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Current Biology 23, 775–781, May 6, 2013 ª2013 Elsevier Ltd All rights reserved
http://dx.doi.org/10.1016/j.cub.2013.03.040
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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: hiro@life.shimane-u.ac.jp
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
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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.
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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
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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
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779
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.
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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
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