Adult neurogenesis in the mushroom bodies of red flour beetles (Tribolium castaneum, Herbst) is influenced by the olfactory environment

Several studies showed adult persisting neurogenesis in insects, including the red flour beetle Tribolium castaneum, while it is absent in honeybees, carpenter ants, and vinegar flies. In our study, we focus on cell proliferation in the adult mushroom bodies of T. castaneum. We reliably labelled the progenies of the adult persisting mushroom body neuroblasts and determined the proliferation rate under several olfactory conditions within the first week after adult eclosion. We found at least two phases of Kenyon cell proliferation in the early adult beetle. Our results suggest that the generation of Kenyon cells during the first three days after adult eclosion is mainly genetically predetermined and a continuation of the developmental processes (nature), whereas from day four on proliferation seems to be mainly dependent on the odour environment (nurture). Considering that the mushroom bodies are linked to learning and memory, neurogenesis in the mushroom bodies is part of the remodelling of neuronal circuits leading to the adaption to the environment and optimization of behaviour.

The ground pattern of the central nervous system is genetically encoded. Following development, when the nervous system first encounters environmental sensory input, it is crucial for the survival of an animal to adapt to the actual conditions and cues. Therefore, the genetically encoded wiring scheme of the nervous system must be altered. This plasticity typically occurs in special time windows of elevated susceptibility to sensory input (critical periods or sensitive phases) 1 .

Identification of newly born cells.
To label adult-born cells, we used the 5-ethynyl-2′-desoxyuridine (EdU) method 75,76 . With this method, we reliably and exclusively labelled the mushroom body neuroblasts and their progeny, while neurogenesis is not present in other areas of the cerebral ganglion of the red flour beetle (Fig. 1A). The neuroblasts were distinguished from their progeny based on their larger size (Fig. 1B).
We confirmed that in T. castaneum, adult-born Kenyon cells usually derive from two neuroblasts (NB) per hemisphere and send their axons into the core of the mushroom body peduncle (PED) and lobes as shown by Zhao and colleagues 60 (Fig. 1C).
The neuronal identity of the EdU labelled cells was verified by demonstrating co-localization with the reporter signal ( Fig. 1D-D") in the neuron labelling EF1-B-DsRed line 77 , as well as immunohistochemical staining against the glia cell marker reversed polarity 78 resulting in no co-localization ( Fig. 1E-E").
Environmental conditions influence adult neurogenesis. The proliferation rates of the mushroom body neuroblasts during the first week after adult eclosion were analysed on a day-to-day basis (Fig. 2). A statistical comparison using Kruskal-Wallis test between both sexes showed no major intersexual difference in the number of newly born Kenyon cells, deriving from a single neuroblast (Supplemental Table 1). Thus, for    all further analysis, both sexes were pooled. Testing for no variation between conditions and ages by means of Scheirer-Ray-Hare test resulted in rejection of the null hypothesis (olfactory conditions: H = 114.899, p = 0.0; ages; H = 655.154, p = 0.0; overall: H = 231.580, p = 0.0). Further, per condition Kruskal-Wallis analysis between ages showed significant differences between cell numbers at different days, which were further analysed by post-hoc analysis using Dunn's multiple comparison test (Fig. 2).
For enrichment of the olfactory environment we chose the beetles' aggregation pheromone 4,8-dimethyldecanal (DMD) 79 and the food-related green-leaf volatile cis-3-hexenol as both are known to elicit antennal responses in EAG recordings 80 and at least DMD causing a clear OR/Orco (TcOR1) dependent behavioural response 81 .
The mushroom body neuroblasts of beetles reared in mixed-sex groups of 20 individuals generate new Kenyon cells within the first four days after adult eclosion. During the first three days of the proliferation phase, the median daily proliferation rate of each neuroblast is 10 to 15 cells, with a reduction on day four (Figs. 2, S1). At day 5 the proliferation rate drops significantly and afterwards no neurogenesis is detectable.
Isolating beetles as pupae lead to a proliferation phase lasting three days after adult eclosion. During this proliferation phase, each neuroblast gives rise to about 10 to 20 cells per day, decreasing on day three and significantly dropping at day four before from day five onwards no neurogenesis is detectable.
Enriching the environment of group-reared beetles with DMD leads to a prolonged proliferation phase of five days. During the first three days of the proliferation phase, the median daily proliferation rate of each neuroblast equals to about 15 to 20 cells, with slight decrease on days four and five. At day 6 the proliferation rate drops significantly and afterwards no neurogenesis is detectable.
Stimulating olfactory-deprived (pupal dsRNA orco injection) group-reared beetles with DMD leads to a proliferation phase lasting four days after adult eclosion. During the first four days each neuroblast's median daily proliferation rate is 10 to 15 cells, with a high variation on day four (Figs. 2, S1). At day 5 the proliferation rate drops significantly and afterwards no neurogenesis is detectable.
Stimulation of group-reared beetles with cis-3-hexenol results in mushroom body neuroblasts generating new Kenyon cells during the first six days after adult eclosion with a median rate of 10 to 15 cells per day, decreasing to about 5 cells at day 8.
Stimulating olfactory-deprived (pupal dsRNA orco injection) group-reared beetles with cis-3-hexenol leads to a proliferation phase lasting four days after adult eclosion. During the first three days of the proliferation phase, the daily proliferation rate per is 10 to about 15 cells, with a significant reduction on day four (Figs. 2, S1) before dropping to zero at day 6.

Antennal responses to cis-3-hexenol and 4,8-dimethyldecanal. Electroantennographical record-
ings (EAG) were used to demonstrate that beetles with an RNAi-mediated systemic knockdown of Orco (dsR-NA orco , test group) differ in their response to the tested odours, compared to beetles injected with sham (DsRed) dsRNA (dsRNA sham , control group).
Stimulation with the three lowest tested concentrations (10 −8 − 10 −6 ) of the beetle's aggregation pheromone DMD leads to no obvious reaction of the antennae ( Starting with a dilution of 10 −5 , a dose-dependent reaction of the antennae is visible in dsRNA sham injected beetles, but not in beetles injected with dsRNA orco . Stimulation with the five lowest tested concentrations (10 −8 − 10 −4 ) of the food volatile cis-3-hexenol leads to no obvious responses. Stimulating with a dilution of 10 −3 causes a slight response in the control group only. Whereas, at the highest tested concentration (10 −2 ) both groups respond (Fig. 3, cis-3-hexenol; Supplemental Figs. S2 and S3), but the peak response of the control group is significantly higher compared to the test group (Fig. 3, cis-3-hexenol). Furthermore, the response onset in the orco knock-down is delayed (Supplemental Fig. S2).

Discussion
Neurogenesis in adult brains is present to varying extents throughout invertebrates and vertebrates 57,[82][83][84][85][86][87][88][89] . Several studies showed adult neurogenesis in the mushroom bodies of hemimetabolous and ametabolous insects such as crickets 56 , cockroaches 90 , and firebrats 91,92 . In holometabolous insects persisting adult neurogenesis in the mushroom bodies was described in the black cutworm Agrotis ipsilon 58 and several beetles, including the red flour beetle T. castaneum 59,60 , while it is absent in the mushroom bodies of the honeybee A. mellifera 54 , the carpenter ant Camponotus floridanus 34 , and the vinegar fly D. melanogaster 55 .
Localization of adult neurogenesis in the mushroom bodies using 5-bromo-2′-deoxyuridine (BrdU) as published for the red flour beetle by Zhao and colleagues 60 gave no reliable results in our hands and was not useful for the comparison of larger experimental groups needed for the current study. Instead, we successfully and reliably labelled mushroom body neuroblasts and their progeny using the EdU method 75,76 . Like BrdU, EdU is a thymidine analogue that is incorporated into the DNA during replication. The major advantage compared to BrdU is the labelling procedure. While BrdU is localized via immunohistochemistry and therefore requiring the DNA to be denaturized to allow binding of the antibody, EdU is labelled by selective direct chemical coupling with an azide-fluorochrome. Avoiding denaturation of the DNA with HCl provides improved overall tissue preservation. By using this reliable labelling method for newly born neurons and their neuroblasts (Fig. 1) we studied the generation of new Kenyon cells in the early adulthood of the red flour beetle T. castaneum under different conditions (Figs. 2, S1) to answer whether this adult neurogenesis depends on olfactory input and if there is a critical period. The activity-dependent remodelling of neuronal circuits during critical periods leads to the adaption to the environment and optimization of behaviour 93 . Previous studies in A. mellifera and D. melanogaster showed that critical periods during which the mushroom body circuitry is remodelled exist in holometabolous insects and that the underlying mechanisms are the refinement of old as well as the growth of new synapses 48-52,54 , but (2020) 10:1090 | https://doi.org/10.1038/s41598-020-57639-x www.nature.com/scientificreports www.nature.com/scientificreports/ not neurogenesis 54,55 . The adult olfactory system of the beetle is first confronted with odour cues directly after adult eclosion.
Rearing beetles in groups of 20 animals of both sexes leads to the generation of new Kenyon cells during the first four days after adult eclosion. This phase is shortened in isolated beetles by one day and prolonged by additional olfactory stimulation. Enriching the olfactory environment of group-reared beetles with the beetles' aggregation pheromone DMD 79 prolongs the proliferation phase by one day, while stimulation with the food odour cis-3-hexenol leads to a prolongation of the proliferation phase for at least four days. This prolonging effect is mostly inhibited in beetles with a significantly reduced perception of the tested odorants (Fig. 3, dsRNA orco ), generated by RNAi mediated knockdown of orco.
A study in crickets already demonstrated that sensory deprivation by isolation results in significantly less neurogenesis among the Kenyon cells of young females, compared to group-reared females 82 . A second study based on these results asked what proportion of neurogenesis is caused by visual and olfactory stimuli 83 and demonstrated that olfactory and visual deprivation is sufficient to decrease neurogenesis in adult crickets, regardless whether reared in groups or isolation. This clearly showed a link between sensory input and adult neurogenesis.
In our study, we used a day-to-day analysis of adult neurogenesis together with the specific manipulation of the Orco dependent olfactory sensory neurons. This allowed us to temporally dissect adult neurogenesis into distinct phases and link them to olfactory activity.
Similarly to the results from crickets 82 , our data suggest that isolation reduces adult neurogenesis. We were able to demonstrate that this reduction is based on a shortened proliferation phase rather than an altered proliferation rate.
It seems that the generation of Kenyon cells during the first three days after adult eclosion is mainly genetically predetermined and a continuation of developmental processes rather than depending on sensory activity.
The shorter proliferation phase in isolated beetles compared to group-reared beetles suggest an influence of social interactions. This is supported by the fact that the proliferation phase after orco knock-down in odour stimulated beetles is still longer than in isolated beetles. Since the beetles were kept under constant darkness, visual stimulation can be excluded, making tactile and gustatory cues the most likely triggers. This is partially in accordance with findings in crickets, were unilateral removal of the antennae, causing the loss of chemosensory as well as mechanosensory antennal input in the ipsilateral hemisphere, lead to less adult neurogenesis in the ipsilateral hemisphere 83 .  www.nature.com/scientificreports www.nature.com/scientificreports/ The significant differences in the proliferation rate within the first three days might indicate an influence of odour stimulation. However, as the results for DMD and cis-3-hexenol are contradicting, we conclude that these differences are very likely artificial.
However, the main and most striking effect of DMD stimulation is the prolongation of the proliferation phase compared to group-reared beetles, which is not present in dsRNA orco injected beetles. Since after a RNAi mediated knockdown of orco the EAG response towards DMD is significantly reduced (Fig. 3) and no longer measurable (Supplemental Fig. S2) and the behavioural response towards DMD is abolished 81 , the prolongation is olfactory induced and driven via activity of the OR/Orco complex.
Interestingly, the potential for a longer capacity of the MB system to produce new Kenyon cells seems to depend on the odour, as the stimulation with the food-related odour cis-3-hexenol leads to a longer proliferation phase when compared to DMD stimulation, with cis-3-hexenol causing an increased proliferation rate even at day eight. This seems to contradict the EAG data that suggest a higher sensitivity towards DMD. However, the lack of responses to cis-3-hexenol at lower concentrations does not necessarily mean that it is not perceived, as the recorded response likely depends on the localisation of the OSNs relative to the recording electrode 94 .
This longer proliferation phase is massively shortened in dsRNA orco injected beetles, clearly demonstrating that this effect is most likely olfactory but not solely driven via the activity of the OR/Orco complex. While after knockdown of orco, the EAG responses to DMD are fully abolished, the EAG responses to cis-3-hexenol show a significantly lower, but still exiting reaction (Fig. 3) with a delayed response onset (Supplemental Fig. S2), which suggests the involvement of another receptor type. As Getahun and colleagues 95 already described in D. melanogaster the best candidates with slower responses are the ionotropic glutamate-like receptors (IR) 26 .
The different effects of DMD and cis-3-hexenol on the duration of the proliferation phase (five vs. eight days) might be explained already on the receptor level, as IRs, unlike ORs, do not exhibit adaptation to longer stimulations 95 . This could mean that continued exposure to high DMD concentration leads to a desensitisation of the OR/Orco complex, causing the shorter proliferation phase. Whereas, the response to cis-3-hexenol seemingly also perceived by IRs persists longer. Furthermore, there is the possibility of different pheromone vs. normal odour processing networks in the antennal lobes that provide the input to the MB as shown among others in Manduca sexta 96 , D. melanogaster 97 , and A. mellifera 98 . However, so far, such separate olfactory processing networks were not described in the red flour beetle and it remains unclear whether they exist.
The EAG responses showed that the reaction of both odorants is significantly reduced in adult female beetles seven days after dsRNA orco injection, corresponding roughly to A4. Besides, we performed immunohistochemical staining against Orco in freshly eclosed (A0) and seven days old beetles (A7) using the cross-reactive Moth-R2 antiserum 26 . This resulted in no immunoreactivity after pupal injection of dsRNA orco (Supplemental Fig. S4) demonstrating the effectivity of the RNAi within the studied ages. Furthermore, the RNAi effect in T. castaneum has already been published to last from weeks to months 99 .
T. castaneum has a life span of up to two years 74 and studies on the origin of the beetle 63 together with a large variety of odorant receptors 26,81 suggest a broad spectrum of potential food sources, which may change over time. Adapting the MB neuronal network via newly born cells could be part of the strategy to cope with ongoing environmental changes. The process might be triggered by changes in the OR repertoire in response to the environmental changes, as speculated by Dippel and colleagues 26 . This could explain why Zhao et al. 60 . occasionally found neurogenesis in 88 days old beetles without stimulation. The involvement of adult proliferation in behavioural adaption to the changing odorant environment has already been suggested in crickets 82,83 . In analogy, a study on the mouse hippocampus demonstrated that adult neurogenesis is not triggered by continued long-term exposure to enriched environments, but by novel complex stimuli 100 .
Considering that the mushroom bodies are linked to learning and memory 39 , adult neurogenesis might contribute to the formation of new odorant memories. As Zhao and colleagues demonstrated, the adult-born Keyon cells send their axons into the core of the mushroom bodies 60 , which were shown to be involved in differential olfactory learning in D. melanogaster 101,102 . Suppression of adult neurogenesis in crickets leads to a significant impairment in olfactory learning and memory 44 , undermining the role of adult-born neurons in learning and memory. Furthermore, studies in vertebrates show that olfactory enrichment leads to increased neurogenesis in the hippocampus and plays a role in olfactory learning [103][104][105][106] .

Methods
Animals. Red flour beetles (Tribolium castaneum, Herbst 1797; Insecta, Coleoptera) of the wild-type strains "San Bernadino" 107 and "black" 108 , as well as the neuron labelling 26,77 transgenic line EF1-B-DsRed (elongation factor1-alpha regulatory region-DsRedExpress; kindly provided by Michalis Averof, Institut de Génomique Fonctionnelle de Lyon, France) were used. The beetles were bred under constant darkness at about 28 °C and 40-50% relative humidity on organic whole grain wheat flour supplemented with 5% dried yeast powder and 0.05% Fumagilin-B (Medivet Pharmaceuticals Ltd., High River, Alberta, Canada) to prevent sporozoan infections 109 . For age determination, freshly eclosed beetles (A0) were collected and kept in mixed-sex groups of 20 in 68 ml Drosophila vials on about 20 g substrate.
For isolation experiments, individuals were separated as pupae into 5 ml glass vials on about 2 g substrate.
Immunohistochemistry and EdU detection. The brains of cold anaesthetized beetles were dissected in PBS (phosphate-buffered saline, 0.01 M, pH 7.4) and fixed in 0.01 M PBS containing 4% paraformaldehyde for 1-2 hours at room temperature or at 4 °C overnight. Fixation was stopped by rinsing 4 × 10 min in PBS supplemented with 0.3% Triton X-100 (PBS-TrX). Afterwards, the brains were transferred into a blocking solution (5% normal goat serum (NGS, Dianova, Hamburg, Germany) in PBS-TrX) and incubated for 3-4 hours at room temperature or overnight at 4 °C. The brains were then incubated with primary antibody solution (PBS-TrX, 2% NGS, concentrations and details see Table 1). After 2-3 days at 4 °C, the antibodies were removed by rinsing 5 × 10 minutes in PBS-TrX. Subsequently, the brains were incubated for 2-3 days at 4 °C in constant darkness with secondary antisera and fluorescent markers (for details see Table 1). Afterwards, the brains were rinsed 5 times in PBS-TrX for 10 min and 2 times in 0,1 M TRIS buffer (pH 8,5; Tris-Base, HCL) and the incorporated EdU was detected using copper-catalysed click-chemistry 76 . Therefore, the brains were incubated for 1 to 2 hours at room temperature in constant darkness in freshly prepared reaction solution (4 mM CuSO 4 , 10 μM azide-fluorochrome (see Table 1), and 500 mM ascorbic acid in 0.1 M TRIS buffer pH 8.5). Afterwards, the brains were embedded in a mixture of glycerol and PBS (80% glycerol, 20% PBS) or Mowiol 110 between two coverslips using a layer of two reinforcing rings as spacers. www.nature.com/scientificreports www.nature.com/scientificreports/ Olfactory stimulation. For enhanced olfactory stimulation we reared 20 freshly eclosed adults in mixed-sex groups in para-film sealed 68 ml Drosophila vials on about 20 g substrate, supplemented with 1 µl pure green-leaf alcohol (cis-3-hexen-1-ol; Sigma-Aldrich) or 1 µl of the beetles aggregation pheromone 4,8-dimethyldecanal 79 in a dilution of 1:1000 in silicone oil (4,8-dimethyldecanal; Trécé Inc., Adair, OK, USA; Silicone oil M 10, Carl Roth, Karlsruhe, Germany), on filter paper (1 cm²).

Orco-knockdown.
Orco dsRNA (dsRNA orco ) and DsRed dsRNA (dsRNA sham ) used as a control were synthesized as previously published 26 . Both dsRNAs were injected into pupae at about 30%-40% of total metamorphosis. The Orco knockdown was verified by immunohistochemistry against Orco (Moth-R2, kindly provided by J. Krieger, University of Hohenheim, Germany) on cryo-sections of antennae as previously published 26 (Supplemental Fig. S4).
Image acquisition and processing. Fluorescent preparations were imaged using a widefield microscope setup (Axio Observer Z1; Carl Zeiss Microscopy, Jena, Germany) and a confocal laser scanning microscope (TCS SP5, Leica Microsystems, Wetzlar, Germany). Confocal image stacks were analysed with Amira 6 graphics software (FEI Visualization Sciences Group, Mérignac Cedex, France).
For further processing, snapshots of single sections and projections generated in Amira were processed (global level adjustments, contrast and brightness optimization) in Adobe Photoshop CC (Adobe Systems, San Jose, CA, USA). Final figure arrangements were performed in Adobe Illustrator CC.
EAG recordings. For electronantennographical recordings, we followed a previously published protocol 80 .
The tests were performed using five female beetles aged seven days after dsRNA injection, corresponding to about A4, with three repeated measurements per animal. The antennal response was recorded at 25 Hz via a custom-build amplifier attached to a data acquisition controller (IDAC-4 A/D converter, Syntech, Hilversum, The Netherlands) using EAG 2000 software (Syntech). During the EAG recordings, the antennae were exposed to a constant flow (~3 l/min) of filtered and humidified air. The green leaf volatile cis-3-hexenol and the beetle's aggregation pheromone DMD 111 were used as test odours. They were presented as 1 s pulse via a stimulus controller (CS-02, Syntech) that looped in the odour diluted in silicone oil from a filter paper impregnated with 20 µl odour sample. Measurements were performed in minute-long intervals with increasing concentrations (10 −8 -10 −2 ). Each repeat was preceded by the measurement of the reaction to DMD (positive control) and silicone oil, which was used as solvent) Data analysis and plotting. Analysis of cell numbers and EAG responses including statistical comparison and plotting was performed using Python (version 3.7.3, Python Software Foundation, www.python.org) based custom scripts. Those scripts utilize the SciPy ecosystems (www.scipy.org) modules SciPy (version 1. To test for significant differences between experimental groups, Scheirer-Ray-Hare 118 test (https://github.com/ jpinzonc/Scheirer-Ray-Hare-Test), Kruskal-Wallis test from SciPy and posthoc analysis by Dunn's multiple comparison from scikit-posthocs with p-value correction using the Holm method 119 were used.
Raw EAG voltage trains exported from EAG 2000 were initially smoothed using robust LOESS (local regression) method from localreg to account for local spikes and subsequently normalized by subtracting the corresponding responses to silicone oil which was used as the solvent to dilute the test odours.
Figures were plotted as vector graphics and final figure arrangements were performed in Adobe Illustrator CC.
Ethics approval and consent to participate. All experiments involving animals were performed in compliance with the guidelines of the European Union (Directive 2010/63/EU). As all experiments were on insects an approval of the study by an ethics committee was unnecessary.

Data availability
Except for the original microscope images/stacks, all data generated or analysed during this study and all analysis scripts are included in this published article [and its Supplemental Information]. The confocal image stacks are available from the corresponding author upon request.