853
The Journal of Experimental Biology 215, 853-862
© 2012. Published by The Company of Biologists Ltd
doi:10.1242/jeb.063925
RESEARCH ARTICLE
Dietary protein quality differentially regulates trypsin enzymes at the secretion and
transcription level in Panulirus argus by distinct signaling pathways
Erick Perera1,*, Leandro Rodríguez-Viera1, Javier Rodríguez-Casariego1, Iliana Fraga2, Olimpia Carrillo3,
Gonzalo Martínez-Rodríguez4 and Juan M. Mancera5
1
Center for Marine Research, University of Havana, Cuba, 2Center for Fishery Research, MINAL, Cuba, 3Faculty of Biology,
University of Havana, Cuba, 4Andalucian Institute of Marine Science, CSIC, Cadiz, Spain and 5Department of Biology, Faculty of
Marine and Environmental Science, University of Cadiz, Spain
*Author for correspondence ([email protected])
Accepted 23 November 2011
SUMMARY
The effects of pelleted diets with different protein composition (fish, squid or soybean meals as main protein sources) on trypsin
secretion and expression were studied in the lobster Panulirus argus. Trypsin secretion was shown to be maximal 4h after
ingestion. At this time, fish- and squid-based diets induced trypsin secretion, as well as up-regulation of the major trypsin isoform
at the transcription level. While fish- and squid-based diets elicited a prandial response, soybean-based diet failed to stimulate the
digestive gland to secrete trypsin into the gastric fluid or induce trypsin expression above the levels observed in fasting lobsters.
In vitro assays showed that intact proteins rather than protein hydrolysates stimulate trypsin secretion in the lobster. However,
the signal for trypsin transcription appears to be different to that for secretion and is probably mediated by the appearance of free
amino acids in the digestive gland, suggesting a stepwise regulation of trypsin enzymes during digestion. We conclude that
trypsin enzymes in P. argus are regulated at the transcription and secretion level by the quality of dietary proteins through two
distinct signaling pathways. Our results indicate that protein digestion efficiency in spiny lobsters can be improved by selecting
appropriated protein sources. However, other factors like the poor solubility of dietary proteins in dry diets could hamper further
enhancement of digestion efficiency.
Key words: Panulirus argus, spiny lobster, protein digestion, digest, regulation, trypsin.
INTRODUCTION
As in most crustacea, trypsin enzymes play a central role in protein
digestion in spiny lobsters (Johnston, 2003; Celis-Gerrero et al.,
2004; Perera et al., 2008a). Some information is available on the
biochemical characterization of spiny lobster trypsins (Galgani and
Nagayama, 1987; Celis-Gerrero et al., 2004; Perera et al., 2008a)
and variation in trypsin activity throughout development and molt
stages has also been reported (Johnston, 2003; Perera et al., 2008b).
However, the effect of diet on these enzymes has been poorly studied
and the time course of trypsin activity after ingestion of different
diets has only recently been reported for one spiny lobster species
(Jasus edwardsii) (Simon, 2009). In addition, virtually nothing is
known about the regulatory mechanisms behind protein digestion
in spiny lobsters.
In Panulirus argus, trypsin is a polymorphic enzyme (Perera et
al., 2008a) and trypsin cDNA showing different expression rates has
been cloned (Perera et al., 2010a). Additionally, trypsin isoforms in
lobster appear to differ in catalytic properties or specificity as
differences in digestion efficiency have been found among three
distinct trypsin phenotypes (Perera et al., 2010b). These features of
lobster trypsins indicate several points at which regulation of their
activity can occur and, thus, makes this species a good model for
studying trypsin regulation in crustacea. Despite the many studies on
trypsin enzymes in other crustaceans, these regulatory mechanisms
are not well known and, in general, limited information is currently
available for non-insect invertebrates (Muhlia-Almazán et al., 2008).
There is also great interest in the commercial growout of wildcaught spiny lobsters (Jeffs and Davis, 2003). Research effort over
the past decades has considerably increased our knowledge about
the nutritional requirements of spiny lobsters (Williams, 2007)
but feeding most species with formulated diets on a least-cost
basis is still a challenge. Spiny lobster growth performance is
relatively poor when they are fed on dry formulated diets unless
high levels of krill meal and/or krill hydrolysate are included
(Smith et al., 2005; Barclay et al., 2006; Cox and Davis, 2009).
It seems that spiny lobsters cannot efficiently use proteins from
common aquafeed ingredients (e.g. fish, squid and soybean
meals) in dry food. This conflicts with the fact that they are
equipped with a wide repertoire of digestive enzymes, especially
proteases (Galgani and Nagayama, 1987; Celis-Gerrero et al.,
2004; Perera et al., 2008a), and with the results of previous
digestibility studies (Ward et al., 2003; Irving and Williams, 2007;
Perera et al., 2010b). Poor protein use could be related to some
impairment in digestive progression when lobsters are fed on
pelleted diets (Simon and Jeffs, 2008; Simon, 2009).
The aim of the present work was to assess the effect of feeding
P. argus with different protein sources on trypsin expression and
secretion. Our results demonstrate that trypsin enzymes are
differentially regulated at the transcription and secretion level by
ingested proteins. Additionally, we provide evidence that intact
proteins, rather than small peptides or free amino acids, are the major
signal eliciting the prandial secretory response in lobster, whereas
THE JOURNAL OF EXPERIMENTAL BIOLOGY
854
E. Perera and others
transcriptional regulation of the major isoform appears to occur by
a distinct mechanism.
MATERIALS AND METHODS
Animals and diets
Spiny lobsters, P. argus (Latreille 1804) (90–150g), were collected
in the Gulf of Batabanó, Cuba, and held under optimal laboratory
conditions at the Center for Marine Research of the University of
Havana as described before (Perera et al., 2005). Lobsters were fed
daily with fresh fish (Opisthonema oglinum) ad libitum and
experiments were carried out after animals had been allowed to adapt
to captive conditions for 2weeks and had fed intensively on the
food offered. Only intermolt specimens [determined according to
Lyle and MacDonald (Lyle and MacDonald, 1983)] were used in
experiments because maximum trypsin activity at this stage has
previously been reported (Perera et al., 2008b). Soybean, fish and
squid meals used as protein sources in diets were prepared as
described earlier (Perera et al., 2010b) and experimental formulated
diets (Table1) were prepared by thoroughly mixing the dry
ingredients with oil and then adding water until the consistency was
that of stiff dough. This was passed through a mincer, dried at 60°C
and broken into a convenient pellet size (~3⫻10mm). Diets were
stored at –20°C until use.
In vivo effects of food proteins on trypsin activity and
expression
It has been shown that the time course of digestive enzymes during
digestion in the spiny lobster J. edwardsii is better reflected in the
gastric fluid, as only secreted enzymes can be measured (Simon,
Table 1. Formulation (g100g–1) of experimental diets
a
Squid meal
Fish meal (herring)a
Soybean mealb
Gelatinb
Whole wheat c
Shark oild
Soybean lecithinb
Cholesterole
Vitamin/mineralf
Vitamin Cb
Vitamin Eb
Dicalcium phosphatec
Calcium carbonatec
Metionineg
Talcb
Total
Squid
Fish
Soybean
44
8
–
8
26.75
1.9
2
2
2
0.1
0.25
1
2
–
2
100
6.5
44
–
8
26.75
0.2
2
2
2
0.1
0.25
1
2
–
5.2
100
–
8
61
8
5.25
6.4
2
2
2
0.1
0.25
1
2
0.1
2
100
Diets were formulated with 45% crude protein, 9% crude fat, 25%
carbohydrates.
Pellets contain 10–12% of water.
a
Prepared at the laboratory as detailed before (Perera et al., 2010b).
b
Commercially available foodstuffs in Cuba.
c
Santa Cruz Fish Feed Factory, Camagüey, Cuba.
d
Fisheries Research Center Laboratory, Havana, Cuba.
e
Wako Pure Chemical Industries Ltd, Osaka, Japan.
f
Premix from DIBAQ-Aquaculture, Segovia, Spain, containing (per kg of
feed): vitamin A 15,000IU, vitamin D3 3000IU, vitamin E 180mg, vitamin
K 15mg, vitamin B1 37.5mg, vitamin B2 37.5mg, vitamin B6 24.75mg,
vitamin B12 0.045mg, vitamin H 1.14mg, D-pantothenic acid 120mg,
nicotinic acid 225mg, vitamin C 300mg, folic acid 11.24mg, inositol
112.5mg, zinc 75mg, selenium 0.3mg, magnesium 86.25mg, copper
2.25mg, manganese 22.5mg, iodine 7.5mg, iron 3mg, cobalt 0.3mg.
g
Merck KGaA, Darmstadt, Germany.
2009). Thus, we first examined the time course of trypsin secretion
in P. argus by sampling the gastric fluid after ingestion. However,
this was possible only when fresh fish were used as food, because
the ingestion of dry pelleted diets resulted in dough with almost no
free gastric fluid. Specimens of P. argus (N8) were starved for
48h, fed with fresh fish (O. oglinum) and sampled every hour for
7h. Gastric fluid samples were obtained through the oral cavity using
disposable insulin syringes as described by Vonk (Vonk, 1960) but
instead of a fire-polished glass cannula we used a needle with a
plastic cannula over the sharp end. Gastric samples were
immediately frozen in liquid nitrogen and then stored at –80°C.
Lobsters were handled with care and samples were rapidly taken
(less than 1min) to avoid excessive stress.
As the hyperglycemic response to stress is well known in
crustaceans (Kleinholz and Little, 1949), in a preliminary trial we
tested the influence of this manipulation on glucose levels in P.
argus hemolymph (N5). Hemolymph was taken from the sinus at
the base of the third walking leg as described before (Perdomo et
al., 2007) and glucose was measured using a clinical RapiGlucoTest (Helfa Diagnostic, Havana, Cuba) according to the
manufacturer’s instructions. This preliminary experiment also
confirmed that trypsin activity in gastric fluid does not vary as a
result of manipulation.
In a second experiment, four groups of five lobsters each were
starved for 48h and fed with three diets named according to the
main protein source they contain (soybean, fish and squid). One
group was left without food and served as a control. After 4h of
ingestion, lobsters were chilled by immersion in ice-cold water and
dissected for digestive gland and gastric fluid extraction. In this
study, digestive gland samples were always taken from the medial
and superior part of the gland. Both samples for activity assays were
immediately frozen in liquid nitrogen and stored at –80°C.
Additional digestive gland samples for expression analysis were
rapidly transferred to RNAlater® Solution (Ambion, Life
Technologies Corporation, Austin, TX, USA), left to stand at 4°C
for 24h, then stored at –20°C.
In vitro effect of protein/hydrolysates on trypsin activity and
expression
For assessing whether digestion end-products or intact proteins can
stimulate trypsin synthesis and/or secretion, the effects of protein (diet)
hydrolysates and bovine serum albumin (BSA) on the digestive gland
were studied in vitro. The incubation medium was Panulirus saline
(PS) solution (Zhainazarov et al., 1997) composed of (in mmoll–1):
460 NaCl, 13 KCl, 14 Na2SO4, 13 CaCl2, 10 MgCl2, 2 glucose and
10 Hepes, but pH was adjusted to 6.6 instead to 7.4, according to the
slightly acidic pH in the digestive gland of crustaceans (Bickmeyer
et al., 2008). The solution was filtered using Whatman cellulose nitrate
membrane filters (0.45m pore size) and aerated at room temperature
(25°C) during the 30min before the experiment.
The hydrolysates were obtained as follows. The formulated diets
(soybean, fish and squid) were shaken in PS (0.5gml–1) for 15min
and then incubated with lobster gastric fluid (3:1) for 4h at room
temperature. The mixtures were then centrifuged at 1000g for 5min
to eliminate insoluble materials. The supernatants were boiled for
5min, in order to abolish enzyme activity and precipitate proteins,
and centrifuged again for 30min under similar conditions. Although
this treatment should leave only free amino acids and peptides in
the supernatants, we filtered the supernatants through Amicon filters
with a cut-off of 10,000Da. The resultant solutions were stored at
–20°C and are referred to as diet hydrolysates. The total amino acid
composition of diets and free amino acids in hydrolysates was
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Dietary proteins regulate trypsin
determined (Instituto de la Grasa analytical service, Sevilla, Spain).
Peptide composition of filtered hydrolysates was assessed by
Tricine-PAGE.
Digestive gland samples (30mg) were taken from 48h starved
lobsters (N5) as described above and washed in 1ml of PS to
remove hemolymph and digestive enzymes from the surface of the
tissue, and then transferred to 1.5ml Eppendorf tubes containing
200l of filtered and aerated PS. Then, 100l of the following
solutions were added to five samples (tubes) from each individual:
soybean hydrolysate, fish hydrolysate, squid hydrolysate, BSA and
PS. After 20min of incubation, digestive glands were transferred
to RNAlater® solution, left to stand at 4°C for 24h and finally
conserved at –20°C. The incubating medium was frozen in liquid
nitrogen and stored at –80°C for determination of the trypsin activity
released.
Trypsin activity assay
Digestive glands were homogenized in reaction buffer (200mmoll–1
Tris HCl pH7.5) and centrifuged at 1000g at 4°C for 30min. Crude
extracts of digestive glands or gastric fluid were diluted with reaction
buffer to measure initial rates of enzyme activity. Trypsin activity
was measured using 1.25mmoll–1 N-benzoyl-DL-arginine pnitroanilide (BApNA) in 200mmoll–1 Tris HCl pH7.5 as described
before (Perera et al., 2008a). One unit of trypsin activity was defined
as the amount of enzyme that catalyzed the release 1mol of pNA
per minute, using the appropriate molar extinction coefficient. Under
our assay conditions, the molar extinction coefficient of pNA was
calculated to be 2.563lmmolcm–1. Trypsin activity was expressed
per ml or per g of tissue as appropriate.
Determination of total soluble protein
Proteins were measured according to Bradford (Bradford, 1976)
using BSA as standard.
Trypsin expression by RT-qPCR
All kits in this study were used following the manufacturer’s
instructions. Total RNA was purified using the NucleoSpin® RNA
II kit (Macherey-Nagel, Düren, Germany) including RNAse-free
DNAse treatment. RNA was quantified by absorbance at 260nm
(A260) and its quality was assessed by its A260⁄280 value and using the
Agilent RNA 6000 Nano Assay Kit on an Agilent 2100 Bioanalyzer
(Agilent Technologies, Santa Clara, CA, USA). cDNAs were
synthesized using the qScriptTM cDNA Synthesis Kit (Quanta
BioSciences, Gaithersburg, MD, USA) and then used as templates
for RT-qPCR on a Mastercycler ep realplex (Eppendorf, Hamburg,
Germany) using PerfeCTaTM SYBR® Green Fast-MixTM (Quanta
BioSciences) in white wells twin.tec real-time PCR plates 96
(Eppendorf). Four previously isolated lobster trypsin cDNAs (Perera
et al., 2010a) were studied: PaTry1a (GenBank accession no.
GU338026), PaTry2 (GU338028), PaTry3 (GU338029) and PaTry4
855
(GU338030). Specific primers (Biomers.net, Ulm, Germany) used
for lobster trypsins were successfully employed before (Perera et al.,
2010a) and are shown in Table2. The efficiency of target amplification
for each primer set was optimized in the previous study by using
4pmoll–1 of primers and 60°C of annealing/extension (Perera et al.,
2010a); thus, these conditions were employed here. By means of
calibration curves (10-fold dilutions, corresponding to cDNA in
reactions from 20ng to 0.2pg) all primer pairs were checked to
produce similar efficiencies, except PaTry4. The reaction volume was
10l, containing 4l cDNA (4ng total cDNA), 5l of PerfeCTa
SYBR Green Fast Mix (2⫻) and 0.5l of each primer. Cycling
conditions were as follows: initial denaturation/activation step for
5min at 95°C, followed by 40 cycles of denaturation (95°C for 30s)
and annealing/extension (60°C for 45s). Control reactions with
DEPC water and RNA instead of cDNA were included to ensure the
absence of contamination or sample genomic DNA. Specificity was
checked by melting curve analysis. Additionally, PCR products were
verified by nucleotide sequencing. All samples were run in duplicate.
Replicate PCR reactions generated highly reproducible results. The
intra-assay coefficient of variation (CV) for Ct values varied from 0
to 0.004 and the inter-assay CV from 0.011 to 0.027.
Standard curves and absolute quantification
Four full-length lobster trypsin cDNAs were amplified as detailed
before (Perera et al., 2010a) and cloned into plasmids using the
TOPO TA Cloning® Kit (Invitrogen Ltd, Paisley, UK). Plasmids
were extracted from Transformed One Shot® TOP10 competent
Escherichia coli cells using the GenEluteTM Five-Minute Plasmid
Miniprep Kit (Sigma-Aldrich, St Louis, MO, USA). Clones
containing inserts of the expected size were identified by PCR
analysis (T3 and T7 primers of TOPO TA Cloning® Kit) followed
by agarose gel electrophoresis, and sequenced from both directions
to ensure their correspondence with previously reported lobster
trypsins (PaTry1 to PaTry4). Plasmids containing the trypsin
variants of interest were quantified using a spectrophotometer.
Absolute calibration curves were constructed using circular plasmid
vectors (pCR®4-TOPO®) containing each of the four full-length
lobster trypsin cDNAs, serially diluted with DEPC water from
3⫻107 to 30 copies. Targets and calibrators showed similar
amplification efficiencies near 100%, in all cases but PaTry4. The
number of transcript molecules was calculated from the linear
regression of the standard curve and analyses were done with the
Mastercycler ep realplex software. Results are expressed as means
+ s.e.m., after being normalized to ng of total RNA.
Pattern of evolution of trypsin enzymes: preliminary
assessment
The software MEGA5 was used for all tests performed (Tamura et
al., 2007). Pairwise comparisons between PaTry1 to PaTry3
nucleotide sequences were carried out to calculate the ratio of non-
Table 2. Primers used in this study for RT-qPCR
Name
PaTry1 F
PaTry2 F
PaTry3 F
All PaTrys R
PaTry4 R
All PaTrys F
Nucleotide sequence
Position*
Direction
5⬘-AACAAGATCGTTGGTGGTGA-3⬘
5⬘-CTGACGCCGAGCCTGGTA-3⬘
5⬘-GGACATCTCCTTCGGCTT-3⬘
5⬘-AGTGACCAGCACAGATAGC-3⬘
5⬘-GTGGATCCAGTGTTCGTCAT-3⬘
5⬘-CCGTGCCCATCGTGTCTGA-3⬘
96–115
115–132
159–176
220–238
689–706
551–569
Forward
Forward
Forward
Reverse
Reverse
Forward
*For primers used for several trypsin variants, numbers correspond to hybridization position on PaTry1. For the other variants, few nucleotide displacements
could occur. F, forward; R, reverse.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
E. Perera and others
synonymous to synonymous substitution (Ka/Ks) as an indicator of
the pattern of evolution (Hurst, 2002) of these enzymes. The best
model of nucleotide substitution was obtained in MEGA5 and
resembled that of Tamura (Tamura, 1992) because of the
transition–transversion and G+C content biases. Then, taking into
account the calculated transition–transversion bias (R1.21) and
applying the Jukes–Cantor correction for multiple substitutions at
the same site, we computed the Ka/Ks ratio for each mature trypsin
pair using the modified Nei and Gojobori method with 1000
bootstraps for variance estimation. Statistical significance of
selection was tested by the Z-test and Fisher’s exact test. A codonbased selection test using the likelihood method was used for
detecting positive selection sites.
Statistical analyses
0.9
Trypsin activity in gastric juice (U ml–1)
856
a
a
a
a
0.8
a,b
0.7
a,b
a,b
0.6
b
0.5
0.4
0
All data were checked for normality and homogeneity of variance
using Kolmogorov–Smirnov and Levene’s tests, respectively, with
P≤0.05. Data from the time course of trypsin activity after ingestion
and data from protein and activity comparison among diets,
hydrolysates and BSA were subjected to one-way ANOVA
(P≤0.05). Expression data were analyzed by two-way ANOVA
(P≤0.05), with diet and trypsin being the two sources of variation.
In all cases, N refers to the number of lobsters used and duplicate
measurements were performed. The Tukey test (P≤0.05) was used
to determine differences among means. The software package
Statistica 7.0 (StatSoft Inc., Tulsa, OK, USA) was used for all tests
performed and figures were generated by OriginPro 8 (OriginLab
Corporation, Northampton, MA, USA).
RESULTS
Trypsin secretion after ingestion
A preliminary trial testing the effects of gastric fluid sampling
procedure on stress level showed that mean glucose levels in
hemolymph did not vary as a result of manipulation (F0.32,
P>0.05, 0.2±0.027mmoll–1), and no variation in trypsin activity
occurred in the gastric fluid within the 7h of sampling in non-fed
lobsters (F1.18, P>0.05, 0.7±0.04Uml–1).
Trypsin activity in the gastric fluid of fasting lobsters was highly
dependent on the protein concentration in the fluid and this
relationship was better described by a straight line [trypsin
activity0.1525(protein concentration)+0.044] with a high
determination coefficient (R20.94). This indicates that 94% of
trypsin activity in the gastric fluid can be explained by proteins from
digestive gland secretion. Dietary proteins remaining in the gastric
chamber after 48h of fasting and non-trypsin enzymes in the gastric
fluid (Perera et al., 2008a) account for the variation observed. Thus,
the time course of trypsin activity in gastric fluid after ingestion
was followed as being indicative of trypsin secretion by the digestive
gland. Trypsin activity in the gastric fluid varied significantly
(F4.16, P≤0.01) after ingestion. Following feeding, trypsin activity
progressively decreased in gastric fluid, reaching significantly
lower levels after 3h (Fig.1). However, between the 3rd and the
4th hour after ingestion a significant enhancement of trypsin activity
was observed (Fig.1), returning rapidly to prefeeding levels.
Afterwards, trypsin activity remained stable until 7h post-feeding.
Effects of dietary protein on trypsin secretion
As trypsin secretion peaked 4h after ingestion, trypsin activity of
the gastric fluid and digestive gland at this time was compared in
P. argus feeding on diets of different protein composition. No
differences were observed in the trypsin activity of digestive glands
between lobsters fed with the different diets and fasting animals
0
1
2
3
4
5
6
Time after ingestion (h)
7
Fig.1. Time course of trypsin activity (means + s.e.m.) in gastric fluid of
Panulirus argus (N8) after ingestion of fresh fish. Differences were found
over time (P≤0.05). Letters above bars indicate statistical differences
according to the Tukey test (P≤0.05). Arrow indicates a pulse of trypsin
secretion from the digestive gland to the gastric fluid after meal ingestion.
(Fig.2A). However a significant reduction (F3.96, P≤0.05) in
protein content of the digestive gland was observed in individuals
ingesting fish and squid diets (Fig.2B). This reduction in soluble
protein in the digestive gland corresponds with the significantly
higher (F8.59, P≤0.05) levels of trypsin activity in the gastric fluid
of these lobsters (Fig.2C). The secretion elicited by fish and squid
diets is comparable to that found after the ingestion of fresh fish
(Fig.1), whereas lobsters ingesting the soybean-based diet showed
no change in trypsin activity in gastric fluid (Fig.2C).
Effect of dietary protein on trypsin expression
Expression was studied by means of RT-qPCR for four trypsin variants
(PaTry1 to PaTry4) previously reported in P. argus (Perera et al.,
2010a). For absolute quantification, standard curves were generated
with plasmids containing the different trypsin cDNAs (Fig.3) and
checked to produce similar amplification efficiencies (E) to their targets
PaTry1 (E0.99, R21.0, slope–3.357), PaTry2 (E0.97, R20.999,
slope–3.401) and PaTry3 (E1.02, R20.999, slope–3.284). PaTry4
was not included in the statistical analysis because there were so few
individuals expressing this transcript in most treatments and because
of the small difference in amplification efficiency (E0.92, R21.0,
slope–3.542) obtained. Significant differences in trypsin expression
were found among diets (F3.10, P≤0.05) and among trypsin variants
(F19.35, P≤0.001). PaTry3 was always found to be the most
abundant transcript (Fig.4). No variation was found in the expression
of PaTry1 and PaTry2 among feeding treatments, whereas the most
abundant variant (PaTry3) significantly increased its expression in
lobsters ingesting fish and squid diets (Fig.4).
Effects of diet hydrolysates and intact proteins on trypsin
secretion in vitro
Under our assay conditions, incubation of lobster digestive glands
with diet hydrolysates (<10kDa) did not produce significant changes
in trypsin secretion into the incubating media (Fig.5A). Digestive
glands incubated with squid diet hydrolysate seemed to have slightly
raised trypsin secretion, but a significant increase above controls could
only be detected for glands incubated with BSA (Fig.5A).
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Dietary proteins regulate trypsin
A
Ct=32.75–3.317 log10(PaTry1 plasmid copies)
Efficiency=0.98, R2=1.000
Ct=36.5–3.299 log10(PaTry3 plasmid copies)
1.4
1.0
Efficiency=0.94, R2=0.999
0.8
0.6
0.4
20
15
PaTry1 plasmid
PaTry2 plasmid
PaTry3 plasmid
PaTry4 plasmid
0.2
Fasting
Total soluble protein in digestive gland
(mg g–1)
Efficiency=1.01, R2=0.999
Ct=34.88–3.477 log10(PaTry4 plasmid copies)
25
1.2
0
120
B
Soybean
Fish
Squid
a
10
5
a
100
1
80
60
b
b
40
2
3
4
5
6
log10(copy number)
7
8
Fig.3. Standard curves for absolute quantification by RT-qPCR of four
trypsin variants in lobster. Data points are means of triplicate
measurements. Equations and amplification efficiencies are shown. Target
trypsins have similar amplification efficiencies to the standards (see Results
for details).
20
0
Fasting
0.9
Trypsin activity in gastric juice
(U ml–1)
Efficiency=1.00, R2=0.999
Ct=33.19–3.370 log10(PaTry2 plasmid copies)
30
1.6
Ct (cycles)
Trypsin activity in digestive gland
(U ml–1 g–1)
1.8
857
Soybean
C
Fish
a
0.8
Squid
a
0.7
0.6
b
Pattern of evolution of trypsin enzymes: preliminary
assessment
Soybean
The Ka/Ks ratio for PaTry1/PaTry2 and PaTry1/PaTry3 pairs was
0.70 and 0.66, respectively, while Ka/Ks for the PaTry2/PaTry3 pair
was 1.03. Both Z-test and Fisher’s exact test of selection gave no
statistical indication of positive selection. However, the maximum
likelihood approach revealed 25 codons evolving under positive
selection (dN–dS>0). From these sites, 72% contain unique
substitutions in PaTry3. Interestingly, 55% of these PaTry3
substitutions occur in important regions for enzyme function
(Table4).
b
0.5
0.4
0.3
0.2
0.1
0
Fasting
Glu+Gln content of soybean hydrolysate was lower and the content
of Arg and Lys was higher than in the other digests (Table3).
Electrophoresis analysis of hydrolysates showed strong differences
in peptide composition between the soybean and the fish/squid
digests (Fig.6).
Fish
Squid
Fig.2. Trypsin activity in the digestive gland (A), total soluble protein
content in the digestive gland (B) and trypsin activity in the gastric fluid (C)
of P. argus (N5) 4h after ingestion. Values are means + s.e.m. Soybean,
fish and squid refer to the major protein source in dry formulated diets.
Differences were found (P≤0.05) in the protein content of the digestive
gland and in trypsin activity in the gastric fluid among diets. Different letters
above the bars indicate statistical differences according to the Tukey test
(P≤0.05).
Effects of diet hydrolysates and intact proteins on trypsin
expression in vitro
PaTry3 was the only trypsin variant induced by dietary stimulation,
resembling the results obtained in vivo. Only squid diet hydrolysate
increased the copy number for PaTry3 transcripts (Fig.5B). Total
amino acid composition of the test diets was similar except for some
differences in Val and Lys content (Table3). Free amino acid
composition differed among diet hydrolysates, reflecting differences
in the digestion of animal vs vegetal protein sources. The Val and
DISCUSSION
Trypsin activity has been positively correlated with digestion
efficiency and growth in fishes (Rungruangsak-Torrissen et al., 2006;
Savoie et al., 2011) but conflicting results have been obtained in
crustacea (Le Vay et al., 1993). The effect of dietary protein on
trypsin activity has been reported in shrimp larvae (Le Moullac et
al., 1994), juveniles (Muhlia-Almazán et al., 2003) and adults (Le
Moullac et al., 1996) but in general, the effect of proteins on trypsin
enzymes has been poorly studied in crustacea in comparison to
vertebrates and other groups of invertebrates.
Effect of dietary proteins on trypsin secretion
Trypsin secretion has been reported in fasting mammals (Konturek
et al., 2003), shrimp (Lehnert and Johnson, 2002) and insects
(Moffatt et al., 1995) [except in batch digesters such as mosquitoes
(Lehane et al., 1995)]. In accordance, a significant amount of trypsin
activity was observed in fasting lobsters. After ingestion of fish,
trypsin activity in the gastric fluid of P. argus dropped gradually,
THE JOURNAL OF EXPERIMENTAL BIOLOGY
*
Trypsin mRNA (copies ng–1 total RNA)
1.8⫻105
1.6⫻105
*
PaTry1
PaTry2
PaTry3
PaTry4
1.4⫻105
1.2⫻105
1.0⫻105
8.0⫻104
6.0⫻104
4.0⫻104
2.0⫻104
0
Fasting
Soybean
Fish
Trypsin activity released from digestive gland
(U ml g–1)
E. Perera and others
1.2
A
a
1.1
1.0
a,b
0.9
0.8
b
0.7
Fig.4. Differential expression (means + s.e.m.) of four trypsin variants
(PaTry1 to PaTry4) in the digestive gland of P. argus (N5) 4h after
ingestion. Soybean, fish and squid refer to major protein sources in dry
formulated diets. Differences were found among diets (P≤0.05) and trypsin
transcripts (P≤0.001). PaTry3 was always significantly more abundant than
PaTry1 and PaTry2, and it is likely to be more abundant than PaTry4 (not
included in statistical analysis because of the small number of individuals
expressing this form of the enzyme). PaTry3 was the only trypsin
responding to the protein quality of the diet. Asterisks above bars indicate
statistical differences among diets for PaTry3, according to the Tukey test
(P≤0.05).
as reported before for the lobster J. edwardsii (Simon, 2009) and
assumed to be due to the drinking of water (Simon and Jeffs, 2008).
In the case of dry diets, food absorbed most of the gastric fluid.
This highlights a great problem with the use of dry diets in P. argus
as proteins must be dissolved before being attacked by proteolytic
enzymes. Recent studies have pointed out that the solubility of dry
diets would determine protein digestion efficiency in lobsters
(Simon, 2009; Perera et al., 2010b).
Previous studies have found different secretory patterns in the
digestive gland of crustaceans: (i) three phases (at 0–15min, 1–2h
and 3.5–5h after a meal) in the lobster Homarus gammarus (Barker
and Gibson, 1977), (ii) two peaks within 6h of feeding in the crayfish
Astacus leptodactylus (Hirsch and Jacobs, 1928) and the shrimp
Litopenaeus vannamei (Muhlia-Almazán and García-Carreño,
2002), and (iii) only one phase of secretion 1–4h after feeding in
the prawn P. semisulcatus (Al-Mohanna et al., 1985). In spiny
lobsters, a significant amount of trypsin enzymes from the digestive
gland have attained the gastric chamber after 4h of ingestion [J.
edwardsii (Simon, 2009); P. argus, this work].
In our study no statistical differences in trypsin activity of the
digestive gland were found in P. argus 4h after ingestion. Studies
on the effects of diet on digestive enzyme activity in crustacea have
yielded contradictory results and this has mainly been attributed to
the use of the digestive gland as the examined tissue. After
collection, glands are usually disrupted to obtain homogenates, in
which stored and secreted enzymes are mixed. In spite of this
limitation, it is interesting to note that the non-significant trend
observed in the digestive gland for trypsin activity indicates more
enzyme secretion in fish- and squid-fed lobsters than in fasting or
soybean-fed specimens. The high amount of trypsin enzymes
remaining in the gland after 4h of digestion could explain the lack
of significance in our results. In contrast, soluble protein content of
the gland significantly decreased after feeding lobsters with fish and
b
b
SB
hydrolysate
F
hydrolysate
0.6
0.5
0.4
0.3
Saline
Squid
Trypsin mRNA (copies ng–1 total RNA)
858
1.2⫻105
B
PaTry1
S
hydrolysate
BSA
a
PaTry2
PaTry3
1.0⫻105
PaTry4
b
b
8.0⫻104
b
b
6.0⫻104
4.0⫻104
2.0⫻104
0
Saline
SB
hydrolysate
F
hydrolysate
S
hydrolysate
BSA
Fig.5. Trypsin activity released (A) and expression (B) of four trypsin
variants (PaTry1 to PaTry4) in isolated digestive gland of P. argus (N5)
after 20min of exposure to diet hydrolysates or bovine serum albumin
(BSA). Values are means + s.e.m. SB (soybean), F (fish) and S (squid)
refer to major protein sources in dry formulated diets used for hydrolysate
preparation (see Materials and methods for details). Differences were found
in trypsin secretion (P≤0.05) and expression among treatments (P≤0.05).
PaTry3 was always significantly (P≤0.001) more abundant than PaTry1 and
PaTry2, and it is likely to be more abundant than PaTry4 (not included in
statistical analysis because of the small number of individuals expressing
this form of the enzyme). PaTry3 was the only trypsin responding to
treatments. Different letters above bars indicate statistical differences
among treatments for PaTry3 according to the Tukey test (P≤0.05).
squid diets (but not with soybean diet), perhaps as a result of
secretion of other enzymes in addition to trypsin. Consequently, by
examining the gastric fluid, we found that soybean-based diets lack
stimulatory capacity in the lobster digestive gland (or some
components of the soybean meal block signal transduction into
secreting cells), while fish and squid diets elicited a secretory
response similar to that observed with fresh fish. These results can
be taken as definite evidence that the nature of ingested protein
affects the released of trypsin enzymes from the digestive gland of
P. argus and that the use of fish and squid proteins in dry feeds is
not limited by the digestive response of lobsters, but probably by
diet solubility hampering digestibility as suggested before (Simon
and Jeffs, 2008; Simon, 2009; Perera et al., 2010b). Conversely, in
adult shrimp, Le Moullac and colleagues found that only casein
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Dietary proteins regulate trypsin
859
Table 3. Amino acid composition of experimental diets and corresponding hydrolysates
Diets (total amino acids, %)
Asp+Asn
Glu+Gln
Ser
His
Gly
Thr
Arg
Ala
Pro
Tyr
Val
Met
Cis
Ile
Trp
Leu
Phe
Lys
Hydrolysates (free amino acids, %)
Squid
Fish
Soybean
Squid
Fish
Soybean
10.24±0.50
1.18±0.14
6.49±0.05
2.86±0.05
10.89±0.48
5.85±0.05
8.46±0.03
9.16±0.05
1.80±0.28
4.21±0.12
2.80±0.18
2.78±1.05
0.97±0.07
4.91±0.13
0.49±0.04
10.45±0.15
5.81±0.05
10.64±0.07
9.97±0.34
1.51±0.36
6.47±0.05
2.82±0.11
12.22±1.03
5.63±0.15
8.53±0.15
9.44±0.28
2.14±0.20
3.90 ±0.22
2.94±0.20
2.20±0.57
0.95±0.05
4.75±0.24
0.35±0.02
10.02±0.30
5.78±0.06
10.39±0.27
12.72±0.43
1.24±0.20
7.56±0.05
2.92±0.19
9.37±0.57
4.92±0.12
8.95±0.21
7.34±0.09
3.28±0.62
4.14±0.01
6.02±0.98
1.89±0.21
1.25±0.05
4.57±0.06
0.43±0.00
9.71±0.14
6.00±0.12
7.69 ±0.23
ND
2.40±0.17
3.17±0.19
7.62±0.54
1.50±0.07
2.25±0.09
14.07±0.94
8.28±0.26
13.26±0.26
11.45±3.55
2.34±0.08
3.48±0.00
0.88±0.01
5.13±0.19
*
10.60±0.73
5.87±0.10
7.69±0.62
ND
1.48±0.05
3.69±0.03
7.43±0.22
1.27±0.08
1.03±0.07
13.86±0.65
7.97±0.41
14.05±0.27
8.22±1.77
2.06±0.22
3.71±0.18
0.71±0.12
5.52±0.11
2.54±0.08
12.68±0.65
6.05±0.00
7.73±0.58
0.51±0.05
0.90±0.02
2.52±0.03
6.23±0.09
1.38±0.07
0.85±0.02
19.43±0.22
6.72±0.07
11.82±1.02
6.75±0.10
0.76±0.23
4.45±0.20
0.86±0.04
5.38±0.08
2.21±0.05
11.32±0.11
7.60±0.12
10.32±0.19
Values (%) are means (±s.e.m.) of triplicate measurements. ND, not detected. *Not determined.
increased trypsin activity, while squid meal and fish soluble
concentrate had no effect (Le Moullac et al., 1996).
Effect of dietary proteins on trypsin expression
Our results indicate that trypsin enzymes in P. argus are also
regulated transcriptionally by dietary proteins, as reported before
for shrimp (Le Moullac et al., 1996; Muhlia-Almazán et al., 2003),
mosquito (Noriega et al., 1994) and rats (Lhoste et al., 1994; Hara
et al., 2000). Interestingly, the most abundant transcript in P. argus
(PaTry3) was the only one for which dietary up-regulation could
be demonstrated while all other trypsins appear to be expressed in
a constitutive fashion. To our knowledge, this is the first time that
different trypsin isoforms have been shown to differ in their
responsiveness to dietary proteins for a crustacean species.
Differences in trypsin isoform expression have been found in
Daphnia magna but in response to protease inhibitors in the diet
(Schwarzenberger et al., 2010). It is noteworthy that in the present
work, RNA extraction was carried out from tissue biopsies with no
regard for the different cell types contained in the digestive gland.
Trypsin synthesis in crustacean digestive gland has been
demonstrated only in the F cells (Lehnert and Johnson, 2002), while
expression values are given herein on a total RNA basis. Therefore,
our results probably underestimate actual trypsin expression in F
cells of P. argus.
Signals for trypsin secretion and expression
Early studies (Hirsch and Jacobs, 1928) revealed that digestive
enzymes in crustaceans are secreted upon feeding but the
mechanism was unknown. In dogs (Meyer and Kelly, 1976) and
humans (Thimister et al., 1996) amino acids and peptides are more
effective than intact proteins in stimulating pancreatic exocrine
secretion. However, trypsin secretion is more stimulated by intact
proteins than by hydrolyzed proteins or amino acids in rats (Green
and Miyasaka, 1983), fishes (Cahu et al., 2004) and insects
(Blakemore et al., 1995; Lehane et al., 1995). In order to study
Table 4. List of positive selected sites in important functional motifs
Fig.6. Tricina-PAGE showing peptide composition of fish (F), squid (S) and
soybean (SB) hydrolysates. Note the absence of detectable amounts of
peptides in the fish and squid hydrolysates, while soybean hydrolysate is
rich in peptides of less than 14kDa. M, markers.
Sites
Change PaTry1/2 to PaTry3
Location
52
54
95
105
107
190
195
206
207
246
W to F
S to F
N to D
A to T
V to P
D to N
G to S
V to E
P to T
N to S
Loop 37
Loop 37
Ca2+ binding site
Near the Ca2+ binding site
Near the Ca2+ binding site
Loop 3
Loop 3
Loop 1
Loop 1
Loop 2
Site numbers start at the first residue of lobster trypsinogens (Perera et al.,
2010a).
Loop 37 is used according to the nomenclature in crayfish trypsin (Fodor et
al., 2005).
Loops 1, 2 and 3 are according to vertebrate nomenclature (Perona and
Craik, 1995; Hedstrom, 1996).
THE JOURNAL OF EXPERIMENTAL BIOLOGY
860
E. Perera and others
the possible signals for trypsin induction/secretion in P. argus, we
performed in vitro assays in which digestive glands were exposed
to an intact protein (BSA) and diet hydrolysates (<10kDa). Our
results show that trypsin secretion did not change upon incubation
with hydrolysates, while 0.01% BSA significantly stimulated
trypsin secretion. Trypsin secretion in insect opaque zone
preparations occurs at concentrations of BSA as low as 0.0001%
(Blakemore et al., 1995). It seems that in P. argus the signals for
trypsin secretion are similar to those in rats, fishes and insects
(intact proteins) and this information has been not available for
crustaceans until now.
Postprandial enzyme secretion in mammals is mostly (70%)
stimulated by cholecystokinin (CCK) through neural pathways
(Konturek et al., 2003). Although the mechanism of action is not
as well understood as in mammals, CCK is also recognized as a
major regulator of trypsin secretion in fishes (Buddington and
Krogdahl, 2004). There is strong evidence for the presence of
CCK-like peptides in crustaceans (van Wormhoudt et al., 1989;
Favrel et al., 1991; Resch-Sedlmeier and Sedlmeier, 1999)
although their role in digestive enzyme secretion has not been
clarified. However, in our in vitro assays BSA was able to elicit
enzyme secretion by acting directly over the digestive gland
(prandial mechanism), suggesting that, as in insects (Lehane et
al., 1995; Noriega et al., 1996; Noriega and Wells, 1999; Lu et
al., 2006; Brandon et al., 2008; Graf et al., 1998), peripheral
signals (neural and endocrine) are not obligatory though may have
modulator roles.
We found that squid diet hydrolysate was able to induce
transcription of the major trypsin (PaTry3) after feeding while intact
proteins like BSA had no effect. This result indicates that induction
signals are different to those for secretion and are probably mediated
by the appearance of free amino acids in the gland. [Note the absence
of a significant amount of small peptides in squid and fish
hydrolysates (Fig.6).] This result suggests a stepwise regulation of
trypsin enzymes during digestion.
Lys, Arg and Met are considered to be the most limiting amino
acids for growth of crustaceans (Akiyama et al., 1992).
Digestibility of these amino acids (and His and Tyr) appears to
be high in the three tested diets (but the soybean diet was
supplemented with free Met; Table1). It is known that these amino
acids largely appear as free amino acids in casein (Savoie, 1994)
and soybean (Henn and Netto, 1998) hydrolysates. No major
disparity was found in the amino acid composition of the squid
and fish hydrolysates. Thus, the difference in their capacity for
stimulating trypsin expression could result from differences in
amino acid concentration. The release of free amino acids from
squid meal far exceeds that from fish meal during early digestion
(Perera et al., 2010b). Previous studies have also suggested a
specific effect of squid proteins on digestive enzymes (Le Moullac
et al., 1996; Perera et al., 2005).
As the current results suggest that free amino acid can increase
trypsin expression in the digestive gland of P. argus (and potentially
protein digestion efficiency), further studies are needed on the
selection of rapidly digestible protein sources. However, we argue
for the inclusion of protein hydrolysates to counteract the poor
digestibility of dietary proteins. The effect of protein hydrolysates
other than in terms of the attractiveness of diets is not yet well
understood.
Preliminary insight into the evolution of trypsins in P. argus
The very marked differences in expression among trypsins in P.
argus encouraged us to perform a preliminary analysis on the pattern
of evolution for three P. argus trypsins (PaTry1 to PaTry3).
Analysis of Ka/Ks for PaTry1/PaTry2 and PaTry1/PaTry3 pairs
suggests purified selection (Ka/Ks<1) although values are unusually
high, whereas neutral selection is suggested by Ka/Ks for
PaTry2/PaTry3 (Ka/Ks≈1). However, because averaging Ka/Ks for
all sites ignores the type of selective pressure that applies to
individual amino acids, we used the maximum likelihood approach
for detecting positively evolving sites in a background of
purifying/neutral selection.
Our results indicate that most sites of P. argus trypsins are under
purifying selection and thus that these proteins are subject to
functional constraints. In addition, most non-synonymous changes
resulted in the substitution of similar amino acids, with a slight
change in charge or hydrophobicity while conserving the volume
of residues. However, it is noteworthy that several positively
evolving sites are within (or in close vicinity to) functionally relevant
motifs (Table4). Loop 37 in crayfish trypsin is known to be
important in hydrophobic interactions with extended subsites of
inhibitors (Fodor et al., 2005) and probably proteinaceous substrates.
In P. argus trypsins, Trp and Ser residues of PaTry1 and PaTry2
loop 37 are both replaced by the more hydrophobic Phe in PaTry3.
In the calcium binding site, Asn residues in PaTry1 and PaTry2 are
replaced by the more charged Asp in PaTry3. Two, one and two
positive selected sites occur within loops 1, 2 and 3, respectively.
These three loops determine trypsin specificity (Hedstrom, 1996).
It is not possible to anticipate the impact of these substitutions on
enzyme activity but one can hypothesize that highly expressed
PaTry3 has distinct functional roles. Although high levels of
expression have been correlated with lower rates of protein evolution
(Drummond et al., 2005), other studies have provided some evidence
for recent adaptive evolution of protein-coding regions in highly
expressed genes (Holloway et al., 2007). Studies on the gene
sequence of lobster trypsins would provide information on regulatory
elements (e.g. cis-acting elements in 5⬘ regions) to support our
findings.
Trypsin isoform sequences from other spiny lobster species are
also required to truly determine whether some trypsins are under
selective pressure at certain sites to undergo adaptive evolution
(or are evolving independently) while other trypsins are evolving
in a concerted fashion, as occurs in Drosophila (Wang et al.,
1999).
ACKNOWLEDGEMENTS
The authors express their gratitude to the crew of the research vessel ʻFelipe
Poeyʼ for their assistance during animal collection. We thank Y. S. Wunderink for
useful comments on sequence evolution mechanisms and E. García for
comments on an earlier version of the manuscript. Comments from reviewers
significantly improved this work. We specially thank the Editor for his support.
FUNDING
This work was supported by grants from the International Foundation for Science
[grant no. A/4306-1] and Agencia Española de Cooperación Internacional/
Asociación Universitaria Iberoamericana de Postgrado (AUIP/AECI). E.P. is a
PhD fellow of AUIP at the University of Cadiz, Spain, within the Program
ʻDoctorado Iberoamericano en Cienciasʼ, whose support is highly appreciated.
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