Journal of Comparative Physiology B

https://doi.org/10.1007/s00360-022-01438-5

ORIGINAL PAPER

Prejuveniles of Mugil liza (Actinopterygii; Fam. Mugilidae) show

digestive and metabolic flexibility upon different postprandial
times and refeeding
Albanesi Camila1 · González‑Castro Mariano1 · López‑Mañanes Alejandra1 

Received: 5 October 2021 / Revised: 30 March 2022 / Accepted: 11 April 2022

© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
Many animals face periods of feeding restrictions implying fasting and refeeding. The determination of digestive/metabolic
and body condition parameters at different times of food deprivation and after refeeding allows to evaluate the postprandial
dynamics, the transition from feeding to fasting and the capacity to reverse digestive and metabolic alterations. In spite of its
physiological importance, studies on estuarine-dependent detritivore fish are lacking. We determined total mass (TM), relative
intestine length (RIL), hepatosomatic index (HSI), digestive enzymes activities in the intestine and energy reserves in liver
and muscle at 0, 24, 72, 144 and 240 h after feeding and at 72 h after refeeding in prejuveniles of Mugil liza (Mugilidae) as
a model species. After feeding, a decrease occurred in: TM (144 h, 25%), RIL (144 h, 23%); amylase and maltase (72 h, 45
and 35%), sucrase (24 h, 40%) and lipase (24 h, 70%) in intestine; glycogen and free glucose (72 h, 90 and 92%) in liver. In
muscle, glycogen (72–144 h) and free glucose (144 h) (170% and 165%, respectively) peak increased; triglycerides decreased
at 24–240 h (50%). After refeeding TM, RIL, carbohydrases activities in intestine, glycogen and free glucose in liver were
recovered. In muscle, glycogen and free glucose were similar to 0 h; lipase activity and triglycerides were not recovered.
Trypsin and APN in intestine, triglycerides in liver, protein in liver and muscle and HSI did not change. The differential
modulation of key components of carbohydrates and lipid metabolism after feeding/refeeding would allow to face fasting and
recover body condition. Our results improve lacking knowledge about digestive and metabolic physiology of detritivore fish.

Keywords  Digestive enzymes · Energy reserves · Feeding · Detritivore fish · Mugil liza

Abbreviations MAL Maltase

MCh Mar Chiquita Coastal Lagoon SUC Sucrase
TL Total length LIP Lipase
ST Standard length TRY​ Trypsin
TM Total mass APN  N-Aminopeptidase
HSI Hepatosomatic index GLY Glycogen
RIL Relative intestine length GLU Free glucose
AMS Amylase TAG​ Triglycerides
PROT Protein
RF Refeeding
Communicated by H.V. Carey.

Senior authors Mariano González-Castro and Alejandra López- Introduction

Mañanes have contributed equally to this work.

* López‑Mañanes Alejandra
mananes@mdp.edu.ar
1
Grupo Fisiología Bioquímica, Integrativa y Adaptativa,
Instituto de Investigaciones Marinas y Costeras (IIMyC),
Universidad Nacional de Mar del Plata CONICET—FCEyN,
Funes 3250, 7600 Mar del Plata, Argentina
The ability to balance acquisition, storage and use of energy
is critical for survival, growth and maintenance of animals
(Secor 2001; Karasov and Douglas 2013; Karasov and
Caviedes-Vidal 2020). The performance of the digestive
system at different levels (e.g., biochemical, morphological)
influences all physiological processes. The extent to which

13
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digestive and metabolic features allow or constraint shifts
in feeding conditions constitutes a central issue in studies
of ecological, digestive and nutrition physiology (Ramirez-
Otarola et al. 2018; López Mañanes et al. 2020; Volkoff and
Rønnestad 2020; Nespolo et al. 2022). Phenotypic flexibility
implies reversible within-individual variations in phenotypic
traits at different levels (e.g., morphological, biochemical
and physiological), which can increase the chances of sur-
vival for animals under different internal and external condi-
tions (Piersman and Drent 2003; Kelly 2012). A differential
modulation of the activity of key digestive enzymes and of
the concentration of energy reserves constitutes an important
strategy at the biochemical level to face temporal variations
of feed restriction in various animals (del Valle et al. 2004,
2006; Li et al. 2008; del Valle and López Mañanes 2008;
Karasov et al. 2011; López Mañanes et al. 2020; Yang et al.
2019; Lallès 2020).
To determine the activity of specific digestive enzymes in
the digestive tract and energy reserves at different times after
feeding allows to establish the temporal dynamics of key
components of carbohydrates, proteins and lipids metabo-
lism over time of feed restriction (Furné et al. 2008; Abol-
fathi et al. 2012; Caruso et al. 2014; Pujante et al. 2018).
Fasting is a component of life history of various species of
animals, including various fish due to environmental factors
or behavioral patterns that limit access to food (Secor and
Carey 2016; Ersminger et al. 2021). Fasting represents an
extreme situation of food deprivation that many animals,
including fish species can suffer both in the field and under
controlled conditions (Zaldúa and Naya 2014; Secor and
Carey 2016; Yang et al. 2019).
Like other animals, fish require different sources of
energy to maintain physical condition and fundamental
processes, such as growth, metabolism and reproduction
(Karasov and Douglas 2013; Steimberg2018; Gilannejad
et al. 2020). However, knowledge of the physiological and
biochemical specifics of digestion remains limited for most
fish (Small et al. 2022). Food deprivation can have nega-
tive effects on fish metabolism and physiology (Shan et al.
2008; Choi et al. 2012; Yang et al. 2019). To survive fasting,
various carnivorous species mobilize energy reserves to face
the energy deficit and maintain vital processes (Salem et al.
2007; Furné et al. 2012; Yang et al. 2019). The concomitant
determination at different times after feeding of digestive
enzymes, energy reserves and morphological traits allows
to establish the transition from feeding to food deprivation
and to evaluate the potential effects of fasting and digestive
and metabolic adjustments at different levels. Furthermore,
when determined after refeeding it allows to evaluate the
capacity of the animal to reverse digestive and metabolic
alterations upon fasting and the digestive and metabolic
flexibility (Tamadoni et al. 2020; Zaefarian et al. 2020).
However, integrative studies on the dynamics of digestive
13
Journal of Comparative Physiology B
and metabolic responses at the biochemical level involved
in the transition from feeding to food deprivation are frag-
mentary in fish (German et al. 2010; Yang et al. 2019; Shen
et al. 2021). Moreover, postprandial dynamics and the effect
of fasting on digestive enzymes and energy reserves stud-
ies available have been focused in herbivores, carnivorous
and omnivores fishes under controlled condition (Day et al.
2014; Yang et al. 2019; Favero et al. 2020; Zhou et al. 2021).
In this context, no studies are available in estuarine-depend-
ent detritivores fish.
In aquatic environments, an important part of metabolic
processes is sustained by terrestrial debris, which is respon-
sible for maintaining the detritivores food chain. Detritivore
fish are a fundamental part of the energy flow in the different
ecosystems and trophic chains (Taylor et al. 2006; Santana
et al. 2015). Members of the family Mugilidae, generally
known as mullets, are coastal marine fishes with a world-
wide distribution, which spend part or even their whole life
cycle in coastal lagoons, lakes and/or rivers and migrating
to the sea to reproduce (González-Castro and Ghasemzadeh
2016). They occupy a unique position in the food web due
to their primarily detritivore (iliophagous) feeding (Blaber
1976; Cardona 2016). The mullet Mugil liza constitutes an
interesting model for fish biochemical, physiological and
ecological research; particularly to study digestive and meta-
bolic flexibility at different levels in an estuarine- dependent
detritivore fish.
The southern population of Mugil liza is distributed from
the state of São Paulo, Brazil (23°S) to Argentina (47°S)
(González-Castro et al. 2012; Whitfield et al. 2012; Mai
et al. 2014; González-Castro and Minos 2016). Mar Chiquita
Coastal Lagoon (MCh) (Buenos Aires Province. Argentina;
37°32´–37°45´ S 57°19’–57°26´ W) is considered since
1996 as a World Biosphere Reserve by UNESCO. M. liza
represents the second most abundant species of mullet in
MCh, in terms of both biomass and abundance (González
Castro et al. 2009a, b). Mugil liza is characterized by a com-
plex diadromous life cycle. Coastal reproductive spawning
occurs in the offshore waters between Santa Catarina and
Paraná, Brazil (28–26°S) (González-Castro et  al. 2011;
González-Castro and Minos 2016; Lemos et al. 2014). The
reproductive events end and neustonic post-larvae are car-
ried from offshore waters to the coast (surf zone) by wind-
generated surface currents (Vieira and Scalabrin 1991;
González Castro et al. 2009a, b). The prejuveniles (< 30 mm
total length; having two anal spines, including the transition
period until the third spine is completely developed) enter
into MCh and inhabit in this environment until reaching its
gonadal maturity (Gonzalez Castro 2007). The entrance
to coastal lagoons or estuaries implies a strong challenge
for the recruiters, with direct repercussions for the survival
of the species resource. Upon recruitment, prejuveniles of
M. liza undergo a series of changes in a crucial period of
Journal of Comparative Physiology B
time, which includes adaptation to detritivore feeding mode
(Castellini et al. 2019; Albanesi et al, 2021a, b). However,
the heterogeneous challenged conditions found by preju-
veniles upon recruitment can lead to irregular periods of
feed restriction to balance additional energy cost besides
digestion and absorption. Feed restriction and short-term
fasting can be an important adaptative strategy of metabolic
adjustment to face multiple stresses in various fish (Lu et al.
2019; Ensminger et al. 2021). In this context, the aim of this
work was to analyze at (i) different times after feeding and
(ii) after refeeding, including: (a) the total mass, relative
intestine length, hepatosomatic index as parameters of physi-
cal conditions; (b) amylase, maltase, sucrase, lipase, trypsin
and APN activities in the intestine and (c) glycogen, free
glucose, triglycerides and protein concentration in energy
storage in late prejuveniles of M. liza. Determinations of
morphological parameters, such as total length, total mass,
relative intestine length and hepatosomatic index, further
enable the understanding how postprandial effects impact
on the organs-morphology and physical condition (Kalhoro
et al. 2018; Albanesi et al. 2021a, b). We hypothesized that
prejuveniles of estuarine-dependent detritivore fish exhibit
differential adjustments at different times after feeding and
after refeeding in digestive and metabolic characteristics and
flexibility at the biochemical and morphological level, which
constitute adaptive strategies to support irregular periods of
food deprivation in the wild.
Materials and methods
Study area
Mar Chiquita Coastal Lagoon (MCh) is located on the
south-west Atlantic in the Buenos Aires province of Argen-
tina (37°32´–37°45´ S 57°19´–57°26´ W). This irregularly
shaped brackish-water lagoon is separated from the sea by a
sandbar with an inlet, which links the lagoon to the ocean.
The depth varies between 0.5 and 3 m. Seawater enters in
the lagoon with the high tides, and the quantity depends on
the direction and intensity of the winds. Freshwater inflow
is from several streams and artificial channels, which con-
tribute abundant quantities of water during the rainy periods
(González-Castro et al. 2009a).
Collection of individuals and sample procedures
Prejuveniles individuals of M. liza were collected (total
length TL: 50.5 ± 5.1 mm, standard length SL: 42.1 ± 4.3 g)
after their recruitment into the Zone II of MCh, according
to González-Castro et al. (2009b), employing a beach seine
net (10 m in length × 1.8 m high; each wing measured 4 m
in length and the cod-end was 3 m in length; the mesh in the
lateral wings was 10 mm, and the mesh in the cod-end was
5 mm) (González-Castro et al. 2009b; Albanesi et al. 2021b).
Water temperature and salinity determined in practical salin-
ity units (psu) were registered using an alcohol thermometer
and a refractometer, respectively. Temperature and salinity
at the time of capture were 13° C and 18 psu. The values of
temperature and salinity in water in the sites of collection
usually ranged from 7 to 27 °C and 5–35 psu, respectively
(Gonzalez Castro et al. 2009b). Fish were taxonomically
identified according to González-Castro et al. (2012).
The animals were transported to the laboratory in water
tanks from the collection site. The aquarium water (170 L)
was aerated and continuously filtered (Michiels et al. 2015;
Albanesi et al. 2021b). Prejuveniles were fed twice a day
(TetraMin food, tropical flakes) ad libitum, salinity was 18
psu and temperature was 20 °C. A value of 20 °C is both,
a temperature usually faced by prejuveniles of M. liza in
the capture site in Mar Chiquita coastal lagoon but also is
approximately the water temperature where coastal spawning
occurs (González-Castro and Minos 2016). In this context,
the temperature of maintenance in the lab did not represent
a stressful temperature for prejuveniles. Furthermore, this
temperature is adequate because facilitates food intake when
food is available (Como et al. 2013; Cardona et al. 2016).
Morphological traits, digestive enzymes activity in intestine
and energy reserves concentration in liver and muscle were
measured immediately (t0) and at 24, 72, 144 and 240 h after
feeding. At 240 h, fish were re-fed and the corresponding
determinations were done after 72 h. Fish were put imme-
diately on ice until loss of consciousness (about 10 min)
and subsequently processed (Wilson et al. 2009; Trushenski
et al. 2012; del Valle et al. 2016; Ma et al. 2019; Albanesi
et al. 2021a, b). Total length (TL) (mm), standard length
(SL) (mm) and total mass (TM) (g) of each mullet were
recorded, employing a digital caliper to the nearest mm and
an electronic balance (0.1 g). The mullet sample was ontoge-
netically determined as prejuveniles (Querimana) according
to Albanesi et al. 2021b. Immediately, for each individual,
the whole intestine, liver and muscle were excised, weighed
and put on ice to be used to prepare homogenates and to
determine hepatosomatic index (HSI %) (liver weight/ total
mass) × 100) and relative intestine length (RIL) (intestine
length/ standard length) × 100). The intestine and liver were
separately homogenized in 50 mM Tris/HCl, pH 7.4 (4 ml
of tissue g­ −1) (CAT 9 120 homogenizer, T10 tool) on ice
(del Valle et al. 2016; Albanesi et al. 2021a, b). The same
procedure was followed for the body muscle although 8 ml
of tissue g −1 was used (del Valle et al. 2016; Albanesi et al.
2021a, b).
This study was conducted following the regulations and
statements of Ethics Committee CICUAL (OCA 1499/12;
FCEyNat, UNMdP, Argentina).
13

Biochemical assays
We measured enzyme activities at optimal assays conditions
(Albanesi et al. 2021a, b) to not underestimate the potential
digestive capacity at the biochemical level for the differ-
ent key nutrients. This approach (i.e., to measure digestive
enzymes activities at optimal assay conditions) is commonly
and generally used in studies about digestive physiology in
fish and in animals in general in both in the wild and under
controlled conditions (Perera et al. 2008; Uddin et al. 2009;
Xiong et al. 2011; Ferreira-Lazarte et al. 2017; Abdel-Taw-
wab et al. 2018) In the intestine of prejuveniles of M. liza in
the wild, digestive enzymes are active (and in some cases,
such as carbohydrases, exhibit similar values) over a wide
range of temperatures from 4 to 45 °C (Albanesi et al. 2021a,
b).
Amylase activity in intestine was measured using
starch (15 mg  ­mL−1) as substrate, according to del Valle
et al. (2016), Asaro et al. (2018), Albanesi et al. (2021a,
b). Briefly, the sample was incubated for 15 min at 30 °C,
in the presence of starch in 50 mM phosphate buffer (pH
7.4); 300 μl of dinitro salicylic acid reagent (Miller 1959)
was added for further incubation for 10  min at 100  °C.
After cooling immediately on ice, the released maltose was
assessed reading absorbance at 540 nm.
Maltase and sucrase activity were determined measur-
ing the glucose released from the specific substrate as we
detailed (del Valle et al. 2016; Asaro et al. 2018; Albanesi
et al. 2021a, b). The sample was incubated during 10 min
at 30 °C with 42 mM of maltose or sucrose in buffer 0.1 M
malate buffer (pH 6, 4). The reaction was stopped with
1.5 mL of a glycemia kit ((glucose oxidase 10 kU L; peroxi-
dase 1 kU; l,4-aminophenazone 0.5 mmol L ­ −1; phosphates
pH 7.0100 mmol ­L , hydroxybenzoate 12 mmol ­L−1) (Wie-
−1
ner Lab Glicemia AA) and further incubated during 5 min at
37 °C. Glucose amount was quantified reading absorbance
at 505 nm of the colored quinone complex (del Valle et al.
2016; Albanesi et al. 2021a, b).
Lipase activity was determined colorimetrically by quan-
tification of p-nitrophenol (pNP) released from p-nitrophe-
nyl-palmitate as we described (Michiels et al. 2013; del
Valle et al. 2016; Albanesi et al. 2021a, b). The sample was
incubated with 0.85 mM pNPP in buffer 50 mM Tris–HCl
pH 8.5 during 5 min at 37° C. The reaction was stopped by
adding 0.5 ml of TCA 0.1% (w/v). The amount of p-nitro-
phenol released was determined by reading the absorbance
at 410 nm.
Trypsin activity was determined using N-α-benzoyl-
dl -arginine-4-nitroanilide (BAPNA) as substrate as we
described (del Valle et  al. 2016; Michiels et  al. 2017;
Albanesi et  al. 2021a, b). The sample was incubated in
1.23 mM of the substrate in 50 mM Tris buffer/HCl pH
9.0/400 mM ­Cl2Ca during 15 min at 45 °C. The reaction
13
Journal of Comparative Physiology B
was stopped by adding 250 μl of 0.1 M KOH and absorbance
was measured at 405 nm.
APN activity was determined using l-alanine-p-nitroan-
ilide (L-Ala pNA) according to (Michiels et al. 2015, 2017;
del Valle et al. 2016; Albanesi et al. 2021a, b). The reaction
was initiated by the addition of substrate (final concentra-
tion 0.33 mM) to a reaction mixture containing the sample
in 50 mM Tris–HCl buffer to pH 7.4. After incubation for
15 min at 45 °C, the reaction was stopped with 0.2 ml of cold
2 M acetic acid and absorbance was determined at 384 nm.
Glycogen was measured as glucose equivalent as previ-
ously described (Pinoni et al. 2013; del Valle et al. 2016;
Albanesi et al. 2021a, b). The corresponding sample was
boiled for 4  min and then incubated in acetate buffer
(pH 4.8) in the presence and absence of 0.2 mg ­ml−1 of
α-amyloglucosidase (Sigma Chemicals) for 2.5 h at 55 °C.
After incubation, samples were centrifuged at 3000 rpm for
15 min. Glucose was quantified in the supernatant using
the commercial kit for enzyme glycemia (Wiener Lab AA).
Released glucose from glycogen hydrolysis was calculated
as the difference between the values obtained with and with-
out α-amyloglucosidase. Free glucose was assessed from
assay without α-amyloglucosidase.
Triglycerides (TAG) were measured by the colorimetric
method of glycerol phosphate oxidase (TAG Wiener-Lab
AA code 861,110,001). An aliquot of the corresponding
sample was incubated with this reactant for 5 min at 37 °C
(Pinoni et al. 2011; Albanesi et al. 2021a, b). The amount of
released glycerol was determined by reading the absorbance
at 505 nm of the colored quinone complex.
The protein concentration was assayed according to Brad-
ford (1976). Bovine serum albumin (0.96 mg × ­ml−1) was
used as standard.
Statistical analysis
Statistical analysis was determined using the Sigma 3.0 pro-
gram for Windows, which automatically performs previous
test of equality of variances and normality. Analysis of vari-
ance (one-way ANOVA) was used to estimate the statistical
significance of the differences and p < 0.05 was considered
significant. ANOVA (Student–Newman–Keuls) was used to
identify differences (Albanesi et al. 2021b).
Results
Morphological traits of prejuveniles of M. liza
at different times after feeding and after refeeding
No changes in TM occurred at 24 and 72 h after feed-
ing (Table 1). At 144 h, TM decreased (about 35%) and
maintained constant up to 240 h. At 3 days after refeeding,
Journal of Comparative Physiology B

Table 1  Total mass (TM), relative intestine length (RIL) and hepato- Digestive enzyme activities in intestine
somatic index (HSI) of prejuveniles of Mugil liza at different times of prejuveniles of M. liza at different
after feeding and after refeeding
times after feeding and after refeeding
TM (g) RIL (%) HIS (%) N

Mean ± SD Mean ± SD Mean ± SD

Amylase and maltase activities at 24 h post-feeding were
t = 0 1.97 ± 0.34 3.13 ± 0.09 1.81 ± 0.65 7
similar at t = 0. Both activities decreased at 72 h (about 45%
t = 24 2.08 ± 0.55 3.04 ± 0.33 1.90 ± 0.49 7
and 35%, respectively) and remained unchanged until to
t = 72 1.59 ± 0.33 2.55 ± 0.30 1.87 ± 0.64 7
240 h. After refeeding, amylase and maltase activities were
t = 144 1.41 ± 0.22* 2.39 ± 0.52* 1.50 ± 0.31 7
similar to t = 0 (Fig. 1a, b). At 24 h after feeding, sucrase
t = 240 1.20 ± 0.29* 2.24 ± 0.20* 1.36 ± 0.31 7
activity was lower than activity at t = 0 (about 40%). This
RF 1.51 ± 0.28 2.93 ± 0.28 2.03 ± 0.51 7
activity remained decreased at 72–240 h. After refeeding,
sucrase activity was similar at t = 0 (Fig. 1c).
RIL (intestine length/standard length) × 100, HSI (liver weight/total Lipase activity decreased 24 h after feeding (about 70%)
mass) × 100 and remained constant until 240  h. After refeeding, the
*
 Represents significantly different from t = 0 (ANOVA, p < 0.05). Val- activity was lower than t = 0 (about 65%) (Fig. 2a). Trypsin
ues are means ± SE for seven individuals. SD standard deviation, N
number of samples, RF refeeding and APN activities did not change at any time after feed-
ing (Fig. 2b, c). After refeeding, both activities were similar
throughout the experimental period (Fig. 2b, c).
individuals TM had returned to control levels (t = 0).
Relative intestine length was similar to t = 0 up to 72 h, Energy reserves in liver and muscle of prejuveniles
but decreased at 144–240 h (about 23%). At 3 days after of M. liza at different times after feeding
refeeding, RIL was similar to t = 0 (Table 1). No differ- and after refeeding
ences were found in the HSI at any time post-feeding or
after refeeding (Table 1). Glycogen and free glucose concentration in liver at 24 h
after feeding was similar to t = 0. Glycogen and free glu-
cose decreased at 72 h post-feeding (about 90% and 92%,

Fig. 1  Amylase (A), maltase (B) and sucrase (C) specific activities in the intestine of prejuveniles of M. liza at different times after feeding and
after refeeding (RF). Asterisk (*) represents significantly different from t = 0 (ANOVA, p < 0.05). Values are means ± SE for seven individuals

Fig. 2  Lipase (A), trypsin (B) and APN (C) specific activities in the intestine of prejuveniles of M. liza at different times post-feeding and after
refeeding (RF). Asterisk (*) represents significantly different from t = 0 (ANOVA, p < 0.05). Values are means ± SE for seven individuals

13

respectively) and maintained constant at 144–240 h. After
refeeding, glycogen and free glucose in the liver increased
reaching values similar to t = 0 (Fig. 3a, b). In muscle, glyco-
gen concentration was increased by 72–144 h (about 170%).
At 240 h, glycogen decreased to values similar to t = 0.
After refeeding, no changes occurred in glycogen concen-
tration (Fig. 3c). Free glucose concentration in muscle was
unchanged at 24–72 but it increased at 144 h after feeding
(about 165%). At 240 h glycogen decreased being similar to
t = 0. After refeeding, no changes occurred in free glucose
(Fig. 3d).
Triglycerides concentration in liver was similar at t = 0
at different times after feeding and after refeeding (Fig. 4a).
In muscle, triglycerides concentration decreased at 24 h
after feeding (about 50%). At 72, 144 and 240 h, concentra-
tion was maintained constant. After refeeding, triglycerides
concentration in muscle was lower than t = 0 (about 50%)
(Fig. 4c). Protein concentration in both, liver and muscle, did
not change at any time after feeding. After refeeding, protein
concentration in liver and muscle was similar to those at
t = 0 (Fig. 4b, d).
Fig. 3  Glycogen and free glucose concentration in liver and muscle
of prejuveniles of M. liza at different times after feeding and after
refeeding (RF). Glycogen (a) and free glucose (b) concentration
13
Journal of Comparative Physiology B
Discussion
Prejuveniles of the fish species used in this study (Mugil
liza) constitutes an adequate model to study digestive and
metabolic flexibility upon feed restriction/refeeding in an
estuarine-dependent detritivores fish. After recruitment in
coastal lagoon or estuaries, such as MCh, prejuveniles of
M. liza face irregular periods of feeding restrictions (Gon-
zalez Castro et al. 2009; Castellini et al. 2019). The intestine
shows an adequate battery of digestive enzymes (amylase,
maltase, sucrase, lipase, trypsin and APN) to perform a com-
plete digestion of dietary substrates from different sources
(Albanesi et al. 2021b). Although detritus is dilute due to
the content of water and inorganic material (Bowen 1983;
Bowen et al. 1995; Wilson et al. 2003) detritus consumed for
prejuveniles of M. liza in the wild contain items from both
vegetable and animal origin, thus exhibiting a balanced com-
position (Botto et al. 2005; Bruno 2014; Bruno et al. 2017).
The digestion of key glycogenic substrates (e.g.,
starch/glycogen) by amylase and membrane-bound
in liver. Glycogen (c) and free glucose (d) concentration in muscle.
Asterisk (*) represents significantly different from t = 0 (ANOVA,
p < 0.05). Values are means ± SE for seven individuals
Journal of Comparative Physiology B
Fig. 4  Triglycerides (TAG) and protein concentration in liver and
muscle of prejuveniles of M. liza at different times post-feeding and
after refeeding (RF). TAG (a) and protein (b) concentration in liver.
disaccharidases in the intestine is a main source of circu-
lating glucose in fish. This is important for maintaining
glucose homeostasis and supporting regular functions and
responses to environmental stresses (Polakof et al. 2011,
2012; Gominho-Rosa et al. 2015; Steimberg 2018; Small
2022). Changes of the activity of specific digestive enzyme
in the intestine could lead to modifications in the diges-
tive capacity for the corresponding dietary substrate. This
approach is largely used to establish which nutrients are
involved in response to internal and external factors (Kara-
sov and Douglas 2013; Sanz et al. 2015; Pujante et al.
2016; Karasov and Caviedes-Vidal 2020). The lower amyl-
ase activity at 72 h after feeding suggests that a decrease
in the amylolytic capacity and therefore for initial steps of
glycogenic carbohydrate digestion are one of the responses
at the biochemical level upon short-term food deprivation
in prejuveniles of M. liza. Postprandial decreases of amyl-
ase activity occurred in the digestive tract of omnivores
and carnivorous fishes (Fúrne et al. 2008; Abolfathi et al.
2012; Caruso et al. 2014).
TAG (c) and protein (d) concentration in muscle. Asterisk (*) repre-
sents significantly different from t = 0 (ANOVA, p < 0.05). Values are
means ± SE for seven individuals
Scarce information is available about characteristics and
modulation by feeding, fasting and refeeding of intesti-
nal membrane-bound disaccharidases such as sucrase and
maltase in fish (Small 2022). In the carnivore fish Salmo
salar L, a decrease in the activity of intestinal maltase occurs
upon food deprivation (Krogdahl et al. 2005). Sucrase and
maltase activities in intestine of prejuveniles of M. liza
(Albanesi et al. 2021b) would correspond to the sucrase-
isomaltase and sucrase-independent maltase–glucoamylase
complex, respectively (Burke 2019; Karasov and Caviedes
Vidal 2020). The lower sucrase and maltase activities sug-
gest that a diminished capacity for final steps of carbohy-
drate digestion is another component of the response to
temporal feed restriction in M. liza. The earlier decrease
in sucrase activity compared to that in maltase suggests
an independent down-regulation of sucrase and maltase
complexes and a modulation of pre-existent enzymes. The
fact that the decrease in membrane-bound disaccharidases
occurred earlier (24–72 h) than that in RIL (144 h), fur-
ther indicates a modulation of pre-existent enzymes activity
13

independently of morphological changes. The values of RIL
are commonly employed in fish as index of digestion effi-
ciency (German et al. 2006; Wagner et al. 2009; Whitfield
et al. 2012; Day 2014). The decrease of the RIL at 144 h
along with the concomitant decrease in TM points out that
this time of food restriction compromises the body condi-
tion of prejuveniles of M. liza. However, since no mortality
occurs, underlying adjustments would allow survival. As
ectotherms, fishes can endure food deprivation over longer
time scales than endothermic animals (German et al. 2010;
Killen et al. 2016; Kristensen et al. 2020). This can be an
advantage for survival within heterogeneous aquatic systems
such as estuaries or coastal lagoons for estuarine-dependent
detritivore fish. Changes in the length of the gut and there-
fore variations in surface area and total volume can affect
both digestion and absorption and to impact body condition
in various fish (Krogdahl et al. 2005; Vidal et al. 2019; Small
et al. 2022). Since no information is available about normal
gut transit time in M. liza, whether changes in this physi-
ological parameter can affect digestive components at the
biochemical level upon feed restriction cannot be discarded.
Gut transit time is another factor that can alter or produce
changes in digestion and absorption upon different condition
in various animals (Karasov and Douglas 2013; da Silva
Reis et al. 2019; Gilannejad et al. 2019).
In the fresh-water detritivores tropical catfish Pterygopli-
chthys disjunctivus, various digestive enzyme activity are
modulated in the intestine by exposure to long term food
deprivation under controlled condition (German et al. 2010).
However, nothing is known about the temporal dynamic on
the response at digestive enzyme in detritivores fish. The
decrease at 24 h in lipase activity in intestine of M. liza
points out that digestive capacity for dietary lipids would
also be diminished along with that for carbohydrates diges-
tion at short-time of food deprivation. The fact that amyl-
ase, sucrase, maltase and lipase activity in intestine were
decreased while no changes occurred in key proteases, such
as trypsin and APN, suggests the occurrence of specific
modulation of carbohydrates and lipids digestion pathways.
The differential responses in M. liza suggests the occurrence
of specific temporal dynamics and mechanisms of regula-
tion of different digestive enzymes. The earlier decrease in
lipase activity compared to that in amylase is contrary to that
previously shown in carnivorous fishes such as the sturgeon
Acipenser naccarii, trout Oncorhynchus mykiss and javelin
goby Synechogobius hasta in which amylase decreased at
shorter time than lipase activity (Furné et al. 2008; Zhou
et al. 2021).
Feed restrictions and short-term fasting can be an
important means of metabolic adjustment and in this way
beneficial to face multiple stresses in various fish (Lu et al
2019; Ensminger et al. 2021). To survive to fasting, sev-
eral species of carnivores, omnivores and herbivores fishes
13
Journal of Comparative Physiology B
mobilize reserves to face the energy deficit and to main-
tain vital processes (Salem et al. 2007; Furné et al. 2012;
Zaldúa and Naya 2014; Yang et al. 2019). The absence
of food along with the decrease of the activity of diges-
tive enzymes, which are a main link between digestion
and absorption, could lead to a limited availability of spe-
cific metabolites for building and maintenance of energy
reserves. In most fish species, liver glycogen is mobilized
to maintain glucose homeostasis during the early stages of
fasting, being the first substrate utilized as energy source
(Chen et al. 2018; Nebo et al. 2018; Lu et al. 2019). This
appears to be also the case for prejuveniles of M. liza. The
decrease of glycogen and free glucose concentration in
the liver at 72 h after feeding suggests their modulation
(increased degradation and mobilization and/or decreased
synthesis). This could be a biochemical strategy to face the
diminished availability of dietary glycogenic substrates. In
the liver of several fish, enzymes involved in the glycogen
synthesis and gluconeogenic pathways are postprandial
modulated at different levels (expression, quantity and /
or activity) (Chen et al. 2017; Conde-Sieira and Soegas
2017; Yang et al. 2019). Nothing is known to the best of
our knowledge in detritivores fish.
In various carnivorous, herbivores and omnivore fishes,
the liver is a major source of metabolites (e.g., glucose) sup-
ply for other tissues such as muscle. The net flux of glucose
can be modify by adjustment of production and utilization
pathways (Enes et al. 2009; Polakof et al. 2012; Nebo et al.
2018). The increase in the glycogen concentration in muscle
of prejuveniles of M. liza concomitant with the decrease in
liver at 72–144 h after feeding, support that also in a detri-
tivore fish, the liver can be a source of glucose for synthesis
processes in muscle. The following decrease in glycogen
concentration in muscle along with the increase in free glu-
cose suggest a later mobilization of this reserve. This could
support the peaked increase in free glucose concentration at
144 h and its potential local utilization. In teleost fish, skel-
etal muscle is a major glucose entry site following a glucose
load via a specific GLUT 4-type glucose transporter whose
membrane activity and abundance appears to be subject to
regulation (Marín-Juez et al. 2013; Yang et al. 2021).
In most adult fish, triglycerides are stored mainly in
liver, muscle, and adipose tissue (Steimberg 2018; Small
2022). Since prejuveniles have not still fat, liver and mus-
cle are involved in triglyceride storage, being the liver the
main site (Albanesi et al. 2021b). The decrease in triglyc-
erides concentration at 24 h after feeding while no changes
occurred in liver, suggests that muscle is the site of lipid
mobilization upon fasting to support the decrease in dietary
substrates available. This could be particular important
to provide energy for key physiological processes such as
movement (Morse 2019). In various fish, lipids constitute a
main source of energy for physiological processes such as
Journal of Comparative Physiology B
Fig. 5  Summary of morphological, digestive and metabolic param-
eters at different times after feeding (a) and after refeeding (b) in
prejuveniles of M. liza. TM total mass, RIL relative intestine length,
HSI hepatosomatic index, AMS amylase, MAL maltase, SUC sucrase,
LIP lipase, TRY​ trypsin, APN N-aminopeptidase, GLY glycogen,
growth, reproduction and movement, including migration
(Tocher 2003; Sandre et al. 2017; Oishi et al. 2020; Albanesi
et al. 2021a).
Studies on fasting and posterior refeeding responses at
the biochemical level in estuarine-dependent detritivores
fish are not available. In carnivores and herbivores fishes,
the recovery after refeeding of digestive enzymes activities
in intestine appears to be differential for specific enzymes
and dependent on species and alimentary habit (Fúrne et al.
2008; Caruso et al. 2014; Day 2014; Babaei et al. 2020; Dar
et al. 2021). The increase of TM of prejuveniles of M. liza
after 72 h of refeeding points out the existence of underly-
ing adjustments leading to recovery of body condition. The
enhancement of amylase, maltase and sucrase activities in
the intestine to values similar to those at t = 0 points out the
reversible modulation of these enzymes after refeeding. A
recovery of carbohydrases activity has been shown in carni-
vore, omnivore and herbivore fishes (Day et al. 2014). The
increase after refeeding of the RIL point out the occurrence
of reversible integrated responses at different levels (e.g.,
morphological, biochemical) in M. liza. Fasting/refeeding, in
various animals, can trigger a coordinated set of response at
different levels (from molecular to morphological) of range
from being relatively modest to significantly large (Secor
2005, 2010; Day et al. 2014; Lo Cascio et al. 2017).
GLU free glucose, TAG​ triglycerides, PROT proteins. Comparisons
were made with respect to the corresponding parameter immediately
after feeding (t = 0) as described in “Materials and methods” and in
“Results”. ↑ , ↓, = indicate increase, decrease and unchanged, respec-
tively
The mechanisms of regulation (i.e., primary chemical
messengers) involved in the modulation of the activity and/
or secretion of digestive enzymes in fish have been scarcely
investigated and are very far from having been elucidated
(Small 2022). In this context, the regulation of digestive and
absorptive processes at the biochemical level in detritivores
fishes is still unknown. In the intestine of Carassius auratus
and Odontesthes bonariensis, ghrelin and nesfatin-1 modu-
late in vitro the expression of sucrase-isomaltase (Blanco
et al. 2017; Bertucci et al. 2018). Further experimental work
is needed to stablish whether these mechanisms are involved
in the up-regulation of disaccharidases after refeeding in
M. liza.
The resulting recovery in digestive capacity for glyco-
genic substrates due to increased carbohydrase would allow
the rebuilding of glycogen reserves and of free glucose con-
tent in liver after refeeding. Since lipase activity in intestine
and triglycerides concentration in muscle was not recovered,
the reverse modulation of key components of glucose home-
ostasis appeared to be enough to recover body condition.
The degradation of enzymatic, mechanical, and structural
proteins could result in the decline of critical tissues and
organs and serious homeostatic imbalances that could even-
tually lead to death (Secor and Carey 2016). This appeared
not to be the case for M. liza. The maintenance of the activity
13

of key proteases in the intestine and of protein concentration
in liver and muscle could be an important factor allowing
survival upon fasting/refeeding.
In summary, the results of the present study show a dif-
ferential and specific modulation of key components of car-
bohydrate and lipid metabolism (Fig. 5) that could represent
adaptive strategies in estuarine dependent detritivores fish
to face periods of irregular feeding inside coastal lagoon or
estuaries upon recruitment.
Acknowledgements  This work was financed by the Consejo Nacional
de Investigaciones Científicas y Técnicas (CONICET) (PIP Nº 112
201301 00009 and PIP 112-201301-00339); the Fondo para la Investi-
gación Científica y Tecnológica (FONCyT) (PICT 2019-1344) and the
Universidad Nacional de Mar del Plata. C. Albanesi was supported by
scholarship from CONICET (Argentina). We want to thank to Agustín
Texeira-Moreira, Damián Castellini, Cristian Di Paolo, Daniel and Juan
Pablo Gaudioso for their help in specimens collect, and also to MCh
authorities/forest guard for logistic support.
Funding  This work was financed by the Consejo Nacional de Investiga-
ciones Científicas y Técnicas (CONICET) (PIP Nº 112 201301 00009
and PIP 112–201301-00339); the Fondo para la Investigación Cientí-
fica y Tecnológica (FONCyT) (PICT 2019–1344) and the Universidad
Nacional de Mar del Plata. C. Albanesi was supported by scholarship
from CONICET (Argentina).
Declarations  of burrowing crabs on C and N sources, control, and transfor-
Conflict of interest  The authors declare no competing/conflicts of in-
terest.
Code availability  Not applicable.
Ethics approval  This study was conducted following the regulations
and statements of Ethics Committee CICUAL (OCA 1499/12; FCEy-
Nat, UNMdP, Argentina).
Consent to participate  Not applicable.
Consent for publication  Not applicable.
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