Analysis of Cucurbitacins in Cucumber plants and Examination of Cucumber Beetle Behavior

By Dominique Hedderich and Kara McDonagh

Abstract

Figure 1. Cucumis Sativus

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.

Introduction

Figure 2. Cucurbitacin

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.

Figure 3. Striped and Spotted Cucumber Beetles

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

Table B. The standard Rf values of cucurbitacins

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.

Table A. The calculated Rf values of examined plant matter

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.

Table C. Standard Concentration using HPLC showed peak areas

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.

Figure 4. Standard curve for estimation of cucurbitacin concentrations in cucumber leaf tissue using HPLC

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.

Acknowledgements

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.

References

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.

 

 

 

  1. Arnold T, Witter A. 2011. Cucurbaticans as kairomones for cucumber beetles.  NSF Chemical Ecology 1: 1-4. []
  2. “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>. []
  3. 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. []
  4. 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. []
  5. Ref: 2 []

Studying Cucurbitacins and Cucumber Beetle Behavior Through TLC and HPLC Anaysis

Abstract

 There were two methods used to analyze the cucurbitacins and cucumber beetle behavior.  The first step to TLC analysis was to make cucumber extracts.  After this, the extracts were used to isolate the cucrbitacins using thin layer chromatography.  Then, marks would be made where ever spots showed up on the TLC plate and observations would be made about what spots on the plates would be consumed by the beetles.  The reason for this experiment was to isolate the terpenes that are feeding stimulants.  

There are less steps in HPLC because the machine does much of the work.  The extractions of cucumber leaves were made and texted on 3 different callibration standards.  The data was used in order to quantify the major form of cucurbitacin in the cucumber extract.  Also, the data was used to see how the HPLC would support or refute the data obtained from the TLC lab. 

Flock of Cucumber Beetle in Cucumber flower

Introduction

For the lab we were able to go to the Dickinson College farm in Carlisle, PA and hand pick sample plant for the experiment. This is a good procedure for taking plant sample so that you know what you have and there are no questions about where it came from and who was the one who retrieved the sample. After deliberation we chose to use the cucumber stem as we felt that it would provide the best results for our project. We chose to do this experiment to see the relationship between the cucurbitacins that come from the cucumber plants and what impact they have on herbivores and others eating the plant. After some research we found that the that the bitter compound found in the cucumber plant is the before mentioned cucurbitacins. This compound which is found in the both the flower and actual body of the plant is thought to be used as a protecting force from herbivores because it is extremely bitter and even can cause sickness to certain animals. The scientific name for this sort of relationship is known as chemical ecology. Chemical ecology is defined as the study of chemicals that are involved in interactions between living organisms. Some examples of the type of chemicals that are studied is this field are venoms, deterrants, phermones, toxins, and attractants. In this same category is chemicals that provide protection for the plant in varying ways like the cucurbitacins do for the cucumber plant. As we continue to talk about the cucurbitacins, we must also talk about their compository-compound which are called terpenes. This compository-compound as stated before creates an extremely high toxicity for many herbivores and animals. These terpenes are extremely unstable which leads to an extremely foul smell that is a deterrent to many animals after entering their nose. “The signals are sent to what is called the primary olfactory area of  the brain. At this point we become aware of the odor. The primary olfactory is part of the lymbic system in the brain, and this probably causes certain odors and tastes to have strong emotional content or may cause recall of past events.1

As in many cases in the world of science there are oddities that defy the common trend and create problems are pretty unique. With the cucumber plant, its main nemesis is a bug called the cucumber beetle. This beetle is attracted by the cucurbitacins and feasts on the cucumber plant as a result. You can tell when the field has been infested by these beetles when the leaves and flowers look amost like swiss chesese with very many small holes.

 

Beetle damage

 These cucumber beetles have two different patterns, either spotted or striped with the most common colors being black and gold. How these beetles operate is that they will wait until the plant begins to mature and will then eat and destroy vital organs in the plant which deter the ability to grow and mature into a fully developed plant. This problem happens all throughout the United States and if the infestation gets bad enough can cripple the market on these cucumber and similar vegetables. These beetles cause numerous short and long term diseases and problems for the plants which can take up to years to get back to good health.

 Finally, during the first experiment we will be use high-pressure liquid chromatography (HPLC)  to separate the different forms of molecules present in the cucurbitacin samples.For the second experiment which will show the cucurbitacins and cucumber beetle behavior experiment, we will use thin layer chromatography (TLC)  to detect whether cucurbitacins are present in the samples of various leaves and blossoms. For this part of the lab, “the sample to be chromatographed was place as a small dot near the nottom of the strip and pencil lines were made on the adsorbent to indicate the orginal position of the sample and the desired length of solvent travel.2

Materials and Methods

To begin the TLC lab we obtained pieces of cucumber and squash leaves, roots, and flowers.  We cleaned and cut 112.5 mg of squash leaves, 107 mg of cucumber flowers, 105.9 mg of squash flowers, and 103.8 mg of cucumber leaves.  These were then added to the drinding tubes.  We grinded the extracts by hand for approximately 5 minutes.  Then 1ml fo extraction solvent (95% chloroform: 5% methanol) was added to each tube.  The tubes were covered and inverted several times.  We then waited for 5 minutes so the tissues could extract.  We set up a thin layer chromatography plate by drawing a thin line 2cm from the bottom of the plate.

Next, using a capalary tube  in the fume hood we applied all the extracts to the TLC plate along the thin line.  We labeled each spot appropriately and proceeded to add 75ml of the “running solvent” chloroform: methonal (95:5) to the chamber, making sure that the level of the solvent would not reach the thin line on the TLC plate.  Then, we carefully lowered the plate, covered the plate and waited till the solvent rose up to within 3-4cm of the top of the plate.   When the solvent reach this point we removed the plates from the jar and quickly drew a line at the solvent front to indicate how far the solvent had traveled.  We then let the plate dry in the fume hood.  After the plate had dried we lighltly marked the spots that were visible in regular light.  From there we used a UV light to ditect invisible compounds and marked those spots as well.  We determined the RF values for each compound using the following chart.

  Rf value
Compound Chloroform Solvent
Cuc E 0.8
Cuc B 0.77
Cuc I 0.72
Cuc L 0.59
Cuc D 0.7
Cuc E-glycoside 0.27

We took pictures of the TLC plate and after 5 days returned to the lab to inspect the plate.  We compared the areas that were consumed by the beetles to the picture we had previously taken and determined the Rf value of the feeding sites. 

For the HPLC lab we weighed out ?mg of cucumber blowwoms and put it into a test tube.  After this we added chloroform to 3/4 of the test tube and inserted one small glass bead.  We used the bead beater to shake the sample for approximately 30 seconds.  We pipetted off the chloroform and transferred the liquid to a clean vial.  Next, we evaporated the chloroform using heated sand and gentle air pressure.  Then, we added 1ml of H2O, followed by 2mL of Hexan and shook the sample.  After we let some time pass in order to seperate the two layers we pippeted off the upper layer (the Hexan).  Then 1mL of 50:50 bethanol:acetate was add to the top of the water layer, we centrifuged the sample and seperated the upper layer.  We evaporated the vial and then labeled it appropraitely.  This vial was then used in the HPLC machines and the resulting data was used, as previously noted, to quantify the major form of cucurbitacin in the cucumber extract and to see how the HPLC would support or refute the data obtained from the TLC lab.

Results and Discussion

Figure 1A Results from HPLC

Figure 1A Results from HPLC

 

Band #

CB Rf value

CL Rf value

SB Rf value

SL Rf value

Observations

1

1.00

1.00

1.00

1.00

bright green – common extract color

2

0.84

0.81

0.79

0.86

yellow or very light green

3

0.72

0.63

0.70

0.74

darker green to bluish fade

4

0.37

0.51

0.53

0.33

UV only

5

       

UV only

6

0.12

0.26

0.23

0.16

 

7

 

0.21

     

8

 

0.09

   

 dark green

Figure 2A TLC Rf Values

The results from both the HPLC and the TLC labs were very interesting.  In Figure 1A we see the peak areas detected in from the results of the HPLC lab.  The results show that the smaller particles have a smaller peak area than the larger particles.  The level of cucrbitacin also goes up along with the concentration level increasing from 0.49mg/L to 2.0mg/L . 

The results of the TLC labs are shown in Figure 2A, here we can see the various Rf values listed for the different samples.  The beetles ate two different spots on our TLC plate, the first was on the cucumber leaves with an Rf value of 0.81, which is most likely compound Cuc E.  The second spot was on the squash leaves at the Rf value of 0.74, which could, according to the chart shown in the Materials and Methods section, be either Cuc b or Cuc I.  It is interesting that the beetles ate from the leaves on both but not from the buds. 

The results also prove that we were able to successfully isolate the cucurbaticins.  In the TLC this is proven because the beetle ate from the highest, and  next to the highest Rf values of both squash and cucumber leaves, showing that the cucurbitacins were isolated and rising with the solvent.  In the HPLC this is proven because of the cucurbitacin levels rising with the concentration levels.

Acknowledgements

We would like to acknowledge Professors Arnold and Witter for their commitment and effort in teaching a class like this for the first time at Dickinson College.  Also, we would like to give a special thanks to both the TAs, who put in several extra hours to help make our labs possible.  Finally, we would like to thank our lab partners and other classmates for being so helpful and making our labs and classes a worthwhile and an enjoyable atmosphere.

References

1.Glaeske, Kevin, and Paul Boehlke. “The American Biology Teacher.” Making Sense of Terpenes: An exploration into Biological Chemistry. National Association of Biology Teachers, 2002. Web. 7 Dec 2011. <http://www.bioone.org/doi/full>.

 2.Durso, Donald. “Automatic Fraction Collector for Chromatographic Separations.” Diss. Purdue University, 1951. Print.

 

  1. Glaeske, Kevin, and Paul Boehlke. “The American Biology Teacher.” Making Sense of Terpenes: An exploration into Biological Chemistry. National Association of Biology Teachers, 2002. Web. 7 Dec 2011. []
  2. Durso, Donald. “Automatic Fraction Collector for Chromatographic Separations.” Diss. Purdue University, 1951. Print. []