Scientific note: behavioral dynamics of pollen storage in Melipona quadrifasciata

Scientific note: behavioral dynamics of pollen storage in Melipona quadrifasciata | 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 Short Report Scientific note: behavioral dynamics of pollen storage in Melipona quadrifasciata Patrícia Miranda-Pinto, Luciana Teresa Dias Cappelini, MICHELLE MANFRINI MORAIS This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2981706/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Nov, 2023 Read the published version in Insectes Sociaux → Version 1 posted 4 You are reading this latest preprint version Abstract Stingless bees are important eusocial insects found in tropical and subtropical regions. They store nectar and pollen from flowers to survive periods of scarcity. During storage, nectar and pollen undergo fermentation processes, resulting in honey and fermented pollen. However, the fermentation process of pollen collected by stingless bees is not well understood. Understanding these processes not only clarifies pollen fermentation in different stingless bee species but also sheds light on the transformation process of pollen. This knowledge can be applied to produce fermented feeds using specific microorganisms. Therefore, this study aimed to observe pollen storage behavior within colonies to understand the fermentation process and aid in the development of fermented feeds for bees. A Melipona quadrifasciata colony from the Experimental Meliponary of the Universidade Federal de São Paulo, Brazil, was observed for 30 days. Daily observations were conducted in the morning and night, and pollen pots were marked with random colors to differentiate them and collect data on worker bee dynamics. Observations showed no consistent patterns in the opening and closing of the pots, with worker bees frequently entering and exiting the open pollen pots. This suggests an ongoing fermentation process involving gas exchange and nutrient flow. The lack of regularity in handling pollen storage in M. quadrifasciata indicates its noteworthy variability. These findings are crucial for advancing meliponiculture techniques and developing fermented protein supplements globally and locally. Stingless bees pollen storage Melipona quadrifasciata fermentation Figures Figure 1 Figure 2 Full Text Stingless bees (Apidae, Meliponini) make up an important group of eusocial insects occurring in tropical and subtropical areas of the planet (Costa and Venturieri 2009) . To survive periods of scarcity, these bees store nectar and pollen collected from floral resources inside cerumen pots inside the colony (Roubik 1982) . Both nectar and pollen undergo fermentation processes during storage, turning into honey and fermented pollen, respectively (Nogueira-Neto 1997) . Although stingless bees share many similarities with Apis mellifera , this diverse group (Roubik 1992) still conceals many particularities that have not yet been explored (Menezes et al. 2013) . The characteristics of the pollen, such as flavor, odor, color, and texture, change considerably after being stored and vary among bee species (Camargo et al. 1992; Souza et al. 2004). A few bee species, such as Tetragonisca angustula and Frieseomelitta varia , produce dry and relatively sweet fermented pollen. However, other meliponines, such as bees from the genus Melipona and Scaptotrigona , produce and store moist and sour pot-pollen (Menezes et al. 2013). It is clear that the dynamics of pollen storage are still unclear, and knowing this dynamic is essential for the investigation of the fermentation process, clarifying questions about fermentation time, moment of inoculation of microorganisms, and consumption of fermented pollen by bees. This study aimed to investigate pollen storage behavior within the colonies of the bee species M. quadrifasciata to obtain information that can aid in understanding its fermentation process. Observations were carried out for 30 days between November and December 2021. An observation colony of M. quadrifasciata from the Experimental Meliponary of the Federal University of São Paulo was used on the Diadema campus (23° 43' 10"). S 46° 37' 39" W). The colony used for observation had a glass top to facilitate internal visualization and, consequently, study data collection. Before the beginning of the observations, the pollen pots in the colony were removed, leaving only one pot to supply the bees' needs with protein and mainly to stimulate the search for external resources by the forager bees to build new pollen pots. During the 30 days of the experiment, observations were made in the morning and at night. In the morning, the pots were observed between 7 am and 10 am (UTC -3), and in the night, between 7 pm and 9 pm (UTC -3). The active pollen pots that had pollen entry and/or exit observed were marked daily with a non-toxic, water-based pen (Posca®), facilitating the observations of the pots and the work dynamics performed by the worker bees (Fig. 1). Thus, each time a new pot was observed with pollen inside (Start), it was randomly marked with a new combination of colors. To differentiate active pollen pots (marked with random colors) from honey pots and empty pots (not considered in this experiment), a yellow marking was used (in these particular pots). In addition to the observations regarding pollen storage dynamics, the pollen emptying process (Finish) was also monitored. For this, each time a pollen pot was emptied, the observation date of what happened was noted (Table 1). According to the results of Graph 1, we see that the pollen pots analyzed during the experiment did not remain closed for long periods. In addition, it was noted in the active pots that they were most of the time open during the morning period. Another important fact to be reported is that after thirty days, the presence of 14 active pots was observed, and at the beginning of the experiment, only one active pot had been left. This fact may have occurred to guarantee the constant supply of essential nutrients provided only by pollen. Stingless bees, like honeybees, adjust their foraging activities (food search) to maximize food intake when floral resources are unavailable (Biesmeijer and de Vries 2001; Maia-Silva et al. 2015). These adjustments, such as the search for more resources such as pollen, are a reaction to changes in external environmental factors and the colony's needs (Eltz et al. 2001; Hofstede and Sommeijer 2006). Since the observations were carried out during spring in Brazil, the increased input of floral resources, mainly pollen (the study's objective), justifies the appropriate period for the study. According to the results, there was no standardization regarding the dynamics of opening and closing the pollen pots of the observed colony (Fig. 2), a fact that happens with A. mellifera (Detry et al. 2020). Honeybees progressively fill the wax alveoli with pollen grains brought by foraging bees, practically in their entirety of the storage space. After filling the alveoli, they receive a layer of honey for a certain period and then are used for consumption by worker bees (Seeley 1982). From the observations of this study, it was evident that there is no regularity between having pots open until filled with pollen brought in from the field by foragers and closing after the pot is at or near full capacity. In addition, as shown in Fig. 2, when we compare two different pots in the same period, we notice that the opening times and the frequency of the work dynamics in the pots differ from one to the other. Our data go opposite to those reported by Nogueira-Neto and Menezes (Nogueira-Neto 1997; Menezes et al. 2013), who indicate that stingless bees close the pots when complete, storing them for approximately two weeks before consumption. As stingless bees are a diverse group, understanding how worker bees work to store pollen inside cerumen pots is highly relevant information for understanding the storage time and transformation of food through fermentation processes and later consumption by individuals of the colony. Few studies report the fermentation dynamics of pollen stored by stingless bees (Menezes et al. 2013). As shown in Fig. 2, the behavior of the workers who supply the pollen pots does not maintain regularity in how they perform the task, as studies show that it occurs in A. mellifera (vanEngelsdorp et al. 2009). In the case of this behavior, for M. quadrifasciata , it was observed that several pots are opened and closed throughout the day or night, regardless of the amount of pollen inside. It can be verified through Graph 1; Table 1, that 18 pots were observed throughout the experiment. Of these, some could be observed over several days, such as pot number two (36 observations - remaining open for 58.4% of the time and 41.6% of the time closed); four (38 observations - remaining open for 89.4% of the time and 10.6% closed) and five (34 observations - remaining for 64.7% of the open time and 35.3% of the closed time). Thus, an interesting and recurrent data in the observations is the way the foraging bees bring pollen and how it is treated, as it is known that during this conditioning, workers add secretions that are assumed to contain beneficial microorganisms (Teixeira et al. 2003). These microorganisms may be involved in the stored food's metabolic conversion, fermentation, and preservation. The transformation of pollen to bee bread has often been postulated to result from microbial action, mainly lactic acid fermentation caused by bacteria and yeasts (Haydak 1958). New observational studies need to be carried out about the pollen storage of stingless bees, but the dynamics of opening and closing the pollen pots may indicate a continuity of the fermentation process where gas exchanges may be occurring and even maintenance of nutrient flow by adding more content between closing and opening pots, as noted. Thus, information on how the workers of stingless bee species work, store and ferment their food is of great value within the scenario of understanding the importance of the microbiota associated with this process to produce alternative sources of food during periods of scarcity. Our observations on the behavior of storage and handling of pollen in the species M. quadrifasciata led us to conclude that there is a lack of regularity in treating this food, contrary to the behavior of A. mellifera . The pollen pots are opened and closed daily, and bee entry and exit behavior are frequent. Studies on the behavioral dynamics of stingless bees in food pots are scarce in the literature. Therefore, this work highlights the importance of data that explain how food storage occurs within colonies. Such data are paramount for advancing techniques that improve meliponiculture nationally and internationally, such as developing fermented protein supplementation. Understanding the storage time and natural occurrence within the colonies is essential for the fermentation of an artificial diet that mimics the natural characteristics of this important food source . Declarations Acknowledgment We thank Marcos Reis for his contribution to the analysis of the results, Tiago Francoy for his important help in the discussion and interpretation of the data, and Leandro Barros de Souza for drawing up Fig. 2. We also thank Capes (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the financial support to carry out this study. References Biesmeijer JC, de Vries H (2001) Exploration and exploitation of food sources by social insect colonies: a revision of the scout-recruit concept. Behav Ecol Sociobiol 49:89–99 Camargo JMF de, Garcia MVB, Eugenio Junior RQ, Castrillon A (1992) Notas prévias sobre a bionomia de Ptilotrigona lurida ( Hymenoptera, Apidae, Meliponinae ): associação de leveduras em pólen estocado. Boletim do Museu Paraense Emílio Goeldi 8:391–395 Costa L, Venturieri GC (2009) Diet impacts on Melipona flavolineata workers ( Apidae, Meliponini) . J Apic Res 48:38–45 Detry R, Simon-Delso N, Bruneau E, Daniel H-M (2020) Specialisation of yeast genera in different phases of bee bread maturation. Microorganisms 8:1789 Eltz T, Brühl, CA, van der Kaars S, Chey VK, Linsenmair K E (2001) Pollen foraging and resource partitioning of stingless bees in relation to flowering dynamics in a Southeast Asian tropical rainforest. Insectes Soc 48:273–279 Haydak MH (1958) Pollen-pollen substitutes-beebread. Am Bee J 98:145–146 Hofstede FE, Sommeijer MJ (2006) Influence of environmental and colony factors on the initial commodity choice of foragers of the stingless bee Plebeia tobagoensis (Hymenoptera, Meliponini). Insectes Soc 53:258–264. https://doi.org/10.1007/s00040-006-0866-9 Maia-Silva C, Hrncir M, da Silva CI, Imperatriz-Fonseca VL (2015) Survival strategies of stingless bees ( Melipona subnitida ) in an unpredictable environment, the Brazilian tropical dry forest. Apidologie 46:631–643 Menezes C, Vollet-Neto A, Contrera FAFL, et al (2013) The role of useful microorganisms to stingless bees and stingless beekeeping. Pot-Honey: A legacy of stingless bees 153–171 Nogueira-Neto P (1997) Vida e criação de abelhas indígenas sem ferrão. In: Vida e criação de abelhas indígenas sem ferrão. p 446 Roubik DW (1982) Seasonality in colony food storage, brood production and adult survivorship: studies of Melipona in tropical forest ( Hymenoptera: Apidae ). J Kans Entomol Soc 789–800 Roubik DW (1992) Ecology and natural history of tropical bees. Cambridge University Press Seeley TD (1982) Adaptive significance of the age polyethism schedule in honeybee colonies. Behav Ecol Sociobiol 11:287–293 Souza RC da S, Yuyama LKO, Aguiar JPL, Oliveira FPM (2004) Valor nutricional do mel e pólen de abelhas sem ferrão da região amazônica. Acta Amazon 34:333–336 Teixeira ACP, Marini MM, Nicoli JR, et al (2003) Starmerella meliponinorum sp. nov., a novel ascomycetous yeast species associated with stingless bees. Int J Syst Evol Microbiol 53:339–343 vanEngelsdorp D, Evans JD, Saegerman C, et al (2009) Colony Collapse Disorder: A Descriptive Study. PLoS One 4:e6481. https://doi.org/10.1371/journal.pone.0006481 Table Table 1 is available in the Supplementary Files section. Graph Graph 1 is available in the Supplementary Files section. Supplementary Files Table1.docx floatimage3.png Graph 1 Behavior of nurse bees when opening and closing the pollen pots according to the period and number of observations during the 30 days of the experiment Cite Share Download PDF Status: Published Journal Publication published 03 Nov, 2023 Read the published version in Insectes Sociaux → Version 1 posted Reviewers agreed at journal 27 May, 2023 Reviewers invited by journal 27 May, 2023 Editor assigned by journal 27 May, 2023 First submitted to journal 25 May, 2023 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2981706","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":204488989,"identity":"9314dc33-e5f4-45f5-a8bd-4284d7ac2d24","order_by":0,"name":"Patrícia Miranda-Pinto","email":"","orcid":"","institution":"Universidade Federal de São Paulo: Universidade Federal de Sao Paulo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Patrícia","middleName":"","lastName":"Miranda-Pinto","suffix":""},{"id":204488990,"identity":"bf03a72b-433d-4e9e-8d47-87bcb60d0d4b","order_by":1,"name":"Luciana Teresa Dias Cappelini","email":"","orcid":"","institution":"Florida International University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luciana","middleName":"Teresa Dias","lastName":"Cappelini","suffix":""},{"id":204488991,"identity":"f5c86c87-f7b3-4452-8811-6dfc5b5b5500","order_by":2,"name":"MICHELLE MANFRINI MORAIS","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-6184-3202","institution":"Universidade Federal de São Paulo - Campus Diadema","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"MICHELLE","middleName":"MANFRINI","lastName":"MORAIS","suffix":""}],"badges":[],"createdAt":"2023-05-25 14:35:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2981706/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2981706/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00040-023-00940-5","type":"published","date":"2023-11-03T15:01:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":37678131,"identity":"2797802f-d2d0-4925-a543-e468b1d16152","added_by":"auto","created_at":"2023-05-30 15:57:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":442305,"visible":true,"origin":"","legend":"\u003cp\u003eMarking of the food pots with a non-toxic Posca® pen using random colors for the active pollen pots and for the honey pots and empty pots, marking in yellow was used. (A) visualization of pollen pots in the morning (B) visualization of pollen pots in the night\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2981706/v1/c3372198643ee31977915945.png"},{"id":37678129,"identity":"c4df84cf-7d35-40de-8071-ec62f9ceacef","added_by":"auto","created_at":"2023-05-30 15:57:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":135175,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentation of the opening and closing dynamics of pollen pots two and four, respectively, observed during the same week in the morning and night periods\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2981706/v1/3dc5aae3a380ae5ada655e3d.png"},{"id":45943168,"identity":"1e30ce2c-7e11-45df-aa60-45927f4d0c2d","added_by":"auto","created_at":"2023-11-06 15:08:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":839192,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2981706/v1/71887854-64c9-4c03-a5f4-0386c8fcaeed.pdf"},{"id":37678128,"identity":"96460258-ccd3-4131-9c0d-954445de0662","added_by":"auto","created_at":"2023-05-30 15:57:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15370,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-2981706/v1/3773934178548d8e63d17bd8.docx"},{"id":37678130,"identity":"6b12b4f5-9b0c-4798-981b-68b466797299","added_by":"auto","created_at":"2023-05-30 15:57:01","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10234,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraph 1 \u003c/strong\u003eBehavior of nurse bees when opening and closing the pollen pots according to the period and number of observations during the 30 days of the experiment\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2981706/v1/97236d2f39d4f44668727fc9.png"}],"financialInterests":"","formattedTitle":"Scientific note: behavioral dynamics of pollen storage in Melipona quadrifasciata","fulltext":[{"header":"Full Text","content":"\u003cp\u003eStingless bees (Apidae, Meliponini) make up an important group of eusocial insects occurring in tropical and subtropical areas of the planet \u003cspan lang=\"EN-US\"\u003e(Costa and Venturieri 2009)\u003c/span\u003e. To survive periods of scarcity, these bees store nectar and pollen collected from floral resources inside cerumen pots inside the colony \u003cspan lang=\"EN-US\"\u003e(Roubik 1982)\u003c/span\u003e. Both nectar and pollen undergo fermentation processes during storage, turning into honey and fermented pollen, respectively \u003cspan lang=\"EN-US\"\u003e(Nogueira-Neto 1997)\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eAlthough stingless bees share many similarities with \u003cem\u003eApis mellifera\u003c/em\u003e, this diverse group \u003cspan lang=\"EN-US\"\u003e(Roubik 1992)\u003c/span\u003e still conceals many particularities that have not yet been explored \u003cspan lang=\"EN-US\"\u003e(Menezes et al. 2013)\u003c/span\u003e. The characteristics of the pollen, such as flavor, odor, color, and texture, change considerably after being stored and vary among bee species (Camargo et al. 1992; Souza et al. 2004). A few bee species, such as \u003cem\u003eTetragonisca angustula\u003c/em\u003e and \u003cem\u003eFrieseomelitta varia\u003c/em\u003e, produce dry and relatively sweet fermented pollen. However, other meliponines, such as bees from the genus \u003cem\u003eMelipona\u0026nbsp;\u003c/em\u003eand \u003cem\u003eScaptotrigona\u003c/em\u003e, produce and store moist and sour pot-pollen (Menezes et al. 2013). It is clear that the dynamics of pollen storage are still unclear, and knowing this dynamic is essential for the investigation of the fermentation process, clarifying questions about fermentation time, moment of inoculation of microorganisms, and consumption of fermented pollen by bees. This study aimed to investigate pollen storage behavior within the colonies of the bee species \u003cem\u003eM. quadrifasciata\u003c/em\u003e to obtain information that can aid in understanding its fermentation process.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eObservations were carried out for 30 days between November and December 2021. An observation colony of \u003cem\u003eM. quadrifasciata\u003c/em\u003e from the Experimental Meliponary of the Federal University of S\u0026atilde;o Paulo was used on the Diadema campus (23\u0026deg; 43\u0026apos; 10\u0026quot;). S 46\u0026deg; 37\u0026apos; 39\u0026quot; W).\u003c/p\u003e\n\u003cp\u003eThe colony used for observation had a glass top to facilitate internal visualization and, consequently, study data collection. Before the beginning of the observations, the pollen pots in the colony were removed, leaving only one pot to supply the bees\u0026apos; needs with protein and mainly to stimulate the search for external resources by the forager bees to build new pollen pots. During the 30 days of the experiment, observations were made in the morning and at night. In the morning, the pots were observed between 7 am and 10 am (UTC -3), and in the night, between 7 pm and 9 pm (UTC -3). The active pollen pots that had pollen entry and/or exit observed were marked daily with a non-toxic, water-based pen (Posca\u0026reg;), facilitating the observations of the pots and the work dynamics performed by the worker bees (Fig. 1). Thus, each time a new pot was observed with pollen inside (Start), it was randomly marked with a new combination of colors. To differentiate active pollen pots (marked with random colors) from honey pots and empty pots (not considered in this experiment), a yellow marking was used (in these particular pots). In addition to the observations regarding pollen storage dynamics, the pollen emptying process (Finish) was also monitored. For this, each time a pollen pot was emptied, the observation date of what happened was noted (Table 1).\u003c/p\u003e\n\u003cp\u003eAccording to the results of Graph 1, we see that the pollen pots analyzed during the experiment did not remain closed for long periods. In addition, it was noted in the active pots that they were most of the time open during the morning period. Another important fact to be reported is that after thirty days, the presence of 14 active pots was observed, and at the beginning of the experiment, only one active pot had been left. This fact may have occurred to guarantee the constant supply of essential nutrients provided only by pollen. Stingless bees, like honeybees, adjust their foraging activities (food search) to maximize food intake when floral resources are unavailable (Biesmeijer and de Vries 2001; Maia-Silva et al. 2015). These adjustments, such as the search for more resources such as pollen, are a reaction to changes in external environmental factors and the colony\u0026apos;s needs (Eltz et al. 2001; Hofstede and Sommeijer 2006). Since the observations were carried out during spring in Brazil, the increased input of floral resources, mainly pollen (the study\u0026apos;s objective), justifies the appropriate period for the study.\u003c/p\u003e\n\u003cp\u003eAccording to the results, there was no standardization regarding the dynamics of opening and closing the pollen pots of the observed colony (Fig. 2), a fact that happens with \u003cem\u003eA. mellifera\u003c/em\u003e (Detry et al. 2020). Honeybees progressively fill the wax alveoli with pollen grains brought by foraging bees, practically in their entirety of the storage space. After filling the alveoli, they receive a layer of honey for a certain period and then are used for consumption by worker bees (Seeley 1982). From the observations of this study, it was evident that there is no regularity between having pots open until filled with pollen brought in from the field by foragers and closing after the pot is at or near full capacity. In addition, as shown in Fig. 2, when we compare two different pots in the same period, we notice that the opening times and the frequency of the work dynamics in the pots differ from one to the other. Our data go opposite to those reported by Nogueira-Neto and Menezes (Nogueira-Neto 1997; Menezes et al. 2013), who indicate that stingless bees close the pots when complete, storing them for approximately two weeks before consumption.\u003c/p\u003e\n\u003cp\u003eAs stingless bees are a diverse group, understanding how worker bees work to store pollen inside cerumen pots is highly relevant information for understanding the storage time and transformation of food through fermentation processes and later consumption by individuals of the colony. Few studies report the fermentation dynamics of pollen stored by stingless bees (Menezes et al. 2013). As shown in Fig. 2, the behavior of the workers who supply the pollen pots does not maintain regularity in how they perform the task, as studies show that it occurs in \u003cem\u003eA. mellifera\u003c/em\u003e (vanEngelsdorp et al. 2009). In the case of this behavior, for \u003cem\u003eM. quadrifasciata\u003c/em\u003e, it was observed that several pots are opened and closed throughout the day or night, regardless of the amount of pollen inside. It can be verified through Graph 1; Table 1, that 18 pots were observed throughout the experiment. Of these, some could be observed over several days, such as pot number two (36 observations - remaining open for 58.4% of the time and 41.6% of the time closed); four (38 observations - remaining open for 89.4% of the time and 10.6% closed) and five (34 observations - remaining for 64.7% of the open time and 35.3% of the closed time).\u003c/p\u003e\n\u003cp\u003eThus, an interesting and recurrent data in the observations is the way the foraging bees bring pollen and how it is treated, as it is known that during this conditioning, workers add secretions that are assumed to contain beneficial microorganisms (Teixeira et al. 2003). These microorganisms may be involved in the stored food\u0026apos;s metabolic conversion, fermentation, and preservation. The transformation of pollen to bee bread has often been postulated to result from microbial action, mainly lactic acid fermentation caused by bacteria and yeasts (Haydak 1958).\u003c/p\u003e\n\u003cp\u003eNew observational studies need to be carried out about the pollen storage of stingless bees, but the dynamics of opening and closing the pollen pots may indicate a continuity of the fermentation process where gas exchanges may be occurring and even maintenance of nutrient flow by adding more content between closing and opening pots, as noted.\u003c/p\u003e\n\u003cp\u003eThus, information on how the workers of stingless bee species work, store and ferment their food is of great value within the scenario of understanding the importance of the microbiota associated with this process to produce alternative sources of food during periods of scarcity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur observations on the behavior of storage and handling of pollen in the species \u003cem\u003eM. quadrifasciata\u003c/em\u003e led us to conclude that there is a lack of regularity in treating this food, contrary to the behavior of \u003cem\u003eA. mellifera\u003c/em\u003e. The pollen pots are opened and closed daily, and bee entry and exit behavior are frequent. Studies on the behavioral dynamics of stingless bees in food pots are scarce in the literature. Therefore, this work highlights the importance of data that explain how food storage occurs within colonies. Such data are paramount for advancing techniques that improve meliponiculture nationally and internationally, such as developing fermented protein supplementation. Understanding the storage time and natural occurrence within the colonies is essential for the fermentation of an artificial diet that mimics the natural characteristics of this important food source\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eWe thank Marcos Reis for his contribution to the analysis of the results, Tiago Francoy for his important help in the discussion and interpretation of the data, and Leandro Barros de Souza for drawing up Fig.\u0026nbsp;2. We also thank Capes (Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior) for the financial support to carry out this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBiesmeijer JC, de Vries H (2001) Exploration and exploitation of food sources by social insect colonies: a revision of the scout-recruit concept. Behav Ecol Sociobiol 49:89\u0026ndash;99\u003c/li\u003e\n \u003cli\u003eCamargo JMF de, Garcia MVB, Eugenio Junior RQ, Castrillon A (1992) Notas pr\u0026eacute;vias sobre a bionomia de Ptilotrigona lurida (\u003cem\u003eHymenoptera, Apidae, Meliponinae\u003c/em\u003e): associa\u0026ccedil;\u0026atilde;o de leveduras em p\u0026oacute;len estocado. Boletim do Museu Paraense Em\u0026iacute;lio Goeldi 8:391\u0026ndash;395\u003c/li\u003e\n \u003cli\u003eCosta L, Venturieri GC (2009) Diet impacts on \u003cem\u003eMelipona flavolineata\u003c/em\u003e workers (\u003cem\u003eApidae, Meliponini)\u003c/em\u003e. J Apic Res 48:38\u0026ndash;45\u003c/li\u003e\n \u003cli\u003eDetry R, Simon-Delso N, Bruneau E, Daniel H-M (2020) Specialisation of yeast genera in different phases of bee bread maturation. Microorganisms 8:1789\u003c/li\u003e\n \u003cli\u003eEltz T, Br\u0026uuml;hl, CA, van der Kaars S, Chey VK, Linsenmair K E (2001) Pollen foraging and resource partitioning of stingless bees in relation to flowering dynamics in a Southeast Asian tropical rainforest. Insectes Soc 48:273\u0026ndash;279\u003c/li\u003e\n \u003cli\u003eHaydak MH (1958) Pollen-pollen substitutes-beebread. Am Bee J 98:145\u0026ndash;146\u003c/li\u003e\n \u003cli\u003eHofstede FE, Sommeijer MJ (2006) Influence of environmental and colony factors on the initial commodity choice of foragers of the stingless bee \u003cem\u003ePlebeia tobagoensis\u0026nbsp;\u003c/em\u003e(Hymenoptera, Meliponini). Insectes Soc 53:258\u0026ndash;264. https://doi.org/10.1007/s00040-006-0866-9\u003c/li\u003e\n \u003cli\u003eMaia-Silva C, Hrncir M, da Silva CI, Imperatriz-Fonseca VL (2015) Survival strategies of stingless bees (\u003cem\u003eMelipona subnitida\u003c/em\u003e) in an unpredictable environment, the Brazilian tropical dry forest. Apidologie 46:631\u0026ndash;643\u003c/li\u003e\n \u003cli\u003eMenezes C, Vollet-Neto A, Contrera FAFL, et al (2013) The role of useful microorganisms to stingless bees and stingless beekeeping.\u0026nbsp;Pot-Honey: A legacy of stingless bees 153\u0026ndash;171\u003c/li\u003e\n \u003cli\u003eNogueira-Neto P (1997) Vida e cria\u0026ccedil;\u0026atilde;o de abelhas ind\u0026iacute;genas sem ferr\u0026atilde;o. In: Vida e cria\u0026ccedil;\u0026atilde;o de abelhas ind\u0026iacute;genas sem ferr\u0026atilde;o. p 446\u003c/li\u003e\n \u003cli\u003eRoubik DW (1982) Seasonality in colony food storage, brood production and adult survivorship: studies of Melipona in tropical forest (\u003cem\u003eHymenoptera: Apidae\u003c/em\u003e). J Kans Entomol Soc 789\u0026ndash;800\u003c/li\u003e\n \u003cli\u003eRoubik DW (1992) Ecology and natural history of tropical bees. Cambridge University Press\u003c/li\u003e\n \u003cli\u003eSeeley TD (1982) Adaptive significance of the age polyethism schedule in honeybee colonies.\u0026nbsp;Behav Ecol Sociobiol 11:287\u0026ndash;293\u003c/li\u003e\n \u003cli\u003eSouza RC da S, Yuyama LKO, Aguiar JPL, Oliveira FPM (2004) Valor nutricional do mel e p\u0026oacute;len de abelhas sem ferr\u0026atilde;o da regi\u0026atilde;o amaz\u0026ocirc;nica.\u0026nbsp;Acta Amazon 34:333\u0026ndash;336\u003c/li\u003e\n \u003cli\u003eTeixeira ACP, Marini MM, Nicoli JR, et al (2003) \u003cem\u003eStarmerella meliponinorum\u003c/em\u003e sp. nov., a novel ascomycetous yeast species associated with stingless bees. Int J Syst Evol Microbiol 53:339\u0026ndash;343\u003c/li\u003e\n \u003cli\u003evanEngelsdorp D, Evans JD, Saegerman C, et al (2009) Colony Collapse Disorder: A Descriptive Study. PLoS One 4:e6481. https://doi.org/10.1371/journal.pone.0006481\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Graph","content":"\u003cp\u003eGraph 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Stingless bees, pollen storage, Melipona quadrifasciata, fermentation","lastPublishedDoi":"10.21203/rs.3.rs-2981706/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2981706/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Stingless bees are important eusocial insects found in tropical and subtropical regions. They store nectar and pollen from flowers to survive periods of scarcity. During storage, nectar and pollen undergo fermentation processes, resulting in honey and fermented pollen. However, the fermentation process of pollen collected by stingless bees is not well understood. Understanding these processes not only clarifies pollen fermentation in different stingless bee species but also sheds light on the transformation process of pollen. This knowledge can be applied to produce fermented feeds using specific microorganisms. Therefore, this study aimed to observe pollen storage behavior within colonies to understand the fermentation process and aid in the development of fermented feeds for bees. A Melipona quadrifasciata colony from the Experimental Meliponary of the Universidade Federal de São Paulo, Brazil, was observed for 30 days. Daily observations were conducted in the morning and night, and pollen pots were marked with random colors to differentiate them and collect data on worker bee dynamics. Observations showed no consistent patterns in the opening and closing of the pots, with worker bees frequently entering and exiting the open pollen pots. This suggests an ongoing fermentation process involving gas exchange and nutrient flow. The lack of regularity in handling pollen storage in M. quadrifasciata indicates its noteworthy variability. These findings are crucial for advancing meliponiculture techniques and developing fermented protein supplements globally and locally.","manuscriptTitle":"Scientific note: behavioral dynamics of pollen storage in Melipona quadrifasciata","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-30 15:56:57","doi":"10.21203/rs.3.rs-2981706/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-05-27T22:00:09+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-05-27T21:56:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-05-27T08:35:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Insectes Sociaux","date":"2023-05-25T10:55:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3e741716-672a-4905-85cc-5849674ade0d","owner":[],"postedDate":"May 30th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-11-06T15:06:04+00:00","versionOfRecord":{"articleIdentity":"rs-2981706","link":"https://doi.org/10.1007/s00040-023-00940-5","journal":{"identity":"insectes-sociaux","isVorOnly":false,"title":"Insectes Sociaux"},"publishedOn":"2023-11-03 15:01:44","publishedOnDateReadable":"November 3rd, 2023"},"versionCreatedAt":"2023-05-30 15:56:57","video":"","vorDoi":"10.1007/s00040-023-00940-5","vorDoiUrl":"https://doi.org/10.1007/s00040-023-00940-5","workflowStages":[]},"version":"v1","identity":"rs-2981706","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2981706","identity":"rs-2981706","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}