DOI: 10.25338/b84p78
title: Growth, osmoregulation, and Ionoregulation of Longfin Smelt (Spirinchus thaleichthys) yolk-sac larvae at different salinities

description: Longfin smelt (Spirinchus thaleichthys) is a threatened anadromous fish
 species that spawns in freshwater to moderately brackish (i.e. 5-10 ppt)
 reaches of the upper San Francisco Estuary and has declined to
 approximately 1% of its pre-1980s abundances. Despite 50+ years of
 population monitoring, the efficacy of 10+ years of conservation efforts
 for longfin smelt remain uncertain due to a limited understanding of how
 the species responds to environmental variation, such as salinity. For
 example, high mortality during larval stages has prevented culture efforts
 from closing the life cycle in captivity. Here, we investigated the
 effects of salinity on longfin smelt yolk-sac larvae. Newly hatched larvae
 from four single-pair crosses were acutely transferred to and reared at
 salinities of 0.4, 5, 10, 20, or 32 ppt. We compared whole-body water and
 sodium ion (Na+) content, notochord length, and yolk-sac volume at 12, 24,
 48, 72, and 96 h post-transfer for each salinity treatment. We found that
 larvae maintained osmotic and ionic balance at 0.4-10 ppt, whereas
 salinities ˃10 ppt resulted in decreased water and increased whole-body
 Na+ content. We also found that larvae grew largest and survived the
 longest when reared at 5 and 10 ppt and that yolk resorption stalled at
 0.4 ppt. Finally, there were significant but small interclutch variations
 in responses to different salinities, with clutch accounting for
 &lt;8% of the variance in our statistical models. Overall, our results
 indicate that longfin smelt yolk-sac larvae likely perform best at
 moderately brackish conditions, thus yielding a mechanism that explains
 their distribution in field surveys and providing key information for
 future conservation efforts.
description: <strong>2.1 Broodstock and Embryo
 Collection</strong> Adult broodstock longfin
 smelt were collected from the wild by the U.S. Fish and Wildlife Services
 Chipps Island trawl with a midwater trawl, the FCCL using a lampara net in
 the Sacramento San Joaquin Delta, and the UC Davis Otolith Geochemistry
 and Fish Ecology Laboratory using an otter trawl in the Alviso Marsh in
 South San Francisco Bay. Broodstock collections were approved under
 California Department of Fish and Wildlife MOU ID: Hobbs_LFS_2021 and
 Specific Use Permit IDs: S-191990002-19, 199-001, and D-0021521915-6. Fish
 were held in 76 L cylindrical carboys during collections and transported
 to the FCCL at the end of each sampling day and held at 10 ppt at 12℃
 until visually gravid and then strip-spawned. A total of four clutches
 from separate single-pair crosses were obtained between January and April
 2019 (see Supplementary Table 1 for parental information) and embryos were
 maintained using methods described in Yanagitsuru et al. (2021).
 Fertilized embryos were held at 12℃ in pre-treated Delta water (0.2 ppt;
 see Supplementary Table 2 for additional water constituents) at the FCCL
 until 5 or 7 days post-fertilization (dpf) when fertilized and
 unfertilized embryos could be differentiated.
 Fertilized embryos were transported to UC Davis fish conservation
 physiology laboratory in conical tubes within coolers chilled with two ice
 packs. The ice packs were separated from direct contact with the tubes
 with a Styrofoam pad to prevent temperatures from cooling too much. By the
 time embryos arrived, water temperatures within tubes were between 11.7
 and 13.2℃. Upon arrival, embryos were placed in 500 mL nylon mesh (100 µm
 mesh size) containers within gently aerated 2-gallon buckets receiving
 flow-through (approximately 4 L h<sup>-1</sup>) freshwater
 (well water; 0.4 ppt; see Supplementary Table 2 for additional water
 constituents). Temperature was lowered gradually to 9℃ at 0.5℃
 h<sup>-1</sup> as this temperature yielded high hatching
 success in a previous study (Yanagitsuru et al., 2021). Embryos were
 exposed to a 12:12 light:dark cycle. Embryos from all clutches were
 maintained in these conditions and checked twice daily between 8:00-9:00
 and 22:00-24:00 to remove any dead embryos (indicated by a milky opaque
 coloration, similar to delta smelt (Tsai et al., 2021), until hatching.
 Temperature (8.3-9.2℃), dissolved oxygen (94.8-99.2%), salinity (remained
 constant at 0.4 ppt), and pH were measured daily with a YSI 556 (YSI,
 Inc., Yellow Springs, OH, USA). Ammonia (total ammonia nitrogen) was also
 measured daily with a salicylate colorimetric water test kit (API,
 Calfont, PA, USA) and remained below detection threshold (&lt;0.25
 ppm) throughout the duration of the experiment (see Supplementary Table 3
 for water parameters). All clutches were monitored for hatching at 8:00
 and between 22:00-24:00. No hatching was observed between 22:00-24:00,
 indicating that all hatching occurred between 24:00-8:00, and thus all
 individuals used for experimentation were between 0 and 10 hours
 post-hatch (hph). <strong>2.2 Salinity
 Exposures</strong> Clutch 1 was used to measure
 survival over time at five different salinities: 0.4 (freshwater), 5, 10,
 20, and 32 ppt. Water of different salinities were made by mixing well
 water with Red Sea Coral Pro salt mix (Red Sea Fish Pharm, Ltd., Herzliya,
 Israel) and stored in heavily aerated and mixed 300 L reservoirs. Between
 8:00-9:00, hatched larvae were divided into five groups of 30 individuals
 and acutely transferred to 500 mL mesh containers in separate, gently
 aerated 2-gallon buckets receiving flow-through water (approximately 4 L
 h<sup>-1</sup>) of one of the five experimental salinities.
 Because yolk-sac larvae do not feed exogenously and to ensure water
 quality issues from excess feed did not affect survival, larvae were left
 unfed. Larvae were monitored every 24 hours thereafter, and dead larvae
 were recorded and removed to determine survival until 192 hours
 post-transfer (hpt). Clutches 2, 3, and 4 were used to
 measure wet and dry mass (used to calculate whole-body water content),
 notochord length, yolk-sac volume, and whole-body sodium ion (Na+) content
 over time post-transfer at the five experimental salinities (methods
 described below). For each clutch, a subset of hatched larvae was
 collected between 8:00-9:00 to gather pre-transfer measurements. All other
 larvae were divided into groups of 100 individuals into 500 mL mesh
 containers within 2-gallon buckets of different salinities as described
 above and left unfed. Because the survival trial indicated that
 substantial mortality began at 96 hpt and because of the limited
 availability of this imperiled species, we only sampled up to 96 hpt.
 Subsets of larvae from each salinity treatment were subsequently sampled
 at 12, 24, 48, 72, and 96 hpt to gather post-transfer measurements.
 Temperature (8.3-9.1℃), dissolved oxygen (93.5-100%), salinity (0.4 ppt:
 0.4 ppt; 5 ppt: 5.0-5.7 ppt; 10 ppt: 9.9-10.5 ppt; 20 ppt: 19.9-20.8 ppt;
 and 32 ppt: 31.6-32.4 ppt), pH (8.2-8.6), and ammonia were also measured
 at this time (Supplementary Table 3). Ammonia remained below detection
 threshold throughout the duration of the experiment.  
 <strong>2.3 Whole Body Water Content and Na+
 Content</strong> For each clutch, five groups of
 10 larvae each (n=15) were sampled pre-transfer and at 12, 24, 48, 72, and
 96 hpt to measure wet and dry masses and further used to calculate
 whole-body water content. Due to high mortality between 48 and 96 hpt in
 the 32 ppt treatment group for all clutches, sample sizes were reduced at
 72 (n=5) and 96 hpt (n=5) for this group. Likewise, clutches 2 and 3
 experienced high mortality in the 20 ppt treatment group before 96 hpt and
 sample sizes were reduced at 96 hpt (n=13; see Supplementary Table 4 for
 all sample sizes). Larvae were pooled for each mass measurement to ensure
 measured masses fell within the working range (minimum load: 10,000 µg;
 repeatability: 50 µg) of the Veritas HPB 625i semi-micro balance (Veritas
 Technologies LLC, Santa Clara, CA, USA) used. Minimum loads were achieved
 by measuring larvae within 1.5 mL centrifuge tubes. Upon collection, each
 group of larvae was euthanized with 0.5 g L<sup>-1</sup>
 tricaine methanesulfonate (MS-222) buffered to pH 8.0 with sodium
 bicarbonate (NaHCO<sub>3</sub>) in accordance with UC Davis
 IACUC protocols and rinsed over a 100 µm nylon mesh screen with 1 mL of
 Millipore water to remove any residual salts on body surfaces. Larvae were
 gently dried by wicking away water with a KimWipe (Kimberly-Clark
 Worldwide, Inc., Roswell, Georgia, United States) and then weighed for wet
 mass. During preliminary testing, consecutive measures of wet mass were
 found to decrease with each repeated measure, suggesting that the samples
 were quickly drying and all wet mass for experimental samples were thus
 measured only once. This procedure was completed within two minutes of
 euthanizing larvae. The same groups of larvae were then dried in a
 convection oven at 60 ℃ over 48 h and weighed for dry mass. We chose 60 ℃
 as this fell within the recommended temperature range by Schmidt et al.,
 (2013) and was a temperature close to that used by a similar study by
 Gallagher et al., (2013). Dry mass was measured three times for each
 sample and the average of masses was used for analyses. Wet and dry masses
 were divided by 10 (number of larvae per group) to calculate average
 individual larval wet and dry masses. Whole-body water content was
 calculated as the difference between wet and dry mass and reported as a
 percentage of wet mass. The same dried groups of larvae
 previously used to measure dry mass were then used to measure whole-body
 Na+ content following modified methods described in Gallagher et al.
 (2013). Larvae were placed in 0.2 mL microcentrifuge tubes and digested in
 10x the wet mass of the pooled group in 1 mol L<sup>-1</sup>
 nitric acid (V/W) at 65 ℃ for one week. Larvae were dissociated with daily
 mixings with a vortex shaker (Scientific Industries, Inc., Bohemia, New
 York, United States) to aid digestion. After digestion, tubes were
 centrifuged to separate the solids and supernatant was analyzed on a
 Sherwood Scientific M360 flame photometer (Sherwood Scientific Ltd.,
 Cambridge, United Kingdom) and standardized to dry mass for whole-body
 Na<sup>+</sup> content. Whole-body Na<sup>+</sup>
 content values were divided by 10 (number of larvae per group) to
 calculate an average individual whole-body Na<sup>+</sup>
 content per sample. <strong>2.4
 Morphometrics</strong> Subsets of larvae (see
 Supplementary Table 4 for sample sizes) were imaged with a Canon EO6 Rebel
 T6 (Canon, Tokyo, Japan) mounted on a Leica S8APO stereomicroscope (Leica
 Microsystems, Chicago, IL, USA) and analyzed for morphometrics with Fiji
 ImageJ software following methods described in Yanagitsuru et al. (2021).
 Whole-body larval images were taken next to an electron microscopy grid
 (200 µm grid, 50 µm bar) to provide a scale for calibrating pixels to mm
 to measure notochord length. Lateral and ventral images of yolk-sacs were
 all taken at 12.8x magnification and calibrated to a separate image of an
 electron microscopy grid at the same magnification to measure yolk-sac
 length, depth, and width. Yolk-sac volume was approximated as an ellipsoid
 (V = <sup>4</sup>/<sub>3</sub>πabc, where V is
 volume, a is half-length, b is half-depth, and c is half-width of the
 yolk-sac). <strong>2.5 Statistical
 Analyses</strong> R 3.6.3 (R Core Team, 2013) was
 used for statistical analyses. Linear models were used to analyze all
 metrics. Wet mass, dry mass, whole-body water content, whole-body Na+
 content, notochord length, and yolk-sac volume were each modeled
 separately as functions of the fixed categorical effects of salinity, time
 post-transfer, clutch ID, and their interactions. Whole-body Na+ content
 was log-transformed to meet the assumptions of normality. Tukey’s multiple
 comparison post-hoc tests were used to examine differences between
 salinities and times post-transfer. Statistical significance was accepted
 at p &lt; 0.05. All values are reported as mean ± SEM unless otherwise
 stated.
description: Some image files were missing and morphometrics were thus unable
 to be measured for those fish.