Part C | 18.4
480 Part C Marine Genomics
val Atlantic cod (Gadus morhua), 2D-PAGE and MS
were used to identify changes in the expression of larval proteins [18.40]. Interestingly, the study found that
the five identified cod larval proteins that were downregulated in the probiotic-treated larvae function in the
immune system of the fish, while two of the four upregulated proteins identified in the probiotic-treated larvae were ˛-actins from skeletal muscle. Sveinsdóttir
et al. [18.40] concluded that probiotic treatment may
have decreased the effect of environmental stress on
the larvae, resulting in increased growth and development as indicated by decreased expression of immune system proteins and increased ˛-actin expression,
respectively.
Proteomics has also proved to be invaluable
for investigating the virulence response of bacterial
pathogens of marine organisms. Although members of
the genus Vibrio are ubiquitous in the ocean, a number of species are pathogenic to sea urchins, oysters,
abalone, and fish, and thus are a major threat to aquaculture operations farming these animals. Vibrio splendidus is known to infect and cause disease in fish
and shellfish, and is responsible for major mortalities of farmed animals [18.41]. The virulence mechanism of this bacterium towards marine invertebrates
is poorly characterized due to the absence of molluscan cell lines, making it necessary to employ an ex
vivo approach to investigate Vibrio splendidus virulence. Consequently, Binesse et al. [18.42] conducted
a study of the extracellular products (ECPs) of the oyster pathogen Vibrio splendidus LGP32 which induce
a strong cytopathic effect in infected oysters. Since
the ECPs of a vsm strain, a metalloprotease deletion mutant of LGP32, were significantly less toxic,
a proteomic analysis of the ECPs of both LGP32 and
the vsm mutant was performed to identify additional
cytotoxic proteins [18.42]. One of the differentially
expressed proteins (VSA1062), found to have 41%
identity to the Bacillus thuringiensis virulence factor
immune inhibitor A precursor (InHA), was identified as
a metalloprotease. The observation that the cytotoxicity
of the ECPs of a vsm-1062 double mutant was similar
to that of the vsm mutant even though metalloprotease
activity could not be detected in the vsm-1062 mutant, led Binesse et al. [18.42] to discard the hypothesis
that the VSA1062 metalloprotease compensated for the
loss of VSM activity in the vsm mutant. Instead, they
proposed that VSA1062 plays a role in bacterial resistance to the oyster immune response in a similar
manner to InHA which functions in antibacterial peptide resistance.
In another study, the cellular and extracellular protein profiles of two Vibrio anguillarum strains, Vibrio anguillarum MN and 3010, were compared by
2D-PAGE and MALDI-TOF/TOF analysis since Vibrio anguillarum MN is 50-fold more virulent than
3010 towards the Japanese flounder Paralichthys olivaceus [18.43]. Two of the nine spots found to be
differentially expressed between the two strains were
identified as the outer membrane protein OmpU and
the metalloprotease PrtV, both of which were highly upregulated in Vibrio anguillarum MN. Since OmpU increases the bacterium’s resistance to bile [18.44], allowing it to colonize the fish digestive system, while PrtV
from closely related Vibrio cholera is cytotoxic and
can degrade extracellular matrix components such as fibronectin, fibrinogen, and plasminogen [18.45], Zhao
et al. [18.43] concluded that these proteins most likely
participate in the virulence response of V. anguillarum.
18.4.2 Reproduction
Aquaculture of commercially important marine animals relies on breeding healthy, fast-growing, highquality animals. Consequently, a great deal of research
and development has been invested in larviculture and
hatchery design. As the aquaculture industry expands,
there is a growing reliance on hatchery-produced seed
for restocking farms. Many hatcheries maintain broodstock animals that are artificially induced to spawn
in relation to a schedule based on the routine of the
farm. Up to now, broodstock animals have either been
obtained from the wild and cared for under optimal
conditions or from what are perceived as being fastgrowing animals produced on the farm and retained for
breeding. However, it is becoming increasingly evident
that a more directed approach to selecting broodstock
for seed production is required in order to ensure production of robust, fast-growing spat.
Some studies have been published that employed
a proteomics approach to address various challenges associated with breeding marine animals for commercial
production. For example, it is necessary to determine
that the gonads of broodstock animals have reached
the appropriate developmental stage in order to induce spawning. The most common approach to this is
visual inspection, which is acceptable for gastropods
such as abalone, but inappropriate for bivalves such as
oysters. Li et al. [18.46] employed surface enhanced
laser desorption/ionization (SELDI) technology (ProteinChip Array System, Ciphergen Biosystems, Fremont, CA, USA) to obtain haemolymph protein profiles
480 Part C Marine Genomics
val Atlantic cod (Gadus morhua), 2D-PAGE and MS
were used to identify changes in the expression of larval proteins [18.40]. Interestingly, the study found that
the five identified cod larval proteins that were downregulated in the probiotic-treated larvae function in the
immune system of the fish, while two of the four upregulated proteins identified in the probiotic-treated larvae were ˛-actins from skeletal muscle. Sveinsdóttir
et al. [18.40] concluded that probiotic treatment may
have decreased the effect of environmental stress on
the larvae, resulting in increased growth and development as indicated by decreased expression of immune system proteins and increased ˛-actin expression,
respectively.
Proteomics has also proved to be invaluable
for investigating the virulence response of bacterial
pathogens of marine organisms. Although members of
the genus Vibrio are ubiquitous in the ocean, a number of species are pathogenic to sea urchins, oysters,
abalone, and fish, and thus are a major threat to aquaculture operations farming these animals. Vibrio splendidus is known to infect and cause disease in fish
and shellfish, and is responsible for major mortalities of farmed animals [18.41]. The virulence mechanism of this bacterium towards marine invertebrates
is poorly characterized due to the absence of molluscan cell lines, making it necessary to employ an ex
vivo approach to investigate Vibrio splendidus virulence. Consequently, Binesse et al. [18.42] conducted
a study of the extracellular products (ECPs) of the oyster pathogen Vibrio splendidus LGP32 which induce
a strong cytopathic effect in infected oysters. Since
the ECPs of a vsm strain, a metalloprotease deletion mutant of LGP32, were significantly less toxic,
a proteomic analysis of the ECPs of both LGP32 and
the vsm mutant was performed to identify additional
cytotoxic proteins [18.42]. One of the differentially
expressed proteins (VSA1062), found to have 41%
identity to the Bacillus thuringiensis virulence factor
immune inhibitor A precursor (InHA), was identified as
a metalloprotease. The observation that the cytotoxicity
of the ECPs of a vsm-1062 double mutant was similar
to that of the vsm mutant even though metalloprotease
activity could not be detected in the vsm-1062 mutant, led Binesse et al. [18.42] to discard the hypothesis
that the VSA1062 metalloprotease compensated for the
loss of VSM activity in the vsm mutant. Instead, they
proposed that VSA1062 plays a role in bacterial resistance to the oyster immune response in a similar
manner to InHA which functions in antibacterial peptide resistance.
In another study, the cellular and extracellular protein profiles of two Vibrio anguillarum strains, Vibrio anguillarum MN and 3010, were compared by
2D-PAGE and MALDI-TOF/TOF analysis since Vibrio anguillarum MN is 50-fold more virulent than
3010 towards the Japanese flounder Paralichthys olivaceus [18.43]. Two of the nine spots found to be
differentially expressed between the two strains were
identified as the outer membrane protein OmpU and
the metalloprotease PrtV, both of which were highly upregulated in Vibrio anguillarum MN. Since OmpU increases the bacterium’s resistance to bile [18.44], allowing it to colonize the fish digestive system, while PrtV
from closely related Vibrio cholera is cytotoxic and
can degrade extracellular matrix components such as fibronectin, fibrinogen, and plasminogen [18.45], Zhao
et al. [18.43] concluded that these proteins most likely
participate in the virulence response of V. anguillarum.
18.4.2 Reproduction
Aquaculture of commercially important marine animals relies on breeding healthy, fast-growing, highquality animals. Consequently, a great deal of research
and development has been invested in larviculture and
hatchery design. As the aquaculture industry expands,
there is a growing reliance on hatchery-produced seed
for restocking farms. Many hatcheries maintain broodstock animals that are artificially induced to spawn
in relation to a schedule based on the routine of the
farm. Up to now, broodstock animals have either been
obtained from the wild and cared for under optimal
conditions or from what are perceived as being fastgrowing animals produced on the farm and retained for
breeding. However, it is becoming increasingly evident
that a more directed approach to selecting broodstock
for seed production is required in order to ensure production of robust, fast-growing spat.
Some studies have been published that employed
a proteomics approach to address various challenges associated with breeding marine animals for commercial
production. For example, it is necessary to determine
that the gonads of broodstock animals have reached
the appropriate developmental stage in order to induce spawning. The most common approach to this is
visual inspection, which is acceptable for gastropods
such as abalone, but inappropriate for bivalves such as
oysters. Li et al. [18.46] employed surface enhanced
laser desorption/ionization (SELDI) technology (ProteinChip Array System, Ciphergen Biosystems, Fremont, CA, USA) to obtain haemolymph protein profiles