From sneaky to bully: reappraisal of male squid dimorphism indicates ontogenetic mating tactics and striking ejaculate transition

From sneaky to bully: reappraisal of male squid dimorphism indicates ontogenetic mating tactics... Abstract The expression of alternative reproductive tactics (ARTs) is often influenced by the environmental cues and intrinsic conditions experienced by each male, and it may be reversible or permanent. In male loliginid squid, ARTs include consorts and sneakers, which differ in behaviour, body size, sperm deposition site, and morphology and functioning of ejaculates. Here, we describe intermediate-sized males in the squid Doryteuthis plei (Blainville, 1823) that produce sneaker-like, consort-like and intermediate ejaculates. In addition, age estimates show that sneakers and intermediate males are younger than consorts. Our findings suggest that ARTs in this species may be ontogenetically expressed. Such a transition between phenotypes requires dramatic anatomical and physiological reorganization of the male reproductive system, not only to produce different-structured spermatophores, but also to generate sperm with distinct behaviour, both of which are compatible with the switch in sperm deposition site. These findings demonstrate that this peculiar mating system may also fit the current predictions of ARTs. INTRODUCTION Sexual selection promotes high variance in male reproductive success (Shuster & Wade, 2003). Under strong competition, males less successful in fighting with dominant males may pursue alternative reproductive tactics (ARTs) to maximize fertilization success (Taborsky, Oliveira & Brockmann, 2008). Such male ARTs often involve not only discontinuous mating behaviour, but also distinct life histories (Gross, 1991), and discontinuous physiological and morphological traits (Moczek & Emlen, 1999), a phenomenon known as intrasexual male dimorphism (Gadgil, 1972). Discontinuous reproductive phenotypes may result from polymorphic genotypes or, more commonly, from polyphenism (Taborsky et al., 2008). In polyphenic species, all males are able to express both phenotypes, morph determination being ultimately influenced by environmental cues and intrinsic conditions experienced by each male (Gross, 1996; Tomkins & Hazel, 2007). Males express the phenotype that maximizes their fitness, which will depend on males’ relative status (e.g. size, age). Males that reach a critical status switch point (e.g. males that grow larger than a critical body size) have higher reproductive success by expressing dominant tactics, whereas those below it have higher fitness pay-offs by adopting sneaking tactics (Gross, 1996). Adoption of conditional ARTs may be irreversible or reversible in life (Taborsky et al., 2008). In horn beetles, morph determination is triggered by resource availability during larval development and it is permanent in the adult. Males above a certain body-size switch point develop horns and adopt dominant tactics, whereas those that do not reach the switch point lack horns and become sneakers (Moczek & Emlen, 1999). In some fishes, morph expression depends on juvenile growth rate. Fast-growing males mature precociously at smaller sizes and become sneakers, whereas slow-growing males delay sexual maturity and employ dominant tactics at larger sizes later in life (Gross, 1984, 1991). In reversible tactics, males may alternate between tactics multiple times by adaptively responding to current conditions and employing different strategies accordingly (Taborsky, 1994; Fischer & Fiedler, 2001). Alternating between tactics may also be ontogenetic, when individuals change tactics only once in life (Mazzoldi et al., 2000; Bleeker et al., 2017). In fishes, ontogenetic tactics are often associated with their indeterminate growth, young and small males benefiting from sneaking until they become older and larger, when it pays to shift to dominant roles (Taborsky, 1994; Bleeker et al., 2017). In loliginid squid, different sized males often adopt ARTs (Hanlon, 1996). Large (consort) males fight with rivals, guard females and mate with females in male–parallel position, depositing spermatophores near the oviduct opening (Fig. 1D, E). Small (sneaker) males, in turn, avoid fighting contests, do not guard females and adopt head-to-head mating, placing spermatophores near the seminal receptacle, below the mouth (Fig. 1A–C; Hanlon, 1998). The presence of two spermatophore attachment sites on the female is thought to promote differences in fertilization timing and success between male morphs. Consort sperm from spermatangia attached near the oviduct opening meet the eggs first as they leave the oviduct (Fig. 1E, G), whereas sneaker sperm only meet the eggs when the female holds the egg capsule near the mouth, before depositing it on the substrate (Fig. 1B, G). Thus, sneakers are assumed to have lower fertilization success than consorts (Iwata, Munehara & Sakurai, 2005; Shashar & Hanlon, 2013), as there are probably fewer unfertilized eggs available to sperm near the seminal receptacle (Hanlon, Maxwell & Shashar, 1997; Buresch et al., 2009). Figure 1. View largeDownload slide Schematic representation of the complex mating system of loliginid squids, which includes alternative mating tactics and distinct spermatophore attachment sites within females. (A) Small sneaker males mate with females in the head-to-head position and place spermatophores near their seminal receptacle. (B) Detail of the female buccal membrane (frontal view). The seminal receptacle (blue star) is located below the mouth, between the left and right ventral arms. (C) Detail of the seminal receptacle (sagittal section) and club-like spermatangia from sneaker males. Sneaker sperm aggregate as a round mass around the spermatangium tip after being released. (D) Large consort males mate with females in the male-parallel position and place spermatophores near the oviduct opening. Consort males may exhibit visual signalling in agonistic contests with rival males (in this case, the ‘lateral flame’, i.e. the flame-like streaks seen unilaterally in each consort male of the figure). (E) Detail of the dissected mantle cavity of females. Consort spermatangia are attached near the oviduct opening (green star). (F) Detail of hook-like spermatangia of consort males attached in the female. In contrast to sneaker sperm, consort sperm rapidly diffuse after release. (G) During egg-laying, consort sperm from spermatangia attached near the oviduct opening (green star) meet the eggs first, as they leave the oviduct, whereas sneaker sperm only meet the eggs when the egg capsule is near the mouth (blue star), before being deposited on the substrate. Figure 1. View largeDownload slide Schematic representation of the complex mating system of loliginid squids, which includes alternative mating tactics and distinct spermatophore attachment sites within females. (A) Small sneaker males mate with females in the head-to-head position and place spermatophores near their seminal receptacle. (B) Detail of the female buccal membrane (frontal view). The seminal receptacle (blue star) is located below the mouth, between the left and right ventral arms. (C) Detail of the seminal receptacle (sagittal section) and club-like spermatangia from sneaker males. Sneaker sperm aggregate as a round mass around the spermatangium tip after being released. (D) Large consort males mate with females in the male-parallel position and place spermatophores near the oviduct opening. Consort males may exhibit visual signalling in agonistic contests with rival males (in this case, the ‘lateral flame’, i.e. the flame-like streaks seen unilaterally in each consort male of the figure). (E) Detail of the dissected mantle cavity of females. Consort spermatangia are attached near the oviduct opening (green star). (F) Detail of hook-like spermatangia of consort males attached in the female. In contrast to sneaker sperm, consort sperm rapidly diffuse after release. (G) During egg-laying, consort sperm from spermatangia attached near the oviduct opening (green star) meet the eggs first, as they leave the oviduct, whereas sneaker sperm only meet the eggs when the egg capsule is near the mouth (blue star), before being deposited on the substrate. In at least two loliginid species (Doryteuthis plei and Heterololigo bleekeri), size-dependent ARTs are also associated with intrasexual dimorphism between sneakers and consorts, including differences in sperm size and behaviour, sperm release strategies, and spermatophore and spermatangium (i.e. everted spermatophore) morphology and functioning (Iwata & Sakurai, 2007; Iwata et al., 2011; Hirohashi & Iwata, 2014; Apostólico & Marian, 2017a, b). For example, sneakers produce small club-like spermatangia and sperm with a clustering behaviour (Fig. 1C), whereas consorts produce large hook-like spermatangia and diffuse rather than aggregative sperm (Fig. 1F). Such profound differences in ejaculate traits have been attributed to the intrinsic characteristics of each sperm deposition site (Hirohashi & Iwata, 2014; Hirohashi et al., 2016; Apostólico & Marian, 2017a, b). Although ARTs are widespread in animals and the mechanisms responsible for their expression have been extensively investigated (Oliveira, Taborsky & Brockmann, 2008), such mechanisms are completely unknown in squids. Squids offer an interesting model for studying ARTs, given the presence of distinct fertilization sites associated with ARTs (Fig. 1), a peculiar condition compared to the model animals generally used to study ARTs. Previously, we demonstrated the occurrence of intrasexual dimorphism in the squid D. plei (Blainville, 1823), showing that sneakers and consorts differ in spermatangia (Fig. 1C, F) and spermatophore morphology and also in gonadal investment (Apostólico & Marian, 2017a). We subsequently used preserved material and videos of in vitro experiments from the same sampling to describe and compare the fine structure and functioning of sneaker and consort ejaculates, showing how differences in spermatophore structure lead to the peculiar dimorphism observed in spermatangia morphology (Apostólico & Marian, 2017b). While further analysing the preserved material and videos from the same sampling, we found five intermediate sized males of D. plei that stored both sneaker-like and consort-like spermatophores simultaneously within the spermatophoric sac. In these previous papers (Apostólico & Marian, 2017a, 2017b), four of the five males were classified as sneakers and one as a consort, based on their body size and on the switch point previously described by Apostólico & Marian (2017a). At first, these cases were treated as probable abnormalities, but further detailed analyses revealed them to be indicative that ARTs in this species may be ontogenetically expressed. Here, we describe in detail the morphology and in vitro functioning of spermatophores from these ‘intermediate’ males. We show that they produce not only sneaker-like and consort-like ejaculates, but also structures with intermediate features between the two morphs, suggesting a sequential production (i.e. from sneaker to consort spermatophores). We also show that sneakers and intermediate males are younger than consorts based on the statoliths extracted during the original sampling. Our findings suggest that expression of ARTs in this species may change with ontogeny, and lead to a dramatic ejaculate transition between morphs that is consistent with change in sperm deposition site. MATERIAL AND METHODS Data collection As detailed elsewhere (Apostólico & Marian, 2017a), we collected 287 mature males of D. plei (88–327 mm in mantle length, ML) from December to February 2014–2016, off São Sebastião Island (São Paulo, Brazil). We classified males as ‘sneakers’ (N = 73; 88–169 mm ML) or ‘consorts’ (N = 209; 170–327 mm ML) based on spermatophore and spermatangia morphology (Apostólico & Marian, 2017a), except for males later revealed to have both sneaker-like and consort-like spermatophores (N = 5; 146, 149, 159, 166 and 176 mm ML), classified herein as ‘intermediate’ males. Spermatophore morphology and in vitro experiments To confirm if sneaker-like and consort-like spermatophores of intermediate males were indeed similar to those of sneakers and consorts, respectively, we sectioned and stained spermatophores of intermediate males, which were preserved in Karnovsky’s fixative (Marian, 2012a) during the original sampling. Light microscopy followed the procedures of Apostólico & Marian (2017b). Based on videos from the original sampling (Apostólico & Marian, 2017b), we also analysed the behaviour of fresh spermatophores from the five intermediate males (including nine sneaker-like, 14 consort-like and 25 intermediate spermatophores) during the spermatophoric reaction (i.e. spermatophore eversion, leading to formation and attachment of the spermatangium; Marian, 2012b). Age estimation We selected 250 males (175 consorts, 70 sneakers, five intermediate males) for age estimates based on statolith growth increments. During the original sampling, we dissected statoliths from fresh animals and fixed them in 70% ethanol for future use. Later, we ground and polished them following the methods of Perez, Aguiar & Santos (2006). We examined statoliths under an Axio Imager M2 Zeiss microscope (1000× magnification, immersion oil) and performed three blind counts of growth increments by a single reader. For each male, we determined age as the mean total number of increments counted, assuming that growth-ring deposition occurs daily in this species (Perez et al., 2006). For details of this method, see Supporting Information S1. Statistical analysis For age estimates, we tested data for normality with the Shapiro–Wilk test and for variance homogeneity with Bartlett’s test. We performed one-way ANOVA, followed by Tukey’s honest significant difference post-hoc analysis, to test for significant differences between sneaker, intermediate and consort males. We also tested the accuracy of blind counts with two indices, the average percentage error (APE) (Beamish & Fournier, 1981), and the coefficient of variation (CV) (Chang, 1982). Acceptable levels of precision for both indices have been conventionally established as values less than 10% (e.g. Schwarz & Perez, 2010). We conducted statistical analyses in R 3.3.2 (R Development Core Team, 2008), and set up the level of significance as P ≤ 0.05 for all analyses. RESULTS Spermatophore morphology Typical sneakers and consorts of D. plei produce only spermatophores with exclusive morphologies (see Apostólico & Marian, 2017b). Intermediate males, however, simultaneously store spermatophores of both types (sneaker-like and consort-like), and also spermatophores with intermediate features of both morphs (hereafter called ‘intermediate spermatophores’) (Fig. 2; see also Supporting Information S2). Sneaker-like spermatophores had a short sperm mass (Fig. 2A), an ejaculatory apparatus tube with a looser arrangement and incomplete loops (Fig. 2B), and a cement body with a cylindrical oral region, a constricted intermediate region and an ellipsoidal aboral region (Fig. 2G). In turn, consort-like spermatophores had a long sperm mass (Fig. 2A), an ejaculatory apparatus tube more convoluted and tightly arranged (Fig. 2F), and a cement body with a wide and conically shaped oral region, a broad intermediate region and a cylindrical aboral region (Fig. 2K). Both morphologies were identical to those of sneakers and consorts (see Apostólico & Marian, 2017b). Also, histological methods (Supporting Information S3) showed that sneaker-like and consort-like spermatophores within the same intermediate male had identical characteristics as regards tunics, membranes and staining affinities to those described for sneakers and consorts, respectively (see Apostólico & Marian, 2017b). Figure 2. View largeDownload slide Spermatophores of ‘intermediate’ males of Doryteuthis plei. All spermatophores were sampled from the same male. (A) Whole spermatophores, showing the transition from typical sneaker-like (top) to typical consort-like structure (bottom). Intermediate morphologies (centrally) encompass a gradual modification on the oral region (i.e. cement body and ejaculatory apparatus), followed by increase in sperm mass. (B–F) Detail of the oral region of spermatophores from A. Notice that the ejaculatory apparatus tube and the cement body gradually change from a sneaker-like (B) to a consort-like morphology (F), with intermediate configurations in between (C–E). (G–K) Detail of the tripartite cement body of the spermatophores from A. Notice the morphological transition from a sneaker-like (G) to a consort-like morphology (K), with intermediate forms in between (H–J). Abbreviations: cb, cement body; cba, cement body aboral region; cbi, cement body intermediate region; cbo, cement body oral region; ea, ejaculatory apparatus; sm, sperm mass. Figure 2. View largeDownload slide Spermatophores of ‘intermediate’ males of Doryteuthis plei. All spermatophores were sampled from the same male. (A) Whole spermatophores, showing the transition from typical sneaker-like (top) to typical consort-like structure (bottom). Intermediate morphologies (centrally) encompass a gradual modification on the oral region (i.e. cement body and ejaculatory apparatus), followed by increase in sperm mass. (B–F) Detail of the oral region of spermatophores from A. Notice that the ejaculatory apparatus tube and the cement body gradually change from a sneaker-like (B) to a consort-like morphology (F), with intermediate configurations in between (C–E). (G–K) Detail of the tripartite cement body of the spermatophores from A. Notice the morphological transition from a sneaker-like (G) to a consort-like morphology (K), with intermediate forms in between (H–J). Abbreviations: cb, cement body; cba, cement body aboral region; cbi, cement body intermediate region; cbo, cement body oral region; ea, ejaculatory apparatus; sm, sperm mass. Meanwhile, intermediate spermatophores had heterogeneous combinations of the two morphologies. They generally had sneaker-like short sperm mass (Fig. 2A), whereas the ejaculatory apparatus and cement body were more variable. While the ejaculatory apparatus tube gradually became more convoluted and with a higher number of complete loops (Fig. 2C–E), the three cement body regions resembled intermediate shapes ranging from typical sneaker-like to a consort-like format (Fig. 2H–J). Further analysis of the spermatophoric sac of one intermediate male also revealed that spermatophores with distinct morphologies were spatially separated within the organ: sneaker-like and consort-like spermatophores were located more anteriorly and more posteriorly in the organ, respectively, while intermediate spermatophores occupied the medial region. This indicates that sneaker-like spermatophores were older than consort-like spermatophores. Spermatophoric reaction The spermatophoric reaction encompasses several stages of spermatophore eversion that ultimately result in the formation of the spermatangium and, subsequently, in sperm release (Fig. 3). In intermediate males, sneaker-like spermatophores (Fig. 2A, B, G) always produced club-like spermatangia (Figs 3A, 4A, left). Consort-like spermatophores (Fig. 2A, F, K), by contrast, always produced hook-like spermatangia (Figs 3B, 4A, right). Such patterns were identical to the spermatophoric reaction and spermatangia formation from typical sneakers and consorts in D. plei (see Apostólico & Marian, 2017b). Moreover, in sneaker-like spermatophores, released sperm aggregated in a spherical mass near the spermatangium (Figs 3A, 4B), while consort-like spermatangia showed intense sperm diffusion (Figs 3B, 4D), also consistent with the typical behaviour of sneaker and consort morphs (see Apostólico & Marian, 2017b). Figure 3. View largeDownload slide Schematic representation and images of the spermatophoric reaction in sneakers, consorts and ‘intermediate’ males of Doryteuthis plei. (A) In sneakers, the spermatophoric reaction (i.e. osmotically mediated process of eversion of the spermatophore tunics and membranes) results in the formation of a club-like spermatangium. Sperm form a dense mass after release from the spermatangium. (B) In consorts, the reaction results in the formation of a hook-like spermatangium, which is completely filled with sperm from the sperm mass. Sperm quickly diffuse after release from the spermatangium. (C) In ‘intermediate’ males, sneaker-like and consort-like spermatophores show the same pattern depicted in A and B, respectively, but ‘intermediate’ spermatophores form a third spermatangium type. Although similar to the consort hook-like type, ‘intermediate’ spermatangia have lower sperm mass content, which does not completely fill the space formed by the inner tunic. Thus, after detachment of the remaining empty case, the spermatangium shrinks. Similarly to sneaker spermatangia, sperm also aggregate in a dense mass around the spermatangium’s tip. (D, E) Spermatangia formation in consort-like and intermediate spermatophores, both from the same intermediate male, illustrating the schematic representations of B and C, respectively. In consort-like spermatophores (D), the sperm mass content completely fills the space created by the inner tunic, whereas in ‘intermediate’ spermatophores (E), the lower sperm mass content does not fill the inflated portion of the inner tunic. Several spermatophore structures were omitted or simplified in A, B and C for clarity. Abbreviations: cb, cement body; it, inner tunic; mm, middle membrane; ot, outer tunic; rec, remaining empty case; sm, sperm mass; spt, spermatangium; spz, released sperm. Figure 3. View largeDownload slide Schematic representation and images of the spermatophoric reaction in sneakers, consorts and ‘intermediate’ males of Doryteuthis plei. (A) In sneakers, the spermatophoric reaction (i.e. osmotically mediated process of eversion of the spermatophore tunics and membranes) results in the formation of a club-like spermatangium. Sperm form a dense mass after release from the spermatangium. (B) In consorts, the reaction results in the formation of a hook-like spermatangium, which is completely filled with sperm from the sperm mass. Sperm quickly diffuse after release from the spermatangium. (C) In ‘intermediate’ males, sneaker-like and consort-like spermatophores show the same pattern depicted in A and B, respectively, but ‘intermediate’ spermatophores form a third spermatangium type. Although similar to the consort hook-like type, ‘intermediate’ spermatangia have lower sperm mass content, which does not completely fill the space formed by the inner tunic. Thus, after detachment of the remaining empty case, the spermatangium shrinks. Similarly to sneaker spermatangia, sperm also aggregate in a dense mass around the spermatangium’s tip. (D, E) Spermatangia formation in consort-like and intermediate spermatophores, both from the same intermediate male, illustrating the schematic representations of B and C, respectively. In consort-like spermatophores (D), the sperm mass content completely fills the space created by the inner tunic, whereas in ‘intermediate’ spermatophores (E), the lower sperm mass content does not fill the inflated portion of the inner tunic. Several spermatophore structures were omitted or simplified in A, B and C for clarity. Abbreviations: cb, cement body; it, inner tunic; mm, middle membrane; ot, outer tunic; rec, remaining empty case; sm, sperm mass; spt, spermatangium; spz, released sperm. Figure 4. View largeDownload slide Spermatangia types and sperm release patterns of Doryteuthis plei. (A) Club-like spermatangium obtained from a sneaker-like spermatophore (left), ‘intermediate’ spermatangium from an ‘intermediate’ spermatophore (central) and hook-like spermatangium from a consort-like spermatophore (right). (B) In sneakers, sperm form a dense mass around the spermatangium’s tip. (C) In ‘intermediate’ spermatophores, sperm aggregate around the spermatangium, similar to sneaker males’ spermatangia. (D) In consorts, sperm quickly diffuse after extrusion from the spermatangium. Scale bar: 1.0 mm (A) and 0.5 mm (B–D). Abbreviations: cb, cement body; sm, sperm mass; spz, spermatozoa. Figure 4. View largeDownload slide Spermatangia types and sperm release patterns of Doryteuthis plei. (A) Club-like spermatangium obtained from a sneaker-like spermatophore (left), ‘intermediate’ spermatangium from an ‘intermediate’ spermatophore (central) and hook-like spermatangium from a consort-like spermatophore (right). (B) In sneakers, sperm form a dense mass around the spermatangium’s tip. (C) In ‘intermediate’ spermatophores, sperm aggregate around the spermatangium, similar to sneaker males’ spermatangia. (D) In consorts, sperm quickly diffuse after extrusion from the spermatangium. Scale bar: 1.0 mm (A) and 0.5 mm (B–D). Abbreviations: cb, cement body; sm, sperm mass; spz, spermatozoa. In intermediate spermatophores, almost all stages coincide with those of consorts (Fig. 3). Differences arise when the sperm mass is transferred to the spermatangium. As the inner tunic is everted, it forms a hook-like shape, with an inflated end, just like in consorts (Fig. 3B–E). However, in contrast to consorts, there is not enough sperm mass to fill this space within the spermatangium (Fig. 3C, E). Consequently, this inflated portion shrinks after detachment from the empty case, resulting in a different morphology from the typical hook-like shape of consorts (Figs 3, 4). Besides the resemblance to hook-like spermatangia (Fig. 4A), a striking feature of intermediate spermatangia was sperm agglomeration near their opening (Figs 3C, 4C), a phenomenon observed in one of the intermediate males. For details of the spermatophoric reaction in intermediate spermatophores, see Supporting Information S4 and S5. Age estimates All males were less than 1 year old, with age estimates ranging from c. 5 to 10 months (Supporting Information S6). One-way ANOVA revealed significant variation among sneakers, consorts and intermediate males (F2,247 = 81.22, P < 0.001) (sneakers, 176.64 ± 18.49 days old; intermediate, 177.67 ± 19.44 days old; consorts, 215.20 ± 23.14 days old). Post-hoc Tukey tests showed that sneakers and intermediate males were not significantly different in age (P = 0.994), but both were significantly younger than consorts (P < 0.001). Blind counts were highly consistent, indicated by extremely low values of 1.61% and 2.18% for APE and CV, respectively. DISCUSSION We report the first record of male squids that simultaneously store sneaker-like, consort-like and intermediate spermatophores. Furthermore, age estimates showed that sneakers and intermediate males have similar age, both being younger than consorts. As discussed below, our data suggest that ARTs are ontogenetically expressed in D. plei, as cumulative evidence indicates that intermediate males represent transitional stages from sneakers to consorts. Data on squid growth and population dynamics indicate that different life pathways may co-occur, i.e. ontogenetic shifts between tactics may not be obligatory. Rejecting the abnormality hypothesis Spermatophores with different morphologies (i.e. sneaker-like, intermediate and consort-like, Fig. 2) simultaneously stored within the same individual could at first be interpreted as abnormalities. However, several lines of evidence reject this hypothesis: (1) Histological techniques confirmed the fine structure of sneaker-like and consort-like spermatophores to be identical to sneaker and consort structures, respectively. Hence, they are not aberrant morphologies. (2) The range of morphologies and their spatial distribution within the spermatophoric sac suggest strongly a transitional phase in spermatophorogenesis from sneaker to consort spermatophores. These lines of evidence also reject that intermediate spermatophores are ‘tentative’ or ‘false’ spermatophores (Nigmatullin, Sabirov & Zalygalin, 2003). All types of spermatophore morphologies produced spermatangia with sperm release, and represent a grade between two spermatangium types naturally found on females, whereas ‘tentative’ and ‘false’ spermatophores are reportedly not used for copulation. The rarity of intermediate males could be explained if their transition occurs very rapidly. Given the range of spermatophore morphologies found within the same individual, this is probably a very brief stage during ontogeny of dimorphic males, with rapid changes in both morphology and physiology. If males do not stop copulating (i.e. using spermatophores) during this transition, evidence of intermediate spermatophores would be lost. Implications of an ontogenetic hypothesis Ontogenetic changes between phenotypes would require profound anatomical and physiological reorganizations of the male reproductive system, not only to produce different spermatophores, but also sperm with distinct behaviour, which would require changes in the biochemical properties of sperm (Hirohashi et al., 2013; Hirohashi & Iwata, 2014). In H. bleekeri, for example, the aggregative behaviour of sneaker sperm is mediated by their own respiratory CO2 (Hirohashi & Iwata, 2014). Sneaker sperm, but not consort sperm, are able to move towards acidic environments (pH-taxis) and aggregate with sibling sperm. Interestingly, flagellar length was proposed to affect this chemical sensitivity, which would explain why only long sperm of sneakers would exhibit such pH-taxis (Iida et al., 2017). In gobies, expression of ARTs is ontogenetic, and the transition from sneaker to parental morphs may also involve changes in several traits, including behaviour, body coloration, size of reproductive organs, ejaculate sperm density and increase in gonadotropin-releasing hormone cells in the forebrain (Immler, Mazzoldi & Rasotto, 2004; Scaggiante et al., 2004; Takegaki, Svensson & Kvarnemo, 2012). However, to the best of our knowledge, this is the first record of sperm with two strikingly different behaviours within the same individual. Sperm aggregation has been hypothesized as a mechanism to avoid dilution and delay sperm release due to the more external deposition site of sneaker spermatophores and to the extended interval between sneaker mating and fertilization (Fig. 1; Hirohashi & Iwata, 2014; Apostólico & Marian, 2017b). Consorts, by contrast, copulate with females when they are about to release their eggs, so rapid and intense sperm diffusion seems more advantageous (Fig. 1; Apostólico & Marian, 2017b). Thus, switching to consort tactics – and hence to the more privileged internal deposition site – would require changes in ejaculate traits. Sperm aggregation would theoretically not be advantageous, given that retaining sperm within the spermatangium would possibly decrease the number of fertilizations in comparison with the diffusion type. Moreover, considering that females may copulate with several consorts, rapid and intense sperm diffusion would outcompete sperm aggregation when the time between mating and egg-laying is short (Buresch et al., 2009). Besides distinct ejaculate morphology and functioning, dimorphic males of D. plei also differ in resource allocation to gonads and somatic growth, probably associated with asymmetrical risk of sperm competition. Sneakers invest relatively more in the spermatophoric complex and testis, whereas consorts invest more in body size (Apostólico & Marian, 2017a; see also Supporting Information S7). So, if sneakers become consorts, they should modify spermatophore production rate and change allocation from testis to somatic growth. This would enable them to grow larger, which is advantageous for consorts, as size might be critical in female guarding and physical contests (Apostólico & Marian, 2017a). In gobies, shifts from sneaking to parental tactics are also associated with a decrease in gonadosomatic index and a change in function of their seminal vesicle (from sperm storage to mucin secretion) (Scaggiante et al., 1999, 2004; Immler et al., 2004). The ontogenetic tactic as one of several possible life-pathways? Our data support lifespan estimates of less than 1 year for Brazilian populations of D. plei (Perez et al., 2006; Barcellos, 2014). Age estimates show that, in our sample, consorts hatched mainly in summer and early autumn, and sneakers mainly in winter. One possible explanation for the existence of two maturation groups in the population – apparently common in D. plei (Perez, Aguiar & Oliveira, 2002; Postuma & Gasalla, 2014) – and age differences between morphs could be related to differences in maturation rate. Males hatched in summer and early autumn could delay sexual maturity until the following summer, thus attaining larger body size at older age and becoming consorts, whereas those hatched in winter could mature precociously over the next summer, at younger age and smaller size, and become sneakers. Different maturation rates are responsible for morph determination in other species, in which male phenotypic expression reflects different life-history pathways and does not change with ontogeny (Gross & Charnov, 1980; Gross, 1982). Data on spermatophore morphology and sperm behaviour of intermediate males, however, do not support the hypothesis of irreversible conditional tactics in D. plei. The coexistence of two maturation groups could also be explained if (at least some) males can mature early in life and continue to grow afterwards (Guerra & Rocha, 1994). In D. plei, growth rates gradually decrease after sexual maturity, but they do not cease during the animal’s life (Barcellos, 2014). Such a growth pattern is compatible with an ontogenetic expression of ARTs, i.e. small males adopting sneaker tactics, but shifting to consort tactics when older and larger. Within this context, one possibility would be an obligatory ontogenetic shift between tactics. All males would mature at c. 6 months old and at small size as sneakers. They would then switch towards consort phenotypes when reaching a certain body size switch point (c. 169 mm ML; Apostólico & Marian, 2017a). However, inferred growth rates of mature males of D. plei (c. 16.5 mm month−1; Barcellos, 2014) do not entirely support this hypothesis, as it does not seem feasible that all sneakers almost double their size in just a few months. If the hypothesis above were rejected, another possibility would concern the co-occurrence of different life-pathways within the population. In the Mediterranean wrasse, for example, environmental conditions influence early growth rates and ultimately the adoption of one of several possible life-pathways. For example, fast-growing males mature as satellites and still grow enough to become nesting males, whereas slower growing males abstain from sexual maturity for longer and become larger nesting males later in life (Alonzo, Taborsky & Wirtz, 2000). A similar strategy with sequential tactics and alternative life-pathways is present in the peacock blenny, although in this case there is no difference in growth rates between individuals following distinct pathways, which are apparently determined by the time between birth and first breeding season (Fagundes et al., 2015). A similar ‘birthdate effect’ (Taborsky, 1998) could affect the expression of tactics in squids. For example, environmental cues such as water temperature and food availability are known to influence squid growth and sexual maturation (Boyle & Rodhouse, 2005), so it would not be surprising if they also regulate morph determination in these molluscs. Distinct oceanographic conditions are present throughout the year in the sampled area (e.g. intrusion of South Atlantic Central Water during summer; Castro et al. 2008). So, based on our sample, we could hypothesize that males hatched during winter may mature precociously as sneakers, but continue to grow and later become smaller consorts, whereas males hatched during summer–early autumn may delay maturation and become the largest consorts later in life, without experiencing a sneaker stage. It is possible that males subjected to different environmental conditions during the year may follow different paths, although further studies are required to determine which underlying mechanisms regulate morph determination in this species. However, the extensive age (Perez et al. 2002) and size (Barcellos, 2014) overlap between maturing and mature males of D. plei support the two-life-pathways hypothesis. This hypothesis may help to explain the two switch point ranges found for D. plei (Apostólico & Marian, 2017a). The 169 mm ML switch point is robust to divide sneakers and consorts based on sperm mass relative length and spermatangia morphology. However, the 205–220 mm ML switch point is apparently not associated with ARTs (see discussion in Apostólico & Marian, 2017a). We now hypothesize that this switch point divides the two consort groups, i.e. those that first develop into sneakers and then become smaller consorts (at c. 169 mm ML), and those that delay maturity and become the largest consorts later in life, possibly after reaching c. 205–220 mm ML. Data on D. plei biology appear to support this hypothesis, since the largest maturing males are usually around 200–230 mm ML (Perez et al., 2002). Finally, we cannot exclude the possibility that expression of ARTs in squids is also regulated by their social context, as demonstrated for some gobies, in which particular social contexts (e.g. absence of dominant males, presence of ripe females) may induce a morph change in sequential ARTs (Immler et al., 2004; Scaggiante et al., 2004; Takegaki et al., 2012). Future experimental studies are needed to test this possibility. Concluding remarks This is the first record of intermediate-sized male squids that produce sneaker-like, consort-like and intermediate ejaculates. The present findings are consistent with the predictions of current theoretical models on ARTs (Gross, 1996; Tomkins & Hazel, 2007). As discussed herein, cumulative evidence indicates that ARTs in D. plei are environmentally cued threshold traits, with the trigger for changing tactics depending on body size (Buzatto, Tomkins & Simmons, 2014). Under an ontogenetic gradient in adoption of ARTs, small males might first benefit from sneaking, and later switch to dominant tactics when their large sizes enable them to monopolize resources and mates (Taborsky, 2008), a prediction that also accommodates the present findings. In loliginid squids, which have two distinct sperm deposition sites associated with ARTs, the size-related benefits from switching to the dominant tactic possibly surpass the costs of the radical transition in ejaculate traits described herein. This finding should stimulate future studies on ART expression and evolution, given that the complexity of this system should put the robustness of current ART models to the test. Moreover, squids may now be used as models for studying reversal of tactic-specific investments in male reproductive traits, in addition to some fishes (Immler et al., 2004; Scaggiante et al., 2004; Takegaki et al., 2012). ACKNOWLEDGEMENTS This paper is part of the first author’s Master’s dissertation in Zoology at the University of São Paulo (USP). We obtained a collecting licence from the Instituto Chico Mendes de Conservação da Biodiversidade ICMBio/SISBIO (Permit Number: 44738). In Brazil, ethics approval is still not required for experimentation with cephalopods by the CONCEA (‘Conselho Nacional de Controle de Experimentação Animal’) but we have carried out this study in accordance with international protocols for the welfare of cephalopods and made all efforts to minimize animal suffering. We appreciate the financial support and grants provided by CAPES (Coordenation for the Improvement of Higher Education Personnel), CAPES/PROEX and CNPq (National Council for Scientific and Technological Development – proc. 477233/2013–9). We thank Dr Alvaro E. Migotto (USP) for assistance during in vitro experimentation, and Dr Bruno Buzatto (University of Southwestern Australia), Dr Glauco Machado (USP) and two anonymous reviewers for the comments that helped to improve the quality of the manuscript. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site S1. Age estimation using statoliths in the squid Doryteuthis plei. S2. Spermatophore morphometry of sneakers, consorts and intermediate males of Doryteuthis plei. S3. Sagittal sections of the cement body of spermatophores from an ‘intermediate’ male of Doryteuthis plei. S4. Still images of in vitro spermatophoric reaction of an ‘intermediate’ spermatophore of Doryteuthis plei. S5. Video of in vitro spermatophoric reaction of an ‘intermediate’ spermatophore of Doryteuthis plei. S6. Number of statolith increments per size class in males of Doryteuthis plei. S7. 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From sneaky to bully: reappraisal of male squid dimorphism indicates ontogenetic mating tactics and striking ejaculate transition

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Abstract

Abstract The expression of alternative reproductive tactics (ARTs) is often influenced by the environmental cues and intrinsic conditions experienced by each male, and it may be reversible or permanent. In male loliginid squid, ARTs include consorts and sneakers, which differ in behaviour, body size, sperm deposition site, and morphology and functioning of ejaculates. Here, we describe intermediate-sized males in the squid Doryteuthis plei (Blainville, 1823) that produce sneaker-like, consort-like and intermediate ejaculates. In addition, age estimates show that sneakers and intermediate males are younger than consorts. Our findings suggest that ARTs in this species may be ontogenetically expressed. Such a transition between phenotypes requires dramatic anatomical and physiological reorganization of the male reproductive system, not only to produce different-structured spermatophores, but also to generate sperm with distinct behaviour, both of which are compatible with the switch in sperm deposition site. These findings demonstrate that this peculiar mating system may also fit the current predictions of ARTs. INTRODUCTION Sexual selection promotes high variance in male reproductive success (Shuster & Wade, 2003). Under strong competition, males less successful in fighting with dominant males may pursue alternative reproductive tactics (ARTs) to maximize fertilization success (Taborsky, Oliveira & Brockmann, 2008). Such male ARTs often involve not only discontinuous mating behaviour, but also distinct life histories (Gross, 1991), and discontinuous physiological and morphological traits (Moczek & Emlen, 1999), a phenomenon known as intrasexual male dimorphism (Gadgil, 1972). Discontinuous reproductive phenotypes may result from polymorphic genotypes or, more commonly, from polyphenism (Taborsky et al., 2008). In polyphenic species, all males are able to express both phenotypes, morph determination being ultimately influenced by environmental cues and intrinsic conditions experienced by each male (Gross, 1996; Tomkins & Hazel, 2007). Males express the phenotype that maximizes their fitness, which will depend on males’ relative status (e.g. size, age). Males that reach a critical status switch point (e.g. males that grow larger than a critical body size) have higher reproductive success by expressing dominant tactics, whereas those below it have higher fitness pay-offs by adopting sneaking tactics (Gross, 1996). Adoption of conditional ARTs may be irreversible or reversible in life (Taborsky et al., 2008). In horn beetles, morph determination is triggered by resource availability during larval development and it is permanent in the adult. Males above a certain body-size switch point develop horns and adopt dominant tactics, whereas those that do not reach the switch point lack horns and become sneakers (Moczek & Emlen, 1999). In some fishes, morph expression depends on juvenile growth rate. Fast-growing males mature precociously at smaller sizes and become sneakers, whereas slow-growing males delay sexual maturity and employ dominant tactics at larger sizes later in life (Gross, 1984, 1991). In reversible tactics, males may alternate between tactics multiple times by adaptively responding to current conditions and employing different strategies accordingly (Taborsky, 1994; Fischer & Fiedler, 2001). Alternating between tactics may also be ontogenetic, when individuals change tactics only once in life (Mazzoldi et al., 2000; Bleeker et al., 2017). In fishes, ontogenetic tactics are often associated with their indeterminate growth, young and small males benefiting from sneaking until they become older and larger, when it pays to shift to dominant roles (Taborsky, 1994; Bleeker et al., 2017). In loliginid squid, different sized males often adopt ARTs (Hanlon, 1996). Large (consort) males fight with rivals, guard females and mate with females in male–parallel position, depositing spermatophores near the oviduct opening (Fig. 1D, E). Small (sneaker) males, in turn, avoid fighting contests, do not guard females and adopt head-to-head mating, placing spermatophores near the seminal receptacle, below the mouth (Fig. 1A–C; Hanlon, 1998). The presence of two spermatophore attachment sites on the female is thought to promote differences in fertilization timing and success between male morphs. Consort sperm from spermatangia attached near the oviduct opening meet the eggs first as they leave the oviduct (Fig. 1E, G), whereas sneaker sperm only meet the eggs when the female holds the egg capsule near the mouth, before depositing it on the substrate (Fig. 1B, G). Thus, sneakers are assumed to have lower fertilization success than consorts (Iwata, Munehara & Sakurai, 2005; Shashar & Hanlon, 2013), as there are probably fewer unfertilized eggs available to sperm near the seminal receptacle (Hanlon, Maxwell & Shashar, 1997; Buresch et al., 2009). Figure 1. View largeDownload slide Schematic representation of the complex mating system of loliginid squids, which includes alternative mating tactics and distinct spermatophore attachment sites within females. (A) Small sneaker males mate with females in the head-to-head position and place spermatophores near their seminal receptacle. (B) Detail of the female buccal membrane (frontal view). The seminal receptacle (blue star) is located below the mouth, between the left and right ventral arms. (C) Detail of the seminal receptacle (sagittal section) and club-like spermatangia from sneaker males. Sneaker sperm aggregate as a round mass around the spermatangium tip after being released. (D) Large consort males mate with females in the male-parallel position and place spermatophores near the oviduct opening. Consort males may exhibit visual signalling in agonistic contests with rival males (in this case, the ‘lateral flame’, i.e. the flame-like streaks seen unilaterally in each consort male of the figure). (E) Detail of the dissected mantle cavity of females. Consort spermatangia are attached near the oviduct opening (green star). (F) Detail of hook-like spermatangia of consort males attached in the female. In contrast to sneaker sperm, consort sperm rapidly diffuse after release. (G) During egg-laying, consort sperm from spermatangia attached near the oviduct opening (green star) meet the eggs first, as they leave the oviduct, whereas sneaker sperm only meet the eggs when the egg capsule is near the mouth (blue star), before being deposited on the substrate. Figure 1. View largeDownload slide Schematic representation of the complex mating system of loliginid squids, which includes alternative mating tactics and distinct spermatophore attachment sites within females. (A) Small sneaker males mate with females in the head-to-head position and place spermatophores near their seminal receptacle. (B) Detail of the female buccal membrane (frontal view). The seminal receptacle (blue star) is located below the mouth, between the left and right ventral arms. (C) Detail of the seminal receptacle (sagittal section) and club-like spermatangia from sneaker males. Sneaker sperm aggregate as a round mass around the spermatangium tip after being released. (D) Large consort males mate with females in the male-parallel position and place spermatophores near the oviduct opening. Consort males may exhibit visual signalling in agonistic contests with rival males (in this case, the ‘lateral flame’, i.e. the flame-like streaks seen unilaterally in each consort male of the figure). (E) Detail of the dissected mantle cavity of females. Consort spermatangia are attached near the oviduct opening (green star). (F) Detail of hook-like spermatangia of consort males attached in the female. In contrast to sneaker sperm, consort sperm rapidly diffuse after release. (G) During egg-laying, consort sperm from spermatangia attached near the oviduct opening (green star) meet the eggs first, as they leave the oviduct, whereas sneaker sperm only meet the eggs when the egg capsule is near the mouth (blue star), before being deposited on the substrate. In at least two loliginid species (Doryteuthis plei and Heterololigo bleekeri), size-dependent ARTs are also associated with intrasexual dimorphism between sneakers and consorts, including differences in sperm size and behaviour, sperm release strategies, and spermatophore and spermatangium (i.e. everted spermatophore) morphology and functioning (Iwata & Sakurai, 2007; Iwata et al., 2011; Hirohashi & Iwata, 2014; Apostólico & Marian, 2017a, b). For example, sneakers produce small club-like spermatangia and sperm with a clustering behaviour (Fig. 1C), whereas consorts produce large hook-like spermatangia and diffuse rather than aggregative sperm (Fig. 1F). Such profound differences in ejaculate traits have been attributed to the intrinsic characteristics of each sperm deposition site (Hirohashi & Iwata, 2014; Hirohashi et al., 2016; Apostólico & Marian, 2017a, b). Although ARTs are widespread in animals and the mechanisms responsible for their expression have been extensively investigated (Oliveira, Taborsky & Brockmann, 2008), such mechanisms are completely unknown in squids. Squids offer an interesting model for studying ARTs, given the presence of distinct fertilization sites associated with ARTs (Fig. 1), a peculiar condition compared to the model animals generally used to study ARTs. Previously, we demonstrated the occurrence of intrasexual dimorphism in the squid D. plei (Blainville, 1823), showing that sneakers and consorts differ in spermatangia (Fig. 1C, F) and spermatophore morphology and also in gonadal investment (Apostólico & Marian, 2017a). We subsequently used preserved material and videos of in vitro experiments from the same sampling to describe and compare the fine structure and functioning of sneaker and consort ejaculates, showing how differences in spermatophore structure lead to the peculiar dimorphism observed in spermatangia morphology (Apostólico & Marian, 2017b). While further analysing the preserved material and videos from the same sampling, we found five intermediate sized males of D. plei that stored both sneaker-like and consort-like spermatophores simultaneously within the spermatophoric sac. In these previous papers (Apostólico & Marian, 2017a, 2017b), four of the five males were classified as sneakers and one as a consort, based on their body size and on the switch point previously described by Apostólico & Marian (2017a). At first, these cases were treated as probable abnormalities, but further detailed analyses revealed them to be indicative that ARTs in this species may be ontogenetically expressed. Here, we describe in detail the morphology and in vitro functioning of spermatophores from these ‘intermediate’ males. We show that they produce not only sneaker-like and consort-like ejaculates, but also structures with intermediate features between the two morphs, suggesting a sequential production (i.e. from sneaker to consort spermatophores). We also show that sneakers and intermediate males are younger than consorts based on the statoliths extracted during the original sampling. Our findings suggest that expression of ARTs in this species may change with ontogeny, and lead to a dramatic ejaculate transition between morphs that is consistent with change in sperm deposition site. MATERIAL AND METHODS Data collection As detailed elsewhere (Apostólico & Marian, 2017a), we collected 287 mature males of D. plei (88–327 mm in mantle length, ML) from December to February 2014–2016, off São Sebastião Island (São Paulo, Brazil). We classified males as ‘sneakers’ (N = 73; 88–169 mm ML) or ‘consorts’ (N = 209; 170–327 mm ML) based on spermatophore and spermatangia morphology (Apostólico & Marian, 2017a), except for males later revealed to have both sneaker-like and consort-like spermatophores (N = 5; 146, 149, 159, 166 and 176 mm ML), classified herein as ‘intermediate’ males. Spermatophore morphology and in vitro experiments To confirm if sneaker-like and consort-like spermatophores of intermediate males were indeed similar to those of sneakers and consorts, respectively, we sectioned and stained spermatophores of intermediate males, which were preserved in Karnovsky’s fixative (Marian, 2012a) during the original sampling. Light microscopy followed the procedures of Apostólico & Marian (2017b). Based on videos from the original sampling (Apostólico & Marian, 2017b), we also analysed the behaviour of fresh spermatophores from the five intermediate males (including nine sneaker-like, 14 consort-like and 25 intermediate spermatophores) during the spermatophoric reaction (i.e. spermatophore eversion, leading to formation and attachment of the spermatangium; Marian, 2012b). Age estimation We selected 250 males (175 consorts, 70 sneakers, five intermediate males) for age estimates based on statolith growth increments. During the original sampling, we dissected statoliths from fresh animals and fixed them in 70% ethanol for future use. Later, we ground and polished them following the methods of Perez, Aguiar & Santos (2006). We examined statoliths under an Axio Imager M2 Zeiss microscope (1000× magnification, immersion oil) and performed three blind counts of growth increments by a single reader. For each male, we determined age as the mean total number of increments counted, assuming that growth-ring deposition occurs daily in this species (Perez et al., 2006). For details of this method, see Supporting Information S1. Statistical analysis For age estimates, we tested data for normality with the Shapiro–Wilk test and for variance homogeneity with Bartlett’s test. We performed one-way ANOVA, followed by Tukey’s honest significant difference post-hoc analysis, to test for significant differences between sneaker, intermediate and consort males. We also tested the accuracy of blind counts with two indices, the average percentage error (APE) (Beamish & Fournier, 1981), and the coefficient of variation (CV) (Chang, 1982). Acceptable levels of precision for both indices have been conventionally established as values less than 10% (e.g. Schwarz & Perez, 2010). We conducted statistical analyses in R 3.3.2 (R Development Core Team, 2008), and set up the level of significance as P ≤ 0.05 for all analyses. RESULTS Spermatophore morphology Typical sneakers and consorts of D. plei produce only spermatophores with exclusive morphologies (see Apostólico & Marian, 2017b). Intermediate males, however, simultaneously store spermatophores of both types (sneaker-like and consort-like), and also spermatophores with intermediate features of both morphs (hereafter called ‘intermediate spermatophores’) (Fig. 2; see also Supporting Information S2). Sneaker-like spermatophores had a short sperm mass (Fig. 2A), an ejaculatory apparatus tube with a looser arrangement and incomplete loops (Fig. 2B), and a cement body with a cylindrical oral region, a constricted intermediate region and an ellipsoidal aboral region (Fig. 2G). In turn, consort-like spermatophores had a long sperm mass (Fig. 2A), an ejaculatory apparatus tube more convoluted and tightly arranged (Fig. 2F), and a cement body with a wide and conically shaped oral region, a broad intermediate region and a cylindrical aboral region (Fig. 2K). Both morphologies were identical to those of sneakers and consorts (see Apostólico & Marian, 2017b). Also, histological methods (Supporting Information S3) showed that sneaker-like and consort-like spermatophores within the same intermediate male had identical characteristics as regards tunics, membranes and staining affinities to those described for sneakers and consorts, respectively (see Apostólico & Marian, 2017b). Figure 2. View largeDownload slide Spermatophores of ‘intermediate’ males of Doryteuthis plei. All spermatophores were sampled from the same male. (A) Whole spermatophores, showing the transition from typical sneaker-like (top) to typical consort-like structure (bottom). Intermediate morphologies (centrally) encompass a gradual modification on the oral region (i.e. cement body and ejaculatory apparatus), followed by increase in sperm mass. (B–F) Detail of the oral region of spermatophores from A. Notice that the ejaculatory apparatus tube and the cement body gradually change from a sneaker-like (B) to a consort-like morphology (F), with intermediate configurations in between (C–E). (G–K) Detail of the tripartite cement body of the spermatophores from A. Notice the morphological transition from a sneaker-like (G) to a consort-like morphology (K), with intermediate forms in between (H–J). Abbreviations: cb, cement body; cba, cement body aboral region; cbi, cement body intermediate region; cbo, cement body oral region; ea, ejaculatory apparatus; sm, sperm mass. Figure 2. View largeDownload slide Spermatophores of ‘intermediate’ males of Doryteuthis plei. All spermatophores were sampled from the same male. (A) Whole spermatophores, showing the transition from typical sneaker-like (top) to typical consort-like structure (bottom). Intermediate morphologies (centrally) encompass a gradual modification on the oral region (i.e. cement body and ejaculatory apparatus), followed by increase in sperm mass. (B–F) Detail of the oral region of spermatophores from A. Notice that the ejaculatory apparatus tube and the cement body gradually change from a sneaker-like (B) to a consort-like morphology (F), with intermediate configurations in between (C–E). (G–K) Detail of the tripartite cement body of the spermatophores from A. Notice the morphological transition from a sneaker-like (G) to a consort-like morphology (K), with intermediate forms in between (H–J). Abbreviations: cb, cement body; cba, cement body aboral region; cbi, cement body intermediate region; cbo, cement body oral region; ea, ejaculatory apparatus; sm, sperm mass. Meanwhile, intermediate spermatophores had heterogeneous combinations of the two morphologies. They generally had sneaker-like short sperm mass (Fig. 2A), whereas the ejaculatory apparatus and cement body were more variable. While the ejaculatory apparatus tube gradually became more convoluted and with a higher number of complete loops (Fig. 2C–E), the three cement body regions resembled intermediate shapes ranging from typical sneaker-like to a consort-like format (Fig. 2H–J). Further analysis of the spermatophoric sac of one intermediate male also revealed that spermatophores with distinct morphologies were spatially separated within the organ: sneaker-like and consort-like spermatophores were located more anteriorly and more posteriorly in the organ, respectively, while intermediate spermatophores occupied the medial region. This indicates that sneaker-like spermatophores were older than consort-like spermatophores. Spermatophoric reaction The spermatophoric reaction encompasses several stages of spermatophore eversion that ultimately result in the formation of the spermatangium and, subsequently, in sperm release (Fig. 3). In intermediate males, sneaker-like spermatophores (Fig. 2A, B, G) always produced club-like spermatangia (Figs 3A, 4A, left). Consort-like spermatophores (Fig. 2A, F, K), by contrast, always produced hook-like spermatangia (Figs 3B, 4A, right). Such patterns were identical to the spermatophoric reaction and spermatangia formation from typical sneakers and consorts in D. plei (see Apostólico & Marian, 2017b). Moreover, in sneaker-like spermatophores, released sperm aggregated in a spherical mass near the spermatangium (Figs 3A, 4B), while consort-like spermatangia showed intense sperm diffusion (Figs 3B, 4D), also consistent with the typical behaviour of sneaker and consort morphs (see Apostólico & Marian, 2017b). Figure 3. View largeDownload slide Schematic representation and images of the spermatophoric reaction in sneakers, consorts and ‘intermediate’ males of Doryteuthis plei. (A) In sneakers, the spermatophoric reaction (i.e. osmotically mediated process of eversion of the spermatophore tunics and membranes) results in the formation of a club-like spermatangium. Sperm form a dense mass after release from the spermatangium. (B) In consorts, the reaction results in the formation of a hook-like spermatangium, which is completely filled with sperm from the sperm mass. Sperm quickly diffuse after release from the spermatangium. (C) In ‘intermediate’ males, sneaker-like and consort-like spermatophores show the same pattern depicted in A and B, respectively, but ‘intermediate’ spermatophores form a third spermatangium type. Although similar to the consort hook-like type, ‘intermediate’ spermatangia have lower sperm mass content, which does not completely fill the space formed by the inner tunic. Thus, after detachment of the remaining empty case, the spermatangium shrinks. Similarly to sneaker spermatangia, sperm also aggregate in a dense mass around the spermatangium’s tip. (D, E) Spermatangia formation in consort-like and intermediate spermatophores, both from the same intermediate male, illustrating the schematic representations of B and C, respectively. In consort-like spermatophores (D), the sperm mass content completely fills the space created by the inner tunic, whereas in ‘intermediate’ spermatophores (E), the lower sperm mass content does not fill the inflated portion of the inner tunic. Several spermatophore structures were omitted or simplified in A, B and C for clarity. Abbreviations: cb, cement body; it, inner tunic; mm, middle membrane; ot, outer tunic; rec, remaining empty case; sm, sperm mass; spt, spermatangium; spz, released sperm. Figure 3. View largeDownload slide Schematic representation and images of the spermatophoric reaction in sneakers, consorts and ‘intermediate’ males of Doryteuthis plei. (A) In sneakers, the spermatophoric reaction (i.e. osmotically mediated process of eversion of the spermatophore tunics and membranes) results in the formation of a club-like spermatangium. Sperm form a dense mass after release from the spermatangium. (B) In consorts, the reaction results in the formation of a hook-like spermatangium, which is completely filled with sperm from the sperm mass. Sperm quickly diffuse after release from the spermatangium. (C) In ‘intermediate’ males, sneaker-like and consort-like spermatophores show the same pattern depicted in A and B, respectively, but ‘intermediate’ spermatophores form a third spermatangium type. Although similar to the consort hook-like type, ‘intermediate’ spermatangia have lower sperm mass content, which does not completely fill the space formed by the inner tunic. Thus, after detachment of the remaining empty case, the spermatangium shrinks. Similarly to sneaker spermatangia, sperm also aggregate in a dense mass around the spermatangium’s tip. (D, E) Spermatangia formation in consort-like and intermediate spermatophores, both from the same intermediate male, illustrating the schematic representations of B and C, respectively. In consort-like spermatophores (D), the sperm mass content completely fills the space created by the inner tunic, whereas in ‘intermediate’ spermatophores (E), the lower sperm mass content does not fill the inflated portion of the inner tunic. Several spermatophore structures were omitted or simplified in A, B and C for clarity. Abbreviations: cb, cement body; it, inner tunic; mm, middle membrane; ot, outer tunic; rec, remaining empty case; sm, sperm mass; spt, spermatangium; spz, released sperm. Figure 4. View largeDownload slide Spermatangia types and sperm release patterns of Doryteuthis plei. (A) Club-like spermatangium obtained from a sneaker-like spermatophore (left), ‘intermediate’ spermatangium from an ‘intermediate’ spermatophore (central) and hook-like spermatangium from a consort-like spermatophore (right). (B) In sneakers, sperm form a dense mass around the spermatangium’s tip. (C) In ‘intermediate’ spermatophores, sperm aggregate around the spermatangium, similar to sneaker males’ spermatangia. (D) In consorts, sperm quickly diffuse after extrusion from the spermatangium. Scale bar: 1.0 mm (A) and 0.5 mm (B–D). Abbreviations: cb, cement body; sm, sperm mass; spz, spermatozoa. Figure 4. View largeDownload slide Spermatangia types and sperm release patterns of Doryteuthis plei. (A) Club-like spermatangium obtained from a sneaker-like spermatophore (left), ‘intermediate’ spermatangium from an ‘intermediate’ spermatophore (central) and hook-like spermatangium from a consort-like spermatophore (right). (B) In sneakers, sperm form a dense mass around the spermatangium’s tip. (C) In ‘intermediate’ spermatophores, sperm aggregate around the spermatangium, similar to sneaker males’ spermatangia. (D) In consorts, sperm quickly diffuse after extrusion from the spermatangium. Scale bar: 1.0 mm (A) and 0.5 mm (B–D). Abbreviations: cb, cement body; sm, sperm mass; spz, spermatozoa. In intermediate spermatophores, almost all stages coincide with those of consorts (Fig. 3). Differences arise when the sperm mass is transferred to the spermatangium. As the inner tunic is everted, it forms a hook-like shape, with an inflated end, just like in consorts (Fig. 3B–E). However, in contrast to consorts, there is not enough sperm mass to fill this space within the spermatangium (Fig. 3C, E). Consequently, this inflated portion shrinks after detachment from the empty case, resulting in a different morphology from the typical hook-like shape of consorts (Figs 3, 4). Besides the resemblance to hook-like spermatangia (Fig. 4A), a striking feature of intermediate spermatangia was sperm agglomeration near their opening (Figs 3C, 4C), a phenomenon observed in one of the intermediate males. For details of the spermatophoric reaction in intermediate spermatophores, see Supporting Information S4 and S5. Age estimates All males were less than 1 year old, with age estimates ranging from c. 5 to 10 months (Supporting Information S6). One-way ANOVA revealed significant variation among sneakers, consorts and intermediate males (F2,247 = 81.22, P < 0.001) (sneakers, 176.64 ± 18.49 days old; intermediate, 177.67 ± 19.44 days old; consorts, 215.20 ± 23.14 days old). Post-hoc Tukey tests showed that sneakers and intermediate males were not significantly different in age (P = 0.994), but both were significantly younger than consorts (P < 0.001). Blind counts were highly consistent, indicated by extremely low values of 1.61% and 2.18% for APE and CV, respectively. DISCUSSION We report the first record of male squids that simultaneously store sneaker-like, consort-like and intermediate spermatophores. Furthermore, age estimates showed that sneakers and intermediate males have similar age, both being younger than consorts. As discussed below, our data suggest that ARTs are ontogenetically expressed in D. plei, as cumulative evidence indicates that intermediate males represent transitional stages from sneakers to consorts. Data on squid growth and population dynamics indicate that different life pathways may co-occur, i.e. ontogenetic shifts between tactics may not be obligatory. Rejecting the abnormality hypothesis Spermatophores with different morphologies (i.e. sneaker-like, intermediate and consort-like, Fig. 2) simultaneously stored within the same individual could at first be interpreted as abnormalities. However, several lines of evidence reject this hypothesis: (1) Histological techniques confirmed the fine structure of sneaker-like and consort-like spermatophores to be identical to sneaker and consort structures, respectively. Hence, they are not aberrant morphologies. (2) The range of morphologies and their spatial distribution within the spermatophoric sac suggest strongly a transitional phase in spermatophorogenesis from sneaker to consort spermatophores. These lines of evidence also reject that intermediate spermatophores are ‘tentative’ or ‘false’ spermatophores (Nigmatullin, Sabirov & Zalygalin, 2003). All types of spermatophore morphologies produced spermatangia with sperm release, and represent a grade between two spermatangium types naturally found on females, whereas ‘tentative’ and ‘false’ spermatophores are reportedly not used for copulation. The rarity of intermediate males could be explained if their transition occurs very rapidly. Given the range of spermatophore morphologies found within the same individual, this is probably a very brief stage during ontogeny of dimorphic males, with rapid changes in both morphology and physiology. If males do not stop copulating (i.e. using spermatophores) during this transition, evidence of intermediate spermatophores would be lost. Implications of an ontogenetic hypothesis Ontogenetic changes between phenotypes would require profound anatomical and physiological reorganizations of the male reproductive system, not only to produce different spermatophores, but also sperm with distinct behaviour, which would require changes in the biochemical properties of sperm (Hirohashi et al., 2013; Hirohashi & Iwata, 2014). In H. bleekeri, for example, the aggregative behaviour of sneaker sperm is mediated by their own respiratory CO2 (Hirohashi & Iwata, 2014). Sneaker sperm, but not consort sperm, are able to move towards acidic environments (pH-taxis) and aggregate with sibling sperm. Interestingly, flagellar length was proposed to affect this chemical sensitivity, which would explain why only long sperm of sneakers would exhibit such pH-taxis (Iida et al., 2017). In gobies, expression of ARTs is ontogenetic, and the transition from sneaker to parental morphs may also involve changes in several traits, including behaviour, body coloration, size of reproductive organs, ejaculate sperm density and increase in gonadotropin-releasing hormone cells in the forebrain (Immler, Mazzoldi & Rasotto, 2004; Scaggiante et al., 2004; Takegaki, Svensson & Kvarnemo, 2012). However, to the best of our knowledge, this is the first record of sperm with two strikingly different behaviours within the same individual. Sperm aggregation has been hypothesized as a mechanism to avoid dilution and delay sperm release due to the more external deposition site of sneaker spermatophores and to the extended interval between sneaker mating and fertilization (Fig. 1; Hirohashi & Iwata, 2014; Apostólico & Marian, 2017b). Consorts, by contrast, copulate with females when they are about to release their eggs, so rapid and intense sperm diffusion seems more advantageous (Fig. 1; Apostólico & Marian, 2017b). Thus, switching to consort tactics – and hence to the more privileged internal deposition site – would require changes in ejaculate traits. Sperm aggregation would theoretically not be advantageous, given that retaining sperm within the spermatangium would possibly decrease the number of fertilizations in comparison with the diffusion type. Moreover, considering that females may copulate with several consorts, rapid and intense sperm diffusion would outcompete sperm aggregation when the time between mating and egg-laying is short (Buresch et al., 2009). Besides distinct ejaculate morphology and functioning, dimorphic males of D. plei also differ in resource allocation to gonads and somatic growth, probably associated with asymmetrical risk of sperm competition. Sneakers invest relatively more in the spermatophoric complex and testis, whereas consorts invest more in body size (Apostólico & Marian, 2017a; see also Supporting Information S7). So, if sneakers become consorts, they should modify spermatophore production rate and change allocation from testis to somatic growth. This would enable them to grow larger, which is advantageous for consorts, as size might be critical in female guarding and physical contests (Apostólico & Marian, 2017a). In gobies, shifts from sneaking to parental tactics are also associated with a decrease in gonadosomatic index and a change in function of their seminal vesicle (from sperm storage to mucin secretion) (Scaggiante et al., 1999, 2004; Immler et al., 2004). The ontogenetic tactic as one of several possible life-pathways? Our data support lifespan estimates of less than 1 year for Brazilian populations of D. plei (Perez et al., 2006; Barcellos, 2014). Age estimates show that, in our sample, consorts hatched mainly in summer and early autumn, and sneakers mainly in winter. One possible explanation for the existence of two maturation groups in the population – apparently common in D. plei (Perez, Aguiar & Oliveira, 2002; Postuma & Gasalla, 2014) – and age differences between morphs could be related to differences in maturation rate. Males hatched in summer and early autumn could delay sexual maturity until the following summer, thus attaining larger body size at older age and becoming consorts, whereas those hatched in winter could mature precociously over the next summer, at younger age and smaller size, and become sneakers. Different maturation rates are responsible for morph determination in other species, in which male phenotypic expression reflects different life-history pathways and does not change with ontogeny (Gross & Charnov, 1980; Gross, 1982). Data on spermatophore morphology and sperm behaviour of intermediate males, however, do not support the hypothesis of irreversible conditional tactics in D. plei. The coexistence of two maturation groups could also be explained if (at least some) males can mature early in life and continue to grow afterwards (Guerra & Rocha, 1994). In D. plei, growth rates gradually decrease after sexual maturity, but they do not cease during the animal’s life (Barcellos, 2014). Such a growth pattern is compatible with an ontogenetic expression of ARTs, i.e. small males adopting sneaker tactics, but shifting to consort tactics when older and larger. Within this context, one possibility would be an obligatory ontogenetic shift between tactics. All males would mature at c. 6 months old and at small size as sneakers. They would then switch towards consort phenotypes when reaching a certain body size switch point (c. 169 mm ML; Apostólico & Marian, 2017a). However, inferred growth rates of mature males of D. plei (c. 16.5 mm month−1; Barcellos, 2014) do not entirely support this hypothesis, as it does not seem feasible that all sneakers almost double their size in just a few months. If the hypothesis above were rejected, another possibility would concern the co-occurrence of different life-pathways within the population. In the Mediterranean wrasse, for example, environmental conditions influence early growth rates and ultimately the adoption of one of several possible life-pathways. For example, fast-growing males mature as satellites and still grow enough to become nesting males, whereas slower growing males abstain from sexual maturity for longer and become larger nesting males later in life (Alonzo, Taborsky & Wirtz, 2000). A similar strategy with sequential tactics and alternative life-pathways is present in the peacock blenny, although in this case there is no difference in growth rates between individuals following distinct pathways, which are apparently determined by the time between birth and first breeding season (Fagundes et al., 2015). A similar ‘birthdate effect’ (Taborsky, 1998) could affect the expression of tactics in squids. For example, environmental cues such as water temperature and food availability are known to influence squid growth and sexual maturation (Boyle & Rodhouse, 2005), so it would not be surprising if they also regulate morph determination in these molluscs. Distinct oceanographic conditions are present throughout the year in the sampled area (e.g. intrusion of South Atlantic Central Water during summer; Castro et al. 2008). So, based on our sample, we could hypothesize that males hatched during winter may mature precociously as sneakers, but continue to grow and later become smaller consorts, whereas males hatched during summer–early autumn may delay maturation and become the largest consorts later in life, without experiencing a sneaker stage. It is possible that males subjected to different environmental conditions during the year may follow different paths, although further studies are required to determine which underlying mechanisms regulate morph determination in this species. However, the extensive age (Perez et al. 2002) and size (Barcellos, 2014) overlap between maturing and mature males of D. plei support the two-life-pathways hypothesis. This hypothesis may help to explain the two switch point ranges found for D. plei (Apostólico & Marian, 2017a). The 169 mm ML switch point is robust to divide sneakers and consorts based on sperm mass relative length and spermatangia morphology. However, the 205–220 mm ML switch point is apparently not associated with ARTs (see discussion in Apostólico & Marian, 2017a). We now hypothesize that this switch point divides the two consort groups, i.e. those that first develop into sneakers and then become smaller consorts (at c. 169 mm ML), and those that delay maturity and become the largest consorts later in life, possibly after reaching c. 205–220 mm ML. Data on D. plei biology appear to support this hypothesis, since the largest maturing males are usually around 200–230 mm ML (Perez et al., 2002). Finally, we cannot exclude the possibility that expression of ARTs in squids is also regulated by their social context, as demonstrated for some gobies, in which particular social contexts (e.g. absence of dominant males, presence of ripe females) may induce a morph change in sequential ARTs (Immler et al., 2004; Scaggiante et al., 2004; Takegaki et al., 2012). Future experimental studies are needed to test this possibility. Concluding remarks This is the first record of intermediate-sized male squids that produce sneaker-like, consort-like and intermediate ejaculates. The present findings are consistent with the predictions of current theoretical models on ARTs (Gross, 1996; Tomkins & Hazel, 2007). As discussed herein, cumulative evidence indicates that ARTs in D. plei are environmentally cued threshold traits, with the trigger for changing tactics depending on body size (Buzatto, Tomkins & Simmons, 2014). Under an ontogenetic gradient in adoption of ARTs, small males might first benefit from sneaking, and later switch to dominant tactics when their large sizes enable them to monopolize resources and mates (Taborsky, 2008), a prediction that also accommodates the present findings. In loliginid squids, which have two distinct sperm deposition sites associated with ARTs, the size-related benefits from switching to the dominant tactic possibly surpass the costs of the radical transition in ejaculate traits described herein. This finding should stimulate future studies on ART expression and evolution, given that the complexity of this system should put the robustness of current ART models to the test. Moreover, squids may now be used as models for studying reversal of tactic-specific investments in male reproductive traits, in addition to some fishes (Immler et al., 2004; Scaggiante et al., 2004; Takegaki et al., 2012). ACKNOWLEDGEMENTS This paper is part of the first author’s Master’s dissertation in Zoology at the University of São Paulo (USP). We obtained a collecting licence from the Instituto Chico Mendes de Conservação da Biodiversidade ICMBio/SISBIO (Permit Number: 44738). In Brazil, ethics approval is still not required for experimentation with cephalopods by the CONCEA (‘Conselho Nacional de Controle de Experimentação Animal’) but we have carried out this study in accordance with international protocols for the welfare of cephalopods and made all efforts to minimize animal suffering. We appreciate the financial support and grants provided by CAPES (Coordenation for the Improvement of Higher Education Personnel), CAPES/PROEX and CNPq (National Council for Scientific and Technological Development – proc. 477233/2013–9). We thank Dr Alvaro E. Migotto (USP) for assistance during in vitro experimentation, and Dr Bruno Buzatto (University of Southwestern Australia), Dr Glauco Machado (USP) and two anonymous reviewers for the comments that helped to improve the quality of the manuscript. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site S1. Age estimation using statoliths in the squid Doryteuthis plei. S2. Spermatophore morphometry of sneakers, consorts and intermediate males of Doryteuthis plei. S3. Sagittal sections of the cement body of spermatophores from an ‘intermediate’ male of Doryteuthis plei. S4. Still images of in vitro spermatophoric reaction of an ‘intermediate’ spermatophore of Doryteuthis plei. S5. Video of in vitro spermatophoric reaction of an ‘intermediate’ spermatophore of Doryteuthis plei. S6. Number of statolith increments per size class in males of Doryteuthis plei. S7. 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Biological Journal of the Linnean SocietyOxford University Press

Published: Mar 1, 2018

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