Plastic sexual expression in the androdioecious barnacle Octolasmis warwickii (Cirripedia: pedunculata).
Barnacles (Crustacea: Thoracica) offer an ideal ground for considering the evolution of reproductive strategies. Darwin (1851) found that barnacles exhibit a diverse array of sexual systems, including simultaneous hermaphroditism, dioecy (coexistence of females and males in a population), and androdioecy (hermaphrodites and males). In both dioecious and androdioecious barnacles, males are always tiny and live attached to conspecific females or hermaphrodites; hence, they are known as dwarf males (see Hoeg, 1995 and Yusa et al., 2010, 2012 for terminology). Compared with hermaphrodites, dwarf males are considered to mature earlier and emphasize male function. Thus, they may increase survivorship to maturity, and may fertilize eggs more efficiently than hermaphrodites, because they are nearer to the fertilization site (Crisp, 1983; Foster, 1983; Yusa et al., 2010; Yamaguchi et al., 2012).
The adaptive significance of this diverse sexual system has been studied both theoretically (Charnov, 1982, 1987; Yamaguchi et al., 2008, 2012, 2013a, b, c) and empirically (Kelly and Sanford, 2010; Yusa et al., 2012; Lin et al., 2015). These studies suggest that the diverse sexual systems of barnacles are an adaptation to mating opportunities. Mating opportunities are constrained by certain environments, such as deep seas or epibiotic habitat. In contrast, little is known about the ecological mechanisms maintaining the sexual systems of barnacles. The mechanisms that allow the persistence of androdioecy (i.e., how hermaphrodite and male sexes are determined) are especially important, because 1) androdioecy is an evolutionary transition phase between hermaphroditism and dioecy (Weeks et al., 2006; Yusa et al., 2012), and 2) this sexual system is believed to be extremely rare in nature (Charlesworth, 1984; Pannell, 2002; Weeks et al., 2006).
Two general hypotheses have been suggested in the determination of hermaphrodite and male sexes in androdioecious barnacles. Gomez (1975) theorized that sexual expression in the balanomorphan barnacle Conopea galeata (Linnaeus, 1771) is genetically determined, because the sex ratio of newly settled individuals is constant (hermaphrodites: dwarf males = 3:1). Callan (1941) suggested that sex determination is environmentally controlled in the pedunculate barnacle Scalpellum scalpellum (Linnaeus, 1767), based on its apparent lack of sex chromosomes. Even a mix of these two mechanisms has been suggested. In S. scalpellum, Svane (1986), in a settlement experiment, found that the proportion of males varies but never exceeds 0.5. Based on this discovery, he suggested that sexual expression is influenced by both genetic and environmental factors in a unique way: one type of larvae is destined to become hermaphrodites, and the other type can choose between male and hermaphroditic expression according to the environment.
Transplanting experiments using newly settled individuals have studied the environmental effects on sexual expression of hermaphroditic barnacles. Yamaguchi et al. (2014) conducted a transplanting experiment in the pedunculate barnacle Poecilasma kaempferi Darwin, 1851, by gluing small individuals to large individuals to study the possible shift in sexual expression of the small animals. They found that the sexual expression of conspecific-attached individuals did not differ from the control individuals that were glued to plastic sticks. Yusa et al. (2013) carried out a similar experiment in the pedunculate barnacle Octolasmis angulata (Aurivillius, 1894). Interestingly, conspecific-attached individuals in that study developed penises earlier, such as dwarf males, than did the control individuals, although dwarf males are not known to exist in O. angulata. These studies suggest that plasticity of sexual expression may differ among species. Except for a one-directional transplanting experiment in conspecific-attached individuals that were experimentally detached and transferred to the petri dish (Hoeg et al., 2015), no study has evaluated the degree of plasticity of sexual expression in androdioecious barnacles.
The androdioecious barnacle Octolasmis warwickii Gray, 1825 has two types of sexual expression (Yusa et al., 2010): 1) as a hermaphrodite, attached directly to a host crab (mainly of the family Portunidae), or 2) as a dwarf male, attached to the scutum of a large hermaphrodite, and maturing as male at smaller sizes than the hermaphrodites. Some "dwarf males" may later become hermaphroditic, but the proportion is small (Yusa et al., 2010). The purpose of this study was to reveal the phenotypic plasticity of sexual expression in this androdioecious barnacle using bidirectional transplanting experiments.
Materials and Methods
Individuals of Octolasmis warwickii were collected from host crabs Portunus pelagicus (Linnaeus, 1758) and Cha rybdis japonica (A. Milne-Edwards, 1861), which were purchased live at a local fish market in Izumisano, Osaka, during regular visits (once or twice a week) from autumn to spring, 2013-2015. In the laboratory, the capitulum lengths of the barnacles were measured to the nearest 0.01 mm under a binocular microscope. Forty individuals smaller than 2.5 mm in capitulum length, which is still immature, were used as "treatment" individuals; 20 individuals larger than 2.5 mm were used as "substrate" individuals, and served as an attachment site for some of the treatment individuals. Irrespective of collection efforts, a low prevalence rate did not permit the use of additional samples for the experiments.
The experiments were conducted when at least one treatment individual was obtained. All of the individuals were carefully detached from the original substrate using fine forceps. Based on the original attachment site, individuals were categorized as either conspecific-attached (attached to conspecific individuals; i. e., juvenile dwarf males) or independent (directly attached to the host crab; i.e., juvenile hermaphrodites). Similarly, there were two treatments in the transplanting experiments regardless of the original attachment sites: conspecific-attached or independent. In the conspecific-attached treatment, the base of the peduncle of each individual was glued to the scutum of a larger conspecific. The larger conspecific was then glued to a plastic stick (1 X 5 X 80 mm long) using a cyanoacrylate adhesive (Toagosei, Tokyo). In the independent treatment, the base of the peduncle was glued directly onto a plastic stick. Therefore, the transplanting was conducted in 4 patterns: 1) from conspecific-attached to conspecific-attached (c-c); 2) from conspecific-attached to independent (c-i); 3) from independent to conspecific-attached (i-c); and 4) from independent to independent (i-i). In nature, barnacles cement themselves to the substrate; thus, experimental detachment and gluing have no apparent side effects (Yusa et al., 2013; Yamaguchi et al., 2014).
The sticks with the glued barnacles were placed at > 5-cm intervals in an aquarium (32 X 19 X 26 cm deep) containing 36 1 of artificial seawater (Premium Salt; Gex, Osaka), which was circulated by air pump and maintained at 21-24 [degrees]C for 22 days. The experimental duration was based on similar experiments conducted in other pedunculate barnacles (Yusa et al., 2013; Yamaguchi et al., 2014), and was sufficient for newly settled individuals to become mature as dwarf males in this species. Barnacles were fed newly hatched larvae (from 1 g of dry cysts) of the brine shrimp Artemia salina (Linnaeus, 1758) (Japan Pet Design, Tokyo) and bread yeast (1 g) (Nisshin Food Company, Tokyo) every 2 days. The aquarium seawater was changed when it looked fouled, even in mid-experiment.
After the experiment, the capitulum length of each barnacle was measured, and all barnacles were fixed with 100% ethanol. Later, they were dissected, during which time the presence or absence of egg masses was checked, and penis length was measured to the nearest 0.01 mm, using a binocular microscope. The growth rate was calculated in daily increments (final capitulum length minus initial capitulum length)/22 days.
For statistical analysis, a generalized linear model with a model selection procedure was used. Initially, original attachment site (independent or conspecific-attached), treatment (independent or conspecific-attached as a result of transplanting), interaction term, and final capitulum length were included as explanatory variables. The response variable was either survival (as a binomial of dead or alive), penis length, growth rate, or presence or absence of an egg mass. The explanatory variables then were sequentially omitted, and the model with the lowest corrected Akaike's Information Criterion (AICc) value was selected as the best model (Burnham et al., 2011). All statistical tests were conducted using JMP version 9 software (SAS, Cary, NC).
The survival rate of Octolasmis warwickii over 22 days was 45% (18/40 individuals). The best model with the lowest AICc was the model with only the intercept, which showed no effects of treatment or other factors on survival.
Concerning male function, the best model indicated that both original site (F = 6.49, P = 0.022) and treatment (F = 32.8, P < 0.001) affected penis length. Specifically, individuals that were transplanted onto a conspecific developed longer penises than did those that were transplanted as independent individuals. Conspecific-attached individuals that were transplanted onto conspecific individuals (c-c) had the longest penises, followed by the i-c individuals, the c-i individuals, and the i-i individuals (Fig. 1). The best model did not include final capitulum length, indicating that body size did not affect penis length within the size range of the experimental individuals.
As for female function, only one (i-c) individual was carrying eggs (n = 5 eggs) in the mantle cavity at the end of the experiment. The best model revealed that capitulum length was chosen as the only explanatory variable, but it had no significant effect on the presence of eggs (likelihood ratio x2 = 2.96, P = 0.09). Original site and treatment variables were not selected in the best model.
The best model on growth rate indicated that both treatment (F = 5.62, P = 0.032) and final capitulum length (F = 43.47, P < 0.001) affected growth. Growth rate was lower in individuals experimentally attached to conspecifics than it was in those directly attached to the substrate (Fig. 2). Growth rate was also lower in the smaller than the larger barnacles.
In this study, the effects of the original attachment site and the transplanted site on penis length, egg production, and growth were investigated in the androdioecious barnacle Octolasmis warwickii. Penis length was greater in the originally conspecific-attached individuals than in the independent individuals. The effect of the original site was expected, because O. warwickii is an androdioecious species, and individuals attached to conspecifics develop male function at a smaller size to become dwarf males (Yusa et al., 2010). Similarly, penis development at smaller sizes in conspecific-attached dwarf males than in independent hermaphrodites has been reported in the congener Octolasmis unguisiformis Kobayashi & Kato, 2003 (Sawada et al., 2015), the pedunculate Alepas pacifica Pilsbry, 1907 (Yusa et al., 2015), and the acorn barnacle Chelonibia patula (Ranzani, 1818) (Crisp, 1983).
The penis was also better developed in the experimentally conspecific-attached individuals than in the independent ones. The effect of the treatment on penis length indicated that hermaphroditic and dwarf male sex expressions are influenced by the environment. Therefore, sexual expression is plastic in Octolasmis warwickii. Likewise, when newly metamorphosed individuals of the pedunculate barnacle Scalpellum scalpellum settled on conspecific hermaphrodites are removed and transferred to a dish, several become hermaphrodites instead of dwarf males (Hoeg et al, 2015).
The egg laying observed in the single i-c individual does not support the prediction that conspecific attachment emphasizes male over female function. However, the egglaying individual was originally an independent, and it would have become a hermaphrodite if we had not changed its position. Hence, the result simply may have reflected the effect of the original attachment site. However, since only one individual laid eggs in this experiment, more data are required to accurately evaluate the effect of original and transplanted sites on female function.
This result also supports the prediction that conspecific-attached individuals grow less than their independent counterparts, because individuals that are attached to the scutum of hermaphrodites should emphasize male function for fertilizing the eggs of the hermaphrodites. The conspecific-attached male also should not grow so large that its mantle aperture is in contact with that of the hermaphrodite. In contrast, independent individuals need to allocate more resources to growth for future reproduction as females, because larger individuals generally tend to carry more eggs in pedunculate barnacles. These include, for example, Lepas anatifera Linnaeus, 1758 (Zann and Harker, 1978), Koleolepas avis (Hiro, 1931) (Yusa et al., 2001), and Scalpellum stearnsi Pilsbry, 1890 (Ozaki et al., 2008).
The results of this study suggest that sexual expression is altered by the environment as a result of changes in allocation of resources to growth and male function (Yusa et al., 2013). Overall, the change in allocation agrees with the predictions made in theoretical work (Charnov, 1982, 1987; Yamaguchi et al., 2008, 2012, 2013a, b, c): that individuals should stress their male allocation when living on a conspecific, and allocate more resources to growth when they live independently (Yusa et al., 2013). However, genetic effects on sex determination cannot be ruled out in our study, because the effect of the original site on penis length was detected. Additional study is needed, such as with smaller individuals, to distinguish the genetic component and post-settlement environmental effects.
It is hypothesized that individuals adjust their sexual expression according to the environment rather than remain genetically fixed if they confront highly variable and unpredictable environments such as those that affect food, survival, or the presence and number of mating partners (Kelly and Sanford, 2010; Yusa et al., 2013; Yamaguchi et al., 2013a, b, c). In several androdioecious barnacles, including Octolasmis warwickii, this theory appears to be the case, as evidenced by their variable adult sizes and mating opportunities according to attachment site (Yusa et al., 2010; Ewers-Saucedo et al., 2015). However, information on male and female reproduction success is still fragmentary, and the survival rate under natural conditions has rarely been estimated in androdioecious barnacles (but see Ewers-Saucedo et al., 2015). A more detailed study evaluating these fitness components is required.
We thank Drs. Keiji Wada, Hiroaki Sato, and Jens T. Hoeg for their constructive discussion and comments. We are also grateful to the people at the Izumisano Fish Market for their help in obtaining the materials, and to the members of our laboratory for their support and encouragement. This work was supported by JSPS Kakenhi (grant no. 15H04416).
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HENDRY WIJAYANTI AND YOICHI YUSA (*)
Faculty of Science, Nara Women's University, Kitauoya-nishi, Nara 630-8506, Japan
Received 10 September 2015; accepted 9 December 2015.
(*) To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
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|Author:||Wijayanti, Hendry; Yusa, Yoichi|
|Publication:||The Biological Bulletin|
|Date:||Feb 1, 2016|
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