Sorghum grain induced disruption of gut amylases in lesser grain weevil Sitophilus oryzae (L)

P.G. Padmaja, B.L.Shwetha, A. Kalaisekar, K. Srinivasa babu, G Shyamprasad and V.A. Tonapi*

ICAR - Indian Institute of Millets ResearchRajendranagar, Hyderabad - 500 030, India

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• The loss of cereal grains during storage is a serious problem worldwide.• Direct-feeding damage by insects reduces

grain weight nutritional value and germination of stored seeds

• Infestations also cause contamination odor and mold damage problems

that reduce the quality of the grain and make it unfit for processing intofood for humans.

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• Worldwide annual post-harvest pest losses have been estimatedat 10% of the total yield,

and weight losses (dry matter loss) in cerealsgenerally range from 0.5 to 17% .

• The stored sorghum is attacked and damaged by a number ofpests that lead to qualitative and quantitative deterioration.

• The weevils are the most important pests, among them Sitophilusoryzae is the major one.

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• Infestation by this weevil begins in the field and carry forward tocause extensive damage in storage.

• Grubs after hatching from the eggs feedinside the kernels.

• Several research avenues are openingup to contain the storage pest menace.

• One such ecofriendly area is managingby way of food grain mediated physiologicaldisturbance in the insect pest.

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α- amylase inhibitors

α - amylase

• Amylases are the most important digestive enzymes of insects that feedexclusively on seed products during larval and/or adult life.

• Stored product insects, live on a polysaccharide-rich diet and aredependent on their α-amylases for digestion.

• α- amylase inhibitors are attractive candidates for the control of seed weevilsas these insects are highly dependent on starch as an energy source.

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Objective

Our study evaluates α-amylase inhibitory activity in seven released sorghum varieties and hybrids in India

Genotype Variety/Hybird

CSV 15 Variety (Rainy)

CSV 24SS Variety (Sweet sorghum)

CSH 16 Hybrid (Rainy)

CSV 26 Variety (Post-rainy)

Phule Suchitra Variety (Post-rainy)

Phule Revathi Variety (Post-rainy)

Phule Vasudha Variety (Post-rainy)

• Reproductive success of S. oryzaereared on different sorghum genotypes

• α - amylase activity of S. oryzae

• α - amylase inhibitory activity of

sorghum grains

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Insect culture

• Adult Sitophilus oryzae were obtained froma laboratory stock culture maintained at

27 ± 1oC,70 % RH, and aphotoperiod of 12:12 (L:D) h

at ICAR-Indian Institute of MilletsResearch,Rajendranagar, Hyderabad, India.

Culture of S.oryzae

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Methodology

• 50g of each sorghum genotype used

• Cohort of 100 newly emerged male-female (1:1) adult weevils wereintroduced

• Left for 15 days for oviposition

• All founding adult insects were removedafter a 15-d oviposition period

• The experiment was conducted in aCompletely Randomized Design (CRD)

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Progeny emergence of S. oryzae

• Samples were left for 45 d for progeny emergence. • Adults that emerged from each genotypes were used to

obtain amylase solutions.

Phule VasudhaCSH 16 CSV 26

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Progeny emergence of S. oryzae

• Higher numbers of progeny were produced in CSV 15. • The number of progeny were significantly lower in the sorghum varieties,

CSV 26 & CSV24SS

Genotype Progeny emergence (No.)

CSV 15 421g

CSV 26 173a

CSV 24SS 185b

CSH 16 230c

Phule Suchitra 282d

Phule Revathi 374e

Phule Vasudha 396f

P <0.001

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Extraction of α- Amylase from S. oryzaeWeevils homogenized at 4oC

(with 20 mM sodium phosphate buffer (pH 7.2) containing 20 mM NaCl and 0.1 mM CaCl2)

Homogenate centrifuged at 10000g for 20 min at 4oC

Ammonium sulphate of the supernatant was increased to 55%, kept for 12h at 40C

Centrifuged & Precipitate was dissolved with the homogenate buffer

Sitophilus oryzae

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α- Amylase activity in S. oryzae3,5-dinitrosalycilic acid (DNS) method

• The enzyme was substituted by aliquots of S. oryzaehomogenate.

• Enzyme dissolved in the sodium phophate buffer was pre-incubated for 15 min at 300C, in the presence of 1% starch as substrate for 20min at 370C.

• The reaction was stopped by the addition of 1ml of DNS reagent to each tube.

• After boiling for 10 min absorbance was read at 530 nm.

One unit of α- amylase activity was defined as one µg of maltose liberatedunder the assay conditions Standard curve was plotted using maltose as standard

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α- amylase activity

Amylase activity levels of theprogeny emerged from thesorghum grains tested weresignificantly different

Genotype α- amylase activityµmoles/min/mg protein

CSV 15 4.63d

CSV 26 4.89c

CSV 24SS 6.20a

CSH 16 5.33b

Phule Suchitra 4.31e

Phule Revathi 3.80g

Phule Vasudha 3.99f

P <0.001

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α- amylase inhibitor activity

Extractionα- amylase inhibitor was extracted from sorghum grains usingFeng et al 1991

Optimization of α- amylase • Aliquotes of inhibitor (50 µg of protein) pre-incubated with • a range of 10, 50, 100, 150, 160, 170, 180, 190,200, 250, 300 µg

S. oryzae total gut protein

The conc. at which the α- amylase brought about the maximum α-amylase inhibitor activity was optimized for all the genotypes individually

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Relationship between amylase activity in S.oryzae and amylase inhibitionin sorghum genotypes

Increasing concentrations of amylase(10-300 µg) produced a correspondingincrease in the per cent amylaseinhibition until 180 µg and thereaftershowed a declining trend in different sorghum grains .

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Relationship between amylase activity in S.oryzae and amylase inhibitionin sorghum genotypes

All the genotypes, expressed linearity to the enzyme extract containing 50 µg of inhibitor protein.

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Genotype Progeny emergence

(No.)

α- a amylase activity

µmoles/min/mg protein

α- amylase inhibitor

activity (%)

CSV 15 421g 4.63d 55g

CSV 26 173a 4.89c 72b

CSV 24SS 185b 6.20a 74a

CSH 16 230c 5.33b 71c

Phule Suchitra 282d 4.31e 67d

Phule Revathi 374e 3.80g 63e

Phule Vasudha 396f 3.99f 61f

P <0.001 <0.001 <0.001

Relationship among progeny emergence, amylase activity and amylase inhibition

CSV 24SS showed maximum α- amylase inhibitory activityDO NOT C

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Progeny emergence Vs amylase inhibitory activity

Negative relationship –Progeny emergence Vs amylase inhibitory activity

r = -0.96

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Conclusion

• From our results it is evident that

amylase inhibition decreases beyond 180µg of amylase production in S.oryzae.

Progeny emergence decreased with increasing amylase inhibition activity.

• Genotypes with more amylase inhibitory activity need to be explored for managing the lesser grain weevil, S. oryzae.

this will help in reducing the pest load by disrupting the digestion

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