Eggs, Eggs Everywhere and Not a Fish to Eat
Larry Buckley, Director
URI-NOAA Cooperative Marine Education and Research Program
The Early Life Dynamics (ELD) research group
at the URI Bay Campus uses biotechnology to study the survival and growth of
marine finfish in their early lifestages. Although marine fish in temperate
climates produce large numbers of very small eggs, most of the fish die within
the first months of life due to predation, starvation, and a variety of other
factors. One female may produce thousands to millions of eggs in a single year.
Most produce small planktonic eggs about 1mm in diameter. Generally, the eggs
are slightly positively buoyant and mixed through the water column, transported
by tides and currents. Other species, like the winter flounder, produce demersal
eggs that are attached to the bottom substrate. Larvae hatch from the eggs within
a period of days to weeks depending upon the species and temperature. The small
(3-5 mm) larvae are planktonic and rely on yolk reserves for a period of days
until they are capable of capturing and digesting planktonic prey. After a few
weeks to several months of rapid growth, the larvae metamorphose to the juvenile
stage when they first begin to resemble the adults.
Mortality is very high during the first months
of life. It is a rare individual that survives to reproduce or to be caught
in a targeted fishery. Many species of marine fishes, including the cod, haddock,
and yellowtail flounder found off the northeast United States, are heavily fished
and abundance levels are low. Reduced catches and restrictions on the fishery
have led to increased interest in the factors that control fish production and
in methods for mass cultivation. The environment is thought to play an important
role in determining growth and survival during the early life stages of fish,
but the linkages and mechanisms have been difficult to establish. Factors such
as temperature, storms, fronts, and eddies are considered critical to feeding
and retention of planktonic larvae in areas favoring rapid growth and survival.
The Early Life Dynamics group uses marine biotechnology to develop methods for
mass cultivation of marine fishes and to establish linkages between the environment
and growth and survival of young fish.
There are three basic approaches that the ELD
group uses to accomplish its mission: Spawning fish in captivity and conducting
experimental studies with embryos, larvae, and juveniles conducted at the National
Marine Fisheries Service (NMFS) Narragansett Laboratory; performing biochemical
and molecular studies of growth and development under controlled conditions;
and conducting field studies. Each of these approaches employs marine biotechnology.
The ELD group pioneered the use of hormone treatments to induce spawning in
marine fish and was among the first laboratories in the world to raise several
marine species including haddock, summer flounder, yellowtail flounder, and
silver hake from captive broodstocks through metamorphosis.
Molecular indicators of growth rate and mortality
risk are developed and calibrated in the laboratory using fish grown under controlled
environmental conditions. Once calibrated and tested, these molecular markers
are used to determine growth rates and mortality risks of fish caught at sea
and to establish linkages among environmental conditions, growth, and mortality.
The development and application of RNA-DNA ratio analysis is illustrative of
the success of this approach.
What can RNA levels in an organism tell us about
growth, nutritional condition, or mortality risk? RNA (ribonucleic acid) is
the class of molecules that translates the information contained in the genetic
code of DNA (deoxyribonucleic acid) into protein and living matter. The three
classes of RNA molecules that carry out protein synthesis are ribosomal RNA
(rRNA), the nucleic acid component of ribosomes; messenger RNA (mRNA), the templates
for individual proteins translated by the ribosomes; and transfer RNA (tRNA),
carriers of specific amino acids to the active ribosomes. Unlike the DNA content
of a cell, which is fixed, RNA levels are actively regulated in response to
the requirements for protein synthesis and the availability of nutrients and
energy. The dynamic nature of RNA regulation provides information on growth
rate, development, and the environmental conditions experienced by an organism.
Based on work with several species reared in
captivity, we were able to demonstrate that the RNA-DNA ratio was higher in
well-fed, fast-growing fish compared to starved or slower-growing fish. Moreover,
we determined that growth of larvae could be estimated from their RNA-DNA ratio
and water temperature. RNA-DNA analysis has provided a reliable and widely used
tool to estimate recent growth of field-caught larvae. Our group, Elaine Caldarone,
Jeanne Burns, and I, is using RNA-DNA analysis to estimate recent growth of
cod and haddock larvae on Georges Bank as part of the US Global Ocean Ecosystems
Dynamics (GLOBEC) Program. Working in collaboration with investigators from
the NMFS Woods Hole Laboratory and other institutions, we have demonstrated
that growth of larvae is food limited at higher temperatures within their range
of tolerance and that growth of larvae in May is highest in waters around 7
degrees C.
Due to the relative abundance of rRNA (75 to
80 percent of total RNA) compared to other classes of RNA and the conservative
nature of DNA, the RNA-DNA ratio primarily reflects changes in rRNA content.
We are examining the utility of selected RNAs and the proteins they code for
(the DNA contains the information used by the cell to synthesize specific proteins)
to provide insight into the effects of the environment on important physiological
processes in fish larvae and juveniles.
Todd Smith, a NOAA-National Research Council
post-doctoral associate with ELD and recent graduate of URI, uses biotechnology
techniques to study juvenile cod, a species that has historically been of enormous
economic importance in New England. Two topics being pursued by Smith are the
use of the RNA-DNA ratio to assess the nutritional condition of juvenile cod
and the effects of changes in diet composition on the activity of digestive
enzymes and the mRNAs that code for these proteins.
Performing nucleic acid assays on larval, juvenile,
or adult fish typically involves homogenizing the whole fish or collecting a
sample of liver or muscle tissue. Either approach requires killing the fish.
But sensitive assays that use a fluorescent dye can measure RNA and DNA in samples
of scales removed from live fish. In cod, for instance, scales are bony plates
embedded in the skin and covered by living tissue. Removing a small number of
scales is not harmful to the fish, and there is sufficient live tissue associated
with the scales to measure the RNA and DNA content. This technique provides
a sensitive, non-invasive means of obtaining estimates of feeding conditions.
Because the process is non-lethal, it can be performed repeatedly on the same
individuals. This may be particularly valuable in studies of rare or endangered
fish species.
Studies of juvenile cod reared under controlled
conditions show that the RNA-DNA ratio in fish changes within a few days of
a change in feeding condition. For example, the RNA-DNA ratio in a well-fed
fish from which food is withheld will begin to decline within two to four days,
indicating that the RNA-DNA ratio obtained from fish scales is a sensitive indicator
of fish feeding condition.
There is a need for the mass culture of fish
for research, stock enhancement, and consumption. Another focus of Smithıs research
is the change in the production of digestive enzymes that occur in larval and
juvenile cod at different stages of their development. Culturing cod and haddock
in captivity, for use as research models and potential aquaculture species,
requires doing so on a relatively large scale. To efficiently culture fish on
this scale, it is necessary to raise them on commercially prepared diets that
will meet all of their nutritional requirements. However, because the physiology,
behavior, and prey of these fish change during their development, the capacity
to digest certain nutrients also changes. It is important to know how digestion
in juvenile cod and haddock is influenced by diet and how well these fish will
grow on commercially prepared diets. Smith has developed a suite of very sensitive
assays for selected digestive enzymes and their mRNAs that should provide insight
into dietary requirements in the culture and nutrition of fish caught at sea.
Tun Liang Ong, a GSO marine scientist who works
in the ELD group, heads up an NSF-funded project that uses selected RNA markers
coupled with risk assessment techniques to assess the mortality risk of cod
larvae. The insurance industry uses risk assessment to estimate life expectancy
and accident rates to set insurance and annuity premiums and to allocate reserves
and dividends. For example, to apply for life insurance coverage, an applicant
provides information on his/her age, gender, personal health history, behavior,
living habits, and family health history. Using that information, the insurance
company calculates the risks of an individual dying at a certain age and establishes
the premium rates. Can a similar approach be used to study and manage living
marine resources? Using Atlantic cod larvae as a model, Ong is studying the
effect of starvation and its relation to predation on mortality risks of marine
fish larvae. A series of laboratory experiments was conducted to measure the
responses of selected markers of starvation to changes in feeding, development,
and growth in Atlantic cod. Our earlier work established that a rapid decline
in a specific ribosomal RNA (18s rRNA) to minimum levels can be used as a marker
for the initial stages of starvation in Atlantic cod larvae. A dramatic increase
in the mRNA levels of the protein metallothionein (MT) was indicative of starvation.
These RNA markers are being used to pinpoint when larvae begin to recover from
starvation after refeeding. The power of estimating mortality risks will greatly
increase if the point of recovery from starvation can be established. Animal
and human studies showed that dietary deprivation leads to an interruption of
protein synthesis and a reduction in the number of ribosomes. Refeeding after
starvation resulted in a significant increase in the levels of rRNAs.
We anticipate that RNA markers of mortality risks,
when used concurrently with RNA-DNA ratio analysis, will provide a description
of the short-term growth, condition, and starvation-related mortality risks
of individual fish larva collected at sea. This information, together with detailed
observation on the physical and biotic environment, should lead to a better
understanding of growth and mortality during the critical early life stages
of larval fish and the effects of the environment on fish production.