# Adult brain neurogenesis does not account for behavioral differences between solitary and social bees

Iulia Antioch, Sarah Larnaudie, Isabelle Lafon, Jean-Marc Devaud, Claire

Rampon, Raphaël Jeanson

► To cite this version:

Iulia Antioch, Sarah Larnaudie, Isabelle Lafon, Jean-Marc Devaud, Claire Rampon, et al. Adult brain neurogenesis does not account for behavioral differences between solitary and social bees. Journal of Insect Physiology, In press, pp.104737. 10.1016/j.jinsphys.2024.104737. hal-04848388

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# Adult brain neurogenesis does not account for behavioral differences between solitary and social bees

Iulia Antioch<fnref n="1" />, Sarah Larnaudie<fnref n="1" />, Isabelle Lafon, Jean-Marc Devaud, Claire Rampon<fnref n="1" />,
Raphaël Jeanson*<fnref n="1" />,†

Centre de Recherches sur la Cognition Animale (UMR5169), Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, France

## ARTICLE INFO

**Keywords:**

Eusociality

Insects

Mushroom bodies

Plasticity

Sociality

## ABSTRACT

In many taxa, increasing attention is being paid to how group living shapes the expression of brain plasticity and behavioural flexibility. In eusocial insects, the lifelong commitment of workers and queens to a reproductive or non-reproductive caste is accompanied by a loss of behavioural totipotency, and often, by the expression of a limited behavioural repertoire in workers due to their specialisation. On the other hand, individuals of solitary species have a broader behavioural repertoire as they have to perform all the tasks themselves. This raises the question of whether solitary and social insects differ in their levels of brain plasticity. One mechanism found in both invertebrates and vertebrates to contribute to brain plasticity is adult neurogenesis. It is a mechanism by which adult-born neurons are generated, differentiated and functionally integrated in the brain circuits during adulthood. In this study, we compared the solitary bee *Osmia bicornis* and the eusocial bee *Apis mellifera*. We focused on the mushroom bodies which are higher-order integration centres in the insect brain. Based on their known behavioural repertoire, our prediction was that both solitary and social bees would exhibit neurogenesis in the brain until the pupal stage, but that this capacity would persist only in adult solitary bees. However, our results do not validate this prediction, as they indicate that no cells are produced in the mushroom bodies or other areas of the adult solitary bee brain.

## 1. Introduction

The transition to eusociality is considered one of the eight major evolutionary transitions (Maynard Smith and Szathmary, 1995). In social groups, eusociality reflects the existence of a reproductive division of labour where the queen (or queens) monopolises reproduction and is assisted by a caste of workers performing non-reproductive tasks. In colonies of highly eusocial insects (e.g., ants and corbiculate bees), workers and queens irreversibly commit for life to distinct castes, which involves the activation of various regulatory gene networks, leading to massive physiological changes (Boomsma and Gawne, 2018). The existence of a reproductive division of labour implies, by definition, that colony members lose their behavioural totipotency defined as the lifelong potential to express the full behavioural repertoire of the population (Crespi and Yanega, 1995). In addition, in many cases, non-reproductive individuals typically specialise in specific tasks (e.g., colony defence, brood care, foraging), even if they can perform all tasks at

different moments of their lives, when division of labour is age-
dependent as in honey bees (Robinson, 1992). This markedly contrasts
with solitary species where females must perform all activities alone,
from nest building to brood care and foraging. In social insects, several
studies have explored how changes in complexity of social organization
correlate with changes in brain structure (Godfrey and Gronenberg,
2019). It has been proposed that living in large social groups may reduce
brain investment, as division of labour among colony members can
lower the cognitive demands on individuals (O'Donnell et al., 2015). In
addition, solitary individuals have to deal on their own with unpredictable
fluctuations in the environment, whereas within societies, responses
to disturbances are shared among individuals. Altogether these
data led to the hypothesis that solitary and social individuals might
differ in their levels of behavioural flexibility, with those in social groups
being more canalized, while offering colonies all the benefits of
specialisation (Jeanson, 2019).

In insects, most comparative studies addressing the link between

<footnote>* Corresponding author.
E-mail address: raphaël.jeanson@univ-tlse3.fr (R. Jeanson).</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>
cognitive abilities, brain architecture and sociality have compared the size of brain areas of interest, often considering the effects of body size on brain allometry (Smith et al., 2010, O'Donnell et al., 2019). Brain structures that have received much attention are the mushroom bodies (MBs), a pair of neuropils located in the dorsal protocerebrum of insects and involved in multisensory integration, associative learning and spatial memory (Zars, 2000, Fiala and Kaun, 2024). Variations in MB connectivity have been related to experience-dependent plasticity, such as changes in cognitive flexibility in honey bees (Cabirol et al., 2017, 2018), expansion of the behavioural repertoire in ants (Seid and Traniello, 2006), and storage of long-term memories in both taxa (Hourcade et al., 2010, Falibene et al., 2015). In addition, a comparative study in wasps showed that the relative volume of MBs was larger in solitary species than in social species (O'Donnell et al., 2019 but see Pahlke et al., 2020). Yet, while volumetric approaches have traditionally been used because they are relatively easy to implement, changes in brain architecture may not always be based on detectable volume changes (Godfrey and Gronenberg, 2019).

Across taxa, an important mechanism promoting brain plasticity is adult neurogenesis which is the formation and integration of new neurons in the adult brain circuits. In mammals, adult-born neurons differ notably from mature ones in their excitability so that their addition to a network, despite in small numbers, may exert a significant impact on neural dynamics and thus on brain function (Snyder and Cameron, 2012). Adult neurogenesis in the insect brain is restricted to the MBs and has been reported in diverse taxa (Diptera, Lepidoptera, Orthoptera, Coleoptera, Dictyoptera) (Cayre et al., 1996). Adult neurogenesis in insects contributes to experience-dependent plasticity in several species (Cayre et al., 1994, 2002, 2007, Dufour and Gadenne, 2006, Scotto-Lomassese et al., 2002). For instance, the manipulation of sensory inputs modulates the proliferation of adult-born cells in the house cricket (Cayre et al., 2002) and in the red flour beetle (Trebels et al., 2020). On the other hand, the abolition of adult neurogenesis impairs olfactory learning in crickets (Scotto-Lomassese et al., 2003). Strikingly, so far, evidence for adult neurogenesis in the brain, and particularly in the MBs, has only come from solitary species (e.g. cockroaches, locusts, crickets, moths: Cayre et al., 1996). Yet, a eusocial species such as the honey bee *Apis mellifera*, constitutes a traditional research model for the study of learning and memory, as it shows impressive cognitive aptitudes such as numerosity, pattern recognition and advanced communication (Howard et al., 2018; Giurfa 2021). Despite these cognitive skills, adult neurogenesis was not found in honey bees (Fahrbach et al., 1995) and their brain lacks the capacity to produce new cells in adulthood, as neuroblasts are eliminated by apoptosis during the pupal stage (Ganeshina et al., 2000). Interestingly, adult neurogenesis was also not found in the other eusocial insect species tested so far, the ant *Camponotus floridanus* (Gronenberg et al., 1996) and the bumble bee *Bombus terrestris* (Guan et al., 2019). The fact that several non-social species of insects can produce adult-born brain cells but that adult neurogenesis is absent in eusocial species led us to hypothesize that social transitions have relaxed the need for adult neurogenesis and that, consequently, this ability has been lost in highly social insects.

In this context, our main objective was to determine whether a sol-
itary bee, *Osmia bicornis*, exhibits adult neurogenesis. The choice of this
species was motivated by the fact that it is used as a pollination aid
(Gruber et al., 2011), which facilitates its installation in orchards in
order to harvest individuals at different stages of development and of
known age, unlike wild solitary bees. In addition, bees of the genus
*Osmia* are among the most extensively studied solitary species, in
particular to evaluate their cognitive abilities. They have been shown to
display navigational skills when searching for food or building materials
(Vicens and Bosch, 2000). *Osmia* are also able to identify the most
rewarding nectar sources based on volatile substances (Howell and
Alarcón, 2007), to integrate multiple sensory cues for mate selection
(Conrad et al., 2010) and to innovate to solve new tasks (Collado et al.,
2021). We also aimed to confirm previous findings on the absence of

adult neurogenesis reported in the honey bee *A. mellifera* (Fahrbach
et al., 1995) by using a different methodological approach. We predicted
that while both solitary and social bees would exhibit neurogenesis in
the brain until the pupal stage (i.e., before emerging as adults), this
capacity would be maintained only in the adults of the solitary species.

**2. Materials and methods**

2.1. Animals

2.1.1. *Osmia bicornis*

This bee species has an annual life-cycle and adults never co-exist with offspring. In the field, the female mates immediately after emergence in early spring and then searches for cylindrical nesting cavities. Once found, she collects pollen from surrounding flowers to form a pollen ball on which she lays an egg before sealing the brood cell with a mud wall. Each female produces a series of brood cells arranged linearly. An adult female is active between about 50 and 70 days. About 30 to 40 days after egg laying, the larva spins a silk cocoon to pupate in late summer. After metamorphosis, the young adult remains in the cocoon where it hibernates and emerges the following spring when the temperature rises (Splitt et al., 2021). Cocoons of *O. bicornis* were provided by the company Horizon (Osmia) (Agen, France) and were kept in a cooled incubator (4 °C) for 5 months to simulate winter diapause. In March 2022, we placed the cocoons in artificial boxes containing cardboard tubes as potential nesting sites, which were installed in an apple orchard in Lavelanet-de-Comminges (France). Regular monitoring of the nesting boxes in the field enabled us to collect adult females (N = 16) that had settled in the nesting tubes for less than 48 h. We also placed cocoons in the lab at room temperature (22 °C) to end overwintering and obtain newly emerged adult females (N = 5). A sample of nesting tubes occupied by bees in the field was collected in July 2022 in order to obtain pupae (N = 6) (white body and coloured eyes, equivalent to the P2-P4 developmental stages of *A. mellifera*, Ganeshina et al., 2000).

2.1.2. *Apis mellifera*

The honey bee *A. mellifera* is a highly eusocial bee species that forms colonies containing a queen and up to 60,000 workers. The queen lays eggs in brood cells in the honeycomb. The worker larva is fed royal jelly for 2 to 3 days, then pollen and honey. After about 6 days, the cell is capped with wax. The larvae spin a cocoon inside the sealed cell and then pupate to undergo metamorphosis. After about 2 weeks, the adult worker bee emerges and leaves the cell. In her first 10 to 15 days, the worker bee remains inside the nest where she performs in-hive tasks such as larval feeding, cell cleaning or food processing. She then shows physiological and behavioural changes that prepares her to leave the hive to forage. Honey bees were collected between March and November 2022 from hives of our experimental apiary located on the campus of the University Paul Sabatier (Toulouse, France). We compared four different groups of bees at different ontogenic stages: pupae, newly emerged workers, nurses (all collected on brood frames) and foragers. We collected young bees (N = 6) that had emerged less than 1 h before (identified by their characteristic greyish body) and nurses (N = 6) identified as workers seen inserting their heads into uncapped brood cells on hive frames. We opened capped brood cells to collect pupae (N = 3) at P2-P4 developmental stages (Ganeshina et al., 2000). Foragers (N = 7) were collected at artificial feeders filled with a 50 % sucrose solution and placed near the hives.

## 2.2. Positive controls for the proliferation-detection method

The incorporation and measurement of nucleoside analogues in
newly synthesized DNA is classically used to detect cell proliferation.
Here we used the 5-ethynyl-2'-deoxyuridine (EdU), a nucleoside
analogue of thymidine which is incorporated into DNA during mitosis
which allows birth-dating and labelling of newborn cells (Chehrehasa
et al., 2009). We generated positive controls for EdU incorporation using the gut, which is a highly proliferative structure (Ward et al., 2008). This allowed us to assess the validity of our proliferation detection method and eliminate the risk that the possible absence of EdU staining in the adult brain was due to methodological problems.

## 2.3. Intracerebral injection of EdU in bees

Adults of both species were chilled on ice and harnessed in custom 3D-printed plastic holders. Their head was immobilized with a drop of wax (Deiberit 502 Hard Sticky Wax, Red) melted with a soldering iron (Antex XS25, 230V). Next, we removed the lens of the median ocellus with a microblade. Through the ocellus hole, we injected a 2 µl solution of 20 mg/mL EdU (EdU Click-488 ROTI®kit for imaging, Carl Roth) dissolved in PBS 1X (Phosphate-Buffered Saline 10X, pH ~ 7, Euro-medex) or of 2 µl solution of PBS 1X using a WPI Sub-Microliter Injection System 10-µl NanoFil syringe (World Precision Instruments). This dose of EdU injected was of the same order of magnitude as that used in a previous study which succeeded in revealing newborn cells in the mushroom bodies of red flour beetle (Trebels et al., 2020). After injection, bees were individually placed in 1.5 mL microtubes (Sarstedt) with a 2 µl droplet of 70 % sucrose solution for 6 h before sacrifice. A small hole was drilled in the lid of the microtube for air circulation. Microtubes with *A. mellifera* were placed in an incubator at 28 °C (Heratherm Thermo Scientific) with a container of water to maintain humidity. Microtubes with *O. bicornis* were left at room temperature. After cooling on ice for 10 min, pupae were carefully injected a 2 µl solution of EdU into the neck. After injection, the pupae were placed in a Petri dish in a 28 °C incubator (Heratherm Thermo Scientific) with a container of water for 6 h.

## 2.4. Dissection and histological preparation of brains and guts

Adult bees were placed on ice until lethargic immobility and placed on the plastic holder. A hole was quickly made in the cuticle between the compound eyes using a microblade. Pupae were placed on ice for 10 min. Both pupae and adults were decapitated with micro-scissors. Heads were introduced to a 1.5 mL tube containing a solution of 4 % formaldehyde (PFA, ThermoScientific, 16 % methanol-free formaldehyde solution) in PBS 1X and placed under agitation at 4 °C for two hours. Then, the brain was removed from the head capsule and placed overnight under agitation at 4 °C in a 1.5 mL microtube filled with 4 % PFA solution. In addition, the last abdominal segment of the abdomen of adults was gently pulled with straight forceps to extract the midgut, which was used as a positive control of EdU incorporation. The gut was stretched on nylon paper (white nylon membrane 0.2 µm, Dutscher) and placed in a 1.5 mL microtube containing 4 % PFA on a shaker overnight at 4 °C. Tissues (brains and guts) were placed in embedding cassettes (Simport™ Scientific M™ Biopsy Processing/Embedding Cassettes) and dehydrated in a graded series of ethanol baths (70 % for 2x 10 min, 90 % for 2x 10 min, 96 % for 30 min, 100 % for 40 min). The tissues were then transferred into Histo-clear II (Electron Microscopy Sciences) for 4 h. The cassettes were then immersed in molten paraffin (Paraplast Plus, Sigma Aldrich) at 62 °C overnight. The next day, the samples were placed in a second paraffin bath for 4 h at 62 °C. We used the TES Valida Paraffin Embedding System Medite adjusted at 64 °C to embed samples in paraffin which were then stored at 4 °C. We used a microtome (HM 355S Automatic Microtome Leica) to cut 5 µm-thick frontal brain sections or longitudinal gut sections that were then transferred with a fine paint-brush to a rectangular Medax warm bath at 38 °C. Sections were mounted on microscope slides (ground 90°, frosted 20 mm) (Thermo-Scientific) and dried on a slide warmer (Premiere Slide Warmer XH-2002) at 40 °C.

## 2.5. EdU and DAPI staining

Tissue sections were placed in two consecutive baths (10 min) of Histo-Clear II for deparaffinization and then transferred to successive baths of ethanol (100 % for 5 min, 90 % for 3 min, 80 % for 3 min). Next, the slides were rinsed in water, placed in phosphate buffer saline (PBS 1X) for 2 x 15 min and then in a slide staining system (Simport Scientific). Slides were immerged in PBST (PBS 1X containing 0.25 % Triton-X 100) for 30 min, and then in the EdU kit 488 solution (Carl Roth). After one rinse in PBS for 15 min, the sections were stained with DAPI (Sigma, DA9542) diluted at 1:6000 in PBS 1X for 30 min, in order to visualize cell nuclei. Because autofluorescence was present in *Osmia* but not *Apis*, we incubated brain sections of adult *Osmia* for 30 min in 0.1 % Sudan Black B (George T. Gurr Ltd.) dissolved in 70 % ethanol, in order to reduce autofluorescence in brain tissue. Then, the slides were rinsed in PBS 1X for 15 min and coversliped using Mowiol (Calbiochem). The slides were then left in the dark, at room temperature for 24 h, before being stored at 4 °C until use.

## 2.6. Fluorescent microscopy and tissue imaging

Serial brain and gut sections were observed under a wide-field fluorescent microscope (Nikon eclipse Ti) at 20x, 40x and 100x objectives, first based on DAPI staining to identify the areas of interest. Photographs of the Kenyon cells present in the calyces of the mushroom bodies, as well as photographs of EdU-labelled (green) cells in the bee guts, were then taken using a digital camera (ORCA-flash 4.0 LT, Hamamatsu C11440). The images were analyzed using the Fiji software (Schindelin et al., 2012). When EdU staining was observed, images for both channels were merged in order to check for co-localization of EdU with the DAPI nuclear signal. Brains of adult bees showing no EdU-labelled cells in the gut were excluded from the analysis (*A. mellifera* – emerging bees: N = 2/6, nurses: N = 1/6, foragers: N = 0/7; *O. bicornis* – emerging bees: N = 0/5, adults: N = 1/16). We also removed from the analysis bees whose brains were not well preserved or sectioned (*A. mellifera* – emerging bees: N = 1/6, nurses: N = 1/6, foragers: N = 2/7; *O. bicornis* – emerging bees: N = 2/5, adults: N = 1/16).

## 3. Results

For each bee, 12 brain sections were analysed for EdU-positive cells in the mushroom bodies (MBs). We found EdU-positive cells in the MBs of all pupae of *O. bicornis* (N = 6; Fig. 1), and *A. mellifera* (N = 3; Fig. 1). This shows that EdU indeed labels dividing cells in the bee brain. However, when looking at adult brains, we detected no EdU-positive cells among Kenyon cells (MB neurons). This was irrespective of the age or social role of individuals in both *O. bicornis* (emerging bees: N = 3; adult bees: N = 14; Fig. 2) and *A. mellifera* (emerging workers: N = 3; nurses: N = 4; foragers: N = 5; Fig. 2). Extending our observations to other regions of the adult bee brain, we also found no EdU-labelled cells (data not shown), despite the clear and consistent EdU staining observed in the guts of these individuals (Fig. 2).

## 4. Discussion

In this study, we tested the prediction that solitary, but not eusocial, adult bees would produce new neurons as a mean to sustain their higher need for brain plasticity related to their expression of a larger behavioural repertoire. We focused on the mushroom bodies as they are the brain area where neurogenesis is maintained in adults of several insect species (Cayre et al., 1996), and because MB exhibit plasticity related with critical functions in experience-dependent plastic behaviours in insects (Zars 2000, Devaud et al., 2007, Withers et al., 2008, Lutz and Robinson 2013). Our results did not validate our prediction, as they indicate that no cells are produced in the MB of solitary bee *Osmia bicornis* during adulthood. Our results are also in line with earlier
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Photographs of frontal brain sections of pupae of *O. bicornis* and *A. mellifera* bees after EdU staining (green) and DAPI coloration (blue). Images on the left were obtained in the mushroom bodies region, and those on the right are an enlargement of Kenyon cells showing the presence of Edu-positive nuclei (arrowheads). Scale bars represent 100 µm (left) and 10 µm (right). AL: antennal lobe, CC: central complex; LCA/CAL: lateral calyx; MCA/CM: median calyx; OL: optic lobe; Pe: peduncle; KC: Kenyon cells.</figcaption></figure>

findings reporting the absence of adult neurogenesis in the social bee *Apis mellifera* (Fahrbach et al., 1995) and *Bombus terrestris* (Guan et al., 2019). Since, to our knowledge, EdU had not been tested in bees so far, we validated its efficiency for labelling newly born cells using pupal brains. In addition, positive staining found in the guts of adult bees provided a validation of the EdU injection and staining techniques, even in the absence of any labelled cell in the adult brains. The ability of EdU to penetrate the bee brain to reach the site of cell proliferation may be questioned, but this approach has been used successfully to label adult-born cells in the brain of the red flour beetle (Trebels et al., 2020). Similarly, the lack of cell proliferation evidenced by the absence of EdU-labelled cells in the adult brains of two butterflies was confirmed by an alternative method based on counting cells in the mushroom bodies (Alcalde Anton et al., 2023). Since the adult solitary and social bees were collected in the field and thus exposed to their natural environment, the absence of adult neurogenesis cannot be attributed to impoverished living conditions, which are known to impair cell proliferation and survival in the adult brain of many animal species (e.g., crayfish:

Sandeman and Sandeman 2000, cricket: Scotto-Lomassese et al., 2003, mouse: van Praag et al., 2000).

Studies on brain plasticity in bees have largely focused on *Apis mellifera*, comparing the neuroanatomical changes associated with the transition from nursing to foraging. Indeed, this ontogenic shift exposes bees to massive changes in their sensory landscape and requires an increased cognitive load imposed by spatial navigation and foraging activities in a constantly changing environment. The increase in MB volume that accompanies behavioural maturation towards foraging involves a change in both the length and branching pattern of Kenyon cell dendrites, but not the addition of new brain cells (Fahrbach et al., 1995, Farris et al., 2001). Thus, in *Apis mellifera*, brain plasticity relies only on changes in connectivity based on the rearrangement of existing neural circuits (Groh and Meinertzhagen 2010). Remodelling of MB synaptic networks occurs through both pruning events that accompany behavioural maturation (Muenz et al., 2015; Cabiol et al., 2017; 2018) and probable addition of new synapses to support long-term memory storage (Hourcade et al., 2010). Likewise, ants, which also exhibit age
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Photographs of frontal brain sections of emerging and mature adult *O. bicornis* females and of emerging workers, nurses and foragers of *A. mellifera*. Brain sections were stained with EdU (green) and DAPI (blue) and images were taken in the mushroom bodies region (left). An enlargement of Kenyon cells is shown (middle) demonstrating the absence of Edu-positive cells. Images on the right show Edu-labelled cells (arrowheads) in the guts of the corresponding animals. Scale bars represent respectively 100 µm (left), 10 µm (middle) and 100 µm (right). AL: antennal lobe; CC: central complex; LCA/CAL: lateral calyx; MCA/CM: median calyx; OL: optic lobe; Pe: peduncle; KC: Kenyon cells.</figcaption></figure>
polyethism, show structural variations in their MBs (Stieb et al., 2010; Falibene et al., 2015). Similarly, the increased cognitive abilities of *Heliconius erato*, as compared to other related butterfly species, are associated with an enlargement of MBs, which also expand with age, not attributed to increased cell proliferation (Alcalde Anton et al., 2023). In the solitary orchard bee *Osmia lignaria*, the size-ratio of the neuropil region to the Kenyon cell region remains constant throughout matura- tion and is similar to that of honey bee foragers. This contrasts with the pattern found in the honey bee, where this ratio increases as workers initiate foraging (Withers et al., 2008). However, very few studies have deployed comparative approaches to identify and dissect potential dif- ferences in neural processes between solitary and social insect species, beyond volumetric measures. The only one that we are aware of is a work on a halictid bee, *Lasioglossum albipes*, whose natural populations are either solitary or eusocial, depending on their geographical location. Bees from these populations were shown to differ in their expression levels of syntaxin 1a, a protein known to mediate synaptic vesicle release (Kocher et al., 2018). Interestingly, this protein appears to be involved in the shift between gregarious and solitary phases in locusts (*Locusta migratoria*) (Chen et al., 2015), suggesting a possibly conserved role in insect phenotypic plasticity related with social interactions.

Adult brain neurogenesis is common in vertebrates including rep-
tiles, fish, birds and mammals (Lindsey and Tropepe, 2006). In these
taxa, the main questions are why neurogenesis is restricted to certain
brain regions, what its roles are and what factors modulate its rate.
Among these factors, the social context plays an important role in cell
proliferation with, for example, dominant individuals generally showing
higher cell proliferation than subordinates as in mammals or fish
(Opendak et al., 2016, Tea et al., 2019). There is also considerable
empirical evidence that exposure to an enriched and complex environ-
ment leads to increased levels of brain neurogenesis (van Praag et al.,
2000). Among the ecological factors, seasonality can also affect the
proliferation and survival of newborn neurons in adults such as lizards
(Delgado-Gonzalez et al., 2011) or birds (Sherry and
MacDougall-Shackleton 2015). Our understanding of neurogenesis in
invertebrates lags far behind that of vertebrates. In the few species
studied, some of the factors listed for vertebrates also play a role in the
modulation of adult neurogenesis such as seasonality (crab: Hansen and
Schmidt 2004) or exposure to new stimuli (red flour beetle: Trebels
et al., 2020). An important question, that remains largely unsolved, is
why some invertebrates exhibit neurogenesis while others do not. Adult
neurogenesis in insects is not marginal, particularly in the cricket where
the production of Kenyon cells in adulthood accounts for 20 % of the
cortex of the mushroom bodies (Malaterre et al., 2002). To date, no
single life-history trait has been associated with the presence or absence
of adult neurogenesis. For example, the existence of cell proliferation in
adulthood is not linked to whether insects show a complete or incom-
plete metamorphosis pattern, since it has been described in species of
both categories (Lindsey and Tropepe, 2006). On the basis of our current
knowledge of Hymenoptera, it seems also reasonable to exclude sociality
as a factor associated with the absence of neurogenesis in the adult
brain. As *Osmia bicornis*, *Apis mellifera* and *Bombus terrestris* are all
members of the order Hymenoptera and the superfamily Apoidea, it can
be speculated that their brain development, including cell death of
mushroom body neuroblasts, follows a similar intrinsic programme and
that no species of this clade would exhibit adult neurogenesis, irre-
spective of the complexity of their social organisation. The general
answer to the question of why neurogenesis persists in adults only in
certain insect taxa still eludes us and the selective pressures responsible
for this property have yet to be identified (Cayre et al., 2002, Lindsey
and Tropepe, 2006). Further efforts should be made in all taxa to engage
in more naturalistic studies to identify the ecological challenges faced by
organisms that correlate with the onset of adult neurogenesis.

Overall, a better understanding of the link between adult brain
plasticity and behavioural flexibility would benefit from a combination
of the rigorous use of more systematic behavioural protocols and

neuroanatomical approaches (Fahrbach and Van Nest, 2016). Stan-
dardized comparisons between solitary and social bees are currently
under way to assess their respective level of behavioural flexibility.
Future comparative work should explore the contribution of other forms
of neuronal plasticity than adult neurogenesis, to explain differences in
behavioural plasticity. Synaptic organisation in mushroom bodies,
where modifications have already been associated with the expression of
behavioural flexibility, is a prime candidate.

5. Ethic statement

This work complies with the current laws in France on the use of
invertebrates in research.

CRediT authorship contribution statement

**Iulia Antioch:** Writing – review & editing, Visualization, Validation, Methodology, Investigation. **Sarah Larnaudie:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. **Isabelle Lafon:** Writing – review & editing, Validation, Resources, Methodology, Investigation. **Jean-Marc Devaud:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Claire Rampon:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Raphaël Jeanson:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Funding

This work was funded by the Centre National de la Recherche Sci-
entifique, University Paul Sabatier, and the Agence Nationale de la
Recherche (grant ANR-21-CE37-0001). IA was supported by a post-
doctoral fellowship from the Fyssen Foundation.

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Acknowledgments

We thank the Domaine des Nozes (Lavelanet-de-Comminges) for
giving us free access to their orchard to install the solitary bees. We
thank Petnoi Petsophonsakul for her technical advices and Kamela
Nikolla and Leslie Lafouasse for their contributions to pilot experiments.
We are grateful to the Fyssen Foundation for awarding a post-doctoral
fellowship to I Antioch. We thank the LITC (Light Imaging Toulouse
CBI) imaging platform of Toulouse TRI (Toulouse Réseau Imagerie) for
their assistance in microscopy. We thank two anonymous reviewers for
their comments and suggestions.

Data availability

Data will be made available on request.

References

Alcalde Anton, A., Young, F.J., Melo-Flórez, L., Couto, A., Cross, S., McMillan, W.O., Montgomery, S.H., 2023. Adult neurogenesis does not explain the extensive post-eclosion growth of Heliconius mushroom bodies. R. Soc. Open Sci. 10, 230755.
Boomsma, J.J., Gawne, R., 2018. Superorganismality and caste differentiation as points of no return: how the major evolutionary transitions were lost in translation. *Biol. Rev.* 93, 28–54.

Cabirol, A., Brooks, R., Groh, C., Barron, A.B., Devaud, J.-M., 2017. Experience during early adulthood shapes the learning capacities and the number of synaptic boutons in the mushroom bodies of honey bees (Apis mellifera). Learn. Mem. 24, 557–562.

Cabirol, A., Cope, A.J., Barron, A.B., Devaud, J.-M., 2018. Relationship between brain plasticity, learning and foraging performance in honey bees. PLoS One 13, e0196749.

Cayre, M., Strambi, C., Strambi, A., 1994. Neurogenesis in an adult insect brain and its hormonal control. *Nature* 368, 57–59.

Cayre, M., Strambi, C., Charpin, P., Augier, R., Meyer, M.R., Edwards, J.S., Strambi, A., 1996. Neurogenesis in adult insect mushroom bodies. J. Comp. Neurol. 371, 300–310.

Cayre, M., Malaterre, J., Scotto-Lomassese, S., Strambi, C., Strambi, A., 2002. The common properties of neurogenesis in the adult brain: from invertebrates to vertebrates. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 132, 1–15.

Cayre, M., Scotto-Lomassese, S., Malaterre, J., Strambi, C., Strambi, A., 2007. Understanding the regulation and function of adult neurogenesis: contribution from an insect model, the house cricket. Chem. Senses 32, 385–395.

Chehrehasa, F., Meedeniya, A.C.B., Dwyer, P., Abrahamsen, G., Mackay-Sim, A., 2009. *EdU*, a new thymidine analogue for labelling proliferating cells in the nervous system. J. Neurosci. Methods 177, 122–130.

Chen, Q., He, J., Ma, C., Yu, D., Kang, L., 2015. Syntaxin 1A modulates the sexual maturity rate and progeny egg size related to phase changes in locusts. Insect Biochem. Mol. Biol. 56, 1–8.

Collado, M.A., Menzel, R., Sol, D., Bartomeus, I., 2021. Innovation in solitary bees is driven by exploration, shyness and activity levels. J. Exp. Biol. 224, jeb232058.

Conrad, T., Paxton, R.J., Barth, F.G., Francke, W., Ayasse, M., 2010. Female choice in the red mason bee, Osmia rufa (L.) (Megachilidae). J. Exp. Biol. 213, 4065–4073.

Crespi, B.J., Yanega, D., 1995. The definition of eusociality. Behav. Ecol. 6, 109–115.

Delgado-Gonzalez, F.J., Gonzalez-Granero, S., Trujillo-Trujillo, C.M., García-Verdugo, J. M., Damas-Hernandez, M.C., 2011. Study of adult neurogenesis in the gallotia galloti lIZID during different seasons. Brain Res. 1390, 50–58.

Devaud, J.-M., Blunk, A., Podufall, J., Giurfa, M., Grünewald, B., 2007. Using local anaesthetics to block neuronal activity and map specific learning tasks to the mushroom bodies of an insect brain. Eur. J. Neurosci. 26, 3193–3206.

Dufour, M.-C., Gadenne, C., 2006. Adult neurogenesis in a moth brain. J Comp Neurol 495, 635–643.

Fahrbach, S.E., Van Nest, B.N., 2016. Synapsin-based approaches to brain plasticity in adult social insects. Curr. Opin. Insect Sci. 18, 27–34.

Fahrbach, S.E., Strande, J.L., Robinson, G.E., 1995. Neurogenesis is absent in the brains of adult honey bees and does not explain behavioral neuroplasticity. Neurosci. Lett. 197, 145–148.

Falibene, A., Roces, F., Rossler, W., 2015. Long-term avoidance memory formation is associated with a transient increase in mushroom body synaptic complexes in leaf-cutting ants. Front. Behav. Neurosci. 9, 84.

Farris, S.M., Robinson, G.E., Fahrbach, S.E., 2001. Experience- and age-related outgrowth of intrinsic neurons in the mushroom bodies of the adult worker honeybee. J. Neurosci. 21, 6395–6404.

Fiala, A., Kaun, K.R., 2024. What do the mushroom bodies do for the insect brain? Twenty-five years of progress. Learn. Mem. 31, a053827.

Ganeshina, O., Schäfer, S., Malun, D., 2000. Proliferation and programmed cell death of neuronal precursors in the mushroom bodies of the honeybee. J. Comp. Neurol. 417, 349–365.

Giurfa, M., 2021. Learning of sameness/difference relationships by honey bees: performance, strategies and ecological context. Curr. Opin. Behav. Sci. 37, 1–6.

Godfrey, R.K., Gronenberg, W., 2019. Brain evolution in social insects: advocating for the comparative approach. J. Comp. Physiol. A 205, 13–32.

Groh, C., Meinerzthagen, I.A., 2010. Brain plasticity in Diptera and Hymenoptera. Frontiers in Biosciences 2, 268–288.

Gronenberg, W., Heeren, S., Hölldobler, B., 1996. Age-dependent and task-related morphological changes in the brain and the mushroom bodies of the ant Camponotus floridanus. J. Exp. Biol. 199, 2011–2019.

Gruber, B., Eckel, K., Everaars, J., Dormann, C.F., 2011. On managing the red mason bee (Osmia bicornis) in apple orchards. Apidologie 42, 564.

Guan, C., Egertová, M., Perry, C.J., Chittka, L., Chittka, A., 2019. Temporal correlation of elevated PRMT1 gene expression with mushroom body neurogenesis during bumblebee brain development. J. Insect Physiol. 116, 57–69.

Hansen, A., Schmidt, M., 2004. Influence of season and environment on adult neurogenesis in the central olfactory pathway of the shore crab, Carcinus maenas. Brain Res. 1025, 85–97.

Hourcade, B., Muenz, T.S., Sandoz, J.-C., Rössler, W., Devaud, J.-M., 2010. Long-term memory leads to synaptic reorganization in the mushroom bodies: A memory trace in the insect brain? J. Neurosci. 30, 6461–6465.

Howard, S.R., Avargués-Weber, A., Garcia, J.E., Greentree, A.D., Dyer, A.G., 2018. Numerical ordering of zero in honey bees. Science 360, 1124–1126.

Howell, A.D., Alarcón, R., 2007. Osmia bees (Hymenoptera: Megachilidae) can detect nectar-rewarding flowers using olfactory cues. Anim. Behav. 74, 199–205.

Jeanson, R., 2019. Within-individual behavioural variability and division of labour in social insects. J. Exp. Biol. 222, jeb190868.

Kocher, S.D., Mallarino, R., Rubin, B.E.R., Yu, D.W., Hoekstra, H.E., Pierce, N.E., 2018. The genetic basis of a social polymorphism in halictid bees. Nat. Commun. 9, 4338.

Lindsey, B.W., Tropepe, V., 2006. A comparative framework for understanding the biological principles of adult neurogenesis. Prog. Neurobiol. 80, 281–307.

Lutz, C.C., Robinson, G.E., 2013. Activity-dependent gene expression in honey bee mushroom bodies in response to orientation flight. J. Exp. Biol. 216, 2031–2038.

Malaterre, J., Strambi, C., Chiang, A.-S., Aouane, A., Strambi, A., Cayre, M., 2002. Development of cricket mushroom bodies. J Comp Neurol 452, 215–227.

Maynard Smith, J., Szathmary, E., 1995. The Major Transitions in Evolution. Oxford University Press, Oxford.

Muenz, T.S., Groh, C., Maisonnasse, A., Le Conte, Y., Plettner, E., Rössler, W., 2015. Neuronal plasticity in the mushroom body calyx during adult maturation in the honeybee and possible pheromonal influences. Dev. Neurobiol. 75, 1368–1384.

O'Donnell, S., Bulova, S., DeLeon, S., Barrett, M., Fiocca, K., 2019. Brain structure differences between solitary and social wasp species are independent of body size allometry. J. Comp. Physiol. A 205, 911–916.

O'Donnell, S., Bulova, S.J., DeLeon, S., Khodak, P., Miller, S., Sulger, E., 2015. Distributed cognition and social brains: reductions in mushroom body investment accompanied the origins of sociality in wasps (Hymenoptera: Vespidae). In: Proceedings of the Royal Society b: Biological Sciences, p. 282.

Opendak, M., Briones, B.A., Gould, E., 2016. Social behavior, hormones and adult neurogenesis. Front. Neuroendocrinol. 41, 71–86.

Pahlke, S., Seid, M.A., Jaumann, S., Smith, A., 2020. The loss of sociality is accompanied by reduced neural investment in mushroom body volume in the sweat bee Augochlora pura (Hymenoptera: Halictidae). Ann. Entomol. Soc. Am. 114, 637–642.

Robinson, G.E., 1992. Regulation of division of labor in insect societies. Annu. Rev. Entomol. 37, 637–665.

Sandeman, R., Sandeman, D., 2000. "Impoverished" and "enriched" living conditions influence the proliferation and survival of neurons in crayfish brain. J. Neurobiol. 45, 215–226.

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Cardona, A., 2012. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682.

Scotto-Lomassese, S., Strambi, C., Aouane, A.C., Strambi, A., Cayre, M., 2002. Sensory inputs stimulate progenitor cell proliferation in an adult insect brain. Curr. Biol. 12, 1001–1005.

Scotto-Lomassese, S., Strambi, C., Strambi, A., Aouane, A., Augier, R., Rougon, G., Cayre, M., 2003. Suppression of adult neurogenesis impairs olfactory learning and memory in an adult insect. J. Neurosci. 23, 9289–9296.

Seid, M.A., Traniello, J.F.A., 2006. Age-related repertoire expansion and division of labor in Pheidole dentata (Hymenoptera: Formicidae): a new perspective on temporal polyethism and behavioral plasticity in ants. Behav. Ecol. Sociobiol. 60, 631–644.

Sherry, D.F., MacDougall-Shackleton, S.A., 2015. Seasonal change in the avian hippocampus. Front. Neuroendocrinol. 37, 158–167.

Smith, A.R., Seid, M.A., Jiménez, L.C., Wcislo, W.T., 2010. Socially induced brain development in a facultatively eusocial sweat bee Megalopta genalis (Halictidae). Proc. R. Soc. B Biol. Sci. 277, 2157–2163.

Snyder, J.S., Cameron, H.A., 2012. Could adult hippocampal neurogenesis be relevant for human behavior? Behav. Brain Res. 227, 384–390.

Splitt, A., Schulz, M., Skórka, P., 2021. Current state of knowledge on the biology and breeding of the solitary bee – Osmia bicornis. J. Apic. Res. 61, 1–17.

Stieb, S.M., Muenz, T.S., Wehner, R., Rössler, W., 2010. Visual experience and age affect synaptic organization in the mushroom bodies of the desert ant Cataglyphis fortis. Dev. Neurobiol. 70, 408–423.

Tea, J., Alderman, S.L., Gilmour, K.M., 2019. Social stress increases plasma cortisol and reduces forebrain brain proliferation in subordinate male zebrafish (Danio rerio). J. Exp. Biol. 222.

Trebel, B., Dippel, S., Schaaf, M., Balakrishnan, K., Wimmer, E.A., Schachtner, J., 2020. Adult neurogenesis in the mushroom bodies of red flour beetles (Tribolium castaneum, Herbst) is influenced by the olfactory environment. Sci. Rep. 10, 1090.

van Praag, H., Kempermann, G., Gage, F.H., 2000. Neural consequences of environmental enrichment. Nat. Rev. Neurosci. 1, 191–198.

Vicens, N., Bosch, J., 2000. Nest site orientation and relocation of populations of the orchard pollinator Osmia cornuta (Hymenoptera: Megachilidae). Environ. Entomol. 29, 69–75.

Ward, K.N., Coleman, J.L., Clinnin, K., Fahrbach, S., Rueppell, O., 2008. Age, caste, and behavior determine the replicative activity of intestinal stem cells in honeybees (Apis mellifera L.). Exp. Gerontol. 43, 530–537.

Withers, G.S., Day, N.F., Talbot, E.F., Dobson, H.E.M., Wallace, C.S., 2008. Experience-dependent plasticity in the mushroom bodies of the solitary bee Osmia lignaria (Megachilidae). Dev. Neurobiol. 68, 73–82.

Zars, T., 2000. Behavioral functions of the insect mushroom bodies. Curr. Opin. Neurobiol. 10, 790–795.