The Biologist (Lima), 2017, 15(1), jan-jun: 109-117.

ORIGINAL ARTICLE / ARTÍCULO ORIGINAL

USE OF MICROALGAE FOR BROODSTOCK CONDITIONING OF MESODESMA

DONACIUM (MESODESMATIDAE)

USO DE MICROALGAS PARA EL ACONDICIONAMIENTO DE REPRODUCTORES DE

MESODESMA DONACIUM (MESODESMATIDAE)

1 Laboratorio de Investigación Acuícola (LIA) del Instituto del Mar del Perú (IMARPE),

sede Ilo, Jr. Mirave 101 Ilo, Moquegua 18601, Perú

2 Departamento Acuicultura, Facultad de Ciencias del Mar, Universidad Católica del Norte, Casilla 117,

Coquimbo 1780000, Chile

Corresponding author. E-mail: szevallos@imarpe.gob.pe (Sheyla Zevallos)

1 2

Sheyla Zevallos & Pedro Toledo

ABSTRACT

Keywords: bivalves – gonadal development – local microalgae – total lipids

Adult Mesodesma donacium were conditioned with the following microalgae diets: D1) Isochrysis galbana (local)

+ Chaetoceros sp. (local) + Phaeodactylum tricornutum (local); D2) I. galbana (local) + Ch. gracilis (commercial) +

Pavlova lutheri (commercial); D3) Chaetoceros sp. (local) + I. galbana (commercial) + P. lutheri (commercial); and

a control (C) with commercial I. galbana, Ch. gracilis, and P. lutheri. M. donacium were fed with a continuous

feeding system (2.0 x 10 cells/mL) and maintained at 16 °C, with constant aeration and daily full water replacement.

Chemical and histological analyses determined gonadal development and total lipid content. Gonadal development

significantly differed as a result of the different diets, with D2 individuals showing the best response at day 44 of

conditioning (56% mature). Total lipid content did not significantly differ between groups, although levels were

higher than ocean-sampled M. donacium. Moreover, temperature did not influence gonadal development or total

lipid content, with no correlation between this factor and dependent variables. To optimize broodstock conditioning

of M. donacium, a continuously administered microalgae diet is recommended under constant temperatures and with

consideration to total lipid content.

ISSN Versión Impresa 1816-0719

ISSN Versión en linea 1994-9073 ISSN Versión CD ROM 1994-9081

109

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The Biologist (Lima)

The Biologist (Lima). Vol. 15, Nº1, jan - jun 2017

RESUMEN

Palabras clave: bivalvos – maduración gonadal – microalgas locales – lípidos totales

Ejemplares adultos de Mesodesma donacium fueron acondicionados bajo condiciones controladas aplicando tres

diferentes mezclas de microalgas: D1) Isochrysis galbana (local) + Chaetoceros sp (local) + Phaeodactylum

tricornutum (local); D2) I. galbana (local) +Ch. gracilis (comercial) + Pavlova lutheri (comercial); D3)

Chaetoceros sp (local) +I. galbana (comercial) + P. lutheri (comercial) y un control (C) con las microalgas

comerciales I. galbana, Ch. gracilis y P. lutheri. Las microalgas fueron suministradas a los adultos de M. donacium

mediante un sistema de alimentación continuo en una concentración de 2,0 x 10 cel/mL, a una temperatura promedio

de 16°C, aireación constante y recambio diario de agua. Los análisis químicos e histológicos determinaron el

desarrollo gonadal y el contenido total de lípidos. El desarrollo gonadal mostró diferencias significativas como

resultado de las dietas suministradas, siendo D2 la que presenta la mejor respuesta a los 44 días de

acondicionamiento (56% maduros). El contenido total de lípidos no presentó diferencia significativa entre los

tratamientos, pero fueron superiores a los evidenciados en medio natural. Además, la temperatura no influyó en el

desarrollo gonadal ni en el contenido total de lípidos, descartando la correlación entre este factor y las variables

dependientes. Para optimizar el acondicionamiento de reproductores de M. donacium, se recomienda utilizar un

sistema de alimentación continuo, manteniendo la temperatura constante y considerando los lípidos totales como

una estrategia de manejo en medio controlado.

assessed for nutritional applications, but only 20

species are currently used in the aquaculture

industry – (Priyadarshani et al., 2012). The

nutritional quality of an alga is a determinant of its

final utilization –(Volkman et al., 1989), with local

microalgae acting as natural food sources

(Borowitzka, 1997).

Microalgae are an important food source in the

commercial breeding of aquatic animals,

especially in larval and juvenile bivalve molluscs,

rotifers use to feed larvae of crustaceans and

marine fish (Borowitzka, 1997); source depends on

the content and nature of its biochemical

constituents, representing the basis for the

formation of new tissue, restoration of those who

have been affected and for normal metabolism of

organisms breeding. A combination of algae as diet

provides more nutritional value because they

contain an assortment of most nutritional

requirements for growth of the larvae, are these

shrimps, molluscs and fish (Álvarez, 1994).

They have reported numerous studies aimed at

analyzing the effect of different diets and

nutritional components on both the gonadal

maturation and larval development in bivalve; as

reporting on the microalgal most commonly use for

Mesodesma donacium (Lamarck, 1818) is a

Lamellibranchia bivalve of commercial interest

that inhabits the mid- and infralittoral zones of

sandy beaches exposed to waves (Segura et al.,

1998), extending from the intertidal zone to a depth

of 15 m (Carré, 2007). This mollusk is distributed

from the Bay of Sechura, Peru (5°S) to the Inio

river mouth located at the extreme southern end of

Chiloé, Chile (43°S) (Alamo & Valdivieso, 1997;

Vargas, 2003; Zaro, 2004; Santelices, 2006). The

commercial extraction of M. donacium and

increasing ocean surface temperatures (Quiroz &

Barriga, 1997) are factors that have contributed to

the decrease and collapse of natural beds of this

bivalve (Segura et al., 1998). In fact, the capture of

M. donacium is banned in Peru.

A potential alternative for recovering populations

of this species is through the incorporation of

broodstock-obtained juveniles into the natural

environment. For this to occur, considerable

consideration should be given to nutritional aspects

(Farías & Uriarte, 2002) and the survival of bivalve

larvae and juveniles (Contreras, 2006). In the last

40 years, hundreds of microalgae have been

INTRODUCTION

110

Zevallos & Toledo

The Biologist (Lima). Vol. 15, Nº1, jan - jun 2017

111

the production of molluscs cultured controlled

were T - Iso and Chaetoceros neogracile

(S.L.VanLandingham, 1968), associated with

elevated levels of total lipid and docosahexaenoic

acid (DHA) in T - Iso and high levels of

carbohydrates, eicosapentaenoic acid (EPA) and

riboflavin in C. neogracile (Farías & Uriarte,

2002).

Farias (2008) reports that within the requirements

lipid are the main energy source and in particular

triglycerides of fatty acids, saturated short chain

are predominantly used for energy purposes, β

oxidation way (Borowitzka, 1997). On the other

hand, the main function of essential fatty acids in

bivalves, as part of membrane phospholipids, is the

regulation of the flow of cellular and subcellular

membranes, and acclimation to different

temperature ranges.

Highlighting reproductive studies Argopecten

purpuratus (Lamarck, 1819) maintained at 16 ° C

and fed with a mixture of microalgaes Isochrysis

galbana (Parke, 1949) and Chaetoceros gracilis

(Pantocsek, 1892), obtaining lipid and gonadal

recoveries achieved with higher percentages of

42.64 % ± 4.68 maturity after the first 10 days in a

period of 80 days reproductive conditioning

(Martınez et al., 2000).

In the case of M. donacium, there is a lack of

information Salgado & Ishiyama (1979) showed

that immature and less mature stages occur June

through August; intermediate stages and full

maturity from August to November; stages of

partial or total evacuation from December to

March, and are gradually descending until July;

stages of gonadal reversal observed from February

to July.

In this context, the aim of the present study was to

evaluate the effect of different mixed local and

commercial microalgae diets on the gonadal

development of broodstock M. donacium.

Collection area and samples

Adult M. donacium were collected from La Punta,

Arequipa, Peru (17º11'S-71º47'W) and transported

to the Laboratory of Mollusk Research at the

Instituto del Mar, Ilo, Peru.

Random samples of 21 specimens were made

before the start of laboratory tests and 29

specimens during the fortnight of December for the

morphometric measurement, determination of

gonadal status and biochemical profile.

Broodstock conditioning

Adult M. donacium (56-84 mm length) were

conditioned in a tank with four 20 L trays (30 x 20

cm) that contained sifted and washed sandy

substrate (0.5 mm diameter, phi 2-1) (Wentworth,

1922). Twenty M. donacium individuals were

placed in each tray. The seawater in the tank was

maintained at an average of 16±1°C and was fully

replaced every 36 h by an air lift pump. The

dissolved oxygen content was kept at 3.61 mL/L,

while salinity was maintained at 34.824 psu.

Dietary trials

Strains of local microalgae were collected from the

surface of the water column at sandy ocean floor

beaches in Vila Vila (Tacna, Peru; 18°06'S-

70°43'W) and La Punta (Arequipa, Peru;

17º11'S–71º47'W) using a 20 µm mesh net. The

microalgae were isolated using either successive

dilutions or dish isolation with a Pasteur pipette

(Almaguer et al., 2004). The microalgae were then

acclimated in dishes with a liquid medium enriched

with F/2 Guillard (1975) for growth and

maintenance for a month. Samples of each

microalgae were sent to the Germplasm Bank for

Aquatic Organisms of the Instituto del Mar, Peru

and were identified as Isochrysis galbana (Parke,

1949), Chaetoceros sp, and Phaeodactylum

trycornutum (Bohlin, 1898).

The commercial microalgae used in the dietary

assays were Ch. gracilis, Pavlova lutheri

(J.C.Green, 1975), and I. galbana. These

microalgae were obtained from the Central

Laboratory for Marine Cultures of the Faculty of

Marine Sciences at the Universidad Católica del

Norte, Coquimbo, Chile.

Three diets were formulated using mixes of local

and commercial microalgae. The diets respectively

used each microalgae in a 35-30-35% proportion:

D1) I. galbana (local) + Chaetoceros sp (local) +

Microalgae for Mesodesma

MATERIALS AND METHODS

The Biologist (Lima). Vol. 15, Nº1, jan - jun 2017

112

Ph. tricornutum (local); D2) I. galbana (local) +

Ch. gracilis (commercial) + P. lutheri

(commercial); D3) Ch. sp (local) + I. galbana

(commercial) + P. lutheri (commercial); and C) a

control using commercial I. galbana, + Ch.

gracilis, + P. lutheri. These microalgae diets were

administered daily using a continuous feeding

system at a concentration of 2.0 x 10 cells/mL.

Analysis of gonadal development

Of the collected M. donacium, eight were measured

and examined under an optic microscope to

determine sex and gonad development. Every 15

days during the dietary trials, all M. donacium

individuals were measured (total length, TL; ± 0.01

mm) and weighed (total weight, TW; and soft

tissue weight, WST; ± 0.001 g). Additionally, the

sex and gonadal development of eight individuals

were determined every 15 days during the dietary

trials through smear analysis under a microscope

using the scale proposed by Buitrón & Perea

(1996). In addition to these determinations, four

individuals were sampled every 15 days and

preserved in 96° ethanol for posterior histological

analyses. The remaining samples were frozen at -

13 °C until determinations of fat content.

Chemical assays

The soft tissues of M. donacium from the different

diets were separated by sex and homogenized. The

moisture content (AOAC, 2000) was determined

for the soft tissue samples of M. donacium by

drying them at 95 °C in a forced air drying oven

(H.W. Kessel) until reaching a constant weight.

Total lipid content was determined with the Soxhlet

Soxtec Fat Extraction System 2045, using 25-50

mL of hexane as a solvent (Foss, 1992).

Statistical analysis

The Chi-squared test was used for categorical data

to compare the proportion of each sex and gonadal

development in function diets of M. donacium

individuals. A one-way analysis of variance

(ANOVA) was used to compare total lipid results,

while a multivariate ANOVA (p = 0.05) was used to

determine the influence of temperature on gonad

development and the percent of total lipids. Data

normality and homocedasity were verified before

analyses, which were performed in the SPSS v.21.0

statistical software. If significant differences were

found, an a posteriori Tukey test was applied.

Zevallos & Toledo

Effect of diet on gonadal development in M.

donacium 2

The Chi-squared test (X = 18.46) demonstrated

significant differences in gonad maturity between

the M. donacium samples (p < 0.05) in relation to

the distinct diets during the reproductive

conditioning. In the D2 group, 60% were classified

in stage 3 (mature) of the reproductive cycle,

followed by the C group with 55%; the D1 group

with 50%; and the D3 group with 10%. Likewise,

the D1 group presented the lower percentage of M.

donacium in stage 4 (20%), followed by D2 and C

(35%), and D3, with 70% of individuals in the

spawning stage (Table 1).

Regarding gonadal development at the beginning

of November, the majority of the broodstock were

spawning (stage 4; 44%), while 25% were in

recovery (stage 5). Over the trial period, gonadal

development was progressive, and by the first and

second halves of December, 56 and 50% of the

conditioned individuals respectively reached

maturity (stage 3) (Table 2).

In November, 38% of the M. donacium sampled

from natural ocean conditions were in stage 3,

while 63% were in stage 4. In December, 100% of

the individuals collected from the ocean were

spawning (stage 4).

Gonadal development of broodstock conditioned

M. donacium 2

The Chi squared test (X = 89.54) demonstrated

significant differences (p < 0.05) in sexual maturity

over the period of broodstock conditioning. At the

beginning of November, 44% of the M. donacium

individuals were spawning (stage 4) and 25% were

in recovery (stage 5). Sampling at day 23

evidenced progressive gonadal development

towards maturity (stage 3), with 56 and 50% of the

broodstock respectively reaching this stage by the

first and second halves of December (Table 2).

The D2 diet favored rapid gonad development,

with 50% of this group reaching maturity (stage 3)

during the first 44 days of broodstock conditioning

(16 ±1°C, 3.61 mL/L dissolved oxygen, 34.82 psu).

This treatment also resulted in the release of

gametes, with 50% of the broodstock spawning

RESULTS

The Biologist (Lima). Vol. 15, Nº1, jan - jun 2017

113

(stage 4), with decreased biomass found in the

individuals sampled during this period. A similar

situation was observed with the D1 group fed a diet

comprised of local microalgae.

3.3 Effect of microalgae diets on the lipid content of

M. donacium

One-way ANOVA indicated that there were no

significant differences between the different

dietary groups in regards to total lipids (p > 0.05).

However, a higher amount of lipids was found in

mollusks of the D3 group during the second half of

December, which coincided with 50% of the

female reaching maturity (stage 3) (Table 3). The

total lipid content of broodstock conditioned M.

donacium decreased over the first 13 days before

evidencing a slight increase at day 40 of the trial.

Due to this, the November total lipid content of

laboratory individuals was very similar to that of

M. donacium sampled from the ocean, the majority

of which were in stage 4 or 5 of sexual

development. In December, the total lipid content

of the broodstock conditioned M. donacium

increased, albeit insignificantly, as compared to

November. However, these values were

significantly higher than the December values of

M. donacium sampled from the ocean. These

observations indicate a correlation between lipid

content and gonadal development (until stage 3) as

a result of the local and commercial microalgae

diets consumed during sexual maturation.

Microalgae for Mesodesma

Table 1. Effect of microalgae diets on gonadal development in Mesodesma donacium.

Reproductive Cycle Stage Total

Diets

Number of individuals

10*

% of diet

50% 20% 30%

100%

Number of individuals 12* 7 1 20

% of diet

60% 35% 5%

100%

Number of individuals 2 14 4 20

% of diet

10% 70% 20%

100%

Number of individuals 11* 7 2 20

% of diet

55% 35% 10%

100%

Total

Number of individuals 35* 32 13 80

% of diet

44%

40%

16%

100%

X2= 18.46

0.05*

* indicates signicant differences (p < 0.05)

Table 2. Stage of gonadal development in Mesodesma donacium over time under controlled conditions (three

treatments and a control).

Reproductive

Cycle Stage

Period of Broodstock Conditioning

15 Nov

28 Nov

13 Dec

25 Dec

08 Jan

Nº

% Nº % Nº % Nº % Nº %

Total

0.0

31.3*

43.8

25.0

100

0.0

37.5*

43.8

18.8

100

0.0

56.3*

43.8

0.0

100

0.0

50.0*

50.0

0.0

100

0.0

43.8

18.8

37.5

100

X2= 89.54

0.05*

* indicates signicant differences

0.05).

The Biologist (Lima). Vol. 15, Nº1, jan - jun 2017

114

Zevallos & Toledo

* indicates signicant differences (p < 0.05).

Fo: test statistic

Diet Period of Broodstock Conditioning

15 Nov 28 Nov 13 Dec 25 Dec 08 Jan

2.86±1.92

1.34±0.47

1.11±0.81

0.46±0.25

0.77±0.54

3.16±1.59

1.18±0.71

0.88±0.40

0.50±0.37

1.44±0.22

0.96±0.68

1.32±0.82

0.64±0.45

0.75±0.64

0.66±0.96

Statistics

1.95±0.76

0.70±0.15

1.89±0.88

1.64±0.71

0.40±0.47

*Sig.

0.89

3.49

p>0.05

1.01

3.49

p>0.05

2.64

3.49

p>0.05

0.95

3.49

p>0.05

2.10

3.49

p>0.05

Table 3. Effect of microalgae diets on total lipid content in Mesodesma donacium.

production under controlled conditions would

require better microalga species as a dietary

foundation.

The microalgae diets given to broodstock M.

donacium influenced gonad development. The D2

diet, composed of the local I. galbana and

commercial Ch. gracilis and P. lutheri, was the

most apt for obtaining the greatest percentage of

mature (stage 3) individuals by day 44 of

broodstock conditioning. Following this point,

gametes were released, resulting in 50% of the

broodstock moving into stage 4 (recovery) and a

consequent decrease in the biomass of M.

donacium individuals. A similar situation was

observed in the D1 group fed a diet composed only

of local microalgae. These results contrast with

those obtained by Martínez et al. (2008), who

demonstrated that adult specimens of A.

purpuratus maintained at 16 °C and fed a mix of

microalgae (I. galbana and Ch. gracilis) and fats

resulted in 42,64 ± 4,68% gonad recovery during

the first 10 days of an 80-day broodstock

conditioning period. Martínez (2000) later

indicated that a diet of mixed microalgae enriched

with unsaturated fats led to better results of sexual

maturity and the number of spawning A.

purpuratus.

Lipid content decreased in the gonads of

broodstock conditioning M. donacium during the

first month, but increased slightly during the

second month. Due to this, the lipid content values

Broodstock conditioning was achieved in adult M.

donacium at 44 days through a continuous feeding

regimen that used mixed microalgae local and

commercial diets, whereas M. donacium in the

natural environment evidenced only 38% in stage 3

and the remaining individuals in stage 4. During

December, 100% of the clams sampled from the

ocean were spawning. These observations

corroborate that reported by Salgado & Ishiyama

(1979) about stage partial or total evacuation

evidenced from December to March, and the

results obtained by FIP (2009) where females

evaluated during November in Tacna presented

more than 80% on high maturity and the onset of

spawning occurred in December 2007; and Tejada

concluded that females collected in Tacna during

December 2010 found mostly spawned and 39% in

gonadal recovery period (Tejada, 2009; 2010).

Considering all of this, the mixed diets used in the

treatments provided positive benefits, more

quickly inducing sexual maturity in laboratory-

maintained clams through microalgae commercial

and local obtained from the littoral region of Peru,

as compared to a purely commercial diet.

Considering all of this, the mixed diets used in the

treatments provided positive benefits, inducing

sexual maturity in laboratory-maintained clams

through microalgae obtained from the littoral

region of Peru. This is in line with Borowitzka

(1997), who indicated that better bivalve

DISCUSSION

The Biologist (Lima). Vol. 15, Nº1, jan - jun 2017

115

were initially similar to those of clams sampled

from the ocean, where the majority of adults were

in stage 4 or 5 of the reproductive cycle. However,

the lipid content of captive adults was significantly

greater than that of ocean samples in December,

although these values were not significantly higher

than those of laboratory individuals in November.

This occurrence was related to the progressive

development of the gonad until reaching stage 3,

which in turn was associated with the distinct diets

consumed during the reproductive cycle. The

assessed diets were primarily composed of local I.

galbana (local) and commercial P. lutheri and Ch.

gracilis. According to Ching & Liang (2001), I.

glabana and P. lutheri are dominated by the fatty

acids 18:1n-9 and 18:4n-3 and 20:5n-3,

respectively. This would indicate that these lipids

are a primary energy source, as also supported by

Farías & Uriarte (2002), Hendricks et al. (2003),

and Farías (2002). Specifically, these authors

found lipids to be a necessary element of the adult

bivalve diet during spawning, as well as during

gamete development since energy reserves are

absorbed during gametogenesis (Martínez, 2000).

Fatty acids also play a role in forming part of the

phospholipids present in cell membranes, aiding in

the regulation of fluidity and acclimatization to

different temperature ranges (Farias, 2008).

The temperature of seawater did not influence the

progress of gonadal development in individuals fed

distinct diets. Likewise, the percentage of total

lipids in M. donacium individuals subject to the

experimental trials was not influenced by

temperature. The recorded temperatures fluctuated

within a range of ± 0.6 °C, values that did not

change the process of broodstock conditioning.

Due to this, the progressive variation in gonadal

development is likely attributable to the various

mixed diets given over the experimental period.

This research was carried out thanks to the

logistical support of the Institute of the Sea of Peru

and its own contribution. The authors are grateful

to all the people who provided their support in the

field and experimental phase of this research.

Microalgae for Mesodesma

ACKNOWLEDGMENT

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Microalgae for Mesodesma

Received January 28, 2017.

Accepted March 2, 2017.