6. SUMMARY Due to long co-evolution of plants and herbivores a vast repertoire of unique bioactive compounds have appeared in plants, some of which have found use in medicines, drugs, antibiotics, insecticides, poisons and scents. Among these, a wide array of defense proteins such as proteinase inhibitors, α-amylase inhibitors and lectins are induced in response to insect attack and injury. Proteinase inhibitors (PIs) are abundantly present in the storage tissues of plants such as seeds and tubers. The synthesis of these PIs is induced under various stress-prone conditions such as mechanical wounding, pathogen attack, pest attack and UV exposure. Some of these plant PIs have been found to be effective in retarding growth and development of insects belonging to orders Lepidoptera and Coleoptera. With the advent of molecular biology it became possible to develop crop plants with specific genes that confer resistance to insects. The transgenic crops, expressing insecticidal genes have been speculated to make a significant contribution in safe, environment friendly practices in sustainable agriculture. However, it is important that appropriate proteins be selected for expression to strengthen plant s defense against the target pests. Proteinase inhibitors show varying degrees of effectiveness as an antimetabolite against different insect species. Some transgenic plants expressing PI genes such as tobacco, potato, Arabidopsis and Brassica have been produced with anti-insect activities against some important insect pest species. In order to design strategies for sustainable use of plant PIs in developing insect tolerant transgenic plants thorough information is required about the interaction of pests and their proteases with a variety of inhibitors. The present study deals with the influence of four serine plant proteinase inhibitors against dipteran insect pest. Four inhibitors selected for the present study were Kunitz type Trypsin inhibitor from soybean (Glycine max), (SBTI), Bowman-Birk type Trypsin-Chymotrypsin inhibitor from soybean (SBBI), Trypsin inhibitor from Lima bean (Phaseolus limensis) (LBTI) and cabbage proteinase inhibitor (CPI). The first three PIs were purchased from Sigma- Aldrich chemicals company Pvt. Limited, USA
whereas the fourth inhibitor was extracted and semi purified from cabbage (Brassica oleracae) in the Insect Physiology Laboratory of the Department of Zoology, G.N.D.U., Amritsar, India. The melon fruit fly, Bactrocera cucurbitae (Coquillett) belonging to order Diptera and family Tephritidae was selected for the present investigation as Tephritid flies are among the most diverse groups of insects with a global distribution, covering tropical, subtropical, and temperate regions and occupy habitats ranging from rainforests to open savanna. Many species of Tephritidae inflict heavy losses on fruit and vegetable crops which include direct loss in yield due to fruit drop and production of injured and disfigured fruits and indirectly with increase in pest control costs, loss of export markets and construction and maintenance of fruit treatment and eradication facilities. The genus Bactrocera is economically significant genus, with about 40 species considered to be important pests and B. cucurbitae is among the most notorious pests with which vegetable growers have to contend. Over 125 species of host plants have been recorded for it, the preferred host being cucurbit and solanaceous fruits. The fruits of cucurbits, of which the melon fly is a serious pest, are picked up at short intervals for marketing and self consumption; therefore, the use of insecticides as a means of controlling this pest has had a limited success, moreover, there are only a few reports available about the successful use of bio-control agents against the melon fruit fly. The melon fruit fly was procured from the infested bitter gourds, Momordica charantia L. collected from the vegetable market of Amritsar city and kitchen gardens in the university campus. The taxonomic characters given by Kapoor (1993) and White & Elson-Harris (1992) were used for the identification and sexing of newly emerged flies from the infested bitter gourds. The laboratory rearing of fruit flies was done according to the needs and requirements of the experiments on natural food or in the artificial diet in an insect culture room with controlled temperature (25±2ºC), relative humidity (60-80%) and photophase (10L : 14D). One inch cubes of pumpkin fruit, Cucurbitae moschata (Mosch) were used for egg laying and rearing of larvae whereas 20% sugar solution and Proteinex was used for feeding the adults. The artificial medium (Gupta et al., 1978) was used for experimental culturing of larvae.
The bio-regulatory effect of four plant PIs was investigated via bioassays (influence on the growth and development) and biochemical assays (activity of enzymes). For assessing the effects on growth and development experiments were performed with first (44-48h old), second (64-72h old) and third (88-96h old) instar larvae. The three larval stages were harvested from egg charged pumpkin pieces after 44, 68 and 92h of the removal of pumpkin pieces from fruit fly cages with adult gravid females. These larvae were washed with distilled water, excess water was soaked with filter paper and the larvae were shifted to culture vials containing medium incorporated with various test concentrations (0-400ppm) of plant PIs. The experimental vials were kept in culture room maintained at conditions mentioned earlier and observed daily for various parameters such as time taken for pupation, number of pupae formed, pupal and larval weight, time taken for emergence of flies and the number of flies emerged. The recorded data was analyzed for larval period, pupal period, total development period, percentage pupation, percentage emergence and larval and pupal weight gain etc. The values for lethal concentrations i.e. LC 40, LC 50 and LC 90 were calculated by Probit analysis. The studies on enzymatic activity in B. cucurbitae were carried out on the second instar larvae treated with LC 40 concentrations of the four inhibitors in artificial diet. The adlibitum feeding to these larvae was permitted for three time intervals (24, 48 and 72h) on both treated and untreated diet. After these three time intervals, the larvae were removed from the experimental vials and estimations were done for activity of various enzymes involved in digestion (proteases), detoxification and antioxidant mechanism (GSTs, esterases and catalase) and development (acid and alkaline phosphatases) according to the standard protocols. The treatment of three larval stages of melon fruit fly with four inhibitors significantly affected growth and development of the fly. The larval period of all the three instars increased under the influence of all the four inhibitors. The maximum prolongation in the larval period in first and third instar was observed with LBTI treatment where the larval period was delayed by 3.12 (32.1%) and 1.79 (66.05%) days at 64 and 256ppm, respectively. In the second instar larvae maximum prolongation (2.47 days) was with SBBI at 50ppm. In the case of CPI the larval period rather decreased in the first instar larvae as compared to control. The pupal period increased under the
influence of all the four inhibitors in all the three instars but the increase was statistically insignificant in most of the cases. The total development period increased in all the immature stages of melon fruit fly and the prolongation ranged from 1.06 days (5.23%) to 4.88 days (24.09%) days when three larval instars were permitted adlibitum feeding on inhibitor treated diets. Maximum prolongation in total development period of first instar (4.88 days, 24.09%) was with SBTI treatment at 200ppm. However, in second instar larvae, LBTI had more detrimental effect as compared to other inhibitors as it delayed the development period by 4.15 days (23.97%) at 256ppm. In the third instar maximum prolongation (2.35 days, 18.77%) in development period was observed with SBBI treatment. The percentage pupation got suppressed under the influence of all the four inhibitors in the three larval instars of melon fruit fly. The maximum suppression in pupation (51.39%) of first instar was with SBBI treatment at 200ppm. In second instar larvae maximum suppression in pupation (61.89%) was observed with SBTI at 100ppm, whereas CPI had the maximum inhibitory effect on the pupation in the third instar larvae (42.31%) at 400ppm. The inhibitory effect of the PIs was also observed on adult emergence from treated larvae. Among the four PIs, maximum inhibitory effect on first instar larvae was with LBTI at 256ppm where the emergence was reduced by 60.72%. In second instar the maximum decline was by 61.90% with SBTI at 200ppm and in the third instar the maximum decrease in percentage of adult emergence was by 56% with CPI treatment at 400ppm. On the basis of adult emergence the LC 50 values were calculated using Probit analysis which was lowest in the case of SBTI where it was 52.76 ppm for first, 112.02 ppm for second and 90.44 ppm for third instar. LC 50 was highest in the case of CPI where it was 383.69, 424.62 and 353.93ppm for first, second and third instar, respectively. The ascending order of lethal concentrations of the four inhibitors was SBTI>LBTI>SBBI>CPI. The larval weight decreased significantly with SBTI when the second instar larvae were permitted feeding on artificial diet containing PIs at LC 40 concentrations for 72h. The proteases activity in the second instar larvae of B. cucurbitae was determined by selecting a series of appropriate substrates and the ph optima for enzymes that can
hydrolyze each substrate was ascertained. For trypsin like (BTpNA-hydrolysing) activity, a broad region of higher activity was identified that ranged from ph 9.0 to 11.0, with an optimum ph 11.0. For chymotrypsin like (BTpNA-hydrolysing) activity the broad region of higher activity ranged from ph 8.0 to 11.0 with a ph optima at 10.5. For the exopeptidase Leucine aminopeptidase (LpNA-hydrolysing), two peaks with higher activity were observed at ph 8.0 and 10.5. Elastase like (SAAPLpNA-hydrolysing) activity showed a maximum rate of SAAPLpNA hydrolysis at ph 7.0 when Tris buffer was used. The studies on enzymatic activity in B. cucurbitae were carried out on the second instar larvae (64-72h) treated with LC 40 concentration of various inhibitors in artificial diet. The ad-libitum feeding to these larvae was permitted for three time intervals (24, 48 and 72h) on treated diet as well as on untreated diet. After these three time intervals, the larvae were removed from the experimental vials and estimations were done for ascertaining the specific activity of four enzymes involved in digestion (Trypsin, chymotrypsin, elastase and leucine aminopeptidase), three in detoxification and antioxidant mechanisms (GSTs, esterases and catalase) and two in regulating development (phosphatases). The LC 40 concentrations of the four inhibitors incorporated in the diet of the 64-72h old larvae resulted in a decrease in specific activity of Trypsin with SBTI, SBBI and CPI at almost all treatment intervals when compared to control. On the other hand, in LBTI treated larvae the enzyme activity after showing a significant increase during the initial treatment interval of 24h decreased to 19.5% of the control after 48h of treatment. The observations made after 72h revealed a non-significant increase in enzyme activity in the 136-144h old larvae. The specific activity of chymotrypsin increased significantly in the larvae of B. cucurbitae treated with SBTI, SBBI and LBTI in all treatment durations. In the larvae treated with CPI the activity of chymotrypsin which was slightly more than control at 24h treatment interval decreased to 31.51% of the control in 112-120h old larvae at 48h treatment interval. Further exposure of the larvae to the inhibitor resulted in a 3 fold increase in enzyme activity in the 136-144h old larvae.
All inhibitors had a stimulatory effect on elastase activity which showed a significant increase when the 64-72h old larvae were treated for three different time intervals. The inhibitors showed varied effects on the activity of leucine aminopeptidase (LAP) in the larvae of the melon fruit fly. When the larvae were treated with SBTI, the specific activity of LAP which was significantly lower than control at 24h increased at 48h and again declined at 72h treatment duration. SBBI treatment resulted in a significant increase in the activity of LAP in the larvae of melon fly at 24h treatment duration where it was 2.99 folds higher than control. At 48h the enzyme activity was slightly less than control but again increased when the larvae were exposed to SBBI for 72h. In the LBTI treated larvae, the activity after showing a significant increase at 24h treatment interval decreased when the larvae were treated with the inhibitor for another 48h. CPI had an inhibitory effect on the LAP activity as it decreased at all treatment durations. Among the detoxification enzymes, a significant suppression in the GSTs activity at all the three treatment intervals was observed under the influence of SBTI and SBBI. In the larvae fed on LBTI treated diet, the enzyme activity which was not much affected during the initial 24h treatment interval, increased significantly when observed after 48h but then declined after 72h treatment duration. A significant increase in enzyme activity was observed with CPI after 24h of treatment of the larvae. But prolongation in the treatment to 48h caused the enzyme activity to decrease to 50% of the control. However, further feeding of the larvae on the inhibitor treated diet for another 24h caused the enzyme activity to increase non-significantly. The observations made for the influence of protease inhibitors on esterases showed an increase in enzyme activity at most of the treatment intervals only with LBTI and CPI. In the SBTI treated larvae the enzyme activity after showing 17% increase at 24h, decreased when the treatment time was prolonged to 72h. On the other hand, SBBI suppressed the enzyme activity when the treatment was given for 48h but further prolongation in treatment time resulted in a significant induction in enzyme activity. The estimations made for catalase activity revealed an increase in enzyme activity upto 48h treatment interval with SBTI and CPI. Thereafter, the enzyme activity got
suppressed in the 136-144h old larvae. Likewise, in LBTI treated larvae, the catalase activity increased at 24h treatment interval but then got suppressed in the 112-120h old larvae. At 72h the enzyme activity was not much different from control. Nevertheless in the SBBI treated larvae the catalase activity which was significantly induced at 24h treatment interval, decreased at 48h, but showed a 31% increase in enzyme activity after 72h of treatment. The acid phosphatase activity was significantly induced when the second instar larvae of B. cucurbitae were treated with all the four inhibitors for 48h. The assay of alkaline phosphatase showed an increase in activity at all three treatment intervals only in SBBI treated larvae. Also, in the LBTI treated larvae an increase in enzyme activity was observed after 48h of treatment, but when the larvae were treated for another 24h the enzyme activity got suppressed (17.5%). Nevertheless in the SBTI and CPI treated larvae the enzyme activity after showing an increase at 24h treatment interval decreased when the larvae were treated with the inhibitor for another 48hrs. The present study clearly highlights the potential of inhibitors in dipteran pest management as all the parameters investigated in B. cucurbitae were adversely influenced under the influence of all the four proteinase inhibitors. The maximum activity as per LC 50 was shown by SBTI followed by LBTI, SBBI and CPI. The adverse effects of the inhibitors on development could be due to the inhibition of the major digestive enzyme trypsin by the PIs. The decrease in trypsin activity was compensated partly by an increase in the activity of other digestive enzyme, elastase and to some extent chymotrypsin but this might have further depleted the level of amino acids leading to a nutritional stress in this dipteran fly. Also the activity of the primary detoxification enzyme, GST, was completely suppressed with SBTI and SBBI. Though an induction was observed in esterses and catalases at some of the treatment intervals yet it was not sufficient to combat the adverse effects of plant proteins on the melon fruit fly. Finally the present study substantially widens up the scope for deployment of inhibitors as potential molecules in pest management. But, further investigations need to be carried out for identifying the genes encoding these inhibitors so as to enable them to be transferred in plants for effective management of fruit flies.