI. Vol. 40. No. 9, pp. 803-R I I, 1994 Copyright C 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910/94 $7.00 + 0.00
Effects of Protease Inhibitor Concentration and Combinations on the Survival, Growth and Gut Enzyme Activities of the Black Field Cricket, Teleogryllus commodus E. P. J. BURGESS,* W. A. LAINGT
C. A. MAIN,*
P. S. STEVENS,*
J. T. CHRISTELLER,?
A. M. R. GATEHOUSE,f
Received 22 October 1993; revised 18 February 1994
Growth and survival responses were determined for the black field cricket Teleogryllus commodus fed 6 different protease inhibitors (SBTI, WGI-1, POT-l, POT-2, BPTI and CpTI), either singly or in combination, at a range of concentrations in diets containing 3 different levels of casein. Several inhibitors caused high levels of mortality at concentrations as low as 0.1% (w/v) on diets with low or moderate casein. In all cases the efficacy of the protease inhibitors in reducing cricket growth was strongly influenced by the level of dietary casein as well as by the concentration of the inhibitor. POT-2 and SBTI were particularly effective. Two-way combinations of protease inhibitors caused significant mortality and reduced growth as effectively as the best single inhibitors at all dietary casein levels. A synergistic effect was obtained from the combination of POT-1 (binds strongly to SAAPLpNA hydrolysing enzyme) and WGI-1 (binds strongly to trypsin). A time-series study using POT-2 showed that this inhibitor caused continual suppression in the activity of the digestive endopeptidases trypsin and SAAPLpNA hydrolysing enzyme, but no reduction in the activity of the exopeptidase leucine aminopeptidase. Increased levels of enzyme inhibition at the higher POT-2 concentration were directly reflected in the feeding trials where greater growth suppression was also observed. Protease inhibitors
Orthoptera
Teleogrylh
commodus
INTRODUCTION
Insect resistance
Digestive protease activity
inhibitors under the control of the constitutive CaMV 35s promoter at levels of approx. l-2% of the total soluble protein, have displayed significantly enhanced levels of resistance to several insect pests (Gatehouse et al., 1992). The black field cricket, Teleogryllus commodus (Walker) (Orthoptera: Gryllidae), is a pest of pasture in New Zealand and Australia (Blank, 1982; Williams et al., 1982). Procedures have been developed for genetically transforming white clover (Trifolium repens) (White and Greenwood, 1987; Voisey et al., 1993, 1994; White and Voisey, 1994), the major pasture legume in New Zealand, and we are currently evaluating protease inhibitors as possible insect resistance factors for this plant. We initially identified and characterized the midgut digestive proteases of crickets, then surveyed a range of protease inhibitors in vitro for their ability to bind with these enzymes (Christeller et al., 1990). These data allowed us to select a smaller range of inhibitors for subsequent testing in vivo. In a further study, we showed that protease inhibitors reduced cricket growth rate when ingested at 0.33% w/v of
Protease inhibitors are proteins or polypeptides which bind with proteolytic enzymes, and, in plants which produce them, are thought to provide a form of natural defense against herbivorous insects (Green and Ryan, 1972; Ryan, 1979; Broadway et al., 1986). Direct evidence for a role of protease inhibitors in providing protection in the field was first reported by Gatehouse et al. (1979) and this trait was initially exploited by conventional plant breeding (Redden et al., 1983). Protease inhibitors constitute a major class of proteins which at present are being used in the development of insect-resistant transgenic crops (Hilder et al., 1987; Johnson et al., 1989; White et al., 1990; Gatehouse et al., 1992; McManus et al., 1994). Plants expressing protease *The Horticulture and Food Research Institute of New Zealand, Private Bag 92169, Auckland, New Zealand. tThe Horticulture and Food Research Institute of New Zealand, Private Bag 11030, Palmerston North, New Zealand. SDepartment of Biological Sciences, University of Durham, DHl 3LE, England. 803
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E. P. J. BURGESS
artificial diets containing casein as the source of nutritive protein (Burgess et al., 1991). To gain a better understanding of the mode of action of protease inhibitors on crickets, the current study determined the effects of altering the dietary concentration of a range of inhibitors on cricket growth rate, survival and digestive enzyme activity when fed in artificial diets containing 0.5, 1 and 3% casein. In addition, the progressive effects of protease inhibitor ingestion on gut protease activity were determined by analysing the digestive enzyme activities of crickets fed POT-2 in a time-series.
MATERIALS Source
AND METHODS
of crickets
Neonate crickets were obtained within 24 h of hatching from eggs in a laboratory colony which had been reared using a method adapted from that of Singh and Charles (1975) as follows: the light-dark regime used in the present colony was 16: 8 h, eggs and first to thirdinstar nymphs were stored at 30°C while later-instar nymphs and adults were kept at 25 f 1°C all nymphs and adults were fed on a diet of whole wheat grain, lucerne meal, rolled oats and dog biscuits (Pal Meaty Bites@‘,Uncle Ben’s of Australia). Experimental
conditions
Experiments were conducted in a controlled temperature room run at 25 f 1°C with a light-dark regime of 16 : 8 h. Crickets were stored individually in clear plastic tissue culture dishes, 23 mm deep and 86 mm in diameter, with vented lids. Each dish was lined with a 70 mm diameter filter paper disc which was moistened daily with water. Diet was provided ad libitum in plugs cut with a cork-borer (8 mm diameter). Random assignment of crickets to treatments, combined with the individual rearing of each cricket in a separate dish, and random positioning of each dish within the experimental room resulted in each cricket being a separate replicate. Base diets
Agar-based artificial diets were made to a recipe based on that of Malone and Wigley (1990) modified to improve cricket feeding, growth and survival as described in Burgess et al. (1991). Base diets with different casein levels (specified below, all with 3% wheatgerm w/v) were made with 80% of total water (by volume) and autoclaved. Ingredients which were not autoclaved were added once the diet had cooled to 70°C. Following this, the remaining 20% of water, containing either protease inhibitor, or additional casein (as controls to match the additions of protease inhibitor) or no additive (plain base diets, termed blank controls), was mixed with aliquots of base diet. To minimize variation between treatments, each of the base diets (at the different casein levels) was made in a single batch so that all treatments
et al.
at a given casein level were identical in all respects except for the additive. Growth rate calculation
Cricket growth during the period from neonate to 6 weeks is exponential, thus the relationship between log weight and time is linear for any given cricket during this time (Burgess et al., 1991). In the present study, relative growth rate was determined for each individual as the slope of this linear relationship, providing a measure of the rate of change of weight relative to initial weight in units of g/g/week. The average relative growth rate for each treatment was calculated as the mean of the rates for all individuals within that treatment. Determination of gut enzyme activities
On termination of each treatment, crickets were coldanaesthetized, dissected and their whole intact guts, which normally contained food, were frozen in liquid nitrogen. Guts were individually homogenized in 1 ml of 0.1 M Tris-HCl buffer, pH 8.0, centrifuged at lO,OOOg, 4°C for 5 min, and the supernatant assayed for enzyme activity as described in Christeller et al. (1990). The gut activity levels of the endopeptidases, SAAPLpNA hydrolysing enzyme and trypsin, and the exopeptidase leucine aminopeptidase (LAP) (expressed on a weight of insect basis) were determined. Efects of protease inhibitor concentration and combinations on cricket survival, growth and digestive enzyme activity Bioassay conditions and design. Protease inhibitor and control treatments were prepared on three base diets with 0.5, 1 and 3% casein w/v, respectively. Each treatment diet was fed to 24 neonate crickets, with the exception of the blank controls, which were fed to 48 crickets on both the 0.5 and 3% casein diets, and to 96 crickets on the 1% casein diet (reflecting a higher number of protease inhibitor treatments on this diet). Crickets were weighed as neonates at the start of the experiment and then at 7 day intervals to an accuracy of f 10,ug. Each treatment was terminated when most crickets in that treatment had grown sufficiently to allow dissection for gut enzyme analysis, or had all died. Protease inhibitors. The protease inhibitors tested were Kunitz soybean trypsin inhibitor (SBTI, purchased from Sigma Chemical Company), potato proteinase inhibitors 1 and 2 (POT-l, Bryant et al., 1976 and POT-2, Melville and Ryan, 1972), wheatgerm trypsin inhibitor 1 (WGI-1, Odani et al., 1986), cowpea trypsin inhibitor (CpTI, Gatehouse et al., 1980) and bovine pancreatic trypsin inhibitor (BPTI, extracted by Dr Lance Smith, personal communication). POT-l, POT-2, WGI-1 and CpTI were purified by the authors according to the published methods referred to above. Protease inhibitor concentration and dietary casein level. Cricket growth is strongly inhibited by selected
protease inhibitors in a diet with a limiting level of protein (Burgess et al., 1991) and the relationship
EFFECTS OF PROTEASE
INHIBITORS
between casein level and growth suggests that approximately half the maximum growth rate would be expected on a diet containing 1% casein. White clover leaves are known to contain 1 to 2% soluble protein (M. McManus, personal communication). For these reasons, a base diet with 1% casein was used in the present experiment to test protease inhibitors at the widest range of concentrations. Inhibitors, or further casein as a control, were added to this base diet at 0.03, 0.1, 0.3, 0.6 and 1% (w/v). On the 0.5 and 3% casein diets fewer protease inhibitors were tested, and only at 3 levels (0. 1, 0.3 and 0.6%). Due to the limited availability of BPTI, CpTI and SBTI, these protease inhibitors were offered at a reduced range of concentrations. BPTI was fed to crickets at only 0.3% in the 0.5% casein diet, and at 0.03, 0.1 and 0.3% on the 1% casein diet, but was not offered on the 3% diet. CpTI and SBTI were offered at up to only 0.3% on the 0.5 and 3% casein diets, but at the full concentration range (up to 1“A) on the 1% casein diet. Protease inhibitor combinations. Protease inhibitors which bind to both trypsin and SAAPLpNA (Nsuccinyl-r_-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide) hydrolysing enzyme extracted from cricket guts (POT-l and POT-2) reduce cricket growth more than those which bind exclusively with either trypsin (WGI-1) or SAAPLpNA hydrolysing enzyme (SBTI) (Burgess et al., 1991). In the present study, to determine the effect of combining protease inhibitors with complementary binding properties, POT-l, a powerful SAAPLpNA hydrolysing enzyme inhibitor with moderate trypsin binding, POT-2, a trypsin inhibitor with some affinity for SAAPLpNA hydrolysing enzyme, and WGI-1, a strong trypsin inhibitor, were fed to crickets in three two-way combinations. Each inhibitor was added at 0.3%, giving a total concentration of 0.6%. EfSects of POT-2
on digestive
enzyme
activity
with time
Pre-conditioning of crickets. To obtain developmentally synchronized crickets large enough to allow dissection of a sample of individuals at the start of the experiment, a cohort of crickets which hatched in the laboratory colony over a 24-h period was collected, stored en masse, and fed standard colony food until 2 weeks old. For 1 week following this, they were pre-conditioned by being fed plain base diet containing 1% casein. Bioassay conditions and design. Five experimental treatments were prepared on a 1% casein base diet as follows: POT-2 or additional casein, added at 0.1 and 0.6% (w/v). and a water only addition (plain base diet as a blank control). Three hundred and ten 3-week-old crickets which had been pre-conditioned as above were randomly selected, individually weighed, and 60 were placed on each of the 5 treatment diets in individual tissue culture dishes (as above). The remaining 10 crickets were weighed, dissected, and their guts stored for enzyme analysis. On days 1, 2, 4, 7, 14 and 21 following this, 10 crickets from
ON T. COMMODUS
805
each diet were randomly chosen, weighed, and gutted. When all crickets had been dissected, guts were individually assayed for enzyme activities. This procedure was repeated with a further replicate of 310 crickets treated identically but collected from the same colony 1 week after the first replicate.
RESULTS Effects of protease inhibitor concentration and combinations on cricket survival, growth and digestive enzyme activity Effect of single protease inhibitors on survival and growth. Over a 6-week period, SBTI, POT-l, POT-2 and BPTI caused dramatic reductions in cricket survival on the low casein (0.5%) diet [Fig. l(a)]. On the 1% casein level diet, SBTI and POT-2 killed nearly all crickets [Fig. l(b)], while on the 3% casein diet, the inhibitors tested had comparatively little effect on survival [Fig. 1(c)l. Treatments were terminated after differing lengths of time ranging from 6 to 12 weeks depending on when crickets in any given treatment had grown large enough to allow dissection. Overall, only 1.5% of crickets fed protease inhibitors in a 0.5% casein diet survived this 12-week period, compared with 39% of blank controls. This figure increased to 15% survival for protease inhibitor treatments on the 1% casein diet compared to 69% of blank controls, and 40% on the high casein diet, on which 71% of controls survived. Abbott’s Formula (Abbott, 1925) was used to calculate corrected mortalities due to protease inhibitors of 96,78 and 44% on low, medium and high casein diets, respectively. On each of the 3 base diets, relative growth rates at each inhibitor concentration were compared using analysis of variance. The standard error of the difference (SED) from each analysis was used to make individual comparisons between growth rates on inhibitor diets and growth rate on the plain base diet (blank control). Bartlett’s Test for Homogeneity of Variances (Miller, 1986) was used to check that the use of a single SED for all comparisons at a given inhibitor concentration was acceptable. On the 0.5% casein diet, in addition to causing mortality, all 5 protease inhibitors which were added at 0.1% (SBTI, WGI-1, POT-l, POT-2 and CpTI) effectively reduced cricket growth when compared to the blank control (P < 0.05) [Fig. 2(a)]. On this diet, increasing inhibitor concentration up to 0.6% (using WGI-1, POT-l and POT-2) did not alter the level of growth reduction achieved. BPTI, which was added at only 0.3%, had a similar effect to the other protease inhibitors. On the 1% casein diet none of the inhibitors significantly reduced growth at 0.03%, but all 6 were effective at 0.1% (P < 0.05) [Fig. 2(b)]. Increasing the concentration from 0.1 to 0.3% caused a slight decrease in growth for most protease inhibitors, but further
806
E. I? J. BURGESS
et al.
increases in inhibitor concentration to 0.6 and 1% (tested for all but CpTI) had no further effect. The growth rate on the 0.6% WGI-1 treatment is, however, not significantly different from that on the blank control diet, proving an exception to this trend. Despite this result, Fig. l(b) shows that all crickets consuming 0.6% WGI-1 had died within 6 weeks. On the 3% casein diet, higher concentrations of
0.6 I C’ --* - ..
0.5
POT-l POT-2
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x f * 0 A 0 ..‘.
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SBTI &Q-J-j POT-l POT-2 BPTI CpTl Casein Pl +P2 Pl +w1 pz+w1
:
. _.
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0.4
0.6
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rJB u 0.6
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>
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. . .
0.0
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0.2
0.4
0.6
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0.0
’
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0.1
I
0.0 0.0
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0.8
0.6
.
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,’
i
0.2
FIGURE 1. Effect of protease inhibitors at a range of concentrations on cricket survival after 6 weeks. (a) 0.5% dietary casein; (b) 1% dietary casein; and (c) 3% dietary casein. Proportion surviving on each of the plain base diets (blank controls) appears in a-c on the y axis, connected to the line representing the casein addition controls. In a, where several treatments resulted in complete mortality (POT-2 and BPTI at 0.3%, POT-2, Pl + P2, PI + Wl and P2 + Wl at 0.6%), symbols have been slightly offset below zero for clarity. N = 24 in all treatments except blank controls which comprised 48 in a and c, and 96 in b. In the key, PI refers to POT-I, P2 to POT-2 and Wl to WGI-1.
0.2
0.4
protease
0.6
0.8 _
inhibitor
(g/l
1.0
_
OOml)
FIGURE 2. Effect of.protease inhibitors at a range of concentrations on cricket growth rate. (a) 0.5% dietary casein; (b) 1% dietary casein; and (c) 3% dietary casein. Growth rate on each of the plain base diets (blank controls) appears in sac on the y axis, connected to the line representing the casein addition controls. Standard errors (SE) were similar for all protease inhibitors at each specified concentration, hence a single pooled estimate of SE is plotted at each level of inhibitor (offset above or below for clarity). In c, it was appropriate to pool SEs for single inhibitors and combinations of inhibitors separately. The pooled SE referring to the combinations is plotted below the SE for single inhibitors at 0.6%. N = 24 in all treatments except blank controls which comprised 48 in a and c, and 96 in b. In the key, Pl refers to POT-l. P2 to POT-2 and WI to WGI-1.
protease inhibitors were needed to reduce cricket growth, and growth reductions were not as great as those observed on diets with less protein. SBTI and WGI-1 were the only inhibitors of the 5 tested found to be effective at 0.1% (although WGI-1 did not reduce
EFFECTS
OF PROTEASE
INHIBITORS
growth at 1%) while CpTI and POT-2 became effective at 0.3%, and POT-l at 0.6% [Fig. 2(c)]. Efect of protease inhibitor combinations on survival and growth. On the 0.5% casein diet, all crickets ingesting protease inhibitor combinations died within 6 weeks [Fig. l(a)], while on the 1% casein diet there were no survivors of the POT-l + WGI-1 treatment, and only 1 and 2 survivors on the POT-l + POT-2 and POT2 + WGI-1 treatments, respectively [Fig. l(b)]. Such high levels of mortality were not observed on the 3% casein diet, where survival was lowest on the POT-2 and POT-l + WGT-I treatments [Fig. l(c)]. At all 3 casein concentrations, the 3 combinations of protease inhibitors, offered at a total concentration of 0.6%, reduced growth as effectively as POT-2, the most powerful single inhibitor, at the same concentration (Fig. 2). Cornell (198 1, p. 24) has provided a mathematical definition of synergy in response to two components of a mixture. The joint effect of the 2 components that are in the proportions x : 1 - x is additive if it can be written 8, .Y+ pz (1 - x) where 8, and /I* are the respective responses for x = 1 (all component 1) and x = 0 (all component 2). Where this relationship does not apply, Cornell defines the joint effect as synergistic. In the present study we define the joint action of inhibitors to be synergistic if the response to a combination of inhibitors is not intermediate between the responses for the two inhibitors separately, where the total inhibitor concentration is held constant. This is a more stringent requirement for synergy than that of Cornell. At all casein levels, the combination of POT-l, which has
TABLE
I. Gut enzyme
activity
Enzymr
Base diet (% casein)
Trypsin
0.5
levels for blank
1.0
controls
Additive, concentration
ON T. COMMODUS
a high binding affinity with cricket SAAPLpNA hydrolysing enzyme, and WGI-1 which strongly inhibits cricket trypsin (Christeller et al., 1990), reduced growth significantly more than either single protease inhibitor at the same total concentration. The growth rate on 0.6% POT-l was always lower than that on 0.6% WGI-1 (P CO.01 on 0.5% casein, P
(plain base diets) and treatments Enzyme activity (nmol/g/min) R (SE)
(%)
807
Blank control WGI-I (0.6)
186 (31) 33 (28)
Blank control WGI-I (0.6)
97 (16) 4.6 (3.8)
which demonstrated
a response*
level Proportion of control activity
N
P value
0.18
13 3
0.05
40 4
0.01
34 23 17 19
0.01 0.01 0.01
0.05 3.0
SAApLpNA hydrolysing enzyme
1.0
3.0
Leucine aminopeptidase
1.0 3.0
*Only treatments
in which
more than
Blank control WGI-I (0.1) WGI-I (0.3) WGI-I (0.6)
112(17) 16.6 (9.1) 28.6 (9.8) 10.5 (3.1)
Blank control POT-l (0.03) POT-I (0.6) Blank control POT-l (0.1) POT-l (0.3)
188 64 9.8 159 11.5 19.0
Blank control CpTI (0.1) Blank control POT-2 (0.1) POT-2 (0.6) Casein (0.6)
857 (123) 1746(192) 692 (88) 1341(147) 1520 (208) 308 (5 1)
2 crickets
survived
are included
(24) (16) (7.5) (20) (3.7) (8.0)
in the table.
0.15 0.26 0.09
0.34 0.05 0.07 0.12
2.0 1.9 2.2 0.45
40 13 4 34 21 19 40 7 34 19 13 16
0.05 0.01 0.01 0.01
0.05 0.05 0.05 0.05
808
E. P. J. BURGESS
treatments (plain base diets) with those on all other treatments (Table 1). Crickets from treatments on the same base diet (with the same level of casein) were compared, but only where more than 2 insects survived to be gutted. This showed that WGI-1, which binds strongly with trypsin in vitro (Christeller et al., 1990) when ingested at concentrations ranging from 0.1 to 1%, reduced gut trypsin activity on all three base diets to values ranging from 0.05 to 0.25 of the activities measured for control insects. POT- 1, which binds to the SAAPLpNA hydrolysing enzyme in vitro, when fed to crickets in medium and high casein diets at concentrations as low as 0.03% and up to l%, decreased SAAPLpNA hydrolysing activity to levels ranging from 0.05 to 0.34 of those of crickets on control diets. The response to POT-I on the low casein diet could not be measured as all crickets died on that treatment. LAP activity was doubled by CpTI at 0.1 on the medium casein diet. POT-2 at 0.1 and 0.6% on the high casein diet increased LAP activity to 1.9 and 2.2 times control levels. LAP activity was decreased by the highest level of casein offered in the experiment, when 0.6% casein was added to a base diet containing 3% casein, reducing activity to 0.45 that of controls. Efects
et al.
PO”/
ca)
of POT-2 on digestive enzyme activity with time
In all treatments, there was a notable decrease in the activity of LAP between the initial sample of crickets taken on day 0, and all following samples, including those taken 24 h later [Fig. 3(c)]. In contrast, over the first 2 days of this experiment, activity of trypsin and SAAPLpNA hydrolysing enzyme increased rapidly on the control and casein addition treatments [Fig. 3(a and b)]. Trypsin levels remained elevated throughout the experiment on the diet with the highest casein level, while on the blank control and 0.1% casein addition diets, trypsin activity decreased after an early peak but remained higher than the day 0 reading. SAAPLpNA hydrolysing activity also gradually diminished after this initial peak. There was a tendency for increased SAAPLpNA hydrolysing enzyme and trypsin activities to be associated with higher casein levels. Ingestion of POT-2 caused an immediate and ongoing depression in the activity of SAAPLpNA hydrolysing enzyme [Fig. 3(b)] and a continuing suppression of trypsin [Fig. 3(a)]. POT-2 has been shown to bind with both of these enzymes in vitro (Christeller et al., 1990). No such effect was demonstrated with LAP [Fig. 3(c)] with which POT-2 has no binding affinity. The depression caused by 0.6% POT-2 was greater than that by 0.1% POT-2 for both enzymes, and this was reflected in a greater growth suppression achieved at the higher concentration, as observed in the previous experiment [Fig. 2(b)]. DISCUSSION
Neonate crickets suffered high levels of mortality and significant growth reductions as a result of ingesting
600
0
21
7
Time
(l&s)
FIGURE 3. Progressive effect of potato protease inhibitor 2 in a 1% casein diet on protease activities within the cricket gut. (a) trypsin activity; (b) SAAPLpNA hydrolysing activity; (c) Ieucine aminopeptidase activity. Within each treatment standard errors (SE) were similar at all sampling occasions, hence a single pooled SE was calculated for each treatment. These are plotted to the right of the corresponding protease level measured on day 21, the final sampling day, with the appropriate symbol to identify which treatment is represented, and are offset for clarity. N = 20 for each data point.
protease inhibitors at low concentrations in low and medium casein level diets. Protease inhibitor toxicity has been shown to increase with diminishing protein quality (Broadway and Duffey, 1988). Cricket growth on white clover is considerably less than that on an artificial diet containing protein at equivalent levels (Burgess et al., 1990, 1991). It is thus likely that protease inhibitors expressed in transgenic white clover will be effective against crickets.
EFFECTS
OF PROTEASE
INHIBITORS
Broadway and Duffey (1986) found that ingestion of SBTI (an inhibitor of lepidopteran trypsin) and POT-2 (which inhibits lepidopteran trypsin and chymotrypsin) at 0.18% dietary wet weight (a dose thought comparable to that found in tomato, Gustafson and Ryan, 1976) caused hyperproduction of trypsin, and inhibition of growth and development in the larval noctuids Spodopteru exigua and Helicoverpa zea (as Heliothis zea). They proposed that the elevated tryptic activity resulting from protease inhibitor ingestion led to a depletion of sulphur-containing amino acids, resulting in a reduction in insect growth. Evidence for this was provided when the anti-nutritional effect of the protease inhibitors was alleviated by supplementation of the diet with methionine. Gatehouse and Boulter (1983) had previously shown that survival of the cowpea bruchid Callosobruchus maculatus was increased by addition of methionine to resistant cowpea meals containing CpTI. Broadway and Duffey (1986) also noted that SBTI and POT-2 caused different patterns of growth inhibition in S. exigua and H. zea, in diets with a range of casein levels. While S. exigua growth was inhibited over the entire range of casein levels (from 0 to 2.4%) that of H. zea was inhibited at low and high protein, but not at the mid-point. They concluded that insect growth was inhibited more at high protein levels because the additional dietary casein induced high levels of tryptic activity, resulting in further depletion of sulphur-containing amino acids. In the study reported here, cricket growth was significantly reduced by protease inhibitor levels as low as 0.1% (w/v) on diets with low and moderate levels of protein which were limiting to growth. In a high protein diet, however, higher protease inhibitor concentrations were required to reduce growth, and protease inhibitors had less effect. Although the addition of casein was shown in the time-series study to somewhat increase the gut activity of both trypsin and SAAPLpNA hydrolysing enzyme in crickets, in contrast to the results of Broadway and Duffey, this was not reflected in increased effectiveness of protease inhibitors at high protein levels. Work with other insects has shown that at higher levels of dietary protein, greater proteolytic activity is induced (Chen, 1978) requiring a higher concentration of inhibitor for deactivation. However, we only observed small changes in proteolytic activity with increased casein in the diet. We suggest that the requirement for higher protease inhibitor levels to saturate inhibition at higher casein concentrations may be due to casein out-competing the protease inhibitors for some of the proteases. Christeller et al. (1990) showed that protease inhibitors could reduce casein hydrolytic activity by a maximum of about 85% in crickets. In the case of POT-l, this reduction could reflect strong inhibition of SAAPLpNA hydrolysing enzyme, weaker inhibition of trypsin and possibly other proteases, and no inhibition of the remaining 15% of proteolytic activity. Where a protease inhibitor has a high binding affinity with a protease, it is unlikely that casein would out-compete the inhibitor
ON 7’. COMMODUS
809
for the enzyme. Casein could, however, successfully compete for binding to weakly inhibited enzymes, and under these conditions, protein digestion by the insect would be dependent on the remaining proteolytic activity of such enzymes. The reduction in effectiveness of protease inhibitors observed at higher dietary casein concentrations could thus be attributable to restoration of digestion by weakly inhibited proteases. In the present study, ingestion of protease inhibitors in some cases resulted in decreased gut activity of endopeptidases. In the first experiment described, the high levels of mortality caused by the ingestion of many of the protease inhibitors prevented determination of enzyme responses to several treatments, but we found that trypsin activity was reduced by WGI-1, and SAAPLpNA hydrolysing activity was reduced by POT1. These results reflect the high levels of in vitro inhibition of trypsin by WGI-1 and of SAAPLpNA hydrolysing enzyme by POT-l (Christeller et al., 1990). In the time-series bioassay, which was conducted over 3 weeks, POT-2 ingestion induced ongoing reductions in both SAAPLpNA hydrolysing enzyme and trypsin activity, also corresponding with in vitro binding. However, the reduced activities contrast with the results of an earlier study, in which neonate crickets ingested 0.33% (w/v) POT-2, POT-l, WGI-1 or SBTI over a 6 week period (Burgess et al., 1991). In the previous study, POT-2 caused increased gut activity levels of SAAPLpNA hydrolysing enzyme, trypsin and LAP, SBTI caused an increase in SAAPLpNA hydrolysing activity, and the other two inhibitors had no significant effect on protease activities. The observed enzyme hyperproduction suggested the operation of a complex system involving a feedback mechanism determining and adjusting enzyme levels within the gut, and was consistent with the model proposed by Broadway and Duffey (1986). The current results were obtained from crickets which were initially 3 weeks old, and were fed POT-2 at 0.1% and 0.6% (w v) for up to 3 weeks. The reduced enzyme activities suggest that the growth reduction in these crickets could have resulted from a simple mechanism whereby POT-2 bound with digestive enzymes, and thus deactivated them within the gut, diminishing the digestive capacity. The differences observed in gut protease activity levels between the earlier trial and this one could be attributed either to the difference in ages of crickets when they first consumed protease inhibitors or, in the case of POT-2, to the difference in duration of the period over which crickets were fed protease inhibitors. The potency of protease inhibitors has been shown to be age-related to some extent in rats, with young and old animals being very much more susceptible (Grant et al., 1993). It is possible that after a further 3 weeks the crickets in the present time-series experiment may have responded to prolonged POT-2 ingestion by demonstrating increased protease activity levels. Further experiments are planned to elucidate the mechanisms involved.
810
E. P. J. BURGESS
In the time-series bioassay, the dramatic decrease in LAP activity in all treatments over the initial 24 h of the experiment may have resulted from the change from mass rearing of crickets to individual rearing, which provided each cricket with greater access to food. The availability of an unlimited supply of diet could then have stimulated the increased production of the endopeptidases SAAPLpNA hydrolysing enzyme and trypsin observed on all the control treatments, and a corresponding reduction in the exopeptidase, LAP. In all experiments, growth was never reduced to below a minimum relative growth rate of about 0.05 to 0.08 g/g/week. The gut contains a mixture of proteases, some of which would not be bound by the protease inhibitor. Consequently, even when susceptible proteases were completely inactivated by the presence of an inhibitor, other proteases would be able to degrade casein to provide amino acids for growth, albeit at a much slower rate. Perhaps the protease inhibitor itself might act as a source of amino acids, being degraded by proteases to which it has no binding affinity. This is supported by the synergistic effect of the POT-l + WGI-1 combination, which exhibits a broader spectrum of inhibition than either single inhibitor. POT-2 was consistently more effective in reducing cricket growth and survival than either POT-l or WGI1. However, when the latter two were combined, they acted synergistically to become as powerful as POT-2. The synergy resulting from this combination on base diets with three different levels of casein would suggest that the transformation of plants with multiple protease inhibitor genes could afford enhanced protection against insect pests, but that a single protease inhibitor such as POT-2, which binds with both trypsin and SAAPLpNA hydrolysing enzyme, may be as potent as this particular synergistic combination with respect to crickets. Johnson et al. (1989) found the trypsin/chymotrypsin inhibitory activity of inhibitor II from both potato and tomato to be more potent in reducing the growth of Manduca sexta than inhibitor I from tomato, which was a powerful inhibitor of chymotrypsin alone. Previous plant transformations with combinations of single resistance genes has shown that transformation of tobacco with the genes for both CpTI and the pea lectin (P-Let) had an additive effect on Heliothis uirescens (Boulter et al., 1990). Transformation of tobacco with a combination of a protease inhibitor from squash seed (CMTI) and a Bacillus thuringiensis var, kurstaki protein (Btk) in the form of a CMTI-Btk HD-1 fusion protein, resulted in enhanced levels of insecticidal activity (Macintosh et al., 1990), compared to the activity of the protease inhibitor or the Bt endotoxin alone. However this result has not been reproduced by other workers using different insecticidal protease inhibitors (Tabashnik et al., 1992; A. M. R. Gatehouse, personal communication). Protease inhibitor expression levels of approx. 1% of the total soluble leaf protein have been detected in tobacco plants transformed with a Bowman-Birk type trypsin inhibitor gene from cowpea, and this signifi-
et al.
cantly reduced survival and feeding damage by the tobacco budworm (Heliothis virescens) (Hilder et al., 1987). The presence of foreign tomato or potato proteinase inhibitor II in transgenic tobacco leaves at > 100 ,ug/g of tissue severely retarded the growth of larval tobacco hornworm (Manduca sexta) which fed on the leaves (Johnson et al., 1989). The results of the present study suggest that if similar expression levels are achieved within white clover, the target plant for pest resistance, cricket growth and survival are likely to be severely affected. REFERENCES Abbott W. S. (1925) A method of computing the effectiveness of an insecticide. J. Econ. Em. 18, 265-267. Blank R. H. (1982) The pest status of Teleogryllus commodus in New Zealand. In Proceedings of the 3rd Australasia Conference on Grassland Invertebrate Ecology (Ed. Lee K. E.), pp. 3340. South Australian Government Printer, Adelaide. Boulter D., Edwards G. A. Gatehouse A. M. R., Gatehouse J. A. and Hilder V. A. (1990) Additive protective effects of different plantderived insect resistance genes in transgenic tobacco plants. Crop Prot. 9, 351-354. Broadway R. M. and Duffey S. S. (1986) Plant proteinase inhibitors: mechanism of action and effect on the growth and digestive physiology of larval Heliothis sea and Spodoptera exigua. J. Insect Physiol. 32, 827-823. Broadway R. M. and Duffey S. S. (1988) The effect of plant protein quality on insect digestive physiology and the toxicity of plant proteinase inhibitors. J. Insect Physiol. 34, 1111&l 117. Broadway R. M., Duffey S. S., Pearce G. and Ryan C. A. (1986) Plant proteinase inhibitors: A defense against herbivorous insects? Entomologia exp. appl. 41, 33338. Bryant J., Green T. R., Gurusaddaiah T. and Ryan C. D. (1976) Proteinase inhibitor II from potatoes; isolation and characterisation of its promoter components. Biochemistry 15, 3418-3424. Burgess E. P. J.. Stevens P. S., Beuning L. L., Christeller J. T. and Laing W. A. (1990) Feeding, survival and growth of the black field cricket, Teleogryllus commodus, on potential model plants for genetic transformation. Proc. 43rd NZ Weed and Pest Contr. Conf... 352-355. Burgess E. P. J., Stevens P. S., Keen G. K., Laing W. A. and Christeller J. T. (1991) Effects of protease inhibitors and dietary protein level on the black field cricket, Teleogryllus commodus. Entomologia exp. appl. 61, 123-130. Chen P. S. (1978) Protein synthesis in relation to cellular activation and deactivation. In Biochemistry of Insects (Ed. Rockstein M.), pp. 145-203. Academic Press, New York. Christeller J. T., Laing W. A., Shaw B. D. and Burgess E. P. J. (1990) Characterisation and partial purification of the digestive proteases of the black field cricket, Teleogryllus commodus (Walker): Elastase is a major component. Insect Biochem. 20, 1577164. Cornell J. A. (1981) Experiments with mixtures: designs, models, and the analysis of mixture data. In Wile,> Series in Probability and Mathematical Statistics. John Wiley & Sons, Inc., U.S.A. Dunnet C. W. (1955) A multiple comparisons procedure for comparing several treatments with a control. J. Am. Slat. Ass. 50, 1096-l 121. Gatehouse A. M. R. and Boulter D. (1983) Assessment of the antimetabolic effects of trypsin inhibitors from cowpea (I’igna ungulatu) and other legumes on development of the Bruchid beetle Callosobruchus maculatus. J. Sci. Fd Agric. 34, 345-350. Gatehouse A. M. R., Gatehouse J. A., Dobie P., Kilminster A. M. and Boulter D. (1979) Biochemical basis of insect resistance in Vigna unguiculata J. Sci. Fd Agric. 30, 9488958. Gatehouse A. M. R., Gatehouse J. A. and Boulter D. (1980) Isolation and characterisation of trypsin inhibitors from cowpea (Vigna uneuiculata). n ~~ , Phvtochemistry , i 19., 751-756.
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cowpea (Vigna unguiculata L. Walp.) I. Analysis of parental, F I, F2, F3 and backcross seed generations. Aust J. agric. Res. 34, 681695. Ryan C. A. (1979) Proteinase inhibitors. In Herbiuores: Their Interaction nith Secondary Plant Merabolites (Eds Rosenthal G. A. and Janzen D. H.), pp. 599618. Academic Press, New York. Singh P. and Charles J. G. (1975) A list of laboratory cultures and rearing methods of terrestrial arthropods in New Zealand. Bulletin 3, Entomological Society of New Zealand. Tabashnik B. E., Finson, N. and Johnson M. W. (1992) Two protease inhibitors fail to synergize Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). J. econ. Em. 85. 2082-2087. Voisey C. R., White D. W. R.. Dudas B., Appleby R. D.. Ealing P. M. and Scott A. G. (1994) Agrobacterium-mediated transformation of white clover using direct shoot organogenesis. PI. C’eII Rep. 13, 309--3 14. Voisey C. R., White D. W. R., McGregor P. G. and Barker B. L. (1993) Transformation of white clover with a gene encoding a lepidopteran-active fi -endotoxin from Bacillus thuringiensis var. berliner 1715. Proc. XVII Int. Grass/. Congr.. New Zealand and Queensland, 1993. 1175-l 176. White D. W. R. and Greenwood D. (1987) Transformation of the forage legume Tr{fo/ium repens L. using binary Agrobacterium rectors. PI. Molec. Biol. 8, 461469. White D. W. R. and Voisey C. R. (1994) Prolific direct plant regeneration from cotyledons of white clover. PI. Ceil Rep. 13, 303-30s. White D. W. R.. McManus M. T.. Ealing P. M. and McGregor P. G. (1990) Expression of modified potato proteinase inhibitor II genes in transgenic plants. J. cell. Biochem. (Suppl.) 14E, 312. Williams P.. Stahle P. P., Gagen S. J. and Murphy G. D. (1982) Strategies for control of the black field cricket, Teleogr)Nus commodus (Walker). In Proceedings of the 3rd Australasia Conference on Grassland Invertebrate Ecology (Ed. Lee K. E.). pp. 33-40. South Australian Government Printer, Adelaide.
Acknou$edgements-Tony Cooper DSIR Physical Sciences, and John Maindonald, Horticulture and Food Research Institute of New Zealand, provided statistical advice and analysis for this study. BPTI was extracted by Dr Lance Smith. Meat Research Institute of New Zealand, Box 617, Hamilton. New Zealand. Funding for this work was provided by the Foundation for Research Science and Technology contract Cl0 248.