Contract Title:
1988-053-03 EXP PARKDALE NOAA COMPARATIVE HATCHERY STUDY
Contract Start Date:
12/1/2012
Contract End Date:
11/30/2013
Title:
E: 157 - Hood River Steelhead Residual monitoring
Description:
The objective of this Work Element is to determine residualism rates in Hood River Steelhead
Background
Hatchery-reared steelhead may adopt one of three life history pathways after release: 1) smolt and migrate to sea 2) sexually mature and remain in freshwater and attempt to spawn naturally, or 3) "residualize" as a non-smolting and non-maturing parr and delay migration or maturation 'decisions' until the subsequent year(s). The physiological 'decision' to mature in freshwater or undergo smoltification and migrate to sea is commonly described as a conditional strategy under polygenic control in which expression of a given phenotype depends on exceeding some genetically determined threshold (Roff 1996). This phenomenon has been thoroughly described in Atlantic salmon where studies have shown that individuals whose size, growth rate and/or energetic condition exceeds some genetically determined threshold are more likely to mature precociously (Myers and Hutchings 1986, Aubin-Horth and Dodson 2004, Pich et al. 2008). A similar threshold mechanism likely determines whether a fish undergoes smoltification or remains as a non-maturing parr as well (Thorpe et al. 1998, Thorpe 2007).
Maturation
In another study in the Methow River basin under BPA project # 1993-056-00 steelhead residualism rates are currently being examined. In this investigation, we are applying the same methods to measure a suite of physiological indicators to predict age-at-maturity in hatchery-reared steelhead approximatley one year prior to spermiation. The hallmark of initiation of maturation in males is activation of the brain-pitutiary-gonad axis (BPG), or more specifically, enhanced production and release of gonadotropin-releasing-hormone (GnRH) by the brain, follicle-stimulating hormone (FSH) and leutinizing hormone (LH) by the pituitary gland and 11-KT by the testis (Campbell et al. 2003, and reviewed by Schulz et al. 2010, Taranger et al. 2010). The activation of the BPG axis causes the rapid proliferation of spermatogonia, the first phase of spermatogenesis that precedes meiosis (Schulz et al. 2010). Antimullerian hormone (AMH) is a growth factor produced in Sertoli cells in the testis that inhibits the differentiation and proliferation of spermatogonia (reviewed in Schulz et al. 2010) and a decline in AMH is required for spermatogenesis to proceed. Thus, initiation of maturation of males can be detected at the very earliest stages, by monitoring several points of the reproductive axis for the following changes: 1) increases in mRNA or protein levels for hypothalamic GnRH, pituitary FSH and LH, and testicular steroidogenesis related genes; 2) decreases in mRNA levels of AMH in the testis; 3) increases in plasma levels of 11-KT; and 4) enhanced proliferation of spermatogonia in the testis. Theoretically the earliest changes should occur at higher levels of the endocrine cascade. Increases in FSH and LH and testicular steroidogenesis related genes and decreases in testicular AMH precede the increase in 11-KT levels, making these parameters useful as a monitoring tool to identify maturing males at earlier time points to identify male steelhead that would mature one year (or less for Winter Run stocks) after release from the hatchery.
Smoltification
Determining which fish are in fact smolts versus immature male or female parr is another essential step in accurately quantifying rates of residualism. Unlike efforts to detect early male maturation 12 months in advance of spermiation, a large portion of the hatchery fish sampled just prior to release are typically at the peak of smolting and physiological indicators at the level of the pituitary and the gill may be employed to detect individuals that are not smolts (see Folmar and Dickhoff 1981, Beckman et al. 1999). Two potential targets are the thyroid axis and the gill Na+, K+ ATPase system. Dickhoff et al. (1982) demonstrated that thyroid hormones are elevated in steelhead at the time of smolting. We propose to measure mRNA for the pituitary hormone, thyroid stimulating hormone (TSH). TSH stimulates activation of the thyroid axis and as part of our transcript analysis for LH and FSH, we will analye pituitary TSH mRNA levels. The up-regulation of gill Na+, K+ ATPase activity is considered a benchmark indicator of smoltification and Ewing et al. (1984) demonstrated that gill Na+, K+ ATPase activity was higher in migrant versus non-migrant steelhead.
Methods
Sample collection
In May 2013 we will sample 300 randomly selected steelhead at Parkdale Fish Facility. Individual fish will be anesthetized with buffered 0.05% tricaine methanesulfonate (MS-222, Argent Chemical Laboratories, Redmond, WA) for blood collection and then euthanized by decapitation. We will use a smolt index with 3 rating categories. A value of "1" will be assigned to fish that have not smolted and still retain the physical characteristics of parr. A value of "2" will be assigned to fish that have not fully smolted, but are beginning to show indications of smoltification such as parr marks beginning to fade and development of silver coloration. A value of "3" will be assigned to fully smolted fish, indicated by complete silver coloration, absence of parr marks, and blackening of the snout, and edges of the dorsal and caudal fin. Prior to blood collection, fish will be weighed (g) to the nearest 0.1 g and measured for fork length (mm). Blood will be collected from the caudal vasculature using heparinized Natelson tubes after severing the caudal peduncle with a single-edge razor blade, stored on ice and then centrifuged at 800 x g for 5 min. Plasma will be removed and stored frozen on dry ice until transport to the laboratory where it is stored at -80 degrees C. Gill arches from each fish will be placed in a solution of sucrose, EDTA, and imidazole according to methods described by Zaugg (1982) and then frozen on dry ice and stored at -80 C for enzyme analysis. Fish will be dissected to identify gender and stage of maturity for males. Paired testes will be removed and weighed to the nearest 0.001 g. One testis will be fixed in 0.5 ml of Histochoice (AMRESCO, Solon, OH) in a 1.5 ml polypropylene microfuge tube for histological analysis. The other testis will be preserved in 0.5 ml RNAlater (Qiagen, Valencia, CA) in a 1.5 ml polypropylene microfuge tube for analysis of mRNAs for genes associated with spermatogenesis. Pituitary glands will be removed and preserved in 0.5 ml RNAlater for analysis of mRNAs for gonadotropin subunits. Tissue samples will be transported to the NWFSC laboratory in Seattle and after one week in RNAlater, the solution will be removed and tissues stored at -80 degrees C. Testis samples will be preserved in Davidson's fixative for two days then transferred to 70% ethanol for long-term storage.
All samples will be analyzed under a sibling contract with the University of Washington.
WE Agreement Type:
Contracted
Deliverable Specification:
1) Sample 300 Hood River Steelhead trout for length, weight, condition factor, gender, gonad weight for GSI in May of 2013. This includes identifying any age-1 precocoius parr males that will be running milt at the time of sampling.
2) Visually assess smolt status on a scale of 1 =parr, 2 = transitional, 3 = smolt.
3) Collect 300 gills for analysis of Na+/K+-ATPase activity (enzymatic indicator of smolt status) in 300 Hood River steelhead (males and females).
4) Collect pituitary glands for measurement of FSH and LH (maturation) and TSH (smolting) and tests for gonadal AMH (maturation) transcript levels in 150 male Hood River steelhead.
5) Collect testes (from pair in deliverable 4 above) from approximately 150 male Hood River steelhead trout for histological evaluation.
6) Measure plasma 11-KT levels in approximately 150 male Hood River steelhead trout.
WSE Effective Budget:
$20,000
% of Total WSE Effective Budget:
28.57%
WSE Completion:
05/31/2013
WSE ID Continued From:
n/a
Finite or Recurring:
Recurring
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