Influence of Cabbage Protease Inhibitor on Larval Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae)

SYMPOSIUM PROCEEDINGS RecentAdvancesin Biotechnology forFood and Fuel November 19–20, 2014 TERI IHC, New Delhi

The Energy and Resources Institute Department of Biotechnology Government of India

Influence of Cabbage Protease Inhibitor on Larval Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) Author 1,2

H arpreet Kaur ,Satw inder K Sohal1, Pushpinder JRup1,and Am andeep Kaur1,3 1

Insect Physiology Laboratory,Zoology Departm ent, G uru N anak Dev U niversity,Am ritsar,Punjab,India 2 The Energy and Resources Institute (TERI), India H abitat Centre,Lodhi Road,N ew Delhi,India 3 Departm ent of Zoology,Dolphin (PG ) College of Life Sciences, Chunni Kalan,Fatehgarh Sahib,Punjab,India

Abstract In the course of evolution, plants have elaborated protective mechanisms which allow them to successfully resist different kinds of unfavourable conditions including attacks by insects and phytopathogenic microorganisms. The most important components of all protective mechanisms are proteinaceous compounds and one class of such compounds is Protease Inhibitors (PI), which act on insect’s digestive enzymes and ultimately affect their normal growth and development. Many PIs have been shown to act as defensive compounds against pest by direct assay or expression in transgenic crop plants, and a body of evidence for their role in plant defence has been accumulated consistently. In order to explore their pest-control potential, the present study was conducted to investigate the effect of protease inhibitor !"#$%&''&()$*+,-.$/&0$ )1$2"$23)$4&!5&)$" $23)$#)4"6$ !782$9:;$Bactrocera cucurbitae (Coquillett) (Diptera: %8)62$4)5)4$" $ protection to crops as an estimated 37% of all crop production is lost worldwide to pests and diseases, with at least 13% lost directly to insects (Gatehouse et al . 1993). The plant diversity of India is subj ect to perpetual onslaught by insect infestation due to tropical and subtropical climate that is prevalent in India. At present, crop protection relies predominantly on the use of environmentally toxic agrochemicals that

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H arpreet Kaur,Satwinder K Sohal,Pushpinder JRup,and Am andeep Kaur

are also deleterious to human health. Also, the large-scale use of synthetic compounds has created an excessively high pressure on the environment resulting in destabilization of the existing ecological balance (Dunaevsky et al . 2005). So, it has become imperative to explore ecofriendly alternatives to combat the 860)%2$=)020$86$23)$>)41? Plants and herbivores have co-evolved over a long period of time and developed defensive and counter-offensive strategies to outwit each other. As a result of these long interactions, today 350,000 species of the world’s terrestrial plants represent a vast repertoire of unique bioactive compounds, some of which have found use as medicines, drugs, antibiotics, insecticides, poisons, and scents (Simpson and Ogorzaly 1995). However, a vast number of these compounds are yet unexplored for their potential to be translated into pest controlling products. A wide array of defence proteins (proteases 8638'82"!0;$B@&#:4&0)$8638'82"!0;$&61$4)%2860.$80$8617%)1$86$!)0="60)$2"$860)%2$&22&%C$86$=4&620? Most storage organs such as seeds and tubers contain 1–10% of their total proteins as Protease Inhibitors (PI) (Ryan 1981). Studies have shown that proteinase inhibitors are induced as a component of many defence cascades produced under various stress-prone conditions such as insect attack, mechanical wounding, pathogen attack, and Ultraviolet (UV) exposure (Tamayo et al . 2000). Studies on the effect of 18)2&!:$=4&62$,-0$"6$23)$1)5)4"=#)62$" $860)%20$/)!)$>!02$!)="!2)1$':$D8=C)$et al . (1954) following an "'0)!5&28"6$23&2$23)$4&!5&)$" $%"6 70)1$9"7!$'))24);$Tribol ium conf usum Jacquelin duVal failed to develop on raw soybeans. They were shown as plant defence proteins in 1972when the induction of PIs in potato and tomato was observed due to wounding and insect herbivory (Ryan 1990). Since then, some investigations have been carried out to ascertain the toxicity of plant PIs towards herbivorous insects. However, these studies have been carried out mainly against insects belonging to orders Lepidoptera and Col eoptera where they have been found to be effective in retarding growth and development of these insects. W ith the advent of molecular biology techniques, it is now possible to develop crop plants with 0=)%8>%$()6)0$23&2$%"6 )!$!)0802&6%)$2"$860)%20?$%&62$%"62!8'728"6$2"$0& );$)658!"6#)62$ !8)614:$=!&%28%)0$86$0702&86&'4)$&(!8%7427!)$/3)!)$ %&6181&2)$=!"2)860$ !"#$58!27&44:$&6:$0:02)#$%&6$')$)E=!)00)1$86$1)08!)1$=4&62$28007)$86$&$28#)$0=)%8>%$ #&66)!$*A"742)!$FGGHI $+3!802)44)!$et al ?$JKKJ.?$A72$82$80$8#="!2&62$23&2$&==!"=!8&2)$=!"2)860$')$0)4)%2)1$ for expression that will strengthen the plant defence in a sustainable manner against the target pest *L8!8$&61$M&%3"4)$FGGN.?$O40";$0"#)$2!&60()68%$=4&620I $2"'&%%"$*P"360"6$et al ?$FGNGI $Q841)!$et al . 1987), potato (Gatehouse et al . 1997), Arabidopsis(De Leo et al . 1998), and Brassica (Rahbe et al . 2003) expressing PI genes have been produced with anti-insect activities against some important pest species. Nevertheless, two maj or points need to be considered before establishing the possibility of using PI ()6)0$86$2!&60()68%$=4&620$&0$&$=!"2)%285)$#)%3&680#I $>!02;$/823$&$(85)6$860)%2$0=)%8)0;$0"#)$8638'82"!0$&!)$ "761$2"$')$) )%285)$&628#)2&'"482)0;$/3)!)&0$"23)!0$&!)$6"2I $0)%"61;$&$(85)6$8638'82"!$%&6$03"/$5&!:86($ degrees of effectiveness as an antimetabolite against different insect species (Gatehouse et al . 1993). There is, therefore, a need to select appropriate inhibitors for the digestive proteases of each pest species. This approach requires a thorough knowledge about the interaction of pest proteases with a variety of inhibitors. Although some work has been done on the PIs to signify their role in pest management, but

Influence ofCabbage Protease Inhibitor on LarvalBactrocera cucurbitae (Coquillett)(Diptera:Tephritidae)

02844$23)!)$80$&$6))1$2"$865)028(&2)$23)0)$8697)6%)0$86$1)=23$"6$23)$7#'!)44&$" $860)%2$=)020$" $&$=&!28%74&!$ crop and on a variety of insect species before designing strategies for sustainable use of plant PIs in developing insect-tolerant transgenic plants. %8&4$ 18)2$#)187#$/823$%&''&()$2!:=086$8638'82"!$"!$18)2$#)187#$/823"72$8638'82"!$&0$%"62!"4?$%8&4$18)2$ medium was prepared according to the formula and methodology suggested by Srivastava (1975) for this !782$9:?

Statisticalanalysis -6$"!1)!$2"$&00)00$23)$!)4&28582:$&61$08(68>%&6%)$" $23)$) )%2$" $+,-$"6$23)$(!"/23$&61$1)5)4"=#)62$" $ larvae of B.cucurbitae, the data were subj ected to statistical analysis such as Analysis of Variance (ANOVA), D)&02$V8(68>%&62$W8 )!)6%)$*DVW.;$V2&61&!1$W)58&28"6$*VW.;$V2&61&!1$X!!"!$*VX.;$&61$V271)62Y 0$Z 2Y $2)02$

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H arpreet Kaur,Satwinder K Sohal,Pushpinder JRup,and Am andeep Kaur

depending on type of data and need of experiment using computer programming SPSS-11for windows. Probit line was drawn for emergence in order to calculate LC40 and LC50 values.

Results and Conclusion Effect ofCPI on developm ent period +&''&()$=!"2)&0)$8638'82"!$08(68>%&624:$03"!2)6)1$23)$4&!5&4$&61$!)#&8686($1)5)4"=#)62$=)!8"1$/3)6$23)$ 4&!5&)$/)!)$(85)6$&1$48'827#$ ))186($"6$+,-$86%"!="!&2)1$&!28>%8&4$18)2?$%&62$*1 R;$JR$[$\?JU;$,$]$K?KF;$6186(0$/)!)$ also observed by Shukla et al . (2005) when they found a reduction in pupation and adult emergence of Hel icoverpa armigera$*Q`'6)!.$ )1$"6$VA6186(;$82$80$%4)&!$23&2$ Serine protease inhibitor from Cabbage (CPI) adversely affect the normal development and metabolism of B.cucurbitae. However, there is a need to explore the mechanism of action of this inhibitor in detail, its interaction with enzyme systems at molecular level so as to facilitate the subsequent transformation of this gene into transgenic plants.

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