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ESTIMATES OF REPRODUCTIVE POTENTIAL AND TIMING IN CALIFORNIA SEA CUCUMBERS PARASTICHOPUS CALIFORNICUS (STIMPSON, 1857) FROM SOUTHEAST ALASKA BASED ON NATURAL SPAWNING.

INTRODUCTION

The California sea cucumber Parastichopus californicus (Stimpson, 1857) is the predominant species of sea cucumber commercially harvested on the North Pacific coast of the United States and Canada and has a wide distribution ranging from Baja California, Mexico, to the Aleutian Islands, AK (Lambert 1997). This deposit-feeding species ingests surface sediments, digests the labile organic fraction, and excretes the inorganic and/or indigestible material (Cameron & Fankboner 1984). The highest densities off. californicus (mean 10 individuals [m.sup.-2]) are found at depths between 30 and 60 m, typically on gravel and vertical rock substrates (Zhou & Shirley 1996).

Life-history characteristics (i.e., slow growth and low recruitment rates) of Parastichopus californicus leave it highly susceptible to overfishing, stimulating recent efforts to develop an aquaculture industry for this species. Maturity is reached at approximately 4 y of age, and adults have been reported to spawn annually in early summer (Cameron & Fankboner 1989). The late age of maturity may result in slow replenishment of reproductive adults to replace harvested animals. Spawned eggs are fertilized in the water column, where embryos develop through a feeding auricularia stage and a nonfeeding doliolaria stage. After 1-4 mo in the plankton, larvae settle to the sea floor and metamorphose into juveniles (Cameron & Fankboner 1986).

With rising international demands for sea cucumber products, Parastichopus californicus commercial fisheries are showing growth in Alaska (Hebert 2014), although harvests have remained relatively stable in British Columbia (Department of Fisheries and Oceans Canada 2017), and have declined drastically in Washington over the last few decades (Carson et al. 2016). Recent observations suggest that individuals in Southeast Alaska (SEAK) are smaller in average size and less abundant in some fishing regions (Clark et al. 2009, Hebert 2014). The exact causes for population changes of P. californicus have not been identified, but predation by sea otters (Larson et al. 2013) and fishing pressure (Anderson et al. 2011) have been suggested. As a result, there is growing interest in improving understanding of stock dynamics and constructing aquaculture programs for this species. Reliable estimates of spawn timing and reproductive output for P. californicus are essential to the development of fishery population models and hatchery protocols to better manage this important marine resource.

Sea cucumber fisheries in Alaska, British Columbia, and Washington generally occur in the fall, and are managed as rotational fisheries to minimize assessment and management costs (Hebert 2014, Carson et al. 2016, Department of Fisheries and Oceans Canada 2017). In Alaska and British Columbia, fishing regions are divided into quadrants that are only harvested every third year, whereas many fishery areas have remained closed in Washington. Commercial fisheries operate under an annual total allowable catch model of 6.4% of total biomass in Alaska and 9.5% in British Columbia. Total allowable catch varies yearly in Washington, and has been maintained close to 0% in many areas (Carson et al. 2016). Other management measures in Alaska and British Columbia include specific quotas for smaller sections of fished areas, as well as participant licensing restrictions that limit the number of participants in the fishery (Hebert 2014, Department of Fisheries and Oceans Canada 2017).

Natural spawning events are difficult to observe in situ; thus, spawn timing is determined using gonad indices (GI) in many marine invertebrates (West 1990). The GI (ratio of gonad to body weight) is assumed to be the highest when animals have fully mature gametes, with natural spawning events occurring soon thereafter (Ebert et al. 2011). Past estimates suggest that Parastichopus californicus populations in British Columbia exhibit peak GI and spawning behavior in June and July (Cameron & Fankboner 1986); however, these data were collected more than three decades ago and spawn timing has not been re-evaluated to determine whether it has shifted over time. Other measures of reproductive potential and their relationship to GI, including histological observations of egg development and gonad lipid content, are also lacking for P. californicus. Estimates of spawning season are essential to management decisions regarding the timing of commercial harvests and broodstock collection for aquaculture (Caddy 2004, Bruckner 2005, Anderson et al. 2011) and need to be validated using other measures of gonad maturation stage.

Reproductive output is difficult to quantify in natural populations of nonaggregating broadcast-spawning marine invertebrates such as Parastichopus californicus, but it directly affects the supply of larvae and juveniles into harvested populations (Fairweather 1991). Typically, reproductive potential is estimated by strip spawning and quantifying the number of eggs recovered from each female; however, strip spawning involves soaking the gonad in a chemical such as dithiothreitol (DTT) which induces germinal vesicle breakdown, a disintegration of the coating around the egg that would be removed naturally by passage through the gonoduct (Courtney 1927, Johnson & Johnson 1950, Smiley 1986). Thus, all eggs are released from the gonad regardless of maturity, potentially yielding substantial portions of nonviable eggs which could overestimate recruitment potential in the wild, particularly in P. californicus which undergoes sequential gamete maturation over a period of 3 y (Smiley 1986). Most published fecundity estimates for P. californicus were derived from strip spawning (e.g., Courtney 1927, Strathmann & Sato 1969, Cameron & Fankboner 1989), although Ren et al. (2016) recently reported estimates from live-spawned individuals.

This study determined spawn timing of Parastichopus californicus in SEAK over three annual cycles based on GI, and then compared GI with gonad lipid content and histological observations of egg development to determine whether GI presents an accurate indication of gonad maturation stage. Total fecundity and viable eggs were estimated from live-spawned individuals, and compared with estimates obtained through strip spawning to determine whether live spawning could improve production in commercial aquaculture of this species.

MATERIALS AND METHODS

Collection and Maintenance

Adult specimens of Parastichopus californicus (n = 56; mean blotted wet mass [+ or -] SD: 142.1 [+ or -] 30.2 g) were collected by the Southeast Alaska Regional Dive Fisheries Association commercial divers in George's Inlet, SEAK (55[degrees] 20' N, 131[degrees] 28' W) at depths of 5-10 m. Animals collected in April 2011, April 2012, June 2012, April 2013, June 2013, and July 2013 were live spawned. Additional collections for other measurements occurred every 2-3 mo from February 2012 to July 2013, for a total of nine collections. Roughly 25-50 live animals were collected at each time point. Live animals were placed in groups of five into plastic bags filled with seawater (7[degrees]C-8[degrees]C), and transported in coolers via air cargo to the University of Alaska Fairbanks, Seward Marine Center. Animals typically arrived in Seward within 15 h of collection. On arrival, live animals were transferred to flow-through seawater aquaria (L x W x H: 0.75 x 0.75 x 1.25 m) at 6[degrees]C. Evisceration rates (expulsion of internal organs, sometimes including gonads) were low (2%-10%). Although evisceration does not kill P. californicus (Fankboner & Cameron 1985), all eviscerated animals were excluded from this study to avoid confounding results with the effects of tissue loss and regeneration.

Gonad Index, Lipid Content, and Egg Developmental Stage

The timing of natural spawning was estimated based on multiple metrics including GI, gonad total lipid content, and histological examination. Gonad index was calculated for 5-10 dissected animals per time point, for a total of 65. Gonad total lipid was measured in these same individuals, except for five females collected in February 2012 which were processed before it was decided to conduct lipid analyses. Histological observations to determine egg developmental stages during peak GI were conducted on a subset of these same animals (n = 28). Gonads selected for histology represent the full range of GI observed, and were collected in April (n = 6), June (n = 9), and August (n = 3) 2012, and April 2013 (n = 10). Histological samples were not collected from November to February when GI were lowest because too little gonad tissue was present to conduct both histology and lipid analyses on the same animal.

One-third of each gonad was preserved in 10% phosphate-buffered formalin for histology, and the rest was frozen at -40[degrees]C for lipid analysis. Females were targeted in this study, yet sex cannot be determined externally. To obtain sufficient replicates per collection time point, animals were sacrificed one by one until a minimum of five females had been dissected. The opportunistic nature of sampling provided variable numbers of individuals for each collection, and as many as 10 females were dissected at some time points. Gonad and body-wall tissues (dermis, connective tissue, and muscle) were blotted and weighed wet to the nearest 0.01 g. The gonad index was calculated as the ratio of gonad to body-wall wet weight, according to the method of Cameron and Fankboner (1986). Large variance in GI in a given month was taken as evidence of spawning season because it suggests that both gravid (high GI) and recently spawned (low GI) individuals were present at that time point.

Gonad samples for lipid content analysis were freeze-dried for 32 h, and then ground before lipid extraction. Lipid extractions were performed using a Dionex Accelerated Solvent Extractor (ASE 200) using two static cycles (5 min each) at 85[degrees]C under [N.sub.2] gas at 1,500 psi according to the method of Dodds et al. (2004). Chromatography-grade dichloromethane was used as the extraction solvent, which was treated with butylated hydroxytoluene at a concentration of 100 mg [L.sup.-1] to prevent lipid oxidation. Before extraction, 0.5 g of dry gonad was mixed with 1.0 g of Chem-tube hydromatrix drying agent (Varian, Inc., Palo Alto, CA). Gonad lipid extracts were concentrated under [N.sup.2] in a TurboVap LV solvent evaporator (Zymark/Biotage, Inc., Charlotte, NC), incubated at 36[degrees]C for 2 h, and then weighed again for gravimetric calculation of milligram lipid per gram wet gonad. Lipid content was determined as per gram gonad wet weight, rather than per gram dry weight because GI was also calculated based on wet weights.

Before histological analysis, preserved gonads were dehydrated in Neoclear. Full strands of gonad tubules were embedded into paraffin wax (Type 6). Eight 5-[micro]m wax sections were cut from different regions of the sample using a microtome (model 820 Mark II; Reicheret-Jung, Vienna, Austria), placed onto glass slides, and then dried for 24 h at 50[degrees]C. Sections were stained with eosin Y solution (1 % alcohol) and modified Harris hematoxylin, according to the method of Galigher and Kozloff (1971). Twenty-five individual tubule cross-sections were examined for each female, following the protocol of Foglietta et al. (2004).

Five stages of oogenesis were identified in histological sections: postspawning (PS), recovery (R), growth (G), advanced growth (AG), and mature (M; Fig. 1). The reproductive status of each female was determined by calculating the individual weighted maturity index (IWMI; Foglietta et al. 2004), where

IWMI = [1 (%PS) + 2(%R) + 3(%G) + 4(%AG) + 5(%M)]/100

The mean proportion of eggs at each stage was determined in each of the 25 histological sections, and then averaged to produce a single value of the IWMI for each female. Values of the IWMI range from 1 (100% PS tubules) to 5 (100% M tubules).

Spawning Methods

Total fecundity and viable eggs per female were determined for both strip-spawned and live-spawned individuals. Previous studies suggest the peak spawning period for Parastichopus californicus occurs in June to July (Cameron & Fankboner 1989). Thus, a subset of animals from the collections in this time frame (April 2011, April 2012, June 2012, April 2013, June 2013, and July 2013) were subjected to spawning trials. Following each field collection, about 25 individuals of unknown sex were randomly assigned to one of two holding aquaria (L x W x H: 2 x 1 x 1 m). Stocking densities were within natural ranges observed in SEAK (~10 individuals [m.sub.-2]; Zhou & Shirley 1996). Depending on the collection date, animals were maintained for 2 days to 12 wk before spawning induction. Water temperatures in aquaria were slowly raised from 8[degrees]C in April to 12[degrees]C in July to mimic natural conditions in SEAK (Weingartner et al. 2009). Aquaria were filled with 20-[micro]m filtered flow-through seawater (6 L [min.sup.-1]) and lined with a 4-cm layer of sand (grain size 500 [micro]m) to facilitate deposit-feeding. Every 5 days, aquaria were supplied with 10 g dry feed that was emulsified in seawater. Feed consisted of equal parts AlgaeMac Protein Plus, AlgaeMac 3050, and Spirulina (Bio-Marine, Inc., Hawthorne, CA). During feeding, water flow was stopped for 4 h to allow emulsified feed to settle onto the bottom of the tank.

Spawning induction was conducted in late spring/early summer 2011, 2012, and 2013 (Fig. 2A). Animals were subjected to temperature shock by transferring them from holding aquaria (12[degrees]C) into sterile spawning aquaria (L x W x H: 0.75 x 0.75 x 1.25 m) containing static 20-[micro]m filtered seawater at 18[degrees]C, and stocked with live Isochrysis spp. at a concentration of 10,000 cells m[L.sup.-1] to simulate a food pulse. For each spawning attempt, between 10 and 25 animals were placed into one of four aquaria. To decrease chances of polyspermy, males were removed from spawning aquaria once they began to spawn. Any sperm that had been released remained in the spawning aquaria to fertilize eggs. Sperm concentrations in spawning tanks were between 10,000,000 and 15,000,000 cells [L.sup.-1]. Spawned eggs were aerated in the spawning aquaria for 1-1.5 h to allow adequate time for fertilization to occur. Fertilized gametes were then rinsed over 710-[micro]m screens that retained gonad tissue and fecal matter, and a 47-[micro]m screen that retained spawned eggs. Eggs were resuspended in 5 L of 20-[micro]m filtered seawater at 16[degrees]C, and then fecundity, viable eggs, and egg size were determined from subsamples of this suspension as described in the following paragraphs. Total blotted wet weights were determined for each spawned female once spawning had ceased.

Animals for strip spawning were collected in June 2013 (N = 9 females, 5 males) and July 2013 (N = 11 females, 5 males) and maintained at 12[degrees]C in flow-through aquaria as before for 4 days before spawning. The strip-spawning method of Maruyama (1980) was followed with minor modifications. Gonads were removed, and female GI were determined as previously described. Female gonads were placed into separate containers containing 850 mL of seawater. Male and female gonads were then cut into approximately 3-mm pieces to allow sperm to be released and eggs to leak out of tubules. To induce germinal vesicle breakdown, 5 mL of 1-M DTT was added to each container. After 20 min, eggs were washed through a 47-[micro]m screen to remove the DTT solution and any remaining fragments of gonad tissue. Eggs were resuspended in 950 mL of seawater. Sperm were pooled from males obtained during the same collection as females, suspended in 20-[micro]m filtered seawater, and added to the egg suspensions from the corresponding females at a concentration of 10,000,000 cells [L.sup.-1]. Sperm-egg contact was allowed for 20 min, and then total fecundity, viable eggs, and egg-size estimates for each female were determined as described in the following paragraphs.

Fecundity, Viable Eggs, and Egg Sizes

Egg suspensions for each female were thoroughly mixed and three 1-mL subsamples were placed on well slides for examination under a compound microscope. Fecundity was calculated by multiplying the average number of eggs from the three subsamples by the total volume (5 L) of the egg suspension. The total number of eggs per gram spawned female wet weight in each treatment aquarium was also recorded. Numbers of viable eggs were calculated for each female by multiplying total fecundity by percent fertilization (absence of the germinal vesicle; Fig. 2B), and then recorded as viable eggs per gram female wet weight in that treatment aquarium. Average egg size was determined for live-spawned females in 2011 and 2013, as well as all strip-spawned females. From each female, 50 fertilized eggs were photographed before initial cell division using a digital camera attached to a compound microscope. Eggs were measured from digital images using Image J software. An average egg size was recorded for each individual female.

Statistical Analysis

All statistical analyses were conducted using R software. Residual and normal Q-Q plots were performed before statistical testing. If data violated analysis of variance (ANOVA) assumptions of normality, homogeneity of variance, and/or linearity, appropriate transformations were applied. Significance levels for all tests were set at [alpha] = 0.05. Body-wall weight, gonad weight, and GI data were log(x + 1) transformed, and three separate two-way ANOVA were performed for the factors month and year, with a month x year interaction term. Gonad index was then compared with other measures of gonad maturation (i.e., lipid gonad and IWMI) using Spearman correlations.

Estimates of total fecundity and viable eggs for live-spawned individuals were each compared among sampling years (2011, 2012, and 2013) using separate one-way ANOVA on untransformed data. Because no differences were detected between years, data from all 3 y were pooled for subsequent comparison with strip-spawned estimates. Pooled fecundity and viable egg estimates were [x.sup.1/3]-transformed, and Student's t-tests were used to determine whether fecundity or number of viable eggs differed between live-spawning and strip-spawning methods. Egg-size data for live-spawned individuals were also pooled among years, and Student's t-tests were again used to determine whether sizes differed between live- and strip-spawned females.

RESULTS

Gonad Indices and Reproductive Timing

Mean female body-wall weight was 126.27 [+ or -] 25.57 g (SD; range: 60.70-214.16 g) and gonad weight was 5.12 [+ or -] 5.96 g (range: 0.05-26.30 g). Mean GI was 3.91 [+ or -] 4.37 (range: 0.72-14.89; Fig. 3). Body-wall weight did not significantly differ among months or years, nor was there a significant month x year interaction (Table 1). Gonad weight and GI were significantly different among months and years, and the month X year interaction terms were also significant (Table 1). Gonad index was significantly higher in April and June than in other months (Fig. 3). Maximum variance in GI occurred in April and June 2012, and in June 2013 (Table 2), and in both years GI was significantly higher in April and June than in other months (Fig. 3).

Gonad Maturation

Mean gonad lipid was 0.031 [+ or -]0.015 mg lipid [g.sub.-1] wet gonad weight (range: 0.002-0.118 mg [g.sub.-1]). The IWMI indicated that none of the samples were dominated by postspawned tubules (IWMI range of 1.00-2.00). Mean IWMI was 3.44 [+ or -] 0.69 (range: 2.24-4.60; Fig. 3). Mean gonad lipid was significantly different among months and years, but the month x year interaction was not significant (Table 1). Mean IWMI was significantly different among months, but not among years (Table 1). Gonad index was moderately correlated with IWMI ([r.sub.s] = 0.47, P = 0.011) and gonad lipid ([r.sub.s] = 0.43, P = 0.024), but IWMI was not correlated with gonad lipid ([r.sub.s] = 0.22, P = 0.271).

Fecundity, Viable Eggs, and Egg Size

At least one spawning was observed in 68% of individuals; however, only males spawned in multiple spawning trials. Before spawning, animals displayed "cobra" behavior, swaying with arched bodies and open oral tentacles (Fig. 2A). Males spawned first, approximately 1 h after placement into spawning aquaria. Females spawned 1.5 h after placement into spawning aquaria, usually after several males had already begun to spawn. Males and females spawned for 0.5-3 h, with relatively constant gamete release when undisturbed.

Neither fecundity in eggs per gram female wet weight (ANOVA; F= 2.145, P = 0.140, df = 2) nor viable eggs per gram female wet weight (ANOVA; F= 0.441, P= 0.649, df = 2) differed significantly between years when animals were live spawned (Fig. 4). Pooled across years, mean fecundity of live-spawned individuals was 2,863 [+ or -] 1,502 [g.sub.-1] and mean number of viable eggs was 2,495 [+ or -] 1,517 [g.sub.-1] (Table 3). When live-spawned fecundity data were pooled among years and compared with results from strip spawning, neither total fecundity (t = 0.422, P = 0.519) nor viable egg production (t = 0.009, P = 0.923) were significantly different between treatments (Fig. 4), although strip spawning was completely unsuccessful in five of the 20 individuals. Strip-spawned female fecundity was highly correlated with GI ([r.sub.-s] = 0.820, P [less than or equal to] 0.001). Egg sizes also differed significantly between spawning methods (t = 1.22, P [less than or equal to] 0.001). Average egg size of all live-spawned females was 169.15 [+ or -] 19.15 [micro]m, compared with 146.53 [+ or -] 10.64 [micro]m in strip-spawned females.

DISCUSSION

Life-history parameters, including timing of spawning, fecundity, and gametogenesis, are important to the understanding of marine invertebrate population dynamics. In this study, various indices suggest spawning of Parastichopus californicus from SEAK occurs from April to June, about 1-2 months earlier than previously observed in British Columbia (Cameron & Fankboner 1986). Gonad indices were smaller in SEAK than in the earlier British Columbia study, suggesting differences in reproductive potential between geographic locations and/or over time. Gonad indices were moderately correlated with gonad lipid and egg development stage. Compared with strip spawning, live spawning yielded comparable estimates of total fecundity, but eggs were larger and variance in the number of eggs produced per female was lower, demonstrating that live spawning could be an improvement to strip spawning for larger scale captive breeding of this species. With the rising international demand for sea cucumber products, these data can inform management and recovery efforts for the SEAK sea cucumber fishery, and support development of stock enhancement aquaculture programs in the Northeast Pacific.

Reproduction in Southeast Alaska

Current U.S. fishery management guidelines for Parastichopus californicus use reproductive information, in part, collected in British Columbia by Cameron and Fankboner (1986) and Clark et al. (2009). The results presented here for populations farther north in SEAK differ somewhat from this earlier study, with evidence that spawning may have occurred between April and June, rather than June and August as in 1982, or July and September as in 1983, according to Cameron and Fankboner (1986). If this apparent difference in timing accurately reflects in situ trends, it may be attributed to a number of factors, including methodology and/or spatial or temporal variation in environmental conditions. With a separation of only 1.35[degrees] latitude between locations, the difference in spawn timing is probably not related to a latitudinal gradient in the timing of seasonal cycles in environmental conditions; indeed, the opposite latitudinal trend would be expected, with more northerly populations spawning later in the year. Differences in spawn timing may represent a longer term temporal shift, perhaps due to changes in oceanographic conditions (e.g., Stabeno et al. 2004, Royer & Grosch 2006). Seawater temperature affects growth and egg development rate, and cues spawning in other holothurian species (Brockington & Clarke 2001, Lester et al. 2007). Average seawater temperatures have increased by 0.12[degrees]C-0.25[degrees]C [decade.sup.-1] in the Gulf of Alaska (Cheung et al. 2015), and may also be increasing earlier in the year, causing a shift in timing of spawning over the last few decades.

Values of GI also differed between SEAK and British Columbia. Peak female GI reached 28-32 in British Columbia in the early 1980s (Cameron & Fankboner 1986), about two times higher than peak values observed here for SEAK (14-16; Fig. 3). Lower GI must indicate higher body-wall weights and/or lower gonad weights in SEAK. Fankboner and Cameron (1985) found that mean body-wall weights in British Columbia in June and July ranged from 300 to 360 g--much higher than that detected here (<150 g). Gonad index is a ratio of gonad to body-wall weight, such that lower values of both GI and body-wall weight must indicate smaller gonad weights as well. Smaller gonads in the present-day SEAK population suggest reduced fecundity and reproductive potential relative to the earlier study. Fluctuations in population size and reproductive potential of several other Alaskan fisheries species, including multiple species of crab (Zheng & Kruse 2006) and most salmon species (Hare et al. 1999), have been linked to decadal shifts in oceanographic conditions in the Gulf of Alaska that occur in conjunction with "cold" and "warm" phases of the Pacific Decadal Oscillation (Stabeno et al. 2004, Sturdevant et al. 2012). Although this link is highly speculative, it is interesting to note that the study by Cameron and Fankboner (1986) occurred during a strong "warm" Pacific Decadal Oscillation phase (Glynn 1988, Masson & Cummins 2007), whereas this study took place during a weak "cold" phase (Pozo Buil & Di Lorenzo 2015). Increased primary production typical of warm years could have contributed to larger body sizes and higher GI in the British Columbia study. Variations in GI, gonad, and body-wall weights may also be due in part to fishing and/or sea otter predation pressure, which target the largest individuals and result in reduced reproductive potential in harvested species (e.g., Anderson et al. 2011, Purcell et al. 2014). Both SEAK and British Columbia commercial sea cucumber dive fisheries have operated since the mid 1980s; therefore, collections in British Columbia occurred before strong fishing pressures, whereas this study took place following three decades of fishing (Bruckner 2005, Anderson et al. 2011).

The low GI and smaller gonads in the SEAK population could indicate lower fecundity and/or reduced egg size. Previous studies report egg diameters of 180-200 [micro]m in strip-spawned Parastichopus californicus (Smiley & Cloney 1985, Cameron & Fankboner 1986), whereas average egg sizes reported here were 165-175 [micro]m in live-spawned individuals and even smaller with strip spawning (mean 146 [micro]m). Egg size does not necessarily impact the quality of offspring (Moran & McAlister 2009); however, reduced egg size may reflect less lipid-provisioning and thus reduced energy reserves in eggs (e.g., Peters-Didier & Sewell 2017). These stored lipids provisioned to the egg are important in the formation of the larval body and successful metamorphosis (e.g., Sewell 2005).

Although GI provides an easy way to estimate the spawning season, the relatively weak correlation with IWMI and gonad lipid suggests it may not be as reliable for marine invertebrates as for many fishes (DeVlaming et al. 1982, West 1990, Ebert et al. 2011). For fish, GI calculations use age and length measurements to adjust for changes in gonad growth over the life span of the animal (Ebert et al. 2011). Holothurians from wild populations cannot be aged, and they tend to contract longitudinal muscles when disturbed such that length measurements are highly variable for a given individual. In addition, use of GI makes the unrealistic assumptions that the body wall is linearly related to gonad weight, and that gonads start developing at a hypothetical weight zero (West 1990). Holothurians do not begin to grow gonads until they reach a certain size; however, current GI calculations used for holothurians do not take this into account (e.g., Cameron & Fankboner 1986, Muthiga et al. 2009). Not surprisingly, the relationships between body-wall weight and GI or gonad weight were not linear. Other studies on holothurians have also shown that GI does not scale linearly with body weight (Drumm & Loneragan 2005, Muthiga 2006).

For GI to be applied as a reliable management tool for predicting reproductive potential in the Parastichopus californicus fishery, calculations should be modified similarly to those proposed for echinoids (Ebert et al. 2011). Specifically, GI calculations should be scaled to body weight at first development of the gonad, and include the slope of the regression between gonad and body-wall weights and a seasonal weight-scaling exponent. Although most P. californicus females are reproductive within 4 y postsettlement (Cameron & Fankboner 1989, Hannah et al. 2012), no study has determined how reproductive status correlates with animal weight. These data are needed to develop a new GI calculation that more accurately estimates P. californicus reproductive potential.

Aquaculture Application and Development

Hatchery production of Parastichopus californicus has been hindered in the past by inefficiencies in the production of viable eggs from multiple females with predictable timing. To maximize GI, broodstock should be collected in February to March for Alaskan populations, to correspond with late stages of egg development. This timing would also correspond with the lowest internal organ masses (Fankboner & Cameron 1985). During shipment, P. californicus often eviscerate internal organs, sometimes including gonads, and then regrow organs from existing energy stores before spawning. Before the remoteness of animal collection sites as well as hatchery facilities in Alaska, high evisceration rates during broodstock transport may be expected. Thus, timing collections to coincide with the period when individuals are already at their minimum visceral mass may be the most efficient approach.

Seasonal seawater temperatures are an important consideration in the development of live-spawning methods for maximizing production of viable eggs and culturing Parastichopus californicus in captivity. In SEAK. peak GI coincided with a seawater temperature increase from about 5[degrees]C-8[degrees]C in 2012, and 8[degrees]C-12[degrees]C in 2013 (Fig. 3). Maximum sustained seawater temperatures in SEAK were as high as 15[degrees]C (Fig. 3), indicating that temperatures in spawning aquaria as high as 15[degrees]C-18[degrees]C are required to ensure at least a 3[degrees]C-5[degrees]C temperature shock (cf., Hamel et al. 1993, Morgan 2000).

Total fecundity and estimates of viable egg production based on live spawning did not significantly differ between years in this study, although there were large variations among individuals within years. Significant differences in fecundity estimates were not observed between the spawning methods, but large variation among strip-spawned individuals may have biased results. Reduced fertilization success combined with the effects of DTT on larval development rates indicate live spawning should be pursued in hatchery culture initiatives. Live-spawning methods have been successful for other sea cucumber species (Chen 2003, Fujiwara et al. 2010, Hu et al. 2010, Ren et al. 2016). Captive spawning of Parastichopus californicus may present an additional challenge, however, in that it yielded just over half as many eggs as Stichopus vastus and Apostichopus japonicus which are cultured in Japan (Chen 2003, Hu et al. 2010).

Life-history information for Parastichopus californicus has been limited, and must continue to be expanded to better manage fisheries and support aquaculture stock enhancement. Large gaps in life-history information include recruitment rates and habitat requirements, mortality rates, and predation rates. Identification of in situ "nursery" areas and environmental requirements for juvenile settlement (e.g., substrate type, food requirements, and temperature) is most urgently needed to support fisheries management and stock enhancement efforts.

ACKNOWLEDGMENTS

We would like thank the Southeast Alaska Regional Dive Fisheries Association and the University of Alaska, Fairbanks, for funding, animal collections, and research space. We would also like to thank Dr. Katrin Iken and Mr. Jeff Hetrick for guidance in this project. The Southeast Alaska Regional Dive Fisheries Association supplied funding and in-kind support for animal collections and research supplies (e.g., aquaria, pipettes, and aquarium heaters). Lipid analysis was supported by the University of Alaska Fairbanks, Gulf of Alaska Applied Research Student Award. Histological analysis was supported by the NOAA-CIFAR Center for Global Change Student Research Grant Competition. Salary support was provided by the Rasmuson Fisheries Research Center. None of the funding sources were involved in the study design, writing of the manuscript, or decision to submit the article for publication.

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CHARLOTTE REGULA WHITEFIELD (*) AND SARAH MINCKS HARDY

College of Fisheries and Ocean Sciences, University of Alaska Fairbanks, P.O. Box 757220, Fairbanks, AK 99775

(*) Corresponding author. E-mail: cmregulawhitefield@gmail.com

DOI: 10.2983/035.038.0118
TABLE 1.
Results of ANOVA tests for differences in female Parastichopus
californicus body-wall wet weight, gonad wet weight, GI, gonad lipid
content, and IWMI over time.

                 df   Mean square   F       P

Body-wall wt.
  Month          6     3,988.54      2.24    0.051
  Year           1    16,221.11      2.95    0.091
  Month x year   6       188.09      6.04    0.174
Gonad wt.
  Month          6       153.82     10.40   <0.001
  Year           1       196.95     13.31   <0.001
  Month x year   6       241.94     16.35   <0.001
GI
  Month          6        93.67     12.60   <0.001
  Year           1        89.66     12.06    0.001
  Month x year   6       110.83     14.91    0.001
Gonad lipid
  Month          5         0.97      3.44    0.016
  Year           1         4.87     17.30   <0.001
  Month x year   5         0.49      1.70    0.203
IWMI
  Month          2         1.83      5.36    0.011
  Year           1         1.13      3.36    0.081

Significant P values are shown in bold.

TABLE 2.
Variance in female Parastichopus californicus GI for each collection
time point.

      February   March   April   May    June    July   August  September

2012  1.44       n.d.    13.56   n.d.   18.80   n.d.   0.07    n.d.
2013  n.d.       n.d.     6.42   n.d.   18.69   0.15   n.d.    n.d.

      October   November   December

2012  n.d.      0.09       0.49
2013  n.d.      n.d.       n.d.

n.d., no data.

TABLE 3.
Estimates of total fecundity and viable eggs per female [+ or -] SD
from live- and strip-spawned individuals.

                  Fecundity                  Viable eggs
                  (eggs [female.sup.-1])     (eggs [female.sup.-1])

Live spawning
  2011 (n = 6)    449,588 [+ or -] 207,875   345,388 [+ or -] 253,113
  2012 (n = 9)    333,996 [+ or -] 150,758   317,943 [+ or -] 145,978
  2013 (n = 11)   279,438 [+ or -] 170,013   269,438 [+ or -] 173,301
Strip spawning    382,749 [+ or -] 814,470   256,138 [+ or -] 551,793
(n = 15)
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Author:Whitefield, Charlotte Regula; Hardy, Sarah Mincks
Publication:Journal of Shellfish Research
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Date:Apr 1, 2019
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