The subgenual organ complex in the cave cricket Troglophilus neglectus (Orthoptera: Rhaphidophoridae): comparative innervation and sensory evolution

Comparative studies of the organization of nervous systems and sensory organs can reveal their evolution and specific adaptations. In the forelegs of some Ensifera (including crickets and tettigoniids), tympanal hearing organs are located in close proximity to the mechanosensitive subgenual organ (SGO). In the present study, the SGO complex in the non-hearing cave cricket Troglophilus neglectus (Rhaphidophoridae) is investigated for the neuronal innervation pattern and for organs homologous to the hearing organs in related taxa. We analyse the innervation pattern of the sensory organs (SGO and intermediate organ (IO)) and its variability between individuals. In T. neglectus, the IO consists of two major groups of closely associated sensilla with different positions. While the distal-most sensilla superficially resemble tettigoniid auditory sensilla in location and orientation, the sensory innervation does not show these two groups to be distinct organs. Though variability in the number of sensory nerve branches occurs, usually either organ is supplied by a single nerve branch. Hence, no sensory elements clearly homologous to the auditory organ are evident. In contrast to other non-hearing Ensifera, the cave cricket sensory structures are relatively simple, consistent with a plesiomorphic organization resembling sensory innervation in grasshoppers and stick insects.

2. Introduction

For several species of insects, the neuronal innervation pattern of legs has been documented with particular emphasis on the complex sensory organs containing numerous sensory neurons [1–4]. Scolopidial sensilla form internal sensory organs distributed over all body segments. They consist of one or more sensory neurons and additional cell types [5–8]. The sensory neurons of scolopidia are activated by stretching of the dendrite and code for a variety of mechanical forces, caused by body tension as well as external stimuli like substrate vibrations or sound [8–12].

The subgenual organ (SGO) is an important scolopidial organ present in the tibia of most insects, which is most sensitive to substrate vibrations [13] but may also respond to airborne sound [14–18]. Among several species of Blattodea and Orthoptera, the SGO is commonly found next to other, closely associated sensory organs also containing scolopidial sensilla [8,19–21]. These additional organs are, for example, the distal organ (DO) [19,22] or the intermediate organ (IO) [23]. Because in most orthopteroid insects more than one sensory organ occurs in the proximal tibia, these are together called the SGO complex [21]. These organs are usually supplied by distinct nerve branches.

Ensifera (the ‘long-horned grasshoppers’) are well studied for their auditory organs (tympanal organs characterized by thin tympanal membranes), located in the foreleg tibia of some taxa [24–26]. In these tympanal organs, specific sets of auditory sensilla occur close to the SGO, which respond to airborne sound with high sensitivity, e.g. in crickets [27–30] and tettigoniids [23,31–33]. In tettigoniids and some related hearing Ensifera, the so-called complex tibial organ consists of the SGO, the IO and the crista acustica (table 1). The tettigoniid crista acustica contains the auditory sensilla [31,32,43], which respond to airborne sound with a specific frequency tuning of individual sensilla [17,44,45]. The auditory sensilla are usually arranged along the proximo-distal leg axis in a characteristical linear array [23,31,43,46,47]. A morphologically similar hearing organ (the tympanal organ) is present in crickets [28,30,48]. However, the homology of tympanal organs in crickets and tettigoniids is not adequately resolved [23,26,49,50].

Besides these tympanate groups, Ensifera also include atympanate groups that completely lack a tegminal (wing) stridulation apparatus for sound production and tympanal ears in the foreleg (table 1) [25,26,51].

The Rhaphidophoridae or ‘cave crickets’ generally lack auditory tympana or vestiges thereof [24,52] and may be a basal group in the ensiferan lineage [25]. These species occupy a great diversity of niches, most including different degrees of specialization to cave life [53]. Troglophilus neglectus uses caves only as a refuge during day time and the winter season, while it forages and reproduces in forests during night time [54,55]. Its mating behaviour includes male-produced vibratory signals and seems to reflect a primitive signalling pattern among Ensifera [56,57]. In accordance with this, the SGO complex of the species consists of the SGO and the IO only [41], and both are sensitive to vibrations transferred to the sensory organs through the leg [58]. The sensory organs respond to airborne sound only of low frequencies at high amplitudes [41]. The IO is anatomically differentiated into a proximal intermediate organ (pIO) and a distal intermediate organ (dIO) with specific arrangements of sensory cells [41]. By contrast, all other atympanate taxa in Ensifera investigated so far include a sensory organ clearly homologous to the crista acustica of tettigoniids [51].

The evolution of this structural diversity in the SGO complex and the sensory functions of the different organs across Ensifera are not resolved. In addition to the SGO, another organ is commonly located distally of the SGO. This is termed the DO (Blattidae, Mantophasmatodea, Phasmatodea, Caelifera) or IO (Ensifera), and these organs are not well characterized in terms of their physiology. Since these organs are common among Orthopteroidea and share the position in the anterior tibia, they may actually be homologous [51]. The physiological function of the IO depends on the presence of a tympanal organ. In tettigoniid species with tympanal hearing organs, the IO may respond to airborne sound (up to 8 kHz) [14,17,59]. In atympanate legs of tettigoniids, the IO is insensitive to sound but sensitive to vibrations with a maximum sensitivity between 600 and 1000 Hz [60]. The DO in cockroaches has been suggested to measure changes in the hemolymphe pressure [22] but may contain vibration-sensitive sensilla [13]. Both DO and IO may differentiate into anatomically distinct, proximal and distal sets of sensilla [22,61–64]. The crista acustica is the main auditory organ in Tettigoniidae [31,44], Haglidae [38] and Anostostomatidae (wetas) [65]. The physiology of the crista acustica homologue in atympanate Ensifera has not been studied in detail but was suggested to be vibration-sensitive, possibly with a different tuning than sensilla in the SGO [40]. A small scolopidial organ, the Nebenorgan [22,35] or accessory organ [37,64], may occur on the posterior side in the leg. In cockroaches, it has been suggested to perceive low-frequency vibrations [22].

With respect to the evolution of sensory organs, it is plausible that the SGO complex in orthopteroid insects ancestrally contained two major organs innervated by the main sensory nerve, the SGO and DO/IO, as these organs are commonly present [51]. In Tettigonioidea, the crista acustica homologue or crista acustica was added. The highly conserved distribution of the crista acusticahomologue in atympanate taxa raises the question if the neuroanatomy of the SGO complex in cave crickets is indeed ‘primitive’ compared to the hearing Ensifera [41] and resembles a plesiomorphic organization prior to the evolution of the crista acustica homologue, or if it may in fact share this sensory homologue to auditory sensilla with other atympanate Ensifera. Remarkably, the five to six neurons in the dIO of T. neglectus occur in a line [41], not unlike the crista acustica.

In tettigonioids, each of the scolopidial organs in the SGO complex is usually innervated by a distinct nerve branch [1,4,31,39,64]. In T. neglectus, the sensory innervation of the SGO complex has not been documented [41]. Here, we analyse innervation patterns of the sensory organs in T. neglectus for their similarity to other tettigonioids. Axonal pathways or innervation patterns of sensory organs are helpful for identification and comparison of neural elements across taxa, as they appear to be rather conserved in evolution [66–70]. Some variability in the innervation pattern can nevertheless be expected between individuals even within one species, as was described from ensiferan SGOs in both tympanate and atympanate species [1,39]. This variability is most probably due to stochastic alterations in axonal pathfinding of sensory neurons during embryogenesis.

In this study, we describe the innervation pattern of the SGO and IO and compare its variability between individuals. The aim is to document whether the pIO and dIO in T. neglectus have a joint or separate innervation, supporting them as a single organ or rather two distinct organs. These data are compared to those from other Ensifera. If separate innervations for the pIO and the dIO are confirmed, this might indicate that the dIO sensilla correspond to the crista acustica homologue of atympanate Ensifera, thus highlighting the presence of shared sensory structures across the atympanate groups of Ensifera.

3. Material and methods

3.1 Animals

Animals were caught in northwestern Slovenia, in a cave in the vicinity of Most na Soči. They were maintained in the laboratory in terraria filled with moss, at room temperature and in constant darkness and fed with dried fish food ad libitum. High humidity in the terraria was maintained by keeping the moss moist.

3.2 Axonal tracing experiments

Retrograde tracing of both nerves 5B1 and 5B2 was carried out to document the neuroanatomical organization of the tibial organ in T. neglectus. The legs were cut off at the proximal femur and mounted in Sylgaard-covered glass dishes (Sylgaard 184, Suter Kunstoffe AG, Fraubrunnen, Switzerland) with insect pins under locust saline [71] (pH=7.2). They were opened ventrally with a piece of a razor blade. Nerves were cut with iridectomy scissors proximal of the femur–tibia joint. In some experiments, only one of the leg nerves 5B1 or 5B2 was filled to show the innervated structures, while in other experiments both were filled to show the whole sensory complex. The cut nerve ends were transferred into a glass capillary filled with 5% CoCl₂ solution (cobalt chloride from Merck, Darmstadt, Germany) dissolved in distilled water [72,73]. The preparations were then incubated for 48 h at 4°C. For visualization of the fills, the cobalt was precipitated by incubating the legs in a solution of 1% ammonium sulfide (Fluka, Buchs, Switzerland) in locust ringer for 10–15 min at room temperature. The legs were rinsed in locust saline and immediately fixed for 60 min in chilled 4% paraformaldehyde (Sigma Chemicals, St Louis, MO, USA) dissolved in phosphate buffer (0.04 mol l⁻¹ Na₂HPO₄, 0.00574 mol l⁻¹ NaH₂PO₄×2H₂O; pH=7.4). The preparations were dehydrated in a graded ethanol series (Carl Roth, Karlsruhe, Germany) for 60 min at each step, and finally cleared in methyl salicylate (Fluka). Overall, 48 leg preparations were of adequate quality to be analysed for this study. All thoracic leg pairs were included. As previously found [41], there were no differences in the sensory neuroanatomy between organs in different leg pairs.

Anterograde tracing of the nerve 5B1 for sensory projections into the central nervous system was carried out. After the animals were anaesthetized with CO₂, they were mounted in a Petri dish ventral side up, using insect pins and a beeswax–colophony mixture. The cuticle was removed anteriorly and proximally in the femur. The nerve was cut and placed into a glass capillary filled with NiCl₂, or Lucifer Yellow (each 5%; Sigma) dissolved in distilled water. After incubation at 4°C for 48 h, the thoracic ganglia were excised and NiCl₂ was precipitated by the saturated solution of rubeanic acid (Merck) in 100% ethanol, by adding three to five drops to 1 ml of saline for 10 min, fixed in Carnoy's solution (1:4 mixture of glacial acetic acid (Sigma) and 100% ethanol) and silver intensified [74]. All preparations were dehydrated in ethanol series (for 10 min in each step) and cleared in methyl salicylate (Sigma). After the morphological analysis of wholemount preparations (see below), preparations of T1 were embedded in epoxy resin (Agar 100 resin kit; Agar scientific, Stansted, UK), serially sectioned in the transverse plane (18 μm) and embedded in Floromount (Sigma).

3.3 Analysis and documentation

The preparations were viewed with an Olympus BH-2 microscope (peripheral innervation) and with a Leica DMRB/E microscope (central projections). Photos of the respective preparations were taken with a Leica DCF-320 camera (2088×1055 pixel) and Zeiss AxioCam MRc camera (1300×1030 pixel), respectively, that was attached to the microscope. Most preparations included here were photographed in series, and stacked pictures were obtained with the freeware program CombineZP (http://www.hadleyweb.pwp.blueyonder.co.uk/CZP/Installation.htm). Sensory organs were documented by drawing with the help of a drawing attachment (Leitz) to a Leitz Dialux microscope (Leitz, Wetzlar, Germany) and later redrawn in ink. Drawings of central projection patterns were made from photomicrographs using a graphic tablet (Wacom, Kazo, Japan) and Adobe Photoshop (Adobe Systems, San Jose, CA, USA). Photomicrographs were adjusted for brightness and contrast using Corel PhotoPaint (Corel, Ottawa, Canada) or Photoshop. Figures were assembled and labelled using CorelDraw 11 (Corel) and Adobe Illustrator (Adobe Systems).

3.4 Nomenclature of leg nerves and nerve branches

The two main leg nerves are referred to as N5B1 (main sensory nerve) and N5B2 (main motor nerve) following Campbell [75]. The nerve branches entering these major nerves in the tibia that ultimately innervate target organs are numbered consecutively (-T1, -T2, etc.) [1].

3.5 Statistical analysis

The statistical analysis was carried out with Prism 4 (GraphPad, San Diego, CA, USA).

4. Results

4.1 The subgenual organ complex in Troglophilus neglectus

The SGO complex and its innervation in T. neglectus were revealed by cobalt tracing of the leg nerves (figure 1). Two nerves enter the femur: N5B1 (main sensory nerve) and N5B2 (main motor nerve). The nerve branches entering both nerves have been numbered in order of appearance from proximal to distal (figures 1a and 2f).

From N5B1, the first dorsal branch (N5B1-T1) innervates tibial sensory hairs (figure 2f). The next branch, N5B1-T2, supplies the SGO, and the nerve branch extending further distally is termed N5B1-T3. This nerve branch, N5B1-T3, innervates the IO. Commonly, three further nerve branches enter N5B1-T3, which all innervate hair sensilla in the tibia (N5B1-T4, -T5, -T6; figure 2f).

4.2 Neuroanatomy and innervation of the intermediate organ

The IO is exclusively innervated by N5B1 (figures 1 and 2). Sensilla in the IO can be divided into the pIO and the dIO (figures 1a–c and 2a–d). The scolopidial sensilla of these two groups lie in close proximity (figures 1 and 2). The dIO contains sensilla with the cell bodies arranged in a line (figure 2a,b). Sensilla of the dIO are located more distally of the pIO (figure 2a–d), and the dendrites of the pIO sensilla point more dorsally than those of the dIO (figure 2c,d). Dendrites of the pIO are attached to the tectorial membrane (figure 2c,d), which also covers the dIO. Somata of the pIO sensilla locate more dorsally than those in the dIO.

There are usually no distinct nerve branches separately innervating the pIO and the dIO (figure 2c,d). In a few cases, some of the pIO axons may form a minor branch before joining the nerve 5B1 (figure 2e). Yet, this innervation does not apply for all pIO sensilla but only the dorsal-most ones, as the ventral pIO sensilla lie at the N5B1-T3 also innervating the dIO (figure 2e). The minor branch was not considered a distinct nerve branch from N5B1 supplying the sensory organ because (i) it was rather short (not longer than the group of sensilla group it supplies) and (ii) it did not innervate the complete set of pIO sensilla. Similar smaller nerve branches are also present within different neuron groups of the SGO in T. neglectus (not shown) and may be commonly formed in scolopidial sensilla arranged along the dorsoventral axis of the leg. No distinct nerves or nerve branches usually exist for the complete set of pIO sensilla, though some variability in the innervation pattern occurs (see below).

4.3 Variability in the sensory innervation branching pattern

We have focused the analysis on N5B1, as this nerve innervates the anterior SGO and the entire IO. The IO consists of two anatomically recognizable sets of sensilla, while the neuronal innervation by N5B1 usually supports it as one unit of sensilla (figures 1 and 2). The most common innervation pattern by N5B1 is here referred to as a ‘consensus innervation’, which was found in 50% of preparations (figure 2f). Most of the variation in the N5B1 branching pattern occurred in the number of nerve branches entering N5B1 on the ventral side, which innervated hair sensilla, but not the scolopidial sense organs (for scolopidial organs, see below). Most commonly, three nerve branches occur to innervate hair sensilla (termed N5B1-T4, -T5, -T6), but a higher number occurred in a few cases (not shown).

In addition, the variability in the innervation pattern was documented for the SGO and the IO. The SGO is usually supplied via N5B1 by N5B1-T2 (figure 1). In some preparations (n=10 from 48), two nerve branches of N5B1 innervate the subgenual neurons (figure 3a). In this situation, the two SGO nerve branches are not shared with the IO. Variability in the innervation pattern is also notable for the IO: while the majority of leg preparations (n=41 from 48) showed innervation by a single nerve branch, N5B1-T3, in some cases (n=7 from 48), two nerve branches joining N5B1-T3 were innervating different parts of the IO. The branch supplying the pIO was in a few preparations longer, running very close to N5B1 (figure 3a). In such cases, a distinct innervation for a subset of the pIO sensilla is clearly present. In an extreme case, two long nerve branches separate from N5B1 and supply sensilla in the pIO as well as the distal pIO and the dIO (figure 3a).

These different innervation patterns from N5B1 were compared and quantified for both the SGO and the IO (figure 3b). Both organs are most commonly innervated by one distinct nerve branch each (figure 3b) (SGO: 79.2% and IO: 85.4%). The number of preparations with two innervating nerve branches was much lower for both organs, and the difference in proportions of innervating nerve branches between the organs is not statistically significant (χ²-test: d.f.=1, χ²=0.6433, p=0.4225). In the majority of preparations, both the SGO and the IO are thus innervated each by a single N5B1 nerve branch.

4.4 Central projections of sensory afferents

Sensory projections of the nerve 5B1 show a similar pattern in all three thoracic segments (figure 4a). The axon bundle enters the segmental ganglion through the slightly anterior part of the leg nerve. After giving off short processes laterally, it bifurcates medially in the neuropile into two largely separate projections. The projections are strictly ipsilateral in all thoracic segments, with the larger anterior projection terminating about 50 μm laterally, and the posterior projection about 100 μm laterally from the midline. Histological sections demonstrate axonal arborizations in the leg nerve root R5iii and the medial ventral association centre (mVAC) neuropile (figure 4b). Processes of the anterior projection are present in the ventral and intermediate parts of the mVAC (figure 4b(i)), while processes of the posterior projection are present in the ventro-lateral part of the mVAC (figure 4b(ii)).