2 Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China;
3 University of Chinese Academy of Sciences, Beijing 100049, China;
4 Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China;
5 Shandong Fu Han Ocean Sci-Tech Co. Ltd., Haiyang 265114, China
The development of planktonic larvae into juveniles is a key stage in the life cycle of many benthic marine invertebrates (Song et al., 2016a), and settlement and metamorphosis are two closely related and crucial processes in the development of larvae (Yang et al., 2015). Rodríguez et al. (1993) defined settlement as a process that starts with the search for a suitable substratum and ends with metamorphosis. Leise and Cahoon (2012) indicated that Metamorphosis can transform a larva into a juvenile, via a process that involves physical, physiological, and behavioral changes, including changes in organs, external morphology, and lifestyle (Leise and Cahoon, 2012).. As a result of these changes and adaptation to the new microhabitat, planktonic larvae exhibit a high mortality rate during settlement and metamorphosis (Song et al., 2016b). Therefore, the settlement and metamorphosis of shellfish have a vital impact on the dynamics, distribution, and development of natural populations (Song et al., 2016c).
Settlement and metamorphosis of marine invertebrates usually occurs when highly developed competent larvae contact environmental inducers (Jackson et al., 2002). External inducers of metamorphosis of shellfish larvae can include biological, chemical, and physical factors (Burke, 1983; Rodríguez et al., 1993; Li et al., 2006; Laimek et al., 2008; Sánchez-Lazo and Martínez-Pita, 2012; Wang et al., 2012). Biological factors primarily include food, adult secretions, and natural competitors and predators. Chemical factors include microbial membranes and related biological secretions, exogenous neurotransmitters and their precursors, metal cations, and substances affecting signal transduction. Physical factors include temperature, salinity, and settlement surface roughness and color.
Rapana venosa (Valenciennes, 1846) is a widespread carnivorous snail and an economically important shellfish in China although it is an invader in aquatic ecosystems in other countries (Song et al., 2016b). Currently, the supply of R. venosa depends mostly on the harvesting of wild resources. In recent years, the intensity of harvesting is increasing, and the decline of wild resources is a serious concern (Pan et al., 2013a). Therefore, it is necessary to carry out artificial breeding, aquaculture, and resource recovery in China and other native countries, whereas population control is urgent in invaded countries. Artificial breeding of R. venosa has been attempted in China since the 1990s (Yuan, 1992; Wei et al., 1999), but the rate of larval metamorphosis is very low (< 1%), and therefore artificial breeding is far from industrialized and seriously restricts the development of an aquaculture industry for R. venosa.
There are many in-depth studies on shellfish larval metamorphosis in species such as the phytophagous red abalone, Haliotis rufescens (Barlow and Truman, 1992; Searcy-Bernal et al., 1992; Biscocho et al., 2018), and the bivalve Crassostrea gigas (Coon et al., 1985; Fitt et al., 1990; Wang et al., 2015). Considerable progress has been made in research on external inducible factors, neuroendocrine regulation, receptors, signal transduction, and mechanism model construction. During metamorphosis, R. venosa changes from phytophagous to carnivorous (Yu et al., 2018), and the process is more complicated than that in phytophagous snails and bivalve shellfish. There are few reports of settlement and metamorphosis of R. venosa. Yang et al. (2015) indicated that chemical cues (e.g., epinephrine, L-3, 4-dihydroxyphenylalanine, and γ-aminobutyric acid) can induce the metamorphosis of R. venosa. Song et al.(2016a, b) studied the metamorphic mechanism of R. venosa at the molecular level. At present, understanding of the settlement and metamorphosis of R. venosa is limited, and further studies are needed, e.g., on the influence of external factors (light intensity and prey). Light is an important environmental factor affecting the metamorphosis of aquatic organisms (Gao et al., 2016) and prey is a key factor in the metamorphosis of marine invertebrate larvae (Li et al., 2006). In addition, it is known that polysaccharides can induce the settlement and metamorphosis of many species of coral larvae (Morse and Morse, 1991, 1996), and oysters, which are a common prey of R. venosa, are rich in polysaccharides (Shi et al., 2015). However, the inducing effect of oyster polysaccharide on gastropods has not been studied.
Therefore, we studied the following: (1) the time of larval settlement and metamorphosis; (2) the search for efficient methods to induce larval metamorphosis; (3) factors affecting larval settlement and metamorphosis (light, prey, and oyster polysaccharides). The aim is to improve the low rate of larval metamorphosis for cultured populations. Research on the settlement and metamorphosis of R. venosa is beneficial to artificial breeding, aquaculture, and resource recovery.2 MATERIAL AND METHOD 2.1 Larval culture and developmental stages
Adult R. venosa broodstock were collected from Laizhou Bay (37°17′7″N, 119°35′10″E) in Shandong Province. Culturing of parental whelks, mating, spawning, hatching, and larval rearing were carried out based on Yang et al. (2007). Planktonic larvae were cultured in 3 m×6 m×1.2 m cement pools with a density of 0.1 ind./mL, temperature of 23–25℃, and salinity of 29.8-31.7. Larvae were fed Isochrysis galbana, Platymonas subcordiformis, and Chlorella vulgaris three times daily (at 5.0×104 cells/mL).
Larval development was monitored using a microscope. The planktonic larval development of R. venosa can be divided into six stages according to the spiral whorl, the shape of the velum, and the organs present: 1 spiral whorl (A, 320–340 μm shell length (SL)), 2 spiral whorls (B, 340–550 μm SL), early 3 spiral whorls (C, 550–780 μm SL), middle and late 3 spiral whorls (D, 780–1 000 μm SL), early 4 spiral whorls (E, 1 000–1 250 μm SL), and middle and late 4 spiral whorls (F, 1 250–1 500 μm SL) (Pan et al., 2013a).2.2 Experimental design 2.2.1 Time of settlement and metamorphosis and substrates
The development of the planktonic larval foot begins at D stage and the development of the foot indicates that the larvae begin to have the ability to settle (Pan et al., 2013a). Therefore, larvae at D, E, and F stages were selected to observe settlement and metamorphosis. During metamorphosis, larvae undergo a feeding transformation from phytophagous to carnivorous (Yu et al., 2018). Therefore, substrates (scallop shell) with an equal number and size of juvenile oysters (Crassostrea gigas, < 10 mm SL) were selected. All experimental larvae (200 individuals in each experimental group) culture was as described in Section 2.1. Two groups of experiments: (1) substrates were placed into three groups of larvae at different stages, and no substrates were used as a control group; (2) in order to understand the effect of scallop shells on larval metamorphosis, different substrates (1: scallop shells with oysters; 2: scallop shells without oysters; 3: no substrate) were tested with F-stage larvae for three days. All the experimental groups provided single-celled algae. Larval development was observed using a microscope. The rates of settlement, metamorphosis, and mortality of larvae were recorded. The experiment was repeated three times for each group.2.2.2 Illumination
F stage larvae were selected for the illumination experiment. Larval culture was as described in Section 2.1. A total of seven illumination conditions were tested: red (R), white (W), blue (B), green (G), yellow (Y), dark (D), and dark 12 h plus white 12 h (DW); at 1 000±100 lx illumination (by luxmeter testo 540, testo AG, Germany)for three days. To improve the metamorphosis rate of larvae, scallop shells with oysters were provided, and these were placed perpendicular to the light source to prevent shading. All the experimental groups provided single-celled algae. The rate of metamorphosis of larvae was recorded and the experiment was repeated three times for each group.2.2.3 Prey
F stage larvae were selected for the prey experiment. Larval culture was as described in Section 2.1. Three species of juvenile shellfish (5 mm SL), C. gigas (Cg), Mercenaria mercenaria (Mm), and Mactra chinensis (Mc), were selected to induce larval settlement and metamorphosis. The experiment was divided into two induction modes. In the feeding group, the R. venosa larvae could directly feed on shellfish. In the isolation group, shellfish were separated from R. venosa larvae by silk sieve (mesh: 0.1 mm); therefore, shellfish secretions could enter the water environment of the R. venosa larvae, but the larvae could not feed on the shellfish. All the experimental groups provided single-celled algae (the control group were provided with single-cell algae, but not juvenile shellfish). The rate of settlement and metamorphosis of larvae was recorded and the experiment lasted ten days. The experiment was repeated three times for each group.2.2.4 Oyster polysaccharide
Oyster polysaccharide, derived from C. gigas (Shi et al., 2015), is a homogeneous glucose polymer. F stage larvae were selected for this experiment. Larval culture was as described in Section 2.1. Different concentrations of oyster polysaccharides (0, 0.1, 1, and 10 mg/L) were provided daily. The rate of settlement and metamorphosis of larvae was recorded and the experiment lasted ten days. The experiment was repeated three times for each group.2.3 Statistical analysis
The data were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene's test). Data related to the time of settlement and metamorphosis and provision of substrates were analyzed using a two-way ANOVA, Tukey's post-hoc test was performed to identify significant differences between groups. Data related to illumination, prey, and oyster polysaccharide were analyzed using a oneway ANOVA; Tukey's post-hoc test was performed to identify significant differences between groups. All statistical computations were conducted using SPSS v. 16.0 software (SPSS Inc., Chicago, IL, USA) and α-values < 0.05 were considered to be statistically significant.3 RESULT 3.1 Metamorphic characteristics of larvae
The foot, shell, velum, and feeding habits of larvae at D, E, and F developmental stages are shown in Fig. 1 and Table 1. These changes in morphology and feeding habits are closely related to larval settlement and metamorphosis. In D and E stages, the growth and development of foot, velum and shell indicated that the zooplankton larvae could not complete the settlement and metamorphosis behavior at these two stages. The foot development of F stage larvae was complete, and when without inducer, larvae would continue to float, only a small number of larvae would settle. When the larva floated too long and did not complete the metamorphosis, there would be a large number of deaths. When providing inducers, larvae would complete the settlement behavior within 24 hours and began the process of metamorphosis.
The morphological changes in the adult shell of R. venosa are shown in Fig. 2. As shown in Fig. 2a, the larvae is in F stage, and is mainly planktonic and feed on unicellular algae. The adult shell has not started to grow, and the shell is dark brown. As shown in Fig. 2b, c, d, during metamorphosis, the larvae are mainly benthic and occasionally planktonic, feeding on unicellular algae and bivalves. The white adult shell begins to grow. As shown in Fig. 2e, when metamorphosis is almost completed, larvae live mainly in the benthos, feeding on bivalves, and not in the plankton. The white adult shell occupies the whole spiral layer. As shown in Fig. 2f, the larvae complete the metamorphosis into juvenile snails, benthic life, feeding on shellfish, and the adult shell is white.3.2 Time of settlement and metamorphosis and provision of substrates
As shown in Fig. 3a & b, scallop shells with oyster can effectively induce larval settlement and metamorphosis. As shown in Fig. 4a, b, c, the settlement rate, metamorphosis rate, and mortality rate of larvae differed significantly among the different stages (F=318.08, P < 0.001; F=168.37, P < 0.001; and F=9.48, P < 0.001, respectively) and different substrates (F=113.41, P < 0.001; F=172.24, P < 0.001; and F=55.80, P < 0.001, respectively). Different stages and substrates had significant interaction effects on the settlement rate, metamorphosis rate, and mortality rate (F=100.86, P < 0.001; F=165.29, P < 0.001; and F=9.01, P < 0.01, respectively). The settlement rate, metamorphosis rate, and mortality rate of larvae with scallop shell substrates were higher than those of control groups (P < 0.001). The settlement rate and metamorphosis rate of F stage larvae were higher than those of D and E stage larvae (P < 0.001); however, the mortality rate of F stage larvae was lower than those of D and E stage larvae (P < 0.05).
As shown in Fig. 4a and b, when scallop shell with oysters was provided, the F stage larvae had high settlement (86.83%) and metamorphosis (36.33%) rates, whereas stage D and E larvae had very low rates of settlement (0, 3.17%, respectively) and metamorphosis (< 1%). When scallop shells were not provided, the metamorphosis rate of F stage larvae was very low (< 1%), while the settlement rate was as high as 24.17%. Settlement and metamorphosis rates were very low in D and E stage larvae (< 1%). The results showed that the larvae began to settle and metamorphose significantly in the F stage, and the scallop shell with oysters had a good effect on the settlement and metamorphosis of larvae.
As shown in Fig. 4c, when scallop shell with oysters was provided, the larvae of stages D and E had higher mortality rates (31.33%, 22.33%, respectively) than that of F stage larvae (9.67%). When scallop shells were not provided, there was no significant difference in mortality among D (6.00%), E (5.33%), and F (5.67%) stage larvae (P < 0.05). There was no significant difference in mortality between scallop shell and control groups in F stage larvae (P < 0.05). The results showed that premature provision of substrates increased larval mortality and only when the larvae reached the F stage can they begin to settle.
As shown in Fig. 4d, at the F stage, the rate of metamorphosis was significantly difference among different substrates (F=166.05, P < 0.05). The rate of metamorphosis in scallop shells with oysters groups was significantly higher than that in scallop shells without oysters groups and no substrate groups (P < 0.05). There was no significant difference (P > 0.05) in the rate of metamorphosis between scallop shells without oysters groups and no substrate groups.3.3 Illumination
Different illumination conditions had a significant effect on the metamorphosis rate of planktonic larvae (F=9.547, P < 0.001). The metamorphosis rate of larvae in dark (46.67%) conditions was significantly higher than that in other illumination (16.11%–23.33%) groups (P < 0.05; Fig. 5). The rate of metamorphosis of planktonic larvae decreased significantly when light was provided, which indicated that darkness was beneficial to the metamorphosis of larvae.3.4 Prey
There were significant differences in the effects of different prey inducers on the settlement rate of planktonic larvae (F=30.13, P < 0.001). Compared with the algae group, the settlement rate of larvae could be significantly increased by the presence of shellfish juveniles (P < 0.05), including shellfish that could be eaten directly by larvae and those that could not be eaten by larvae (Fig. 6a).
As shown in Fig. 6b, the effects of different prey inducers on the larval metamorphosis rate were significantly different (F=34.56, P < 0.001). The metamorphosis rates of Cg-F (35.67%), Mm-F (28.00%), and Mc-F (27.00%) were significantly high than those of Cg-I (14.33%), Mm-I (7.33%), and Mc-I (7.67%), respectively (P < 0.05). Group Cg-F had the highest rate of metamorphosis,