Research on Direct Effects of PIs on Bees

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A number of studies have now been published describing the effects of PI ingestion on adult worker honeybees in terms of toxicity and changes in bee gut protease activity levels.14-17 There has also been some unpublished work carried out on the effects of PI ingestion on olfactory learning behavior, which is a significant component of foraging behavior in adult bees.18,19 However there have been no published studies of the direct effects of PIs on larvae or reproductive adult bees, or on their effects on other aspects of adult bee performance such as development or foraging activity in the field. Published studies on PI effects at the whole colony level are also lacking.

Belzunces et al14 found that the Bowman-Birk soybean trypsin inhibitor (BBI) fed to foraging (older) honeybees at dose levels of 1, 0.1, 0.01 or 0.001 mg/g of sugar syrup had no effect on bee survival over 4 days. However trypsin activity levels in foraging bees fed three different doses of BBI in syrup for 3.5 days were significantly different from those in control bees. The lowest BBI dose (0.001mg/ g) resulted in a slight but significant increase in trypsin activity, while the two other doses (0.1 and 1 mg/g) resulted in significant reductions in activity. In vitro tests in which enzyme extracts from control bee guts were incubated with BBI at a range ofconcentrations showed an 80% reduction in non-specific protease activity and a 100% reduction in trypsin activity.

In our own studies on the direct effects of PIs on bees we have used newly-emerged adult bees,15-17 because ingestion of a PI expressed in pollen is most likely to affect an adult bee via a disruption of protein digestion, and because it is only during these first few days of adulthood that honeybees consume and need to digest significant amounts of this food. We found that, when fed to these young bees, four different serine endopepti-dase inhibitors have a dose-dependent effect on bee longevity and that protease activity levels in the midgut of these bees are significantly altered by many of the PI treatments.15-17

We have found that bovine pancreatic trypsin inhibitor (BPTI) and soybean Kunitz trypsin inhibitor (SBTI, or SKTI) significantly reduce the longevity of bees fed these PIs ad lib in sugar syrup at 10, 5 or 1 mg/ml, but not at 0.1 or 0.01 mg/ml.15,16 We also determined the in vivo activity levels of three midgut endopeptidases (trypsin, chymot-rypsin and elastase) and the exopeptidase leu-cine aminopeptidase (LAP) at two time points: the 8th day after emergence and when

75% of bees had died. LAP activity levels increased significantly in bees fed with either inhibitor at all concentrations. At day 8, bees fed BPTI at all concentrations had significantly reduced levels of trypsin, chymotrypsin and elastase. At the time of 75% mortality, bees fed BPTI at each concentration had reduced trypsin levels, but only those fed the inhibitor at the highest dose had reduced chymotrypsin or elastase activity. At both time points, only bees fed SBTI at the highest concentration had lowered trypsin, chymotrypsin and elastase activities. These results suggest that the observed reductions in bee longevity at the higher PI dose levels are in fact the result of a disruption in their ability to digest protein. We may also speculate that the increased levels of LAP represent some kind of compensatory mechanism to make up for the loss of proteolytic function in the gut.

We obtained very similar results with bees fed potato proteinase inhibitor I (PI-I) and potato proteinase inhibitor II (PI-II).17 For these experiments newly-emerged bees were fed different doses of the PIs in either sugar syrup (2 or 0.1 mg/ml) administered ad lib, or in a pollen-based food (10 or 2 mg/ g) which was replaced with control food 8 days from the start of the experiment. As in our previous experiments we determined the in vivo activities of trypsin, chymotrypsin, elastase and LAP at two time points: at day 3 (Fig. 8.3) and at day 8. Enzyme activities were significantly lower at day 8 than at day 3, except for elastase which did not change. Potato PI-II significantly reduced the activity of all endopeptidases at both time points, regardless of the dose level or the medium in which the inhibitor was administered. Potato PI-I acted in a similar manner, except that 0.01% potato PI-I in syrup had no effect on bees. There was no consistent trend in changes in LAP activity. Bees fed either inhibitor at 1% in pollen or at 0.2% in syrup had significantly reduced lifespans, with the effect of the pollen treatment being greater than the syrup treatment (Fig. 8.4). Survival of bees fed potato PI-I or potato PI-II at 0.2% in pollen or 0.01% in syrup did not differ from the controls.

Girard et al21 have conducted tests of short- and long-term toxicity of BBI, oryzacystatin I (OC-I) and chicken egg white cystatin to honeybees. In the short-term test, 15-day-old worker bees were supplied with 11 ^g of PI each over a period of 24 hours, and then given control syrup. None of the treatments resulted in significant bee mortality at 24, 48 or 96 hours. In the long-term test, 2-day-old bees were given a continuous supply of syrup with 26 ^g/ml PI added and their longevity recorded. There was considerable variability in bee longevity in this test, but no significant effects could be attributed to the ingestion of these PIs at this low concentration and bees taken from the long-term test at 15-16 days had levels of midgut proteolytic activity that did not differ from the controls. Sandoz19 conducted further long-term tests with SBTI, OC-I, BBI and a mixture of OC-I and BBI fed continuously to 2-day-old bees at concentrations of 1, 0.1 or 0.01 mg/ml. Significant bee mortality occurred only for bees fed SBTI, BBI or the OC-I/BBI mixture at the highest dose level.

We conclude from these studies that different PIs have similar effects on adult honey bees and that low doses of PIs in pollen will probably not cause mortality in bees, but may cause changes in proteolytic enzyme activity in the gut. Transgenic rice plants with a potato PI-II gene directed by a wound-inducible promoter have been shown to be effective against the pink stem borer when the gene is expressed at levels equivalent to 0.5 to 2% of total soluble leaf protein.22 Furthermore, transgenic tobacco leaves expressing potato PI-II as 0.22 to 0.65% of total soluble protein significantly affected growth rates of larval green loopers.23 Levels of inhibitor expression in pollen have not yet been measured for pest-resistant transgenic PI-pla detailed studies of gene expression in transgenic pollen have involved only reporter genes such as GUS and luciferase thus far, with expression levels being recorded in terms of enzyme activity rather than as a percentage of total protein (see for instance references 24-27). Therefore, the potential exposure of bees to PIs in the field cannot yet be estimated

Fig. 8.3. Effects of two potato PIs (potato PI-I and potato PI-II), administered at different doses in either sugar syrup or pollen-food, on honeybee digestive peptidase activities after 3 days. A) Trypsin activity levels; B) chymotrypsin activity levels; C) elastase activity levels; D) leucine aminopeptidase (LAP) activity levels.

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Pot PI-I Pot Pl-ll

§^0.01% In Syrup ^0.2% in Pa lien 0,2% in Syrup m 1% in Pollen

Pot Pl-ll

§^0.01% In Syrup ^0.2% in Pa lien 0,2% in Syrup m 1% in Pollen

Pot Pl-ll accurately. However, if it is assumed that PI expression levels in pollen may be similar to leaf levels (i.e. 0.22 to 2%), and given that bees died when fed potato PI-I or potato PI-II17 at a level equivalent to 4% of total protein (the 1% pollen treatment) but survived when fed these PIs as 0.81% of their total protein (the 0.2% pollen treatment), it would appear that bees are not likely to be exposed to lethal levels of inhibitor in the pollen of pest-resistant PI-transgenic plants.

The impact of exposure to sub-lethal doses of PIs on adult honeybees is not yet known, but some studies of one component of foraging behavior, olfactory learning, have been carried out with bees that have consumed PIs. Addition of cowpea trypsin inhibitor (CpTI) at 1, 5 or 10 ^g/ml to the reward syrup offered in a conditioned proboscis extension assay significantly reduced the ability of bees to learn this response.18 In contrast, addition of BBI or cystatin at the same concentrations did not affect short- or long-term learning ability in 15-day-old bees. Furthermore, the learning performances of bees that had been fed ad lib with syrup containing 26 mg/ml of either OC-I or BBI for about 13 days prior to the proboscis extension assay were unaltered by this treatment.21 When bees were fed with SBTI, OC-I, BBI or a OC-I/BBI

Inhibitor Concentration^

Days

Fig. 8.4. Effects of two potato PIs, PI-I and PI-II, administered at different doses in either sugar syrup or pollen-food, on survival of adult honeybees. A) Survival of bees fed potato PI-I; B) survival of bees fed potato PI-II.

mixture at 1, 0.1 or 0.01 mg/ml for 15 days prior to testing, their learning ability was significantly impaired only with the 1 mg/ml BBI treatment.19

Recent work in our laboratory with bumblebees has shown that their responses to PIs may differ somewhat from those of honeybees. We fed worker adult bumblebees with BPTI, SBTI, potato PI-I and potato PI-II at 5 different dosage levels (10, 5, 1, 0.1 and 0.01 mg/g in pollen-food). As bumblebee adults consume pollen throughout their lives, they were provided with these diets ad lib. Even with this high consumption of PI, only potato PI-I had a significant, dose-dependent effect on bee longevity (Table 8.2). Bumblebees fed with potato PI-II also displayed a dosage effect, with bees receiving high doses having significantly shorter lifespans than those receiving the lower doses. However, poor survival of the control bees in the PI-II trial meant that none of the PI-treated bees had significantly shortened lifespans. BPTI and SBTI apparently had no effect on bumblebee survival.

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