From an Inquiry-Guided Project to a CURE in General Biology: Testing Repellent Effects of Essential Oils and a Parasitoid Wasp Against Callosobruchus maculatus.

Joseph W. Felts, Davidson County Community College

Location: North Carolina

Abstract

Agricultural pests damage and destroy substantial amounts of crops worldwide, as well as post-harvest stores yearly. Depending on the economic status and geographic location, anywhere from 25% to 60% of crops are lost each year to pests. In the developing areas of the world, the rates at which crops are lost combined with increasing population size, the development of pesticide resistance, and the inability to purchase newer synthetic pesticides leaves crops and stored materials vulnerable to such pests. One economically important example is the Cowpea Weevil, Callosobruchus maculatus. As a pest, C. maculatus is the main source for wide-spread damage to stores of pulse (lentils, dry peas, beans and chickpeas) crops leading to as much as 60% loss in some cases (Radha and Susheela 2014). As a means to finding alternatives to expensive, synthetic, environmentally damaging pesticides in controlling the weevil, the use of aromatic plant extracts that act as either natural insecticides or repellents that prevent damage to local community stores has been and is still being explored. In addition to these extracts (essential oils), other control methods, such as the use of parasitoid wasps or other predators/parasitoids of pests, are being explored.

The purpose of the CURE described below, is to continue this research into the use of both essential oils and parasitoid wasps as potential biological controls against C. maculatus. In particular, the project seeks to examine and determine the effects of using a combination of the two control methods (Essential Oils + Parasitoid) against the weevil. With the utilization of a guided inquiry project initially, students build a foundation and transition to an authentic research experience using C. maculatus, essential oils, and the parasitoid Anisopteromalus calandrae as a model system to develop and test hypotheses regarding these bio-control measures.

Student Goals

  1. Students will learn to help design an experiment that aims to advance knowledge regarding methodology to control Cowpea Weevils that could be applied in low-to-no cost scenarios.
  2. Students will learn to collaborate in determining Experimental Design, the equitable sharing of project responsibilities, and communication of results to others, including producing and presenting a group scientific poster.
  3. Students will be able to conduct an appropriately sized literature review and contribute to the growth of a research library regarding both species that can be utilized by future General Biology students.

Research Goals

  1. Determine the efficacy of using essential oils and the parasitoid wasp, Anisopteromalus calandrae, as bio-control agents for Callosobruchus maculatus.
  2. Determining if cowpea weevils (Callosobruchus maculatus) can detect the recent presence of parasitoid wasps using a body-wash and whether this can be applied as a biological control method?

Context

This CURE was designed within the framework of an Introductory General Biology major's course of approximately 180 total students, after it was expanded to include students in a fellow faculty member's course. The project begins with a guided inquiry based project focused on cowpea weevils (Callosobruchus maculatus), also referred to as bean beetles, and learning experimental design. We then use that foundation to transition to an actual authentic research experience. The guided-inquiry is a edited version of "Using bean beetles to teach experimental design and experimental variables" (D'Acosta and Schlueter, 2014) as well as other materials from the Bean Beetle Website. The CURE utilizes C. maculatus, essential oils and a parasitoid wasp (Anisopteromalus calandrae) to produce a host-parasitod model system to test repellency and associated hypotheses. The use of a parasitoid wasp closely follows from the non-consumptive effects predators can have on prey species, but in this case, it is a parasitoid on reproductive success (i.e. survival of offspring). The CURE was developed with a community college biology course and student population in mind. The guided inquiry provides the basic knowledge and skills that students need in a CURE based project, and allows for more equitable experiences for community college students whose university peers are already taking part in such projects. It is also low cost compared to other high profile CUREs taking place at research universities and some larger community colleges. The needed time for the CURE could be very variable, but my goal is to keep this to less than a half-semester (after the guided-inquiry is complete) and to utilize a second CURE that covers biotechnology and antibiotic resistance.

Target Audience:Introductory
CURE Duration:Half a term

CURE Design

This CURE has a dual focus: 1) an applied research aspect in one area (essential oils as repellents) and a basic research scenario utilizing the parasitoid wasp and beetle avoidance behavior, in the other. Both areas have the potential to guide and provide more focused directions for future research. In application, students will focus on determining the effects of cheaper, environmentally safer essential oils as repellents. The potential use of the parasitoid wasp as a bio-control measure against of C. maculatus is also explored. Pursuing the wasp effect utilizes the life-history traits (avoidance behaviors) of the model organism and also opens the door to the pursuit of non-lethal effects parasitoids may have on host species, similar to the non-consumptive effects predators have on prey. Testing hypotheses related to those two areas potentially move research into biological control forward to the next areas that are unexplored. Answers regarding host (C. maculatus) behavior towards essential oils and A. calandrae, in addition to the roles they play in decisions, may be used to make better decisions about controlling the current buchrid beetle being investigated. Increased knowledge in these two areas can then be used to mitigate the use of more economically expensive and environmentally degrading control measures (pesticides) while at the same time increasing the amount of pulse (bean) stores in economically disadvantaged areas of the world.

Core Competencies: Analyzing and interpreting data, Asking questions (for science) and defining problems (for engineering), Planning and carrying out investigations
Nature of Research:

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓
Research Goal 1: Determine the efficacy of using essential oils as bio-control agents for Callosobruchus maculatus.
Research Goal 2: Can cowpea weevils (Callosobruchus maculatus) detect the recent presence of parasitoid wasps and can this be applied in the use of bio-control?.


Student Goal 1: Students will help design an experiment that aims to advance knowledge regarding methodology to control Cowpea Weevils that could be applied in low-to-no cost scenarios.

1) Students will use choice-chambers to design experiments using essentials oils to determine their use as effective bio-control agents. 2) Students will determine the correct type of data to collect and the proper statistical analysis within the framework of an introductory biology course.

1) Students will use choice-chambers to design experiments using body-wash of parasitoid wasp to determine any avoidance behaviors.



Student Goal 2: Students will learn to collaborate in determining Experimental Design, the equitable sharing of project responsibilities, and communication of results to others.

1) Students will utilize Moodle Workshop Functionality to work in collaborative groups online and in-person to a) develop, b) design, and c) critique ("peer-review") each groups design. 2) Students will negotiate, then reach a consensus and determine the equitable sharing of project responsibilities, 3) Properly communicate the results and conclusions in either written and/or oral presentations via professional scientific poster.



Student Goal 3: Students will be able to conduct an appropriately sized literature review and contribute to the growth of a research library regarding both species that can be utilized by future General Biology students.

1) Each student must complete the "ProQuest Research Companion" Learning Modules and submit assessment. 2) Each student must contribute 2 papers via reference software Mendeley that apply to the research topic, with annotations to group "paper pool" and answers to reviewing literature framework questions. 3) Students that acquire peer-reviewed, research articles will contribute them to course research library.


Instructional Materials

Example Lab Schedule

Example Semester Lab Schedule CUREnet.pdf (Acrobat (PDF) 60kB Mar11 19)

Pre-lab Homework for Experimental Design Jigsaw Activity.

BIO 111 Exp Design homework jigsaw.pdf (Acrobat (PDF) 54kB Feb17 19)


Individual Group Packets for Experimental Design Jigsaw. Includes Inquiry-guided part of project.

Exp Design Lab Group_A.pdf (Acrobat (PDF) 1.1MB Feb17 19)

Exp Design Lab Group_B.pdf (Acrobat (PDF) 1.1MB Feb17 19)

Exp Design Lab Group_C.pdf (Acrobat (PDF) 1.1MB Feb17 19)

Exp Design Lab Group_D.pdf (Acrobat (PDF) 1.1MB Feb17 19)

Quantitative Reasoning Skills

I use the MathBench Bio modules to develop and reinforce quatitative reasoning skills in my courses. I time their use as to have intorudction and using t-tests overlap with the week students conduct their first choice-chamber experiments. The MathBench BIO modules can be accessed at the following MathBench Website

Mendeley Reference Software

For building personal research library. The free software can be downloaded on the Mendeley Website

Annotations on Mendeley (Acrobat (PDF) 325kB Jul17 18)

Introduction to Parasitoids Lab

This lab introduces students to parasitoids and sets the stage for the first research lab using essential oils.

Introduction to Parasitoids Lab_Final.pdf (Acrobat (PDF) 653kB Mar10 19)

Essential Oils and Bean Beetle Lab

Testing EO Methods_Updated.pdf (Acrobat (PDF) 211kB Mar10 19)

Parasitoid Wasp Body Wash Lab

Method Parasitoid Body Wash_Updated.pdf (Acrobat (PDF) 300kB Mar10 19)


The following files are used after students begin building their research library and help with their review and analysis of the papers.

Figure Facts Template (Acrobat (PDF) 46kB Mar5 18)

Research Article Submission Form Example

Google Research Article Submission Form (Acrobat (PDF) 100kB Jul17 18)

Choice Chambers

Assessment

Students will be assessed using three different pre/post test assessments. Students will be given the Test of Scientific Literacy Skills Test (TOSLS), the Statistical Reasoning in Biology Concept Inventory, and the Experimental Design First Year Undergraduate Concept Inventory. The TOSLS is available at this link (Gormally et al., 2012), but both Concept Inventories must be obtained from the Q4B (Questions for Biology) Group at the University of British Columbia.

Students will be assessed via their completion of the following ProQuest Research Companion Activity.

Proquest Assessment (Acrobat (PDF) 272kB Jul17 18)

Students will be assessed via the production of a group professional, conference level scientific poster. It will be presented to their lab section.

Example of Student Poster Fall 2018 followed by rubric.

Bean Beetle - Essential Oil Student Poster_ Fall 2018.pdf (Acrobat (PDF) 480kB Feb17 19)

BIO 111 Group Scientific Poster Rubric.pdf (Acrobat (PDF) 79kB Feb18 19)

Students also maintain a lab notebook.

Instructional Staffing

As this is being constructed in a community college environment, and as such, the instructional staffing resources can vary widely, application of the generalities presented here should be applied and revised as needed. Lab technician/coordinator should be up to date on all current procedures for the rearing the insect species involved. As one species is completely dependent on the other in diet, one should be confident in rearing beetles and having an adequately sized population that could sustain a parasitoid wasp colony. Lab coordinators should also be familiar enough with the design of the CURE to be able to oversee labs if no one else is available to do so. Make sure lab coordinators have up to date supply lists and are comfortable with any prep work prior to beginning the CURE. I suggest providing the entire set-up protocol and supply list at least 3 months before work is scheduled to begin. In other situations, community college instructors do not have access to other instructional staff as at universities and in those situations, one should take care to inform all those involved with the CURE should be given adequate instruction and information for its successful implementation.


Author Experience

Joseph W. Felts, Davidson County Community College

I have designed this CURE to be unique among community colleges in that it is constructed by transitioning from a guided inquiry-based project using cowpea weevils (Callosobruchus maculatus) and resources available on BeanBeetles.org to a Course-based Research Experience in a first semester General Biology course. This allows for the teaching of the process of science and experimental design prior to research based activities. The CURE tests the repellent effects of essential oils against the cowpea beetle. It also utilizes a parasitoid wasp in a host-parasitoid model to test for repellent effects of the detection of the wasp using a body-wash. By providing an inquiry guided approach that transitions to a full CURE, allows the teaching and application of concepts and skills many first semester biology students lack and will provide a more equitable starting point for community college students with their non-transfer peers.

Advice for Implementation

1) Before beginning this CURE, whether you use the Inquiry-Guided project or not, my first piece of advice: be sure that you have plenty of C. maculatus cultures already producing offspring and for several weeks before you begin.

2) Have a variety of essential oils from which students can choose. I use the same company for all the oils we use to remove any confounding factors of different manufacturers. If you have access to a chemistry lab and distillation equipment, you can also produce your own oils from local flora. Which is an area I wish to pursue once I have all the equipment.

3) If you wish to use parasitoid wasps, you will need to contact the USDA to get an APHIS permit (if you use A. calandrae) to obtain the wasps. I obtained my wasps from the USDA lab in Kansas. If you wish to utilize a different species, be sure to check all requirements for obtaining and culturing them.

4) You will need to have choice-chambers for the experiments. I obtained mine from Carolina Biological Supply. Note* If you use these chambers, do not get acetone on the plastic.

5) When inoculating Whatman filter paper with essential oil solutions (we currently are testing 5% solutions in acetone), sterilize lab top surfaces and place solution on filter paper outside the chamber on lab table. Then let air dry prior to placement in chamber. If not, you run the risk if the oil drying and causing the paper to become "glued" to the plastic.


Iteration

Students are able to choose their inquiry based project, where many learn, fail and repeat their experiments after they have modified them. If the research based lab experiences, students are able to choose which essential oils, combinations of essential oils, etc. Student's must trouble shoot any time there is disagreement over which side a beetle has chosen if still within the insertion point. Students have to replicate each choice-chamber and sometimes modify the numbers they use if for example there were too few beetles because of a bad culture. Currently students are considering changes in the parasitoid lab because initial results showed no avoidance behavior or males towards the body wash but marginally significant results from females (0.10

0.05). Students are trying to develop ways to increase the concentration of cuticle chemicals.

Using CURE Data

Data collected from this CURE will be stored annually on Mendeley Data with Metadata including all student participants. Any other publication will either result in student co-authors, or a large acknowledgements sections, depending on the journal to which the manuscript is to be submitted. If anyone is interested in keeping a national data bank via Google, please contact me.

Resources

D'Costa A, Schlueter M. 2014. Using bean beetles to teach experimental design and experimental variables – Bean Beetles. www.beanbeetles.org. [accessed 2018 Feb 12]. https://www.beanbeetles.org/protocols/experimental-design/.

Blumer LS, Beck CW. Bean Beetles – A Model Organism for Inquiry-based Undergraduate Laboratories. https://www.beanbeetles.org/. [accessed 2018 Feb 12]. https://www.beanbeetles.org/.

Bean Beetle Handbook.pdf (Acrobat (PDF) 2.4MB Feb18 19)