Biological activity of Dolichos biflorus L. trypsin inhibitor against lepidopteran insect pests
Amarjit K Nath*, Reena Kumari, Shilpa Sharma & Heena Sharma
Department of Biotechnology, Dr Yashwant Singh Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India
Received 03 February 2014; revised 17 July 2014
Protease inhibitors confer resistance in plants against insect pests by inhibiting larval gut proteases. Cultivars of Dolichos biflorus were screened for their inhibitory activity against midgut proteases of Pieris brassicae larvae. Seed extracts of developing and germinating seeds of HPK4 cultivar inhibited larval gut proteases of Spodoptera littoralis efficiently. Neonate larvae of P. brassicae fed on cabbage leaf discs coated with 0.025-2.50 mg protein (seed extract) resulted in 10-80% larval mortality and significantly reduced leaf area eaten and faecal matter as compared to control. The treated larvae had 40% less soluble proteins per mg faecal matter and there was similar decline in midgut proteases of treated larvae (@ 2.5 mg protein) compared to untreated ones after 5 days. The LC50 and LT50 value was calculated to be 1.05 mg/leaf disc and 4.8 days (2.5 mg protein), respectively for neonate larvae of P. brassicae. Significant reduction in egg hatching (75%) was observed in egg mass treated with 5.3 mg of crude inhibitor protein of mature seeds. This could be due to the inhibition of proteases involved in the hydrolysis of egg chorion proteins. The studies demonstrated the insecticidal activity of D. biflorus seed extracts.
Keywords: Biopesticides, Horsegram, Insect pests, Kulattha, Midgut protease, Pieris brassicae, Protease inhibitor, Spodoptera littoralis
In plants, protease inhibitors (PIs) are one of the important defense strategies to combat phytophagous insects and microorganisms. The defense capabilities of plant protease inhibitors rely on inhibition of proteases present in insect guts causing reduction in the availability of amino acids necessary for their growth and development and are highly specific for a particular class of digestive enzymes1. However, insects have shown enough flexibility to switch the proteinase composition of their guts to overcome a particular PI expressed in the transgenic plants2. Insects belonging to order Lepidoptera and Coleoptera can overexpress the existing gut proteases or induce the production of new types that are insensitive to the introduced PIs and thereby overcome the deleterious effect of PI ingestion. This might be a contributing factor to the decreased effectiveness of the PIs expressed in transgenic plants3,4. Therefore, to achieve an effective pest control strategy it is important to employ different insecticidal proteins.
In search of new molecules active against insects, several plant species, especially those from the Fabaceae family, stand out as insecticidal protein producers.
Among the effects caused by Fabaceae on insects are:
reduction in oviposition, digestive enzymes, growth interruption, malformations and increase in larval mortality5,6. A considerably high amount of PIs in seeds has been the subject of speculation as to whether these inhibitors have any role in the control of proteolysis during seed development and germination.
Keeping in view the above, we investigated the biological activity of trypsin inhibitor from developing and germinating seeds of Dolichos biflorus L. HPK4 cultivar towards lepidopteran pests.
Materials and Methods
Materials—Seeds of fifteen Dolichos biflorus cultivars (Table 1) were procured from the Department of Plant Breeding, CSKV, Palampur (H.P.), India. The larvae of Spodoptera littoralis and eggs of Pieris brassicae were collected from the university fields. The chemicals viz., bovine pancreas trypsin and BApNA (α-Benzoyl-DL-arginine-p-nitroanilide) were procured from Sigma Aldrich (USA). Other chemicals were procured from SRL Pvt. Ltd. (India) and Merck (Germany).
*Correspondence:
Phone: 91 1792 252369; Fax: 91 1792 252844 E-mail: amarjitnath@yahoo.com
Abbreviations: BApNA, α-Benzoyl-DL-arginine-p-nitroanilide; TUI, trypsin units inhibited.
Extraction of trypsin inhibitor—The collected seeds were ground to make a fine powder. The seed flour was defatted with acetone (1:10 W/V), air dried and 500 mg of defatted seed flour was extracted in 10 ml of 0.1 M sodium phosphate buffer (pH7.5). To study the inhibitor activity in developing seeds, the seeds of D. biflorus HPK4 cultivar were sown in glass house. After flowering, flowers were tagged and the pods were harvested (11:00 A.M.) at different days after flowering (3-60 DAF). The samples were stored at 70ºC till use. Pods and seed coats were removed from the developing seeds and cotyledons were crushed and defatted with acetone. In case of mature seeds, the seeds were hard and hence, were ground and flour defatted. To measure trypsin inhibitor activity in germinating seeds, seeds of HPK4 cultivar of D. biflorus (the seed extract of which showed maximum trypsin inhibitory activity) were sterilized by soaking in ethanol for 1 min and washed repeatedly with sterile distilled water. They were soaked in distilled water for 24 h at 35-37C and transferred on to thick layers of moist filter paper kept in Petri plates. Germination was carried out at room temperature and moisture was maintained by wetting filter paper with distilled water. Seed coat, radicle, plumule and cotyledons were separated at different intervals (0-10 days). Cotyledons (500 mg) were crushed, defatted with acetone, air dried and extracted in 10 ml of extraction buffer. The extracts were centrifuged at 10000 rpm for 30 min at 4ºC and the trypsin inhibitor activity was estimated in the supernatants as described by Deepika and Nath7.
Extraction and assay of midgut trypsin-like protease—Actively growing 4th instar larvae of P. brassicae and S. littoralis were fixed on wax plate with paper pins and dissected. Midguts were removed and homogenized in chilled 0.1 M phosphate buffer pH 7.6 with glass rod. The homogenate was centrifuged at 10000 rpm for 5 min at 4C and supernatant was used as source of trypsin instead of bovine trypsin. Trypsin inhibition assay was done as described by Deepika et al.8.
To measure the inhibitor activity against gut protease, optical density of the test and control were measured at 410 nm against blank using a UV/VIS spectro-photometer. The decline in optical density (OD) of 0.01 OD per minute at 410 nm as compared to control was taken as one trypsin unit inhibited (TUI). The inhibitor activity was expressed as TUI/mg protein.
Estimation of total soluble protein—Total soluble protein in seed extracts was estimated as described by Lowry et al.9.
Feeding bioassays—Cabbage leaf discs, 5 cm in diameter were coated with different concentrations (0.025, 0.05, 0.5, 1.01 and 2.50 mg) of crude inhibitor protein prepared from mature seeds (harvested at 60 DAF) of HPK4 cultivar in treatment. Neonate larvae of P. brassicae (10 in number) were fed on treated leaf discs, in control, same numbers of larvae were fed on leaf discs coated with buffer and the experiment was replicated thrice. Larvae were fed fresh leaves coated with inhibitor for 10 days, percent leaf area consumed, faecal matter and mortality was recorded after every 24 h. To calculate LC50 (lethal concentration required to kill 50 percent of test population) and LT50 (time required to kill 50% of test population) value, percent mortality in each treatment was recorded everyday and corrected by Abbott’s formula10. The data on percent mortality of larvae fed on different concentrations of crude inhibitor protein was subjected to probit analysis and LC50 value was calculated. Crude inhibitor protein (2.5 mg) extracted from mature seeds of HPK4 cultivar was coated on cabbage leaf discs and immediately hatched larvae were fed on treated leaf discs. Reduction in larval weight and protein content in faecal matter of treated larvae was observed as compared to control after 24 h. Similarly, LT50 at 2.50 mg dose of crude protein/disc was calculated. Midgut protease was extracted from 2nd instar larvae fed on leaf discs coated with 2.50 mg of crude inhibitor protein extract and trypsin activity was estimated in both treatment and control (fed without inhibitor).
Effect of crude inhibitor on egg hatching of P. brassicae—Freshly laid eggs of P. brassicae along with leaves were collected from the university fields.
Leaves with eggs on their surface were placed in Petri plates covered with moist filter paper. Eggs were coated with 5.3 mg of crude inhibitor protein, in control they were coated with 0.1 M phosphate buffer.
After 20 min, excess of inhibitor protein was drained.
The eggs were allowed to hatch at room temperature and observed for emergence of larvae. The experiment was done in triplicate.
Statistical analysis—All the laboratory experiments were carried out thrice with duplicates for each replication. Complete Randomized Design (CRD) was applied to the data obtained in experiments involving screening of seed extracts of
D. biflorus cultivars for inhibitory activity of on midgut protease of P. brassicae, inhibitory activity of crude protein extracts of HPK4 cultivars during seed development and germination on midgut protease of S. littoralis. The feeding bioassays experiments were also laid out in a CRD factorial11. The experiments were conducted in triplicate and LC50 and LT50 values were calculated12. Student’s t-test was applied to the data pertaining to the effect of crude inhibitor extract of HPK4 cultivar on egg hatching of P. brassicae, as the student’s t-distribution is applicable for smaller sample size (<30). In the above mentioned experiment a paired t-test was used for testing the mean difference of the samples13.
Results and Discussion
Crude trypsin inhibitor from seed extracts of all the cultivars under study showed inhibition of gut protease of P. brassicae indicating its insecticidal potential. The HPK4 cultivar showed maximum inhibition (2980.0 ± 0.06 TUI/per g seed flour) and HPKC-131 showed minimum inhibition (740.0 ± 1.01 TUI/per g seed flour) of midgut protease (Table 1). Variations for midgut protease inhibition were reported among cultivars of cow pea, highest inhibition of 66% against midgut protease of Helicoverpa larvae was in P-256, and lowest 22% in Pusa Pragati variety14. Further experiments were carried out using HPK4 cultivar as it exhibited the highest inhibitory activity. Crude
inhibitor protein prepared from developing seeds harvested at (21, 27, 33, 39, 45, 51 and 60 DAF) showed inhibitory activity against gut proteases of S. littoralis thereby indicating its insecticidal potential (Table 2). Maximum inhibition of gut proteases of S. littoralis was observed in seeds harvested at 60 DAF (3021.00 TUI/ g fresh seed weight). In germination experiment, maximum inhibitory activity against gut proteases was observed in seed extract prepared from dry seeds (2956.00± 0.06 TUI/g seed wt.) followed by 24 h soaked seeds (2970.00±0.04 TUI/g cotyledon wt.).
Minimum inhibitor activity was observed in cotyledons of 5 days old germinating seeds (1590.00±0.48 TUI/g fresh weight) and the results were significant at 5%
level of significance (Table 3). The decline in inhibitory activity coincided with decline in total soluble protein content during seed germination. It decreased from 105 mg/g dry seed wt. on soaking seeds for 24 h to 101 mg/g cotyledon wt. During seed germination it further decreased, and on 10th day it reduced to 73.9 mg/g cotyledon wt. This decline could be due to mobilization and enzymatic degradation of
Table 1—Inhibitory activity of seed extracts of Dolichos biflorus cultivars on midgut protease of Pieris brassicae. Cultivars Trypsin Inhibitor activity
(TUI/g seed flour weight) Specific activity (TUI/mg protein) HPKC-130 1938.5± 0.14e 18.28 ± 0.02cd Himganga 2048.5± 1.75c 19.32 ± 0.05c DHG-3 1762.5 ± 0.49g 17.62 ± 0.01cd HPKC-129 1954.5 ± 1.45d 18.26 ± 0.02cd HPKC-107 1529 ± 1.73j 15.76 ± 0.04de HPKC-112 1190.5 ± 2.03k 12.02 ± 0.09f HPKC-137 805.0 ± 0.69n 8.05 ± 0.06h HPKC-115 1702 ± 1.16h 16.68 ± 0.06d
HPKC-2 1072 ± 0.74l 10.61 ± 0.20g
HPKC-111 1926.5 ± 1.76f 18.52 ± 0.09c HPKC-114 1633.5 ± 1.15i 16.66 ± 0.07d
VLG-1 913.0 ± 1.17m 8.86 ± 0.06h
HPK4 2980 ± 0.06a 27.59 ± 0.02a
DHG-2 2734 ± 0.88b 25.31 ± 0.04b
HPKC-131 740.0 ± 1.01o 7.55 ± 0.12
Data represents mean values ± standard error of six values. Values in the same column followed by a similar superscript letters are not significantly different at P ≤0.05.
Table 2—Inhibitory activity of seed extracts of developing seeds of Dolichosbiflorus HPK4 cultivar on midgut protease of
Spodoptera littoralis Day after
flowering Trypsin inhibitor activity
(TUI/g seed weight) Specific activity (TUI/mg protein)
21 1556 ± 1.76e 19.24 ± 0 .09e
27 1976 ± 2.40d 21.95 ± 0 .07d
36 2508 ± 5.13c 25.3 ± 0.02c
42 2728 ± 3.75b 26.48 ± 0.05b
60 3021 ± 1.52a 27.92 ± 0.09a
Data represents mean values ± standard error of six values. Values in the same column followed by a similar superscript letters are not significantly different at P ≤0.05.
Table 3—Inhibitory activity of germinating seed extracts of Dolichos biflorus HPK4 cultivar on midgut protease of
Spodoptera littoralis. Day after
germination Trypsin inhibitor activity
(TUI/g fresh seed weight) Specific activity (TUI/mg protein) Dry seed 2970 ± 0.04a 28.69 ± 0.71a
0* 2956 ± 0.06b 28.42 ± 0.42a
1 2736 ± 0.09c 27.69 ± 0.89a
2 2390 ± 0.12d 24.74 ± 1.24b
3 1938 ± 0.05e 20.4 ± 0.79c
4 1610 ± 0.23f 17.4 ± 1.67d
5 1590 ± 0.48g 17.5 ± 1.37d
*(24 h after soaking)
Data represents mean values ± standard error of six values. Values in the same column followed by a similar superscript letters are not significantly different at P ≤0.05.
proteins including protease inhibitors in seeds during germination15. Several protease inhibitors have been reported to exhibit inhibitory activity against insect proteases16. Inhibition of gut proteases of S. frugiperda larvae by purified trypsin inhibitor from Poecilanthe parviflora and Crotalaria pallida has been reported17,18. Trypsin inhibitor purified from the seeds of Albizzia kalkora and Cassia obtusifolia showed inhibitory activity against larval midgut proteases of P. rapae19,20. In the present study, feeding bioassays indicated first instar larvae of P. brassicae to be sensitive to trypsin inhibitor present in crude seed extract. Larvae fed on leaf discs coated with different concentrations (0.025, 0.05, 0.5, 1.01 mg and 2.50 mg) of crude inhibitor protein extracted from
the mature seeds (60 DAF) of HPK4 cultivar showed 10, 20, 30, 40 and 80 percent mortality, respectively after 5 days of feeding (Fig. 1). The calculated LC50
for crude inhibitor protein of HPK4 cultivar was 1.05 mg with 95% confidence limit. The LT50 value with 2.5 mg/disc crude protein extract was 4.8 days. The percent leaf area eaten and faecal matter in all treatments was significantly less as compared to the control (Table 4 & 5). This may be due to protein starvation of the larvae from essential amino acids.
Reduction in larval weight, 38.80 percent decline in gut trypsin and total soluble protein in faecal matter was observed in P. brassicae larvae fed on leaf disc coated with 2.5 mg of inhibitor protein as compared to control (Table 3 & 4). Reduction in soluble protein
Fig. 1—Pieris brassicae larvae fed on cabbage leaf disc. (a) Control after five days of feeding; (b) Treatment, fed on 1.01 mg inhibitor protein after 5 days; (c) Control after 5 days of feeding; and (d) Treatment, fed on 2.50 mg inhibitor protein after 5 days.
Table 4—Effect of different concentrations of Dolichos biflorus HPK4 cultivar trypsin inhibitor on percent leaf area eaten and faecal matter of Pieris brassicae larvae
Concentration of
trypsin inhibitor Per cent leaf area eaten Faecal matter (mg)
1st day 2nd day 3rd day 4th day 5th day 1st day 2nd day 3rd day 4th day 5th day Control 11.83
±1.56a 8.63
±0.02a 16.43
±1.04a 27.10
±0.15a 30.10
±0.20a 4.66
±0.03a 9.33
±0.17a 15.30
±0.21a 20.0
±0.21a 27.0
±0.1a 0.025 mg 10.70
±0.64b 6.03
±1.36b 15.80
±1.18b 21.90
±0.12b 23.45
±0.14b 4.33
±0.42b 8.00
±0.5b 13.33
±0.01b 15.66
±0.11b 20.66
±0.2b 0.05 mg 10.60
±0.10b 4.66
±0.22c 12.03
±0.28d 20.86
±0.39c 22.50
±0.06c 4.33
±0.24b 7.66
±0.0bc 12.66
±0.03c 14.33
±0.33c 19.00
±0.4c
0.50 mg 9.29
±10.08c 8.24
±0.00a 5.60
±0.12c 5.16
±0.15d 4.26
±0.06d 4.00
±0.58b 7.00
±0.0b 8.66
±0.12d 10.33
±0.23d 12.33
±1.2d
1.01 mg 7.79
±0.12d 3.30
±0.39d 3.43
±0.11e 4.89
±0.02e 3.43
±0.10e 1.66
±0.00c 3.33
±0.0d 6.33
±0.31e 9.00
±0.21e 9.0
±0.3e
2.50 mg 3.80
±0.66e 1.21
±0.60e 1.61
±0.25f 3.31
±0.14f 0.97
±0.25f 1.33
±0.17c 1.33
±0.2e 2.0
±0.21f 3.33
±0.00f 1.66
±0.14f
Mean 9.00 5.34 9.15 15.87 14.19 3.38 6.11 9.72 11.94 14.86
Effect SE CD (0.05) SE CD (0.05)
C 0.47 0.950 0.34 0.68
D 0.43 0.867 0.31 0.617
C×D 1.062 2.125 0.76 1.513
Data represents mean values ± standard error of six values. Values in the same column followed by a similar superscript letters are not significantly different at P ≤0.05.
content in faecal matter could be due to less intestinal aberration and per cent leaf area eaten in treatment as compared to control. Dorrah21 reported effect of different concentrations (0.1, 0.2, and 0.5%) of soybean trypsin inhibitor on the survival rate of S. littoralis larvae to be 4, 3 and 12% reduction, respectively in larval survival. The calculated LC50 for dietary soybean isolate (SBI) was reported to be 0.58 mM at 95%
confidence limits. Significant reduction in body wt. as compared to control was also observed in Ostrinia nubilalis larvae fed on diets containing 2% SBI22. Similar reduction was observed in S. litura fed on diet supplemented with 0.2 and 0.5% of SBI23. Insect bioassay of purified Bowman-Birk protease inhibitor from D. biflorus against Helicoverpa armigera resulted in 68% decline in larval weight after 7 days of feeding on artificial diet containing inhibitor24. P. brassicae larvae fed on leaf discs coated with trypsin inhibitor purified from local bean cultivar (150 µg per leaf disc) showed inhibition of growth25; and 100 % mortality when fed on leaf discs coated with 134 µg of purified trypsin inhibitor from Albizzia lebbeck26. Hilder et al.27 reported 50 % reduction in the biomass of Spodoptera litura larvae fed on transgenic leaves expressing 3-5 µg CpTi per g leaf weight. Transgenic tobacco plants expressing trypsin inhibitor gene resulted in increased mortality, reduced insect growth, and reduced plant damage in Heliothis virescens, Helicoverpa zea, S. littoralis and Manduca sexta28-30. However, Nandi et al.31 reported normal growth and development of H.
armigera larvae fed on transgenic tobacco expressing soybean trypsin inhibitor gene. In the above study, 5.3 mg crude trypsin inhibitor prepared from mature seed extract resulted in 75 % reduction of hatching of freshly laid egg in P. brassicae. This could be due to
the binding of crude protein inhibitor to the choroin of the eggs during embryonic period (48-72 h) and inhibition of proteases involved in the hydrolysis of egg choroin protein. Of several proteases tested for enhancing the rate of egg hatching, the egg shell associated proteases have been reported to significantly (P ≤0.001) enhance Luclia cuprina egg hatchiing up to 70%. The commercial proteases chymotrypsin and trypsin have also significantly enhanced (P ≤0.01) egg hatching by 36 and 44%, respectively, compared to untreated control. Further, addition of phenyl methyl sulphonyl fluoride, an inhibitor of serine proteases significantly reversed (P ≤0.001) this egg hatching enhancement32. The SDS-PAGE electrophoresis of purified crystalline chorionic layer of Leptinotarsa decemlineata showed it to be proteinaceous in nature, composed of 3-5 polypeptides with molecular weight ranging 28-60 KDa33.
Conclusion
The trypsin inhibitor present in crude extracts of developing and germinating seeds was found to inhibit gut proteases of lepidopteran larvae. The protease inhibitors expressed and produced in higher amounts in the agronomically important crops may lead to development of resistance against a variety of polyphagous insects. This eco-friendly strategy may help in effectively reducing the rampant use of toxic pesticides without compromising on controlling the pest population.
Acknowledgement
Financial assistance provided by the University Grants Commission, Govt. of India, New Delhi is gratefully acknowledged.
Table 5—Effect of Dolichos biflorus HPK4 cultivar trypsin inhibitor on growth of Pieris brassicae larvae and soluble protein content in faecal matter
Concentration of
trypsin inhibitor Weight of larvae (mg) Protein content (µg)
1st day 2nd day 3rd day 4th day 5th day 1st day 2nd day 3rd day 4th day 5th day
Control 1.74
±0.01a 4.33
±0.05a 10.33
±0.02a 15.66
±0.51a 24.60
±0.05a 131.5
±0.27a 137.9
±0.45a 144.5
±0.05a 155.3
±2.62a 163.2
±4.85a Treatment
(2.5mg) 1.71
±0.02a 3.80
±0.05a 7.30
±0.00b 11.00
±0.42b 19.60
±0.03b 121.6
±0.20b 117.9
±1.32b 108.6
±6.43b 98.4
±2.00b 59.76
±1.3b
Mean 1.72 4.08 8.83 13.3 22.16 126.5 127.9 126.3 126.8 111.48
Effect SE(m) CD(0.05) 0.443 0.927 0.703 1.466 .994 2.074
SE(m) CD(0.05) 1.859 1.793 1.359 2.83 1.922 4.01 C
D C×D
Data represents mean values ± standard error of six values. Values in the same column followed by a similar superscript letters are not significantly different at P ≤0.05.
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