BMC Zoology(2022) 7:8
Sugiura et al. BMC Zoology
https://doi.org/10.1186/s40850-022-00109-w

Open Access

RESEARCH

Morphological differences in tardigrade
spermatozoa induce variation in gamete
motility
Kenta Sugiura1 , Kogiku Shiba2 , Kazuo Inaba2

and Midori Matsumoto1*

Abstract
Background: Fertilization is an event at the beginning of ontogeny. Successful fertilization depends on strategies for
uniting female and male gametes that developed throughout evolutionary history. In some species of tardigrades,
investigations of reproduction have revealed that released spermatozoa swim in the water to reach a female, after
which the gametes are stored in her body. The morphology of the spermatozoa includes a coiled nucleus and a species-specific-length acrosome. Although the mating behaviour and morphology of tardigrades have been reported,the motility of male gametes remains unknown. Here, using a high-speed camera, we recorded the spermatozoon
motilities of two tardigrades, Paramacrobiotus sp. and Macrobiotus shonaicus, which have longer and shorter spermatozoa, respectively.
Results: The movement of spermatozoa was faster in Paramacrobiotus sp. than in M. shonaicus, but the beat frequencies of the tails were equal, suggesting that the long tail improved acceleration. In both species, the head partconsisting of a coiled nucleus and an acrosome did not swing, in contrast to the tail. The head part of Paramacrobiotus
sp. spermatozoa swung harder during turning; in contrast, the tail of M. shonaicus moved more widely than the head.
Finally, after mating, the spermatozoa that reached the female aggregated around the cloaca while waiting to enter
her body in both tested species.
Conclusions: This study provides results for the first observations and analyses of individual spermatozoon motility
in tardigrades. A comparison of the spermatozoon movements of the two tardigrades suggested that the motilitiesof the male gametes were affected by morphological differences, where the longer spermatozoa swam faster and theshorter ones showed more stable swimming. Swimming was mainly induced by tail movement, but the long headof Paramacrobiotus sp. spermatozoa might be especially important for turning. In addition, observations of matedfemale cloacae suggested that the head parts of the spermatozoa were required for aggregation around the cloaca ofa mated female.
Keywords: Tardigrade, Mating, Spermatozoa, Motility, Fertilization, Reproduction, Paramacrobiotus sp, Macrobiotus
shonaicus, Imaging, Morphology*Correspondence: mmatsumo@bio.keio.ac.jp
1
Department of Biosciences and Informatics, Faculty of Scienceand Technology, Keio University, 3‑14‑1 Hiyoshi, Kohoku, Yokohama,
Kanagawa 223‑8522, Japan
Full list of author information is available at the end of the article

BackgroundFertilization is an important event in the production of
the next generation in amphimictic animals. Female andmale gametes meet, and then ontogeny begins. Modes offertilization are generally divided into two types: internal and external fertilization. In the internal mode, theunion of gametes occurs inside the female’s body. In© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Sugiura et al. BMC Zoology(2022) 7:8

contrast, in the external mode, fertilization begins in theenvironment. In both cases, the behavioural strategies
of spermatozoa for reaching eggs show diversity and are
required to increase the rate of successful fertilization[1]. Tardigrades, often called water bears, are aquatic
animals in which females lay their eggs in their moulted
exuvia or directly into the environment [2]. In relation
to the former scenario, fertilization is thought to occur
externally in Isohypsibius dastychi (Pilato, Bertolani &Binda, 1982) because male ejaculation and female oviposition are simultaneous processes [3, 4], but in Pseudobiotus megalonyx (Thulin, 1928) and Ursulinius nodosus(Marray, 1907), internal fertilization is presumed because
ejaculation occurs before egg deposition, with spermatozoa enter in the oviduct via cloaca opening [5–9]. On
the other hand, in species that have a spermatheca andlay their eggs freely, internal fertilization is expected
because there is a spermatheca connected to the oviductbetween the cloaca and ovary [9–12]. However, observations of egg laying in Paramacrobiotus sp. and Macrobiotus shonaicus (Stec, Arakawa and Michalczyk, 2018)

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indicated that fertilization begins after eggs are laid into
the environment [13, 14]. Moreover, observations ofmating behaviours have revealed that the spermatozoaof the two species are first ejaculated into the environment, after which they swim to the mating female andare stored in her spermatheca; then, oviposition occurs[13, 15]. The morphologies of the spermatozoa have been
well reported not only in these two species but also inother species belonging to the same family, Macrobiotidae, and demonstrated that the spermatozoa have a tail,an inflated or rod-shaped mid-piece, a coiled nucleus andan acrosome [10, 14, 16–20] (Fig. 1C, D). Morphologicalanalyses and comparisons have been performed, indicating that the length of the acrosome is extremely variable
among genera and suggesting that the functions of the
spermatozoa are affected by morphological characters.
However, information on the motility of tardigrade spermatozoa is lacking.
Here, we observed the spermatozoon motilities of
two tardigrades, Paramacrobiotus sp. and M. shonaicus, which are the species the mating behaviours andFig. 1 Observations of the ejaculated spermatozoa A&B The ejaculated spermatozoa were recorded. Arrow heads indicate the released
spermatozoa. C, D Schemas of spermatozoa of Paramacrobiotus sp. and M. shonaicus, respectively. E Time lapse images of spermatozoa swimming.
Scale bars: μm. Abbreviations a, m, n, t and tt indicate acrosome, mid-piece, nucleus, tail and tuft, respectively

Sugiura et al. BMC Zoology(2022) 7:8

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Fig. 2 Tracks, speeds and beats of the spermatozoa A The longest (black lines) and shortest (dotted lines) swimming tracks of the respective
species. "x" indicates the start position. B The swimming speeds of the spermatozoa. C The beat frequency of tails. *: p < 0.01 in the Mann–Whitney
U-test, n.s.: no significant difference

spermatozoon morphologies have been confirmed [13,14]. According to morphometric research, the spermatozoa of Paramacrobiotus sp. and M. shonaicus had longer
and shorter acrosomes, respectively; thus, we examined
and compared their movement as affected by morphological variation.

Results
Observation and tracking of ejaculated spermatozoa

Ejaculated tardigrade spermatozoa in a droplet within
the 0.5 μm gap under cover slips were observed under
an inverted-phased microscope with a stroboscopic
flash and a high-speed camera (Fig. 1A, B). The recorded
movies demonstrated the detailed motilities of the spermatozoa. Each spermatozoon included a head part(an acrosome and a nucleus) and a tail (Movies S1, S2,
Fig. 1C-E). The gametes swam by leading with the midpiece (Movie S3).
Most of the released spermatozoa were able to reacha female during the observation period, but some could
not. The trajectories of the spermatozoa that reached afemale were straight and/or curved (Fig. 2A, Movie S3).
The swimming speeds were 259.3 μm/sec and 207.6 μm/
sec in Paramacrobiotus sp. and M. shonaicus, respectively (Fig. 2B), and the spermatozoa of the former
moved significantly faster (n > 25, Mann–Whitney U-test,p < 0.01). The beat frequency of the tails during swimming was 49.5–52.5 Hz on average (Fig. 2C) and did not
differ significantly between species (n > 26, Mann–Whitney U-test, p < 0.1).
Obtaining coordinates of the motilities

The motilities of the head parts and tail parts were analysed using Bohboh software (Bohboh Soft, Tokyo, Japan)(Movie S3). From the obtained coordinates, trajectoriesof the mid-piece, head and tail were visualized (Fig. 3). In
addition, normalized waveforms and curvatures corresponding to the length were calculated (Fig. 3, Movie S3).
To compare the amplitude between the head and thetail, the transition of the curvature correlating to the distance from the jointing point was obtained. In the comparison, the head parts trembled less than the tail parts

Sugiura et al. BMC Zoology(2022) 7:8

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Fig. 3 Spermatozoa trajectories Obtained trajectories, normalized waveforms and curvatures of the heads and tails in the respective species.
“Straight” and “Turn” indicate a spermatozoon swimming in a straight line and turning, respectively. Coloured lines of obtained trajectories correlate
to waveforms in right panels(Fig. 4, F-test, p < 0.01). In addition, the obtained data
showed differences in amplitude between the tested
species. The head curvatures of the two species showedsignificant differences in 63.6% of the compared positions(1–3, 6–7, and 9–10 μm from the base, F-test, p < 0.01). In
the comparison of the tails, the curvatures were different

Sugiura et al. BMC Zoology(2022) 7:8

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Fig. 4 Spermatozoon motility comparison between parts and species The curvatures of spermatozoa of Paramacrobiotus sp. (left) and M. shonaicus(right)in the 12–16 μm position (26.3% of compared positions,
F-test, p < 0.01).
Comparison of motility curvatures

The obtained curvatures were categorized according to
the direction of swimming (in a straight line or turning),
and a comparison of motility curvatures in the two swimming patterns showed intraspecies differences in movement. In Paramacrobiotus sp., 61.0% (36 positions/59
positions in total) of the head regions were significantly
curved during turning compared with swimming in astraight line; in addition, the positions were concentrated
in the range 13–48 μm (Fig. 5). The tail parts were morecurved in 6 of the 31 positions (19.4%). In contrast to theresults in Paramacrobiotus sp., 3/13 positions (23.1%) of
the head parts and 12 out of 21 positions in total (57.1%)of the tail parts were widely curved during turning in M.
shonaicus (Fig. 5).
Spermatozoa around the female’s cloaca

Fifteen minutes after ejaculation, the cloacae of the matedfemales were observed with scanning electron microscopy (SEM). The observations revealed that numerous
spermatozoa aggregated around the cloaca and the leg IV(Fig. 6).

Discussion
In this study, we performed the first detailed observations of the motilities of tardigrade spermatozoa. The
observations clearly revealed that the spermatozoa swamwith the mid-piece in the front position and moved in astraight line and/or turned. Additionally, the swimming
speeds in two species were measured. The spermatozoa
of Paramacrobiotus sp. were faster than those of M. shonaicus. The spermatozoa of the genus Paramacrobiotus
were generally longer than those of the genus Macrobiotus; moreover, the extraordinary head length of spermatozoa in the genus Paramacrobiotus was considered toenhance spermatozoon movements [19]. Our results support this hypothesis.
The curvature values of the tails were significantly
larger than those of the heads in both species. The results
indicated that the movements were mainly induced by
the tails. In other aquatic animals, for example, ascidiansand sea urchins, although the head parts are not as longas those in tardigrades, the swimming of the spermatozoa is induced by tail movement [21]. The tails, called flagella, are formed by axonemes, and the configuration is
widely conserved [22]. Axonemes have been observed in
the spermatozoa of some tardigrade species; in addition,
the constructions were similar to those in other animals[10, 19, 23–27]. Unfortunately, ultrastructural information for the tested species Paramacrobiotus sp. and M.
shonaicus is lacking, but the formation of the axonemeis also thought to be conserved in the species, suggesting that tail motility is induced by the same mechanism.
In addition, the motilities of each part were significantly
greater in Paramacrobiotus sp. than in M. shonaicus, suggesting that the longer heads and tails of Paramacrobiotus sp. spermatozoa enhanced their movement [14].

Sugiura et al. BMC Zoology(2022) 7:8

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Fig. 5 Spermatozoon movement comparison between swimming in a straight line and turning Green and purple boxplots represent thecurvatures of spermatozoa swimming in a straight line and turning, respectively. Yellow highlighted positions show significant differences in their
movements (F-test, p < 0.01)

Fig. 6 Spermatozoon aggregation SEM photos of the cloaca of a mated female. The claws of legs IV are visible. Arrow heads indicate the cloaca.
Scale bars: μm

Sugiura et al. BMC Zoology(2022) 7:8

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In a comparison of the trajectories of the spermatozoa, the tails of the turning spermatozoa in M. shonaicus
curved more dramatically than when the spermatozoa
were swimming in a straight line, indicating that thedetermination of direction depended on the tail’s movements. On the other hand, the extraordinarily longhead parts of Paramacrobiotus sp. spermatozoa showed
increased curvature during turning, suggesting that thedirection, i.e., whether turning or swimming in a straight
line, was affected by longer parts than the tail. According to our results, the acrosome is more flexible than the
coiled nucleus because the tip part of Paramacrobiotus
sp. spermatozoa swung widely. In contrast, the flexible
component of M. shonaicus spermatozoa is short; therefore, the head parts did not curve as much as those in
Paramacrobiotus sp., allowing more stable swimming.
Most spermatozoa reached the female’s cloaca in a few
seconds. However, the spermatozoa remained aggregatedaround the cloaca for 15 min after mating, suggestingthat they waited to enter the spermatheca of the female.
The correlation of spermatozoon and egg morphologies in Macrobiotidae was suggested [26]; for example,
eggs of species that have longer acrosomes are generally
conical. On the other hand, eggs of species with shorter
acrosomes are usually ornamented and often have an
inverted-goblet shape. Generally, the shapes of gametes
should have some functional significance for efficientand certain reproduction. A previous study hinted at theexistence of gamete morphological interference at thephase of fertilization in some species [14]. Although thisstudy focused only on male gametes, the comparisonsof motility in relation to length might provide insight
into the morphological differentiation that affects oneof the functions of spermatozoa. To reveal the factors
affecting and benefits of morphological variation in tardigrade spermatozoa, we believe it is important to accumulate observations and knowledge in many species. Asa related species, Mesobiotus spp. could support next
studies because the morphology of its spermatozoon
was intermediate between the genus Paramacrobiotus
and Macrobiotus [28]. In future investigations, if similar
knowledge is accumulated for various species as is donein this study, evolutionary history can be inferred basedon species differentiation in gamete features.

differences between swimming directions suggested thatthe long head of Paramacrobiotus sp. spermatozoa affects
turning. In addition, SEM observations of mated female
cloacae indicated that the released male gametes aggregate around the cloaca at least 15 min after mating.

Conclusions
In summary, we performed detailed observations ofswimming spermatozoa of tardigrades. A comparisonof movements between spermatozoa with longer versus
shorter acrosomes demonstrated differences in swimming speeds, waveforms and curvatures, indicating thatthe morphological differences of the spermatozoa intardigrades influence their movements. The waveform

Analyses of spermatozoon motility

Methods
Tardigrade culture conditions and specimen preparation

Two tardigrades, namely, Paramacrobiotus sp. TYO strain
and M. shonaicus, were used for this investigation. Thesetardigrades were provided by Dr. Takekazu Kunieda and
Dr. Kazuharu Arakawa, respectively. The culture conditions were the same as those described in Sugiura et al.
[13]. The tardigrades were placed in plastic dishes containing 1.2% agar gel (Nacalai Tesque, Kyoto, Japan) topped
with mineral water (Volvic) and then kept in the dark at
20℃. The rotifer Lecane inermis and the green alga Chlorella vulgaris (Recenttec KK, Tokyo, Japan) were added tothe culture as food sources. The water was changed every
3–5 days, and the dishes were renewed every 2–3 weeks.
Sample preparations for mating observations also followed
Sugiura et al. [13]. Females in stage 3–4 that showed tightly
packed ovary were isolated in a separate culture for at least
one week, and males with testes were separated into a different culture to prevent unexpected mating. Observations
until this step were performed under a stereomicroscope(SZX10, Olympus, Tokyo, Japan).
Recording motilities of the spermatozoa

A pair of tardigrades, a female and a male, was selected
to observe mating. The specimens were suspended in a
droplet of the mineral water placed within a 0.5 μm gap
created by a U-shaped silicon sheet (AS ONE, Osaka,
Japan) between 18 mm × 18 mm and 50 mm × 24 mmcover slips (Matsunami Glass Ind., Ltd., Osaka, Japan).
The behaviours of the tardigrades were observed under
an inverted-phased microscope (IX71, Olympus, Tokyo,
Japan) with a 20 × objective lens. The field was illuminated with a laboratory-made red stroboscopic LED
lamp (620–630 nm, Power LED, Edison, Taiwan). The
swimming motility of the released spermatozoa wasvideo recorded using a high-speed charge-coupled device(CCD) camera (HAS-U2, DITECT, Tokyo, Japan) at 150
frames per second (fps) [29].
The coordinates of the spermatozoon was obtained bytracking the mid-piece as an indicator in ImageJ software[30], and the speed of movement (velocity curvilinear:
VCL [29]) was calculated from the moving distance andthe frames of the video on R software [31]. Computerassisted sperm analysis (CASA) was mainly performed
with Bohboh software (Bohboh Soft, Tokyo, Japan) [29].

Sugiura et al. BMC Zoology(2022) 7:8

The spermatozoa were divided to a head part (including a nucleus and an acrosome) and a tail part, and thecoordinates of the two regions were obtained from thebase (junction with the mid-piece) to tip of each part ineach one micrometre position over 10 sequential frames.
Normalized waveforms and curvatures were determinedwith the software and visualized with ImageJ or R [30,31]. The beat frequency was calculated by counting thewave peaks in the 5 μm position of the tails and dividing
the count by the elapsed time. Motility was categorized
as swimming in a straight line or turning. Distinction of
the nucleus and acrosome was performed according tomorphometrical data available in Sugiura and Matsumoto [14] because of limited resolution. Specifically, thenucleus was defined as 0–21 μm and 0–11 μm in Paramacrobiotus sp. and M. shonaicus, respectively, and theacrosome was defined as the remaining region.
Statistical comparisons

Statistical comparisons were performed with a Mann–
Whitney U-test or an F-test in R [31], using the “wilcox.
exact” and “var.test” functions, respectively.
SEM observations

SEM observations were performed as described in Sugiura and Matsumoto [14]. Fifteen minutes after mating,eight females of each species were dehydrated in 100%
ethanol for 3 h and then soaked in tertial-butyl alcohol
overnight. After being lyophilized with a JFD-320 device(JEOL, Tokyo, Japan), the samples were transferred onto
aluminium stubs. The samples were then sputter-coatedwith gold and observed with a JSM 6510 (JEOL) scanningelectron microscope.
Abbreviations
CASA: Computer-assisted sperm analysis; CCD: Charge-coupled device;
FPS: Frames per second; SEM: Scanning electron microscopy; VCL: Velocity
curvilinear.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s40850-​022-​00109-w.
Additional file 1.
Additional file 2.
Additional file 3.
Additional file 4.
Acknowledgements
We thank to Dr. Takekazu Kunieda (University of Tokyo), Dr. Kazuharu Arakawa(Keio University) and Naoko Ishii (Keio University) for providing us the tardigrades we used. We also thank to Dr. Atsushi C. Suzuki (Keio University) for
supporting the SEM experiments. We would like to thank to Dr. Ryuji Yanase,

Page 8 of 9

Dr. Kei Jokura, Dr. Yu Sato, Dr. Misa Shibata, Dr. Nana Terauchi, Sayaka Yamaguchi (University of Tsukuba) and Osamu Horiguchi (Okinawa Institute of Scienceand Technology) for supporting our study.
Authors’ contributions
KeS performed all experiments and wrote the manuscript. KoS improved themethods of experiments. All authors designed the study and improved themanuscript. The authors read and approved the final manuscript
Funding
This study was supported in part by KAKENHI Grant-in-Aids from the Japan Society for the Promotion of Science (JSPS) and Grant-in-Aid for Scientific Researchin Innovative Areas –Platforms for Advanced Technologies and Research
Resource “Advanced Bioimaging Support” for JSPS Fellow PD (JP18J21345 and20D-19C-010-L08 to KeS), and a grant for Research Project from Research and
Education Center for Natural Science (Keio University) to MM and KeS.
Availability of data and materials
The datasets used and/or analyzed during the current study are available fromthe corresponding author.

Declarations
Ethics approval and consent to participate
No need
Consent for publication
No needCompeting interests
The authors declare that they have no competing interests.
Author details
1
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3‑14‑1 Hiyoshi, Kohoku, Yokohama, Kanagawa 223‑8522,
Japan. 2 Shimoda Marine Research Center, University of Tsukuba, 5‑10‑1
Shimoda, Shizuoka 415‑0025, Japan.
Received: 1 July 2021 Accepted: 18 January 2022

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