Baobao Xue, Delong Meng, Hao Li, Wei Liang, Donghong Niu,Jiale Li,＊, Heding Shen,＊＊

aKey Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai, 201306, China

bShanghai Collaborative Innovation for Aquatic Animal Genetics Breeding, Shanghai, 201306, China

cNational Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, 201306, China

Keywords:

Sinonovacula constricta

Biological zero

Gonads

Development

Histology

ABSTRACT

The biological zero of gonadal development and the effective accumulated temperature of one and two-year-old razor clams Sinonovacula constricta (Lamarck 1818) were studied in Sanmen County, Zhejiang Province, China between April 2016 to October 2017, using a new variety named “Shen-Zhe No. 1”. The biological zero was determined by using the rule of effective accumulated temperature and the linear regression method, which was confirmed using a histological analysis of the full gonadal development cycle. The results showed that the biological zero of gonadal reproduction of 1-year-old and 2-year-old S. constricta was the same: 20.8 °C. When the temperature reached 20—21 °C, analysis of the histological sections indicated that more than half of the S. constricta individuals initiated gonadal development. The average effective accumulated temperature over two years was 806 d °C. This study contributes to our understanding of the regulation and control of gonadal maturation, suggesting that changes in the spawning and breeding season during one-year of farming using the new variety of razor clam could provide an economic benefit to industrialized large-scale spat production. The results demonstrated that the razor clam S. constricta is a typical clam of the accumulative temperature mature oviposition type. In addition, although S. constricta does not display hypothermy, it requires a lower temperature for gonadal maturity and spawning.

1.Introduction

The razor clam

Sinonovacula constricta

(Lamarck 1818), belonging to Mollusca (Bivalvia: Veneroida: Solecurtide), is a commercially important variety of bivalve that has rapidly increased in terms of yield and is cultured in the intertidal zones and estuarine waters of China and Japan(Suzuki et al., 2008; Yanagita, Enomoto, & Yamamoto, 1991). It is extensively aquafarmed in China and other countries with 823,024 tons valued at $1,351,405 USD harvested in 2016 (FAO, 2018, p. 56). It is one of the four major clams in Chinese aquaculture, which also includes

Crassostrea gigas

,

Ruditapes philippinarum

, and

Tegillarca granosa

(Wang,1993, p. 397). It has been cultured for more than 400 years in the Zhejiang and Fujian Provinces, China (Shen, Liu, Fang, & Li, 2013). The cultivation of

S. constricta

has also been carried out in Shandong,Jiangsu, Guangdong, and Guangxi Provinces in China. The production of razor clams is gradually increasing through China, with a culture areas of 54,578 hmin 2017 (SOA, 2018).

S. constricta

is a filter-feeding shell fish and its growth and survival are affected by the water quality of the surrounding environment. The rapid development of the aquaculture industry has caused a variety of environmental problems, including water quality deterioration and contamination (Hargreaves, 1998; Huang, Guo, & Huang, 2010; Wu,Lam, Mackay, Lau, & Yam, 1994), biological invasion (Naylor, Williams,& Strong, 2001), drug residues (Grave, Hansen, Kruse, Bangen, & Kristoffersen, 2008; Schnick, 2001), and aquatic diseases (Bondad-Reantaso et al., 2005; Meyer, 1991). However, a new breeds of

S. constricta

were selected to overcome environment and culturing problems, resulting in improved growth and survival rates (Li et al., 2017).Several studies related to the physiology of

S. constricta

have been published (Fan, Pan, Ma, & Dong, 2002; Pan, Fan, Ma, & Dong, 2002).Studies revealed that cathepsin L, C, and B genes (Niu et al., 2013a,2013b, 2014), insulin-like peptide genes (Niu et al., 2016b), and complement C3 genes (Peng et al., 2016, 2017), play significant roles in the innate immunity of the razor clam. In addition, studies related to polymorphic microsatellite loci (Niu, Li, & Liu, 2008), the mitochondrial DNA genome (Niu, Li, Feng, & Liu, 2010), significant genetic differentiation (Wang, Niu, & Li, 2012), single nucleotide polymorphism (SNP)markers (Feng et al., 2012), and developmental transcriptome analysis(Niu et al., 2016a), have been reported. The results of these studies have provided the theoretical basis and technological support for germplasm resource protection and the identification of a cryptic variety of

S. constricta.

The success of the spat culture (larvae attached to a surface) is fundamental for the availability of adult

S. constricta

. However, immature gonads has significantly affected the economic benefit of industrialized cultures of

S. constricta.

An important environmental factor for gonadal development of marine invertebrates is temperature (Deridovich, Motavkin, Evdikimov, & Khotimchenko, 1998, pp. 23—78; Sreedevi, Uthayakumar, Jayakumar, & Ramasubramanian, 2014). Mann produced the concept of biological zero, meaning a temperature below which a biological or physiological process does not occur (Mann &Stuart, 1935, pp. 595—597). Indeed, many studies of biological zero related to reproduction in bivalves have been published, such as in

Ostrea virginica

(Loosanoff, 1939; Loosanoff & Davis, 1952),

Argopecten irradians

(Zhou, 1991),

R.philippinarum

(Liang, Yan, Xia, & Zhang,2007), and

Scapharaca subcrenata

(Wang, Wang, & Du, 2011). Few studies have been published on the reproduction of

S. constricta

(Chang,2007, p. 109). Razor clams undergos cooling and spawning and, like other low-temperature oviposition bivalves, they can spawn at low temperatures. Therefore, the breeding season of the northern sea is earlier than that in the southern sea. However, no studies have considered the biological zero and the effective accumulated temperature of gonadal development in

S. constricta

.The purpose of this study was to establish the biological zero and the effective accumulated temperature for gonadal development for the new breed of

S. constricta

, Shen-zhe No. 1, using the rule of effective temperature summation and histological techniques. The results will produce theoretical support for the process of normalized culture of this new breed of razor clam, contributing to largescale parent production and breeding of spat.

2.Materials and methods

2.1.Materials and cultivation

The new breed of razor clam

S. constricta

“Shen-zhe No. 1” was used in this experiment, which studied razor clams at two ages: 1 and 2 years old. The morphometric measurement data of the two age groups are shown in Table 1. The samples were collected from an aquatic farm in Sanmen County, Zhejiang Province, China. According to the different ages, razor clams with artificially promoted mature gonads were separated into two groups and placed in two 30 mindoor ponds with a layer of mud at the bottom that was more than 30 cm thick (Fig. 1). They were cultured separately at a density of 120 razor clams and 100 razor clams per m, respectively (Xue et al., 2017). The different age groups of

S. constricta

were cultured separately in the same pond to avoid the potentially confounding effects of diet, salinity, dissolved oxygen and other environmental conditions. The seawater of the parent clam culture was then filtered, and the clams were provided with aeration and three species of microalgae,

Chaeroeeros moelleri

,

Isochrysis galbana

and

I. Zhangjiangensis

, which were used as food for

S. constricta

(Ran et al.,2017). In the study,

S. constricta

were cultured in indoor ponds with dissolved oxygen more than 5 mg/L, and seawater with a specific salinity from 10.42 to 12.85.

Table 1The morphometric measurement data of two samples of the razor clam S. constricta.

Fig. 1.The top view of heating system indoors. A: valve; B: drainage port; C:steam pipe; D: screen.

2.2.Calculating biological zero

Utilizing indoor heating until the culture spawned, we recorded the water temperature during the artificial heating process in the indoor ponds at 2:00, 8:00, 14:00, and 20:00 every day and the total gonadal developmental. The average daily water temperature was calculated according to the monitoring schedule. We used the following formula to calculate the biological zero:

K

=

H

(

T

-

t

) (He & Zhang, 1983, pp.133—144). The accumulated temperature was represented by matching linear heating curves

L

and

L

(Fig. 2).

K

is the accumulated temperature;

H

is the developmental duration of the gonads;

T

is the seawater temperature in the indoor ponds; and

t

is the biological zero of gonad development. The values hand hare the days when the biological zero of gonad development was attained. The

K

values were equal, therefore

S

=

S

(Fig. 2),

S

and

S

are the accumulated temperatures.

Fig. 2.The accumulated temperature of the two groups during the experiment.Note: h1 and h2 are the time that gonadal development began; hS is the sum days to attain spawning; “t” is the biological zero; L1 and L2 are the matched artificial heating curves; S1 and S2 are the accumulated temperatures.

In other experiment, we measured the temperature at 2:00, 8:00,14:00, and 20:00 in the four shrimp ponds in situ, each of which had an area of 1.334 ha, and cultured 1-year-old juvenile

S. constricta

at 5000 grains per kilogram (approximately 750,000 individuals). The time(days) and average temperature were recorded from germination to gonadal maturity.

2.3.Histological studies

From April 2017 to October 2017, 20 specimens were taken at 5-day intervals for gross examination and histological studies in an early stage of the experiments. In the medium-term experiments, equal numbers of

S. constricta

were taken every 15 days. After August 2017, however, 20 samples of

S. constricta

were collected each week. The gonad tissue from each razor clam was excised using a transverse cut from the visceral mass in the foot or near the digestive tract. If the mature gonads reached into the visceral mass of the foot, a second transverse cut was made a few millimeters below the first (Steele & Mulcahy, 2000). The resulting gonad tissue was fixed in Bouin's solution, and then dehydrated through a series of increasing ethanol concentrations. The dehydrated samples were cleared using different alcohol gradients and embedded in paraffin following a standard procedure (Lango-Reynoso, Chávez-Villalba,Cochard, & Pennec, 2000). Sections of 5—6 μm were cut, mounted on glass slides, and stained using Groat's Hematoxylin and Eosin (Humason, 1962). Each histological section was examined and photographed under an Olympus CX41 microscope (Olympus Corporation, Japan) to distinguish the sex and stage of gonadal development. The stage of the gonadal development of

S. constricta

was determined according to the reproduction cycle reported by Castilho Westphal et al. (2013) and Ivell(1979): Stage I (initiation), Stage II (development), Stage III (maturation), Stage IV (spawning), and Stage V (exhaustion).

2.4.Statistical analyses

Data was expressed as the mean ±S.D. The average daily water temperature was calculated according to the monitoring schedule. The total days from the onset of heating to spawning was recorded. Drawing was perfomed with EXCEL and analyses were conducted using SPSS(SPSS Inc., Chicago, IL, USA) 20.0 software. The significance level for all analyses was set to

P＜

0.05.

3.Results

3.1.The biological zero of gonad development of razor clams in 2016

The time taken to spawn via artificial heating stimulation of the parents was 107 and 119 days, respectively. We based the daily mean water temperature of testing for each group on the matched artificial heating curves

L

and

L

(Fig. 3), as follows:

L

:

Y

=0.1296

X

+20.821,

R

=0.9198,

L

:

Y

=0.111

X

+20.44,

R

=0.9466, where

Y

is the seawater temperature,

X

is the time of induced maturity, and

R

represents fitness. According to the accumulated temperature formula, the statistical calculation showed that the biological zero for gonad development of razor clams in 2016 was 20.6 °C, and the effective accumulated temperature was 780.7 d °C.

Fig. 3.Seawater temperature exchange of artificial heating and matched curve in 2016. Note: L1 is the matched artificial heating curve for pond 1; L2 is the matched artificial heating curve for pond 2. Y is the seawater temperature, X is the time of induced maturity and R2 is the coefficient of determination Fitness.

3.2.The biological zero of gonad development of razor clams in 2017

The time taken to spawn via artificial heating stimulation of the parents was 116 and 130 days, respectively. We based the daily mean water temperature of testing for each group on the matched artificial heating curves

L

and

L

(Fig. 4), as follows:

L

:

Y

=0.1253

X

+20.7500,

R

=0.9388,

L

:

Y

=0.1066

X

+20.300,

R

=0.9488, where

Y

is the seawater temperature,

X

is the time of induced maturity, and

R

represents fitness. According to the accumulated temperature formula, the statistical calculation showed that the biological zero for gonad development of razor clams in 2017 was 21.0 °C, and the effective accumulated temperature was 824.5 d °C. The average biological zero for the two years was 20.8 °C, and the average effective accumulated temperature was 806 d °C.

Fig. 4.Seawater temperature exchange of artificial heating and matched curve in 2017. Note: L3 is the matched artificial heating curve for pond 3, L4 is the matched artificial heating curve for pond 4. Y is the seawater temperature, X is the time of induced maturity and R2 is the coefficient of determination Fitness.

3.3.Demonstration of biological zero using histopathological observation

The appearance of follicles when the temperature ranged from 18 °C to 19 °C indicated the initiation of gonad formation in the new breed of

S. constricta

as the production of reproductive cells results from the differentiation of follicle walls. At the start of thermal culture, the visceral mass was occupied by very thick connective tissue. At this stage,gonads were so undeveloped that their sex could not be easily determined (Fig. 5-A, 5-A). The histological observation showed that more than half of the 1-year-old and 2-year-old

S. constricta

had follicles and initiated development of their gonads when the temperature ranged from 20 to 21 °C. Meanwhile, the original reproductive cells in the follicle walls entered the active fission phase, as indicated by their constant increase in numbers. The follicular walls of the female were discontinuous and had a monolayer oogonium (Fig. 5). The follicular walls of the male comprised spermatogonium and a few primary spermatocytes. At this stage, the gonad development of 2-year-old razor clams was further advanced than that of the 1-year-old clams. Thus, when the temperature was 20 °C—21 °C, the gonads started to develop, which agreed with the calculated biological zero values of 20.6 °C and 21.0 °C.

3.4.Demonstration of effect of the accumulated temperature in nature

Table 2 shows that gonad maturation required 146.75 days in nature,and the average effective accumulative temperature was 813.6 d ⋅°C compared to 802.6 d ⋅°C in the indoor ponds. The temperature difference between the average natural temperature and the biological zero was 5.6 °C while the value of the effective accumulative temperature was approximately equal compared with 813.6 d ⋅°C. This data from the natural culture demonstrated the credibility of the experimental data.

Table 2Data of the days and average temperature from germination to maturity of the gonads of S. constricta in the four shrimp ponds.

Fig. 5.Different gonad development in 1-year-old and 2-year-old razor clams. A1: Before the initiation stage of one-year-old razor clams; A2: before the initiation stage of two-year-old razor clams; B1:development stage of razor clams (♀); B2: development stage of two-year-old razor clams (♀); C1:development stage of one-year-old razor clams (♂);C2: development stage of two-year-old razor clams(♂). CT: Connective tissue; FO: Follicle; RE: Reproduction Epithelium; OG: Oogonium; OO: Oocyte; SG:Spermatogonia; SO: Spermatocyte; SA: Spermatid.Scale bars, 50 μm. Note: The subscript “1” indicates one-year-old S. constricta; subscript “2” indicates twoyear-old S. constricta.

3.5.The cycles of gonadal development

The histological observations showed that spawning and spermiation were successfully initiated when the effective accumulative temperature reached 824.5 d⋅ °C.

Stage I:

The presence of follicles indicated the initiation of gonadal formation in

S. constricta

, because the production of reproductive cells results from the differentiation of follicles walls. The fairly thick connective tissue occupied almost all of the gonadal tissue and the follicles gradually formed along the connective tissue. The shape of the follicles was irregular; the original reproductive cells in the follicle walls entered the activity fission phase, as indicated by their constant increase in numbers (Fig. 5-A).

Stage II:

The number of follicles rapidly increases, and the original reproductive cells continued to differentiate. A few reproductive cells appeared and a rapidly increasing number of gonocytes was observed. In the female, the number of oogonia decreased, and the number of oocytes gradually increased, with a few free oocytes observed (Fig. 5-B). In the male, a thick layer of spermatophores was observed, and spermatocytes appeared along the follicular walls,together with a large number of sperm cells in the follicular cavity(Fig. 5-C).

Stage III:

Follicles were occupied by free oocytes and spermatozoa.In the female, oocytes were round or oval (Fig. 6-A); in the male,spermatocyte and spermatozoa were arranged in characteristic bundles (Fig. 6-B).

Fig. 6.Histological observations of the gonad development cycle of S. constricta. A: Maturation stage (♀); B: Maturation stage (♂); C: Spawning stage (♀); D:Spawning stage (♂). M: Mature oocyte; SG: Spermatogonia; SO: spermatocyte; SP: Sperm; RO: Residual oocytes; RS: Residual sperm. Scale bars, 50 μm. Note: The subscript “1” indicates one-year-old S. constricta; the subscript “2” indicates two-year-old S. constricta.

Stage IV:

Obvious spawning activity was observed in the histological sections (Fig. 6). Especially after spawning, the follicles became atrophied and the density of the oocytes and spermatozoa in the gonads rapidly decreased because mature gametes are released, and residual oocytes or spermatozoa occupied most of the space. Some follicle walls were fractured.

Stage V:

The follicle becomes a large cavity, appearing nearly transparent; the follicles and follicles walls appear hollow or have disappeared (Fig. 6-C and 6-D). In the female and male, the quantity of oocytes and spermatozoa is markedly decreased, and any remaining follicles are inside large spaces. In these follicles, only a few residual oocytes and spermatozoa are present.

4.Discussion

Environmental factors, especially temperature, play a dominant role in the reproductive cycle of marine invertebrates (Mackie & Ansell,1993). In the process of industrialized production, gonadal maturation can be accelerated or postponed, and the sperm and egg-spawning periods can be controlled by adjusting the seawater temperature, as informed by combining knowledge of the biological zero and effective accumulated temperature for gonadal development of

S. constricta

. The inhibitory effect of high salinity on the growth rate appears to be common among marine bivalve, such as,

Crassostrea hongkongensis

(Huo et al., 2014) and

S. constricta

(Ran 2017). We revealed that the biological zero was nearly equal for the new variety of

S. constricta

at 1 and 2 years of age. Thus, we suggest that 1-year-old razor clams could serve as parents. This could not only reduce production costs and, enhance their economic value. Meanwhile, our results also provide the theoretical foundation to accelerate ripening of parent razor clams and encourage large-scale production of this new breed of

S. constricta

.The gametogenic cycle can be influenced by several environmental conditions, such as temperature, salinity (Boisset et al., 2007; Lannan,1981), and levels of nutrition, which differ between localities (Chavezvillalba et al., 2002). Previous studies confirmed that the biological zero of gonadal development is 7.8 °C, 10.1 °C, 14—16 °C and 17.0 °C for

A. irradians

(Zhou, 1991),

R. philippinarum

(Liang 2007),

C. gigas

(Mann,1979), and

S. subcrenata

(Wang 2011) respectively. Thus, the temperature of gonadal development is species-specific. The experiments described in the present study were designed to decrease the interference of dissolved oxygen and nutritional factors, and the temperature control experiments were designed to induce the maturation of

S. constricta

at different ages, which were carried out in a concrete pond over a continuous period of two years. Using the equation for the accumulated temperature and the linear function relationship allowed us to calculate the average biological zero of two ages of the clams,which was 20.8 °C. At the same time, histological observations verified that gonadal development was induced at 20—21 °C. We noted that the males matured earlier than the females (Figs. 4 and 5), which agreed with the results reported by Loosanoff (1952). The 1 and 2-year-old razor clams had the same biological zero for gonadal development,20.8 °C, which was higher than that of other mollusks. However, to shorten the time to induce maturity earlier, we believe that the new breed of

S. constricta

could be used as a parent razor clam at 1 year old,when they are about 7 cm in shell length and weigh 22.7 g per one individual, and could naturally mature if transferred to Guangdong and Fujian Provinces where the seawater temperature is higher.Indoor heating could also effectively shorten the gonadal maturation period of

S. constricta

, allowing them to spawn one month earlier compared with spawning in natural conditions. Our indoor heating experiment resulted in an average effective accumulated temperature of 802.6 d⋅ °C for gonad development of

S. constricta

. However, in natural conditions, the average effective accumulated temperature was 811.5 ± 8.0 d⋅ °C. Given that the error in determining spawning is no more than three days, the value of the effective accumulated temperature is approximately equal between indoor and natural conditions.The process of heating indoors requires more than 3 months from the beginning of gonad maturation; however, because of the smooth and steady trend of natural temperatures, the production cost might be decreased. A rapid increase in temperature could decrease the period of gonad maturation, and even if the gonads mature prematurely, spawning and metamorphosis into larvae is successful. Variations in temperature and salinity have effects on body water, crude protein content,metabolic capability, and digestive enzyme activity of

S. constricta

(Lv,Liu, Chen, Duo, & Shen, 2013; Zhu et al., 2003). Furthermore, high temperature and salinity can cause spat death of

S. constricta

(Wang,Cao, & Yan, 2006). However, the temperature or salinity of both shrimp ponds and the intertidal zone are too high for intermediate culture(bivalve breeding from spat to juveniles).Gonad development of marine mollusks is not only influenced by endogenous factors, such as sex steroids, 17b estradiol (E), gonadotropins, and testosterone (Nels, 2012; Yan et al., 2011), and the estrogen receptor (Enmark et al., 1997; Ni, Zeng, & Ke, 2013), but also by multiple exogenous factors (Ran 2017; Shen 2013). Studies have reported that at a suitable temperature, the metabolism of shell fish is enhanced,which has a positive effect on breathing, excretion, movement, ingestion, absorption, digestion, germination, gonadal development, and reproduction (Wang, 2008, pp. 302—334; Wei, 1995, pp. 224—258; Wu,1999, p. 175). The formation and development of the gonads depends on the accumulation of nutrients derived from microalgae. However, 1 and 2-year-old

S. constricta

were reared under the same conditions, such as,temperature, microalgae, and salinity, and their effective accumulated temperature was identical, allowing them to simultaneously induce spawning and spermiation.

5.Conclusion

The biological zero of gonadal reproduction of the two groups (1 and 2-year-old

S. constricta

) was the same, 20.8 °C, as calculated using the rule of effective accumulative temperature. Meanwhile, histological examinations confirmed that the biological zero was between 20 °C and 21 °C. Furthermore, this study showed a clear cycle of gonadal development and reproduction. Therefore, artificial heating culturing of 1-year-old

S. constricta

as parent razor clams could be used for the industrialized production of spat

.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The authors followed all applicable international, national, and/or institutional guidelines for the care and use of animals.

Acknowledgments

The authors thank the Sanmen Aquatic Comprehensive Breeding Base for assistance with hatchery and farm operations. This work was supported by the National High-tech R&D Program (863 Program) (863 Program) of China [grant number 2012AA10A400], the Shanghai Universities First Class Disciplines Project of Fisheries, and the Shanghai Universities Knowledge Service Platform [grant number ZF1206].