Abstract
In the current study, the soybean trypsin–chymotrypsin inhibitor (Bowman–Birk Inhibitor, SBBI) was tested against Bactrocera cucurbitae (Coquillett), a major pest of cucurbit crops. Bioassays conducted using different concentrations (12.5, 25, 50, 100 and 200 ppm) revealed a detrimental effect of the inhibitor on the growth and development of the second instar larvae of the melon fruit fly. SBBI prolonged the larval and total development period and reduced the percentage pupation and emergence. Enzymatic assays of proteases conducted at three time intervals using the LC40 (59 ppm) concentration of SBBI showed an inhibitory effect on trypsin activity, whereas an increase was observed in the activity of chymotrypsin, elastase and leucine aminopeptidase. Among the enzymes involved in detoxification, antioxidant and general metabolism, an increase was observed in the activity of catalases, and acid and alkaline phosphatases at most treatment intervals. The activity of esterases was induced only with prolonged treatment whereas that of glutathione S-transferases was suppressed in larvae treated with SBBI. The findings revealed the potential of SBBI to disrupt the growth of the melon fruit fly.
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Introduction
The melon fruit fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae), is distributed throughout many parts of the world, but India is considered its native home. It damages over 81 plant species and plants belonging to the family Cucurbitaceae are preferred most (Allwood et al. 1999). The extent of losses varies between 30 and 100%, depending on the cucurbit species and the season (Singh et al. 2000). Also, in warm climates, the fly remains active throughout the year on one or the other host. The fruits of cucurbits, of which the melon fly is a serious pest, are picked at short intervals for marketing and self-consumption. The export of these fruits and vegetables is severely affected by the stringent quarantine laws aimed at preventing the entry of melon flies in areas where it does not occur.
Plants naturally possess proteins that include lectins, arcelins and inhibitors of alpha amylases and proteases of insect pests. These proteins are induced in response to injury caused by herbivore attack. Among the different plant proteins, protease inhibitors found abundantly in seeds work by inhibiting the activity of the digestive proteases, thereby impairing the development of the insect.
Soybean trypsin–chymotrypsin inhibitor (SBBI) is a protease inhibitor (PI) which is a member of the Bowman–Birk-type family. Bowman–Birk-type inhibitors are double-headed small polypeptides (8 kDa) that bind simultaneously and independently to two separate proteinase molecules such as trypsin and chymotrypsin (Bode and Huber 1992). SBBI has been reported to significantly retard growth and development and affect enzyme activity of the legume borer, Helicoverpa armigera (Hübner); honey bee, Apis mellifera L.; tobacco budworm, Heliothis virescens (Fab.); and aphid parasite, Aphelinus abdominalis (Dalman) (Johnston et al. 1995; Pham-Delègue et al. 2000; Azzouz et al. 2005). However, its effect on the melon fruit fly has not been reported. Therefore, it was proposed to explore the potential of plant protease inhibitor, SBBI in the management of melon fruit fly, B. cucurbitae. In addition, studies were carried out to ascertain the activities of the enzymes involved in digestion and detoxification mechanisms in order to understand the biochemical responses occurring in the insect body in response to the protease inhibitor.
Materials and methods
Rearing of insects
The melon fruit flies were reared according to the procedure described by Gupta et al. (1978). Adult flies were kept in wire mesh cages (Rescholar equipment; L45 X B45 X H50 cm) and were provided with Protinex (Pfizer India) and 20% sugar solution as food. The pumpkin fruit, Cucurbita moschata Duch was provided for oviposition and also served as natural food for larvae. The stock cultures of flies were maintained in an insect culture room under regulated temperature (25 ± 2 °C), relative humidity (70–80%) and photoperiod (L10: D14).
Bioassay
Fresh pumpkin pieces were kept in wire mesh cages having approximately 100 gravid females of B. cucurbitae for 6–8 h. Exposed pumpkin pieces were removed from the cages and dissected after 64 h for harvesting the second instar larvae. The harvesting was done in saline water, and the larvae were washed in distilled water before shifting them into culture vials (25D X 100L mm) containing culture medium with or without (control) soybean trypsin–chymotrypsin inhibitor (SBBI, Sigma–Aldrich Chemicals Pvt. Ltd.). The artificial diet used in the bioassay was prepared according to the methodology suggested by Srivastava (1975) for this fruit fly.
Treatment consisted of different SBBI concentrations (12.5, 25, 50, 100 and 200 ppm) fed to the second instar larvae. The experimental vials were kept in the culture room for observations. Larval period, pupal period, total development period, percentage pupation and percentage emergence were recorded. There were six replications with ten larvae in each replication for each concentration, and each experiment was repeated twice.
In order to assess the effect of the inhibitor on pupal weight, the second instar larvae were permitted ad libitum feeding on medium with LC40 concentration (59 ppm) of SBBI till pupation and the pupal weights were measured. There were ten larvae in each vial, and six replications were used for control and treatment.
Determination of pH optima for proteolytic enzymes
To determine pH optima of various proteolytic enzymes, aliquots of insect extracts were incubated in the ratio of 1:1 v/v with Tris-HCl buffers at pH 7.0, 8.0, 9.0, 9.5, 10.0, 10.5 and 11.0 for 10 min before monitoring the enzyme activities as described below.
Proteolytic enzyme assays
In these assays, SBBI was incorporated into the artificial diet as described above at a concentration of 59 ppm (LC40). The second instar (64–72 h) larvae were released on both the treated and the control diet for periods of 24, 48 and 72 h and then collected for analysis. Larval extracts were prepared by whole-body homogenization in 0.15 M NaCl. The homogenates were centrifuged at 14,000 g for 10 min at 4 °C. The resulting supernatants were pooled and frozen at −20 °C for future use. These extracts were assayed for proteolytic activity of four substrate-specific proteolytic enzymes (trypsin, chymotrypsin, elastase and leucine aminopeptidase).
Trypsin, chymotrypsin, elastase and leucine aminopeptidase (LAP) activities were assayed using benzoyl-arginine-p-nitroanilide (BApNA), benzoyl-tyrosine-p-nitroanilide (BTpNA), succinyl-alanine-alanyl-prolyl-leucine-p-nitroanilide (SAAPLpNA) and leucine-p-nitroanilide (LpNA), respectively, in a total volume of 175 µl in a 96-well plate (Christeller et al. 1990, 1992). The extract was incubated for 5 min with appropriate buffer at optimum pH (1:1 v/v) at 30 °C. The reaction was initiated with the addition of 50 µl of 1 mM of BApNA, BTpNA, SAAPLpNA or LpNA, each dissolved in dimethyl sulfoxide (DMSO) in 100 µl of solution. The reaction was monitored at 405 nm using a Model 680 X R plate reader (Bio-Rad Lab. Ltd), and activity was calculated. Blanks were run in each case by replacing extract with distilled water.
Activity of detoxification enzymes
The second instar (64–72 h) larvae were placed on control or treated (59 ppm SBBI) diets for periods of 24, 48 and 72 h. The larvae were harvested after specified treatment period and were assayed for activity of five enzymes involved in antioxidant and detoxification mechanisms, i.e., catalase, glutathione S-transferases, esterases, and acid and alkaline phosphatases. There were six replications for each experiment. The procedure of Katzenellenbogen and Kafatos (1971) was used for extraction and measuring esterase activity, using 1% homogenate prepared in 0.1 M sodium phosphate buffer (pH 6.5) and 1 mM α-naphthyl acetate as substrate. The absorbance was recorded at 540 nm, and 0.01 M α-naphthol was used as standard. Catalase activity was estimated according to the protocol given by Bergmeyer (1974), and 0.05 M potassium phosphate buffer (pH 7.0) was used for preparing 5% homogenates, and 0.05% solution of hydrogen peroxide (30%) was taken as substrate. A decrease in absorbance was monitored at 240 nm for 5 min at 1-min interval. Phosphatase (acid and alkaline) activities were determined by the method of Mc Intyre (1971) with 2% homogenates prepared in 0.05 M sodium acetate buffer (pH 5.0) and 0.05 M Tris buffer (pH 8.0) for acid and alkaline phosphatases, respectively. Sodium α-naphthyl phosphate (0.5 mM) was used as substrate for both acid and alkaline phosphatases. Absorbance at 540 nm was recorded with a Systronics UV–VIS spectrophotometer. As a standard, α-naphthol (0.01 M) was used for both acid and alkaline phosphatase. The estimation of glutathione S-transferase (GST) activity was done by following the methodology of Chien and Dauterman (1991) using 2% homogenates prepared in 0.1 M sodium phosphate buffer (pH 7.6). Distilled water was used for preparing 50 mM reduced glutathione (GSH) as substrate, and the activity was estimated by monitoring the absorbance for five min at 340 nm at 25 °C.
Statistical analysis
In order to assess the effect of SBBI on the growth and development of larvae of B. cucurbitae, the data were subjected to statistical analysis, i.e., analysis of variance (ANOVA) using SPSS 10.0 computer program. If the variable was significant, Tukey’s multiple range test was used for pairwise comparison of difference for mean separation (p < 0.05). Significance of the effect of the soybean trypsin–chymotrypsin inhibitor on activity of various digestive, detoxification and antioxidant enzymes of B. cucurbitae at three exposure intervals was determined by t tests.
Results
Bioassays
Soybean trypsin–chymotrypsin inhibitor (Bowman–Birk type) had an adverse effect on the larval and pupal development of melon fruit fly, B. cucurbitae, as was evident by prolongation in the larval and pupal development time of the second instar larvae when reared on SBBI-incorporated artificial diet (Table 1). Maximum prolongation of the larval stage was observed at 50 ppm concentration where the larval period was extended significantly (F 5,29 = 9.80, p < 0.01) by 2.47 days. By comparison, the pupal development time was shorter for the same (50 ppm) treatment. SBBI affected the pupal weight which was reduced significantly (p < 0.01) from 11.86 ± 0.29 mg/pupa (mean ± SD) in the control to 8.34 ± 0.28 mg/pupa when the larvae were treated with LC40 concentration.
SBBI had an inhibitory effect on the pupation and emergence rates of melon fruit fly which decreased with treatment. Maximum decrease in pupation rate was at 50 ppm where it declined to 65.96% of the control (Fig. 1). Percentage emergence too reduced to half of that in the control (52.63%) at 50 ppm, but further increase in PI concentration resulted in an increase in emergence, although it remained lower than the control (Fig. 2).
Enzyme assay
The analysis of proteases in the second instar larvae of B. cucurbitae showed a broad region of higher activity that ranged from pH 9.0 to 11.0 for trypsin-like enzyme (BApNA-hydrolyzing), with an optimum pH 11.0 (Table 2). For chymotrypsin-like activity (BTpNA-hydrolyzing), the broad region of higher activity ranged from pH 8.0 to 11.0 with a pH optimum at 10.5. For elastase-like activity (SAAPLpNA-hydrolyzing), two peaks with higher activity were observed at pH 8.0 and 10.5. For the exopeptidase, leucine aminopeptidase (LpNA-hydrolyzing), the maximum rate of LpNA hydrolysis was observed at pH 7.0 when Tris buffer was used.
SBBI treatment given to the second instar larvae of B. cucurbitae had an inhibitory effect on the activity of trypsin which decreased significantly (p < 0.01) at all the treatment intervals (Fig. 3). Conversely, the activity of elastase increased drastically at all the treatment durations and the increase was highly significant (p < 0.01). The maximum increase was observed after 72-h exposure where a 7.8-fold increase in activity was observed (Fig. 3). The activity of both chymotrypsin and LAP after showing 4.0-fold and 2.8-fold increase, respectively, after 24-h exposure, decreased and was comparable to the control at 48 h. Further exposure of the larvae resulted in a significant increase in the enzyme activities of chymotrypsin and LAP in the 136–144-h-old larvae (Fig. 3).
Among the various antioxidant and detoxification enzymes, SBBI significantly inhibited (p < 0.01) the GST activity of treated larvae at all three time intervals compared with the control. The inhibition was maximum after 72 h of treatment where the enzyme activity decreased to 86.25% of that in the control (Fig. 4). The esterase activity was suppressed during the initial treatment interval of 48 h, but increased significantly in the 136–144-h-old larvae after 72 h (Fig. 4). The activity of catalase was induced with treatment except at 48 h where it was found to decrease as compared to the control (Fig. 4). The activity of acid phosphatase in the larvae was induced during the initial treatment with SBBI for 48 h, but after 72 h the enzyme activity decreased nonsignificantly as compared to the control (Fig. 4).The alkaline phosphatase activity increased at all the three treatment intervals with maximum increase observed at 48 h (Fig. 4).
Discussion
The results showed detrimental effects of soybean Bowman–Birk inhibitor (SBBI) on larval and pupal development, percent pupation and adult emergence of melon fruit fly, B. cucurbitae. A significant prolongation of the second instar larvae development was observed when the larvae were fed on artificial diet containing SBBI at various concentrations. Similar findings were also observed by Steffens et al. (1978) when they tested another inhibitor from soybean, SBTI (soybean trypsin inhibitor, Kunitz type) and showed delayed pupation in European corn borer, Ostrinia nubilalis (Hübner) by 6 days. SBTI (soybean trypsin inhibitor) also increased larval period of Spodoptera litura (Fab.) and sugarcane borer, Diatraea saccharalis (Fabricius), as was observed by Mc Manus and Burgess (1995) and Pompermayer et al. (2001), respectively. Soybean trypsin inhibitor (Kunitz type) also prolonged larval period (36.6%) and remaining development period (21.4%) of the second instar larvae of melon fruit fly (Kaur et al. 2009). The prolongation of both larval period and remaining development period observed in the present studies may be due to depletion in the amino acids required for normal growth and inhibition of major digestive proteases like trypsin.
A significant inhibitory effect on percentage pupation and adult emergence of melon fruit fly was observed under the influence of SBBI as both parameters showed a significant decrease. In a previous study, Kaur et al. (2009) had observed a reduction in pupation and adult emergence of melon fruit fly, when they treated the second instar larvae with SBTI. SBTI also reduced pupation and adult emergence of H. armigera and Spodoptera exigua (Hübner) (Shukla et al. 2005). The pupal weight also showed a decrease under the effect of SBBI, and this may be one of the reasons for reduced pupation as the larvae could not reach a threshold weight and hence could not enter pupation. These results are in agreement with the findings reported by Pompermayer et al. (2001) in D. saccharalis and Telang et al. (2003) in H. armigera.
Digestive proteases catalyze the release of peptides and amino acids from dietary protein, and they are found most abundantly in the midgut region of the insect digestive tract (Jongsma and Bolter 1997). Dipterans generally use serine proteases to digest proteins and have alkaline midguts (Gomes et al. 2005). Serine PIs constitute an important component of the plant defense system by inhibiting digestive proteases in the insect midgut, leading to inadequate digestion and absorption of essential amino acids, arrested growth and eventually to death by starvation (Giri et al. 2003).
Results of the present investigation showed that there is a complete inhibition of trypsin activity. Mc Manus and Burgess (1995) had previously reported that another soybean inhibitor, SBTI, was most effective at retarding the trypsin-like activity in larvae of cotton cutworm, S. litura. Insects use two strategies to defend themselves against PIs, either there is overproduction of PI-sensitive proteases (Broadway and Duffey 1986) or they restore the proteolytic activity by production of PI-insensitive proteases (Bolter and Jongsma 1995). In the present study, there is an increase in the activity of chymotrypsin under treatment with soybean trypsin–chymotrypsin inhibitor. This could be the first defense strategy of the insect against SBBI by overproduction of an enzyme which is otherwise present in very low quantities in the larvae. Secondly, there is an increase in the activity of PI-insensitive proteases like LAP and elastase. The increase in the activity of these proteases could be due to an alteration in the expression of other proteases to circumvent the negative effects of PIs as reported by Jongsma and Bolter (1997).
The activity of growth and detoxification enzymes of B. cucurbitae was assayed during the present investigation to assess the role of these enzymes in combating the stress produced by SBBI in the larvae. Among the various detoxification enzymes studied, the activity of GST was inhibited significantly at all feeding intervals. GST belongs to the class of transferases, a diverse family of enzymes found ubiquitously in aerobic organisms. GSTs are one of the most important and efficient xenobiotic detoxification system in all animals (O’Brien and Tew 1996; Sivori et al. 1997), but a significant suppression in GST activity leads to the conclusion that the SBBI may have interfered in the GST-mediated detoxification of xenobiotics. Congruent to the present findings, Kaur et al. (2009) had also observed a decrease in GST activity under the influence of another inhibitor from soybean (SBTI) in the second instar larvae of B. cucurbitae. The activity of esterases too was suppressed initially but was induced with prolonged treatment. Esterases are known to participate in the regulation of juvenile hormone titer (Whitmore et al. 1972; Newitt and Hammock 1980) and in post-embryonic differentiation (Kapin and Ahmad 1980). Esterases are also known to play a role in insect resistance to insecticides (Zhao et al. 1996) and to allelochemicals (Lindroth 1989). An induction in the activity of esterase with prolonged treatment indicates an adaptation at the biochemical level to counteract the stress caused by the protease inhibitor. The activity of catalase, an oxido-reductase enzyme, was induced both after 24 h and 72 h of feeding the larvae on SBBI-incorporated diet. Catalase is known to convert toxic H2O2, produced during metabolism and oxidative stress, to water and molecular oxygen (De Duve and Baudhuin 1966; Mannaerts and Van Veldhoven 1993). The induction in the activity of catalase indicates its role in the defense of the insect against PI.
Phosphatases include two major classes depending upon their pH optima viz. acid phosphatases (AcP) and alkaline phosphatases (AkP). They catalyze the hydrolytic cleavage of phosphoric acid esters and have been studied generally during embryonic and post-embryonic development. They play an important role in the metabolism of nucleotides, carbohydrates, phospholipids and proteins (Rockstein 1956; Chaubey and Bhatt 1987). In the present study, the activity of both acid and alkaline phosphatases was significantly induced with treatment. The implication of phosphatases in the breakdown of nucleic acids and hydrolysis of phosphate esters suggests that the increase in their activity observed after treatment with protease inhibitors could be a metabolic response of the insect for increased energy required for overproduction of digestive enzymes necessary for its survival.
The observations made in the present study showed that the larvae of melon fruit fly performed better at higher concentrations than at lower concentrations when compared to the control. These studies reflect the ability of the insect to sense high levels of the inhibitor which could be detrimental to its survival and, therefore, might have responded by making rapid metabolic adjustments according to the quantity of the dietary inhibitors. The adverse effects of the inhibitor on the growth of B. cucurbitae at lower concentrations could be due to increased susceptibility of the enzyme trypsin-like serine protease whose activity levels were found to be much higher as compared to other proteases at their optimum pH. Similar findings have been reported in the sheep blow fly, Lucilia cuprina (Wiedemann) (Casu et al. 1994, 1996). Also the increase in the activities of other proteases particularly chymotrypsin and elastase could be due to a feedback mechanism operating that might have led to hyper-production of proteases which in turn led to depletion of essential amino acids and generated a metabolic stress in the insect body. However, survival of the insect at higher concentrations has possible consequences in generating resistant populations which needs to be assessed.
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The authors gratefully acknowledge use of the services and facilities provided by Guru Nanak Dev University, Amritsar, India to conduct the research work successfully.
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Kaur, H., Kaur, A., Kaur, A.P. et al. Assessment of soybean inhibitor as a biopesticide against melon fruit fly, Bactrocera cucurbitae (Coquillett). J Plant Dis Prot 124, 445–451 (2017). https://doi.org/10.1007/s41348-017-0108-6
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DOI: https://doi.org/10.1007/s41348-017-0108-6