Int. J. Aquat. Biol. (2023) 11(1): 41-49

ISSN: 2322-5270; P-ISSN: 2383-0956

Journal homepage: www.ij-aquaticbiology.com

Original Article
Hormonal failure and osmoregulatory disruption in laboratory food-deprived Caspian

kutum, Rutilus frisii larvae during brackish water challenge

Mohammad Mohiseni

Department of Environmental Science and Fisheries, Lorestan University, Khorramabad, Iran.

Article history:

Received 19 December 2021

Accepted 10 December 2022

Available online 2 5 February 2023

Keywords:

Starvation

Osmoregulation

Caspian kutum

Salinity

Abstract: Caspian Kutum, Rutilus frisii, is a valuable species in the Caspian Sea basin. With the

aim of restocking, the Iranian Fisheries Organization (IFO) annually released millions of Caspian

kutum larvae into the estuaries of the Caspian Sea. This study was conducted to evaluate the effects

of starvation on fish during Caspian seawater (CSW) adaptation. Caspian kutum larvae (0.5±0.1 g)

were divided into two groups; one was considered a control fed ad libitum during the experiment and

another group was left food deprived. Both groups were continuously exposed to the CSW challenge

(13 ppt) for 7 days and sampling was done on the second, third, fourth and seventh days after

initiation of the CSW challenge. Different physiological factors, including hormones (cortisol, T3,

and T4), gill Na+/K+-ATPase activity, whole body glucose and protein, gill protein, body moisture,

and seawater preferences, were analyzed in each sampling time. The results showed that although

fed larvae can successfully overcome the physiological changes imposed by the CSW challenge, the

starved fish indicated significant failures in the most measured parameters and eventually

demonstrated significantly lower salinity preferences. Therefore, it can be concluded that starvation

may negatively affect the success of CSW adaptation. Since physiological impairment during the

CSW adaptation period is directly related to the effectiveness of the restocking program, more studies

about the feeding condition of Caspian kutum larvae pre and post-releasee and the nutritional status

of recipient rivers are suggested.

Introduction
Rutilus frisii (Kamensky, 1901) is an important

commercial fish in Iran, with a wide distribution from

north to south, and its main population is on the southern

coast of the Caspian Sea (Ebrahimi and Ouraji, 2012;

Hasanpour et al., 2016; Eagderi et al., 2022). Having a

migratory anadromous habit, Caspian kutum migrates to

the rivers and lagoons of the southern Caspian Sea for

spawning. The spawning season of Caspian kutum is

from late March to mid-May. During reproduction, they

spawn on aquatic weeds, gravel, and sandy substrates

(Bastami et al., 2012). Because of the severe decline in

the annual catch of this species during the 1970s and

1980s due to the demolition of their natural spawning

substrates, overfishing, and other factors, IFO launched a

restocking project in 1984 (Salehi, 2008). In the

restocking centers, larval rearing is performed in the

earthen ponds until a releasing weight is around 1 g. Then,

Correspondence: Mohammad Mohiseni DOI: https://doi.org/10.22034/ijab.v11i1.1430

E-mail: mohiseni.m@lu.ac.ir DOR: https://dorl.net/dor/20.1001.1.23830956.2023.11.1.6.6

the fingerlings are released into the rivers that carry them

toward the Caspian Sea (Jafari et al., 2009).

During downstream migration, changes in

morphology and physiology will occur to successfully

prepare the fish for seawater entry (Lerner et al., 2007).

This transformation is driven by a change in

environmental factors and mediated by significant

alteration in specific hormones, including thyroid

hormones, cortisol, growth hormone, and insulin-like

growth factor I (McCormick, 2001). Cortisol has a critical

role in hypoosmoregularoty capacity through the

development and proliferation of gill chloride cells

(mitochondria-rich cells) and upregulating of Na+/K+-

ATPase (NKA) expression (Madsen et al., 1995). The

activation of these processes implies an increased energy

requirement that eventually can alter gill energy

metabolism and whole organism energy partitioning.

Integral in the seawater adaptation is a reduction in

Mohiseni / Hormonal failure and osmoregulatory disruption in food-deprived Caspian kutum

glycogen supply, change in body lipids, and depletion of

energy stores. This will make the juveniles vulnerable to

starvation during the early phase of seawater adaptation

(Stefansson et al., 2009). Starvation is known to affect

hypo-osmoregulatory ability in many fish species,

including rainbow trout (Jürss et al., 1983), Mozambique

tilapia (Jürss et al., 1984), Arctic charr (Aas-Hansen et al.,

2003), sea bream (Polakof et al., 2006) and Atlantic

salmon (Stefansson et al., 2009).

There are several reports which investigated and

described the important factors affecting the reproduction

of the Caspian kutum broodstock (Nikoo et al., 2010;

Shafiei Sabet et al., 2010), growth and production of

fingerlings in the hatcheries (Afraei et al., 2010; Ouraji et

al., 2011; Samarin et al., 2011). The effect of different

salinities and fingerling sizes on the survival of the

Caspian Kutum is also reported (Enayat Gholampoor et

al., 2011; Hosseini et al., 2012). Despite the important

role of normal and successful hypo-osmoregulation

during the seawater adaptation in further survival and

fitness of marine life stage, to the best of our knowledge,

there is no information about the effects of starvation on

salinity adaptation of Caspian kutum fingerling. The

present study investigated the behavioral and

physiological changes in the starved Caspian kutum

larvae during Caspian seawater CSW challenges.

Material and Methods

The Caspian kutum larvae (n=180, 0.5±0.1 g body

weight) were provided from Shahid Rajaee, the center of

fish reproduction (Sari, Iran) and transferred to the 300-L

tank and maintained in normal condition (pH=7.3;

temperature=18-20°C and oxygen around saturation

level) for two weeks (Mohiseni et al., 2017). The larvae

were fed 3% of their body weight three times a day on the

starter diet for rainbow trout (BioMar, France). Daily

water exchange during adaptation and also experimental

periods was 30%. After the acclimation period, larvae

were divided into fed and feed-deprived groups (with

three replications), and both groups were transferred to

the brackish water (13 ppt) simultaneously. The first

group was fed ad libitum during the CSW challenge. The

experimental salinity was made by mixing evaporated

full-strength Caspian Seawater with dechlorinated tap

water. The salinity challenge was done (randomly) for

both groups for 7 days continuously, and sampling was

done on the second, third, fourth, and seventh days after

initiation of the CSW challenge.

The whole-body cortisol was measured according to

Peterson and Booth (2010) with minor modifications.

Briefly, Caspian kutum larvae were dried with a paper

towel and weighed before extraction. The sample was

then homogenized (Buch and Holm homogenizer,

Denmark) in PBS, and aliquots of ethyl ether were added

and vortex for 1 min. The samples were then centrifuged

and frozen immediately at -20°C, and an unfrozen portion

was transferred to a fresh tube for ethyl ether evaporation

under nitrogen. The remaining extract was stored at -20°C

until ready for Enzyme-linked immunosorbent assay

(ELISA). Cortisol was measured with Monobind, a

cortisol assay kit (USA). Thyroid hormone extraction was

done based on Mukhi et al. (2005). Triiodothyronine (T3)

and thyroxine (T4) were measured using Pishtazteb,

ELISA assay kits (Iran).

Gill arches were collected for Gill Na+/K+-ATPase

(NKA) activity. It was determined following McCormick

(1993) developed for microplates. The ouabain-sensitive

hydrolysis of adenosine triphosphate is enzymatically

coupled to the oxidation of nicotinamide adenine

dinucleotide, which is directly measured in a microplate

reader. Glucose was determined by enzymatic-

colorimetric test (Moss and Henderson, 1999). Total

protein concentrations were analyzed using the Bradford

method with bovine serum albumin as standard

(Bradford, 1976). To determine whole-body moisture, 5

larvae were dried with a paper towel and then weighed

(based on mg) precisely. Afterward, the larvae were dried

until full dehydration at 60˚C (approximately 72 h) and

then weighed. The difference between wet and dried mass

was considered whole-body moisture and reported as a

percentage (Moustakas et al., 2004).

The seawater (SW) preferences were evaluated based

on Lerner et al. (2007). Briefly, the SW preference tank

was constructed as two parallel chambers of Styrofoam

connected by a PVC bridge. Two chambers were filled

with Freshwater (FW) and CSW (13 ppt) separately. Each

chamber was filled just below the bridge. 10 fish from

both groups were transferred into the FW chamber and

allowed to acclimate for 2 h. The level of FW was then

elevated until the connection between the two chambers

was formed. Afterward, the fish activity was videotaped

from above the chambers for 1 h. The videotape was

analyzed for the presence of fish in CSW at 30 s intervals

for 45 min and presented at the percent of fish in CSW.

Trials were conducted in three replications from each

experimental group.

Int. J. Aquat. Biol. (2023) 11(1): 41-49

All datasets were statistically analyzed by

Independent Sample t-test. Pearson correlation was also

used to determine correlated factors. All statistical

analyses were performed using IBM SPSS Statistics for

Windows (Version 19) at the significance level of

P<0.05. The results were reported as mean±SE.

Results

Hormonal changes in both fed and starved groups are

illustrated in Figure 1. Cortisol levels tend to increase

over time and reach its maximum level at the end of the

experiment in fed fish, while the pattern of the hormonal

change in starved fish showed a significant reduction 7

days after the salinity challenge (P<0.05). Similarly, T3

was also increased during the salinity challenge in fed fish

and showed a significant difference with starved fish most

of the time (P<0.05). The level of T4 in starved fish

remained almost unchanged during the experiment and

showed significant reduction at 2, 3, and 7 days after the

challenge (P<0.05). T3/T4 ratio was consistently higher

in fed treatment throughout the experiment, with

significant differences on 3, 4, and 7 days after the

challenge (P<0.05).

Gill NKA activity was altered in response to the

salinity challenge in both groups. The enzyme activity

was consistently increased for fed fish over time, despite

the transient elevation of enzyme level in starved fish

until the 4th day. The enzyme activity dropped

significantly at the end of the experiment (Table 1). A

similar trend was observed for glucose in both treatments,

with a recorded significant decrease at 2, 4, and 7 days of

salinity challenge for the starved group (P<0.05).

Although the whole-body protein was approximately

unchanged in starved fish, the recorded values for the fed

group indicated a slight increment and were higher than

the starved group all the time, with a significant

difference on the 7th day (P<0.05).

The gill protein was elevated to the maximum level in

both groups 3 days after the challenge, but the recorded

value for fed was higher (P<0.05). The gill protein

decreased in both groups, but the reduction rate for

starved fish was significantly higher than for fed fish

(P<0.05). The whole-body moisture in the starved group

tended to decrease over time, but it remained largely

stable in fed fish throughout the experiment, resulting in

these fish having significantly higher levels on 3, 4, and

7 days after the challenge than those in starved fish

(P<0.05).

The starved fish were first observed in seawater

immediately after the formation of the aqueous bridge,

but with time advancement, the percentage of fish in

seawater significantly decreased (about 92%) and

A B

Figure 1. Hormonal change (A: Cortisol, B: T3, C: T4 and D: T3/T4 ratio) during seawater challenge in fed and starved Caspian kutum. (*) and

(**) show a significant difference between fed and starved fish at the same time at P<0.05 and P<0.01, respectively. Dissimilar small letters show
the differences among different times for Fed and dissimilar capital letters show differences among different times for the Starved groups (P<0.05).

Mohiseni / Hormonal failure and osmoregulatory disruption in food-deprived Caspian kutum

reached the minimum (5.22%) at the end of the

experiment (Fig. 2A, B). On the other hand, fed fish

displayed obvious latency (about 12.5 min) to enter CSW,

but their CSW preference behavior was increased until 30

min and remained stable toward 45 min. Furthermore, the

results exhibited that the most measured osmoregulatory

parameters were significantly correlated (P<0.05) in fed

fish, whereas a few correlated parameters were found for

starved fish (Table 2).

Discussion

This study revealed that starvation may lead to endocrine

disruption in the CSW adaptation period in Caspian

kutum. Cortisol is a mineralocorticoid hormone in teleost

and has a direct role in osmoregulatory change and

seawater adaptation in anadromous, downstream

migratory juvenile fish (McCormick, 2001; Nemova et

al., 2021). Cortisol is also participating in energy-

providing through glycogenolysis for normal metabolism

and stress response (Laiz-Carrion et al., 2002). In the

current study, whole-body cortisol levels significantly

increased over time in fed fish. A prolonged cortisol

increase is reported during smolting and successful

seawater adaptation in anadromous fish (McCormick,

Factor Time (day) Fed Starved P-value

Gill Na+K+ATPase (NKA) (µmol ADP/mgPr/h)

2 26.97±3.02 a 27.99±3.42 0.834

3 43.33±3.55 a 33.32±1.51 0.06

4 43.41±4.84 a 41.94±5.72 0.854

7 70.88±10.85 b 38.61±2.06 0.043*

Whole body Glucose (mg/dl)

2 72.95±4.75 56.93±2.26 B 0.048*

3 81.5±26.91 41.21±13.3 AB 0.15

4 105.01±16.06 57.9±9.98 AB 0.03*

7 108.88±0.71 39.89±17.31 A 0.025*

Whole body protein (mg/dl)

2 3.08±0.17 a 3.11±0.13 0.88

3 3.84±0.97 a 2.89±0.11 0.39

4 5.04±0.38 b 3.67±0.42 0.072

7 4.69±0.57 ab 3.03±0.11 0.047*

Gill protein (mg/dl)

2 2.19±0.21 1.61±0.08 A 0.062

3 3.34±0.24 2.17±0.17 B 0.018*

4 2.87±0.16 1.48±.08 A 0.002**

7 2.84±0.13 1.21±0.07 A 0.033*

Whole body moisture (%)

2 69.53±8.02 62.14±2.76 B 0.433

3 66.93±2.05 53.13±1.51 A 0.006**

4 73.28±2.65 60.99±1.85 B 0.046*

7 70.98±2.95 52.33±0.97 A 0.004**

Values are means±standard error; (*) shows a significant difference between two groups at P<0.05 and (**) shows a significant

difference at P<0.01. Dissimilar small letters show the differences among different times for Fed and dissimilar capital letters show

differences among different times for the Starved groups (P<0.05).

Table 1. Gill and whole-body parameters change after different times of salinity challenge in fed and starved Caspian kutum.

Figure 2. Seawater preferences quantity (A) and pattern (B) for fed and starved fish during seawater challenge. (*) indicate significant differences

from the fed group at the same time.

Int. J. Aquat. Biol. (2023) 11(1): 41-49

2001; McCormick et al., 2005; Lerner et al., 2007;

Mancera and McCormick, 2019). Cortisol also has a

promotive effect on the development and proliferation of

gill chloride cells, which is directly connected to an

increase in gill NKA activity (Madsen et al., 1995;

McCormick et al., 2008). The activation of this process

involves an elevation in energy requirement that

apparently could alter the gill energy metabolism (Laiz-

Carrion et al., 2005). There was a significantly positive

correlation between cortisol levels and NKA activity in

the fed group. Therefore, the significant decrease in

cortisol levels due to starvation may explain this study's

significant reduction in gill NKA activity.

Thyroid hormones failed to increase during the CSW

challenge in the starved Caspian kutum fish. Thyroxin

(T4) and triiodothyronine (T3) are the principal thyroid

hormones involved in the development and growth of

fishes (Peter and Peter, 2009). T4 is the main secreted

prohormone by the thyroid gland in teleost. Further

enzymatic outer ring deiodination will transform the less

potent T4 into the bioactive thyroid hormone (T3). In

fishes, thyroid hormones have a fundamental role in

different physiological processes, including somatic

growth, metamorphosis, parr-smolt transformation,

bioenergetics, and reproduction (Arjona et al., 2010).

Several studies have shown that thyroid hormones

regulate basal and active metabolic rates in different

tissues of teleost (Narayansingh and Eales, 1975; Pavlidis

et al., 1997; Aas-Hansen et al., 2003; López-Bojórquez et

al., 2007; Jarque and Piña, 2014; Tovo-Neto et al., 2018;

Deal and Volkoff, 2020). Several studies have also

reported the involvement of thyroid hormones during

salinity acclimation. Prolonged T4 treatment led to an

increase in the number of chloride cells and gill NKA

activity in Atlantic salmon (Madsen and Korsgaard,

1989). Physiological levels of T3 and T4 have also

increased chloride cell size and gill NKA activity in

Mozambique tilapia (Peter et al., 2000). Accordingly, we

found a significant positive correlation between NKA

activity and T3 levels only for the fed group.

The liver metabolism may be enhanced during CSW

adaptation because of its direct involvement in

glycogen/glucose turnover in fish. This process will make

the glucose available to provide energy requirements for

the osmoregulatory phenomenon in different tissues,

especially the gill and kidney (Sangiao-Alvarellos et al.,

2003). Food deprivation resulted in changes in hepatic

energy metabolism, as reported in several teleosts

(Sangiao-Alvarellos et al., 2005; Stefansson et al., 2009;

Costas et al., 2011). These included: (1) elevation of

glycogenolysis and gluconeogenesis rate that can be

attributed to the increased plasma cortisol concentration,

Cortisol T3 T4 T3/T4 Na
+K+

ATPase
Glucose Body

protein

Gill

protein

Body

moisture

Salinity

preference
Cortisol

Fed 1 .663* .177 .764** .675* .626* .319 -.330 .719** .178

Starved 1 -.019 -.328 .105 -.186 -.514 -.004 -.354 .489 .327

Fed .663* 1 .101 .857** .673* .430 .764** .034 .696* .718*

Starved -.019 1 .317 .817** .052 -.155 -.194 .063 -.502 .100

Fed .177 .101 1 .043 .128 .213 .094 .819** .312 .436

Starved -.328 .317 1 -.112 .223 .587* .223 -.147 -.179 -.187

T3/T4

Fed .764** .857** .043 1 .839** .488 .487 .004 .685* .697*

Starved .105 .817** -.112 1 .085 -.336 -.270 -.070 -.421 .217

Na+K+ ATPase

Fed .675* .673* .128 .839** 1 .372 .366 .013 .477 .818**

Starved -.186 .052 .223 .085 1 .370 .510 -.108 .058 -.508

Glucose

Fed .626* .430 .213 .488 .372 1 .131 -.032 .295 .004

Starved -.514 -.155 .587* -.336 .370 1 .331 -.281 -.024 -.597

Body protein

Fed .319 .764** .094 .487 .366 .131 1 .176 .486 .691*

Starved -.004 -.194 .223 -.270 .510 .331 1 -.321 .313 -.445

Gill protein

Fed -.330 .034 .819** .004 .013 -.032 .176 1 -.182 .800**

Starved -.354 .063 -.147 -.070 -.108 -.281 -.321 1 -.249 .197

Body moisture

Fed .719** .696* .312 .685* .477 .295 .486 -.182 1 .674*

Starved .489 -.502 -.179 -.421 .058 -.024 .313 -.249 1 -.313

Salinity preference

Fed .178 .718* .436 .697* .818** .004 .691* .800** .674* 1

Starved .327 .100 -.187 .217 -.508 -.597 -.445 .197 -.313 1

*. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed).

Table 2. Pearson correlation among different osmoregulatory factors during seawater challenge in fed and starved Caspian kutum.

Mohiseni / Hormonal failure and osmoregulatory disruption in food-deprived Caspian kutum

(2) increased liver capacity for glucose transferring, and

(3) decreased concentration of the plasma triglyceride and

protein. Cortisol is one of the most important factors

during salinity adaptation and has a special role in

chloride cell proliferation and development. Chloride

cells are the main site of NKA that makes a driving force

for monovalent ion secretion via gill arcs (Madsen et al.,

1995; Evans et al., 2005). According to the results, the

cortisol and NKA levels were increased a time-dependent

and correlated manner, whereas both factors finally failed

to surge in the fasting group during the seawater

challenge i.e. gluconeogenesis is the common role of

cortisol action in the liver. Cortisol is responsible for

glycogen fraction and glucose production. Accordingly,

we found a significant correlation between body cortisol

and glucose of Caspian kutum fish only in the fed group.

Osmoregulatory adaptation during the CSW challenge

is one of the most energy-consuming processes in aquatic

animals (Madsen et al., 2015). Various tissues prefer

specific energy resources to overcome the major changes

due to the CSW challenges. The excess energy

requirement of the liver and brain is mainly based on

carbohydrates, while amino acids and lactate are more

important in the gills and kidneys. Therefore, a significant

decrease in the whole body and gills protein content in

food-deprived fish in the current study was in agreement

with the previous studies (Sangiao-Alvarellos et al., 2005;

Polakof et al., 2006). On the other hand, the protein

content of the body and gills of fed larvae remained

approximately unchanged during different times after the

CSW challenge.

During the first hours after CSW challenges, the

drinking rate is elevated constantly due to the water loss

via gills epithelia. In this process, the water is absorbed

from the intestine along with excess divalent ion

secretions. Excess monovalent ions will further be

secreted via gills through the efficient actions of chloride

cells. This process keeps the hydromineral balance and

blood osmolality within the normal range (Marshall and

Bryson, 1998; Webb et al., 2001; Grosell, 2010). It seems

that fed larvae successfully kept their body moisture

during the CSW adapting periods while starved larvae,

probably due to the failures of different osmoregulatory

factors, would not be able to ameliorate water loss during

the salinity challenge. The body moisture was, therefore,

constantly decreased over time and reached the lowest

level at the end of the experiment.

Voluntary movement into CSW is a complete

organism response dependent on the exact integration

between physiological and developmental cues. The

external environment can alter the timing and quality of

response (Lerner et al., 2007). Based on the results, there

was a behavioral instability in starved larvae, they were

observed in CSW immediately after the formation of the

aqueous bridge, but their presence in CSW was

subsequently reduced and reached the minimum at the

end of the recording. Fed larvae showed more stability in

CSW entrance, and their presence in CSW gradually

increased and reached the maximum. Disruption of CSW

preference is probably related to the physiological failure

of the starved larvae in CSW adaptation. Several studies

have also emphasized on negative effects of food

deprivation on the seawater adaptation phase for different

species (Aas-Hansen et al., 2003; Taylor and Grosell,

2006; Stefansson et al., 2009; Costas et al., 2011).

Conclusion

This study showed that starving had potentially impaired

effects on the osmoregulatory fitness of Caspian kutum

larvae. Key factors related to the CSW adaptation and

cortisol, NKA, and thyroid hormones failed due to

starvation, and larvae could not overcome imposed

changes during the CSW challenge. Since the Caspian

kutum larvae were released mainly in the small size (0.5-

1 g) by IFO through an annual restocking program, the

feeding condition of larvae should be considered and

monitored before and after their release into the rivers.

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