We will discus about the MATERIALS AND METHODS of Insecticidal Activity of Datura metel Linn. Against the red flour beetle, Tribolium castaneum (Hbst.) adult Step by Step.
MATERIALS AND METHODS
In the tropical and subtropical countries, post harvest application on plant crop pest defense technologies still under development is also prevalent in these times. But many of the techniques used are not scientific and it hampers conservation of some endangered species of plants. It needs more materials to make sure the cropland free from pest infestation.
By isolation of active principles from these plants, it could be possible to reduce the volume of utilization of plant materials; moreover the rapid disappearance of tropical forests and other important areas of vegetation have meant that it is essential to have access to methods which lead to the rapid isolation and identification of bio-active natural products for this purpose.
Interest in native plant as potential source of new antibiotics, anticancer agent, pesticides and other pharmaceutical agent has been rewarded by the discovery of unexpected diversity of new natural products. Since plant may contain hundreds or even thousands of metabolites, this is currently a resurgence of interest in the vegetable kingdom a possible source of new lead compounds for introduction into screening programs. After development of multimedia techniques natural resource have been used to be the potential source for safe, biodegradable and more beneficial drugs, remedies or pesticides for a sustainable environment on the planet. Insect, mites, algae or even micro-organism have also been subjected to yield active compound in this regard. But plants are the most suitable source for such an interesting propagation in the field of pesticide technology while some plants in different parts of the world are considered toxic and some are used in the traditional medicine. A literature search on the title plant offered some essential openings this species bears repellent, and toxicological properties which is subjected to go through screening and then isolation, purification and structure elucidation of essential constituents to develop natural non-hazardous biodegradable pesticides.
The approach adopted to obtain an exploitable pure plant constituent involves interdisciplinary work in botany, zoology, pharmacology, chemistry and toxicology as described by Hostettman et al. (1995).
Selection of plant materials
In order to arrive at useful compounds in the shortest possible time, careful selection of plant material is obviously very important. Random collection is one method but it is more judicious to base the selection on certain criteria. By way of illustration, plants used in traditional medicine are more likely to provide pharmacologically active compounds (Huxtable, 1992). Similarly, folk use of toxic plants could be taken with desirable output.
In this investigation different parts of D. metel have been collected for the presence of toxic, as well as, bio-active constituents since the plant is well known as a medicinal plant and also considered to contain toxic constituents. Incase of very small plants, incase of very small plants, such as herbs, shrubs, grass, etc. normally the whole plant is subjected for extraction, because the distribution of constituents generally not very too much. Being a large timber plant, the distribution of compounds in different parts of this plant is obviously different. The presence of constituents in the heart-wood may disappear in the leaves; similarly constituents in the roots may not be the same that present there in the fruits.
Preparation of plant materials for extraction
The fresh leaves of D. metel were collected from plant by following way-
Leaves: After collection of leaves of D. metel were spread out to dry without heaping the material together. It was done under the shade avoiding direct sunshine.
After dry well the plant leaves were powdered in a grinder machine avoiding excess heat during grinding.
Seed: After collection of seed of D. metel were dried without direct sunshine. After dry well seed were powdered in a grinder machine avoiding excess heat during grinding.
Chemical extraction of the collected materials
There are basically two methods for extracting compounds from plant materials. Which one to choose, depends on whether the aim is to extract the more polar compounds (especially glycosides) which are present in the cell vacuole, or to obtain the less polar aglycones present on the surface of the plant, in aerial parts heartwood or roots.
In the present on study one solvent was selected to extract three different parts of D. metel separately. The ground dried material, viz. leaves, fruit-shell, stem were extracted with sufficient amount of chloroform (500 × 1500ml × 3times) for each of the items. Separate extracts have been collected by the cool method after 72 hours of plunging for each of the material. Extract, thus, obtained are filtered and concentration on a rotary evaporator at 40°c and only as residue is left and kept in a refrigerator after labeling.
For each of the items one solvent was used, and the amount of materials were recorded deducing the amount of the extract afforded in extraction for some conveniences in preparing doses for the molluscicidal tests. The pathway for the extraction, in detail, used in this investigation is given in the above figure.
Selection of test organism:
To carry on tests for insecticidal properties of the extracts of the title species D. metel, T. castaneum was selected as test organism, because it is an easy cultivable and noble laboratory animal. Moreover, it is an important stored grain pest in a wide variety of cereal products. The life history made these insects as popular choice as test insects for biological studies. It is also easy to culture this insect in large numbers and require no sophisticated equipment for maintenance.
Collection of test insects
T. castaneum used in the present experiment were taken from the stock cultures of Crop Protection and Toxicology Laboratory, University of Rajshahi, Rajshahi-6205, Bangladesh and reared as subculture to be used in the experimentation.
Culture of test insects
Mass cultures were maintained in plastic containers (1200 ml) and sub-culture in beakers (100 ml) with the food medium. The beakers were kept in an incubator at 30°c ± 0.5°c without light and humidity control. Each container and beaker contained 250 g and 150 g of food respectively. About 200 adults in each container and 100 adults in each beaker were introduced. The cultures were checked in regular intervals and eggs and larvae were separated to increase properly. A crumpled filter paper was placed in side each container and beaker for easy movement of the beetles. The containers and beakers were covered with pieces of muslin cloth tightly fixed with the help of rubber bands to avoid possible escape of the beetles.
Preparation of the food medium
The whole wheat flour was used as the food medium for the insect species. The flour was sterilized at 120°c for 6 hours in an oven. A standard mixture of whole wheat flour with powdered dry yeast in a ratio of 19:1 (Park and Frank, 1994; Park, 1962; Zyromska-Rudzka, 1966; Khalequzzaman et al. 1994) was used as food medium throughout the experimental period. Both the flour and the powdered dry yeast were sterilized at 60°c for six hours in an oven. Food was not used until at least 15 days after sterilization to allow its moisture content to equilibrate with the environment (Khan, 1981).
Collection of eggs
About 500 beetles were placed in a 500ml beaker containing food medium. The beaker was covered with a piece of cloth and kept in an incubator at 30°c ± 5°c. In regular interval the eggs were collected by sieving the food medium by two sieves of 500 and 250 mesh separating the adults and eggs respectively following the methods of Khan and Selman (1981). Eggs were then transferred to Petri dish (50 mm in diameter) and incubated at the same temperature.
Collection of newly hatched larvae
After 3-5 days, larvae hatched out in described conditions. Newly hatched larvae were then collected with a fine pointed camel hair brush and then shifted to the fresh food medium for culture. The larvae are yellowish white in color and long cylindrical shape. It appears 1 mm in length after hatching and become 6-7 mm at maturation.
Collection of mature larvae
Most larvae had six instars as reported by good (1936). The larval instars were determined by counting the number of exuviate (larval skin) deposited in the food medium according to Good (1936). Two days old larvae was considered as first instars larva while second, third, fourth, and fifth instars larvae were considered on fourth, seventh, tenth and thirteenth day from hatching respectively. Depending on these days according to larval instars sixteen days old larva have been considered as a mature larva. Larval cultures were maintained in an incubator in the same procedure at 30°c ± 5°c without light and humidity control. The food medium was replaced by three days interval to avoid conditioning by the larvae (Park, 1934).
Collection of adults
A huge number of beetles were thus reared to get a regular supply of the newly formed adults. When sufficient adults produced in the sub-cultures, they were collected from the food medium. For this purpose some pieces of filter paper were kept inside the beaker on the food. Adults crawled upon the paper and then the paper was taken out with a forceps. Beetles were than collected in a small beaker (100 ml) with the help of a fine camel- hair brush.
Bioassays for activity of the collected extracts
Crucial to any investigation of plants with biological activities is the availability of suitable bioassays for monitoring the required effects. In order to cope with the number of extracts a high sample throughout is necessary. The test systems should ideally be simple, rapid, reproducible, and inexpensive. If active principles are only present low of concentration in the crude extract then bioassay is to be high enough sensitive for their detection.
Another factor of special relevance to plant extract is the solubility of the sample. Finding a suitable system can pose problems. For the selection of bioassays to employ in research on plant constituents, the first step is to choose suitable target organisms. The complexity of the bioassay has to be designed as a function of the facilities and resources available.
Bioassay with residual film/surface film experiments
All extracts were diluted with the solvents in which they were extracted and the actual amount of extracted matter in a dose was recorded. The application of dose was carried out by residual film method (Busvine, 1971). For each dose one ml of mixture was dropped on a Petri dish (90 mm) in such a way that it made a uniform film over the Petri dish. Then the Petri dishes were air-dried leaving the extraction on it. The actual extract present in one ml mixture was calculated and dividing the value by the area of the Petri dish the dose per square centimeter was calculated.
After drying 10 red flour beetles (3-5 days old) were released in each Petri dish with 3 replications. A control batch was also maintained with the same number of insects after preparing the Petri dish by applying and evaporating the solvent only. The treated beetles were placed in the incubator at the same temperature as reared in stock cultures and the mortality of the beetles were counted after 24 and 48 hours of treatment.
This is also one basic application method for doses of toxic substances to any insect population. The test material has been dissolved in an organic solvent with a certain concentration to apply to a Petri dish of known surface area. After application being volatile the solvent evaporates out immediately simple with the atmospheric temperature. Thus the ingredient goes to make film on the surface of the Petri dish. Released insects within this captivity might have contact with the substance distributed evenly on the floor. However, being covered with the upper lid of the Petri dish there could have a captive environment with the extract distributed even in the air inside and may cause promptly if there is any volatile bioactive principles in the test material.
Preparation of doses with the crude extracts for the surface film test
A general concentration for each of the extracts was selected as 10 mg/2ml as the stock dose for surface film application to make other successive doses by serial dilution to give for pet. Ether leaf extract 1.528, 0.764, 0.382, 0.191, 0.096 and 0.048 mg cm-2 chloroform seed extract 2.038, 1.0186, 0.509, 0.255, 0.128 and 0.064 mg cm-2 pet. Ether extract of seed 1.528, 0.764, 0.382, 0.191, 0.095 and 0.048 mg/cm2 concentrations.
Application of doses in the surface film test
To conduct surface film activity test 50 mm Petri dishes were taken for all doses and their replications. One ml of the doses were poured into the lower part of the Petri dish and allowed them to dry out. Being volatile the solvent was evaporated out within a few minutes. Ten insects were released in each of the treated Petri dish. A control experiment by applying the only solvent into the Petri dish was also set at the same time under the same condition.
Observation of mortality
Completing the all the arrangements treated Petri dishes were placed in a secured place at room temperature. The whole experiment was observed from time to time and mortality was observed by every 12 h and the data was recorded. A simple microscope was used to check each and every beetle by tracing natural movement of its organism. In some cases hot needle was taken closer to the bodies (without movement) to confirm death. Attention was also paid to recovery of the insects if occurred.
The mortality records of the residual film experiments done
T. castaneum adult was corrected by the Abbott”s (1925) formula:
Pr = Corrected mortality (%)
Po = Observed mortality (%)
Pc = Control mortality (%)
Then mortality percentages were subjected to analysis according to finney (1947) and (1971) by using a software developed in the Department of Agricultural Environment Science, University of Newcastle upon Tyne, U.K. the dose –mortality relationship was expressed as a median lethal dose (LD50).
Dose mortality effect on T. castaneum larvae
Effect of toxicity of the extracts collected from D. metel against larvae of T. castaneum was assessed by observing their chronic action on any stage of the beetle’s life span. The selected food medium (1 g of whole wheat flour in a vial for each dose) was treated with different doses of the extracts to release selected number of larvae in each of the units. Changes in all the developmental stages were observed from time to time including mortality. Any sort of anomaly in their growth was observed.