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Supplementary information

A novel silkworm infection model with fluorescence imaging using transgenic

Trichosporon asahii expressing eGFP

Yasuhiko Matsumoto1, Hideki Yamazaki1, Yusuke Yamasaki1, Yuki Tateyama1,

Tsuyoshi Yamada2, Takashi Sugita1

1Department of Microbiology, Meiji Pharmaceutical University, 2-522-1, Noshio, Kiyose,

Tokyo 204-8588, Japan.

2Teikyo University Institute of Medical Mycology, 359 Otsuka, Hachioji, Tokyo 192-

0395, Japan.

*Address correspondence to: Dr. Yasuhiko Matsumoto, Department of Microbiology,

Meiji Pharmaceutical University, 2-522-1, Noshio, Kiyose, Tokyo 204-8588, Japan, Tel:

+81-42-495-8745, e-mail: ymatsumoto@my-pharm.ac.jp.

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0.1 1 10 100 1000 100000

50

100

Injected cell number (cells / larva)

Surv

ival

(%)

190619 Matsumoto Y

190927 Yamazaki H

191031 Yamazaki H

191107 Yamazaki H

191130 Yamazaki H

191120 Yamazaki H

Experiment 1Experiment 2Experiment 3Experiment 4Experiment 5Experiment 6

103 104 105 106 107 108

Supplementary Figure 1. Killing of silkworms by T. asahii in a dose dependent manner Survival of the silkworms at 48 h was monitored. Six independent experiments were performed and plotted by non-linear regression using Prism 8 (GraphPad Software, LLC, San Diego, CA, USA, https://www.graphpad.com/scientific-software/prism/).

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AMPH-B FLCZ VCZ

0.1 1 100.0

0.5

1.0

AMPH-B (µg/g larva)

Surv

ival

0.1 1 10 1000.0

0.5

1.0

FLCZ (µg/g larva)

Surv

ival

0.1 1 100.0

0.5

1.0

VCZ (µg/g larva)

Surv

ival

Supplementary Figure 2. Determination of ED50 values of antifungal drugs T. asahii cells (1-5 x 106 cells) were injected into the silkworm hemolymph, and various concentrations of the antifungal agents (50 µl) dissolved in saline were injected immediately afterwards into the silkworm hemolymph. The doses were created by 4-fold serial dilutions. To determine the ED50 values, 5 or 6 silkworms were injected with each dose of the antifungal agents. Survival of the silkworms at 48 h was monitored. The ED50 values were calculated from combined data of 4-5 independent experiments by simple logistic regression model using Prism 8 (GraphPad Software, LLC, San Diego, CA, USA).

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A

Surv

ival

(%)

Time (h)

WTSaline

B

0

20

40

60

80

100

0 20 40 60

eGFP-Tg

n = 5

37˚C

1 10 100 10000

50

100

Injected cell number (cells / larva)

Surv

ival

(%)

WTeGFP-Tg

104 105 106 107

Supplementary Figure 3. Killing of silkworms by injection of T. asahii WT and transgenic T. asahii expressing eGFP (A) Saline, T. asahii WT cells (2.1 x 106 cells), or T. asahii JCM2466 eGFP-Tg strain (2.2 x 106 cells) were injected into the silkworm hemolymph. Survival of the silkworms at 37°C was monitored. n = 5/group. (B) Survival of the silkworms at 48 h was monitored and plotted by non-linear regression using Prism 8 (GraphPad Software, LLC, San Diego, CA, USA, https://www.graphpad.com/scientific-software/prism/).

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ASaline

WT

eGFP-Tg

B

10 µm

1

10

100

0 1 2 3106

107

108

Viab

le c

ell n

umbe

r in

hem

olym

ph(C

FU/m

l)

WT eGFP-Tg

NS

Supplementary Figure 4. Morphology and viable cell number of T. asahii WT and transgenic T. asahii expressing eGFP in silkworm hemolymph Saline, T. asahii WT cells (2.1 x 106 cells), or T. asahii JCM2466 eGFP-Tg strain (2.2 x 106 cells) were injected into the silkworm hemolymph. Silkworm hemolymph was collected at 24 h after injection and observed with a microscope (A). Scale bar, 10 µm. The colony-forming units (B) were calculated. Statistically significant differences between groups were evaluated using Student t-test. NS: not significant, P >0.05.

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PCR Genome

WT

eGFP-Tg

WT

eGFP-Tg

Supplementary Figure 5. Full-length gels of Figure 5C.