Marine Biology

, Volume 157, Issue 4, pp 899–905

Effects of temperature and food availability on growth and reproduction in the neustonic pedunculate barnacle Lepas anserifera

Original Paper

DOI: 10.1007/s00227-009-1373-0

Cite this article as:
Inatsuchi, A., Yamato, S. & Yusa, Y. Mar Biol (2010) 157: 899. doi:10.1007/s00227-009-1373-0

Abstract

To elucidate the life history of neustonic animals, growth and reproductive patterns were investigated in the hermaphroditic pedunculate barnacle Lepas anserifera in field and laboratory experiments in Wakayama, western Japan from 2006 to 2008. The effects of temperature (19, 24 or 29°C) and food availability (once or twice a week) on growth and reproduction were also studied in the laboratory. The barnacles grew and matured rapidly, especially in the field: individuals on the average grew from 3 mm to more than 12 mm in capitulum length within 15 days and some were brooding. High temperature and high food availability resulted in greater growth. High temperature also resulted in earlier maturation of both testes and ovaries, whereas the effect of food availability was less clear. The rapid growth and maturation, together with earlier maturation at higher temperatures, may be an adaptation to ephemeral floating objects to which they attach.

Introduction

The neuston are organisms that live on or just under the water surface. In marine environments, most of them live in open seas and are difficult to access. Therefore, their ecology has been studied rarely, although they are classified as one of the major categories of marine communities together with the nekton, plankton and benthos. It is believed that the neustonic animals generally grow and mature rapidly since most of them are “obligatory rafters” that utilize ephemeral floating objects (Thiel and Gutow 2004, 2005).

Temperature and food availability are two important factors that are likely to affect various life history traits in many marine invertebrates (for some crustacean examples, see Emmerson 1980; Poleck and Denys 1982; Suprayudi et al. 2002). However, they might have different effects on neustonic animals. The survival of the neustonic animals that rely on ephemeral floating objects is inevitably affected by the duration of floating. Thus, environmental factors that affect the floating duration are more likely to affect the life histories of the neuston than other factors. Natural floating objects such as plant or animal materials are expected to decompose and sink earlier at high temperatures (Thiel and Gutow 2004). On the other hand, food availability is less likely to affect the longevity of the floating objects. Thus, we expect that temperature is more likely to affect the life history traits, especially the age at maturity, than food availability, with higher temperature likely resulting in early maturation.

Species of the pedunculate barnacle Lepas (Cirripedia: Pedunculata) are typical members of the neuston, attaching to floating objects such as driftwood, macroalgae or pumice stone. Their rapid growth (reaching the adult size within 1–2 months) has been inferred from the sizes of individuals attaching to buoys or ships floating for a known period (Evans 1958; Skerman 1958; MacIntyre 1966; Green et al. 1994; Yusa and Abe 1996; Thiel and Gutow 2005). Patel (1959) examined the influence of temperature on molting and reproduction of L. anatifera in short laboratory experiments, but longer-term examinations of growth rates and size-dependent reproduction in Lepas spp. under defined environmental conditions are lacking. Such information is important for understanding the evolution of life histories of marine neuston (Bieri 1966; Thiel and Gutow 2005) and of pedunculate barnacles in general, where only limited information has been available (Kaufmann 1965; Lewis and Chia 1981; Jeffries et al. 1985; Green et al. 1994; Ozaki et al. 2008).

Lepas anserifera is one of the commonest neustonic animals distributed worldwide between 40°N and 40°S (Darwin 1851; Anton 1949; Bieri 1966; Jones 1967; Newman 1971; Yusa and Abe 1996). As in L. anatifera (Patel 1959), fully grown adults are simultaneous hermaphrodites, with well-developed testis and ovary. They can mate as males almost all the time except for just after molting and as females for ~1 day after molting (our personal observations). At the end of the period of mating as females, they lay egg masses inside the mantle cavity, and their ovaries become transparent. Then, they brood the embryos for 4–5 days (at 25°C), after which they release their larvae at the nauplius I stage almost simultaneously with their molts. At that time, their ovaries become fully developed again.

We reared individuals of L. anserifera both in the field and in laboratory to collect information on growth and reproductive patterns in a typical neustonic animal. In particular, we were interested to observe whether they show rapid growth and maturation. Moreover, we reared individuals at different temperatures and food levels in the laboratory, from early juveniles considered to have settled recently (<3 mm in capitulum length) to investigate the effects of temperature and food availability. We hypothesized that higher temperature would result in more rapid maturation both as males and as females than higher food availability.

Materials and methods

Materials

Lepas anserifera used in the laboratory experiment were collected from floating objects such as driftwood or plastic materials around Tanabe Bay, Wakayama, Japan (33°42′N, 135°20′E) in August 2006. The water temperature at the collection was 28°C. Individuals <3 mm in capitulum length were used. They were considered to have settled recently because the cyprid of L. anserifera is ~1.3 mm, and the capitulum grows a maximum of 0.8 mm day−1 in this area in summer (Yusa and Abe 1996). Individuals of 1.2–16.9 mm in capitulum length, collected in the same area in July 2008 (water temperature being 26°C), were used in the field experiment. In both experiments, individuals were reared afloat in the original small groups, attaching to the original substratum. They were individually identified by recording their positions and sometimes with the aid of markings drawn on the shell plates using paint markers. Predators such as the polychaete Amphinome rostrata and the nudibranch Fiona pinnata (Bieri 1966; Thiel and Gutow 2005) were carefully removed before the experiments.

Laboratory experiment

In the laboratory experiment, L. anserifera were reared under four different experimental treatments: 19, 24 and 29°C at a high food level, and 24°C at a low food level, for 50 days from 10 August to 30 September 2006. The three levels of water temperatures approximately correspond to the average temperature in May, and the lowest and the highest temperatures in August in this area, respectively. In this area, L. anserifera do not occur during winter (December–April), and ovigerous individuals are found in June–October (our personal observations). Forty-one or 42 individuals of L. anserifera were randomly assigned to one of the four experimental treatments. At the beginning of the experiment (day 0), there were no significant differences in the capitulum length among different temperature treatments (ANOVA; F2, 101 = 0.056, P = 0.95) or between different food treatments (t-test; df = 68, t = 1.08, P = 0.29). Two 20 l tanks were used for each treatment. Unfiltered running sea water was supplied at a rate of ~300 ml min−1, and the tank water was aerated by air pumps and circulated by water pumps. At first, a fine mesh covered the water outlet of each tank to keep food (hatchlings of the brine shrimp) inside each tank, but it was removed on day 17 due to fouling of the water. After that, there was no apparent water fouling in any tank. The water was kept within ±1°C of the established temperature with a heater and a cooling device, except for the 19°C tank where the water was accidentally lowered to 11°C between days 32 and 34.

Individuals of L. anserifera in each tank were fed with newly hatched larvae of the brine shrimp Artemiasalina (Patel 1959) in proportion to their number and size. Based on results of preliminary feeding experiments, individuals of <5 mm capitulum length were given 25 mg of dry brine shrimp cysts (Tetra Japan, Tokyo) each, and larger ones were given 1 × [capitulum length (mm)]2 mg each, which were much beyond the daily consumption of the barnacles and sufficient for their growth and reproduction. The brine shrimp cysts were directly put into the tank instead of making them hatch in another aquarium since the larvae hatched over several hours in the experimental tank, allowing the barnacles to feed over a long period. Most of the cysts soon floated to the water surface and gathered along the tank wall without being washed away and hatched there. Although gradually decreasing in number, the hatchlings remained in the tank for 1–2 days. To change the food availability, individuals at the high food level were fed twice a week, and those at the low food level were fed once a week. A preliminary experiment using the same lot of the brine shrimp cysts showed that there was no significant difference in the hatching rate among 19, 24 and 29°C (each temperature with three replicates; F2, 8 = 1.34, P = 0.33 in ANOVA).

We measured the capitulum length of each individual and determined the day when the individual showed the first mating behavior as a male and the day when it began to develop the ovary. Capitulum length was defined as the maximum length from the lower edge of the scutum to the upper tip of the tergum. This length was measured to the nearest 0.05 mm by calipers about once a week. The day at first mating as a male was determined by observing individuals for four consecutive days per week. One to five focal individuals were placed, together with a conspecific that was receptive as a female, for 30 min in a shallow aquarium (30 × 20 × 4.5 cm deep) filled with still water 2–3 cm deep from the same rearing tank as the focal individuals. The water temperatures did not change much from the original settings, and no food was given during the observation. All observations were made between 11:00 and 18:00 h. When a focal individual stretched out its penis to the conspecific, it was regarded as being mature as a male. Individuals ≥5 mm in capitulum length were examined up to day 30, and since a 5.00 mm individual showed mating behavior on day 30, after that all individuals were examined except for those already judged as being mature as males. The size at maturity as a male for each individual was defined as the capitulum length on the day nearest to first mating.

Individuals used as female mating partners were chosen from stock individuals. They were collected either at the same time as the experimental individuals or later beached on Banshozaki Cape in September 2006. They were placed in 20 l tanks and fed with brine shrimp larvae given ad libitum. Since individuals of L. anserifera mate as females just after the molt, each stock individual was reared in a cone-shaped mesh net (maximal radius = 32 mm, depth = 80 mm) to check the presence of a molt. Individuals having molted 1 or 2 days before the experiment and having observed to mate as females with other adult individuals were used as female mating partners.

The day at first developing the ovary was determined by observing the peduncle of each individual 4 days a week. When mature, the ovary is orange and visible through the epidermis of the peduncle (Patel 1959). The size at maturity as a female, i.e., capitulum length on the day nearest to the day of first developing an ovary, was determined.

On day 50, all individuals were fixed in ~100% ethanol. They were later dissected and checked for the presence or absence of the brooded embryos under a stereomicroscope.

Field experiment

In the field experiment, driftwood with 78 individuals of L. anserifera was attached to a buoy that was tethered ~10 m off the coast of Banshozaki Cape at the mouth of Tanabe Bay. Natural settlement of L. anserifera and L. anatifera sometimes occurred on this and nearby buoys, but not on the driftwood during the exposure, which lasted 41 days (16 July–27 August 2008). Hourly water temperature during this period, measured at 5 m depth at the mouth of Tanabe Bay, ranged from 23.3 to 29.4°C, with the average being 27.2°C (S. Serizawa, personal communications). The individuals were covered with a net (10 × 8 mm mesh) to protect from large predators. No artificial food was given to the individuals.

Individuals attached to the buoy were collected, brought to the laboratory, and their capitulum lengths were measured by calipers about once a week. At each measurement, ~20% of the individuals were fixed in 10% formalin on and after day 15. The remaining individuals were returned immediately. On day 42, all individuals were fixed in formalin. They were later dissected and checked for the presence or absence of the egg mass or a ripe ovary under a stereomicroscope.

Data and statistical analyses

In the field experiment, data on the individuals that were lost during the experiment were included up to the last census just before their loss, as the major causes appeared to be dislodgement and mechanical damage. In the laboratory experiment, the last data collected on individuals that eventually died were excluded because water fouling (up to day 17) might have affected their growth and reproduction as well as survival.

The effects of temperature (three levels) and food availability (two levels) were tested separately. To secure independence of the data, only final capitulum lengths were statistically compared among treatments in the growth analysis. For statistical analyses of continuous variables, a Student’s t-test (for two levels) or ANOVA (for three levels) was primarily used, with JMP IN 5.1.1 J (SAS Institute Inc.). The data were transformed to the common logarithm when the residuals were not normally distributed. When the transformation did not satisfy normality of the residuals, a Mann–Whitney U-test (for two levels) or Kruskal–Wallis test (for three levels) was used. In these cases, the U or H values were approximated by χ2-distribution (default of JMP). For statistical analyses of proportion data, a likelihood-ratio test was used. In all cases, when there was a significant difference among three levels, either a Tukey’s HSD (for data analyzed by ANOVA) or a Bonferroni correction (for other data) was used as post hoc tests.

Results

Laboratory experiment

Unfortunately, many individuals of Lepas anserifera died in all experimental treatments probably because of water fouling up to day 17 (Table 1). However, only a few individuals died thereafter, and 29–42% of them survived the 50-day observation period. There was no significant difference in the survival rate among different temperature treatments (likelihood-ratio test; df = 2, χ2 = 0.0066, P = 1.00) or between different food treatments (likelihood-ratio test; df = 1, χ2 = 1.52, P = 0.22).
Table 1

Number of Lepas anserifera individuals surviving in the laboratory experiment

Treatment

Days

Survival rate to final day

0

5

15

22

29

36

43

50

29°C (high food)

41

33

27

19

15

14

13

12

0.293 A

24°C (high food)

42

32

18

15

13

13

12

12

0.286 A

24°C (low food)

41

37

25

21

20

18

18

17

0.415

19°C (high food)

41

37

23

18

16

15

12

12

0.293 A

For the effect of temperature, values followed by the same capital letter within a column are not significantly different (likelihood-ratio test with Bonferroni correction)

The capitulum showed an almost steady growth under all treatments (Fig. 1). However, growth rate of the capitulum differed among treatments, and the best growth was attained at 29°C, followed by 24°C at the high food level, 24°C at the low food level, and then 19°C. No significant difference in capitulum length was found between 29 and 24°C (at the high food level) after 50 days, but that of individuals reared at 19°C was significantly smaller than that of those reared at higher temperatures (Fig. 1; Tukey’s HSD). When reared at 24°C, the capitulum lengths of individuals reared at the low food level were significantly shorter than those at the high food level (t-test; df = 27, t = 2.71, P < 0.05).
Fig. 1

Changes in mean ± SE capitulum length of Lepas anserifera individuals reared at different temperatures and food levels. Data points are slightly offset to show SE bars. For the effect of temperature, signs followed by the same capital letter are not significantly different (Tukey’s HSD). For the effect of food level, signs followed by asterisks are significantly different (t-test)

All individuals reared at 29°C matured as males (i.e., showing mating behavior as males) by day 30, while 67% of individuals reared at 24°C at the high food level matured as males in 50 days (Table 2). Only 38% of individuals reared at 19°C matured as males in 50 days. The effect of rearing temperature on the proportion of individuals maturing as males was significant (likelihood-ratio test; df = 2, χ2 = 15.55, P < 0.001). A post hoc test showed that there was no significant difference between 24 and 19°C, but the proportion at 29°C was significantly larger than those in other treatments (Table 2). Likewise, the effect of rearing temperature on the size at maturity among mature individuals was significant (ANOVA; F2, 26 = 4.15, P < 0.05). However, day at maturity as males was not significantly different among 29, 24 and 19°C (Kruskal–Wallis test; df = 2, χ2 = 3.43, P = 0.18).
Table 2

Proportion of individuals matured and size and day at maturity as males in Lepas anserifera

Treatment

Number observed

Number showing male mating behavior

Proportiona

Size as capitulum length in mm (mean ± SE)b

Age in days (mean ± SE)c

29°C (high food)

14

14

1

A

6.61 ± 0.30

AB

29.5 ± 0.5

A

24°C (high food)

12

8

0.67

B

7.44 ± 0.80

A

31.8 ± 3.2

A

24°C (low food)

19

10

0.53

 

5.63 ± 0.46

 

32.8 ± 1.1

 

19°C (high food)

13

5

0.38

B

5.02 ± 0.15

B

38.4 ± 5.1

A

For the effect of temperature, values followed by the same capital letter within a column are not significantly different (a likelihood-ratio test with Bonferroni correction; b Tukey’s HSD; c Kruskal–Wallis test)

The effect of food availability on the proportion of individuals matured as males was not significant (likelihood-ratio test; df = 1, χ2 = 0.60, P = 0.44). Similarly, no significant differences were detected either in size at maturity (t-test; df = 16, t = 2.08, P = 0.054) or day at maturity as males (Mann–Whitney’s U-test; df = 1, χ2 = 0.98, P = 0.32) (Table 2).

No individuals laid eggs within 50 days of rearing. However, individuals maturing as females (i.e., individuals with ripe ovaries) were found for the first time on day 16 at 29 and 24°C at the high food level. All individuals reared at 29°C matured as females up to day 35, whereas only 53% of individuals reared at 24°C at the high food level matured up to day 50 (Table 3). No individuals developed their ovaries at 19°C. The effect of temperature on the proportion of individuals that matured as females was significant (likelihood-ratio test; df = 2, χ2 = 31.95, P < 0.001). Similarly, the day at maturity among mature individuals was greater at 24°C than at 29°C (t-test; df = 17, t = −2.42, P < 0.05), and the size at maturity was larger at 24°C than at 29°C (t-test; df = 17, t = −3.77, P < 0.01).
Table 3

Proportion of individuals matured and size and day at maturity as females in Lepas anserifera

Treatment

Number observed

Number with ripe ovary

Proportiona

Size as capitulum length in mm (mean ± SE)b

Age in days (mean ± SE)c

29°C (high food)

11

11

1

A

6.49 ± 0.41

A

26.9 ± 2.1

A

24°C (high food)

15

8

0.53

B

8.03 ± 0.51

B

37.3 ± 4.1

B

24°C (low food)

21

5

0.24

 

6.55 ± 0.52

 

33.4 ± 3.4

 

19°C (high food)

12

0

0

C

 

 

For the effect of temperature, values followed by the same capital letter within a column are not significantly different (a likelihood-ratio test with Bonferroni correction; bt-test)

Only 24% of the individuals matured as females up to day 50 at the low food level (Table 3). This proportion was lower than that at the high food level, but the difference was not significant (likelihood-ratio test; df = 1, χ2 = 3.31, P = 0.07). Food availability significantly affected the size at maturity (t-test; df = 11, t = 3.54, P < 0.01), but not the day at maturity (t-test; df = 11, t = 0.65, P = 0.53).

Field experiment

The number of individuals that survived decreased at an almost constant rate in the field experiment (Table 4). Excluding individuals dissected to check the reproductive state, 31% of individuals <5 mm in capitulum length at the beginning, and 44% of initially ≥5 mm individuals survived until day 42, respectively (Table 4). Individuals <5 mm long (mean ± SE = 3.09 ± 0.19 mm) grew rapidly, reaching 12.45 ± 0.54 mm on day 15 (Fig. 2). After that, their growth slowed and finally reached 16.26 ± 0.49 mm on day 42. Individuals initially ≥5 mm long showed slower growth, with the final size similar to the initially smaller individuals.
Table 4

Number of Lepas anserifera individuals surviving in the field experiment

Capitulum length at beginning

Days

Natural survival rate to final daya

0

8

15

24

28

37

42

<5 mm

38

20

13 (3)

9 (1)

8 (1)

7 (2)

5 (5)

0.31

≥5 mm

40

33

33 (8)

23 (5)

18 (3)

10 (2)

7 (7)

0.44

The number of dissected individuals is shown in parenthesis

aExcluding dissected individuals, calculated by multiplying survival rates between days 0 and 8, between days 8 and 15, etc

Fig. 2

Changes in mean ± SE capitulum length of Lepas anserifera individuals in the field experiment. Data points are slightly offset to show SE bars. Numerals besides the plot represent the numbers of individuals examined

Ovigerous individuals were already found on day 15 in those initially <5 mm as well as ≥5 mm long (Table 5). By the end of the experiment, most individuals either had brooded or had a ripe ovary, irrespective of the initial size.
Table 5

Reproductive states of Lepas anserifera individuals dissected during the field experiment

Capitulum length at beginning

Reproductive state

Days

15

24

28

37

42

<5 mm

Immature

     

Ripe ovary

2

   

2

Brooding

1

1

1

2

3

≥5 mm

Immature

 

1

  

1

Ripe ovary

1

2

  

3

Brooding

7

2

3

2

3

Discussion

The laboratory experiment showed that Lepas anserifera can survive between 19 and 29°C. In addition, few barnacles died when the temperature of the tanks set at 19°C was accidentally lowered to 11°C, indicating that L. anserifera can survive temporarily at this low temperature. The surface temperature in the areas where L. anserifera distributes (between 40°N and 40°S) rarely drops below 10°C in winter, or rises above 30°C in summer (National Astronomical Observatory of Japan 2007). Therefore, the temperature tolerance of L. anserifera revealed in this study is within the normal range they experience in the field. In the temperate species L. anatifera, Patel (1959) showed that all individuals died at 34–36°C and none reproduced at 30°C. In this study, L. anserifera reproduced at water temperatures that averaged 27.2°C in the field, and individuals reared at 29°C in the laboratory developed ovaries. Lepas anserifera may, therefore, be more tolerant of high temperatures than L. anatifera.

The field experiment confirmed previous reports on the rapid growth and early maturation in L. anserifera (Hoek 1907; Evans 1958; Yusa and Abe 1996) as well as in other Lepas spp. (Anton 1949; MacIntyre 1966; Green et al. 1994; Yusa and Abe 1996; Tsikhon-Lukanina et al. 2001; Thiel and Gutow 2005). Other pedunculates, however, generally show slower growth and maturation (e.g., reaching 17 mm within 1 year in the littoral pedunculate Pollicipes polymerus: Lewis and Chia 1981; becoming ovigerous within 170 days in the deep-sea pedunculate Poecilasma kaempferi: Green et al. 1994). On the other hand, Octolasmis cor, living on the gills of large crabs, became ovigerous within 15 days after settlement (Jeffries et al. 1985). The rapid growth and maturation in Lepas spp., as well as those of the symbiotic pedunculates such as O. cor, appear to be an adaptation to the ephemeral nature of substrata that they utilize.

In the laboratory experiment growth and maturation was delayed compared with the field experiment. Barnacles reared at either 29 or 24°C in the laboratory grew more slowly than in the field (average 27.2°C). The amount of food given each time was sufficient for daily consumption in the laboratory experiment. However, factors such as low frequency (twice a week even at the high food level), lack of food variety (only brine shrimp hatchlings were given, although smaller organisms such as ciliates might have served as additional food items especially for small individuals) or reduced food intake (due to the lack of wave action) might be responsible for the slower growth in the laboratory. Nevertheless, the general growth and reproductive patterns appeared to be normal in the laboratory.

In the laboratory experiment, maturation as males was earlier than that as females, either in terms of ovarian development or egg laying. In other words, they have a tendency of protandric simultaneous hermaphroditism (Bauer 2006), first mature as males and then become simultaneous hermaphrodites. Earlier maturation as males than as females has been reported in other barnacles (Chelonibia patula: Crisp 1983; Poecilasma kaempferi: Green et al. 1994; Chthamalus malayensis: Yan et al. 2006). This is congruent with a general prediction of sex allocation theories in hermaphrodites that small individuals should first function as males, rather than as females, when mating in limited, often small groups such as in barnacles (Charnov 1987; Angeloni et al. 2002; Yamaguchi et al. 2008).

Judging from the proportion of mature individuals in the different treatments, low temperature delayed the maturation of both male and female functions. This tendency was not reflected in the days to maturity as males, but the proportion of individuals maturing may be a better index of maturity in the population than the number of days as several individuals did not reach maturity and hence were excluded to calculate the days. In the same way, size at maturity was also affected by the proportion of maturing individuals. Thus, although the size at maturity as females was larger at 24°C than 29°C, this was affected by the fact that several small, immature individuals were excluded from the analysis in the 24°C treatment.

Water temperature is likely to affect how long floating objects remain near the ocean surface (Thiel and Gutow 2004), whereas food availability is not. In addition, age at maturity under various environmental conditions is generally considered to be an adaptive life history trait (Stearns and Koella 1986). Therefore, earlier maturation at high temperatures is likely to have evolved in response to the ephemeral nature of objects to which L. anserifera attaches. However, other reasons for rapid growth at high temperatures (e.g., high metabolic rates) are possible, and we must also consider the possibility that more extreme food conditions (but those encountered in nature) might have lead to clearer responses to food availability. Further study is necessary to fully elucidate the life history patterns and their adaptive significance in L. anserifera. Also, it would be interesting to study if other neustonic pedunculates such as Dosimafascicularis show similar responses to temperature and food availability, because they can make floats themselves (Darwin 1851) and do not rely on the longevity of floating objects.

Acknowledgments

We thank K. Wada, H. Sato, the editor and reviewers for their valuable comments, Y. Yamamoto, K. Okita, S. Urano, Y. Kitano, M. Matsumura and S. Serizawa for technical advice and help. The authors declare that they have no conflict of interest.

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Ayano Inatsuchi
    • 1
  • Shigeyuki Yamato
    • 2
  • Yoichi Yusa
    • 1
  1. 1.Faculty of ScienceNara Women’s UniversityNaraJapan
  2. 2.Seto Marine Biological LaboratoryKyoto UniversityWakayamaJapan

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