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Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2
Please cite this article in press as: Hirohashi et al., Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.03.040 Current Biology 23, 1–7, May 6, 2013 ª2013 Elsevier Ltd All rights reserved
http://dx.doi.org/10.1016/j.cub.2013.03.040
Report Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2 Noritaka Hirohashi,1,2,* Luis Alvarez,3 Kogiku Shiba,4 Eiji Fujiwara,5 Yoko Iwata,6 Tatsuma Mohri,7 Kazuo Inaba,4 Kazuyoshi Chiba,2 Hiroe Ochi,2 Claudiu T. Supuran,8 Nico Kotzur,3 Yasutaka Kakiuchi,2 U. Benjamin Kaupp,3 and Shoji A. Baba2 1Oki Marine Biological Station, Education and Research Center for Biological Resources, Shimane University, 194 Kamo, Okinoshima-cho, Oki, Shimane 685-0024, Japan 2Graduate School of Humanities and Sciences, Ochanomizu University, 2-2-1 Otsuka, Tokyo, 112-8610, Japan 3Department of Molecular Sensory Systems, Center for Advanced European Studies and Research (CAESAR), 53175 Bonn, Germany 4Shimoda Marine Research Center, University of Tsukuba, 5-10-1 Shimoda, Shizuoka 415-0025, Japan 5Documentary Channel Co. Ltd., Kawaguchi, Saitama 333-0844, Japan 6Atmosphere and Ocean Research Institute, University of Tokyo, Kashiwa, Chiba 277-8564, Japan 7Section of Individual Researches, National Institute for Physiological Sciences, Okazaki, 444-8585, Japan 8Universita degli Studi di Firenze, Polo Scientifico, Dipartimento di Scienze Farmaceutiche, 50019 Florence, Italy
Summary Behavioral traits of sperm are adapted to the reproductive strategy that each species employs. In polyandrous species, spermatozoa often form motile clusters, which might be advantageous for competing with sperm from other males [1]. Despite this presumed advantage for reproductive success [2, 3], little is known about how sperm form such functional assemblies. Previously, we reported that males of the coastal squid Loligo bleekeri produce two morphologically different euspermatozoa that are linked to distinctly different mating behaviors [4]. Consort and sneaker males use two distinct insemination sites, one inside and one outside the female’s body, respectively. Here, we show that sperm release a self-attracting molecule that causes only sneaker sperm to swarm. We identified CO2 as the sperm chemoattractant and membrane-bound flagellar carbonic anhydrase as its sensor. Downstream signaling results from the generation of extracellular H+, intracellular acidosis, and recovery from acidosis. These signaling events elicit Ca2+-dependent turning behavior, resulting in chemotactic swarming. These results illuminate the bifurcating evolution of sperm underlying the distinct fertilization strategies of this species. Results Sperm from Sneaker Males Exhibit Chemotactic Swarming We tested whether squid sperm form motile clusters by drawing sperm suspensions into capillary tubes. Within 3 min, sneaker, but not consort, sperm swarmed and formed a regularly striped pattern along the capillary axis (Figure 1A;
*Correspondence: [email protected]
Movie S1 available online). The pattern was transient, with a peak at about 4 min, although motility appeared unchanged (Figure S1A). To ascertain that swarming is sneaker-spermspecific, a mixture of sneaker and consort sperm, each labeled with a different mitochondrial dye, was introduced into the tube. Independent of the dye, only sneaker sperm formed swarms (Figure 1B, one dye combination shown). Swarming did not involve impaired motility or binding of sperm to each other; rather, each cell in the swarm swam independently (Figure 1C). We tested whether swarming was caused by a chemical factor using a filter assay (Figure 1D). In brief, two chambers were separated by a filter that allowed only small molecules to pass. A diluted sperm suspension was added to the lower chamber. Then, we tested whether sperm swarmed near the filter while gradually adding sperm to the upper chamber. Only sneaker sperm migrated toward the upper chamber (Figures 1E and 1F), suggesting that swarming is mediated by a chemical factor produced by sperm in the upper chamber. Notably, when consort or starfish (Asterina pectinifera) sperm were placed in the upper chamber, sneaker sperm were also attracted, suggesting that the attractant is a molecule generated by sperm themselves (Figure 1E). Sperm Chemotaxis is Mediated by CO2 Other known chemoattractants, such as cAMP for the amoeba Dictyostelium discoideum [5], dicarboxylic acids for fern spermatozoids [6], L-tryptophan for abalone sperm [7], and L-aspartate, sugars, or amino acids for bacteria [8], were inactive in our test system (Figure S1B). Finally, we observed that CO2 attracted sneaker, but not consort, sperm (Figures 1G and S2A; Movie S2). Thus, we reasoned that chemotactic swarming is mediated by sperm-emitted respiratory CO2 or other molecules generated by the carbonate system. In artificial seawater (ASW), several chemical reactions interconnect the molecules that make up the carbonate system. [9]: CO2 + H2 O!HCO32 + H + CO2 + OH 2 !HCO32 CO23 2 + H + !HCO32 HCO32 + OH 2 !CO23 2 + H2 O To dissect this complex system, we developed caged carbonate (Figures S2B–S2D) to sculpture gradients of bicarbonate, H+, and CO2. Single sperm were studied while swimming in ASW containing 200 mM caged carbonate. Upon photolysis (0.1 s), only sneaker sperm were swarming to the irradiated area (Figure 1H; Movie S3) without a significant decrease in their swimming speed (before the flash, 166 6 3 mm/s, n = 85; after the flash, 163 6 3 mm/s, n = 102), thus ruling out trapping effects. Swarming was maximal about 20 s after release and lasted for at least 60 s (Figure 1I). When 10 mM HEPES was present, swarming was abolished, indicating that changes in the pH of the medium (pHe) are essential for swarming. We calculated the spatiotemporal distribution of CO2, bicarbonate, and H+. After release, the predicted relative change in [H+] was large (Figure 1J). Because released H+ are buffered, their relative concentration decreased abruptly within the first 5 s to values similar to those predicted for CO2 (relative
Please cite this article in press as: Hirohashi et al., Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.03.040 Current Biology Vol 23 No 9 2
Figure 1. Chemotactic Swarming of Squid Sperm in Response to CO2 (A) Top (0 min) and bottom (3 min) panels show first and last images of capillaries containing 107 sperm/ml (green). Middle panels show time course. (B) Selective swarming by sneaker sperm (red) mixed with consort sperm (green). (C) Swimming paths of individual sperm after one, five, and ten frames; 30 frames = 1 s. (D) A diagram of the filter assay that does not allow sperm passage between the two chambers. (E) A normalized number of swim-up sperm in the lower chamber before (black columns) and 5 min after (gray columns) the addition of sperm to the upper chamber (mean 6 SEM; n = 3). S, sneaker; C, consort; sf, starfish. (F) Representative swim-up sneaker sperm before (left) and after (right) the addition of sperm to the upper chamber. Scale bars represent 200 mm. (G) Rapid swarming to a CO2 bubble by sneaker, but not consort, sperm. Scale bars represent 200 mm. (H) Sperm suspended into seawater containing 200 mM BCMACM-caged carbonate were stimulated by photolysis (0.1 s) of the caged compound. Photographs show a superposition of all frames 5 s before (top) and 5 s after (bottom) UV illumination (middle). (I) Changes in sperm dispersion around the center of the flash upon carbonate release (black dashed line; n = 8). Seawater was either buffered by 10 mM HEPES (+) or not (2). Experiments were carried out with sneaker (S) or consort sperm (C). Scale bars represent 200 mm. Error bars represent a 95% confidence interval. (J) Numerically calculated concentration profiles of H+, CO2, and bicarbonate after the release of carbonate in the presence or absence of 10 mM HEPES. The inset shows an H+ profile at t = 0.
Please cite this article in press as: Hirohashi et al., Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.03.040 Sperm Navigation by CO2 Sensing 3
Figure 2. Membrane-Bound Carbonic Anhydrase as a CO2 Sensor (A) End-point capillary swarming assay in the presence of CA inhibitors; zinc chelator, 1,10-Phenanthroline (PNT), or acetazolamide (ATZ). The scale bar represents 200 mm. (B and C) Immunoblots of whole sperm extracts. Live sperm from consort (C) or sneaker (S) males were treated with or without proteinase K before extraction. (C) Immunolocalization of sperm carbonic anhydrase with or without proteinase K treatment before fixation. The staining in the absence of the primary antibody is shown as a control (Cont.). The scale bar represents 10 mm. (D) Measurements of O2 consumption, CO2 emission, and extracellular and intracellular acidosis of sperm in the presence (red) or absence (gray) of 2 mM acetazolamide. Aquatic [O2] and [CO2] were simultaneously measured. (E) Absence of pHi homeostasis in sneaker sperm against acidosis. Quantification of CO2 emission (top) and extracellular (middle) and intracellular (bottom) acidosis relative to O2 consumption in sneaker (S) or consort (C) spermatozoa in the presence (red) or absence (gray) of 2 mM ATZ. O2 consumption was monitored simultaneously for all measurements (mean 6 SEM; n = 3).
changes of w190% for H+ and w90% for CO2). In contrast, the predicted change in [HCO32] was low (w12% after 5 s), indicating that bicarbonate does not mediate sperm swarming. This was further supported by experiments with HEPES (Figure 1J). Assuming that H+ were buffered by HEPES, the [CO2] would not change substantially after a flash (relative change w1.5%). In contrast, the [HCO32] increased to values similar to those obtained without HEPES (w7% after 5 s). In summary, our simulations predict that swarming depends on CO2 and/or H+ gradients but not on a bicarbonate gradient (Figures 1I and 1J). Flagellar Carbonic Anhydrase Serves as a CO2 Sensor Carbonic anhydrases (CAs) serve as CO2 sensors in many cellular systems [10, 11]; therefore, we tested CA inhibitors
(e.g., acetazolamide (ATZ) and 1,10-Phenanthroline) and observed an inhibitory effect on swarming by these compounds (Figure 2A and S3A–S3C). A full-length complementary DNA encoding a CA from both sneaker and consort testes was cloned and revealed high similarity to membrane-anchored CA isoforms (Figures S3D and S3E). We generated an antibody against a synthetic peptide to confirm protein expression in both sperm types. Western blots of whole-cell extracts identified a w31.7 kDa band (the calculated molecular mass of cloned CA is 28.8 kDa) (Figure 2B). The flagella of sneaker and consort sperm were stained by the antibody (Figure 2C), and staining was diminished by treating live sperm with proteinase K, indicating that the CA localizes to the cell surface (Figures 2B and 2C). We also examined CO2 metabolism and found that sneaker and consort sperm converted respiratory
Please cite this article in press as: Hirohashi et al., Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.03.040 Current Biology Vol 23 No 9 4
Figure 3. An Extracellular pH Gradient Establishes Chemotactic Swarming (A) The spatiotemporal formation of a pHe gradient coincides with the development of the swarm. Ratiometric measurements with BCECF-dextran (left panels) were performed to measure the pHe. Right panels represent bright-field images of the same experiment. (B) Absence of swarm formation in buffered seawater. (C) Sperm chemotaxis toward acid-loaded pipettes. Sneaker and consort sperm exhibit acidotaxis but with different sensitivities. (D) Absence of pHi homeostasis in sneaker sperm against acidosis. (E) Intracellular acidosis or alkalosis evokes a positive or negative chemotactic response in buffered seawater. These responses were observed in the acidic range (pH 5.0–6.0), which mimics swarming conditions. Representative results of three independent experiments are shown. Scale bars represent 200 mm.
CO2 to H+ and HCO32 and, thereby, lowered the pHe (Figures 2D and 2E). These results suggest that CA activity is involved in chemotactic swarming. However, if sneaker and consort sperm command over a similar inventory of CA isoforms, why do only sneaker sperm display chemotaxis? An Extracellular H+ Gradient Causes Swarming Considering the complex CO2-HCO32-H+ equilibrium rapidly established by CA, any of these molecules might serve as chemoattractant. We hypothesized that sneaker sperm detect H+ gradients by which swarming is enabled. We measured pHe using a pH-sensitive dye during swarming. Acidification of the medium started near the swarm center and expanded during swarm growth, which produced an H+ gradient at the swarm boundary (Figure 3A). When the formation of the pHe gradient was attenuated by pH-buffered ASW (10 mM Tris or HEPES), no swarming was observed (Figure 3B). Finally, when an acid-loaded pipette was inserted into a sperm suspension, both sneaker sperm (>pH 5.0) and consort sperm (>pH 4.0) showed a chemotactic response to acid (acidotaxis) and kept swarming near the pipette for some time (w30 min; Figures 3C andS4; Movie S4). Sperm did not respond to a pipette loaded with 50 mM bicarbonate (pH 8.0) (data not shown), further demonstrating that HCO32 itself is not an attractant. Why do consort sperm show acidotaxis but not CO2 taxis? First, the sensitivity of acid detection is w1 pH unit higher in sneaker than in consort sperm (Figure 3C). Moreover, only sneaker sperm lowered their intracellular pH (pHi) concomitantly with pHe (Figures 2D and 2E), suggesting that only sneaker sperm are equipped with an H+ transport system that conveys CO2 taxis. To explore this hypothesis, we
examined pHi homeostasis at various pHe using buffered ASW. Both sneaker and consort sperm were similar in maintaining their pHi against alkalosis. However, only consort sperm showed pHi homeostasis against acidosis (Figure 3D). When sperm swim up or down an H+ gradient, the changes in pHe are reflected by changes in pHi. To test whether pHi changes mediate chemotactic responses, sperm were placed in buffered ASW (with a pH of 8.0 or 6.0); then, a pipette filled with 1 M sodium acetate (NaAc)-soaked agarose gel (also with a pH of 8.0 or 6.0) was inserted. Because NaAc readily crosses the plasma membrane and causes cytoplasmic acidosis, the pHi changed according to the swimming direction; sperm swimming toward the pipette acidified, and those swimming away recovered from cytoplasmic acidosis at constant pHe. Sperm from sneaker males, but not consort males (data not shown), showed directional movements toward the pipette when pHe was adjusted to 6.0 (Figure 3E). Conversely, when a pipette loaded with ammonium chloride (pH 5.0) (an alkalosis-inducing agent) was placed in ASW at pH 5.0, sneaker sperm swam away from the pipette; i.e., they showed chemorepellent behavior (Figure 3E). These results suggest that pHe changes also affect pHi, thereby facilitating directional movement to establish and maintain the swarm formation. Recovery from Intracellular Acidosis Evokes Calcium-Dependent Turning Responses The looping swimming path of sperm from sea urchins [12–14] and ascidians [15] while approaching the chemoattractant source alternates between low curvature (runs) and high curvature (turns). Ca2+ influx controls this swimming pattern. L. bleekeri sperm also require extracellular Ca2+ for swarming
Please cite this article in press as: Hirohashi et al., Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.03.040 Sperm Navigation by CO2 Sensing 5
Figure 4. Recovery from Acidosis Evokes a Calcium-Dependent Turn Episode (A) Two-dimensional swimming paths near the acid-loaded pipette (pH 4.0) in the presence or absence of extracellular Ca2+. A representative swimming path in the presence of Ca2+ is shown (right box). (B) Sperm swimming paths (bottom; 1 min) and quantification of the sperm density along the swarm (top). The border zone of the swarm is defined in the red box. L and R indicate the left and right boundary, respectively. The scale bar represents 100 mm. (C) Representative swimming paths, classified into four different patterns of in or out direction combinations of swimming sperm in the border zone. (D) Frequency of appearance of the four categories of the paths classified in (C). Each bar represents the mean 6 SEM (n = 3). (E) Change in curvature of the swimming path upon intracellular release of caged Ca2+. Shown are the representative cases of individual sneaker sperm. The inset shows a representative swimming path before (dark blue), during (red), and after photolysis (light blue). (F) Swimming paths (1 s) of sperm in Ca2+-free seawater (pH 8.0) in the vicinity of a Ca2+-loaded pipette. Sperm were pretreated in acidic (pH 5.0) or control (pH 8.0) seawater. Sneaker sperm frequently tumbled (red) when pretreated at pH 5.0. The scale bar represents 100 mm. (G) A model of the CO2 metabolic and signaling pathway in squid sperm. Respiratory CO2 emitted from self and sibling sperm is hydrated by the flagellummembrane-bound carbonic anhydrase (CA) into bicarbonate and H+. Only sneaker, and not consort, sperm take up extracellular H+ that is generated, resulting in intracellular acidification. When sperm swim along a descending CO2/H+ gradient, recovery from acidification evokes a Ca2+ influx and changes the flagellar waveform.
(Figures 4A and S3A). We analyzed the swimming path of those sperm entering the border zone (between the L and R lines) of the swarming region (Figure 4B). Sperm approaching a swarm and entering the border zone by crossing the L line maintain straight swimming paths; approximately 80% of these sperm joined the swarm. In contrast, sperm leaving a swarm make frequent turns and w90% of those sperm returned to the swarm (Figures 4C and D). These results demonstrate that sperm swarming is driven by chemotaxis; however, contributions from chemokinesis or trapping cannot be entirely disregarded.
Analysis of two-dimensional swimming paths showed that the chemotactic response consisted of a turning motion followed by a period of straight swimming toward the chemical source (Figure 4A inset). We asked whether the increase of path curvature (turn) is initiated by an increase of internal [Ca2+] ([Ca2+]i). Unfortunately, we failed to image changes in flagellar [Ca2+]i, because the flagellum of squid sperm frequently beated out of focus, making the quantification of [Ca2+]i unreliable. Moreover, loading sperm with Ca2+ dyes severely attenuated the CO2 response (Movie S5). Therefore, we took alternative approaches to show that Ca2+ is involved.
Please cite this article in press as: Hirohashi et al., Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.03.040 Current Biology Vol 23 No 9 6
First, photolysis (30 ms) of caged Ca2+ (o-nitrophenyl-EGTA, AM) loaded into sperm evoked a rapid motor response. Shortly after the flash, curvilinear swimming was interrupted by a turn, followed by return to straight swimming (Figure 4E). Second, sperm preincubated in acidic ASW (pH 5.0) were placed in Ca2+-free ASW (pH 8.0) in front of a pipette loaded with ASW containing normal [Ca2+]. Both types of sperm, regardless of preincubation conditions, exhibited mostly straight swimming in Ca2+-free ASW. However, only sneaker sperm that were preincubated in an acidic medium displayed turning episodes in the vicinity of the Ca2+-loaded pipette (Figure 4F). These results suggest that, in sneaker sperm, recovery from acidosis elicits a Ca2+ influx that triggers a turn episode (Figure 4G). Discussion Some coastal squids in the loliginidae employ alternative mating tactics [4, 16, 17]. Large males engage in male-male agonistic bouts and the winner—usually the largest squid— forms a temporary consortship with a female, mates her in parallel position, and guards her from other males while she lays egg fingers. These consort male deposit spermatophores around or in the oviduct opening in the mantle cavity of the female. In contrast, small males do not engage in agonistic bouts, but rather use sneaker mating tactics, such as mating head-to-head and darting spermatophores at an external location near the sperm storage organ (the seminal receptacle). When females extrude an egg capsule from their oviduct, consort sperm have first access to the egg capsules (akin to internal fertilization) and fertilize a large number of eggs. Then, females hold the egg capsule in their arms as they approach the spawning substrate, and, at this time, sperm from sneaker males have access to the egg capsules as well (akin to external fertilization). We previously reported a tight link between sperm phenotype and mating tactics [4]. However, it is not known whether mating tactics depend on the presence of rivals during mating [18] or switched ontogenetically during the life cycle. Nonetheless, squid loliginid sperm are unique in that internal and external fertilization coexist during a single spawning episode. Previously, we reported that sneaker sperm are w50% longer than consort sperm [4]. Although no such clear difference of within-species dimorphic eusperm had been reported, sperm size differences among closely related species have been interpreted as sperm competition [19–21]. However, the landscape of fertilization might have driven the evolution of sperm in L. bleekeri [4]. In this study, we show that sneaker sperm form motile assemblies and stay close to the spermatophore (Movie S6). We also show that CO2 causes sperm from sneaker males, but not consort males, to form assemblies. From an ecological point of view, retaining the ejaculates at the site of egg deposition enhances the chances of fertilization, because mating and egg laying are temporally independent [22, 23]. Thus, it will be intriguing to study why only sneaker sperm acquired the swarming trait in light of both postcopulatory sexual selection [24] and natural selection [2]. In addition, sneaker sperm might sense CO2 released from eggs, and fertilization by sneaker sperm might rely on CO2 chemotaxis toward the egg [25]. The carbonate system is important for many organisms and cells. CO2/acid detection by several sensory systems [10, 26–31] and the central nervous system [32] may share common mechanisms with CO2 taxis in squid sperm in terms
of intracellular acidosis via transmembrane H+ currents [27] and Ca2+ responses after recovery from an acid challenge called ‘‘off-response’’ [28]. However, the complexity of coupled chemical equilibria between chemical species and across cell membranes has largely been neglected. It is inherently difficult to trace down the active agent and to predict the consequences of altering one component or the other. Moreover, the ion channels and transport systems that mediate pH changes and generate the Ca2+ response are unknown. A unique, voltage-gated Na+/H+ exchanger [33] and an H+-selective channel [34] have been identified in the sperm flagellum. Moreover, a pH-sensitive K+ channel (KSper) [35–37] and a pH-sensitive Ca2+ channel (CatSper) [38] transduce changes in pHi into voltage and Ca2+ responses. Additional studies are required to precisely identify and delineate the sequence of signaling events during CO2 taxis or acidotaxis in sperm. We identified a plasma-membrane-anchored CA; however, soluble CAs might also play a role in CO2 taxis, because CO2 can diffuse across the cell membrane and soluble CAs have been identified as components of cellular acidification [11, 39]. Isoform-specific inhibitors of CAs are required in order to dissect the contribution of intra versus extracellular CAs to CO2 metabolism. Finally, a novel caged carbonate, synthesized for this study, represents a powerful tool for unravelling the molecular pathways of CO2/H+ sensation not only in sperm but also in olfactory, gustatory, or neuronal systems. Supplemental Information Supplemental Information contains Supplemental Experimental Procedures, four figures, and six movies and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2013.03.040. Acknowledgments We thank Victor D. Vacquier for his critical reading and comments on the manuscript. We thank H. Mieda, F. Nakaya, T. Nishigaki, K. Moriwaki, N. Sato, C. Ohkami, and R. Pascal for their technical assistance. This work was supported by a Narishige Zoological Science Award, the Research Institute of Marine Invertebrates, the Yamada Science Foundation, a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a grant from the Japanese Association for Marine Biology to N.H. Received: November 5, 2011 Revised: January 28, 2013 Accepted: March 14, 2013 Published: April 11, 2013 References 1. Fisher, H.S., and Hoekstra, H.E. (2010). Competition drives cooperation among closely related sperm of deer mice. Nature 463, 801–803. 2. Foster, K.R., and Pizzari, T. (2010). Cooperation: the secret society of sperm. Curr. Biol. 20, R314–R316. 3. Higginson, D.M., and Pitnick, S. (2011). Evolution of intra-ejaculate sperm interactions: do sperm cooperate? Biol. Rev. Camb. Philos. Soc. 86, 249–270. 4. Iwata, Y., Shaw, P., Fujiwara, E., Shiba, K., Kakiuchi, Y., and Hirohashi, N. (2011). Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours. BMC Evol. Biol. 11, 236. 5. Kimmel, A.R., and Parent, C.A. (2003). Dictyostelium discoideum cAMP Chemotaxis Pathway. Sci. STKE 2003, cm1. 6. Brokaw, C.J. (1957). Electro-chemical orientation of bracken spermatozoids. Nature 179, 525. 7. Riffell, J.A., Krug, P.J., and Zimmer, R.K. (2002). Fertilization in the sea: the chemical identity of an abalone sperm attractant. J. Exp. Biol. 205, 1439–1450.
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http://dx.doi.org/10.1016/j.cub.2013.03.040
Report Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2 Noritaka Hirohashi,1,2,* Luis Alvarez,3 Kogiku Shiba,4 Eiji Fujiwara,5 Yoko Iwata,6 Tatsuma Mohri,7 Kazuo Inaba,4 Kazuyoshi Chiba,2 Hiroe Ochi,2 Claudiu T. Supuran,8 Nico Kotzur,3 Yasutaka Kakiuchi,2 U. Benjamin Kaupp,3 and Shoji A. Baba2 1Oki Marine Biological Station, Education and Research Center for Biological Resources, Shimane University, 194 Kamo, Okinoshima-cho, Oki, Shimane 685-0024, Japan 2Graduate School of Humanities and Sciences, Ochanomizu University, 2-2-1 Otsuka, Tokyo, 112-8610, Japan 3Department of Molecular Sensory Systems, Center for Advanced European Studies and Research (CAESAR), 53175 Bonn, Germany 4Shimoda Marine Research Center, University of Tsukuba, 5-10-1 Shimoda, Shizuoka 415-0025, Japan 5Documentary Channel Co. Ltd., Kawaguchi, Saitama 333-0844, Japan 6Atmosphere and Ocean Research Institute, University of Tokyo, Kashiwa, Chiba 277-8564, Japan 7Section of Individual Researches, National Institute for Physiological Sciences, Okazaki, 444-8585, Japan 8Universita degli Studi di Firenze, Polo Scientifico, Dipartimento di Scienze Farmaceutiche, 50019 Florence, Italy
Summary Behavioral traits of sperm are adapted to the reproductive strategy that each species employs. In polyandrous species, spermatozoa often form motile clusters, which might be advantageous for competing with sperm from other males [1]. Despite this presumed advantage for reproductive success [2, 3], little is known about how sperm form such functional assemblies. Previously, we reported that males of the coastal squid Loligo bleekeri produce two morphologically different euspermatozoa that are linked to distinctly different mating behaviors [4]. Consort and sneaker males use two distinct insemination sites, one inside and one outside the female’s body, respectively. Here, we show that sperm release a self-attracting molecule that causes only sneaker sperm to swarm. We identified CO2 as the sperm chemoattractant and membrane-bound flagellar carbonic anhydrase as its sensor. Downstream signaling results from the generation of extracellular H+, intracellular acidosis, and recovery from acidosis. These signaling events elicit Ca2+-dependent turning behavior, resulting in chemotactic swarming. These results illuminate the bifurcating evolution of sperm underlying the distinct fertilization strategies of this species. Results Sperm from Sneaker Males Exhibit Chemotactic Swarming We tested whether squid sperm form motile clusters by drawing sperm suspensions into capillary tubes. Within 3 min, sneaker, but not consort, sperm swarmed and formed a regularly striped pattern along the capillary axis (Figure 1A;
*Correspondence: [email protected]
Movie S1 available online). The pattern was transient, with a peak at about 4 min, although motility appeared unchanged (Figure S1A). To ascertain that swarming is sneaker-spermspecific, a mixture of sneaker and consort sperm, each labeled with a different mitochondrial dye, was introduced into the tube. Independent of the dye, only sneaker sperm formed swarms (Figure 1B, one dye combination shown). Swarming did not involve impaired motility or binding of sperm to each other; rather, each cell in the swarm swam independently (Figure 1C). We tested whether swarming was caused by a chemical factor using a filter assay (Figure 1D). In brief, two chambers were separated by a filter that allowed only small molecules to pass. A diluted sperm suspension was added to the lower chamber. Then, we tested whether sperm swarmed near the filter while gradually adding sperm to the upper chamber. Only sneaker sperm migrated toward the upper chamber (Figures 1E and 1F), suggesting that swarming is mediated by a chemical factor produced by sperm in the upper chamber. Notably, when consort or starfish (Asterina pectinifera) sperm were placed in the upper chamber, sneaker sperm were also attracted, suggesting that the attractant is a molecule generated by sperm themselves (Figure 1E). Sperm Chemotaxis is Mediated by CO2 Other known chemoattractants, such as cAMP for the amoeba Dictyostelium discoideum [5], dicarboxylic acids for fern spermatozoids [6], L-tryptophan for abalone sperm [7], and L-aspartate, sugars, or amino acids for bacteria [8], were inactive in our test system (Figure S1B). Finally, we observed that CO2 attracted sneaker, but not consort, sperm (Figures 1G and S2A; Movie S2). Thus, we reasoned that chemotactic swarming is mediated by sperm-emitted respiratory CO2 or other molecules generated by the carbonate system. In artificial seawater (ASW), several chemical reactions interconnect the molecules that make up the carbonate system. [9]: CO2 + H2 O!HCO32 + H + CO2 + OH 2 !HCO32 CO23 2 + H + !HCO32 HCO32 + OH 2 !CO23 2 + H2 O To dissect this complex system, we developed caged carbonate (Figures S2B–S2D) to sculpture gradients of bicarbonate, H+, and CO2. Single sperm were studied while swimming in ASW containing 200 mM caged carbonate. Upon photolysis (0.1 s), only sneaker sperm were swarming to the irradiated area (Figure 1H; Movie S3) without a significant decrease in their swimming speed (before the flash, 166 6 3 mm/s, n = 85; after the flash, 163 6 3 mm/s, n = 102), thus ruling out trapping effects. Swarming was maximal about 20 s after release and lasted for at least 60 s (Figure 1I). When 10 mM HEPES was present, swarming was abolished, indicating that changes in the pH of the medium (pHe) are essential for swarming. We calculated the spatiotemporal distribution of CO2, bicarbonate, and H+. After release, the predicted relative change in [H+] was large (Figure 1J). Because released H+ are buffered, their relative concentration decreased abruptly within the first 5 s to values similar to those predicted for CO2 (relative
Please cite this article in press as: Hirohashi et al., Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.03.040 Current Biology Vol 23 No 9 2
Figure 1. Chemotactic Swarming of Squid Sperm in Response to CO2 (A) Top (0 min) and bottom (3 min) panels show first and last images of capillaries containing 107 sperm/ml (green). Middle panels show time course. (B) Selective swarming by sneaker sperm (red) mixed with consort sperm (green). (C) Swimming paths of individual sperm after one, five, and ten frames; 30 frames = 1 s. (D) A diagram of the filter assay that does not allow sperm passage between the two chambers. (E) A normalized number of swim-up sperm in the lower chamber before (black columns) and 5 min after (gray columns) the addition of sperm to the upper chamber (mean 6 SEM; n = 3). S, sneaker; C, consort; sf, starfish. (F) Representative swim-up sneaker sperm before (left) and after (right) the addition of sperm to the upper chamber. Scale bars represent 200 mm. (G) Rapid swarming to a CO2 bubble by sneaker, but not consort, sperm. Scale bars represent 200 mm. (H) Sperm suspended into seawater containing 200 mM BCMACM-caged carbonate were stimulated by photolysis (0.1 s) of the caged compound. Photographs show a superposition of all frames 5 s before (top) and 5 s after (bottom) UV illumination (middle). (I) Changes in sperm dispersion around the center of the flash upon carbonate release (black dashed line; n = 8). Seawater was either buffered by 10 mM HEPES (+) or not (2). Experiments were carried out with sneaker (S) or consort sperm (C). Scale bars represent 200 mm. Error bars represent a 95% confidence interval. (J) Numerically calculated concentration profiles of H+, CO2, and bicarbonate after the release of carbonate in the presence or absence of 10 mM HEPES. The inset shows an H+ profile at t = 0.
Please cite this article in press as: Hirohashi et al., Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.03.040 Sperm Navigation by CO2 Sensing 3
Figure 2. Membrane-Bound Carbonic Anhydrase as a CO2 Sensor (A) End-point capillary swarming assay in the presence of CA inhibitors; zinc chelator, 1,10-Phenanthroline (PNT), or acetazolamide (ATZ). The scale bar represents 200 mm. (B and C) Immunoblots of whole sperm extracts. Live sperm from consort (C) or sneaker (S) males were treated with or without proteinase K before extraction. (C) Immunolocalization of sperm carbonic anhydrase with or without proteinase K treatment before fixation. The staining in the absence of the primary antibody is shown as a control (Cont.). The scale bar represents 10 mm. (D) Measurements of O2 consumption, CO2 emission, and extracellular and intracellular acidosis of sperm in the presence (red) or absence (gray) of 2 mM acetazolamide. Aquatic [O2] and [CO2] were simultaneously measured. (E) Absence of pHi homeostasis in sneaker sperm against acidosis. Quantification of CO2 emission (top) and extracellular (middle) and intracellular (bottom) acidosis relative to O2 consumption in sneaker (S) or consort (C) spermatozoa in the presence (red) or absence (gray) of 2 mM ATZ. O2 consumption was monitored simultaneously for all measurements (mean 6 SEM; n = 3).
changes of w190% for H+ and w90% for CO2). In contrast, the predicted change in [HCO32] was low (w12% after 5 s), indicating that bicarbonate does not mediate sperm swarming. This was further supported by experiments with HEPES (Figure 1J). Assuming that H+ were buffered by HEPES, the [CO2] would not change substantially after a flash (relative change w1.5%). In contrast, the [HCO32] increased to values similar to those obtained without HEPES (w7% after 5 s). In summary, our simulations predict that swarming depends on CO2 and/or H+ gradients but not on a bicarbonate gradient (Figures 1I and 1J). Flagellar Carbonic Anhydrase Serves as a CO2 Sensor Carbonic anhydrases (CAs) serve as CO2 sensors in many cellular systems [10, 11]; therefore, we tested CA inhibitors
(e.g., acetazolamide (ATZ) and 1,10-Phenanthroline) and observed an inhibitory effect on swarming by these compounds (Figure 2A and S3A–S3C). A full-length complementary DNA encoding a CA from both sneaker and consort testes was cloned and revealed high similarity to membrane-anchored CA isoforms (Figures S3D and S3E). We generated an antibody against a synthetic peptide to confirm protein expression in both sperm types. Western blots of whole-cell extracts identified a w31.7 kDa band (the calculated molecular mass of cloned CA is 28.8 kDa) (Figure 2B). The flagella of sneaker and consort sperm were stained by the antibody (Figure 2C), and staining was diminished by treating live sperm with proteinase K, indicating that the CA localizes to the cell surface (Figures 2B and 2C). We also examined CO2 metabolism and found that sneaker and consort sperm converted respiratory
Please cite this article in press as: Hirohashi et al., Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.03.040 Current Biology Vol 23 No 9 4
Figure 3. An Extracellular pH Gradient Establishes Chemotactic Swarming (A) The spatiotemporal formation of a pHe gradient coincides with the development of the swarm. Ratiometric measurements with BCECF-dextran (left panels) were performed to measure the pHe. Right panels represent bright-field images of the same experiment. (B) Absence of swarm formation in buffered seawater. (C) Sperm chemotaxis toward acid-loaded pipettes. Sneaker and consort sperm exhibit acidotaxis but with different sensitivities. (D) Absence of pHi homeostasis in sneaker sperm against acidosis. (E) Intracellular acidosis or alkalosis evokes a positive or negative chemotactic response in buffered seawater. These responses were observed in the acidic range (pH 5.0–6.0), which mimics swarming conditions. Representative results of three independent experiments are shown. Scale bars represent 200 mm.
CO2 to H+ and HCO32 and, thereby, lowered the pHe (Figures 2D and 2E). These results suggest that CA activity is involved in chemotactic swarming. However, if sneaker and consort sperm command over a similar inventory of CA isoforms, why do only sneaker sperm display chemotaxis? An Extracellular H+ Gradient Causes Swarming Considering the complex CO2-HCO32-H+ equilibrium rapidly established by CA, any of these molecules might serve as chemoattractant. We hypothesized that sneaker sperm detect H+ gradients by which swarming is enabled. We measured pHe using a pH-sensitive dye during swarming. Acidification of the medium started near the swarm center and expanded during swarm growth, which produced an H+ gradient at the swarm boundary (Figure 3A). When the formation of the pHe gradient was attenuated by pH-buffered ASW (10 mM Tris or HEPES), no swarming was observed (Figure 3B). Finally, when an acid-loaded pipette was inserted into a sperm suspension, both sneaker sperm (>pH 5.0) and consort sperm (>pH 4.0) showed a chemotactic response to acid (acidotaxis) and kept swarming near the pipette for some time (w30 min; Figures 3C andS4; Movie S4). Sperm did not respond to a pipette loaded with 50 mM bicarbonate (pH 8.0) (data not shown), further demonstrating that HCO32 itself is not an attractant. Why do consort sperm show acidotaxis but not CO2 taxis? First, the sensitivity of acid detection is w1 pH unit higher in sneaker than in consort sperm (Figure 3C). Moreover, only sneaker sperm lowered their intracellular pH (pHi) concomitantly with pHe (Figures 2D and 2E), suggesting that only sneaker sperm are equipped with an H+ transport system that conveys CO2 taxis. To explore this hypothesis, we
examined pHi homeostasis at various pHe using buffered ASW. Both sneaker and consort sperm were similar in maintaining their pHi against alkalosis. However, only consort sperm showed pHi homeostasis against acidosis (Figure 3D). When sperm swim up or down an H+ gradient, the changes in pHe are reflected by changes in pHi. To test whether pHi changes mediate chemotactic responses, sperm were placed in buffered ASW (with a pH of 8.0 or 6.0); then, a pipette filled with 1 M sodium acetate (NaAc)-soaked agarose gel (also with a pH of 8.0 or 6.0) was inserted. Because NaAc readily crosses the plasma membrane and causes cytoplasmic acidosis, the pHi changed according to the swimming direction; sperm swimming toward the pipette acidified, and those swimming away recovered from cytoplasmic acidosis at constant pHe. Sperm from sneaker males, but not consort males (data not shown), showed directional movements toward the pipette when pHe was adjusted to 6.0 (Figure 3E). Conversely, when a pipette loaded with ammonium chloride (pH 5.0) (an alkalosis-inducing agent) was placed in ASW at pH 5.0, sneaker sperm swam away from the pipette; i.e., they showed chemorepellent behavior (Figure 3E). These results suggest that pHe changes also affect pHi, thereby facilitating directional movement to establish and maintain the swarm formation. Recovery from Intracellular Acidosis Evokes Calcium-Dependent Turning Responses The looping swimming path of sperm from sea urchins [12–14] and ascidians [15] while approaching the chemoattractant source alternates between low curvature (runs) and high curvature (turns). Ca2+ influx controls this swimming pattern. L. bleekeri sperm also require extracellular Ca2+ for swarming
Please cite this article in press as: Hirohashi et al., Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.03.040 Sperm Navigation by CO2 Sensing 5
Figure 4. Recovery from Acidosis Evokes a Calcium-Dependent Turn Episode (A) Two-dimensional swimming paths near the acid-loaded pipette (pH 4.0) in the presence or absence of extracellular Ca2+. A representative swimming path in the presence of Ca2+ is shown (right box). (B) Sperm swimming paths (bottom; 1 min) and quantification of the sperm density along the swarm (top). The border zone of the swarm is defined in the red box. L and R indicate the left and right boundary, respectively. The scale bar represents 100 mm. (C) Representative swimming paths, classified into four different patterns of in or out direction combinations of swimming sperm in the border zone. (D) Frequency of appearance of the four categories of the paths classified in (C). Each bar represents the mean 6 SEM (n = 3). (E) Change in curvature of the swimming path upon intracellular release of caged Ca2+. Shown are the representative cases of individual sneaker sperm. The inset shows a representative swimming path before (dark blue), during (red), and after photolysis (light blue). (F) Swimming paths (1 s) of sperm in Ca2+-free seawater (pH 8.0) in the vicinity of a Ca2+-loaded pipette. Sperm were pretreated in acidic (pH 5.0) or control (pH 8.0) seawater. Sneaker sperm frequently tumbled (red) when pretreated at pH 5.0. The scale bar represents 100 mm. (G) A model of the CO2 metabolic and signaling pathway in squid sperm. Respiratory CO2 emitted from self and sibling sperm is hydrated by the flagellummembrane-bound carbonic anhydrase (CA) into bicarbonate and H+. Only sneaker, and not consort, sperm take up extracellular H+ that is generated, resulting in intracellular acidification. When sperm swim along a descending CO2/H+ gradient, recovery from acidification evokes a Ca2+ influx and changes the flagellar waveform.
(Figures 4A and S3A). We analyzed the swimming path of those sperm entering the border zone (between the L and R lines) of the swarming region (Figure 4B). Sperm approaching a swarm and entering the border zone by crossing the L line maintain straight swimming paths; approximately 80% of these sperm joined the swarm. In contrast, sperm leaving a swarm make frequent turns and w90% of those sperm returned to the swarm (Figures 4C and D). These results demonstrate that sperm swarming is driven by chemotaxis; however, contributions from chemokinesis or trapping cannot be entirely disregarded.
Analysis of two-dimensional swimming paths showed that the chemotactic response consisted of a turning motion followed by a period of straight swimming toward the chemical source (Figure 4A inset). We asked whether the increase of path curvature (turn) is initiated by an increase of internal [Ca2+] ([Ca2+]i). Unfortunately, we failed to image changes in flagellar [Ca2+]i, because the flagellum of squid sperm frequently beated out of focus, making the quantification of [Ca2+]i unreliable. Moreover, loading sperm with Ca2+ dyes severely attenuated the CO2 response (Movie S5). Therefore, we took alternative approaches to show that Ca2+ is involved.
Please cite this article in press as: Hirohashi et al., Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.03.040 Current Biology Vol 23 No 9 6
First, photolysis (30 ms) of caged Ca2+ (o-nitrophenyl-EGTA, AM) loaded into sperm evoked a rapid motor response. Shortly after the flash, curvilinear swimming was interrupted by a turn, followed by return to straight swimming (Figure 4E). Second, sperm preincubated in acidic ASW (pH 5.0) were placed in Ca2+-free ASW (pH 8.0) in front of a pipette loaded with ASW containing normal [Ca2+]. Both types of sperm, regardless of preincubation conditions, exhibited mostly straight swimming in Ca2+-free ASW. However, only sneaker sperm that were preincubated in an acidic medium displayed turning episodes in the vicinity of the Ca2+-loaded pipette (Figure 4F). These results suggest that, in sneaker sperm, recovery from acidosis elicits a Ca2+ influx that triggers a turn episode (Figure 4G). Discussion Some coastal squids in the loliginidae employ alternative mating tactics [4, 16, 17]. Large males engage in male-male agonistic bouts and the winner—usually the largest squid— forms a temporary consortship with a female, mates her in parallel position, and guards her from other males while she lays egg fingers. These consort male deposit spermatophores around or in the oviduct opening in the mantle cavity of the female. In contrast, small males do not engage in agonistic bouts, but rather use sneaker mating tactics, such as mating head-to-head and darting spermatophores at an external location near the sperm storage organ (the seminal receptacle). When females extrude an egg capsule from their oviduct, consort sperm have first access to the egg capsules (akin to internal fertilization) and fertilize a large number of eggs. Then, females hold the egg capsule in their arms as they approach the spawning substrate, and, at this time, sperm from sneaker males have access to the egg capsules as well (akin to external fertilization). We previously reported a tight link between sperm phenotype and mating tactics [4]. However, it is not known whether mating tactics depend on the presence of rivals during mating [18] or switched ontogenetically during the life cycle. Nonetheless, squid loliginid sperm are unique in that internal and external fertilization coexist during a single spawning episode. Previously, we reported that sneaker sperm are w50% longer than consort sperm [4]. Although no such clear difference of within-species dimorphic eusperm had been reported, sperm size differences among closely related species have been interpreted as sperm competition [19–21]. However, the landscape of fertilization might have driven the evolution of sperm in L. bleekeri [4]. In this study, we show that sneaker sperm form motile assemblies and stay close to the spermatophore (Movie S6). We also show that CO2 causes sperm from sneaker males, but not consort males, to form assemblies. From an ecological point of view, retaining the ejaculates at the site of egg deposition enhances the chances of fertilization, because mating and egg laying are temporally independent [22, 23]. Thus, it will be intriguing to study why only sneaker sperm acquired the swarming trait in light of both postcopulatory sexual selection [24] and natural selection [2]. In addition, sneaker sperm might sense CO2 released from eggs, and fertilization by sneaker sperm might rely on CO2 chemotaxis toward the egg [25]. The carbonate system is important for many organisms and cells. CO2/acid detection by several sensory systems [10, 26–31] and the central nervous system [32] may share common mechanisms with CO2 taxis in squid sperm in terms
of intracellular acidosis via transmembrane H+ currents [27] and Ca2+ responses after recovery from an acid challenge called ‘‘off-response’’ [28]. However, the complexity of coupled chemical equilibria between chemical species and across cell membranes has largely been neglected. It is inherently difficult to trace down the active agent and to predict the consequences of altering one component or the other. Moreover, the ion channels and transport systems that mediate pH changes and generate the Ca2+ response are unknown. A unique, voltage-gated Na+/H+ exchanger [33] and an H+-selective channel [34] have been identified in the sperm flagellum. Moreover, a pH-sensitive K+ channel (KSper) [35–37] and a pH-sensitive Ca2+ channel (CatSper) [38] transduce changes in pHi into voltage and Ca2+ responses. Additional studies are required to precisely identify and delineate the sequence of signaling events during CO2 taxis or acidotaxis in sperm. We identified a plasma-membrane-anchored CA; however, soluble CAs might also play a role in CO2 taxis, because CO2 can diffuse across the cell membrane and soluble CAs have been identified as components of cellular acidification [11, 39]. Isoform-specific inhibitors of CAs are required in order to dissect the contribution of intra versus extracellular CAs to CO2 metabolism. Finally, a novel caged carbonate, synthesized for this study, represents a powerful tool for unravelling the molecular pathways of CO2/H+ sensation not only in sperm but also in olfactory, gustatory, or neuronal systems. Supplemental Information Supplemental Information contains Supplemental Experimental Procedures, four figures, and six movies and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2013.03.040. Acknowledgments We thank Victor D. Vacquier for his critical reading and comments on the manuscript. We thank H. Mieda, F. Nakaya, T. Nishigaki, K. Moriwaki, N. Sato, C. Ohkami, and R. Pascal for their technical assistance. This work was supported by a Narishige Zoological Science Award, the Research Institute of Marine Invertebrates, the Yamada Science Foundation, a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a grant from the Japanese Association for Marine Biology to N.H. Received: November 5, 2011 Revised: January 28, 2013 Accepted: March 14, 2013 Published: April 11, 2013 References 1. Fisher, H.S., and Hoekstra, H.E. (2010). Competition drives cooperation among closely related sperm of deer mice. Nature 463, 801–803. 2. Foster, K.R., and Pizzari, T. (2010). Cooperation: the secret society of sperm. Curr. Biol. 20, R314–R316. 3. Higginson, D.M., and Pitnick, S. (2011). Evolution of intra-ejaculate sperm interactions: do sperm cooperate? Biol. Rev. Camb. Philos. Soc. 86, 249–270. 4. Iwata, Y., Shaw, P., Fujiwara, E., Shiba, K., Kakiuchi, Y., and Hirohashi, N. (2011). Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours. BMC Evol. Biol. 11, 236. 5. Kimmel, A.R., and Parent, C.A. (2003). Dictyostelium discoideum cAMP Chemotaxis Pathway. Sci. STKE 2003, cm1. 6. Brokaw, C.J. (1957). Electro-chemical orientation of bracken spermatozoids. Nature 179, 525. 7. Riffell, J.A., Krug, P.J., and Zimmer, R.K. (2002). Fertilization in the sea: the chemical identity of an abalone sperm attractant. J. Exp. Biol. 205, 1439–1450.
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