June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Research Paper

DESI-IMS coupled with triple quadrupole mass spectrometry demonstrated
the capability of NMN imaging in Shelfordella lateralis tissue
1

1

1

1

1

1

Zinat Tamannaa , Takumi Sakamoto , Md. Al Mamun , Soho Oyama , A.S.M. Waliullah , Ariful Islam ,
1

1,2

4,5

4,6

4

Md. Mahamodun Nabi , Shuhei Aramaki , Eisuke Tasaki , Yuki Mitaka , Mamoru Takata ,
1,2

1,2

4

1,2,3

Tomohito Sato , Tomoaki Kahyo , Kenji Matsuura , Mitsutoshi Setou*

1

Department of Cellular & Molecular Anatomy, Hamamatsu University School of Medicine,1‒20‒1 Handayama, Higashi-Ku, Hamamatsu, Shizuoka 431‒3192, Japan
2
International Mass Imaging Center, Hamamatsu University School of Medicine,1‒20‒1 Handayama, Higashi-Ku, Hmamatsu, Shizuoka 431‒3192, Japan3
Department of Systems Molecular Anatomy, Institute for Medical Photonics Research, Preeminent Medical Photonics Education &Research Center, 1‒20‒1 Handayama, Higashi-Ku, Hamamatsu, Shizuoka 431‒3192, Japan
4
Laboratory of Insect Ecology, Graduate School of Agriculture, Kyoto University,
Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606‒8502, Japan
5
Department of Life and Food Sciences, Graduate School of Science and Technology, Niigata University,
8050 Ikarashi 2-no-cho, Nishi-ku, Niigata 950‒2181, Japan
6
Department of Entomology, 2143 TAMU, Texas A & M University, College Station, Texas, 77843‒2143, USA
Abstract

Imaging mass spectrometry (IMS) has become an indispensable analytical tool for visualizing molecular

distributions in biological tissues. However, the detection of many important biomolecules, such as nicotinamide mononucleotide (NMN) in tissue by IMS is still challenging due to their lower abundance. Although the sensitivity of widelyused IMS instruments, desorption electrospray ionization (DESI) utilizing a time-of-ﬂight (TOF) mass analyzer, is not
suﬃcient to detect NMN in tissues, the recently developed DESI coupled with a triple-quadrupole (TQ) provides about ten
times higher sensitivity than the DESI-Q-TOF. In this study, we ﬁrst screened six insect species for NMN content using
ultra-performance liquid-chromatography-tandem mass spectrometry (UPLC-MS/MS). Interestingly, Shelfordella lateralis
showed signiﬁcantly higher NMN content (15.5 mg/100 g of dry weight) than ﬁve other insect species used in this study.
After screening, we tried to observe the distribution of NMN in S. lateralis using both DESI-Q-TOF and DESI-TQ, andonly the DESI-TQ successfully detected the NMN in S. lateralis. DESI-TQ revealed prominent distributions of NMN in theabdomen of S. lateralis in selective reaction monitoring mode. To the best of our knowledge, this is the ﬁrst report of NMN
imaging in animal tissue applying IMS. Our study will contribute to the extended application of IMS for NMN imaging in
mammalian tissues in the future. Furthermore, NMN imaging may help researchers to study nicotinamide adenine dinucle＋

otide (NAD ) metabolism in living organisms, which has signiﬁcant implications for medical research and diagnosis.
Key words: IMS, NMN, DESI-TQ, DESI-Q-TOF, UPLC-MS/MS, Shelfordella lateralis*Corresponding author
Mitsutoshi Setou

Introduction

Department of Cellular & Molecular Anatomy, Hamamatsu
University School of Medicine, 1‒20‒1 Handayama,

Imaging mass spectrometry (IMS) has been increasingly
used to visualize biomolecules due to its high sensitivity
and label-free nature. This technology has made it possible

Higashi-ku, Hamamatsu, Shizuoka 431‒3192, Japan
Tel: ＋81‒53‒435‒2086, Fax: ＋81‒53‒435‒2468

to reveal the identity and distribution of the speciﬁc mole1)
cule and the localization of the molecule in organs . Nowa-

E-mail: setou@hama-med.ac.jp

Received: August 16, 2023. Accepted: January 11, 2024.

days, imaging with desorption electrospray ionization(DESI), in combination with quadrupole-time-of-ﬂight

Epub April 19, 2024.
DOI: 10.24508/mms.2024.06.001
2

June 2024

Medical Mass Spectrometry Vol. 8 No. 1(Q-TOF) mass spectrometry, has been gaining much popu-

insects as a potential source of calories and protein . In

larity for visualizing molecular distribution on a sample
2)
surface under ambient conditions . DESI-Q-TOF has

traditional folk medicine and Chinese medicine, the whole22)

body extracts of several bees, wasps, ﬂies, butterﬂies,
moths, cockroaches, beetles, and other insects act as antivi-

shown the capability of visualizing spatial distributions of

21)
ral, antibacterial, and anti-cancer agents . While insects

molecules with diverse chemical natures, such as small
3)
4‒6)
7)
molecules , free fatty acids , phospholipids , lipid bio8)

9)

are consumed as food in various cultures around the world10)

marker , active compounds , and other metabolites . Oneof the signiﬁcant advantages of DESI-IMS is that it requires

and are known to be rich in protein and nutrients, there is
not adequate knowledge or research about their NMN con-a minimal sample preparation than traditional mass spectrometry, such as matrix-assisted laser desorption ionization

tent.

mass spectrometry (MALDI-MS) . Recently, DESI com-Cinnamomum verum J. Presl bark, a medicinal plant, using
23)
liquid chromatography mass spectrometry (LC-MS) .

Recently, our group has explored high NMN content in

11)

bined with triple quadrupoles (TQ) mass spectrometry has
emerged and oﬀers advantages over DESI-Q-TOF. The TQ

Applying the same method, we ﬁrst screened NMN content
in six insect species in this study, namely, the Turkestanmass analyzer consists of three quadrupole mass ﬁlters,
while DESI-Q-TOF consists of a single quadrupole mass ﬁl-cockroach Shelfordella lateralis, the ﬁeld cricket Gryllus
bimaculatus, the damp-wood termite Hodotermopsis sjost-ter. These three quadrupole mass ﬁlters make DESI-TQ
more speciﬁc for precursor-to-product ion transitions of theedti, the black soldier ﬂy Hermetia illucens, the Japanesetargeted compound analysis . This allows DESI-TQ to
exhibit higher speciﬁcity and excellent sensitivity in selec12)

subterranean termite Reticulitermes speratus, and the giant
mealworm Zophobas atratus. Finally, we employed both
DESI-Q-TOF and DESI-TQ to observe the distribution oftive reaction monitoring (SRM) mode than other high-reso13)

lution mass analyzers .

NMN in S. lateralis bodies, which showed the highest

Despite the signiﬁcant improvements in the sample

NMN content among the other species under this study.

preparation and instrumentations of IMS, detecting andimaging of many biologically important molecules in tissue
is still challenging. For example, nicotinamide mononucle-

Materials and Methods

otide (NMN), a well-known vital precursor of nicotinamide

We purchased the standard β-nicotinamide mononucleotide (β-NMN) from Tokyo Chemical Industry Co.

Chemicals and reagents＋

adenine dinucleotide (NAD ), has been found diﬃcult to be
detected by IMS. The analytical detection of NMN is chal-(Tokyo, Japan). LC/MS-grade formic acid (99%), acetonitrile (ACN), methanol, ethanol, and ultra-pure water were14)

lenging due to its physicochemical characteristics . NMN
is a nucleotide containing a pyridine base. It is an acid- andwater-soluble nucleotide found in all living cells as a coen-

obtained from Fujiﬁlm Wako Pure Chemical Corporation,
Ltd. (Osaka, Japan). Ammonium format solution was pur-zyme. Increasing NAD levels improves insulin sensitivity,
reverses mitochondrial dysfunction, and enhances lifes-

chased from Kanto Chemical Co., INC. (Tokyo, Japan).
Sodium formate was obtained from Sigma-Aldrich Co.

＋

15)

pan . When taken orally, NMN is quickly absorbed andLLC (St. Louis, MO, USA) and, leucine enkephalin from. NAD availability is essential for17)
slowing the aging process . Oral nicotinamide supplemen-

Waters Corporation (Milford, MA, USA).

tation within the daily acceptable upper threshold is an eﬃ-

Sample preparation for the quantiﬁcation of NMN

cient strategy to raise NAD levels and inﬂuence blood lipid

Two types of termite species (H. sjostedti, and R. speratus) were used in this study. H. sjostedti were collected ini-

＋16)

＋

converted into NAD＋

18)composition . Thus, NMN has important applications intially from logs from brown-rotted Luchu pine wood (Pinus

19)

medicine and healthcare , but the chemical synthesis of
20)

NMN is complex . Therefore, it is worth exploring a richluchuensis) in the forest of Amami Oshima Island in Japan.

source of natural NMN and its imaging in medical research.
Unlike other natural products, insects have received com-

And the colonies of R. speratus were collected from logs
from brown-rotted cedar wood (Cryptomeria japonica) inparatively little attention in imaging studies as sources ofmodern medicines . Insects can provide proteins forthe forest in Kyoto, Japan. The termite colonies were maintained at the Laboratory of Insect Ecology, Kyoto Univer-human use, and people in many parts of the world consume

sity, at about 25°
C under dark. The Individuals of H. sjost-21)

3June 2024

Medical Mass Spectrometry Vol. 8 No. 1

edti were randomly extracted from each of the six colonies

40°
C throughout the measurement. The electrospray ioniza-(colony code: KM022, KM024, KM025, KM103, KM109,
TMK091). And the individuals of R. speratus were ran-tion (ESI) source was used on the mass spectrometer, andthe analysis was performed in positive ion mode. The capil-

domly extracted from each of the three colonies (colony

lary voltage was set to 3.5 kV, the cone voltage to 22 V andthe source temperature was 150°
C. The desolvation tem-

code: MT616D, MT617E, and MT619G). We bought theperature was ﬁxed at 500°
C and the desolvation gas ﬂow

cockroach S. lateralis from Rakuten Group, Inc., Tokyo,
Japan, and the freeze-dried G. bimaculatus from Leaf Cor-rate was 600 L/h. The cone and nebulizer gas ﬂows were set
to 150 L/h and 7.0 Bar, respectively. Mobile phase A con-poration, Gunma, Japan. The two other insect species, dried

sisted of 0.3% formic acid in ACN, while mobile phase B

H. illucens, and dried Z. atratus, were purchased from GEX
Corporation, Osaka, Japan, and Sanko-Tokai Co., Ltd.,

was acetonitrile/100 mM ammonium formate (20:80, v/v).
The ﬂow rate used for elution was 0.6 mL/min. The follow-

Osaka, Japan, respectively.
All the samples were kept in a −80°
C freezer until
freeze dried treatment. The frozen insect samples were

ing setup was used to perform the gradient elution proce-freeze-dried (Freeze Dryer FD-1000, EYELA, Bunkyo,
Tokyo, Japan) and ground (Wonder Crusher WC-3 L, Osaka

2.5 to 4.5 min, 100% B; 4.5 to 4.6 min, 100% to 20% B; 4.6

Chemical Co. Ltd., Kita, Osaka, Japan) to form a powder.
The resulting insect powder (each about 5 g) was respec-

7 min. An aliquot of 5μL of the prepared sample wastively vacuum-sealed (Impulse Vacuum Sealer V301G-

constant temperature of 10°
C. The SRM transition from m/z

10WK, FUJIIMPULSE Co. Ltd., Toyonaka, Osaka, Japan)

335.1 to m/z 123.0 using a collision energy of 10.0 eV wasand kept in −20°
C freezer until use for analyses.

used for NMN quantiﬁcation.

Standard sample preparationacquire and analyze the UPLC-MS/MS data, and, TargetLynx software (Waters, version 4.1) was used for the quan-dure: 0 to 0.5 min, 20% B; 0.5 to 2.5 min, 20% to 100% B;
to 7.0 min, 20% B. The whole duration of the run time wasinjected into the system while the autosampler was kept at a

A MassLynx software (Waters, version 4.1) was used to
For standard sample preparation, an analytical balance(AUY220, SHIMADZU, Kyoto, Japan) was used to weight
the NMN standards. Then, 25% ethanol was used to dis-titative measurement. The statistical analysis was carriedout using GraphPad Prism (version 8.42).

solve the NMN standard. The prepared NMN standard was

Sample preparation for DESI-IMS analysis

then serially diluted to control solutions at concentrations

After collecting the samples, the whole body of the cockroach was embedded overnight by a super cryo-embeddingof 500, 200, 100, 50, and 20 ng/mL for calibration curve
preparation.

medium in the middle of a cryomold and frozen at −20°
C.

Insect extract preparation for UPLC-MS/MS

The frozen cockroach samples were then mounted on a

For each sample, approximately 50 mg of powder was

sample holder using the optimal cutting temperature (OCT)compound (Sakura Finetek Japan, Tokyo, Japan). The tis-weighed three times. Then 25% ethanol was added (40μL/

sues were sectioned sagittally at 20μm thickness at −20°C

mg of the sample) and extracted for one minute on ice witha mechanical homogenizer (HK-1, AS ONE, Osaka, Japan)

by a cryostat system (CM1950; Leica Biosystems, Wetzlar,

at 9000 rpm. The homogenized insect extracts were thenGermany). Then the sections were mounted on glass slides

centrifuged for 10 minutes at 3000 rpm in 4°
C. Then the(Matsunami, Osaka, Japan) and stored at −80°
C. Before
DESI-IMS analysis, the slices were air-dried.

supernatant was ﬁltered through a 0.42μm ﬁlter, and thecleared sample was transferred to LC-MS vials.

DESI-Q-TOF analysis

UPLC-MS/MS analysis

DESI-IMS analysis was performed in positive ion modeusing DESI source attached to a Q-TOF mass analyzer

ACQUITY (UPLC H-Class PLUS, Waters) coupled witha triple quadrupole mass spectrometer (Xevo TQ-XS,(Xevo G2-XS Q-TOF, Waters). Before the measurements,
sodium formate solution (500μM) in 2-propanol: water

Waters) was used to analyze the insect extracts. An Intrada
Amino Acid column (3μm, 3.0×100 mm, Imtakt) was car-(90:10, v/v) was used to calibrate the mass spectra externally, and leucine enkephalin solution (500μM) was used

ried out for chromatographic separation, which was kept at
4

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

to set up the detector. A solvent pump (ACQUITY UPLC

Results

Binary Solvent Manager, Waters) was used to deliver thespray solvent (98:2 methanol/water, v/v) at a ﬂow rate ofIn this study, we ﬁrst conducted a quantiﬁcation study of
NMN in six diﬀerent insect species S. lateralis, G. bimacu-

2μL/min. A 2D sampling stage was equipped with a DESI

latus H. sjostedti, H. illucens, R. speratus, and Z. atratus bysource to scan the designated area with a scan rate of 200μLC/MS (Fig. 1b). As seen in Fig. 1a, a linear calibration

m/sec, and the pixel size was 100μm × 100μm. The DESI

curve (Y＝9.3056X−154.914, R＝0.99) of NMN standards
was observed in the range of 20‒500 ng/mL. Chromato-source conditions were optimized using the following

grams showed the clear detection of NMN standard at eachpoint (Fig. S1a‒e).

parameters: a nebulizing nitrogen gas pressure of 0.4 MPa,a capillary voltage of 4.0 kV, a spray impact angle of 70°,a

A Chromatogram of NMN (SRM transition at m/z 335.1
to m/z 123.0) was observed with high intensities in S. later-

sampling cone of 50, and an inlet temperature of 120°C.
The setup for mass resolution, mass window, and collisionenergy were at 20000, 0.02 Da, and 4.00 V. The mass spec-

alis, G. bimaculatus (Fig. 1b, Fig. S2a‒b), H. sjostedti, H.
illucens (Fig. 1b, Fig. S3c‒d), and R. speratus (Fig. 1b, Fig.

tra were collected using a mass range of m/z 100 to 1000 of

S4e). On the other hand, NMN was not detected in the species of Z. atratus (Fig. 1b, Fig. S4f). The average concen-

the 1000 peaks with the highest intensities. The data werethen further processed at the range of m/z 300 to 400 to
detect the ion images of lower intensities.
DESI-Q-TOF data were acquired and processed using the

tration of NMN in S. lateralis, G. bimaculatus, H. sjostedti,

MassLynx software (Waters, version 4.1). And, HDI Imag-and 0.13 mg/100 g (dry weight) respectively. S. lateralis

ing software (Waters, version 1.4) was used to analyze theshowed signiﬁcantly higher NMN content than all otherion images.

species in this study (p＜0.0001) (Fig. 2). G. bimaculatus
showed the second highest content of NMN, which was sig-

DESI-TQ analysis

niﬁcantly higher than H. illucens, R. speratus, and Z. atra-

H. illucens, and R. speratus were 15.5, 6.93, 3.01, 0.143,

A Xevo TQ-XS equipped with a DESI source (Waters)tus (p＜0.005) (Fig. 2).
For the imaging study, we ﬁrst performed DESI-Q-TOF

has been employed for imaging in SRM mode. A syringe
pump delivered the DESI-IMS solvent (MeOH/H2O, 98/2,

analysis in the whole body of S. lateralis. But we could notv/v) at a ﬂow rate of 2μL /min. For NMN imaging, thedetect NMN in the entire body of S. lateralis (Fig. S5a), we＋
could only detect NMN at m/z 335.1 [M＋H] in NMN stan-

DESI source conditions were optimized using the following
parameters: N2 nebulizing gas pressure of 0.8 Mpa; capil-

dard (20μg/mL) (Fig. S5a). In this analysis, NMN was notlary voltage of 0.85 kV (Positive); MS source temperaturedetected with other adducts in both NMN standard and S.

of 150°C; sampling cone voltage of 20 V. The distances

lateralis (Fig. S5b).

between the emitter tip and the surface, the mass inlet andthe surface, and the mass inlet were roughly 2, 6, andNext, we performed DESI-TQ in the SRM mode to see
the distributions of NMN in the sagittal sections of whole-

0.5 mm, respectively. The identiﬁed areas on the glass slide

body S. lateralis. NMN (SRM transition of m/z 335.1 to m/

were scanned at a scan rate of 10 scan/sec. The pixel sizez 123.0) was successfully detected in almost entire regions

was set as 50μm × 50μm for tissue and 100μm × 100μm
for standard. A collision energy of 20 eV was used forof the S. lateralis section (Fig. 3b). To observe theregion-speciﬁc distribution of this molecule in S. lateralis,

NMN transition (m/z 335.1 to m/z 123.0). The SRM dwell

the histological images were overlaid on the ion image

time was set as 0.0016 sec/pixel. At least two serial sections(Fig. 3c). Interestingly, NMN was notably higher in someof the cockroach sample were analyzed under the same
experimental conditions. Following the measurement, tis-

speciﬁc parts of the S. lateralis abdomen, possibly in the
midgut. For further conﬁrmation of the NMN assignment,
pure NMN solution (1μg/mL) and extract from dry S. lat-sue sections were stained with hematoxylin and eosin(H&E) for histological analysis.
The ion image of DESI-TQ data was acquired and pro-eralis were spotted on the same slide and measured subse-

cessed using the updated version of MassLynx (Waters, ver-transition were clearly detected for pure NMN (Fig. 3d) andsion 4.2) software and HDI Imaging (Waters, version 1.6)

the S. lateralis extract (Fig. 3e).

quently. As expected, the ion images at the same NMN

software.
5June 2024

Fig. 1.

Medical Mass Spectrometry Vol. 8 No. 1

Calibration curve of NMN standards (a) and representative chromatogram of NMN obtained from six diﬀerent
species of insects (b).

Discussion

times higher sensitivity than the DESI-Q-TOF. In this study,
DESI-TQ successfully detected NMN in S. lateralis (Fig.

For the ﬁrst time, in this study, we demonstrated the
NMN imaging in biological tissue employing DESI-TQ in3), which could be attributed to its improved sensitivity. It
is to be noted that the sensitivity of DESI-TQ is inﬂuenced

SRM mode that showed higher sensitivity and excellent
selectivity than DESI-Q-TOF. Detection of NMN in tissue

by various factors, including dwell time. Dwell time refers

is challenging by IMS since this molecule is very labile andreadily undergoes in-source fragmentation under standard

to the duration the mass spectrometer spends analyzing aelectrospray ionization settings . DESI-Q-TOF is typically

Increasing dwell time leads to higher sensitivity as the
instrument has more time to accumulate ions from the sam-particular ion in a particular spot on the sample surface.

24)

used for untargeted analysis where the target ion is mixed
with myriads of other ions and could not be detected. In our
experience, DESI-TQ provides about ten to a thousandple. However, longer dwell times can compromise spatial
resolution. This is because the instrument spends more time6June 2024

Fig. 2.

Medical Mass Spectrometry Vol. 8 No. 1

Comparison of NMN content among diﬀerent
species of insects.
Diﬀerent letters indicate a signiﬁcant diﬀerence (p＜0.01) among diﬀerent species. One-way ANOVA was
performed followed by Tukeyʼs multiple comparison
tests among diﬀerent species of insects. All the data arerepresented as mean±SD. The same insect samples wereprepared in triplicate.

at each spot, resulting in a larger area being sampled, which
may reduce the ability to discriminate features at a ﬁner
spatial scale. Therefore, ﬁnding the optimal balance

Fig. 3.

between dwell time, sensitivity, and spatial resolution isrecommended.

Distribution of NMN in the whole body of S.
lateralis by DESI-TQ.
H&E images of S. lateralis section (a). Representative
DESI-IMS ion image for NMN (SRM transition at m/z
335.1 to m/z 123.0) in S. lateralis section (b), NMN
standard (d), and S. lateralis extract (e). Transparent
overlaid images between H&E and ion images for NMN
in S. lateralis (c).

The analysis of insects, such as S. lateralis, by IMS presents inherent challenges. Ideally, freezing the sample immediately after sacriﬁcing the animal or insect is crucial toprevent post-mortem degradation of certain analytes. However, the delicate anatomical structure of S. lateralis poses
diﬃculties in cryo-sectioning fresh-frozen samples without

dried the sample immediately after cryo-sectioning to minimize the NMN delocalization. Previously MALDI-IMS wasusing an embedding medium. To address this issue, we
employed a super cryo-embedding medium for the entire

used for peptide imaging in formalin-ﬁxed and paraﬃn-em-

body of S. lateralis. This approach facilitates cryo-section-25)

bedded (FFPE) S. lateralis . However, FFPE samples are
not suitable for NMN imaging since they require deparaf-

ing without compromising the mass spectral quality, thanks
to the mediumʼs monomeric property. It is important to notethat freezing samples using cryo-embedding medium

ﬁnization and extensive washing procedure.

requires a longer time compared to direct freezing, this pro-

(7 min run per injection) to screen and quantify NMN in
23)
plant extract using UPLC-MS/MS . SRM has been a gold

Our group recently developed a rapid SRM method

cedure may impact the imaging of certain biomolecules
susceptible to rapid post-mortem degradation. Fortunately,

standard for quantiﬁcation studies. We employed the same

there is no reported literature on the post-mortem degradation of NMN, supporting the compatibility of NMN imag-

method in our current study and found a signiﬁcantly
higher content of NMN in S. lateralis compared to otherspecies of insects. Cockroaches are one of the most suc-

ing in frozen and embedded samples.
One of the challenges in NMN imaging is the potential
delocalization during sample preparation. In our experi-

cessful and diverse insects in traditional and Chinese medi-

ence, NMN delocalization was observed when tissue sec-various natural raw foods, including edamame, broccoli,tion was stored in the freezer before analysis. This is

cucumber seed, cucumber peel, cabbage, avocado, tomato,

because NMN is highly soluble in water. In our study, wemushroom, beef, and shrimp. Among these, edamame

26)

cine . Previously Miles et al. investigated NMN content in7

June 2024

Medical Mass Spectrometry Vol. 8 No. 1(0.47‒1.88)
28)
mg/100 g . Considering the moisture content (60‒65%) ,showedthehighestcontentofimaging in tissue by DESI-TQ and revealed S. lateralis as a

NMN

27)

rich source of NMN, speciﬁcally in the abdomen. We

the NMN content in edamame should be approximately
1.18‒4.7 mg/100 g dry weight. In contrast, cockroaches

believe that, our method will help to develop other animal

have an NMN content of 15.5 mg/100 g dry weight (Fig. 2),

imaging in the ﬁeld of biomedical research. In the future,
NMN imaging could aid in understanding age-related

about 3 to 13 times higher than edamame. This is the ﬁrst

changes in NAD metabolism and its potential role instudy that revealed the surprisingly higher content of NMN

human health.

＋

in insects. Cockroach extracts have been used for centuries

Acknowledgment

to treat aches, pains, inﬂammation, and even chronic heart

This work was supported by the Cabinet Oﬃce, Government of Japan, Moonshot Research and Development Pro-29)

failure . The gut microbial metabolites of cockroach is avaluable source of new bioactive compounds with therapeu26)

tic promise for human health . Our study also indicatedthe promising use of this insect, particularly in pharmaceu-

gram for Agriculture, Forestry and Fisheries (funding
agency: Bio-oriented Technology Research Advancementticals. A recent study found that milk from cockroach species had a higher potential for nutrition than milk from typi-

Institution) Grant number JPJ009237 (to K.M.), JSPS
KAKENHI (Grant Number JP18H05268 to K.M.), and30)

cal mammals . As a result, it is thought of as a potential

JSPS KAKENHI (Grant Number JP23H69332 to K.M.).

new superfood that might be made accessible for ingestion
shortly. It is to be noted that cockroaches carry food-borne

Conﬂicts of Interest

pathogens that may transmit diseases to humans . There31)

The authors declare no conﬂict of interest.

fore, more research is required to focus on the sanitation
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