Digestive system development and study of acid and alkalineprotease digestive capacities using biochemicaland molecular approaches in totoaba (Totoaba macdonaldi)larvae

Mario A. Galaviz . Lus M. Lopez . Alejandra Garcıa Gasca .

Carlos Alfonso Alvarez Gonzalez .

Conal D. True . Enric Gisbert

Received: 27 March 2014 / Accepted: 11 May 2015 / Published online: 19 May 2015

� Springer Science+Business Media Dordrecht 2015

Abstract The present study aimed to describe and

understand the development of the digestive system in

totoaba (Totoaba macdonaldi) larvae from hatching to

40 days post-hatch (dph) from morphological and

functional perspectives. At hatch, the digestive system

of totoaba was undifferentiated. The anus and the

mouth opened at 4 and 5 dph, respectively. During

exogenous feeding, development of the esophagus,

pancreas, liver and intestine was observed with a

complete differentiation of all digestive organs.

Expression and activity of trypsin and chymotrypsin

were observed as early as at 1 dph, and increments in

their expression and activity coincided with changes in

food items (live and compound diets) and morpho-

physiological development of the accessory digestive

glands. In contrast, pepsin was detected later during

development, which includes the appearance of the

gastric glands between 24 and 28 dph. One peak in

gene expression was detected at 16 dph, few days

before the initial development of the stomach at

20 dph. A second peak of pepsin expression was

detected at day 35, followed by a peak of activity at

day 40, coinciding with the change from live to

artificial food. Totoaba larvae showed a fully mor-

phologically developed digestive system between 24

and 28 dph, as demonstrated by histological observa-

tions. However, gene expression and activity of

alkaline and acid proteases were detected earlier,

indicating the functionality of the exocrine pancreas

and stomach before the complete morphological

development of the digestive organs. These results

showed that integrative studies are needed to fully

understand the development of the digestive system

from a morphological and functional point of views,

since the histological organization of digestive struc-

tures does not reflect their real functionality. These

results indicate that the digestive system of totoaba

develops rapidly during the first days post-hatch,

especially for alkaline proteases, and the stomach

All authors contributed equally to the study.

M. A. Galaviz (&) � L. M. Lopez � C. D. TrueFacultad de Ciencias Marinas, Universidad Autonoma de

Baja California (UABC), PO Box 76, 22860 Ensenada,

BC, Mexico

e-mail: mgalaviz@uabc.edu.mx

A. Garcıa Gasca

Centro de Investigacion en Alimentacion y Desarrollo,

Unidad Mazatlan en Acuicultura y Manejo Ambiental,

Avenida Sabalo Cerritos s/n, 82010 Mazatlan, Sinaloa,

Mexico

C. A. Alvarez Gonzalez

Laboratorio de Acuicultura Tropical DACBIOL-UJAT,

Carr. Villahermosa-Cardenas Km 0.5, Bosques de Saloya,

Villahermosa, Tabasco, Mexico

E. Gisbert

Institut de Recerca i Tecnologia Agroalimentaries. IRTA-

Sant Carles de la Rapita, Crta. Poble Nou km 5.5,

43540 Sant Carles de la Rapita, Spain

123

Fish Physiol Biochem (2015) 41:1117–1130

DOI 10.1007/s10695-015-0073-6

becomes functional between 20 and 24 dph allowing

the weaning process to begin at this age.

Keywords Fish larvae � Totoaba macdonaldi �Digestive system � Ontogeny � Proteases � Geneexpression

Introduction

Totoaba (Totoaba macdonaldi) is an endemic fish

from the Gulf of California and is considered one of

the largest members of the Sciaenidae family (Cis-

neros-Mata et al. 1997). This species has been

included in the list of endangered species (CITES

2005; Bobadilla et al. 2011), and important efforts

have been focused on its restocking through repro-

duction in captivity (True et al. 1997) or proper

management of totoaba broodstock (Lopez et al.

2006). Research has led to a complete technological

package for rearing production; however, larval

production is quite irregular with variable results

depending on the facilities, egg batches and/or rearing

procedures. The latter difficulty of rearing procedures

can in part be attributed to poor larval nutrition. With

this in mind, studying the digestive physiology with

integrative methods is essential, especially during the

larval period which is considered to be the bottleneck

for providing quality seed for aquaculture proposes.

The ontogeny of the digestive system of marine fish

larvae has been studied during the last two decades,

providing a valuable tool to better understand the

digestive physiology of larvae and has been used to

establish feeding protocols to optimize mass larval

rearing (Ueberschar 1993; Gisbert et al. 2008; Zam-

bonino-Infante et al. 2008; Lazo et al. 2011). The

development of the digestive tract and associated

organs has been well documented for a number of

marine and freshwater species (Verreth et al. 1992;

Segner et al. 1994; Pena et al. 2003; Gisbert et al.

2004; Mai et al. 2005; Chen et al. 2006; Garcıa-Gasca

et al. 2006; Rønnestad and Morais 2008; Zambonino-

Infante et al. 2008; Galaviz et al. 2011). However, only

few studies combine molecular and biochemical

procedures to describe the relationship between the

transcription of a digestive enzyme and its corre-

sponding enzymatic activity, as reported for spotted

sand bass Paralabrax maculatofasciatus (Alvarez-

Gonzalez et al. 2008, 2010), winter flounder Pleu-

ronectus americanus (Douglas et al. 1999; Murray

et al. 2004, 2006), bullseye puffer Sphoeroides

annulatus (Garcıa-Gasca et al. 2006), Atlantic salmon

Salmo salar (Rungruangsak-Torrissen et al. 2006),

turbot Scophthalmus maximus (Chi et al. 2013) and

Asian seabass Lates calcarifer (Srichanun et al. 2013).

Studies integrating the development of the digestive

system with expression or activation of digestive

enzymes during development of larvae are scarce

(Peres et al. 1998; Cahu et al. 2004; Garcıa-Gasca et al.

2006; Gisbert et al. 2009; Galaviz et al. 2011, 2012).

Most of the information during the development of the

larvae has been generated based on studies dealing

with the anatomical and histological organization of

the digestive system or the quantification of expres-

sion or activity of digestive enzymes (pancreatic and

intestinal). However, integrative studies provide a

more clear representation of the events occurring in

the morpho-physiological development of the diges-

tive system and so can provide valuable data for

establishing optimum feeding protocols that improve

larval rearing.

The aim of the present study was to understand and

describe for the first time the larval digestive

physiology of T. macdonaldi by measuring the

expression and activity for three of the major digestive

proteases: trypsin, chymotrypsin and pepsin and to

relate them with the development of the digestive

system and the feeding regimen. This information

provides a better understanding of the digestive

system function during early stages of life and can

be used for improving actual rearing procedures.

Materials and methods

Eggs and larval fish rearing

Fertilized totoaba eggs were obtained from a captive

broodstock kept in two separate groups (30 fish per

group; 25–30 kg in weight, sex ratio male vs fe-

male = 2:1) held at the marine finfish hatchery of the

Facultad de Ciencias Marinas, Universidad Autonoma

de Baja California, Mexico. Gonadal maturation in

adult totoaba was induced using photothermal control

to simulate natural seasonal cycles, and fish were

induced to ovulate and spermiate using [des-Gly10,

D-Ala6]-LHRH ethylamine acetate salt hydrate

1118 Fish Physiol Biochem (2015) 41:1117–1130

123

(SIGMA�). Fertilized (floating) eggs were collected

28–36 h after spawning and further treated with

100 ppm formalin for 1 h, rinsed and stocked at a

density of 100 eggs L-1 in 2200-L cone bottom tanks

with 24 �C seawater recirculated at a rate of

1.5–2 L min-1 through a fluidized bed biofilter, UV

sterilizer and foam fractionator. Eggs hatched ap-

proximately 20 h after incubation. Yolk sac larvae

were transferred using beakers, stocked in ten 100-L

rearing tanks at a density of 30 individuals L-1 and

cultured using the same environmental conditions as

in the process of incubation. Beginning at 4 days post-

hatch (dph), larvae were fed three times per day

(08:00, 12:00 and 18:00 h). Feeding consisted of live

preys, starting with rotifers (Brachionus plicatillis) at

4 dph, followed by Artemia metanauplii at 20 dph

(Salt Creek Inc, Salt Lake City, UT, USA) enriched

with a commercial emulsion (Bio-Marine Algamac

3050TM) at a concentration of 0.6 g L-1. Artemia

nauplii were supplied at a concentration of 5 nauplii

mL-1 from 18 dph until 34 dph. At 30 dph, the

amount of live food was reduced and a combination of

enriched Artemiametanauplii and microdiet (Otohime

Japanese Marine Weaning Diet, Red Mariculture;

protein 52.1 %, lipid 16.3 %, ash 11.2 %, particle size

200–1410 lm) was supplied. Weaning was completed

at 34 dph, when live preys were no longer supplied.

Fish were fed the microdiet from 34 to 40 dph when

the trial ended.

Sampling method

Totoaba larvae (n = 50–100 depending on their size)

were randomly sampled from the rearing tanks using a

200-lm dip net. Sampling was conducted before

morning feeding. Samples were collected daily from

hatching to 6 dph, then every 2 days until 20 dph and

thereafter every fourth day until the end of the study at

40 dph. After sampling, larvae were killed by an

anesthetic overdose (tricaine methanesulfonate—MS

222), rinsed with distilled water to remove excess salts

and stored at -70 �C for biochemical analyses and

RNA later � (Ambion, Life sciences) for the molecular

work. Additional samples (n = 30 larvae) were col-

lected to measure larval size and for histological

analysis. Average total length (mm) was calculated by

measuring 10 larvae under a dissecting microscope

using a digital camera and software Pax-it version 6

(Mis Inc, USA).

Histology

Larvae used for histology were fixed in 2%

paraformaldehyde for 24 h at 4 �C, then washed,

dehydrated in a graded series of ethanol, cleared and

embedded in paraffin. Sagittal sections (5 lm) were

obtained with a conventional microtome (Leica- RM

2125 RT), deparaffined, rehydrated and stained with

hematoxilin–eosin (H&E). Histological sections were

viewed under a light microscope and photographed

with an Infinity digital camera using the PAXcam2

software (Pax-it version 6).

Enzymatic activity assays

Biochemical quantification of digestive proteases was

conducted by means of spectrophotometric methods

using three different pools of larvae per sampling point

(biological replicates). Pools of larvae were composed

of 50 specimens from hatching to 10 dph, 30 larvae

from 12 to 20 dph and 10 larvae from 25 to 40 dph.

Because of the difficulties associated with dissecting

very small larvae, whole body homogenates were used

in larvae younger than 16 dph. After this age, the

digestive system was dissected individually removing

the whole digestive system from the esophagus to the

posterior intestine; this was done by removing the

head and tail section of the larvae on a glass slide

supported on a frozen mini-table. Each sample was

homogenized with a tissue grinder (VWRTM pellet

mixer) into 1 mL of ice-cold distilled water (4 �C) andcentrifuged using a Biofuge primo R Heraeus at

14,000g for 30 min at 4 �C, and supernatants were

stored at -70 �C until further analyses.

Trypsin (EC 3.4.21.4) activity was determined

according to Erlanger et al. (1961), using BAPNA (N-

a-Benzoyl-DL-arginine-4-nitroanilide) as substrate.

The mixtures were incubated at 37 �C, and the

absorbance of the final products was measured at

410 nm. The reaction was stopped by adding 30 %

acetic acid.

Chymotrypsin (EC 3.4.21.1) activity was measured

according to Hummel (1959), using the modification

of Applebaum et al. (2001) and using BTEE (N-

benzoyl-L-tyrosine ethyl ester) as substrate. The

mixtures were incubated at 37 �C, and the absorbanceof the reaction products was measured at 256 nm. For

trypsin, one unit of enzyme activity was defined as

1 lmol p-nitroanilide released per minute, using a

Fish Physiol Biochem (2015) 41:1117–1130 1119

123

molar extinction coefficient of 8.8 for trypsin, while

for chymotrypsin, one unit of activity was defined as

1 lmol of BTEE hydrolyzed per minute, using an

extinction coefficient of 964 mL/lg/cm.

Acid protease (pepsin; EC 3.4.23.1) activity was

determined as described by Sarath et al. (1989), using

2 % hemoglobin as substrate. Enzyme crude extracts

and their substrate were incubated at 37 �C, and the

absorbance of the reaction products was measured at

280 nm. One unit of enzyme activity was defined as

1 lg tyrosine released per minute, using the molar

extinction coefficient of 0.005 mL/lg/cm. The ac-

tivity of acid and alkaline proteases in crude extracts

was determined using the following equations: Total

activity (units mL-1) = [Dabs reaction final volume

(mL)]/[MEC time (min) extract volume (mL)]; speci-

fic activity (units mg protein-1) = total activity/sol-

uble protein (mg), where Dabs represents the increasein absorbance at a determined wavelength and MEC

represents the molar extinction coefficient for the

product of the reaction (mL/lg/cm). The soluble

protein level in crude enzyme extracts was determined

according to the Bradford (1976) method using bovine

serum albumin as a standard. All assays were carried

out in triplicate (methodological replicates).

Gene expression

Quantification of gene expression related to the selected

digestive enzymes was performed according to Garcıa-

Gasca et al. (2006). Briefly, total RNAwas isolated using

Trizol� reagent (Invitrogen) followed by DNAse I

treatment. cDNA synthesis was performed at 45 �Cwith

5 lg of total RNA, M-MLV reverse transcriptase

(Promega) and random primers. The absence of genomic

DNA contamination was confirmed by performing the

same reaction without reverse transcriptase. Initial PCR

amplifications were completed using degenerated pri-

mers for trypsin, chymotrypsin and pepsin precursors

obtained by the alignment of available sequences from

several marine fish species. Expected PCR products for

trypsinogen, chymotrypsinogen and pepsinogen genes

were 314, 477 and 450 bp, respectively. Primers for

totoaba 18S rRNA gene were designed to render a

product of 443 bp. This genewas used as internal control

for quantitative PCR (qPCR) analysis (Table 1). Purified

PCRproductswere ligated into apGEM-Tcloningvector

(Promega). E. coli DH5a competent cells (Invitrogen)

were transformed by heat shock, and plasmid extraction

was performed by alkaline lysis. Bidirectional sequenc-

ing was carried out using labeled T7/SP6 universal

primers and a LICOR IR2 DNA sequencer. Sequence

analysis was performed using the National Center for

Biotechnology Information (NCBI) Basic Local Align-

ment Search Tool (BLAST) program. The sequences

obtained were submitted to GenBank (Table 1).

Trypsinogen-, chymotrypsinogen- and pepsinogen-

specific primers were designed using the Primer3

software to perform qPCR (Table 1). PCR products

were 179, 169, 173 and 154 bp for trypsin, chy-

motrypsin, pepsin and 18S rRNA, respectively

(Table 1). qPCR was performed with a SmartCycler

(Cepheid) using SYBR GREEN� (Invitrogen) under

the following PCR conditions: 95 �C for 2.5 min, and

40 cycles at 95 �C for 30 s, 60 �C for 30 s and 72 �Cfor 30 s. Dilution series of cDNA amplified with

trypsin, pepsin and 18S rRNA primers were used to

construct a standard curve for each gene. Standard

curves were calculated by linear regression analysis

using threshold cycle (CT) values and log copy

numbers (log Co) obtained from the serial dilution

analysis. The copy numbers (Co) of unknown samples

were calculated as follows: Co = a ? [b 9 *CT],

where a = y intercept and b = slope of the standard

curves. The normalized Co of trypsinogen, chy-

motrypsinogen and pepsinogen for each sample was

determined by dividing the Co of each gene by the Co

of 18S rRNA, and each normalized sample was

divided by the internal calibrator at 1 dph.

Statistical analysis

Enzymatic expression and activity data were analyzed

using one-way ANOVA (data previously checked for

normality and homogeneity of variance), and the

Tukey test was used for multiple comparisons with a

significance level of P\ 0.05. All statistics were

conducted using Sigma-Stat 11.0 for Windows (Sig-

ma-Plot� 11.0, USA).

Results

Larva of totoaba

The increase in wet weight (mg) and total length (cm)

of T. macdonaldi is shown in Fig. 1. During the

1120 Fish Physiol Biochem (2015) 41:1117–1130

123

rearing period, growth in body weight was slow from 1

to 16 dph; however, body weight increased exponen-

tially from 18 dph until the end of the experiment (dph

40). Growth in total length was steady during the first

16 days and then increased exponentially from 18 dph

until the end of experiment.

Histological development of the digestive system

At hatching (2.58 ± 0.03 mm TL), totoaba larvae

showed a homogeneous acidophilic yolk, surrounded

by a syncytial layer of squamous cells. The yolk sac

contained a single vacuole located at the posterior part

of the yolk sac, corresponding with the oil globule that

dissolved during the paraffin embedding process

(Fig. 2a). At this stage, the intestine was lined by a

simple layer of columnar cells. Differentiation of a

rudimentary digestive tube was already distinguish-

able as a straight tube running dorsally to the yolk sac

and closed to the exterior, since the anus and mouth at

this stage were not yet formed.

Between 1 and 2 dph (3.28 ± 0.09 mm TL), the

yolk sac volume was reduced by a half, whereas the oil

globule was only reduced by a third of its original size

from hatching. Stratified squamous epithelium corre-

sponding to the pharynx could be distinguished

connecting the short esophagus with the intestine,

whereas the posterior region of the intestine was evident

and bent in a 90� angle (Fig. 2b). The basophilic

cytoplasm of the exocrine pancreas was homogeneous,

and zymogen granules and pancreatic ducts were not

yet apparent. The pancreatic acinar cells resembled the

Fig. 1 Mean wet weight

(mg ± SD, n = 30, filled

circle) and total length

(cm ± SD, n = 10, filled

square) of totoaba larvae

during cultured

experimental conditions

Table 1 Gene-specific primers for qPCR and GenBank accession numbers for totoaba trypsin, chymotrypsin and pepsin nucleotide

sequences

Gene Primer name Primer sequence Product size (bp) GenBank accession number

Trypsin TmTryp-F

TmTryp-R

50-ACCCGCTGTCTGATCTCTGGAT-30

50-AGGAGTCTTTGCCTCTCTCGACAA-30179 HM754480

Chymotrypsin TmChymo-F

TmChymo-R

50-CGCTCACTGTAACGTCAGGACCTA-30

50-GGTGGACAACTTGATGAGGGAGAT-30169 HM754481

Pepsin TmPepsin-F

TmPepsin-R

50-CTCTGACGATGTTGTGCCAGTCTT-30

50-CAGAGGTCAGAGGGATCCAGGTAA-30173 HM754482

18S rRNA Tm18s-F

Tm18s-R

50-CTGAACTGGGGCCATGATTAAGAG-30

50-GTCTTCGAACCTCCGACTTTCGTT-50154 HM754483

Fish Physiol Biochem (2015) 41:1117–1130 1121

123

hepatocytes in shape, and they also displayed a

spherical nucleus. At 3 dph (3.34 ± 0.1 mm TL), the

yolk did not appear homogeneous and was formed by

small acidophilic yolk platelets surrounded by hepatic

tissue located in the anterior region of the abdominal

cavity close to the heart cavity. The oil globule was still

visible and, similar to the yolk, was also surrounded by

round-shaped hepatocytes with central-located nuclei.

At this stage, the mouth opened, the buccopharyngeal

cavity and esophagus elongated, and the first goblet

cells appeared. The esophageal mucosa could be seen

as lined by a simple cuboidal epithelium surrounded by

a thin layer of connective and muscular tissue in

differentiation (Fig. 2c, d).

Between the fourth and fifth dph (3.37 ± 0.16 mm

TL) (Fig. 3a, b), remnants of the yolk sac and oil

globule were still visible surrounded by hepatic tissue.

The esophageal mucosa started to fold and the

muscular layer surrounding it increased in thickness.

The intestine folded and coiled, whereas the intestinal

valve appeared as a constriction of the intestinal

mucosa dividing the intestine in two regions, the pre-

valvular (anterior) and post-valvular (posterior) intes-

tine. Exocrine pancreatic acinar cells containing

eosinophilic zymogen granules were visible and

grouped in rosette patterns around central canals that

anastomosed with large pancreatic ducts.

Between 6 (3.54 ± 0.13 mm TL) and 12 dph

(4.09 ± 0.20 mm TL), the digestive tract grew in

length and complexity. The folding (transversal folds)

of the esophagus increased, as did the number of

goblet cells lining the esophageal epithelium and the

thickness of the circular and longitudinal layers of

muscular fibers that surrounded the esophageal mu-

cosa. In addition, the folding of the intestinal mucosa

increased although the folding level of the posterior

intestine was more prominent than that of the anterior

region of the intestine (Fig. 3c). Acidophilic supranu-

clear inclusions were present in the enterocytes of the

posterior intestine in larvae aged 16–18 dph. Active

pinocytosis was evident at the base of microvilli of the

enterocytes except for those near the anus. During this

period, the liver increased in size and achieved its

globular shape. Hepatocytes did not show lipid

inclusions and retained a polygonal shape with central

nuclei and slight eosinophilic cytoplasm (Fig. 3d).

During the period between 20 (6.90 ± 0.29 mm

TL) and 24 dph (10.52 ± 0.12 mm TL), the most

Fig. 2 Sagittal sections of larvae at days 0 (a), 2 (b), 3 (c, d) post-hatch. bc buccopharynx; e esophagus; eye; i intestine; l liver; mfmuscular fibers; m mouth; n notochord; og oil goblet; ys yolk sac. Hematoxylin & eosin staining

1122 Fish Physiol Biochem (2015) 41:1117–1130

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relevant histological event was the appearance of

clusters of undifferentiated cuboidal cells between the

esophagus and the anterior intestine and posteriorly to

the swim bladder (Fig. 4a, b). These clusters of

cuboidal cells would develop into gastric glands

arranged along numerous longitudinal folds and

surrounded by a thin layer of circular musculature

and connective tissue at 26–28 dph (13.0 ± 0.41 mm

TL) (Fig. 5a, b). Gastric glands were composed of a

single type of secretory cells devoid of microvilli on

their apical border and lining their base with a simple

cubic epithelium. The wall of the glandular stomach

was composed of mucosa, lamina propia-submucosa,

thin muscularis and serosa layers. The pyloric sphinc-

ter appeared as an epithelial fold that separated the

stomach from the anterior intestine. Stomach devel-

opment was coupled with the appearance of lipid

inclusions in hepatocytes that occupied most of the

cell’s cytoplasm and displaced the nucleus to the

periphery of the cell. During this period, the intestine

continued to grow in length. The size of longitudinal

and transversal folds of the intestinal mucosa also

continued to grow, although the anterior intestine only

presented small transversal (villi) folds in contrast to

the posterior region that contained both types of

mucosal folds. From this age to the end of the study,

the stomach increased in size and complexity by

means of an increase in the size and number of

mucosal folds and gastric glands, but no relevant

histology was observed (Fig. 5c). Pyloric caeca that

formed part of the most anterior region of the intestine

appeared between 32 and 36 dph (18.49 ± 0.23 mm

TL) as fingerlike projections, and they grew in size and

number by 40 dph (20.09 ± 1.43 mm TL) (Fig. 5d).

Histologically, pyloric caeca were similar to the

anterior intestine, in that they were lined by a simple

columnar epithelium with prominent microvilli and

surrounded by a thin layer of connective and muscular

tissue.

Alkaline proteases

Trypsinogen mRNA expression was barely detected

prior to 5 dph; however, a distinct increase was

Fig. 3 Sagittal sections of larvae at days 4 (a), 5 (b), 16 (c) and18 (d) post-hatch. a anus; ai anterior intestine; bc buccopharynx;e esophagus; eye; ep exocrine pancreas; h hearth; ga gill arch;

l liver; mi medium intestine; m mouth; n notochord; og oil

goblet; pd pancreatic duct; pi posterior intestine; r rectum; sb

swim bladder; ys yolk sac. Hematoxylin & eosin staining

Fish Physiol Biochem (2015) 41:1117–1130 1123

123

evident between 5 and 6 dph, which was concomitant

to the completion of yolk sac absorption and the onset

of larvae with rotifers. Trypsinogen expression levels

increased again at 16 dph, 2 days before the change

from rotifers to Artemia nauplii. Maximum trypsino-

gen expression levels were detected at 36 dph

(P[ 0.038), and at this time, larvae were fed with

the microdiet (Fig. 6a). Regarding enzyme quantifi-

cation, trypsin-specific activity was detected as early

as 1 dph (0.39 ± 0.05 mU mg protein-1) and re-

mained low during the following days; however, a

significant increase in specific activity was observed at

12 dph (1.06 ± 0.20 mU mg protein-1; P\ 0.001).

After that, trypsin-specific activity decreased at

16 dph (0.39 ± 0.01 mU mg protein-1), increased

again at 25 dph (0.99 ± 0.17 mU mg protein-1;

P\ 0.001), and then tended to decrease during the

subsequent days but not in a statistically significant

manner (P[ 0.05). The maximum level of activity of

trypsin was observed at 40 dph (1.83 ± 0.05 mU mg

protein-1) when larvae were fed with the microdiet

(Fig. 6b).

Chymotrypsinogen gene expression was barely

detected at 1 dph (yolk sac larvae), and after this

age, two peaks of expression were evident. The first

peak was observed at 16 dph, 2 days before the

changing in the feeding sequence from rotifers to

Artemia nauplii, and the second peak of gene expres-

sion was observed at 36 dph, when larvae were fed

with the microdiet (Fig. 6c). After that, expression

levels of chymotrypsinogen decreased until the end of

the experiment at 40 dph. Specific activity of chy-

motrypsin was detected at 1 dph (0.18 ±

0.08 mU mg protein-1), showing a significant in-

crease at 12 dph (2.57 ± 0.38 mU mg protein-1)

(P\ 0.001) which coincided with the feeding of

rotifers enriched with fatty acids and acidophilic

supranuclear inclusions present in the enterocytes of

the posterior intestine. No changes in chymotrypsin-

specific activity were found between 12 and 36 dph;

however, chymotrypsin-specific activity increased at

36 dph (1.75 ± 0.95 mU mg protein-3; P\ 0.001),

coinciding with the end of the transition from Artemia

to the microdiet (Fig. 6c).

Acid protease

Pepsinogen gene expression was not detected until

16 dph, 4 days before the histological differentiation

of the stomach and the observation of the first gastric

glands (Fig. 6d). Maximum expression levels were

detected at 36 dph (P\ 0.05), coinciding with the

Fig. 4 Sagittal sections of larvae at day 24 (a, b). ai anterior intestine; e esophagus; ep exocrine pancreas; l liver; pd pancreatic duct; piposterior intestine; sb swim bladder; s stomach. Arrows indicate appearance of gastric glands. Hematoxylin & eosin staining

1124 Fish Physiol Biochem (2015) 41:1117–1130

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complete histological development of the stomach.

Expression levels decreased at 36 dph and remained

constant until the end of the experiment at 40 dph.

Additionally, pepsin-specific activity was detected

between 18 and 20 dph (0.20 ± 0.07 U mg pro-

tein-1), showing a peak at 24 dph (2.16 ±

1.17 U mg protein-1; P\ 0.05) and coinciding with

the differentiation of the gastric glands and the

stomach. After this age, pepsin activity decreased

and remained constant between 28 and 32 dph,

increasing again at the end of experiment at 40 dph

(Fig. 6e).

Discussion

The development of the digestive system in totoaba

larvae presented similarities with other marine fish

species such as gilthead sea bream Sparus aurata

(Sarasquete et al. 1995), spotted seabass P. maculato-

fasciatus (Pena et al. 2003), California halibut Par-

alichthys californicus (Gisbert et al. 2004), bullseye

puffer S. annulatus (Garcıa-Gasca et al. 2006), white

seabass Atractoscion nobilis and spotted rose snapper

Lutjanus guttatus (Galaviz et al. 2011, 2012), among

others. In totoaba, the folding of the intestinal mucosa

and differentiation of enterocytes, as well as the

morphogenesis of the buccopharynx, esophagus and

rectum, occurred between 3 and 5 dph, coinciding

with the opening of the mouth and anus (4 dph) and

the onset of exogenous feeding. According to Segner

et al. (1994), the differentiation of enterocytes indi-

cates that the intestine is suitable for absorption of

nutrients at the beginning of the exogenous feeding.

The first days of development in marine fish larvae

are critical, particularly when the yolk sac is reab-

sorbed and exogenous feeding starts. At the onset of

exogenous feeding, larvae mainly depend on

Fig. 5 Sagittal sections of larvae at days 28 (a, b), 32 (c), 40(d). ai anterior intestine; e esophagus; ep exocrine pancreas; gg

gastric glands; l liver; mi medium intestine; pc pyloric caeca; pi

posterior intestine; s stomach. Arrows indicate appearance of

gastric glands. Hematoxylin & eosin staining

Fish Physiol Biochem (2015) 41:1117–1130 1125

123

pancreatic digestive enzymes for nutrient digestion

such as trypsin, chymotrypsin, carboxypeptidases

among others. Therefore, studies on the physiology

of the digestive system, as well as feeding patterns and

nutritional capabilities of fish larvae, have been of

great importance to understand the progress of

digestive enzymes during larval development (Dıaz-

Lopez et al. 1997; Zambonino-Infante and Cahu 2001;

Alvarez-Gonzalez et al. 2008; Gisbert et al. 2008,

2009; Galaviz et al. 2011). This implies that the

moment of appearance of the digestive enzymes is

genetically programmed in all vertebrates. As in the

Fig. 6 Gene expression and enzymatic activity of trypsin,

chymotrypsin and pepsin during totoaba larviculture (mean ± SD,

n = 3). a Trypsin mRNA expression in larvae relative to 0 dph.

b Trypsin activity in larvae. c Chymotrypsin mRNA expression in

larvae relative to0 dph.dChymotrypsin activity in larvae.ePepsinmRNA expression in larvae relative to 0 dph. f Pepsin activity inlarvae. Asterisks indicate significant differences in expression or

activity levels of the three digestive enzymes

1126 Fish Physiol Biochem (2015) 41:1117–1130

123

rest of vertebrates, the moment of appearance of the

digestive enzymes is genetically programmed, but the

amount of activity is the net result of the interaction

between gene expression and a number of other

factors, which may greatly affect enzyme production,

such as the amount and composition of available food

and feeding patterns. Changes in metabolism, nutri-

tional requirements and feeding habits taking place in

most fish species from the larval stages to the adult

state are also evidenced as quantitative and qualitative

changes in the profile of their digestive enzymes

(Gisbert et al. 2013).

Themorphological differentiation of the stomach in

totoaba larvae was observed at 20 dph as an extension

of the esophagus; however, the complete development

of gastric glands was not observed until a few days

later, at 24–28 dph. According to Baglole et al. (1997),

the development of gastric glands in marine fish larvae

is considered the last event in the development of the

digestive system, and some authors have suggested

that their presence indicates the end of the larval

period and the onset of the juvenile stage (Sarasquete

et al. 1995; Pena et al. 2003; Galaviz et al. 2011). In the

present study, the complete development of the

stomach and the differentiation of the gastric glands

were observed at 28 dph, whereas the activity of the

acid protease (pepsin) was detected earlier, starting at

18–20 dph, with a peak at 24 dph, which indicated

their functionality, before the complete differentiation

of the stomach, including the formation of cardiac and

pyloric sphincters. Another explanation for premature

acid protease activity is the potential presence of

catepsins that have intracellular activity in several

tissues, and this could have occurred because whole

larvae were processed as pools for preparation of

enzymatic assays (Lazo et al. 2007). Time shifts

between pepsin activity and morphological develop-

ment of gastric glands have also been observed in

other species such as California halibut P. californicus

(Alvarez-Gonzalez et al. 2006), white seabass A.

nobilis (Galaviz et al. 2011), spotted rose snapper L.

guttatus (Galaviz et al. 2012) and Asian sea bass L.

calcarifer (Srichanun et al. 2013). The time sequence

of gastric gland appearance and the maturation of the

stomach vary among families, species and their

reproductive guilds (Falk-Petersen 2005; Trevino

et al. 2011). In the present study, an increase in

pepsinogen gene expression was first observed at

14–16 dph, while the increase in pepsin activity was

first observed at 20–24 dph, coinciding with the

formation of the stomach as an extension of the

esophagus, showing a relationship between the syn-

thesis of the precursor and the production of pepsin.

Similar results were observed in winter flounder

Pseudopleuronectes americanus (Douglas et al.

1999), white seabass A. nobilis (Galaviz et al. 2011),

spotted rose snapper Lutjanus guttatus (Galaviz et al.

2012) and Asian seabass L. calcarifer (Srichanun et al.

2013). These results, together with the appearance of

gastric glands, suggest a fully functional stomach

between 24 and 28 dph.

Pancreatic enzyme synthesis and secretion appear

to be particularly sensitive to food deprivation and

dietary composition in teleost larvae, and consequent-

ly, pancreatic enzyme activity provides a reliable

biochemical marker of larval fish development and

condition. The pancreatic secretory process matures

during the first 3 or 4 weeks after hatching in

temperate marine fish larvae (Gisbert et al. 2013).

The expression and activity of both trypsin and

chymotrypsin were detected at the time of hatching

and increased in a fluctuating fashion during larval

development. Few studies have detected alkaline

protease activity during embryonic development,

indicating that trypsin-like enzymes seem to be

functional before exocrine pancreas become function-

al. These enzymes’ functionality may occur because

they are involved in protein hydrolysis in the yolk

(Sveinsdottir et al. 2006), providing energy to the yolk

sac embryo before exogenous feeding (Alvarez-

Gonzalez et al. 2008). Expression and activity of

trypsin and chymotrypsin prior to the first feeding

suggest that the activity of these enzymes is derived

from genetically preprogrammed expression and not

from the first exogenous feeding, since these proteases

are involved in yolk protein cleavage during embryo-

genesis and the lecitotrophic stage (Zambonino-

Infante and Cahu 2001; Sveinsdottir et al. 2006;

Alvarez-Gonzalez et al. 2008; Gisbert et al. 2009;

Galaviz et al. 2011). In the present study, trypsinogen

and chymotrypsinogen expression levels increased

from 4 dph onwards, after the onset of exogenous

feeding. Peaks in gene expression were detected

concomitantly with changes in the type of food (live

prey and microdiet) administered to larvae and

preceded those of specific enzyme activity. Gene

expression increased with larval development starting

between 4 and 6 dph when the larvae opened the

Fish Physiol Biochem (2015) 41:1117–1130 1127

123

mouth and were fed live food (rotifers), showing two

peaks of maximum expression levels of trypsin and

chymotrypsin at 16 and 34 dph, while enzymatic

activity showed maximum levels at 40 dph for trypsin

and 34 dph for chymotrypsin, suggesting that the

pancreas was fully functional after 34 dph. The

efficient synthesis and secretion of pancreatic zymo-

gen granules plays an important role in the hydrolysis

of food proteins and the activation of other enzymes

(Hjelmeland and Jørgensen 1985, Galaviz et al. 2012).

Similar results were observed in other marine fish

larvae such as bullseye puffer S. annulatus (Garcıa-

Gasca et al. 2006), California halibut P. californicus

and spotted snapper P. maculatofasciatus (Alvarez-

Gonzalez et al. 2006, 2008) and Dentex dentex

(Gisbert et al. 2009). In the present study, the peaks

of trypsin and chymotrypsin expression preceded

those of specific activity and generally coincided with

changes in food supply. Thus, protein digestion in fish

larvae generally starts at the time of hatching and is

due to the contribution of pancreatic enzymes such as

trypsin and chymotrypsin. During the following days,

activity levels of these alkaline enzymes increase due

to the maturation of the digestive system, especially

the exocrine pancreas (Garcıa-Gasca et al. 2006;

Alvarez-Gonzalez et al. 2008; Galaviz et al. 2011).

In conclusion, thepresent results indicated that totoaba

larvae showed a fully morphologically developed diges-

tive system at 28 dph, as demonstrated by histological

observations. However, gene expression and activity of

alkaline and acid proteases were detected earlier, indi-

cating the functionality of the exocrine pancreas and

stomach before the complete morphological develop-

ment of the digestive organs. These results demonstrate

that integrative studies are needed to fully understand

digestive development since thehistological organization

of digestive structures does not reflect their real func-

tionality. The contribution of histological, gene expres-

sion and proteases activity analyses suggested that at this

earlier time (24–28 dph), larvae should be able to digest

inert food and absorb nutrients. Based on these results,

the onset of the weaning period (currently performed

between 30 and 32 dph) could be conducted at earlier

ages, almost certainly between 24 and 28 dph, although

further rearing trials are needed.

Acknowledgments This work was supported by the National

Council for Science and Technology (CONACyT-SAGARPA) of

Mexico (S2011-08-164673) and an internal project by

Autonomous University of Baja California (UABC) Mexico.

The authors are grateful to Rubı Hernandez-Cornejo (CIAD-

Mazatlan) and Deyanira Rodarte (UABC) for technical assistance.

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