Updated: September 2020

Lab 1: Arthropod Collection

& Identification

Student
Guide
Page Contents
3 Introduction
4 -- Taxonomy
5 -- Arthropods
6 -- Insects
7 -- Review Key Concepts
Lab Activity
8 -- Pre-Lab Questions
9 -- Part 1: Arthropod Collection
10 -- Part 2: Arthropod Identification
11 -- Database Entry Checklist
12 -- Post-Lab Questions
13 -- Extension Opportunities
14-15 -- Arthropod Collection Forms

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The Wolbachia Project: Discover the Microbes

Within! was developed by a collaboration of
scientists, educators, and outreach specialists.
It is directed by the Bordenstein Lab at
Vanderbilt University.

https://www.vanderbilt.edu/wolbachiaproject

Figures created with BioRender.com.

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 2

 Introduction

In this activity, you will embark on an expedition to collect arthropods from local fauna, the
animals present in a particular region, habitat, or time. You should review the scientific
method, think about creating a hypothesis and discuss a sampling scheme for determining
which organisms (individual life forms such as plants, animals, and bacteria) may or may not
harbor the bacterial endosymbiont, Wolbachia. An endosymbiont is an organism that lives
within the body or cells of another organism; Wolbachia is one of the most successful
endosymbionts on the planet and infects up to 60% of all arthropods. Sampling locations
should be coordinated with your group and could include your home, nearby nature park,
schoolyard, or carefully selected habitats that differ in temperature, location, etc. We
recommend using this exercise to practice designing an experiment and creating a
hypothesis. Some example hypotheses:
• 60% of the Culex mosquito species collected in Nashville, TN are Wolbachia positive.
• There is no difference between the Wolbachia infection prevalence of Coccinellidae
(ladybird beetles) in the spring and fall seasons.
• Ants nesting in the soil are Wolbachia-positive, but tree-dwelling ants are not.

After arthropods are preserved in ethanol or rubbing alcohol, you will identify each specimen
to taxonomic order and create an entry in the Wolbachia Project Database. This will enable
further exploration of new questions, such as:

• Why do species in some insect groups have few or no endosymbionts while insect species
in other groups have these microorganisms in every species so far examined?
• Suppose you find that a collection of ants nesting in soil have Wolbachia, whereas the ants
that live in the trees above do not. Does this indicate that soils in some way contribute to
how Wolbachia transmits itself among species of ants? Can this hypothesis be supported
if both soil-dwelling and tree-dwelling ants have Wolbachia?

Without knowing the identity of the arthropod host these ideas may never be explored, and
certainly could not be tested. Therefore, it is essential to (i) upload a high-quality picture for
each arthropod that will be screened for Wolbachia, (ii) provide clear and concise
observation notes in the database, and, if possible, (iii) include DNA sequence data.

Recommended Resources

iNaturalist (https://www.inaturalist.org/) is a crowdsourced species identification app that is

used to record biodiversity observations and help naturalists identify organisms in their local
environment. The software will suggest options for identifying your arthropod and, after
submission, citizen scientists will provide feedback on the classification.

OpenStax (https://openstax.org/) is a free online textbook. Chapter 28.6 of the Biology 2e

textbook provides an overview of the phylum Arthropoda, including descriptions for each of
the subphyla discussed in this lab activity.
• https://openstax.org/books/biology-2e/pages/28-6-superphylum-ecdysozoa-arthropods

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 3

 Taxonomy

Taxonomy is the classification of organisms based on relatedness. Developed by Swedish

botanist Carl Linnaeus in the 1700’s, scientists across the world use taxonomy to organize all
living things in a hierarchical structure. Because there are over one million named species of
arthropods, it can be difficult to classify beyond order based solely on visual observation;
therefore, DNA sequence similarity is often used alongside morphology (form and structure)
and ecology (the study of the relationships between organisms and their physical
environment) to determine classification. The scientific name for an organism is based on
binomial nomenclature, a unique two-word name combining the genus and species. It is
written in italics and the genus is always capitalized. This name can be shortened by only
including the first initial of the genus, as shown in the table below.

Domain
Kingdom
Phylum
Class
Order
Family
Genus
Species

Domain * Eukarya Eukarya Bacteria

Kingdom Animalia Animalia Eubacteria
Phylum Chordata Arthropoda Firmicutes
Class Mammalia Insecta Bacilli
Order Primates Lepidoptera Bacillales
Family Hominidae Nymphalidae Listeriaceae
Genus Homo Danaus Listeria
Species Homo sapiens Danaus plexippus Listeria monocytogenes
Abbreviated name H. sapiens D. plexippus L. monocytogenes
* The above classification is based on the three-domain system of taxonomy (Archaea, Bacteria, Eukarya).
However, increasing evidence supports a two-domain system. First proposed as the eocyte hypothesis by
James A. Lake and colleagues in 1984 (doi: 10.1073/pnas.81.12.3786), Eukarya are included with the Archaea.

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 4

 Arthropods

Arthropods, the most diverse and abundant animals on earth, are characterized as
invertebrates with a hard covering called an exoskeleton, segmented body, and jointed
appendages. They represent about 85% of all described animal species and there are an
estimated 10 quintillion (10,000,000,000,000,000,000) individual arthropods inhabiting the
planet on any given day. If the services these animals provide everyday (for free) suddenly
disappeared, humans would soon disappear. They clean water, pollinate flowers that
produce about 1/3 of the food we eat, break down waste, and decompose plants and
animals. Given their importance, why are they so little appreciated and poorly understood?
Perhaps the single largest factor is their small size. Most of the one million described species
are less than 1/3” long, the size of this line:
This entire world of small creatures exists literally under your feet.

Kingdom: Animalia

Phylum: Arthropoda

Subphylum: Hexapoda

Class: Insecta

Order: Hemiptera

The phylum Arthropoda is classified into five subphyla (plural of subphylum, a taxonomic
category that ranks below phylum and above class):
• Chelicerata: spiders, scorpions, mites, ticks, horseshoe crabs, and relatives
• Crustacea: crabs, lobsters, crayfish, shrimps, krill, barnacles, prawns, woodlice, and
relatives
• Hexapoda: insects, springtails, and relatives
• Myriapoda: millipedes, centipedes, and relatives
• Trilobita: trilobites (extinct)

What is the difference between an arthropod, spider, insect, or bug? Taxonomy. True bugs
belong to the order Hemiptera and include bedbugs, assassin bugs, and stinkbugs. All bugs
belong to the class Insecta, which are characterized by three body segments (head, thorax,
abdomen) and six legs. Spiders, on the other hand, belong to the class Arachnida and have
two body parts (cephalothorax and abdomen) and eight legs. All insects and arachnids
belong to the phylum Arthropoda.

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 5

 Insects

Most arthropods are insects, members of class Insecta and subphylum Hexapoda. Hexa and
poda both originate from Latin (from Greek) meaning six and those having feet, respectively.
All insects have three body segments.

• The head features conspicuous

eyes, mouthparts, and antennae
• The thorax contains wings and legs
• The abdomen contains the
reproductive organs, where many of
the endosymbiotic bacteria reside.

Of course, it is common for there to be exceptions to the body plan. Many insects lack wings
or legs (particularly in the larval stage), some are eyeless, etc. These features allow for
classification into groups. Order is the most familiar taxonomic grouping. Common examples
are:
• Order: Blattodea – cockroaches and termites
• Order: Coleoptera – beetles
• Order: Diptera – flies
Coleoptera
• Order: Hemiptera – true bugs
• Order: Hymenoptera – bees, wasps and ants
• Order: Lepidoptera – moths and butterflies
• Order: Mantodea – praying mantids
Hemiptera
• Order: Odonata – dragonflies and damselflies
• Order: Orthoptera – grasshoppers and crickets
• Order: Phasmatodea – stick insects and leaf insects
• Order: Phthiraptera – biting and sucking lice
Orthoptera
• Order: Siphonaptera – fleas

Notice that most, but not all, insect orders end with the suffix “-ptera.” Ptera is the plural
form of the Ancient Greek pterón, which means wing or feather. Wings are one of the most
conspicuous ways orders of insects are differentiated.

For the purpose of this lab, it is recommended to study smaller species because they are
easier to dissect and preserve better. Larger insects rot more quickly, thus degrading DNA
and making it more difficult to examine bacterial endosymbionts.

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 6

 Review Key Concepts

1. Define the following keywords.

• Arthropod:

• Binomial nomenclature:

• Ecology:

• Endosymbiont:

• Exoskeleton:

• Fauna:

• Morphology:

• Organism:

• Subphylum:

• Taxonomy:

2. Use the word bank to label the images below.

Abdomen Arachnida Head Thorax
Abdomen Cephalothorax Insecta Wings

Tarantula Asian Fruit Fly (male)

3. List at least three morphological differences between the above organisms.

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 7

 Pre-Lab Questions

Read through the entire protocol and answer the questions below.

1. Why is it important to take pictures and label each arthropod?

2. Are spiders classified as arthropods? Are they insects? Why or why not?

3. This charismatic arthropod has many common names throughout the English-speaking
world. Based on its taxonomic classification, which name is most accurate?

4. Can you hypothesize why some insect groups might have microorganisms associated with
them?

5. Use an online taxonomic key to complete the chart.

The pillbug represents an example where you may not be able to confidently identify genus/species.
Therefore, classify down to the most reliable taxonomic category.

Kingdom Animalia
Phylum Arthropoda
Class Insecta Alphaproteobacteria
Order Diptera
Family Armadillidiidae
Genus ? Drosophila Wolbachia
Species Drosophila
?
melanogaster
Scientific
? W. pipientis
name
Common Yellow fever
Pillbug N/A
name mosquito

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 8

 Part 1: Arthropod Collection

Preparation
1. State your hypothesis, collection method, and materials needed.
2. Review the arthropod collection plan with your instructor. Avoid arthropods that are
poisonous, venomous, and/or sting. If you have a history of allergic reactions to bites
or stings, consult with your teacher prior to this lab.

Hypothesis

Collection Method

Materials Needed

Arthropod Collection
Note 1.1: If unable to 3. Document each arthropod in its natural habitat by taking pictures and recording
capture a good image of
the arthropod, you may specific Observation Data in the Arthropod Collection Form at the end of this lab
draw a sketch or take a activity. Fill out as much information as possible – note specific details about the
picture after it has been collection location, seasonal conditions, and arthropod behavior. Complete one form
stored in alcohol for at
least 24 hours. per specimen. (See Note 1.1)
4. Collect each arthropod according to the specific protocol detailed above.
Note 1.2: If the arthropod 5. Preserve each specimen by transferring to a small tube or vial of alcohol. This can be
is moving too quickly to 80% or greater ethanol (ethyl alcohol) or over-the-counter rubbing alcohol
preserve in alcohol, first
slow it down by placing
(isopropanol or isopropyl alcohol). (See Note 1.2)
into a collection jar at 6. Carefully label each tube to correlate with the ID number from the Arthropod
cold temperature: Collection Form. Make sure the cap is tightly sealed as the alcohol can wipe off the
refrigerator (20 min) or
freezer (10 minutes). label. For extra reassurance (optional), place each individual tube in a plastic Ziploc
bag along with a labeled notecard.
7. Store in the freezer overnight, or until ready to proceed with the lab.

LAB 1: ARTHROPOD IDENTIFICATION 9

 Part 2: Arthropod Identification

Materials per Group

q Insect collection q Transfer pipette (eye dropper) q Computer or mobile device
q Gloves, 2 pair q Small squirt bottle of 80% ethanol q Field guides (optional)
q 2 safety goggles q Paper towels
q Bent probe and/or forceps q Colored pencils
q 2-3 Petri dishes (100 x 15 mm or q Dissecting microscope (or
150 x 20 mm) stereoscope)

Note 1.3: Ethanol

Arthropod Identification
preserves DNA for future 1. Remove the arthropod and place in a petri dish. Carefully squirt enough ethanol into
molecular studies, such the dish to properly cover. (See Note 1.3)
as PCR.
2. Use forceps or a probe to carefully position the arthropod.
Note 1.4: If you were 3. Capture morphology with digital pictures and/or hand drawn illustrations. Make large
unable to take a picture sketches using colored pencils to correctly portray the color. Use a dissecting
of the arthropod in its microscope, if needed, to visualize fine details. (See Note 1.4)
natural habitat, you may
quickly and carefully 4. If removed, immediately place your arthropod back into ethanol.
remove from alcohol. 5. Record a written observation of each specimen on the Arthropod Collection Form.
Place on a solid color
Include specific notes about body segmentation (e.g., head, thorax, and abdomen for
background, such as a
paper towel, for best insects), number of legs, presence of wings and antennae, etc.
results. 6. Use one or more of the following resources to identify each arthropod at least to
Order. (See Note 1.5)
Note 1.5: When using • iNaturalist: https://www.inaturalist.org/
online keys, answer only
the questions that you • Insect Identification: https://www.insectidentification.org/
are confident in. • Discover Life: https://www.discoverlife.org/20/q?guide=Insect_orders
Compare the possible
identification with your
• Field Guides (check with your local library)
notes and images. 7. Clean up your lab station.

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 10

 Database Entry Checklist

After completing the DNA Extraction, create an

entry in The Wolbachia Project Database and
record observation and protocol notes. A
comprehensive guide is located under the
Resources tab.

https://wolbachiaprojectdb.org/

Database fields to complete

q Picture
q Photo credit
q Location
q Collection date
q Captive/cultivated
q Observations
q Putative identification

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 11

 Post-Lab Questions

1. Complete an Arthropod Collection Form for each of your specimens.

2. Research the habitat and life of your identified arthropods. Record notes in the chart below.

Arthropod 1 Arthropod 2

#_________________ #_________________

Common name

Habitat
(trees, soil, etc.)

Diet

Life cycle

Geographic location

Interesting facts

3. Does your research match the actual collection site and observed behaviors? Why or why not?

4. Were you able to identify down to order? Genus and species? If not, why?

5. Based on your research of each arthropod, do you expect them to be Wolbachia-infected?

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 12

 Extension

1. Taxonomy, like all of science, is constantly evolving to accommodate new discoveries and scientific data.
Research the following classification systems and provide an overview of each in the form of a short essay or
PowerPoint/Google Slides presentation. Conclude with a personal recommendation of the best system.
• Two- empire system
• Three-domain system
• Two-domain system

2. Research the five subphyla of Arthropoda. Prepare a PowerPoint or Google Slides presentation summarizing
key characteristics and representative taxa of each group.

3. Insects are fundamental to life as we know it. Select one of the general categories below and provide a
comprehensive review of their contribution and importance.
• Agriculture
• Ecology
• Economy
• Forensics
• Food
• Medicine

4. Create an EOL Biodiversity Card Deck featuring at least 10 taxa related to your hypothesis. If you are only
studying one specific organism, such as Culex pipiens mosquitoes, you may broaden the scope to include ten
different mosquito species of your local environment.
• EOL Biodiversity Cards: https://education.eol.org/card_resources
• Example card decks: https://education.eol.org/card_maker

5. Research or design two arthropod traps: a trap that is designed to only capture one arthropod class, and
another arthropod trap that can trap multiple arthropod classes. What are the similarities and differences
between the traps? Write a protocol for how to build one of your traps, then build it at home and test it.
Write a report on what you observed, and if your trap was successful.

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 13

 Arthropod Collection Form

Collector name
Arthropod ID
Initials and number

Collection date

Location
description

Latitude
Observation Data

Longitude

Season

Temperature

Habitat Terrestrial / Aquatic / Urban / Other

Arthropod
observation
notes

Arthropod
Arthropod Identification

description

Arthropod order

Scientific name

Common name
iNaturalist Record #
Only submit to iNaturalist if you
took a picture of the live
arthropod in its natural habitat.

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 14

 Arthropod Collection Form

Collector name
Arthropod ID
Initials and number

Collection date

Location
description

Latitude
Observation Data

Longitude

Season

Temperature

Habitat Terrestrial / Aquatic / Urban / Other

Arthropod
observation
notes

Arthropod
Arthropod Identification

description

Arthropod order

Scientific name

Common name
iNaturalist Record #
Only submit to iNaturalist if you
took a picture of the live
arthropod in its natural habitat.

LAB 1: ARTHROPOD COLLECTION & IDENTIFICATION 15