ISSN 1347-7048

つくば生物ジャーナル
生物学類

Tsukuba Journal of Biology
Vol.22 No.7 July 2023
www.biol.tsukuba.ac.jp/tjb

つくば生物ジャーナル
Tsukuba Journal of Biology
Vol.22 No.7 July 2023

www.biol.tsukuba.ac.jp/tjb

Abstracts of Graduation ResearchPresentations, July 2023
July 7th 2023

College of Biological Sciences
University of Tsukuba

表紙画

Cover art
天野 史子

表紙画の解説Amano FumikoExplanation of the cover art：

テーマは「切手」。モチーフは卒業生を対象に行なったアンケートの結果から選びました。
切手を貼れば日本中、世界中に手紙を届けることができます。筑波大学での学びや研究もまた、私たちを新たな世界へ連れて行ってくれることでしょう。

The theme is "postage stamps". The motif was chosen from the results ofa survey conducted on graduates.
Stamps can be used to send letters to all over Japan and the world. The
study and research at University of Tsukuba will also take us to a newworld.

Graduation research presentation in Biology
生物学類卒業研究発表会（2023年度春）
July 7th, (11:30-12:40)
at 総合研究棟A110-Program-

11:30–11:35
Greetings from English course faculty, Louis Irving John
11:35–11:50
Presenter: Park Kiwon
ASD-like phenotypes and brain abnormalities in Usp15 KO mice
11:50–12:05
Presenter: Tawan Polsilapa
Optimisation of Glucose Concentration in Mixotrophic Cultivations of a
Lichen Phycobiont Elliptochloris subphaerica
12:05–12:20
Presenter: Cerilles Tanisha Anne Sarmiento
Cold Stress-induced Oxidative Stress in Corals
12:20–12:35
Presenter: Lee Jennifer Jaime
Investigating the influence of positively valenced decorative visuals on
knowledge gain and risk perception
12:35–12:40
Closing remarks from the Dean Kentaro Nakano

We look forward to your participation
どなたでもご参加いただけます。
皆様のご来場をお待ちしております。
Contact: gradtex@biol.tsukuba.ac.jp

つくば生物ジャーナル Tsukuba Journal of Biology (2023) 22(7), 1Ⓒ2023 筑波大学生物学類

ASD-like phenotypes and brain abnormalities in Usp15 KO micePark Kiwon（筑波大学 生物学類）

指導教員：鶴田文憲（筑波大学 生命環境系）
temperature for 2 hours. Fluorescence images were obtainedusing a fluorescence microscope (Leica Microsystems
THUNDER 3D Cell Culture) and analyzed by FIJI ImageJsoftware.

Introduction
Autism spectrum disorder (ASD) is a neurodevelopmental
disease defined by significant social, communication, andbehavior challenges. Accumulating evidence suggests thatgenetic defect is the major cause of ASD. Ubiquitin carboxyterminal hydrolase 15 (Usp15) is one of the candidate genes
identified by the whole-exome sequencing of ASD patients.
Previous studies have found that Usp15 regulates autistic
behavior and balance of excitatory/inhibitory (E/I) synapses.
However, the mechanism of how the absence of Usp15 leads toautistic behavior and abnormal brain structure remains
unknown. In this study, I reported the differences in brain
development and behavioral patterns between wild-type (WT)and Usp15 knockout (KO) mice. I found that Usp15 KO mice
exhibit morphological abnormalities in the cerebral cortex andhippocampus, altered microglial state, and ASD-like behavior.
These findings suggest that a defect in the Usp15 gene is
implicated in early brain development and the manifestation of
behavioral patterns associated with ASD.

Results & Discussion
Previously we identified that Usp15 KO mice show autistic
behavior by a three-chamber test. To assess detailed ASD-likebehavior in Usp15 KO mice, I performed additional behavior
tests. In the marble burying test, Usp15 KO mice buriedsignificantly fewer marbles than WT mice. In the nest-building
test, none of the Usp15 KO mice could build a proper nest
resulting in significantly lower nesting scores in Usp15 KO micecompared to the WT mice. These results indicate that Usp15 KOmice exhibit additional ASD-like behavior else than impaired
sociability. Then I focused on the structural change of the brain
from one of the most common autistic phenotypes, cortex
thinning. Quantification of cortex thickness in the WT and
Usp15 KO mice at postnatal (P) 21 and P28 with the brain
sections immunostained by DAPI showed that the cerebral
cortex is significantly thinner in Usp15 KO mice brains at P28.
Next, I focused on the hippocampus which is known to behighly involved in ASD. I found that Usp15 KO mice exhibit
abnormal dentate gyrus (DG) structures, which weresignificantly shorter in Usp15 KO mice compared to WT at P28.
This result is in line with previous studies that have reported
abnormal DG development is involved in the expression of ASDlike behavior. These data demonstrate that a loss of the Usp15
gene causes abnormal brain formation.
As it has been reported that microglia are associated with
ASD and our finding supports the idea that impaired microglia
are involved in the mechanism of ASD-like behavior and brain
abnormalities. I therefore next quantified the number ofmicroglia to identify potential molecular mechanisms.
Quantification of the GFP signal at the cerebral cortex andhippocampus in the Usp15+/+, CX3CR1 GFP+/- mice and Usp15/-, CX3CR1 GFP+/- mice at P28 showed that the density of
microglia is significantly decreased in Usp15-/-, CX3CR1 GFP+/-.
However, the expression level of Iba1, which is a marker ofreactive microglia, was higher in Usp15 KO mice. Thus, theseresults indicate that Usp15 KO mice exhibit a decreased
number of microglia in the cerebral cortex and hippocampus
and impaired microglial states, which may contribute toimbalanced synapses and cortex thinning. Taken together, myresults suggest that the absence of Usp15 induces a decreased
number of microglia and impaired microglial states that lead to
ASD-like behavior and significant differences in brain
morphology in Usp15 KO mice.

Material & Methods
1. Mice
Male and female WT and Usp15 KO mice at 4-5 weeks of age
were used for behavior experiments. Usp15 +/+, CX3CR1 GFP +/mice and Usp15 -/-, CX3CR1 GFP +/- mice were generated bycrossing Usp15 +/- mice and CX3CR1 GFP +/- or CX3CR1 GFP +/+mice for microglial quantification. All mice used in this study
were backcrossed with the C57BL/6J background.
2. Behavior tests
For the marble burying test, a total of twenty marbles were
evenly arranged on 5cm-thick bedding in the plastic cage and 4week-old mice were placed in the cage for 30 minutes. The
number of marbles buried more than 75% were counted. For thenest-building test, the same size and weight of nesting material
were placed in the cage with 4-week-old mice for 24h. Nests
were scored based on the weight of intact nesting material andthe shape of a nest.
3. Immunohistochemistry
The brains were perfused with PBS and fixed by 4% PFA/PBS
overnight. After 30% sucrose infiltration, the samples wereembedded in OTC compound and sliced at a 40-µm thickness.
The brain sections were washed in PBS and permeabilized with
0.4% TritonX-100 in PBS, then blocked with 5% bovine serum
albumin (BSA) and 0.4% Triton X-100 in PBS for 1 hour.
Sections were incubated with primary antibodies diluted in a
blocking solution at 4°C overnight. Sections were washed in
PBS containing 0.4% TritonX-100 and incubated withsecondary antibodies diluted in a blocking solution at room1

つくば生物ジャーナル Tsukuba Journal of Biology (2023) 22(7), 2Ⓒ2023 筑波大学生物学類

Optimisation of Glucose Concentration in Mixotrophic Cultivationsof a Lichen Phycobiont Ellipitochloris subphaerica
Tawan Polsilapa （筑波大学 生物学類）

指導教員：Ishida Ken-Ichiro (筑波大学 生命環境系)

1. Introduction

2.4. Observation of Lipid Accumulation: Cells collected on day

The transition towards utilizing renewable resources for

14 were stained with Nile-red dye at a ratio of 1:100 andbioenergy has sparked considerable interest, with a particularobserved under a fluorescent microscope.

focus on microalgae known for their high lipid content. Among
microalgae, Botryococcus braunii, a trebouxiophycean green

3. Results & Discussion

alga, is one of such microalgae and known for its high production

3.1. Growth Characteristics: E. subphaerica did not present anyof extracellular long-chain hydrocarbons, comprising up to 86%

significant differences in growth rate among the conditions, Figof its total dry weight (Borowitzka, 2018). However, B. braunii

1 (p = 0.8912 - 0.9941). Mixotrophically cultured green algae

grow very slowly in culture, which raises the cost of hydrocarbon

suchproduction.

pyrenoidosa has been

Elliptochloris subsphaerica, a close relative of B. braunii, is

Chlorella

as

known

to

utilisea phycobiont of a lichen. In a previous study conducted at themonosaccharides

suchlaboratory to which I am affiliated, E. subsphaerica exhibitedas glucose fortheirfast growth (3.5 g 𝐿−1 DCW in 14 days) and significant oil

growth (Zhang el. al.,accumulation (30% of DCW) when cultured in medium X with2014). On the contrary,

2% glucose (Shen, 2021). However, E. subsphaerica did not

my result indicates thatthrive in GTY medium, also containing 2% glucose (Shen, 2021),

E. subphaerica is not able to use glucose in mixotrophic

prompting questions about the role of glucose in its cultivation.

condition but utilises alternative substances as its carbon sourceThis experiment aims to identify the best glucose

in the medium X, which differs from many previous reports.

concentration for cultivating E. subphaerica with high biomass

Moreover, E. subsphaerica achieved a high biomass yield (2.55 -and lipid ratio. The effects of glucose concentrations in medium

3.94 g 𝐿−1 DCW) and cell density (27.2 million cells mL-1) overX ranging from 0% to 4% on growth characteristics and lipid

two weeks using a glucose-free medium. This suggests potential

accumulation were assessed.

cost reduction in large-scale cultivation of E. subsphaerica forbioenergy, reducing the dependence on glucose.

2. Material & Methods

3.2. Lipid Analysis:

2.1. Culture media & Conditions: Five medium X with glucose

Lipid droplets (LD) were observed in all conditions, with noconcentrations of 0, 10, 20, 30, & 40 g 𝐿−1 each, along with asignificant difference (p > 0.9999) related to growth rate.

constant rate of other substances were prepared. Cells were

Optimising LD accumulation and identifying unknown lipid

inoculated at a concentration of 1𝑥106 cells per 𝑚𝐿, under theconstituents are crucial for utilizing E. subphaerica as a viable

light intensity at 250𝜇 𝑚𝑜𝑙 𝑚−2 𝑠 −1 at 21◦𝐶, 100 rpm.

resource for oil production. If the significant portion of the

2.2. Cell Growth: The cellular growth was monitored biweekly,unidentified

with observations taken once every two days via manual cellhydrocarbons (Shen, 2021), it would further enhance its

count by C-Chip (F01). Dry Cell Weight (DCW) was measured

potential as a valuable source for hydrocarbon production.

accumulated

lipid

at the start and end of each experiment. Cells were harvested bycentrifugation, froze at -80◦𝐶, and freeze-dried overnight.

References

2.3. Statistical Analysis: The analysis of data was carried out by

Borowitzka,M.A.(2018).Biologyofmicroalgae.23–72

Prism 9 software. The statistical significance was determined byZhangetal.(2014).BioresourceTechnology.173,52–58

analysis of variance (ANOVA). A P value < 0.05 was considered

Shen. Y. (2021). Tsukuba Journal of Biology.

as statistically significant.

2(approximately

80%)つくば生物ジャーナル Tsukuba Journal of Biology (2023) 22(7), 3Ⓒ2023 筑波大学生物学類

Cold Stress-induced Oxidative Stress in Corals
Tanisha Cerilles（筑波大学 生物学類）

指導教員：Sylvain Agostini（筑波大学 生命環境系）

Introduction:
Coral bleaching, characterized by the loss of symbiotic algae
from corals, is primarily associated with elevated watertemperatures, known as hot stress induced bleaching. However,cold water can also induce coral bleaching, impacting various
physiological processes (Higuchi et al., 2015). Extensive
research has focused on the mechanisms of coral bleachingunder heat stress, highlighting the association between
oxidative stress and subsequent bleaching events (DeSalvo et al.,2008). On the other hand, cold stress bleaching has been lessstudied compared to heat stress bleaching, resulting in a limited
understanding of the mechanisms and impacts involved(Higuchi et al., 2015). Similar stress responses are observed inhigher plants under cold stress to those reported for coralbleaching under hot stress, including disrupted photosynthesis
and oxidative stress (Wang et al., 2017). The accumulation ofreactive oxygen species (ROS) can cause damage to cellularcomponents. These parallels between corals and higher plants
suggest a common underlying mechanism for cold stressinduced bleaching. The purpose of this study is to investigate
whether oxidative stress occurs during cold-induced coralbleaching.
Lipid peroxidation (LPO) and total antioxidant capacity(TAC) assays were used to quantify oxidative stress in coldstressed corals. Trial experiments confirmed reliable andreproducible quantification of LPO and TAC in the coral host.

cold stress and control conditions (p= 0.0032, p= 0.03016, p=
0.04179, respectively). However, no significant results wereobserved for protein biomass, zooxanthellae density.

Figure 1: Lipid peroxidation levels as shown as MDA concentrations (A) and total
antioxidant capacity (B) of corals exposed to short term cold stress.

Discussion:
Reduced photosystem efficiency is commonly observed in both
hot stress-induced bleaching (Hoegh-Guldberg, 1999) and cold
stress-induced bleaching (Roth et al., 2012).. The significant
difference in total antioxidant capacity indicates compromised
antioxidant enzyme generation, such as superoxide dismutase(Higuchi et al., 2015). This limited capacity contributed to ROS
accumulation and increased lipid peroxidation levels, indicatingoxidative damage. This imbalance in reactive oxygen species(ROS) production and scavenging, resulting in oxidative stressand cellular damage, aligns with findings for higher plants
under cold stress (Hasanuzzaman et al., 2020). Overall,
impaired PSII function under cold stress reduced
photosynthesis, increased ROS, and compromised antioxidant
defences, leading to oxidative damage. This shows thatmechanisms of cold stress induced bleaching are similar to thoseunder hot stress for hermatypic corals.

Material & Methods:
A total of 12 microcolonies of P. heronensis were randomly
selected. The microcolonies were then divided into two
conditions: control condition(n=6) and cold stress(n=6), and thenincubated for 48 hours.
In the control condition, the temperature was kept constantat 20°C throughout the entire 48-hour duration. In the cold
stress group, the temperature was initially maintained at 20°C
for 24 hours. Using chilling circulators, the temperature was
gradually lowered from 20°C to 15°C over a span of 5 hours,with a decrease of 1°C per hour. The temperature was thenmaintained at a constant 15°C for an additional 24 hours.
Following the experimental period, a dark adaptation of 30
minutes was conducted on the microcolonies to measure thefluorescence (Fv/Fm). The physiological and biochemical
performances of both the coral host and symbionts were
measured.

References:
Ashraf, M., Harris, P. (2013). Photosynthesis Under Stressful Environments: An
Overview. Photosynt., 2(51), 163-190.
DeSalvo, M. K., Voolstra, C. R., Sunagawa, S., Schwarz, J. A., Stillman, J. H.,
Coffroth, M. A., ... & Medina, M. (2008). Differential gene expression during
thermal stress and bleaching in the Caribbean coral Montastraea faveolata.
Molecular Ecology, 17(17), 3952-3971.
Higuchi, T., Agostini, S., Casareto, B. E., Suzuki, Y., Yuyama, I. (2015). The
Northern Limit Of Corals Of the Genus Acropora In Temperate Zones Is
Determined By Their Resilience To Cold Bleaching. Sci Rep, 1(5).
Hoegh-Guldberg. “Climate Change, Coral Bleaching and the Future of the World’s
Coral Reefs.” Marine and Freshwater Research 50, no. 8 (1999): 839–66.
Hasanuzzaman, M., Bhuyan, M. H. M. B., Parvin, K., Bhuiyan, T. F., Anee, T. I.,
Nahar, K., … & Fujita, M. (2020). Regulation Of Ros Metabolism in Plants
Under Environmental Stress: a Review Of Recent Experimental Evidence.
IJMS, 22(21).
Li, C., Xu, S., Wang, R., Wang, R., & Liang, Z. (2017). Cold stress-induced
chloroplast damage mediates oxidative stress-induced antioxidant defense inleaves of tea plant (Camellia sinensis). Frontiers in Plant Science, 8, 1-13.
Roth, Melissa S., Ralf Goericke, and Dimitri D. Deheyn. “Cold Induces AcuteStress but Heat Is Ultimately More Deleterious for the Reef-Building Coral
Acropora Yongei.” Scientific Reports 2 (February 2, 2012).
https://doi.org/10.1038/srep00240.

Results:
Photosynthetic efficiency, lipid peroxidation levels, and total
antioxidant capacity showed a significant difference between

3つくば生物ジャーナル Tsukuba Journal of Biology (2023) 22(7), 4Ⓒ2023 筑波大学生物学類

Investigating the influence of positively valenced decorative visuals on knowledge gain andrisk perception
Jennifer Lee（筑波大学 生物学類） 指導教員：Matthew Wood（筑波大学 生命環境系）
Introduction
Decorative pictures in multimedia material are visuals aimed atincreasing aesthetic appeal rather than providing additional
information and context,1 and have been shown to influencecognitive processes and emotional affect.1,2,3 Affect refers to theexperience of an emotional state separate from mood, and is a
key component of attitude4. Affect can be measured along thedimensions of activation and valence.1 Valence is the force of
attraction or aversion to a subject and is often described as ascale from positive to negative,5 while activation is the degree ofintensity of the emotion.
Several studies have found positive affect to be beneficial tohuman engagement, learning, and behavior. For example,
adding a positively valenced decorative picture closely related tothe subject material led to decreased irrelevant thinking andimproved learning.1 However, these findings are not consistent,
and in another study, adding a decorative visual to a fact list
decreased knowledge gain and attitude, and increased perceived
risk.6In this study, we ask the question: do positively valenced
decorative visuals influence knowledge gain and risk
perception?

Results and Discussion
Internal reliability scores of the risk perception and positive
attitude questions were acceptable (α=0.810 and 0.860,
respectively), confirming the validity of the scales used in thisstudy.
The positive visual used in the PosV group was rated as
significantly more positive than the neutral visual in the NeutV
group(x̄PosV=5.483(SD=1.056),x̄NeutV=4.806(SD=1.167),
U=357.000, p <0.05), confirming the influence of the visual onparticipant affect. However, this did not translate to a similareffect in knowledge, risk perception, and positive attitude scores(Hknowledge=3.950 (p =0.139), Hrisk=3.259 (p=0.195), Hpositivity=3.575(p=0.167)).
These results suggest that the positive affect elicited from a
positively valenced visual did not influence knowledge gain andperceived risk towards the subject depicted in the visual. This
contrasts with previous studies suggesting positive affect from avisual improves knowledge gain and decreases perceived risk. It
should also be noted that although participants demonstrated adifference in positive affect when asked only about the visual,the difference in affect did not extend to their attitude towardsbats.
It is important to note, however, that effect sizes were low(around f=0.12 for all tests), and achieved statistical power wasapproximately 0.19, well below the minimum acceptable 0.8.
Therefore, there is an unacceptably high risk that these results
constitute a false negative. To reduce the possibility of a Type IIerror and confirm the lack of significant difference between PosV,
NeutV, and NoV groups, a greater sample size of upwards of 750
participants in total is required according to a priori estimates.
Additionally, this study could be further improved by theaddition of more difficult questions evaluating participants’knowledge gain. In the current study, the majority ofparticipants across all groups were able to correctly answer all
knowledge questions, resulting in an inability to conclusively
determine a difference between groups.

Material & Methods
We performed a 3 x 1 between-groups comparative study withpicture type as the independent variable and knowledge gain,
perceived risk, and positive attitude as dependent variables.
Picture types consisted of positive visual (PosV), neutral visual(NeutV), and no visual (NoV).
Participants were shown an information sheet concerning
Myotis bats and their interaction with humans. PosV and
NeutV groups each contained one picture of a bat. Pictures wererelevant to the content and questions in the survey, but did notprovide any additional information. Positive and neutral visuals
were determined from a preliminary ranking study of nine
photos by 28 independent volunteers.
A total of 128 participants were recruited during the study;
28 for the preliminary photo ranking, 98 for the main study. Twoparticipant’s data were removed after data screening.
In the main study, participants were randomly assigned to
one of the picture types, presented with the information sheet,
and asked questions to evaluate their understanding of theinformation, their perceived risk of bats and diseases they may
carry, and positive feelings and attitudes after looking at the
information sheet and picture. NoV group respondents were notasked about a picture. All surveys were conducted online using
Google Forms.

References[1] S. Schneider, J. Dyrna, L. Meier, M. Beege, G. D. Rey, J Educ Psychol.
110, 233–249 (2018).
[2] M. Mikheeva, S. Schneider, G. D. Rey, Journal of Articles in Supportof the Null Hypothesis. 18 (2021).
[3] S. Schneider, S. Nebel, G. D. Rey, Learn Instr. 44, 65–73 (2016).
[4] J. S. Black et al., Organizational Behavior (OpenStax, ed. 1, 2019).
[5] V. Shuman, D. Sander, K. R. Scherer, Front Psychol. 4 (2013).
[6] D. Flemming, U. Cress, S. Kimmig, M. Brandt, J. Kimmerle, Front
Commun (Lausanne). 3 (2018).

4