Macroinvertebrates are readily available at the undergraduate level and provide an indicator of stream quality. DNA barcoding is the use of a sequence comparison of a specific region of the DNA that allows for classification of the organism. In most undergraduate courses, the discussion of genetic identification and stream quality assessment remain separated concepts. However, joining these lectures into a three-part laboratory learning module could bridge this gap and prepare students for a real-world application. During the first week of the proposed activity, students perform taxonomic identification. The following week, they perform a DNA isolation. In the last week, they use polymerase chain reaction and electrophoresis, possibly with a lecture examining a barcode for an example of the final outcome. In our research, we have determined effective methods that will allow all three sections to be completed in three three-hour undergraduate lab sessions, with minor modifications. Furthermore, the data we collected showed 54% efficiency. The methods we outline introduce new techniques and skills that prepare students for next-level education or employment and attempt to integrate ecological or environmental analysis with genetic analysis and DNA extraction techniques, making this lab series worth exploring at the undergraduate level.

Introduction

In college introductory-level biology, students are expected to retain and connect a wide range of concepts and techniques that will provide the foundation for further study of the biological sciences (Momsen et al., 2010). The second-semester General Biology course at St. Vincent College begins with the study of genetics, progresses through plant and fungal biology, and ends the semester with study of environmental and ecological science. Although the students are given a good overview of all topics covered, a common complaint of the class is the disconnection between concepts covered early in the semester and ecological study made at the end of the semester. The objective of developing the current learning module is to provide a means of aligning the study of genetics early in the semester with the stream quality assessment discussed later in the semester. In order to achieve this goal, the learning module uses the macroinvertebrates involved in biological assessment of stream quality with the isolation and analysis of the genetic data.

In the biological assessment of stream quality, many different types of organisms can be used, but their ease of capture, life cycle length, and diversity of niches occupied in the stream make macroinvertebrates an ideal group of organisms for the study of stream quality. In the present study, three critical families – caddisflies, mayflies, and stoneflies – have been selected for use in analysis of stream quality in a southwestern Pennsylvania stream that has been impacted by abandoned mine drainage and agricultural waste (Figure 1). Although these organisms work well for assessment of stream quality, classification of the organisms to the species level can be difficult for students not well experienced in taxonomic classification of insects. The backbone of all life is deoxyribose nucleic acid (DNA). The sequence of specific regions of the DNA can provide a mechanism by which an organism can be identified at the species level. One method to do this is DNA barcoding. This technique uses DNA isolated from the macroinvertebrates and uses polymerase chain reaction (PCR) to amplify a specific region of the DNA. This barcode pattern is species specific and visually aids in species identification. For macroinvertebrates, the cytochrome c oxidase I (COI) gene is commonly used in DNA barcoding (Pfrender et al., 2010; Carew et al., 2013). The present study seeks to use the process of DNA barcoding as a mechanism to allow students to understand the connection between genetic species identification and stream quality assessment.

Figure 1.

Sample images of common macroinvertebrates found in the streams of southwestern Pennsylvania, including the stonefly, identifiable by the two long antenna-like tails; mayfly, identifiable by the three (in some species only two) long, antenna-like tails and feathery gills on the abdomen; midge larvae, identifiable by cylindrical, slightly curved, segmented bodies with distinct head and prolegs; and caddisfly, identifiable by its curved body with abdominal gills and hooks at the dorsal end.

Figure 1.

Sample images of common macroinvertebrates found in the streams of southwestern Pennsylvania, including the stonefly, identifiable by the two long antenna-like tails; mayfly, identifiable by the three (in some species only two) long, antenna-like tails and feathery gills on the abdomen; midge larvae, identifiable by cylindrical, slightly curved, segmented bodies with distinct head and prolegs; and caddisfly, identifiable by its curved body with abdominal gills and hooks at the dorsal end.

Methods

Site Description

All macroinvertebrate samples were collected during the summer of 2015 from Four Mile Run in Latrobe, Pennsylvania, near the property of Saint Vincent College. This stream suffers from infiltration by abandoned mine runoff and agricultural waste. Biological assessment of the stream has been carried on over the past 30 years and the stream is commonly used for an introduction to stream assessment in the General Biology II lab. All equipment required for completing the lab activity is presented in Table 1.

Table 1.
Required equipment for completion of the laboratory.
Equipment or MaterialPurpose
Dissecting microscope with digital camera Identification classification of the macroinvertebrates 
Tweezers, razor blades, 1.5 mL tubes Preparation of samples for DNA isolation 
55° water bath Incubation with proteinase K 
Centrifuge for 1.5 mL tube Isolation of DNA (must be able to spin at 12,000 × g) 
Various micropipettes (10 µL, 200 µL, 1000 µL) DNA isolation and PCR reaction setup 
PCR primers Can be ordered from a variety of sources 
Thermocycler (PCR machine) Required for PCR amplification 
Nanodrop spectrophotometer Quantification of the DNA; any spectrophotometer with UV capabilities can be used 
Microwave Preparation of the agarose gel 
Agarose gel electrophoresis rig Analysis of the PCR reactions by electrophoresis 
UV light source Visualization of the DNA on the gel (perhaps consider a gel doc system) 
Equipment or MaterialPurpose
Dissecting microscope with digital camera Identification classification of the macroinvertebrates 
Tweezers, razor blades, 1.5 mL tubes Preparation of samples for DNA isolation 
55° water bath Incubation with proteinase K 
Centrifuge for 1.5 mL tube Isolation of DNA (must be able to spin at 12,000 × g) 
Various micropipettes (10 µL, 200 µL, 1000 µL) DNA isolation and PCR reaction setup 
PCR primers Can be ordered from a variety of sources 
Thermocycler (PCR machine) Required for PCR amplification 
Nanodrop spectrophotometer Quantification of the DNA; any spectrophotometer with UV capabilities can be used 
Microwave Preparation of the agarose gel 
Agarose gel electrophoresis rig Analysis of the PCR reactions by electrophoresis 
UV light source Visualization of the DNA on the gel (perhaps consider a gel doc system) 

Experimental Procedure

The methods provided below are divided into three lab sessions, which are expected to take three hours each. Lab 1 provides the background for the use of macroinvertebrates and identification of macroinvertebrates using anatomical characteristics (see Figure 1 and Table 2). Lab 2 isolates the DNA using the organisms identified in the first session. Finally, Lab 3 amplifies the COI region of the DNA samples collected and analyzes the PCR products using gel electrophoresis.

Table 2.
Common macroinvertebrates found in streams near St. Vincent College.
PhylumClassOrderCommon NameFeeding MethodTolerance
Arthropoda Insecta Ephemeroptera Mayfly Collector Intolerant 
Scraper Intolerant 
Plecoptera Stonefly Predator Intolerant 
Shredder Intolerant 
Trichoptera Caddisfly Net spinners (collector) Moderate 
Stone case building (shredder) Intolerant 
Coleoptera Beetle Adult (collector) Moderate 
Larvae (collector) Moderate 
Odonata Dragonfly Predator Moderate 
Damselfly Predator Moderate 
Hemiptera Water strider Predator Tolerant 
Megaloptera Dobson fly Hellgrammite (predator) Moderate 
Diptera Crane fly Shredder Moderate 
Midge Gathering collector Tolerant 
Crustacean Decapoda Crayfish Scavenger Moderate 
Amphipoda Scud Scavenger Moderate 
PhylumClassOrderCommon NameFeeding MethodTolerance
Arthropoda Insecta Ephemeroptera Mayfly Collector Intolerant 
Scraper Intolerant 
Plecoptera Stonefly Predator Intolerant 
Shredder Intolerant 
Trichoptera Caddisfly Net spinners (collector) Moderate 
Stone case building (shredder) Intolerant 
Coleoptera Beetle Adult (collector) Moderate 
Larvae (collector) Moderate 
Odonata Dragonfly Predator Moderate 
Damselfly Predator Moderate 
Hemiptera Water strider Predator Tolerant 
Megaloptera Dobson fly Hellgrammite (predator) Moderate 
Diptera Crane fly Shredder Moderate 
Midge Gathering collector Tolerant 
Crustacean Decapoda Crayfish Scavenger Moderate 
Amphipoda Scud Scavenger Moderate 

Lab 1

The first lab begins with a review of macroinvertebrate identification and importance in stream ecology and toxicology. This section should include classification of the organisms for the assessment of stream quality and ecological niche occupied by the organisms present in the stream (Table 2). The identification of the macroinvertebrates can be completed using macroinvertebrates that are collected fresh from the stream or that have been collected previously and preserved in ethanol. Either way, the goal of this portion of the lab is to allow students to gain some experience in the traditional taxonomic identification methods. The method outlined in the current learning module is given in this manner because of the arrangement of the current lab at St. Vincent College, where the molecular genetics section of the lab occurs before the stream water analysis, which necessitates the use of preserved samples. The students should be instructed to photograph with a dissecting microscope to aid in identification of macroinvertebrates by general taxonomic features. The images will be used to allow the students to share the characteristics of the insects for identification of the taxonomy group. Images can be used by students in preparing a report from the lab, assessing the students' ability to properly identify the macroinvertebrates. For the identification, several published keys are available to assist students, such as those from the Stroud Institute (https://stroudcenter.org/macros/key/), Missouri Department of Conservation (https://nature.mdc.mo.gov/discover-nature/field-guide), and U.S. Geological Survey (https://water.usgs.gov/wsc/assessment.html). In addition to the taxonomic family, the macroinvertebrates will be characterized on the basis of their ecological niche (Table 2), including different classes based on feeding mechanism or use of the stream. Using these classifications for the macroinvertebrate samples, students will be able to understand the quality of the stream water and potential contaminants present. Additionally, the use of tolerant vs. intolerant classification may be helpful for assessment of the stream (Table 2).

Lab 2

Students will be instructed to begin the process immediately upon starting the lab; time for discussion will occur during the incubation and centrifugation periods. Additionally, if time is limited, the organism preparation (Table 3) and first two steps of the DNA isolation (Table 4) can be completed by the instructor before beginning the lab. The methods provided give the students an opportunity to complete the DNA isolation and understand the different chemicals involved and their roles in the isolation process. Alternatively, a number of DNA isolation kits are available that could be used to save time, including DNeasy Blood and Tissue kit (Qiagen, Germantown, Maryland), NucleoSpin Tissue XS Kit (Macherey-Nagel, Bethlehem, Pennsylvania), and QuickExtract DNA Extraction Solution (Lucigen Corporation, Middleton, Wisconsin).

Table 3.
Instructions for organism preparation, second lab session.
StepInstructions
Thaw preserved samples. 
Use disposable razor blade to chop the organism into 6–8 pieces. 
Place into a 1.5 mL tube labeled with the date of harvest and extraction and organism type. 
StepInstructions
Thaw preserved samples. 
Use disposable razor blade to chop the organism into 6–8 pieces. 
Place into a 1.5 mL tube labeled with the date of harvest and extraction and organism type. 
Table 4.
Instructions for DNA extraction and quantification, second lab session.
StepInstructions
Add 1 μL TNES buffer (50 mM Tris pH 7.5, 0.44 M NaCl, 100 mM EDTA, 0.5% SDS) and 350 μg proteinase K (17.5 μL of 20 μg/μL). 
Incubate at 55°C for 1 hour in a water bath. 
Add 167 μL saturated 6 M NaCl and shake for 15 seconds. 
Centrifuge at 13,200 × g for 30 minutes. 
Transfer supernatant to clean, labeled micro-centrifuge tube. 
Add 600 μL of cold 95% ethanol and invert gently to mix. 
Centrifuge at 13,200 × g for 30 minutes. 
Drain supernatant and dispose. 
Wash with 100 μL of cold 75% ethanol and invert to mix. 
10 Centrifuge at 13,200 × g for 30 minutes. 
11 Remove supernatant and allow to air dry. 
12 Once dried, add 50 μL of nuclease free water (NFW) and pipette up and down to dissolve the pellet. 
StepInstructions
Add 1 μL TNES buffer (50 mM Tris pH 7.5, 0.44 M NaCl, 100 mM EDTA, 0.5% SDS) and 350 μg proteinase K (17.5 μL of 20 μg/μL). 
Incubate at 55°C for 1 hour in a water bath. 
Add 167 μL saturated 6 M NaCl and shake for 15 seconds. 
Centrifuge at 13,200 × g for 30 minutes. 
Transfer supernatant to clean, labeled micro-centrifuge tube. 
Add 600 μL of cold 95% ethanol and invert gently to mix. 
Centrifuge at 13,200 × g for 30 minutes. 
Drain supernatant and dispose. 
Wash with 100 μL of cold 75% ethanol and invert to mix. 
10 Centrifuge at 13,200 × g for 30 minutes. 
11 Remove supernatant and allow to air dry. 
12 Once dried, add 50 μL of nuclease free water (NFW) and pipette up and down to dissolve the pellet. 

DNA quantification. The NanoDrop 2000 (Thermo Scientific, Wilmington, Delaware) spectrophotometer was used for quantification of the DNA. Alternatively, any spectrophotometer with the ability to measure in the UV light range may be used for quantification. To begin the procedure with the NanoDrop, clean twice with 2 μL of nuclease-free water (NFW) and zeroed with 2 μL NFW. Measure each sample of DNA twice, using 2 μL of sample. Confirm that readings are within 10% of each other. The target A260:A280 ratio is 1.7:1.9, but higher ratios may be observed. For the quality of the DNA, this ratio will reflect the purity of the DNA, since proteins and other contaminants increase the A280 nm and lowering the ratio of the sample.

Lab 3

Polymerase chain reaction. PCR was run with 1 μL DNA per sample (assuming between 50 ng/μL and 500 ng/μL concentration). For the DNA barcoding using the COI locus, primer sequences are provided in Table 5. A 24 µL of primer and enzyme mix is prepared as shown in Table 6. The reactions were run on a standard thermocycler using the conditions shown in Table 7. While the PCR is running, pour an agarose gel with 1.5 g of agarose powder per 100 mL 1× TAE solution with 1 μL ethidium bromide added. Alternatively, nontoxic gel stains such as Sybr-Safe (Thermo Scientific) may be used to reduce waste-handling costs. Allow the gel to set for 15–20 minutes before loading. Place gel into appropriate gel electrophoresis rig and cover with 1 × TAE solution.

Table 5.
PCR primer information: macroinvertebrate barcoding primers.a
Primer NameSequence
LCO1490-Invert COI 5′-TGT AAA ACG ACG GCC AGT CAA CAA ATC ATA AAG ATA TTG G-3′ 
HC02198-Invert COI 5′-CAG GAA ACA GCT ATG ACT AAA CTT CAG GGT GAC CAA AAA ATC A-3′ 
Primer NameSequence
LCO1490-Invert COI 5′-TGT AAA ACG ACG GCC AGT CAA CAA ATC ATA AAG ATA TTG G-3′ 
HC02198-Invert COI 5′-CAG GAA ACA GCT ATG ACT AAA CTT CAG GGT GAC CAA AAA ATC A-3′ 
Table 6.
PCR master mix components.
Stock ConcentrationVolume per ReactionFinal Concentration
dH2O *** 17.25 μL *** 
5× Buffer 10× 2.5 μL 1× 
MgCl2 50 mM 0.25 μL 0.5 mM 
dNTPs 2 mM 1 μL 80 μM 
Primer 1 20 μM 1.25 μL 1 μM 
Primer 2 20 μM 1.25 μL 1 μM 
Taq 5 units/μL 0.5 μL 0.1 Units/μL 
Stock ConcentrationVolume per ReactionFinal Concentration
dH2O *** 17.25 μL *** 
5× Buffer 10× 2.5 μL 1× 
MgCl2 50 mM 0.25 μL 0.5 mM 
dNTPs 2 mM 1 μL 80 μM 
Primer 1 20 μM 1.25 μL 1 μM 
Primer 2 20 μM 1.25 μL 1 μM 
Taq 5 units/μL 0.5 μL 0.1 Units/μL 
Table 7.
PCR cycling conditions.
Step of Reaction°CStage Length (min:sec)
Activation of enzyme 94 10:00 
Amplification cycles 94 00:45 
46 00:45 
72 00:45 
 35 Cycles 
Final extension 72 10:00 
Storage  4 40:00 
Step of Reaction°CStage Length (min:sec)
Activation of enzyme 94 10:00 
Amplification cycles 94 00:45 
46 00:45 
72 00:45 
 35 Cycles 
Final extension 72 10:00 
Storage  4 40:00 

Add 5 μL of 6× blue loading dye (40% sucrose, 0.1 M EDTA, 0.5% SDS, 0.05% bromophenol blue) to each reaction. Load 10 μL of each dye-sample mixture into each well on the gel, leaving the first and last column empty for 3 μL of green 100 bp marking dye (Genemate Quanti Marker 100bp+; BioExpress, Kaysville, Utah). Run gels for 45 minutes on 100 V. Photograph gels under UV light and discard. The band size of each reaction can be used for identification of the organism classification. The final PCR image with two of the seven viable results noted and the date of the PCR run included is shown in Figure 2.

Figure 2.

Gel electrophoresis results for the analysis of DNA barcoding for the sample collected in summer 2015 from Four Mile Run.

Figure 2.

Gel electrophoresis results for the analysis of DNA barcoding for the sample collected in summer 2015 from Four Mile Run.

Results & Discussion

This learning module offers novel ways of combining techniques and skills in the areas of ecology, taxonomic identification, and genetic analysis. Students can improve their laboratory skills of taxonomic identification, microscopy, DNA isolation and purification, and PCR. The methods described here were compiled and completed by the first author (M.J.) as an undergraduate student in the summer of 2016. Working through the procedures over the summer, M.J. was able to modify the methods to their current form to allow the completion of the session over the limited three-hour timeframe. Additionally, she was able to give insight into limitations of introductory-level students that must be addressed in completing the learning module. In particular, there is a need to include clear descriptions of the components of the PCR and specific chemicals used for the reaction. Finally, students in the introductory-level course will need to be instructed on the meaning of the PCR products and how this procedure will represent different species of macroinvertebrates. The success rate for the current learning module was 54% positive results for the macroinvertebrates collected (Table 8). This means that if at least four insects are provided to each lab group, it is expected that two of the four will provide working data for the students.

Table 8.
Success rate for completion of learning module by undergraduate student in the summer 2016.
Total samples run 47 
Total samples with only one organism/sample 39 
Total samples successful 21 
Percentage of success 54% 
Total samples run 47 
Total samples with only one organism/sample 39 
Total samples successful 21 
Percentage of success 54% 

Some modifications may be needed to make these procedures fit into three hours per lab. Macroinvertebrate collection is not included in the procedure and may occur as a separate lab, depending on stream availability. For DNA isolation, organism preparation and proteinase K digestion can be accomplished within three hours, but quantification may need to be completed outside of this time limit. However, if quantification is considered a critical portion of the lab, preparation of the organisms must be completed before students start the lab. PCR may not be completed in the three-hour slot and may occur as a separate lab session or may be loaded and/or run outside of the classroom.

The current learning module is developed for an introductory-level undergraduate biology laboratory, but simple modification may allow for development of an upper-level curriculum. For example, DNA sequencing could be added to this procedure, and complete DNA barcoding could be included for upper-level classes. Additionally, for environmental science courses, the use of macroinvertebrates in preserved collections may allow the further identification of a broader diversity of macroinvertebrates. Overall, the flexibility and the integration of concepts presented in this learning module provide a unique opportunity for the advancement of introductory biology laboratory investment at the undergraduate level.

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