Embryonic Developmental Progression in Lake Trout (Salvelinus namaycush) (Walbaum, 1792) and Its Relation to Lake Temperature

J. Great Lakes Res. 31:187–209 Internat. Assoc. Great Lakes Res., 2005

Embryonic Developmental Progression in Lake Trout (Salvelinus namaycush) (Walbaum, 1792) and Its Relation to Lake Temperature Jeffrey D. Allen1,*, Glenn K. Walker2, Jean V. Adams1, S. Jerrine Nichols1, and Carol C. Edsall1 1USGS-Great

Lakes Science Center 1451 Green Road Ann Arbor, Michigan 48105 2Department

of Biology Eastern Michigan University Ypsilanti, Michigan 48197 ABSTRACT. Developmental progression of lake trout (Salvelinus namaycush) embryos was examined with light and scanning electron microscopy. From this examination, key developmental stages were described in detail. The key developmental stages were then applied to individual lake trout egg lots incubated in constant temperatures of 2, 4, 6, 8, and 10°C. We used Belehradek’s, Thermodynamic, and Power models, and also developed the Zero model to determine stage specific developmental rates of lake trout eggs for each background temperature. From the models, hatch dates and staging were predicted for temperature regimes from Lake Superior (1990–91) and Lake Huron (1996–97). Based on the existing lake temperature data and the observed spawning dates, the Zero and the Power models predict that post peak spawning may contribute significantly to overall recruitment success for these years. INDEX WORDS: Salvelinus namaycush, lake trout, development, embryo, scanning electron microscopy, temperature.

INTRODUCTION During the 1950s, the combined effects of sea lamprey Petromyzon marinus (Linnaeus, 1758) predation, over fishing, and habitat and water quality degradation nearly drove the lake trout Salvelinus namaycush (Walbaum, 1792) to extinction. In an attempt to rebuild the adult lake trout populations to historic levels, intensive culture and stocking of the remaining strains began in all of the Great Lakes. With the development of an effective lampricide, tight harvest restrictions, and improvements in habitat and water quality, adult lake trout population levels began to increase. It was hoped that the increase in adult populations would lead to a naturally reproducing and sustainable lake trout fisheries. In Lake Superior, success was achieved in the 1980s when lake trout were able to re-establish natural reproduction to the levels necessary for effective recruitment of the progeny into the adult *Corresponding

population. This was attributed to reduced adult mortalities due to sea lamprey predation (Selgeby et al. 1995). Outside Lake Superior, recruitment success remained relatively elusive in the lower lakes, with only a few areas showing natural reproduction and limited recruitment (Cornelius et al. 1995, Elrod et al. 1995, Eshenroder et al. 1995, Hansen et al. 1995, Holey et al. 1995). At first, failures during early embryonic development were attributed to high levels of environmentally persistent chemicals in the adults and eggs. These chemicals were thought to impact recruitment through increased egg mortality and a greater incidence of developmental deformities (Stauffer 1979, Spitsbergen et al. 1991, Walker et al. 1991, Mac and Schwartz 1992, Mac et al. 1993). However, continued reductions in contaminant loadings in the adults due to bans and restrictions of persistent chemicals did not lead to a substantial increase in successful reproduction and recruitment. Since the mid-1990s, the continued lack of recruitment success in most of the lakes has been attributed to Early Mortality Syn-

author. E-mail: [email protected]

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drome (EMS) and the role of thiamine and thiaminase in the food web (Brown et al. 1998, Honeyfield et al. 1998, Fitzsimons et al. 1999). Whatever the cause, the main failure in restoring self-sustaining stocks in the lower lakes is still thought to occur during the first year of life. Overall, the timing of reproduction, embryonic development, hatching, and swim-up plays a key role in successful recruitment during the first year of life. However, little data exist because of the difficulty of sampling during the winter months when embryonic development and hatching occurs. Because of this, little is known about how environmental conditions influence hatch and survival. Modeling time scale developmental progression of lake trout embryos and applying these data to lake temperature data may help delineate critical factors involved in successful recruitment of fry. There were two specific goals of this study 1. Describe in detail key developmental stages of lake trout development. 2. Model embryonic developmental progression, using lake temperature data, leading to a predictive tool for staging and hatching. MATERIALS AND METHODS Embryonic Development Eggs used for detailed stage descriptions were collected in the fall of 1997 from spawning shoals off Sturgeon Bay, WI by the Wisconsin Department of Natural Resources. Pooled milt from several ripe males was added to eggs from individual females. The samples were stirred with a feather and set aside for 15 minutes, after which they were washed with fresh lake water and allowed to water-harden. Each egg lot was then transferred into separate 568ml plastic containers and transported within 24 hours of fertilization to the USGS-Great Lakes Science Center (Ann Arbor, MI). Temperature was maintained at 3–5°C. At the laboratory, each lot was separated into individual Heath incubation trays. The eggs were maintained in a flow-through system of well water at 7°C until hatching. Ten to twenty eggs were sampled daily for histological and Scanning Electron Microscopy (SEM) examination. Embryonic development was classified using established general intervals of ontogeny (Balon 1980, Kimmel et al. 1995).

Scanning Electron Microscopy (SEM) Embryos were fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.4) at 4°C for 1 hour, after which the chorion was removed with watchmaker forceps. The embryos were rinsed in three changes of fresh buffer and post-fixed in 1% buffered osmium tetroxide for 1 hour at room temperature. Samples were dehydrated through a graded series of ethanol and critical-point drying using liquid carbon dioxide. The embryos were mounted on aluminum stubs with colloidal graphite, vacuum dried overnight, sputter-coated with gold, and examined with an Amray 1820 I SEM at 10 kV. Light Microscopy Embryos were fixed in 10% neutral acetate buffered formalin at 4°C and stored at room temperature until processing. Immediately before processing, the chorion was removed using watchmaker forceps. Processing involved rinsing in filtered well water, dehydration through a graded ethanol series, and infiltration with L.R. White embedding media. Embryos were placed in gel capsules containing fresh embedding media and left overnight at 4°C. Blocks were hardened at 60 ± 1°C, trimmed, and sectioned. Sections were floated on glass slides, adhered to the slide by drying for two minutes in a microwave oven, then stained with an aqueous solution of 1% sodium borate and 1% toluidine blue. Slides were rinsed with reverse osmosis water, dried in a microwave oven, and mounted in Permount. Egg Development at Different Water Temperatures The data used for staging came from unpublished data obtained from Tom Edsall collected in 1972/73. The eggs used for monitoring development at different temperatures were Marquette brood stock from Marquette State Fish Hatchery (Marquette, MI). Eggs were collected, fertilized, and held overnight. The next day they were transported to the Jordan River National Fish Hatchery (Elmira, MI) and later to the USGS-Great Lakes Science Center, where they were placed into five different constant temperature profiles: 2, 4, 6, 8, and 10°C. At the Ann Arbor facility, eggs, water temperature, and dissolved oxygen levels were monitored daily. Ten eggs from each temperature

Embryonic Developmental Progression profile were examined daily, staged, and returned to the original tank. Data Analysis We used three existing models to determine stage specific developmental rates of lake trout eggs (Hamel et al.1997). Power: D = aT b

(1)

D = a(T − t0 )b

(2)

Belehradek’s:

Thermodynamic: 1 + exp D=

1   ∆H L  1 −   R  T K   1/ 2t

(3)

K 1   ∆H A  1 ρ(25°C ) exp  −   R  298 K   298

We then fitted a fourth model, forcing the developmental rate to be zero at 0°C. We refer to this as the Zero model. Zero:  T  D = a  b − T 

c

(4)

We modeled the developmental rate of lake trout eggs, D, as the fraction of development (from spawning to stage) per day observed for eggs held at different background temperatures. In Equations 1, 2, and 4, T is temperature in degrees Celsius, and a, b, and c are constants. In Equation 3, the Thermodynamic model was used as described by Hamel et al. (1997). Here K is temperature in degrees Kelvin, R = 1.987 cal⋅deg–1⋅mol–1 (Universal Gas Constant), and the constants were ∆ HL = change in enthalpy associated with low temperature inactivation of an enzyme (cal⋅mol–1), T1/2t = Kelvin temperature where enzyme is half active and half low-temperature inactive, ρ(25°C) = developmental rate at 25°C, and ∆ HA = enthalpy of activation of the reaction catalyzed by the rate controlling enzyme (cal⋅mol –1 ). We used nonlinear regression (SYSTAT) to estimate the parameters a, b, c, ∆ HL, T 1/2t, ρ (25°C), and ∆ H A. Equations 1 through 4 were then used to calculate developmental staging

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and timing of hatch using lake temperature and spawning data obtained from: 1) Lake Superior spawning reef at Gull Island Shoal (Bronte et al. 1995), and 2) Lake Huron spawning reef at Six Fathom Bank (Savino et al. 1999). RESULTS Embryonic Development and Staging Embryonic development was examined, and detailed descriptions of 11 stages were developed. Results from the staging data can easily be grouped into developmental Milestones. Milestone 1 includes spawning, fertilization, cleavage, and blastulation, which relates to cellular proliferation. Milestone 2 includes epiboly, gastrulation, and early neurulation, which relates to the organism laying down the general body plan. Milestone 3 includes segmentation and neurulation, which relates to refinement of the body plan. Milestone 4 includes pharyngular processes which structures cranial-facial development, organogenesis, and development until hatch. Milestone 1 Stage 1—Fertilization to early blastula Cleavage follows a meroblastic, telolecithal, discoidal pattern [Fig. 1(a), (c)]. At approximately the 32-cell stage, the cell mass region itself has begun differentiating into three distinct layers: the nonmarginal blastomeres, deep cells, and the marginal blastomeres [Fig. 1(a), (b), (c)]. The marginal blastomeres (those closest to the yolk), continue to undergo incomplete division [Fig. 1(c)] and maintain cytoplasmic connections, with the yolk cell region further developing the proteolytic zone [Fig. 1(b)]. Stage 2—Early to late blastula Important aspects in the transition from early to middle blastula are shown in Figure 1(a′), where the enveloping layer and the deep cells become more distinguished. Changes can be seen in the marginal blastomeres, which begin to collapse into the yolk cell [Fig. 1(b), (b′)]. This development begins the formation of the multinucleated yolk syncytial layer (YSL). During the transition between middle to late blastula, the epithelial sheet forms a single well-distinguished layer with tight junctions. The marginal blastomeres have completely col-

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Embryonic Developmental Progression TABLE 1.

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List of abbreviations.

List of Abbreviations 4 fourth ventricle am axial mesoderm az active zone bc brachet’s cleft bd blastodisc br brain rudiment bv blood vessel c cerebellum cm cytoplasmic membrane cp cleavage plain ct cartilage d deep cell de desmosomes e ectoderm ea embryo anlage ed endoderm el enveloping layer ep epiblast ept eye pigment erc ectodermal ridge cell es embryonic shield fb forebrain g gill ga gill arch gl ganglion layer gr germ ring grc germ ring closure h head hb hindbrain ht heart hyp hypoblast inl inner nuclear layer int intestine ipl inner plexiform layer l lens la lamella lgi lower gastrointestinal lm lower mandible lp lipid pillow lrc layer of rods and cones

lv lvr m mb mcm mrb n nc ne nfa nmb nt o oc olp onl opl opp opt otc otv ov pe pf pm pz s sac sag sp st t tb tbs tc ugi um y yc ysl

lens vesicle liver macula midbrain mesenchymal cell migration marginal blastomere nucleus notochord neuroectoderm nerve fiber area nonmarginal blastomere neural tube operculum optic cup olfactory pit outer nuclear layer outer plexiform layer optic primordium optic tectum otic capsule otic vesicle optic vesicle pigment epithelium pectoral fin paraxial mesoderm proteolytic zone somite sacculus sagitta spleen stomach tegmentum tailbud transverse brain subdivision telencephalon upper gastrointestinal upper mandible yolk region yolk cell yolk syncytial layer

FIG. 1. MILESTONE 1. STAGE 1—FERTILIZATION TO EARLY BLASproteolytic zone; y = yolk region (see Table 1 for TULA (Cross sections). (a) Light micrograph of the complete list of abbreviations). blastodisc and lipid pillow, showing incomplete STAGE 2 – EARLY TO LATE BLASTULA. (a′′) Light micrograph of early blastula showing division in the marginal blastomeres, complete divienveloping layer, deep cells, and the marginal sion in non-marginal blastomere cells, and location blastomeres (bar = 50 µm). (b′′) Light micrograph of the deep cells, lipid pillow, proteolytic zone, and of deep cells associated with the developing YSL yolk region (bar = 50 µm). (b) Light micrograph of (bar = 10 µm). (c′′) Light micrograph of mid-stage the proteolytic zone between marginal blastomeres blastula showing deep cells, and yolk syncytial and the lipid pillow/ yolk region (bar = 10 µm). (c) layer (bar = 25 µm). Cm = cytoplasmic membrane; Light micrograph of two marginal blastomeres de = desmosomes; d = deep cell; el = enveloping showing the incomplete cell division (bar = 10 µm). layer; nmb = nonmarginal blastomeres; n = bd = blastodisc; cp = cleavage plain; d = deep cell; nucleus; ysl = yolk syncytial layer (see Table 1 for lp = lipid pillow; mrb = marginal blastomeres; n = complete list of abbreviations). nucleus; nmb = nonmarginal blastomeres; pz =

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Embryonic Developmental Progression lapsed into the proteolytic zone, forming the YSL [Fig. 1(b′), (c′)]. As the blastula stage progresses, cell division continues, and the ratio of cytoplasm to nuclear volume in the deep cells decreases [Fig. 1(a), (a′), (b′), (c′)]. Milestone 2 Stage 3—Germ ring and embryonic shield to 1⁄4 epiboly The blastula begins to flatten down on top of the yolk cell and migrate vegetally [Fig. 2(a), (b)]. This vegetal migration of the blastula is termed epiboly. The area in front of the marginal epithelial cells appears to become active, possibly indicating the extension of microvilli into the yolk cell, which is the process that drives epiboly [Fig. 2(b)]. As the blastula flattens, the independently motile deep cells [Fig. 2(c)] form a thickening around the margin of the blastodisc termed the germ ring [Fig. 2(a), (b), (c)]. This is where the deep cells from the epiblast (cells closest to the epithelial sheet) and those from

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the hypoblast (cells closest to the margin) form two distinct layers. Bracket’s cleft marks this separation of the epiblast and the hypoblast. As epiboly continues, the cells of the epiblast and hypoblast begin to converge around the circumference of the germ ring to form the embryonic shield [Fig. 2(d)]. This signifies the beginning of gastrulation. Stage 4 – 1⁄2 epiboly to 3⁄4 epiboly Figure 2 (a′), (b′), (c′), (d′) details the processes occurring as the developing embryo reaches 3⁄4 epiboly. Here, convergence of the epiblast and hypoblast, along with continuation of epiboly, has extended the embryonic anlagen caudally [Fig. 2(a′)]. Figure 2(b′) shows a cross section through the germ ring [Box B’ of Fig. 2(a′)], detailing the epiblast, hypoblast, and Bracket’s cleft in the cellular region, and the large YSL and the active zone in the yolk region. The active zone is thought to contain the microtubule and microfilament array which draws the epiblast down the yolk cell. Figure 2(c′)

FIG. 2. MILESTONE 2. STAGE 3—GERM RING AND EMBRYONIC the neuroectoderm and paraxial mesoderm germ SHIELD to 1⁄4 EPIBOLY. (a) Light micrograph of layer formation (bar = 50 µm). (d′′) SEM showing the animal view during the beginning of epiboly cross section of the early differentiation of the when the blastula flattens down on top of the yolk germ layers: ectoderm, neuroectoderm (neural cell and begins to migrate vegetally (bar = 1 mm). tube), axial mesoderm (chordamesoderm, or noto(b) SEM of the animal view showing the beginchord), paraxial mesoderm (adaxial or somites), ning of epiboly where the surface of the epithelial and endoderm ( bar = 20 mm). Am = axial mesosheet has become active, extending microvilli into derm; az = active zone; bc = Brachet’s cleft; e = the yolk cell (bar = 10 µm). (c) SEM of the animal ectoderm; ea = embryo anlage; ed = endoderm; ep view, after the epithelial sheet has been removed, = epiblast; gr = germ ring; h = head; hp = showing the motile deep cells of the germ ring hypoblast; pm = paraxial mesoderm; ne = neu(bar = 30 µm). (d) SEM of the animal view showroectoderm; and ysl = yolk syncytial layer (see ing the embryonic shield (bar = 100 µm). Az = Table 1 for complete list of abbreviations). active zone; d = deep cells; el = enveloping layer; STAGE 5—BLASTOPORE CLOSING TO es = embryonic shield; gr = germ ring; yc = yolk BLASTOPORE CLOSED. (a″) Dorsal view, showing that the germ ring is 90% closed (bar = 1 mm). cell (see Table 1 for complete list of abbrevia(b″) Dorsal view, depicting end of Stage V. Optic tions). vesicles, primary brain vesicles; forebrain (prosSTAGE 4—1⁄2 EPIBOLY TO 3⁄4 EPIBLOY. (a′′) Dorsal view, showing the embryonic anlage extending encephalon), midbrain (mesencephalon), and posteriorly. The box regions B′′, C′′, and D′′ correhindbrain (rhombencephalon) are visible. There is spond to Figures b′′, c′′, and d′′ (bar = 1 mm). (b′′) further refinement of the notochord, neural tube, Cross section through germ ring showing epiblast, and somites (bar = 1 mm). (c ″ and d ″) SEM of hypoblast, bracket’s cleft, YSL, and the active zone germ ring closure (bar = 100 mm. Br = brain rudiarea of epiboly (bar = 50 µm). (c′′) Frontal section, ment; fb = forebrain; grc = germ ring closure; hb showing transition from the germ ring to embryo = hindbrain; mb = midbrain; ov = optic vesicles; anlage. The germ ring shows ectoderm, hypoblast, and s = somite (see Table 1 for complete list of and bracket’s cleft, and the embryo anlage, shows abbreviations).

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FIG. 3. MILESTONE 3. STAGE 6—TAIL FREE OF YOLK-SAC. (a) Dorsal view, at the end of stage 6 showing the transverse brain subdivision between the mid/hind brain border; primary brain rudiments, forebrain (prosencephalon), midbrain (mesencephalon), and hindbrain (rhombencephalon); and the tail bud lifting off the yolk sac (bar = 1 mm). (b) Scanning electron image of tail bud formation and cellular

movement in support of tail formation (bar = 100 µm). Fb = forebrain; hb = hindbrain ; mb = midbrain; otv = otic vesicle; S = somite; tb = tailbud; tbs = transverse brain subdivision (see Table 1 for complete list of abbreviations). STAGE 7—FIN-FOLD. (a′′) Dorsal view, showing the head region at the beginning of this stage. The mid/hind brain border is becoming more refined,

Embryonic Developmental Progression shows a frontal plane section through the transitional zone where the germ ring converges into the embryonic anlagen [Box C′ of Fig. 2(a′)]. It shows the transition between the epiblast, hypoblast, and Bracket’s cleft of the germ ring, and the neuroectoderm and paraxial mesoderm of the embryonic anlagen. Figure 2(d′) represents the trunk region in the growing embryonic anlagen [Box D′ of Fig. 2(a′)]. This region shows the neuroectodermal cells forming the precursors of the neural tube; the axial mesoderm (the chordamesoderm), which is the primordium of the notochord; the paraxial mesoderm (adaxial cells), which are the precursors of the somites, the ectodermal layer; and some endodermal cells. Stage 5—Blastopore closing to blastopore closed As gastrulation continues, epiboly and convergence close the germ ring around the yolk cell. This progression is shown in Figures 2(a″), (b″), (c″) and (d″). Figures 2(c″) and (d″) shows SEM images of the germ ring closure. The embryonic anlagen is well defined at this stage, with differentiation beginning in the head region [Fig. 2(b″)]. The optic primordium becomes visible at the beginning of this stage [Fig. 2(a″)], and by the end of this stage, the formation of optic vesicles can be seen [Fig. 2(b″)]. Also at the beginning of this stage, the brain rudiment is visible [Fig. 2(a″)]. The primary brain vesicles, forebrain (prosencephalon), midbrain (mesencephalon), and hindbrain (rhombencephalon) are formed by the end of blastopore closure [Fig. 2(b″)]. The area of otic vesicle development becomes visible and further refine-

the cerebellum becoming distinct, and expansion of the fourth ventricle. Distinction between the optic tectum, the tegmentum and the telencephalon, the otic capsule has formed and optic cup and lens vesicle formation continues. Pectoral fin rudiments become apparent (bar = .5 mm). (b′′) SEM showing a cross section of the tail. Development at this stage showing that borders are becoming more refined between somites, the neural tube, and notochord (bar = 100 µm). 4 = fourth ventricle; c = cerebellum; lv = lens vesicle; nc = notochord; nt = neural tube; oc = optic cup; opt = optic tectum; otc = otic capsule; s = somite t = tegmentum; tc = telencephalon; tbs = transverse

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ment of the notochord, neural tube, and somites occur [Fig. 2(b″)]. Milestone 3 Stage 6—Tail free of yolk-sac From blastopore closure until tail bud formation, the greatest developmental progression can be seen in brain differentiation and eye formation. The primary brain rudiments, prosencephalon, mesencephalon, and rhombencephalon are formed by the beginning of this stage. Within the brain the transverse brain subdivision between the mid/hind brain border becomes defined with the beginning of hind brain ventricle formation [Fig. 3(a)]. Within the optic vesicle region, the lens vesicles begin to form. The tail bud lifts off the yolk sac as gastrulation continues to form the tail [Fig. 3(b)]. Stage 7—Fin fold The progressive development of the head and brain region continues. The mid/hind brain border becomes more refined, with the cerebellum becoming distinct and expansion of the fourth ventricle [Fig. 3(a′)]. Within the mesencephalon, distinction between the optic tectum and the tegmentum becomes apparent. The telencephalon of the prosencephalon becomes prominent [Fig. 3(a′)]. The otic capsule has formed, and optic cup and lens vesicle formation continues [Fig. 3(a′)]. Borders become more refined between somites, the neural tube, and notochord, as shown by the SEM image of tail development [Fig. 3(b′)].

brain subdivision (see Table 1 for complete list of abbreviations). STAGE 8 – VITELLINE VENATION AND LIGHT EYE PIGMENT. (a”) Dorsal view, showing head development which includes refinements in the cerebellum, optic tectum, tegmentum, telencephalon, and pectoral fin development (bar = .5 mm). (b ″ ) Sagittal view showing the progression of eye development, light eye pigmentation, and inflation of the fourth ventricle (bar = .5 mm). 4 = fourth ventricle; c = cerebellum; ept = eye pigment; opt = optic tectum; otc = otic capsule; pf = pectoral fin; t = tegmentum; tbs = transverse brain subdivision; tc = telencephalon (see Table 1 for complete list of abbreviations).

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Embryonic Developmental Progression Stage 8 –Vitelline venation and light eye pigment There is further brain refinement between the cerebellum, optic tectum, tegmentum, and telencephalon [Fig. 3(a″), (b″)]. Optic cup and lens development continues, and light eye pigmentation is evident [Fig. 3(a″), (b″)]. Pectoral fin development is clearly visible [Fig. 3(a″)]. Milestone 4 Stage 9—Complete eye pigment The head has lifted off the yolk sac by this stage and several neural-crest-cell-derived developmental processes are occurring. These include pharyngeal arch, jaw, gill, and operculum development. In Figure 4(b), lower and upper mandible differentiation has begun, and the olfactory pit is clearly visible. Figure 4(c), (d) shows the progression of gill and operculum development. Pectoral fin development is shown in Fig. 4(c), (e) with mesenchymal and ectodermal ridge cell migration evident. Liver development is indicated in Fig. 4(a). Stage 10—Head and body pigment Edsall’s unpublished data included the stage, head and body pigment. When examining embryo development it was clear that this was a gradual progression and interpretation as to the actual stage endpoint would be difficult. Stage 11 – Hatching At hatching [Fig. 5(a)] metamorphosis of many of the organ systems has advanced towards completion. Sensory system development, particularly of the optic and auditory tissue is shown in Figure

FIG. 4.

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5(b), (c). Formation of the eight tissue layers in the retina has been completed and is detailed in Figure 5(b). Examination of the sacculus [Fig. 5(c)] shows how advanced auditory tissue development has progressed. Development of gill arches, blood vessels, and lamellae are shown in Figure 5(d) while progression of tissue development in the digestive system is shown in Figure 5(e), detailing size and position of the stomach, intestine, spleen, and liver. Egg Development at Different Water Temperatures Figure 6 summarizes staging distribution patterns, and is a good way to visualize how development progresses under different constant temperature regimes. By Stage 11 (hatch) the eggs raised under the different constant temperatures appear to show distinct temporal separations, with those raised at higher temperatures requiring fewer days to hatch than those raised at lower temperatures. Table 2 summarizes the data from Figure 6 by stage and temperature. It lists the number of observations, the mean number of days, and the 95% confidence interval of the mean, for that stage and constant temperature profile. Data Analysis The models were run using 2, 4, 6, 8, and 10°C constant temperatures for each stage. Figure 7 compares each model to the data for stage 11. The models are very similar between the 4 to 10°C ranges. From the 2 to 4°C range the Thermodynamic and the Power models diverge substantially. Between the 0 and 2°C range the Belehradek’s and Zero models diverge but the Zero and Power models converge. The Zero model assumes the develop-

MILESTONE 4.

STAGE 9—COMPLETE EYE PIGMENT. (a) Dorsal view of stage 9 showing eye pigmentation, midbrain, cerebellum, fourth ventricle, and pectoral fin development. At this stage the early liver is visible (bar = 1 mm). (b) SEM of the ventral head region showing that the head has lifted off the yolk sac and lower and upper mandible differentiation has begun. The olfactory pit is clearly visible (bar = 100 µm). (c) SEM of the dorsal head region showing progression of gill and operculum development. The box D corresponds to Figure (d)

(bar = 100 µm). (d) SEM of the dorsal head region showing the gill region of development (bar = 10 µm). (e) SEM of the dorsal head region showing pectoral fin development with mesenchymal and ectodermal ridge cell migration evident. 4 = fourth ventricle (bar = 100 µm). c = cerebellum; erc = ectodermal ridge cell; g = gill; ht = heart; lvr = liver; lm = lower mandible; m = midbrain; mcm = mesenchymal cell migration; o = operculum; olp = olfactory pit; pf = pectoral fin; um = upper mandible (see Table 1 for complete list of abbreviations).

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Milestone 4

Embryonic Developmental Progression ment is halted when temperatures reach freezing. Belehradek’s model predicted that the development rate at freezing is about 70% that at 2°C. We believe that development is halted or nearly halted at 0°C (Hamel et al. 1997), thus the Thermodynamic model and Belehradek’s model overestimated the development rate at freezing temperatures (Fig. 7). Both the Power model and the Zero model predict halted development at 0°C, but the Power model underestimated the development rate for eggs held at 2°C (Fig. 7), so we used the Zero model to estimate hatching dates based on lake temperature data. Tables 3, 4, 5 and 6 give the estimated parameters, the asymptotic standard error (ASE) and the R2 values for each model and stage. Stage 3 consistently gave the lowest R2 values ranging from 0.191 to 0.196. Eggs undergoing the transition between Stage 2 (early to late blastula) and Stage 3 (Germ ring and embryonic shield to 1⁄4 epiboly) can appear to be viable but arrested in development. They usually show no further developmental progression for a very long time, eventually becoming opaque, signifying death. Lake temperature regimes were obtained at the Gull Island Shoal Complex, Lake Superior (Bronte et al. 1995). Bronte et al. (1995) reported that the peak spawning date occurred on 17 October 1990. With conservative estimates that spawning could last up to 30 days, 02 October 1990 was used as the early spawning date, and 01 November 1990 was used as the late spawning date. Figure 8 shows temperature data for Lake Superior and the Zero model calculated staging for early, peak, and late spawning dates. Tables 7, 8, and 9 show stage specific model predictions for the early, peak, and late spawning dates. The Zero model predicted that the degree to which Lake Superior cooled would cause a significant delay in hatch between the peak and

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FIG. 6. Summary of staging distribution patterns. Secondary y-axis is the frequency of embryos examined. late spawning lake trout. Hatch for early spawners would occur 10 December 1991 after 69 days incubation (Table 7), hatch for peak spawners would occur 08 March 1991 after 142 days incubation (Table 8), and hatch for the late spawners would occur 06 May 1991 after 185 days incubation (Table 9). Hatching for the early spawning group

FIG. 5. MILESTONE 4. STAGE 11—HATCH. (a) Sagittal view of Stage development (bar = 100 µm). bv = blood vessel, ct 11 showing regions detailed in box b corresponds= cartilage, ga = gill arch, gl = ganglion layer, inl ing to Figure (b), box c correspondsing to Figure = inner nuclear layer, int = intestine, ipl = inner (c), box d correspondsing to Figure (d), and box e plexiform layer, l = lens, la = lamella, lrc = layer correspondsing to Figure (e) (bar = 1 mm). (b) of rods and cones, lvr = liver, m = macula, nfa = Frontal section through the eye showing differennerve fiber area, opl = outer plexiform layer, onl = tiation of the retina (bar = 100 µm). (c) Frontal outer nuclear layer, pe = pigment epithelium, sac section through of the Sacculus (bar = 100 µm). = sacculus, sag = sagitta, sp = spleen, st = stom(d) Frontal section through the gill arches (bar = ach, y = yolk region (see Table 1 for complete list 100 µm). (e) Frontal section of the gut region, of abbreviations). showing spleen, stomach, intestine, and liver

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TABLE 2. Summary of data presented in Figure 1 by Stage and Temperature giving the number of observations, the mean number of days, and the 95% confidence interval of the mean. Stage 11 11 11 11 11 10 10 10 10 10 9 9 9 9 9 8 8 8 8 8 7 7 7 7 7 6 6 6 6 6 5 5 5 5 5 4 4 4 4 4 3 3 3 3 3 2 2 2 2 2 1 1 1 1 1

Temp 10 8 6 4 2 10 8 6 4 2 10 8 6 4 2 10 8 6 4 2 10 8 6 4 2 10 8 6 4 2 10 8 6 4 2 10 8 6 4 2 10 8 6 4 2 10 8 6 4 2 10 8 6 4 2

N 53 60 64 59 26 28 32 19 13 23 71 75 61 57 66 34 84 133 168 129 32 12 50 31 6 17 2 23 70 72 18 66 37 54 73 3 19 27 35 37 35 32 92 109 191 46 44 26 39 68 11

Mean 51.1 64.8 81.1 108.2 150.5 32.6 41.1 55.9 81.5 109.2 29.5 36.3 51.6 72.3 92.9 22.1 28.7 39.9 51.4 76.9 17.7 23.4 28.6 40.3 58.8 14.1 17.0 25.0 35.3 51.0 14.9 18.3 23.3 28.1 41.6 15.0 15.3 16.5 22.5 32.8 16.4 16.8 25.0 23.4 30.8 7.9 8.3 10.7 14.8 17.2 6.2

–95% 50.7 63.9 79.0 105.2 145.5 31.1 40.4 55.4 81.2 108.0 28.5 35.6 51.0 70.6 91.2 21.4 28.0 39.0 50.6 75.2 16.8 22.9 27.7 39.3 58.4 13.2 17.0 26.5 34.6 49.4 13.6 17.7 21.3 27.0 39.0 15.0 14.1 16.0 21.6 31.7 14.3 14.2 22.5 21.1 28.6 7.4 7.5 8.8 12.8 18.8 4.9

95% 51.5 65.7 83.1 111.2 155.5 34.1 41.8 56.3 81.9 110.3 30.5 37.1 52.3 73.9 94.7 22.7 29.4 40.7 52.3 78.5 18.5 23.9 29.6 41.4 59.3 14.9 17.0 23.5 36.1 52.6 16.2 19.0 25.4 29.3 44.2 15.0 16.5 17.1 23.4 33.9 18.5 19.3 27.5 25.7 33.1 8.5 9.0 12.6 16.8 15.5 7.5

9 24 24

6.0 8.6 13.2

6.0 7.7 8.6

6.0 9.4 17.7

FIG. 7. Compares Stage 11 non-linear calculations for Belehradek’s, Thermodynamic, Power, and Zero models.

would occur around 4°C as the lake was still in its cooling phase (Table 7). Hatching for the peak spawning group would occur during the coldest portion of the lake temperature cycle (Table 8). The late spawning group would hatch after the lake had begun its warming phase (2.33°C)(Table 9). Lake temperature regime data were obtained for Six Fathom Bank, Lake Huron (Savino et al. 1999). They reported that spawning began in mid-October. Based on this, 15 October 1996 was considered the early spawning date. Again using the 30 day spawning period, peak spawning was estimated to occur on 29 October 1996, and late spawning was estimated to occur on 13 November 1996. Figure 9 TABLE 3. Estimated parameters, asymptotic standard error (ASE) and the R2 values by Stage for Power model. Stage 1 2 3 4 5 6 7 8 9 10 11

a 0.088 0.049 0.032 0.022 0.015 0.009 0.007 0.006 0.004 0.004 0.003

ASE 0.009 0.005 0.002 0.001 0.001 0.000 0.001 0.000 0.000 0.000 0.000

b 0.377 0.511 0.348 0.576 0.650 0.912 0.943 0.847 0.902 0.905 0.778

ASE 0.060 0.048 0.033 0.026 0.024 0.020 0.038 0.015 0.023 0.032 0.015

R2 0.356 0.398 0.191 0.832 0.789 0.914 0.850 0.875 0.884 0.927 0.931

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TABLE 4. Estimated parameters, asymptotic standard error (ASE) and the R2 values by Stage for Thermodynamic model. Stage 1 2 3 4 5 6 7 8 9 10 11

∆HL 10.855 26.335 0.138 0.136 0.135 0.135 0.100 0.100 0.079 0.079 0.015

ASE 1.642 . . . 0.006 . 0.005 . 0.003 . 0.003

T1/2t 3.395 1.848 0.070 0.037 0.036 0.027 0.027 0.027 0.027 0.027 0.027

ASE . . 0.012 0.002 . 0.001 . 0.001 . 0.001 .

ρ(25°C) 10.300 2060.963 17.670 17.670 17.670 17.762 17.762 18.649 18.649 20.723 20.723

ASE . 293.693 . . . . . . . . .

∆HA –11510.055 –15229.269 –11931.809 –18003.257 –20039.089 –25965.495 –22159.031 –24428.335 –23892.485 –23987.422 –20001.472

ASE 1745.128 1332.118 1034.768 929.684 677.837 413.885 823.227 334.212 478.571 653.164 317.688

R2 0.338 0.389 0.193 0.759 0.791 0.943 0.853 0.903 0.906 0.943 0.944

TABLE 5. Estimated parameters, asymptotic standard error (ASE) and the R 2 values by Stage for Belehradek’s model. g 0.0815 0.026 0.00479 0.029 0.00181 1.49E-06 3.25E-04 2.27E-08 2.22E-07 5.55E-06 2.57E-05

TABLE 6. model.

Estimated parameters, asymptotic standard error (ASE) and the R2 values by Stage for Zero

Stage 1 2 3 4 5 6 7 8 9 10 11

a 22.452 0.296 0.0623 40.977 0.0591 0.0463 0.0492 0.0306 0.0257 0.0239 0.0152

ASE 0.064 0.034 0.016 0.005 0.003 0 0.001 0 0 0 0

ASE 17.432 0.555 0.009 . 0.008 0.003 0.012 0.001 0.002 0.003 0.001

t0 –0.348 –1.98 –7.589 8.22E–01 –5.109 –14.464 –6.416 –21.242 –17.464 –11.966 –10.804

ASE 3.311 3.988 12.632 4.20E-01 3.26 6.196 5.973 8.324 9.054 6.645 3.837

b ASE 2.41E+06 . 48.672 141.68 14.217 4.199 4.89E+05 2.50E+05 16.027 2.415 13.526 0.726 16.77 3.69 13.811 0.506 15.126 1.068 15.683 1.537 15.32 0.896

shows temperature data for Lake Huron and the Zero model calculated staging for peak, and late spawning dates. Table 10 and 11 show stage specific model predictions for the peak and late spawning dates. Temperature profiles for Lake Huron did

h 0.407 0.732 0.959 4.54E-01 1.358 3.395 1.869 4.231 3.623 2.805 2.192

c 0.377 0.455 0.222 0.576 0.427 0.489 0.541 0.475 0.509 0.526 0.438

ASE 0.295 0.43 0.965 7.10E-02 0.431 1.027 0.844 1.289 1.372 1.019 0.487

R2 0.356 0.4 0.194 0.836 0.797 0.945 0.857 0.904 0.907 0.946 0.946

Stage 1 2 3 4 5 6 7 8 9 10 11

ASE 0.06 0.173 0.053 0.026 0.043 0.03 0.099 0.019 0.034 0.046 0.024

R2 0.356 0.398 0.196 0.832 0.799 0.942 0.858 0.903 0.902 0.943 0.947

not extend back far enough to predict development and hatching for early spawning fish. The Zero model predicted that the cooling rate of Lake Huron would not cause as a significant delay in hatch between peak and late spawning lake trout

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FIG. 8. Lake Superior temperature profile and Zero model hatching predictions for Early, Peak, and Late spawning dates.

as in Lake Superior. Hatch for peak spawners would occur 29 January 1997 after 92 days incubation (Table 10), and hatch for the late spawners would occur 12 March 1997 after 119 days incubation (Table 11). Hatching for the peak spawning group would occur during the cooling phase at around 1.94°C (Table 10). Hatching for the late

TABLE 7.

Stage 11 10 9 8 7 6 5 4 3 2 1 0

spawning group would occur during the coldest portion of the lake temperature cycle (0.97°C)(Table 11). Figure 10 and Figure 11 show Lake Superior and Lake Huron temperature profiles, and Stage 11 peak (Fig. 10) and Late (Fig. 11) development rates. It is used to illustrate how lake specific timing of spawning, in combination with differences in the rates of cooling, degree and extent of cooling, and degree and extent of warming can affect egg hatching. Table 12 summarizes Zero model calculations by lake and spawning period. It shows how lake temperature profiles in combination with the timing of spawning can affect stage progression and hatch. In Lake Superior and Lake Huron we determined that timing of spawning and lake temperature dynamics have a slight affect on completion of the first two milestones. The third milestone is moderately affected, but the completion of the fourth milestone can be significantly affected by timing of spawning and lake temperature dynamics. DISCUSSION Our Zero model shows the degree to which changes in lake water temperature affects embryonic development. Developmental progression begins as the lake trout egg is fertilized and falls into the interstitial spaces of the substrate. This progression will last for several months, during which time the reef will undergo significant changes in physical and environmental conditions and predator/prey

Lake Superior stage specific model predictions for the Early spawning date.

Date 09/12/90 08/11/90 03/11/90 26/10/90 20/10/90 16/10/90 18/10/90 15/10/90 17/10/90 09/10/90 07/10/90 02/10/90

Power Temp 3.89 7.25 7.53 8.32 8.84 9.14 8.99 9.22 9.07 9.62 9.74 10.02

Days 68 37 32 24 18 14 16 13 15 7 5 0

Lake Superior Early Thermodynamic Belehradek’s Date Temp Days Date Temp Days 11/12/90 3.84 70 10/12/90 3.93 69 09/11/90 6.94 38 08/11/90 7.25 37 04/11/90 7.41 33 03/11/90 7.53 32 26/10/90 8.32 24 26/10/90 8.32 24 20/10/90 8.84 18 20/10/90 8.84 18 16/10/90 9.14 14 16/10/90 9.14 14 16/10/90 9.14 14 17/10/90 9.07 15 14/10/90 9.29 12 15/10/90 9.22 13 16/10/90 9.14 14 16/10/90 9.14 14 09/10/90 9.62 7 09/10/90 9.62 7 07/10/90 9.74 5 07/10/90 9.74 5 02/10/90 10.02 0 02/10/90 10.02 0

Date 10/12/90 09/11/90 04/11/90 26/10/90 20/10/90 16/10/90 17/10/90 14/10/90 16/10/90 09/10/90 07/10/90 02/10/90

Zero Temp 3.93 6.94 7.41 8.32 8.84 9.14 9.07 9.29 9.14 9.62 9.74 10.02

Days 69 38 33 24 18 14 15 12 14 7 5 0

Embryonic Developmental Progression TABLE 8.

Stage 11 10 9 8 7 6 5 4 3 2 1 0

Stage 11 10 9 8 7 6 5 4 3 2 1 0

Lake Superior stage specific model predictions for the Peak spawning date.

Date 13/04/91 01/12/90 24/11/90 15/11/90 07/11/90 02/11/90 03/11/90 31/10/90 01/11/90 24/10/90 22/10/90 17/10/90

TABLE 9.

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Power Temp 1.03 4.73 5.62 5.85 7.02 7.64 7.53 7.84 7.74 8.50 8.67 9.07

Days 178 45 38 29 21 16 17 14 15 7 5 0

Lake Superior Peak Thermodynamic Belehradek’s Date Temp Days Date Temp Days 20/01/91 0.52 95 23/01/91 0.07 98 07/12/90 4.01 51 05/12/90 4.12 49 28/11/90 4.92 42 28/11/90 4.92 42 16/11/90 5.94 30 16/11/90 5.94 30 07/11/90 7.02 21 07/11/90 7.02 21 03/11/90 7.53 17 03/11/90 7.53 17 02/11/90 7.64 16 03/11/90 7.53 17 31/10/90 7.84 14 31/10/90 7.84 14 01/11/90 7.74 15 01/11/90 7.74 15 24/10/90 8.50 7 24/10/90 8.50 7 22/10/90 8.67 5 22/10/90 8.67 5 17/10/90 9.07 0 17/10/90 9.07 0

Date 08/03/91 05/12/90 28/11/90 16/11/90 08/11/90 04/11/90 03/11/90 30/10/90 01/11/90 24/10/90 22/10/90 17/10/90

Zero Temp 0.12 4.12 4.92 5.94 7.25 7.41 7.53 7.94 7.74 8.50 8.67 9.07

Days 142 49 42 30 22 18 17 13 15 7 5 0

Zero Temp 2.33 0.14 1.52 3.93 4.92 5.62 6.06 5.85 6.13 6.94 7.02 7.74

Days 185 97 57 38 26 22 19 13 16 7 5 0

Lake Superior stage specific model predictions for the Late spawning date.

Date 24/05/91 17/03/91 23/12/90 07/12/90 27/11/90 21/11/90 21/11/90 17/11/90 18/11/00 09/11/90 07/11/90 01/11/90

Power Temp 4.43 0.23 2.52 4.01 5.10 6.06 6.06 6.10 6.13 6.94 7.02 7.74

Days 204 136 52 36 26 20 20 16 15 8 6 0

Lake Superior Late Thermodynamic Belehradek’s Date Temp Days Date Temp Days 04/03/91 0.09 122 16/03/91 0.21 134 14/01/91 0.17 73 15/01/91 0.37 74 30/12/90 2.00 58 30/12/90 2.00 58 11/12/90 3.84 39 10/12/90 3.93 38 28/11/90 4.92 26 28/11/90 4.92 26 24/11/90 5.62 22 23/11/90 5.78 21 21/11/90 6.06 19 21/11/90 6.06 19 18/11/90 6.13 16 17/11/90 6.10 15 18/11/90 6.13 16 18/11/90 6.13 16 09/11/90 6.94 7 09/11/90 6.94 7 07/11/90 7.02 5 07/11/90 7.02 5 11/01/90 7.74 0 01/11/90 7.74 0

dynamics. The grouping of the stages into milestones greatly facilitates the discussion on the effects of lake temperature dynamics on development progression. Survival of the embryo between milestones relies on the completion of important endpoints as described below. Milestone 1 Milestone 1 begins with fertilization and finishes after the formation of the late blastula (Stage 2). The endpoint of this milestone is a blastula, ready to initiate embryonic gene activation and a func-

Date 06/05/91 07/02/91 29/11/90 10/12/90 28/11/90 24/11/90 21/11/90 15/11/90 18/11/90 09/11/90 07/11/90 01/11/90

tioning YSL. This milestone is completed within 7 to 8 days post-fertilization (Table 12). Thus, predation, egg quality, and environmental and physical properties within the interstitial spaces of the substrate probably have a more significant effect on recruitment success during this milestone than temperature on developmental progression. The formation of the marginal blastomeres and the beginning of YSL formation were observed very early, around the 32-cell blastula. A functioning YSL is critical for the production of microtubules and microfilaments for use in epiboly (Kimmel et al.

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Allen et al. to deep cells (Nieuwkoop center) (Fekany et al. 1999, Feldman et al. 1998, Larabell et al. 1997). If the YSL becomes ineffective at transferring these large molecular weight proteins from the yolk cell into the deep cells, development may cease or cell fates and developmental organization may become altered. Thus, the chokepoints to successful recruitment occurring during Milestone 1 include an inability: to activate the machinery for division, develop a functioning YSL, and activate the embryonic gene (mid-blastula transition).

FIG. 9. Lake Huron temperature profile and Zero model hatching predictions for Peak and Late spawning dates.

1995). It also is the source of nutrition to the growing blastula and the mechanism for transfer of large molecular weight proteins between the yolk cell/marginal blastomere complex and the inner cells of the blastula. These proteins are believed to establish early dorsal ventral polarity and specify endoderm, dorsal and ventral mesoderm, and organizer (embryonic shield, Spemann organizer) fates TABLE 10.

Stage 11 10 9 8 7 6 5 4 3 2 1 0

Milestone 2 Milestone 2 begins with the initiation of epiboly (Stage 3) and completes after closure of the blastopore (Stage 5). After the initiation of epiboly, the germ ring had closed between day 15 and day 22 (Table 12). Again, predation, egg quality, and environmental and physical properties are more likely to affect recruitment success. Milestone 2 includes the important developmental processes of gastrulation and early neurulation. The process involves the formation of the germ ring and the convergence of hypoblast cells along it, resulting in a thickening called the embryonic shield. Studies in zebrafish (Danio rerio, Hamilton) have shown that the embryonic shield contains the gastrula organizer, a region where cells induced by the yolk cell, secrete protein products specifying dorsal fate to the embryo (Feldman et al. 1998, Sampath et al. 1998). In zebrafish interplay between these dorsalizing and ventralizing signals establishes protein gradients that determine the early embryonic fate map to the

Lake Huron stage specific model predictions for the Peak spawning date.

Date 27/1/97 20/12/96 12/12/96 1/12/96 23/11/96 17/11/96 17/11/96 13/11/96 14/11/96 6/11/96 4/11/96 29/10/96

Power Temp 2.72 4.47 4.84 5.33 5.89 6.42 6.42 6.63 6.32 7.14 6.69 8.28

Days 90 52 44 33 25 19 19 15 16 8 6 0

Lake Huron Peak Thermodynamic Belehradek’s Date Temp Days Date Temp Days 31/1/97 1.56 94 30/1/97 1.87 93 26/12/96 4.28 58 25/12/96 4.27 57 18/12/96 4.47 50 17/12/96 4.60 49 4/12/96 5.27 36 3/12/96 5.39 35 24/11/96 5.72 26 23/11/96 5.89 25 19/11/96 6.26 21 19/11/96 6.26 21 17/11/96 6.42 19 17/11/96 6.42 19 14/11/96 6.32 16 13/11/96 6.63 15 15/11/96 6.11 17 14/11/96 6.32 16 6/11/96 7.14 8 6/11/96 7.14 8 4/11/96 6.69 6 4/11/96 6.69 6 29/10/96 8.28 0 29/10/96 8.28 0

Date 29/1/97 23/12/96 16/12/96 3/12/96 24/11/96 19/11/96 17/11/96 12/11/96 15/11/96 6/11/96 4/11/96 29/10/96

Zero Temp 1.94 4.13 4.62 5.39 5.72 6.26 6.42 6.34 6.11 7.14 6.69 8.28

Days 92 55 48 35 26 21 19 14 17 8 6 0

Embryonic Developmental Progression TABLE 11.

Stage 11 10 9 8 7 6 5 4 3 2 1 0

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Lake Huron stage specific model predictions for the Late spawning date.

Date 4/4/97 14/1/97 5/1/97 21/12/96 12/12/96 5/12/96 4/12/96 30/11/96 30/11/96 21/11/96 19/11/96 13/11/96

Power Temp 1.13 3.45 3.62 4.33 4.84 5.25 5.27 5.47 5.47 5.89 6.26 6.63

Days 142 62 53 38 29 22 21 17 17 8 6 0

Lake Huron Peak Thermodynamic Belehradek’s Date Temp Days Date Temp Days 3/3/97 0.81 110 7/3/97 1.17 114 20/1/97 3.21 68 19/1/97 3.03 67 10/1/97 3.55 58 10/1/97 3.55 58 26/12/96 4.28 43 25/12/96 4.27 42 13/12/96 4.76 30 13/12/96 4.76 30 8/12/96 5.18 25 8/12/96 5.18 25 5/12/96 5.25 22 5/12/96 5.25 22 1/12/96 5.33 18 30/11/96 5.47 17 1/12/96 5.33 18 1/12/96 5.33 18 22/11/96 5.89 9 22/11/96 5.89 9 19/11/96 6.26 6 19/11/96 6.26 6 13/11/96 6.63 0 13/11/96 6.63 0

Date 12/3/97 17/1/97 8/1/97 24/12/96 13/12/96 8/12/96 5/12/96 28/11/96 1/12/96 21/11/96 19/11/96 13/11/96

Zero Temp 0.97 3.34 3.90 4.13 4.76 5.18 5.25 5.52 5.33 5.89 6.26 6.63

Days 119 65 56 41 30 25 22 15 18 8 6 0

cells of the hypoblast during gastrulation. As in Milestone 1, interfering with these protein signal gradients, may cause development to cease or alter cell fates and developmental organization of the body plan. The differentiated notochord becomes extremely important for further development. In zebrafish, the notochord establishes signal gradients between the ectoderm, endoderm, and mesoderm that establish the dorsal-ventral, anterior-posterior, and medial-lateral axis of the embryo (Fisher et al.

1996, Rodaway et al. 1999, Sampath et al. 1998, Warga and Nuesslein-Volhard 1999). Interplay between these signals results in a continuous refinement of morphogenetic movements, resulting in greater commitment to a particular cell fate. The endpoint during this period of development is the closure of the blastopore with the basic structuring of the body plan complete. Chokepoints to successful recruitment occurring during Milestone 2 include an inability to: activate

FIG. 10. Lake Superior and Lake Huron temperature profiles showing Zero model, Stage 11, Peak development rates.

FIG. 11. Lake Superior and Lake Huron temperature profiles showing Zero model, Stage 11, Late development rates.

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TABLE 12. Zero model calculations for Lake Superior and Lake Huron by lake and spawning period. Zero Model Lake Superior Early Peak Late Milestone Stage Days Days Days 4 11 69 142 185 10 38 49 97 9 33 42 57

Lake Huron Peak Late Days Days 92 119 55 65 48 56

3

8 7 6

24 18 14

30 22 18

38 26 22

35 26 21

41 30 25

2

5 4 3

15 12 14

17 13 15

19 13 16

19 14 17

22 15 18

1

2 1 0

7 5 0

7 5 0

7 5 0

8 6 0

8 6 0

embryonic gene transcription, develop microtubule and microfilament motors, activate deep cell motility, and establish dorsalizing and ventralizing protein gradients for determining cellular fate (which is key in establishing the normal body plan). Milestone 3 Milestone 3 begins as the tail bud forms and lifts off the yolk sac (Stage 6), and it completes with vitelline venation and light eye pigment (Stage 8). There is moderate potential for thermal modification during this milestone, resulting in completion of this milestone between 24 to 41 days (Table 12). During the beginning of this phase of development, the posterior body lifts off the yolk sac and continues development through a dual process of tissue restricted domains associated with the continuation of gastrulation and unique cellular movements (Kanki and Ho 1997). Also important during this milestone is the continued specification between endoderm and mesoderm. Endoderm derivatives will eventually form the digestive system and respiratory structures. Mesoderm derivatives will form elements of the circulatory system, digestive system, somites, muscles, and cartilage. The greatest changes can be seen in embryo length and brain and eye differentiation. By the completion of this milestone, the brain capsule has inflated and has differ-

entiated five regions, the notochord is completely formed, and myogenesis has begun. Chokepoints to successful recruitment occurring during milestone 3 include inability to form mesoderm and endoderm derivatives, gastrulation defects in the tail, and defects in brain differentiation. Milestone 4 Milestone 4 begins as the head lifts off the yolk sac (Stage 9) and ends with the completion of hatch (Stage 11). The progression of this stage can be substantially modified by spawning date and lake temperature profiles, completion ranging between 69 and 185 days. Several neural-crest-cell-derived developmental processes are occurring during this milestone. These include pharyngeal arch, jaw, gill, and operculum development (Baker et al. 1997, Hu and Helms 1999, Schilling et al. 1996). Also important are the formation of the digestive system, further refinements in the circulatory system, formation of the liver and integration with the vasculature system, and further development of the brain and eye. Chokepoints to successful recruitment occurring during Milestone 4 include cranial-facial deformities consistent with defects in neural crest migration, failures in vasculature development, and brain and neural development. All of these processes seem to be particularly sensitive to the effects of environmental contaminants during this developmental period (Dong et al. 2002, Teraoka et al. 2002, Toomey et al. 2001). Temperature Development Relationships The information used to build our models of development was based solely on those embryos that were alive on any given day that they were examined for stage of development. Developmental rate predictions from the Zero model for stage 11 were constrained to temperatures between 0 and 15°C, 0 ≤ T < b. For temperatures below freezing, development was assumed to be halted, i.e., D = 0 when T < 0. We built a model of developmental rate based on observations at fixed temperatures and applied it to embryos in an environment of changing temperatures. In order to do this we had to assume that the developmental rate at any given stage depended only on the current temperature and not on previously experienced temperatures. This assumption is invalid if, for example, early development within a certain temperature range results in faster (or

Embryonic Developmental Progression slower) development at later stages. For model simplicity we also assumed that development rate was constant at a given temperature regardless of the current developmental stage. This assumption is invalid if, for example, early development is faster (or slower) than later development. The extent to which these assumptions affect model prediction is unknown, but could be explored further through detailed examination of developmental rates over varying temperature regimes. For Lake Superior, Bronte (1995) gave a modal hatching date of 28 May 1991. The Zero model predicted Early hatching on 10 December 1990 (Table 7), Peak hatching to occur on 08 March 1991 (Table 8), and Late hatching to occur on 06 May 1991 (Table 9). For Lake Huron, Savino (1999) gave an estimate of mid-April for hatching to begin. The Zero model predicted Peak hatching 29 January 1997 (Table 10), and Late hatching to occur on 12 March 1997 (Table 11). The Zero model estimated hatch to occur earlier then the stated observations. One possibility is that hatching dates observed by Bronte and Savino may represent eggs that were spawned greater than 30 days past the peak spawning dates. From this the Zero model predicts that eggs spawned on 14 November 1991, on Gull Island Shoal Complex, Lake Superior would result in a modal hatch date of 28 May 1991. The water temperature for Lake Superior on 14 November 1991 was 5.65°C. For Lake Huron peak spawning, the Zero model predicts spawning would have to occur on 26 November 1996, for peak hatching on 15 April 1997. The water temperature for Lake Huron on 26 November 1996 was 6.02°C. Casselman (1995) stated that late spawned lake trout eggs come from older females and survive better than those spawned earlier. The model was developed using the Marquette brood stock. Genetic differences between this strain and the Lake Superior and Lake Huron strains might also account for the observed differences between model predictions and observed hatch dates. Horns (1985) found that hatching dates varied up to 18 days between different lake trout strains raised under the same conditions. If successful hatch correlates to post peak spawning, then the lake trout population may be of a strain acclimated to spawning around 6°C. The timing of spawning can significantly alter the timing of hatch. The models of the relationship between temperature and embryological development presented in this study can be used to predict the timing of hatch from spawning observations and temperature profiles. This becomes important in de-

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termining factors important to increased hatch and fry survival. Based on timing of spawning and lake temperature dynamics, hatching can occur while the lake was still in its cooling phase, during the coldest portion of the lake temperature cycle, or during the warming phase. This in combination with seasonal changes in predatory activities on the eggs and fry, and food availability for the fry could profoundly affect fry survival and recruitment success. ACKNOWLEDGMENTS This research was supported by USGS-Great Lakes Science Center. T. Edsall provided egg development/temperature data. D. Wilcox and R. Quintal provided editorial assistance. Lake Superior water temperature data was provided by Charles R. Bronte. Lake Huron data were provided by Jacqueline Savino. References to trade names or manufacturers does not imply U.S. Government endorsement. This article is contribution 1320 of the USGS Great Lakes Science Center. REFERENCES Baker, C.V.H., Bronner-Fraser, M., Douarin, N., and Teillet, M.A. 1997. Early- and late-migrating cranial neural crest cell populations have equivalent developmental potential in vivo. Development 124: 3077–3087. Balon, E.K. 1980. Early Ontogeny of the Lake Charr, Salvelinus (Cristivomer) Namaycush. In Charrs. Salmonid fishes of the genus Salvelinus, ed. E. Balon, pp. 485–561. The Hague (Netherlands): W. Junk. Bronte, C.R., Selgeby, J.H., Saylor, J.H., Miller, G.S., and Foster, N.R. 1995. Hatching, dispersal, and bathymetric distribution of age-0 wild lake trout at the Gull Island Shoal complex, Lake Superior. J. Great Lakes Res. 21 (Suppl. 1):233–245. Brown, S.B., Fitzsimons, J.D., Palace, V.P., and Vandenbyllaardt, L. 1998. Thiamine and Early Mortality Syndrome in Lake Trout. In Early life stage mortality syndrome in fishes of the Great Lakes and Baltic Sea, eds. G. McDonald, J.D. Fitzsimons, and D.C. Honeyfield, Am. Fish. Soc. Symp. 21:18–25. Casselman, J.M. 1995. Survival and development of lake trout eggs and fry in eastern Lake Ontario—in situ incubation, Yorkshire Bar, 1989–1993. J. Great Lakes Res. 21 (Suppl. 1):384–399. Cornelius, F.C., Muth, K.M. and Kenyon, R. 1995. Lake Trout rehabilitation in Lake Erie: a case history. J. Great Lakes Res. 21 (Suppl. 1):65–82. Dong, W., Teraoka, H., Yamazaki, K., Tsukiyama, S., Imani, S., Imagawa, T., Stegeman, J.J., Peterson, R.E., and Hiraga, T. 2002. 2,3,7,8-Tetrachlorodibenzo-p-dioxin toxicity in the zebrafish

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