The effects of differing dietary soybean flour treatments on response of third instar (30 – 40 mg)
1. Introduction
During the last 50 years, worldwide use of synthetic insecticides to control insect pests has led to both insecticide resistance and environmental problems (Roush and Tabashnik, 1990).
Soybean based diets are common artificial diets for
Leguminous seeds, such as those from soybean, are protected against herbivores, including insects, by anti-nutritional defence proteins- trypsin inhibitors, which are found in the seeds and raw flour made from the seeds. Levels of these plant protease inhibitors are high in legume seeds, comprising 1-5% of total protein (Macintosh et al., 1990; Sastry et al., 1987) Trypsin inhibitors of the Kunitz type (Kunnitz, 1945a; Kunnitz, 1945b) are single chain polypeptides (~ 20 kDa) that act on target serine proteases in the gut forming a 1:1 complex. Perhaps the best known of these inhibitors is Kunitz soybean trypsin inhibitor (KSTI).
Midgut serine protease activity has been found in a wide variety of Lepidopteran pests, including
KSTI and other potent trypsin inhibitors are also known to have synergistic effects with delta endotoxins of
In addition to Bt toxin, non-specific esterases in Australian
We conclude that detection of spinosad and pyrethroid (bifenthrin) resistance in
2. Materials and methods
2.1. Insects
The
2.2. Rearing methods
2.3. Diets for bioassays
For pyrethroid and spinosad bioassays, three forms of the artificial diet were prepared. The standard diet (described above) incorporating dry roasted soybean flour, a second diet incorporating raw soybean flour or a third diet in which 450 g soybean flour was first boiled for 4 min in 1500 ml of water before being incorporated with rest of diet ingredients (A, C and D) above.
2.4. Insecticides and bioassay
Insecticides used were technical grade bifenthrin (Crop Care) and spinosad (Dow), dissolved and serially diluted in acetone.
The larval bioassay procedure utilised topical application, similar to that recommended by the Entomological Society of America (Anon, 1970). Technical-grade material was dissolved in acetone and 5 - 6 serially diluted concentrations were prepared. Three replicates of 10, 30-40 mg third instar larva were treated at each dose by applying 1 μl of solution to the dorsal thorax of each with a microapplicator (Hamilton).
Following treatment, the larvae were maintained individually at 25 ± 10C in bio-assay trays (Bio-BA-1280©, C-D International, Inc., Pittman, N.J. USA, 609-5832-2392) and supplied with adequate diet. Mortality was assessed at 48 hours and 72 hours after treatment for bifenthrin and spinosad respectively. Larvae were considered dead if they were unable to move in a coordinated manner when prodded with a blunt probe. There was no control mortality. Data were analysed by Probit Analysis (Finney, 1970) and resistance factors calculated from the ratio of the resistant strain LD50 and the LD50s / susceptible strain LD50 and the LD99.9s.
3. Results
3.1. Spinosad
Log dose probit data for spinosad bioassays are shown in Table 1. The treatment of soybean flour in the three rearing diets made no significance to spinosad toxicity in the spinosad susceptible strain. On a diet made from dry roasted soybean flour, the spinosad resistant strain was found to be 62 and 378 fold resistant to spinosad at the LD50 and the LD99.9 levels respectively. The field strain was 7 and 131 fold resistant at the LD50 and the LD99.9 levels respectively. Bioassays on the raw soybean flour diet and boiled soybean flour, however, failed to detect resistance, with the LD50 and LD99.9 levels in the resistant and susceptible strains not being significantly different.
3.2. Bifenthrin
Data for bifenthrin are shown in Table 2. The treatment of soybean flour in the three rearing diets again resulted it no significant effect to bifenthrin toxicity in the pyrethroid susceptible strain. In the pyrethroid resistant strain, high levels of resistance to bifenthrin were recorded from larvae reared and bioassayed on the diet prepared with dry roasted soybean flour. Resistance factors were 284 and 320 fold at the LD50 and the LD99.9 levels respectively. Larvae from the field strain were 37 and 154 fold at the LD50 and the LD99.9 levels respectively. Low levels of bifenthrin resistance (13.3 and 10.4 fold at the LD50 and the LD99.9 levels respectively) were detected in the bifenthrin resistant strain on a raw soybean flour diet, but significant resistance was not detected in the field strain. Significant bifenthrin resistance was not detected in either the field or pyrethroid resistant strain when soyflour was boiled prior to incorporation into the
Strain | Soybean flour treatment | χ2 | Slope | LD50 (95 %fiducial limits) | Resistance factor | LD99.9 (95 %fiducial limits) | Resistance factor |
Susceptible | Dry roasted | 0.5 | 3.1 | 0.29 (0.16 – 0.34) | 1.0 | 1.6 (0.17 – 3.1) | 1.0. |
Spin-R | Dry roasted | 0.43 | 2.1 | 10.0 (13.0– 21.0) | 62 | 605 (153 – 2390) | 378 |
Breeza Field | Dry roasted | 5.8 | 1.5 | 1.9 (1.1- 3.2) | 7 | 210 (45 – 910) | 131 |
Susceptible | Raw | 0.45 | 3.0 | 0.28 (0.15 – 0.34) | 1.0 | 1.5 (0.19 – 3.2) | 1 |
Spin-R | Raw | 0.5 | 3.2 | 0.29 (0.16 – 0.33) | 1.0 | 1.7 (0.14 – 3.2 | 1.1 |
Breeza Field | Raw | 1.2 | 3.1 | 0.29 (0.06 – 0.14) | 1.0 | 1.8 (0.19 – 3.2) | 1.2 |
Susceptible | Boiled | 0.4 | 3.2 | 0.28 (0.16 -0.33) | 1.0 | 1.5 (0.2 - 3.3 | 1 |
Spin-R | Boiled | 1.1 | 3.0 | 0.29 (0.14 – 0.37) | 1.0 | 1.8 (0.25 – 4.0) | 1 |
Breeza Field | Boiled | 0.6 | 3.0 | 0.3 (0.13 – 0.39) | 1.1 | 1.7 (0.16 – 3.2) | 1.1 |
Strain | Soybean flour treatment | χ2 | Slope | LD50 (95 %fiducial limits) | Resistance factor | LD99.9 (95 %fiducial limits) | Resistance factor |
Susceptible | Dry roasted | 1.5 | 3.8 | 0.03 (0.01 – 0.05) | 1 | 0.125 (0.08 – 1.6) | 1 |
Pyr-Resistant | Dry roasted | 0.62 | 4.6 | 8.5 (6.7 – 10.1) | 284 | 40.0 (31 -66) | 320 |
Breeza Field | Dry roasted | 1.9 | 1.9 | 1.1 (0.7 – 1.9) | 37 | 19.2 (6.6 – 56) | 154 |
Susceptible | Raw | 1.5 | 3.8 | 0.03 (0.01 – 0.05) | 1 | 0.11 ( 0.07 – 1.6) | 1 |
Pyr-Resistant | Raw | 0.8 | 3.6 | 0.4 (0.1 – 0.6) | 13.3 | 1.30 (0.5 – 4.2) | 10.4 |
Breeza Field | Raw | 1.1 | 3.5 | 0.06 (0.01 – 0.07) | 2 | 0.15 (0.05 – 1.6) | 1.2 |
Susceptible | Boiled | 1.5 | 3.6 | 0.03 (0.01 – 0.05) | 1 | 0.120 (0.06 – 1.9) | 1 |
Pyr-Resistant | Boiled | 1.1 | 3.0 | 0.12 (0.08 – 0.47) | 4 | 0.5 (0.20 – 3.5) | 4.3 |
Breeza Field | Boiled | 0.7 | 3.3 | 0.04 (0.01 - 0.07) | 1 | 0.14 (0.07 – 1.9) | 1.1 |
4. Conclusion
The data show that treatment of soybean flour in the preparation of
The protease inhibitors present in soybean seeds, including the KSTI-like inhibitors, are reversibly denatured and require prolonged heating at high temperatures to bring about irreversible denaturation (Kunitz, 1948). KSTI is highly resistant to thermal and acidic denaturation (Roychaudhuri, 2003). Thus any resistance to xenobiotics that is conferred by enhanced serine hydrolases (i.e. esterases) could be compromised by the presence of KSTI-like inhibitors in the diet.
A previous study (Gunning & Moores, 2010), showed that a raw soybean flour diet prevented detection of non-specific esterase mediated resistance to
Given that KSTI is remarkably resistant to thermal denaturation and that denaturation is readily reversible at lower temperatures, it is likely that failure to detect resistance on a diet made from boiled flour is due inadequate denaturation of KSTI and/or other trypsin inhibitors. We observed that a soybean flour/ water mix boiled at 40oC, which is far below the temperature required to permanently denature KSTI (Kunitz, 1948). It is also possible that boiling the soybean flour in water solubilises the tyrpsin inhibitors.
KSTI and other trypsin inhibitors are known to have synergistic effects with delta endotoxins of
Non-specific esterases, derived from cell adhesion proteins, are serine hydrolases with an ability to sequester, pyrethroids, spinosad and Bt toxins in Australian
Thi
Acknowledgments
The authors would like to thank, Industry and Investment NSW for supporting this work. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.
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