Effects of Trypsin Inhibitors on the Growth of Manduca sexta

By Lea Serres

AbstractThis study was performed in order to test how growth of Manduca sexta was altered by the trypsin inhibitor from Glycine max (soybean). The control group (n=50) was

fed artificial medium with no trypsin inhibitor added, whereas the two variable groups had added trypsin

inhibitor to the medium. Measurements were taken on the fifth, tenth and fifteenth days of larval development.

Data were then analyzed. For Group 1, day 5, the average length was 1.4 cm. On day 10 it was 3.8 cm, and on day 15 it was 5.9 cm. In Group 2, day 5 growth

averaged to 1.2 cm, day 10 to 2.9 cm, and day 15 to 5.0 cm. In Group 3, day 5 growth averaged out to 1.1 cm, day 10 to 2.2 cm, and day 15 to 4.5 cm. The standard

deviation for Group 1 averaged out to 0.35, in Group 2, 0.54, and in Group 3, 0.25. There was a significant

difference in the data.

Knowledge Base• The larval stage of the life cycle of

Manduca sexta generally lasts approximately 18 days

• Trypsin Inhibitors are chemicals that reduce the amount of trypsin (a chemical that allows for healthy growth) produced in an organism

• Trypsin- a protease that breaks down proteins in the digestive system. Trypsin is necessary for the uptake of proteins in an organism, which allows for healthy growth.

Trypsin inhibitor complex

Manduca sexta

Literature Review

Z. Madar• The trypsin inhibitor had an effect on the

organisms tested by the fourth and fifth days, but the most prominent effects of the chemical occurred around the seventh and tenth days

Reinecke, John P., Buckner, J.S., Grugel, S.R.• Most rapid growth occurred approximately 10-12

days after hatching of larvae compared to the means of the rest of the larval stages

Need

• Pesticides dangerous to humans, the environment and often cause crop damage

• Can cause damage to the nervous and reproductive systems, and cause disruption to hormone and immune function

• Trypsin Inhibitors slow production of trypsin in organisms, impairing growth

• Preferable (in terms of that there are less dangers) to pesticides to rid crops of pests in a safer way

Purpose

The purpose of this experiment was to investigate the effects of various

concentrations of Glycene max (soybean) trypsin inhibitors on the growth of

Manduca sexta larvae.

Null Hypothesis

The Null Hypothesis, or H(o), states that the means of growth in Group 1, or the control group will not equal the means of growth in

Group 2 and Group 3, or the variable groups.

Alternative Hypothesis

The Alternative Hypothesis, or H(a), states that the means of growth in Group 1, or

the control group, will not equal the means of growth in Group 2 and Group 3, but

rather that the means of growth in Group 3 will be the lowest, and the means of

growth in Group 1 will be the highest.

Effects of Trypsin Inhibitor on the Growth Rate of Manduca sexta

50 Tobacco Hornworms

Control Group Variable 1

50 Tobacco Hornworms

50 Tobacco Hornworms

Variable 2

2 grams artificial diet per day

2 grams soybean trypsin inhibitor added

to food

1 gram soybean trypsin inhibitor added to food

2 grams artificial diet per day

2 grams artificial diet per day

no soybean trypsin inhibitor added to food

Worms kept at temperature of 26 degrees Celsius

20 day trials run (larval stage of metamorphosis) through all groups. Larvae were measured in centimeters on the 5th, 10th, and 15th days of

development

The Effects of the Glycine max Trypsin Inhibitor on Manduca sexta

The Effects of the Glycene max Trypsin Inhibitor on Manduca Sexta

0

1

2

3

4

5

6

7

Group 1 Group 2 Group 3

Day Measured

Gro

wth

in

Ce

nti

me

ters

Day 5

Day 10

Day 15

In the above graph, growth in time intervals of five days is expressed. Significant difference was shown between groups. For group 1, day 5, the average length was 1.4 cm. On day 10 it was 3.8 cm, and on day 15 it was 5.9 cm. In group 2, day 5 growth averaged to 1.2 cm, on day 10, 3.8 cm, and on day 15, 5.9 cm. In group 3, day 5 growth averaged out to 1.1 cm, day 10, 2.2 cm, and day 15, 4.5 cm.

The Effects of the Glycine max Trypsin Inhibitor on Manduca sexta

The Effects of the Glycine max Trypsin Inhibitor on Manduca sexta

0

1

2

3

4

5

6

7

Day 5 Day 10 Day 15

Day Measured

Gro

wth

in

Ce

nti

me

ters

Group 1 0

Group 2 0

Group 3 0

In the above graph, a better visual is given of the growth rate of the hornworms. In Group 1, the most significant growth was between days 5 and 11, whereas in Groups 2 and 3, the most significant growth was displayed between days 10 and 15.

Discussion

• In Group 2, the growth of the hornworms on average was approximately 0.7 cm less than the hornworms in group 1, and the growth of the hornworms on average in Group 3 was approximately 0.9 cm less than the hornworms in group 1

• Trypsin inhibitor had an effect on the growth of the larvae

• Growth not significant enough to be used as pest control on crops (would just result in slightly smaller pests unless trypsin inhibitor used in larger quantities)

Conclusion

The data supports of the alternate hypothesis, and refutes the null

hypothesis. It showed a deficiency of growth in group 2 (v1), and a more

prominent reduction of growth in group 3 (v2) when compared to the control group.

The hornworms that did not ingest the Glycine max trypsin inhibitor generally had

healthier growth than those that were exposed to the chemical.

Limitations

An imperfection to this study, could have been the number of trials performed, and the number of larvae per group. My data and results could have been enhanced,

had larger sample sizes been studied, and had more trials been performed.

Measuring of the artificial hornworm diet more accurately could have also

enhanced the data quality.

Bibliography• Z. Madar. Kinetics of native and modified Bowman-Birk soya-bean trypsin inhibitor on growth and enzyme

activities of the chick pancreas. January 21, 1979. Br. J. Nutr. Volume 42, page 121.• Reinecke, John P., Buckner, J.S., Grugel, S.R. Life Cycle of Laboratory Reared Tobacco Hornworms,

Manduca Sexta, a Study of Development and Behavior, Using Time Lapse Cinematography. Bio Bull 158. 129- 140

• Kalinova, Hoskovec, Unelius, Hansson. Detection of Sex Peromone Components in Manduca sexta. Oxford Journals. Chemical Senses 26. 1175- 1186

• Safranek, Louis, Williams, Carroll M. Critical Weights for metamorphosis in the Tobacco Hornworm: Manduca sexta. Bio Bull. 167. Pages 555- 567.

• Nijhout, Frederik H., Williams, Carroll M. Metamorphosis in the Tobacco Hornworm, Manduca sexta: Cessation of Juvenile Hormone Secretion as a Trigger for Pupation. January 25, 1974. J. Exp. Biol. 61. Pages 493- 501.

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• Pimentel, D., H. Acquay, M. Biltonen, P. Rice, M. Silva, J. Nelson, V. Lipner, S. Giordano, A. Horowitz, and M. D'A-mare. "Environmental End Economic Costs of Pesticide Use." Bioscience 42 (1992): 750-760.

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• Hirata, Toshifumi. Izumi, Shunsuke. Tsuji, Seiichiro. A Protein with the GTP- Binding and Trypsin Inhibitory Activities from Glycine max. 1999. Bioscience, Biotechnology, and Biochemistry. Volume 63, Number 10. Pages 1816-1818.

• Laskowski, R.A., Hutchinson, E.G., Michie, A.D., Wallace, A.C., Jones, M.L. & Thornton, J.M. Protein Identification. 1997. Trends Biochem. Sci. Volume 22. Pages 488- 490.