The gut microbiome plays an essential role in the health of many organisms, including insects. Here we report initial findings of bacteria present in the caterpillar midgut of the cabbage looper, Trichoplusia ni, grown in lab conditions on artificial diet and identified using biochemical tests. We identified one species previously reported as part of the gut microbiota of Trichoplusia ni and three species not previously reported. Our results support the need for multiple types of bacterial identification when looking at gut microbiomes, with the most confidence in identification being when multiple tests are in agreement

Barding, Erin E

Childs, Naomi

Gray, Bethany

Scoggins, Robert

Smith, Margaret (Meg)

English

North Georgia College & State University: Digital Commons

Papers & Publications: Interdisciplinary Journal of Undergraduate 
Research 
Volume 8 Article 4 
2021 
Gut Microbiota of the Cabbage Looper, Trichoplusia ni 
Naomi Childs 
University of North Georgia 
Bethany Gray 
University of North Georgia 
Robert Scoggins 
University of North Georgia 
Margaret (Meg) Smith 
University of North Georgia 
Erin E. Barding 
University of North Georgia 
Follow this and additional works at: https://digitalcommons.northgeorgia.edu/papersandpubs 
 Part of the Biology Commons 
Recommended Citation 
Childs, Naomi; Gray, Bethany; Scoggins, Robert; Smith, Margaret (Meg); and Barding, Erin E. (2021) "Gut 
Microbiota of the Cabbage Looper, Trichoplusia ni," Papers & Publications: Interdisciplinary Journal of 
Undergraduate Research: Vol. 8 , Article 4. 
Available at: https://digitalcommons.northgeorgia.edu/papersandpubs/vol8/iss1/4 
This Article is brought to you for free and open access by the Center for Undergraduate Research and Creative 
Activities (CURCA) at Nighthawks Open Institutional Repository. It has been accepted for inclusion in Papers & 
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Institutional Repository. 
Gut Microbiota of the Cabbage Looper, Trichoplusia ni 
Acknowledgments 
We would like to thank the University of North Georgia (UNG) Department of Biology. In addition, we are 
grateful for assistance from Marissa Fidler, Dr. Dobroslawa Bialonska, Andrew Shirley, and Ava Shearer. 
This article is available in Papers & Publications: Interdisciplinary Journal of Undergraduate Research: 
https://digitalcommons.northgeorgia.edu/papersandpubs/vol8/iss1/4 
Childs, et al. | 17
 Papers & Publications, vol. 8.1
Abstract: The gut microbiome plays an essential role in the health of many organisms, including 
insects. Here we report initial findings of bacteria present in the caterpillar midgut of the cabbage 
looper, Trichoplusia ni, grown in lab conditions on artificial diet and identified using biochemical 
tests. We identified one species previously reported as part of the gut microbiota of Trichoplusia 
ni and three species not previously reported. Our results support the need for multiple types of 
bacterial identification when looking at gut microbiomes, with the most confidence in identifica-
tion being when multiple tests are in agreement.
ß
Organisms are colonized by a large community of microbes early in life, and microbes that colonize the digestive tract are collectively called the gut microbiota (Cenit et al. 2014). The re-
lationship between the gut microbiota and their host has been shaped by 
millions of years of coevolution, but the complexity and function of this 
relationship is still being determined (Engel and Moran 2013, Cenit et al. 
2014). The gut microbiome plays an essential role in the health of many 
organisms, including insects (Tang et al. 2012, Engel and Moran 2013). 
Studies have shown that insect gut microbiota provide nutrients, detoxify 
harmful secondary metabolites, and contribute to the development and 
functioning of the immune system (Tang et al. 2012, Pennington et al. 
2017). Studies have also shown, especially in lepidopterans, that large 
components of the gut microbiome are colonized from food (Hammer 
et al. 2017). So if we can understand the species in the gut and what 
function they play, we may be able to manipulate them to encourage 
population growth of beneficial insects and discourage pests. Therefore 
knowledge of the role of gut microbiota in insects has many potential 
future applications in fields such as medicine, agriculture, and ecology 
(Engel and Moran 2013). For example, secondary plant compounds and 
environmental contaminants have been shown to change the gut micro-
biome of insect larvae as well as increase larval mortality (Nunez-Mejia et 
al. 2016, Pennington et al. 2017). And while we are far from application 
of this knowledge, identifying the taxonomic composition of bacteria 
in the gut of lepidopteran larvae is a relevant first step in understanding 
the reciprocal relationship between the environment and life histories of 
both microbes and host. 
There has been much debate about how gut microbes should be 
identified. Traditional culturing techniques are biased because many 
microbes cannot grow in standard lab conditions (Broderick et al. 2004). 
In response to this limitation, direct sequencing of genes from microbes 
has been employed. However, given the level of horizontal gene transfer 
Gut Microbiota of the Cabbage Looper, 
Trichoplusia ni
Naomi Childs, 
Bethany Gray, 
Robert Scoggins, 
Erin Barding, PhD, 
and Meg Smith, PhD
University of North Georgia
18 | Childs, et al.
Microbiome of T.ni
among bacterial species, this type of method 
may inaccurately identify species present in 
the gut (Riley and Lizotte-Waniewski 2009). 
Instead, some have proposed the use of the Core 
Genome Hypothesis (CGH) to identify bacterial 
species (Riley and Lizotte-Waniewski 2009). 
This hypothesis suggests that species should 
be identified based on a core set of metabolic 
housekeeping and informational processing 
genes which are shared by all members of the 
same species. Based on this species concept, it 
might be more accurate to identify species based 
on biochemical tests of their essential metabolic 
functions such as carbon utilization, amino acid 
synthesis, and salt tolerance (Biolog 2016).
Here we report initial findings of the 
bacteria identified in the caterpillar midgut of 
Trichoplusia ni, a common lepidopteran also 
known as the cabbage looper, grown in lab con-
ditions on artificial diet and identified using 
biochemical tests that assess metabolic functions. 
Materials and Methods
Insect culture
Trichoplusia ni was reared in the lab following 
standard practices (Strand 1989). Larval 
stages were fed a cornmeal based artificial diet 
(Southland Products Cabbage Looper Diet) ad 
lib and maintained in a Percival environmen-
tal chamber under constant conditions (27 °C, 
16L:8D, 20% relative humidity). 
Bacterial Isolation
Five caterpillars in the 5th instar, as determined 
by head capsule width (McEwen and Hervey 
1962), were haphazardly selected for midgut 
dissection. All caterpillars were starved for 4 
hours prior to dissection. After separation from 
both the foregut and hindgut, the midgut was 
surface sterilized with a cotton swab soaked in 
70% ethanol. Then the mid-gut was smeared on 
a tryptic soy agar (TSA) plate. The plates were 
incubated for 48 hours at 25 degrees Celsius 
under aerobic conditions. After the 48 hour in-
cubation period, the developing colonies were 
visually identified by general colony morphology 
and were isolated onto separate TSA plates. After 
two days, the TSA plates were visually inspected 
to verify that the colonies were correctly isolated 
and appeared to be pure cultures. If the colonies 
were not pure as evidenced by colonies of 
varying morphologies on the same plate, they 
were further isolated. To confirm the visual in-
spection of colony purity, all bacterial colonies 
were also Gram stained using standard tech-
niques and visually assessed for shape. Purity was 
indicated as all bacteria having the same Gram 
staining results and bacterium shape.
Bacterial Identification
Bacteria were identified using GENIII 
MicroPlates (Biolog, Inc.) read automatically by 
a plate reader and compared to bacterial species 
Figure 1. Representative images of bacterial colonies. (A) TSA plate with multiple colonies growing 48 hours after smearing of a single 
caterpillar cut. (B-H) Pure isolates on blood agar plates of each of the 7 different bacterial types initially isolated from T. ni midguts.
Childs, et al. | 19
 Papers & Publications, vol. 8.1
data in the Biolog database. Bacterial identifica-
tion by this method requires that colonies grow 
in the presence of iron, so pure colonies were 
taken from TSA plates and grown on blood agar 
plates for 24 hours at 25 degrees Celsius prior 
to analysis. These colonies were then identified 
with the GENIII MicroPlates following man-
ufacturer instructions (Biolog 2016). Biolog 
results were compared to the Gram-staining 
results to verify that they were consistent.
Results
Seven visually distinct colonies were isolated 
from the midgut of T. ni (Figure 1). Of those 
seven distinct colonies (Figure 1B-F), four were 
identified using the Biolog system: Achromobacter 
spanius, Cellulomonas fimi, Paenibacillus pasaden-
ensis, and Bacillus subtilis (Table 1). All species 
identifications via Biolog had a probability of > 
50% and were also confirmed with Gram stains 
(Figure 2). The four identified genera were a 
mixture of aerobes and facultative anaerobes 
(Table 1). 
Discussion
Four genera of bacteria in the gut of 
Trichoplusia ni were identified, including 
Achromobacter, Paenibacillus, Cellulomonas, and 
Bacillus (Table 1), and 3 isolates could not be 
identified by these methods. This small number 
of major bacterial genera is consistent within 
findings from bacterial communities in the 
midgut of other leaf-feeding lepidopteran cater-
pillars (Engel and Moran 2014, Xia et al. 2017, 
van der Hoeven et al. 2008). 
We recovered a genus, Achromobacter that 
belongs to a family Alcaligenaceae, which was 
previously reported as part of the gut microbial 
community in organisms reared on artificial 
diet with bacteria identified through sequencing 
(Pennington et al. 2017). In contrast, the genera 
of Bacillus, Paenibacillus and Cellulomonas 
species have not previously been reported to be 
present in the gut of T. ni as far as the authors 
know. However, all three of these species have 
been recovered from other lepidopterans, such 
as Helicoverpa amiga (Priya et al. 2012) and 
Hofmannophila pseudospretella (Shannon et al. 
2001).
We failed to recover two genera that 
are common residents of insect midguts, 
Clostridium and Enterococcus (Tang et al. 2012, 
Pennington et al. 2017). However, we believe 
this may be, in part, due to the limitations of 
Table 1: Identification and descriptive features of the four microbial isolates from T. ni midguts
Probability Colony 
Description
Picture Gram 
Staining
Shape
Achromobacter spanius 75.7% Grey gel Fig. 1B Negative Rod
Cellulomonas fimi 50.9% Green-yellow gel Fig. 1C Positive Rod
Paenibacillus pasadenensis 53.5% Yellow gel Fig. 1C Positive Cocci
Bacillus subtillus 54.2% Brown-beige border growth Fig. 1E Positive Rod
Figure 2. Representative images of Gram staining. (A) Gram negative (B) Gram positive
20 | Childs, et al.
Microbiome of T.ni
the Gen III microplates. These plates can only 
recover aerobic or facultative anaerobic species, 
those bacteria that can grow in the presence of 
oxygen. Clostridium sp. are obligate anaerobes 
and would not have grown in our lab condi-
tions if present. Enterococcus species tend to be 
facultative anaerobes that potentially we could 
have cultured. We may not have recovered 
Enterococcus at all, or a species among the three 
bacteria that we were unsuccessful in identifying. 
It is not completely unexpected that our 
results do not perfectly match previous studies, 
even with the limitations of which species we 
can culture. T. ni gut microbiome composi-
tion likely varies among populations given 
the evidence that insect gut microbiomes are 
generally thought to be colonized from the envi-
ronment (Hammer et al. 2017, Engel and Moran 
2013, Priya et al. 2012). Populations feeding on 
different food may have different bacterial gut 
residents, a pattern seen in other species (Priya 
et al. 2012). Collectively, these results highlight 
the dynamic reciprocal relationship between the 
host gut and the microbiome dwelling therein. 
The host food sources and microenvironment of 
the gut affects which bacteria can live there, and 
likely the bacteria affect the host life history in 
return. Understanding how the specific bacterial 
species we identified affect the host life history 
in T. ni is a future direction for research in this 
system. 
Additionally, results may vary between 
studies because the methods used for bacterial 
identification, sequencing or biochemical, vary 
among studies. Our results support the need for 
multiple types of bacterial identification when 
looking at gut microbiomes, with the most con-
fidence in identification being when multiple 
tests are in agreement. With confidence in 
bacterial species identification, future research 
could then include functional studies to un-
derstand how members of the gut microbiome 
impact the life history of the host.
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Contributor Bios
Naomi Childs received her undergraduate degree 
in Biology from the University of North Georgia 
in 2018. With a primary interest in animal phys-
iology, she hopes to further her education at the 
University of Georgia to eventually practice as 
a Doctor of Veterinary Medicine. Bethany Gray 
graduated in Spring 2019 with a BS in Secondary 
Education and a focus in Biology. She is eager to 
teach young students about the joys of science. 
Robbie Scoggins is also a recent graduate of the 
Biology program at the University of North 
Georgia. He is currently a chemistry teacher at 
Pepperell High School in Lindale Georgia and 
applying to masters programs in the southeast. 
Drs. Barding and Smith are both Associate 
Professors of Biology at the University of North 
Georgia–Dahlonega campus.
Acknowledgements
We would like to thank the University of North 
Georgia (UNG) Department of Biology. In 
addition, we are grateful for assistance from 
Marissa Fidler, Dr. Dobroslawa Bialonska, 
Andrew Shirley, and Ava Shearer.


Gut Microbiota of the Cabbage Looper, \u3ci\u3eTrichoplusia ni\u3c/i\u3e

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Gut Microbiota of the Cabbage Looper, \u3ci\u3eTrichoplusia ni\u3c/i\u3e

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