Worker Emergence Following Fungal Fruiting May Facilitate Symbiont Acquisition in a Fungus-Growing Termite

Worker Emergence Following Fungal Fruiting May Facilitate Symbiont Acquisition in a Fungus-Growing Termite | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Worker Emergence Following Fungal Fruiting May Facilitate Symbiont Acquisition in a Fungus-Growing Termite Masaru Hojo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9135322/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Fungus-growing termites depend on symbiotic Termitomyces fungi; establishing this association is a critical step during colony foundation. However, a seasonal relationship between early colony development and fungal reproductive phenology in natural populations remains poorly resolved. In this study, the timing of colony foundation behavior in the fungus-growing termite Odontotermes formosanus on Okinawa Island was examined by integrating long-term observations of field alate dispersal, controlled rearing experiments, and fungal fruiting records. Alates occurred within mature nests from the middle of March, but opportunistic field records spanning eight years showed that nuptial flights started only from April and were consistently associated with heavy rainfall before sunset. In incipient colonies established from alate pairs (n = 793), the first foraging workers predominantly began appearing 11–13 weeks after colony initiation. Such a developmental schedule predicts peak worker emergence from July to August for colonies founded during the main swarming period. In contrast, fruiting of two Japanese Termitomyces fungi began earlier during the season and mostly preceded worker emergence. These results suggest a consistent temporal ordering, which likely increases the probability of founding workers encountering abundant fungal spores when collecting plant substrate for constructing a fungus comb. Such ordering may also buffer symbiont acquisition against interannual climatic variations. These findings highlight the processes by which seasonal behavioral timing structures the establishment phase of an obligate insect–fungus mutualism. fungus farming mushroom social insect symbiosis Termitomyces intermedius Termitomyces sp. Type A Figures Figure 1 Introduction In termites, reproductive alates (winged imagos), which later become primary queens and kings post-colony foundation, are produced annually within parental colonies and dispersed by swarming flights (Nutting 1969 ; Lepage and Darlington 2000 ). After landing, alates shed their wings and walk on the ground searching for sexual partners. The pairs formed then walk together to seek suitable sites for laying the foundations of a new colony (Bordereau and Pasteels 2011 ), in which the primary queen and king care for their offspring until worker castes develop (Chouvenc 2019 ). Fungus-growing termites of the subfamily Macrotermitinae (Blattodea: Termitidae) maintain an obligate mutualism with basidiomycete species of the genus Termitomyces , which are cultivated in nests as the primary food source and help digest lignocellulose for the host termites (Sands 1969 ; Poulsen et al. 2014 ; Schalk et al. 2021 ; Ahmad et al. 2022 ). Successful establishment of this symbiosis is a significant step during the initial phase of colony foundation (Noble et al. 2000 ; Aanen et al. 2002 ; Wisselink et al. 2020 ). The first worker caste in fungus-growing termites typically appear above the ground several months after colony initiation following nuptial flights (Cheng et al. 2007 ; Koné et al. 2011 ). These early workers are responsible for the foraging plant materials and establishing an initial fungal garden (fungus comb). In mature colonies, Termitomyces produce conspicuous above-ground fruiting bodies and release large numbers of spores into the environment (Sands 1969 ; Bignell 2000 ; Aanen 2006 ; Koné et al. 2011 , 2018 ). As fungal symbionts must be acquired from the surroundings, the temporal relationship between worker emergence and seasonal spore availability may be a key determinant of successful colony establishment (Sands 1960 ; Sieber and Leuthold 1981 ; Johnson et al. 1981 ; Sieber 1983 ; Han and Bordereau 1992; Tian et al. 2009 ; Connétable et al. 2012 ; Mitchell 2020 ; Anwar et al. 2020 ). Despite its potential importance, empirical data linking worker emergence timing and fungal phenology within natural populations remain scarce. Previous work has suggested such relationships based primarily on fungal phenology and limited observations of colony development under laboratory conditions (Koné et al. 2011 ). Direct behavioral observations are nearly impossible in sterile workers produced by incipient colonies in natural populations because initial colonies develop underground and remain cryptic. The critical behavioral question is whether the timing of worker surface activity aligns with the environmental availability of fungal propagules. In this study, the seasonal relationship between alate dispersal, early colony development, and Termitomyces fruiting was investigated in the fungus-growing termite Odontotermes formosanus on Okinawa Island, Japan, an introduced species with a geographically restricted distribution (Hojo 2019). The hypothesis that fungal reproduction precedes worker emergence in incipient colonies was tested by combining long-term field observations with controlled rearing experiments. Materials and Methods Alate observation and sampling On Okinawa Island, O. formosanus is distributed mainly around Naha and Urasoe Cities near the Shuri District (Hojo 2019). This species constructs numerous small soil mounds that serve as exit points for alate dispersal during the season (Fig. S1). These mounds were inspected weekly from March to July each year from 2018 to 2025. In the presence of alates, individuals were collected directly from these mounds and transported to the laboratory for colony initiation experiments. When nuptial flights were observed, rainfall data between 15:00 and 19:00 h for the same day were obtained from hourly precipitation records provided by Naha Meteorological Station of the Japan Meteorological Agency (https://www.data.jma.go.jp/stats/etrn/index.php?prec_no=91&block_no=47936&year=&month=&day=&view=). Pairing initiation and first worker emergence After natural dealation in the laboratory, each male–female alate pair was introduced into an individual plastic Petri dish (90 × 20 mm) containing purchased red soil as a substrate. Each pair immediately burrowed into the soil and initiated nesting behavior. Colonies were maintained in a dark room at 25 ± 3 °C, and development was monitored weekly. The timing of first worker emergence was operationally defined as the initial above-ground appearance of termite trails on the soil surface, indicating worker foraging activity for fungus comb materials and Termitomyces spores. Colonies in which no trails appeared within 6 months post-pairing were excluded. A total of 793 alate pairs were employed for colony initiation experiments. Field survey of Termitomyces fruiting bodies The species Termitomyces sp. Type A and Termitomyces intermedius are known fungal symbionts of O. formosanus on Okinawa Island (Katoh et al. 2002; Hojo 2019; Hojo and Shigenobu 2019). To document seasonal fruiting, O. formosanus habitats in Naha and Urasoe Cities were surveyed daily during the main fruiting period, June–August, each year from 2018 to 2025. The in-field presence and date of emergence of fungal fruiting bodies were recorded. Results Seasonal presence of alates in mature colonies O. formosanus alates were observed within mature nests beginning from March through July, between 2018 and 2025, during repeated inspections (Table 1). The earliest detection ranged from mid-March to late April across years, and the latest extended to early July in a few cases. Interannual variations were evident, with alates retained longer within nests during years when conditions suitable for swarming (e.g., heavy rainfall) were delayed. Opportunistic records of nuptial flights Nuptial flights were recorded opportunistically on 16 occasions between 2018 and 2025 (Table S1). All observed swarming events occurred only from April onward, across the eight-year observation period; the recorded dates ranged from early April to early July. All events coincided with rainfall between 15:00 and 19:00 h; however, causal inferences could not be established as monitoring was opportunistic. Meteorological records indicated that in most events, rainfall exceeded 5 mm (Table S1). However, these records represent only confirmed occurrences and not swarming frequency, as observations were based on intermittent monitoring. Rainfall data may not precisely reflect precipitation at the exact locations where swarming events were recorded. Timing of worker emergence in incipient colonies Incipient colonies established from alate pairs under controlled rearing conditions (n = 793) produced surface-visible workers after a variable developmental period. Worker emergence, defined as the confirmed construction of the first surface-reaching gallery, occurred between 9 and 20 weeks after colony initiation (Fig. 1). The distribution was prominently concentrated between 11 and 13 weeks, which together accounted for a majority of colonies, at 194, 218, and 213 pairs, respectively. The median time-to-worker emergence was 12 weeks with an interquartile range of 11–13 weeks, encompassing 79.0% of colonies. Earlier emergence at 9–10 weeks (n = 23) and later emergence at ≥16 weeks (n = 43) were comparatively rare. Seasonal fruiting periods in symbiotic fungi Fruiting bodies of the symbiotic fungi on Okinawa Island were recorded annually during the study period (Table 2). Termitomyces sp. Type A produced fruiting bodies mainly from early July through August, whereas Termitomyces intermedius fruited primarily from late June through July, with occasional extensions into early August. During each year, the first recorded fruiting date of Termitomyces sp. Type A occurred later than that of T. intermedius . Interannual variations in the onset and duration of fruiting periods were observed between the two species, but seasonal ranges were broadly consistent across years. Discussion This study demonstrates a consistent temporal ordering of key events during early colony establishment in O. formosanus . Although alates were present within nests from mid-March (Table 1), nuptial flights were observed only from April onward (Table S1 ), and workers started appearing at the soil surface predominantly from July, 11–13 weeks after colony initiation (Fig. 1 ). In contrast, fruiting in the symbiotic fungi occurred earlier in the season, beginning in mid-June, with peak fruiting often occurring before the peak of worker emergence (Table 2). Overall, these findings indicate that, in this system, fungal reproduction generally precedes worker emergence. Opportunistic records of nuptial flights collected over eight years indicate that swarming in O. formosanus in Okinawa Island begins no earlier than April and often continues into May or later in years when suitable environmental conditions are delayed (Table S1 ). Given the distribution of worker emergence times observed (Fig. 1 ), colonies formed by alates dispersing in late April or May are expected to produce surface-active workers primarily from late July to early August. Colonies established following later swarming events, such as in June (Table S1 ), may exhibit even later worker emergence. These late temporal patterns further support the conclusion that emergence typically occurs after, rather than before, the seasonal availability of fungal fruiting bodies. This seasonal pattern differs from the expectation proposed by Koné et al. ( 2011 ), who suggested that worker emergence should coincide closely with the period of maximal spore release from fungal fruiting bodies. In the present study, however, peak fruiting often preceded the peak of worker emergence, indicating that strict temporal coincidence may not be required for successful symbiont acquisition in O. formosanus. Such a temporal sequence suggests a simple but powerful ecological mechanism underlying symbiosis establishment. By the time workers begin emerging above ground for foraging, fungal fruiting bodies have often already released spores into the environment. Early workers are therefore likely to simultaneously encounter plant material suitable for substrate fungus comb construction and viable fungal symbionts. Transporting these materials into the nest would allow the establishment of a fungus comb without requiring precise synchronization between termite development and fungal reproduction. Even in those years when fruiting occurs relatively late, colonies founded by alates dispersing in early summer are expected to produce workers after the onset of fruiting, maintaining this functional sequence. From an evolutionary perspective, the decoupling of termite developmental timing from fungal reproductive phenology may enhance the robustness of mutualism under variable climatic conditions. Rather than relying on tightly synchronized life cycles, O. formosanus colonies appear to exploit a seasonal window in which fungal spores predictably precede worker foraging. Such a strategy represent a general feature of fungus-growing termite systems in seasonal environments and may reduce the risk of symbiont acquisition failure. Declarations Declarations Conflict of Interest The author declares no conflict of interest. Ethical Approval This study did not involve human participants or vertebrate animals. Termites were collected and maintained for behavioral observation in accordance with local regulations. Consent to Participate and Consent for Publication Not applicable. Funding This work was supported by JSPS KAKENHI Grant Numbers 15K07798 and 23K05262, and Hokuto Foundation for Bioscience. Author Contribution M.H. conceived the study, conducted field observations and laboratory experiments, analyzed the data, and wrote the manuscript. Acknowledgement I thank Drs. Takeshi Arakawa and Gaku Tokuda for permission to use his equipment throughout the study. Thanks are also due to Haruhiko Fujii (facility staff of Sueyoshi Park ‘Mori no Ie Minmin’) and Dr. Shuji Shigenobu for help about field sampling. This work was financially supported by a JSPS KAKENHI (No.15K07798 and No.23K05262) to M.H. provided by the Ministry of Education, Culture, Sports, Science and Technology, Japan. This work was also financially supported by Hokuto Foundation for Bioscience. Data Availability The datasets generated during the current study are available from the corresponding author on reasonable request. References Aanen DK (2006) As you reap, so shall you sow: coupling of harvesting and inoculating stabilizes the mutualism between termites and fungi. Biol Lett 2:209–212. https://doi.org/10.1098/rsbl.2005.0424 Aanen DK, Eggleton P, Rouland-Lefèvre C, Guldberg-Frøslev T, Rosendahl S, Boomsma JJ (2002) The evolution of fungus-growing termites and their mutualistic fungal symbionts. 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(2009) Incipient Colony Development and Biology of Odontotermes formosanus (Shiraki) and O. hainanensis (Light) (Isoptera: Termitidae). J Agr Urban Entomol 26:147–156. https://doi.org/10.3954/1523-5475-26.3.147 Wisselink M, Aanen DK, van 't Padje A (2020) The longevity of colonies of fungus-growing termites and the stability of the symbiosis. Insects 11(8):527. https://doi.org/10.3390/insects11080527 Tables Table 1 Period of alate presence beneath mounds on Okinawa Island Year Period 2018 19 April-5 June 2019 22 April-25 May 2020 30 March-15 June 2021 29 March-4 June 2022 16 March-11 May 2023 22 March-12 May 2024 29 March-10 May 2025 13 March-11 July Table 2 Periods of flush in Termitomyces fruiting bodies observed on Okinawa Island Year Termitomyces sp. Type A Termitomyces sp. Type B 2018 1 August-8 August 26 June-30 July 2019 22 July-9 August 1 July-31 July 2020 5 July-15 August 29 June-25 July 2021 5 July-3 August 17 June-15 August 2022 8 July-22 July 28 June-23 July 2023 16 July-31 July 23 June-10. August 2024 24 June-7 August 19 June-2 August 2025 18 July-27 July 19 June-2 August Additional Declarations No competing interests reported. Supplementary Files FigS1.pptx TableS1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 19 Mar, 2026 Reviewers invited by journal 19 Mar, 2026 Editor assigned by journal 19 Mar, 2026 Submission checks completed at journal 19 Mar, 2026 First submitted to journal 16 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Worker emergence was operationally defined as the first visualization of termite trails on the soil surface. Workers from most colonies appeared between 11 and 13 weeks after colony initiation\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9135322/v1/c741455346dcd68fad6ae0e3.png"},{"id":105323689,"identity":"b8336321-1f65-415a-bd7f-3b5c8cfe14a5","added_by":"auto","created_at":"2026-03-24 18:05:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":603274,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9135322/v1/15aafe9b-dc6e-4407-81cb-0b3cb99bbac9.pdf"},{"id":105323688,"identity":"269a18c8-8967-4309-ace5-4ff49e823734","added_by":"auto","created_at":"2026-03-24 18:05:45","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":770294,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.pptx","url":"https://assets-eu.researchsquare.com/files/rs-9135322/v1/81a7e8a12aee86d34896017b.pptx"},{"id":105323687,"identity":"9ae1edb3-3ba8-4281-ad7f-264593e78809","added_by":"auto","created_at":"2026-03-24 18:05:45","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10694,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9135322/v1/e4b3d124519871008ef896ba.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Worker Emergence Following Fungal Fruiting May Facilitate Symbiont Acquisition in a Fungus-Growing Termite","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn termites, reproductive alates (winged imagos), which later become primary queens and kings post-colony foundation, are produced annually within parental colonies and dispersed by swarming flights (Nutting \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Lepage and Darlington \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). After landing, alates shed their wings and walk on the ground searching for sexual partners. The pairs formed then walk together to seek suitable sites for laying the foundations of a new colony (Bordereau and Pasteels \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), in which the primary queen and king care for their offspring until worker castes develop (Chouvenc \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFungus-growing termites of the subfamily Macrotermitinae (Blattodea: Termitidae) maintain an obligate mutualism with basidiomycete species of the genus \u003cem\u003eTermitomyces\u003c/em\u003e, which are cultivated in nests as the primary food source and help digest lignocellulose for the host termites (Sands \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Poulsen et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Schalk et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ahmad et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Successful establishment of this symbiosis is a significant step during the initial phase of colony foundation (Noble et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Aanen et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wisselink et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The first worker caste in fungus-growing termites typically appear above the ground several months after colony initiation following nuptial flights (Cheng et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Kon\u0026eacute; et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These early workers are responsible for the foraging plant materials and establishing an initial fungal garden (fungus comb).\u003c/p\u003e \u003cp\u003eIn mature colonies, \u003cem\u003eTermitomyces\u003c/em\u003e produce conspicuous above-ground fruiting bodies and release large numbers of spores into the environment (Sands \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Bignell \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Aanen \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kon\u0026eacute; et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As fungal symbionts must be acquired from the surroundings, the temporal relationship between worker emergence and seasonal spore availability may be a key determinant of successful colony establishment (Sands \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1960\u003c/span\u003e; Sieber and Leuthold \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Johnson et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Sieber \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Han and Bordereau 1992; Tian et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Conn\u0026eacute;table et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mitchell \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Anwar et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Despite its potential importance, empirical data linking worker emergence timing and fungal phenology within natural populations remain scarce. Previous work has suggested such relationships based primarily on fungal phenology and limited observations of colony development under laboratory conditions (Kon\u0026eacute; et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Direct behavioral observations are nearly impossible in sterile workers produced by incipient colonies in natural populations because initial colonies develop underground and remain cryptic.\u003c/p\u003e \u003cp\u003eThe critical behavioral question is whether the timing of worker surface activity aligns with the environmental availability of fungal propagules. In this study, the seasonal relationship between alate dispersal, early colony development, and \u003cem\u003eTermitomyces\u003c/em\u003e fruiting was investigated in the fungus-growing termite \u003cem\u003eOdontotermes formosanus\u003c/em\u003e on Okinawa Island, Japan, an introduced species with a geographically restricted distribution (Hojo 2019). The hypothesis that fungal reproduction precedes worker emergence in incipient colonies was tested by combining long-term field observations with controlled rearing experiments.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAlate observation and sampling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn Okinawa Island, \u003cem\u003eO. formosanus\u003c/em\u003e is distributed mainly around Naha and Urasoe Cities near the Shuri District (Hojo 2019). This species constructs numerous small soil mounds that serve as exit points for alate dispersal during the season (Fig. S1). These mounds were inspected weekly from March to July each year from 2018 to 2025. In the presence of alates, individuals were collected directly from these mounds and transported to the laboratory for colony initiation experiments. When nuptial flights were observed, rainfall data between 15:00 and 19:00 h for the same day were obtained from hourly precipitation records provided by Naha Meteorological Station of the Japan Meteorological Agency (https://www.data.jma.go.jp/stats/etrn/index.php?prec_no=91\u0026amp;block_no=47936\u0026amp;year=\u0026amp;month=\u0026amp;day=\u0026amp;view=).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePairing initiation and first worker emergence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter natural dealation in the laboratory, each male\u0026ndash;female alate pair was introduced into an individual plastic Petri dish (90 \u0026times; 20 mm) containing purchased red soil as a substrate. Each pair immediately burrowed into the soil and initiated nesting behavior. Colonies were maintained in a dark room at 25 \u0026plusmn; 3 \u0026deg;C, and development was monitored weekly. The timing of first worker emergence was operationally defined as the initial above-ground appearance of termite trails on the soil surface, indicating worker foraging activity for fungus comb materials and \u003cem\u003eTermitomyces\u003c/em\u003e spores. Colonies in which no trails appeared within 6 months post-pairing were excluded. A total of 793 alate pairs were employed for colony initiation experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eField survey of \u003cem\u003eTermitomyces\u003c/em\u003e fruiting bodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe species \u003cem\u003eTermitomyces\u003c/em\u003e sp. Type A and \u003cem\u003eTermitomyces intermedius\u003c/em\u003e are known fungal symbionts of \u003cem\u003eO. formosanus\u003c/em\u003e on Okinawa Island (Katoh et al. 2002; Hojo 2019; Hojo and Shigenobu 2019). To document seasonal fruiting, \u003cem\u003eO. formosanus\u003c/em\u003e habitats in Naha and Urasoe Cities were surveyed daily during the main fruiting period, June\u0026ndash;August, each year from 2018 to 2025. The in-field presence and date of emergence of fungal fruiting bodies were recorded.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSeasonal presence of alates in mature colonies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eO. formosanus\u003c/em\u003e alates were observed within mature nests beginning from March through July, between 2018 and 2025, during repeated inspections (Table 1). The earliest detection ranged from mid-March to late April across years, and the latest extended to early July in a few cases. Interannual variations were evident, with alates retained longer within nests during years when conditions suitable for swarming (e.g., heavy rainfall) were delayed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpportunistic records of nuptial flights\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNuptial flights were recorded opportunistically on 16 occasions between 2018 and 2025 (Table S1). All observed swarming events occurred only from April onward, across the eight-year observation period; the recorded dates ranged from early April to early July. All events coincided with rainfall between 15:00 and 19:00 h; however, causal inferences could not be established as monitoring was opportunistic. Meteorological records indicated that in most events, rainfall exceeded 5 mm (Table S1). However, these records represent only confirmed occurrences and not swarming frequency, as observations were based on intermittent monitoring. Rainfall data may not precisely reflect precipitation at the exact locations where swarming events were recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTiming of worker emergence in incipient colonies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIncipient colonies established from alate pairs under controlled rearing conditions (n = 793) produced surface-visible workers after a variable developmental period. Worker emergence, defined as the confirmed construction of the first surface-reaching gallery, occurred between 9 and 20 weeks after colony initiation (Fig. 1). The distribution was prominently concentrated between 11 and 13 weeks, which together accounted for a majority of colonies, at 194, 218, and 213 pairs, respectively. The median time-to-worker emergence was 12 weeks with an interquartile range of 11\u0026ndash;13 weeks, encompassing 79.0% of colonies. Earlier emergence at 9\u0026ndash;10 weeks (n = 23) and later emergence at \u0026ge;16 weeks (n = 43) were comparatively rare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeasonal fruiting periods in symbiotic fungi\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFruiting bodies of the symbiotic fungi on Okinawa Island were recorded annually during the study period (Table 2). \u003cem\u003eTermitomyces\u003c/em\u003e sp. Type A produced fruiting bodies mainly from early July through August, whereas \u003cem\u003eTermitomyces intermedius\u003c/em\u003e fruited primarily from late June through July, with occasional extensions into early August. During each year, the first recorded fruiting date of \u003cem\u003eTermitomyces\u003c/em\u003e sp. Type A occurred later than that of \u003cem\u003eT. intermedius\u003c/em\u003e. Interannual variations in the onset and duration of fruiting periods were observed between the two species, but seasonal ranges were broadly consistent across years.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates a consistent temporal ordering of key events during early colony establishment in \u003cem\u003eO. formosanus\u003c/em\u003e. Although alates were present within nests from mid-March (Table\u0026nbsp;1), nuptial flights were observed only from April onward (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and workers started appearing at the soil surface predominantly from July, 11\u0026ndash;13 weeks after colony initiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, fruiting in the symbiotic fungi occurred earlier in the season, beginning in mid-June, with peak fruiting often occurring before the peak of worker emergence (Table\u0026nbsp;2). Overall, these findings indicate that, in this system, fungal reproduction generally precedes worker emergence.\u003c/p\u003e \u003cp\u003eOpportunistic records of nuptial flights collected over eight years indicate that swarming in \u003cem\u003eO. formosanus\u003c/em\u003e in Okinawa Island begins no earlier than April and often continues into May or later in years when suitable environmental conditions are delayed (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Given the distribution of worker emergence times observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), colonies formed by alates dispersing in late April or May are expected to produce surface-active workers primarily from late July to early August. Colonies established following later swarming events, such as in June (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), may exhibit even later worker emergence. These late temporal patterns further support the conclusion that emergence typically occurs after, rather than before, the seasonal availability of fungal fruiting bodies.\u003c/p\u003e \u003cp\u003eThis seasonal pattern differs from the expectation proposed by Kon\u0026eacute; et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), who suggested that worker emergence should coincide closely with the period of maximal spore release from fungal fruiting bodies. In the present study, however, peak fruiting often preceded the peak of worker emergence, indicating that strict temporal coincidence may not be required for successful symbiont acquisition in \u003cem\u003eO. formosanus.\u003c/em\u003e Such a temporal sequence suggests a simple but powerful ecological mechanism underlying symbiosis establishment. By the time workers begin emerging above ground for foraging, fungal fruiting bodies have often already released spores into the environment. Early workers are therefore likely to simultaneously encounter plant material suitable for substrate fungus comb construction and viable fungal symbionts. Transporting these materials into the nest would allow the establishment of a fungus comb without requiring precise synchronization between termite development and fungal reproduction. Even in those years when fruiting occurs relatively late, colonies founded by alates dispersing in early summer are expected to produce workers after the onset of fruiting, maintaining this functional sequence.\u003c/p\u003e \u003cp\u003eFrom an evolutionary perspective, the decoupling of termite developmental timing from fungal reproductive phenology may enhance the robustness of mutualism under variable climatic conditions. Rather than relying on tightly synchronized life cycles, \u003cem\u003eO. formosanus\u003c/em\u003e colonies appear to exploit a seasonal window in which fungal spores predictably precede worker foraging. Such a strategy represent a general feature of fungus-growing termite systems in seasonal environments and may reduce the risk of symbiont acquisition failure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclarations\u003c/h2\u003e\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe author declares no conflict of interest.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthical Approval\u003c/strong\u003e \u003cp\u003eThis study did not involve human participants or vertebrate animals. Termites were collected and maintained for behavioral observation in accordance with local regulations.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Participate\u003c/strong\u003e \u003cp\u003e \u003cb\u003eand Consent for Publication\u003c/b\u003e Not applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by JSPS KAKENHI Grant Numbers 15K07798 and 23K05262, and Hokuto Foundation for Bioscience.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.H. conceived the study, conducted field observations and laboratory experiments, analyzed the data, and wrote the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eI thank Drs. Takeshi Arakawa and Gaku Tokuda for permission to use his equipment throughout the study. Thanks are also due to Haruhiko Fujii (facility staff of Sueyoshi Park \u0026lsquo;Mori no Ie Minmin\u0026rsquo;) and Dr. Shuji Shigenobu for help about field sampling. This work was financially supported by a JSPS KAKENHI (No.15K07798 and No.23K05262) to M.H. provided by the Ministry of Education, Culture, Sports, Science and Technology, Japan. This work was also financially supported by Hokuto Foundation for Bioscience.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAanen DK (2006) As you reap, so shall you sow: coupling of harvesting and inoculating stabilizes the mutualism between termites and fungi. Biol Lett 2:209\u0026ndash;212. https://doi.org/10.1098/rsbl.2005.0424\u003c/li\u003e\n\u003cli\u003eAanen DK, Eggleton P, Rouland-Lef\u0026egrave;vre C, Guldberg-Fr\u0026oslash;slev T, Rosendahl S, Boomsma JJ (2002) The evolution of fungus-growing termites and their mutualistic fungal symbionts. Proc Natl Acad Sci U.S.A 99(23):14887\u0026ndash;14892. https://doi.org/10.1073/pnas.222313099\u003c/li\u003e\n\u003cli\u003eAanen DK, de Fine Licht HH, Debets AJM, Kerstes NAG, Hoekstra RF, Boomsma JJ (2009) High symbiont relatedness stabilizes mutualistic cooperation in fungus-growing termites. Science 326:1103\u0026ndash;1106. https://doi.org/10.1126/science.1173462\u003c/li\u003e\n\u003cli\u003eAhmad F, Yang G, Zhu Y, Poulsen M, Li W, Yu T, Mo J (2022) Tripartite symbiotic digestion of lignocellulose in the digestive system of a fungus-growing termite. 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In: Krishna K, Weesner FM (eds) Biology of termites, vol 1. Academic Press, New York, pp 495\u0026ndash;524\u003c/li\u003e\n\u003cli\u003eSchalk F, Gostinčar C, Kreuzenbeck NB, Conlon BH, Sommerwerk E, Rabe P, Burkhardt I, Kr\u0026uuml;ger T, Kniemeyer O, Brakhage AA, Gunde-Cimerman N, de Beer ZW, Dickschat JS, Poulsen M, Beemelmanns C (2021) The termite fungal cultivar \u003cem\u003eTermitomyces\u003c/em\u003e combines diverse enzymes and oxidative reactions for plant biomass conversion. mBio 12(3):e03551-20. https://doi.org/10.1128/mbio.03551-20\u003c/li\u003e\n\u003cli\u003eSieber R (1983) Establishment of fungus comb in laboratory colonies of\u003cem\u003e Macrotermes michaelseni \u003c/em\u003eand\u003cem\u003e Odontotermes montanus\u003c/em\u003e (Isoptera, Macrotermitinae). Insectes Soc\u003cem\u003e \u003c/em\u003e30:204\u0026ndash;209\u003c/li\u003e\n\u003cli\u003eSieber R, Leuthold RH (1981) Behavioural elements and their meaning in incipient laboratory colonies of the fungus-growing Termite \u003cem\u003eMacrotermes michaelseni \u003c/em\u003e(Isoptera: Macrotermitinae). Insectes Soc 28:371\u0026ndash;382. https://doi.org/10.1007/BF02224194\u003c/li\u003e\n\u003cli\u003eTian W-J, Ke Y-L, Zhuang T-Y \u003cem\u003eet al\u003c/em\u003e. (2009) Incipient Colony Development and Biology of \u003cem\u003eOdontotermes formosanus\u003c/em\u003e (Shiraki) and \u003cem\u003eO. hainanensis \u003c/em\u003e(Light) (Isoptera: Termitidae). J Agr Urban Entomol 26:147\u0026ndash;156. https://doi.org/10.3954/1523-5475-26.3.147\u003c/li\u003e\n\u003cli\u003eWisselink M, Aanen DK, van \u0026apos;t Padje A (2020) The longevity of colonies of fungus-growing termites and the stability of the symbiosis. Insects 11(8):527. https://doi.org/10.3390/insects11080527\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"491\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"2\" valign=\"bottom\" style=\"width: 454px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Period of alate presence beneath mounds on Okinawa Island\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003eYear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 406px;\"\u003e\n \u003cp\u003ePeriod\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 406px;\"\u003e\n \u003cp\u003e19 April-5 June\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 406px;\"\u003e\n \u003cp\u003e22 April-25 May\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e2020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 406px;\"\u003e\n \u003cp\u003e30 March-15 June\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 406px;\"\u003e\n \u003cp\u003e29 March-4 June\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 406px;\"\u003e\n \u003cp\u003e16 March-11 May\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 406px;\"\u003e\n \u003cp\u003e22 March-12 May\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 406px;\"\u003e\n \u003cp\u003e29 March-10 May\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e2025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 406px;\"\u003e\n \u003cp\u003e13 March-11 July\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"491\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"bottom\" style=\"width: 491px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Periods of flush in \u003cem\u003eTermitomyces\u003c/em\u003e fruiting bodies observed on Okinawa Island\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003eYear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003e\u003cem\u003eTermitomyces\u003c/em\u003e sp. Type A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 234px;\"\u003e\n \u003cp\u003e\u003cem\u003eTermitomyces\u003c/em\u003e sp. Type B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003e1 August-8 August\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 234px;\"\u003e\n \u003cp\u003e26 June-30 July\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003e22 July-9 August\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 234px;\"\u003e\n \u003cp\u003e1 July-31 July\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e2020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003e5 July-15 August\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 234px;\"\u003e\n \u003cp\u003e29 June-25 July\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003e5 July-3 August\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 234px;\"\u003e\n \u003cp\u003e17 June-15 August\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003e8 July-22 July\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 234px;\"\u003e\n \u003cp\u003e28 June-23 July\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e2023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003e16 July-31 July\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 234px;\"\u003e\n \u003cp\u003e23 June-10. August\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003e24 June-7 August\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 234px;\"\u003e\n \u003cp\u003e19 June-2 August\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 65px;\"\u003e\n \u003cp\u003e2025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003e18 July-27 July\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 234px;\"\u003e\n \u003cp\u003e19 June-2 August\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-insect-behavior","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joir","sideBox":"Learn more about [Journal of Insect Behavior](http://link.springer.com/journal/10905)","snPcode":"10905","submissionUrl":"https://submission.nature.com/new-submission/10905/3","title":"Journal of Insect Behavior","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"fungus farming, mushroom, social insect, symbiosis, Termitomyces intermedius, Termitomyces sp. Type A","lastPublishedDoi":"10.21203/rs.3.rs-9135322/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9135322/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFungus-growing termites depend on symbiotic \u003cem\u003eTermitomyces\u003c/em\u003e fungi; establishing this association is a critical step during colony foundation. However, a seasonal relationship between early colony development and fungal reproductive phenology in natural populations remains poorly resolved. In this study, the timing of colony foundation behavior in the fungus-growing termite \u003cem\u003eOdontotermes formosanus\u003c/em\u003e on Okinawa Island was examined by integrating long-term observations of field alate dispersal, controlled rearing experiments, and fungal fruiting records. Alates occurred within mature nests from the middle of March, but opportunistic field records spanning eight years showed that nuptial flights started only from April and were consistently associated with heavy rainfall before sunset. In incipient colonies established from alate pairs (n\u0026thinsp;=\u0026thinsp;793), the first foraging workers predominantly began appearing 11\u0026ndash;13 weeks after colony initiation. Such a developmental schedule predicts peak worker emergence from July to August for colonies founded during the main swarming period. In contrast, fruiting of two Japanese \u003cem\u003eTermitomyces\u003c/em\u003e fungi began earlier during the season and mostly preceded worker emergence. These results suggest a consistent temporal ordering, which likely increases the probability of founding workers encountering abundant fungal spores when collecting plant substrate for constructing a fungus comb. Such ordering may also buffer symbiont acquisition against interannual climatic variations. These findings highlight the processes by which seasonal behavioral timing structures the establishment phase of an obligate insect\u0026ndash;fungus mutualism.\u003c/p\u003e","manuscriptTitle":"Worker Emergence Following Fungal Fruiting May Facilitate Symbiont Acquisition in a Fungus-Growing Termite","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 18:05:40","doi":"10.21203/rs.3.rs-9135322/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"13654411283194567193796035382984622134","date":"2026-03-19T14:47:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-19T14:37:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-19T09:37:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-19T09:37:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Insect Behavior","date":"2026-03-16T08:44:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-insect-behavior","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joir","sideBox":"Learn more about [Journal of Insect Behavior](http://link.springer.com/journal/10905)","snPcode":"10905","submissionUrl":"https://submission.nature.com/new-submission/10905/3","title":"Journal of Insect Behavior","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3a1cdefd-fadc-4e93-b242-ef57a55902ee","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-24T18:05:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 18:05:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9135322","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9135322","identity":"rs-9135322","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}