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Citation: Ian, E.; Chu, X.; Berg, B.G.
Brain Investigation on Sexual
Dimorphism in a Gynandromorph
Moth. Insects 2022,13, 284. https://
doi.org/10.3390/insects13030284
Academic Editors: Sylvia Anton and
Romina B. Barrozo
Received: 11 February 2022
Accepted: 11 March 2022
Published: 14 March 2022
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insects
Article
Brain Investigation on Sexual Dimorphism in a
Gynandromorph Moth
Elena Ian *, Xi Chu and Bente Gunnveig Berg
Department of Psychology, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway;
xi.chu@ntnu.no (X.C.); bente.berg@ntnu.no (B.G.B.)
*Correspondence: elena.ian@ntnu.no
Simple Summary:
The noctuid moth, Helicoverpa armigera, is one of the globally most damaging
agricultural pest insects. Generally, exploration of the male- and female-specific neural architecture
underlying its reproductive behavior is crucial for developing biological and environment-friendly
alternatives to the traditional pest control management. In this study, we utilized the opportunity to
uncover putative sex differences in H. armigera by comparing details in the brain anatomy between the
male and female hemispheres in one gynandromorphic individual. The methods included synapsin
immunostaining, confocal microscopy, and the digital reconstruction of several brain areas involved
in processing input about odor and vision, respectively. The results demonstrated sex-specific
arrangements applying to distinct olfactory neuropils, including not only the primary olfactory center,
the antennal lobe, but also higher order levels involved in odor-associated memory formation.
Abstract:
The present study was dedicated to investigating the anatomical organization of distinct
neuropils within the two brain hemispheres of a gynandromorphic moth of the species Helicoverpa
armigera. High quality confocal imaging of a synapsin immuno-stained preparation combined with
three-dimensional reconstructions made it possible to identify several brain structures involved in
processing odor input and to measure their volumes in the male and female hemispheres. Thus,
in addition to reconstructing the antennal lobes, we also made digital models of the mushroom
body calyces, the pedunculus, and the vertical and medial lobes. As previously reported, prominent
sexual dimorphism was demonstrated in the antennal lobes via the identification of a male-specific
macroglomerular complex (MGC) and a female-specific complex (Fc) in each of the two brain
hemispheres of the gynandromorph. Additionally, sex-specific differences were found in volume
differences for three other neuropil structures—the calyces, pedunculus, and vertical lobe. The
putative purpose of larger volumes of three mushroom body neuropils in females as compared to
males is discussed.
Keywords: gynandromorph moth; olfaction; antennal lobe; confocal images; digital reconstruction
1. Introduction
Dimorphic individuals, in which the bodies consist of both female and male parts,
were observed before the term gynandromorphism was coined and described by Rudolfi
for the moth species, Gastropacha quercifolia, in 1825. Though this phenomenon is more
commonly reported in insects than in other animal classes, the estimated occurrence of
insect gynandromorphs is about 0.01 to 0.05%. Gynandromorphism has been observed
in several insect orders including Lepidoptera, Hymenoptera, Coleoptera, Diptera, and
Orthoptera, but its occurrence across orders is not even [
1
]. Several thousands have been
reported in Lepidoptera in comparison with 90 in Hymenoptera; however, this fact could
be due to easily recognizable sexual dimorphic features in the former order [2].
Lepidoptera (moths and butterflies) have a female heterogametic sex chromosome
system, with most females having a WZ constitution while males are ZZ [3]. The cause of
Insects 2022,13, 284. https://doi.org/10.3390/insects13030284 https://www.mdpi.com/journal/insects
Insects 2022,13, 284 2 of 12
bilateral gynandromorphism arises from an event in mitosis during a very early stage in
the development of the organism. When the juvenile organism grows through a series of
cell divisions, a genetic error in the chromosomes leads to one of the dividing cells splitting
its sex chromosomes aberrantly, resulting in two dissimilar daughter cells, one having the
chromosomes that cause female development and the other the chromosomes that cause
male development.
While external dimorphism appears as morphological features like size, form, and
color, internal dimorphism is reflected by gender-specific reproductive organs and brain
anatomy. In our lab, the Chemosensory lab at the Norwegian University of Science and
Technology (NTNU), an emerged moth of the species, Helicoverpa armigera, proved to be a
bilateral gynandromorph, with a clear external segregation of the male and female appear-
ances on the left and right side, respectively. In addition, internally observed bilateral brain
structures gave us a unique opportunity to uncover putative sexual dimorphic traits of the
moth brain, making a comparison of the gynandromorphs male and female hemispheres.
In the moth brain, the antennal lobe (AL) stands out as a prominent sexually dimorphic
neuropil. Functionally, the moth AL, also called the primary olfactory center of the insect
brain, plays a role in processing chemo-sensory information. The AL consists of spherical
units, glomeruli, which are segregated into several groups based on their morphological
and functional specificities [
4
]. In H. armigera, two recent studies performed a global-wide
comparison between female and male ALs to identify sexually dimorphic and isomorphic
glomeruli [
5
,
6
]. Three-dimensional reconstruction of the AL glomeruli revealed four dis-
tinct groups in both sexes. The most prominent sexual dimorphism in the moth species
is demonstrated by the macroglomerular complex (MGC). In H. armigera males, there are
three glomeruli grouped into the MGC and processing input about female-produced com-
ponents [
7
]. In females, a small assembly of enlarged glomeruli, possibly being comparable
with the MGC units in males, has been observed in several moth species: Bombyx mori [
8
],
Manduca sexta [
9
], and Heliothis virescens [
10
]. In H. armigera, five enlarged glomeruli were
identified as female-specific units and belonging to the female complex (Fx; [6]).
Generally, the largest group of glomeruli constitutes a category of so-called ordinary
glomeruli (OG). In H. armigera, the OGs demonstrate high homology, though the numbers
of glomeruli in males and females are different; in the H. armigera male there are
64–65 OGs
and in female 66 [
6
]. The main task of these glomeruli is to process plant odor information.
Additionally, there are also some reports about their involvement in thermal and mechanical
stimuli processing as well [5,11].
Another category of glomeruli is the so-called posterior complex (PCx), a cluster of
dorso-posteriorly located units. The function of this group is not yet defined, though it
was shown in several male heliothine species that one of the PCx glomeruli receives input
from two co-located distinct types of pheromone sensory neurons [
7
]. In addition, calcium
imaging results also demonstrated pheromone-evoked responses in the PCx glomeruli
(personal observations). The previous anatomical investigation of the AL in H. armigera
males and females showed sexual dimorphism in PCx, including a different number of
glomeruli: ten in males and nine in females [
6
]. The fourth glomerular category consists of
a single large unit, the labial pit organ glomerulus (LPOG), receiving input from sensory
neurons tuned to carbon dioxide [
12
]. The former investigations of H. armigera, showed
that the LPOG volume is larger in females than in males [6].
Whether other neuropils than the primary olfactory center may also have a sex dif-
ference is still an open question. Thus, in the present study, we examined the H. armigera
gynandromorph with respect to putative anatomical differences in the central brain areas of
the two hemispheres. Brain regions including those for olfactory and visual processing were
selected, as both olfactory and visual inputs are fundamental for moth navigation [
13
,
14
].
Comparative quantitative analysis of the digital neuropil reconstructions was performed
on the optical lobes (OL), the anterior optic tubercle (AOTu), and the mushroom bodies
(MB) including the calyces, lobes, and the pedunculus. The results demonstrate that the sex
Insects 2022,13, 284 3 of 12
differences are not only present in the AL, but also in some of the protocerebral neuropils
in the higher brain centers.
2. Materials and Methods
2.1. Insects
Helicoverpa armigera pupae (Lepidoptera; Noctuidae, Heliothinae), delivered by Keyun
Bio-pesticides (Henan, China), were reared in climate chambers (Refritherm 200 and 6E,
Struers-Kebolab, Albertsund, Denmark, or Binder KBF 720, Tuttlingen, Germany) at
24 ◦C
and 70% air humidity on a 14:10 h light/dark cycle. The individual used in this study
was one day old. According to Norwegian law of animal welfare, there are no restrictions
regarding the experimental use of Lepidoptera.
2.2. Immunostaining
The preparation procedure was described in detail elsewhere [
14
,
15
]. In short, the
brain was dissected in Ringer’s solution (in mM: 150 NaCl, 3 CaCl
2
, 3 KCl, 25 sucrose, and
10 N-tris (hydroxymethyl)-methyl-2-amino-ethanesulfonic acid, pH 6.9) and immediately
transferred into a Zinc–Formaldehyde fixative [
16
] at room temperature overnight. The
brain was then washed in HEPES-buffered saline (HBS, 8
×
30 min), and subjected to a
permeabilization step (60 min incubation with a fresh mixture of 20% DMSO and 80%
methanol) before being washed 3
×
10 min in a Tris-HCL buffer (0.1 M, pH 7.4). After
pre-incubation in 5% normal goat serum (NGS, Sigma–Aldrich, St. Louis, MO, USA) in
0.1 M
phosphate-buffered saline (PBS, pH 7.2) containing 0.3% Triton X-100 (PBT), the
brain was incubated for 6 days at 4
◦
C in the primary antibody, SYNORF1 (dilution 1:25 in
PBT containing 1% NGS). Following rinsing in PBT 8
×
30 min, the brain was incubated
for
5 days
at 4
◦
C with Alexa Flour 647 conjugated goat-anti-mouse secondary antibody
solution (Invitrogen, Eugene, OR; dilution 1:300 in PBT with 1% NGS). After washing
4×30 min
in PBT and 2
×
30 min in PBS, the brain was dehydrated in an increasing
ethanol series (50%, 70%, 90%, 95%, and 100% (2
×
), 10 min each). Then, the brain was
transferred to a mixture of methyl salicylate and ethanol (1:1) for 10 min and after that
cleared completely in methyl salicylate for at least 1 h. Finally, the brain was mounted in
a Permount mounting medium (Electron Microscopy Sciences) between two coverslips,
separated by spacers.
2.3. Confocal Image Acquisition
The immunostained brain was imaged dorso-frontally by using a confocal laser
scanning microscope (LSM 800 Zeiss, Jena, Germany) equipped with a Plan-Neofluar
20×/0.5 objective
. The sample was excited with a HeNe laser at 653 nm and the fluorescent
emission passed through a 650 nm long-pass filter at a voxel size of 0.62
×
0.62
×
2
µ
m.
The confocal images shown in this study were edited in ZEN 2.3 (blue edition, Carl Zeiss
Microscopy GmbH, Jana, Germany).
2.4. Digital Reconstruction and Volumetric Analysis
The high-quality confocal stack allowed for making 3D visualizations based on the
auto-fluorescence signals without an immunostaining application. A complete image of
the raw confocal stack of the gynandromorph moth was used for making digital recon-
structions of the relevant brain structures by means of the AMIRA software (AMIRA 6.0,
Visage Imaging, Fürth, Germany). The brain areas were manually demarcated by using
a segmentation editor for all three spatial planes with a consequent wrap-tool to obtain
full neuropil volumes. Surface models of the segmented areas were constructed, and their
volumes were measured by the material statistics function in the segmentation editor.
2.5. Glomerular Naming
In correspondence with the previous classification of antennal-lobe glomeruli in
the male and female heliothine moth, we grouped the units in four assemblies: (1) the
Insects 2022,13, 284 4 of 12
male/female specific complex, (2) the ordinary glomeruli (OG), (3) the posterior complex
(PCx), and (4) the labial pit organ glomerulus (LPOG) [
5
,
6
]. For the male specific MGC, the
previously established names, Cu, Dmp and Dma were used. For the female counterpart,
namely, the female complex, Fx, the three relevant glomeruli were named, Fx1–Fx3. Since
the OGs in the male and female hemispheres could not be identified with respect to ho-
mologies, they were numbered without any correspondence. The OGs in the male part
were named O
m
1–O
m
66 and in the female part O
f
1–O
f
66. For the PCx units, the names
P
m
1–P
m
9 were used for the glomeruli in the male hemisphere and P
f
1–P
f
9 for the glomeruli
in the female hemisphere. The LPOG unit was named L
m
and L
f
for male and female
halves, respectively. In addition to the four above-mentioned glomerular assemblies, we
created a new one, the ventroposterior complex (VPx), which was selected out of the OGs
and named Vm1–Vm5 and Vf1–Vf5 in males and females, respectively.
2.6. Neuropil Volume Analyses
As gynandromorphic moths are rare, the analyses of the neuropile volume were con-
ducted based on the comparison of data from one gynandromorphic brain and a male
genotype representative brain. We used the hemispheric ratio to clarify the volumetric
difference of corresponding neuropils between the two hemispheres. Thus, in the represen-
tative brain, the hemispheric ratio of a neuropil was computed by comparing the volume of
corresponding neuropils in the left and right hemisphere. For the gynandromorphic brain,
this ratio was calculated by comparing the neuropil volumes of corresponding structures
in the male and female halves. For example, the hemispheric ratio of the vertical lobe in
the gynandromorphic brain was calculated as the increase rate of vertical lobe volume in
the female hemisphere relative to the male hemisphere. To determine whether the hemi-
spheric ratios of neuropils in the gynandromorphic brain were different from those in the
representative brain, we defined a threshold (T) based upon the hemispheric ratio of eight
corresponding neuropils in the representative brain at a 10% significance level. We also
compared the mean hemispheric ratios (across 8 neuropils) to a theoretically symmetrical
brain, in which the hemispheric ratio should be 0, by using a one-sample ttest. In addition,
to investigate whether the hemispheric ratio was related with the neuropil function, we
generated a hierarchical cluster analysis by using the centroid clustering method to obtain
an overview of the linkage of all hemispheric ratios from, totally, 16 neuropils, including 8
in the representative brain and 8 in the gynandromorphic brain. All probabilities given are
two-tailed. SPSS, version 25, was used for the statistical analysis.
3. Results
3.1. Anatomical Comparison between Female and Male Hemisphere in a Bilateral
Gynandromorphic Brain
Digital reconstruction of the prominent and easily recognizable brain areas allowed a
comparison of anatomical traits and volumes of the paired neuropils of two hemispheres
in a bilateral gynandromorphic brain. We reconstructed six protocerebral neuropils in each
hemisphere: the mushroom body calyces, the pedunculus, the vertical and medial lobes, the
anterior optic tubercle and the central body. In addition, each antennal-lobe glomerulus and
the three optical lobes, medulla, lobula, and lobula plate, were digitally labeled. Figure 1
visualizes the confocal sections and the detailed reconstruction of the labeled neuropils,
where the male brain parts are presented in green shades and the corresponding female
neuropils in magenta shades.
Insects 2022,13, 284 5 of 12
Figure 1.
Anatomical organization of a bilateral gynandromorphic brain in H. armigera. (
A
) Confocal
images of both hemispheres in the bilateral gynandromorphic brain. The two optical sections contain
slices from a dorsal (depth: 57.98
µ
m, top image) to ventral (depth: 129.34
µ
m, bottom image).
From the insect’s perspective, right hemisphere: male half; left hemisphere: female half. (
B
) Three-
dimensional reconstruction (dorsal view) of selected neuropils in the gynandromorphic H. armigera
brain. Each neuropil is in a shade of green/magenta, representing the male (green) and the female
(magenta) hemispheres, respectively. AL: antennal lobe; AOTu: anterior optic tubercle; CB: central
body; Ca: calyces; Lo: lobula; LoP: lobula plate; Me: medula; ML: medial lobe; Ped: pedunculus; VL:
vertical lobe; OL: optical lobe.
3.2. Proposed Dimorphic Neuropils in Heliothine Moths
The gynandromorphic sample with bilateral asymmetry provided a unique oppor-
tunity to compare morphological details of putative dimorphic brain structures between
the female and male parts. We selected eight brain compartments with distinguishable
boundaries, including three optical lobe neuropils (medulla, lobula and lobula plate) and
five protocerebral neuropils (mushroom body calyces, pedunculus, vertical lobe, medial
lobe, and anterior optic tubercle). The volume of each compartment was quantified based
on two digital reconstruction datasets (Figure 2), one originating from the previously
established representative brain [
14
], and the other from the bilateral gynandromorphic
brain. The calculated hemispheric ratios were based on the volumes of paired neuropils
within one reconstruction dataset. In the representative brain, the hemispheric ratio of a
distinct neuropil was computed based on the volume of the neuropil in the left and right
hemisphere, respectively. For the gynandromorphic brain, this ratio was calculated based
on the neuropil volumes between the male and female halves. We defined a threshold
(T) based upon the hemispheric ratio across eight neuropils in the representative brain
at a 10% significance level (T = 10.73%). It was demonstrated that, in the representative
male moth brain, none of the neuropil pairs had a hemispheric ratio above the threshold
(Figure 2B,G); however, in the gynandromorphic brain, three neuropil pairs exceeded the
hemispheric ratio threshold, i.e., the mushroom body calyces, vertical lobes, and pedunculi.
In the female hemisphere, each of these three neuropils made up a larger volume than their
counterparts in the male hemisphere (Figure 2D). The spatial organization of these three
neuropil pairs are highlighted in Figure 2H.
Insects 2022,13, 284 6 of 12
Figure 2.
Comparison of distinct neuropil volumes in female and male hemispheres. (
A
) Volumes
of 8 selected neuropils from both hemispheres in the representative male brain. (
B
) Volume ratios
of these neuropils. The dashed line illustrates the hemispheric ratio threshold (T), computed from
the hemispheric ratio in the representative brain at a 10% significance level. (
C
) Volumes of 8
selected neuropils from the female and male hemispheres in the bilateral gynandromorphic brain.
(
D
) Hemispheric ratios of these structures. The dashed line illustrates the same kind of threshold
(T) as shown in panel B. Columns in yellow show the 3 neuropils exceeding the threshold (T).
(E) Comparison
of the presumed hemispheric ratio (= 0, grey dash line) and the actual hemispheric
ratio in the gynandromorphic and the representative brain, respectively. (
F
) Hierarchical clustering
based on the hemispheric ratio of 16 neuropil pairs from the representative brain (indicated by R) and
the gynandromorphic brain (indicated by G), including 8 left vs. right hemispheric neuropil pairs and
8 male vs. female hemispheric neuropil pairs (N = 16). Each neuropil was color-coded by the value of
its hemispheric ratios. (
G
,
H
) 3D reconstructions of the 8 neuropiles in both brains. The neuropils
having higher hemispheric ratios than the threshold (T) are marked in yellow. *, one-sample t test,
compared with the hemispheric ratio in a theoretically symmetrical brain (0%), p< 0.05.
Theoretically, in a wildtype moth brain, the volume of corresponding neuropils in
the two hemispheres should be identical, which implies a hemispheric ratio of zero. To
confirm the significance of the different volumes shown in the gynandromorphic brain, we
compared the mean ratio of all eight neuropil pairs to 0. The one-sample ttests determined
that the hemispheric ratios in the gynandromorphic brain were larger than 0 (t(7) = 2.97,
Insects 2022,13, 284 7 of 12
p= 0.02
), whereas the corresponding data in the representative brain were comparable with
0 (t(7) = 1.33, p= 0.23, Figure 2E). To further reveal the putative association between the
neuropil pairs, we performed a hierarchical clustering test upon the hemispheric ratios
in all 16 neuropil pairs, where 8 of them were collected from the male representative
brain (R) and the other 8 pairs were from the gynandromorphic brain (G). Due to the
restricted gynandromorphic brain sample size, the hemispheric ratio between each of the
eight neuropil pairs in two brains was treated as an independent sample (N = 16). The test
resulted in four clusters (Cluster I–IV, Figure 2F). Interestingly, the neuropil pairs in Clusters
I and II, which demonstrated low hemispheric ratios, were mostly vision-associated brain
areas. Almost all the neuropil pairs in Clusters III and IV, on the other hand, which had
relatively high hemispheric ratios, were related to memory and learning.
3.3. Antennal Lobes: Glomerular Identification and Clustering
Comparison of the two ALs in the gynandromorphic brain clearly demonstrated a
sexual dimorphism in the form of an assembly of sexually dimorphic glomeruli located
at the antennal-nerve entrance. These sex-specific units made up the MGC and the Fx
in the male and female hemisphere, respectively. As in a ‘normal’ brain, the additional
AL glomeruli formed several segregated clusters forming seemingly sexually isomorphic
glomeruli (Table 1). Generally, the male and female ALs of H. armigera are reported to
consist of 67 glomeruli in both male and female parts. While the OGs, PCx, and LPOG
are common categories previously described in both males and females, we classified a
new cluster of glomeruli located ventro-posteriorly in the AL, the ventro-posterior complex
(VPx). These glomeruli, positioned at the border between the AL and the protocerebrum,
form a distinct group based on their atypical size, form, and compilation.
Table 1.
Categories and numbers of glomeruli in H. armigera, identified from the present and previous
studies. MGC: macroglomerular complex; OG: ordinary glomeruli; PCx: posterior complex; VPx:
ventroposterior complex; LPOG: labial pit organ glomerulus.
Gynandromophic
H. armigera [5] [17]
Male AL Female AL Male AL Female AL Male AL Female AL
MGC/Fx 3 3 3 5 3 3
OG 47 50 66 a66 a61 b61 b
PCx 11 9 10 9 - -
VPx 5 4 - - - -
LPOG 1 1 1 1 1 1
Total 67 67 80 81 65 65
athe OG in [5] include the VPx. bthe OG in [17]) include the PCx and VPx.
Three-dimensional reconstructions of the AL glomeruli allowed for making an overall
comparison between the male and female halves; however, since it was not possible to
define homologous glomeruli, the glomerular numbers on each side do not correspond to
each other.
3.3.1. Sex Specific Areas: MGC and Fx
In the bilateral gynandromorphic brain, the sex-specific AL glomeruli, the MGC and
the Fx, were comparable to the corresponding glomerular structure in the normal male and
female brain, respectively. The Fx in the gynandromorph sample, was identified as three
enlarged glomeruli located at the area equivalent to the MGC (Figure 3).
Insects 2022,13, 284 8 of 12
Figure 3.
Three-dimensional reconstruction of the male macroglomerular complex (MGC) and the
female complex (Fx). The MGC includes three units: the cumulus (Cum), the dorsomedial posterior
(Dmp) and dorsomedial anterior (Dma) glomeruli. The Fx includes three enlarged glomeruli, having
a similar location with the MGC.
3.3.2. Ordinary Glomeruli
The ordinary glomeruli comprise the largest AL cluster. In the gynandromorph brain,
there were 47 units in the male hemisphere and 50 in the female (Figure 4). In a previous
study of the AL glomeruli in H. armigera, the majority of OGs in normal males and females
were found to be homologous [
6
]. In the present investigation, it was not possible to
perform such a detailed analysis. Therefore, the OG numbers for the two brain hemispheres
listed here are not in correspondence. The total numbers of OGs identified in the ALs of the
gynandromorph, 47/50, were reduced as compared to the quantity previously reported,
66/66 [
5
]. This is probably due to several factors, such as the classification of the new
glomerular group, VPx, and the poorer staining quality of the glomerular units.
Figure 4.
Three-dimensional reconstruction of the ordinary glomeruli in the male and female parts of
the gynandromorph brain: dorsal view (
top
) and ventral view (
bottom
). The colors and numbers of
the glomeruli in the two antennal lobes do not correspond to homologous units.
Insects 2022,13, 284 9 of 12
3.3.3. Posterior Complex Glomeruli
Located posteriorly to the MGC/Fx in the gynandromorph, was the posterior complex
(PCx) including 11 glomeruli in the male AL and 9 in the female (Figure 5). The number-
labeling in the male and the female PCx does not correspond to homologous glomeruli. A
prominent feature of the PCx is an enlarged glomerulus, number 1, located most dorsally.
The calculated volume of this glomerulus in the male and female parts is notably different:
68.6 µm3and 49.2 µm3, respectively.
Figure 5.
Three-dimensional reconstruction of the posterior complex in the male and female ALs of
the gynandromorph: dorsal view (top) and ventral view (bottom).
3.3.4. Labial Pit Organ Glomerulus (LPOG), and Ventroposterior Complex (VPx)
In this study, the glomeruli belonging to the VPx were described as a part of the
ordinary glomeruli in the previous studies [
5
,
6
,
17
] and are presented here together with
the labial pit organ glomerulus.
The LPOG was recognized in both the male and female AL of the gynandromorph. The
volumes of the male and female LPOGs were distinct: 12.5
µ
m
3
and 14.3
µ
m
3
, respectively.
In the proximity to the LPOG, the ventrally located AL cluster, VPx, was localized. This
glomerular assembly comprised four glomeruli in the female AL and five in the male
(Figure 6). Among these enlarged glomeruli, V1 and V2 displayed a similar spatial location
across the two ALs, indicating homology. The remaining VPx glomeruli in the male and
female hemispheres seemed to be non-homologous.
Insects 2022,13, 284 10 of 12
Figure 6.
Three-dimensional reconstruction of the ventroposterior glomeruli (VPx) and the labial pit
organ glomerulus (LPOG) in the male and female antennal lobe of the gynandromorph.
4. Discussion
In the present study, we investigated the brain of a gynandromorphic H. armigera
moth to uncover putative sex-specific neuropil regions. Comparison of the primary ol-
factory center, the AL, in normal males and females of this species has been conducted
previously [5,6]
; however, since neuropil volumes in different moth brains may depend on
age, and pre- and post-eclosure experience, such comparisons across different individuals
could reflect inter-individual variations rather than sex-specific distinctions. Thus, the
gynandromorphic sample used in this study provided a remarkable opportunity to explore
the putative sex differences in the same brain, where both the male and female hemispheres
developed under the same conditions.
Confocal imaging and subsequent 3D reconstruction of individual brain regions in
the gynandromorphic sample allowed us to make a precise volume comparison between
several pairs of olfactory neuropils in the male and female hemispheres including the
antennal lobe, calyces, pedunculus, and lobes of the mushroom bodies. In addition, we
also examined the optical lobes regarding possible sex differences.
The most prominent sex-specific feature in the moth brain is the MGC of the male
AL [
5
,
18
]. A previous study on a gynandromorphic moth of the species, Agrotis ipsilon
(Hufnagel), demonstrated morphological differences within the brain and reproductive
system [
18
]. Here, however, the ALs in both the male and female parts were incompletely
developed in comparison with the typical male/female genotype. Thus, the AL of the
A. ipsilon’s
gynandromorphic brain included ordinary glomeruli in both hemispheres but
no female-specific glomeruli in the female hemisphere and only a partially developed MGC
in the male hemisphere. In our study on the gynandromorphic H. armigera, on the other
hand, both ALs seemed to be completely developed. The anatomical organization in the
two brain hemispheres was in full correspondence with the ‘normal’ male and female
individuals, respectively. This included the presence of five glomerular assemblies in both
hemispheres, the MGC/Fx, OGs, PCx, LPOG, and VCx.
As shown in the results, the arrangement of glomerular assemblies in the two brain
halves of the gynandromorphic brain were comparable, with one exception—a sex-specific
group formed by the MGC and Fx in the male and female hemispheres, respectively. This
is in full agreement with previous data on this species reporting about a sex-specific group
of glomeruli located at the antennal-nerve entrance into the AL [5,6,17].
Concerning the largest group of glomeruli, the OGs, comparison between the ALs
of the gynandromorph H. armigera and a normal type showed a significant difference. In
this study, there was a total number of 47 and 50 OGs in the male and female hemispheres,
respectively; however, in the previous studies on H. armigera, [
5
,
6
], the respective numbers
were 66 and 61 [
5
,
6
]. The relatively large difference between the numbers of identified
glomeruli could be due to the different staining procedures applied. While only synapse-
associated protein immunostaining was used in the present study, the former investigations
Insects 2022,13, 284 11 of 12
combined multiple staining methods including two distinct immunostaining techniques,
anterograde staining of the sensory axons, and retrograde staining of AL output neurons.
The previous reports therefore allowed a staining quality probably visualizing distinct
glomeruli in the deep parts of the AL-glomeruli that might not be recognized in this study.
Moreover, the definition of the VCx glomeruli as a separate group in the present study,
which were previously included in the OGs, leads to a lower number as well.
In addition to the anatomical difference in one part of the male and female AL of the
gynandromorph, we found volumetric distinctions between the protocerebral neuropils
in the two hemispheres. Altogether, the high resolution of the confocal images made it
possible to reconstruct three optical lobe neuropils (medulla, lobula, and lobula plate) and
five protocerebral neuropils (MB calyces, pedunculus, vertical lobe, medial lobes, and the
AOTu). This contrasts with the fact that none of the corresponding neuropil pairs labeled in
the representative brain had hemispheric ratios above the threshold. The three MB neuropil
pairs exceeding the hemispheric ratio threshold in the gynandromorphic brain were the
calyces, vertical lobe, and pedunculus.
Interestingly, the female hemisphere in the gynandromorph demonstrated enlarged
volumes of neuropils within the MBs, a higher order region for olfactory memory formation.
Like in other insects, the MBs of the moth are formed by several morphological types of
Kenyon cells (KC) having distinct anatomical traits within the Ca and the MB lobes [
19
,
20
].
Notably, the KCs are assumed to play a role in adjusting odor representation according
to experience [
21
]. The difference between the volumes of certain MB compartments as
obtained in this study may indicate the involvement of distinct circuits for processing
biologically relevant signals in males and females, respectively. While the male moth
mostly orients for detecting and finding a suitable conspecific female, the female has
to recognize suitable host plants for oviposition—a task involving appropriate behavior
according to the ecological surroundings. This may be reflected in a bigger number of the
KCs in the female, leading to the ability to identify, learn, and remember a large amount of
potentially suitable plant odors. Therefore, the different volumes of the MB structures in
the two hemispheres of the gynandromorph may be related to an increased number of KCs
in the female part satisfying the special requirements during oviposition. While volumetric
differences between the MB structures of male and female moths have not been previously
reported, sex-specific projection patterns within the calyces are well documented. Thus, in
males, projection neurons (PNs) originating from the MGC and the OGs are reported to
terminate within distinct areas of the calyces whereas females seem to have no organized
patterns of PNs within this neuropil structure [14,22,23].
In conclusion, the data from the gynandromorph moth investigated here, not only
confirmed the previous findings about sexual dimorphism in the primary olfactory center
of the moth brain, the AL, but also in the higher centers like the calyces, pedunculus,
and vertical lobe of the mushroom body. It is worth noting that there is a limitation
in our conclusion, in particular that the statistical analyses were conducted in only two
individual brains, due to the rareness of a gynandromorphic brain; however, the difference
between the male and female brains we revealed in the gynandromorphic brain sample
is representative.
Author Contributions:
Conceptualization, X.C., B.G.B. and E.I.; methodology, X.C. and E.I.; software,
E.I.; validation, X.C., E.I. and B.G.B.; formal analysis, E.I. and X.C.; investigation, E.I. and X.C.;
writing—original draft preparation, E.I.; writing—review and editing, B.G.B. and X.C.; visualization,
E.I. and X.C.; project administration, B.G.B.; funding acquisition, B.G.B. All authors have read and
agreed to the published version of the manuscript.
Funding:
This research was funded by the Research Council of Norway, grant number 324379 to
B. G. Berg. https://www.forskningsradet.no/en/ (accessed on 10 February 2022).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Insects 2022,13, 284 12 of 12
Data Availability Statement: Data sharing is not applicable to this article.
Conflicts of Interest: The authors declare no conflict of interests.
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