
Brain volume increase and neuronal plasticity underly predator-induced morphological defense expression in Daphnia longicephala
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Download PDF Article Open access Published: 15 June 2021 Brain volume increase and neuronal plasticity underly predator-induced morphological defense expression in Daphnia longicephala A
Graeve1 na1, I Ioannidou1 na1, J Reinhard2, D. M. Görl1, A Faissner2 & …LC Weiss1 Show authors Scientific Reports volume 11, Article number: 12612 (2021) Cite this article
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Subjects EcophysiologyEvolutionary ecologySynaptic plasticity AbstractPredator-induced phenotypic plasticity describes the ability of prey to respond to an increased predation risk by developing adaptive phenotypes. Upon the perception of chemical predator
cues, the freshwater crustacean Daphnia longicephala develops defensive crests against its predator Notonecta spec. (Heteroptera). Chemical predator perception initiates a cascade of
biological reactions that leads to the development of these morphological features. Neuronal signaling is a central component in this series, however how the nervous system perceives and
integrates environmental signals is not well understood. As neuronal activity is often accompanied by functional and structural plasticity of the nervous system, we hypothesized that
predator perception is associated with structural and functional changes of nervous tissues. We observe structural plasticity as a volume increase of the central brain, which is independent
of the total number of brain cells. In addition, we find functional plasticity in form of an increased number of inhibitory post-synaptic sites during the initial stage of defense
development. Our results indicate a structural rewiring of nerve-cell connections upon predator perception and provide important insights into how the nervous system of prey species
interprets predator cues and develops cost–benefit optimized defenses.
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Phenotypic plasticity describes the ability of an organism with a given genotype to adapt its phenotype in response to changing environmental conditions1. One well-known example of
phenotypic plasticity is inducible defenses. Here, the presence of predators, indicated by chemical cues (kairomones), induces defensive features in the prey, which make them less
susceptible to predation. The freshwater crustacean Daphnia is a prime example of predator-induced defenses2, which range from morphological features such as helmets in D. cucullata3, neck
teeth in D. pulex4 or crests in D. longicephala5 to changes in life history6,7 or adaptive behavior8. Interestingly, morphological defenses are not only expressed in a predator-density
specific manner, but rather they are fine-tuned to the predation risk, so that also the conspecific density is decisional for the degree of defense expression9. Such condition-dependent
adjustments require a nervous system capable to decipher different chemical cues from the heterogeneity of many environmental factors and transfer this into a cost–benefit optimized
phenotype. Daphnia perceive chemical cues via the antennules, upon which a signaling cascade involving cholinergic, dopaminergic, glutamatergic and GABAergic components is initiated, which
ultimately leads to the transformation of the undefended into the defended phenotype10,11,12,13,14,15. It is still unknown how the nervous system detects and integrates environmental
information when the Daphnia brain morphologically appears rather simple (Fig. 1A). It is subdivided into three main neuropils: protocerebrum, deutocerebrum and tritocerebrum16. The
protocerebrum receives direct visual input from the compound eye via the optic ganglia (lamina and tecta) and chemosensory input from the antennules via the deutocerebrum16. Via the
circumoral connectives, the protocerebrum is connected to the tritocerebrum. This consists of two ganglia inferior to the esophagus that are transversally connected by the tritocerebral
commissures. These two neuropils connect with the mandibular ganglia and extend into the rope-ladder-like nerve cord innervating the body and appendages16. Between these three neuropils, the
protocerebrum comprises the largest part of the brain and is discussed to be centrally involved in the analysis and interpretation of environmental sensory signals17. Such environmental
signal integration is often associated with changes in form and function of nervous systems. While changes in form are referred to as structural brain plasticity, changes in function are
referred to as functional plasticity18. Structural plasticity has been reported in vertebrates and invertebrates, e.g. rats living in complex environments show larger brains with larger
cells and more dendritic fields than rats raised in homogeneous, uncomplex environments19. Tadpoles of the leopard frog develop smaller and narrower brains upon an emerging predator
threat20. Similarly, different insect spec
ies have been found to undergo structural brain plasticity associated with learning, food search and novel tasks21,22,23,24. In honeybees certain brain regions, i.e. the mushroom bodies in
the protocerebrum, experience anatomical reorganization associated with labor division due to differences in foraging experience25. Drosophila develop larger brain structures in diversified
environments than in isolation26. All these structural changes are discussed to be cost–benefit optimized so that the neuronal structures become as large as needed in order to be highly
effective for the given task. At the same time brain size is limited as nervous tissues are energetically demanding27. In contrast to structural brain plasticity, functional brain plasticity
affects the nervous system´s function by altering the abundance and reactive strength of neuronal connections28. Neuronal connections are e.g. made at chemical synapses. These consist of a
presynaptic bouton with the active zone (AZ), responsible for the regulated release of neurotransmitters, and the postsynaptic density (PSD), representing the neurotransmitter reception
apparatus. These synaptic sites are intermitted by the synaptic cleft into which neurotransmitters are released. Neuronal activity can either trigger neurotransmitter release at excitatory
synapses, increasing the activity of the postsynaptic neuron, or at inhibitory synapses, reducing neuronal activity29. Functional plasticity by means of differences in postsynaptic receptor
abundance has been shown in the avian brain during the filial imprinting process30 and in mammals experiencing enriched environments19,31. As space is the factor limiting the number of
synaptic connections in one neuron32, only an enlargement of the neuronal surface area can increase synaptic connections. The surface area can increase through an enlarged cell volume and
when many cells enlarge their volume, the overall neuropil mass will increase and is therefore a component of structural plasticity18. Thus, structural and functional brain plasticity are
closely related as the formation of new neuronal connections requires space19 and may therefore be able to shape neuropil form and size. Especially inhibitory synapses are important with
regard to the control and regulation of overall neuronal excitability within neuronal networks of cognitive processes33,34. The scaffolding protein gephyrin is a central component of the
inhibitory nervous system, as it anchors GABAA and glycine receptors at the PSD35. In addition, this protein is involved in post-Golgi transport of the respective receptors to the PSD36.
Taken together, gephyrin is a good indicator of functional plasticity, as it indicates activity changes of the inhibitory PSD by receptor shuffling.
Figure 1Schematic overview of experimental procedures. (A) Schematic frontal view of a Daphnia brain displaying the optic ganglia: composed of lamina and tecta, and pc/dc-complex. (B) Points in
time of D. longicephala induction. Predator exposure started in the 3rd or 5th instar, respectively. When exposed in the 3rd instar, individuals were sampled at 0 h, 24 h and 48 h. To
validate our findings in a different instar, animals were also exposed in the 5th instar; here we focussed on the target stages i.e. 0 h and 24 h. Controls were performed alongside the
respective treatment but without predators. (C) Measurement of morphological defence parameters. We measured the crest height as the distance between the upper eye margin and the rostral
extension of the head (yellow line). (D) Dissected brain mount stained with DAPI. We counted the cells of the pc/dc-complex (yellow). (E) 3D projection of a brain mount from confocal stacks
for volumetrics created with MorphoGraphX Version 1.1.1280-CellAtlas (available from https://www.mpipz.mpg.de/MorphoGraphX/software). Only the pc/dc-complex (insert) was used to determine
the brain volumes. (A, D, E) Arrows depict the anterior and ventral (arrow points out of the drawing pane) orientation. Abbreviations: CH, crest height; ce, compound eye; la, lamina; tc:
tecta, pc/dc, pc/dc-complex; tcn, tritocerbral neuropil.
Full size imageHere, we investigated structural and functional brain plasticity underlying predator perception in Daphnia longicephala. As cognition is a central task of the protocerebrum37, we focused on
the proto- and deutocerebrum (pc/dc-complex), which are indistinct in Daphnia. We determined pc/dc-complex volume together with a reorganization of neuronal circuitry by determination of the
number of neuronal cells. To exclude that alterations in pc/dc-complex volume are a result of the predator induced overall body growth and the crest, we also determined the volume of the
optic ganglia (OG). Additionally, we investigated cells expressing the anchoring protein gephyrin in pc/dc-complexes of predator-exposed and control D. longicephala. In order to exclude an
age-dependent effect, we conducted the experiment at two different developmental points in time. We demonstrate that there is structural and functional plasticity independent of
developmental age associated with predator perception.
Material and methods Daphnia longicephala cultureD. longicephala clone LP1 (Lara-Pond, Australia) was cultured in artificial Daphnia medium (ADaM (Klüttgen et al. 1994)) in 1 L beakers (Weck®, Germany) containing 20–25 age-synchronized
individuals under constant day:night conditions (16 h:8 h) at 20 ± 1 °C. Animals were fed ad libitum with the algae Acutodesmus obliquus. The beakers were cleaned every 48 h. Half of the
medium was exchanged weekly.
Predator cultureBackswimmers (Notonecta spec.) were collected from the ponds of the Ruhr-University Botanical Gardens. Animals were maintained in 1 L beakers (WECK®, Germany) filled with charcoal-filtered
tap water under standardized conditions (20 ± 1 °C with 16 h:8 h light:dark cycle). They were fed regularly ad libitum with Daphnia spec.
Experimental conditionsAll animals of the culture and the experiments were maintained in ADaM and reared at 20 °C ± 0.1 °C in incubators at constant light with a 16 h:8 h day:night cycle.
Experimental setupWe collected 14 h (± 1 h) old D. longicephala from age-synchronized cultures and reared 30 animals per 1 L beaker. Once the animals reached the 3rd instar i.e., after the successful
completion of two molting cycles, they were transferred into predator exposure or control conditions. For the predator exposure, net cages (mesh size 100 µm) containing one Notonecta spec.
were placed into 1 L beakers. Notonectids were fed with five D. longicephala per day to ensure continuous kairomone exposure. The experimental D. longicephala were located outside of the net
cage, which prevented predators from feeding on the test specimens. Predator exposure was conducted for 48 h until D. longicephala reached the 5th instar. Control treatments were cultivated
accordingly, but without predators. Control and predator-exposed specimens were collected at explicit points in time and treatment from the moment of predator exposure i.e. 0 h, 24 h, and
48 h. Per point in time and treatment, we collected 20 to 30 D. longicephala (Fig. 1B, red). The complete experimental set-up was repeated 9 times. Since D. longicephala is responsive to
predator cues also at later stages15, we also conducted experiments where predator exposure started in the 5th instar (Fig. 1B, green) focusing on the relevant points in time i.e. 0 h and 24
h and replicated this 3 times.
Measurement of defensive featuresWe measured defense expression at above-described points in time using a SZX16 stereomicroscope (Olympus, Japan) equipped with a TSO digital-camera (TSO-KST 6000849) controlled by the
software Vidmess Version 3.0 (TSO Thalheim Spezialoptik GmbH, Germany). We measured the crest height from the upper margin of the compound eye to the distal height of the crest (Fig.
1C).
ImmunohistochemistryAnimals were fixed using 4% PFA (formaldehyde (J.T. Baker) diluted in phosphate-buffered saline (PBS) 0.1 M; pH 7.4) for 5 min. Then, we dissected the pc/dc-complex, still attached to the
optic ganglia and compound eye (prospectively referred to as ‘brain mounts’) using fine forceps. We used the dark pigmented compound eye as a visual guide to facilitate working with the
small and unpigmented neuronal tissue. Brain mounts were transferred to poly-L-lysine coated object slides (VWR, Germany). All subsequent incubation steps were performed in a humidified
chamber and on an orbital shaker (neoLab®, Germany) at 15 rpm and 4 °C. Brain mounts were post-fixed for 10 min in 4% PFA and rinsed with PBS 3*5 min. Fixed brain mounts were blocked with 5%
goat normal serum (Dianova, Germany) diluted in PBS-TX 0.1% (PBS, 0.1 M; pH 7.4 with 0.05% Triton-X 100; Sigma Aldrich, Germany) for 3 h. Subsequently, we applied the primary antibody
anti-gephyrin raised in mouse (Cat. No. 147011; Synaptic Systems, Germany; RRID:AB_887717; 1:100 in PBS-TX 0.1%). Samples were incubated overnight and then washed with PBS-TX 0.1% 3*10 min.
The secondary antibody anti-mouse Alexa488 (Dianova, Germany; RRID:AB_2338845) was applied (1:250 diluted in PBS-TX 0.1%) and incubated for 2 h. Brain mounts were rinsed 3*10 min with PBS-TX
0.1%, mounted in DAPI-Vectashield (Vector Laboratories, USA) for nuclei staining and preservation. Slides treated with secondary antibody only and DAPI-Vectashield served as negative
controls (Fig. S1A, B). In order to confirm that gephyrin is associated with inhibitory glycinergic neurons in Daphnia, we performed a double staining using the anti-gephyrin antibody and an
anti-glycine receptor (GlyR) antibody raised in rabbit (Cat. No. 146008; Synaptic Systems, Germany; RRID:AB_2636914) (1:50 diluted in PBS-TX 0.1%). This antibody binds to the glycine
receptor α1-subunit. Detection was performed with the secondary antibody anti-rabbit594 (1:250) (Dianova, Germany; RRID:AB_2307325) on brain mounts of adult D. longicephala. Every step was
performed as described above. Antibody validation is described in the supplement (Fig. S2).
Confocal imagesWe acquired image stacks of the D. longicephala brain mounts using a confocal laser scanning microscope (SP5II, Leica Microsystems, Wetzlar, Germany) and Leica cLSM (SP8, Leica Microsystems,
Germany). Image stacks were acquired with a 40× water immersion objective, a 63× and a 100× oil immersion objective. Step intervals were chosen between 0.5 and 0.8 µm, according to nuclear
size ensuring the recording of all cells in one stack. Figures were composed and labelled in Photoshop CS 4 (Adobe Systems, San Jose, CA). Brightness, saturation, and contrast were
optimized; no further image editing was conducted.
Cell counting and volumetric measurement in pc/dc-complexes and OG3D projections of captured image stacks were composed in ImageJ 1.52n (Fiji)38. The total number of nerve cells (identified with the nuclear marker
2-[4-(Aminoiminomethyl)phenyl]-1H-Indole-6-carboximidamide hydrochloride (DAPI) and the number of gephyrin-labelled cells in the pc/dc-complex were counted manually in 3D projections using
the ‘multipoint tool’ (Fig. 1D). To determine the pc/dc-complex and OG volume, we used cLSM image stacks of the brain mount DAPI staining and created tif-stacks with Fiji for MorphoGraphX
import39 (Fig. 1E). Here, we used the ´Gaussian Blur` with a rate of 1 µm in each direction. Using ´Marching Cubes Surface` we created a mesh surface with a cube size of 1 µm. Since we aimed
to measure only the pc/dc-complex or OG volume respectively, we removed the surrounding parts of the brain mount using the ´pixel edit` tool (Fig. 1E insert). Each stack was saved as
ply-file before conversion into stl-files using MeshLab40. These files were required for the analysis in Blender, an open-source 3D graphics and animation package. The stl-files were opened
in Blender and the volume was calculated with the ‘3D printing’ and ‘volume’ function as described in Horstmann et al.41 (Fig. 1E). OG volume was only measured in individuals that were
exposed from the 3rd to the 5th instar.
Statistical analysesWe tested for normal distribution using a Shapiro–Wilk test. Only crest height, and brain volume followed a normal distribution, so that all other data was log-transformed. To analyze crest
expression in line with structural and functional plasticity across three instars starting from the third to the fifth instar we used a one-way analysis of variance (ANOVA) with instar as
factor. By this we determined age-dependence of the measured parameters (i.e. crest height, brain volume, OG volume, log(#gephyrin-labelled cells), log(#DAPI-labelled cells),
log(#gephyrin/#DAPI-labelled cells) individually per treatment (control; induced), as the interaction of stage and instar was irrelevant to our hypothesis. Post hoc comparisons to determine
differences between individual instars were performed using a Tukey Test. We then determined differences between control and predator-induced groups using a Student’s t-test for each
developmental instar and adjusted the significance threshold for multiple testing using the alpha-correction. The analysis of the control experiment using a later instar only comprised two
instars, so that we investigated for differences between instar and treatment using a Student’s t-test. Levels of significance were again adjusted for multiple testing.
Data is displayed untransformed. Statistics were performed in STATISTICA 14 (Statsoft inc.) plots were made in R using the ggplot2 package42,43.
ResultsInducible defensesCrests are constant across instars in the control group but grow significantly larger in the predator-induced treatment. Across instars, defenses grow significantly, so that we find a
significantly increased crest height in predator-exposed animals after 48 h (5th instar) compared to 0 h (3rd instar, Fig. 2A; Tables S1, S2). Within this time, crests increased 1.2-fold in
the predator exposed treatment and remain constant in size in the control treatment (1.0×).
Figure 2Morphological defense expression in D. longicephala exposed to predators in the 3rd instar. (A) Parameters were directly determined at 0 h (3rd instar), and subsequently monitored after 24 h
(4th instar), and 48 h (5th instar). Crest height shows a tendency towards an increase in predator-exposed D. longicephala after 24 h (P = 0.061; t = − 1.91; df = 57). After 48 h
predator-exposed individuals have a significantly increased crest height compared to controls (P = 0.01; t = − 2.64; df = 68). Alpha corrected levels of significance: *P ≤ 0.017; ***P ≤
0.0003. (B) Predator exposure started in the 5th instar and parameters were directly determined at 0 h (5th instar), and subsequently after 24 h (6th instar). Crest height is significantly
increased after 24 h predator exposure (P ≤ 0.001; t = −4.18; df = 44). Alpha corrected levels of significance: ***P ≤ 0.0005; Table S1.
Full size imageWe find a tendency towards an increased crest expression in predator-exposed animals compared to controls after 24 h (4th instar; (P = 0.061; t = − 1.91; df = 57) and a significantly
increased crest height after 48 h (5th instar; P = 0.01; t = − 2.64; df = 28, Table S3). Individuals that were exposed to predator cues in the 5th instar also showed an increased crest
expression after 24 h predator exposure (P ≤ 0.001; t = 4.18; df = 44; Fig. 2B; Tables S4, S5).
Structural plasticity in the pc/dc-complexVolumetric changes of the pc/dc-complexWe observed increasing pc/dc-complex volumes in both treatments with age. Naïve, control individuals showed a mean volume of 249,348.70 µm3 ± 70,955.12 in the 3rd instar and a mean volume of
353,098.40 ± 142,285.70 µm3 in the 5th instar (Fig. 3A, Tables S1, S2). In predator-exposed animals pc/dc-complex volume increased significantly from 247,623.70 µm3 ± 97,067.05 µm3 in the
3rd instar to 451,761.40 µm3 ± 146,165.9 µm3 in the 4th instar and to 565,640.5 µm3 ± 340,501.1 µm3 in the 5th instar (Fig. 3A; Tables S1, S2). We did not observe volumetric changes in
pc/dc-complexes of naïve individuals from the 5th to the 6th instar (Fig. 3B, Table S4). Predator-exposed D. longicephala showed an increase in pc/dc-volumes after 24 h (mean 5th instar:
310,848.2 µm3 ± 122,657 µm3; mean 6th instar: 424,134.2 µm3 ± 43,543.65 µm3; Fig. 3B, Table S5). When animals were exposed to the predator in the 3rd instar, no significant difference
between control and predator-exposed D. longicephala pc/dc-complex volumes was observed at 0 h (Table S3). Twenty-four hours later, pc/dc-complex volume in predator-exposed individuals was
significantly larger than in 24 h controls with a mean of 451,761.40 ± 146,165.90 µm3 (Fig. 3A, Table S3). In 48 h predator-exposed D. longicephala, the volume was also significantly
increased compared to the control (mean induced: 565,640.50 µm3 ± 340,501.1; Fig. 3A, Table S3). During this time the pc/dc volume increases 1.4-fold in control animals, and 2.2-fold in
predator exposed animals. Accordingly, among individuals exposed in the 5th instar, we detected a significant increase in pc/dc-complex volumes of predator-exposed animals compared to the
control after 24 h (mean control: 350,290.4 µm3 ± 81,781.9 µm3, mean induced: 424,134.0 µm3 ± 43,543.70; Fig. 3B; Tables S4, S5).
Figure 3Structural plasticity underlying morphological defense expression (A) 24 h predator exposure starting from the 3rd instar, significantly induced an increase in pc/dc-complex volume compared
to the controls (P = 0.011; t = 2.73; df = 28). This increase was also observed after 48 h predator-exposure (P = 0.014; t = − 2.59; df = 33). (A.1, A.2, A.3). We detected no significant
differences in OG volume between treatments. We observed a significant increase in OG volume between instar 3 and instar 5 in both treatments (control ANOVA: F(2;13) = 9.63 P = 0.003 Tukey:
P = 0.002; induced ANOVA: F(2;14) = 12.07 P = 0.001 Tukey: P ≤ 0.001). Alpha corrected levels of significance: * P ≤ 0.017; (B) When exposed to predators from the 5th instar onwards, the
pc/dc-complex volume shows a tendency towards an increase after 24 h predator exposure compared to the control (P = 0.049; t = − 2.19; df = 12) and to the 5th instar (P = 0.036; t = −
2.42; df = 10) Alpha corrected levels of significance: *P ≤ 0.025. (C) We detected no significant differences in nerve cell numbers between treatments. We found a tendency towards an
increased nerve cell number in naïve individuals in the 5th instar compared to the 3rd instar, when the animals were exposed to predators from the 3rd instar onwards (ANOVA: F(2;98) = 2.64 P
= 0.077 Tukey: P = 0.076) Alpha corrected levels of significance: * P ≤ 0.025. (D) When exposed to predators from the 5th instar onwards, we find no differences in nerve cell numbers
between treatments or instars. Alpha corrected levels of significance: *P ≤ 0.017.
Full size imageVolumetric changes of the OGControl and predator-exposed D. longicephala show an increase in OG volume with increasing age so that animals in the 5th instar have significantly larger OG than animals in the 3rd instar
(mean control 3rd instar: 75,837.76 µm3 ± 21,080.22 µm3, mean control 5th instar: 226,191.2 µm3 ± 63,729.89 µm3; mean induced 3rd instar: 89,783.36 µm3 ± 20,623.36 µm3, mean induced 5th
instar: 259,253.9 µm3 ± 81,789.8 µm3; Fig. 3A; Tables S1, S2). We observed no significant differences in OG volume between control and predator-exposed individuals at any point in time (Fig.
3A; Tables S3, S4, S5). Optic ganglia of control specimens increase by 2.9-fold and of predator induced specimens by 2.88-fold over the developmental stages.
Nerve cell numbers in thepc/dc-complex
We detected a tendency towards an increased number of cells in the pc/dc-complex of naïve D. longicephala from 734.86 ± 180.13 cells in the 3rd to 903.18 ± 319.73 cells in the 5th instar
(Fig. 3C; Tables S1, S2). Across developmental stages the number of cells increase 1.23-fold irrespective of the treatment. Between control and predator-exposed specimens, we did not detect
differences in nerve cell numbers at any point in time (Fig. 3C, D; Tables S3, S4).
Functional plasticity in the pc/dc-complexCharacterization of synaptic compositionCells labelled with anti-gephyrin and anti-GlyR antibodies were found throughout the whole pc/dc-complex tissue (Fig. S3, Fig. 4A–C). We found gephyrin in the cytoplasm and on the surface of
the cell soma (Fig. 4D–F). GlyRs are limited to the cell surface in colocalization with gephyrin (Fig. 4G–I).
Figure 4Co-localization of gephyrin and glycine receptor (GlyR). (A) Merged display of anti-gephyrin and anti-GlyR staining pattern. Both antibodies are identified within the pc/dc-complex.
Anti-GlyR staining colocalized with the anti-gephyrin antibody staining. Not all gephyrin-labelled cells show markings of anti-GlyR (white arrow heads). (B) Gephyrin is found in the cell
soma and on the cell surface evenly distributed within the pc/dc-complex. (C) GlyRs are evenly distributed in the pc/dc-complex and the staining pattern shows the typical receptor spots.
A–C: Scale bar 50 µm. (D) Merged display of anti-gephyrin and anti-GlyR staining on the cellular level. Anti-gephyrin staining is found throughout the whole cytoplasm, while anti-GlyR
staining is limited to concentrated spots on the cell surface (double arrow heads). Inset is magnified in G. (E) Staining pattern of gephyrin on the cellular level displayed in green. Inset
is magnified in H. (F) Staining pattern of anti-GlyR on the cellular level displayed in red. Inset is magnified in I. D–F: Scale bar 10 µm. (G) Magnifying inset (displayed in D) of the
concentrated Glycin receptors (red) in colocalization with gephyrin labelled cells (green). (H) Magnifying inset displayed in (E) of gephyrin labelled cells (green). (I) Magnifying inset
(displayed in F) of concentrated Glycin receptors (red). G–H: Scale bar 5 µm. Images were created with ImageJ 1.52n (Fiji, available from https://fiji.sc/)38.
Full sizeimageReorganization of inhibitory synaptic sites
In control animals the number of gephyrin-labelled cells did not change over time (Fig. 5A; Tables S1, S2). In individuals that we exposed to predators from the 3rd instar onwards, the
number of gephyrin-labelled cells increased significantly from a mean of 352.62 ± 105.41 cells (0 h) to 440.62 ± 138.76 cells within 24 h (Fig. 5A; Tables S1, S2). In 48 h predator exposed
specimens the number of gephyrin-labelled cells remained constant in comparison to 24 h exposed specimens (Fig. 5A; Tables S1, S2). At 0 h no significant difference in the number of
gephyrin-labelled cells was detected between the control and predator-exposed group (Fig. 5A; Table S3). After 24 h predator exposure individuals showed a significantly increased number of
gephyrin-labelled cells compared to the control (Fig. 5A; Table S1). After 48 h predator exposure no significant difference in the number of gephyrin-labelled cells was detected between the
two treatments (Fig. 5A; Table S1). In individuals that we exposed from the 5th instar onwards the number of gephyrin-labelled cells in predator-exposed D. longicephala was significantly
increased in comparison to the control after 24 h predator exposure (Fig. 5B, Table S4).
Figure 5Functional plasticity underlying morphological defense expression. (A) When exposed to predators from the 3rd instar onwards, the number of gephyrin-labelled cells is significantly increased
after 24 h predator-exposure compared to the control (P = 0.013; t = − 2.56; df = 57) and to the 3rd instar (ANOVA: F(2;97) = 3.12 P = 0.049 Tukey: P = 0.039). (B) When exposed to
predators from the 5th instar onwards, the number of gephyrin-labelled cells is significantly increased after 24 h predator exposure compared to the control (P ≤ 0.001; t = − 3.63; df =
44). (C) The number of gephyrin-labelled cells per nerve cells is significantly increased compared to the control after 24 h predator exposure, when the animals were exposed to predators
from the 3rd instar onwards (P = 0.011; t = − 2.65; df = 57). (D) When exposed to predators from the 5th instar, the ratio of gephyrin-labelled cells/nerve cells is significantly increased
compared to the control after 24 h predator exposure (P ≤ 0.001; t = − 3.41; df = 44). (A, C) Alpha corrected levels of significance: *P ≤ 0.017. (B, D) Alpha corrected levels of
significance: **P ≤ 0.005; ***P ≤ 0.0005. Boxplots show median (middle line), 25th to 75th percentiles (box) and whiskers show highest and lowest value within 1.5× the interquartile range
(1.5× below the 25th percentile and 1.5× above the 75th percentile), respectively. Dots indicate outliers > 1.5× the interquartile range.
Full size imageAt the beginning of the experiment and prior to predator exposure (0 h), we detected no significant differences between the gephyrin-labelled cells per absolute cell number in the two
treatments (Fig. 5C; Table S3). After 24 h predator-exposed individuals showed a significantly higher ratio than controls (Fig. 5C; Table S3). 48 h after initial predator exposure no
significant differences between the treatments were detected (Fig. 5C; Table S3).
The ratio of gephyrin-labelled cells/neurons in individuals that we exposed from the 5th instar onwards, was significantly increased in predator-exposed individuals compared to controls
after 24 h predator exposure as well (Fig. 5D; Table S5).
DiscussionInducible defense expressionWhen we expose D. longicephala to predators in the 3rd instar, we observe a tendency towards larger crests in the 4th instar and significant crest expression in the 5th instar. We observe a
similar result when we expose the animals in the 5th instar, upon which crests are also significantly expressed 24 h later. This indicates that predator exposure gradually induced crest
expression within a short time frame of 24 h, so that defenses become effective in a fast manner as reported previously15.
Structural plasticity in the pc/dc-complexVolumetric changes ofthe pc/dc-complex and the OG
As the main cognitive functions are performed by the protocerebrum, we focused on the pc/dc-complex. The mean volume of naïve D. longicephala pc/dc-complexes increases 1.4-fold over the
observed instars showing the highest volume in the 6th instar with ~ 350,000 µm3. Larvae of the fruit fly Drosophila show a total brain volume of ~ 800,000 µm3 44 and adult flies even ~ 80
Mio µm3 45, with a body size similar to that of adult D. longicephala. When exposed to predators for 24 h and 48 h, D. longicephala showed significantly larger pc/dc-complex volumes than the
respective controls. We detected this predator-induced volumetric increase independent of the Daphnias’ age. Therefore, the growth of the morphological defense is accompanied with the
growth of the pc/dc-complex. Our results show that the volume of predator-induced animals’ pc/dc-complexes more than doubles (2.2-fold) within the first 48 h of predator exposure from ~
250,000 µm3 to a volume of ~ 565,000 µm3. This means that predator-exposed individuals show a 60% larger pc/dc-complex compared to controls after 48 h.
In Drosophila, different brain structures have been observed to increase in response to diversified environments that are e.g. enriched with food vials, odor sources and conspecifics. The
Drosophila central brain is probably best comparable to the pc/dc- complex in Daphnia, and this structure increased its volume by 7.5% when encountering complex environments for 19 days46.
The largest volumetric increase of 21% was observed in the calyx, a part of the mushroom bodies26. The volumetric increase of the Daphnia pc/dc-complex is three times as high as the largest
volumetric increase in Drosophila brains. Furthermore, the increase in the Daphnia pc/dc-complex appears much faster and reflects a strong structural plasticity in a very short time. This
may be an important factor when coping with predators, as the defenses need to be expressed in due time so that strong and rapid structural plasticity is probably selected for.
Mechanistically, such a high degree of volumetric expansion can only be achieved if either the absolute number of cells, the cell size or intercellular distances increase. Larger
intercellular distances provide additional space for cell- cell connections in the form of dendrites that expand the cell surface. It is reasonable to suggest that the basis of such novel
cell–cell connections is established during the first 24 h of predator perception. Since we detected no volumetric changes in the OG between the observed treatments, the functional
plasticity observed in the pc/dc-complex has to be a result of predator-exposure. This supports the hypothesis that the pc/dc-complex plays a central role in inducible defense
expression.
Nerve cell numbers in the pc/dc-complexThe 1.4-fold pc/dc-complex volume increase that we observe with age and the 2.2 fold increase that we observe with predator exposure must originate from some kind of gain in neuronal tissue.
Therefore, we determined the overall number of cells, irrespective of their type, in the pc/dc-complex. With increasing instars, we detected a 1.2-fold increase in cell numbers within the
pc/dc-complex. Cell numbers increased from ~ 750 cells in the 3rd instar to ~ 900 cells in the 6th instar. So, with age the animals obtain more cells in this central complex. As the animals
grow continuously throughout life, it is possible that the number of cells in the brain continues to increase. In fact, a life-long addition of new neurons, e.g. in the olfactory pathways of
insects47 and crustaceans48,49,50, has already been reported. Similarly, the continuous increase in body size requires the addition of new receptor neurons to cope with the increased
surface area of the body, the length of the appendages or the size of the compound eye51,52,53. This probably also leads to the observed volumetric increase in the OG with increasing age
(i.e. ~ 2.9-fold increase in control and predator exposed D. longicephala). The 1.23-fold increase in the number of brain cells in the pc/dc-complex lies within the range of the volumetric
changes across instars (1.4-fold), so that there may be a direct correlation between volume and cell number. However, this correlation only holds true when comparing successive instars.
Between control and predator-exposed animals, we did not observe that the 2.2-fold increase in brain volume is due to an increase in the cell number (i.e. only 1.23-fold). Therefore, the
predator-induced increase in pc/dc-complex volume must rely on other factors, e.g. less dense brain tissues and/or larger neurons in predator-exposed D. longicephala. In fact, in rats it has
already been reported that an enriched and demanding environment leads to increased brain volume based on reduced cell density and increased cell size but the number of neurons in the brain
tissue was not affected (reviewed in32). It can be speculated that larger intercellular clefts provide more space for structures like dendrites and synapses, while bigger cell bodies
provide a larger surface area on which more synapses can attach. This has been observed in the visual cortex of rats19. Individuals exposed to a complex environment had larger neurons,
allowing the attachment of 20% more synapses per neuron than rats from less complex environments. It was hypothesized that a larger neuron size may improve cellular stability and efficiency.
Likewise, larger cells may also be required to ensure the metabolic support of more extensive dendritic fields19. As synaptic density remains unaltered in plastic brain tissue32, an
increased neuron size is necessary for the attachment of additional synapses. Therefore, the here observed increase in pc/dc-complex volume is not correlated with an increase of cell nu
mbers but must depend on an increase in inter-neuronal connections. We hypothesize that there is a predator-induced rewiring of inter-neuronal connections i.e. through the development of new
dendrites, increasing intercellular distances and thus the overall volume. Likewise, we anticipate that neuron size is increased, which enables the attachment of more synapses and enhances
cell–cell stability. This may contribute to the formation of a dedicated neuronal circuitry that is associated with predator perception.
Functional plasticity in thepc/dc-complexReorganization of inhibitory synaptic sites
In order to investigate potential remodeling and/or emergence of a dedicated cell circuitry, we focused on the number and distribution of inhibitory cell synapses indicated by the
scaffolding protein gephyrin. The staining pattern of gephyrin shows intensive markings in the cytoplasm and on the surface of the cell soma, which is typical for this antibody and explained
by the fact that gephyrin is involved in post-Golgi transport and cell surface delivery of GlyRs36. In contrast, GlyRs are observed with a typical dot-like staining pattern on the cell
membranes. Gephyrin was found in colocalization with GlyRs, and all cells that stained with the anti-GlyR antibody showed a colocalization with the gephyrin antibody. This colocalization
clearly demonstrates that also in Daphnia, gephyrin is the anchoring protein for receptors like GlyRs at inhibitory PSDs. The fact that we observe gephyrin-labelled cells that do not
co-localize with GlyRs indicates that gephyrin also anchors other inhibitory receptors like the GABAA receptor known from other species33. Unfortunately, we so far did not find a specific
GABAAR antibody to test for this co-localization and therefore this awaits further investigation.
Both, gephyrin as well as GlyRs are homogeneously distributed throughout the pc/dc-complex. We did not observe any kind of neuropil concentric localization of gephyrin that would point to a
specialized brain area. This pattern maintained also throughout predator exposure. However, after 24 h of predator exposure, the number of gephyrin-labelled cells in the pc/dc-complex is
significantly increased compared to controls. As the number of neurons is stable between treatments, the ratio of gephyrin-labelled cells per neuron is also significantly higher in
predator-exposed individuals after 24 h. This coincides with the onset of crest development and is again age independent. An increased number of cells expressing gephyrin indicates the
presence of more inhibitory synaptic connections, as gephyrin anchors and clusters GABAARs and GlyRs at the postsynaptic site35.
Our findings show that the number of cells possessing inhibitory synapses in pc/dc-complexes of predator-exposed D. longicephala increases in line with the morphological defense expression.
It is possible that glycinergic signals play a role in the pathway underlying predator perception. Controlling the excitability of nerve cells is central to ensuring normal brain function54,
and increased inhibition could thus lead to a more directional excitability that focuses on crucial functions. Hence, adapted inhibition patterns in the Daphnia brain might be a central
element of predator perception. While the involvement of glycinergic responses is still unknown, the involvement of GABAergic signaling in predator perception has been demonstrated in D.
pulex responding to fish and Chaoborus predation13. It is very likely that the emergence of additional GABAARs occurs upon the perception of predator cues. Functionally, GABAARs are a
central component of synaptic plasticity by ensuring that glutamatergic N-methyl-D-aspartate receptors (NMDARs) are predominantly activated during high frequency synaptic transmission
only55. Especially, NMDARs play a fundamental role in long-term potentiation (LTP) and long-term depression (LTD), two common forms of synaptic plasticity responsible for fostering cell–cell
connections56,57. It is therefore possible that such mechanisms play a role in deciphering and determining the predation risk amongst the diversity of environmental cues.
We only observe this increase in gephyrin-labelled cells in the instar that coincides with the onset of defense expression i.e. 24 h of predator exposure. In the later stage, we do not see
differences in the number of gephyrin-labelled cells in comparison to the control group. However, although defense expression persists in the following instar i.e. after 48 h predator
exposure, we detect no further changes in gephyrin abundance between control and predator-exposed individuals, which is an effect of a slight and not significant increase of the total number
of nerve cells in the controls. With the increase in the total number of cells, also the proportion of gephyrin-labelled cells increases. Even if this increase is not significant, it
diminishes the differences between the predator-induced and control groups in 5th instar specimens and is a result of the gradual growth of the animals. Nevertheless, the number of
gephyrin-labelled cells remains at a constant level, so that there are no differences between 4th and 5th instar predator exposed specimens. We hypothesize that there is a dedicated rewiring
event during the initial phase of predator exposure. Once this rewiring is accomplished, this new neuronal circuitry is manifested, indicated by the unchanged number of gephyrin-labelled
cells of 48 h exposed specimens. It remains to be determined whether this pattern will persist at later stages and will require a longer observation period in future experiments.
Taken together, the increase in gephyrin expression supports our hypothesis that putatively larger cells enable the enrichment of synaptic sites. Thus, the increased gephyrin abundance might
not only indicate a higher number of cells possessing inhibitory synapses but also more inhibitory synapses per neuron within pc/dc-complexes of predator-induced individuals. By this, the
directional excitability might be further enhanced as the activation of inhibitory receptors has also been found to interfere with glutamatergic inputs if the respective synapses are located
on the same dendritic branch58,59. The increase or reduction of activity at synapses is achieved by adjustments of the postsynaptic response, like modulation of receptor availability at the
post synaptic site60,61. Highly mobile GABAAR forms allow trafficking of inhibitory receptors, which has been postulated to be important for the modulation and expression of various forms
of synaptic plasticity56,62. Additionally, GABAAR mediated transmission itself can be modified as a result of plasticity in inhibitory synapses63. In this regard, evidence for the potential
modification of GABAARs has been found in form of increasing receptor numbers56,64.
Although we focused on the rewiring of the inhibitory nervous system, inhibition and excitation act in concert to form directed and dedicated neuronal signal patterns. It is thus very likely
to suggest that there is also a rewiring of excitatory synapses accompanying the signal integration of predator perception.
In conclusion, our findings suggest three mechanisms that are involved in signal integration of predator-specific chemical cues in the Daphnia pc/dc-complex: 1.) An increase in the
pc/dc-complex volume provides space for more cell–cell connections 2.) The formation of more inhibitory synaptic connections that control cellular activity, increasing directionality of
neuronal signaling 3.) The rewiring of a neuronal circuitry that is associated with predator perception. Taken together, our study provides new insights into how organisms interpret
environmental challenges and transform these into phenotypic adaptations.
Data availabilityData associated with this manuscript is given in the supplements.
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AcknowledgementsWe thank Dr. Martin Horstmann for supporting 3D Daphnia brain constructions and volume measurements. Additionally, we acknowledge Sabine Kindermann for excellent technical
support.
FundingOpen Access funding enabled and organized by Projekt DEAL. This work was supported by the Friedrich-Ebert-Foundation and the German Science Foundation WE 6019/2-1.
AuthorinformationAuthor notes
These authors contributed equally: A Graeve and I Ioannidou.
Authors and Affiliations Department of Animal Ecology, Evolution and Biodiversity, Ruhr-University Bochum, Universitätsstrasse 150, 44780, Bochum, Germany
A Graeve, I Ioannidou, D. M. Görl & LC Weiss
Department of Cell Morphology and Molecular Neurobiology, Ruhr-University Bochum, Universitätsstrasse 150, 44780, Bochum, Germany
J Reinhard & A Faissner
AuthorsA GraeveView author publications You can also search for this author inPubMed Google Scholar
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J ReinhardView author publications You can also search for this author inPubMed Google Scholar
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ContributionsL.C.W. conceived, and L.C.W., J.R., A.F. designed the experiment, I.I., A.G., D.M.G. carried out the labwork, A.G., I.I., L.C.W. analyzed the data, A.G. and L.C.W. drafted the manuscript,
L.C.W., A.G.; J.R., A.F. contributed to the final version. All authors gave final approval for publication and agree to be held accountable for the work performed therein.
Corresponding author Correspondence to LC Weiss.
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About this articleCite this article Graeve, A., Ioannidou, I., Reinhard, J. et al. Brain volume increase and neuronal plasticity underly predator-induced morphological defense expression in
Daphnia longicephala. Sci Rep 11, 12612 (2021). https://doi.org/10.1038/s41598-021-92052-y
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DOI: https://doi.org/10.1038/s41598-021-92052-y
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