The Age and Growth of One Population of Diaphus watasei (Jordan & Starks, 1904) in the South China Sea

Abstract

We estimated, for the first time, the age of Diaphus watasei (Jordan & Starks, 1904) in the South China Sea (SCS) based on otolith microstructure. According to one-way ANOVA, differences were not observed between the sexes with regard to standard length, body mass, or age. Based on 137 specimens, the sex ratio and relationship between standard length and body mass was 1.32:1 (male/female) and W = 0.0000433L^2.78 (r² = 0.923), respectively. The von Bertalanffy model was fitted as L_t = 171.38 [1 − exp(−0.00206(t − 3.82))], r² = 0.645 (n = 92), which indicated a maximum growth rate of 0.356 mm/day. The speculated birth date of the 92 specimens of D. watasei occurred across almost all months of the year.

1. Introduction

Biomass assessments of mesopelagic fishes suggest global reserves of 2.0–19.5 Gt [1,2,3,4]. Lanternfishes (Myctophidae) constitute the dominant component of mesopelagic fishes, though most individuals are small and short-lived [1,5,6,7]. They exhibit diel vertical migration—descending at dawn and ascending at dusk—which allows them to inhabit deep waters by day and surface layers by night. This behavior links epipelagic and deep-sea ecosystems and contributes to the “biological carbon pump” by transporting organic matter and nutrients to deeper layers [5,8].

Within Myctophidae, the genus Diaphus (commonly referred to as “headlight fishes”) is the largest, comprising 77 recognized species [9,10,11]. Species in this genus share one pair of supraorbital luminous organs, a unique feature distinguishing them from other mesopelagic fishes [9,10,12,13]. Fisheries’ resource surveys have shown that Diaphus species are distributed worldwide, exhibiting high abundances in low-latitude regions [5,7,14,15]. Among lanternfishes, Diaphus species have received more attention due to their economic value, especially in the Indian Ocean, where they have been commercially exploited [16,17]. Diaphus watasei is notable for its abundance and economic value. It is one of the few mesopelagic fishes utilized for human consumption. It is processed as a regional snack in central and southern Japan [15,16]. Studies have provided preliminary insights into its length–weight relationships, sex, reproduction, and feeding habits across the East China Sea, South China Sea (SCS), and Indian Ocean [15,18,19,20,21]. In the SCS, age estimation remains lacking, though it is essential for stock assessment, population dynamics, and effective fisheries management [22].

Otoliths are part of the inner ear of a fish. Otoliths are characterized by a stable chemical composition and a rigid structure. They have been applied widely in age determination. Otoliths are regarded to be the earliest calcified tissue formed during embryonic development in osteichthyans [23]. Following core formation, otolith growth proceeds through daily deposition of calcium carbonate and protein, producing continuous growth increments visible in frontal and sagittal sections [10,23]. The age of a fish is determined by counting the total number of daily growth increments in the otolith [23]. Among the three otolith pairs (lapillus, astericus, and sagitta), the sagitta is the largest and has been used effectively for age identification in lanternfishes [10,24,25]. The otolith microstructure of the sagittal otolith intuitively displays the daily growth increments, and the otolith is divided into distinct zones based on morphological differences between these increments [10,18,24,25,26,27].

Located at the junction of the Western Pacific Ocean and Indian Ocean, the SCS is a critical biogeographic hub. Research on mesopelagic fishes in this region has increased in recent years, but data on the age and growth of D. watasei are limited [10,17,18,19,20,21]. In this study, we complemented existing research by analyzing the microstructure of the sagittal otolith of D. watasei to provide additional estimates of age and growth. Our results could: (i) support future fisheries development of D. watasei; and (ii) contribute to understanding the mesopelagic fish community in the SCS.

2. Materials and Methods

2.1. Fish Collection

Specimens of D. watasei in the SCS were collected on 17 June 2015 via a time-series station, and the location (19°31.11′ N to 19°53.38′ N, 113°53.45′ E to 114°68.58′ E) is shown in Figure 1. The R/V “Nan Feng” (1537 t GT; length = 66.7 m; width = 12.4 m; draught and 4.8 m) maintained a towing speed of 1.9–2.0 m/s for 1 h. During the trawling process, the research vessel made reasonable small-scale movements within the operation area. A bottom shrimp trawl (mesh = 50 mm) was used, covering an area of 110.00 m × 87.16 m. To reduce the effect of currents and ensure that the trawl depths remained relatively constant, the PI44 monitoring system (Kongsberg Maritime, Kongsberg, Norway) was used. Three bottom-trawl operations were conducted, and the operation details are shown in

Table 1. Trawl sampling information.

Sample	D/M/Y	Starting Location	Finishing Location	Trawl Depth	Towing Speed
Time		Lat. (N)/Long. (E)	Lat. (N)/Long. (E)	(m)	(m/s)
04:20–05:20	17/06/2015	19°45.16′/114°08.11′	19°33.54′/113°58.92′	487–490	2.0
10:46–11:46	16/06/2015	19°42.30′/114°20.24′	19°31.11′/113°53.45′	550–560	1.9
20:50–21:50	17/06/2015	19°53.38′/114°40.75′	19°52.15′/114°68.58′	465–470	1.9

. Due to the non-uniform topography of the investigation area, which exhibits variations in depth, the sampling depths for all fish specimens differed slightly. All specimens were collected and stored at −19 °C before being transferred to an onshore laboratory at the end of the research cruise.

2.2. Fish Biology

Specimens of D. watasei with intact morphology (not compressed or torn apart by the trawling process) were collected from the catch. After thawing, the standard length (L) of D. watasei was measured to the nearest 1 mm, and the body mass (W) was measured to the nearest 0.01 g. All trawled D. watasei individuals had lost scales to varying degrees. To reduce inter-specimen error, all samples were descaled. Hence, the D. watasei used in our study were scaleless. Afterwards, macroscopic observations of the gonads provided information for sex identification [10,26].

2.3. Otolith Acquisition and Age Estimation

Sagittal otoliths were extracted, cleaned of adhering tissue, numbered, and stored in 2.0-mL tubes with 75% ethanol. Before polishing, they were rinsed with distilled water and dried at 60 °C for 30 min. The left otolith was selected preferentially. If the left otolith was damaged, then the right otolith was used. Otoliths were embedded in epoxy resin (EpoHeat^®; Buehler, Lake Bluff, IL, USA) using plastic molds and hardened while minimizing air bubbles. Polishing along the sagittal plane was performed progressively with sandpapers of grit sizes 400, 800, and 1200 using a grinder (MetaServ 250; Buehler).

Classification of otolith zones followed the system described by Gigarosov and Ovcharov, and refined by Hosseini et al., Zhang et al., and Tian et al. [10,25,27,28]. The otolith microstructure in lanternfishes is thought to reflect developmental transitions associated with egg hatching, metamorphosis, and juvenile growth, corresponding to the central zone (CZ), middle zone (MZ), and external zone (EZ), respectively [25,27,28]. Daily growth increments were counted outward from the primordium, starting from the first clearly visible ring. The CZ contained very narrow increments until an abnormally wide ring marked the beginning of the MZ. The MZ was characterized by daily growth increments that were noticeably wider than those in the CZ, with variable width and depth and a turbid background, ending at a distinct color boundary. The EZ exhibited clearer, evenly spaced increments. Counts extended to the otolith margin, with the outermost edge taken as the final increment.

Each otolith was independently read three times by each of the two trained readers. For each reader, the three counts were considered consistent if they differed by less than 5% from their mean, and the average of these three counts was then used as that reader’s final estimate. If the three counts differed by more than 5% from their mean, the otolith was re-read. The two readers’ mean values were subsequently compared, and only those otoliths for which the two estimates differed by <5% were retained for analysis. Final age estimates were reported as whole numbers [10,25,29]. The 5% consistency criterion followed previous studies [10,25]. A plot detailing the age bias was used to evaluate potential counting errors between the two readers in their daily age determinations [30], and no systematic age bias was found.

2.4. Selection and Application of Our Model

Differences between sexes were analyzed using one-way ANOVA. All available specimens were included in the analysis of the relationship between W and L and were subsequently used for otolith preparation to obtain age information, although a small number of specimens could not be aged due to difficulties in otolith extraction or damage. Furthermore, to demonstrate more directly the relationship between sex and standard length, specimens were grouped at 10-mm intervals by length. The relationship between the theoretical value of L and W of fish was represented by the equation:

$$W=a{L}^b$$

(1)

where W is body mass, L is standard length, a is the intercept, and b is the slope of the equation. Both a and b are obtained by transforming logarithmic expression (log W = log a + b·log L). The equidistant growth mode of the population is determined by b. That is: if b = 3, it is isometric growth; if b < 3, then negative allometric growth is denoted; if b > 3, it is positive allometric growth [31]. Determination of the ordinate was by the mean body mass of all specimens in the body length interval.

The relationship between daily growth increments in the CZ and MZ was ascertained using the following equation:

N_MZ/N_CZ

(2)

where N_MZ and N_CZ are the numbers of daily growth increments for the CZ and MZ, respectively [10,25,32].

The growth of D. watasei was assessed using the von Bertalanffy (VB) growth model:

L_t = L_∞ [1 − exp(−k(t − t₀)])

(3)

where L_t is the actual length at age t, L_∞ is the theoretical asymptotic length, t is expressed in days, t₀ is the theoretical length at the age when L = 0, and k is the growth coefficient.

The growth rate equation was obtained by modification of the VB equation:

dL/dt = L_∞ × k × exp[−k(t − t₀)]

(4)

where dL/dt is the growth rate.

The speculated spawning time for individual D. watasei was reliant upon the age determination and capture date. The month of spawning for D. watasei collected in the SCS was determined by aggregating the timing of spawning for all specimens.

3. Results

3.1. Body Size

A total of 137 individuals (78 males and 59 females) of D. watasei were collected. A pattern diagram was drawn based on the luminescent glands and body morphology of D. watasei (Figure 2) [33]. D. watasei possesses a distinct ventral nasal photophore, which is approximately triangular in shape (with minor intraspecific variation in shape and size). The posterolateral photophores are located below the lateral line. Within the size groups of D. watasei, males were more abundant than females in the standard-length range 110–140 mm, with an average male-to-female ratio of approximately 1.72:1 (Figure 3). The length distributions of males and females exhibited a normal pattern. Analyses of the variability in standard length and body mass between males and females were done separately, and the results did not reveal significant differences (p > 0.05;

Table 2. Standard length and body mass of Diaphus watasei in the South China Sea (n = 137). The three data groups represent males, females, and all individuals combined, respectively. The data represent specimens collected on 17 June 2015 from a time-series station.

Sex	Number	Standard Length Range (mm)	Mean Standard Length (±SD; mm)	Body Mass Range (g)	Mean Body Mass (±SD; g)
Male	78	91–140	116 (±11)	11.85–40.80	23.37 (±6.25)
Female	59	80–147	114 (±12)	7.47–44.22	23.49 (±7.20)
All	137	80–147	115 (±11)	7.47–44.22	23.42 (±6.65)

). Therefore, as no significant differences were found between sexes, all specimens from the SCS were combined and analyzed together in the subsequent analyses. The overall male/female ratio was 1.32:1. The standard length was mainly concentrated in the range 100–130 mm.

The standard length of all D. watasei ranged from 80 to 147 mm (mean ± SD = 115 ± 11 mm), and body mass ranged from 7.47 to 44.22 g (mean ± SD = 23.42 ± 6.65 g). The relationship between standard length and body mass of D. watasei (Figure 4) was calculated as:

W = 0.0000433L^2.78, r² = 0.923

(5)

where the 95% confidence limit (CL) for the coefficients a and b was determined separately. The 95% CL for a ranged from 0.0000160 to 0.0000706. The 95% CL for b ranged from 2.65 to 2.90.

3.2. Otolith Microstructure

Ninety-two otoliths from various length groups were successfully obtained, sequentially polished, and had their daily growth increments counted for the assessment of the age of D. watasei. The CZ was a nearly circular transparent area that recorded juvenile development with the lightest-colored daily growth increments (Figure 5). The MZ area reflects the metamorphic stage of growth. Among the three regions, the MZ had the largest difference in width among daily growth increments, with random abnormal protrusions or color deepening in some places. There were individual differences in the size and shape of the MZ. The abnormality of otoliths in the MZ affected the precipitation shape of the daily growth increments in the EZ. However, naked-eye observation suggested that the shape of the otoliths of specimens of D. watasei was nearly identical. Daily growth increments in the EZ were densely deposited and most regular. The daily growth increments of the CZ, MZ, and EZ were counted separately, and the age of each specimen was obtained by adding up these three parts: CZ: 20–37 days (mean ± SD = 26.8 ± 3.8), MZ: 8–14 days (mean ± SD = 11.2 ± 1.5), and EZ: 273–657 days (mean ± SD = 496.8 ± 68.8). These results are shown in

Table 3. Otolith microstructure counts of Diaphus watasei in the South China Sea (n = 92; sexes combined). The data groups represent the counts of daily growth increments in the central zone, middle zone, external zone, and the total of these three zones. The data represent specimens collected on 17 June 2015 from a time-series station.

Otolith Microstructure	Daily Growth Increments Counting Range	Mean Value (±SD)
Central zone	20–37	26.8 (±3.8)
Middle zone	8–14	11.2 (±1.5)
External zone	273–657	496.8 (±68.8)
Total	304–687	534.5 (±68.9)

. No significant differences in daily growth increments were found between sexes in any of the three regions or for the whole otolith (one-way ANOVA, p > 0.05). Therefore, all results were reported for the combined sexes. The ratio of daily growth increments between the MZ and CZ (N_MZ/N_CZ) ranged from 0.24 to 0.60, with an average of 0.43 ± 0.08.

3.3. Age and Growth

Ninety-two specimens of D. watasei were analyzed. A high level of agreement was observed between the two readers’ otolith ages readings, with most specimens closely aligned along the line of equality and no systematic deviation observed, as illustrated in Figure 6. The estimated age of all D. watasei (sexes combined) ranged from 304 to 687 mm (mean ± SD = 534.5 ± 68.9). The youngest individual (304 days) corresponded to the shortest standard length (80 mm). The oldest individual (687 days) had a standard length of 135 mm. The individual with a standard length of 136 mm corresponded to an age of 669 days, as shown in Figure 7.

The VB growth curve (Figure 7) fitted to the age against standard length (L) was:

L_t = 171.38 [1 − exp(−0.00206 (t − 3.82))], r² = 0.645

(6)

The equation for growth rate was:

dL/dt = 171.38 × 0.00206 × exp[−0.00206 (t − 3.82)]

(7)

According to the growth-rate model, D. watasei exhibited its highest growth rate during the larval stage, reaching a maximum of 0.356 mm/day (Figure 8). As the fish approached the theoretical limit of the standard length (171.38 mm), the growth rate became increasingly closer to zero.

3.4. Spawning Period

Estimation of the spawning period of D. watasei was conducted by referencing the capture dates of the samples and calculating the daily growth increments of otoliths retrospectively. The speculated birth dates of the 92 specimens of D. watasei occurred across all months of the year, with the sole exception of June (Figure 9).

4. Discussion

4.1. Sex Ratios

We estimated the overall sex ratio of males to females to be 1.32. This value is similar to the reported values of 1.30 in the East China Sea but differs from the value of 1.63 reported off the southwest coast of India [15,19]. There have been differences among surveys in different regions. However, there is no doubt that, for D. watasei, males were more numerous than females. For most species of mesopelagic fish, there are usually more females than males, which ensures that there are sufficient numbers of offspring to replenish the population [1,5,15]. For example, the sex ratio for fish also belonging to the genus Diaphus has been reported to be 0.23 for D. brachycephalus, 0.90 for D. garmani, and 0.8 for D. chrysorhynchus [10,15]. Sassa et al. reported average oocyte counts of 1327 in D. garmani, 7243 in D. chrysorhynchus, and 23,172 in D. watasei, indicating higher fertility in D. watasei females. In this study, males dominated at a standard length of 90–150 mm, compared with 125–145 mm in the East China Sea population [18]. The occurrence of a male-biased sex ratio in D. watasei may be influenced by multiple factors, such as environmental conditions (e.g., temperature and depth) and sex-specific habitat preferences. It is also hypothesized that the higher proportion of males serves an adaptive reproductive function by ensuring sufficient fertilization opportunities for the highly fecund females, thereby maintaining population stability.

4.2. Biology and Growth

For fishes belonging to the family Myctophidae, the standard length of most species is <100 mm, with only a very few species reaching the extreme length of ≥130 mm [1,10,15,21,33]. D. watasei is notable among Myctophidae for its relatively large body size. It is one of the few edible-sized lanternfishes and plays an important predatory role in deep-sea ecosystems. The maximum standard length of D. watasei in our study was 147 mm, which is shorter than the longest record of 170 mm in the Fishbase, and similar to the standard length in the East China Sea and Indian Ocean [15,17,18]. The relationship between the standard length and body mass of D. watasei in the SCS is W = 0.0000433L^2.78, suggesting that D. watasei has negative allometric growth in the SCS.

A survey by Zhang and Guo in the East China Sea classified D. watasei into “summer” and “winter” groups. Their survey revealed a pattern of negative allometric growth, with b values of 2.044 in summer and 1.750 in winter [18]. A study by Baby et al. [34] indicated that the D. watasei population along the southwest coast of Kerala (India) exhibited isometric growth (b = 3.02). Integrating our findings with those of Zhang and Guo and Baby et al. suggests a latitudinal trend in which D. watasei becomes increasingly slender in body shape from lower to higher latitudes. This trend, which has also been observed in D. brachycephalus (b = 2.88 in the SCS vs. 3.39 in the Atlantic Ocean) [10,35], may be attributed to environmental differences such as temperature, food availability, and metabolic constraints. In warmer and nutrient-rich low-latitude waters, faster growth rates and greater reproductive investment may lead to relatively deeper-bodied individuals. In cooler or less productive environments, selective pressures favor more streamlined body forms that minimize energy expenditure during diel vertical migration [1,16,28].

According to our study, the known lifespan of D. watasei reached up to 1.8 years (687 days), exceeding that reported for individuals in the East China Sea population (608 days) [18]. Compared with other documented species such as D. brachycephalus (168 days), Ceratoscopelus warmingii (298 days), and Myctophum asperum (400 days), D. watasei is considered to be a long-lived lanternfish [10,18,25,36]. Daily growth increments in the CZ are thought to represent the time it takes for fertilized eggs to hatch, which suggests an incubation period of 20–37 days for D. watasei [10,18,25,36,37,38]. Variation in the incubation time among species is widespread: researchers have documented 16–26 days for D. brachycephalus, 22–32 days for Benthosema pterotum, and 14–40 days for C. warmingii [10,25]. The causes of MZ formation are controversial. Substantial morphological differences are observed between the MZ and the CZ/EZ. The MZ shows increased pigmentation, blurred margins, and wider daily growth increments. These features are commonly interpreted as the result of habitat transitions occurring during the juvenile stage, reflecting the process of metamorphic development [10,18,36,37]. Takagi et al. suggested that at this stage juvenile fish complete metamorphosis, a view that is similar to those of Zhang et al. and Han et al. [10,25,33]. For D. watasei, the daily growth increments of the MZ ranged from 8 days to 14 days (mean = 11 days), similar to that of D. brachycephalus (12 days to 20 days, mean = 16 days) [10]. The MZ:CZ ratio is an auxiliary indicator of early developmental timing and nutritional transition—from yolk-dependent to externally supplemented feeding—providing insight into the early life history of mesopelagic fishes. The MZ:CZ ratio for D. watasei in the SCS was 0.43, which is similar to the reported value of 0.48 in the East China Sea [18]. In comparison, the ratio has been recorded as 0.79 for D. brachycephalus and 0.54 for B. pterotum [18,28]. This suggests that D. watasei experiences stronger ecological pressure during the post-hatching metamorphic stage, possibly arising from food acquisition challenges or drastic changes in habitat depth among the compared populations.

Through the VB growth curve, we predicted an average maximum size of ~171 mm for D. watasei based on the otolith microstructure of 92 individuals. This value is close to the known maximum individual length of 170 mm according to Fishbase. Sebastine et al. used a mathematical model to extrapolate the results for D. watasei from the Kerala coast with a limiting body length of 150.6 mm [17]. Using otolith analyses, Zhang and Guo predicted a maximum length of 162.6 mm for D. watasei in the East China Sea [18]. This is an interesting finding because these differences may be related to variations in marine environments, but also to different sampling methods at various depths and the escape ability of fish during trawling. For other mesopelagic fish, growth is also thought to vary from region to region. The growth of fish is directly related to differences in latitude and marine geography, and can be used as an indicator of the biogeographical characteristics of specific regions [5,7,39,40]. Notably, compared with other species, the decline in growth rate with age in D. watasei appeared to be slower. However, the absence of the largest and smallest individuals, together with the lack of their otolith information, makes the fitting and analyses of the VB growth curve less robust. This limitation has been highlighted consistently in previous studies, but filling the gap in biological information for juvenile and exceptionally large individuals will be a key focus for future research on D. watasei.

4.3. Predicted Spawning Time

Very few reports have provided information on D. watasei spawning [15,18]. We referenced the capture dates of specimens and calculated the daily growth increments in otoliths retrospectively. Our data indicated that the birth dates of the D. watasei specimens collected in the SCS spanned almost the entire year. Although our results showed a lack of spawning information for this species in June, this may be directly related to the limited sampling. Sassa et al. inferred a spawning period of D. watasei in the East China Sea by assessing the gonadosomatic index, and suggested that it begins in June [15]. Information on year-round spawning of the East China Sea population has been supplemented [18]. Upon comparison with our study, an interesting phenomenon was revealed. The SCS population had fewer hatchings from May to August whereas, in the East China Sea, June to August has been reported to be the peak period [18]. Apart from regional differences, we must consider the objective condition that both surveys were limited by sample size, which might also have been influenced by different age groups of fish forming separate schools. The peak spawning period for some species is likewise thought to be concentrated in autumn and winter, such as that for C. warmingii (November to January of the following year) and M. asperum (October to June of the following year). Jun et al. suggested that spawning in winter may be beneficial to larval growth [25,36].

Our samples were collected continuously over a 24-h short-term survey, encompassing individuals born at various times throughout the year. How this species recognizes conspecifics and recruits its juveniles remains a critical scientific quest meriting in-depth investigation. Given that lanternfish generally lack strong long-distance migratory capacity, D. watasei is unlikely to exhibit large-scale horizontal migration, suggesting that the surveyed area may serve as its potential spawning ground [5]. The species exhibits a relatively stable population distribution in the ECS and the SCS. Nevertheless, the dispersal of juveniles may be significantly influenced by periodic upper- and mid-layer oceanic currents. Under such conditions, gene flow between populations in these two regions remains plausible, and they may constitute a single genetic population [32]. During the southwest monsoon period in the SCS (May to September), seawater from the Java Sea and the southern SCS flows northeastward through the waters off Vietnam. Part of this water merges into the Kuroshio Current south of Taiwan Island, while another portion enters the ECS via the Taiwan Strait. In contrast, during the northeast monsoon period (November to March of the following year), surface wind stress drives some Kuroshio water into the SCS through the Bashi Strait, while coastal water from the ECS flows southward through the Taiwan Strait, forming a persistent southwestward-flowing current along the western margin of the SCS. D. watasei may serve as an indicator of potential connectivity for individual exchange and population replenishment between the ECS and the SCS. Notably, our sampling area, located near the Bashi Strait and Taiwan, lies within a region strongly influenced by the Kuroshio Current. The key to addressing the aforementioned scientific challenges lies in acquiring a sufficient number of juvenile specimens. However, due to limitations in the efficacy of current sampling techniques, only a small number of early life stage individuals of D. watasei have been collected, with those having a standard length below 80 mm being especially rare, a limitation that is also prevalent in studies of other mid- to upper-layer pelagic fish species. Consequently, elucidating the population replenishment mechanisms of D. watasei should be prioritized in future research.

Based on the literature, we suggest that the population characteristics of D. watasei in the SCS and East China Sea are highly similar, including age, sex, and body length [18]. Another limitation of our study lay in the representativeness of sampling conducted on a single day. Therefore, more comprehensive investigations of the same species across additional regions of the Western Pacific should be done. Also, comparative analyses among the East China Sea, broader Western Pacific, and Indian Ocean should be carried out to examine variations in age, growth, and distribution patterns as well as their underlying influencing factors. In addition, the scarcity of juvenile D. watasei specimens (standard length < 80 mm) presents major challenges for the estimation of age and growth. Therefore, collection of juvenile fish and related biological studies should also be prioritized as part of future research efforts.

5. Conclusions

We provided, for the first time, an otolith-based assessment of age and growth for D. watasei in the SCS. We presented key information on its sex ratio, length–weight relationship, age, and growth and speculated about the spawning period. Our findings highlight its deep-water residency, relatively large body size, and long lifespan compared with those of other lanternfishes.