DOI: 10.5061/dryad.cjsxksn4s
title: Structural plasticity of olfactory neuropils in relation to insect diapause

description: 1. Many insects that live in temperate zones spend the cold season in a
 state of dormancy, referred to as diapause. As the insect must rely on
 resources that were gathered before entering diapause, keeping a low
 metabolic rate is of utmost importance. Organs that are metabolically
 expensive to maintain, such as the brain, can therefore become a liability
 to survival if they are too large. 2. Insects that go through diapause as
 adults generally do so before entering the season of reproduction. This
 order of events introduces a conflict between maintaining low metabolism
 during dormancy, and emerging afterwards with highly developed sensory
 systems that improve fitness during the mating season. 3. We investigated
 the timing of when investments into the olfactory system are made by
 measuring the volumes of primary and secondary olfactory neuropils in the
 brain as they fluctuate in size throughout the extended diapause
 life-period of adult Polygonia c-album butterflies. 4. Relative volumes of
 both olfactory neuropils increase significantly during early adult
 development, indicating the importance of olfaction to this species, but
 still remain considerably smaller than those of non-diapausing
 conspecifics. However, despite butterflies being kept under the same
 conditions as before the dormancy, their olfactory neuropil volumes
 decreased significantly during the post-dormancy period. 5. The opposing
 directions of change in relative neuropil volumes before and after
 diapause dormancy indicate that the investment strategies governing
 structural plasticity during the two life-stages could be functionally
 distinct. As butterflies were kept in stimulus-poor conditions, we find it
 likely that investments into these brain regions rely on
 experience-expectant processes before diapause and experience-dependent
 processes after diapause conditions are broken. 6. As the shift in
 investment strategies coincide with a hard shift from pre-mating season to
 mating season, we argue that these developmental characteristics could be
 adaptations that mitigate the trade-off between dormancy survival and
 reproductive fitness.
description: <b>Species and rearing</b> The
 Comma butterfly <i>Polygonia c-album</i> (Linné, 1758) has a
 large distribution area, spanning from Great Britain and northern Africa
 in the west, to Japan in the east. It is a facultatively multivoltine
 species, having more than one generation per year in areas where the
 summer season is sufficiently long, and undergo diapause as imago. Uniform
 daylength, especially if shorter than 13 hours, favours the developmental
 path leading to diapause readiness, while increasingly longer days (e.g.
 L:D 12:12 --&gt; 22:2) experienced during larval development favours
 the path leading to directly reproducing adults, especially when
 accompanied with increasingly higher temperatures (e.g. 17°C --&gt;
 23°C). <i>P. c-album </i> is considered a
 polyspecialist with regards to host plant utilisation, and olfaction is
 likely to play an important role during the search for suitable
 oviposition sites as mated females are known to show attraction towards
 host plants based solely on olfactory cues (Schäpers et al.
 2015). A cohort of <i>P. c-album</i>
 females, derived from a lab population originating in the Norfolk region
 of the United Kingdom and provided by the company World Wide Butterflies,
 was used in this study. Larvae from approximately 30 egg-laying females
 were reared on fresh cuts of stinging nettles <i>Urtica
 dioica</i> under a light and temperature regime promoting the
 development of diapausing adults (12:12 L:D, 17°C). Pupae were removed
 from their pupation sites two days after pupation, sexed by inspection of
 the genital slits, and placed in paper lined cups covered with mesh nets
 until adult eclosion. <b>Conditions during
 treatment</b> One day old adults were transferred
 to mesh net cages (50cm*50cm*50cm) in a secluded room with a neutral
 olfactory environment. The room was under a 6:18 L:D light cycle, and a
 dynamic temperature cycle peaking at 27°C during the end of the light
 period, and dropping down to 20°C during the dark period. Cages were
 equipped with feeding stations consisting of a plastic cup with a dish
 sponge and freshly mixed 25% sugar water. Feeding stations were regularly
 exchanged in order to prevent growth of algae and fermentation of the
 sugar as to avoid non-standardized variations in gustatory and olfactory
 conditions during the time spent in flight cages.
 Butterflies were kept in the flight cages for 14 days before the
 diapause dormancy climate conditions were initiated, and again for two
 weeks after the dormancy conditions were terminated. Throughout the period
 when dormancy conditions were active, the butterflies were kept in plastic
 cups covered with mesh nets (secured with rubber bands) and placed in a
 cardboard box which in turn was kept in a sheltered area on the roof of
 the Zoology Department of Stockholm University during the winter of
 2018-2019. All butterflies were placed on the underside
 of the mesh net, inside the cups, as this position seems to increase
 diapause survival rates (Christer Wiklund, Stockholm University, 2018.07,
 personal communication). The animals were regularly checked on during the
 dormancy period and individuals which had dropped off from the net were
 gently put back into their original position if still alive at the time of
 discovery. <b>Sampling </b>
 Butterflies were sampled at five different time points: 1) 1 day
 after adult eclosion, 2) immediately before the onset of dormancy
 conditions, at 14 days after adult eclosion, 3) during the mid-stage of
 the diapause dormancy period, approximately two months after dormancy
 conditions were initiated, 4) at the end of diapause dormancy,
 approximately four months after dormancy conditions were initiated, and 5)
 two weeks after dormancy conditions were terminated and butterflies had
 been returned to indoor flight cages. It should be
 noted that <i>P. c-album </i>butterflies generally take flight
 within minutes and start feeding within hours after diapause dormancy
 climate conditions are terminated, and are ready to mate within a few days
 up to one week (M. E., unpublished observation).
 <b>Dissection and sample preparation</b>
 Butterflies were decapitated using micro scissors, and their
 heads were fixated overnight at 4°C in a 4% paraformaldehyde
 (Sigma-Aldrich, Steinheim, Germany) solution. Heads were washed in
 phosphate buffered saline (Sigma-Aldrich, Steinheim, Germany) containing
 2,5% Triton-X (Sigma-Aldrich, Steinheim, Germany) (PBStx) 4x15 min before
 the brains were dissected out. Brain samples were incubated in a 1:20
 solution of primary antibody targeting synapsin (3C11 anti-SYNORF1,
 Developmental Studies Hybridoma Bank, Iowa City, IA, USA) and PBStx with
 0,5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) over 3
 days at 4°C on a shaker at low setting. Samples were then washed 5x1h
 before incubation in secondary antibody (AlexaFluor 488 (Life
 technologies, Eugene, OR, USA) 1:500 in PBStx 0,5% BSA) for 3 days at the
 same conditions as for primary incubation. Stained samples were washed
 4x1h in PBStx, 1x1h in PBS, and treated for optical clearing in Omnipaque
 (GE Healthcare AS, Oslo, Norge), firstly in a 1:1 solution of
 Omnipaque:PBS for 24h and then stored in pure Omnipaque for a minimum of
 24h. As clearing in Omnipaque eliminates the need for sample dehydration
 in ethanol and usage of methyl salicylate, the tissue shrinking associated
 with such protocols (Bucher et al. 2000; Smolla et al. 2014) is avoided,
 allowing for more accurate volume measurements. Omnipaque is an odourless
 and benign liquid commonly used in clinical treatments, making it safe and
 easy to work with. <b>Confocal scanning and
 reconstruction</b> Cleared samples were
 whole-mounted in omnipaque on glass slides with custom-made 0,5 mm metal
 spacers and were optically sectioned using a Zeiss LSM 780 META (Zeiss,
 Jena, Germany) scanning confocal laser microscope. Images with a
 resolution of 1024 x 1024 pixels were obtained with a 10x air objective.
 Each section had a thickness of approximately 3 mm, resulting in image
 stacks of about 100 sections per sample. 3D
 reconstruction and extraction of volumetric measurements (in
 mm<sup>3</sup>) from antennal lobes, mushroom body calyces,
 and whole central brain regions (optic lobes were excluded from analysis)
 were performed by using the native segmentation, volume rendering, and
 surface reconstruction tools in the Thermo
 Scientific<sup>TM</sup> AMIRA<sup>TM</sup> (v.
 2019.3) image processing software.  
description: Samples that were physically damaged during technical processing
 were excluded from analysis, as were those that did not render high
 quality images during confocal scanning, or had physical abnormalities
 such as divergent number of calycal cups. In cases where only one of a
 paired neuropil was discarded for the previously stated reasons, the
 intact neuropil volume was duplicated as to achieve total neuropil volumes
 comparable to fully intact samples. As paired neuropils are known to be
 symmetrical between hemispheres (Galizia et al. 1998; el Jundi et al.
 2009; Eriksson et al. 2019) both neuropils were discarded in cases where
 the volume difference was greater than 10% within a pair. The latter
 phenomenon is to be expected in cases where the neurolemma was ruptured
 before samples were fully fixated, allowing damaged tissues to expand post
 mortem.