Current Biology

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Correspondence A D E

How female squid

inseminate their
eggs with stored
00:00:24
sperm B

Yoko Iwata1,*, Noriyosi Sato2, F

Noritaka Hirohashi2,
Takashi Kasugai3, Yoshiro Watanabe1, 200 µm 50 µm 30 µm
and Eiji Fujiwara4

0 100 200 300 400 500 600

G

No. of sperm per egg

00:00:27
How sperm reach ova after mating
is one of the central questions in C
reproductive biology. Many species
copulate and store sperm in female
reproductive organs until spawning
[1]. The way females use stored
sperm is closely associated with
sperm competition and cryptic female 00:00:35 A B C D E Field
Batch ID
choice. However, it is difficult to
observe the process of fertilization H J
Ventral side A-I
in natural spawning, as fertilization
usually occurs in some ‘hidden
place’ within the female’s body. Here, BME
we report the fertilization process
NJ
of a squid using a glass plate as BK
a spawning substratum, enabling
observation within the female arm BMA
Egg
crown where the sperm storage organ I
is located and where fertilization may Opening
occur. Additionally, we detail the SR
distribution of sperm around newly OJ BME
spawned eggs. Our observations
reveal that: the female places her A-IV
sperm-storage organ (seminal Dorsal side
receptacle) over an egg held within
her arm crown and inseminates the
eggs one-by-one during attachment Figure 1. Spawning behavior and sperm distribution in eggs of the Japanese pygmy squid
to the spawning substratum; sperm Idiosepius paradoxus.
pass through a pathway within the (A) Female squid bends all arms ventrally and directs her funnel into her arms to collect egg jelly
as spawning commences. Scale bar = 5 mm. (B) An egg (red arrow) passes through the funnel
jelly layers surrounding an egg; and
into the arm crown. (C) After being caught in the arms, the female immediately attaches the
such direct insemination behavior jelly-coated egg to a seagrass blade. Thread-like spermatangia attached to the female head are
and the pathway through the egg jelly visible (A–C captured from Video S1). (D) Lateral view of a spawned egg using differential interfer-
enables a female squid to externally ence contrast microscopy. (E) Distribution of sperm nuclei (stained blue) around the chimney-like
fertilize her eggs using relatively few structure within egg-jelly layers. (F) Close-up of egg-jelly layers (left), sperm heads (yellow arrow)
sperm. This study is the first to reveal observed within egg jelly (middle), and sperm cell collected from spermatophores (right) under
normal (above) and UV light with Hoechst staining (below). (G) Number of sperm cells per egg in
the fertilization process using stored
six egg batches: A–E collected in captivity; Field = field-collected eggs. (H) View within the female
sperm, under female control. arm crown during egg-attaching behavior; the blue arrow indicates the position of the seminal
Some coastal squid species have receptacle (resembling a white spot). (I) Female places the seminal receptacle over the egg (H,
one or more seminal receptacles I, images captured from Video S2). (J) Arrangement of the seminal receptacle and egg jelly dur-
located on the buccal membrane ing egg-attaching behavior. A-I, arm I; A-IV, arm IV; BK, beak; BMA, buccal mass; BME, buccal
surrounded by their arms. Male membrane; SR, seminal receptacle. OJ and NJ: egg-jelly layers secreted by the oviducal and
nidamental glands, respectively.
squid transfer packages of sperm
(spermatophores) to the female body see Supplemental Information). After An intriguing characteristic of the
near her seminal receptacle (white spermatophore attachment, sperm squid reproductive system, unlike
arcuate spermatangia attached to the migrate to the seminal receptacle(s) those in other copulating species [1],
female head are visible in Video S1, where they are stored until spawning. is that the sperm reservoir is located

R48 Current Biology 29, R39–R49, January 21, 2019 © 2018 Published by Elsevier Ltd.
Current Biology

Magazine

apart from the oviduct [2]. Although as sperm distribution and number might enable the discharge of existing
this characteristic is common in many (23–283 sperm, 72 ± 61 cells, N = 18; sperm, so that the seminal receptacles
squid species, exactly how sperm Figure 1G). We next filmed female can be filled with sperm of new males
stored within the seminal receptacles spawning activity within the arm in some later copulations, therefore
reach the ova during spawning has crown — an activity typically obscured affecting egg paternity in subsequent
long remained a mystery. by the female’s body being adpressed spawning cycles. Although further
We describe the spawning to the spawning substratum (seagrass studies are required to address the
behavior of the Japanese pygmy leaf) — through aquarium glass walls. possibility of female control on the
squid Idiosepius paradoxus, having During egg-attaching behavior, the amount of sperm release, our present
a single seminal receptacle on female placed the opening of her study demonstrates that females
the buccal membrane [3]. During seminal receptacle over the egg, then play a fundamental role in sperm–egg
spawning, females bend all of their pressed the buccal mass against the encounter after copulation.
arms ventrally, direct their funnel egg (Figure 1H–J, Video S2).
into their arms and collect egg jelly Our observations reveal that female SUPPLEMENTAL INFORMATION
secreted from the nidamental gland pygmy squid actively inseminate their
through the funnel (Figure 1A). An eggs one-by-one when attaching them Supplemental information contains two
egg, coated with jelly matrix secreted to the substratum. The fertilization videos and experimental procedures and
from the oviducal gland, then passes process might be common among can be found at https://doi.org/10.1016/j.
through the funnel and is collected cephalopod species that have their cub.2018.12.010.
by the arms (Figure 1B). Females seminal receptacle(s) located on the
immediately start attaching the egg to buccal area, such as in cuttlefishes REFERENCES
the spawning substratum by pushing and loliginid squids [2]. The very
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(Figure 1C, Video S1). Females repeat study is observed more frequently in 2. Hanlon, R.T., and Messenger, J.B. (2018).
this behavioral sequence and spawn internal fertilizers than external ones. Cephalopod Behaviour (second edition).
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Atmosphere and Ocean Research
nuclei, morphologically consistent over the number of sperm released,
Institute, The University of Tokyo, 5-1-5
with sperm heads (Figure 1F), were and the difference of released Kashiwanoha, Kashiwa, Chiba 277-8564,
concentrated around this chimney- sperm number possibly depends Japan; 2Oki Marine Biological Station,
like structure within the egg jelly on how full the seminal receptacle Shimane University, 194 Kamo, Okinoshima,
(Figure 1E). No unfertilized eggs is. Alternatively, if intentional sperm Oki, Shimane 685-0024, Japan; 3Port of
were observed; each jelly contained ejection from a sperm storage organ Nagoya Public Aquarium, 1-3 Minato-machi,
Minato-ku, Nagoya, Aichi 455-0033, Japan;
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Documentary Channel Co. Ltd. 851-1
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those in egg-jelly morphology as well [10]. An excess release of sperm *E-mail: [email protected]

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