| Search the Collection|Browse Subjects|Services|Library Information|Community |News & Events | |
|
|
|
|
|
|
|
|
||||
|
|||||||
Type of Document Dissertation Author Luckenbach, John Adam, URN etd-11012004-212652 Title Breeding Biotechnology, Sex Determination, and Growth in Southern Flounder, Paralichthys lethostigma Degree PhD Graduate Program Zoology Advisory Committee
Advisor Name Title John Godwin Committee Co-Chair Russell J. Borski Committee Co-Chair Edward J. Noga Committee Member Harry V. Daniels Committee Member Keywords
- aromatase gene expression
- induction of gynogenesis
- teleost fish
- aquaculture
- environmental sex determination
- gonads
- sex differentiation
Date of Defense 2004-10-29 Availability unrestricted Abstract LUCKENBACH, JOHN ADAM. Breeding Biotechnology, Sex Determination, and Growth in Southern flounder, Paralichthys lethostigma. (Under the direction of Dr. John Godwin and Dr. Russell J. Borski).
Southern flounder (Paralichthys lethostigma) support valuable, but declining US fisheries. This species is therefore a strong candidate for aquaculture to mitigate fishing
impacts and stabilize seafood supply. Because female flounder reach substantially larger
sizes than males, all-female culture is desirable for commercial aquaculture. Hence, a thorough understanding of sexual development, its timing and regulation by temperature
is essential for optimization of flounder aquaculture.
To better understand ovarian and testicular development in southern flounder,
structural and cellular sex-distinguishing markers were studied using histological
methods. We found that histologically discernible sex differentiation occurs in southern
flounder at ~75-120 mm TL and that early differentiation is considerably delayed relative
to its Japanese congener, P. olivaceus. High (28°C) and low (18°C) water temperatures,
produced a higher proportion of males (96% and 78% males, respectively). The sex ratio
at a mid-range (23°C) temperature was not different from 1:1. This suggests that
southern flounder possess a temperature sensitive mechanism of sex determination.
Growth was also affected by temperature with the temperature that maximized females
inducing better growth.
Aromatase cytochrome P450 (P450arom) is responsible for estrogen biosynthesis
and plays a critical role in ovarian differentiation. We cloned ovarian P450arom and
developed a qRT-PCR for assessment of early sex differentiation. The deduced amino
acid sequence for southern flounder P450arom is very similar to P450arom in other teleosts.
Comparison of P450arom intron sequences of fish within and between different
populations revealed substantial inter-individual variation that may affect sex
determination responses. Ovary and spleen exhibited high levels of P450arom mRNA,
while P450arom was weakly detected in testis, brain, and gill. Gonads sampled from wild
flounder spanning the period of sex differentiation initially exhibited a low level of
P450arom gene (fish 40-55 mm TL) followed by bifurcation into two clearly segregated groups beginning at ~65 mm TL. Through histology we show high and low P450arom
mRNA relates to ovarian and testicular differentiation, respectively. This work imparts a
powerful tool for better understanding mechanisms of sex determination and rapidly
defining conditions that influence sex.
Effective methods for induction of diploid gynogenesis in southern flounder are
needed to initiate all-female culture. To test methods for inducing gynogenesis in this
species, four treatments, named for their expected outcome, were employed: haploid,
diploid, triploid, and gynogenetic diploid. Diploid gynogenesis was induced by
activating egg development with UV-irradiated flounder sperm (70 J/cm2) and then ?cold shocking? the eggs to prevent second polar body extrusion. Control treatments omitted
one or more of these steps to separately assess the effectiveness of UV irradiation and
cold shock. Haploid larvae exhibited abnormal external morphology while diploid,
gynogenetic diploid, and triploid larvae appeared normal. Erythrocyte nuclear sizes for
treatment groups corresponded to predicted ploidy (triploid>diploid>haploid) and did
not differ between normal diploids and gynogenetic diploids, suggesting that our
procedures were successful. To eliminate any possible paternal genetic contribution
during gynogenesis, activation of flounder eggs with striped mullet (Mugil cephalus) sperm was also tested. Induction of diploid gynogenesis was successful when flounder eggs were
fertilized with irradiated mullet sperm and cold shocked. This work provides procedures for induction of diploid gynogenesis in southern flounder and validates a method for verification of ploidy in larval fishes.
These studies establish the schedule of southern flounder sex differentiation based
on results of gonadal histology and patterns of P450arom gene expression. We also
demonstrate that sex determination in this species is temperature sensitive. This
knowledge, along with methods for induction of gynogenesis, can be utilized for creation
of all-female southern flounder populations for aquaculture. These findings may also
benefit fisheries management by providing methods for sex assessment and guiding stock
enhancement.
Files
Filename Size Approximate Download Time (Hours:Minutes:Seconds)
28.8 Modem 56K Modem ISDN (64 Kb) ISDN (128 Kb) Higher-speed Access etd.pdf 4.74 Mb 00:21:56 00:11:16 00:09:52 00:04:56 00:00:25