Laguna (2021) 28: 37-53

ISSN 2185-2995

Seasonal variation in Terrestrial cellulose assimilation ofbenthic invertebrates inhabiting wetlands in Osaka Bay, Japan
Wen Liu1 , Yu Umezawa2, Soma Kato2 and Kazumi Matsuoka3,4*

Abstract: It has become much clearer that benthic invertebrates are involved in terrestrial organic
matter (TerrestrialOM) decomposition in wetlands since the discovery of endogenous carbohydratehydrolases (CHs, e.g., cellulase) in various invertebrates during recent decades. Because thebreakdown of high molecular TerrestrialOMs is essential to the circulation of carbon between theterrestrial and marine ecosystems, the level of TerrestrialOMs decomposed by benthic invertebrates
should be estimated correctly to evaluate the true TerrestrialOM decomposition capability of a wetland.

To estimate invertebrates’ contribution to the wetland, both the levels of relevant enzymatic

activities (capability) and stable isotope signatures (performance) and their relationship must be
investigated. Also, seasonal variation should be taken into consideration to increase the accuracyof the estimation. Here we firstly used the CMC Plate Assay to confirm that 8 dominant benthic
invertebrate species that inhabit Onosato River Estuary (ORE) and Kishiwada Artificial Tidal

Flat (KATF), two wetlands facing Osaka Bay, possess cellulase activities. Except for Cerithidea
moerchii, all of these invertebrates showed obvious seasonal variations in cellulase activities andstable isotope signatures, but the variation of enzymatic activities did not always correspond tothat of the carbon and nitrogen isotope ratio (δ13C and δ15N). In addition, variation in δ13C of theinvestigated invertebrates was much larger than that of their candidate food source, namely the
sedimentary organic matter (SOM) in the sediments and particulate organic matter (POM) in thesea, indicating the existence of selective feeding habits of the invertebrates, or biasing in theirprocessing of TerrestrialOM. Furthermore, the carbon isotope ratio and C/N ratio of the SOM/POM in

ORE and KATF corresponded to the δ13C value of the invertebrates that inhabited each wetland.

Additional cellulose-feeding experiments using Cerithidea moerchii showed that the δ13C did notdecrease significantly after the cellulose consumption, suggesting that the degradation product(glucose in this case) is not assimilated to its body tissues. In summary, this study revealed thataquatic invertebrates possessing CHs could use TerrestrialOMs as one of their food sources. Seasonal

1

Graduate School of Global Environmental Studies, Kyoto University, Kyoto 606-8371, Japan.

2

Department of Environmental Science on Biosphere, Tokyo University of Agriculture and Technology, Tokyo,183-8509, Japan.

3

C/O Institute for East China Sea Research, Nagasaki University, Nagasaki 850-2213, Japan.

4

Osaka Museum of Natural History, Osaka 546-0034.

* Corresponding Author

受付日：2021 年 6 月 19 日，受理日：2021 年 11 月 9 日，WEB 掲載日：2021 年 12 月 31 日

37Wen Liu・Yu Umezawa・Soma Kato・Kazumi Matsuoka

variations of enzymatic activity and carbon and nitrogen isotope ratios could indicate whether
animals were more/less dependent on TerrestrialOMs, but were insufficient to reveal the true amount of
OMs consumption by aquatic invertebrates.

Terrestrial

Key words: Benthic invertebrates, cellulase

as measurements by the tetrazolium method (Jue andIntroduction

Lipke, 1985) or Somogyi-Nelson Method (Green et al.,Wetlands are transitional areas between terrestrial and1989), are very useful tools to quantify the enzymatic

marine ecosystems. In additional to local hydrophytic

activities of polysaccharides decomposition, and thus

vegetation, the large amounts of TerrestrialOM that flow into estimate the consumption of TerrestrialOM. However,from hinterlands make wetlands big carbon sinks that

estimations of the TerrestrialOM consumption by enzymatic

sometimes even surpass terrestrial forests as carbon

activities are made under the assumptions that thesinks (Sudip et al., 2005; Scholz, 2011). It was at firstorganisms possessing these enzymes use them all theempirically shown that wetlands play an importanttime, as well as that the TerrestrialOM supply at the localityrole in breakdown of high molecular TerrestrialOMs intoof the inhabitant is constantly sufficient, which might not

low molecular nutrients that benefit marine organisms

always be true for every species of benthic invertebrate(Hatakeyama, 2019), and more recently endogenousand each wetland environment.

carbohydrate hydrolase (CH) (e.g., cellulase) genes have

In the present study, we focused on cellulose, which isbeen found in many aquatic invertebrates and proven to

the most abundant TerrestrialOM, and its catabolic enzyme,

Our recent studies summarized benthic invertebrates

the cellulase activity (the capability of decomposing

be responsible for this process (Tanimura et al., 2012).

namely cellulase, to investigate the relationship between

that have been proven to date to have endogenous

cellulose/TerrestrialOM) and the stable isotope signature (thecellulase genes, and/or cellulase activity (Liu et al.,assimilation level of TerrestrialOM). If these two indexes2021). Nevertheless, those studies are not able to answer

are in accord, it means that the cellulase activity could

the most-often asked questions from ecologists, such

represent the real amount of consumption of TerrestrialOM,as “do benthic invertebrates consume more TerrestrialOMand thus could be used to roughly estimate the amount(e.g., cellulose) than microorganisms?” or “what are theof TerrestrialOM consumed by benthic invertebrates. In

differences between wetlands with different benthic

addition, our previous studies (not published) showed

invertebrate species and populations?”. To answer thesethat both the cellulase level and stable isotope signatures

questions, quantification of the TerrestrialOM consumption

could differ according to the season. Therefore, weof benthic invertebrates will be essential.

collected all target benthic invertebrates as far as possible

Stable isotope analysis has some advantages forin all four seasons (May, July, October, and December,revealing food web structures. Recent stable isotope2020). Another attempt made in the present study isa n a lys e s h ave p r ov id e d ev id e n c e t h a t b e nt h ic

that we cultured one of the dominant invertebrates,

invertebrates inhabiting wetlands assimilate TerrestrialOMs

Cerithidea moerchii, in an environment in which only

as their food source (Kikuchi and Wada 1996; Doi

pure cellulose served as the food source, and investigatedet al., 2005; Kasai et al., 2005; Antonio et al., 2010;

the relationship between the amount of cellulose

Kawaida et al., 2019), but the level of assimilation of

consumption and the C and N isotope variation. Names

OMs differed between species and locations (Kanaya etof species in this article basically follow WoRMS (World

al., 2007; Liu et al., 2014). Thus, stable isotope analyses

Register of Marine Species; https://www.marinespecies.

are insufficient to quantify exactly how much TerrestrialOMorg).

is consumed/decomposed by each benthic invertebrate.

On the other hand, reducing sugar measurements, such
38

Seasonal variation in terrestrial cellulose assimilation of benthic invertebrates inhabiting wetlands in Osaka Bay, Japan

Table 1 Sampling site, sampling date and corresponding information
for the plate-assay (Fig. 2) of benthic invertebrates collected in ORE andKATF.

S p e c ie s

S a m p lin g sit e

Cerithidea moerchii

ORE A

Batillaria multiformis

KATF A

Tegula argyrostoma

ORE C
KATF B

Cellana nigrolineata

ORE C

Acanthopleura japonica

KATF B

Capitulum mitella

ORE D

Chiromantes dehaani

ORE B

Fistrobalanus kondakovi

ORE D

S a m p lin g Da t e
May 26th
July 30th
October 16th, 2020
July 30th
October 16th
December 18th, 2020
July 30th
October 16th
December 18th, 2020
May 26th
October 16th
December 18th, 2020
July 30th
October 16th, 2020
May 26th
October 16th
December 18th, 2020
May 26th
July 30th
October 16th
December 18th, 2020
October 16th
December 18th, 2020

c

d
e
f

g

h

collected, of which two were detritus feeders, two wereTarget benthic invertebrates were collected from ORE

scraping feeders, and two were filter feeders. Gastropod

and KATF, both located near Osaka Bay, Osaka, Japan

Cerithidea moerchii (A. Adams, 1855, (WoRMs, sameon May 26 , July 30 , October 16 , and December

below)) (Mollusca) were collected from a reed field (Fig.

th18 , as shown in Table 1. All maps were acquired from1c, site A). Crab Chiromantes dehaani (H. Mile Edwards,

th

the Geospatial Information Authority of Japan (Fig. 1)

1853) (Arthropoda) were captured on a muddy field (Fig.

(https://www.gsi.go.jp/). The area of the natural wetland

1c, site B) using a shovel to turn over their hiding nests.

ORE is 0.021 km . The area near the north shore has

Limpet Cellana nigrolineata (G. B. Sowerby, 1839)

2

sandy mud sediment, while the area near the south(Mollusca) and gastropod Tegula argyrostoma (Gmelin,

has sandy sediment. Dominant vegetation in ORE is1791) (Mollusca) were collected on the quay on the west

Phragmites australis, Zoysia sinica var. nipponica,side (Fig. 1c, site C). Goose barnacle Capitulum mitella

Lysimachia mauritiana, Calystegia soldanella, etc.

(Linnaeus, 1758) (Arthropoda) and acorn barnacle(Biodiversity Center, Ministry of the Environment,

Fistrobalanus kondakovi (Tarasov and Zevina, 1957)

2007). The area of the artificial wetland KATF is 0.05

(Arthropoda) were detached from the dents of the quay

km2. Sediment in KATF is sandy. Dominant vegetation

close to the mouth of the river (Fig. 1c, site D). At KATF,

in KATF is Phragmites australis (majority), Glehnia

we only found 3 species of dominant macro-benthos, ofwhich one was a detritus feeder and two were scraping

littoralis, Tetragonia tetragonioides, Calystegia

sol d a n ell a, etc. ( K ish iwad a Sh i zen Tomonoka ifeeders. Gastropod Batillaria multiformis (Lischke, 1869)

Higatahozen Kenkyu Gurupu, 2009). In 2020, the(Mollusca) was collected in the sandy shore (Fig. 1d, sitehighest tide level of these 2 wetlands was 473 cm (March

A). Gastropod T. argyrostoma and chiton Acanthopleura

10 ), the lowest was 256 cm (February 11 ), and thethbAgency). At ORE, 6 dominant macro-benthos were

1. Samplingthamean tide level was 376.9 cm (Japan Meteorological

Material and Methodsth

P la t e
A ssa yjaponica (Lischke, 1873) (Mollusca) were collected onst39Wen Liu・Yu Umezawa・Soma Kato・Kazumi Matsuoka
135º10′0″

145º0′0″

135º0′0″135º20′0″34º30′0″

Osaka BayKishiwada
Artificial Tidal Flat

40º0′0″Kansai
AirportOnosato River
Estuary35º0′0″100 km34º20′0″aKishiwada
Artificial Tidal Flat

135º15′0″

Onosato River Estuary

D

C

5 km

b

135º21′40″

1A

34º22′30″

2

B

B
1

50 m

c

A

34º28′40″d

50 m

Fig. 1 Sampling sites.

a, Location of Osaka Bay, shown in dashed squares. Latitude and longitude of this area, as wellas the scale bar are shown in the margin of the map, same below. b, Location of ORE and KATF
in the east coast of Osaka Bay. c, Detailed map of ORE. Sampling sites of benthic invertebrates

and sediments (SOM) are shown by letters, while sampling sites of seawater (POM) are shown in
numbers. d, Detailed map of KATF. Sampling sites are shown in the same way as in ORE.

Table 2 Carbon and nitrogen concentrations, carbon and nitrogen stable isotope
signatures and carbon/nitrogen ratios of SOM and POM in ORE and KATF.
Date
July 30th, 2020
SOM (Sediment)
October 16th, 2020

Wetlandδ15N (‰) δ13C (‰)

Sampling site

N (%)

C (%)

ORE

Fig.1c, A(Reed field)

C/N

0.10

0.87

3.9

-26.5

10.6

ORE

Fig.1c, A(Reed field)

0.13

1.50

3.0

-28.6

13.6

KATF

Fig.1d, A(Sandy field)

0.03

0.22

7.5

-20.1

7.4

N (mg/L) C (mg/L)
May 26th, 2020

POM (seawater)

July 30th, 2020

ORE

ORE

ORE
October 16th, 2020
KATF

Fig.1c, 1(River mouth)

0.03

0.23

7.4

-23.6

9.4

Fig.1c, 2(Upper stream)

0.04

0.41

6.1

-24.4

12.1

Fig.1c, 1(River mouth)

0.43

3.20

7.0

-22.2

8.6

Fig.1c, 2(Upper stream)

0.18

1.57

5.6

-24.6

10.0

Fig.1c, 1(River mouth)

0.29

2.35

7.4

-23.5

9.5

Fig.1c, 2(Upper stream)

0.12

1.10

5.8

-25.1

10.9

Fig.1d, 1(Quay west side)

0.07

0.43

9.3

-21.2

7.3

40

Seasonal variation in terrestrial cellulose assimilation of benthic invertebrates inhabiting wetlands in Osaka Bay, Japanthe quay on the west side of the wetland (Fig. 1d, siteLtd). The cellulase activity was defined as the quantity of

before they were transported to the lab and dissected.

power as glucose generated by each mg of the digestiveB). All benthic invertebrate samples were stored on ice

reducing sugar (in micrograms) with the same reducing

Seawater and surface sediment were collected near the

gland protein in 1 minute (μg glucose/min・mg protein)sampling sites in both wetlands (Table 2, sampling site)(Nelson, 1944).

to investigate the characteristics of POM as potential
food sources for the targeted species.

4. Stable isotope analysis

2. Plate Assay

4.1.1. Benthic invertebrates

4.1. Pre-treatments of the samples

One percent carboxymethyl cellulose (CMC) was added

Foot muscle from C. moerchii, B. multiformis, T.

to an agar plate for the cellulase activity assay. For C.

argyrostoma, and C. nigrolineata, mantles from A.

and A. japonica, digestive glands were separated as thepeduncle muscle from C. mitella, and working leg

japonica and F. kondakovi (near the basal plate),

moerchii, B. multiformis, T. argyrostoma, C. nigrolineata
sample for the assay. For C. mitella and F. kondakovi,

muscles from C. dehaani were isolated. No delipidation

the whole body except their exoskeleton was used.

process was applied because these parts contain very less

The C. dehaani were first frozen at −20℃ for 2 hours

lipids and have little individual differences. All samples

before their digestive glands were separated. All samples

were lyophilized to avoid ammonia volatilization by

were washed with 70% ethanol and dried first, and thenusing a vacuum drying oven (DRV320DA, ADVANTEC)

homogenized with scissors before they were applied toconnected to a cold trap (DRT140FB, ADVANTEC)

the agar plate. After a 24-hour incubation, 1mM Tris-

and a diaphragm vacuum pump (MD4C, Vacuubrand)

HCl buffer (pH = 7.5) containing 1% Congo Red wasfor over 1 day until the weight of the samples stabilized.

added to stain the CMC for 2 hours. Then, 1M NaCl was

Dried samples were powdered using a mortar andadded to de-stain unbound Congo Red in the agar plate.

pestle, and transferred to 2-ml glass screwtop storage

Cellulase activity was shown as a halo around the sample

bottles that were pre-heated at 550℃ for 3 hours.

where CMC was decomposed.

Approximately 0.3–0.6 mg of each sample (whichcontained 80 ~ 100 μg of nitrogen) was weighed inside

3. Cellulase activity quantification

a tin-capsule (SA76981101, SANTIS) by using an ultra-

Digestive glands were separated from C. moerchii, C.

micro-electronic balance (XP2U, METTLER TOLEDO),
molded into a small pellet and kept dry until analyses.

dehaani, C. nigrolineata, T. argyrostoma, B. multiformis,and A. japonica into a 1.5 ml tube and 0.5 ml Tris-

HCl buffer (pH = 7.5) was added. After the sample

4.1.2. Sediment organic matter (SOM)

min, it was centrifuged at 8,000 × g for 5 min, and themesh to remove plant fragments, shells, and coarse sand,of the digestive glands. The protein concentration of theApproximately 10 mg of sediment sample was weighed

was homogenized (MH-1000, AS ONE Corp.) for 1

Dried sediment samples were sieved through a 300-μm

supernatant was transferred to a new tube as the extract

and then homogenized with an agate mortar and pestle.

extracts was measured by the Bradford method (Bradford,

inside a silver capsule (SA76981105, SANTIS) by using

1976). Fifty micrograms of the extract were transferred

an ultra-micro-electronic balance, and acidified withto a new tube and diluted to 50 μl by adding Tris-HCl

2–3 drops of 1N HCl to remove CaCO3. More drops ofbuffer (pH = 7.5). Fifty microliters of 1% CMC (in1N HCl were added to the sample collected from KATF

Tris-HCl, pH = 7.5) was then added to start the enzyme

because numerous CO2 bubbles appeared. Then, samples

reaction. After incubation at 25℃ for 30 minutes, 100 μl

were transferred onto a 60℃ hot plate (C-MAG HP7,

Somogyi reagent was added and the mixture was heated

IKA) and heated for 5 hours to remove remaining HCl

at 100℃ for 10 minutes. After the heating, the tube

and water. Finally, SOM samples were packed into cubes

was cooled down, 200 μl of Nelson reagent was added,

with tin disks (SA999902, SANTIS) as described above.

and the absorbance at 660 nm was measured by using aspectrometer (NanoPhotometer®N60, WakenBtech Co.,
41

Wen Liu・Yu Umezawa・Soma Kato・Kazumi Matsuoka

4.1. 3. Particulate organic matter (POM)

alanine (δ13C = −19.6‰, δ15N = 13.7‰, SI Science Inc.)

Five liters of seawater from each sampling site were

was also analyzed to check the accuracy of the regression

using a suction pump (DA-30S, ULVAC Inc.). Seawater

for both δ13C and δ15N of the samples.

transferred to the lab and filtered within the same day by

curve. The precision of the analysis was under ± 0.1‰

samples were mixed well before they were applied tothe suction filter system until the glass filter was clogged

5. Cellulose-feeding experiment

Aldrich Co.) was pre-heated at 450℃ for 3 hours toethanol, 10 individuals of C. moerchii were moved to apressure was fixed at under 0.02 MPa to prevent largeheight) containing 500 ml autoclaved artificial seawaterwith POM. The glass filter (φ=47mm, GF/F, Sigma-

After the surface of shells was cleaned with 70%

remove remaining organic matter before use. Vacuum

plastic culture tank (20 cm length, 20 cm width, 20 cm

size particles from penetrating the glass filter. Collected

previously poured into it (Fig. 5a). An approximately 100

POM was then transferred to a plastic container and

cm2 wide stone higher than the surface of the seawater

fumigated with 12 N–HCl for 1 day to remove inorganic

was placed in the tank as a resting scaffold (since C.

carbon, and then transferred to a vacuum desiccatormoerchii seems to avoid seawater by climbing reeds inwith NaOH pellets and allowed to stand for 1 week

natural wetlands). Cerithidea moerchii were fed withto remove HCl vapor. The filter was dried again on alaboratory paper tissue (NIPPON PAPER CRECIA CO.,hotplate at 60℃ for 2 hours to completely remove water

LTD, δ 13C = −23.4‰, δ 15N = 4.1‰ analyzed in theand remaining HCl. Margins of the glass filter without

present study), which was cut into same-size pieces (eachPOM were removed. The remaining glass filter was cut

piece weighed approximately 32.8 mg). After 20 days, ato one-quarter to one-third of the original size to adjust

total of 18 pieces of paper tissue had been provided andthe nitrogen concentration, and packed with tin-disks

consumed by C. moerchii, and three individuals were

(SA999902, SANTIS).

randomly sampled. After 44 days, an additional ninepieces of paper tissue had been eaten by the remaining

4.2. Elemental analysis and mass spectrometry

seven individuals of C. moerchii, and another three were

Pre-treated samples were then applied to an Organic

randomly sampled. For the control group, instead of

Elemental Analyzer (Flash 1112, Thermo Fisher

artificial seawater, sediment heated at 600℃ for 3 hours

Scientific) and an Isotope Ratio Mass Spectrometer

was spread in the bottom of the tank, and no paper tissue(Delta plus XP, Thermo Scientific Inc.) via a Universal

was provided until Day 44. All sampled C. moerchii wereContinuous Flow interface (Conflo III, Thermo Fisher

dissected and their carbon and nitrogen isotope ratios

Scientific). The carbon concentration (shown as C (%) in

were analyzed as described above in 4.1.1.

SOM and C (mg/L) in POM) and nitrogen concentration(shown as N (%) in SOM and N (mg/L) in POM) were

6. Statistical analysis

nitrogen stable isotope ratio (δ N) were calculated usingver.22. Levene’s test was used before one-way ANOVAδ 13C, δ 15N = (R sample/R standard −1) × 1000 (‰), whereof each invertebrate species collected in ORE and KATF

measured, and the carbon stable isotope ratio (δ C) andStatistical analyses were carried out using IBM SPSS,

13

15

the following equation:

was applied to check the differences in cellulase activities

the term R denotes the ratios of C/ C or N/ N, and
13

12

15

among seasons. If ANOVA showed significance, Tukey

14

Vienna Pee Dee Belemnite and atmospheric nitrogen

Scheffe Post-Hoc analysis was applied. In addition, sincewere used as standards for carbon and nitrogen isotopes,
respectively.

we only had data of July and October for A. japonica in

KATF, bivariate correlation analysis (Pearson correlation

As the working standard, alanine (δ C = −19.6‰, δ N
13coefficients, 2-tailed) was applied instead. Bivariate

15= 8.7‰, SI Science Inc.) was regularly analyzed to checkcorrelation analysis was also applied in the cellulose-

the accuracy of the analysis and construct a regression

feeding experiment to verify the differences of δ 13C

curve between the stable isotope ratio and the amount ofand δ15N values between cellulose-feeding groups andsample for the calibration of mass-dependent fluctuation

control groups.

of stable isotopes values (Umezawa, 2020). In addition,
42

Seasonal variation in terrestrial cellulose assimilation of benthic invertebrates inhabiting wetlands in Osaka Bay, Japan

a

b

d

f

c

g

e

h

Fig. 2 Plate assay of benthic invertebrates.

Eight species of benthic invertebrates (5 species from ORE, 2 species from KATF,

1 species from both sites) were investigated for their cellulase activities. Digestive
glands or the whole body of each species were placed in a well made in a 1% CMC-

containing agar plate. Congo Red was used to stain the CMC after the enzymatic
reaction. The halos around each well mean no CMC remained, representing thecellulase activity. Corresponding information between the plate assay letters andspecies is summarized in Table 1.

of C and N, the highest concentration of C (%) and N

Results(%) of SOM in ORE was shown in October. The N

1. Plate Assay

concentration (%) of SOM in ORE was about 4.3 times

For purposes of discussion, we categorized all collectedlarger than that in KATF in October, and the carbon

invertebrates into three feeding habit groups: detritus

concentration (%) in ORE was about 6.8 times larger thanfeeder, scraping feeder and filter feeder. As shown in Fig.

that in KATF in the same month. POM showed the same2, all species showed strong cellulase activity.

tendency: the highest concentration of both C (mg/L) andN (mg/L) in ORE (in both river mouth and upper stream)

2. Composition differences and the seasonal variation

were shown in July. The C (mg/L, river mouth) in ORE

of SOM and POM between Onosato River Estuary

was about 5.5 times higher than that in KATF, and the N(ORE) and Kishiwada Artificial Tidal Flat (KATF)(mg/L, river mouth) in ORE was about 4.1 times higher

As shown in Table 2, we investigated the carbon andthan that in KATF. In addition, the δ13C values of both

nitrogen concentrations in SOM (C (%), N (%)), carbon

SOM and POM in ORE were much smaller than those in

and nitrogen concentration in POM (C (mg/L), N (mg/L)),

KATF (8.5‰ variation in SOM, 2.4–3.9‰ variation incarbon and nitrogen isotopes ratio (δ C, δ N) as well
13POM). The C/N ratio in ORE was much higher than that15

as carbon/nitrogen ratio (C/N) of both SOM and POM

in KATF. In ORE, the lowest C/N ratio in SOM (10.6)

in ORE and KATF. SOM was sampled in both July andwas found in July, and the lowest C/N ratio in POM (8.6)

October in ORE, but only in October in KATF. POM

was found in the same month.

was sampled in May, July and October in ORE, but onlyin October in KATF. δ13C and δ15N showed no significant

3. Seasonal variation in cellulase activity and stableand δ N showed a 0.9‰ difference between July and3.1. Onosato River Estuary

isotope ratio of benthic invertebrates

seasonal variation in ORE: δ C of SOM showed a 2.1‰,
13

15

October, while δ C of POM showed a maximum of 1.4‰

The stable isotope ratios of benthic invertebrates

13

difference in the river mouth, and δ15N of POM showed

differed among feeding habit groups (Fig. 3). Usually,among May, July and October. As for the concentration

C. dehaani) are lower than those of scraping feeders (i.e.,a maximum of 0.5‰ difference (in the upper stream)

the δ13C values of detritus feeders (i.e., C. moerchii and43

Wen Liu・Yu Umezawa・Soma Kato・Kazumi MatsuokaabFig. 3 Carbon and nitrogen stable isotope signatures and cellulase activity level of the benthic
invertebrates in ORE.

a. Carbon and nitrogen stable isotope signatures. Ellipse of each color shows the range ofthe seasonal variation of carbon (horizontal axis) and nitrogen (vertical axis) of each benthic
invertebrate species. Four shapes represent the sampling seasons (Triangle: May; Rhomboid:
July; Square: October; X mark: December; same in each species). Values are means ± SD.

b. Cellulase activities. The cellulase activity was defined as the reducing sugar produced inunit time (min) and unit tissue (mg protein) that have the same reducing power of unit weight
glucose (μg). Values are means ± SD. p values of Post Hoc analysis (Scheffe) are shown

between adjacent seasons.

T. argyrostoma and C. nigrolineata). Among detritus

range of δ13C value (−11.9 to −15.3‰), but the variation

narrow range (−17.5 to −17.7‰) throughout the wholelowest δ13C values of C. nigrolineata and T. argyrostoma

feeders, the δ C value of C. moerchii stayed in a low andbetween individuals in each season was small. Also, the13year. Both the δ13C value (−14.5 to −16.1‰) and δ15N

were both shown in winter (December). The δ13 C

value (11.3 to 12.2‰) of C. dehaani, another dominantvalues of filter feeders (i.e., C. mitella and F. kondakovi)

As scraping feeders, C. nigrolineata and T. argyrostoma

feeders, but the δ15N value of the filter feeders was much

detritus feeder, were higher than those of C. moerchii.

showed no big differences compared to those of detritus

showed higher δ13C and δ15N values compared to thehigher (14.6–15.5‰ in C. mitella and 15.4–16.9‰ in

detritus feeders. Tegula argyrostoma showed a very wide

F. kondakovi). The filter feeders also showed a large
44

Seasonal variation in terrestrial cellulose assimilation of benthic invertebrates inhabiting wetlands in Osaka Bay, Japanab

Fig. 4 Carbon and nitrogen stable isotope signatures and cellulase activity level of thebenthic invertebrates in KATF.

a. Carbon and nitrogen stable isotope signatures. b. Cellulase activities. Legends are thesame as for Fig. 3, except p values of A. japonica are for 2-tailed t test.

variation of δ13C values between individuals (especiallywith a large deviation among individuals, and showedwhich was not seen in the other two feeding habit groups.

activity in December.

C. mitella in December and F. kondakovi in October),the highest activity in October, and slightly decreased

The cellulase activities of four investigated invertebrates(C. moerchii, C. dehaani, C. nigrolineata, and T.

3.2. Kishiwada artificial tidal flat

C. moerchii showed the highest level of cellulase activity

in this tidal f lat throughout the year, namely, B.

C. dehaani was kept at a high level from May to Octoberin Fig. 4, overall, the δ13C value and δ15N value of theseargyrostoma) all showed seasonal variation. For example,

We found only three dominant species of invertebrates

in May, and the lowest in July. The cellulase activity ofmultiformis, T. argyrostoma, and A. japonica. As shown(slightly dropped in July), but suddenly decreased tothree species were higher than those invertebrates

almost zero in December. For C. nigrolineata, thethat lived in ORE. Nevertheless, seasonal variation ofhighest cellulase activity was shown in May, it decreased

the stable isotope ratio could still be observed in eachin October, and further decreased in December but

species. For B. multiformis, the lowest δ13C value wasstill remained at half the activity level of May. Tegula

shown in December, with a large deviation amongargyrostoma showed the lowest cellulase activity in May

individuals. The δ13C value in July was close to the45Wen Liu・Yu Umezawa・Soma Kato・Kazumi Matsuoka

a

c

b

Fig. 5 Cellulose-feeding experiment.

a. Scheme of the culturing system for cellulose-feeding group. b. Carbon stable isotope

ratio between cellulose-feeding group (black bars) and control group (white bars). Values aremeans ± SD. p values are for 2-tailed t test between the cellulose-feeding groups and control
groups. c. Nitrogen stable isotope ratios, legends are the same as for b.

average value in December, with much smaller deviation.

4. Cellulose-feeding experiment

range of δ13C value of B. multiformis at KATF was

feeding group did not significantly decrease compared toT. argyrostoma in October). Tegula argyrostoma living

below) or 44 (p = 0.91) days. Similarly, Fig. 5c showed

The highest δ13C value was shown in October. The

As shown in Fig.5b, the δ13C value of the cellulose-higher than that of the scraping feeders at ORE (except

the control group after 20 (p = 0.71, 2 tailed t test, samein KATF showed exactly the same pattern of seasonalthat the δ15N value of the cellulose-feeding group was notvariation of δ13C and δ15N, but the values were higher

significantly increased at day 20 (p = 0.69) or day 44 (p =than those in ORE.

0.53).

For the cellulase activities, it seems that the higher

the cellulase activities were, the higher the δ13C value

5. Statistical analysis

japonica, however, the lowest cellulase activity andcellulase activities of each invertebrate, as well as the ppattern appeared in October.

only) are shown in Fig. 3b and Fig. 4b. The results of thebecame in B. multiformis and T. argyrostoma. For A.

The p values of Post Hoc analysis between seasons’highest δ C value were found in July and the opposite

values of bivariate correlation analysis (for A. japonica

13

t test between the δ13C and δ15N values of the cellulosefeeding group and control group are shown in Fig.5.
46

Seasonal variation in terrestrial cellulose assimilation of benthic invertebrates inhabiting wetlands in Osaka Bay, Japanratios compared to marine phytoplankton and freshwater

Discussion

phytoplankton (Hayami et al., 2019). In addition, theBefore any investigation of the relationship between

fact that the carbon and nitrogen concentrations (mg/the seasonal variations of cellulase activity and isotopeL) in POM in July both suddenly increased almost 14-

ratios, we first validated the cellulase activity in eachfold compared to those in May indicated a large marine-

collected invertebrate species by plate assay. As shown

origin organic matter (C/N ratio = 8.6) input in thatin Fig. 2, all species showed strong activity. The plateseason. In KATF, the carbon and nitrogen concentrations

assay could not distinguish between whether thesein SOM (0.22% carbon, 0.03% nitrogen) and POM

activities came from the invertebrates themselves or(0.43 mg/L carbon, 0.07 mg/L nitrogen) were lower

symbiotic microorganisms in their digestive system.

than those in ORE (0.87–1.5% carbon and 0.1 ~ 0.13%

Nevertheless, previous studies revealed a variety ofnitrogen in SOM, 0.23–3.2 mg/L carbon and 0.03–0.43

aquatic invertebrates, especially molluscs and arthropods,

mg/L nitrogen in POM, respectively), suggesting thatpossessing endogenous cellulase (Watanabe and Tokkuda,organic matters used as the food resources for benthic

2001; Tanimura et al., 2014; Liu et al., 2021). In theorganisms in SOM and POM might be limited in KATF.

present study, we added several drops of 1mM NaN3 to

In addition, the δ13C values of SOM and POM in KATF

each sample to suppress the activity of microorganisms(SOM: −20.1‰; POM: −21.2‰) were both higher thansuch as bacteria during the enzymic reaction, indicating

those in ORE (SOM: −28.6‰; POM: −23.5 to −25.1‰),

that the detected cellulase activity was more likely fromand the C/N ratios in KATF (SOM: 7.4; POM: 7.3) were

the invertebrates themselves. With either origin of thelower than those of ORE (SOM: 13.6; POM: 9.5~10.9),

cellulase, however, these invertebrates are capable ofindicating that KATF not only had limited total organic

decomposing Terrestrial cellulose, and thus they are very

matter, it also lacked TerrestrialOM inputs. This lack oflikely to be responsible for utilizing TerrestrialOMs.

Terrestrial

OM might have been because this small tidal flat

et al., 1999; Ward et al., 2013), and the composition of

from the land, and thus had no riverine TerrestrialOM inputs

SOM and POM usually contain TerrestrialOMs (Mishima

was newly reclaimed in the middle of the sea, isolated

OMs will affect the carbon and nitrogen isotope

as well as limited hinterland for hydrophyte vegetation.

Terrestrial

ratios of SOM and POM (Ogawa et al., 1994; Wada et

In contrast, the Onosato River in ORE carries TerrestrialOMal., 1984; Wada et al., 1987). Normally, δ13C of Terrestrial(such as leaves, which can be seen by the eye in the POM

C 3 plants ranged from −23 to −33‰, δ C of marine

samples), which accumulates in the sediment near the13

phytoplankton ranged from −18 to −21‰, and δ C ofriver mouth.

13

Terrestrial

C4 plants or seagrass ranged from −8 to −15.5‰,

As shown in Fig. 3, in ORE, the δ 13C values of C.

Hayami et al., 2019). Thus, in Japan which is dominatedmoerchii and C. dehaani (both detritus feeders) were

SOM and POM, the lower their δ C value will be.

(scraping feeders), which is reasonable because theby C 3 plants, the higher the Terrestrial OMs contents inlower than those of T. argyrostoma and C. nigrolineataI13

We investigated the seasonal variation of carbon andsediment in ORE contains a large amount of TerrestrialOM

nitrogen isotope ratios in ORE. The results showed that(δ 13 C = −26.5 to −28.6‰, Table 2). Because C.

most of the time, the δ13C value of both SOM and POM

moerchii maintained its δ13C value low in all seasons,in ORE was lower than −22‰, and it did not fluctuate

it is considered to assimilate more TerrestrialOMs than the

OM content was lower. On the other hand, thewe consider the low δ13C value to be evidence of morevery much across seasons (Table 2), indicating that theTerrestrial

other investigated invertebrates. In the present study,

δ13C values suggested that the TerrestrialOM content in POM

Terrestrial

OM assimilation. But normally, the δ13C value

was smaller than that in the SOM, which was supported

will not be much different from that of an animal’s(normally, the C/N ratio of Terrestrial C3 plants and soils is

Fry, 2006, sometimes 2–3‰, Busst and Britton, 2016;

by the fact that the C/N ratio of the POM was lower

food source (trophic enrichment factor (TEF) = 0–1‰,

13–25, while that of marine phytoplankton is 8–10.5;

Umezawa et al., 2018). In ORE, however, a difference

Boutton, 1991;Yoneyama, 2008), since previous studiesin δ13C between potential food sources (SOM) and C.

revealed that Terrestrial plant-origin OMs have higher C/Nmoerchii were large (approx. 10‰), which remains to47

Wen Liu・Yu Umezawa・Soma Kato・Kazumi Matsuoka

be explained. One of the possibilities is the existence ofprovided), but somehow was not assimilated by theirfocused on four gastropod species inhabiting anothereach C. moerchii individual consumed the same amountunidentified food source(s) in ORE. Our previous study

body tissue. Note here that this experiment assumed thatnatural wetland in the east coast of Japan revealed thatof cellulose, which might not be true. Since we sampledthe δ C values of the gastropods were positively related

them randomly, there was a chance that we accidently

13

to the cellulase activities, indicating a relationshipsampled the individuals that consumed less cellulose.

between the δ 13C value and TerrestrialOM assimilation

However, a previous study using grapsid crabs feeding(Liu et al., 2014). The δ13C value of C. moerchii inon mangrove leaf litter has raised some other possibilities

and the microphytobenthos (MPB) investigated in thefeeding experiment data (Bui and Lee, 2014). First,

the previous study was approximately −18 to −19‰,that should also be considered when looking at ourprevious study had a δ13C value of around −18‰, andthe time frame of 44 days might not be long enoughwere considered to be the most likely food source forfor C. moerchii to show measurable isotopic change in

C. moerchii. Another previous study in Obitsu River,their muscle tissue. Secondly, the food source that C.

Tokyo Bay also showed that C. rhizophorarum (samemoerchii primarily consumes in the field may have a

as C. moerchii) had a δ13C value of −18.8 ± 0.2‰, close

similar δ13C value to that of the tissue paper. In fact, C.

to the MPB (−19.8 ± 0.3‰) in the same region (Kon et

moerchii often resides in or adjacent to reed fields belowal., 2012). In the present study, we only investigated thewetlands. If reed litter dominated their diet before theisotope ratio of SOM and POM as a total. The MPB in

feeding experiment, the tissue paper provided as food

ORE probably have a similar δ13C value to these previous

in the experiment may not have produced a change

studies, and thus might be the main food source of C.

in their body δ 13C value. Thirdly, the TEF of the C.

moerchii. However, it is hard to consider that C. moerchii

moerchii consuming tissue paper was extremely high

had a selective feeding habit to differentiate high δ C(c.a. 5‰), and similar to the difference in δ13C between

13

value foods from SOM and/or POM, which needs further

C. moerchii and tissue paper, resulting in a “concealing”

investigation, and in this case, it is difficult to explainof any change in the δ13C value even if tissue paper was

why there would be a need to possess cellulase activity.

assimilated. Newly designed future experiments such

Another possible explanation is that C. moerchii (as well

as extending feeding period, changing target animals,

as other investigated invertebrates in the present study)

and using tissue paper with a higher δ13C value than thatmight have an isotope biasing or fractionation in theirof C. moerchii body tissues will be required to inspect

processing of food. The product of cellulose digestion

these possibilities. On the other hand, the δ15N values

is glucose, and it can be used in respiration pathways(7.8–10.2‰) of C. moerchii in ORE were lower than inrather than other biochemical synthesis pathways. There

the previous study (10–13‰, Liu et al., 2014), whichmight be some unknown bias of metabolizing a particular

could be ascribed to the lower δ15N value of POM in the

carbon isotope (13C) in glucose and finally releasing it

present study (ORE, 5.6–7.4 ‰) compared to the δ15N

as carbon dioxide. However, testing this hypothesis willvalue of POM (about 9 ‰) in the previous study. Thisrequire a series of experiments, probably using isotope

result suggests that C. moerchii from these two studies

techniques, to follow carbon in the metabolic pathways

consume similar amount of MPB.

of these invertebrates. Instead, we have conducted a

As for the cellulase activity, we thought at first that thecellulose-feeding experiment in the present study, and thecellulase level would correspond to the seasonal variation

by C. moerchii (average 59 mg cellulose/individual after

thinking, higher cellulase activity means more cellulose

result showed that after cellulose was consumed (eaten)of the organisms’ carbon isotope ratio, because generally

20 days, average 101 mg cellulose/individual after 44

decomposition which could leads to more cellulosedays), the δ C value of C. moerchii had a trend, but did(TerrestrialOM) assimilation. But there were quite a large

13

not significantly decrease compared to that of the control

number of exceptions in the present study. C. moerchii,group (Fig. 5b). This is strong evidence that the cellulose

for example, showed the highest level of cellulase

was consumed by C. moerchii (we observed that theactivity in May, the same season when the lowest δ13C

paper tissue was eaten by C. moerchii every time it wasvalue was shown. However, in October, C. moerchii
48

Seasonal variation in terrestrial cellulose assimilation of benthic invertebrates inhabiting wetlands in Osaka Bay, Japan

showed the highest δ13C value but the cellulase activity

when we visited ORE in winter. If no food is taken in

OM input in July might deposit in the sediment andassimilated body parts, which would cause increased

was quite low. As described above, the marine-origin

anymore during the winter, they might start to consume

increase its δ C value, causing the decrease of cellulase

δ13C and δ15N values, but since this phenomenon depends

13

activity and the increase of δ13C and δ15N values of theon the species, further investigation is required.

detritus feeders living on it from July to October.

As scraping feeders, C. nigrolineata and T. argyrostoma

Chiromantes dehaani is another detritus feeder

showed higher δ13C and δ15N values compared to the−16.1‰) and δ N value (11.3 to 12.2‰) of C. dehaani

microphytobenthos that include heterotrophic ones

investigated in ORE. Both the δ 13C value (−14.5 todetritus feeders, possibly because they consume more

15

were higher than those of C. moerchii. Previous studies(e.g., Chlamydomonas, Blifernez-Klassen et al, 2012;

based on stomach/fecal content analysis or direct

Actinophrys, Sakaguchi and Suzaki, 1999) probably

observations revealed that aquatic crabs consume awith higher δ13C and δ15N ratio living on the quay andsignificant amount of leaf litter (Odum and Heald,

breakwater. Interestingly, T. argyrostoma showed a very

1972: Poon et al., 2010; Nordhaus et al., 2011). Anotherwide range of δ13C values (−11.9 ~ −15.3‰), but theprevious study in China showed that C. dehaani thatvariation between individuals in each season was small.

inhabit a salt marsh dominated by Phragmites australisThis might because all individuals of T. argyrostoma(reed, C 3 plant) have a significant lower δ C valuechange their feeding habit similarly to each other in

13(−22.67 ± 0.58‰) than that (−16.07 ± 0.63‰) of thedifferent seasons. Also, the lowest δ13C values of C.

same species that inhabit a nearby salt marsh dominatednigrolineata and T. argyrostoma were both found inby Spartina alterniflora (cordgrass, C4 plant) (Qin et al.,December. This might be because 1) in winter, the2010). These results are strong evidence that C. dehaani

production of microalgae on the quay was decreased

assimilates TerrestrialOM, resulting in the decrease of δ C(Sutherland et al., 2013) so the scraping feeders who rely

13

value. In the case of C. dehaani in ORE, the seasonalon them have to use TerrestrialOMs as an alternative foodvariation of cellulase activity was basically synchronizedsource. There seems to be a large input of TerrestrialOMwith the variation of δ C value: the cellulase activity wasduring this month because POM in the upper stream

13

maintained at a high level from May to October (Fig. 3),

showed the lowest δ 13C value (−25.1‰) then among

while the δ C value decreased accordingly. The cellulase

the values throughout the year, or 2) microphytobenthos

13

activity suddenly decreased to close to zero in December,is their main food source but the δ 13 C value ofand the δ13C value of C. dehaani then increased to almost

microphytobenthos decreased in winter. The reason forthe highest level of the year. Chiromantes dehaani wasthis is that the microphytobenthos have a larger isotopic

known to consume marine-origin microphytoplankton

fractionation of dissolved inorganic carbon (DIC) uptake

according to a previous study (Kon et al. 2012). Similar

in winter, when they have low productivity, as well as

to the values in C. moerchii, the δ C values in C. dehaanithat the low temperature will increase the dissolution

13

in the present study were quite high (−14.5 to −16.1‰),of atmosphere CO2 and thus decrease the δ13C value

indicating that C. dehaani might consume both TerrestrialOMof seawater DIC (Macleod and Barton, 1998; Finlay,

and marine-origin microphytoplankton, but assimilate2004). For cellulase activities, it seems that the higher

the latter more, supported by the high δ N values of C.

the activities of C. nigrolineata and T. argyrostoma

15

dehaani in ORE (11.2 to 12.6‰) compared to the values

are, the higher the δ13C value becomes, which is veryin the study in China (7.54 ± 0.21‰ in C3 plant-dominantpuzzling. One of the possibilities is that, in May, C.

salt marsh (Qin et al., 2010). However, the lowest δ13C

nigrolineata and T. argyrostoma mainly assimilated

value and cellulase activity shown in winter could also

microphytobenthos, and high cellulase activity was

be ascribed to their habit of hibernation. Previous studies

used for decomposing more cellulose but most of

revealed that starvation could result in an increase of

the products (i.e., glucose) were used to synthesize

δ13C and δ15N values in chironomid larvae (Doi et al.,ATP for microphytobenthos-searching activities. In

2007) and salt marsh snail (Kurata and Kikuchi, 2001).

winter, cellulase activity decreases because the whole

Chiromantes dehaani individuals were all hidden in nests

metabolism was decreased by the low temperature.
49

Wen Liu・Yu Umezawa・Soma Kato・Kazumi Matsuoka

Instead of being used to synthesize ATP, producedon marine-origin OMs, unlike other detritus feeders

in body tissues, which cannot be acquired much fromfed on sea lettuce (Ulva spp.) several times in KATF.

glucose was bypassed to synthesize biochemicals usedliving in ORE. We have observed that B. multiformis

microphytobenthos. However, these are all speculations

According to Matsuo et al., 2009, Ulva spp. in Osakawith no support from previous studies, and thus remain

Bay have δ 13C value around 10.5‰ and δ 15N value

to be further investigated.

around 9.0‰, which is quite close to October’s values

The filter feeders (i.e., C. mitella and F. kondakovi)of B. multiformis in KATF. Our previous studies showed

showed high δ N values, which might be becausethat in addition to cellulase, B. multiformis also has

15of that they assimilate zooplankton in POM as their

mannanase and laminarinase activities, which could be

food source. For C. mitella, the δ C value was quite

used to decompose the sea lettuce (Liu et al., 2014).

13

low, close to that of C. moerchii, indicating they might

Compared to the T. argyrostoma that inhabited ORE, T.

also assimilate TerrestrialOMs. Also, both C. mitella andargyrostoma living in KATF showed exactly the same

F. kondakovi showed a large variation of δ C values

pattern of seasonal variation of δ13C and δ15N, but the13

between individuals (especially C. mitella in December

values were higher. For A. japonica, although the δ15N

and F. kondakovi in October), which was not seen in thevalue (14.4–15.0‰) was similar to that of its related

other two feeding habit groups. This might be because

species (C. nigrolineata) in ORE (13.2–14.0‰), the δ13C

these two water filtering invertebrates cannot movevalue was much higher (−11.4 to −12.2‰) compared toonce they are grounded on the quay and breakwater.

that of C. nigrolineata (−14.7 to −16.4%). The 2 scraping

The POM composition should have no big differences

feeders in KATF seemed to assimilate less TerrestrialOMs

within such a small area, but microturbulence in narrowcompared to the same/related species in ORE, possibly

spaces between stones of quays and breakwaters might

because of the shortage of TerrestrialOMs.

cause unevenness of POM distribution, causing theRegarding the cellulase activities, the higher thevariation of δ13C values. Previous studies showed that an

cellulase activities were, the higher the δ 13C value

in phytoplankton in a close water range (Yoshioka et al.,results might indicate that TerrestrialOMs are acting as

approximately 2–4‰ isotope fractionation could appear

became in B. multiformis and T. argyrostoma. These

1994), and phytoplankton with small size and short life

an alternative food source of these invertebrates forcycle could receive more influence (Montoya, 2007), andrespiration without assimilating them for making

thus accordingly might cause the variation of δ C values

body tissues during the time they could not find high13of C. mitella and F. kondakovi.

nutritional value foods (i.e., foods with high δ13C andKishiwada Artificial Tidal Flat (KATF) is a newlyδ 15 N value). For A. japonica, however, the lowest

reclaimed land close to ORE, but with no river inputs

cellulase activity and highest δ 13C value was foundand remarkably less vegetation, which we thought toin July and the opposite pattern appeared in October,make it valuable for comparison to ORE. As describedindicating that A. japonica consumed and assimilated

above, the total organic matter content is limited, andmore TerrestrialOMs in in October, possibly because of themost of its organic matter is of marine origin. We foundlimited planktonic and benthic microalgae production

only three species of invertebrates that inhabit thiscaused by the dropping of water temperature.

tidal flat throughout the year, namely, B. multiformis,

T. argyrostoma, and A. japonica. As shown in Fig. 4,

Conclusion

overall, the δ13C value and δ15N values of these three
species were higher than those of invertebrates thatAll dominant invertebrates inhabiting wetlands aroundlive in ORE, which might because of the shortage ofOsaka Bay, including detritus feeders, scraping feeders

OMs. Nevertheless, seasonal variation of the stableand filter feeders, were found to have cellulase activity.

Terrestrial

isotope ratio could still be observed in each species. For

Further investigations are needed to determine whether

B. multiformis, the range of δ13C value was even higher

the cellulases are endogenous or of symbiotic origin.

than that of the scraping feeders (except T. argyrostoma

Nevertheless, these invertebrates have the capability ofin October), indicating that their feeding habit reliedutilizing Terrestrial plant-origin cellulose. In addition, the50

Seasonal variation in terrestrial cellulose assimilation of benthic invertebrates inhabiting wetlands in Osaka Bay, Japanhigh δ13C value and low C/N ratio of SOM and POM

to trace the metabolic pathway of the decomposed/

which might cause the higher δ13C and δ15N values ofaquatic invertebrates in carbon cycling between land andin KATF indicate that KATF lacks TerrestrialOMs inputs,

consumed TerrestrialOM to reveal the true role played by

the invertebrates inhabit KATF compared to ORE. Thosesea.

results suggested that the isotope ratio (especially δ C)
13

could be used as an indicator of the assimilation level

Acknowledgementsof TerrestrialOMs. Seasonal variations of δ13C value were

found in almost all investigated invertebrates, indicating

We thank Dr. So Ishida, Osaka Museum of Naturalthat their level of assimilation of TerrestrialOM changes

History, for classifying the benthic invertebrates andWe tried to quantify the cellulase activity in orderinhabitants and to D. Ken'ichiro Ishii for his kind support

throughout the year.

providing valuable information about invertebrate

to estimate how much TerrestrialOMs the invertebrates

in the field survey. We also express our sincere thanks

consume, but the results showed that the pattern of theto four reviewers who provided valuable suggestions

seasonal variation of cellulase activity didn’t always

and comments which are very useful for improving

match the seasonal variation pattern of the stable isotope

the manuscript. This work was partly supported by theratio, which means that we cannot simply apply the

Research Fund for Rehabilitation and Creation of Osaka

cellulase activity to estimate how much TerrestrialOMs (inBay Area in 2019 and 2020 (No. 020007).

this case, cellulose) these invertebrates are consuming.

This is an unexpected result. From another point of view,

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