Introduction
Stalked and acorn barnacles (Cirripedia, Thoracica) are important members of marine communities from the rocky intertidal zone to offshore habitats. Barnacles are the most successful fouling animals because of their prolific settlement and wide global distribution (Clare & Matsumura, Reference Clare and Matsumura2000; Aldred & Clare, Reference Aldred and Clare2008; Maréchal & Hellio, Reference Maréchal and Hellio2011). The barnacle life cycle comprises six nauplius instars followed by a non-feeding, settling cyprid instar. The sole purpose of the cyprid stage is to locate, explore and attach on a suitable substratum, from where a subsequent complex metamorphosis takes place. Cyprid attachment and metamorphosis is also called ‘cyprid settlement’ (Clare & Matsumura, Reference Clare and Matsumura2000; Harder et al., Reference Harder, Thiyagarajan and Qian2001a; Franco et al., Reference Franco, Aldred, Cruz and Clare2016). For barnacles, attachment refers to the cementation and fixation of a cyprid onto a surface (Høeg et al., Reference Høeg, Maruzzo, Okano, Glenner and Chan2012; Maréchal et al., Reference Maréchal, Matsumura, Conlan and Hellio2012; Spremberg et al., Reference Spremberg, Høeg, Buhl-Mortensen and Yusa2012; Dreyer et al., Reference Dreyer, Olesen, Dahl, Chan and Høeg2018), whereas metamorphosis denotes the subsequent sequence of morphological, physiological and biochemical events that transform a permanently attached cyprid into a sessile juvenile (Thiyagarajan, Reference Thiyagarajan2010; Maréchal et al., Reference Maréchal, Matsumura, Conlan and Hellio2012; Maruzzo et al., Reference Maruzzo, Aldred, Clare and Høeg2012). Understanding factors governing the settlement of sessile marine invertebrates is important in elucidating mechanisms determining species distribution, aquaculture and control of biofouling. The larval settlement of benthic marine invertebrates influenced by various physical, chemical and biological factors has been extensively reviewed (Morse et al., Reference Morse, Duncan, Hooker, Baloun and Young1980; Hadfield, Reference Hadfield1986; Fraschetti et al., Reference Fraschetti, Giangrande, Terlizzi and Boero2003).
Various exogenous and endogenous factors influence cyprid settlement and ultimately affect the recruitment and distribution of barnacles (Rittschof et al., Reference Rittschof, Forward, Cannon, Welch, McClary, Holm, Clare, Conova, McKelvey, Bryan and van Dover1998; Clare & Matsumura, Reference Clare and Matsumura2000; Anil et al., Reference Anil, Khandeparker, Desai, Baragi and Gaonkar2010; Thiyagarajan, Reference Thiyagarajan2010). The exogenous factors include current flow (Rittschof et al., Reference Rittschof, Branscomb and Costlow1984), tidal height (Olivier et al., Reference Olivier, Tremblay, Bourget and Rittschof2000), larval supply (Olivier et al., Reference Olivier, Tremblay, Bourget and Rittschof2000; Tremblay et al., Reference Tremblay, Olivier, Bourget and Rittschof2007), surface contour, free energy, substratum types (Rittschof et al., Reference Rittschof, Branscomb and Costlow1984; O'Connor & Richardson, Reference O'Connor and Richardson1994), adult extracts (Dineen & Hines, Reference Dineen and Hines1992, Reference Dineen and Hines1994; Khandeparker et al., Reference Khandeparker, Anil and Raghukumar2002), settlement-inducing protein complex (Matsumura et al., Reference Matsumura, Nagano and Fusetani1998), cyprid footprint(Clare et al., Reference Clare, Freet and McClary1994; Clare & Matsumura, Reference Clare and Matsumura2000), natural biofilms (Olivier et al., Reference Olivier, Tremblay, Bourget and Rittschof2000; Tremblay et al., Reference Tremblay, Olivier, Bourget and Rittschof2007), temperature and salinity (Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2002a, Reference Thiyagarajan, Harder and Qian2003a). Temperature and salinity are two important ecological factors that regulate the survival, duration of larval development, recruitment and settlement of many marine invertebrate larvae (Pechenik et al., Reference Pechenik, Wendt and Jarrett1998). The effects of these two factors on the settlement of barnacle cyprids vary according to species (Dineen & Hines, Reference Dineen and Hines1992, Reference Dineen and Hines1994; Kon-Ya & Miki, Reference Kon-Ya and Miki1994; Anil & Khandeparkar, Reference Anil and Khandeparkar1998; Nasrolahi et al., Reference Nasrolahi, Pansch, Lenz and Wahl2012).
The endogenous factors determine the physiological condition of cyprids and largely refer to the amount of energy reserves and cyprid age (Miron et al., Reference Miron, Walters, Tremblay and Bourget2000; Olivier et al., Reference Olivier, Tremblay, Bourget and Rittschof2000; Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2002a; Tremblay et al., Reference Tremblay, Olivier, Bourget and Rittschof2007). The amount of energy reserves in cyprids is strongly determined by nauplius feeding history (Miron et al., Reference Miron, Walters, Tremblay and Bourget2000; Harder et al., Reference Harder, Thiyagarajan and Qian2001b; Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2002a, Reference Thiyagarajan, Harder and Qian2002b; Thiyagarajan, Reference Thiyagarajan2010). The triacylglycerols (TAG) to sterol content ratio (TAG/ST) (Tremblay et al., Reference Tremblay, Olivier, Bourget and Rittschof2007), TAG to DNA ratio (Miron et al., Reference Miron, Walters, Tremblay and Bourget2000; Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2002b), RNA/DNA ratio (Anil et al., Reference Anil, Desai and Khandeparkar2001) and cyprid major protein (CMP) (Shimizu et al., Reference Shimizu, Saikawa and Fusetani1996) all serve as indicators of cyprid energy reserves. Thus, cyprid energetic factors play a key role in determining their substratrum selection and settlement. Settlement success depends on cyprid age, in addition to energy reserves (Pechenik et al., Reference Pechenik, Rittschof and Schmidt1993; Harder et al., Reference Harder, Thiyagarajan and Qian2001b; Head et al., Reference Head, Berntsson, Dahlström, Overbeke and Thomason2004). In general, old cyprids respond more readily to settlement cues than newly moulted ones (Rittschof et al., Reference Rittschof, Branscomb and Costlow1984; Pechenik et al., Reference Pechenik, Rittschof and Schmidt1993; Satuito et al., Reference Satuito, Shimizu, Natoyama, Yamazaki and Fusetani1996, Reference Satuito, Shimizu and Fusetani1997; Aldred et al., Reference Aldred, Alsaab and Clare2018). Some marine invertebrate larvae have a pre-competent stage (Morse et al., Reference Morse, Duncan, Hooker, Baloun and Young1980; Hadfeld et al., Reference Hadfeld, Carpizo-Ituarte, del Carmen and Nedved2001; Maréchal et al., Reference Maréchal, Matsumura, Conlan and Hellio2012). During the pre-competent stage, the larvae are incapable of settlement and metamorphosis even in the presence of an appropriate and potent settlement inducer. Marine larvae tend to develop through a pre-competent period until they attain competence to settle under appropriate conditions (Morse et al., Reference Morse, Duncan, Hooker, Baloun and Young1980; Hadfeld et al., Reference Hadfeld, Carpizo-Ituarte, del Carmen and Nedved2001; Hodin et al., Reference Hodin, Ferner, Ng, Lowe and Gaylord2015).
A pedunculate barnacle Capitulum mitella (Linnaeus, 1758), formerly assigned to the genus Pollicipes (Leach, 1817) and of high economic value, is a dominant intertidal cirripede (Lee et al., Reference Lee, Shim and Kim2000). Capitulum mitella is distributed from Korea through India to the West Pacific Ocean. It extensively settles on mid- and high-intertidal coasts of exposed rocky crags with strong wave action in the south of the Changjiang River estuary in China. However, the heavy exploitation of C. mitella has led to increased fishery pressure and declining populations in recent years.
Although there are numerous studies on acorn barnacle species, there are fewer studies on larval development and settlement of stalked barnacle species (Moyse, Reference Moyse and Southward1987; Molares et al., Reference Molares, Tilves and Pascual1994; Lee et al., Reference Lee, Shim and Kim2000; Franco et al., Reference Franco, Aldred, Cruz and Clare2016, Reference Franco, Aldred, Cruz and Clare2017). We have been using C. mitella as an intertidal, pedunculate barnacle model for biology and ecology research. Within the last two decades, we have dealt with the ecology, reproduction, gametogenesis, embryonic development and culture conditions of C. mitella (Lin et al., Reference Lin, Qiu and Qi1994, Reference Lin, Xu, Rao and Cheng2002; Zhang et al., Reference Zhang, Rao, Lin and Cheng2009; Rao et al., Reference Rao, Lin, Zhang, Chen and Xu2010). The effects of diet, larval density, temperature and salinity on nauplius survival and development were investigated. In our laboratory, C. mitella nauplii have been cultured until their cyprid stage with a high survival rate. However, C. mitella cyprids cannot attach and metamorphose into juvenile barnacles without drug induction, and they cannot continue their life cycle under laboratory conditions.
The C. mitella cyprid morphology has been described in detail (Rao & Lin, Reference Rao and Lin2014) and cyprids have been successfully induced to metamorphose into juvenile barnacles after artificial exposure to juvenile hormone III (JH- III) (Lin & Rao, Reference Lin and Rao2017). Nevertheless, the effects of various factors on settlement remain unknown. To understand the effects of extrinsic (i.e. water temperature and salinity) and intrinsic (i.e. cyprid age) factors on the metamorphosis of C. mitella, we stored C. mitella cyprids at room temperature (24–26°C) and low temperature (7°C) and then compared the effects of cyprid age and storage temperature on C. mitella cyprid metamorphosis. Furthermore, we examined the effects of salinity and temperature on the metamorphosis and survival of C. mitella cyprids. Our study mainly aimed: (1) to clarify whether the settlement response of C. mitella cyprids is age-dependent and C. mitella cyprids have a pre-competent phase and to determine the suitable storage temperature and duration of cyprids and (2) to understand how salinity and temperature affect the metamorphosis of this species and to establish a suitable salinity and temperature range for cyprid metamorphosis. This study may provide a basis for the settlement biology, recruitment ecology and aquaculture of this species.
Materials and methods
Cyprid culture
Adult Capitulum mitella specimens were collected in September in the intertidal zone of Dinghai, Fuzhou, Fujian, China (26°16′N 119°48′E). The cyprids were reared according to the method established by Rao & Lin (Reference Rao and Lin2014). Each batch of egg masses obtained from the mantle cavity of adults (>10 individuals) was cultured in aerated filtered seawater (FSW) with 28–30mg l−1 salinity at 24–26°C in the dark. The seawater was changed every day. Nauplii I were collected after hatching via positive phototaxic behaviour and transferred to plastic barrels. The larval culture at a density of 1 larva ml−1 was maintained at 24–26°C. Nauplii I–nauplii III were fed with Dicrateria zhanjiangensis at a density of 5 × 104 cells ml−1, and nauplii IV–VI were fed with D. zhanjiangensis at a density of 5 × 104 cells ml−1 and Platymonas subcordiformis at a density of 104 cells ml−1. Half of the larval culture seawater was replaced daily. These algae were semi-continuously grown in f/2-medium in batch culture at 24–26°C and at 14 h light:10 h dark photoperiod. After ~9–11 days, nauplii VI moulted into cyprids, and the culture was filtered through a series of plankton mesh filters. The cyprids were transferred to freshly filtered seawater. Batches of newly moulted cyprids were referred to as 0-day old.
Settlement bioassays
Bioassays were carried out in glass dishes (60 mm diameter) with 5 ml filtered seawater (30 mg l−1 salinity, except in experiment 2). In metamorphosis experiments the seawater contained 1 μg ml−1 JH-III, in survival experiments the seawater contained no drug. About 20 healthy cyprids were added to each dish and incubated at room temperature (24–26°C) (except in experiment 3) in the dark. The assays were run as static experiments with four replicates by using different batches of cyprids. The dishes were examined and evaluated for dead cyprids and metamorphosed juveniles under a dissecting microscope at 24 h intervals for a period of 7 days.
Per cent metamorphosis was expressed as a percentage of metamorphosed juveniles relative to the initial number of cyprids at the time (on day 4, on day 5, on day 6, on day 7). Per cent mortality of cyprids was expressed as a percentage of dead cyprids relative to the initial number at the end of the experiment (on day 7). The development duration (in days) from cyprid to juvenile was taken to be the time for the first appearance of metamorphosed juveniles (Lin & Rao, Reference Lin and Rao2017).
Experiment 1: Effects of age and storage temperature on metamorphosis
The same batch of 0-day cyprids was separated into two groups and held in filtered seawater at room temperature (24–26°C) and 7°C separately in the dark. The seawater for the cyprids held at room temperature was changed every other day. The cyprids at two temperatures were sampled on day 0 (0d), day 1 (1d), day 3 (3d), day 5 (5d), day 7 (7d), day 10 (10d) and day 14 (14d) for the bioassays.
Experiment 2: Effects of salinity on metamorphosis and survival
The effect of salinity on cyprid metamorphosis and survival was determined at 15, 20, 25, 30, 35, 40 and 45 mg l−1 salinity levels. Depending on the desired salinity level, seawater was either diluted with deionized water or increased by the addition of seawater crystals. The 3d–5d cyprids stored at room temperature were used to perform the assays.
Experiment 3: Effects of temperature on metamorphosis and survival
The effect of temperature on cyprid metamorphosis and survival was determined at the following temperatures: 18°C ± 1°C, 21°C ± 1°C, 24°C ± 1°C, 27°C ± 1°C, 30°C ± 1°C, 33°C ± 1°C and 36°C ± 1°C. Each treatment was kept in separate temperature-controlled incubators. The 3d–5d cyprids stored at room temperature were used to perform the assays.
Data analysis
The data were analysed by using SPSS 21.0 for Windows. The data of per cent metamorphosis and per cent mortality were inverse arcsine-transformed prior to statistical analysis. The normality and homogeneity of variance were examined using Shapiro–Wilk test and Levene test, respectively. Since the analysis of variance of complex experiments is robust to non-normality, the importance of the failure of data in the normality test (Shapiro–Wilk test) was ignored (Underwood, Reference Underwood1997; Thiyagarajan et al., Reference Thiyagarajan, Harder, Qiu and Qian2003b). The effects of cyprid age, salinity and temperature on metamorphosis and mortality at the end of the experiment (on day 6 or day 7) were evaluated using one-way ANOVA. Statistical significance was determined by post-hoc investigation via Tukey test when relevant (P < 0.05). Comparison data between the same age groups at two storage temperatures were analysed by using Student's t-test. A P value of less than 0.05 was considered significant. All data were presented as the mean ± SD by Student's t-test.
Results
Effects of age and storage temperature on the Capitulum mitella cyprid metamorphosis
The per cent metamorphosis for cyprids stored at two temperatures is presented in Figure 1A, B, respectively.
(A) Effect of age on the per cent metamorphosis of Capitulum mitella cyprids stored at room temperature (24–26°C) over a 6-day period. (B) Effect of age on the per cent metamorphosis of Capitulum mitella cyprids stored at 7°C over a 7-day period. Different small letters on the columns refer to significant difference in terms of the per cent metamorphosis of different cyprid ages (P < 0.05).
1d–14d cyprids stored at room temperature (Figure 1A) were competent to metamorphose except for 0d cyprids. The per cent metamorphosis was the highest for 3d cyprids (98.81%), but decreased gradually with age. The per cent metamorphosis at the end of the experiment (on day 6) was significantly influenced by age (ANOVA, df = 5, F = 20.28, P = 0.000001). Also, the per cent metamorphosis of 3d cyprids was not significantly different from that of 5d cyprids (92.86%) (P = 0.29), but had a significant difference with all other treatments (P < 0.05). The per cent metamorphosis of 5d cyprids did not have a significant difference with that of 7d and 10d cyprids (84.52 and 82.14%, respectively) (P ≧ 0.16). The per cent metamorphosis of 7d and 10d cyprids did not have a significant difference with that of 1d cyprids (64.29%) (P ≧ 0.06), but showed a significant difference with that of 14d cyprids (53.57%) (P < 0.05). The development duration of 1d–10d cyprids was relatively short at only 4 days, and the development duration of 14d cyprids was longer for 5 days. The results showed that 1d–14d cyprids stored at room temperature could metamorphose, but 0d cyprids were not competent to metamorphose. The optimum age of cyprid metamorphosis was 3–5 days.
Only 1d–7d cyprids stored at 7°C (Figure 1B) were competent to metamorphose, whereas 10d cyprids did not metamorphose. The per cent metamorphosis at the end of the experiment (on day 7) was significantly influenced by age (ANOVA, df = 3, F = 35.04, P = 0.000003). A maximum metamorphosis of 70.24% for 3d cyprids and 55.59% for 1d cyprids was observed. No significant difference was observed between the per cent metamorphosis of 3d and 1d cyprids (P = 0.09). A significant difference between 5d (36.90%) and 7d (19.05%) cyprids was observed. The development duration of different age of cyprids was 4 days.
In comparison with the per cent metamorphosis of cyprids at different storage temperatures with the same age, a significant difference between 7°C and room temperature (P < 0.01) was observed except for 1d cyprids (Student's t-test, P = 0.22). The per cent metamorphosis of cyprids with the same age was higher at room temperature than at 7°C.
Effect of salinity on the metamorphosis and survival of C. mitella cyprids
The per cent metamorphosis and per cent mortality of cyprids at different salinity levels are presented in Figure 2A, B, respectively.
(A) Effect of salinity on the Capitulum mitella cyprid metamorphosis over a 7-day period. (B) Effect of salinity on the Capitulum mitella cyprid mortality on day 7. Different small letters on the columns refer to significant difference in terms of the per cent metamorphosis and per cent mortality at different salinities (P < 0.05).
The cyprids could complete metamorphosis at 20–45 mg l−1 salinity, but did not metamorphose at 15 mg l−1 salinity. Salinity significantly affected the per cent metamorphosis at the end of the experiment (on day 7) (ANOVA, df = 5, F = 63.49, P = 0.00000). At 25, 30 and 35 mg l−1 salinity levels, the per cent metamorphosis was high (91.57, 95.99 and 94.45%, respectively) (no significant differences, P ≧ 0.91). The per cent metamorphosis at 20 and 40 mg l−1 salinity levels (44.69 and 67.50%, respectively) was not significantly different (P = 0.08). The per cent metamorphosis at 45 mg l−1 salinity was the lowest at only 7.50%, and with significant difference from other treatments (P = 0.00). At 25–35 mg l−1 salinity levels, the development duration was relatively short at only 4 days. At 10 and 40 mg l−1 salinity, the duration was longer for 5 days, and at 45 mg l−1 salinity, the duration was the longest for 6 days.
The cyprid mortality on day 7 was influenced by salinity (ANOVA, df = 6, F = 9.86, P = 0.000003). At 20–45 mg l−1 salinity, the per cent mortality was low, and no significant difference was observed (P ≧ 0.50). At 15 mg l−1 salinity, the per cent mortality was up to 36.91%. The per cent mortality of 15 mg l−1 salinity had a significant difference with all other treatments (P < 0.05).
The results showed that at the 20–45 mg l−1 salinity range, the cyprids could complete metamorphosis, and the optimum salinity range for metamorphosis was 25–35 mg l−1.
Effect of temperature on the metamorphosis and survival of C. mitella cyprids
The per cent metamorphosis and per cent mortality of cyprids at different temperatures are presented in Figure 3A, B, respectively.
(A) Effect of temperature on Capitulum mitella cyprid metamorphosis over a 7-day period. (B) Effect of temperature on Capitulum mitella cyprid mortality on day 7. Different small letters on the columns refer to significant difference in terms of the per cent metamorphosis and per cent mortality at different temperatures (P < 0.05).
The cyprids were capable of metamorphosis at a temperature range of 21–36°C. The per cent metamorphosis at the end of the experiment (on day 7) was significantly influenced by temperature (ANOVA, df = 5, F = 51.06, P = 0.00000). At 24, 27 and 21°C, the per cent metamorphosis was up to 99.0, 98.75 and 93.33% respectively, and no significant differences were observed among the three (P ≧ 0.07). At 30 and 33°C, the per cent metamorphosis was 87.50 and 86.25% respectively (no significant difference with 21°C) (P ≧ 0.39). The per cent metamorphosis was the lowest at 36°C, and only 33.7% with significant difference from other temperature treatments (P = 0.00). The development duration was relatively the shortest at only 3 days at 30°C. At 24, 27, 33 and 36°C, the development duration was 4 days. At 21°C, the development duration was the longest at 5 days. In addition, the cyprids could also metamorphose at 18°C. Metamorphosis completion would take 8–9 days, and the ultimate per cent of metamorphosis was up to 76.51%.
In all temperature treatments, the cyprid mortality was relatively low, and the per cent mortality at the end of the experiment (on day 7) fluctuated slightly at a range of 2.50–10.00%. The highest percentage mortality was 10.00% at 36°C. The mortality was not significantly influenced by temperature (ANOVA, df = 6, F = 1.95, P = 0.12).
The results showed that the metamorphosis of cyprids had a wide temperature tolerance range of 18–36°C and that 21–33°C was the optimum temperature range of metamorphosis.
Discussion
In barnacles, two processes are involved during natural cyprid settlement, namely, attachment and metamorphosis (Clare & Matsumura, Reference Clare and Matsumura2000; Lagersson & Høeg, Reference Lagersson and Høeg2002). Notably, metamorphosis generally occurs after attachment, and it may commence without prior attachment under certain circumstances (Clare et al., Reference Clare, Thomas and Rittschof1995; Yamamoto et al., Reference Yamamoto, Kawaii, Yoshimura, Tachibana and Fusetani1997). Although acorn barnacles may attach and metamorphose in the laboratory, the attachment and metamorphosis of some stalked barnacles (for example Pollicipes and Capitulum mitella) in the laboratory remains challenging (Kugele & Yule, Reference Kugele and Yule1996; Lin & Rao, Reference Lin and Rao2017). Most of C. mitella cyprids have been successfully induced to metamorphose into juveniles without attachment after artificial exposure to JH-III, and only a few cyprids can attach and metamorphose into juveniles (Lin & Rao, Reference Lin and Rao2017). Therefore, in the present study, the metamorphosis was referred to as the conversion of the unattached and attached cyprids by shedding of thoracopodal exuvia into the pelagic and sessile juveniles with JH-III induction (Lin & Rao, Reference Lin and Rao2017).
Effects of cyprid age and storage temperature on C. mitella cyprid metamorphosis
Some marine invertebrate larvae undergo a pre-competent stage, during which they develop the ability to respond to settlement cues (Morse et al., Reference Morse, Duncan, Hooker, Baloun and Young1980; Hodin et al., Reference Hodin, Ferner, Ng, Lowe and Gaylord2015). Maréchal et al. (Reference Maréchal, Matsumura, Conlan and Hellio2012) confirmed that young cyprids (0-day old) of Amphibalanus amphitrite do not appear to be competent to settle. This observation seems to be the case for C. mitella in which young cyprids (0-day old) are also not competent to metamorphose. Similarly, C. mitella cyprids have a pre-competent phase, suggesting that C. mitella cyprids may require a period of competence acquisition. Upon acquisition of competence, cyprids readily respond to settlement inducers. However, some discrepancies in studies are likely due to differences in larval culture and bioassay methodology (Pechenik et al., Reference Pechenik, Rittschof and Schmidt1993; Clare et al., Reference Clare, Thomas and Rittschof1995; Kitamura & Nakashima, Reference Kitamura and Nakashima1996; Satuito et al., Reference Satuito, Shimizu, Natoyama, Yamazaki and Fusetani1996, Reference Satuito, Shimizu and Fusetani1997; Franco et al., Reference Franco, Aldred, Cruz and Clare2016).
Cyprid age is critically important to cyprid settlement and juvenile growth (Pechenik et al., Reference Pechenik, Rittschof and Schmidt1993; Miron et al., Reference Miron, Walters, Tremblay and Bourget2000; Anil et al., Reference Anil, Khandeparker, Desai, Baragi and Gaonkar2010; Thiyagarajan, Reference Thiyagarajan2010). Amphibalanus amphitrite cyprids have an age-dependent settlement response. The old cyprids respond more readily to settlement cues and generally settle at higher rates than the newly moulted ones (Rittschof et al., Reference Rittschof, Branscomb and Costlow1984; Pechenik et al., Reference Pechenik, Rittschof and Schmidt1993; Satuito et al., Reference Satuito, Shimizu, Natoyama, Yamazaki and Fusetani1996, Reference Satuito, Shimizu and Fusetani1997). Recently, the quantitative measurement of Aldred et al. (Reference Aldred, Alsaab and Clare2018) showed clearly that older cyprids move less over the substratum and settle faster. However, the storage time of cyprids in these studies is short, that is, 1–6 days (Pechenik et al., Reference Pechenik, Rittschof and Schmidt1993; Satuito et al., Reference Satuito, Shimizu and Fusetani1997; Harder et al., Reference Harder, Thiyagarajan and Qian2001b; Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2002a). Evaluating the relationship between cyprid age and settlement can take a long time. Cyprids have been stored up to 10–15 days (Kitamura & Nakashima, Reference Kitamura and Nakashima1996; Satuito et al., Reference Satuito, Shimizu, Natoyama, Yamazaki and Fusetani1996; Maréchal et al., Reference Maréchal, Matsumura, Conlan and Hellio2012). Satuito et al. (Reference Satuito, Shimizu, Natoyama, Yamazaki and Fusetani1996) reported that A. amphitrite cyprids exhibit two distinct age-related phases (phase 1 and phase 2) during settlement at a prolonged cyprid period. Maréchal et al. (Reference Maréchal, Matsumura, Conlan and Hellio2012) and Kitamura & Nakashima (Reference Kitamura and Nakashima1996) also supported this conclusion. However, the peak settlement age of cyprids varies at 2–10 days (Kitamura & Nakashima, Reference Kitamura and Nakashima1996; Satuito et al., Reference Satuito, Shimizu, Natoyama, Yamazaki and Fusetani1996; Maréchal et al., Reference Maréchal, Matsumura, Conlan and Hellio2012). Our results showed that the 14-day duration of C. mitella cyprids also presented two age-related phases during metamorphosis and the metamorphosis peak occurred on day 3 for two storage temperatures.
The physiological condition of cyprids depends strongly not only on cyprid age, but also on the amount of energy reserves (Miron et al., Reference Miron, Walters, Tremblay and Bourget2000; Olivier et al., Reference Olivier, Tremblay, Bourget and Rittschof2000; Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2002a; Tremblay et al., Reference Tremblay, Olivier, Bourget and Rittschof2007). Cyprid larvae with high energy reserves generally show high settlement, thus, the level of energy reserves in cyprids may be reflected in the level of settlement success (Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2002b; Tremblay et al., Reference Tremblay, Olivier, Bourget and Rittschof2007). Cyprid energy reserves such as TAG/DNA ratio (Miron et al., Reference Miron, Walters, Tremblay and Bourget2000), RNA/DNA ratio (Anil et al., Reference Anil, Desai and Khandeparkar2001) and CMP (Satuito et al., Reference Satuito, Shimizu, Natoyama, Yamazaki and Fusetani1996) decreases over time. The amount of energy reserves drops below the final threshold level where cyprids possess insufficient energy to accomplish metamorphosis into juvenile individuals (Lucas et al., Reference Lucas, Walker, Holiand and Crisp1979; Anil et al., Reference Anil, Desai and Khandeparkar2001). However, Harder et al. (Reference Harder, Thiyagarajan and Qian2001b) reported that cyprids with similar lipid content show remarkably different rates of attachment, suggesting that the metamorphic success of cyprids is jointly affected by the lipid content of cyprids and their age. In the current study, the metamorphosis of C. mitella cyprids peaked at 3–5 days, and then decreased progressively with age. Furthermore, metamorphosis rate on day 14 still remained at a relatively high level (>50%). Thus, the metamorphic competence of C. mitella cyprids could remain for a long time. The amount of cyprid energy reserves depends on the nauplius feeding history, specifically on algal food quantity and quality (Anil et al., Reference Anil, Desai and Khandeparkar2001; Harder et al., Reference Harder, Thiyagarajan and Qian2001b; Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2002b). In the current study, our results illustrated that the chosen algae in the larval culture of C. mitella were a suitable diet, given that they have sufficient energy reserves and high nutritional value to support and ensure the completion of cyprid metamorphosis.
Low-temperature (6°C) storage is a routine method used to age cyprids in bioassays. Pechenik et al. (Reference Pechenik, Rittschof and Schmidt1993) reported that storage temperature (6°C) slightly affects the settlement rates; however, they only compared 3-d and 5-d cyprids. Kitamura & Nakashima (Reference Kitamura and Nakashima1996) indicated that the ability to settle decreases quickly with age when reared at 25°C vs that at 6°C. Satuito et al. (Reference Satuito, Shimizu, Natoyama, Yamazaki and Fusetani1996) reported that the per cent settlement of cyprids stored at 5°C is similar for all ages and the settlement rate for cyprids stored at 25°C decreases if stored for more than 3 days. Thus, the settlement ability in these studies is retained for a long period at a low temperature. However, Maréchal et al. (Reference Maréchal, Matsumura, Conlan and Hellio2012) compared the effects of two storage temperatures on settlement relative to cyprid age and reported conflicting results. Their results suggest that A. amphitrite cyprids stored at 6 and 23°C have similar settlement trends (with a maximum settlement rate on day 10), but cyprids stored at 23°C have a remarkably higher settlement rate than that at 6°C (Maréchal et al., Reference Maréchal, Matsumura, Conlan and Hellio2012). Our observation was consistent with that of Maréchal. The metamorphosis rate for 3-day C. mitella cyprids was the highest at two storage temperatures, but was consistently higher for cyprids stored at room temperature than those stored at 7°C. Clearly, the metamorphosis ability of C. mitella cyprids was retained for a longer period at room temperature than at a low temperature.
Preventing cyprid settlement is essential in bioassays because A. amphitrite cyprids readily settle even onto untreated substrata. Rittschof et al. (Reference Rittschof, Branscomb and Costlow1984) stored cyprids in the dark at 6°C until use. Since then, cyprid storage at a low temperature (4–8°C) is the most prevalent technique in bioassays (Pechenik et al., Reference Pechenik, Rittschof and Schmidt1993; Harder et al., Reference Harder, Thiyagarajan and Qian2001a, Reference Harder, Thiyagarajan and Qian2001b; Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2002a; Head et al., Reference Head, Berntsson, Dahlström, Overbeke and Thomason2004). However, the effect of low temperature storage on the settlement and survival of cyprids has been poorly studied (O'Connor & Richardson, Reference O'Connor and Richardson1994; Harder et al., Reference Harder, Thiyagarajan and Qian2001b; Maréchal et al., Reference Maréchal, Matsumura, Conlan and Hellio2012). This approach is questioned due to the observed cyprid abnormalities at a low temperature (Satuito et al., Reference Satuito, Shimizu, Natoyama, Yamazaki and Fusetani1996). Kitamura & Nakashima (Reference Kitamura and Nakashima1996) suggested that storing cyprids at a low temperature is unsuitable for studies on natural settlement in A. amphitrite. Satuito et al. (Reference Satuito, Shimizu, Natoyama, Yamazaki and Fusetani1996) emphasized that A. amphitrite cyprids are adversely affected by storage at a low water temperature, with many larvae having an extended thorax. In the present study, many C. mitella cyprids stored at 7°C for 9–10 days showed an extended thorax and antennules. This result is attributed to C. mitella being a tropical and subtropical species. The annual average temperature in the sea area of Dinghai, Fuzhou, Fujian, China is 19.2°C, and the water temperature during the breeding period is 19.5–27.2°C (Lin et al., Reference Lin, Qiu and Qi1994). Thus, a low temperature may result in deleterious effects on C. mitella cyprids. We observed in the present study that C. mitella cyprids could live for more than 30 days at room temperature, although the amount of available lipid gradually decreased, and their activity declined.
Clearly, storage temperature and time have a marked effect on cyprid metamorphosis. Consequently, storing cyprids at a low water temperature appeared unsuitable for C. mitella. A time of 3–5 days seemed to be ideal for cyprid storage at room temperature because the cyprids were strongly competent to metamorphose under such conditions.
Effects of salinity and temperature on C. mitella cyprid metamorphosis and survival
The effects of salinity on barnacle larval development and settlement are widely studied (Dineen & Hines, Reference Dineen and Hines1992, Reference Dineen and Hines1994; Kon-Ya & Miki, Reference Kon-Ya and Miki1994; Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2003a; Nasrolahi et al., Reference Nasrolahi, Pansch, Lenz and Wahl2012). The response to salinity greatly varies with species selectivity, correlating with their distribution range. Cyprid settlement responses peak in the presence of conspecific adult settlement factor at meso-polyhaline salinities for A. eburneus (Dineen & Hines, Reference Dineen and Hines1994) and at mesohaline salinities for A. improvisus (Dineen & Hines, Reference Dineen and Hines1992), thereby coinciding with the adult distribution in the field. Amphibalanus amphitrite cyprids can settle in a wide range of salinity (14–52 mg l−1) with a peak settlement between 22 and 35 mg l−1; they can survive at salinities of 9 and 65 mg l−1 but fail to settle (Kon-Ya & Miki, Reference Kon-Ya and Miki1994). The present results showed that C. mitella cyprids could complete metamorphosis in a wide range of salinity levels (20–45 mg l−1). At 15 mg l−1 salinity, the cyprids could survive but failed to metamorphose. Furthermore, the optimum salinity range for metamorphosis was 25–35 mg l−1. Temperature has well-documented effects on barnacle cyprid settlement (Kon-Ya & Miki, Reference Kon-Ya and Miki1994; Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2002a, Reference Thiyagarajan, Harder and Qian2003a; Nasrolahi et al., Reference Nasrolahi, Pansch, Lenz and Wahl2012). Barnacle species exhibit tolerance to different environmental temperatures during settlement. Amphibalanus amphitrite cyprids can settle at a wide temperature range of 15–37°C, and show high settlement rate at 20–30°C; the cyprids can survive at 10°C but fail to settle (Kon-Ya & Miki, Reference Kon-Ya and Miki1994). The present results showed that C. mitella cyprids could complete metamorphosis at a wide temperature range of 18–36°C and the optimum temperature range for metamorphosis was 21–33°C. Both salinity and temperature influence the development speed and duration of barnacle larvae. The duration of larval development is shortened with the optimum range of salinity and temperature (Qiu & Qian, Reference Qiu and Qian1999; Anil et al., Reference Anil, Desai and Khandeparker2000; Franco et al., Reference Franco, Aldred, Cruz and Clare2017). Similarly, the metamorphosis duration of C. mitella cyprids was relatively shorter at a suitable range of salinity and temperature than that at low or high salinity and low temperature. Low temperature and inappropriate salinity lead to low assimilation of energy and the need for additional energy reserves to regulate osmotic pressure (Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2003a).
Amphibalanus amphitrite is a dominant fouling organism that inhabits the rocky intertidal habitats in tropical and subtropical waters. Similar to A. amphitrite cyprids, the metamorphosis and survival of C. mitella cyprids can tolerate a wide spectrum of salinity and temperature, which is related to the environmental conditions and lifestyle during the breeding period in the field. Capitulum mitella mainly inhabits the exposed rock crevices of middle and high intertidal coasts. Under natural conditions, the cyprids generally attach and complete metamorphosis on the stalks of adults. The previous results showed that C. mitella cyprids have a long metamorphosis duration lasting up to 4–5 days (Lin & Rao, Reference Lin and Rao2017). Thus, not only do the adults undergo drastic salinity and temperature changes after suffering from precipitation, constant air exposure, relatively long periods of drying out and temperature differences between day and night, but also the settled cyprids face a similar pressure during and after attachment and metamorphosis.
Barnacles inhabiting the intertidal zone are exposed to extreme physical conditions during low tides. Intertidal barnacles can tolerate various extreme factors by means of morphological, behavioural and physiological adaptations (Anil et al., Reference Anil, Khandeparker, Desai, Baragi and Gaonkar2010). For the settlement of intertidal barnacles, following attachment, the metamorphosing cyprids face various external challenges such as predation, heat or desiccation, mechanical removal by wave action or being groomed away by the host (parasites) and extreme daily variations in temperature and salinity. In contrast to intertidal barnacles, subtidal barnacles are adapted to relatively stable environmental conditions and a narrow tolerable range of salinity and temperature. For Balanus trigonus, the tolerable range of salinity and temperature at the time of settlement is 26–34 mg l−1 and 18–28°C respectively (Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2003a). Therefore, barnacle species from intertidal habitats are more salinity and temperature tolerant than subtidal species.
Pollicipes pollicipes is a pedunculated species, is commercially important and is distributed on the Atlantic coast of Europe and North Africa, between 48°N (UK, France) and 15°N (Dakar, Senegal) (Barnes, Reference Barnes1996; Cruz et al., Reference Cruz, Castro and Hawkins2010). This species is abundant on very exposed rocky shores, ranging from the shallow subtidal to mid-intertidal zone. Pollicipes pollicipes and C. mitella are each other’s closest relatives and form a monophyletic group, that is, the family Pollicipedidae. The settlement of P. pollicipes cyprids on artificial substratum is null or negligible, but they show strong preference for settlement on conspecific adults (Kugele & Yule, Reference Kugele and Yule1996; Franco et al., Reference Franco, Aldred, Cruz and Clare2016). Franco et al. (Reference Franco, Aldred, Cruz and Clare2016) investigated the effects of several factors on P. pollicipes settlement on conspecifics. Their results showed that a few different effects of factors on settlement exist between P. pollicipes and C. mitella. The un-aged (0-day old) P. pollicipes cyprids show strong metamorphosis ability, suggesting that P. pollicipes cyprids do not undergo a pre-competent stage. At a salinity range of 20–40 mg l−1, the suitable salinity for the metamorphosis of P. pollicipes cyprids is slightly higher (30–40 mg l−1) than that of C. mitella (25–35 mg l−1). The optimal temperature for the metamorphosis of P. pollicipes cyprids is considerably lower (17–20°C) than that for C. mitella (21–33°C). These discrepancies might originate from different adult distribution and experimental conditions, considering that P. pollicipes adults are mainly distributed in mid- and low-intertidal zones, the adults are normally found in their natural habitat with high salinity (33–37 mg l−1) and low temperatures (14–20°C) during the breeding season (Franco et al., Reference Franco, Aldred, Cruz and Clare2016, Reference Franco, Aldred, Cruz and Clare2017).
Our previous results (Zhang et al., Reference Zhang, Rao, Lin and Cheng2009) showed that (1) C. mitella embryos can develop at 18–36°C, and the optimum temperature for embryonic development is 24–33°C; (2) the nauplii can develop at 21–33°C, and the optimum temperature for nauplius development is 24–30°C. Clearly, the temperature range of C. mitella cyprid metamorphosis is similar to that for its embryonic development. This temperature range of cyprid metamorphosis and embryonic development is closely correlated with the reproductive characteristics and habitat of C. mitella adults. Under natural conditions, fertilized eggs complete their development in the mantle cavity until hatching, and cyprids commonly attach and metamorphose on the stalks of adults. Developing embryos and metamorphosing cyprids suffer from the same air exposure and heat as the adults. Therefore, the embryonic development and cyprid metamorphosis of C. mitella can occur at a wide temperature range. However, the temperature range for the nauplius development is slightly narrower than that for the cyprid metamorphosis. The nauplii are planktonic in the sea where the temperature change is smaller than at the intertidal coast. Compared with the adults growing in the intertidal zone and cyprids metamorphosing on the stalks of adults, the nauplius larvae of C. mitella may adapt to a relatively more stable environment.
In conclusion, compared with the subtidal barnacle B. trigonus cyprids that tolerate a narrower range of salinity and temperature during settlement (Thiyagarajan et al., Reference Thiyagarajan, Harder and Qian2003a), the intertidal barnacle A. amphitrite and C. mitella cyprids can tolerate a wider range of salinity and temperature at the time of settlement (Kon-ya & Miki, Reference Kon-Ya and Miki1994; O'Connor & Richardson, Reference O'Connor and Richardson1994; Anil & Khandeparkar, Reference Anil and Khandeparkar1998). In comparison with the warmer-water barnacle P. pollicipes, the tropical and subtropical C. mitella has a higher and wider temperature tolerance at the time of settlement. Therefore, an adequate range of salinity and temperature for cyprid settlement correlates with the distribution location, habitat environment and lifestyle of barnacle species.
Financial support
This work was financially supported by the cooperation project on production and education of university of Fujian province, China (2018N5007).
