Papers - GOTOH Ayako
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Establishment of a rapid, cost-effective, and accurate method for assessing insect sperm viability. International journal
Mika Takeshima, Ayako Gotoh
Journal of insect physiology 104682 - 104682 2024.7
High-quality sperm cells are crucial to reproductive success for both males and post-mating females in animals. Sperm viability, defined as the proportion of viable sperm cells, is used as a sperm quality index and this method has provided new insights into research on reproductive strategies. However, current staining protocols could potentially underestimate viability due to cell damage caused by cell treatments such as high dye concentration and long time for post-mounting. In this study, we established a method that enables rapid sperm viability assessment, has low sperm cell toxicity, and provides precise results regardless of operator expertise, and cost-effective using sperm cells from an ant, Crematogaster osakensis (Hymenoptera). First, to shorten the time for observation of a sufficient number of sperm cells, the volume per field of view was increased by height elevation between the glass slide and the coverslip, thereby we increased the number of sperm cells in a field of view. Second, to reduce sperm cell toxicity, we optimized the minimum dye concentration and incubation time using acridine orange (AO) and Hoechst in addition to SYBR14 and propidium iodide (PI), which has been used in most previous studies. We determined the optimal protocol to be 1 µg/mL AO and 150 µM PI without incubation. Besides, we automated counting sperm cells with ImageJ software and combined with manual correction for more accurate results. We employed the improved method for sperm samples from mealworm beetles (Tenebrio molitor) and silkmoths (Bombyx mori). This method, established through our study, will advance research on reproductive strategies, including sperm competition and sperm quality maintenance in females.
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Ayako Gotoh
Molecular Reproduction and Development 91 ( 2 ) 2024.2
Authorship：Lead author, Last author, Corresponding author Publisher：Wiley
Abstract
Ant queens can maintain a large number of sperm cells for over a decade after mating at the beginning of their adult lives until they die. This sperm storage ability is prominent; however, the cellular mechanisms involved remain unclear. Sperm cells are maintained in the female sperm storage organ—the spermatheca—which supplies a suitable environment for sperm cells. To reveal the molecular basis of the long‐term sperm storage mechanisms in ant queens, protein profiles enriched in the spermathecal fluid relative to the hemolymph were identified in Lasius japonicus using data‐independent acquisition‐based quantitative proteomics technology. Proteins related to the extracellular matrix, antioxidants, metabolic pathways, proteases, chaperones, and with uncharacterized functions were especially abundant with higher log ratio values in the spermathecal fluid relative to the hemolymph. These enriched proteins were shared with highly expressed genes previously detected by transcriptome analyses of the spermatheca in queens of Crematogaster osakensis that belong to a different subfamily than L. japonicus. It is likely that the ability for long‐term sperm storage evolved early in the ant lineage. Therefore, the common proteins identified in these two ant species are possibly crucial for this ability.DOI： 10.1002/mrd.23733
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Evolution of meconium removal from cocoons in ants
A. Gotoh, F. Ito, R. Mizuno, Y. Shimamoto, K. Kinomura, E. Katsura, R. Hashim
Insectes Sociaux 2023.8
Publisher：Springer Science and Business Media LLC
DOI： 10.1007/s00040-023-00926-3
Other Link： https://link.springer.com/article/10.1007/s00040-023-00926-3/fulltext.html
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Near-anoxia induces immobilization and sustains viability of sperm stored in ant queens Reviewed
Ayako Gotoh, Mika Takeshima, Ken-ichi Mizutani
Scientific Reports 13 ( 1 ) 2023.3
Authorship：Lead author, Corresponding author Publisher：Springer Science and Business Media LLC
Abstract
After copulation, insect females store sperm in a spermatheca for some duration until fertilization. At the beginning of their adult lives, ant queens can preserve numerous viable sperm cells from copulation for over ten years. However, the key factors influencing long-term sperm storage have not been identified. Here we show that the spermathecal environment is nearly anoxic, which induces sperm immobilization. Furthermore, mitochondrial respiratory inhibitors suppress sperm motility, suggesting that sperm immobilization may be caused by a shortage of ATP generated from only glycolysis under near-anoxic conditions. Sperm immobilization is not induced by acidification via glycolytic metabolism because the spermathecal fluid is not acidic. Finally, we show that artificial anoxic conditions rather than aerobic conditions sustain viable sperm cells. Therefore, near-anoxia is a key factor influencing long-term sperm storage in ant queens. The viability of sperm cells under artificial anoxia, however, is lower than that of those dissected immediately from queens. Moreover, the immotile sperm cells under more than 4 h of anoxia do not begin swimming after aerobic exposure, unlike those under anoxic conditions for less than 2 h. This finding indicates that factors other than anoxia are also necessary for long-term sperm preservation.DOI： 10.1038/s41598-023-29705-7
Other Link： https://www.nature.com/articles/s41598-023-29705-7
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Ayako Gotoh
bioRxiv 2022.11
Authorship：Lead author, Corresponding author Publisher：Cold Spring Harbor Laboratory
Ant queens can maintain a large number of sperm cells for over a decade after mating at the beginning of their adult lives until they die. This ability is prominent because sperm cells cannot maintain their fertilization ability long after ejaculation in animals; however, the cellular mechanisms remain unclear. Sperm cells are maintained in the female sperm storage organ, the spermatheca, which supplies a suitable environment for sperm cells. To reveal the molecular basis of the long-term sperm storage mechanisms in ant queens, protein profiles enriched in the spermathecal fluid relative to the hemolymph were identified inLasius japonicususing mass spectrometry-based proteomics. Proteins related to the extracellular matrix, antioxidant, metabolic pathways, proteases, and with uncharacterized functions were enriched in the spermathecal fluid relative to the hemolymph. These enriched proteins were shared with highly expressed genes previously detected by transcriptome analyses of the spermatheca in queens ofCrematogaster osakensisbelonging to a different subfamily thanL. japonicus. It is considered that the ability for long-term sperm storage has evolved in the early ant lineage; therefore, the common proteins identified in the two ant species are crucial for this ability.
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Ayako Gotoh, Mika Takeshima, Ken-ichi Mizutani
bioRxiv 2022.9
Publisher：Cold Spring Harbor Laboratory
Abstract
Insect females store sperm in a spermatheca after copulation for some duration to use it later for fertilisation. At the beginning of their adult lives, ant queens can preserve numerous viable sperm cells from copulation for over ten years. However, the key factors influencing long-term sperm storage have not been identified. Here we show that the spermathecal environment is near anoxic, which induces sperm immobilisation. Furthermore, mitochondrial respiratory inhibitors suppress sperm motility, suggesting that sperm immobilisation may be caused by a shortage of ATP generated from only glycolysis under near anoxic condition. Sperm immobilisation is not induced by acidification via glycolytic metabolism because the spermathecal fluid is not acidic. Finally, we show that artificial anoxic conditions rather than aerobic conditions sustain viable sperm cells. Therefore, near-anoxia is a key factor influencing long-term sperm storage in ant queens. The viability of sperm cells under artificial anoxia, however, is lower than of those dissected immediately from queens. Moreover, the immotile sperm cells under more than 4 h of anoxia do not begin swimming after aerobic exposure, unlike those that were in anoxic conditions for less than 2 h. This indicates that factors other than anoxia are also necessary for long-term sperm preservation. -
Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae Reviewed
NAOTO IDOGAWA, AYAKO GOTOH, SHIGETO DOBATA
Zootaxa 5105 ( 2 ) 253 - 268 2022.3
Publisher：Magnolia Press
The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, “aphaenogastroid” body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a “pheidoloid” body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25°C. We discuss possible functions of the tubercles of queen larvae based on previous studies.
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Caste differentiation of spermatheca and organs related to sperm use and oviposition in the honeybee, Apis mellifera
Ayako Gotoh, Ken Sasaki
Apidologie 52 ( 1 ) 262 - 271 2021
Joint Work
© 2020, INRAE, DIB and Springer-Verlag France SAS, part of Springer Nature. Striking queen–worker dimorphism associated with each task is a key feature of highly eusocial insects, including Apis mellifera. The present study histologically examined the developmental process of the internal reproductive organs including spermatheca, valve-fold in the vagina, semi-circular muscle surrounding the common oviduct, and abdominal ganglia in honeybee queens and workers. During the pupal stage, queens showed an increased spermathecal reservoir, development of the tracheal network surrounding the spermathecal reservoir, and elongation of the spermathecal gland. Compared with queens, these developmental processes were never observed during the pupal stage in workers. Moreover, development of the valve-fold and semi-circular muscle was aborted, and they became rudimentary at the middle pupal stage in workers. Morphological caste differences in the abdominal ganglia were observed from the prepupal stages, showing that the most posterior ganglion was fused with the anterior ganglia in queens but not in workers.
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A. Gotoh, S. Shigenobu, S. Shigenobu, K. Yamaguchi, S. Kobayashi, F. Ito, K. Tsuji
Insect Molecular Biology 27 212 - 220 2018.4
Joint Work
© 2017 The Royal Entomological Society In insects, seminal fluid proteins that are produced by male accessory glands and transferred to females during mating have key functions in sperm competition and sperm physiology that lead to male reproductive success. In ants, male reproductive success also depends on the longevity of sperm stored in the queen's spermatheca because their sexual offspring are usually produced only after a prolonged storage period. We identified genes that were up-regulated in the male accessory glands relative to the bodies of Crematogaster osakensis to characterize the reproductive molecules associated with male reproductive success in ants. We found novel genes that had no hits in a homology search and that were predominantly expressed in the accessory glands. These reproductive proteins may have evolved under rapid positive selection for reproductive success in the species. Furthermore, we discovered that three spermatheca-specific genes of C. osakensis queens were also enriched in the accessory glands relative to the bodies of males. These genes may be important for maintaining the sperm quality continuously from ejaculation by males to prolonged storage by queens. This research provides crucial information about the molecular mechanisms of sperm maintenance and sexual selection in ants, and also insight into the evolution of reproductive strategies in insects.
DOI： 10.1111/imb.12364
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Journey of sperms from production by males to storage by queens in Crematogaster osakensis (Hymenoptera: Formicidae).
Gotoh A, Furukawa K
Journal of insect physiology 105 95 - 101 2018.2
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Transcriptome profiling of the spermatheca identifies genes potentially involved in the long-term sperm storage of ant queens.
Gotoh A, Shigenobu S, Yamaguchi K, Kobayashi S, Ito F, Tsuji K
Scientific reports 7 ( 1 ) 5972 2017.7
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Social structure of the polygynous ant, Crematogaster osakensis
A. Gotoh, M. Dansho, S. Dobata, Y. Ikeshita, F. Ito
Insectes Sociaux 64 123 - 131 2017.2
Joint Work
© 2016, International Union for the Study of Social Insects (IUSSI). The colony structures of social insects vary greatly among species. In ant societies, the number of queens within a colony is variable during colony maturation. We investigated the social structure of a polygynous ant Crematogaster osakensis in a series of field and laboratory experiments. First, the initial colonies headed by multiple queens were found in the field. In laboratory experiments, queens that were artificially cohabited after their nuptial flight harmoniously co-existed even in the presence of newly emerged workers, suggesting that mated queens of this species can establish their colony cooperatively as primary polygynous colonies. In addition, the mating frequency of a queen was typically more than one, estimated from the sperm number stored in field-collected males and queens, and from genetic relatedness among daughters of lab-reared monogynous colonies. Second, our assessment of genetic relatedness in a mature field colony of this species revealed that dealated queens, as well as workers and alates, were relatives. The number of developed oocytes identified reproductive skew in two of five field-collected nests. Moreover, under laboratory conditions, the most fertile egg layer altered over 3 months of observation. Based on these lines of evidence, we propose that in C. osakensis, polygynous foundresses might either be unrelated and subsequently be replaced by daughter queens of particular foundresses, or be related in the first place.
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Degeneration patterns of the worker spermatheca during morphogenesis in ants (Hymenoptera: Formicidae).
Gotoh A, Billen J, Hashim R, Ito F
Evolution & development 18 ( 2 ) 96 - 104 2016.3
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Vestigial spermatheca morphology in honeybee workers, Apis cerana and Apis mellifera, from Japan
Gotoh Ayako, Ito Fuminori, Billen Johan
APIDOLOGIE 44 ( 2 ) 133 - 143 2013.3
Joint Work
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Histological study of the spermatheca in three thelytokous parthenogenetic ant species, Pristomyrmex punctatus, Pyramica membranifera and Monomorium triviale (Hymenoptera: Formicidae)
Gotoh Ayako, Billen Johan, Tsuji Kazuki, Sasaki Tomonori, Ito Fuminori
ACTA ZOOLOGICA 93 ( 2 ) 200 - 207 2012.4
Joint Work
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Testes degeneration in ants: a histological study of Gnamptogenys bicolor
Allard Diane, Ito Fuminori, Aikawa Yohsuke, Gotoh Ayako, Billen Johan
ACTA ZOOLOGICA 92 ( 4 ) 372 - 376 2011.10
Joint Work
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Thelytokous parthenogenesis by queens in the dacetine ant Pyramica membranifera (Hymenoptera: Formicidae).
Ito F, Touyama Y, Gotoh A, Kitahiro S, Billen J
Die Naturwissenschaften 97 ( 8 ) 725 - 728 2010.8
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Evolution of specialized spermatheca morphology in ant queens: insight from comparative developmental biology between ants and polistine wasps.
Gotoh A, Billen J, Hashim R, Ito F
Arthropod structure & development 38 ( 6 ) 521 - 525 2009.11
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Comparison of spermatheca morphology between reproductive and non-reproductive females in social wasps.
Gotoh A, Billen J, Hashim R, Ito F
Arthropod structure & development 37 ( 3 ) 199 - 209 2008.5
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A. Gotoh, F. Ito
Insectes Sociaux 55 98 - 104 2008.2
Joint Work
Investigation of reproductive strategies of ants in the subfamily Ponerinae is important for understanding of the evolution of social structure and of the significance of caste dimorphisms. The biology of species with mated and egg-laying workers (gamergates) has been studied for many species, however, little attention has been paid to species that reproduce via alate queens only. We investigated the seasonal cycle of changes in the colony structure of Pachycondyla chinensis reproduced by alate queens in western Japan, and found the following novel biological characteristics of this species. P. chinensis showed a remarkable caste dimorphism in ovariole numbers: workers had no ovaries while queens had 18 to 36 ovarioles in their ovaries. The nesting system seemed to be polydomous: 266 of 400 nests collected were queenless. The number of queenless nests increased during the reproductive season. Among the 134 queenright nests, 38 had several mated-queens without significant differences in ovary activation and the remaining 96 nests were monogynous. During winter to early spring, most nests were polygynous. After alate production, most of the old queens seemed to die or be expelled and replaced by new queens. Virgin dealated queens were often found and they seemed to have laid eggs. © 2008 Birkhaeuser.