Exploring Compound Eyes in Adults of Four Coleopteran Species Using Synchrotron X-ray Phase-Contrast Microtomography (SR-PhC Micro-CT)

Abstract

Compound eyes in insects are primary visual receptors of surrounding environments. They show considerable design variations, from the apposition vision of most day-active species to the superposition vision of nocturnal insects, that sacrifice resolution to increase sensitivity and are able to overcome the challenges of vision during lightless hours or in dim habitats. In this study, Synchrotron radiation X-ray phase-contrast microtomography was used to describe the eye structure of four coleopteran species, showing species-specific habitat demands and different feeding habits, namely the saproxylic Clinidium canaliculatum (Costa, 1839) (Rhysodidae), the omnivorous Tenebrio molitor (Linnaeus, 1758) and Tribolium castaneum (Herbest, 1797) (Tenebrionidae), and the generalist predator Pterostichus melas italicus (Dejean, 1828) (Carabidae). Virtual sections and 3D volume renderings of the heads were performed to evaluate the application and limitations of this technique for studying the internal dioptrical and sensorial parts of eyes, and to avoid time-consuming methods such as ultrastructural analyses and classic histology. Morphological parameters such as the area of the corneal facet lens and cornea, interocular distance, facet density and corneal lens thickness were measured, and differences among the studied species were discussed concerning the differences in lifestyle and habitat preferences making different demands on the visual system. Our imaging results provide, for the first time, morphological descriptions of the compound eyes in these species, supplementing their ecological and behavioural traits.

Full text

Citation: Giglio, A.; Vommaro, M.L.;
Agostino, R.G.; Lo, L.K.; Donato, S.
Exploring Compound Eyes in Adults
of Four Coleopteran Species Using
Synchrotron X-ray Phase-Contrast
Microtomography (SR-PhC Micro-CT).
Life 2022,12, 741. https://doi.org/
10.3390/life12050741
Academic Editor: Dmitry L. Musolin
Received: 12 April 2022
Accepted: 15 May 2022
Published: 17 May 2022
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4.0/).
life
Article
Exploring Compound Eyes in Adults of Four Coleopteran
Species Using Synchrotron X-ray Phase-Contrast
Microtomography (SR-PhC Micro-CT)
Anita Giglio 1, * , Maria Luigia Vommaro 1,† , Raffaele Giuseppe Agostino 2,3 , Lai Ka Lo 4
and Sandro Donato 2, 5, †
1Department of Biology, Ecology and Earth Science, University of Calabria, Via Bucci, Arcavacata di Rende,
87036 Cosenza, Italy; marialuigia.vommaro@unical.it
2Department of Physics and STAR-LAB, University of Calabria, Via Bucci, Arcavacata di Rende,
87036 Cosenza, Italy; raffaele.agostino@fis.unical.it (R.G.A.); sandro.donato@fis.unical.it (S.D.)
3Consiglio Nazionale delle Ricerche, Istituto di Nanotecnologia (Nanotec)—UoS Cosenza, Via Bucci,
Arcavacata di Rende, 87036 Cosenza, Italy
4Animal Evolutionary Ecology Group, Institute for Evolution and Biodiversity, University of Münster,
48149 Münster, Germany; lo@uni-muenster.de
5Istituto Nazionale di Fisica Nucleare, Division of Frascati, Via Fermi, 54, Frascati, 00044 Rome, Italy
*Correspondence: anita.giglio@unical.it; Tel.: +39-098-449-2982
† These authors contributed equally to this work.
Abstract:
Compound eyes in insects are primary visual receptors of surrounding environments. They
show considerable design variations, from the apposition vision of most day-active species to the
superposition vision of nocturnal insects, that sacrifice resolution to increase sensitivity and are able to
overcome the challenges of vision during lightless hours or in dim habitats. In this study, Synchrotron
radiation X-ray phase-contrast microtomography was used to describe the eye structure of four
coleopteran species, showing species-specific habitat demands and different feeding habits, namely
the saproxylic Clinidium canaliculatum (Costa, 1839) (Rhysodidae), the omnivorous Tenebrio molitor
(Linnaeus, 1758) and Tribolium castaneum (Herbest, 1797) (Tenebrionidae), and the generalist predator
Pterostichus melas italicus (Dejean, 1828) (Carabidae). Virtual sections and 3D volume renderings of
the heads were performed to evaluate the application and limitations of this technique for studying
the internal dioptrical and sensorial parts of eyes, and to avoid time-consuming methods such as
ultrastructural analyses and classic histology. Morphological parameters such as the area of the
corneal facet lens and cornea, interocular distance, facet density and corneal lens thickness were
measured, and differences among the studied species were discussed concerning the differences
in lifestyle and habitat preferences making different demands on the visual system. Our imaging
results provide, for the first time, morphological descriptions of the compound eyes in these species,
supplementing their ecological and behavioural traits.
Keywords:
beetle; brain; cornea; microtomography; morphology; ommatidia; optical lobe; rendering;
virtual sectioning; visual system
1. Introduction
The application and advantages of microtomography (micro-CT) in entomology pro-
vide a significant improvement step for collecting data on the insect anatomy. This method
avoids artefacts resulting from invasive dissections, followed by relatively time-consuming
fixing and physical tissue slicing, required for image analyses under light and electron
microscopy. Indeed, micro-CT has proven to be useful for virtual dissections, 3D reconstruc-
tion and morphological descriptions of the head [
1
,
2
], muscles [
2
–
4
], brain [
5
], digestive [
6
,
7
]
and reproductive [
8
–
11
] systems, as well as insect fossils [
12
–
15
]. Moreover, Synchrotron
radiation X-ray phase-contrast microtomography (SR-PhC micro-CT) allows the use of
Life 2022,12, 741. https://doi.org/10.3390/life12050741 https://www.mdpi.com/journal/life

Life 2022,12, 741 2 of 15
high-resolution imaging coupled with segmentation, for 3D morphological analyses with
high image contrast-to-noise ratios in biological tissues, and does not require the use of
contrast agents, even in samples with weak X-ray absorption [
16
–
19
]. In recent decades, it
has been applied as a non-invasive technique to observe external and internal anatomical
structures of living insects [
20
,
21
], and specimens immersed in ethanol after fixation [
2
,
6
]
or embedded in amber [22].
In insects, compound eyes, which are paired structures located on the left and right
sides of the head, contain a species-specific number of light-sensitive units named omma-
tidia [
23
,
24
]. Each ommatidium consists of two main components: a lens unit (consisting of
an external corneal facet and a crystalline cone lens), which collects and focusses incoming
light, and the rhabdom, which absorbs and transduces focussed light. The quantity of
light available and the balance between resolution and sensitivity are crucial factors that
define the structure and size of compound eyes, as well as their spatial resolving power [
25
].
The large variety of ecological niches occupied by insects explains the variability of the
eye structure, which differs greatly in different visual tasks (detecting food, predator and
partner recognition) across habitats; therefore, the selected eye design should reflect the
lifestyle and behaviour of each species [
26
–
30
]. For example, visual hunters [
27
,
31
] and
flying insects [
32
–
34
] have large compound eyes, advantageous in the search for food and
partners, while species living in low light conditions show a reduction in the number of
ommatidia, as observed in cave-adapted species belonging to Carabidae [
35
], Leiodidae [
36
]
and Curculionidae [37].
Light and electron microscopy techniques have been largely applied to define the struc-
ture and function of insects’ eyes [
24
,
38
–
40
], as well as the selective pressures that impact
acuity from ecological and evolutionary perspectives [
41
]. X-ray tomographic images of
insects’ eyes have been reported in Ephemeroptera [
42
] or as secondary information in anal-
yses focusing on the head structure [
1
,
43
], brain anatomy [
5
,
44
–
46
] or general anatomy of
miniature insects [
7
]. However, there is a lack of studies applying this technique to analyse
the morphological variations of compound eyes. Thus, the aim of this study was to indicate
a new application of SR-PhC micro-CT for investigating compound eyes in insects. Virtual
sections and 3D renderings of the head were performed in four coleopteran species, inhab-
iting different habitats and with different ecological roles, i.e., (a)
Clinidium canaliculatum
(Costa, 1839) (Rhysodidae), a saproxylic beetle, which feeds on wood-decomposing fungi
in coniferous forests—listed as a vulnerable species in the red list of the International
Union for Conservation of Nature (IUCN) [
47
,
48
]; (b)
Tenebrio molitor
(Linnaeus, 1758) and
Tribolium castaneum (Herbst, 1797) (Tenebrionidae), pests of stored grain and cosmopolitan
in distribution [
49
]; and (c) Pterostichus melas italicus (Dejean, 1828) (Carabidae), a generalist
predator, inhabiting pastures, open forests, forest edges and agricultural land [
50
], well
known as a bioindicator of exposure to agrochemicals [
51
–
53
]. The study was designed
to provide a proof that high-resolution images of compound eyes can be obtained using
SR-PhC micro-CT as an exploratory alternative to invasive and time-consuming techniques.
To the best of authors’ knowledge, this is the first comparative study on insect compound
eyes using this technique and addresses the lack of information in the literature on the eyes
of the investigated species.
2. Materials and Methods
2.1. Insects
Clinidium canaliculatum specimens were hand-collected under rotten pine bark in the
Sila National Park (39
◦
21
0
16.79
00
N, 16
◦
37
0
57.64
00
E, Monte Spina 1550 m a.s.l., San Giovanni
in Fiore, Calabria, Southern Italy) in May 2021. Adults of P. m. italicus were collected
from their natural habitat in an olive grove (39
◦
59
0
07.56
00
N, 16
◦
15
0
32.64
00
E, 1202 m a.s.l.,
San Marco Argentano, Calabria, Southern Italy) using pitfall traps (plastic jars 200 mL in
volume containing fruit as an attractant), in October 2019. In the laboratory, beetles of both
species were identified by using dichotomous keys [54] and separated by gender.

Life 2022,12, 741 3 of 15
Tenebrio molitor specimens were obtained from a laboratory stock population main-
tained at the Morphofunctional Entomology Laboratory, Dept. of Biology, Ecology and
Earth Science, University of Calabria. Mealworm beetles were reared at 60% relative hu-
midity, under a natural photoperiod and room temperature (23
±
2
◦
C), with an ad libitum
diet of organic wheat meal and fruit.
Specimens of T. castaneum, belonging to the strain Croatia 1 (CRO1), were collected
and isolated from a wild population in Croatia [
55
], and reared under laboratory conditions
over generations. Adult beetles, kept in plastic boxes, were fed with heat-sterilised (75 ◦C
for at least 24 h) organic wheat flour with 5% brewer’s yeast powder, and reared at 30
◦
C,
70% humidity and with a 12:12 h light:dark cycle.
2.2. Sample Preparation
Males and females from each species were anaesthetised in a cold chamber at 4
◦
C
for three minutes and prepared as indicated in [
6
]. Briefly, beetles were fixed in 2.5%
glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer, at pH 7.4 (PBS;
Electron Microscopy Sciences), overnight at 4
◦
C, washed with PBS and dehydrated in a
graded ethanol series. The following number of individuals was used for each species: one
male and one female for C. canaliculatum and P. m. italicus, and 2 males and 2 females for
T. molitor and T. castaneum.
2.3. Phase-Contrast Micro-Computed Tomography (PhC micro-CT) and Data Acquisition
To digitally reconstruct the three-dimensional internal anatomy of beetles, we used a
Synchrotron radiation X-ray phase-contrast micro-computed tomographic (SR-PhC micro-
CT) imaging technique. Tomographic acquisitions were performed at the SYRMEP beam-
line of the Elettra synchrotron facility in Trieste (Italy), in the “white-beam” configuration
mode, i.e., illuminating the sample with polychromatic X-ray radiation [
56
,
57
]. A storage
ring-bending magnet produces the X-ray beam, available at the beamline in the energy
range from 8.5 to 40 keV. To compensate for beam hardening effects, we filtered the X-ray
beam for low energy components using 1.0 mm of Silicon, thus resulting in an average
energy of around 20 keV. Considering the natural divergence of the X-rays produced by
the source, the beam cross-section at the sample position (22.5 m away from the source) is
150 mm (horizontal)
×
5 mm (vertical). The imaging system consisted of a water cooled
Hamamatsu sCMOS detector (with sensors providing 2048
×
2048 pixels each, with a
size of 6.5
µ
m
×
6.5
µ
m), coupled optically with a GGG (Gd3Ga5O12:Eu) scintillator, and
utilising a set of optical lenses that enabled the setting of different magnification levels.
We employed the GGG scintillator with a 17
µ
m thick sensitive layer to acquire im-
ages of C. canaliculatum and T. castaneum, while for P. m. italicus and T. molitor, we used a
GGG with a sensitive layer with a thickness of 45
µ
m. Tomographic images were recon-
structed from 1800 evenly spaced projections, spanning over 180 degrees, and collected
in continuous rotation mode. Projection images were obtained in the propagation-based
phase-contrast regime [
18
,
58
], setting a propagation distance between the sample and the
detector. The propagation distance was set to optimise the signal-to-noise ratio in the near-
field regime, once the pixel size had been set [
59
]. Phase-contrast effects emerging from the
free-space propagation result in an enhanced contrast arising at the boundaries between
details with different compositions (the so-called edge-enhancement). For P. melas italicus,
the optical magnification was set to 2.4, resulting in a pixel size of 2.7
µ
m
×
2.7
µ
m and a
lateral field of view of 5.5 mm
×
5.5 mm. The exposure time was set to 250 ms/projection
and the sample-to-detector distance was 150 mm. Four vertical scans were needed to image
the full length of the sample. For T. molitor, the optical magnification was set to 1.6, resulting
in a pixel size of 4.0
µ
m
×
4.0
µ
m and a lateral field of view of nearly 8.1 mm
×
8.1 mm.
The exposure time was set to 150 ms/projection and the sample-to-detector distance was
250 mm. Four vertical scans were needed to image the full length of the sample.
For C. canaliculatum and T. castaneum, the optical magnification was set to 4.3, resulting
in a pixel size of 1.5
µ
m
×
1.5
µ
m and a lateral field of view of nearly 3.1 mm
×
3.1 mm.

Life 2022,12, 741 4 of 15
The exposure time was set to 200 ms/projection and the sample-to-detector distance was
100 mm. Four and two vertical scans were needed to image the full length of C. canaliculatum
and T. castaneum, respectively.
2.4. Computer-Based 3D Reconstruction and Segmentation
Image reconstruction was performed with a GPU-based filtered back-projection algo-
rithm (applying a Shepp–Logan filter), using the SYRMEP Tomo Project (STP) software
suite [
60
]. Before image reconstruction, projections were further processed using a phase-
retrieval filter, based on the homogeneous transport of intensity equation (TIE-Hom) [
61
],
obtaining a higher signal-to-noise ratio at the cost of a loss of edge-enhancement signal [
62
].
The filter parameter,
δ
/
β
, was tuned to effectively regulate the amount of smoothing, as
usually used in experimental practice. For the four specimens, we set
δ
/
β
= 400. After
processing, the final CT reconstruction yields a 3D map which is substantially proportional
to the linear attenuation coefficient of the sample [
63
,
64
]. Volume renderings of different
sections of the beetles were performed using the scientific visualisation software Drishti [
65
]
and Avizo®3D.
2.5. Image Analyses and Measurements
Morphometric measurements on 2D virtual slices were assessed with the open-source
software ImageJ [
66
] on digitised images and processed as mean
±
standard deviation.
For each species, the following measurements were taken: the area of the corneal facet
lens and cornea, interocular distance, facet density and corneal lens thickness (Figure 1).
To define the differences in the total surface between the cornea and ommatidia facet
lenses, the measures were also performed on the segmentation of the lens by using the
“Generate Surface” and “Surface Area/Volume” modules of the Avizo software. The area
of the corneal facet lens surface was measured as
π
d
2
/4 (d = diameter of facet). The
interocular distance, calculated on the volume rendering of the head, was measured as the
frontal distance between the inner edges of both eyes, at the level of the central row of the
ommatidium. The facet density (mm
−2
) was calculated as the ratio between the number of
ommatidia (n) and the surface area of the cornea. The ommatidial axis was taken as a line
through the midpoint of the rhabdom and the corneal lens, and the interommatidial angle
was measured from line drawings by two continuous ommatidia on 2D virtual sections.
Life 2022, 12, x FOR PEER REVIEW 4 of 15
of view of nearly 8.1 mm × 8.1 mm. The exposure time was set to 150 ms/projection and
the sample-to-detector distance was 250 mm. Four vertical scans were needed to image
the full length of the sample.
For C. canaliculatum and T. castaneum, the optical magnification was set to 4.3, result-
ing in a pixel size of 1.5 µm × 1.5 µm and a lateral field of view of nearly 3.1 mm × 3.1 mm.
The exposure time was set to 200 ms/projection and the sample-to-detector distance was
100 mm. Four and two vertical scans were needed to image the full length of C. canalicu-
latum and T. castaneum, respectively.
2.4. Computer-Based 3D Reconstruction and Segmentation
Image reconstruction was performed with a GPU-based filtered back-projection al-
gorithm (applying a Shepp–Logan filter), using the SYRMEP Tomo Project (STP) software
suite [60]. Before image reconstruction, projections were further processed using a phase-
retrieval filter, based on the homogeneous transport of intensity equation (TIE-Hom) [61],
obtaining a higher signal-to-noise ratio at the cost of a loss of edge-enhancement signal
[62]. The filter parameter, δ/β, was tuned to effectively regulate the amount of smoothing,
as usually used in experimental practice. For the four specimens, we set δ/β = 400. After
processing, the final CT reconstruction yields a 3D map which is substantially propor-
tional to the linear attenuation coefficient of the sample [63,64]. Volume renderings of dif-
ferent sections of the beetles were performed using the scientific visualisation software
Drishti [65] and Avizo® 3D.
2.5. Image Analyses and Measurements
Morphometric measurements on 2D virtual slices were assessed with the open-
source software ImageJ [66] on digitised images and processed as mean ± standard devi-
ation. For each species, the following measurements were taken: the area of the corneal
facet lens and cornea, interocular distance, facet density and corneal lens thickness (Figure
1). To define the differences in the total surface between the cornea and ommatidia facet
lenses, the measures were also performed on the segmentation of the lens by using the
“Generate Surface” and “Surface Area/Volume” modules of the Avizo software. The area
of the corneal facet lens surface was measured as πd2/4 (d = diameter of facet). The inter-
ocular distance, calculated on the volume rendering of the head, was measured as the
frontal distance between the inner edges of both eyes, at the level of the central row of the
ommatidium. The facet density (mm−2) was calculated as the ratio between the number of
ommatidia (n) and the surface area of the cornea. The ommatidial axis was taken as a line
through the midpoint of the rhabdom and the corneal lens, and the interommatidial angle
was measured from line drawings by two continuous ommatidia on 2D virtual sections.
Figure 1.
Drawing shows morphometric measurements on compound eye:
1
corneal surface,
2
corneal
facet lens thickness 3corneal facet surface area and 4interommatidial angle.
3. Results
The complete series of virtual sections and 3D reconstructions of the heads for each
analysed species allowed us to describe the external morphology of the eyes and their
internal dioptrical and sensorial parts (Figures 2–6).

Life 2022,12, 741 5 of 15
Life 2022, 12, x FOR PEER REVIEW 5 of 15
Figure 1. Drawing shows morphometric measurements on compound eye: 1 corneal surface, 2 cor-
neal facet lens thickness 3 corneal facet surface area and 4 interommatidial angle.
3. Results
The complete series of virtual sections and 3D reconstructions of the heads for each
analysed species allowed us to describe the external morphology of the eyes and their
internal dioptrical and sensorial parts (Figures 2–6).
The compound eye of P. melas (Figures 2A–E and 6A) has a hemispherical curved
area of 6.9 × 105 µm2 and a 2000 n/mm2 density of facets (Table 1; Figure 2A,B).
Figure 2. Phase-contrast micro-CT analysis of Pterostichus melas italicus head. Volume renderings of
lateral view (A), segmented corneal (B), frontal (dorsal view) (C) and cross (D) sections. Virtual 2D
cross section (E) showing the compound eyes connecting to the cerebrum (cr) through the optical
lobe (ol). a: axones; bl: basal lamina; c: cornea; cu: cuticle; cx: central complex; cc: crystalline cones;
cz: clear zone; cocr: circumocular ridge; e: compound eye; f: facet; la: lamina; lb: labium; lbp: labial
palp; lo: lobula; m: mandible; me: medulla; ms: muscles; mx: maxilla; mxp: maxillary palp; oe: oe-
sophagus; p: pedicellum; r: rhabdoms; sc: scape; t: tentorial bridge. Bar: 500 µm.
The adjacent ommatidia are covered by a regular biconvex corneal facet lens, having
a thickness of 64.9 ± 6.07 µm (N = 13), while the interocular distance was estimated at
approximately 2.73 mm (Table 1). We estimated approximatively 1380 ommatidia. Virtual
Figure 2.
Phase-contrast micro-CT analysis of Pterostichus melas italicus head. Volume renderings of
lateral view (
A
), segmented corneal (
B
), frontal (dorsal view) (
C
) and cross (
D
) sections. Virtual 2D
cross section (
E
) showing the compound eyes connecting to the cerebrum (cr) through the optical
lobe (ol). a: axones; bl: basal lamina; c: cornea; cu: cuticle; cx: central complex; cc: crystalline
cones; cz: clear zone; cocr: circumocular ridge; e: compound eye; f: facet; la: lamina; lb: labium; lbp:
labial palp; lo: lobula; m: mandible; me: medulla; ms: muscles; mx: maxilla; mxp: maxillary palp;
oe: oesophagus; p: pedicellum; r: rhabdoms; sc: scape; t: tentorial bridge. Bar: 500 µm.
The compound eye of P. melas (Figures 2A–E and 6A) has a hemispherical curved area
of 6.9 ×105µm2and a 2000 n/mm2density of facets (Table 1; Figure 2A,B).
The adjacent ommatidia are covered by a regular biconvex corneal facet lens, having
a thickness of 64.9
±
6.07
µ
m (N = 13), while the interocular distance was estimated
at approximately 2.73 mm (Table 1). We estimated approximatively 1380 ommatidia.
Virtual sections (Figures 2E and 6A) and 3D reconstructions (Figure 2C–D) highlight the
clear zone, characterised by a high level of X-ray attenuation (bright pixels), between the
upper crystalline cone layer and the underlying layer (rhabdom), both of which have
lower attenuation. The dioptric apparatus is covered by the basal lamina. The axons
are connected to the optic lobe, clearly distinguishable from the distal part in the lamina,
medulla and lobula, connected to the cerebrum (Figure 2C–E). The interommatidial angle
was 4.09 ±0.66◦(N = 13).

Life 2022,12, 741 6 of 15
Table 1. Morphological parameters of studied species measured on 2D slices and volume renderings of beetles’ heads.
Species N. of Ommatidia Corneal Facet
Surface Area (µm2)
Total Surface of
Facets a(µm2)
Cornea Surface b
(µm2)Facet Density cCorneal Facet Lens
Thickness (µm)
Interocular
Distance (mm) Head Size d(mm)
Tribolium castaneum 92 (1.02 ±0.14) ×103(9.40 ±1.31) ×1049.0 ×1041022 22.8 ±0.97 0.43 0.7–0.7
Tenebrio molitor 440 (1.97 ±0.26) ×103(8.67 ±1.13) ×1058.2 ×105536.6 38.7 ±1.03 1.85 2.68–2.8
Pterostichus melas italicus 1380 (0.50 ±0.07) ×103(6.96 ±0.99) ×1056.9 ×1052000 64.9 ±1.68 2.73 3.39–3.45
Clinidium canaliculatum 70 (0.29 ±0.06) ×103(2.09 ±0.56) ×1044.5 ×1041555 50.94 ±0.63 0.79 0.97–1.0
The values are expressed as mean
±
standard deviation and the measured structures are named as indicated in Figure 1.
a
corneal facet surface area x number of ommatidia;
b
data from
Avizo software; cnumber of ommatidia estimated/surface (mm2) of cornea; dlength–width: measured from clypeus apex to neck base and between apices of eyes, respectively.

Life 2022,12, 741 7 of 15
Life 2022, 12, x FOR PEER REVIEW 7 of 15
Figure 3. Phase-contrast micro-CT analysis of C. canaliculatum head. Volume renderings of the head.
The lateral view (A) shows the flattened area of the cornea (brown) covering the ommatidia, which
are visible through the virtual cuticle removal (B). Frontal (dorsal view) (C) and cross (D) sections
highlight compound eyes’ internal structures. Virtual 2D slice of the cross-section (E) showing the
compound eyes connecting to the cerebrum (cr) through the optical lobe (ol). a: axones; bl: basal
lamina; c: cornea; cu: cuticle; cc: crystalline cones; cocr: circumocular ridge; cz: clear zone; e: com-
pound eye; m: mandible; ms: muscles; mx: maxilla; o: ommatidium; oe: oesophagus; p: pedicellum;
r: rhabdoms; sc: scape; t: tentorium. Bar: 250 µm.
Comparing the 2D virtual sections of the compound eyes (Figure 6), two different
structures can be distinguished. Indeed, both P. melas and C. canaliculatum (Figure 6A,B)
show higher attenuation in the layer corresponding to the clear zone, interposed between
the crystalline cones and the rhabdom layer. In contrast, in T. molitor and T. castaneum
(Figure 6C,D), as no clear zone is present, the difference in attenuation between the differ-
ent layers is not evident. Moreover, the eyes in tenebrionid beetles are characterised by
the lowest facet density and the highest facet surface area (Table 1).
Figure 3.
Phase-contrast micro-CT analysis of C. canaliculatum head. Volume renderings of the head.
The lateral view (
A
) shows the flattened area of the cornea (brown) covering the ommatidia, which
are visible through the virtual cuticle removal (
B
). Frontal (dorsal view) (
C
) and cross (
D
) sections
highlight compound eyes’ internal structures. Virtual 2D slice of the cross-section (
E
) showing the
compound eyes connecting to the cerebrum (cr) through the optical lobe (ol). a: axones; bl: basal
lamina; c: cornea; cu: cuticle; cc: crystalline cones; cocr: circumocular ridge; cz: clear zone; e: com-
pound eye; m: mandible; ms: muscles; mx: maxilla; o: ommatidium; oe: oesophagus; p: pedicellum;
r: rhabdoms; sc: scape; t: tentorium. Bar: 250 µm.
The volume renderings and virtual sections of the C. canaliculatum head (
Figure 3A–E
)
highlight an ocular elliptic flattened area of 4.5
×
10
4µ
m
2
and a measured thickness of
50.94 ±2.73 µm
(N = 19) (Table 1). The cornea is smooth and the external facets of the
corneal lens marking the position of the ommatidia are indistinguishable in both males and
females. However, the virtual cuticle removal from the head shows a cluster of 70 omma-
tidia (Figure 3B), corresponding to the area of (2.09
±
0.56)
×
10
4µ
m
2
(
N = 15
), which is
smaller than the surface area of the cornea, revealing a facet density of 1555 n/mm
2
. More-
over, crystalline cones and rhabdoms show low attenuation if compared to the intermediate
clear zone (retina), which is clearly defined by the difference in attenuation (Figure 6B).
Rhabdoms are lined by the basal lamina and axons are visible in the virtual renderings and
2D sections of the eyes (Figure 3C–E), connecting with the cerebrum. The interommatidial
angle was 7.36 ±1.25◦(N = 8).

Life 2022,12, 741 8 of 15
Life 2022, 12, x FOR PEER REVIEW 8 of 15
Figure 4. Phase-contrast micro-CT analysis of T. molitor head. Volume renderings of head showing
lateral view (A), segmented cornea (B), frontal (dorsal view) (C) and cross (D) sections. (E) Virtual
2D slice of the cross-section showing the compound eyes connecting to the cerebrum (cr) through
the optical lobe (ol). a: axones; bl: basal lamina; c: cornea; cu: cuticle; cx: central complex of cerebrum;
cc: crystalline cones; cocr: circumocular ridge; d: deuterocerebrum; e: compound eye; f: facet; g:
gena; la: lamina; lb: labium; lo: lobula; m: mandible; me: medulla; ms: muscles; mx: maxilla; mxp:
maxillary palp; oe: oesophagus; p: pedicellum; r: rhabdoms; sc: scape; t: tentorium. Bar: 500 µm.
Figure 4.
Phase-contrast micro-CT analysis of T. molitor head. Volume renderings of head showing
lateral view (
A
), segmented cornea (
B
), frontal (dorsal view) (
C
) and cross (
D
) sections. (
E
) Virtual
2D slice of the cross-section showing the compound eyes connecting to the cerebrum (cr) through the
optical lobe (ol). a: axones; bl: basal lamina; c: cornea; cu: cuticle; cx: central complex of cerebrum;
cc: crystalline cones; cocr: circumocular ridge; d: deuterocerebrum; e: compound eye; f: facet; g: gena;
la: lamina; lb: labium; lo: lobula; m: mandible; me: medulla; ms: muscles; mx: maxilla; mxp: maxillary
palp; oe: oesophagus; p: pedicellum; r: rhabdoms; sc: scape; t: tentorium. Bar: 500 µm.
The compound eyes of both tenebrionid species are dorsoventral extended and cover a
large part of the lateral head. They exhibit a characteristic bilobed shape, due to a protrusion
of the strongly expanded gena in the anterior eye field (Figures 4A and 5A). In
T. molitor
, the
eyes consist of 440 regular facets, 50
±
3.2
µ
m (N = 7) in diameter in both males and females;
the cornea covers a surface area of 8.2
×
10
5µ
m
2
, for a density of facets of approximatively
537 n/mm
2
. The volume renderings and virtual sections (Figures 4C–E and 6C) showed
a corneal lens with a thickness of 38.7
±
4.83
µ
m (N = 22) and an interocular distance of
approximately 1.85 mm (Table 1). The area of crystalline cones is brighter (i.e., shows a
higher attenuation) (Figure 6C), in contrast to the underneath layers (rhabdom) lined by the
basal lamina, where a clear zone is not present. The axons converged towards the optic lobe
(Figure 4D,E), which is divided in the lamina, medulla and lobula. The interommatidial

Life 2022,12, 741 9 of 15
angle was 6.89
±
1.02
◦
(N = 14). The facets in the eye of T. castaneum (Figure 5A) are 92 in
both males and females. The volume renderings and virtual sections (Figure 5C–E) show a
corneal lens with a thickness of 22.8
±
3.07
µ
m (N = 10). The compound eye surface area
reaches 9.0
×
10
4µ
m
2
, with a facet density of 1022 n/mm
2
, and the interocular distance
reaches about 0.43 mm (Table 1). The area of the crystalline cones shows a higher level of
attenuation than the rhabdom layer below, flats on the basal lamina. The axons converged
towards the optic lobe connected to the cerebrum (Figure 5D,E). The lamina, medulla and
lobula are also distinguishable. The interommatidial angle was 12.99 ±1.2◦(N = 9).
Life 2022, 12, x FOR PEER REVIEW 9 of 15
Figure 5. Phase-contrast micro-CT analysis of T. castaneum head. Volume renderings of head show-
ing lateral view (A), segmented cornea (B), frontal (dorsal view) (C) and cross (D) sections. Virtual
2D cross-section (E), showing the compound eyes connecting to the cerebrum (cr) through the opti-
cal lobe (ol). a: axones; an: antenna; bl: basal lamina; c: cornea; cu: cuticle; cb: central body; ca: calyx;
cc: crystalline cones; cocr: circumocular ridge; e: compound eye; f: facet; g: gena; la: lamina; lb: la-
brum; lp: labial palp; lo: lobula; m: mandible; me: medulla; ms: muscles; mx: maxilla; mxp: maxillary
palp; oe: oesophagus; r: rhabdoms. Bar: 250 µm.
Figure 5.
Phase-contrast micro-CT analysis of T. castaneum head. Volume renderings of head showing
lateral view (
A
), segmented cornea (
B
), frontal (dorsal view) (
C
) and cross (
D
) sections. Virtual 2D
cross-section (
E
), showing the compound eyes connecting to the cerebrum (cr) through the optical
lobe (ol). a: axones; an: antenna; bl: basal lamina; c: cornea; cu: cuticle; cb: central body; ca: calyx; cc:
crystalline cones; cocr: circumocular ridge; e: compound eye; f: facet; g: gena; la: lamina; lb: labrum;
lp: labial palp; lo: lobula; m: mandible; me: medulla; ms: muscles; mx: maxilla; mxp: maxillary palp;
oe: oesophagus; r: rhabdoms. Bar: 250 µm.

Life 2022,12, 741 10 of 15
Life 2022, 12, x FOR PEER REVIEW 10 of 15
Figure 6. Phase-contrast micro-CT analysis, virtual 2D cross-sections of compound eyes in P. melas
(A), C. canaliculatum (B), T. molitor (C) and T. castaneum (D). a: axones; bl: basal lamina; c: cornea; cc:
crystalline cones; cocr: circumocular ridge; cz: clear zone; r: rhabdoms. Bar: 150 µm (A,C), 50 µm
(B,D).
4. Discussion
The high resolution of the beetle virtual dissections obtained under SR-PhC micro-
CT analyses was useful to observe the head in transversal, sagittal and frontal planes, and
the 3D reconstructions have the advantage of facilitating the rotation of the sample on all
axes. Moreover, the contrast between the different tissues allowed us to distinguish the
complex internal structures inside the head capsule, moving within the 2D image stacks,
or by cutting into the 3D models as rendered by the Drishti and Avizo software. Scanning
(SEM) and transmission (TEM) electron microscopy analyses and histology have been
largely used to study the external morphology and ultrastructure of insects [24,67,68],
mainly to describe the sensorial equipment involved in detecting biotic and abiotic stimuli
from environments [69–74], including the compound eyes [39,75,76]. However, these
methods are limited for scanning the external surface, or require a high number of sam-
ples for ultrastructure and histological analyses [68]. Volume renderings of the compound
eyes for each species analysed in our study provided adequate morphological information
on the internal dioptric apparatus and sensorial parts with a low number of specimens.
This is very useful for the study of vulnerable species such as C. canaliculatum. Moreover,
the differences in attenuation obtained from the virtual sections allowed us to identify two
basic types of compound eyes, according to whether or not the receptor layer and the
dioptric apparatus appear separated, that characterise the superposition eyes of P. melas
and the apposition structure of both T. molitor and T. castaneum. Although the analysed
species differed in size, no differences were found in the quality of the resulting datasets
in terms of detail visibility, confirming SR-PhC micro-CT as a useful tool to study the in-
ternal anatomy of miniature insects [5] such as C. canaliculatum and T. castaneum, as well
as the nervous system and the optical lobe [77].
Our results also indicated that the suitable quality of the morphological data pro-
cessed by SR-PhC micro-CT means the technique has high potential for application in eco-
logical studies. The analysed models were four coleopteran species, which live in low light
conditions, but with species-specific habitat demands. Variations in the structural charac-
teristics of the compound eyes recorded in the studied species, such as the facet diameters,
interommatidial angle and the number of ommatidia, were good indicators of the differ-
ences in behaviour, lifestyle and habitat preference. The superposition eyes, that lack pig-
ment separating the cornea from rhabdomeres, are more sensitive to light because they
permit all photoreceptors to use the corneal dioptric apparatus [24,76]. We found this
Figure 6.
Phase-contrast micro-CT analysis, virtual 2D cross-sections of compound eyes in P. melas (
A
),
C. canaliculatum (
B
), T. molitor (
C
) and T. castaneum (
D
). a: axones; bl: basal lamina; c: cornea;
cc: crystalline cones; cocr: circumocular ridge; cz: clear zone; r: rhabdoms. Bar: 150
µ
m (
A
,
C
),
50 µm (B,D).
Comparing the 2D virtual sections of the compound eyes (Figure 6), two different
structures can be distinguished. Indeed, both P. melas and C. canaliculatum (Figure 6A,B)
show higher attenuation in the layer corresponding to the clear zone, interposed between
the crystalline cones and the rhabdom layer. In contrast, in T. molitor and T. castaneum
(Figure 6C,D), as no clear zone is present, the difference in attenuation between the different
layers is not evident. Moreover, the eyes in tenebrionid beetles are characterised by the
lowest facet density and the highest facet surface area (Table 1).
4. Discussion
The high resolution of the beetle virtual dissections obtained under SR-PhC micro-CT
analyses was useful to observe the head in transversal, sagittal and frontal planes, and the
3D reconstructions have the advantage of facilitating the rotation of the sample on all axes.
Moreover, the contrast between the different tissues allowed us to distinguish the complex
internal structures inside the head capsule, moving within the 2D image stacks, or by
cutting into the 3D models as rendered by the Drishti and Avizo software. Scanning (SEM)
and transmission (TEM) electron microscopy analyses and histology have been largely
used to study the external morphology and ultrastructure of insects [
24
,
67
,
68
], mainly
to describe the sensorial equipment involved in detecting biotic and abiotic stimuli from
environments [
69
–
74
], including the compound eyes [
39
,
75
,
76
]. However, these methods
are limited for scanning the external surface, or require a high number of samples for
ultrastructure and histological analyses [
68
]. Volume renderings of the compound eyes
for each species analysed in our study provided adequate morphological information on
the internal dioptric apparatus and sensorial parts with a low number of specimens. This
is very useful for the study of vulnerable species such as C. canaliculatum. Moreover, the
differences in attenuation obtained from the virtual sections allowed us to identify two
basic types of compound eyes, according to whether or not the receptor layer and the
dioptric apparatus appear separated, that characterise the superposition eyes of P. melas
and the apposition structure of both T. molitor and T. castaneum. Although the analysed
species differed in size, no differences were found in the quality of the resulting datasets in
terms of detail visibility, confirming SR-PhC micro-CT as a useful tool to study the internal
anatomy of miniature insects [
5
] such as C. canaliculatum and T. castaneum, as well as the
nervous system and the optical lobe [77].
Our results also indicated that the suitable quality of the morphological data pro-
cessed by SR-PhC micro-CT means the technique has high potential for application in

Life 2022,12, 741 11 of 15
ecological studies. The analysed models were four coleopteran species, which live in low
light conditions, but with species-specific habitat demands. Variations in the structural
characteristics of the compound eyes recorded in the studied species, such as the facet
diameters, interommatidial angle and the number of ommatidia, were good indicators of
the differences in behaviour, lifestyle and habitat preference. The superposition eyes, that
lack pigment separating the cornea from rhabdomeres, are more sensitive to light because
they permit all photoreceptors to use the corneal dioptric apparatus [
24
,
76
]. We found this
structure in P. m. italicus, a generalist predator in the food web of agroecosystems [
50
],
which is active over a broader intensity range and adjusts the sensitivity of its eyes to the
different levels of environmental brightness. Facet density in P. m. italicus was found to be
the highest among the described species, depending on the size and spacing of the omma-
tidia, and in accordance with the visual resolution requirements of a predatory lifestyle [
78
].
As tiny lenses are thought to deliver poor acuity because of diffraction, the high number
of narrow-diameter facets increases light sensitivity and visual resolution in the visually
challenging lifestyles of species such as P. m. italicus, which can be considered a visual
hunter, according to previous studies on carabid beetles [27–29].
The external morphology of the eyes of C. canaliculatum is consistent with the 3D image
of the orbital grooves shown in a previous study performed using SEM techniques [
79
].
Although the species has been indicated as anophthalmic [
79
], the SR-PhC micro-CT
analyses revealed that the structure, previously considered to be non-functional because
of the absence of facets, shows the typical sensorial area of a functioning superposition
eye. However, the ommatidia are spread apart, occupying in total a lower surface area
than that covered by the overlying cornea. C. canaliculatum is an obligate saproxylic
species, inhabiting the rotten wood of mountain forests in central and southern Italy and
Greece [
47
,
48
,
80
]. Thus, it probably needs a larger lens to increase the light incidence angle
and achieves sufficient contrast sensitivity by increasing light transmittance crystalline
cones in low light conditions [
25
,
81
]. We speculate that the cornea of the transparent cuticle
increases the sensitivity of the eyes to photons for detecting the surrounding environment,
as an adaptation to life in dim light conditions. Furthermore, C. canaliculatum shows the
thickest lens in proportion to the head size among the described species, which requires
further studies to clarify whether the eye is functional and to what extent.
Tenebrio molitor and T. castaneum live in food storage depots that occasionally offer a low
illumination level. However, previous electrophysiological studies indicated that T. molitor
is enabled to discriminate various wavelengths from visible to ultraviolet radiation [
82
–
84
].
Although there are no physiological or behavioural studies on the spectral sensitivity of
T. castaneum, virtual sections and 3D renderings highlighted the typical structure of the
light-adapted apposition eyes, which enable orientating at low light intensities in both the
tenebrionid species. Moreover, T. molitor is among the described species, the one with the
lowest facet density, and the ommatidium is indeed characterised by a larger surface in
proportion to the head size.
In conclusion, this is the first study focusing on the use of SR-PhC micro-CT to
describe the compound eye morphology in insects, and to our knowledge, this is also
the first evidence of structured compound eyes in C. canaliculatum. Moreover, our results
indicated that this is a useful non-destructive technique for investigating vulnerable, rare
or difficult-to-collect species included on the IUCN red list—such as C. canaliculatum—
affected by intensive forest management leading to deadwood reduction [
48
], and allows
for additional analyses to be provided using low numbers of specimens. Some size-
dependent limitations of structures were found for the reconstruction of smaller sensorial
cells, such as the rhabdom reaching the cone, pigment and retinula cells. This method
allows measurements of morphological parameters such as interocular distance, the density
of facets, the thickness of the cornea and the number of ommatidia, which is useful in
future interspecific comparative studies for understanding how different lifestyles and
eye and brain morphology have co-evolved, under the selective pressure of biotic (food,
predators) and abiotic (light) factors. Furthermore, conventional techniques adopted for the

Life 2022,12, 741 12 of 15
investigation of the eye, such as retinal dissection and histology, show several limitations in
small specimens, such as T. castaneum, and are not applicable. In contrast, SR-PhC micro-CT
allows morphological analysis by providing a high degree of detail, even in small species.
However, our findings showed that the resolution and image quality of this technique
make it a useful and reliable tool to describe the dioptric apparatus in situ and the general
organization of the sensorial structure, without any deformation due to the manipulation
requested for microscopic analyses.
Author Contributions:
Conceptualization, A.G., M.L.V. and S.D.; formal analysis, A.G., M.L.V. and
S.D.; funding acquisition, A.G. and R.G.A.; investigation, A.G., M.L.V., L.K.L. and S.D.; methodology,
A.G., M.L.V. and S.D.; supervision, A.G.; writing—original draft, A.G.; writing—review and editing,
A.G., M.L.V., R.G.A., L.K.L. and S.D. All authors have read and agreed to the published version of
the manuscript.
Funding:
This research was funded by “AIM: Attraction and International Mobility”-PON R&I
2014–2020 Regione Calabria; “Progetto STAR 2”—(PIR01_00008)—Italian Ministry of University and
Research; and Progetto Foreste Vetuste Ente Parco Nazionale della Sila.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors are grateful to Elettra Sincrotrone Trieste for providing access to the
SYRMEP beamline and the SYRMEP beamline staff members, especially Giuliana Tromba, for the
help in performing the computed microtomography experiment, and Antonio Mazzei for the field
collection of C. canaliculatum specimens. The authors thank Joachim Kurtz for supplying T. castaneum
specimens from the population reared in his lab.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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