# Sensory pathway in aquatic basal polyneoptera:
Antennal sensilla and brain morphology in stoneflies

Silvana Piersanti, Manuela Rebora, Gianandrea Salerno, Simon Vitecek, Sylvia Anton

► To cite this version:

Silvana Piersanti, Manuela Rebora, Gianandrea Salerno, Simon Vitecek, Sylvia Anton. Sensory pathway in aquatic basal polyneoptera: Antennal sensilla and brain morphology in stoneflies. Arthropod Structure and Development, 2024, 79, pp.101345. 10.1016/j.asd.2024.101345. hal-04576060

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# Sensory pathway in aquatic basal polyneoptera:
Antennal sensilla and brain morphology in stoneflies

Silvana Piersanti, Manuela Rebora, Gianandrea Salerno, Simon Vitecek, Sylvia Anton

► To cite this version:

Silvana Piersanti, Manuela Rebora, Gianandrea Salerno, Simon Vitecek, Sylvia Anton. Sensory pathway in aquatic basal polyneoptera: Antennal sensilla and brain morphology in stoneflies. Arthropod Structure and Development, 2024, 79, pp.101345. 10.1016/j.asd.2024.101345. hal-04576060

HAL Id: hal-04576060

https://hal.science/hal-04576060v1

Submitted on 24 May 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Sensory pathway in aquatic basal polyneoptera: Antennal sensilla and
brain morphology in stoneflies

<figure><img src="image_3.png" /></figure>

Silvana Piersanti<sup>a, *</sup>, Manuela Rebora<sup>a</sup>, Gianandrea Salerno<sup>b</sup>, Simon Vitecek<sup>c, d</sup>, Sylvia Anton<sup>e</sup>

ARTICLE INFO

Article history:

Received 11 November 2023

Received in revised form

27 February 2024

Accepted 27 February 2024

Available online 16 March 2024

Handling Editor: B. Beltz

Keywords:

Aquatic insects

Plecoptera

Ontogenesis

Ultrastructure

Neuroanatomy

Evolution

A B S T R A C T

Aquatic insects represent a great portion of Arthropod diversity and the major fauna in inland waters. The sensory biology and neuroanatomy of these insects are, however, poorly investigated. This research aims to describe the antennal sensilla of nymphs of the stonefly *Dinocras cephalotes* using scanning electron microscopy and comparing them with the adult sensilla. Besides, central antennal pathways in nymphs and adults are investigated by neuron mass-tracing with tetramethylrhodamine, and their brain structures are visualized with an anti-synapsin antibody. No dramatic changes occur in the antennal sensilla during nymphal development, while antennal sensilla profoundly change from nymphs to adults when switching from an aquatic to an aerial lifestyle. However, similar brain structures are used in nymphs and adults to process diverging sensory information, perceived through different sensilla in water and air. These data provide valuable insights into the evolution of aquatic heterometabolous in-sects, maintaining a functional sensory system throughout development, including a distinct adaptation of the peripheral olfactory systems during the transition from detection of water-soluble chemicals to volatile compounds in the air. From a conservation biology perspective, the present data contribute to a better knowledge of the biology of stoneflies, which are very important bioindicators in rivers.

© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/).

**1. Introduction**

Freshwater ecosystems are an often neglected trove of biodi-
versity. Their fauna includes aquatic insects – many diverse insect
species that spend at least one stage of their life cycle in the aquatic
habitat. Most of them move to the terrestrial realm in their adult
stages to disperse and procreate, thus reducing competition with
the young stages. Aquatic insects may comprise up to 80% of the
freshwater fauna but are decidedly under-investigated in many
aspects of their biology (Dijkstra et al., 2014). Considering that more
than 50 separate freshwater colonizations and evolutionary radia-
tions must have taken place within the insect clade, the

heterogeneous group of aquatic insects is a favourable target for
studying how ecological constraints shape evolutionary trajectories
of physiological adaptations (Crespo, 2011; Dijkstra et al., 2014).

All terrestrial insects with aquatic nymphs had to develop new
physiological mechanisms for osmoregulation or gas exchange in
the aquatic stages (Eisenbeis and Wichard, 2010), and also for
dealing with the constraints of water as a medium for sensory
ecology (Rebora et al., 2010; 2019). Aquatic insects encompass
species belonging to holometabolous orders, such as Trichoptera, as
well as species belonging to hemimetabolous orders, whose
development lacks a complete metamorphosis, such as Odonata,
Plecoptera and Ephemeroptera. In Holometabola, the required
changes from nymphs to adults can be easily achieved through
metamorphosis (Mysore et al., 2011). On the contrary, gradual
ontogenetic changes, e.g. in antennal sensilla and/or central pro-
cessing, must be expected in nymphal stages and between nymphs
and adults among the aquatic species that lack a pupal stage,

<footnote>ª Dipartimento di Chimica, Biologia e Biotecnologie, University of Perugia, Via Elce di Sotto 8, 06123, Perugia, Italy</footnote>

<footnote>ᵇ Dipartimento di Scienze Agrarie, Alimentari e Ambientali, University of Perugia, Borgo XX Giugno 74, 06121, Perugia, Italy</footnote>

<footnote>ᶜ QUIVER, WasserCluster Lunz –Biologische Station, Dr.-Carl-Kupelwieserpromenade5, 3293, Lunz am See, Austria</footnote>

<footnote n="4">⁴ Institute of Hydrobiology and Aquatic Ecosystem Management, University of Natural Resources and Life Sciences, Gregor-Mendel-Straße 33, 1180, Vienna, Austria</footnote>

<footnote n="5">⁵ IGEPP, INRAE, Institut Agro, University of Rennes, 2, rue André Le Nôtre, 49045, Angers Cedex 01, France</footnote>

<footnote>* Corresponding author.

*E-mail addresses:* silvana.piersanti@unipg.it (S. Piersanti), manuela.rebora@unipg.it (M. Rebora), gianandrea.salerno@unipg.it (G. Salerno), simon.vitecek@wcl.ac.at (S. Vitecek), sylvia.anton@inrae.fr (S. Anton).</footnote>
belonging to the hemimetabolous insect orders of Palaeoptera, Polyneoptera and Condylognatha. Therefore, studies on the peripheral sensory structures of the antennae, as well as on the central processing of the incoming information into the brain in aquatic insects can significantly contribute to our understanding of insect adaptation and evolution of sensory biology and brain plasticity (Crespo, 2011; Dijkstra et al., 2014).

Only a few studies have investigated the sensory ecology of Ephemeroptera (mayflies), Odonata (dragonflies and damselflies), Plecoptera (stoneflies) or Trichoptera (caddisflies) and primarily focused on the terrestrial adults (Rebora et al., 2019). Like in other insects, the olfactory system of aquatic insects consists of antennal receptor neurons housed in cuticular sensilla situated on the antennae and projecting to the primary olfactory centre, the antennal lobe. The antennal lobe usually consists of globular sub-units, the glomeruli, which play an important role in odour coding. Conversely, in some species like dragonflies (Piersanti et al., 2020; Rebora et al., 2013) or aphids (Kollmann et al., 2010), supposed to rely less on olfactory cues, the antennal lobe is less well-structured without distinguishable glomeruli, and thus aglomerular. Nevertheless there is clear evidence for the use of olfaction in these insects (Badji et al., 2021; Piersanti et al., 2014; Rebora et al., 2012).

In this context, the aim of the present study is to provide baseline data on a largely neglected group of aquatic insects, the stoneflies (Plecoptera), belonging to the highly diverse clade of Polyneoptera together with the better-known Orthoptera (Misof et al., 2014; Tihelka et al., 2021; Trautwein et al., 2012; Wipfler et al., 2019). Stoneflies are characterised by aquatic nymphs that – depending on the group – occupy different ecological niches and live as shredders, grazers, detritivores or predators, mostly in upper stretches of rivers (Graf et al., 2009). The most impressive stoneflies in Europe are the large predators of the Perlidae and Perlodidae families that spend a long time, often years, in fast-running waters, feeding on aquatic insects and other macroinvertebrates (Graf et al., 2009). In contrast, most stonefly adults do not feed and only in some species do they occasionally feed on small amounts of pollen, fungal spores and detritus (Rúa and Tierno De Figueroa, 2013).

Plecoptera antennal sensilla have been investigated in detail in adult Perlidae (Rebora et al., 2016), where the most represented sensilla are (i) sensilla trichodea, whose internal structure reveals a mechanosensory function; (ii) sensilla chaetica, with an apical pore, with an internal structure revealing a typical gustatory function; (iii) porous pegs, representing single-walled olfactory sensilla; (iv) digitated pegs with hollow cuticular spoke channels representing double-walled olfactory sensilla; (v) pegs in pits for which a thermo-hygrosensory function is hypothesized. Adult stonefly antennae can perceive olfactory cues (Rebora et al., 2017; Neupert et al., 2022), while nymphs' ability to respond to chemicals in water has been demonstrated in behavioural observations (see review in (Rebora et al., 2019), but data on the antennal sensilla are fragmentary and rather dated (Kapoor, 1985, 1987, 1991). In addition, the neuroanatomy of these insects has never been investigated to the best of our knowledge. In the literature, an aglomerular antennal lobe is reported for stoneflies (Schachtner et al., 2005), but this is wrongly deduced from data on Odonata and Ephemeroptera (Strausfeld, 1998).

Significant changes must be associated with the transition from the aquatic habitat of immature stoneflies to the terrestrial adults' habitat. Mechanoreception in water and air can be very different, especially for insects living in fast running water such as stoneflies. Odorants in an aquatic environment are water-soluble molecules, while air-borne odorants are generally hydrophobic, highly volatile molecules (Ache and Derby, 1985). In insects with aquatic larvae, there is thus a dramatic qualitative change in sensory cues compared to other heterometabolous insects, where life style and

behaviourally relevant cues do not change substantially between nymphs and adults, apart from sex pheromone detection. Accordingly, in locusts and cockroaches, numbers of olfactory sensilla and associated receptor neurons increase gradually during development, and in the antennal lobe only minor changes, such as increasing numbers of glomeruli or small changes in spatial arrangements of glomeruli, occur (Anton et al., 2002; Chambille and Pierre Rospars, 1985).

In hemimetabolous aquatic insects, we expect a dramatic change of sensory equipment when passing from nymphal life in streams to terrestrial adult life, in spite of a potentially similar brain structure of mature nymphs and adults owing to the heterometabolous ontogenesis.

To test this hypothesis, in the present study we investigated the stonefly *Dinocras cephalotes* (Curtis, 1827) (Plecoptera: Perlidae), an aquatic basal polyneopteran, to provide: 1) a quantitative description of antennal sensilla in different nymphal stages, 2) a mapping of the central target areas of antennal axons in nymphs and adults with a neuronal tracer, and 3) a first attempt for a description of general brain anatomy in nymphs and adults. For comparison with the adult sensilla, we refer to the description reported by Rebora et al. (2016, 2017).

## 2. Materials and methods

### 2.1. Insects

Adults of *D. cephalotes* (Plecoptera, Perlidae) for neuroanatomical stainings were collected on the banks of the Nera River (Vallo di Nera, Perugia) in June 2023. Only a few insects could be captured due to unusual climate conditions (n = 12), and they were highly variable in size (from 18 to 32 mm). Nymphs of the same species (n = 60) were collected in the same river from autumn to winter 2022. Because the development in Plecoptera can encompass a variable number of stages and moult (Iannilli et al., 2002), the total length of the specimens was measured, and immature insects were grouped into four size classes: small (S), nymphs with a length of 5–7 mm, medium (M), nymphs with a length of 8–11 mm, large (L), nymphs with a length of 12–15 mm, and extra-large (XL), nymphs longer than 15 mm.

In the laboratory, insects were kept in polystyrene refrigerated boxes with moist paper and no food at 17 ± 2 °C until they were used. Only nymphs of at least 5 mm in length were used for the research because of technical difficulties connected with the small dimensions of earlier instars and, more importantly, because smaller nymphs represent a small proportion of the immature stage life (only a few weeks of the three years of the aquatic life (Iannilli et al., 2002)).

### 2.2. Scanning electron microscopy

To describe the sensilla on the flagellum, nymphal antennae were observed under scanning electron microscopy (SEM). Antennae of nymphs belonging to the four classes above reported (S, M, L, XL) were dissected from anaesthetised specimens (5 nymphs for each class) and fixed for 12 h in 2.5% glutaraldehyde in cacodylate buffer (Electron Microscopy Sciences) pH 7.2. Ten fixed antennae for each class were repeatedly rinsed in the same buffer and then dehydrated using ascending ethanol gradients (20%, 50%, 70%, 80%, 95%, 100%), followed by drying in an oven at 40 °C for three days. The morphology of the samples was analysed by field emission scanning electron microscopy FE SEM LEO 1525 (ZEISS, Oberkochen, Germany). The specimens were deposited on aluminium supports using adhesive tape. Before the analysis, the samples were covered with a thin layer of chromium (8 nm) using a
belonging to the hemimetabolous insect orders of Palaeoptera, Polyneoptera and Condylognatha. Therefore, studies on the peripheral sensory structures of the antennae, as well as on the central processing of the incoming information into the brain in aquatic insects can significantly contribute to our understanding of insect adaptation and evolution of sensory biology and brain plasticity (Crespo, 2011; Dijkstra et al., 2014).

Only a few studies have investigated the sensory ecology of Ephemeroptera (mayflies), Odonata (dragonflies and damselflies), Plecoptera (stoneflies) or Trichoptera (caddisflies) and primarily focused on the terrestrial adults (Rebora et al., 2019). Like in other insects, the olfactory system of aquatic insects consists of antennal receptor neurons housed in cuticular sensilla situated on the antennae and projecting to the primary olfactory centre, the antennal lobe. The antennal lobe usually consists of globular sub-units, the glomeruli, which play an important role in odour coding. Conversely, in some species like dragonflies (Piersanti et al., 2020; Rebora et al., 2013) or aphids (Kollmann et al., 2010), supposed to rely less on olfactory cues, the antennal lobe is less well-structured without distinguishable glomeruli, and thus aglomerular. Nevertheless there is clear evidence for the use of olfaction in these insects (Badji et al., 2021; Piersanti et al., 2014; Rebora et al., 2012).

In this context, the aim of the present study is to provide baseline data on a largely neglected group of aquatic insects, the stoneflies (Plecoptera), belonging to the highly diverse clade of Polyneoptera together with the better-known Orthoptera (Misof et al., 2014; Tihelka et al., 2021; Trautwein et al., 2012; Wipfler et al., 2019). Stoneflies are characterised by aquatic nymphs that – depending on the group – occupy different ecological niches and live as shredders, grazers, detritivores or predators, mostly in upper stretches of rivers (Graf et al., 2009). The most impressive stoneflies in Europe are the large predators of the Perlidae and Perlodidae families that spend a long time, often years, in fast-running waters, feeding on aquatic insects and other macroinvertebrates (Graf et al., 2009). In contrast, most stonefly adults do not feed and only in some species do they occasionally feed on small amounts of pollen, fungal spores and detritus (Rúa and Tierno De Figueroa, 2013).

Plecoptera antennal sensilla have been investigated in detail in adult Perlidae (Rebora et al., 2016), where the most represented sensilla are (i) sensilla trichodea, whose internal structure reveals a mechanosensory function; (ii) sensilla chaetica, with an apical pore, with an internal structure revealing a typical gustatory function; (iii) porous pegs, representing single-walled olfactory sensilla; (iv) digitated pegs with hollow cuticular spoke channels representing double-walled olfactory sensilla; (v) pegs in pits for which a thermo-hygrosensory function is hypothesized. Adult stonefly antennae can perceive olfactory cues (Rebora et al., 2017; Neupert et al., 2022), while nymphs' ability to respond to chemicals in water has been demonstrated in behavioural observations (see review in (Rebora et al., 2019), but data on the antennal sensilla are fragmentary and rather dated (Kapoor, 1985, 1987, 1991). In addition, the neuroanatomy of these insects has never been investigated to the best of our knowledge. In the literature, an aglomerular antennal lobe is reported for stoneflies (Schachtner et al., 2005), but this is wrongly deduced from data on Odonata and Ephemeroptera (Strausfeld, 1998).

Significant changes must be associated with the transition from the aquatic habitat of immature stoneflies to the terrestrial adults' habitat. Mechanoreception in water and air can be very different, especially for insects living in fast running water such as stoneflies. Odorants in an aquatic environment are water-soluble molecules, while air-borne odorants are generally hydrophobic, highly volatile molecules (Ache and Derby, 1985). In insects with aquatic larvae, there is thus a dramatic qualitative change in sensory cues compared to other heterometabolous insects, where life style and

behaviourally relevant cues do not change substantially between nymphs and adults, apart from sex pheromone detection. Accordingly, in locusts and cockroaches, numbers of olfactory sensilla and associated receptor neurons increase gradually during development, and in the antennal lobe only minor changes, such as increasing numbers of glomeruli or small changes in spatial arrangements of glomeruli, occur (Anton et al., 2002; Chambille and Pierre Rospars, 1985).

In hemimetabolous aquatic insects, we expect a dramatic change of sensory equipment when passing from nymphal life in streams to terrestrial adult life, in spite of a potentially similar brain structure of mature nymphs and adults owing to the heterometabolous ontogenesis.

To test this hypothesis, in the present study we investigated the stonefly *Dinocras cephalotes* (Curtis, 1827) (Plecoptera: Perlidae), an aquatic basal polyneopteran, to provide: 1) a quantitative description of antennal sensilla in different nymphal stages, 2) a mapping of the central target areas of antennal axons in nymphs and adults with a neuronal tracer, and 3) a first attempt for a description of general brain anatomy in nymphs and adults. For comparison with the adult sensilla, we refer to the description reported by Rebora et al. (2016, 2017).

## 2. Materials and methods

### 2.1. Insects

Adults of *D. cephalotes* (Plecoptera, Perlidae) for neuroanatomical stainings were collected on the banks of the Nera River (Vallo di Nera, Perugia) in June 2023. Only a few insects could be captured due to unusual climate conditions (n = 12), and they were highly variable in size (from 18 to 32 mm). Nymphs of the same species (n = 60) were collected in the same river from autumn to winter 2022. Because the development in Plecoptera can encompass a variable number of stages and moult (Iannilli et al., 2002), the total length of the specimens was measured, and immature insects were grouped into four size classes: small (S), nymphs with a length of 5–7 mm, medium (M), nymphs with a length of 8–11 mm, large (L), nymphs with a length of 12–15 mm, and extra-large (XL), nymphs longer than 15 mm.

In the laboratory, insects were kept in polystyrene refrigerated boxes with moist paper and no food at 17 ± 2 °C until they were used. Only nymphs of at least 5 mm in length were used for the research because of technical difficulties connected with the small dimensions of earlier instars and, more importantly, because smaller nymphs represent a small proportion of the immature stage life (only a few weeks of the three years of the aquatic life (Iannilli et al., 2002)).

### 2.2. Scanning electron microscopy

To describe the sensilla on the flagellum, nymphal antennae were observed under scanning electron microscopy (SEM). Antennae of nymphs belonging to the four classes above reported (S, M, L, XL) were dissected from anaesthetised specimens (5 nymphs for each class) and fixed for 12 h in 2.5% glutaraldehyde in cacodylate buffer (Electron Microscopy Sciences) pH 7.2. Ten fixed antennae for each class were repeatedly rinsed in the same buffer and then dehydrated using ascending ethanol gradients (20%, 50%, 70%, 80%, 95%, 100%), followed by drying in an oven at 40 °C for three days. The morphology of the samples was analysed by field emission scanning electron microscopy FE SEM LEO 1525 (ZEISS, Oberkochen, Germany). The specimens were deposited on aluminium supports using adhesive tape. Before the analysis, the samples were covered with a thin layer of chromium (8 nm) using a
Q150 T ES (Quorum, Laughton, UK) sputter coater for 25 s. Observations were carried out using an In-lens detector at 5 kV. The number and distribution of the sensilla on five flagellomeres, located alternatively at the base (Px), at the tip (Dx), and in the medial part (Me) of the antennal flagellum (Fig. 1), were evaluated using the software Adobe Photoshop 2020. The flagellomeres in these three areas were considered representative samples of the whole flagellum. Sensilla are named as in previous publications (Kapoor, 1985, 1987, 1991; Gaino and Rebora, 1999; Neagu, 2008),

except for the types described here for the first time. One-way analysis of variance (ANOVA) was used to compare, for each antennal area, the number of each type of sensilla among the four developmental stages considered. The Tukey test was used for multiple comparisons among means.

## 2.3. Neuroanatomy

The brain structure of *D. cephalotes* nymphs and adults was

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Antennal morphology of *D. cephalotes* nymphs observed with scanning electron microscopy (SEM). **a** Antenna of a S nymph constituted of scape (Sc), pedicellum (Pe), and a very long flagellum (Fl). **b** straight hair (SH) sensillum trichodeum; note the thin blunt tip (arrow and inset) with no pores and extending into the following segment to form an overlapping sensory structure between flagellomeres **c** thin hair (TH) sensillum trichodeum; note the smooth blunt tip with no pores (inset) **d-e** coniform sensillar complex (CSC); several cuticular digitations (D) arise from a socket (So) to converge in a cone. The cuticle is indented but without pores (inset). **f** uniporous coeloconic peg (PC), characterized by ridges (Ri), converging in a visible apical pore (P); **g** nonporous coeloconic peg (NC) visible on the antennal surface but deeply inserted in a pit (arrow); **h** campaniform sensillum (CS) with visible molting pore (MP); **i** leaf sensillum (LS), set in a hole and constituted by a small base (arrow) that separates into two extensions with a leaf shape (L) No pores are visible on the smooth cuticle of the leaves. **Px**, basal part; **Me**, medial part; **Dx** distal part of the antennal flagellum.</figcaption></figure>
Q150 T ES (Quorum, Laughton, UK) sputter coater for 25 s. Observations were carried out using an In-lens detector at 5 kV. The number and distribution of the sensilla on five flagellomeres, located alternatively at the base (Px), at the tip (Dx), and in the medial part (Me) of the antennal flagellum (Fig. 1), were evaluated using the software Adobe Photoshop 2020. The flagellomeres in these three areas were considered representative samples of the whole flagellum. Sensilla are named as in previous publications (Kapoor, 1985, 1987, 1991; Gaino and Rebora, 1999; Neagu, 2008),

except for the types described here for the first time. One-way analysis of variance (ANOVA) was used to compare, for each antennal area, the number of each type of sensilla among the four developmental stages considered. The Tukey test was used for multiple comparisons among means.

## 2.3. Neuroanatomy

The brain structure of *D. cephalotes* nymphs and adults was

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Antennal morphology of *D. cephalotes* nymphs observed with scanning electron microscopy (SEM). **a** Antenna of a S nymph constituted of scape (Sc), pedicellum (Pe), and a very long flagellum (Fl). **b** straight hair (SH) sensillum trichodeum; note the thin blunt tip (arrow and inset) with no pores and extending into the following segment to form an overlapping sensory structure between flagellomeres **c** thin hair (TH) sensillum trichodeum; note the smooth blunt tip with no pores (inset) **d-e** coniform sensillar complex (CSC); several cuticular digitations (D) arise from a socket (So) to converge in a cone. The cuticle is indented but without pores (inset). **f** uniporous coeloconic peg (PC), characterized by ridges (Ri), converging in a visible apical pore (P); **g** nonporous coeloconic peg (NC) visible on the antennal surface but deeply inserted in a pit (arrow); **h** campaniform sensillum (CS) with visible molting pore (MP); **i** leaf sensillum (LS), set in a hole and constituted by a small base (arrow) that separates into two extensions with a leaf shape (L) No pores are visible on the smooth cuticle of the leaves. Px, basal part; Me, medial part; Dx, distal part of the antennal flagellum.</figcaption></figure>
visualized with a monoclonal antibody against the *Drosophila vesicle-associated protein synapsin 1* (SYNORF1, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA). Central projections of antennal sensilla were traced with Microruby solution (tetramethylrhodamine dextran with biotin, 3000 MW, lysine-fixable, D-7162; Molecular Probes, Eugene, OR, USA).

Adults and nymphs of different sizes were immobilized in adapted plastic tubes to allow the head to protrude. The head was then fixed with dental wax (Surgident, Heraeus Kulzer Inc., New York, NY USA). For antennal backfills, a glass capillary with a diameter adapted to the diameter of the antenna was filled with concentrated Microruby in phosphate buffer solution (PBS) and slipped over the cut antenna. The insect preparations were then kept in a moist chamber in the dark at 18 °C for 7–48 h. Then the insect heads were opened with a small blade, and the heads were immersed in 4% EM-grade formaldehyde solution (Fisher Scientific, Illkirch, France) in PBS for 1–3 h. Brains were subsequently dissected in PBS and post-fixed overnight in 4% EM-grade formaldehyde. All brains were submitted to anti-synapsin immunostaining as described earlier (Piersanti et al., 2020). Briefly, after rinsing and pre-incubation with normal goat serum (NGS), brains were incubated with the anti-synapsin 1 antibody (1:25 in PBS with 0.5% Triton X and 2% NGS for 3–5 days). Then incubation with the secondary antibody for 2–3 days was done (1:250 in PBS with 1% NGS; Alexa-Fluor-488-conjugated anti-mouse IgG; Invitrogen, Thermo Fisher Scientific, France). After rinsing and dehydration, brains were immersed in methyl salicylate for clarification and subsequently mounted in Permount (Fisher Scientific, Illkirch, France) on aluminium slides between two cover glasses.

Images were acquired with a PlanFluor objective (10 × /NA 0.3) on a Nikon A1 confocal microscope with excitation wavelengths of 488 and 561 nm. Detection filters were set at 525 nm for the Alexa dye and at 595 nm for Microruby. Confocal image stacks were recorded at 1024 × 1024 pixels with a 4x frame average and a step size of 2 or 3 µm. Stacks were transferred to Fiji software (ImageJ, version 2.0.0, National Institutes of Health, Bethesda, ML, USA) and individual sections were selected for visualization. TIFF files of stacks were saved and transferred to Amira 3.1.1 software (Visualization Sciences Group, Mérignac, France) for 3D reconstructions. 3D models were employed to visualize and qualitatively characterize brain and antennal lobe morphology. We obtained 5 well-stained preparations from adults, and between 1 and 6 good preparations in the different nymphal stages.

# 3. Results

## 3.1. Antennal morphology

The antenna of the nymphs of *D. cephalotes* is composed of a scape, a pedicel, and a long flagellum consisting of many segments, shorter and thicker at the base (Px) and longer and thinner at the tip (Dx), gradually changing their shape in the middle portion of the antenna (Mx) (Table 1, Fig. 1a). The total length of the antenna increases during development, from 4 mm in S nymphs to 9 mm in XL nymphs (Table 1). The number of antennal flagellomeres increases during development, from 48 ± 0.6 in S nymphs to 77 ± 1.8 in XL nymphs (Table 1), but also the dimensions (width and length) of the flagellomeres increase during development (Table 1). The first flagellar segment, the meriston (Kapoor 1985), is formed by the fusion of several flagellomeres in the proximal area of the antenna, and is very different from the subsequent ones; it is smooth and lacks sensory structures on its surface.

## 3.2. Sensilla on the antennal flagellum

The same seven types of sensilla have been observed along the flagellum of all investigated nymphal classes.

### 3.2.1. Sensilla trichodea (SH and TH)

Two types of sensilla trichodea are distributed all over the flagellar surface, in all investigated nymphal classes (Table 2).

i) Straight hairs (SH) (Fig. 1b), 40–50 µm in length and approximately 2.5 µm in width at their base, tapering towards the tip and lacking pores. These sensilla are located relatively regularly spaced around the distal margin of each flagellomere and protrude into the proximal section of the following flagellomere (Fig. 2). SH sensilla are present in all nymphal classes and are the most abundant type of sensilla on the antenna; their number increases through the development, particularly in the proximal and medial area of the antenna (Px and Mx) their number in XL nymphs is significantly larger than in smaller nymphs (L, M and S) (Table 2).

ii) Thin hairs (TH) (Fig. 1c), less than 20 µm in length and approximately 1 µm in width at their base, tapering towards the tip and lacking pores (Fig. 1c and inset). TH sensilla are irregularly and sparsely interspersed among the SH and protrude like SH into the proximal section of the following flagellomere (Fig. 2). TH sensilla are present in all nymphal classes but less numerous than SH sensilla, and particularly scarce in the medial and distal area (Mx, Dx); no significant numerical differences among nymphal classes were found in any of the examined flagellomeres (Fig. 2, Table 2).

### 3.2.2. Coniform sensillar complexes (CSC)

The CSCs comprise 10–15 digitations of variable length (10–15 µm) that are set in individual sockets and converge to a conoid structure (Fig. 1d). Each CSC is located in a shallow indentation. Six to eight CSCs are located around the distal portion of each flagellomere (Fig. 2). No pores are visible on the surface of the individual digitations (Fig. 1e), but their cuticle is irregularly indented by small dints (Fig. 1e inset). These sensilla are numerous in the medial (Mx) and distal (Dx) parts of the antenna (Fig. 2 and Table 2), and sometimes they are absent in the proximal flagellomeres close to the meriston. The number of these sensilla increases through development: both in the medial (Mx) and proximal part (Px), their number in XL nymphs is significantly larger than in S nymphs. M and L nymphs have intermediate numbers of CSCs (Fig. 2 and Table 2).

### 3.2.3. Sensilla coeloconica (PC and NC)

Two types of sensilla coeloconica are distributed over the flagellar surface in the medial and distal parts of the antenna but are completely absent in the Px part (Table 2).

i) Uniporous coeloconic pegs (PC), are small conic pegs (length around 1.5 µm, width around 1.5 µm at the base) located in a pit; they bear 14–16 ridges and an apical pore (Fig. 1f).

ii) Nonporous coeloconic pegs (NC), are spherical pegs (width around 1 µm) located in a deep pit so that they do not project beyond the antennal surface. NC cuticle is irregular without detectable pores on the surface (Fig. 1g). NCs are the third in number on the antenna, after SH and CSC, while PCs are very rare (Fig. 2 and Table 2). There are no differences in the number
visualized with a monoclonal antibody against the *Drosophila vesicle-associated protein synapsin 1* (SYNORF1, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA). Central projections of antennal sensilla were traced with Microruby solution (tetramethylrhodamine dextran with biotin, 3000 MW, lysine-fixable, D-7162; Molecular Probes, Eugene, OR, USA).

Adults and nymphs of different sizes were immobilized in adapted plastic tubes to allow the head to protrude. The head was then fixed with dental wax (Surgident, Heraeus Kulzer Inc., New York, NY USA). For antennal backfills, a glass capillary with a diameter adapted to the diameter of the antenna was filled with concentrated Microruby in phosphate buffer solution (PBS) and slipped over the cut antenna. The insect preparations were then kept in a moist chamber in the dark at 18 °C for 7–48 h. Then the insect heads were opened with a small blade, and the heads were immersed in 4% EM-grade formaldehyde solution (Fisher Scientific, Illkirch, France) in PBS for 1–3 h. Brains were subsequently dissected in PBS and post-fixed overnight in 4% EM-grade formaldehyde. All brains were submitted to anti-synapsin immunostaining as described earlier (Piersanti et al., 2020). Briefly, after rinsing and pre-incubation with normal goat serum (NGS), brains were incubated with the anti-synapsin 1 antibody (1:25 in PBS with 0.5% Triton X and 2% NGS for 3–5 days). Then incubation with the secondary antibody for 2–3 days was done (1:250 in PBS with 1% NGS; Alexa-Fluor-488-conjugated anti-mouse IgG; Invitrogen, Thermo Fisher Scientific, France). After rinsing and dehydration, brains were immersed in methyl salicylate for clarification and subsequently mounted in Permount (Fisher Scientific, Illkirch, France) on aluminium slides between two cover glasses.

Images were acquired with a PlanFluor objective (10 × /NA 0.3) on a Nikon A1 confocal microscope with excitation wavelengths of 488 and 561 nm. Detection filters were set at 525 nm for the Alexa dye and at 595 nm for Microruby. Confocal image stacks were recorded at 1024 × 1024 pixels with a 4x frame average and a step size of 2 or 3 µm. Stacks were transferred to Fiji software (ImageJ, version 2.0.0, National Institutes of Health, Bethesda, ML, USA) and individual sections were selected for visualization. TIFF files of stacks were saved and transferred to Amira 3.1.1 software (Visualization Sciences Group, Mérignac, France) for 3D reconstructions. 3D models were employed to visualize and qualitatively characterize brain and antennal lobe morphology. We obtained 5 well-stained preparations from adults, and between 1 and 6 good preparations in the different nymphal stages.

# 3. Results

## 3.1. Antennal morphology

The antenna of the nymphs of *D. cephalotes* is composed of a scape, a pedicel, and a long flagellum consisting of many segments, shorter and thicker at the base (Px) and longer and thinner at the tip (Dx), gradually changing their shape in the middle portion of the antenna (Mx) (Table 1, Fig. 1a). The total length of the antenna increases during development, from 4 mm in S nymphs to 9 mm in XL nymphs (Table 1). The number of antennal flagellomeres increases during development, from 48 ± 0.6 in S nymphs to 77 ± 1.8 in XL nymphs (Table 1), but also the dimensions (width and length) of the flagellomeres increase during development (Table 1). The first flagellar segment, the meriston (Kapoor 1985), is formed by the fusion of several flagellomeres in the proximal area of the antenna, and is very different from the subsequent ones; it is smooth and lacks sensory structures on its surface.

## 3.2. Sensilla on the antennal flagellum

The same seven types of sensilla have been observed along the flagellum of all investigated nymphal classes.

### 3.2.1. Sensilla trichodea (SH and TH)

Two types of sensilla trichodea are distributed all over the flagellar surface, in all investigated nymphal classes (Table 2).

i) Straight hairs (SH) (Fig. 1b), 40–50 µm in length and approximately 2.5 µm in width at their base, tapering towards the tip and lacking pores. These sensilla are located relatively regularly spaced around the distal margin of each flagellomere and protrude into the proximal section of the following flagellomere (Fig. 2). SH sensilla are present in all nymphal classes and are the most abundant type of sensilla on the antenna; their number increases through the development, particularly in the proximal and medial area of the antenna (Px and Mx) their number in XL nymphs is significantly larger than in smaller nymphs (L, M and S) (Table 2).

ii) Thin hairs (TH) (Fig. 1c), less than 20 µm in length and approximately 1 µm in width at their base, tapering towards the tip and lacking pores (Fig. 1c and inset). TH sensilla are irregularly and sparsely interspersed among the SH and protrude like SH into the proximal section of the following flagellomere (Fig. 2). TH sensilla are present in all nymphal classes but less numerous than SH sensilla, and particularly scarce in the medial and distal area (Mx, Dx); no significant numerical differences among nymphal classes were found in any of the examined flagellomeres (Fig. 2, Table 2).

### 3.2.2. Coniform sensillar complexes (CSC)

The CSCs comprise 10–15 digitations of variable length (10–15 µm) that are set in individual sockets and converge to a conoid structure (Fig. 1d). Each CSC is located in a shallow indentation. Six to eight CSCs are located around the distal portion of each flagellomere (Fig. 2). No pores are visible on the surface of the individual digitations (Fig. 1e), but their cuticle is irregularly indented by small dints (Fig. 1e inset). These sensilla are numerous in the medial (Mx) and distal (Dx) parts of the antenna (Fig. 2 and Table 2), and sometimes they are absent in the proximal flagellomeres close to the meriston. The number of these sensilla increases through development: both in the medial (Mx) and proximal part (Px), their number in XL nymphs is significantly larger than in S nymphs. M and L nymphs have intermediate numbers of CSCs (Fig. 2 and Table 2).

### 3.2.3. Sensilla coeloconica (PC and NC)

Two types of sensilla coeloconica are distributed over the flagellar surface in the medial and distal parts of the antenna but are completely absent in the Px part (Table 2).

i) Uniporous coeloconic pegs (PC), are small conic pegs (length around 1.5 µm, width around 1.5 µm at the base) located in a pit; they bear 14–16 ridges and an apical pore (Fig. 1f).

ii) Nonporous coeloconic pegs (NC), are spherical pegs (width around 1 µm) located in a deep pit so that they do not project beyond the antennal surface. NC cuticle is irregular without detectable pores on the surface (Fig. 1g). NCs are the third in number on the antenna, after SH and CSC, while PCs are very rare (Fig. 2 and Table 2). There are no differences in the number
<table><caption>**Table 1**
Antennal size of *D. cephalotes* during nymphal development.</caption><thead><tr><th></th><th>S nymphs (n = 5)</th><th>M nymphs (n = 5)</th><th>L nymphs (n = 5)</th><th>XL nymphs (n = 5)</th></tr></thead><tbody><tr><td>Antenna total length (mm)</td><td>4.0 ± 0.0</td><td>5.6 ± 0.2</td><td>7.0 ± 0.0</td><td>9.0 ± 0.0</td></tr><tr><td>N° flagellomeres</td><td>48.0 ± 0.6</td><td>60.0 ± 1.1</td><td>73.2 ± 1.9</td><td>77.0 ± 1.8</td></tr><tr><td>Px</td><td></td><td></td><td></td><td></td></tr><tr><td>Flagellomere length (µm)</td><td>42.9 ± 1.2</td><td>46.2 ± 1.3</td><td>48.5 ± 2.1</td><td>60.8 ± 1.0</td></tr><tr><td>Flagellomere width (µm)</td><td>175.0 ± 4.6</td><td>208.8 ± 6.5</td><td>197.1 ± 6.7</td><td>295.2 ± 6.5</td></tr><tr><td>Mx</td><td></td><td></td><td></td><td></td></tr><tr><td>Flagellomere length (µm)</td><td>87.9 ± 2.5</td><td>93.6 ± 2.2</td><td>91.2 ± 3.0</td><td>102.1 ± 2.7</td></tr><tr><td>Flagellomere width (µm)</td><td>95.4 ± 3.6</td><td>107.6 ± 3.4</td><td>108.2 ± 2.5</td><td>141.7 ± 4.2</td></tr><tr><td>Dx</td><td></td><td></td><td></td><td></td></tr><tr><td>Flagellomere length (µm)</td><td>111.4 ± 1.8</td><td>116.1 ± 0.8</td><td>121.3 ± 2.5</td><td>120.8 ± 1.5</td></tr><tr><td>Flagellomere width (µm)</td><td>49.0 ± 1.3</td><td>48.5 ± 1.0</td><td>55.2 ± 2.7</td><td>60.3 ± 3.1</td></tr></tbody></table>

S (small) nymphs with a length of 5–7 mm, M (medium) nymphs with a length of 8–11 mm; L (large) nymphs with a length of 12–15 mm, and XL (extra-large) nymphs longer than 15 mm. Px, proximal part of the antennal flagellum; Me, medial part of the antennal flagellum; Dx, distal part of the antennal flagellum.

<table><caption>**Table 2**
Number (mean ± SE) and distribution of the seven sensilla types on the antennal flagellum of *D. cephalotes* nymphs during development.</caption><thead><tr><th></th><th>Sensilla type</th><th>S nymphs (n = 5)</th><th>M nymphs (n = 5)</th><th>L nymphs (n = 5)</th><th>XL nymphs (n = 5)</th></tr></thead><tbody><tr><td rowspan="6">Px</td><td>SH</td><td>38.4 ± 2.0 b</td><td>40.0 ± 0.9 b</td><td>50.4 ± 5.2 b</td><td>72.4 ± 3.3 a</td></tr><tr><td>TH</td><td>4.2 ± 1.2</td><td>3.2 ± 1.0</td><td>5.8 ± 1.5</td><td>4.6 ± 1.9</td></tr><tr><td>CSC</td><td>5.6 ± 1.2 b</td><td>10.0 ± 0.5 ab</td><td>7.6 ± 0.8 ab</td><td>12.2 ± 1.8 a</td></tr><tr><td>PC</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>NC</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>LS</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="6">Me</td><td>CS</td><td>3.4 ± 1.2</td><td>2.6 ± 1.2</td><td>1.4 ± 0.9</td><td>1.4 ± 0.7</td></tr><tr><td>SH</td><td>34.2 ± 1.6 b</td><td>39.6 ± 1.7 b</td><td>35.8 ± 1.9 b</td><td>48.0 ± 1.5 a</td></tr><tr><td>TH</td><td>1.0 ± 0.6</td><td>0.6 ± 0.4</td><td>1.6 ± 0.7</td><td>1.4 ± 0.5</td></tr><tr><td>CSC</td><td>16.6 ± 1.8</td><td>18.8 ± 0.8</td><td>19.4 ± 0.4</td><td>15.6 ± 0.4</td></tr><tr><td>PC</td><td>2.2 ± 0.7</td><td>3.0 ± 0.7</td><td>3.4 ± 1.0</td><td>1.6 ± 0.8</td></tr><tr><td>NC</td><td>15.8 ± 2.0 b</td><td>21.0 ± 1.3 ab</td><td>18.4 ± 1.5 ab</td><td>25.0 ± 2.1 a</td></tr><tr><td rowspan="6">Dx</td><td>LS</td><td>11.4 ± 1.3</td><td>14.4 ± 1.1</td><td>13.2 ± 1.2</td><td>13.8 ± 0.6</td></tr><tr><td>CS</td><td>7.4 ± 0.4</td><td>9.0 ± 0.6</td><td>8.6 ± 0.9</td><td>7.8 ± 0.5</td></tr><tr><td>SH</td><td>22.4 ± 1.6</td><td>26.0 ± 1.8</td><td>23.2 ± 2.7</td><td>26.8 ± 2.1</td></tr><tr><td>TH</td><td>1.0 ± 0.3</td><td>0.2 ± 0.2</td><td>1.6 ± 0.7</td><td>0.8 ± 0.2</td></tr><tr><td>CSC</td><td>18.0 ± 0.5</td><td>18.4 ± 0.2</td><td>18.4 ± 0.2</td><td>18.6 ± 0.2</td></tr><tr><td>PC</td><td>3.2 ± 0.8</td><td>2.6 ± 0.2</td><td>3.4 ± 0.8</td><td>1.8 ± 0.8</td></tr><tr><td rowspan="3">Dx</td><td>NC</td><td>13.2 ± 1.8 b</td><td>17.4 ± 1.7 ab</td><td>13.8 ± 2.6 b</td><td>22.8 ± 1.9 a</td></tr><tr><td>LS</td><td>10.2 ± 1.2</td><td>12.4 ± 1.3</td><td>11.6 ± 2.0</td><td>9.8 ± 1.1</td></tr><tr><td>CS</td><td>7.2 ± 0.4</td><td>7.8 ± 0.6</td><td>10.4 ± 2.0</td><td>9.0 ± 0.6</td></tr></tbody></table>

S (small) nymphs with a length of 5–7 mm, M (medium) nymphs with a length of 8–11 mm; L (large) nymphs with a length of 12–15 mm, and XL (extra-large) nymphs longer than 15 mm.

SH, straight hair; TH, thin hair; CSC, coniform sensillar complex; PC, uniporous coeloconic peg; NC, nonporous coeloconic peg; LS, leaf sensillum; CS, campaniform sensillum. Px proximal, Me medial, Dx distal part of the antennal flagellum (5 flagellomeres for each part). For each antennal part, the number of each type of sensillum was compared among the four developmental stages considered; different letters along each row show significantly different numbers, if no letters are reported, data are not significantly different at *P* < 0.05 (One-way ANOVA, Tukey test).

and distribution of PC sensilla among the nymphal size classes, while NC sensilla are significantly more numerous in XL nymphs than in S nymphs. M and L nymphs have intermediate numbers of NC sensilla (Fig. 2 and Table 2).

### 3.2.4. Campaniform sensilla (CS)

CSs are elliptical indentations in the cuticle with a length of about 8 µm, which extend along the longitudinal axis of the antenna (Fig. 1h). CSs are more numerous in Mx and Dx than in Px flagellomeres (Fig. 2 and Table 2). There are no differences in the number and distribution of these sensilla among the nymphal size classes (Fig. 2 and Table 2).

### 3.2.5. Leaf sensilla (LS)

They are tiny sensilla composed of a small base that projects into two lanceolate, leaf-shaped projections less than 3 µm long and are set in a pit. No pores are visible on the smooth cuticle of the projections (Fig. 1i). These sensilla are scattered on the surface of each flagellomere in the distal half of each flagellomere, sometimes

arranged around the entire circumference. LS are absent in the proximal part of the antenna (Px), including the meriston (Fig. 2 and Table 2). There are no differences in the number and distribution of these sensilla among the nymphal size classes (Fig. 2 and Table 2).

### 3.3. Brain and antennal lobe structure in nymphs

The nymphal brain of *D. cephalotes* is relatively compact. The optic lobes are situated close to the other parts of the protocerebrum and consist of lamina, medulla and a small lobula. The central body and the mushroom bodies appear relatively small compared to the brain volume and the mushroom bodies seem to lack calyces (Fig. 3). Antennal lobes appear relatively big and contain more or less clearly delimited elongated glomeruli in the anterior part, whereas the posterior part of the AL is aglomerular (Figs. 3 and 4). The dorsoventral diameter of the antennal lobe increases during development from around 100 µm in S nymphs to 170–200 µm in XL nymphs. Among the glomeruli of the AL, the biggest is situated anteriorly and the smallest most posteriorly. The
<table><caption>**Table 1**
Antennal size of *D. cephalotes* during nymphal development.</caption><thead><tr><th></th><th>S nymphs (n = 5)</th><th>M nymphs (n = 5)</th><th>L nymphs (n = 5)</th><th>XL nymphs (n = 5)</th></tr></thead><tbody><tr><td>Antenna total length (mm)</td><td>4.0 ± 0.0</td><td>5.6 ± 0.2</td><td>7.0 ± 0.0</td><td>9.0 ± 0.0</td></tr><tr><td>Nº flagellomeres</td><td>48.0 ± 0.6</td><td>60.0 ± 1.1</td><td>73.2 ± 1.9</td><td>77.0 ± 1.8</td></tr><tr><td>Px</td><td></td><td></td><td></td><td></td></tr><tr><td>Flagellomere length (µm)</td><td>42.9 ± 1.2</td><td>46.2 ± 1.3</td><td>48.5 ± 2.1</td><td>60.8 ± 1.0</td></tr><tr><td>Flagellomere width (µm)</td><td>175.0 ± 4.6</td><td>208.8 ± 6.5</td><td>197.1 ± 6.7</td><td>295.2 ± 6.5</td></tr><tr><td>Mx</td><td></td><td></td><td></td><td></td></tr><tr><td>Flagellomere length (µm)</td><td>87.9 ± 2.5</td><td>93.6 ± 2.2</td><td>91.2 ± 3.0</td><td>102.1 ± 2.7</td></tr><tr><td>Flagellomere width (µm)</td><td>95.4 ± 3.6</td><td>107.6 ± 3.4</td><td>108.2 ± 2.5</td><td>141.7 ± 4.2</td></tr><tr><td>Dx</td><td></td><td></td><td></td><td></td></tr><tr><td>Flagellomere length (µm)</td><td>111.4 ± 1.8</td><td>116.1 ± 0.8</td><td>121.3 ± 2.5</td><td>120.8 ± 1.5</td></tr><tr><td>Flagellomere width (µm)</td><td>49.0 ± 1.3</td><td>48.5 ± 1.0</td><td>55.2 ± 2.7</td><td>60.3 ± 3.1</td></tr></tbody></table>

S (small) nymphs with a length of 5–7 mm, M (medium) nymphs with a length of 8–11 mm; L (large) nymphs with a length of 12–15 mm, and XL (extra-large) nymphs longer than 15 mm. Px, proximal part of the antennal flagellum; Me, medial part of the antennal flagellum; Dx, distal part of the antennal flagellum.

<table><caption>**Table 2**
Number (mean ± SE) and distribution of the seven sensilla types on the antennal flagellum of *D. cephalotes* nymphs during development.</caption><thead><tr><th></th><th>Sensilla type</th><th>S nymphs (n = 5)</th><th>M nymphs (n = 5)</th><th>L nymphs (n = 5)</th><th>XL nymphs (n = 5)</th></tr></thead><tbody><tr><td rowspan="6">Px</td><td>SH</td><td>38.4 ± 2.0 b</td><td>40.0 ± 0.9 b</td><td>50.4 ± 5.2 b</td><td>72.4 ± 3.3 a</td></tr><tr><td>TH</td><td>4.2 ± 1.2</td><td>3.2 ± 1.0</td><td>5.8 ± 1.5</td><td>4.6 ± 1.9</td></tr><tr><td>CSC</td><td>5.6 ± 1.2 b</td><td>10.0 ± 0.5 ab</td><td>7.6 ± 0.8 ab</td><td>12.2 ± 1.8 a</td></tr><tr><td>PC</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>NC</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>LS</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="6">Me</td><td>CS</td><td>3.4 ± 1.2</td><td>2.6 ± 1.2</td><td>1.4 ± 0.9</td><td>1.4 ± 0.7</td></tr><tr><td>SH</td><td>34.2 ± 1.6 b</td><td>39.6 ± 1.7 b</td><td>35.8 ± 1.9 b</td><td>48.0 ± 1.5 a</td></tr><tr><td>TH</td><td>1.0 ± 0.6</td><td>0.6 ± 0.4</td><td>1.6 ± 0.7</td><td>1.4 ± 0.5</td></tr><tr><td>CSC</td><td>16.6 ± 1.8</td><td>18.8 ± 0.8</td><td>19.4 ± 0.4</td><td>15.6 ± 0.4</td></tr><tr><td>PC</td><td>2.2 ± 0.7</td><td>3.0 ± 0.7</td><td>3.4 ± 1.0</td><td>1.6 ± 0.8</td></tr><tr><td>NC</td><td>15.8 ± 2.0 b</td><td>21.0 ± 1.3 ab</td><td>18.4 ± 1.5 ab</td><td>25.0 ± 2.1 a</td></tr><tr><td rowspan="6">Dx</td><td>LS</td><td>11.4 ± 1.3</td><td>14.4 ± 1.1</td><td>13.2 ± 1.2</td><td>13.8 ± 0.6</td></tr><tr><td>CS</td><td>7.4 ± 0.4</td><td>9.0 ± 0.6</td><td>8.6 ± 0.9</td><td>7.8 ± 0.5</td></tr><tr><td>SH</td><td>22.4 ± 1.6</td><td>26.0 ± 1.8</td><td>23.2 ± 2.7</td><td>26.8 ± 2.1</td></tr><tr><td>TH</td><td>1.0 ± 0.3</td><td>0.2 ± 0.2</td><td>1.6 ± 0.7</td><td>0.8 ± 0.2</td></tr><tr><td>CSC</td><td>18.0 ± 0.5</td><td>18.4 ± 0.2</td><td>18.4 ± 0.2</td><td>18.6 ± 0.2</td></tr><tr><td>PC</td><td>3.2 ± 0.8</td><td>2.6 ± 0.2</td><td>3.4 ± 0.8</td><td>1.8 ± 0.8</td></tr><tr><td rowspan="3">Dx</td><td>NC</td><td>13.2 ± 1.8 b</td><td>17.4 ± 1.7 ab</td><td>13.8 ± 2.6 b</td><td>22.8 ± 1.9 a</td></tr><tr><td>LS</td><td>10.2 ± 1.2</td><td>12.4 ± 1.3</td><td>11.6 ± 2.0</td><td>9.8 ± 1.1</td></tr><tr><td>CS</td><td>7.2 ± 0.4</td><td>7.8 ± 0.6</td><td>10.4 ± 2.0</td><td>9.0 ± 0.6</td></tr></tbody></table>

S (small) nymphs with a length of 5–7 mm, M (medium) nymphs with a length of 8–11 mm; L (large) nymphs with a length of 12–15 mm, and XL (extra-large) nymphs longer than 15 mm.

SH, straight hair; TH, thin hair; CSC, coniform sensillar complex; PC, uniporous coeloconic peg; NC, nonporous coeloconic peg; LS, leaf sensillum; CS, campaniform sensillum. Px proximal, Me medial, Dx distal part of the antennal flagellum (5 flagellomeres for each part). For each antennal part, the number of each type of sensillum was compared among the four developmental stages considered; different letters along each row show significantly different numbers, if no letters are reported, data are not significantly different at *P* < 0.05 (One-way ANOVA, Tukey test).

and distribution of PC sensilla among the nymphal size classes, while NC sensilla are significantly more numerous in XL nymphs than in S nymphs. M and L nymphs have intermediate numbers of NC sensilla (Fig. 2 and Table 2).

### 3.2.4. Campaniform sensilla (CS)

CSs are elliptical indentations in the cuticle with a length of about 8 µm, which extend along the longitudinal axis of the antenna (Fig. 1h). CSs are more numerous in Mx and Dx than in Px flagellomeres (Fig. 2 and Table 2). There are no differences in the number and distribution of these sensilla among the nymphal size classes (Fig. 2 and Table 2).

### 3.2.5. Leaf sensilla (LS)

They are tiny sensilla composed of a small base that projects into two lanceolate, leaf-shaped projections less than 3 µm long and are set in a pit. No pores are visible on the smooth cuticle of the projections (Fig. 1i). These sensilla are scattered on the surface of each flagellomere in the distal half of each flagellomere, sometimes

arranged around the entire circumference. LS are absent in the proximal part of the antenna (Px), including the meriston (Fig. 2 and Table 2). There are no differences in the number and distribution of these sensilla among the nymphal size classes (Fig. 2 and Table 2).

### 3.3. Brain and antennal lobe structure in nymphs

The nymphal brain of *D. cephalotes* is relatively compact. The optic lobes are situated close to the other parts of the protocerebrum and consist of lamina, medulla and a small lobula. The central body and the mushroom bodies appear relatively small compared to the brain volume and the mushroom bodies seem to lack calyces (Fig. 3). Antennal lobes appear relatively big and contain more or less clearly delimited elongated glomeruli in the anterior part, whereas the posterior part of the AL is aglomerular (Figs. 3 and 4). The dorsoventral diameter of the antennal lobe increases during development from around 100 µm in S nymphs to 170–200 µm in XL nymphs. Among the glomeruli of the AL, the biggest is situated anteriorly and the smallest most posteriorly. The
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Distribution of antennal sensilla on the flagellomeres of *D. cephalotes* nymphs. **a** distribution of sensilla on one flagellomere. **b** five flagellomeres at the base of the antennal flagellum (Px); **c** five flagellomeres in the medial part of the antennal flagellum (Me), **d** five flagellomeres in the distal part of the antennal flagellum (Dx). In b-d the seven types of flagellar sensilla are indicated with different colors according to their morphology to show their number and distribution.</figcaption></figure>

three most anterior glomeruli in the AL increased in volume from 16 to $32 \times 10^3 \mu m^3$ in S nymphs to larger glomeruli ($58–138 \times 10^3 \mu m^3$) of similar size in M, L and XL nymphs.

### 3.4. Antennal projections to the brain in nymphs

The prominent antennal nerve enters the antennal lobe posterior-laterally and many fibres seem to massively innervate the posterior, aglomerular part of the antennal lobe. The anterior elongated glomeruli of the antennal lobe are also innervated by receptor neurons in all investigated nymph stages (Fig. 4). We did not observe any antennal nerve projections to the suboesophageal ganglion in our preparations of nymphs.

### 3.5. Structure of the adult brain and antennal projections

The adult brain structure of *D. cephalotes* is very similar to that of the nymph brain, but seems slightly more compact (Fig. 5a-d).

Optic and antennal lobes protrude less laterally from the central brain in adults than in nymphs. The central body and mushroom bodies seem to occupy slightly more space in adults than in nymphs, but appear still relatively small. Additionally, mushroom bodies in adults, like in nymphs, also lack calyces (Fig. 5). The dorso-ventral diameter of the adult antennal lobe is similar to the diameter in XL nymphs with $180–200 \mu m$. Also, central antennal projections in adults show similar target regions within the antennal lobe, as in nymphs, innervating elongated anterior glomeruli and the posterior aglomerular region (Fig. 5e). Anterior elongated glomeruli have, however, a larger volume in adults than those in nymphs with $125–290 \times 10^3 \mu m^3$. In one preparation, we observed also several axons entering the suboesophageal ganglion (not shown).

## 4. Discussion

Our data demonstrate that the sensory equipment of the
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Distribution of antennal sensilla on the flagellomeres of *D. cephalotes* nymphs. **a** distribution of sensilla on one flagellomere. **b** five flagellomeres at the base of the antennal flagellum (Px); **c** five flagellomeres in the medial part of the antennal flagellum (Me), **d** five flagellomeres in the distal part of the antennal flagellum (Dx). In b-d the seven types of flagellar sensilla are indicated with different colors according to their morphology to show their number and distribution.</figcaption></figure>

three most anterior glomeruli in the AL increased in volume from 16 to $32 \times 10^3 \mu m^3$ in S nymphs to larger glomeruli ($58–138 \times 10^3 \mu m^3$) of similar size in M, L and XL nymphs.

### 3.4. Antennal projections to the brain in nymphs

The prominent antennal nerve enters the antennal lobe posterior-laterally and many fibres seem to massively innervate the posterior, aglomerular part of the antennal lobe. The anterior elongated glomeruli of the antennal lobe are also innervated by receptor neurons in all investigated nymph stages (Fig. 4). We did not observe any antennal nerve projections to the suboesophageal ganglion in our preparations of nymphs.

### 3.5. Structure of the adult brain and antennal projections

The adult brain structure of *D. cephalotes* is very similar to that of the nymph brain, but seems slightly more compact (Fig. 5a-d).

Optic and antennal lobes protrude less laterally from the central brain in adults than in nymphs. The central body and mushroom bodies seem to occupy slightly more space in adults than in nymphs, but appear still relatively small. Additionally, mushroom bodies in adults, like in nymphs, also lack calyces (Fig. 5). The dorso-ventral diameter of the adult antennal lobe is similar to the diameter in XL nymphs with $180–200 \mu m$. Also, central antennal projections in adults show similar target regions within the antennal lobe, as in nymphs, innervating elongated anterior glomeruli and the posterior aglomerular region (Fig. 5e). Anterior elongated glomeruli have, however, a larger volume in adults than those in nymphs with $125–290 \times 10^3 \mu m^3$. In one preparation, we observed also several axons entering the suboesophageal ganglion (not shown).

## 4. Discussion

Our data demonstrate that the sensory equipment of the
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Anatomy of the nymph brain including the antennal lobe of *D. cephalotes*. a Three-dimensional reconstruction of an L nymph brain with main discernible neuropil areas. b Individual optical sections at different anterior-posterior positions, stained with anti-synapsin 1 antibody. c Three-dimensional reconstruction of the right antennal lobe of the same brain as in a, showing glomeruli in the anterior part. d Individual optical sections at different anterior-posterior positions, stained with anti-synapsin 1 antibody. The depth starting from the anterior surface is provided in each image in b and d. AL antennal lobe, CB central body, La lamina, Lo lobula, MB mushroom body, Me Medulla, orientation is indicated by d dorsal, l lateral, and p posterior.</figcaption></figure>

antennae, i.e. sensillum types, differs markedly between stonefly nymphs and adults. The general brain anatomy, on the other hand, is quite similar during the two phases of the life cycle, despite the considerable differences in lifestyle and habitat. This also applies to the antennal lobe projection pattern formed by the sensory axons. During the different stages of nymphal development investigated, both antennal sensilla types and brain morphology remain similar.

## 4.1. Antennal sensilla

Nymphs of *D. cephalotes*, likewise adults (Rebora et al., 2016), have very long antennae, approximately half of the body length, with a thin flagellum consisting of many flagellomeres. Since flagellomere numbers increase during nymphal development, the total number of sensilla increases from the young to the old nymphal stages. In addition, straight hairs (SH), coniform sensillar complex (CSC) and nonporous pegs (NC) increase their numbers per flagellomere during nymphal development. Apart from that, antennal sensilla do not show dramatic changes during nymphal development, and they are similar to those previously described in nymphs of other stonefly species ((Kapoor, 1985, 1987, 1991); see review in (Gaino and Rebora, 1999)), while they completely differ from the adult antennal sensilla previously described (Rebora et al., 2016).

Trichoid sensilla of *D. cephalotes* nymphs (straight and thin hairs SH and TH) show the typical structure of short bristles, one of the most common types of mechanoreceptors in insects (see review in (Keil, 1997)). Because of their position, forming a crown, whose tips directly touch the adjacent flagellomere, these sensilla probably play a proprioceptive role (Tuthill and Azim, 2018). Similar bristles have been described in other stonefly nymphs (Kapoor, 1985; Neagu, 2008) and other aquatic insects, such as mayflies (Gaino and Rebora, 1998) and Odonata (Piersanti and Rebora, 2018). Such sensilla are completely different from the much longer

(100–150 µm instead of less than 50 µm in nymphs) mechanoreceptors covering the flagellum of adult *D. cephalotes*, probably activated by direct touch, or from filiform hairs on dragonfly nymphs (Gaino and Rebora, 2001) able to perceive water movements (Rebora et al., 2004). They also differ from sensilla chaetica with a touch and taste function, which are numerous on the adult flagellum of *D. cephalotes* (Rebora et al., 2016) but are not present in the nymphs. On the other hand, coniform sensilla (CS) detecting cuticle deformation are widespread mechanoreceptors along the antennae of *D. cephalotes* nymphs, while they have not been described in adults (Rebora et al., 2016).

Two types of putative chemoreceptors (Altner, 1977; Steinbrecht, 1997), are widespread along the antennae of *D. cephalotes* nymphs. The most numerous are the CSC, which have not been reported in adults. Their external and internal ultrastructure has been accurately described in *Paragnetina media* (Kapoor, 1987). In our observations, no pores are visible under SEM on the digitation surface, but in *P. media* pores have been found under TEM, suggesting a chemoreceptive function (Kapoor, 1987).

The second putative chemoreceptors on the antennae are the uniporous coeloconic pegs (PC), also reported in *P. media* nymphs (Kapoor, 1991), but more accurate ultrastructural investigations are necessary to define the actual role of these sensilla. In any case, CSCs and uniporous pegs on the nymphal antennae are very different from single and double-walled olfactory sensilla reported in adult antennae, i.e. multiporous and digitated pegs respectively (Rebora et al., 2016, 2017).

In the nymphal flagellum of *D. cephalotes* we also described two small sensilla with an uncertain function. The nonporous coeloconic pegs (NC) are quite numerous and their external shape suggests a thermo-hygroreceptive role, as in other stonefly species (Kapoor, 1985, 1991; Neagu, 2008). Two similar sensilla, interpreted as thermo-hygroreceptors, have been described at the antennal tip
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Anatomy of the nymph brain including the antennal lobe of *D. cephalotes*. a Three-dimensional reconstruction of an L nymph brain with main discernible neuropil areas. b Individual optical sections at different anterior-posterior positions, stained with anti-synapsin 1 antibody. c Three-dimensional reconstruction of the right antennal lobe of the same brain as in *a*, showing glomeruli in the anterior part. d Individual optical sections at different anterior-posterior positions, stained with anti-synapsin 1 antibody. The depth starting from the anterior surface is provided in each image in *b* and *d*. AL antennal lobe, CB central body, La lamina, Lo lobula, MB mushroom body, Me Medulla, orientation is indicated by *d* dorsal, *l* lateral, and *p* posterior.</figcaption></figure>

antennae, i.e. sensillum types, differs markedly between stonefly nymphs and adults. The general brain anatomy, on the other hand, is quite similar during the two phases of the life cycle, despite the considerable differences in lifestyle and habitat. This also applies to the antennal lobe projection pattern formed by the sensory axons. During the different stages of nymphal development investigated, both antennal sensilla types and brain morphology remain similar.

## 4.1. Antennal sensilla

Nymphs of *D. cephalotes*, likewise adults (Rebora et al., 2016), have very long antennae, approximately half of the body length, with a thin flagellum consisting of many flagellomeres. Since flagellomere numbers increase during nymphal development, the total number of sensilla increases from the young to the old nymphal stages. In addition, straight hairs (SH), coniform sensillar complex (CSC) and nonporous pegs (NC) increase their numbers per flagellomere during nymphal development. Apart from that, antennal sensilla do not show dramatic changes during nymphal development, and they are similar to those previously described in nymphs of other stonefly species ((Kapoor, 1985, 1987, 1991); see review in (Gaino and Rebora, 1999)), while they completely differ from the adult antennal sensilla previously described (Rebora et al., 2016).

Trichoid sensilla of *D. cephalotes* nymphs (straight and thin hairs SH and TH) show the typical structure of short bristles, one of the most common types of mechanoreceptors in insects (see review in (Keil, 1997)). Because of their position, forming a crown, whose tips directly touch the adjacent flagellomere, these sensilla probably play a proprioceptive role (Tuthill and Azim, 2018). Similar bristles have been described in other stonefly nymphs (Kapoor, 1985; Neagu, 2008) and other aquatic insects, such as mayflies (Gaino and Rebora, 1998) and Odonata (Piersanti and Rebora, 2018). Such sensilla are completely different from the much longer

(100–150 µm instead of less than 50 µm in nymphs) mechanoreceptors covering the flagellum of adult *D. cephalotes*, probably activated by direct touch, or from filiform hairs on dragonfly nymphs (Gaino and Rebora, 2001) able to perceive water movements (Rebora et al., 2004). They also differ from sensilla chaetica with a touch and taste function, which are numerous on the adult flagellum of *D. cephalotes* (Rebora et al., 2016) but are not present in the nymphs. On the other hand, coniform sensilla (CS) detecting cuticle deformation are widespread mechanoreceptors along the antennae of *D. cephalotes* nymphs, while they have not been described in adults (Rebora et al., 2016).

Two types of putative chemoreceptors (Altner, 1977; Steinbrecht, 1997), are widespread along the antennae of *D. cephalotes* nymphs. The most numerous are the CSC, which have not been reported in adults. Their external and internal ultrastructure has been accurately described in *Paragnetina media* (Kapoor, 1987). In our observations, no pores are visible under SEM on the digitation surface, but in *P. media* pores have been found under TEM, suggesting a chemoreceptive function (Kapoor, 1987).

The second putative chemoreceptors on the antennae are the uniporous coeloconic pegs (PC), also reported in *P. media* nymphs (Kapoor, 1991), but more accurate ultrastructural investigations are necessary to define the actual role of these sensilla. In any case, CSCs and uniporous pegs on the nymphal antennae are very different from single and double-walled olfactory sensilla reported in adult antennae, i.e. multiporous and digitated pegs respectively (Rebora et al., 2016, 2017).

In the nymphal flagellum of *D. cephalotes* we also described two small sensilla with an uncertain function. The nonporous coeloconic pegs (NC) are quite numerous and their external shape suggests a thermo-hygroreceptive role, as in other stonefly species (Kapoor, 1985, 1991; Neagu, 2008). Two similar sensilla, interpreted as thermo-hygroreceptors, have been described at the antennal tip
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Examples of antennal receptor neuron projections from mass fills in *D. cephalotes* nymphs. Antennal afferences were stained with microruby (red) and projections to the anterior and posterior parts of the antennal lobe of the four nymph classes of *D. cephalotes* are shown. Background staining with anti-synapsin 1 antibody in green. Note the similarity in projection patterns between the different nymphal stages at the anterior and posterior level of the antennal lobe. Orientation is indicated by d dorsal, and 1 lateral.</figcaption></figure>

of Odonata nymphs (Rebora et al., 2007a, 2019), where they
probably allow searching for residual water or moist refugia when
the ponds where insects live dry out (Piersanti et al., 2007; Rebora
et al., 2007b). Thermo-hygroreceptors in the form of a round pit
hosting a small peg are reported also in adult *D. cephalotes*, and
their internal structure is described in detail (Rebora et al., 2016). A
more accurate description of the internal structure of the nonpo-
rous coeloconic pegs on the nymphal flagellum, as well as an
electrophysiological investigation, are necessary to shed light on
the actual function of these sensilla.

Another sensillum with an uncertain function in *D. cephalotes*

nymphs is the leaf sensillum (LS), which has never been described
in Plecoptera. It is visible in images published from nymphs of other
species (Neagu, 2008), but has not been found in adults (Rebora
et al., 2016). The relative abundance of these sensilla on the fla-
gellum suggests a significant sensory role. Their very small
dimension, less than 3 µm, seems to exclude a mechanoreceptive
function (see review in (Keil, 1997)), while the apparent absence of
pores not necessarily precludes a chemosensory function in water,
where a thin cuticle could allow diffusion of chemicals eluted, as in
the olfactory organs of decapod crustaceans called aesthetascs
(Derby et al., 2016; Hallberg et al., 1992; Tuchina et al., 2014;
Zacharuk et al., 1971).

4.2. Brain morphology and antennal pathway

The brain of *D. cephalotes* has a similar structure as other basal
insects, such as dragonflies (Rebora et al., 2013; Piersanti et al.,
2020). It shows three optic neuropils, an antennal lobe with elon-
gated glomeruli in the anterior part, a relatively small central body,
and mushroom bodies without calyces. We did not observe any
antennal nerve projections to the suboesophageal ganglion in our
preparations of nymphs, but this might be due to insufficient
migration time, as connectives between the supra- and sub-
oesophageal ganglia are very long.

This basic brain structure does not change between the inves-
tigated nymphal stages and the adult. Compared to the brain of the
paleopteran dragonfly *Libellula depressa*, the stonefly brain is more
compact and the structure is even more similar between nymphs
and adults (Piersanti et al., 2020). Whereas dragonflies have aglo-
merular antennal lobes, a few elongated glomeruli are present in
the anterior part of the *D. cephalotes* antennal lobe and receive
antennal afferent sensory neurons in both nymphs and adults. We
speculate that these glomeruli receive input from olfactory receptor
neurons and possibly thermo-hygroreceptors. In other insects, such
as cockroaches, ants and *Drosophila melanogaster*, specific
glomeruli, sometimes with an elongated shape, and situated at the
periphery of the antennal lobe, have been shown to receive input
from thermo-hygroreceptors (Frank et al., 2017; Mizunami et al.,
2016; Nishino et al., 2003; Ruchty et al., 2010). The posterior
aglomerular projections in the *D. cephalotes* AL may receive the
numerous mechanosensitive receptor neurons and could corre-
spond either to the aglomerular parts of the AL in Orthoptera or to
the antennal mechanosensory and motor centre described in many
other insects, such as moths and flies (Homberg et al., 1988; Ito
et al., 2014). Even though the sensillar equipment changes pro-
foundly between nymphs and adults, the ratio between chemore-
ceptive and mechanoreceptive sensilla seems similar and might
explain the very similar organization of the antennal sensory
neuropil. Even though sensory information detected in water and
in air is fundamentally different, especially chemosensory inputs
(Crespo, 2011), the same brain structures seem to process the
respective information in stonefly nymphs and adults, similarly to
the situation in dragonflies (Piersanti et al., 2020).

The central body and the mushroom bodies are rather small in
both nymphs and adults of *D. cephalotes*, and the mushroom bodies
lack calyces, similar to dragonflies (Piersanti et al., 2020). Contrary
to the small central body and mushroom bodies, sensory neuropils
are, however, relatively bigger as compared to the entire brain
volume than in *L. depressa*, where the central body and mushroom
bodies occupy a larger proportion of the brain in both nymphs and
adults (Rebora et al., 2013; Piersanti et al., 2020).

The presence of elongated antennal lobe glomeruli receiving
receptor neuron afferences reminds of the structures described in
other Polyneoptera such as Orthoptera, specifically in basal Tetti-
goniidae, such as the cave weta *Macropathus acanthocera*, or
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Examples of antennal receptor neuron projections from mass fills in *D. cephalotes* nymphs. Antennal afferences were stained with microruby (red) and projections to the anterior and posterior parts of the antennal lobe of the four nymph classes of *D. cephalotes* are shown. Background staining with anti-synapsin 1 antibody in green. Note the similarity in projection patterns between the different nymphal stages at the anterior and posterior level of the antennal lobe. Orientation is indicated by d dorsal, and 1 lateral.</figcaption></figure>

of Odonata nymphs (Rebora et al., 2007a, 2019), where they
probably allow searching for residual water or moist refugia when
the ponds where insects live dry out (Piersanti et al., 2007; Rebora
et al., 2007b). Thermo-hygroreceptors in the form of a round pit
hosting a small peg are reported also in adult *D. cephalotes*, and
their internal structure is described in detail (Rebora et al., 2016). A
more accurate description of the internal structure of the nonpo-
rous coeloconic pegs on the nymphal flagellum, as well as an
electrophysiological investigation, are necessary to shed light on
the actual function of these sensilla.

Another sensillum with an uncertain function in *D. cephalotes*

nymphs is the leaf sensillum (LS), which has never been described
in Plecoptera. It is visible in images published from nymphs of other
species (Neagu, 2008), but has not been found in adults (Rebora
et al., 2016). The relative abundance of these sensilla on the fla-
gellum suggests a significant sensory role. Their very small
dimension, less than 3 µm, seems to exclude a mechanoreceptive
function (see review in (Keil, 1997)), while the apparent absence of
pores not necessarily precludes a chemosensory function in water,
where a thin cuticle could allow diffusion of chemicals eluted, as in
the olfactory organs of decapod crustaceans called aesthetascs
(Derby et al., 2016; Hallberg et al., 1992; Tuchina et al., 2014;
Zacharuk et al., 1971).

4.2. Brain morphology and antennal pathway

The brain of *D. cephalotes* has a similar structure as other basal
insects, such as dragonflies (Rebora et al., 2013; Piersanti et al.,
2020). It shows three optic neuropils, an antennal lobe with elon-
gated glomeruli in the anterior part, a relatively small central body,
and mushroom bodies without calyces. We did not observe any
antennal nerve projections to the suboesophageal ganglion in our
preparations of nymphs, but this might be due to insufficient
migration time, as connectives between the supra- and sub-
oesophageal ganglia are very long.

This basic brain structure does not change between the inves-
tigated nymphal stages and the adult. Compared to the brain of the
paleopteran dragonfly *Libellula depressa*, the stonefly brain is more
compact and the structure is even more similar between nymphs
and adults (Piersanti et al., 2020). Whereas dragonflies have aglo-
merular antennal lobes, a few elongated glomeruli are present in
the anterior part of the *D. cephalotes* antennal lobe and receive
antennal afferent sensory neurons in both nymphs and adults. We
speculate that these glomeruli receive input from olfactory receptor
neurons and possibly thermo-hygroreceptors. In other insects, such
as cockroaches, ants and *Drosophila melanogaster*, specific
glomeruli, sometimes with an elongated shape, and situated at the
periphery of the antennal lobe, have been shown to receive input
from thermo-hygroreceptors (Frank et al., 2017; Mizunami et al.,
2016; Nishino et al., 2003; Ruchty et al., 2010). The posterior
aglomerular projections in the *D. cephalotes* AL may receive the
numerous mechanosensitive receptor neurons and could corre-
spond either to the aglomerular parts of the AL in Orthoptera or to
the antennal mechanosensory and motor centre described in many
other insects, such as moths and flies (Homberg et al., 1988; Ito
et al., 2014). Even though the sensillar equipment changes pro-
foundly between nymphs and adults, the ratio between chemore-
ceptive and mechanoreceptive sensilla seems similar and might
explain the very similar organization of the antennal sensory
neuropil. Even though sensory information detected in water and
in air is fundamentally different, especially chemosensory inputs
(Crespo, 2011), the same brain structures seem to process the
respective information in stonefly nymphs and adults, similarly to
the situation in dragonflies (Piersanti et al., 2020).

The central body and the mushroom bodies are rather small in
both nymphs and adults of *D. cephalotes*, and the mushroom bodies
lack calyces, similar to dragonflies (Piersanti et al., 2020). Contrary
to the small central body and mushroom bodies, sensory neuropils
are, however, relatively bigger as compared to the entire brain
volume than in *L. depressa*, where the central body and mushroom
bodies occupy a larger proportion of the brain in both nymphs and
adults (Rebora et al., 2013; Piersanti et al., 2020).

The presence of elongated antennal lobe glomeruli receiving
receptor neuron afferences reminds of the structures described in
other Polyneoptera such as Orthoptera, specifically in basal Tetti-
goniidae, such as the cave weta *Macropathus acanthocera*, or
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Anatomy of the adult brain and antennal projections from mass fills in *D. cephalotes* stained with anti-synapsin 1 antibody. **a** Individual optical sections through the protocerebrum. **b** Individual optical section through the optic lobes. **c** Three-dimensional reconstruction of the adult brain with main discernible neuropil areas. **d** Individual optical section through the antennal lobe. **e** Projections of antennal receptor neurons into the adult antennal lobe, stained through mass fills with microruby (red). The depth starting from the anterior surface is provided in each image in **e**. AL antennal lobe, CB central body, La lamina, Lo lobula, MB mushroom body, Me Medulla, orientation is indicated by **d** dorsal, **l** lateral, and **p** posterior.</figcaption></figure>

bushcrickets (Ignell et al., 2001). The orthopteran antennal lobe is, however, more clearly separated from other brain structures compared to the antennal lobe of *D. cephalotes*, and an antennal mechanosensory and motor centre can be discerned (Ignell et al., 2000). The Orthopteran brain differs also from the stonefly brain, through the presence of prominent calyces of the mushroom bodies. As the calyces of the mushroom bodies are generally associated with higher olfactory and visual processing and learning and memory functions (Couto et al., 2023; Farris and Schulmeister, 2011; Menzel, 2001), it would be interesting to investigate these aspects of stonefly behaviour, both in nymphs and adults. An important role of olfaction in stonefly larvae could be expected because of the well-structured antennal lobe and its massive innervation, as well as the olfactory responses recorded by electroantennography in adults (Rebora et al., 2017; Neupert et al., 2022). Surely the higher number of olfactory sensilla in stoneflies as compared to dragonflies, both at adult and immature stages (Rebora et al., 2008, 2016; Piersanti et al., 2020), could explain a larger AL, and perhaps the more well-structured glomeruli. These findings are in line with the hypothesis that glomerular olfactory centres are a plesiomorphic character of the tetraconate brain, and a loss of antennal lobes or glomerular substructures in several paleopteran insects are apomorphic (Schachtner et al., 2005). At the

same time, the glomerular antennal lobe of Plecoptera, here described for the first time, suggests that the aglomerular AL of Paleoptera could be not related to their aquatic lifestyle, as previously hypothesized (Schachtner et al., 2005; Strausfeld et al., 2009; Rebora et al., 2019) but, most likely, to the excellent vision, exactly as evidenced in primates where the deterioration of the olfactory repertoire occurred concomitant with the acquisition of full trichromatic colour vision (Gilad et al., 2004). On this account, it is interesting to consider that aquatic Coleoptera can show numerous microglomeruli, no glomeruli, or can even lack an antennal lobe, but different anatomical features of the AL do not clearly correlate with lifestyle and behaviours when comparing different Coleoptera species (Kollmann et al., 2016).

Physiological olfactory responses of adult stonefly antennae have been measured using electroantennographic recordings not only in *D. cephalotes* (Rebora et al., 2017), but also in *Zelandoperla fenestrate* (Neupert et al., 2022), evidencing responses to common volatiles such as different acids, ketones and alcohols. Interestingly, wing-reduced lineages of *Z. fenestrate*, showed reduced responses to several investigated compounds, with a slower time course than full-winged lineages (Neupert et al., 2022). It would, therefore be highly interesting to investigate the brain structure in these different lineages.
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Anatomy of the adult brain and antennal projections from mass fills in *D. cephalotes* stained with anti-synapsin 1 antibody. **a** Individual optical sections through the protocerebrum. **b** Individual optical section through the optic lobes. **c** Three-dimensional reconstruction of the adult brain with main discernible neuropil areas. **d** Individual optical section through the antennal lobe. **e** Projections of antennal receptor neurons into the adult antennal lobe, stained through mass fills with microruby (red). The depth starting from the anterior surface is provided in each image in **e**. AL antennal lobe, CB central body, La lamina, Lo lobula, MB mushroom body, Me Medulla, orientation is indicated by **d** dorsal, **l** lateral, and **p** posterior.</figcaption></figure>

bushcrickets (Ignell et al., 2001). The orthopteran antennal lobe is, however, more clearly separated from other brain structures compared to the antennal lobe of *D. cephalotes*, and an antennal mechanosensory and motor centre can be discerned (Ignell et al., 2000). The Orthopteran brain differs also from the stonefly brain, through the presence of prominent calyces of the mushroom bodies. As the calyces of the mushroom bodies are generally associated with higher olfactory and visual processing and learning and memory functions (Couto et al., 2023; Farris and Schulmeister, 2011; Menzel, 2001), it would be interesting to investigate these aspects of stonefly behaviour, both in nymphs and adults. An important role of olfaction in stonefly larvae could be expected because of the well-structured antennal lobe and its massive innervation, as well as the olfactory responses recorded by electroantennography in adults (Rebora et al., 2017; Neupert et al., 2022). Surely the higher number of olfactory sensilla in stoneflies as compared to dragonflies, both at adult and immature stages (Rebora et al., 2008, 2016; Piersanti et al., 2020), could explain a larger AL, and perhaps the more well-structured glomeruli. These findings are in line with the hypothesis that glomerular olfactory centres are a plesiomorphic character of the tetraconate brain, and a loss of antennal lobes or glomerular substructures in several paleopteran insects are apomorphic (Schachtner et al., 2005). At the

same time, the glomerular antennal lobe of Plecoptera, here described for the first time, suggests that the aglomerular AL of Paleoptera could be not related to their aquatic lifestyle, as previously hypothesized (Schachtner et al., 2005; Strausfeld et al., 2009; Rebora et al., 2019) but, most likely, to the excellent vision, exactly as evidenced in primates where the deterioration of the olfactory repertoire occurred concomitant with the acquisition of full trichromatic colour vision (Gilad et al., 2004). On this account, it is interesting to consider that aquatic Coleoptera can show numerous microglomeruli, no glomeruli, or can even lack an antennal lobe, but different anatomical features of the AL do not clearly correlate with lifestyle and behaviours when comparing different Coleoptera species (Kollmann et al., 2016).

Physiological olfactory responses of adult stonefly antennae have been measured using electroantennographic recordings not only in *D. cephalotes* (Rebora et al., 2017), but also in *Zelandoperla fenestrate* (Neupert et al., 2022), evidencing responses to common volatiles such as different acids, ketones and alcohols. Interestingly, wing-reduced lineages of *Z. fenestrate*, showed reduced responses to several investigated compounds, with a slower time course than full-winged lineages (Neupert et al., 2022). It would, therefore be highly interesting to investigate the brain structure in these different lineages.
### 4.3. Stonefly sensory ecology in water vs air

Most studies on stonefly behavioural ecology have been performed on nymphs, because of their long life in water and their behaviour as top-predator within the lotic macroinvertebrate food web (Tierno de Figueroa et al., 2015; Tierno De Figueroa and López-Rodríguez, 2019). Adult Plecoptera have a short lifespan, many species do not feed at all, or only occasionally, and the only aspect of their behaviour deeply investigated is reproduction, in particular the employment of intersexual vibrational communication to facilitate mate encounters, evaluation, copulation, guarding etc (Tierno De Figueroa and López-Rodríguez, 2019). In this context, the differences between the sensory equipment of antennal sensilla in nymphs and adults of *D. cephalotes*, could be simply related to the evident difference in adult and nymph ecology. In adults, olfactory sensilla, gustatory sensilla or mechanoreceptors would be very important for mate finding, recognition and evaluation, both in encounter sites constituted by riparian vegetation or river stones (Jáimez-Cuéllar and De Figueroa, 2005; Rebora et al., 2016). In nymphs, mechanoreceptors that seem to play a proprioceptive role, could be probably related to the indirect perception of currents in the fast river streams, where these insects typically live. The ability to perceive water movements, directly or indirectly by proprioceptive perception of antennal movements, is probably relevant in water also to discriminate between prey and non-prey hydrodynamic cues, as suggested by some behavioural experiments using mayfly larvae and dummies (Peckarsky and Wilcox, 1989), and similar to what is described for sit-and-wait predatory dragonfly nymphs (Rebora et al., 2004). The putative olfactory receptors of nymphs, namely the CSC and the uniporous coeloconic pegs, are probably relevant for prey perception, as suggested by behavioural investigations (Williams, 1987) and some personal observations.

## 5. Conclusions

It seems that in hemimetabolous insects, both in the Paleoptera model *L. depressa* (Piersanti et al., 2020) and in the Polyneoptera model *D. cephalotes*, the general morphology of the brain and the sensory circuitry remain quite similar during development in spite of the substantial differences in antennal sensilla between nymphs and adults and their different environment. This suggests that the same brain centres are able to process highly diverging information provided through different sensory structures adapted to perceive different cues in water and air. Completely different are the results obtained in the unique study on the development of the brain in holometabolous aquatic insects, according to our knowledge, which describes the complete transformation of the sensory neuropils in the mosquito *Aedes aegypti* (Mysore et al., 2011) and references therein), whose larvae possess very few chemoreceptive sensilla on the antennae (Zacharuk and Blue, 1971), and a brain without recognizable antennal lobe. This difference is in line with the need for developmental plasticity in hemimetabolous insects, necessary to maintain functionality throughout the development, when the lack of a pupal stage does not allow metamorphic changes in the nervous system.

## Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

## CRediT authorship contribution statement

Silvana Piersanti: Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Project

administration, Investigation, Conceptualization. **Manuela Rebora:** Writing – review & editing, Validation, Resources, Investigation, Conceptualization. **Gianandrea Salerno:** Writing – review & editing, Visualization, Validation, Formal analysis, Data curation. **Simon Vitecek:** Writing – review & editing. **Sylvia Anton:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Investigation, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

We thank Antonello Sotgiu for his technical support. We thank Giorgia Carboni Marri, Noemi Covarelli and Giulia Petroni and for their contribution to insect collection and morphological observations.

## References

* Ache, B.W., Derby, C.D., 1985. Functional organization of olfaction in crustaceans. *Trends Neurosci.* 35–36.

* Altner, H., 1977. Insect sensillum specificity and structure: an approach to a new typology. In: Le Magnen, J., Mac Leod, P. (Eds.), Olfaction and Taste VI. Information Retrieval, London, pp. 295–303.

* Anton, S., Ignell, R., Hansson, B.S., 2002. Developmental changes in the structure and function of the central olfactory system in gregarious and solitary desert locusts. *Microsc. Res. Tech.* 56, 281–291.

* Badji, C.A., Sol-Mochkovitch, Z., Fallais, C., Sochard, C., Simon, J.-C., Outreman, Y., Anton, S., 2021. Alarm Pheromone Responses Depend on Genotype, but Not on the Presence of Facultative Endosymbionts in the Pea Aphid *Acyrthosiphon pisum*. *Insects* 12, 43.

* Chambille, I., Pierre Rospars, J., 1985. Neurons and identified glomeruli of antennal lobes during postembryonic development in the cockroach *Blaberus cranifer* burm. (dictyoptera: blaberidae). *Int. J. Insect Morphol. Embryol.* 14, 203–226.

* Couto, A., Young, F.J., Atzeni, D., Marty, S., Melo-Flórez, L., Hebberecht, L., Monllor, M., Neal, C., Cicconardi, F., McMillan, W.O., Montgomery, S.H., 2023. Rapid expansion and visual specialisation of learning and memory centres in the brains of Heliconiini butterflies. *Nat. Commun.* 14, 4024.

* Crespo, J.G., 2011. A review of chemosensation and related behavior in aquatic insects. *J. Insect Sci.* 11, 62–39.

* Derby, C.D., Kozma, M.T., Senatore, A., Schmidt, M., 2016. Molecular Mechanisms of Reception and Perireception in Crustacean Chemoreception: A Comparative Review. *Chem. Senses* 41, 381–398.

* Dijkstra, K.-D.B., Monaghan, M.T., Pauls, S.U., 2014. Freshwater biodiversity and aquatic insect diversification. *Annu. Rev. Entomol.* 59, 143–163.

* Eisenbeis, G., Wichard, W., 2010. Wasserhaushalt, Osmo- und lonenregulation sowie Exkretion. In: Dettner, K., Peters, W. (Eds.), Lehrbuch Der Entomologie. Spektrum Akademischer Verlag, Heidelberg, pp. 127–163.

* Farris, S.M., Schulmeister, S., 2011. Parasitoidism, not sociality, is associated with the evolution of elaborate mushroom bodies in the brains of hymenopteran insects. *Proc. R. Soc. A* B 278, 940–951.

* Frank, D.D., Enjin, A., Jouandet, G.C., Zaharieva, E.E., Para, A., Stensmyr, M.C., Gallio, M., 2017. Early Integration of Temperature and Humidity Stimuli in the *Drosophila* Brain.

* Gaino, E., Rebora, M., 1998. Ultrastructure of the antennal sensilla of the mayfly *Baetis rhodani* (Pictet) (Ephemeroptera: Baetidae). *Int. J. Insect Morphol. Embryol.* 27, 143–149.

* Gaino, E., Rebora, M., 1999. Larval antennal sensilla in water-living insects. *Microsc. Res. Tech.* 47, 440–457.

* Gaino, E., Rebora, M., 2001. Apical antennal sensilla in nymphs of *Libellula depressa* (Odonata: Libellulidae). *Invertebr. Biol.* 120, 162–169.

* Gilad, Y., Wiebe, V., Przeworski, M., Lancet, D., Pääbo, S., 2004. Loss of Olfactory Receptor Genes Coincides with the Acquisition of Full Trichromatic Vision in Primates. *PLoS Biol.* 2, e5.

* Graf, W., Lorenz, A., de Figueroa, J.M.T., Lücke, S., López-Rodríguez, M.J., Davies, C., 2009. Distribution and Ecological Preferences of European Freshwater Organisms: Volume 2 Plecoptera. Pensoft Publishing.

* Hallberg, E., Johansson, K.U.I., Elofsson, R., 1992. The aesthetasc concept: Structural variations of putative olfactory receptor cell complexes in crustacea. *Microsc. Res. Tech.* 22, 325–335.

* Homberg, U., Montague, R., Hildebrand, J., 1988. Anatomy of antenna-cerebral pathways in the brain of the sphinx moth *Manduca sexta*. *Cell Tissue Res.* 254, 255–281.

* Iannilli, V., Fochetti, R., de Figueroa, J.T., 2002. Life cycle of *Dinocras cephalotes*
### 4.3. Stonefly sensory ecology in water vs air

Most studies on stonefly behavioural ecology have been performed on nymphs, because of their long life in water and their behaviour as top-predator within the lotic macroinvertebrate food web (Tierno de Figueroa et al., 2015; Tierno De Figueroa and López-Rodríguez, 2019). Adult Plecoptera have a short lifespan, many species do not feed at all, or only occasionally, and the only aspect of their behaviour deeply investigated is reproduction, in particular the employment of intersexual vibrational communication to facilitate mate encounters, evaluation, copulation, guarding etc (Tierno De Figueroa and López-Rodríguez, 2019). In this context, the differences between the sensory equipment of antennal sensilla in nymphs and adults of *D. cephalotes*, could be simply related to the evident difference in adult and nymph ecology. In adults, olfactory sensilla, gustatory sensilla or mechanoreceptors would be very important for mate finding, recognition and evaluation, both in encounter sites constituted by riparian vegetation or river stones (Jáimez-Cuéllar and De Figueroa, 2005; Rebora et al., 2016). In nymphs, mechanoreceptors that seem to play a proprioceptive role, could be probably related to the indirect perception of currents in the fast river streams, where these insects typically live. The ability to perceive water movements, directly or indirectly by proprioceptive perception of antennal movements, is probably relevant in water also to discriminate between prey and non-prey hydrodynamic cues, as suggested by some behavioural experiments using mayfly larvae and dummies (Peckarsky and Wilcox, 1989), and similar to what is described for sit-and-wait predatory dragonfly nymphs (Rebora et al., 2004). The putative olfactory receptors of nymphs, namely the CSC and the uniporous coeloconic pegs, are probably relevant for prey perception, as suggested by behavioural investigations (Williams, 1987) and some personal observations.

## 5. Conclusions

It seems that in hemimetabolous insects, both in the Paleoptera model *L. depressa* (Piersanti et al., 2020) and in the Polyneoptera model *D. cephalotes*, the general morphology of the brain and the sensory circuitry remain quite similar during development in spite of the substantial differences in antennal sensilla between nymphs and adults and their different environment. This suggests that the same brain centres are able to process highly diverging information provided through different sensory structures adapted to perceive different cues in water and air. Completely different are the results obtained in the unique study on the development of the brain in holometabolous aquatic insects, according to our knowledge, which describes the complete transformation of the sensory neuropils in the mosquito *Aedes aegypti* (Mysore et al., 2011) and references therein), whose larvae possess very few chemoreceptive sensilla on the antennae (Zacharuk and Blue, 1971), and a brain without recognizable antennal lobe. This difference is in line with the need for developmental plasticity in hemimetabolous insects, necessary to maintain functionality throughout the development, when the lack of a pupal stage does not allow metamorphic changes in the nervous system.

## Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

## CRediT authorship contribution statement

Silvana Piersanti: Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Project

administration, Investigation, Conceptualization. **Manuela Rebora:** Writing – review & editing, Validation, Resources, Investigation, Conceptualization. **Gianandrea Salerno:** Writing – review & editing, Visualization, Validation, Formal analysis, Data curation. **Simon Vitecek:** Writing – review & editing. **Sylvia Anton:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Investigation, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

We thank Antonello Sotgiu for his technical support. We thank Giorgia Carboni Marri, Noemi Covarelli and Giulia Petroni and for their contribution to insect collection and morphological observations.

## References

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* Altner, H., 1977. Insect sensillum specificity and structure: an approach to a new typology. In: Le Magnen, J., Mac Leod, P. (Eds.), Olfaction and Taste VI. Information Retrieval, London, pp. 295–303.

* Anton, S., Ignell, R., Hansson, B.S., 2002. Developmental changes in the structure and function of the central olfactory system in gregarious and solitary desert locusts. *Microsc. Res. Tech.* 56, 281–291.

* Badji, C.A., Sol-Mochkovitch, Z., Fallais, C., Sochard, C., Simon, J.-C., Outreman, Y., Anton, S., 2021. Alarm Pheromone Responses Depend on Genotype, but Not on the Presence of Facultative Endosymbionts in the Pea Aphid *Acyrthosiphon pisum*. *Insects* 12, 43.

* Chambille, I., Pierre Rospars, J., 1985. Neurons and identified glomeruli of antennal lobes during postembryonic development in the cockroach *Blaberus cranifer* burm. (dictyoptera: blaberidae). *Int. J. Insect Morphol. Embryol.* 14, 203–226.

* Couto, A., Young, F.J., Atzeni, D., Marty, S., Melo-Flórez, L., Hebberecht, L., Monllor, M., Neal, C., Cicconardi, F., McMillan, W.O., Montgomery, S.H., 2023. Rapid expansion and visual specialisation of learning and memory centres in the brains of Heliconiini butterflies. *Nat. Commun.* 14, 4024.

* Crespo, J.G., 2011. A review of chemosensation and related behavior in aquatic insects. *J. Insect Sci.* 11, 62–39.

* Derby, C.D., Kozma, M.T., Senatore, A., Schmidt, M., 2016. Molecular Mechanisms of Reception and Perireception in Crustacean Chemoreception: A Comparative Review. *Chem. Senses* 41, 381–398.

* Dijkstra, K.-D.B., Monaghan, M.T., Pauls, S.U., 2014. Freshwater biodiversity and aquatic insect diversification. *Annu. Rev. Entomol.* 59, 143–163.

* Eisenbeis, G., Wichard, W., 2010. Wasserhaushalt, Osmo- und lonenregulation sowie Exkretion. In: Dettner, K., Peters, W. (Eds.), Lehrbuch Der Entomologie. Spektrum Akademischer Verlag, Heidelberg, pp. 127–163.

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* Gaino, E., Rebora, M., 1998. Ultrastructure of the antennal sensilla of the mayfly *Baetis rhodani* (Pictet) (Ephemeroptera: Baetidae). *Int. J. Insect Morphol. Embryol.* 27, 143–149.

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* Gaino, E., Rebora, M., 2001. Apical antennal sensilla in nymphs of *Libellula depressa* (Odonata: Libellulidae). *Invertebr. Biol.* 120, 162–169.

* Gilad, Y., Wiebe, V., Przeworski, M., Lancet, D., Pääbo, S., 2004. Loss of Olfactory Receptor Genes Coincides with the Acquisition of Full Trichromatic Vision in Primates. *PLoS Biol.* 2, e5.

* Graf, W., Lorenz, A., de Figueroa, J.M.T., Lücke, S., López-Rodríguez, M.J., Davies, C., 2009. Distribution and Ecological Preferences of European Freshwater Organisms: Volume 2 Plecoptera. Pensoft Publishing.

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