2. MATERIALS and METHODS Menadione Is Known for Its Chemosterilent Activity Against Dysdercus (Magdum Et Al

2. MATERIALS AND METHODS Menadione is known for its chemosterilent activity against Dysdercus (Magdum et al. 2001). In the present study, experiments are designed to understand the mechanism of action of Menadione as a chemosterilent on Dysdercus. This will help in exploring its potentiality as an insecticide and is indeed a positive aspect which will balance all our future needs. 2.1 The test compound:

Figure 2: Structure of Menadione ( 2-Methyl-1,4 naphthoquinone )

Menadione, is a synthetic naphthoquinone having IUPAC nomenclature as 2 methyl, 1,4- naphthoquinone. Menadione used in present work is procured by Research Laboratory Industries (RL), India and has following physico-chemical properties mentioned by PubChem, NCBI.

Physico-chemical properties (PubChem, NCBI):

IUPAC name: 2 methyl, 1,4-naphthoquinone

Molecular formula: C11H8O2 Formula weight: 172.18 Melting point: 105oC-107oC Appearance: yellow colored powder Stability: Stable, may be light sensitive

Toxicity: ORL-MUS LD50-500mg/kg, IFR-MUS- 50mg/kg Storage temperature: Store at room temperature Soil sorption coefficient: 370, with moderate mobility in soil

2.2 The test insect “Dysdercus cingulatus”

The red cotton bug, Dysdercus cingulatus (Heteroptera), has been used in the present study as a test insect. Sensitivity to morphogenetic compounds and ease of rearing in the laboratory makes the red cotton bug, the insect of choice for investigation. Moreover, it is a pest of important cash crop – cotton and also attacks okra. It is the most familiar pest of cotton in India. The insect accomplishes its task of predation when the cotton bolls are green and then hibernates in their abode by the time the commencement of cotton picking starts. The cultivator doesn’t realize that the Dysdercus is the actual culprit for the bad crop production. Harm done by insect can be realized after inspecting the growing cotton and the loss can only be found after examining the ginned seed. The net result of this predation involves loss of 1/3rd lint and almost 50% of seeds. Genus Dysdercus includes many species such as Dysdercus similis, Dysdercus koenigii, Dysdercus cingulatus, Dysdercus suturellus and many more.

Figure No.3: Showing the test organism “Dysdercus cingulatus”- a red cotton bug.

2.2.1. Breeding season:

Dysdercus cingulatus is not only a serious pest of cotton; but is also found to infest many plants and trees like Sorgham, Hibiscus species (Okra etc.) Abutilon, Aznara, Sterculia, Baobol and Kopak tree. Life cycle includes five nymphal stages. Both nymphs as well as adults bear piercing and sucking type of mouth parts (Proboscis) and are sap suckers. The adult male stainers are 10-12 mm in length, and the females are 13-15 mm in length, making them larger than males. Its breeding season is from July to November and February to April. During winters and summers the population is relatively low due to unfavorable temperature conditions (Magdum et al. 2001).

2.2.2. Life cycle:

The life cycle (Figure No. 4) of Dysdercus gets completed in 30-45 days. The pest is active throughout the year and passes winters in adult stage. The adults mates after 2-3 days and after which the male and female separates. The male shows mortality after the mating and female lays eggs two to three times in a group at an interval of 3-4 days. First lay comprises of 40-50 eggs and then the quantity drastically reduces for the second and third laying. It is observed that the eggs are laid under the moist crevices of soil near the plant in the field where as in captivity eggs are laid at the edge of soil and wall of the rearing jar/container.

2.2.3. Eggs:

The eggs are yellow, glistening and oval in shape. It measures 1.2 mm in length. Incubation period for the eggs is around 7 -8 days. For progressive development in Laboratory, fertilized eggs require favorable conditions

2.2.4. Embryonic development:

Normal embryonic development in Dysdercus cingulatus requires warm and moist environmental conditions.

2.2.5. Nymphal development:

There are five nymphal stages in the life cycle of Dysdercus cingulatus. First instar nymphs were initially cream in color gradually changing into light orange, which becomes dark

15 orange after 24 hours. These nymphs do not feed and thus congregate near empty shells or in the inner wall of jar at bottom. Sometimes these were found beneath the cotton. After a span of 5-7 days the first instar nymph moults into second instar nymphs which were bigger and brighter than the first instar nymphs. They are more active and were seen moving all over the container. Subsequently, within 15-20 days, nymphs further moulted to third, fourth and fifth instar at an interval of 5-7 days. These nymphs were voracious feeders, red in color. All the nymphal stages resembled each other morphologically except their measures. The size of nymph determines its age. Fifth instar nymphs were largest among all the stages which moulted to adults in next 5 days. Mating is observed after 2-3 days of adult emergence.

2.2.6. Ratio of female and male:

The nymphs emerged from the same batch of eggs laid by a single female did not show any noticeable ratio of male and female, although there were occasions when numbers of males were found to be higher than females or vice-versa. It has not been observed that either only males or females emerge from the same batch of eggs. Thus, female to male ratio is variable and unpredictable.

Figure No. 4. Life cycle of Dysdercus cingulatus. A. adult mating pair (larger is female and smaller is male). B. eggs around the edge of soil and the wall of plastic jar. C. First instar nymphs. D. Second instar nymph. E. Third instar nymph. F. Fourth instar nymph. G. Fifth instar nymph.

2.3 Rearing of Dysdercus cingulatus under laboratory conditions:

Insects were collected from the cotton field of Rahuri, Ahmednagar. The adult male and female were separated by observing the size; males are smaller (10-12 mm) as compared to females (13-15mm). These were placed in pairs in the rearing jar.

Insect rearing jars were filled one-third with wet moist soil forming a layer at the bottom. Each insect rearing jar contained 4 pairs of adult insects. The mouth of these jars was closed by tying a muslin cloth over it (Figure No. 5 and 6). They were fed on soaked cotton seeds. The moist cotton was also placed in the jar to maintain the moisture and provide them water. Dysdercus cingulatus in captivity were maintained at 28ºC ± 2ºC with a photoperiod of 12 hours. These insects showed normal mating patterns and the fertilized eggs obtained from such pair’s required favorable conditions for their progressive development.

The best period for rearing and maintenance of culture as per the climatic conditions of Nashik was between November to April. The month of May being very hot and dry and June being beginning of monsoon, makes the rearing and maintenance difficult. Cold weather and fungal infections to cotton seeds due to high moisture content during monsoon, decreased the number of insect in the colony.

Figure No.5: showing the Laboratory based rearing of Dysdercus cingulatus in the plastic jars containing moist soil and cotton seeds.

Figure No. 6: Showing the nymphs (N) of Dysdercus cingulatus in the jar with soil (S) and cotton seeds (CS) during laboratory rearing.

2.4 Acute toxicity assessment: To assess acute toxicity of Menadione on Dysdercus cingulatus, 1mg/ml Stock solution of Menadione in acetone was prepared. Varied methods were tried for treating the insects. Initially injection method was used. It delivers the precise dosage but has disadvantage of leakage of haemolymph and along with some loss of test solution. in these small insects. It was also seen that feeding could not ensure accurate supply of test compound in insect as consumption of food is variable between individuals. In the same way treatment of food (cotton seeds or okra pods) or even supplying Menadione through drinking water (soaked cotton) is practically difficult as it needs large amount of test compound and thereby leads to more of wastage. Thus, requisite amount of test solution were topically applied to ventro- lateral abdominal region near 4th and 5th tergum of Dysdercus cingulatus with the help of Hamilton microlitre syringe (Figure No. 7). Wings were held carefully during treatment. Topical treatment was preferred over injection or through feeding as this method delivers a precise amount of test solution, thereby ensuring accuracy of doses.

Figure No.7. Hamilton syringe used for the topical treatment of fifth instar and adult of Dysdercus cingulatus.

To begin with investigation on effect of Menadione, LD50 was calculated. In view of finding the LD50 value for Menadione, different concentrations ranging from 0.2 µg to 1.4 µg were tested against fifth instar nymph and adult Dysdercus cingulatus. The experiment was conducted in 3 replicates of control and treated. The insects were subjected to different concentrations of Menadione in a set of ten insects per group. Freshly moulted 24-36 hour fifth instar nymphs and adults from the culture were selected and treated topically with varied concentrations of Menadione at the ventro-lateral abdominal region close to the fourth and fifth terga of 24-36 hrs fifth instar nymph by carefully holding legs were treated. Similarly, in case of adults the wings were held carefully while treating. The treated insects were placed back into the jar after the evaporation of acetone. Mortality of insects in jar was observed and noted daily for the period of 72 hours. Dead insects were taken out from the jar and noted.

The LD50 data for fifth instar nymph and adult Dysdercus cingulatus was calculated by using Least Squares [Lognormal Distribution] Probit Analysis of Biostat 2009 software.

After calculating LD50 value, sublethal doses were calculated and experiments were set to study mode of action of Menadione in adults.

2.5 Mode of action of a synthetic naphthoquinone, Menadione in adult Dysdercus cingulatus:

To elucidate the mode of action of this synthetic naphthoquinone: Menadione, Dysdercus cingulatus were treated topically with sublethal doses of Menadione (0.5, 0.75 and 1µg) using Hamilton syringe. Under this experimental setup, Menadione treated adult male and female Dysdercus cingulatus were dissected in Ringer’s solution on second, fourth and sixth day after treatment i.e. three day old insect, five day old insect and seven day old insect respectively. This was done in the view of the fact that in Dysdercus cingulatus, mating and first gonadal cycle gets completed in a week after emergence. Therefore, keeping this aspect in mind, the observations were made for one week after treating 24- 36 hrs old freshly molted adults. The legs and wings of Dysdercus cingulatus were removed. Later the organism is pinned alive on a wax plate and different organs, such as gonads, fat body and brain were removed after dissection in Ringer’s solution (Figure No. 8). These organs were separated out, labeled (Figure No.9) and then processed further as per the histological and biochemical protocols

Figure No.8: Showing the pinned (P) Dysdercus cingulatus on a wax plate so as to dissect it and excise the different organs. In this figure the red colored testis (T) are visible to be excised out.

Figure No. 9: Showing the labelled eppendorf containing the excised organs of control and treated Dysdercus cingulatus in an eppendorf rack, to be processed further for histological and biochemical analysis.

2.5.1 Histological studies:

To evaluate the effectiveness of Menadione as a reproductive inhibitor and to understand the effect of its toxicity caused during the development of gonads, histopathological details of gonads and brain was done. A set of control of same age was also maintained. To understand the impact, comparison was done between control and treated insects.

2.5.1.1 Histopathological analysis of gonads and brain :

Treated and control insects were dissected in Insect Ringer solution on second, fourth and sixth day after treatment and processed further for histological analysis. A. Processing and embedding of tissue: The ovaries, testes and brain were fixed in aqueous Bouin’s solution for 14-16 hrs, transferred to 70% alcohol and dehydrated in alcohol series 70%, 80%, 90% for half an hour each and in 100% for an hour, followed by a solution of 100% alcohol and Xylene (1:1) for 15 min. Infiltration was done at 60°C in mixture of Xylene and Paraffin wax (1:1) and then in pure Paraffin wax for 30 min. Tissues were then embedded in paraffin wax, whose 5 micron thick microtome sections were cut into a ribbon. This ribbon was taken onto the glass slide which was lubricated using glycerin and albumin solution in 1:1 ratio. These slides containing the section were warmed slightly to straighten the creases of ribbon containing tissue. B. Staining procedure of Haematoxylin/Eosin for gonads: The sections were processed in Xylene (2 changes) for 10 min each first, then in Acetone for 5 min, followed by an downgrade rehydration Alcohol series for 5 min each. All the slides were then stained with Ehlich’s haematoxylin stain for 2-3 minutes and rinsed with tap water. Again the slides were stained with Eosin for 10 min, rinsed with water, followed by a dip in Acid-Alcohol (99ml 70% Alcohol and 1ml HCl). This was then subjected to dehydration alcohol series (upgrade) for 5 min each. The slides were then kept in 100% Alcohol for 10 minutes; two changes were given, followed by two changes of Xylene for 10 minutes each, finally the slides were mounted with DPX and observed under compound light microscope

C. Staining procedure of PAS for Brain:

The sections were processed in Xylene (2 changes) for 10 min each first, then in acetone for 5 min, followed by a down grading alcoholic series for 5 min each. The slides were then stained in 0.5% Periodic Acid by pouring it over the slides and keeping it for 10-15 min. The slides were then rinsed under tap water by pouring the water dropwise over the slides. Later, the slides were stained with Schiff’s reagent for 20-25 min and then rinsed with running tap water for 5-7 min. followed by the counterstaining of sections with Haematoxylin, by keeping the slides in it for 1 min. Then the slides were washed, air dried and then mounted with DPX.

2.5.2 Biochemical studies: To understand and establish the mode of action of Menadione in Dysdercus cingulatus, it is further intended to study biochemical aspect. In doing so here and attempt is made to correlate effect of test compound on energetic and metabolic state of cell and organism as a whole. For this carbohydrate, proteins, RNA and DNA are quantified in experimental and control group.

2.5.2.1. Effect on total carbohydrate content:

Like higher organism even in insects simple sugars like Glucose acts as substrate which oxidizes and releases energy. These simple sugars in the form of glycogen and triglycerides in the adipocytes the main cells of fat body are present. This is used to release energy in response to the energy demand of insects. Trehalose is synthesized in response to energy needs through glycogen breakdown by glycogen phosphorylases in fat body. Breaking of glycogen removes glucose unit. This is promoted by adipokinetic hormone (also called hypertrehalosenic hormones, HTH) produced by Neuro-secretory cells of corpora cardiac, part of ring gland of insects together with the prothoracic gland and corpora allata. Similarly in the brain galactose is formed from blood glucose. Glycogen is also mobilized for the production of trehalose and sugar alcohols under stressing conditions of temperature and drought. Glycogen is also found in eggs of insects. It is synthesized in the ovary and testis from glucose, which is imported from the hemolymph

24 after hydrolysis of trehalose. Thus, glucose is important metabolite among the carbohydrates in insects. Considering all the above it was proposed to observe changes in total carbohydrate content specially glucose in Menadione treated insects along with control group. For this total glucose content in Fat body, gonads and brain were studied quantitatively. Haemolymph was not taken into consideration due to its very low volume in adults

A. Extraction of carbohydrate content:

After treatment with different sublethal doses of Menadione the insects were dissected on different post treatment days (PTD) viz. 2, 4 and 6 in chilled insect ringer’s solution. The gonads, fat body and brain of male and female were separated and homogenized separately by using eppendorf and pestle in ice cold 10% Perchloric acid. The homogenate was allowed to stand still for 10 minutes in ice cold conditions and then centrifuged at 3000rpm for 15 minutes in ice cold conditions. Supernatant was taken out in fresh eppendorf and pellet was again subjected to centrifugation using 5% ice cold Perchloric Acid. Supernatants of both the centrifugation cycles were pooled out and used for carbohydrate estimation as, in the presence of Perchloric acid, proteins were insoluble and carbohydrates were solubilized and hydrolyzed (Plummer, 1987).

B. Estimation of carbohydrate: I. Principle: The carbohydrate present in sample in the presence of Concentrated Sulphuric Acid

(H2SO4) are dehydrated and converted into 5-(hydroxy-methyl)-2- Furaldehyde, which reacts with Anthrone to give green-blue colour. II. Reagents: a. Glucose stock standard: 100 mg of glucose was dissolved in 100 ml of water in a standard flask. b. Working standard solution: 10 ml of the stock was diluted to 100 ml. 1.0 ml of this solution contains 100μg of glucose. c. Anthrone reagent: 0.2% Anthrone was dissolved in ice cold concentrated Sulphuric Acid. Prepared fresh before use

III. Procedure: Total carbohydrate content in different tissues was estimated using Anthrone reagent described by Hedge and Hofreiter, 1962. a. The aliquots of five different concentrations (20, 40, 60, 80 & 100µg) of standard solution were prepared in duplicates along with blank. b. The volume was made 1ml in each test tube. c. The tubes carrying test tubes of unknown carbohydrate content were also processed along with the tubes of standard glucose. d. To each test tube 4 ml of Anthrone reagent was added. e. All the tubes were heated for 8 minutes in boiling water bath and then cooled rapidly. f. The intensity of the green color developed is measured at 630nm. g. A calibration curve on a graph paper was prepared by plotting the glucose concentration on x-axis and absorbance at 630nm on the y-axis. The concentration of the sugar in the sample from the calibration curve was then computed.

2.5.2.2. Effect on total protein content: Proteins are essential metabolites involved in all the physiological activities. Literature survey unmasked the fact that in insect, proteins are produced in fatbody, travelled haemolymph and then are sequestered by gonads. Thus it was thought worthwhile to observe the changes if any in total protein contents in treated insects in comparison to control group. This will help in analyzing further the mode of action of Menadione. A. Isolation of protein fraction: The dissected gonads, brain & fat body of treated and control insects were homogenized separately in ice cold insect saline (0.67 % Potassium Chloride) using eppendorf and pestle for five minutes at room temperature. Homogenized sample was subjected to centrifugation at 3000 rpm for 10 minutes and the resulting supernatant was used for protein estimation.

B. Protein estimation: I. Principle: Total protein levels were estimated according to Lowry method (Lowry et al., 1951), using Bovine Serum Albumin (BSA) as standard protein. The chemistry involves reaction of

26 peptide bonds with copper ions in alkaline conditions and also the reaction of aromatic amino acids present with Folin reagent, which reduces it to an unstable blue coloured complex. The color so formed is measured by using colorimeter at 660 nm. II. Reagents: a. BSA Stock Solution: 1mg/ml stock solution of protein was prepared in distilled water. b. Reagent A: 2% Sodium Carbonate in 0.1N Sodium Hydroxide c. Reagent B: 0.5% Copper Sulphate in 1% Sodium- Potassium Tartarate d. Reagent C: 49ml reagent A + 1ml of reagent B. freshly prepared just before experiment. e. Folin-Phenol: diluted to 1:1 III. Procedure: a. The dilutions were made from the BSA stock solution to make different concentrations (200µg, 400µg, 600µg, 800µg and 1mg) in duplicate along with blank. b. The volume was made 1 ml in each test tube. c. The tubes caring unknown were also processed along with the tubes of standard glucose. d. To each test tube 5 ml of freshly prepared reagent C was added, followed by the addition of 500µl of Folin-Phenol. e. All tubes were vortexed immediately and were kept in dark for 30 minutes. f. The intensity of the blue color developed is measured at 660 nm. g. A calibration curve on a graph paper was prepared by plotting the standard BSA concentration on x-axis and absorbance at 660nm on the y-axis. The concentration of the total protein in the sample from the calibration curve was then computed.

2.5.2.3. Effect on total Nucleic Acid content: Nucleic acids, Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA), are universal in all living forms and are negatively charged macromolecules that carry the complete genetic makeup of each and every cell. Nucleic acids are the master macro molecules controlling all the morphological development and metabolic activities of insects either directly or indirectly.

Large morphological deformities were noted when 5th instar nymphs were treated with various sublethal doses of Menadione. Thus to establish co-relation between variation in nucleic acid if any and development in Dysdercus cingulatus it was thought wise to quantify DNA and RNA in treated and control group.

Nucleoproteins are soluble in solution of high ionic strength but are insoluble in solution of low ionic strength; this property makes DNA and RNA insoluble in pure water. This property of nucleoprotein was further used for extraction and estimation of RNA by Orcinol reagent. Similarly DNA was extracted by Tri-Chloroacetic Acid and quantitative estimation was made by using Di-Phenyl Amine Reagent

A. Extraction of RNA content: The gonads, fat body and brain of treated and control male and female were separated and homogenized separately using eppendorf and pestle in 10% chilled Buffered Saline {0.15M

Sodium Chloride (NaCl) + 0.015M Sodium tri Citrate (Na3 Citrate)}. This solution is then subjected to centrifugation at 5000rpm for 20 minutes at cold conditions (4oC) and supernatant was used for the estimation of RNA (Raksheskar, 2012).

B. Estimation of RNA: I. Principle: RNA was quantified by the method of Orcinol reaction. This is a general reaction for pentoses and depends upon the formation of furfural when pentose is heated with concentrated Hydro chloride (HCl), Orcinol reacts with the furfural in the presence of Ferric Chloride as a catalyst to give a green colored compound.

II. Reagents: a. RNA stock solution: 0.5mg/ml stock solution of RNA was prepared using RNA powder (HiMedia) in Saline Citrate. b. Saline Citrate: 0.15M Sodium Chloride (NaCl )+ 0.015M Tri Sodium Citrate

(Na3 Citrate)

c. Orcinol reagent (0.5gms Hydrous Ferric Chloride FeCl3.6H2O in 500 ml of Conc. Hydro Chloric acid (HCl) + 17.5 ml of 6% w/v Orcinol in alcohol)

III. Procedure: a. The dilutions were made from the RNA stock solution to make different concentrations (200µg, 400µg, 600µg, 800µg and 1mg) in duplicate along with blank (only Saline Citrate, no RNA). b. The total volume in each test tube was made upto 1 ml. c. The tubes of unknown sample and blank along with the tubes of standard glucose were processed further d. To each test tube 3 ml of Orcinol reagent was added. e. All the test tubes were kept for incubation at 100oC for 20 minutes in water bath. f. The tubes were cooled down under running water tap and the intensity of the green color developed was measured at 665 nm. g. A calibration curve on a graph paper was prepared by plotting the standard RNA concentration on x-axis and absorbance at 665 nm on the y-axis. The concentration of the total RNA in the sample from the calibration curve was than computed. (Plummer, 1987) C. Extraction of DNA content: After treating the insects with different concentrations of Menadione, the insects were dissected on different post treatment days (PTD) viz. 2, 4 and 6 in chilled insect ringer’s solution. The gonads, fat body and brain of male and female were separated and homogenized separately with eppendorf and pestle in 5% Tri Chloroacetic Acid (TCA) at 90oC, this step degrades nucleic acid, mainly to soluble nucleotides, but leaves protein intact and denatured in pellet. This solution is then centrifuged at 5000rpm for 20 minutes and supernatant was used for the estimation of DNA (Singh et al., 2010).

D. Estimation of DNA: I. Principle: The DNA was quantified by the methods of Schneider 1957, using Di Phenyl Amine reagent. 2’ deoxyribose of purine generated from the degraded DNA only reacts with Di Phenyl Amine in acidic medium and gave blue colour. II. Reagents: a. DNA stock solution: The 1mg/ml stock solution was prepared using DNA powder (HiMedia) in Saline citrate

b. Saline Citrate: 0.15M Sodium Chloride (NaCl) + 0.015M (Trisodium Citrate) Na3 Citrate. c. Di phenyl amine Reagent: - 5 gm of Diphenyl amine (DPA) + 500ml Glacial

Acetic Acid + 13.75 ml Conc. Sulphuric Acid (H2SO4).

III. Procedure: a. The dilutions were made from the DNA stock solution to make different concentrations (200µg, 400µg, 600µg, 800µg and 1mg) in duplicate. b. Blank (saline citrate, i.e DNA absent) along with the test tubes of unknown samples were also processed. c. The total volume in each test tube was made upto 1 ml. d. Six ml of Di phenyl amine Reagent was added to all the tubes and the tubes were incubated at 100oC for 15 minutes in water bath. e. All the test tubes were cooled and the intensity of the blue color developed was measured at 600 nm. f. A calibration curve on a graph paper was prepared by plotting the standard DNA concentration on x-axis and absorbance at 600nm on the y-axis. The concentration of the total DNA in the sample from the calibration curve was than computed. Analysis of data: Total concentration of carbohydrate, protein, and nucleic acids was estimated by using a formula: Conc. of sample = O.D. of sample/O.D. of standard × conc. of standard The results were expressed in mg/ml or µg/ml. All the biochemical results of total carbohydrate, protein, RNA and DNA were systematically tabulated and have been represented graphically.