Download The antennal sensory function in the oldest pterygote insects: an

Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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The antennal sensory function in the oldest pterygote insects: an
ultrastructural overview
M. Rebora, S. Piersanti and E. Gaino
Department of Cellular and Environmental Biology, University of Perugia, Via Elce di Sotto 1, 06100 Perugia, Italy
Paleoptera (Odonata and Ephemeroptera) represent the oldest pterygote insects. In consideration that antennae are one of
the main site of not-visual insect perception, ultrastructural investigations under SEM and TEM have been recently
performed on the flagellum of species belonging to several families of Odonata and Ephemeroptera, to clarify the sensory
function of the antennae in Paleoptera. These antennae appear very reduced and are constituted by scape, pedicel and an
aristate flagellum, mainly monoarticulated in Ephemeroptera and composed of 1-4 flagellomeres in Odonata. Several
sensory structures have been identified on the ventro-lateral side of the flagellum in both orders with two main possible
functions: thermo-hygroreception and olfaction. Studies on the sensory biology of these aquatic insects can contribute to
clarify interesting aspects of their biology. In addition, studies on Paleoptera sensilla light into the evolution of insect
sensory abilities.
Keywords: sensory biology; thermo-hygroreceptors; olfactory receptors; aquatic insects; Paleoptera; Odonata;
Ephemeroptera
1. Introduction
Odonata (damselflies and dragonflies) and Ephemeroptera (mayflies) constitute an important component of the
freshwater lentic and lotic ecosystems. Both these insect orders develop their larvae in water while the winged adults fly
over streams, lakes and small ponds. Dragonflies and damselflies are predators, both as larvae and adults, and many of
them are protected in Europe as threatened species, mainly for environmental degradation. Mayflies larvae are algae
grazers or detritivorous while the adults do not feed and are short-lived. Many species are important bio-indicators used
in European and American monitoring protocols for freshwater ecosystems.
Traditionally, Ephemeroptera and Odonata have been classified as Paleoptera (old wings), based on their inability to
fold the wings over the abdomen. The remainders of the pterygote insects, able to fold their wings over the abdomen
with the inner surfaces facing the latter, constitute the large clade Neoptera (new wings). Together with their inability to
fold the wings over the abdomen, Odonata and Ephemerotera share other common features such as big eyes and short,
reduced antennae. For this reason these insects have always been considered mainly visual dependent and many studies
have been published on their visual abilities while other sensory modalities have been disregarded.
In consideration that antennae are one of the main sites of insect perception, we decided to perform ultrastructural
investigations under SEM and TEM on the antennal flagellum of some Ephemeroptera and Odonata species [1-4] to
clarify the sensory function of these appendixes.
This paper reviews such data and reports some additional investigations on other Odonata and Ephemeroptera
species belonging to the Italian fauna. This in order to lay the basis for further investigations on the not-visual sensory
abilities of these old insects.
2. Material and Methods
Mayfly adults of Rhithrogena semicolorata (Curtis, 1834) - Electrogena lateralis (Curtis, 1834) – Ecdyonurus venosus
(Fabricius, 1775) (Heptageniidae), Habrophlebia eldae Jacob & Sartori, 1984 (Leptophlebiidae), Baetis rhodani (Pictet
1843) - Cloeon dipterum (Linnaeus, 1761) (Baetidae), Caenis luctuosa (Burmeister) (Caenidae), Siphlonurus lacustris
(Eaton) (Siphlonuridae), Serratella ignita (Poda, 1771) (Ephemerellidae), Ephemera danica Müller 1764
(Ephemeridae), dragonfly adults of Libellula depressa Linnaeus - Sympetrum striolatum (Charpenter, 1840)
(Libellulidae), Onychogomphus forcipatus (Linnaeus, 1761) (Gomphidae), Aeshna cyanea Müller 1764 (Aeshnidae),
Somatochlora metallica Van Der Linden, 1825 (Cordulidae), Cordulegaster boltonii Donovan 1807 (Cordulegastridae)
and damselfly adults of Coenagrion puella Linnaeus 1756 - Ischnura elegans (Van Linden,1820), (Coenagrionidae),
Platycnemis pennipes (Pallas, 1761) (Platycnemidae), Lestes barbarus Fabricius 1798 - Lestes viridis (Vander Linden
1825) (Lestidae), Calopteryx virgo Linnaeus 1758 - Calopteryx haemorrhoidalis (Van der Linden,1825
(Calopterigidae), were obtained in the laboratory from mature larvae. Mayfly and dragonfly larvae were collected in the
Nera River (Perugia, Central Italy) and in a natural pond in Lisciano Niccone (Perugia, Central Italy) during the spring
2007-2008. In the laboratory, the larvae were kept in plastic containers with water, detritus and flora from the collecting
site at 25±2°C, LD12:12 h. Dragonfly larvae were fed ad libitum with plankton.
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Antennal flagella were dissected from anaesthetised specimens and fixed for 12 hours in 2.5% glutaraldehyde in
cacodylate buffer, pH 7.2.
For scanning electron microscopy (SEM) analysis, the fixed material, repeatedly rinsed in the same buffer, was then
dehydrated by using ethanol gradients, followed by critical-point drying in a critical-point dryer CPD 030 Bal-Tec (BalTec Union Ltd., Balzers, Liechtenstein). Specimens were mounted on stubs with silver conducting paint, sputter-coated
with gold-palladium in a sputterer Emitech K550X (Emitech, Ashford, England), and observed with a Philips XL30
(Philips, Eindhoven, Netherlands), at an accelerating voltage of 18kV. For SEM observations of the inner cuticular wall
of Odonata antennae, the flagella were longitudinally sectioned with a razor blade and cleaned with KOH-solution.
These flagella were then dehydrated in a graded ethanol series, dried in an oven and glued onto the SEM specimen
supports in order to allow the observation of the inner side of the antenna. The specimens were then sputter-coated with
gold-palladium and observed with a Philips XL30 (Philips, Eindhoven, Netherlands), at an accelerating voltage of
18kV.
For transmission electron microscopy (TEM), the fixed antennal flagella were repeatedly rinsed in cacodylate buffer
and post-fixed for 1 hour at 4 °C in 1% osmium tetroxide in cacodylate buffer. Afterward the material was repeatedly
washed in the same buffer, dehydrated by using ethanol gradients and finally embedded in an Epon-Araldite mixture
resin. Ultrathin sections, cut on a Leica EM UC6 ultracut (Leica Microsystem GmbH, Wien, Austria), were collected
on formvar-coated copper grids, stained with uranyl acetate and lead citrate, and examined with a Philips EM 208
(Philips, Eindhoven, Netherlands).
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Fig. 1. Odonata (a-c) and Ephemeroptera (d-f) antennae under SEM. (a) Libellula depressa antenna consisting of a scape (S), a
pedicel (P) and a flagellum constituted of 4 segments. Arrow points out the sensilla on the latero-ventral side of the flagellum; (b)
Lestes viridis antenna consisting of a scape (S), a pedicel (P) and a monoarticulated flagellum (F); arrow points out the sensilla on the
latero-ventral side of the flagellum; (c) Detail of the latero-ventral side of the flagellum of Sympetrum striolatum showing sensilla
located in pits (arrow); (d) Habrophlebia eldae antenna consisting of a scape (S), a pedicel (P) and a monoarticulated flagellum (F);
(e) Siphlonurus lacustris antenna showing the pedicel (P) and the flagellum (F) constituted of numerous segments; (f) Detail of the
latero-ventral side of the flagellum of Cloeon dipterum showing sensilla located in pits (arrow) in its proximal portion.
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Fig. 2. Odonata (a-b, d-f) and Ephemeroptera (c, g, h) flagellar sensilla under SEM. (a-c) Coeloconic sensilla of Libellula depressa
(a), Ischnura elegans (b) and Rhithrogena semicolorata (c) showing pores (P) on their cuticular surface; (d) Openings of the deep
cavities in Libellula depressa; (e) Inner cuticular wall vision of the flagellum of Sympetrum striolatum showing the shape of the
convoluted deep cavities hosting sensilla styloconica (possible thermo-hygroreceptors); (f) Type-1 (T1) and type-2 (T2) sensilla
styloconica located inside the deep cavities in a sectioned convoluted cavity (cleaned with KOH-solution) in the antenna of
Onichogomphus forcipatus; (g, h) Possible thermo-hygroreceptors of Siphlonurus lacustris (g) and Habrophlebia eldae (h) showing
a more or less conical shape.
3. Results
The antennal flagellum in Anisoptera shows a smooth surface and is constituted of 2-4 segments (Fig. 1a) while in
Zygoptera it is monoarticulated and shows cuticular scales (Fig. 1b). In both suborders, the flagellum show sensilla
located in pits on its latero-ventral side (Fig. 1c).
Ephemeroptera have a monoarticulated flagellum (Fig. 1d), which is segmented in some families such as
Siphlonuridae (Fig. 1e). The flagellar surface has a texture of cuticular scales (Fig. 1f). As in Odonata, the latero-ventral
side of the flagellum shows sensilla located in pits (Fig. 1f).
In both the insect Orders the sensory structures are more numerous in the proximal portion or in the more proximal
segments of the flagellum.
In Odonata (dragonflies and damselflies) and in Ephemeroptera the sensilla located on the latero-ventral side of the
flagellum are represented by porous sensilla similar to olfactory sensilla (Figs. 2a-c) and by aporous sensilla whose
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structure recalls that of thermo-hygroreceptors (Figs. 2f-h). The porous sensilla in Odonata are pegs located inside pits
(Figs. 2a,b) while in Ephemeroptera they are lobe-shaped pegs, located on the cuticle surface between the scales (Fig.
2c). The possible thermo-hygroreceptors in Odonata are sensilla styloconica (with a sensory peg or cone located on a
protrusion or stylus emerging from the cuticle) located inside deep cavities visible from the outside of the antenna as
simple openings (Fig. 2d). These cavities are more or less developed and can be very deep and convoluted, especially in
Anisoptera (Fig. 2e). Inside the cavities it is possible to distinguish between two kinds of sensilla styloconica, type-1
and type-2 (Fig. 2f). In Ephemeroptera the possible thermo-hygroreceptors are located on the flagellar surface and
show a more or less conical shape (Figs. 2g,h).
Odonata porous sensilla appear innervated by three unbranched neurons, whose outer dendritic segments enter the
peg (Figs. 3a-c). At the base of the peg, the dendrite sheath becomes very thin and tends to disappear, leaving only some
traces in the peg (Fig. 3a). The cuticle of the peg shows wide pore-like structures at the base of which the continuity of
the cuticle is interrupted by actual pores (Fig. 3b). The space between the pores and the dendrites is narrow and is filled
with a material of medium electron-density in which pore tubules are visible (Fig. 3b).
Mayfly porous sensilla are innervated by three neurons whose outer dendritic segments enter the peg (Figs. 3d-f).
These three neurons are enveloped by the dendrite sheath (Fig. 3f) that opens at the entrance into the peg, leaving the
dendrites immersed in the sensillum liquor (Figs. 3d,e). In the peg, the neurons are in contact with the outside by pores
present in the cuticle (Figs. 3d,e). Pore tubules are located among pores and dendrites in the sensillum liquor (Fig. 3e).
No socket is visible at the insertion of the peg (Fig. 3d).
In Odonata, type-1 sensilla styloconica are innervated by four unbranched neurons whose outer dendritic segments
are enveloped by the dendrite sheath (Figs. 4a,c). All four dendrites enter the peg, but only three of them reach the apex
of the sensillum (Figs. 4b,c); the fourth ends beneath it, where the dendrite sheath is interrupted. The accessory cells are
well developed and, around the outer dendritic segments, form long microvilli delimiting numerous electron-lucid
vesicles (some species, such as Onichogomphus forcipatus, show an astonishing number of vesicles) (Fig. 4c). At the
tip of the cone, the cuticle of the peg is interrupted and a cap-like structure made of amorphous material covers the apex
of the dendrites (Figs. 4a and inset,b). From the interrupted cuticle, cuticular fingers emerge to cover the cap-like
structure (Figs. 4a and inset,b). No socket and no pores connecting the dendrites with the outside are visible. Type-2
sensilla styloconica are innervated by three unbranched neurons enveloped by three accessory cells (Figs. 4d-f))
producing an abundant electron-dense secretion which is interspersed among their microvilli (Figs. 4d, f). The three
outer dendritic segments enter the stylus enveloped by the dendrite sheath. One of the three dendrites stops short of the
cone where only two dendrites, closely adherent to each other, are visible (Fig. 4e); these are enveloped by a tightly
adherent dendrite sheath which is in close contact with the surrounding cuticle (Fig. 4e). In cross section, the cuticle
appears solid in its proximal portion, while it shows round clefts in its distal portion (Fig. 4e). No socket and no
connection of the dendrites with the outside are visible.
In mayflies the possible thermo-hygroreceptors are innervated by four neurons (Fig. 4i), two of which are
unbranched and enter the peg (Fig. 4g,h), while the others stop before entering the peg and one of them is branched.
The outer dendritic segments are enveloped by a thick dendrite sheath (Figs. 4g, i). At the base of the peg, the dendrite
sheath fuses with the peg cuticle that penetrates deeply inside the sensillum (Fig. 4g). In the peg the dendrites are in
close contact with the thick cuticle that shows irregular small clefts (Fig. 4h). There is no connection of the dendrites
with the outside in the peg. No socket is evident at the insertion of the peg. In some cases the small pegs are innervated
by only two unbranched neurons; the outer dendritic segments of these neurons enter the peg, whose internal structure is
like that previously described in the small pegs innervated by four neurons.
Both in Odonata and Ephemeroptera antennal flagellum campaniform sensilla are occasionally visible (Figs. 3g,h).
The dendrite stops at the base of the cuticle and forms a tubular body in its apical portion (Figs. 3g,h). Above the
dendrite, the cuticle is in contact with the dendrite sheath of the tubular body by the socket septum.
No difference in the flagellar sensilla number has been so far identified between males and females both in Odonata and
in Ephemeroptera.
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Fig. 3. Odonata (a-c) and Ephemeroptera (d-f) porous coeloconic sensilla under TEM; Odonata (g) and Ephemeroptera (h)
campaniform sensilla under TEM. (a) Longitudinal section showing the dendrites (D) entering the peg; the dendrite sheath becomes
very thin and leaves only some traces at the base of the peg (arrow head). Note the pore-like structures on the peg cuticle (arrows);
(b) Cross section at the level of the peg. Note the dendrites (D) without dendrite sheath and the pore-like structures (arrows) on the
cuticle, together with pore tubules (PT); (c) Cross section at the level of the outer dendritic segments (D) enveloped by the dendrite
sheath (DS). Note the accessory cells (AC); (d) Longitudinal section of a peg showing three dendrites (D) immersed in the sensillum
liquor; the dendrite sheath (DS) opens at the entrance of the peg leaving the dendrites in contact with the outside by pores (arrow) on
the peg cuticle; (e) Cross section of the peg showing dendrites (D), pore tubules (PT) and pores (arrow); (f) Cross section of the outer
dendritic segments (D) enveloped by a thick dendrite sheath (DS). AC, accessory cell; (g,h) Longitudinal sections showing the
tubular body (TB) at the apex of the dendrite (D). The dendrite sheath (DS) of the tubular body is in contact with the cuticle by the
socket septum (SS).
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Fig.4. Odonata (a-f) and Ephemeroptera (g-i) possible thermo-hygroreceptors. (a) Longitudinal section of the peg of a type-1
sensillum showing the dendrites (D) enveloped by the dendrite sheath (DS) entering the peg. AC, accessory cells, CC, ciliary
constriction. In the inset the apical region of the peg shows the dendrite (D) with the cap-like structure (Ca) surrounded by cuticular
fingers (CF); (b) Cross section of a type-1 sensillum at the level of the apical portion of the peg showing the three dendrites (D) with
the cap-like structure (Ca) surrounded by cuticular fingers (CF); (c) Cross section of a type-1 sensillum at the level of the four outer
dendritic segments (OD) with the dendrite sheath (DS) surrounded by numerous electron-lucid vesicles (V); (d) Longitudinal section
of the peg of a type-2 sensillum showing the dendrites (D), wrapped by the dendrite sheath (DS), entering the cone. AC, accessory
cells; (e) Cross section of a type-2 sensillum at the level of the cone showing the two outer dendritic segments (D) entering the peg,
enveloped by the dendrite sheath (DS) in close contact with the surrounding cuticle with round clefts (arrow) in its distal portion; (f)
Cross section of a type-2 sensillum at the level of the outer dendritic segments (OD) wrapped by the dendrite sheath (DS) and the
accessory cells (AC); (g) Longitudinal section of the peg showing the peg cuticle (C) that penetrates deeply into the sensillum and
fuses with the dendrite sheath (DS) enveloping the dendrites (D); (h) Cross section of a peg showing two dendrites (D) in close
contact with the peg cuticle (C); note the irregular clefts of the cuticle (arrow); (i) Cross section at the level of the four outer dendritic
segments (D) enveloped by the dendrite sheath (DS) and the accessory cells (AC).
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4. Discussion
On the ventro-lateral side of the antennal flagellum of Ephemeroptera and Odonata, sensilla have been identified in all
the examined species representatives of the main Italian families [1-4 and the data presented in this paper].
Notwithstanding the differences in shape and location, these sensilla share relevant common features in the two insect
orders: they are more concentrated on the proximal portion of the unarticulated flagella, or on the proximal segments in
the articulated ones; most of them tend to be short, closely adherent to the flagellar surface, located inside pits, and can
be classified as sensilla coeloconica (= pit peg). The presence of coeloconic sensilla in Paleoptera antennae is
particularly interesting owing to the ancient origin of this kind of sensilla in insects [5,6].
The external and internal cuticular morphology of the flagellar sensilla of both Odonata and Ephemeroptera is
compatible with interpreting them as single-walled olfactory sensilla [7,8] and thermo-hygroreceptors [see review in 911]. Occasionally campaniform sensilla are visible on the dorsal antennal surface, and presumably senses the stretching
movements of the flagellum.
The importance of hygroreceptors perceiving changes in humidity is obvious in both Insect Orders being aquatic
insects: they must detect humidity gradient in air to find the adequate laying places. It is well known that Odonata and
Ephemeroptera can “see” the presence of water, as many aquatic insects, on the basis of the horizontally polarized light
reflected from the water surface [12-14]. Indeed, the ventral region of the eye in many aquatic insects is sensitive to the
polarization of light in the visible and/or ultraviolet spectral ranges [15,16]. Polarised light reflected by water can aid
the orientation of these insects from a distance where other cues are still ineffective [17]. Evidently, as reported by
Corbet [18], in Odonata the more general cues are detected visually, and the final, more specific cues by other sensory
modalities. At a closer range, indeed, hygroreception could have an important role in Odonata and Ephemeroptera
habitat selection and oviposition.
Insect hygroreceptors are typically associated with thermoreceptors in peculiar sensilla mainly located on the
antennae [see review in 9-11]. As far as thermoreceptors are concerned, the ability to perceive changes in temperature
is fundamental in Odonata, which rely on direct sunshine for their thermoregulation [19], and in the fragile adult
mayflies, able to perform mating flights only in a peculiar temperature range [14].
The importance of olfactory receptors in Paleoptera is more questionable. Indeed neuroanatomical studies on insect
brain have prompted researchers to hypothesize that the extant paleopteran insects are probably all anosmic with respect
to airborne odours; indeed, they lack glomerular antennal lobes, which typically receive olfactory receptor neurons in
Neoptera [20,21]. A recent neuroanatomical study on the ground plan of the insect mushroom body suggested that the
simplicity of these structures in mayflies may not be entirely attributable to a primitive character state, but rather related
to the highly derived lifestyle of Ephemeroptera, with aquatic larvae and short non-feeding adults, in which olfaction is
redundant [22]. Olfaction could be considered redundant also in Odonata, where vision plays an important role for the
adult behaviour [18], but our results suggest that mayflies, dragonflies and damselflies could perceive odours and could
use these ability for some aspects of their biology.
To understand the function of the antennal sensilla of paleopterans, further molecular, electrophysiological,
neuroanatomical and behavioural studies are in progress. Such studies on paleopteran sensory abilities could add new
important data to trace phylogenetic relationships between the two insect orders and could shed light onto the evolution
of insect olfaction, a research field so far in a fairly initial phase.
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