Single Species and Variable Environments but Different Fish? A Case Study of the Gray Rockfish Sebastes glaucus (Sebastidae, Scorpaeniformes) in the Northern Sea of Okhotsk

1. Introduction

Zavyalov Island (Figure 1) is one of the largest islands (area ca. 116 km²) in the Taui Bay, northern Sea of Okhotsk (Northwestern Pacific, Russia) being 21.5 km long and 4 to 7.5 km wide [1]. Meanwhile, its ichthyofauna, especially of the desalinated lagoon in the northern part of the island, has remained poorly understood until recently. According to the results of two expeditions in the summer of 2019 and 2025, it has been found that over 20 marine fish species occur in the waters of this island [2]. Among them, one of the most abundant is the gray rockfish Sebastes glaucus Hilgendorf, 1880.

This species is endemic to the North Pacific, occurring mainly along the Asian coast (the Sea of Japan, the Sea of Okhotsk and the Bering Sea, the Pacific waters of Japan, the Kuril Islands and eastern Kamchatka). In the north, its range is limited by the Bering Sea, in the south by the Pacific waters of Japan, Iwate Prefecture (northern Honshu). In the Sea of Japan, this species is distributed as far as the Tatar Strait to the north, and as far as the Peter the Great Bay along the mainland coast and to Toyama Bay along the insular coast in the south [3,4,5]. The range of this species is limited to the east by the waters of the Attu Island of the Aleutian Archipelago [6,7].

In most areas, S. glaucus is relatively scarce [8,9,10], reaching its maximum abundance in the northern Sea of Okhotsk [11,12]. It is likely that it may play a certain role in trophic chains, serving as food for fin whales [5]. It is considered an important target of fishing in the northern Sea of Okhotsk [11,12,13] or is considered as a promising target of longline/rod and line, net and trawl coastal fishing in other parts of the range [8,9,14,15,16,17,18,19,20].

The biology of this species has been poorly studied [21,22]. Information on its distribution and basic biological characteristics, although published in a number of papers (mostly in Russian that are hardly accessible to foreign readership) are quite disparate [8,9,10,13,14,15,16,17,23,24,25], while karyological and molecular genetic characteristics of this species and its parasite fauna is still missing. Furthermore, no attempts have yet been made to assess the impact of the varying habitat conditions of this species on the morphology of the axial skeleton, the age, and growth of its individuals, including using scales and otoliths.

The purpose of this publication is to provide the comparative morphological, ecological, biological, karyological, genetic, and parasitological characteristics of the gray rockfish Sebastes glaucus from the desalinated lagoon and adjacent sea waters of Zavyalov Island (northern Sea of Okhotsk) under different environmental conditions.

2. Materials and Methods

2.1. Brief Description of Desalinated Lagoon

The desalinated lagoon is formed by two merging spits (Figure 2), which once separated the lagoon from the sea. Apparently, the lagoon communicates with the sea through pebble spits, since it, like the surrounding sea, is characterized by tidal processes. The length of the spit is about 600 m, the maximum depth according to the echo sounder is 24 m. The salinity during the study period in the surface layer ranged from 22.6 to 25.0 psu, at a depth of about 20 m it was from 28.5 to 31.0 psu, which is significantly lower than the salinity in the adjacent sea areas, which in July is 31.2-31.4 psu at the surface and 32.6-33.0 psu near the bottom [1]. The water temperature during the study period was from 13.5 to 14.2 °C at the surface and from 4.9 to 8.0 °C at a depth of about 20 m, which is significantly higher than that in the sea, which in July is 9.0-10.5 °C at the surface and from -1.0 to 0 °C near the bottom [1]. Fucus thickets and mussel fouling of various ages were observed in the shallow part of the lagoon. Compared to the surrounding marine areas, the species composition of the fish fauna in the lagoon is depleted and includes, in addition to gray rockfish, five more species, i.e. Myoxocephalus stelleri (Cottidae) and Gadus chalcogrammus (Gadidae) in its deep-water areas, as well as Hadropareia middendorfii (Zoarcidae), Porocottus minutus (Cottidae), Alectrias alectrolophus (Stichaeidae) and Opisrocentrus ocellatus (Stichaeidae) in the coastal zone [2].

2.2. Data Collection

Data on S. glaucus were collected in a desalinated lagoon in the north of the Zavyalov Island from July 8 to 11, 2025, and in adjacent areas of the sea on July 7 and 12–16, 2025 (see Figure 1 and Figure 2). Stationary nets 30–50 m long, 1.9–3.0 m high, with mesh size 2.4–2.8 cm, were used as fishing gear. The caught gray rockfishes were subjected to biological analysis, which was carried out using generally accepted methods [26,27] and included measuring fork length (FL) and body weight, determining sex, and the stage of gonadal maturity, as well as the diet composition. The gonadal maturity stage of individuals was examined at the macroscopic level. 23 specimens (16 females and 7 males) from the desalinated lagoon and 79 specimens (29 females, 40 males, and 10 immature individuals) from the adjacent sea area were analyzed. Fork length was measured with a ruler with an accuracy of 1 mm, and body weight was measured on electronic scales with an accuracy of 0.05 g. The sizes of the otoliths (maximum length and height) were measured using a mechanical caliper with an accuracy of 0.1 mm, while their weight was determined using a Digital Mini Scale TL-series (Original Equipment Manufacturer, China) with an accuracy of 0.001 g.

2.3. Morphology

Seven specimens from the desalinated lagoon (ZIN No. 57453, 16.4–22.0 cm standard length - SL), as well as nine individuals collected in the Taui Bay and neighbor areas (northern Sea of Okhotsk), deposited in the collections of the Institute of Biological Problems of the North of the Far Eastern Branch of the Russian Academy of Sciences (IBPN FEB RAS not catalogued, SL 6.7–30.8 cm) and ZIN RAS (Nos. 34363, 37002, 43917, 52836; SL 6.2–18.3 cm) were subjected to comparative morphological analysis. To compare the external morphology of lagoon and marine fish, eight morphometric characters were examined: the number of rays in the first (D1) and second (D2) dorsal fins, the anal fin (A), the number of the lateral line pores (L.l.p.), the number of gill rakers on the 1st gill arch (Sp.br.), the number of trunk (Vert. abd.) and caudal (Vet. caud.) vertebrae, and the total number of vertebrae (Vert. tot.). The statistical significance of the differences between the compared morphological characters was assessed using the Student’s t-test. Photographs of the external appearance of specimens were obtained using digital cameras on smartphones of various models. Photographs of small-sized otoliths were taken using a Neoscan N80 computer microtomograph (Neoscan, Belgium), while those of larger ones were taken using a professional Nikon SMZ 25 stereomicroscope equipped with a specialized Nikon DS-RI2 digital camera (Nikon, Japan).

2.4. Biological Data Processing, Preparation of Otoliths and Age Determination

The relationship between fork length (FL, cm) and body weight (W, g) was described by the equation: W = a × FLᵇ [28,29,30], where “a” is the linear coefficient and “b” is the exponent. The latter was used to describe three-dimensional growth, which is characterized as isometric when b = 3.0, negative allometric (hypoallometric) when b < 3.0, and positive allometric (hyperallometric) when b > 3.0 [31].

To determine the growth rate of juvenile S. glaucus, the average annual growth increments on the scales of immature individuals (FL<26.0 cm) were calculated using the back-calculation method and direct proportionality formula [32]. In addition to determining the age, the processing of the collected scales included measuring the greatest radius of the scale and the annual zones, with the end of the zones being defined as the area of closely spaced or flattened sclerites (Figure 3). The measurements were carried out using the AmScope ToupView 3.1 software (ToupTek Photonics, China) based on images obtained with an OMAX XTL-3400 stereoscopic microscope and an A35100U digital camera (OMAX, Republic of Korea) at 30x magnification. In total, scales were examined from 32 specimens from the sea and 20 specimens from desalinated lagoon (Table S1). Statistical analysis of the results was performed using MS Excel (Microsoft Corporation, USA).

Otoliths (sagittae) were extracted from freshly caught fish. Age of fish in catches was determined by counting annual increments on sectioned otoliths (Figure 4). Otoliths were prepared using the ‘break and burn’ method, which has proven effective for long-lived fish species [33], following the recommendations for age determination of species of the genus Sebases [34]. Thin sections for large specimens and cross-sections for small- and medium-sized fish were prepared by grinding the burnt otolith fracture from both sides or one side respectively using aluminium-oxide- or silicon-carbide-coated grit of 0.1–0.9 µm (Buehler, Chicago, USA). Crystalbond 509 resin (Aremco Products, NY, USA) was used to secure the otolith during grinding. Growth zones were counted on photographs of otolith sections, which were coated with glycerine and imaged using a Motic SMZ-143 microscope camera (Motic, Hong Kong, China).

Age was determined for 74 fish captured in the sea, comprising 39 males, 28 females, and 7 juveniles. In the desalinated lagoon sample, age was determined for 20 fish, including 7 males and 13 females.

To compare growth parameters between males and females, the FL/Age ratio was used across different size classes. For groups with normal distribution, Student’s t-test was applied for small samples; in cases of deviation from normality, the Mann–Whitney U-test was used. Model selection for describing the growth of S. glaucus in the studied samples was based on the Akaike Information Criterion (AIC) [35]. Linear, power, Gompertz, logistic functions, and the von Bertalanffy growth equation (VBGE) were compared.

2.5. Genetics

Eleven S. glaucus specimens from the waters off Zavyalov Island (Taui Bay of the northern Sea of Okhotsk) were genotyped (Table 1). Variability of nucleotide sequences was analyzed for the following mtDNA fragments: the COI gene with a length of 1115 base pairs (bp), the tRNA-Thr – control region containing the genes tRNA threonine (Thr), tRNA proline (Pro) (119-122 bp) and 5’-part of the control region (388 bp). Data from GenBank (www.ncbi.nlm.nih.gov) were used for comparison based on the mtDNA of three specimens of S. glaucus, as well as other species of the genus Sebastes, used as an outgroup for rooting phylogenetic trees. The nucleotide sequences of the sequenced mtDNA regions were deposited in GenBank (see Table 1).

Genomic DNA was isolated from muscle tissue using a standard method that included tissue lysis with 1% SDS in the presence of proteinase K (0.2 mg/mL) and protein purification with phenol [36]. PCR and DNA sequencing were carried out in accordance with the conditions described earlier [37]. The DNA was sequenced using an ABI Prism 3500XL Genetic Analyzer (Applied Biosystems, USA) at the IBPN FEB RAS (Magadan, Russia).

MEGA 12 [38] was used for nucleotide sequence alignment, assessment of genetic marker variability, and calculation of genetic distances under a maximum likelihood model accounting for heterogeneity in substitution rates. Optimal substitution models were selected using the BIC criterion, and maximum likelihood phylogenetic trees were inferred with 10 000 bootstrap replicates for node support estimation. Branch nodes with bootstrap support ≥ 70% were considered reliable [39]. Estimates of haplotype diversity, genetic distances, and phylogenetic trees were obtained without accounting for deletions.

2.6. Karyology

Five specimens of the species used for molecular genetic analysis were karyotyped (see Table 1). The samples included a male and a female from the sea waters, and a male with two females from the desalinated lagoon of the Zavyalov Island. A chromosomal suspension was prepared according to the protocol by [40], which was applied to slides and stained for 25 minutes with a 4% solution of azure-eosin (Giemsa, Merck, Germany) in 0.067 M Sørensen phosphate buffer (pH 6.8).

Analysis of metaphase plates was performed using an Axio Imager.Z2 microscope. The images of the best metaphases were recorded with an AxioCam HR camera using the AxioVision software (Carl Zeiss MicroImaging GmbH, Germany). 170 metaphase plates from sea specimens and 200 plates from the lagoon specimens were analyzed. The study was carried out using the equipment of the Department of Electron and Light Microscopy at the A.V. Zhirmunsky National Scientific Center for Marine Biology, Far Eastern Branch of the Russian Academy of Sciences (Vladivostok, Russia).

Chromosome identification was carried out according to the criteria by [41]. Metacentrics were classified as bi-armed chromosomes, while subtelocentrics and acrocentrics with a very short or visually indistinguishable second arm were classified as uni-armed chromosomes.

2.7. Parasites

When collecting and processing parasitological material, standard methods were used [42,43]. Publications by [44,45] were used to identify parasites. The preparations were examined using a light microscope Mikmed-2 (var. 2) (JSC “LOMO”, Russia).

The study used traditional indicators of host infection by parasites, including the extent of infection (EI), i.e. the proportion of infected fish in the sample under study (%) and the intensity of infection (II), i.e. the extreme values (min–max) of the number of parasites in infected fish (inds.).

The data on the EI of S. glaucus with metacercariae of trematodes were obtained through visual assessment of 23 specimens from the desalinated lagoon and 73 specimens caught outside it in the coastal zone of Zavyalov Island. Data on II are the result of counting metacercariae from 7 specimens of the gray rockfish caught in the desalinated lagoon.

3. Results

3.1. Morphology

The gray rockfish from the desalinated lagoon had all the diagnostic characters typical of the species Sebastes glaucus Hilgendorf, 1880. The presence of more than 12 spines in the dorsal fin and more than 10 trunk vertebrae, combined with the absence of supraorbital spines, indicates their belonging to the subgenus Sebastodes. The presence of 8–9 branched anal fin rays, along with the absence of spines on the top of the head, a wide interorbital space, and the characteristic dark, greenish-brown coloration, allows for identification of this species as Sebastes glaucus.

A comparison of the main morphometric characters of specimens from desalinated lagoon of the Zavyalov Island and the marine waters of the Taui Bay of the Sea of Okhotsk showed no differences in most of them (Table 2). Nevertheless, individuals from the lagoon significantly differed from those inhabiting the sea areas by lower average values of the number of the lateral line pores (45.8 vs. 48.4) and the total number of vertebrae (28.6 vs. 29.0).

In addition, rockfishes inhabiting the lagoon were characterized by a high frequency of abnormalities in the structure of the axial skeleton. The latter included spinal curvature, partial scoliosis of some vertebrae, deformation of pterygiophores and fin rays, as well as loss of part of the latter (Figure 5). Five out of seven lagoon specimens studied had from one to five (a total of 13) such anomalies. At the same time, no such anomalies were found in fish collected in sea areas of the Taui Bay.

The general outline of the sagittal otoliths of both lagoon and marine specimens of S. glaucus is similar and can be characterized by a fusiform shape with a distinctly pronounced, large, broad and, as a rule, pointed rostrum, and short or poorly defined antirostrum (Figure 6).

Throughout the lifecycle, gray rockfish otoliths retain their basic shape and proportions, with the ratio of the otolith length to its height being approximately 2. However, the shape of the otoliths of S. glaucus from the sea areas of the Taui Bay is characterized by high individual variability, which clearly exceeds the age-related one. Thus, there are differences in the length of the rostrum, the degree of prominence of the anirostrum, and, accordingly, the depth and shape of the excisura major, the shape of the cauda, as well as the degree of development of the lobes on the dorsal and ventral edges of the otolith. At the same time, an analysis of the relative size of the otoliths (the ratio of otolith length to body length) revealed no differences between individuals from the sea areas and those from the desalinated lagoon, i.e. for marine areas, the value was 0.0387 ± 0.0029, while for the lagoon it was 0.0383 ± 0.0042 (Mann–Whitney U-test, p = 0.952). Meanwhile, the absolute sizes of the otoliths in the lagoon were smaller (10.13 ± 0.63 mm vs. 10.74 ± 2.70 mm in the sea), which corresponds to the smaller size of the fish. The correlation between the fork length of the fish (FL) and the length of the otoliths (OL) in the lagoon was weaker (R² = 0.516) compared to individuals from adjacent sea areas (R² = 0.954). In the meantime, the equations describing the relationships between the specified parameters also differed noticeably, i.e. for lagoon fish, FL = 35.56 OL – 93.92, while for the adjacent sea, FL = 31.18 OL – 50.75.

3.2. Biological Characteristics

The biological condition of S. glaucus in the lagoon and the sea differed significantly. In the lagoon, fish with a length of 22.3-31.9 cm (mean 26.2 ± 0.6 cm, modal class 23-28 cm) and a body weight of 154-456 g (mean 264.8 ± 19.9 g, modal classes 180-320 g) were recorded in catches. In the sea, rockfish sizes were 9.7-44.0 cm (mean 28.3 ± 1.0 cm, modal classes 15-16 and 30-38 cm) and 13-1440 g (mean 520.6 ± 43.3 g, modal classes 50-100 and 450-900 g) respectively. Length-weight relationships (LWR) in both samples also differed. Thus, linear coefficients “a” and exponent “b” of LWR equation were 2.23 × 10^-2 and 2.860 for the lagoon fishes and 7.85 × 10^-3 and 3.239 for the sea ones respectively.

The catches in the lagoon were dominated by females (69.6%) and juveniles were absent, while males were more numerous (52.6%) in the sea and juvenile fish accounted there for 11.5%. In the lagoon, immature and maturing individuals formed the bulk of catches, while in the sea, they consisted of immature (40.8%), maturing (33.5%) and mature (25.7%) fish.

Both in the lagoon and in the sea, the gray rockfish exhibited a clearly defined sexual dimorphism in size, but in both areas, it had a different direction. In the lagoon, males were significantly larger than females, i.e. the average fork length and weight were 27.6 ± 1.1 cm and 311.1 ± 33.2 g versus 25.8 ± 0.7 cm and 250.0 ± 22.3 g respectively. In the sea, the corresponding figures of males averaged 29.0 ± 1.2 cm and 534.2 ± 56.1 g versus 32.1 ± 1.3 cm and 670.8 ± 66.6 g in females.

3.3. Age and Growth of Juveniles Based on Scale Readings

According to the results of back calculations (Table S2), the maximum increments in the length of S. glaucus were observed in the first year of life and amounted to 4.0–6.5 cm. Subsequently, over the next 10 years, a synchronous decline of growth rate to 0.8–1.1 cm per year was observed in both lagoon and sea samples, with the growth rate of lagoon fishes being lower. Particularly significant differences were observed in young individuals aged up to 8 years. Thus, the average increment in the length of gray rockfish from the desalinated lagoon during this period of life was 1.8 cm, while that from sea areas was 10% greater (2.0 cm).

The calculated growth of immature S. glaucus was best described by a logarithmic equation (Figure 7), which is reflected in the high values of the coefficient of determination (R²). The equation for sea and lagoon fishes, respectively, is as follows:

FL = 69.387 ln(x) + 38.511 with R² = 0.963 and

FL = 62.581 ln(x) + 33.015 with R² = 0.970, where FL is fork length (cm) and x is age (years).

3.4. Age and Growth Based on Otolith Readings

According to the obtained data, the age of male S. glaucus in sea catches ranged from 11 to 32 years, with a mean age of 20.7 ± 0.9 years. The age of females ranged from 12 to 35 years, with mean 21.7 ± 1.0 years. Juveniles were aged from 10 to 14 years, with a mean of 12.6 ± 0.5 years. In the desalinated lagoon sample, male age ranged from 19 to 31 years (mean 23.9 ± 1.3 years), and female age ranged from 19 to 28 years (mean 23.4 ± 0.7 years).

In all size classes, the ratio of fork length to age did not show significant differences between males and females (Table S3). Therefore, growth was subsequently analyzed jointly for specimens in the sample.

Comparison of age between fish of the same size class from the sea and the desalinated lagoon revealed that fishes were significantly younger in the sea than in lagoon within the 220–260 mm FL class, while in other size classes, the differences were not significant (Figure 8A). In a similar comparison based on body weight classes, sea fishes were consistently younger than in the lagoon across all classes, with significant differences found in 3 out of 4 groups (Figure 8B).

To select the model best describing the growth, we compared Akaike Information Criterion (AIC) values. The comparison showed that the von Bertalanffy growth equation (VBGE) provided the best fit for the studied species (Table S4). The growth curves described by this equation differed between the sea and desalinated lagoon samples (Figure 9). The VBGE parameters for sea fishes were L∞ = 46.29 cm, k = 0.076 y^-1, t₀ = 6.38 y with R² = 0.9, while fordesalinated lagoon fishes, these parameters were L∞ = 554.5, k = 0.002 y^-1, t₀ = -1.45 y with R² = 0.6.

Based on data on mtDNA sequences, phylogenetic trees were reconstructed (Figure 10), with the species S. schlegelii at their base. This is followed by the lineages of the other species of the outgroup and the S. glaucus cluster, which is differentiated into two subclusters and three separate lineages. Most of the branching nodes have high confidence (≥ 70%).

3.5. Genetics

The following mtDNA variability parameters have been identified in S. glaucus. The COI gene contained 28 nucleotide substitutions (13 transitions, 15 transversions), 12 phylogenetically informative sites, and out of 371 amino acid residue sites ten were variable. The tRNA-Thr – control region fragment contained 15 nucleotide substitutions (four transitions, 11 transversions), 14 informative sites, and three sites with single-nucleotide deletions. According to data on variable sites, S. glaucus had nine haplotypes for the COI gene, and seven haplotypes for the tRNA--Thr –control region. The average values of the genetic distances for the COI gene and the tRNA-Thr –control region were between S. glaucus and the species of the outer group 1.8-5.20 and 4.34-9.50%, within the S. glaucus species 0.49 and 0.99% respectively (Table 3).

3.6. Karyology

The karyotype of the sea specimens of S. glaucus (Figure 11A) contained 48 chromosomes (2n) and was represented by two metacentrics (pair 1) and 46 subtelocentrics and acrocentrics (pairs 2–24). The number of chromosome arms (NF) was 50. In the desalinated lagoon individuals (Figure 11B), corresponding characteristics were as follows: 2n = 48, all chromosomes were subtelocentric and acrocentric (pairs 1–24), NF = 48. On karyograms, subtelocentric and acrocentric chromosomes were arranged in a common series in descending order of their sizes. The diploid number of chromosomes (2n) was stable in all studied individuals. No differences were found in 2n and NF between males and females in either of the studied samples.

Parasites

A study of S. glaucus from the desalinated lagoon of Zavyalov Island showed its high infection with metacercariae of trematodes. The metacercariae capsules, stained with black pigment, were round (0.5–0.8 mm) or oval in shape (2.5–1.7 × 1.6–0.8 mm). The parasites were distributed throughout the fish body (oral and gill cavities, eyes, muscles, skin, and subcutaneous tissue). The prevalence of metacercaria invasion was 100%, the intensity was 151-1,009 specimens. On the surface of some fish, the pigmented capsules merged into an almost solid black “crust”. 167 metacercariae were found in the eyes of a single S. glaucus specimen (Figure 12). An external examination of a sample of the gray rockfishes from the coastal waters of Zavyalov Island revealed no such metacercariae.

4. Discussion

4.1. Morphology

The morphology of otoliths in members of the genus Sebastes has species-specific features [46] and is therefore often used for species identification [47,48]. Based on the study of the morphology of otoliths in 42 different Sebastes species [49], there were identified nine otolith morphotypes that formed under the influence of predominantly ecological and biogeographical factors. The otoliths of S. glaucus from the waters off the Zavyalov Island that we studied correspond well in shape to morphotype VIII [49], which, in addition to this species, is also characteristic of S. elongatus, S. rosaceus, S. mystinus, and S. constellatus. Moreover, in terms of otolith morphology, the gray rockfish turned out to be most similar to the latter species, which is distributed in the waters of California (USA) and Baja California (Mexico).

The relationship between the size of rockfishes (Sebastes spp.) and their otoliths is tight and is well described by a linear function [50]. An analysis of the relationship between the FL of 30 species of rockfishes of the genus Sebastes and the length of their otoliths (OL) showed that the coefficients “a” and “b” in the equation (FL = a × OL – b) range from 19.91 to 47.46 and from -32.97 to 170.11 respectively [51]. The coefficients of the relationships we obtained between the size of the gray rockfish and its otoliths, both in lagoon and sea individuals, fall within the ranges specified above for members of the genus Sebastes from the waters of the northeastern Pacific.

The statistically significant morphological differences identified in the lagoon population of the gray rockfish appear to be determined by the characteristics of its habitat. However, they may also be the result of the small sample size effect. Therefore, increasing the number of fish studied is an important task.

The number of the lateral line pores of fish from the lagoon sample is slightly lower than that of their sea counterparts, but it does not exceed the known variability for the species as a whole, i.e. from 17 [6,52] to 54 [53,54]. It is impossible to assess the significance of this feature without additional research.

The difference in the smaller number of vertebrae in fish from the lagoon sample appears to be more significant, since all morphological descriptions indicate that S. glaucus has 29–30 vertebrae [6,7,52,53,54]. If the latter is true, the reduction in the number of vertebrae to 28 in lagoon fish appears significant and may be a sign of the formation of a separate form within this species. According to [55], the variability in the total number of vertebrae among members of the Sebastidae family is low. In most species, it is absent or does not exceed 1 vertebra. Exceptions include S. hubbsi, S. longipinnis, S. norvegicus, S. mentella, and S. viviparus, for which this figure is 2, as well as S. fasciatus and S. owstoni, whose number of vertebrae varies by 3. In 38 specimens of S. glaucus, Barsukov [55] found variability of 1 vertebra (29–30). Since all subsequent researchers of this species likely relied on the data provided by [55], studying this trait using larger and geographically more distant samples appears to be relevant.

The number of anomalies in the axial skeleton of lagoon fish is noticeably higher than the frequency of this phenomenon in rockfishes as a whole. Among members of the family, based on 1,715 specimens studied, the number of individuals with abnormalities in the structure of the spine and unpaired fins is about 10% [56]. Currently, it is not possible to determine the cause of the high number of skeletal abnormalities in lagoon sample. The most likely factors negatively affecting skeletal formation in this case appear to be the development of juveniles in conditions of reduced salinity, as well as hyper-intensive parasitic infection at early stages of the lifecycle.

4.2. Biological Characteristics

The size composition of S. glaucus in various parts of its range has been fairly well studied (Table S5), but these data are difficult to compare with each other, as they were obtained using different fishing gear, in different seasons/years, and, moreover, have different completeness. Nevertheless, it can be quite definitely stated that the gray rockfish from the waters of Zavyalov Island (both in the lagoon and in the sea) is significantly smaller than those in the western Bering Sea and in the Pacific waters of southeastern Kamchatka and the northern Kurils. At the same time, in terms of average length, it is similar to individuals from the waters of the western Kamchatka, but in terms of body weight, it is one of the smallest representatives of the species, which is likely due to the more severe conditions in the northern the Sea of Okhotsk.

A comparison of the exponent “b” in the LWR equations for fish from the lagoon and the sea (Table S6) showed that in the former area, gray rockfish demonstrates negative allometric growth, while in the sea, its individuals exhibited positive allometric growth. The identified differences in the value of the exponent “b” indicate that the fish in the lagoon are in poorer condition [30]. Overall, this species exhibits growth that is close to isometric in the Pacific waters of southeastern Kamchatka and the northern Kuril Islands [8,9], and allometric growth in the western Bering Sea, both positive [23] and negative [25]. A comparison of the parameters in the LWR equations for gray rockfish shows significant differences at the intraspecific level, which may be related to the fish belonging to different populations, the characteristics of their physiological state at different times, the degree of stomach fullness and gonadal maturity, sex, and the presence of diseases [57,58]. Since our studies in the lagoon and adjacent sea areas off the Zavyalov Island were conducted at the same time, the differences in the condition of the gray rockfish are most likely due to variations in the degree of parasitic load and food availability.

The sex ratio in the catches of S. glaucus in most areas is characterized by a numerical predominance of males [18]. In the southwestern Sea of Okhotsk, they account for 68% of the total fish number [14]; in the northern Sea of Okhotsk — 65% [15]; in Avacha Bay, eastern Kamchatka — 69.5% [16]; and in the Pacific waters of southeastern Kamchatka and the northern Kurils — 66.1% [8,9]. However, there are exceptions to this rule. Thus, in the waters of western Kamchatka, an almost equal sex ratio was observed, while in the waters of the Commander Islands, females predominated – 59% [16]. Furthermore, there is evidence [24] that in the Taui Bay (the northern Sea of Okhotsk), the sex ratio of S. glaucus is subject to interannual dynamics, with the proportion of males in catches varying from year to year within the range of 46.1–60.7%. In the sea areas of the Zavyalov Island, males slightly predominated in the catches (52.6%), which is consistent with the data from the latest study. In the lagoon, by contrast, females predominated (69.6%). Based on the short period of observation, it is currently impossible to determine the cause of such differences.

The published information on sexual dimorphism in the size of S. glaucus is rather limited and contradictory. Panchenko [14] and Kondratiev [15] did not find significant differences in the growth rates of males and females based on net catches in the southwestern and northern Sea of Okhotsk, respectively. Slightly larger body sizes were observed in 12–15-year-old males in trawl catches in the western Bering Sea [23] and, in general, in angling catches off the South Kuril Islands [17]. Orlov and Tokranov [8,9] also reported that males of S. glaucus are larger than females in trawl catches in the Pacific waters of the northern Kurils and southeastern Kamchatka. Partially, sexual dimorphism in the size of this species may be due to the selectivity of fishing gear, which, depending on the sizes of mesh or hooks, may selectively remove larger or smaller individuals from the harvested population. Nevertheless, the use of the same fishing gear in the waters off the Zavyalov Island revealed larger males in the lagoon and females in the sea, which currently lacks a reasonable explanation and requires further research.

4.3. Age and Growth of Juveniles Based on Scale Readings

A comparison of the calculated growth of immature S. glaucus from the desalinated lagoon and adjacent sea waters of Zavyalov Island showed that marine fish exhibited a significantly higher growth rate. According to the observed data (see Table S1), gray rockfish with FL 21.0–22.0 cm showed a sharp increase in body weight compared to the previous size class (almost twice as much). This is most likely due to the onset of sexual maturation. Therefore, S. glaucus from sea waters becomes sexually mature when it reaches a length of 21.5–24.5 cm and a weight of 180–210 g at the age of 10–12+ years (based on scales), while the slow-growing fish from desalinated lagoon matures at a later age (14–16+ years, based on scales) when it reaches approximately the same fork length but has a lower body weight (150–180 g).

Overall, the growth of immature S. glaucus (largely in terms of weight) in the desalinated lagoon is significantly slower compared to that of fish in the sea coastal area, which may be due to the limited forage resources in the small, isolated water body, especially for larger, older fish. In addition, the slower growth rate in the lagoon may be partly due to heavy parasite infestation.

4.4. Age and Growth Based on Otolith Readings

The data obtained in this study on the longevity of S. glaucus significantly exceed previously reported values. The maximum age of the species in Kamchatka waters was previously estimated 26 years [19,23], in the southern Sea of Okhotsk it was estimated as 14 yeas [14], while in Taui Bay (northern Sea of Okhotsk) it did not exceed 20 years [13,15,24,59,60]. This discrepancy is most likely due to methodological differences. Some earlier studies [14,15] involved scales, while in later studies, otoliths were apparently not sectioned, and age was determined directly on burnt cross-sections. It has been shown for other long-lived members of the genus Sebastes that such an approach to otolith processing fails to account for narrow marginal growth zones, resulting in a significant age underestimation [34].

Statistical analysis revealed no significant differences in growth rates between males and females, which is consistent with previous studies that also reported no pronounced sexual dimorphism in growth for S. glaucus [23].

Comparison of the lagoon and sea fish showed that specimens from the lagoon grow more slowly. When grouped by body length, significant age differences were observed only in single size class (22.0–26.0 cm). This may be due to the presence of vertebral anomalies in some lagoon individuals, which affect body length and obscure age-related differences. Likely for the same reason, the VBGE curve for the lagoon sample resembles a linear function, with a low coefficient of determination (R² = 0.6) and a biologically unrealistic asymptotic length (L∞ = 554.5 cm). When comparing the age by body weight, the differences were more pronounced and significant across most groups, confirming slower growth in the lagoon.

The reduced growth rate of lagoon fish may be related to both a poorer forage resources in the isolated water body and a high parasitic load (100% infestation with trematode metacercariae). It has been shown in many fish species that parasitic infection leads to a reallocation of energy resources from somatic growth to immune response [61]. Moreover, heavy eye infestation with metacercariae can impair foraging ability, leading to reduced food intake and consequently further growth retardation [62,63,64].

4.5. Genetics

In studies of the genus Sebastes, mtDNA analysis has been successfully used to determine intra- and interspecific relationships and to study the demographic history of species and their populations [37,65,66,67,68]. The molecular genetic data obtained in this study confirm that mtDNA markers are an informative tool for assessing intraspecific differentiation of S. glaucus.

A comparative analysis of individuals of the species representing different ecological and geographical groups revealed their differences both in conservative (COI gene) and highly polymorphic (tRNA Thr – control region) DNA fragments. The mtDNA sequences contain changes that mark lagoonal (a total of 11 nucleotide substitutions) and marine (4 substitutions and 3 deletions) individuals from the Zavyalov Island waters, as well as five nucleotide substitutions common to both groups. These common nucleotide changes distinguish Taui Bay S. glaucus (Sea of Okhotsk) from those inhabiting the Pacific waters of the Aleutian Islands and Hokkaido Island.

The S. glaucus sample is characterized by significant haplotype variability. For 14 individuals, there are seven to nine haplotypes, none of which is common to different fish groups, while each group (lagoonal, marine from Zavyalov Island waters, marine from the Aleutian Islands and from Hokkaido Island) has specific haplotypes. Within the species, a relatively high nucleotide variability has been detected, i.e. the values of genetic distances reach 1.83 and 2.48%, respectively, for the COI gene and the tRNA-Thr - control region. A considerable contribution to genetic heterogeneity is made by the gray rockfish from the desalinated lagoon of Zavyalov Island, which differs from marine individuals of adjacent sea areas by an average of 0.80% and 1.86%, respectively, and from individuals from off the Aleutian Islands and Hokkaido by 1.1% and 2.14%, while the differences between marine fishes from significantly distant habitats (Taui Bay vs. Aleutian Islands + Hokkaido) account for 0.97 and 0.69%.

A number of species in the genus Sebastes have been recorded to have levels of genetic variability comparable to the data obtained. In particular, significant intraspecific differentiation of the tRNA-Thr - control region was detected in S. aleutianus from the British Columbia waters (1.86%; Table 3), S. taczanowskii from Peter the Great Bay in the Sea of Japan (1.40%; [66]), S. thompsoni from the Pacific Ocean and Sea of Japan coasts off Honshu Island (3.45%; [65]).

The structure of phylogenetic trees indicates intraspecific variability in S. glaucus associated with its habitat. The haplotypes of lagoon and adjacent sea fishes from off the Zavyalov Island form two independent subclusters, which combine with the haplotypes of individuals from the Aleutian Islands and Hokkaido Island into a single cluster of the species. The high estimates of the probability of branching nodes confirm the reliability of the data obtained.

4.6. Karyology

Cytogenetic studies of the genus Sebastes are few in number [69,70,71,72]. In particular, out of more than 100 species of the genus, only 13 have been karyotyped [73,74].

A study of the chromosome set of S. glaucus (2n = 48, NF = 50) revealed its correspondence to the karyotypic organization typical for most members of the genus, characterized by the presence of two medium-sized bi-armed chromosomes and 46 uni-armed chromosomes [69,70]. As with other Sebastes species [70], the gradual reduction in the size of subtelocentric and acrocentric chromosomes in S. glaucus does not allow for the identification of marker chromosomes in this group.

The results of our study are consistent with the notion of high karyotypic conservatism in most members of the genus Sebastes, although exceptions are also known. Thus, in S. hubbsi, unlike in S. glaucus and other studied species, a Robertsonian fusion was recorded, which led to a reduction in 2n to 46 while the NF remained unchanged at 50 [69]. In contrast, in S. schlegelii, the change affected the number of chromosome arms (2n = 48, NF = 50–52) (Arai, 2011), which indicates the involvement of another chromosomal rearrangement in the evolution of the karyotypes of the genus Sebastes, i.e. possibly a pericentric inversion.

A comparative analysis of marine and lagoonal S. glaucus showed that in lagoonal individuals, while maintaining the diploid number (2n = 48), there was a reduction in the number of chromosomal arms (NF = 48). Unlike in marine fish, their karyotype lacks metacentrics. Most likely, this is a consequence of structural rearrangement of chromosomes in one of the pairs, which occurred as a result of pericentric inversion. The stable nature of this rearrangement is confirmed by its presence in specimens caught both in 2019 and 2025.

The fixation of the chromosomal rearrangement in the lagoon group is likely due to spatial isolation, which has limited gene flow from the marine population. Similar phenomena have been described for other species. For example, in Arctic cod Arctogadus glacialis (Gadidae) from Arctic fjords, intraspecific chromosomal polymorphism (6 karyotype variants) has been detected, associated with the presence of B chromosomes, changes in the distribution of heterochromatin, and the localization of rDNA clusters [75]. This indicates that local environmental conditions influence genome organization.

At the same time, all the individuals we studied from the lagoon were 100% infected with metacercariae of trematodes. The metabolic products of the helminths induce unstable chromosomal aberrations with loss of genetic material [76,77]. The high parasite load likely increased the overall level of genomic instability in these fish. However, the detected pericentric inversion is a balanced rearrangement that is atypical for mutagenesis induced by helminths. Consequently, the isolation from the marine population played a decisive role in its fixation.

4.7. Parasites

Since this study was based on the formalin-preserved fish, difficulties in taxonomic identification of the parasites were encountered. Nevertheless, there were evaluated that S. glaucus was infested by metacercariae of trematodes belonging to the genera Liliatrema Gubanov, 1953 and Cryptocotyle Lühe, 1899 (Trematoda: Plagiorchiida: Opisthorchiidae), which are common intestinal and stomach parasites of fish–eating birds in the Sea of Okhotsk basin [78,79,80]. The metacercariae of these trematodes are common, often abundant parasites of various marine fish in the Sea of Okhotsk and the Taui Bay, in particular [45,80,81,82,83]. In S. glaucus from the Taui Bay, metacercariae of trematodes of the genera Liliatrema and Cryptocotyle have not been previously detected.

The high infestation of S. glaucus in the lagoon with metacercariae of trematodes can be explained by the fact that five species of seabirds are nesting on the Zavyalov Island, and, in addition, migratory birds pass along its coast [1]. Undoubtedly, they periodically visit the lagoon, and since many species among them are the definitive hosts of trematodes of the genera Liliatrema and Cryptocotyle, this water body becomes infested with these trematodes. The lagoon has no obvious connection to the sea, so a high concentration of parasites forms in it. In the coastal waters of the island, such conditions do not exist, since a rather strong North Okhotsk Current flows along the northern and southern shores of the island [1], and it is likely that most of the parasitological material is carried away from the shore into the sea. Perhaps this circumstance explains the absence of these metacercariae in gray rockfish in the coastal areas of Zavyalov Island.

A prerequisite for the development of trematodes is the presence in the water body of various mollusks which are their first intermediate hosts. For the genus Liliatrema, this host is not yet known [45], while for Cryptocotyle in the Taui Bay, it is the gastropod Littorina sitkana [80]. This species was found in the diet of S. glaucus from the lagoon in this study. In the sea, the diet of S. glaucus consists mainly of euphausiids, gelatinous organisms (jellyfish, ctenophores, salps), and fish [8,9,11,15,21] (, while its diet does not include mollusks, which are intermediate hosts for parasites.

5. Conclusions

Thus, lagoonal and marine S. glaucus specimens from the waters off Zavyalov Island demonstrate significant differences in several morphological characters, size, age and growth rate, chromosome morphology and the number of chromosome arms, the degree of infection with metacercariae of trematodes, and also show significant genetic differentiation.

A factor negatively affecting the formation of the skeleton of S. glaucus in the desalinated lagoon is the early ontogenesis occurring under conditions of reduced salinity, as well as intensive infection with metacercariae of trematodes already at the early stages of the lifecycle. Some differences in the results of age and growth studies based on scales and otoliths may be due to the fact that otoliths, when used to study of Sebastes spp., provide more accurate and reliable age estimates compared to scales, especially for older fish [84,85]. In addition, age estimates by different readers may differ [86]. Nevertheless, using both hard structures, unambiguous conclusions were drawn regarding the lower growth rates of fish in the lagoon compared to the adjacent sea areas. The poorer condition of the fish and the slower growth rates in the lagoon may be due to the higher temperature, the limited forage resources of the isolated water body, and the high parasite load. The influence of temperature on the development and growth of juvenile rockfishes of the genus Sebastes, as well as the duration of their pelagic phase and the size at which they settle to the bottom, is well known [87,88,89]. The diet of the gray rockfish usually consists of euphausiids, gelatinous organisms (jellyfish, ctenophores, salps), and fish [8,9,11,15,21], in the absence of which it has to switch to feeding in the lagoon on atypical prey items as benthic shrimps, hermit crabs, and gastropods. The results of parasitological studies make it possible to diagnose the so–called “black-spotted disease” in individuals from the lagoon, one of the most well-known parasitic diseases of fish, which is easily recognized by external examination [90] and can cause body deformity, curvature of the spine, destruction of body integuments and musculature in juvenile fish, and is also one of the powerful factors regulating their abundance [43,91,92].

Genetic differentiation between lagoon and marine individuals may be due to the influence of a number of factors, which include spatial isolation, leading to limited gene flow; an increased role of genetic drift due to a reduction in the effective size of the lagoon population; and the diversifying selection against on the background of differences in ecological conditions (temperature, salinity, hydrological regime) between the desalinated lagoon and the sea. The karyotype of marine S. glaucus from the Taui Bay is typical for the genus Sebastes. Lagoon individuals are similar to most studied species of the genus in terms of their diploid chromosome number (2n = 48), but they differ from them, as well as from marine individuals, in chromosome morphology and the number of chromosome arms (NF = 48 vs NF = 50). The observed differences appear to be due to a pericentric inversion that arose and became fixed in the lagoon group under the conditions of an isolated water body.

Despite the wide range of studies conducted, a number of questions remain unresolved: (1) is the gray rockfish population in the lagoon self-sustaining and self-reproducing, (2) how long has it existed in the lagoon, and (3) how did this species get into the lagoon from the sea? Based on the presence in the lagoon catches of both small immature and large mature fish, as well as significant genetic differentiation between marine and lagoonal individuals, the latter may well represent an independent self-reproducing population. Judging by the age composition in the catches (from 9 to 32 years), the lagoon population may have existed for quite a long time. Several hypotheses can be proposed regarding the penetration of S. glaucus into the lagoon from the adjacent sea. One of the sources of fish dispersal between different water bodies is provided by birds, which can carry fish eggs and juveniles on their feet, feathers, in their crop and excrements [93,94]. Female gray rockfishes release larvae in the northern Sea of Okhotsk in June–July at a depth of 2–3 m [18]. For acclimation, naturalization and maintaining a sufficient population level of S. glaucus in the lagoon, the transfer of its offspring by birds must be regular. Therefore, this scenario does not seem very realistic. Since the lagoon is connected to the sea via spits and its depth is more than 20 m, there is a possibility that not only S. glaucus larvae but also other marine fish observed in the lagoon may enter it with the water coming from the sea [2]. It is also possible that early juveniles of the gray rockfish, which develop in the coastal zone, could be carried into the lagoon by storms. Although such storms occur relatively rarely in summer, their frequency during this period is about 5% [95].

If the gray rockfish population in the lagoon turns out to be self-reproducing and largely isolated from external gene flow, the biological, morphological, and genetic characteristics of the fish that make it up should probably be considered as the initial stages of speciation according to the dispersalist model [96]. If this population is maintained, in the long term it could evolve into a separate species adapted to living in a small, isolated water body with reduced salinity. A striking example of such an evolutionary process is the Kildin cod Gadus morhua kildinensis, which inhabits Lake Mogilnoye on Kildin Island in the Barents Sea [97]. Thus, further monitoring of the lagoon population of S. glaucus on the Zavyalov Island appears to be extremely necessary.