An Addition to the Toolkit: An Ethogram for Trichoplusia ni
Logan Pearson1, Chloe Meewes2, Sydney Spencer1, Angela Ayala1, Brad Bailey3, Erin E. Barding1, Ryan Shanks1,*, and Margaret Smith1
1Department of Biology, University of North Georgia, 82 College Circle, Dahlonega, GA 30597. 2Biomedical Sciences, University of Alabama, 1720 University Blvd, Birmingham, AL 35294. 3Department of Mathematics, University of North Georgia, 82 College Circle, Dahlonega, GA 30597. *Corresponding author.
eBio, No. 10 (2024)
Abstract
Animal behaviors are a consequence of complex neurobiological mechanisms and environmental interactions and thus can be used to understand these processes. However, we can only study the diversity of animal behaviors in this way if there is a diversity of ethograms across many species. Therefore, we developed an ethogram for Trichoplusia ni, the Cabbage Looper, which is well studied in other areas but for which an ethogram did not previously exist. Additionally, we demonstrate the usefulness of this ethogram by manipulating a single environmental variable, the presence of another T. ni individual, and demonstrate that some aspects of behavior change while others do not. With the addition of another individual, the type and frequency of behaviors is not altered, but the location where organisms spend time and the distance traveled does change. Therefore, this work rigorously describes T. ni behaviors and uses these descriptions to establish a foundation for behavioral studies in this organism and in comparison to others.
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2024 eBio 10:1–11
An Addition to the Toolkit: An Ethogram for Trichoplusia ni
Logan Pearson1, Chloe Meewes2, Sydney Spencer1, Angela Ayala1, Brad Bailey3,
Erin E. Barding1, Ryan Shanks1,*, and Margaret Smith1
Abstract - Animal behaviors are a consequence of complex neurobiological mechanisms and environmental
interactions and thus can be used to understand these processes. However, we can only study
the diversity of animal behaviors in this way if there is a diversity of ethograms across many species.
Therefore, we developed an ethogram for Trichoplusia ni, the Cabbage Looper, which is well studied
in other areas but for which an ethogram did not previously exist. Additionally, we demonstrate the
usefulness of this ethogram by manipulating a single environmental variable, the presence of another
T. ni individual, and demonstrate that some aspects of behavior change while others do not. With the
addition of another individual, the type and frequency of behaviors is not altered, but the location
where organisms spend time and the distance traveled does change. Therefore, this work rigorously
describes T. ni behaviors and uses these descriptions to establish a foundation for behavioral studies
in this organism and in comparison to others.
Introduction
Behavior can vary among organisms due to a combination of genetic, environmental,
learned, social, cultural, neurological, and physiological factors (Gérard et al. 2022, London
2017, York 2018). The complex interplay between these factors contributes to the diversity
of behaviors observed among animals. To begin to tease apart the complexities, model organisms
in a lab are useful because they can be used to study individual aspects of behavior
in a controlled environment. However, to understand the full spectrum of animal behaviors,
we must use a variety of models that reflect the diversity of animal life. For example, insect
models can be particularly useful to understand the link between neurological changes and
clearly defined behaviors (Chen and Hong 2018, Kravitz and Hernandez 2015, McClellan
and Montgomery 2023, Steinbeck et al. 2020).
Trichoplusia ni (Hübner) (Cabbage Looper) (Lepidoptera: Noctuidae) has been studied
for a variety of reasons, including its role as an agricultural pest and as part of a variety of
host-parasite models (Burke and Strand 2014, Gordon and Strand 2009, Kang et al. 1996).
Thus it has established research resources, including a commercially available source of
material, basic protocols for lab culture, a sequenced genome (Chen et al. 2018), established
cell lines (Fu et al. 2018, Maghodia et al. 2020), and well described anatomy and life history
(McEwen and Hervey 1960, Shorey et al. 1962). However, currently, there is a paucity of
literature describing detailed behavioral analysis of Lepidoptera in a controlled lab setting.
To add to the toolkit available for T. ni research, we aim to develop T. ni as a behavioral
model. As is typical of lepidopterans, T. ni has 4 developmental stages (egg, larva, pupa
and adult), and thus provides unique and defined developmental windows in which one can
study behaviors. Also, they also have well-defined nervous systems that could provide a
1Department of Biology, University of North Georgia, 82 College Circle, Dahlonega, GA 30597.
2Biomedical Sciences, University of Alabama, 1720 University Blvd, Birmingham, AL 35294. 3Department
of Mathematics, University of North Georgia, 82 College Circle, Dahlonega, GA 30597.
*Corresponding author - (Ryan.Shanks@ung.edu)
Associate Editor: Holly Boettger-Tong, Wesleyan College
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model that allows mechanistic questions to be answered (Caveney and Donly 2002, Fuchs
et al. 2014, Gallant et al. 2003, Liu et al. 2023, Malutan et al. 2002, McLean et al. 2005,
Tang et al. 2019). Neuroscience investigations can use behavioral alterations as an endpoint
measurement of change. Development of a comprehensive ethogram that captures
the intricate behavioral range of model organisms is a key starting point for these types of
studies. To our knowledge, T. ni has no comprehensive ethogram established. Therefore,
this study establishes a foundational ethogram for T. ni larvae and describes its usefulness
in a lab setting with a comparison of these behaviors in single and paired animal paradigms.
Materials and Methods
Research population
The research population was taken from a general population of T. ni kept in constant
culture at 25°C with a 16:8 hr light:dark cycle. Adults were fed a 10% sucrose solution,
and all larvae were fed ad lib on cornmeal-based artificial diet (Southland Incorporated).
Individuals were separated from the general population in the egg stage and reared in pairs.
After hatching, the research individuals in the population were staged every other day, and
instar was determined based on head capsule width (McEwen and Hervey 1960). Behavior
was observed only in larvae on the first day of their fifth instar, and only one of the pair
reared together was used regardless of the behavioral paradigm tested.
Behavioral arena and recording
To document larval behavior, a total of 20 video recordings of a single-animal paradigm
(n = 20 individuals) and 20 videos of a paired-animal paradigm (n = 40 individuals) were
recorded for 30 min by an HX-WA03 Panasonic camera. The behavioral arena was the
bottom of a pipette-tip box with the dimensions 12.5 cm by 10.2 cm (Fig. 1A). The singleanimal
paradigm was defined as one individual in the arena (Fig. 1B), and the paired-animal
paradigm was defined as two individuals in the arena simultaneously (Fig. 1C). In both
paradigms, the arena had no food included. Different larvae were recorded for each video.
Recordings were stopped at 30 min because it was empirically determined that no new behaviors
were exhibited after 30 min. Dark purple cardstock was placed underneath the arena
to prevent glare as the recordings were taken inside the incubator during the light cycle.
The camera was suspended above the arena during recording, and the distance between the
camera and the arena was approximately 30 cm. The arena floor was sanded to roughen the
plastic surface. The walls of the arena were not sanded, and petroleum jelly was applied
along the top edges using a sterile cotton swab to prevent the individuals from crawling
out of the arena. In addition, the pipette box was cleaned with ethanol between each use to
prevent any residual cues from previous trials.
Depending on the behavioral paradigm being recorded, the larvae were placed in different
areas of the arena (Fig. 1B and C). For the single-animal behavioral paradigm, the
larva was placed in the center of the arena at the beginning of the recording (Fig. 1B). For
the paired-animal paradigm, each larva was initially placed in opposing quadrants known as
“native” for each animal (digitally labeled A and B in Fig. 1C and D). The quadrant of the
opposing animal was termed “non-native” for each animal. There were also 2 other quadrants
labeled “neutral”, where no organism was initially placed. For both the single-animal
and paired-animal paradigm, the quadrant in which the animals spent the most time was
referred to as the “primary” quadrant (Fig. 1B and D).
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Ethogram development
The examination of behavior took place once the recordings were taken, with independent
coders coding each video. Collaboratively, the coders developed a list of behaviors and
their associated descriptions for each paradigm. This baseline ethogram was used to independently
record the frequency of each behavior exhibited in 5-min bins over 30 min. Extending
the time beyond 30 min did not reveal additional behaviors or unique frequencies.
Two coders watched videos independently and named, defined, and documented behaviors
exhibited by the individual larva (Table 1, Supplemental Data). The coders then compiled
their list of behaviors into a master list that was used to code several more videos. To
test the quality of the definitions, the list of behaviors was given to additional coders who
were not directly involved in the development of the ethogram to independently identify
behaviors. All behaviors unable to be accurately coded were redefined in the ethogram. This
process was iterated until there was an average intraclass correlation coefficient (ICC) of
0.89 between new coders.
Figure 1. Behavioral arena for coding behavior for single (one animal) and paired (two animals)
paradigms. (A) Arena dimensions for all behavioral experiments. (B) The single behavioral paradigm
started with the larva in the center of the arena. Distance and time were measured for each quadrant
with the primary quadrant representing the one with the greatest value for distance and time. (C)
The paired behavioral paradigm started with the larvae (A and B) in opposing quadrants. The starting
quadrant for A (native) is the non-native quadrant for B and visa-versa. Quadrants where no
larva started were considered neutral. (D) Distance and time were measured for native, non-native,
and neutral quadrants. The primary quadrant represents the one quadrant with the greatest value for
distance and time for each larva individually.
Table 1. Trichoplusia ni ethogram
Type of
behavior
Behavior Description Visual
aid
Locomotion Inchworm Continuous movement whereby the individual’s prolegs and true legs
remain suctioned to the surface and slide toward and away from each other
while its body arches upward and stretches out. Additionally, any range of
that arch is considered inchworm behavior. (See Supplemental File 1, available
online at http://eaglehill.us /ebio-036-Shanks-s1.mp4.).
Vertical
movement
Individual latches both its true legs and prolegs onto the petroleum jelly
covered the edges of the arena. (See Supplemental File 2, available online
at http://eaglehill.us /ebio-036-Shanks-s2.mp4.).
Still Individual exhibits no movement; it is still for at least 5s. (See supplemental
File 3, available online at http://eaglehill.us /ebio-036-Shanks-s3.mp4.).
Elimination Frass Excretion of feces while the individual is in the arena; larval frass is circular
and light brown in color. If the individual releases 1 pellet, record that as 1
frass behavior. (See Supplemental File 4, available online at http://eaglehill.
us /ebio-036-Shanks-s4.mp4.).
Emesis Individual expels fluid from mouth; larval vomit is a green fluid-like
substance. (See Supplemental File 5, available online at http://eaglehill.us /
ebio-Shanks-s5.mp4.).
Non-
Locomotion
Head movement
Individual’s prolegs and true legs remain attached to the surface of the arena
while only its head moves (the area from the tip of its head to the area right
before its true legs). (See Supplemental File 6, available online at http://
eaglehill.us /ebio-Shanks-s6.mp4.).
Upward
extension
The individual’s prolegs are suctioned to the surface of the arena and it
brings its anterior body upward where it holds a still position as seen in the
picture. Individual remains still in this position for longer than 1 second.
(See Supplemental File 7, available online at http://eaglehill.us /ebio-
Shanks-s7.mp4.).
Searching Individual’s prolegs suction to the ground while its thorax extends upward
and moves from one direction to another (can move L-R or R-L); during
this movement, the individual’s true legs are not attached to the surface of
the arena. (See Supplemental File 8, available online at http://eaglehill.us /
ebio-Shanks-s8.mp4.).
Engagement Wall
engagement
An individual engages with the walls of the arena by latching its true legs
onto the petroleum jelly covered surface and maintaining its proleg grip on
the bottom surface of the arena. (See Supplemental File 9, available online
at http://eaglehill.us /ebio-Shanks-s9.mp4.).
Body
engagement
Individuals engage with its body by keeping prolegs on the surface and
bringing head and true legs backwards/forwards to a part of its body. (See
Supplemental File 10, available online at http://eaglehill.us /ebio-Shankss10.
mp4.).
Frass
interaction
Individuals interact with their frass by nudging their frass with their head.
(See Supplemental File 11, available online at http://eaglehill.us /ebio-
Shanks-s11.mp4.).
Sociability 1-Sided
interaction
Any part of individual A or B contacts any body part of individual A or B.
(See Supplemental File 12, available online at http://eaglehill.us /ebio-
Shanks-s12.mp4.).
2-Sided
interaction
Individual A or B physically contacts the other individual and this elicits a
response whereby the other individual uses its head to push A or B away
from itself. (See Supplemental File 13, available online at http://eaglehill.us
/ebio-Shanks-s13.mp4.).
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Behavioral coding
Once the ethogram was established with strong inter-relater reliability, new coders
analyzed 30-min recordings of the individual and paired behavioral paradigms. They were
provided with the established ethogram, to which they had not been previously exposed.
The coders then tallied each time a behavior was displayed according to the terms and definitions
they were given. All behaviors were coded in 5-min bins.
Time and locomotion
To further understand the locomotion patterns of the larvae studied in both paradigms,
the arena was divided into quadrants (Fig. 1). The time spent and total distance traveled in
each quadrant were measured. The locomotion distance was measured with a ruler by tracing
the movement of the individual using a sheet of transparency paper, and the amount of
time spent in each quadrant was tracked for each 5-min bin (Fig . 1B and D).
Data analysis
For tallies of individual behaviors, high-occurrence behaviors were defined as those
exhibited 10 or more times in a 30-min recording. Low occurrence behaviors were defined
as those exhibited less than 10 times in a 30-min recording. Independent sample t-tests were
performed to determine if the frequency of high-occurrence behaviors differed between single-
animal and paired-animal paradigms. Similarly, time and distance traveled in different
quadrants, and frequency of one-sided vs two-sided interactions were analyzed with t-tests.
Duration of time spent in the primary quadrant across time intervals and paired paradigms
were analyzed with ANOVA. Statistical analyses were done in R (v. 4.2.2) and SPSS (v.
29.0). In all graphs, error bars represent the standard error ( SE).
Results
Ethogram
To develop this ethogram for T. ni, we collected behavioral data from 20 single-animal
videos and 20 paired-animal videos. We only recorded the larvae in the first day of the fifth
instar for consistency and visual purposes (Table 1, Supplemental Data).
Figure 2. Coded larval behaviors defined in the ethogram measured over 30 minutes for both the single
(dark columns) and paired (light columns) behavioral paradigms. (A) High-occurrence behaviors (B)
Low-occurrence behaviors. Error bars represent ± standard error (SE).
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Behavior
Using the ethogram, coders tallied behaviors exhibited during 30-min recordings. We
were able to distinguish the high-occurrence behaviors (Fig. 2A) from low-occurrence behaviors
(Fig. 2B). There was no statistical difference in the frequency of high-occurrence
behaviors between single-animal and paired-animal paradigms (inchworm: t58 = 0.02, P =
0.98; searching: t58 = 0.98, P = 0.33; wall engagement: t58 = 1.70, P = 0.09). The scarcity
of low-occurrence behaviors did not warrant statistical analysis of single-animal and paired
animal paradigms (Fig. 2B).
Figure 3. Coded larval average time (A and B) and average distance traveled (C and D) in 30 minutes
for both the single (dark columns) and paired (light columns) behavioral paradigms. * indicates P <
0.001. Error bars represent ± SE.
Figure 4. Average time spent in the primary quadrant for larvae in both the single (dark triangle and
solid linear trend line) and paired behavioral paradigms (light triangles and dashed linear trend line)
shown in 5-minute time intervals for the 30-minute coding perio d. Error bars represent ± SE.
A. B.
0
200
400
600
800
1000
1200
1400
Native quadrant Neutral
quadrants
Non-native
quadrant
Average time (s)
0
200
400
600
800
1000
1200
1400
Primary quadrant Other quadrants
Average time (s)
C. D.
0
50
100
150
200
250
Native quadrant Neutral
quadrants
Non-native
quadrant
Average distance (cm)
*
*
*
0
50
100
150
200
250
Single Paired
Average distance (cm)
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Time and distance
Time and distance traveled in different quadrants was measured for both single-animal
and paired-animal paradigms. The single-animal paradigm revealed more time spent in 1
quadrant compared to the other 3 quadrants (Fig. 3A, t38 = 6.8, P < 0.001). The paired-animal
paradigm also showed that there was an effect of quadrant on time spent in the quadrant
(Fig. 3B, F(2,117) = 11.26, P < 0.001). A post-hoc Tukey’s test indicated that individuals spent
less time in the non-native (P < 0.001) quadrant than either the native or neutral quadrants.
There was no statistical difference in the total distance traveled by individuals in the
single-animal paradigm versus paired-animal paradigm (Fig. 3C, t58 = 0.89, P = 0.37). In
contrast, for the paired-animal paradigm, (Fig. 3D), there was a significant difference in
the average distance traveled in different quadrants (F(2,117) = 12.23, P < 0.001). A post-hoc
Tukey’s test indicated that individuals spent significantly more time in neutral quadrants
than either their native quadrant (P < 0.001) or non-native quadrant (P < 0.001). This increase
is attributed to the fact there is twice as much space in the neutral quadrants, yet
remains notable given the decreased time spent in the non-native quadrant only.
Time interval vs duration in primary quadrant
The duration of time spent in the primary quadrant across time intervals was also examined
(Fig. 4). For the single-animal paradigm, there was no significant difference between
the 5-min time intervals and time spent in a primary quadrant versus other quadrants as
indicated by a regression line slope not statistically significantly distinguishable from 0 (F(5)
= 0.921, P = 0.339). In contrast, for the paired-animal paradigm, individuals decreased the
amount of time they spent in their primary quadrant over time as indicated by a statistically
significant negative slope of the regression line ( F(5) = 5.81, P = 0.017).
Interactions
Lastly, 2 novel behaviors (one-sided interactions and two-sided interactions) occurred
only in the paired-animal paradigm recordings (Table 1, Fig. 5). One-sided interactions were
significantly more frequent than two-sided interactions (t78 = 2.77, P = 0.007).
Figure 5. One-sided and twosided
interactions measured between
larvae in the paired-animal
paradigm over 30 minutes.
* indicates P < 0.05. Error bars
represent ± SE.
0
1
2
3
4
5
6
7
One-sided
interaction
Two-sided
interaction Average occurrence
*
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Discussion
Ethogram
Establishing a well-defined ethogram is fundamental to understanding animal behavior
(Okuyama et al. 2013). With clear operational definitions, this ethogram was created to
give other researchers the opportunity to use a straightforward system to answer additional
questions (Xu et al. 2012). In addition to these behaviors, methodology for how to track and
time the movement of animals within a defined arena provide a template for future studies
(Fig. 1, Table 1, Supplemental Data). Whilst many researchers have investigated specific
behaviors of interest, a more inclusive description of behaviors allows for a more integrative
and cross-disciplinary approach (Collie et al. 2020, McLellan and Montgomery 2023,
Okuyama et al. 2013, Peric-Mataruga et al. 2017, Suszczynska et al. 2017).
Behavior
To illustrate the application of the operational definitions in the ethogram, the behaviors
were coded with the addition of a single variable that compares a single larva’s behaviors
alone and with an additional larva. High occurrence behaviors show no statistical difference
with this single environmental variable we exampled (single vs. paired), yet this may
provide an interesting comparison to the effect of different environmental variables such
as food availability and temperature (Fig. 2A). Similarly, while low frequency behaviors
hold no validity statistically due to the low number of coded events, there were trending
differences that may hold biological meaning (Fig. 2B). Therefore, the importance of these
recorded events lies beyond any statistical findings. This ethogram and the example data
analysis clearly defined all possible behaviors so that future studies may alter environmental,
social, or developmental variables or compare behaviors between species in a more
wholistic approach. For example, the upward extension of the head provided little measurable
difference between coded behaviors within either the single or paired behavioral paradigms,
yet this behavior may be drastically altered if food were introduced into a competitive
environment in which two animals shared the arena. However, for the ability to conduct
future comparative and meta-analysis, it will be important that all behaviors regardless of
their statistical significance be documented.
Location and distance
As with behavior, measures of location and distance traveled provide an important
measurement of neurobiobehavioral alterations. This can be used to measure responses
to the introduction or removal of environmental cues and gauge whether they are appetitive
or non-appetitive. Individuals in both the single and paired behavioral paradigms had
unique location and distance patterns of behavior. These patterns of behavior provide a
unique baseline data set to investigate alterations in these behaviors including investigations
of motivation, competition, and social interactions. For example, the addition of
frass or a potential food source in one area of the arena may alter the primary quadrant
for an individual in a single behavioral paradigm. In a paired behavioral paradigm these
additions may alter the duration that individuals display in a primary quadrant over time
(Fig. 4). Interestingly, the presence of an additional organism did not impact the distance
traveled by individuals, yet individuals in both the single and paired behavioral paradigms
did have unique location and locomotion patterns (Fig. 3). Individuals in the paired behavioral
paradigm did not travel longer distances in their native quadrant; however, they
spent more time in the native quadrant (Fig. 3). The added individual B in the paired paraeBio
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digm could help explain why individual A spent more time in the native quadrant whilst at
the same time traveled less in said quadrant (Fig. 1 and 4). Therefore, it is the combination
of both time spent and distance traveled together that is important to consider. Additionally,
by binning the duration spent in specific quadrants, unique trends were observed in
the individuals of the single and paired behavioral paradigms (Fig. 4). Manipulation of
this simple variable (paired vs. single) alters behavior in some measurable and meaningful
ways (location, Fig. 3) yet has little effect on other behaviors (high-occurrence, Fig. 2).
This highlights the usefulness of establishing an ethogram using a controlled laboratory
environment in which an arena of a defined size is used.
Interaction
Individuals in the paired-animal paradigm exhibited seemingly aggressive one-sided
and two-sided interactions. This is not unusual, even to the point of cannibalism, in both
eusocial and solitary insects where its function is used to defend territories, establish
social hierarchies, and compete for food (Bowen et al. 2008, Collie et al. 2020, Dial and
Adler, 1990, Kemp 2000, Semlitsch and West 1988, Tang et al. 2016, Yack et al. 2001, Zago-
Braga and Zucoloto 2004, Zhou et al. 2016). While T. ni is not a eusocial insect, it can
live in groups, like other lepidopterans, and this may impact behavior (Daly et al. 2012).
Figure 5 documents significantly more occurrences of one-sided interactions compared to
two-sided interactions in the paired-animal behavioral paradigm. The role these interactions
have in the establishment of a potential social hierarchy in T. ni remains unclear, but
this foundational data opens the door to investigations of how size, developmental stage,
and fitness relate to the establishment of potential dominant and submissive individuals
in a population. Furthermore, these investigations relate to more broad questions of how
social interactions lead to the evolution of important survival behaviors in a group setting.
The combined study of interactions and other behaviors in this ethogram serves as a
valuable tool for researchers to further investigate the ecological, physiological, and behavioral
aspects of T. ni, aiding in the understanding of its biology and ecology. It could
open the door to investigations of more broad behavioral notions such as learned helplessness,
usually studied in vertebrate models (Maier and Seligman 2017), or other cued
social interactions that usually depend on multiple sensory inputs in insects as well as
the underlying neural mechanisms dictating these behaviors (Chen and Hong 2018). The
analysis of behavioral data in this ethogram can be used as a reference of a standard social
interaction amongst T.ni and instigate the study of the underlying neural mechanisms
causing these interactions to occur. Understanding the neural basis of T. ni behavior can
shed light on the neural circuits, sensory processing, and neurophysiological processes
involved in not only insect but more complex animal behavior. This research provides a
foundation for future studies on T. ni and other lepidopteran species, with the potential to
uncover novel insights into the neural mechanisms underlying insect behavior and inspire
future research in neuroethology.
Acknowledgments
We thank University of North Georgia’s Department of Biology and Center for Undergraduate
Research and Creative Activities for their financial support for this project. We greatly appreciate
the animal care and technical assistance provided by Shaswat Patel, Laura-Kate Gleaton, and Maria
Fernanda Sanchez Acosta.
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