Fig: 2 Danaus genutia Fig: 1 Graph is plotted taking “Duration of Treatment
in Hours” on X-axis vs. “Percentage of Treatment” on
Y-axis.

ISOLATION OF BIOINSECTICIDES FROM THE STRIPED TIGER BUTTERFLY, Danaus genutia (Lepidoptera: NYMPHALIDAE)

***Arunava Das, Chandan Mithra, R.Revanna, A.R.V. Kumar, and K. Chandrashekara

Department of Entomology,

College of Agriculture

(South Block), University of Agriculture Sciences,

Gandhi Krishi Vigyana Kendra, Hebbal, Bangalore-65, India.

A plant grows in a highly competitive environment. It is continually being threatened by other plants encroaching upon the space from which it draws it’s sustenance, by microorganisms, by insects, avian, reptilian or mammalian herbivoures. In order to survive, each plant must draw upon a complex of defenses, which may be physical, such as Spines or Leathery Leaves or Chemical.These defenses are passive, coming into action only when the plant is attacked which repel insects or the herbivoures that attempt to feed on them.Plants protect themselves from insect attack by providing a multitude of repellent chemicals including toxic and bitter alkaloids and tannins. Non-specialist insects and other animals that attempt to feed on the plant may be repelled, injured through severe gastrointestinal malfunction or even killed. The cabbage, spinach, mustard etc. are well known for their mustard pungent flavor that deters most animals from consuming it. Even most medicinal plants like Cynanchum collialatum of the family Asclepedeciae have tannins or alkaloids like “cardenolides” that repel most animals and insects. Most of these plants have a basic enzyme “Myrosinase” which react with these specific tannins to produce the toxic metabolite, which repels insects and animals. But caterpillars of few insects are known to tolerate these toxic alkaloids (ex: caterpillar of Striped Tiger Butterfly-Danaus genutia) and in turn sequester the toxic alkaloids into their tissues making themselves non-palatable to their enemies. We hypothesize the analysis of possible roles of toxic alkaloids isolated from these butterflies as bio-insecticides.

Keywords- Toxic, Tannins, Alkaloids, Myrosinase, Asclepedeciae.

Email: arunavadas1983@yahoo.co.in , arunava_er@yahoo.com

1 P.G. Student, M.Sc. Biochemistry, CMR Institute of Management Studies, Bangalore University, Bangalore (2005-2007)

2 P.G. Student, M.Sc. Biotechnology, I Sem., Bangalore University, Bangalore (2006-2008)

3 Associate Professor, College of Agriculture, Mudigeri, Karnataka

4 Associate Professor, Department of Entomology, College of Agriculture, UAS, GKVK, Hebbal, Bellary Road, Bangalore

5 Associate Professor, Department of Entomology, College of Agriculture, UAS, GKVK, Hebbal, Bellary Road, Bangalore. & Principal Investigator, Butterfly Park (DBT), Bannerghata National Park, Bangalore.

*** Main presenter at Pondicherry University, JNU and IISc

INTRODUCTION:

Bio-insecticides are a group of insecticides that are derived from Biological origin like plants, microbes, insects, lower amphibians etc. They act only on Target Organisms like pests which cause widespread damage to plants and crops without affecting other spheres of life like grazing mammals, birds etc. They can also reduce major environmental hazards caused by Chemical Pesticides.

They posses some general features:

· Have a narrow target range and a specific mode of action.

· Are slow acting but highly effective.

· Have relatively critical application times.

· Suppress, rather than eliminate a pest population.

· Have limited field persistence and a short Self-life.

· Are safer to human and the environment than conventional chemical ones.

· Present no Residue problems.

The proteins of one insect can either be processed or directly utilized to kill the target organisms.

There are a large number of sources which are discussed below:

Plants: (e.g. Azadiracta indica – Neem)

Other medicinal plants having potentially toxic alkaloids (Alkaloids are mainly Cardenolides: formed by condensation of Progelenone or progesterone with Acetate, normal sugars or unbranched Aldohexoses, have 23 carbon atoms and a Lactone ring) belonging to the families like Apocynaceae, Asclepiadeceae, Brassicaceae, Celestraceae, Euphorbiaceae, Liliaceae, Moraceae, Ranunculaceae, Scrophularaciae, Sterculiaceae and Tiliaceae.

Genetically Modified

(a) Bacteria: Bacillus thuringiensis

(b) Fungi: Actinomycetes, Streptomyces

(c) Viruses: Vascular Arbascular Mycorhizae

(d) Protozoans

Insects: Butterflies belonging to the Order: Lepidoptera, Family: Nymphalidae and Sub-family: Dannaid are known to feed on medicinal plants having toxic alkaloids in their sap and cells. Therefore these butterflies in their caterpillar stage sequester the toxic compounds from their host plants and stored them in their tissues, exhibiting the warning color which gives an indication that these colored caterpillars are not palatable to their natural predators. Even there are reports of permanent damage to the predators who try to feed on them with severe GI tract disorders leading to death or inhibition of Protein Synthesis. Their excreta analysis has revealed that they do not excrete the toxic alkaloids. Hence, like butterflies are fascinating examples of insects storing poisons. One of these butterflies (brightly colored ones) belonging to the Sub-family: Dannaid is Danaus genutia (STRIPED TIGER) being widely available in all urban gardens. (Evans 1932)

The natural chemical metabolites produced by plants may be either acutely or chronically toxic to other animals. These acutely toxic metabolites may be either aversive or repellant, or they may be neutral or attractive (Provenza et al. 1991) A plant grows in a highly competitive environment. It is continually being threatened by other plants encroaching upon the space from which it draws it’s sustenance, by microorganisms, by insects, avian, reptilian or mammalian herbivoures. In order to survive, each plant must draw upon a complex of defenses, which may be physical, such as Spines or Leathery Leaves or Chemical (Rosenthal and Janzen 1979). These defenses are passive, coming into action only when the plant is attacked which repel isects or the herbivoures that attempt to feed on them. The plants might even produce chemicals that exude in soil and inhibit the growth of microorganisms and effective growth of competitive plants, a phenomenon known as Allelopathy. Many predators like Butterflies and Grasshoppers (Brower L.P. 1967, Whittaker and Feeny 1967) are however known to tolerate or circumvent the physical and chemical defenses of these plants and sequester the toxic compounds produced by the plants in their own defense purposes. Certain of these chemicals are amino acids and therefore certainly not utillized as their nutrition and are therefore stored (Rosenthal and Bell 1979).

A great deal of discussion has surrounded the particular concept of Plant Toxins and their relation with other predatory organisms that prey on these plants.

Palatability is extremely difficult to define in terms of biological processes ionvolved in food selection. Odors alone can produce such amarked response that many individuals will refuse to sample the item. Scientists studying the palatibility in insects can accurately measure responses such as refusal to feed, reduced growth rate and disrupted maturity.

Consideration of palatibility or unpalatability of foods generally leads to consideration of aversions. A striking example of aversion in nature is that of the larvae of Monarch Butterflies (Danaus plexippus) and Striped Tiger (Danaus genutia). These larvae feed exclusively on leaves of Milkweed plants (Asclepias curassavica and Cynanchum collialatum, respectively ) from which they absorb and sequester considerable amounts of toxic cardiac glycosides. This led Lincoln Pierson Brown of University of Florida 1967 to rear monarch butterfly larvae on Milkweed and allow them to be eaten by Blue Jays. The birds which consumed these butterflies first vomitted and then became violently sick. Brower also went on to analyse these compounds sequestered by the Monarch larvae and found 10 different cardenolides, the most harmful and toxic of them are Calactin and Calotropin; the total amount of toxin concentration in the body of each butterfly is several times higher than the amount necessary to kill a cat or a small dog. He also experimented on the Grasshopper (Poikilocerous bufonius) which feeds solely on plants of Asclepiadeceae family. When this grasshopper is attacked by a potential predator, it can defend itself by ejecting a spray from it’s poison gland which has the same composition as that of Cardiac Glycosides.

Insects as source of Drugs, Enzymes and Bioactive Compounds: Nowadays insects and some lower amphibians like frogs are being used as potential sources of drugs, enzymes, anti-microbial compounds and other bioactive compounds. The same concept can be extended to search for new insecticide molecules because they repel their natural enemies by chemical defenses they procure from the plants from which they acquire nutrition, well documented by scientists world over. Most of the attempts have centered on the idea of isolating and charecterizing the cardenolides derived from these insects or plants, however this is the first such attempt to study and analyze the effect of cardenolide containing extract as a source of potential Bio-insecticides

OBJECTIVE:

Most of the attempts have centered on the idea of isolating and charecterizing the cardenolides derived from these insects or plants, however this is the first such attempt to study and analyze the effect of cardenolide containing extract as a source of potential Bio-insecticides.There are a large number of pests for the cruciferous crops like cabbage, cauliflower and also mustard plant. One of the major pests creating widespread leaf damage to both these plants are the larvae of Diamond Black Moth (DBM). Instead of spraying harmful chemical insecticides and pesticides, we can similarly spray Bio-insecticides, which will be as effective as their predecessors as well as absolutely safe when these plants are used for human consumption. We wish to analyse the possible effects of toxic alkaloids isolated from these butterflies as bio-insecticides.

MATERIALS & METHODS:

All works reported here have been carried out under strict laboratory conditions at the Department of Entomology, College of Agriculture, UAS, GKVK. The methodology of rearing the test organism and Bioassay methods are outlined below:

Rearing Method of Danaus genutia: Danaus genutia viable eggs are collected (around 20-15) and incubated them in special boxes with fresh leaves (each leaf is washed properly, trimmed, maintaining the size of 5cm. x 2cm surface area, wrapping the end with wet cotton and alluminiun foil) of Cynanhcum colialatum till they hatch. Every 24 hours, the leaf is replaced with a fresh one. One egg should be kept per box. After 2-3 days, they hatch into caterpillars and for next 10-15 days, till they go to pupation, we had to every 24hrs. change the leaves of Cynanhcum colialatum (only when fully eaten) keeping them under constant observation and noting their life cycle data. After 2-3 days in pupae they hatch into adult butterflies.

Preparation of Extract: First allow the butterflies to die and keep them in refrigeration for 4-5 days for drying. Weigh around 1.032gms of butterfly (whole body) and grind them in a pre-chilled mortar and pestle with liquid nitrogen into a fine powder. Then solubilize the powder with 95% pre chilled ethanol in a paste. Filter out with the help of a double-layered muslin cloth. Allow to evaporate to minimum and then Serial Dilute.

Take the filtrate/ extract and dilute with distilled water to get 3%, 2%, 1%, 0.5%, 0.25%, and 0.125% as shown:

33.3ml. Extract + 66.7ml Distilled Water (d/w) = 3% concentration

22.2 ml. Extract + 77.8 ml. d/w = 2% concentration

18.5 ml. extract + 81.5 ml. d/w = 1% concentration

13.9 ml. extract + 86.1 ml. d/w = 0.5% concentration

13.9 ml. extract + 86.1 ml. d/w = 0.25% concentration

13.9 ml. extract + 86.1 ml. d/w = 0.125% concentration

Leaf not sprayed with Extract = Control

Bioassay: Take washed & trimmed (maintaining the same surface area as for Cynanchum leaf) Mustard leaves with intact petiole and moisten ends with cotton and alluminium foil. For each experiment 3 sets of Petri plates or replicas should be taken. In each of the Petri plate one trimmed Mustard leaf sprayed with the said concentration of Extract should be taken. We used Potter’s Spray Tower to spray the leaves with the corresponding concentration. After that the leaves are dried in open air, 10 DBM larvae in each of them are released. All the 3 replicas of a particular concentration are kept together and observed every 24 hours for 7 days. Also a Control was kept in which the leaf was not sprayed with Extract.

OBSERVATION AND RESULTS:

Each day (after every 24 hours) the Mortality Rate of larvae was observed for each of the replicas and for each of the concentration. When leaves were fully eaten up, they were changed with new leaves. This time leaves were not sprayed as this would lead to overdosing of the larvae. The mortality rates were calculated and Corrected Percentage Mortality was worked out as follows:

Calculation of Percent Mortality Rate: Workout the per cent mortality for each replication and workout corrected per cent mortality and then take the mean per cent mortality of three replications of each concentration and express in graph.

1. Percentage Mortality = (No. of Larvae dead / Total no. of Larvae put) x 100

2. Corrected Percentage Mortality Rate

= [(% Mortality in Treatment – % Mortality in Control) /

(100 – % Mortality of Control)] X 100

HOURS MORTALITY RATE

3%

2%

1%

0.5%

0.25%

0.125%

24

10.37

41.11

30.74

7.40

14.44

17.04

48

17.40

51.85

34.07

37.41

14.44

24.07

72

22.5

56.67

30.83

14.17

38.33

20.83

96

16.19

68.99

24.44

14.76

39.58

18.47

120

16.19

68.99

24.44

19.52

42.91

23.23

144

27.3

68.99

24.44

19.52

42.91

23.23

168

27.3

68.99

24.44

19.52

42.91

23.23

Table 1: CORRECTED PERCENTAGE MORTALITY

At the end of 168 hours, i.e., after 7 days, 3% Extract showed a maximum of 27.3% of Corrected Mortality and a minimum of 10.37% of Corrected Mortality. Similarly, 2% Extract showed a maximum of 68.99% of Corrected Mortality and a minimum of 41.11% of Corrected Mortality.

1% Extract showed a maximum of 34.07% of Corrected Mortality and a minimum of 24.44% of Corrected Mortality. 0.5% Extract showed a maximum of 37.41% of Corrected Mortality and a minimum of 7.40% of Corrected Mortality.

0.25% Extract showed a maximum of 42.91% of Corrected Mortality and a minimum of 14.44% of Corrected Mortality. 0.125% Extract showed a maximum of 24.07% of Corrected Mortality and a minimum of 17.04% of Corrected Mortality. This clearly shows that 2% Extract has given the best results followed by 0.25% Extract. Overall, as we can see from the graph below, there has been uniformity in the curves obtained from 2% and 1% Extracts. Therefore our next step is obtaining more of the 2% and 0.25% Extracts which have given the Lethal Doses for larvae mortality such that we can now concentrate on purifying the cardenolides and applying them directly for field trials. We also wish to test the potency of these potential bio-insecticides on higher group of pests like beetles, borers etc.


GRAPH:

A Graph is plotted taking “Duration of Treatment in Hours” on X-axis vs. “Percentage of Treatment” on Y-axis, we got, please see Fig:1.


DISCUSSION:

Thus, we showed that the Butterfly Extract contains toxic alkaloids & cardenolides which have the capacity to kill the larvae of the DBM, the pest of spinach. As expected results were interpreted and calculated and it was seen that the 2% concentration of extract showed the best results i.e., 68.99% mortality followed by 0.25% concentration of extract which showed 42.91% mortality. The data obtained from the graph shows the best results in 2% Extract, but the 3% extract has shown a diminished mortality rate because the larvae did not feed on such a higher concentration of extract sprayed leaf. Our next step is to procure more of 2% and 0.25% Extracts which have given the Lethal Doses for larvae mortality and subject them to purification procedures of Dialysis and HPLC for separation, concentrating and characterization of the toxic alkaloids such that we can proceed to Field Trials. But due to acute shortage of Butterfly eggs our experiment has come to a standstill but will begin as soon as we collect some 20-25 eggs of Striped Tiger. We also wish to test the potency of these potential bio-insecticides on higher group of pests like beetles, borers etc. Thus, we are able to show that the cardenolides present in the butterfly extract can be used as a potential Bio-insecticide. Insects can be an economical source of extracting bio-insecticides. Moreover, they are small in size, have a very short life span or cycle, have small body size and hence can easily be handled as Models of experiments. Also they are widely available and performing experiments on them would prove to be a very cost-effective process. Thus, insects are the best “Biochemists”. Our work as a part of Chemical Ecology will help to show the importance of conserving Nature and it’s Natural Resources, so that we can continue to reap such benefits over and over again. As had been discussed earlier, most of the research in this area has been in isolating and characterizing the cardenolides either directly from plants or from the insects; however our’s has been the first such attempt to study and analyze the effect of these toxic cardenolides present in the butterfly extract on common pests and their prospect as a potential Bio-insecticide.

ACKNOWLEDGEMENT:

My sincere thanks to Dr. K. Chandrasekhara for guiding me in the project. Special thanks also to my team members Dr. Revanna, Dr. Murali, Dr. Jayappa, Dr. A.R.V. Kumar and Mr. Chandan. Also my regards to my parents and my respected Teachers.

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