Aquaculture 541 (2021) 736835

Contents lists available at ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Nutritional contribution of seaweed Ulva lactuca single-cell detritus and

microalgae Chaetoceros calcitrans to the growth of the Pacific oyster
Crassostrea gigas
Alexia Omont a, Clara Py a, b, Julián Gamboa-Delgado c, Héctor Nolasco-Soria a,
Milton Spanopoulos-Zarco d, Alberto Peña-Rodríguez e, *
a
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Instituto Politécnico Nacional 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23096, Mexico
b
Université de Bordeaux, 2 rue du Professeur Jolyet, 33120 Arcachon, France
c
Universidad Autónoma de Nuevo León (UANL), Facultad de Ciencias Biológicas, San Nicolás de los Garza, N.L. 66455, Mexico
d
Universidad Autónoma de Baja California Sur (UABCS), Carr. Transpeninsular al sur Km 5.5, Col. Mezquitito, La Paz, B.C.S. 23080, Mexico
e
CONACYT – Centro de Investigaciones Biológicas del Noroeste, S.C., Instituto Politécnico Nacional 195, La Paz, B.C.S. 23096, Mexico

A R T I C L E I N F O A B S T R A C T

Keywords: The Pacific oyster Crassostrea gigas (Thunberg, 1793) is the most cultivated bivalve in the world. Nonetheless, the
Seaweed detritus massive production of microalgae as feed represents a substantial cost during laboratory production stages. The
Oyster growth use of single cell detritus (SCD) from seaweed Ulva lactuca, cultivated in fish farm effluents, has been proposed as
Microalgae substitution
an alternative to microalgae Chaetoceros calcitrans in oyster culture, with the aim of reducing microalgae pro­
Feed contribution
duction costs. Seaweed meal was subjected to an acid and enzymatic digestion process to obtain SCD particles
smaller than 20 μm to feed the oysters. Five levels of SCD (w:w) replacing microalgae were evaluated: 0, 25, 50,
75, and 100% in a feeding assay of 5 weeks. At the end of the experiment, growth parameters, condition index,
enzymatic activity in the digestive gland (amylase, protease, lipase, and aminopeptidase) were analysed. In
addition, during the course of the experiment, the stable isotope ratio of nitrogen (δ15N) was analysed at natural
abundance levels in both feed sources and in the mantle tissue of oysters reared on different feeding regimes.
Contribution to growth was estimated using an isotope mixing model. Better growth (74 to 94% dry weight gain)
and condition indexes (81–75) were observed in oysters fed on experimental regimes having from 0 to 50%
substitution of microalgae with SCD, showing no significant differences among them. Oysters under the latter
treatments also showed similar enzymatic activity for amylase, alkaline-protease, and lipase. At higher substi­
tution levels of microalgae (75–100%), oysters presented lower growth (13 to 34% dry weight gain) and poor
condition indexes (<60); 100% of SCD also elicited higher amylase, alkaline-protease, and lipase activities,
whereas (Leu) aminopeptidase N activity was lower. The digestive capacity of lipase was improved in oysters fed
with 50 and 75% levels of SCD. Isotopic equilibrium for nitrogen (δ15N) was reached by day 14 in the 50%
substitution treatment. Metabolic turnover rates of nitrogen decreased (0.15 to 0.08 day− 1) whereas elemental
half times in tissue increased (4.1 to 8.2 days) with higher microalgae substitution. Oysters under treatment with
50% microalgae substituted by SCD incorporated similar amounts of dietary nitrogen and dry matter from the
microalgal biomass as from SCD to meet the nitrogen requirement for oyster growth. In conclusion, results
suggest that SCD from U. lactuca can substitute up to 50% of microalgae C. calcitrans without modifying C. gigas
productivity.

1. Introduction million tonnes with an average increase of 3.3% each year and valued at
6.8 billion USD (FAO, 2019). In intensive oyster aquaculture, massive
Oysters Crassostrea sp. represent almost 25% of the total marine production of microalgae is essential for their use as feed during
aquaculture production; in 2017, production reached more than 5.7 broodstock conditioning and larvae and juvenile rearing (Ponis et al.,

* Corresponding author.
E-mail address: apena@cibnor.mx (A. Peña-Rodríguez).

https://doi.org/10.1016/j.aquaculture.2021.736835
Received 9 March 2021; Received in revised form 22 April 2021; Accepted 25 April 2021
Available online 28 April 2021
0044-8486/© 2021 Elsevier B.V. All rights reserved.
A. Omont et al.
2003; Uchida and Murata, 2002). Nonetheless, it has been identified also
as highly technical, labour intensive and as the most restrictive,
expensive, and unpredictable step of oyster farming (Carboni et al.,
2016; Coutteau and Sorgeloos, 1992; Tanyaros and Chuseingjaw, 2014).
Therefore, live microalgae substitutes have been explored to find the
best alternatives to reduce operating costs and improve the efficiency of
bivalve seed production (Ponis et al., 2003; Rivero-Rodríguez et al.,
2007; Tanyaros and Chuseingjaw, 2014). During the past decade, most
microalgae substitution studies focused on the use of microalgal con­
centrates also called algal paste (Carboni et al., 2016; Knuckey et al.,
2006; McCausland et al., 1999; Southgate et al., 2016), dried microalgae
(Yang et al., 2017), yeast (Tanyaros et al., 2016a; Yang et al., 2017),
bacteria (Yang et al., 2017) or microencapsulated feed (Badillo-Salas
et al., 2009; Knauer and Southgate, 1999; Wang et al., 2016), which
have met with limited success either due to cost of production, their
physical properties or their nutritional content (McCausland et al., 1999;
Schiener et al., 2016). But, other economic alternatives have been
studied such as shrimp waste concentrates (Tanyaros et al., 2016b),
dried seaweed (Cardoso et al., 2019; Rato et al., 2018), or seaweed
detritus (Carboni et al., 2016; Peña Rodríguez et al., 2020; Tanyaros and
Chuseingjaw, 2014).
The culture of seaweed is a growing industry worldwide (Makkar
et al., 2016). In 2017, seaweed production reached 31.8 million tonnes
with an estimate value of 11.8 billion USD (FAO, 2019). Their expanding
popularity for human and animal nutrition is a result of their high
nutritional value due to favourable levels of amino acids, fatty acids,
minerals, vitamins, carotenoid pigments, and bioactive compounds (Qiu
et al., 2018). Another conspicuous advantage is the low nutrient input
required for seaweed culture, since they are primary producers (Neori
et al., 2004; Ortiz et al., 2006). Due to their adequate size (Carboni et al.,
2016), single-cell detritus (SCD) from seaweed has been proposed as
alternative feed for bivalve molluscs (Uchida, 1996), the relatively low
production cost and less required labour pave a way to add value to
underdeveloped marine resources (Uchida and Numaguchi, 1998). SCD
from seaweed has been obtained through a multitude of methods
involving chemical and enzymatic digestion, pH manipulation, and
bacterial fermentation (Carboni et al., 2016). SCD has shown to be a
promising feeding item that can yield superior sources of proteins and
oils to be used in formulated feeds (Harel et al., 2007). Species of the
genus Ulva (Chlorophyta) dominate eutrophic environments and are
largely responsible for “green tides” in many regions of the world
(Castelar et al., 2014). Its high nutrient uptake capacity, wide distribu­
tion and easy culture management make them good candidates for waste
water bioremediation in aquaculture (Macchiavello and Bulboa, 2014).
Thus, the abundant biomass of Ulva, genus with fast growing species
(Castelar et al., 2014; Makkar et al., 2016), including Ulva lactuca, from
either natural productivity or aquaculture origin, have been widely
studied to explore valuable utilizations (Dominguez and Loret, 2019;
Macchiavello and Bulboa, 2014; Neori et al., 2004). Detrital macroalgae
such as U. lactuca have been identified to contribute partially to C. gigas
growth in estuarine sites (Marín Leal et al., 2008; Riera and Richard,
1996) and their derived SCD has been suggested for evaluation as partial
substitute of microalgae in C. gigas feeding (Peña Rodríguez et al., 2020).
However, the ingestion of other feed materials by oysters is not
necessarily used as nutrient for somatic growth (Liu et al., 2016). To
understand the contribution of feed sources to growth in individuals,
changes in carbon and nitrogen stable isotope ratios have been used as a
non-hazardous and non-invasive technique (Mazumder et al., 2016).
The determination of isotopic values in feeding sources and animal
consumers can provide important information about assimilation effi­
ciency, nutrients uptake, and turnover rate of dietary components
(Gamboa-Delgado and Le Vay, 2009). Therefore, the present study
engaged in using natural nitrogen stable isotope values and digestive
enzymatic activities to determine the optimal percentage of microalgae
substitution with U. lactuca SCD in C. gigas oyster spat to improve oyster
aquaculture.
2
Aquaculture 541 (2021) 736835
2. Material and methods
2.1. Seaweed and single cell detritus production
Green seaweed U. lactuca was produced in an integrated culture
system with longfin yellowtail Seriola rivoliana conducted at CIBNOR (La
Paz, BCS, Mexico). The culture system consisted in a closed recirculation
system between two tanks of 5000-L capacity, one tank for fish and the
other with seaweed for water bioremediation. After 2 weeks of poly­
culture, the seaweed biomass was collected, washed with fresh water
and oven-dried at 50 ◦ C during 24 h. Dried algae were blended, sieved
through a 350-μm mesh and preserved under dry and dark conditions at
4 ◦ C.
Single cell detritus (SCD) was produced as described by Pérez
Camacho et al. (2004), with some modifications. Briefly, 280 g of dried
powder of U. lactuca were rehydrated in 7-L distilled water (1:25). First,
HCl acid treatment (pH 1.5) proceeded for 4 h at 40 ◦ C under agitation.
Then the pH was adjusted to 5 with NaOH, and cellulase (Celluzyme®,
ENMEX S.A. de C.V., Mexico, containing carboxymethyl cellulose ac­
tivity of 90,000 U ⋅ g− 1, and β-glucanase activity of 1500 U ⋅ g− 1) was
added at 5% w/w of seaweed. Enzymatic digestion was allowed under
continuous agitation (120 rpm) for 24 h at 55 ◦ C. SCD produced was
filtered through a 20-μm mesh and maintained at 4 ◦ C for the week.
Every week, SCD production was renewed to preserve feed quality and
avoid bacterial contamination.
Every week, 100 mL of filtrate was oven-dried for 24 h at 50 ◦ C to
obtain dry weight and yield. Its degradation was assessed by daily cell
count using a Malassez cell under an optical microscope, which also
allowed observing potential contamination by bacteria. In addition,
filtered SCD was dried and coated with gold by ionization at 40 mA for
35 s (Desk.11, Denton Vacuum Inc., Moorestown, NJ, USA), and elec­
tronic microscopy photography (Hitachi s-3000 N High Technologies,
Japan) was taken at 5000 magnifications to estimate the mean size of
SCD particles. Electronic microscopy picture was analysed with the
ImageJ® software; scale was adjusted with “Analyse/Set Scale” com­
mand, according to the microscopic photography scale. Then image was
modified as following: “Image/Type/8-bit”, “Image/Adjust/Bright­
ness&Contrast” with Auto parameter, “Adjust/Threshold” with the pa­
rameters default, B&W and dark background, “Process/Binary/
FillHoles”. Finally, particle size was determined by the command
“Analyse/Analyse Particles/” and SCD area values were exported into
Excel file and the particle diameter was estimated.
2.2. Experimental trial
Oysters C. gigas were kindly donated by the Marimex del Pacifico
farm (La Paz, BCS, Mexico), raised in 1000-L tank and fed daily with the
microalgae Chaetoceros calcitrans (75 ± 2.4 mg ⋅ L− 1) for one week under
laboratory conditions prior to the experimental trial in the aquaculture
nutrition laboratory of CIBNOR. The microalgae C. calcitrans was pro­
vided by the laboratory of phytoplankton at CIBNOR. Raw seaweed
material, SCD and microalga were analysed for total lipids (Barnes and
Blackstock, 1973) and total proteins (Ebeling, 1968), using N-protein
factor of 5.65 for the seaweed and SCD as recommended by Shuuluka
et al. (2013) and factor 4.78 for C. calcitrans as suggested by Lourenço
et al. (2004).
The experiment was conducted over a 5-week period. Feeding re­
gimes (treatments) consisted in dry weight substitutions of microalgae
C. calcitrans at 0, 25, 50, 75 and 100% with U. lactuca SCD (respectively,
D0, D25, D50, D75, D100). A total of 200 oysters, of an average live
weight (LW) of 3.4 ± 0.3 g (avg. dry weight of 0.09 ± 0.01 g) and an
average length (L) of 3.8 ± 0.4 cm, were randomly distributed in groups
of 10 organisms and placed in plastic mesh bags in 70-L tanks (4 bags per
treatment). Each tank was filled with marine water filtered through a 1-
μm mesh and sterilized through UV light and provided with continuous
aeration (≥ 4.0 mg ⋅ L− 1 of dissolved oxygen), temperature (21.2 ±
A. Omont et al. Aquaculture 541 (2021) 736835

0.7 ◦ C), and pH control (7.6 ± 0.6). During the feeding trial, 80% water
exchange was performed every day.
At the end of the feeding trial, live oysters were weighed and
measured to determine final live weight (FLW) and final shell length
(FL). Then, oysters were dissected and separated from the shell and dried
at 90 ◦ C for 4 h to obtain the final dry weight (FDW). Dry weight gain (D-
WG) and specific growth rate (SGR) were calculated as described in the
footnote of Table 1. Empty shells were also weighed after removing
excessive water and mucus with absorbent paper to obtain the condition
index. Initial condition index (82.9 ± 16.1) and final condition index
(CI) (equation in Table 1) were calculated according to Hawkins and
Rowell (1987) with the internal shell cavity’s capacity derived from the
gravimetric techniques of Lawrence and Scott (1982) as suggested
standard method for the evaluation of bivalve condition (Crosby and
Gale, 1990).
reaction mixture, the absorbance was measured at 540 nm. A lipase unit
has been defined as the enzyme amount required to hydrolyse 1 μmol of
β-naphthyl caprylate per minute, using a β-naphthol standard curve.
Alkaline protease activity was modified from Kunitz (1947), using
casein as substrate, at pH 7.5, and 60 min incubation at 25 ◦ C. The
absorbance was measured at 280 nm. A protease unit has been defined
as the enzyme amount required to release 1 μmol of tyrosine per minute,
using a tyrosine MEC of 1290.
(Leu) Aminopeptidase N activity was determined according to Mar­
oux et al. (1973), using L-leucine-p-nitroanilide as substrate, at pH 7.5,
25 ◦ C and 10 min incubation. The absorbance was measured at 405 nm.
An aminopeptidase unit has been defined as the enzyme amount
required to hydrolyse 1 μmol of L-leucine-p-nitroanilide per minute,
using a p-nitroaniline MEC of 9500.

2.4. Stable isotope analysis

2.3. Digestive enzymatic activity

For the isotopic determinations, three oysters from every tank were
At the end of the experiment, 3 oysters per treatment were collected
randomly sampled before feeding on experimental days 0, 7, 14, 21, 28,
and dissected to isolate the digestive gland. Digestive glands were
and 35. Animals were dissected to isolate the mantle tissue. Samples of
weighed and stored at − 80 ◦ C for further enzymatic activity analyses for
macroalgae U. lactuca, U. lactuca SCD, microalgae C. calcitrans, and
lipase, amylase, alkaline protease, and (Leu) aminopeptidase N.
oyster mantle tissue were desiccated (60 ◦ C until constant weight) and
Digestive glands were placed in a vial; then, 10 volumes (w:v) of
ground manually in a mortar to obtain a fine powder (250 μm).
distilled water (4 ◦ C) were added, and the mixture homogenized in a
Individual samples from 900 to 1100 μg were packed into tin cups
fastprep-24 (MP Biomedicals, Sta. Ana, CA, USA) for 2 cycles of 30 s at
(D1008; Elemental Microanalysis Ltd., Okehampton, UK). Elemental
30,000 g. Then, samples were centrifuged at 15,000 g for 15 min at 5 ◦ C.
nitrogen content and isotopic analyses were conducted as described in
The supernatant was recovered to measure the digestive enzymatic ac­
Gamboa-Delgado and Le Vay (2009), using a PDZ Europa Scientific
tivities. During handling, samples were kept in ice to prevent enzyme
Roboprep elemental analyzer coupled to a PDZ Europa Hydra 20/20
degradation.
stable isotope ratio mass spectrometer (Crewe, UK). At natural abun­
Total protein content was performed on each sample according to
dance levels, isotopic results are expressed in delta notation (δ), which is
Bradford (1976), at 595 nm absorbance, using bovine albumin as stan­
defined as per mil (‰) deviations from the δ15N values of the standard
dard. Specific activities and digestive capacities of all enzymes were
reference material (atmospheric nitrogen). Repeated measurements of a
expressed in units per milligram of protein (U ⋅ mg− 1 protein) and units
calibration standard indicated that instrument’s precision (SD) was
per gram of digestive gland (U ⋅ g− 1 digestive gland, DG), respectively.
0.08‰. Relative proportions of dietary nitrogen contributing to oyster
All measurements were performed for each sample in quadruplicate.
growth and originating either from U. lactuca SCD or microalgae were
Amylase activity was determined according to Vega-Villasante et al.
estimated using a 2-source, 1-isotope mixing model (Phillips and Gregg,
(1993), using starch as substrate, at pH 7.5 and with 5 min incubation at
2001). Estimation of isotopic discrimination factors (Isotopic difference
25 ◦ C. After dinitrosalicylic acid (DNS) treatment, the absorbance was
between the consumer and the diet) increases the output accuracy of the
measured at 550 nm. An amylase unit has been defined as the enzyme
mixing models and, in the current study, control values were taken from
amount required to release 1 μmol of glucose equivalent per minute,
the isotopic differences between oysters fed only with microalgae (D0)
using a glucose standard curve.
and only U. lactuca SCD (D100) after isotopic equilibrium was reached.
Lipase activity was determined following the method described by
Estimated relative dietary nitrogen incorporation values from both
Nolasco-Soria et al. (2018) using β-naphthyl caprylate as substrate, at
sources are expressed as means and their truncated 95% confidence
pH 7.5, and incubation time of 10 min at 25 ◦ C. After clarification of the
intervals. To obtain an estimate of the relative dry matter contribution to
growth from U. lactuca SCD and microalgae, dietary nitrogen contri­
Table 1
butions were corrected for differing elemental concentrations (N) using
Zootechnical parameters of the Pacific oyster C. gigas after 5-weeks of feeding on
the equation proposed by Fry (2006). Isotopic changes throughout the
regimes having different proportions of U. lactuca SCD and C. calcitrans.
feeding trial were monitored and values were introduced in an expo­
Diet FLW (g) FDW (g) D-WG (%) SGR (% CI nential model (Hesslein et al., 1993) to obtain the magnitude of the
day− 1)
isotopic rate of change in relation to growth (k) and the metabolic
D0 5.7a ± 0.6 0.17a ± 94.a ± 21.9 1.9a ± 0.3 81.3a ± turnover rate (m). In the Eq. (1), Cn represents the isotopic value of the
0.02 5.5
consuming animal at equilibrium with the new feed, C0 the isotopic
D25 5.6a ± 0.6 0.17a ± 89.3a ± 1.8a ± 0.3 78.9a ±
0.02 19.2 6.4 value at the beginning of the test and C the isotopic value at time t. By
D50 5.9a ± 0.6 0.16a ± 79.4a ± 1.7a ± 0.3 75.0a ± integrating the parameters k and m in EQ. (2) (MacAvoy et al., 2005),
0.01 17.2 5.2 the residency halftime (t50) was estimated, which is the time necessary
D75 5.2bc ± 0.12b ± 34.2b ± 9.6 0.8b ± 0.2 59.8b ± for half of the body tissue to resemble the isotopic signature of the new
0.4 0.01 9.7
D100 4.8c ± 0.4 0.10 ± 0.01 13.4c ± 8.5 0.3c ± 0.2 50.9b ±
diet.
7.9 Hesslein et al. (1993):
Values are given as mean ± SD (n = 10). Different superscripts in the column C = Cn + (C0 − Cn )e− (k+m)t.
(1)
indicate a significant difference determined by Tukey’s test (P < 0.05). FLW,
final live weight; FDW, final dry weight; D-WG, dry weight gain; SGR, specific MacAvoy et al. (2005):
growth rate; CI, condition index. ln2
D-WG (%) = [FLW (g) – ILW (g)]/[ILW (g)] × 100 t50 = (2)
m+k
SGR (% ⋅ day− 1) = [ln FDW – ln IDW]/days of experiment × 100
CI = [FDW (g)/(FLW(g) - Shell weight (g)) × 1000

3
A. Omont et al. Aquaculture 541 (2021) 736835

2.5. Data analysis

Statistical analyses were performed using the R software version

3.3.0. All data were analysed for normal distribution with the Shapiro-
test and for homoscedasticity with the Bartlett-test and transformed if
necessary. Data were subjected to a one-way ANOVA, followed, if
applicable, by a Tukey’s multiple comparison test (95% confidence).
The proportions of nutrients (dietary nitrogen) established in the diets
(expected frequencies) were compared with the proportions of nutrients
estimated in mantle tissue (observed frequencies) by means of Chi-
square goodness of fit tests. Parameter m in Eq. (1) was estimated by
means of iterative non-linear regression.

3. Results
Fig. 2. U. lactuca SCD estimated particle size distribution.
3.1. Feed composition and detritus microscopy
average daily increase in live weight ranged between 51.4 and 40.0 mg ⋅
After filtration, each SCD production batch yielded from 60 to 69%,
day− 1, and from 0.8 and 0.3 mg ⋅ day− 1 on a dry weight basis, the
an average dry weight of 324 ± 18 mg ⋅ mL− 1, and average cell count of
average daily length increase ranged between 0.086 and 0.057 mm ⋅
3.5 ± 0.5 × 107 cells ⋅ mL− 1, which remained stable during the 7 days of
day− 1.
cold storage. Optical microscopy confirmed the integrity of SCD until
day 7. Electronic microscopy showed the effect of the detritus formation
3.3. Digestive enzymatic activity
process on U. lactuca tissue (Fig. 1). U. lactuca powder presented an
array of non-uniform particles ranging in size from 100 to 250 μm
The protein content of the digestive gland of oysters fed on diets
whereas the SCD contained 87.9% of its particles with a size between 1
containing different SCD proportions ranged from 1.4 to 2.8 mg ⋅ mL− 1,
and 10 μm (Fig. 2).
whereas for the control treatment the protein content was 2.1 ± 0.3 mg ⋅
The protein and lipid contents of U. lactuca powder were 21.2 ± 0.2%
mL− 1, these values were not significantly different (P > 0.34). Similarly,
and 0.97 ± 0.03%, respectively, whereas SCD contained 25.6 ± 0.5%
no significant differences (P > 0.28) were observed in the specific ac­
protein and 8.8 ± 0.8% lipids. Finally, the microalga C. calcitrans
tivities and digestive capacity of alkaline protease, (Leu) aminopepti­
depicted 19.3 ± 0.3% protein and 15.9 ± 0.5% lipids.
dase N, and amylase in oysters fed with regimes D25, D50, and D75
compared to the control treatment (Fig. 3). Moreover, D50 and D75
3.2. Oyster productivity induced an increment in digestive capacity of lipase in oyster’s digestive
gland (3.1–3.4 U ⋅ g− 1 DG) compared to the control (1.5 ± 0.2 U ⋅ g− 1
Oyster survival was 100% and final length ranged between 4.0 and DG). For the regime D100, the specific enzymatic activities of alkaline
4.6 cm without significant differences among treatments (P > 0.58). The protease, (Leu) aminopeptidase N, and lipase were significantly higher
final live weight (FLW), final dry weight (FDW), dry-weight gain (D- (0.15 ± 0.02, 0.68 ± 0.02, 0.13 ± 0.04 U ⋅ mg− 1 protein, respectively)
WG), specific growth rate (SGR), and condition index (CI) did not pre­ and significantly lower for amylase (0.85 ± 0.25 U ⋅ mg− 1 protein) than
sent significant differences (P > 0.05) in treatments receiving 25% and in the control treatment (0.09 ± 0.01, 0.46 ± 0.04, 0.07 ± 0.02, 6.05 ±
50% of U. lactuca SCD (D25 and D50) and compared to the control (D0) 1.81 U ⋅ mg− 1 protein, respectively) (P < 0.05). Finally, the digestive
(Table 1). Among these treatments, the average daily increase of live capacity of amylase was reduced significantly (P < 0.01) in oysters fed
weight for D0, D25, and D50 ranged between 62.8 and 71.4 mg ⋅ day− 1, with D100 (11.0 ± 2.0 U ⋅ g− 1 DG) compared to the control (136.1 ±
the average daily increase of dry weight remained between 2.0 and 2.3 24.1 U ⋅ g− 1 DG).
mg ⋅ day− 1, and the average daily increase of length ranged between
0.17 and 0.23 mm ⋅ day− 1. Oysters fed on regimes D75 and D100 pre­
sented a significant reduction in all zootechnical parameters when
compared to those obtained in the control treatment (P < 0.006). The

Fig. 1. Electronic microscopy photography (Hitachi s-3000NHigh Technologies, Japan) at 5000 magnification of Ulva lactuca (A) and U. lactuca SCD (B).

4
A. Omont et al. Aquaculture 541 (2021) 736835

Fig. 3. Enzymatic digestive specific activity (left) and digestive capacity (right) of protease, aminopeptidase, lipase, and amylase in the Pacific oyster C. gigas
digestive gland after 5-weeks fed different proportions of U. lactuca SCD and C. calcitrans. Different superscripts on the columns indicate significant differences as
determined by Tukey’s test (P < 0.05) (n = 3).

3.4. Contribution of nutrients to growth and elemental residence time in
the oyster mantle
U. lactuca SCD and C. calcitrans microalgae showed distant δ15N
values (9.31 ± 0.24 and 3.18 ± 0.19‰, respectively). Both feed sources
had a rapid influence on δ15N values in oysters under all treatments
(Fig. 4). Throughout the experiment, the isotopic values of the oyster
mantle were distributed between the isotopic values of the SCD of
U. lactuca and of the microalga C. calcitrans according to the level of
inclusion of both in the feeding regimes; that is, the higher the inclusion
of seaweed detritus, the closer the isotopic value of oysters was to the
isotopic value of the macroalgal detritus (Fig. 4). Oysters fed on diets D0,
D25, and D100 reached isotopic equilibrium on day 21, whereas those
on the D50 feeding regime reached it earlier (14 days). Animals on the
D75 feeding regime reached isotopic equilibrium later (28 days).
The estimated rates of nitrogen metabolic renewal ranged from

5
A. Omont et al. Aquaculture 541 (2021) 736835

Table 3
Estimated relative proportions of total dry matter and dietary nitrogen supplied
from U. lactuca SCD and C. calcitrans, and contributing to the growth of oyster
C. gigas as indicated by a 2-source,1-isotope mixing model (mean ± CI, n = 5).
Diet Expected Observed in oyster mantle

Min Mean Max

Nitrogen
D25
Microalgae 75 a 79.1 95.8 b 100.0
SCD 25 0.0 4.2 20.9
D50
Microalgae 50 49.4 55.2 60.9
SCD 50 39.1 44.8 50.6
D75
Microalgae 25 11.5 22.4 33.4
SCD 75 66.6 77.6 88.5
Dry mattera
Fig. 4. Changes in nitrogen stable isotope values (per mil) in mantle of Pacific
D25
oyster C. gigas (n = 3) reared on feeding regimes with different proportions of
Microalgae 78.9a 84.3 96.3b 100.0
U. lactuca SCD and C. calcitrans. Lines represent predicted values generated by SCD 21.1 0.0 3.7 15.7
the model of Hesslein et al. (1993) and show the best fit to observed data. D50
Arrows represent isotopic discrimination factors between both feeding sources Microalgae 55.5 58.7 58.0 57.5
and oysters fed C. calcitrans and U. lactuca SCD. SCD 44.5 42.5 42.0 41.3
D75
Microalgae 29.3 16.2 24.5 29.7
0.080 to 0.148 per day and were lower with the regimes containing a SCD 70.7 70.3 75.5 83.8
higher level of inclusion of U. lactuca SCD (Table 2). On the contrary, the
residence halftimes, t50 values, in the oyster tissue consistently increased Superscripts indicate significant differences between expected and mean
observed dietary contributions.
with the inclusion level of U. lactuca SCD, starting with values of 4.1 ±
DM, dry matter; Max., maximum; Min., minimum.
0.7 days for D0 until reaching the double for D100. a
Total dry matter contributions were estimated after correcting nitrogen
The results of the isotopic mixing model indicated that the oysters concentrations measured in both food sources using the equation proposed by
incorporated significantly higher amounts of nitrogen from the micro­ Fry (2006).
algae than from U. lactuca SCD under the D25 feeding regime (Table 3).
At the end of the experiment, oysters in treatment D25 incorporated increment in protein content (+45%) and lower in lipid content (2-fold
95.8% nitrogen from the microalgae, significantly higher (P < 0.001) increment). According to Hou et al. (2015), the hydrolysis during SCD
compared to the expected proportion of nitrogen (75%). In contrast, no production allows releasing sugars, ash, and other components in the
significant differences were found between the observed and expected liquid fraction and the major part of protein recovery is obtained in the
nitrogen contributions in treatments D50 and D75 (P > 0.17). Although solid fraction. Thus, the filtration process at 20 μm applied in the present
the contribution values corrected for elemental concentration indicated study might have separated the protein content in the solid fraction,
that oysters under feeding regime D25 incorporated nutrients mostly which would be further useful in aquafeeds for other organisms. On the
derived from microalgae (96.3% on a dry matter-basis), a higher contrary, U. lactuca SCD, 20 μm-filtered, as reported by Peña Rodríguez
incorporation of dry matter was observed from U. lactuca SCD (42.0 and et al. (2020), also resulted in a higher increment in protein content
75.5%) in oysters fed regimes D50 and D75, respectively. The latter (+33%) and lower in lipid content (2-fold increment), which could be
proportional contributions were not significantly different from ex­ associated to the bacterial fermentation step, which was not applied in
pected contribution values (P > 0.25). the present study.
The analysis of electronic microscopy imaging allowed evaluating
4. Discussion the effect of the SCD production process in whole seaweed meal. In
previous studies, using different seaweed species and SCD process, all
4.1. Feed composition and SCD microscopy detritus sizes range was below 20 μm (Peña Rodríguez et al., 2020; Pérez
Camacho et al., 2007, 2004; Tanyaros and Chuseingjaw, 2014; Uchida
The content of crude protein in raw seaweed material was within the and Murata, 2002). Tanyaros and Chuseingjaw (2014) reported only
range previously reported (Marsham et al., 2007). Moreover, the filtrate 20% of particles with a diameter below 10 μm using a blending and
part of U. lactuca SCD contained 21% more protein than the raw material shaking mechanic process. However, in this study, the acid and enzy­
and resulted in almost a 9-fold increment in lipid content. Fermented matic digestions allowed reducing the seaweed powder to reach more
U. lactuca obtained by Felix and Brindo (2014) resulted in a higher than 87% of particles with a size range between 1 and 10 μm. The latter
range is similar to the cell diameter of C. calcitrans (3–9 μm) (Olenina
Table 2 et al., 2006). Pérez Camacho et al. (2004) demonstrated that applying
Growth rates (k), estimated nitrogen metabolic rates (m), and half-times (t50) in acid treatment before enzymatic digestion in Laminaria saccharina SCD
mantle tissue of Pacific oyster C. gigas (n = 3) fed different proportions of helped to increase 24.8% the proportions of particles having a diameter
U. lactuca SCD and C. calcitrans. less than 10 μm. Initial acid treatment hydrates the organic structures of
Diet k (day− 1) m (day− 1)a t50 (days) r2 the seaweed dried particles, transforms insoluble salts into soluble, and
a a c produces general hydrolysis of complex polysaccharides, which pre­
D0 0.0188 ± 0.003 0.148 ± 0.026 4.1 ± 0.7 0.82
D25 0.0181a ± 0.003 0.103b ± 0.013 5.7b ± 0.7 0.87 pares the particles for subsequent treatment (Pérez Camacho et al.,
D50 0.0166a ± 0.003 0.097c ± 0.025 6.1b ± 1.6 0.74 2004). Moreover, it has been observed that algal particles are associated
D75 0.0083b ± 0.002 0.095c ± 0.032 6.7ab ± 2.3 0.66 with many bacterial cells (Uchida et al., 1997) but, for the use in oyster
D100 0.0035c ± 0.002 0.080d ± 0.001 8.2a ± 0.1 0.98 culture, absence of bacterial activity in detritus is preferable for SCD
Different superscripts indicate significant differences (p < 0.05). preparation (Pérez Camacho et al., 2004; Uchida and Murata, 2002). In
a
m Values were estimated from expected values fitted to observed values this study, no bacterial activity was observed during the 7 days of SCD
using the exponential equation proposed by Hesslein et al. (1993).

6
A. Omont et al.
storage. This bacterial absence was potentially caused by the acid
treatment and the avoidance of fermentation during the SCD produc­
tion, which led to favourable conditions for oyster productivity. More­
over, regarding the SCD process used in this study, avoiding
fermentation treatment, would contribute to the reduction of production
costs for oyster feed at nursery stages. Finally, U. lactuca biomass, either
from natural productivity or to improve profitability of by-products
from sustainable aquaculture to produce SCD, is a practical and reli­
able feed source to be use as an alternative in aquafeed (Carboni et al.,
2016).
4.2. Oyster productivity
The development of high-quality artificial diets to substitute micro­
algae in industrial oyster farming is crucial to meet the feed demand and
continue to improve survival and growth rates (Wang et al., 2016).
Indeed, bivalves under severe nutritional stress allocate all available
energy to survival (Rato et al., 2018). Rato et al. (2018) demonstrated
that unfed C. gigas showed a high mortality rate (72%) after 11-weeks;
whereas when fed 100% of dry Ulva rigida, the physiological condition
was affected, but survival was conserved. In a previous study, substi­
tution of 100% microalgae with SCD caused a high percentage of sur­
vival in oysters C. gigas fed detritus of U. lactuca (94%) after 17 days of
experiment (Peña Rodríguez et al., 2020). In contrast, up to 50%
replacement of microalgae with Porphyra haitanensis SCD led to a sig­
nificant reduction in Crassostrea belcheri survival (Tanyaros and Chu­
seingjaw, 2014). In the present study, oysters fed with a partial or total
substitution of microalgae with U. lactuca SCD retained 100% survival
after 35 days of experiment without significant differences compared to
the control treatment. Considering oyster growth parameters, substitu­
tion of up to 50% of microalgae with U. lactuca SCD resulted in equiv­
alent shell length, live and dry weight, and specific growth rate
compared to the feeding regime receiving only microalgae. The same
observations on growth have been made with juvenile Callionymus bel­
cheri fed with Porphyra haitanensis SCD as 50% substitute of microalgae
(Tanyaros and Chuseingjaw, 2014). In clams, daily rations of SCD from
L. saccharina of 2%, 4%, and 6% induced increasingly lower final live
weights, as compared to clams fed with microalgae (Pérez Camacho
et al., 2004). Pérez Camacho et al. (2007) demonstrated that there is an
additive effect when SCD is supplemented with 10% of live phyto­
plankton, and growth rates obtained were similar to those achieved with
100% of live phytoplankton. In view of the industrial processing, none of
these studies considered the condition index, a parameter that estimates
apparent health and commercial quality of oysters (Orban et al., 2004).
After 42 days of starvation, the condition index in oysters decreased
significantly from 75 to 58, whereas the condition index in fed oysters
ranged between 75 and 82 (Zhang and Li, 2006). Acarli et al. (2011)
defined that oysters in good physiological condition and an acceptable
commercial quality have a condition index between 65 and 90. Rato
et al. (2018) were able to substitute 25% of live microalgae with un­
processed meal of U. rigida, and observed similar nutritional quality and
physiological condition as that observed in broodstock C. gigas oysters
fed 100% live microalgae. In this study, SCD from U. lactuca allowed
substituting 50% of microalgae with a condition index of up to 75, which
seem to be appropriate in oyster hatcheries. On the other hand, the use
of multiple live microalgae strains has proved to promote a better
development of oyster compared to mono-algal diets (Ronquillo et al.,
2012), therefore, the present study shows the potential of SCD U. lactuca
as practical component in the oyster diet.
4.3. Digestive enzymatic activity
Feed digestion and assimilation depend on the enzymatic activity in
the digestive gland and the process is closely related to feed composition
(Peña Rodríguez et al., 2020). Digestive protease activity in C. gigas is
low and has been demonstrated in digestive gland tubules, having
7
Aquaculture 541 (2021) 736835
higher activity at the end of digestion (Bouchaud-Camou et al., 1983).
Chymotrypsin and, in lower proportion, trypsin activity showed higher
activity in oyster fed with U. lactuca SCD, as compared to animals fed
microalgae (Peña Rodríguez et al., 2020). In this study, alkaline protease
presented higher specific activity in oysters fed 100% U. lactuca SCD
compared to other treatments. The latter might be related to the higher
protein content of U. lactuca SCD as compared to microalgae. Moreover,
in bivalves, protein turnover rates are enhanced by aminopeptidase
activity (Donald et al., 2001). In this study, (Leu) aminopeptidase N
activity was higher in oysters fed 100% SCD and reflected lower nitro­
gen (protein) turnover rate and lower growth compared to other treat­
ments. Previous studies have indicated that higher protease and
peptidase activities were related to better dietary protein and higher
growth in oysters (Liao et al., 2020; Yang et al., 2017); nevertheless, in
the present study, protein might have been used as energy for mainte­
nance to preserve vital functions, as concluded by Rato et al. (2018) in a
study with oysters fed 100% dry U. rigida. In C. gigas, at low glycogen
supplies, the main contributors to energy output, and during scarce food
periods, energy needs can be completed with protein and lipid degra­
dation (Liu et al., 2010; Whyte et al., 1990). Indeed, in this study, the
decrease in amylase activity, which contrasted with the increase of
lipase activity at higher levels of U. lactuca SCD inclusion in the feed
regimes, contributes to confirm this hypothesis. Higher amylase activity
is a result of cellular growth due to better food availability (Huvet et al.,
2003). Lower amylase activity has also been reported during dietary
substitution with Ulva sp. in oysters (Peña Rodríguez et al., 2020; Rato
et al., 2018) possibly due to the presence of alpha-amylase inhibitors in
seaweeds (de Oliveira et al., 2009). The enzymatic hydrolysis during
SCD production, release high amounts of monosaccharides (Hou et al.,
2015), that may also explain the significant reduction of amylase ac­
tivity in D100 treatment. A high availability of sugars, may affect car­
bohydrate metabolism and reflect a lower growth as suggested in pearl
oysters (Yang et al., 2018).
On the other hand, the modification of lipid metabolism through
higher lipase activity has been observed in C. gigas fed with dry U. rigida
(Rato et al., 2018), and in other aquatic invertebrates fed Ulva sp.
(Elizondo-Reyna et al., 2016; Omont et al., 2019). Such changes have
been attributed to the lipid quality of diets (Peña Rodríguez et al., 2020).
Actually, U. lactuca contains half the proportion and quantity of highly
unsaturated fatty acids than C. calcitrans (Méndez-Martínez et al., 2018;
Ortiz et al., 2006), which may increase the lipase activity as a way to
adapt to lipid nutritional changes, as observed in Daphnia pulex (Kous­
soroplis et al., 2017). In this study, the higher digestive capacity of lipase
combined with digestive enzymatic activities allowed oysters fed up to
50% of U. lactuca SCD to reach similar growth as oysters fed 100%
microalgae, suggesting good organ performance and good health status.
4.4. Contribution of nutrients to growth and elemental residence times in
oyster mantle
Energy storage and utilization in bivalves are closely correlated to
the quality of diet (Anjos et al., 2016) and it is well known that δ15N
values of bivalves depend on the origin and incorporation of food (Kang
et al., 1999; Savoye et al., 2003).
Dietary nitrogen is important for tissue growth (Liu et al., 2016;
Powell et al., 2002), as observed in the present study, significant vari­
ations were observed in the δ15N values (2.6 to 8.8‰) in the mantle of
oysters fed the different experimental regimes. The 2.6‰ value for δ15N
corresponds to oysters fed with 100% C. calcitrans, close to the value
(2.97‰) observed by Liu et al. (2016) after feeding oyster with micro­
algae. The 8.8‰ value corresponded to oysters fed 100% SCD. Such
values match the range of observed δ15N values in oysters cultured in
natural environment, e.g., 6.6 to 13.6‰ (Kang et al., 2009; Marín Leal
et al., 2008). However, the isotopic values (δ15N) of microalgal biomass
are strongly influenced by the culture media and tend to be low or even
negative (i.e., depleted in 15N) (Le Vay and Gamboa-Delgado, 2011).
A. Omont et al.
Isotopic values in animal tissue change due to growth and/or metabolic
turnover, and if the diet is constant, such values tend to reach an equi­
librium that reflects the isotopic profile of food (Hesslein et al., 1993).
Thus, in the present study, nitrogen isotopic signatures in oyster tissue
reflected the δ15N values of U. lactuca SCD and C. calcitrans. The
significantly different values (respectively, 9.31 ± 0.24 and 3.18 ±
0.19‰) allowed using the isotopic model to estimate nutritional con­
tributions. The same range of difference between macroalgae and
microalgae have been observed in previous studies (Kang et al., 1999;
Yokoyama and Ishihi, 2003). Nitrogen turnover rates (m) in oysters were
higher in animals fed only microalgae; which produced shorter nitrogen
half-times in the mantle tissue (t50 = 4.1 d) than those observed in oyster
fed only detritus (8.2 d). According to MacAvoy et al. (2005), fast tissue
turnover, caused by growth (k) and metabolic tissue replacement in
animals, causes shorter half-times in tissue. Since growth was not
significantly different for oysters fed D25 and D50 compared to control
D0, the significantly lower turnover rates in these treatments indicated a
lower metabolic tissue replacement and, thus, the significantly higher
half-time. Moreover, the low growth observed in oysters fed on regimes
with 75% and 100% SCD suggests that animals, under the effect of these
treatments, reached isotopic equilibrium through tissue catabolic turn­
over and not through biomass accretion. These observations contrast
with previous studies made in P. vannamei shrimps, where a longer half-
time was associated to higher metabolic rates caused by higher growth
rates (Gamboa-Delgado et al., 2013, 2011). Martínez del Rio and Wolf
(2005) considered that feed having low protein quality, particularly
essential amino acid deficiency, leads to greater ingestion and higher
catabolism of amino acids to respond to protein requirements; in
consequence, higher discrimination factors would be observed. In this
study, a negative nitrogen discrimination factor (∆15N) was observed
between oysters and SCD, indicating a relative depletion of the heavier
isotope (15N), which occurs when nutrients (and their isotopes) are
selectively metabolized and incorporated (Gamboa-Delgado et al.,
2011). In the C. gigas oyster, selection efficiency for N increases as a
response of depleting food quality or quantity, whereas selection effi­
ciency for C remains unchanged (Bayne, 2009). After using the isotopic
mixing models to estimate the relative proportions of nitrogen from
microalgae and U. lactuca SCD incorporated in mantle tissue of oysters,
in feeding regime D25, microalgae-derived nitrogen was preferentially
used (as compared to SCD) to promote growth. This observation might
be related to the presence of a higher concentration and availability of
microalgal cells in this feeding regime. As SCD levels increased in
treatments D50 and D75, a similar contribution of dietary nitrogen to
growth was estimated from both feeding regimes. The same pattern was
observed in terms of dry matter incorporation. C. calcitrans had a slightly
lower nitrogen content and, thus, the assimilation of dry matter had to
be greater than that estimated for SCD (which contained more N), to
reach the determined nitrogen contribution. The results showed that
growth and survival in oyster fed with 50% U. lactuca SCD substitution
of microalgae C. calcitrans seemed to be associated with an optimal
adjustment of the assimilation of both feed sources.
5. Conclusion
The use of a simple method of chemical and enzymatic digestion to
produce SCD based on renewable natural sources, like U. lactuca
seaweed, represents an alternative to reduce the need for microalgae as
food during indoor oyster production stages. U. lactuca SCD could sub­
stitute up to 50% of C. calcitrans microalgae, promoting an oyster pro­
duction with good survival and growth rates, apparent optimal health
and commercial quality. In the future, scaling up these findings could
lead to confirm that microalgae substitution with U. lactuca SCD allows
for a significant reduction in production costs and, thereby, improves
oyster farming practices.
8
Aquaculture 541 (2021) 736835
Funding
This work was supported by the Aquaculture program of CIBNOR
and by CONACYT-Mexico (Grant 894930)
Author statement
All data contained in this manuscript are original and were obtained
by the authors. The manuscript has not been previously published or
submitted for publication elsewhere. All authors have contributed
significantly, and the manuscript has been reviewed and approved by all
the authors. The authors also declare that there are no conflicts of
interest.
Declaration of Competing Interest
None.
Acknowledgements
The authors are grateful to CIBNOR staff: Patricia Hinojosa-Baltazar,
José D. Barajas-Frías, Pablo Ormart-Castro, Ariel Cruz-Villacorta, and
Ernesto Goytortua-Bores for all the facilities and technical support. We
also thank Marimex del Pacifico for the oyster donation.
References
Acarli, S., Lok, A., Yildiz, H., Scientific, T., 2011. Comparative growth, survival and
condition index of flat Oyster, Ostrea edulis (Linnaeus 1758) in Mersin Bay, Aegean
Sea, Turkey article. Kafkas Üniversitesi Vet. Fakültesi Derg. 17, 203–210.
Anjos, C., Baptista, T., Joaquim, S., Mendes, S., Matias, A.M., Moura, P., Simoes, T.,
Matias, D., 2016. Broodstock conditioning of the Portuguese oyster (Crassostrea
angulata, Lamarck, 1819): influence of different diets. Aquac. Res. 1–20. https://doi.
org/10.1111/are.13213.
Badillo-Salas, C.E., Valenzuela-Espinoza, E., González-Gómez, M.A., Pares-Sierra, G.,
Ley-Lou, F., Garcia-Esquivel, Z., 2009. Comparative growth of Pacific oyster
(Crassostrea gigas) postlarvae with microfeed and microalgal diets. Aquac. Int. 17,
173–186. https://doi.org/10.1007/s10499-008-9189-3.
Barnes, H., Blackstock, J., 1973. Estimation of lipids in marine animals and tissues:
detailed investigation of the sulphophosphovanillin method for “total” lipids. J. Exp.
Mar. Biol. Ecol. 12, 103–118. https://doi.org/10.3923/pjbs.2004.2182.2186.
Bayne, B.L., 2009. Carbon and nitrogen relationships in the feeding and growth of the
Pacific oyster, Crassostrea gigas (Thunberg). J. Exp. Mar. Biol. Ecol. 374, 19–30.
https://doi.org/10.1016/j.jembe.2009.04.003.
Bouchaud-Camou, E., Lebesnerais, C., Lubet, P., Lihrmann, I., 1983. Dynamique et
enzymologie de la digestion chez l’huitre Crassostrea gigas. In: IFREMER (Ed.), Bases
Biologiques de l’aquaculture. Montpellier, pp. 75–96.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
72, 248–254. https://doi.org/10.1016/0003-2697(76)90527-3.
Carboni, S., Clegg, S.H., Hughes, A.D., 2016. The use of biorefinery by-products and
natural detritus as feed sources for oysters (Crassostrea gigas) juveniles. Aquaculture
464, 392–398. https://doi.org/10.1016/j.aquaculture.2016.07.021.
Cardoso, C., Gomes, R., Rato, A., Joaquim, S., Machado, J., Gonçalves, J.F., Vaz-Pires, P.,
Magnoni, L., Matias, D., Coelho, I., Delgado, I., Castanheira, I., Matos, J., Ozório, R.,
Bandarra, N., Afonso, C., 2019. Elemental composition and bioaccessibility of
farmed oysters (Crassostrea gigas) fed different ratios of dietary seaweed and
microalgae during broodstock conditioning. Food Sci. Nutr. 7, 2495–2504. https://
doi.org/10.1002/fsn3.1044.
Castelar, B., Reis, R.P., dos Santos Calheiros, A.C., 2014. Ulva lactuca and U. flexuosa
(Chlorophyta, Ulvophyceae) cultivation in Brazilian tropical waters: recruitment,
growth, and ulvan yield. J. Appl. Phycol. 26, 1989–1999. https://doi.org/10.1007/
s10811-014-0329-z.
Coutteau, P., Sorgeloos, P., 1992. The use of algal substitutes and the requirement for live
algae in the hatchery and nursery rearing of bivalve molluscs: an international
survey. J. Shellfish Res. 11, 118–128.
Crosby, M.P., Gale, L.D., 1990. review and evaluation of bivalve condition index
methodologies with a suggested standard method. J. Shellfish Res. 9, 233–237.
Dominguez, H., Loret, E.P., 2019. Ulva lactuca, A source of troubles and potential riches.
Mar. Drugs 17, 1–20. https://doi.org/10.3390/md17060357.
Donald, K.M., Hawkins, A.J.S., Smerdon, G.R., 2001. Transcript analysis of the genes
encoding aminopeptidase N and alanine aminotransferase, two enzymes involved in
protein turnover, in the Pacific oyster, Crassostrea gigas. Comp. Biochem. Physiol.
Part B 128, 459–467.
Ebeling, M.E., 1968. The Dumas method for nitrogen in feeds. J. Assoc. Off. Anal. Chem.
51, 766–770. https://doi.org/10.1093/jaoac/51.4.766.
Elizondo-Reyna, E., Medina-González, R., Nieto-López, M.G., Ortiz-López, R., Elizondo-
González, R., Powell, M.S., Ricque-Marie, D., Cruz-Suárez, L.E., 2016. Consumption
A. Omont et al.
of Ulva clathrata as a dietary supplement stimulates immune and lipid metabolism
genes in Pacific white shrimp Litopenaeus vannamei. J. Appl. Phycol. 28, 3667–3677.
https://doi.org/10.1007/s10811-016-0889-1.
FAO, 2019. FAO yearbook. Fishery and Aquaculture Statistics, Rome. https://doi.org/
10.1109/BMEI.2010.5639447.
Felix, N., Brindo, R.A., 2014. Evaluation of raw and fermented seaweed, Ulva lactuca as
feed ingredient in giant freshwater prawn Macrobrachium rosenbergii. Int. J. Fish.
Aquat. Stud. 1, 199–204.
Fry, B., 2006. Stable Isotope Ecology, Springer. ed. Journal of Chemical Information and
Modeling, New York. https://doi.org/10.1017/CBO9781107415324.004.
Gamboa-Delgado, J., Le Vay, L., 2009. Natural stable isotopes as indicators of the relative
contribution of soy protein and fish meal to tissue growth in Pacific white shrimp
(Litopenaeus vannamei) fed compound diets. Aquaculture 291, 115–123. https://doi.
org/10.1016/j.aquaculture.2009.03.012.
Gamboa-Delgado, J., Peña-Rodríguez, A., Ricque-Marie, D., Cruz-Suárez, L.E., 2011.
Assessment of nutrient allocation and metabolic turnover rate in Pacific white
shrimp Litopenaeus vannamei co-fed live macroalgae ulva clathrata and inert feed:
dual stable isotope analysis. J. Shellfish Res. 30, 969–978. https://doi.org/10.2983/
035.030.0340.
Gamboa-Delgado, J., Rojas-Casas, M.G., Nieto-López, M.G., Cruz-Suárez, L.E., 2013.
Simultaneous estimation of the nutritional contribution of fish meal, soy protein
isolate and corn gluten to the growth of Pacific white shrimp (Litopenaeus vannamei)
using dual stable isotope analysis. Aquaculture 380–383, 33–40. https://doi.org/
10.1016/j.aquaculture.2012.11.028.
Harel, M., Clayton, D., Bullis, R.A., 2007. Feed Formulation for Terrestral and Aquatic
Animals (US20070082008A1).
Hawkins, C.M., Rowell, T.W., 1987. The importance of cleansing in the calculation of
condition index in the soft-shell clam,Mya arenaria (L.). J. Shellfish Res. 6, 85–88.
Hesslein, R.H., Hallard, K.A., Ramlal, P., 1993. Replacement of sulfur, carbon, and
nitrogen in tissue of growing broad whitefish (Coregonus nasus) in response to a
change in diet traced by 34S, 13C, and 15 N. Can. J. Fish. Aquat. Sci. 50, 2071–2076.
Hou, X., Hansen, J.H., Bjerre, A.B., 2015. Integrated bioethanol and protein production
from brown seaweed Laminaria digitata. Bioresour. Technol. 197, 310–317. https://
doi.org/10.1016/j.biortech.2015.08.091.
Huvet, A., Daniel, J.-Y., Quéré, C., Dubois, S., Prudence, M., Van Wormhoudt, A.,
Sellos, D., Samain, J.-F., Moal, J., 2003. Tissue expression of two a -amylase genes in
the Pacific oyster Crassostrea gigas. Effects of two different food rations. Aquaculture
228, 321–333. https://doi.org/10.1016/S0044-8486(03)00323-5.
Kang, C.K., Sauriau, P., Richard, P., Blanchard, G.F., 1999. Food sources of the infaunal
suspension-feeding bivalve Cerastoderma edule in a muddy sandflat of Marennes-
OlQon Bay, as determined by analyses of carbon and nitrogen stable isotopes. Mar.
Ecol. Prog. Ser. 187, 147–158.
Kang, C.K., Choy, E.J., Hur, Y.B., Myeong, J.I., 2009. Isotopic evidence of particle size-
dependent food partitioning in cocultured sea squirt Halocynthia roretzi and Pacific
oyster Crassostrea gigas. Aquat. Biol. 6, 289–302. https://doi.org/10.3354/ab00126.
Knauer, J., Southgate, P.C., 1999. A review of the nutritional requirements of bivalves
and the development of alternative and artificial diets for bivalve aquaculture. Rev.
Fish. Sci. 7, 241–280. https://doi.org/10.1080/10641269908951362.
Knuckey, R.M., Brown, M.R., Robert, R., Frampton, D.M.F., 2006. Production of
microalgal concentrates by flocculation and their assessment as aquaculture feeds.
Aquac. Eng. 35, 300–313. https://doi.org/10.1016/j.aquaeng.2006.04.001.
Koussoroplis, A.M., Schwarzenberger, A., Wacker, A., 2017. Diet quality determines
lipase gene expression & lipase/esterase activity in Daphnia pulex. Biol. Open 6,
210–216. https://doi.org/10.1242/bio.022046.
Kunitz, M., 1947. Crystalline soybean trypsin inhibitor: Ii. General properties. J. Gen.
Physiol. 30, 291–310. https://doi.org/10.1085/jgp.30.4.291.
Lawrence, D.R., Scott, G.I., 1982. The determination and use of condition index of
oysters. Estuaries 5, 23–27. https://doi.org/10.2307/1352213.
Le Vay, L., Gamboa-Delgado, J., 2011. Naturally-occurring stable isotopes as direct
measures of larval feeding efficiency, nutrient incorporation and turnover.
Aquaculture 315, 95–103. https://doi.org/10.1016/j.aquaculture.2010.03.033.
Liao, Y., Cai, C., Yang, C., Zheng, Z., Wang, Q., Du, X., Deng, Y., 2020. Effect of protein
sources in formulated diets on the growth, immune response, and intestinal
microflora of pearl oyster Pinctada fucata martensii. Aquac. Reports 16, 100,253.
https://doi.org/10.1016/j.aqrep.2019.100253.
Liu, A., Mazumder, D., Dove, M.C., Lai, T.S., Crawford, J., Sammut, J., 2016. Stable
isotope analysis of the contribution of microalgal diets to the growth and survival of
pacific oyster Crassostrea gigas (Thunberg, 1979) Larvae. J. Shellfish Res. 35, 63–69.
https://doi.org/10.2983/035.035.0108.
Liu, W., Li, Q., Gao, F., Kong, L., 2010. Effect of starvation on biochemical composition
and gametogenesis in the Pacific oyster Crassostrea gigas. Fish. Sci. 76, 737–745.
https://doi.org/10.1007/s12562-010-0274-y.
Lourenço, S.O., Barbarino, E., Lavín, P.L., Lanfer Marquez, U.M., Aidar, E., 2004.
Distribution of intracellular nitrogen in marine microalgae: calculation of new
nitrogen-to-protein conversion factors. Eur. J. Phycol. 39, 17–32. https://doi.org/
10.1080/0967026032000157156.
MacAvoy, S.E., Macko, S.A., Arneson, L.S., 2005. Growth versus metabolic tissue
replacement in mouse tissues determined by stable carbon and nitrogen isotope
analysis. Can. J. Zool. 83, 631–641. https://doi.org/10.1139/z05-038.
Macchiavello, J., Bulboa, C., 2014. Nutrient uptake efficiency of Gracilaria chilensis and
Ulva lactuca in an IMTA system with the red abalone Haliotis rufescens. Lat. Am. J.
Aquat. Res. 42, 523–533. https://doi.org/10.3856/vol42-issue3-fulltext-12.
Makkar, H.P.S., Tran, G., Heuzé, V., Giger-Reverdin, S., Lessire, M., Lebas, F., Ankers, P.,
2016. Seaweeds for livestock diets: A review. Anim. Feed Sci. Technol. 212, 1–17.
https://doi.org/10.1016/j.anifeedsci.2015.09.018.
9
Aquaculture 541 (2021) 736835
Marín Leal, J.C., Dubois, S., Orvain, F., Galois, R., Blin, J.L., Ropert, M., Bataillé, M.P.,
Ourry, A., Lefebvre, S., 2008. Stable isotopes (δ13C, δ15N) and modeling as tools to
estimate the trophic ecology of cultivated oysters in two contrasting environments.
Mar. Biol. 153, 673–688. https://doi.org/10.1007/s00227-007-0841-7.
Maroux, S., Louvard, D., Baratti, J., 1973. The aminopeptidase from hog intestinal brush
border. Biochim. Biophys. Acta 321, 282–295. https://doi.org/10.1016/0005-2744
(73)90083-1.
Marsham, S., Scott, G.W., Tobin, M.L., 2007. Comparison of nutritive chemistry of a
range of temperate seaweeds. Food Chem. 100, 1331–1336. https://doi.org/
10.1016/j.foodchem.2005.11.029.
Martínez del Rio, C., Wolf, B.O., 2005. Mass-balance models for animal isotopic ecology.
Physiol. Ecol. Adapt. Feed. Vertebr. 141–174.
Mazumder, D., Wen, L., Johansen, M.P., Kobayashi, T., Saintilan, N., 2016. Inherent
variation in carbon and nitrogen isotopic assimilation in the freshwater macro-
invertebrate Cherax destructor. Mar. Freshw. Res. 67, 1928–1937. https://doi.org/
10.1071/MF15180.
McCausland, M.A., Brown, M.R., Barrett, S.M., Diemar, J.A., Heasman, M.P., 1999.
Evaluation of live microalgae and microalgal pastes as supplementary food for
juvenile pacific oysters (Crassostrea gigas). Aquaculture 174, 323–342. https://doi.
org/10.1016/S0044-8486(99)00018-6.
Méndez-Martínez, Y., García-Guerrero, M.U., Lora-Vilchis, M.C., Martínez-Córdova, L.R.,
Arcos-Ortega, F.G., Alpuche, J.J., Cortés-Jacinto, E., 2018. Nutritional effect of
Artemia nauplii enriched with Tetraselmis suecica and Chaetoceros calcitrans
microalgae on growth and survival on the river prawn Macrobrachium americanum
larvae. Aquac. Int. 26, 1001–1015. https://doi.org/10.1007/s10499-018-0264-0.
Neori, A., Chopin, T., Troell, M., Buschmann, A.H., Kraemer, G.P., Halling, C.,
Shpigel, M., Yarish, C., 2004. Integrated aquaculture: rationale, evolution and state
of the art emphasizing seaweed biofiltration in modern mariculture. Aquaculture
231, 361–391. https://doi.org/10.1016/j.aquaculture.2003.11.015.
Nolasco-Soria, H., Moyano-López, F., Vega-Villasante, F., del Monte-Martínez, A.,
Espinosa-Chaurand, D., Gisbert, E., Nolasco-Alzaga, H.R., 2018. Lipase and
phospholipase activity methods for marine. In: Methods in Molecular Biology. New
York, pp. 139–167. https://doi.org/10.1007/978-1-4939-8672-9.
Olenina, I., Hajdu, S., Edler, L., Wasmund, N., Busch, S., Göbel, J., Gromisz, S.,
Huseby, S., Huttunen, M., Jaanus, A., Kokkonen, P., Ledaine, I., Niemkiewicz, E.,
2006. Biovolumes and Size-Classes of Phytoplankton in the Baltic Sea, HELCOM
Baltic Sea Environment Proceedings. Baltic Marine Environment Protection
Commission - Helsinki Commission. doi:HELCOM Balt.Sea Environ. Proc. No. 106,
p. 144.
de Oliveira, M.N., Ponte Freitas, A.L., Urano Carvalho, A.F., Tavares Sampaio, T.M.,
Farias, D.F., Alves Teixeira, D.I., Gouveia, S.T., Pereira, J.G., de Castro Catanho de
Sena, M.M., 2009. Nutritive and non-nutritive attributes of washed-up seaweeds
from the coast of Ceará. Brazil. Food Chem. 115, 254–259. https://doi.org/10.1016/
j.foodchem.2008.12.004.
Omont, A., Quiroz-Guzman, E., Tovar-Ramirez, D., Peña-Rodríguez, A., 2019. Effect of
diets supplemented with different seaweed extracts on growth performance and
digestive enzyme activities of juvenile white shrimp Litopenaeus vannamei. J. Appl.
Phycol. 31, 1433–1442. https://doi.org/10.1007/s10811-018-1628-6.
Orban, E., Di Lena, G., Masci, M., Nevigato, T., Casini, I., Caproni, R., Gambelli, L.,
Pellizzato, M., 2004. Growth, nutritional quality and safety of oysters (Crassostrea
gigas) cultured in the lagoon of Venice (Italy). J. Sci. Food Agric. 84, 1929–1938.
https://doi.org/10.1002/jsfa.1896.
Ortiz, J., Romero, N., Robert, P., Araya, J., Lopez-Hernández, J., Bozzo, C., Navarrete, E.,
Osorio, A., Rios, A., 2006. Dietary fiber, amino acid, fatty acid and tocopherol
contents of the edible seaweeds Ulva lactuca and Durvillaea antarctica. Food Chem.
99, 98–104. https://doi.org/10.1016/j.foodchem.2005.07.027.
Peña Rodríguez, A., Morales Alvarado, G., Elizondo González, R., Mendoza Carrión, G.,
Tovar Ramírez, D., Escobedo-fregoso, C., 2020. Seaweed single cell detritus effects
on the digestive enzymes activity and microbiota of the oyster Crassostrea gigas.
J. Appl. Phycol. https://doi.org/10.1007/s10811-020-02167-4.
Pérez Camacho, A., Salinas, J.M., Fuertes, C., Delgado, M., 2004. Preparation of single
cell detritus from Laminaria saccharina as a hatchery diet for bivalve molluscs. Mar.
Biotechnol. 6, 642–649. https://doi.org/10.1007/s10126-004-2901-z.
Pérez Camacho, A., Salinas, J.M., Delgado, M., Fuertes, C., 2007. Use of single cell
detritus (SCD) produced from Laminaria saccharina in the feeding of the clam
Ruditapes decussatus (Linnaeus, 1758). Aquaculture 266, 211–218. https://doi.org/
10.1016/j.aquaculture.2006.12.033.
Phillips, D.L., Gregg, J.W., 2001. Uncertainty in source partitioning using stable isotopes.
Oecologia 127, 171–179. https://doi.org/10.1007/s004420000578.
Ponis, E., Robert, R., Parisi, G., 2003. Nutritional value of fresh and concentrated algal
diets for larval and juvenile Pacific oysters (Crassostrea gigas). Aquaculture 221,
491–505. https://doi.org/10.1016/S0044-8486(03)00075-9.
Powell, E.N., Bochenek, E.A., Klinck, J.M., Hofmann, E.E., 2002. Influence of food
quality and quantity on the growth and development of Crassostrea gigas larvae: A
modeling approach. Aquaculture 210, 89–117. https://doi.org/10.1016/S0044-
8486(01)00891-2.
Qiu, X., Neori, A., Kim, J.K., Yarish, C., Shpigel, M., Guttman, L., Ben Ezra, D.,
Odintsov, V., Davis, D.A., 2018. Evaluation of green seaweed Ulva sp. as a
replacement of fish meal in plant-based practical diets for Pacific white shrimp,
Litopenaeus vannamei. J. Appl. Phycol. 30, 1305–1316. https://doi.org/10.1007/
s10811-017-1278-0.
Rato, A., Joaquim, S., Tavares, T.G., Martins, Z.E., Guedes, A.C., Pereira, L.F.,
Machado, J., Matias, A.M., Gonçalves, J.F.M., Vaz-Pires, P., Magnoni, L.J., Ozório, R.
O.A., Matias, D., 2018. Viability of dietary substitution of live microalgae with dry
Ulva rigida in broodstock conditioning of the Pacific oyster (Crassostrea gigas). Biol.
Open 7, 1–10. https://doi.org/10.1242/bio.035923.
A. Omont et al.
Riera, P., Richard, P., 1996. Isotopic determination of food sources of Crassostrea gigas
along a trophic gradient in the estuarine bay of Marennes-Oleron. Estuar. Coast.
Shelf Sci. 42, 347–360. https://doi.org/10.1006/ecss.1996.0023.
Rivero-Rodríguez, S., Beaumont, A.R., Lora-vilchis, M.C., 2007. The effect of microalgal
diets on growth, biochemical composition, and fatty acid profile of Crassostrea
corteziensis (Hertlein) juveniles. Aquaculture 263, 199–210. https://doi.org/
10.1016/j.aquaculture.2006.09.038.
Ronquillo, J.D., Fraser, J., McConkey, A.J., 2012. Effect of mixed microalgal diets on
growth and polyunsaturated fatty acid profile of European oyster (Ostrea edulis)
juveniles. Aquaculture 360–361, 64–68. https://doi.org/10.1016/j.
aquaculture.2012.07.018.
Savoye, N., Aminot, A., Tréguer, P., Fontugne, M., Naulet, N., Kérouel, R., 2003.
Dynamics of particulate organic matter δ 15 N and δ 13C during spring
phytoplankton blooms in a macrotidal ecosystem (Bay of Seine, France). Mar. Ecol.
Prog. Ser. 255, 27–41. https://doi.org/10.3354/meps255027.
Schiener, P., Atack, T., Wareing, R.A., Kelly, M.S., Hughes, A.D., 2016. The by-products
from marine biofuels as a feed source for the aquaculture industry: a novel example
of the biorefinery approach. Biomass Convers. Biorefinery 6, 281–287. https://doi.
org/10.1007/s13399-015-0190-6.
Shuuluka, D., Bolton, J.J., Anderson, R.J., 2013. Protein content, amino acid composition
and nitrogen-to-protein conversion factors of Ulva rigida and Ulva capensis from
natural populations and Ulva lactuca from an aquaculture system, in South Africa.
J. Appl. Phycol. 25 (2), 677–685. https://doi.org/10.1007/s10811-012-9902-5.
Southgate, P.C., Beer, A.C., Ngaluafe, P., 2016. Hatchery culture of the winged pearl
oyster, Pteria penguin, without living micro-algae. Aquaculture 451, 121–124.
https://doi.org/10.1016/j.aquaculture.2015.09.007.
Tanyaros, S., Chuseingjaw, S., 2014. A partial substitution of microalgae with single cell
detritus produced from seaweed (Porphyra haitanensis) for the nursery culture of
tropical oyster (Crassostrea belcheri). Aquac. Res. 47, 2080–2088. https://doi.org/
10.1111/are.12662.
Tanyaros, S., Sujarit, C., Jansri, N., Tarangkoon, W., 2016a. Baker’s yeast as a substitute
for microalgae in the hatchery rearing of larval and juvenile tropical oyster
(Crassostrea belcheri, Sowerby 1871). J. Appl. Aquac. 28, 35–46. https://doi.org/
10.1080/10454438.2016.1163312.
Tanyaros, S., Tarangkoon, W., Klomkleing, T., 2016b. Evaluation of flocculated
concentrates from intensive shrimp pond water as a substitute for microalgal
concentrates in the nursery culture of juvenile oyster (Crassostrea belcheri). Int.
Aquat. Res. 8, 149–160. https://doi.org/10.1007/s40071-016-0130-5.
10
Aquaculture 541 (2021) 736835
Uchida, M., 1996. Formation of single cell detritus densely covered with bacteria during
experimental degradation of Laminaria japonica Thalli. Fish. Sci. 62, 731–736.
Uchida, M., Murata, M., 2002. Fermentative preparation of single cell detritus from
seaweed, Undaria pinnatifida, suitable as a replacement hatchery diet for unicellular
algae. Aquaculture 207, 345–357. https://doi.org/10.1016/S0044-8486(01)00792-
X.
Uchida, M., Numaguchi, K., 1998. Method for Preparing Algal Detritus
(US005801050A).
Uchida, M., Nakata, K., Maeda, M., 1997. Introduction of detrital food webs into an
aquaculture system by supplying single cell algal detritus produced from Laminaria
japonica as a hatchery diet for Artemia nauplii. Aquaculture 154, 125–137.
Vega-Villasante, F., Nolasco, H., Civera, R., 1993. The digestive enzymes of the pacific
brown shrimp Penaeus californiensis I. Properties of amylase activity in the digestive
tract. Comp. Biochem. Physiol. 106B, 547–550.
Wang, Q., Yang, C., Du, X., Liu, X., Sun, R., Deng, Y., 2016. Growth performance and
biochemical composition of juvenile pearl oyster Pinctada martensii fed on artificial
diets. Aquac. Int. 24, 995–1005. https://doi.org/10.1007/s10499-015-9966-8.
Whyte, J.N.C., Englaf, J.R., Carswell, B.L., 1990. Biochemical composition and energy
reserves in Crassostrea gigas exposed to different levels of nutrition. Aquaculture 90,
157–172.
Yang, C., Hao, R., Deng, Y., Liao, Y., Wang, Q., Sun, R., Du, X., Yang, C., Hao, R.,
Deng, Y., Wang, Q., Jiao, Y., Du, X., 2017. Effects of protein sources on growth,
immunity and antioxidant capacity of juvenile pearl oyster Pinctada fucata
martensii. Fish Shellfish Immunol. 67, 411–418. https://doi.org/10.1016/j.
fsi.2017.06.037.
Yang, C., Hao, R., Du, X., Deng, Y., Sun, R., Wang, Q., 2018. Metabolomics responses of
pearl oysters (Pinctada fucata martensii) fed a formulated diet indoors and cultured
with natural diet outdoors. Front. Physiol. 9, 944. https://doi.org/10.3389/
fphys.2018.00944.
Yokoyama, H., Ishihi, Y., 2003. Feeding of the bivalve Theora lubrica on benthic
microalgae: Isotopic evidence. Mar. Ecol. Prog. Ser. 255, 303–309. https://doi.org/
10.3354/meps255303.
Zhang, Z., Li, X., 2006. Evaluation of the effects of grading and starvation on the
lysosomal membrane stability in pacific oysters, Crassostrea gigas (Thunberg) by
using neutral red retention assay. Aquaculture 256, 537–541. https://doi.org/
10.1016/j.aquaculture.2006.02.002.