Change in malate dehydrogenase and alpha amylase activities in Rubus fruticosus and Valeriana jatamansi treated granary weevil, Sitophilus granarius

Poor storage conditions provide favorable environment to stored grain pests for their growth. The bio-pesticides are the best alternatives to synthetic pesticides. Present study was conducted to compare toxicity of Rubus fruticosus and Valeriana jatamansi against granary weevil, Sitophilus granarius and subsequent changes in enzyme activity responsible for grain damage. In current research 5 g of R. fruticosus fruit and V. jatamansi rhizome powders were tested separately against S. granarius, in 50 g wheat whole grains for seven days in comparison with the control. The enzymatic activity of malate dehydrogenase and α-amylase was observed in the cellular extracts of S. granarius. The insects were crushed and homogenized in phosphate-buffer solution and centrifuged at 10000 rpm for 5 minutes. For the enzymatic measurement supernatant was tested; the spectrophotometer was adjusted at 340 nm. The reagents were mixed and incubated at 25 °C for five minutes. The cuvettes were placed in the experimental and reference sites of spectrophotometer and recorded the change in absorbance for 3-4 minutes. There was 5.60% and 14.92% reduction in the activity of malate dehydrogenase in R. fruticosus and V. jatamansi, treated insects, respectively. The alpha amylase enzyme activity was 6.82% reduced and 63.63% increase in R. fruticosus and V. jatamansi, treated insects, respectively. Present study addresses that both plant powders are effective against granary weevil by altering enzyme activities so both the plant powders can be used as bio-pesticides against the stored grains pests.


Introduction
Cereals are main source of human diet with production exceeding 2100 million tons annually (Shewry, 2007). Bulk of harvested crop (50%) is lost during storage (Fornal et al., 2007). The poor storage conditions provide favorable environment for the growth of stored grain pests (Stoll, 2000). Insect pests of cereals are responsible for reduction in quantity and quality of food grains (Udo, 2011). The weight losses in stored products are mainly associated with activities of insect pests (Arlian, 2002). According to an estimate, annual yield losses due to insect pest are 20-40 percent in overall agricultural products (FAO, 2018). About 1000 insect pests have been reported in harvested products worldwide (Atwal and Dhaliwal, 2008). Many strategies have been adopted to preserve cereals from pest infestations; including pesticides, bio pesticides, repellants, fumigants and biological control measures (Germinara et al., 2008).
Synthetic insecticides are widely used against insect pests (Tapondjou et al., 2005). These pesticides cause environmental pollution and develop resistant insect pests and high mammalian toxicity (Al-Moajel, 2000). Development of bio-insecticides has been an alternative strategy to use of synthetic pesticides (Hashim and Devi, 2003;Meena et al., 2006). In Asia a number of plant products have been traditionally used for protection of stored cereals (Isman, 2000). Plants derivative pesticides are renewable, biodegradable and have low mammalian cytotoxicity (Isman, 2008). Digestive enzymes convert complex biomolecules to simplest one (Erturk, 2006). Any disturbance in enzyme activity of insects is fatal for their survival (Shekari et al., 2008). Dehydrogenases are important tools for the investigation of insect metabolic activities during the course of development (Dickinson and Sullivan, 1975). Terra and Ferreira (2005) confirmed that Amylases are obligatory to digest carbohydrates in insects.

Sampling area
The infested wheat grains with S. granarius were randomly collected from wheat stores of Mansehra and reared in the laboratory. Local wheat, Triticum aestivum was used for rearing of insect. The wheat used in the experiment was cleaned from insects by fumigation with Aluminum sulphate. Fresh, mature and unripen fruits of R. fruticosus were collected from Shatey, Mansehra. The rhizomes of V. jatamansi were collected from Kund Bangla, Mansehra, Pakistan ( Figure 1).

Rearing of insects
Three hundred adult S. granarius were grown in 2-liter jar (14×13.6 cm) having 750 g purified wheat grains at a controlled temperature of 37 ± 2 °C and relative-humidity 60 ± 10% (Mehmood, 2007). Each rearing jar was covered with muslin cloth for aeration.

Preparation of plant powders
Mature and unripened fruits of R. fruticosus and rhizome of V. jatamansi were collected, rinsed with distilled water and air dried in the laboratory at room temperature of 20 °C. Dried plant materials were grounded by electrical blender (Rehman et al., 2009). The resulting powders were sieved, using 40 mesh screens and stored in cool and dry place to maintain efficacy.

Determination of malate dehydrogenase activity
The enzymatic activity of malate dehydrogenase (MDH) was observed in the cellular extract of S. granarius after treatment with R. fruticosus and V. jatamansi powders and compared with control. The experimental insects were crushed and homogenized in phosphate buffer (pH 7.0) solution using a mortar and pistol. Homogenates were centrifuged (Force 1624 microcentrifuge, Edison, NJ, USA) at 10000 rpm for 5 minutes. For the enzymatic measurement of malate dehydrogenase, supernatant was tested in spectrophotometer adjusted at 340 nm. The following solutions were added into the cuvette (1.4 mL Phosphate buffer (0.1M), 0.04 mL Cis-Oxaloacetic acid (0.006M) and 0.02 mL NADH (0.00375M). The reagents were mixed and incubated at 25 °C for five minutes. The cuvettes were placed in the experimental reference sites of spectrophotometer (Perkin Elmer lambd 25 UV/Visible, double-beam, Spectrophotometer, USA) for 2-3 minutes to achieve temperature equilibration, and pippeted 0.005 mL extract of insects into experimental cuvette, mixed it well and monitored the reaction for 2-3 minutes. The oxaloacetate and NADH is converted into L-Malate and NAD + by the enzymatic action of Malate dehydrogenase: MDH Oxaloacetate + NADH → L-Malate + NAD + The measurement of MDH activity was based on the rate of decline in the absorbance at wave length (λ) of 340 nm, subsequent to the oxidation of NADH. One enzyme unit is defined as the amount of enzyme which can catalyze the conversion of one µmole of oxaloacetate to malate per minute at 25 °C. The number of enzyme units per mL of cellular extract was calculated by using the Formula 1:

Determination of alpha amylase activity
Enzymes dilutions were prepared using Sodium Phosphate (Na 3 PO 4 ) 0.02 M buffers at 6.9 pH for each strain separately in eppendorf tubes and kept in ice box. One gram of dinitosalicylic acid as color reagent was mixed in 20 mL of 2M sodium hydroxide (NaOH) and slowly added 30 g sodium potassium tartrate tetrahydrate and made final volume to 100 mL. One percent starch was dissolved in Sodium phosphate buffer (0.02M) having 0.006M sodium chloride (NaCl). 500 µL of 1% starch solution was mixed with 20 µL of cellular extract in triplicates. One ml of the salicylic acid solution was added to each reaction mixture and boiled for five minutes. The reacted samples were cooled to room temperature and added 10 µL of distilled to each tube (making 6.66 time dilution). The absorbance of diluted reaction mixture was measured at 540 nm and compared with standard curve of maltose for the determination of µ-moles of maltose liberated in the reaction. A unit of amylase is defined as the amount of enzyme which can liberate one µ-moles of maltose from starch in one minute (Worthington and Worthington, 2011). The activity of enzyme was calculated as follows (Formula 2):

moles of maltose liberated per minute Enzyme activity U mL
Volume of enzyme added mL = (2)

Results
The reduction in malate dehydrogenase activity for R. fruticosus and V.jatamansi against S. granarius revealed a decrease in the levels of enzyme by 5.60% and 14.92% respectively, compared with that of control (Table 1).
The activity level of amylase in the extract of R. fruticosus and V. jatamansi treated insects showed 6.82% decrease and 63.63% increase respectively. The results represented a significant decrease in the α-amylase activity in V. jatamansi treated insects while a modified activity was observed in R. fruticosus treated S. granarius (Table 2).

Discussion
This study was carried out from May-September, 2011 to assess the efficacy of two plant powders R. fruticosus and V.jatamansi against S. granarius and changes in the enzyme activities. Both the plant powders were collected from different localities of Mansehra Pakistan and were used as treatment against the granary weevil.
In the present study the malate dehydrogenase enzyme activity in R. fruticosus and V. jatamansi treated S. granarius was reduced by 5.60% and 14.92% respectively, compared with that of control. In the previous studies of Hamadah et al. (2010) the alterations in the dehydrogenase activity of the desert locust Schistocerca gregaria was observed, the wild plant Fagonia bruguieri changed the enzyme activity +0.5% in the early-aged nymphs at the lower concentration level of petroleum ether extract along the nymphal instar but the strongest enhancing effect was exhibited in the mid-aged nymphs.
In the present study the α-amylase enzyme activity in R. fruticosus and V. jatamansi treated S. granarius 6.82% decrease and 63.63% increase respectively. Earlier Nehad et al. (2008) reported that activity of amylase was decreased after 24h of plant extract treatment. Mehrabadi et al. (2011) reported Inhibitory effect of plants extracts of A. Siberia, P. harmala, and T. vulgaris against C. maculatus showed the reduced amylase activity 19.22%, 4.58%, and 7.22% respectively. Ahmad et al. (2019) used different plant powders against T. castaneum infesting stored grains. Allium sativum and Zingeber officinale were more effective resulting into 15 time's higher adult mortality. The Azadirachta indica seed powder against the beetle showed better control at lowest (1% w/w) and the highest doses (5% w/w).