By Dominique Hedderich and Kara McDonagh
The cucurbitacin and cucumber beetle behavior biology lab were used to inspect two types of cucumber beetles, spotted and striped, using a dissecting scope. The second portion of this lab was used to test cucurbitacin levels in cucumber and squash samples. Extracts were made from the leaves, roots, and flower parts of cucumber and squash plants. The samples were run through two tests: thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC). The TLC test was used to measure the different cucurbitacins levels in the blossoms, leaves, and flowers of cucumber and squash plants. The HPLC test was used to quantify the amount of cucurbitacin B in cucumber and squash leaves. The plate from the TLC test was then put in the beetle enclosure and left for five days. It was hoped that this would confirm the presence of cucurbitacins in squash and cucumber plant parts, yet the beetles ate little off of the plate. We then referred to the standard Rf values of cucurbitacins, and were able to calculate the levels of cucurbitacins in the plants based off of the TLC plate measurements. The different parts of the plants contained different types of cucurbitacin, including cucurbitacin B, E, and L. Using the HPLC test, cucurbitacin concentrations in a sample of 100mg was determined. The equation: concentration of Cuc B. = mg cucurbitacin / g plant matter bore the results that there was 0.093mg/mL of cucurbitacin B found in the cucumber leaf sample and in the squash leaf sample, the concentration of cucurbitacin B was 0.0566 mg/mL.
This lab examined cucurbitacins in cucumber and squash plants, as well as cucumber beetle behavior. The cucumber is a common vegetable. It has three varieties: slicing, pickling and burpless. The cucumber is classified as a taxonomic group, originating in India. It belongs to the kingdom Plantae. Both cucumbers and squash are from the family Cucurbitaceae. The plants observed in this lab is the Cucumis sativus. They produce bitter tasting compounds known as cucurbitacins. There are many different types of cucurbitacin including A, B, C and more. Cucurbitacins protect the plant from being eaten, and are harmful to most herbivores. The cucurbitacin is an example of a terpene, it is made from isoprene units.
Many herbivores cannot tolerate the cucurbitacins, yet some insects from the genus Diabrotica thrive off of the terpene. It stimulates compulsive feeding and is not harmful to the insects. 1
Cucumber beetles come from the phylum arthropoda, which is the largest and most diverse animal taxon. They can be classified into two types, striped and spotted, and have the common features of other beetles. The striped beetle is one quarter of an inch long with three black stripes down its back. The spotted beetle is also generally one quarter of an inch long, with approximately twelve spots on its back. 2 The female is often much larger than the male, and the spotted cucumber beetle is the larger of the two insects. 3 The cucumber beetle usually appears when the temperatures begin to warm in early to mid spring. The adult beetles primarily feed on the leaves, blossoms and extremities of the plant. The female cucumber beetles lay their eggs in the cracks of the dirt surrounding the base of the plant and the larvae feed on the roots as they begin to grow and develop. The beetles are also known to release pathogens which can kill crop plants. Bacterial Wilt is caused by cucumber beetles as they eat a portion of the plant, and then through either fecal matter or infected mouth parts, they spread the bacteria into the damaged leaves. One leaf usually wilts, soon followed by the rest of the plant. Insecticides 4 have proven to solve the problem of Bacterial Wilt. 5 This lab was used to determine the levels of cucurbitacin in squash and cucumber plants. The different plant parts were tested to see which the cucumber beetles ate most, signifying large concentrations of cucurbitacins.
Materials and Methods
The beetles were inspected using a dissecting scope. Anatomical features such as thorax, abdomen, antennae, mandible and maxilla were identified. It was seen that the banded beetle was smaller than the spotted beetle. The beetles were then released into the beetle enclosure to be used later to test part two of the lab.
Extracts of cucumber and squash were made using 100 milligrams from cucumber leaves, roots and flowers that were then placed into separate grinding tubes. The samples were ground at high speed for five minutes. For both the squash and cucumber leaf samples, liquid nitrogen was added. The leaves froze instantly and were then very easily mashed with the grinding ball. A 95% chloroform: 5% methanol solvent was then added to each tube, shaken and sat for five minutes. A thin layer chromatography (TLC) plate was prepared, with a light line drawn across the bottom of the plate, marking where the extracts were to be placed, three centimeters above the bottom of the plate. 20ul of each extract was then placed along the line on the bottom of the plate and marked. After this, the plate was put in 75 milliliters of ‘running solvent,’ a chloroform: methanol (95:5) solution. The plate sat for approximately twenty minutes, and during this twenty minutes, the solvent began to creep up the plate. Once the solvent nearly reached the top of the TLC plate, it was removed and the results were visible. Lines and marks were noted with a pencil. Using a UV light, any compounds that had not been visible to the eye were detected. The plate was then put in the beetle enclosure for five days. After this time period, the plates were to be removed and examined. They should show visible markings signifying the beetles ate the cucurbitacins.
To begin the second part of the lab, 100 milligrams of cucumber leaves were weighed and put into a test tube. Chloroform was then added to the vial along with a glass bead. The bead beater was then used to shred the sample. The chloroform was pipeted off and put into a clean vial. Using light air pressure, the chloroform, in the new vial was evaporated, leaving a thin layer of dried plant material. Next, one milliliter of water was added as well as two milliliters of hexane. The layers were then left to separate and the top hexane layer was pipeted off. Only the water layer remained. One milliliter of 50:50 butanol: ethyl acetate was then added to the water and the vial was put into the centrifuge to be shaken for one minute. The layers were again separated and the pipet was used to move the upper layer of the butanol acetate into yet another vial. It was then evaporated in sand, under light air pressure. The sample was then taken to the HPLC machine. The process was set up using certain requirements for proper analysis of the cucumber leaves. Standards were run for 0.5 mg/mL, 1.0 mg/mL, and 2.0 mg/mL as well as the unknown sample. Use the standards to determine the concentration of the cucumber leaves. ((Hedderich, Dominique; “Lab Manual.”))
Results and Discussion
After five days in the cucumber beetle enclosure, the beetles showed to have eaten little off of the plates. Therefore, it was difficult to determine whether the samples contained high levels of cucurbitacins. In order to find out, we calculated the Rf values of the marks from each of the four tissues using the formula:
Distance Traveled/ Solvent Front Distance. We then compared them to the table below that contained the standard Rf values for differing types of cucurbitacin compounds.
It was determined that the squash leaves contained the Cucurbitacin L, the cucumber leaves contained Cucurbitacin B, the cucumber blossoms potentially contained Cucurbitacin E, and the squash blossoms contained Cucurbitacin L. With the given information it was determined that the squash leaf contained the greatest amounts of cucurbitacin and it showed the brightest color on the TLC plate.
After running the HPLC sample through the machine, we were able to determine the amount of cucurbitacin B in the cucumber and squash leaves. To find the equation, a scatter plot was formulated using the standards that had been run through the machine. An equation was then generated from this scatter plot.
Using the equation, it was determined that the concentration of cucurbitacin in cucumber leaves was 0.093mg/mL. (source)
y= 787.92x – 73.2 The equation was solved and it showed the result 0.093.
In examination of the squash leaves, the same system was used: standards were recorded, a graph was created, and an equation developed. It was determined that the concentration of cucurbitacin B in squash leaves was 0.0566 mg/mL.
Though it was determined that there was a large amount of cucurbitacin B in the cucumber leaves, the cucumber beetles, did not responded accurately to the TLC plates. They ate little of any of the samples. This may be the result of putting too many TLC plates in the beetle enclosure for examination. The ratio of beetles to cucurbitacin may have been too high. However, nothing difinitive can be said. We were also able to determine levels of different cucurbitacins in the different parts of plants.
Unfortunately, this research did not give concrete results. Although we were able to calculate the Rf values and therefore estimate the differing types of cucurbitacins in each part of the plant, the cucumber beetles did not eat any part of the TLC plate. This could be due to a reason of factors that will need to be researched. In the second part of the lab, we were able to determine that the concentration of cucurbitacin B in cucumber leaves was 0.093 mg/mL and in squash leaves it was 0.0566 mg/mL. This lab proved that there are high amounts of cucurbitacins found in both cucumbers and squash.
Kara and I would like to thank our professors Thomas Arnold and Amy Witter for their aid and guidance in the research for this study. We would also like to thank the Dickinson College Farm for supplying us with the insects and plant matter used in this research.
Arnold T, Witter A. 2011. Biology Cucurbaticins and cucumber beetle behavior. NSF Chemical Ecology 1: 1-3.
Arnold T, Witter A. 2011. Cucurbaticans as kairomones for cucumber beetles. NSF Chemical Ecology 1: 1-4.
Arnold T, Witter A. 2011. Terpenes. NSF Chemical Ecology 1: 1-4.
Cabrera, Nora; Walsh, Guillermo Cabrera. 2004. Diabrotica calchaqui, a New Species of Luperini (Coleoptera:Chrysomelidae: Galerucinae), From Argentina. Annals of the Entomological Society of America, 97 (5) : 889-896.
Cucumber Beetles | University of Kentucky Entomology. Learning, Discovery, Service in the College of Agriculture. N.p., n.d. Web. 5 Dec. 2011. <http://www.ca.uky.edu/entomology/entfacts/ef311.asp>.
Hedderich, Dominique. 2011. Lab Manual: HPLC of Cucurbitacins Isolated from Cucumber Plants.
Schroder, Robert F; Martin, Phyllis A. W.; Athanas, Michael M. 2001. Effect of a Phloxine B-Cucurbitacin Bait on Diabroticite Beetles (Coleoptera: Chrysomelidae). Entomological Society of America, 94 (4): 892-897.
- Arnold T, Witter A. 2011. Cucurbaticans as kairomones for cucumber beetles. NSF Chemical Ecology 1: 1-4. [↩]
- “Cucumber Beetles | University of Kentucky Entomology.” Learning, Discovery, Service in the College of Agriculture. N.p., n.d. Web. 5 Dec. 2011. <http://www.ca.uky.edu/entomology/entfacts/ef311.asp>. [↩]
- Cabrera, Nora; Walsh, Guillermo Cabrera. 2004. Diabrotica calchaqui, a New Species of Luperini (Coleoptera:Chrysomelidae: Galerucinae), From Argentina. Annals of the Entomological Society of America, 97 (5) : 889-896. [↩]
- Schroder, Robert F; Martin, Phyllis A. W.; Athanas, Michael M. 2001. Effect of a Phloxine B-Cucurbitacin Bait on Diabroticite Beetles (Coleoptera: Chrysomelidae). Entomological Society of America, 94 (4): 892-897. [↩]
- Ref: 2 [↩]