On the Morphological Adaptations of the Thorax of Sphagniana sphagnorum (Ensifera, Tettigoniidae) that Enable Complex

Stridulation via the Forewings

By

Sara-Jane Gutierrez

A thesis submitted in conformity with the requirements for the degree of Master of Science Ecology and Evolutionary Biology

University of Toronto

©Copyright by Sara-Jane Gutierrez 2015

ii

On the Morphological Adaptations of the Thorax of Sphagniana sphagnorum (Ensifera, Tettigoniidae) that Enable Complex Stridulation

via the Forewings

Sara Jane Gutierrez

Master of Science

Ecology and Evolutionary Biology University of Toronto

2015

Abstract

Males of the flightless tettigoniid Sphagniana sphagnorum generate a complex two-spectrum

signal via stridulation, using acoustically adapted forewings (tegmina). Complex-wave rapidly

decaying pulses in trains, made upon the distal half of the file, have a broad-band (17-25 kHz)

spectrum; short, spaced, ultrasonic and strongly sinusoidal 35 kHz pulses are emitted as trains over

the file's proximal half. I studied how S. sphagnorum tegminal morphology enables the production

of these two different spectra with the same forewings. The storage of elastic energy in the scraper

is hypothesized to play a key role in driving the scraper over the file at a necessary tooth-contact

rate. Scraper morphology indicates structural adaptations enabling storage of elastic energy. Wax

loading tested the role of scraper-pit flexibility in scraper movement. Sound levels in resonant

spectra were more substantially affected than those associated with broadband generation.

Morphology and alignment of pteralia during tegmino-tegminal stridulation is also investigated.

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Acknowledgements

Glenn my experience as your graduate student has been both nurturing and transformative. I began this long

journey with naiveté but under your guidance I discovered that I lacked a true comprehension of the complexities

pertaining to the natural world. Having taken many phenomena for granted you instilled in me the necessity for

thoughtful observation, critical thought, and the importance of asking “why” – why has nature selected for a

given structure or behaviour? Your insistence on asking “why”, or “imagining it as it isn’t”, has become a part

of my daily habits. I find myself asking such questions for any matter that may arise, from contemplating simple

questions to musing the big mysteries of life. In short you have transformed me into a philosopher that studies

musical bugs on the side. I am forever grateful to you for having given me the opportunity to be your last graduate

student, and for never having given up on me even after I gave up on myself. I owe much of the person I have

become over these past few years to you. Also, I’d like to thank both you and Dita for your openness and

hospitality, you have made me feel like family. Glenn, you have been a great friend, father, and mentor.

Dr. Angela Lange and Dr. Darryl Gwynne thank you for having formed a part of my committee and for having

had the patience to help me move forward with my thesis. Your support and guidance have been invaluable

throughout my tenure as a graduate student.

Caroline Bouilly, Susan Dixon, and Cindy Short thank you for the support, encouragement, knowledge, and

opportunities you have provided me over the past few years. Working alongside the three of you has been a

rewarding experience for which I am very grateful; you truly made my time here at UTM that much more

worthwhile.

Andrew Veglio thank you for designing and fabricating the wax-heating device – that little machine I owe all

of my successful scraper pit experiments to.

To my family (Mama, Papa, Mari, and Mikael) where would I be without you? You are all my number one fans

and largely the reason that I am where I am today. Thank you for always providing me with encouragement and

the belief that I can do anything. I love you all dearly.

Sara-Jane

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Table of Contents

Abstract ii Acknowledgements iii Table of Contents iv List of Figures vi List of Tables vii List of Terms and Abbreviations viii Chapter 1: Introduction Sphagniana sphagnorum: an overview of the only endemically Canadian katydid 1

Type Locality and Distribution 1 An Introduction to the Song of Sphagniana sphagnorum 1 Elastic Resonant Stridulation 3 Objectives and Research Questions 4

Chapter 2: Materials and Methods

Location of Study Animal Collection 5 Generator Morphology 5 Sound Analysis 5 Wax Experiment 6 Statistical Analysis 7

Chapter 3: Results Morphology of the Mesothoracic Wings of Sphagniana sphagnorum 8

Regions of the Forewings 8 Forewing Positioning 9 The File and Scraper 10 From Scraper to Mirror: Truss-Like Basal Posterior Margin and Pedestal of Forewing 11

Statistical Analysis of Wax Experiments on Right Forewing 12

Experimental Procedure: Scraper Pit Obstruction 12 Control Procedure: Stiffening of Intervein Membrane 13

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Morphology of the Mesothoracic Pteralia and Tergum of Sphagniana sphagnorum 14

Proximal and Distal Median Plates (m and m’) 14 The Second Axillary (2Ax) 15 The First and Fourth Axillaries (1Ax and 4Ax) 16 The Third Axillary (3Ax) 17 The Internal Thoracic Terga 18

Morphology of the Mesothoracic Pleuron of Sphagniana sphagnorum 19

Chapter 4: Discussion An Overview of the Stridulatory Mechanism in Sphagniana sphagnorum 20

Flight Mechanics in Dissosteira carolina 20 Stridulating Mechanism: Putting it All Together 21 Scraper Pit Experiments 24

Future Directions 26

Scraper Pit Obstruction Experiments 26 Muscles of the Axillary Inflection 26

Literature Cited 28

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List of Figures

Figure 1: Junior Woodsy collection site with stridulating male on black spruce 31

Figure 2: Sound analysis at increasing time resolution 32

Figure 3: Scanning electron images of file and scraper 33

Figure 4: Schematic illustration demonstrating elastic resonant stridulation 34

Figure 5: Margins, areas, and structures of the forewings 35

Figure 6: Lateral and dorsal view of forewings and associated structures 36

Figure 7: Major regions of the forewings 37

Figure 8: Edge-on views of right forewing basal margin and scraper pit 38

Figure 9: Interaction of pedestal and wing base sclerites 39

Figure 10: Wax placement on right forewing 40

Figure 11: Bar graph showing effect of wax on dBSPL of mode 41

Figure 12: Bar graph showing difference of dBSPL between modes 42

Figure 13: Dorsal view of (right) wing base sclerites 43

Figure 14: Lateral view of pleural pteralia in left forewing 44

Figure 15: Thoracic morphology of D. carolina 45

Figure 16: Mesolateral view of (right) wing base sclerites 46

Figure 17: Excised 2Ax and 3Ax 47

Figure 18: Dorsal view of mesotergum 48

Figure 19: Articulation between forewing and PWP, and PlR and its associated structures 49

Figure 20: Posterior view of mesothoracic cavity 50

Figure 21: Elements of the pleuron 51

Figure 22:Phragmata 52

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List of Tables

Table 1: Morphological and behavioural attributes important to sound production 53

viii

List of Terms and Abbreviations Veins:

A – anal C – costa CuA – cubitus anterior M – median Sc – radius

Wing Base Sclerites:

Ba – basalare m – proximal median plate m’ – distal median plate Sa – subalare 1Ax – first axillary 2Ax – second axillary 3Ax – third axillary 4Ax – fourth axillary

Tergum and Pleura:

1Ph – first phragma 2Ph – second phragma 3Ph – third phragma Aw – prealar arm ANP – anterior notal process CxP – coxal process Scl – scutellum Sct – principal part of scutum sct – posterior lateral scutum subdivisions PlA – pleural apophysis PlR – pleural ridge PWP – pleural wing process

Stridulatory Apparatus:

File – thickened Cu2 vein with ventral finger-like projections; located on left forewing Harp – radiating speculae located on both forewings Mirror – radiating speculae of right forewing Scraper Ridge – region of scraper on anal angle of right forewing that engages with file teeth Vesitgial File – non functional file located on right forewing

Sound Analysis:

dBSPL – sound pressure levels measured in decibel pascals root mean squared (dB Pa rms)

New Terms Presented in Thesis: AxInf – axillary inflection created by fusion and inflection of 1Ax and 4Ax AxInf-A – tergopleural muscle group that attaches to the AxInf , possibly functioning as mesothoracic stabilizers AxInf-B - tergopleural muscle group that attaches to the AxInf , possibly functioning as mesothoracic stabilizers IVM – intervein membrane at the proximal region of forewing inbetween Sc and CuA + M Median Arch – distal angle of m that is positioned dorsally in a resting or flexed wing; pedestal loads atop this region during stridulation Median Arch Loading Point – crevice on the inner surface of the pedestal in which the median arch accomodates Pedestal – concave base of truss that loads atop m when flexed Pit Vein – eccentric anal vein that occurs behind scraper ridge and ahead of scraper pit; vein is pushed on during to movement to deform scraper pit PlA-α2 – dorsal surface of PlA that receives AxInf-A from its attachment site on the AxInf Sc Arch – arch created at base of vein to accomodate dorsal region of 2Ax-δ Sc Cradle – small groove to accomodate ball-like base of Sc Scraper Linkage – anal angle region of right forewing that serves as a continuum between the scraper ridge, pit vein, and pit Scraper Pit – deep depression behind the scraper ridge and pit vein that deforms to store elastic energy Truss – brace-like structure located on both basal margins of either forewing,

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created by the converging and twisting anal veins 2Ax-α – trianguloid phlange of the 2Ax 2Ax-β – smooth rounded surface of 2Ax that pivots about tergum during stridulation 2Ax-γ – eccentric ridge of 2Ax 2Ax-δ – conical protruberance of 2Ax 2Ax-δ notch – small indentation in PWP to accomodate conical protruberance of 2Ax 3Ax-dH – dorsal head of 3Ax 3Ax-mb – main body of 3Ax 3Ax-vAA – ventral anterior arm of 3Ax 3Ax-dPA – dorsal posterior arm of 3Ax 3Ax-vIL – ventral inner leg of 3Ax 3Ax-vOL – ventral outer leg of 3Ax 4Ax-hook – lateral region of 4Ax that tapers and points posteriorly to form a hook that engages ventral region of 3Ax in a flexed forewing

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Chapter 1: Introduction

Sphagniana sphagnorum: an overview of the only endemically Canadian katydid

Type Locality and Distribution

Sphagniana sphagnorum (F. Walker, 1869), previously Metrioptera, is the name given to the only

katydid (Tettigoniidae) endemic to Canada (Vickery & Kevan, 1983). The type locality is Martin

Falls on the Albany River in the Hudson Bay Lowlands of Northern Ontario. The original collector

of the holotype was George Barnston. He was a Hudson Bay Company fur trader and naturalist

stationed in Fort Albany, who would later go on to donate the specimen to the British Museum of

Natural History (Brown & Van Kirk, 1982).

This flightless species of tettigoniid is distributed across Canada’s northwest, from the Quebec

border to the Yukon, in black spruce (Tsuga) and tamarack (Larix) bogs (Figure 1) (Vickery &

Kevan, 1983): hence its common name ‘the bog katydid’. Throughout the summer, males of this

species perch themselves with heads skyward (Figure 1, inset) on the stunted black spruce and

tamarack trees of these bogs, stridulating day and night.

An Introduction to the Song of Sphagniana sphagnorum

As with other Ensiferans, sound generation by S. sphagnorum is produced via tegmino-tegminal

stridulation; the forewings (tegmina) that initially arose as organs of flight have been secondarily

modified to favor sound production for the purpose of acoustic communication (Gwynne, 2001).

In tegmino-tegminal stridulation, one forewing – usually the right – exhibits a sclerotized ridge

that occupies the edge of the anal region; the other – usually the left – bears, on its ventral surface,

a row of sclerotized projections which protrude from the thickened Cu2 vein (Gwynne, 2001).

These two structures are respectively referred to as the scraper and the file. During stridulation the

scraper sweeps across the file and the shocks imparted to the forewings are conveyed via the veins

to much thinner regions of the forewing cuticle – mirror and harp cells – modified for oscillation

and sound radiation (Morris, 2008).

2

S. sphagnorum males differ from other stridulating katydids in that they produce two distinct

spectra by different stroking behavior patterns– these patterns termed the audio mode and the

ultrasonic mode – all while using the same forewing equipment (Morris, 1970; Morris & Pipher,

1972; Morris, 2008). While sound may be produced on both the to (closure) and the fro (opening)

movement of the forewings in acoustic katydids (Morris, 2008), the major sound output in S.

sphagnorum is generated during forewing closure (in contrast to a minor output produced during

opening). The non-resonant spectrum of the audio mode is a broad band of frequencies

approximately 25 kHz in width, when measured -15dB down from its peak (Morris, 2008); the

band’s greatest intensity occurs in the high audio around 18 kHz (Figure 2D) (Morris, 1970; Morris

& Pipher, 1972; Morris, 2008). In contrast, the spectral profile of the ultrasonic mode indicates an

approach to a resonant system, wherein a distinct high intensity peak is observed around 35 kHz

(Figure 2E) (Morris, 1970; Morris & Pipher, 1972; Morris, 2008). The base of the ultrasonic

spectra (Figure 2E) appears broad, however this is due to the sounds produced by the opening

movement of the forewings having been incorporated into the calculations for the spectral output

(Morris, 2008).

The ability of S. sphagnorum to alternate between these two modes is partly explained by the

anatomy of the file. As Morris & Pipher (1972) previously determined, the file of S. sphagnorum

contains approximately 150-180 teeth that gradually change in shape and density from proximal

to distal end (Figures 3A and C-E); near the wing base the teeth are broader and more densely

packed. The distal region, responsible for the production of the audio mode, comprises the majority

of the file. The smaller proximal region, consisting of approximately 60 file teeth (Morris & Pipher,

1972; Morris, 2008), is responsible for generating the ultrasonic mode. The individual pulses

produced by the scraper sweeping across the file in these non-overlapped regions are rather

different for each mode. For a pulse in the audio mode (Figure 2D inset), each tooth strike creates

a complex and rapidly decaying waveform – resulting in the broadband spectra obtained for this

mode. A pulse in the ultrasonic mode (Figure 2E inset) requires a uniformly timed tooth-strike

series that produces a sinusoid wave-form that decays slowly, thus creating the resonant-like

ultrasonic spectra characteristic of S. sphagnorum.

Figure 2 (adapted from Morris, 2008) illustrates, with increasing levels of time resolution (A-E),

a song sample analysis of a male S. sphagnorum individual. The alternation between audio and

ultrasonic mode can be noted in A and the difference in amplitude between the two is immediately

3

evident. While only the audio mode of the calling song is discernible to human ears, the ultrasonic

mode, having a greater amplitude, is in fact much more intense (Morris, 1970; Morris & Pipher,

1972; Morris, 2008).

The sound produced by one cycle of tegminal movement (i.e. phonatome) is comprised of major

and minor pulse trains. Depending on whether it is the audio (Figure 2B) or ultrasonic mode

(Figure 2C), either >18 or >4 major pulses respectively, will occur in succession. These major

pulse trains are followed by a succession of minor pulses (putative return of the scraper along the

file), completing the phonatome for each mode wherein either mode lasts approximately 25ms.

Elastic Resonant Stridulation

The major pulse trains in the ultrasonic phonatome are remarkable as there is a period of downtime

between each pulse during which no teeth are being struck (and yet the animal is still able to

achieve a relatively high frequency tooth strike rate). This observation, first noted by Morris &

Pipher (1972), is of particular consequence as it implies no movement between the file and scraper

is occurring. The model that Morris & Pipher (1972) proposed to understand the underlying

interaction, which they believed to be a form of elastic energy storage, came to be known as elastic

resonant stridulation (ERS) (Morris, 2008). ERS postulates that a flexible scraper wedges itself

behind a file tooth to effectively prevent the scraper from advancing, all while the forewings

proceed with their movement. As the forewings continue closing, the flexible scraper is distorted

backwards, storing elastic energy in its cuticle. At some critical point of bending the scraper is

dislodged from behind the restraining tooth and is catapulted along the file over several successive

teeth until it wedges itself once again. Figure 4 illustrates ERS based on the anatomy of

Arachnoscelis n. sp. (Montealegre-Z et al., 2006).

The use of elastic energy storage is widely observed amongst arthropods (Dickinson and Lighton,

1995; Bennet-Clark, 1997; Haas et al., 2000; Burrows et al., 2008; Zack et al., 2009) as a means

to improve muscle performance (Higham and Irschick, 2013). In the case of S. sphagnorum, the

muscles of the thoracic cavity, which power movement of the wings, contract slowly to store elastic

energy in the theoretically deformable scraper; being deformed maximally, the scraper releases

springing forward much faster than would have been possible if the muscles, and thereby velocity

4

of the closing wings, alone were driving it (Patek et al., 2011). Improved muscle performance that

enables rapid action is therefore afforded by an engine (i.e., muscles in the thoracic cavity), an

amplifier (i.e., the deformable region of the scraper), and a tool (i.e., the scraper) (Patek et al.,

2011).

Using high-speed video, Montealegre-Z et al. (2006) demonstrated that ERS is likely involved in

the calling song of at least three Tettigoniid species, S. sphagnorum being among these. Frame-

by-frame analysis of these videos demonstrate that during ultrasonic stridulation, the forewings

are nearly paused during the in-between-pulse downtime, actual sound emission only coincides

with obvious forewing movement (Morris, 2008). The insight gained from these high-speed videos

is in agreement with the model proposed by Morris & Pipher (1972).

Objectives and Research Questions

A purpose of my thesis is to expand on the understanding that both Morris & Pipher (1972) and

Montealegre-Z et al. (2006) present in relation to elastic scrapers and stridulation in Tettigoniids,

with particular reference to S. sphagnorum.

While the work by Montealegre-Z et al. (2006) supports the likelihood of an elastic scraper, it does

not set out to critically understand where and how the elasticity of the scraper is being enabled. To

address this, I studied the three-dimensional qualities of the scraper and its surrounding structures

to gain further understanding of scraper behaviour during stridulation. In doing so I soon realized

that the interaction between these entities is orchestrated by the complex coordination of the

forewings, wings that are in turn supported by precise alignment of the articulating and structural

pteralia of the mesothorax.

This thesis is founded on morphology. It aims at explaining forces within the stridulatory organ

and how these are adaptively translocated to the deformable scraper and sound-radiating speculae;

in addition I touch upon how mesothoracic skeletal elements, sclerites and muscles, which

originally evolved to power flight, have evolved to effect sound generation.

5

Chapter 2: Methods

Location of Study Animal Collection

Studies were conducted in 2012 and 2013 with specimens from black-spruce sphagnum bogs

located approximately 14 km west of Upsala (Ontario, Canada). The principal collecting site (Jr.

Woodsy) is south of the Trans-Canada highway latitude 4908.035' N, longitude 9046.213' W;

elevation 1525 feet. An unpaved road provides access to pipelines and crosses the study bog as a

sandy causeway. Good populations of singers occur to either side of this causeway, east and west.

The bog is widely open with relatively well-spaced stunted spruce and a few small tamarack (<3m).

The bog surface is a carpet of sphagnum moss and Ericaceae spp., rising and falling in uneven

hummocks with standing water collecting in low spots.

Generator Morphology

Specimens were examined with a dissection microscope under 70 % alcohol, using fine forceps,

microscissors, and minuten pins. Translocation of forces through the frame of the tegmina, starting

from the scraper-tooth contact, was inferred from form: thickness-thinness, flexibility-stiffness,

opacity-transparency; movement/bending/juxtaposition was tested by probing and manipulation

under the microscope, using both partially cleared (with KOH) and freshly dead specimens.

Sound Analysis

For recording, male insects were placed singly in aluminum screen cylindrical cages. A

microphone (GRAS ¼ inch condenser) was clamped with its tip 10 cm away, normal to the plane

of the dorsum. Microphone output went to a Photon 2 (LDS dactron) digitizer, sampling at 192000

ks/s. Ambient temperature varied between 20°C-to-24°C.

Fourier transforms were calculated using the application RTPro Photon (Version 6.3307, LDS

Dactron). This application could be calibrated to obtain sound level measures allowing comparison

of the effects of scraper pit obstruction (see below). Calibration was by means of a 1 kHz, 94dB

standard source (B&K Type 4230) to give sound pressure level (dBSPL) re 2E-5 dB Pa rms.

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In making a sound-level reading using RTPro (of one mode or the other, unwaxed or waxed) three

time samples, for each mode, were obtained at different locations within a song recording and

averaged. For each time sample an FFT (Fast Fourier Transform) was calculated and from the

resulting power spectral density display, a cursor read the most intense spectral peak as a y-axis

value of dB Pa rms. Each time sample was of 4096 points (points being values describing the

waveform), chosen centred upon the major train. The minor trains were excluded as much as

possible. A Hanning window was applied and this mathematically emphasizes the central over the

onset and ending values.

Wax Experiments

A film of low melting point resin wax [®Veet] was applied to wing surfaces, in particular the

scraper pit (Figures 10A and B) and the intervein membrane (IVM) (Figures 10C and D), using a

fabricated wax-heating device (WH). A resistor (277 ohms) served as a heating tip. A standard

wall-adapter power supply, rated at 12V, supplied power to the tip. The leads forming the tips were

¼ watt carbon film; one lead formed a loop acting as reservoir of the beaded wax; the other

wrapped around the resistor body taking up its dissipated heat.

In 2012 six live adult male insects, whose calling song was already recorded (see Sound Analysis

above) with forewings in a natural state, were chilled briefly in a freezer. They were then restrained

on a small piece of foam using angled insect pins. Under a dissecting microscope tegminal overlap

was reversed with flexible forceps and low-melting point wax applied to the right anal forewing

region by dipping the hot tip of the WH in the wax and dragging it lightly over the scraper-pit in a

wiping motion. The result was a thin film of wax (Figures 10A and B), rather than a bead, filling

the depression behind the scraper ridge and hypothetically causing this tegminal region to be

stiffened and have reduced flexibility. Over the next few days each insect was recorded again,

singing with the wax in place. In 2013 another six specimens were similarly recorded then re-

recorded after wax application to the scraper pit. These 2013 specimens were immobilized for wax

application using a brief application of carbon dioxide gas.

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In 2013 three control specimens were recorded both in a natural state and with wax placed (under

identical conditions as experimental) on the wing membrane in between the proximal angle created

by the Sc and CuA + M veins of the right forewing (Figures 10C and D).

Statistical Analysis

Statistical analysis was with SPSS version 20, with guidance from Laerd Statistics

(statistics.laerd.com). A two-way repeated measures ANOVA was run to determine the effect of

the presence or absence of wax in the scraper pit (for the experimental procedure) or IVM (for the

control procedure) on dBSPL between the audio and ultrasonic mode. I wished to test whether

reducing flexibility of the pit region – its stiffness – affected the ultrasonic mode more than the

audio, i.e., that a flexible scraper is more critical for production of the high-Q (resonant) ultrasonic

pulses than that of the low-Q (non-resonant) audio pulses. Post hoc analysis with Bonferroni

adjustment was used for the analysis of the control procedure.

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Chapter 3: Results

Morphology of the Mesothoracic Wings of Sphagniana sphagnorum

S. sphagnorum is a flightless1 tettigoniid. All wings except those of the adult male forewings are

vestigial. The abbreviated – brachypterous – forewings (Figure 1, inset) of the adult male are

acoustically adapted, having evolved to favor sound production – tegmino-tegminal stridulation –

rather than flight. The modifications that enable forewing sound generation in adult male S.

sphagnorum individuals are described below.

Regions of the Forewings

I adopt here the usual convention of aspect terms applied relative to an extended – not a flexed –

wing. Terminology of margins and areas is presented in figure 5. The posterior margin is further

subdivided by the anal angle (scraper location in right wing) and the cubito-anal emargination into

basal, anal, distal margins. Three areas are referenced: costal, mediocubital and anal. The

combined vein M + CuA which borders the harp anteriorly is a convenient, arbitrarily chosen,

boundary distad for delimiting the anal region.

The forewings of S. sphagnorum overlap, left anal region atop right, both canted and cantilevered

above the dorsum (Figure 6). The anal region of each forewing is roughly triangular in shape

(Figures 5 and 6B) and bounded on three sides by: (1) the truss (discussed below) of the basal

posterior margin, (2) the M + CuA vein, and (3) the anal posterior margin (Figure 5) – to where

the CuA vein separates and runs toward the cubito-anal emargination in the posterior margin. The

anal region of each forewing is central to the stridulatory mechanism of S. sphagnorum as it

contains the principal sound-generating structures, namely the scraper, scraper pit, file, and

radiating membranes – the speculae: harp, mirror (Figure 6). Anterior to the M + CuA vein in each

forewing, the mediocubital and costal areas (Figure 5) drape down laterally against the pleuron

1 Both sexes of S. sphagnorum exhibit two morphs: brachypterous and macropterous. Brachypterous individuals are almost exclusive in a typical population; however, high densities and limited food resources may trigger the presence of macropterous individuals in the next generation (Vickery & Kevan, 1983). Macropterous individuals have the ability to fly and therefore exploit nearby environments with access to abundant resources.

9

(Figure 6A). Thick flexible cuticle and a non-directional network of veins – archedictya –

characterize this draped region of the forewings (Figure 6A). This unstiffened, usually flaccid, area

does not apparently contribute to sound production; instead it serves other purposes: 1) its lack of

stiffness enables sound dampening, to actively prevent its role as a potential radiator, and 2) it

manages short-circuiting by baffling the upper surfaces of the radiating membranes from their

lower surfaces; this latter purpose allows for much improved efficiency and louder acoustic signals

(Fletcher, 1992).

Forewing Positioning

When at rest the forewings are folded closely upon the thorax with the overlapping left wing

covering the entire anal surface of the underlying right wing; the basal posterior margins of both

wings are overhung by the projecting posterior edge of the pronotum. In a relaxed individual, the

apex and basal posterior margin of each forewing are in the same plane.

On the other hand, during stridulation the forewings become both tented and forwardly tilted –

thus positioning the apex of each forewing in a plane slightly above each basal posterior margin.

The abdomen is lowered and its segments shifted relative to each other to form an upwardly

concave floor to the subtegminal cavity (Figure 1, inset). Another postural change, forward tipping

of the pronotum, angles the posterior edge of the pronotum upwards uncovering the basal posterior

margins of the forewings. As the wings close and open they alternate between partial flexion and

partial extension2, enabling the scraper to sweep to and fro across the file. How distally, or

proximally, the scraper engages along the file, will determine how much of the left forewing is

covered by the right. During ultrasonic stridulation the scraper is located within the file half

proximad to the wing base and therefore the wings are more overlapped relative to their position

during audio stridulation, wherein the wings are furthest apart and the scraper is within the file half

distad to the wing base (Morris & Pipher, 1972).

2 Full forewing extension in brachypterous adult males of S. sphagnorum is rarely observed since such individuals are unable to fly and therefore have no need for maximal forewing extension.

10

The File and Scraper

The anal angle (Figure 5), which demarcates the transition between the basal and anal posterior

margins, bears the sclerotized scraper ridge (Figure 3B), the component which inserts between file

teeth. Behind this ridge is the scraper linkage (Figure 5) that is characterized as a planar region, a

“scraper shelf” (Montealegre-Z et al., 2006) of cuticle, which is interrupted by the pit vein (Figure

7). This vein is anchored basad, its distal termination coinciding with the anal margin roughly

where the scraper ridge interacts with the file. When the ridge is pushed against the vein by a file

tooth, via the action of the closing wings, it presumably causes elastic distortion of the walls of the

deep concavity located just behind the shelf. This concavity is termed here the scraper pit (Figures

7, 8A and B).

The scraper pit is an oval depression, immediately behind the scraper ridge, and is a widely

observed feature of tettigoniid acoustic tegmina. This region has not been treated as a unitary wing

component. For example, in previous articles by Montealegre-Z et al. (2006) and Montealegre-Z

(2012) the scraper pit region has been vaguely identified as an elastic region behind the scraper

notable due to the absence of dorsal or ventral cuticle, respectively.

From a dorsal perspective, the absence of dorsal cuticle implies that the scraper pit appears to be a

concavity, as is the case for S. sphagnorum (and likely all other Tettigoniids that produce the major

pulse trains on the closing – to – movement). Interestingly the absence of ventral cuticle indicates

that, from a dorsal perspective, the region behind the scraper appears as a convexity. This latter

case is representative of Ischnomela gracilis, a reverse stridulator whose major pulse trains occur

on the opening – fro – movement (Montealegre-Z, 2012). This observation suggests that the

convexity visible on the dorsal cuticle of the forewing potentially deforms as it is pushed against

during the opening of the forewings.

I hypothesize the scraper pit to be the critical site of deformable cuticle, enabled by its

topographical location and shape, to store the necessary elastic energy for sinusoid ultrasonic

pulses such as those made by S. sphagnorum.

11

From Scraper to Mirror: Truss-Like Basal Posterior Margin and Pedestal of Forewing

During forewing closure, the scraper and file push against each other producing strong shear forces

(Josephson, 1985). These forces must be directed precisely so as to avoid unwanted buckling of

the forewing, which could otherwise cause error in the contact between scraper and file.

The evolution of a truss (Figure 6B) strengthens the basal posterior wing margins of both

forewings by providing them with structural stability. Morphologically, the truss converts the basal

region just anterior to the files (both functional and vestigial) into a beam, formed by the

converging and twisting of the anal veins; this braces the region between the wing base and anal

angle of each forewing (Figure 6B). Structurally it sets a rafter-like framework about which the

stridulating forewings can move.

The proximal end of the truss, which associates with the wing base sclerites, becomes a widened

trianguloid base with a deeply sclerotized concavity, that I call here the pedestal (Figure 6B, 8C,

and 9A). This pedestal is of particular interest as its interaction with the modified median plate is

almost solely responsible for functionally positioning the wings above the midline axis of the body

so enabling to and fro forewing movement.

Because of its attachment to the dorsal head of the 3Ax (Figure 9B), the pedestal is able to load

onto the curved distal edge of the proximal median plate during stridulation (Figure 9A); this edge,

which will be discussed in further detail in the upcoming section, has been termed the median

arch. The interaction between these two separate structures is stabilized by the insertion of the

pointed median arch into a small notch located within the pedestal (Figure 9C); this notch of the

pedestal is termed here the median arch loading point.

Distally the truss differs slightly between the two forewings, which is expected as the function of

each forewing also differs. On the left, the truss is well defined beside the length of the heavily

sclerotized file. On the right, the truss is also well defined along the length of the vestigial file,

however it stops abruptly behind the scraper pit and pit vein as that region, hypothetically, requires

deformation. In both the left and right forewings, the truss is outlined by the file and vestigial file,

respectively – these two structures (i.e., the truss and [vestigial] file), working in combination,

both function to support the integrity of the transverse cuticle and direct the forces occurring

between the file and scraper to the oscillating speculae and deformable scraper.

12

The proximal end of either file appears to float within the surrounding cuticle (Figures 6B and 7),

it is not anchored in the same way basally as it is distally. It is likely that when the file and scraper

push against each other the unanchored regions of both files are displaced slightly basad, sliding

into each respective harp causing the harp to buckle longitudinally (evident in the left wing of

figure 6B) thus sending it into vibration, possibly along with the mirror of the right forewing.

Statistical Analysis of Wax Experiments on Right Forewing

For a visual representation of the statistical results for both experimental and control procedures

refer to figures 11 and 12.

Experimental Procedure: Scraper Pit Obstruction

A two-way repeated measures ANOVA was run to determine the effect of the presence or absence

of wax in the scraper pit (Figures 10A and B) on dBSPL between the audio and ultrasonic mode.

Analysis of the studentized residuals showed there was normality, as assessed by a Shapiro-Wilk

test of normality and no outliers, as assessed by no studentized residuals greater than ± 3 standard

deviations. There was sphericity for the interaction term, as assessed by Mauchly's test of

sphericity (p > 0.05). Data are mean ± standard deviation, unless otherwise stated. There was a

statistically significant interaction between wax and mode on dBSPL, F(1, 11) = 189.409, p <

0.0005, partial η2 = 0.945. Therefore, simple main effects were run.

dBSPL was statistically significantly different before the placement of wax in the scraper pit

(80.111 ± 0.449 dBSPL) compared to after the placement of wax in the scraper pit (76.777 ± 0.869

dBSPL) during the audio mode, F(1, 11) = 20.135, p = 0.001, partial η2 = 0.647, a mean difference

of 3.334 (95% CI, 1.699 to 4.970) dBSPL. Similarly, dBSPL was statistically significantly

different before the placement of wax in the scraper pit (92.783 ± 0.905 dBSPL) compared to after

the placement of wax in the scraper pit (78.587 ± 1.645 dBSPL) during the ultrasonic mode, F(1,

11) = 97.637, p < 0.0005, partial η2 = 0.889, a mean difference of 14.195 (95% CI, 11.033 to

17.357) dBSPL.

13

dBSPL was statistically significantly different in the audio mode (80.1110 ± 0.449 dBSPL)

compared to the ultrasonic mode (92.7825 ± 0.905 dBSPL) before wax was placed in the scraper

pit, F(1, 11) = 314.678, p < 0.0005, partial η2 = 0.996, a mean difference of 12.672 (95% CI,

11.099 to 14.244) dBSPL. On the contrary, dBSPL was not statistically significantly different in

the audio mode compared to the ultrasonic mode after wax was placed in the scraper pit F(1, 11)

= 2.984, p = 0.112, partial η2 = 0.213.

Control Procedure: Stiffening of Intervein Membrane

A two-way repeated measures ANOVA was run to determine the effect of the presence or absence

of wax on wing membrane in between the proximal angle created by the Sc and CuA veins of the

right forewing (Figures 10C and D) on dBSPL between the audio and ultrasonic mode. Analysis

of the studentized residuals showed that there was normality, as assessed by the Shapiro-Wilk test

of normality and no outliers, as assessed by no studentized residuals greater than ± 3 standard

deviations. There was sphericity for the interaction term: Mauchly's test of sphericity (p > 0.05).

Data are mean ± standard deviation, unless otherwise stated. There was no statistically significant

interaction between wax and mode on dBSPL, F(1, 2) = 0.000, p = 0.989, partial η2 < 0.000.

Therefore, main effects were run.

dBSPL was not statistically significantly different before or after the placement of wax on IVM,

F(1, 2) = 0.832, p = 0.458, partial η2 = .294. However, dBSPL was statistically significantly

different between the audio and ultrasonic modes F(1, 2) = 2967.867, p < 0.000, partial η2 = 0.99.

Post hoc analysis with a Bonferroni adjustment revealed that there was an increase in dBSPL from

80.393 ± .639 dBSPL in the audio mode to 92.777 ± 0.477 dBSPL in the ultrasonic mode, a

statistically significant increase of 12.403 (95% CI, 11.424 to 13.383) dBSPL, p < 0.0005.

14

Morphology of the Mesothoracic Pteralia and Tergum of Sphagniana sphagnorum

Embedded within membrane and linking the pterothorax to the wings, are the median plates and

axillary sclerites. The axillaries are four small articulating wing sclerites (1Ax, 2Ax, 3Ax, 4Ax)

positioned dorsally between the edge of the tergum and the base of the wing veins; distal to these

are two median plates, proximal (m) and distal (m’), (Figure 13). In combination with the basalare

(Ba) and subalare (Sa) (Figure 14), to be discussed in the upcoming section, these sclerites,

collectively termed pteralia, function to transmit forces, created by muscular strain in the thorax,

to the wings (Dudley 2000).

Proximal and Distal Median Plates (m and m’)

As with Acrididae (Figure 15A), the mesothoracic wing base of S. sphagnorum possesses two

median plates: the proximal median plate (m) and the distal (m’). m, is a trianguloid sclerite, the

larger of the two (Figure 13B), and of particular importance to the stridulatory mechanism of S.

sphagnorum.

The three edges of m are labelled a, b, and c (Figure 13C). In an extended forewing, the proximal

edge, a, movably adjoins to the distolateral edge of the 2Ax (2Ax-). As the wing is flexed this

adjoining region buckles ventrad, allowing for the proximodorsal surface of m to lean against the

distolateral edge of the 2Ax- (Figures 9A and 16). In this position, m becomes perpendicular to

the plane of the forewing and thus the median arch, the angle between the edges b and c (Figure

13C), becomes positioned dorsally (Figure 16). As previously mentioned, when m leans against

the 2Ax-, as is the case when the forewings are in a flexed position, it enables the forewings to

load themselves onto the median arch via the pedestal (Figure 9A), thus enabling to and fro

forewing movement about the midline axis of the body.

The proximal end of edge c, of m, articulates with the distal end of the 3Ax-mb (Figure 13C); the

importance of this association is that the 3Ax-dH fuses to the ventral edge of the pedestal in the

forewing (Figure 9B). The continuity, afforded by the 3Ax, between m and the forewing enables

the pedestal to load atop the median arch and consequentially allows for stridulation to occur.

15

The Second Axillary (2Ax)

The most dramatic feature of the dorsal wing base in the mesothorax of S. sphagnorum is the large

and heavily sclerotized 2Ax. It is positioned centrally in the membrane (Figure 13) and links the

mesothorax to the forewings through its interaction with the other pteralial and pleural elements

which concentrate around it (Figures 13 and 14); as a result, the 2Ax is critical in orchestrating

proper wing alignment during stridulation. The important role of this axillary in stridulation is

highlighted not only by its heavy sclerotization, but also by its many modifications. The

modifications are characterized by four distinct regions: α, β, γ, and δ (Figure 17A), which interact

directly with all other mechanical elements (Figures 9B, 13, 14, 16 and 18) of the mesothorax

critical to stridulatory movement. These modifications will be described in detail below. Note that

the presence of the 2Ax in Acridids (Figures 15A, B and E) is far less dramatic as its primary

function is to act as a fulcral point that enables up and down wing movement necessary in flight

(Snodgrass, 1929).

As mentioned previously, the distolateral edge of 2Ax-β articulates with the mesal edge of the

proximal median plate; this movable connection, along with the flexor action of the 3Ax (discussed

below), is central to the stridulatory mechanism as it enables the wing to load mesally over the

mesothorax. The distolateral edge of 2Ax-β (Figure 17A), about which the proximal median plate

creases in a flexed forewing, is flattened so as to allow the proximal median plate to press against

it without distortion. The heavily sclerotized and smooth rounded shape of β is highly adaptive to

prevent potentially destructive friction occurring in a stridulating individual as 2Ax-β presses and

pivots against the lateral tergum of the mesothorax (Figure 18).

Extending off of β, the anteriorly angled ridge γ (Figure 17A) provides stability to the forewing

when it loads upon the median arch. Such stability is provided by the interaction of γ with the

remaining axillaries (laterally fused 1Ax-4Ax, and 3Ax) that afford a crude locking mechanism.

First, when the forewings are flexed, the proximal end of γ slips into the anteriorly angled

invagination (Figure 16) created by the fusion and inflection of the 1Ax and 4Ax. Second, both

arms of the 3Ax (Figure 17B) come together about the axis of its main body to tuck under the upper

half of γ (Figures 9A and 16), ensuring that the 2Ax, and consequently m, remain in position by

stabilizing γ from behind. Combined, the insertion of γ into the inflection created by the 1Ax and

16

4Ax in addition to the 3Ax tucking behind the upper region of γ, both help to support and stabilize

the mesally loaded wing thus preventing unwanted slipping during stridulation.

Anterior of β the forward pointing trianguloid flange α (Figure 17A) emerges. In a flexed forewing,

the ventrally positioned edge of α is supported by the main body of the 1Ax which itself is upwardly

reinforced by the anterior notal process (ANP) (Figures 16 and 18) of the mesothoracic tergum. By

resting atop the 1Ax, and consequentially the ANP, α provides upright support to the mesal surface

of the 2Ax; furthermore its wide and flat trianguloid surface area pushes against the tergum also

supporting the mesally loaded wing.

Lastly, in a flexed forewing, the lateral surface of the 2Ax, horizontal to γ, is the external conical

protuberance δ (Figure 17A). This protuberance fits securely into the circular opening created by

the pleural wing process (PWP), subcostal vein (Sc), and 3Ax (Figure 14). A posteriorly located

notch of the PWP (Figure 19B inset) accommodates the anterior region of δ (Figure 14); the

proximal end of the subcostal vein arches – which I have termed here the arch of Sc – to

accommodate the dorsally positioned region of δ (Figure 19A); the main body of the 3Ax curves

posteriorly (Figure 14B) to accommodate the posterior region of δ. The elements that create this

circular opening enable the 2Ax to pivot freely about its axis

While no muscles insert onto the 2Ax, δ must closely associate to the action of the Sa as both of

these structures are not only in close proximity but also deeply embedded in the same surrounding

membrane (Figure 14A). The Sa and Ba have multiple functions. In a flexed forewing, their

associated muscles contract simultaneously to enable wing extension; once extended the Sa

functions in supination whereas the Ba in pronation. Furthermore, in ensiferan stridulation the Ba

functions to elevate the forewing (i.e. anterior tilt) whereas the Sa functions to lower it (Pfau and

Koch, 1994)

The First and Fourth Axillaries (1Ax and 4Ax)

The 1Ax and 4Ax movably attach to the lateral edge of the mesothoracic tergum, wherein the 1Ax

is positioned directly atop the ANP (Figure 16). In S. sphagnorum, these two axillaries are highly

modified to facilitate mesally – not lateral – oriented wing movement.

17

The 1Ax is posteriorly elongated such that it can fuse to the anteriorly elongated 4Ax. The fusion

of the posterior and anterior extensions of the 1Ax and 4Ax, respectively, invaginate internally

creating an inflection (Figures 16 and 20) that extends one-third of the way down into the

mesothoracic tergum, between the first and second phragma (Ph), just above the PlA2-α (Figures

21B and C). This inflection, created by the fusion of both the 1Ax and 4Ax, I term here the axillary

inflection (AxInf). The AxInf is a compound apodeme that serves as the insertion point for two

groups of tergopleural muscle: AxInf-A and AxInf-B; these muscles attach to the PlA2-α (Figures

21B and C) and the PlR2 (Figure 21C), respectively. These muscles, which are absent in relevant

literature (such as La Greca, 1947; Matsudda, 1970), likely function as stabilizer muscles, creating

ridgity in the mesothorax to prevent collapse from the action of the to movement in stridulation

(see discussion).

Aside from the previously mentioned action of the 1Ax to sit atop the ANP thus supporting the load

of α from the 2Ax (Figure 16) another interesting feature of S. sphagnorum’s 1Ax is that it appears

to be laterally extended as evidence in figures 13A and B. In macropterous individuals (capable of

flight) it is likely an important enabler of the hinging action against the lateral tergal edge that

allows for up and down wing movement. Without this lateral extension the heavily sclerotized

ANP (Figure 18) would likely inhibit the hinging movement of the 1Ax.

The lateral region of the 4Ax however is also quite remarkable as it is characterized by a tapering

that results in a posteriorly pointed hook (Figures 13B and 14C) that latches on to the ventrally

positioned region (in a relaxed or flexed forewing) of the 3Ax (Figure 14). This hook is important

to the stridulatory mechanism of S. sphagnorum – and likely all stridulating katydids – as it

prevents the forewing from full extension and thus allows it to be mesally oriented above the

mesothoracic tergum (see discussion).

The Third Axillary (3Ax)

The 3Ax is a robust and well-developed axillary in S. sphagnorum. Its main function has shifted

from the primary role of flexion to that of providing a continuum, between the thorax and forewing

(Figure 14B), that enables stridulation. As such it is a multifaceted axillary with multiple

appendages and projections (Figure 17B) that facilitate intricate movement.

18

In a resting or flexed forewing, the main body (mb) of the third axillary extends dorsoventrally

(Figure 16) with the dorsal region ending in a peg-like head – termed here the dorsal head (dH) –

(Figure 17B) that attaches directly to the forewing by fusing to the ventral edge of the pedestal

(Figure 9B). As previously mentioned, the pedestal is able to load atop the median arch of the

proximal median plate through the guidance of the 3Ax. At its most ventral end, the 3Ax forks into

two legs (Figure 17B), termed here the ventral inner leg (vIL) and ventral outer leg (vOL). The

vIL is the longer of the two (Figure 17B) allowing the hook of the 4Ax to grasp it (Figure 14B).

The distal region of the 3Ax-mb movably attaches to the proximal half of edge c of m (Figure 13C).

Because the 3Ax is the flexor sclerites it will inherently determine the position of m through this

attachment – if the wings are rested or flexed m will too be flexed, leaning against the 2Ax-β (Figure

9A); if the wings are extended m will too be extended (Figure 13). As previously mentioned, the

connection between the 3Ax and m is crucial to the stridulatory mechanism of S. sphagnorum –

and likely all other stridulating katydids – because it directly enables the mesal orientation of the

forewing by loading the pedestal atop the median arch of m.

In the middle of the main body there are two phlanges (Figure 17B), they are the ventral anterior

arm (vAA) and dorsal posterior arm (dPA). In a flexed forewing, the dPA and vAA come into

close contact about the axis of mb allowing the dPA to tuck behind the upper half of the 2Ax-γ

(Figures 9B and 16A) while the dorsal edge of the vAA supports this same region but just anterior

to the dPA (Figure 16B). In combination, the two arms of the 3Ax help reinforce and support the

forming framework of the forewing that loads upon m to enable tegmina-tegminal stridulation.

The Internal Thoracic Terga

S. sphagnorum has three inflected cuticular sheets (Figure 22), phragma pairs (Ph), two of which

are well developed. The 1Ph occurs on the anterior edge of the mesotergum, while the 2Ph occurs

on the anterior edge of the metatergum. The vestigial 3Ph is barely visible (Figure 22B) and occurs

on the posterior edge of the metatergum. Dorsal longitudinal muscles only run between 1Ph and

2Ph (Figure 22A). Because brachypterous S. sphagnorum are flightless, and the hindwings

vestigial, the second pair of dorsal longitudinal muscles running between 2Ph and 3Ph are absent

as evidenced in figure 22A.

19

Morphology of the Mesothoracic Pleuron of Sphagniana sphagnorum

The structural elements of the thoracic pleura include the pleural ridge (PlR), the pleural apophysis

(PlA), and the pleural coxal process (CxP). In the pterothorax, the pleural wing process (PWP) and

the two epipleural sclerites, the Ba and Sa, can also be observed. The Ba and Sa are located below

the wing and above the pleural wall lying anterior and posterior, respectively, to the PWP.

The PlR is a highly sclerotized ridge that braces the pleuron between the wing – in the pterothorax

– and the leg (Snodgrass, 1935). Dorsally the PlR becomes the PWP, a projection that supports the

loading of the wing, usually via the 2Ax. The PlA is the internal arm of the ventral PlR, providing

a trianguloid surface area for muscle attachment, in addition to providing a site for association with

the sternal apophysis. The CxP, located ventrally below the PlA, forms a monocondylic joint with

the coxal segment of the leg enabling multiple planes of limb movement. Wing extension occurs

through the action of the muscles, inserted on both epipleural sclerites, contracting simultaneously.

In an already extended wing, the Ba and Sa correspondingly allow for pronation and supination.

A notable modification to the PWP in S. sphagnorum is an anteriorly angled (Figure 21A) and less

sclerotized (Figures 14 and 19B) extension of the PWP. This extension forms a c-shaped groove

(Figure 19B, inset) – termed here the Sc cradle – that supports the ball-like base of the subcostal

vein (Figure 19A) during stridulatory movement of the forewings. At the base of the PWP

extension, and posterior to it, a small indentation – the 2Ax-δ notch – can be observed (Figure

19B). As previously mentioned, this notch helps support the conical protuberance (δ) emerging

from the lateral side of the 2Ax (Figures 14B and C).

The PlA is the internal arm of the ventral PlR. The dorsal edge of the PlA of S. sphagnorum has

an angled oval surface area, termed PlA2-α (Figure 21). This modification to the PlA provides an

attachment site for the previously mentioned muscle group AxInf-A, which inserts onto the AxInf.

Anteriorly, S. sphagnorum has a single large and horizontally elongated Ba (Figure 14). In its

center, the Ba is weakly sclerotized in relation to its periphery. Whilst the Ba is large and

horizontally elongated, the Sa is small and conically shaped (Figure 14A). The Sa is located

posterior to the PWP; it is closely associated with 2Ax-δ as they are both enveloped within the

same membrane.

20

Chapter 4: Discussion

An Overview of the Stridulatory Mechanism in Sphagniana sphagnorum

Much like a machine made up of tiny interlocking and interacting pieces, the mesothoracic

elements of S. sphagnorum must move and align into place when going from rest to stridulation.

In this section I summarize how the mesothoracic elements, which originally evolved to power

flight, fall into place to enable stridulation by S. sphagnorum. In order to understand the novel

structural aspects of stridulation I briefly compare the body plan of S. sphagnorum to Dissosteira

carolina, a non-ensiferan orthopteran. In doing so I hope to shed light on how katydids, S.

sphagnorum included, must make use of the same mesothoracic flight equipment, albeit

importantly modified, to be capable of stridulation [and possibly flight, as is the case with most

stridulating katydids].

Flight Mechanics in Dissosteira carolina

To understand what modifications are required to enable stridulation, one must know the basic

mechanics of flight in non-ensiferan orthoptera. Here I discuss pteralial elements and flight

muscles described for D. carolina as found in Snodgrass (1929).

D. carolina, like all Orthoptera, contain a combination of pteralia at each wing base. Below the

wing at its base, anterior and posterior to the PWP and along the dorsal pleural edge, are the

basalares (1Ba and 2Ba) and subalare (Sa), respectively (Figure 15B). The basalares provide the

insertion for both the first and second pronator-extensor muscles of the wing; the subalare,

provides the site of attachment for the supinator-extensor of the wing (Snodgrass, 1929). As their

name would suggest, both of these sclerites are responsible for extending the wing from a position

of rest; this occurs when all muscle groups contract, creating a pull and subsequent distortion to

the pleural thoracic cuticle resulting in full wing extension. Once the wings are extended, these

“steering muscles” (Walker et al., 2014), associated with either sclerite, contract to respectively

pronate or supinate the wing during flight. Because these muscles attach directly to the wing base

to move the wing they are termed direct flight muscles.

21

On the dorsal surface of the wing base, four discrete axillary sclerites are observable (Figure 15A).

The 1Ax and relatively smaller 4Ax (Figure 15C) are separate hinging plates that articulate

proximally with the tergal edge and distally with the 2Ax and 3Ax (Figure 15A). The weakly

sclerotized, relative to S. sphagnorum, 2Ax, which movably adjoins to the distal edge of the 1Ax,

pivots upon the PWP (Figures 15B and E), functioning as the fulcral region of the wing during

movement (Snodgrass 1929). The 3Ax, movably connects to the distal edge of the 4Ax, and is the

flexor sclerite (Snodgrass 1929): it serves as the insertion site of the flexor muscle of the wing; in

the case of D. carolina this is the only axillary that has muscle attached to it. When compared to

the pteralia of D. carolina, the pteralia S. sphagnorum, appear more robust.s

Up and down powering flight movements of the wings occur through the action of indirect muscles

deforming the thoracic cuticle (Snodgrass, 1929). To enable a downstroke, the following muscles

must be activated: tergocoxal, tergosternal, pleurotergal, and oblique tergal (Josephson and

Halverson, 1971). Combined, these downstroke muscles are referred to as dorsoventral muscles

for simplicity. Conversely, the upstroke is enabled by the activation of the dorsal longitudinal

muscles running between the 1Ph and 2Ph, and the 2Ph and 3Ph. These antagonistic muscle groups

reciprocate to power continued flight via synchronous muscle contractions.

Stridulating Mechanism: Putting it All Together

When flexed, or at rest, the forewings of S. sphagnorum are closed, closely enveloping the thorax,

left anal area atop right. Once stridulation is initiated the forewings elevate above the tergum with

each pedestal of the forewings loading atop the arch of the median plate. Loading each forewing

onto its respective median plate requires precise maneuvering of the robust 3Ax, which acts as a

continuum between the thorax and wing. Maintaining the wing precisely loaded atop the median

arch requires strength and hence the 3Ax is significantly more robust in a stridulating katydid such

as S. sphagnorum, when compared to the 3Ax found in an orthopteran (like Dissosteira) one that

only flies (and therefore only requires the 3Ax to flex the wing back into a resting position).

Once loaded, the scraper will sweep across the file in to and fro movements and the enlarged and

heavily sclerotized 2Ax will press against the lateral wall of the tergum (Figure 18) throughout the

rhythmic stridulatory motions. The continuous to and fro movement occurs about the midline axis

22

of the body wherein the wings are furthest apart (i.e. distal file engaged) during the audio mode

and closest (i.e. proximal file engaged) during the ultrasonic mode. The eccentric ridge (2Ax-β),

which points anteriorly, inserts into the invagination created by the AxInf and helps secure the

wings in place to create stability, along with the vAA and dPA of the 3Ax that tuck behind it,

throughout the powerful stridulatory movements.

According to Josephson & Halverson (1971) the to and fro movements of the wings during

stridulation are powered by the same muscles that enable flight. During stridulation the direct

flight muscles (i.e., first and second pronator-extensor, and depressor-extensor) contract to power

the opening fro movement, and the combined action of the indirect flight muscles (i.e.,

mesothoracic dorsoventrals and the first pair of dorsal longitudinals) contract to power the closing

to movement of the wings.

In S. sphagnorum, the 4Ax hook is a simple, yet elegant, mechanism that allows for the use of

flight muscles in both flight and stridulation. When the direct flight muscles contract, the hook of

the 4Ax, which latches onto the 3Ax, prevents the wing from full extension. However, by

disengaging the hook from the ventral 3Ax, the wing can be deployed for flight (albeit not in a

brachypterous individual). The action of the engaged 4Ax hook changes the angle of work on the

wings thus allowing for to and fro wing movement in stridulation rather than up and down wing

movement used during flight.

The 4Ax is greatly enlarged in S. sphagnorum, and perhaps in all other stridulating tettigoniids. Its

increased size accommodates not only the lateral hook but also the anterior projection that fuses

with the posteriorly enlarged 1Ax. This fusion invaginates into the mesothoracic cavity, forming

the AxInf to provide an insertion site for muscles that are necessitated during stridulation.

Interestingly, the surface, PlA2-α (Figure 21), that receives the muscles of the AxInf is not present

in non-ensiferan orthoptera (such as D. carolina), as is evidenced from figure 15E, simply because

these species do not stridulate and hence do not require an AxInf and corresponding muscles.

Further, from the research on axillary sclerites in crickets done by Thakare (1969), and functional

morphology of cricket stridulation by Pfau and Koch (1994) the AxInf does not appear to occur in

these ensiferans either.

The action of the AxInf can be inferred by pulling on the region where the muscles would otherwise

attach and contract. It was thus noted that contraction of muscles attached to the AxInf would cause

23

the wing to extend; the evolution of this inflection to function as an insertion site for wing opening

muscles is curious, as the direct flight muscles readily enable such a function. I believe that the

muscles attached to the AxInf may be acting as stabilizers to maintain the structural integrity of the

thoracic tergum, thus preventing it from collapsing during the to movement which involves the

isometric3 contraction of both the dorsal longitudinal and dorsoventral muscles.

Stabilizer muscles are defined as non-primary [movement] muscles that contribute to the integrity

of a given structure by co-contraction of an agonist muscle with its antagonist (Sangwan et al.,

2014). In the case of S. sphagnorum the powerful to, or closing, action of the wings should create

considerable unwanted compression and tension in certain regions of the tergum, risking it to

buckle. A preventive and stabilizing measure would then be to dissipate the compression via co-

contraction of the AxInf muscles, which would otherwise serve to open the wings if the indirect

flight muscles were inactive.

Lastly, the morphology of each forewing must reflect the wing’s function in translocation of the

compression-tension forces that arise when the forewings close in the more powerful to motion.

The trusses (Figure 6B) provide a strong foundation against which both wings can push without

unwanted buckling. These trusses can direct compression forces precisely to where the cuticle is

modified to deform adaptively, in the case of S. sphagnorum these locations occur at: (1) the end

of the eccentric pit vein (Figure 7) which would allow for deformation of the scraper pit once

pushed on, and (2) the proximal unanchored base of each file (both functional and vestigial) in

either forewing (Figure 6), which would slide into the harp membrane and send it into oscillation.

These trusses (and the strongly thickened and buttressed file-vein of the left tegmen), would

prevent stridulation from occurring in the escapement manner peculiar to crickets. The escapement

mechanism of crickets requires a flexible file (Bennet-Clark and Bailey, 2002), a file that bends to

release and advance the scraper one tooth at a time, making one specular oscillation per advance.

Katydid wings are incapable of transverse bending in the plane of the trusses: they confine bending

to the region adjoining the scraper.

3 Note that in flight these two muscle groups are antagonist and hence their simultaneous activity would in fact be isometric contraction.

24

Scraper Pit Experiments

The intention of the scraper pit experiments, wherein a concavity behind the scraper shelf was

filled with resin wax, was to determine whether this region was indeed responsible for the storage

of elastic energy and consequently the main driver of increased decoupled (i.e. scraper speed

dissociated from the speed of the closing wing in order to increase its own speed) scraper speeds

sweeping across the file. By filling the scraper pit to prevent its role as an elastic energy store, one

would expect reduced speed in the decoupled scraper: the pit cuticle would no longer be amplifying

the power of the scraper. Raised scraper speeds result in increased striking force of a file tooth

(Xiao et al, 2013) ergo slowing the speed results in reduced striking force. On this basis, in the

scraper pit experiment both modes are expected to show reduced sound levels. But to the extent

that elasticity is more important for the ultrasonic mode than the audio, the ultrasonic mode should

show a more substantial reduction in dBSPL.

The results from my experiments support the hypothesis that the scraper pit is the region of elastic

energy storage as there is a statistically significant (p < 0.0005) interaction between mode and the

presence of wax in the scraper pit. Specifically, a drop in dBSPL for each mode is noted when the

scraper pit is filled with wax (Figure 11). On average the dBSPL in ultrasonic mode, without

scraper pit obstruction, is 92.78; after scraper pit obstruction it drops significantly (p < 0.0005) by

14.2 to a final dBSPL of 78.59. Similarly, the average dBSPL in audio mode without the scraper

pit obstruction is 80.11; after scraper pit obstruction it drops significantly (p = 0.001), albeit less

so than ultrasonic mode, by 12.67 to a final dBSPL of 76.78. These results support the hypothesis

because there is an overall drop in dBSPL as the striking force of the plectrum has been

compromised due to a drop in scraper speed resulting from reduced elastic storage. As evidenced

by the given p-values, while there is a statistically significant drop in both modes, the ultrasonic

mode (p < 0 .0005) is significantly more affected than the audio mode (p = 0.001).

Initially, I believed that elastic storage was important only in the ultrasonic mode of S.

sphagnorum’s song as a high scraper speed is essential to achieve the ultrasonic pure-tone observed

in this species spectral output. However once the results were analyzed one realizes that scraper

speed, resulting from elastic storage, must also be significantly important in the audio mode of the

song. This importance of scraper speed for both modes is also alluded to in Table 1, originally

published in Montealegre-Z et al. (2006).

25

Table 1 shows that the observed tooth strike rate (TSR), and consequently the carrier frequency

(fc), for each mode is not accounted for by the closing wing velocity (CWV) but rather by the

scraper velocity (SV). For the ultrasonic mode, which has an fc of 34.01.2, the TSR calculated

with CWV (i.e., TSR1) is 5642.0 teeth/s compared to the TSR calculated with SV (i.e., TSR2) which

is 33000 teeth/s; alternatively, the audio mode, which has an fc of 17.20.7, TSR1 is 3959.7 teeth/s

compared to the TSR2 which is 17172.1 teeth/s (Montealegre-Z et al, 2006). For each mode

observed, TSR2 is akin to the observed fc as opposed to TSR1.

Furthermore, the statistical analysis illustrates (Figure 12) that prior to scraper pit obstruction the

dBSPL between each mode differs significantly (p < 0.0005), with a mean difference of 12.67;

however after scraper pit obstruction, the dBSPL between each mode no longer differs

significantly (p = 0.012). The fact that there is no longer a statistical significance in the dBSPL

between each mode is of importance as it supports that the scraper pit obstruction experiment

effectively reduced the speed of the scraper and therefore its speed becomes coupled to (i.e. a result

of) the CWV. This result is telling as it relays similar results obtained from Table 1 in Montealegre-

Z et al. (2006): whereas as the values for TSR2 of each mode are very different (mean difference

for TSR2 between each mode is 15,827.9 teeth/s) those of TSR1 are similar (mean difference for

TSR1 between each mode is 1682.3 teeth/s).

The results of the control experiment, wherein the resin wax was placed on the IVM of the right

forewing, confirms the significance of the data obtained for the experimental procedure. The IVM,

a region that plays no role in scraper speed, showed no significant interaction (p = .989) between

mode and the presence of wax. Furthermore, similar to the experimental procedure, after the

placement of wax in the IVM of the control procedure a significant ( p < 0.0005) difference of

12.403 dBSPL is observed between the audio and ultrasonic modes.In the experimental procedure,

prior to application of wax in the scraper pit, a significant (p < 0.0005) difference of 14.195 dBSPL

is observed between the audio and ultrasonic modes. This significant difference between dBSPL

in each mode is also noted in figure 2A wherein the amplitude of the ultrasonic mode is almost

twice as large as that of the audio. The minor decrease in mean difference (-1.792 dBSPL) of the

control procedure (12.403 dBSPL), relative to that of the experimental procedure (14.195 dBSPL),

prior to application of wax in the scraper pit may be a result of amplified dampening effects of the

resin wax placed in the mediocubital region (i.e. the baffling region) of the right forewing.

26

Future Directions

Scraper Pit Obstruction Experiments

The scraper pit obstruction experiments may have shown other effects besides those of scraper

speed on sound pressure levels. One might expect to see reductions in ultrasonic-mode pulse

durations as a result of reduced scraper energy input. Due to various constraints, time and access

to specimens included, detailed analysis of output spectra for each mode before and after scraper

pit obstruction was deferred.

But such information might shed light onto how resonance vs noresonance (i.e. pure-tonality in

ultrasonic mode versus broad-band frequencies in audio mode) arises as a result of scraper speed.

It might also help to explain why Montealegre-Z et al. (2006) state that an elastic scraper is only

required for katydids producing pure-tones above 40 kHz, clearly excluding S. sphagnorum. This

so-called cut off likely does not take into account the fact that S. sphagnorum (uniquely in this

respect) is using the same file to produce both resonance and nonresonance. The size of the file

available to S. sphagnorum’s audio mode is perhaps reduced, when compared to a single-mode

stridulatory species that can employ an entire file vein for its song. The two-mode constraint likely

contributes to the use of an elastic scraper in both modes, as evidenced by the data provided in my

scraper pit experiments in addition to the scraper velocities provided in table 1 of Montealegre-Z

et al. (2006). To be clear, if S. sphagnorum were to comply with the 35 kHz cut-off the file would

have to be dedicated only to one mode. Therefore the case for S. sphagnorum presents itself as a

new subset of rules wherein an elastic scraper is necessary, even in audio mode, if the file is not

dedicated solely to one mode.

Muscles of the Axillary Inflection

In this thesis a previously undescribed modification of the 1Ax and 4Ax is mentioned. It is noted

that these two axillaries are both enlarged and fused to inflect into the mesothoracic cavity to

provide as a site for muscular attachment. While I have inferred the action of the so-called

‘stabilizer muscles’ it is necessary to confirm their actual function and what their function, if any,

would be in an individual capable of flight. Furthermore, with individuals capable of flight, one

needs to discover what changes occur to release the hook of the 4Ax from the ventral base of the

27

3Ax, so as to enable a position for flight by allowing for full wing extension. This is something

not yet put forward in scientific literature.

28

Literature Cited

Bennet-Clark, H.C. (1997). The mechanics and the control of song frequency in the cicada

Cyclochila australasiae. The Journal of Experimental. Biology 200, 1681-1694.

Bennet-Clark, H.C. and Bailey, W.J. (2002). Ticking of the clockwork cricket: the role of the

escapement mechanism. The Journal of Experimental. Biology. 205, 613-625.

Brown, J.S.H and Van Kirk, S.M. (1982). Barnston, George. Dictionary of Canadian Biography.

11 University of Toronto/Universite Laval.

http://www.biographi.ca/en/bio/barnston_george_11E.html

Burrows, M., Shaw, S.R. and Sutton, G.P. (2008). Resilin and chitinous cuticle form a composite

structure for energy storage in jumping by froghopper insects. BMC Biology. 6 (1), 41.

Dickinson, M.H., and Lighton, J.R. (1995). Muscle efficiency and elastic storage in the flight

motor of Drosophila. Science. 268(5027), 87-90.

Dudley, R. (2002). The biomechanics of insect flight: form, function, evolution. New Jersey:

Princeton University Press. 496p.

Fletcher, N.H. (1992). Acoustic systems in biology. New York: Oxford University Press. 352p.

Gwynne, D. (2001). Katydids and bush crickets: reproductive behavior and evolution of

Tettigoniidae. Ithaca: Cornell University Press. 317p.

Haas, F., Gorb, S., and Blickhan, R. (2000). The function of resilin in beetle wings. Proceedings

of the Royal Society of London. Series B: Biological Sciences. 267(1451), 1375-1381.

Higham, T.E., and Irschick, D.J. (2013). Springs, steroids, and slingshots: the roles of enhancers

and constraints in animal movement. Journal of Comparative Physiology. 183, 583-595.

29

Josephson, R.K. (1985). The mechanical power output of a Tettigoniid wing muscle during

singing and flight. Journal of Experimental Biology. 117, 357-368.

Josephson, R.K., and Halverson, R.C. (1971). High frequency muscles used in sound production

by a katydid. I. Organization of the motor system. Biological Bulletin. 141(3), 411-433.

La Greca, M. (1947). Morfologia funzioale dell’articolazione alare degli Ortoterri. Archivo

Zoologico Italiano. 32, 271-327.

Matsuda, R. (1970). Morphology and evolution of the insect thorax. Ottawa: Entomological

Society of Canada. 431p.

Montealegre-Z. (2012). Reverse stridulatory wing motion produces highly resonant calls in a

neotropical katydid (Orthoptera: Tettigoniidae: Pseudophyllinae). Journal of Insect Physiology.

58(1), 116-124.

Montealegre-Z, F., Morris, G.K., and Mason, A.C. (2006). Generation of extreme ultrasonics in

rainforest katydids. The Journal o Experimental Biology. 209(24), 4923-4937.

Morris, G.K. (1970). Sound analyses of Metrioptera sphagnorum (Orthoptera: Tettigoniidae).

The Canadian Entomologist. 102(3), 363-368.

Morris, G.K. (2008). Size and carrier in the bog katydid, Metrioptera sphagnorum (Orthoptera:

Ensifera, Tettigoniidae). Journal of Orthoptera Research. 17(2), 333-342.

Morris, G.K. and Pipher, R.E. (1972). The relation of song structure to tegminal movement in

Metrioptera sphagnorum (Orthoptera: Tettigoniidae). The Canadian Entomologist. 104(7), 977-

985

Patek, S.N., Dudek, D.M., and Rosario, M.V. (2011). From bouncy legs to poisoned arrows:

elastic movements in invertebrates. The Journal of Experimental Biology. 214, 1973-1980.

30

Pfau, H.K. and Koch, U.T. (1994). The functional morphology of singing in the cricket. The

Journal of Experimental Biology. 195, 147-167.

Sangwan, S., Green, R.A., and Taylor, N.F. (2014). Characteristics of stabilizer muscles: a

systematic review. Physiotherapy Canada. 66(4), 348-358.

Snodgrass, R.E. (1929). The thoracic mechanism of a grasshopper, and its antecedents.

Washington: Smithsonian Institution. 111p.

Snodgrass, R.E. (1935). Principles of insect morphology. New York: McGraw Hill Book

Company. 667p.

Thakare, V.K. (1969). On the axillary sclerites and their role in the mechanism of flexion and

extension of the wings in the Indian field cricket Gryllus bimaculatus Deg. (Gryllidae:

Orthoptera). Acta Zoologica. 50(3), 257-270.

Vickery, V.R., and Kevan, D.K.M. (1983). A monograph of the orthopteroid insects of Canada

and adjacent regions. Ste. Anne de Bellevue, Quebec: Lyman Entomological Museum and

Research Laboratory. 1462p.

Walker, S.M., Schwyn, D.A., Moso, R., Wicklein, M., Müller, T., Doube, M., Stamponi, M.,

Krapp, H.G., and Taylor, G.K. (2014). In vivo time-resolved microtomography reveals the

mechanics of the blowfly flight motor. PLOS Biology. 12(3), 1-12

Xiao, H., Chiu, C-W., Zhou, Y., He, X., Epstein, B., and Liang, H. (2013). The mechanical

forces in katydid sound production. Journal of Applied Physics. 114 (164908), 1-7

Zack, T.I., Claverie, T., and Patek, S.N. (2009). Elastic energy storage in the mantis shrimp’s fast

predatory strike. The Journal of Experimental Biology. 212, 4002-4009.

31

Figures

Figure 1: ‘Junior Woodsy’ sphagnum bog collection site near Upsala, ON, Canada. Typical environment

of S. sphagnorum; present conifers include stunted black spruce (Tsuga) and tamarack (Larix) (Jack pine

in foreground is not bog typical). Male individuals perch themselves with head skywards (inset) on black

spruce branches, stridulating day or night. Females are usually found on the moss carpet, however when

they go in search of a stridulating male they climb and mate atop the black spruce branches.

32

Figure 2: Sound analysis at increasing time resolution of S. sphagnorum. A, an approximately 2s sound

sample displaying three complete ultrasonic and two complete audio modes. The ultrasonic mode has a

larger amplitude than that of the audio. B, increased time resolution to show two phonatomes created

throughout the audio mode, each lasting around 25ms. C, increased time resolution to show two phonatomes

created during the ultrasonic mode, each lasting around 25ms. D, spectral profile for broadband audio mode

showing a bandwidth of approximately 25kHz and greatest intensity in the high audio around 18kHz. Inset

shows a single pulse (see ** in B) created by a single scraper-tooth strike, note the complex and rapidly

decaying wave form. E, spectral profile for a [trend towards pure-tone] ultrasonic mode with a high intensity

peak around 35kHz. The apparent wide base is an artifact resulting from the opening minor trains (see ÈfroÈ

in C) incorporated into the spectral output FFT calculations. Inset shows a one pulse (see * in C), a train of

waves created by multiple tooth engagements (imperfection observed in C* may represent missed teeth);

the pulse exhibits a slowly decaying wave form. Reproduced from Glenn K. Morris. JOR 2008; 17: 333-

342. © 2008 Orthopterists’ Society.

33

Figure 3: Scanning electron images of the file (A, C-E) and scraper (B) of S. sphagnorum. A, shows the

entire file. B, dorsoanterior aspect of scraper with ridge and pit vein. C, distal file. D, mesal file. E, proximal

file. The file teeth become broader and more densely packed from most distal (C) to most proximal (E).

Scale in μm. Adapted from Glenn K. Morris and Robert E. Pipher. Can. Entomol. 1972; 104:977-985.

© 1972 Entomological Society of Canada

A B

C D E

Ridge

Pit Vein

34

Figure 4: Schematic cross-section of file (right wing) and scraper (left wing) based on anatomy of

Arachnoscelis n. sp. illustrating hypothetical elastic resonant stridulation that creates the sinusoidal pulse

observed in B. A, degree of scraper bending, while being lodged behind tooth 22, prior to being released

once it can bend no further; 0.0764mm is the length that the scraper will travel down the file before lodging

once again behind tooth 29. B, once the scraper dislodges from tooth 22 it will slip across teeth 23-28,

creating one wave per tooth strike (i.e. green arrows). The red arrows point to the waves created by free

decay after scraper has stopped striking teeth. Reproduced from Fernando Montelagre-Z et al. J. Exp. Biol.

2006; 209:4923-4937. © 2006 by The Company of Biologists Limited.

35

Figure 5: Margins, areas, and structures of the (right) forewing as seen in male S. sphagnorum individuals.

The naming of these margins is in relation to a wing in full extension. The base is proximal to the body

whereas the apex is most distal. The left forewing is identical with exception of the absence of both the

mirror and scraper; also, the file of the left forewing is fully functional and large, not vestigial as observed

in this figure.

Abbreviation: vf, vestigial file.

vf

36

Figure 6: Labelled structures of male S. sphagnorum forewings with left wing overlapping right. A, right-

side view of forewings. B, dorsal view of forewings.

Vein Abbreviations: CuA, anterior cubitus; M, median; R, radius; Sc, subcosta; C, costa.

A

B

37

Figure 7: Major regions of the (right) underlying scraper forewing showing the two speculae mirror and

harp, the truss of anal veins surmounting the pedestal and the eccentric pit vein that lies behind the scraper

ridge. The mirror is bounded posteriorly by an immensely broadened and thickened vein, a ‘bar’ that

perhaps stabilizes that side of the radiator during oscillatory movement.

Vein Abbreviations: Sc, subcosta; CuA, anterior cubitus; M, median.

38

Figure 8: Edge-on views of basal margin of right (scraper) wing of male S. sphagnorum. A, ventral

convexity of scraper pit. B, scraper pit slightly deformed to show dorsally concave pit surface. C,

relationship of pedestal, truss, and scraper. Pedestal and truss occur on both left and right forewings.

A B

C

39

Figure 9: Interaction of the anal arm pedestal and

the surrounding wing base sclerites in male S.

sphagnorum forewings. A, dorsal view of right wing

base, in situ, with pedestal loaded atop median arch

B, mesolateral view of left wing base to show

interaction of 3Ax with pedestal and 2Ax. C, excised

pedestal and 3Ax-dPA of the right forewing to show

loading point of pedestal atop median arch, and

secondary* attachment site to the 3Ax (*primary

attachment occurs with the fusion of the 3Ax-dH at

the ventral edge of the pedestal).

Abbreviations: 2Ax and 3Ax, second and third axillaries; m, proximal median plate; dH, dorsal head; mb,

main body; vOL, ventral outer leg; vIL, ventral inner leg; dPA, dorsal posterior arm. Greek letters (α, β,

and γ) designate specified regions of 2Ax.

A B

C

40

Figure 10: A, dorsal view of forewings showing artificial overlap of right wing atop left to see

placement of wax in the scraper pit for the experimental procedure (i.e. scraper pit obstruction). B,

image in A magnified. C, side view of scraper (right) forewing showing placement of wax in IVM

(Sc and CuA + M) for the control procedure. D, image in C magnified.

A B

C D

41

Figure 11: Bar graph showing the effect of wax placement in the scraper pit (experimental, EXP,

procedure) and in the IVM (Sc and CuA + M) (control, CTR, procedure) on dB SPL of both the ultrasonic

and audio modes of S. sphagnorum. dB SPL measurements were taken both before (BF) and after (AFT)

wax was placed in designated regions. The effect of wax was statistically significantly different (p < 0

.0005) for both modes (see asterisks); however, the effect of wax on the ultrasonic mode was more

significant (p < 0.0005) than that of the audio (p = .001). The effect of wax in the experimental procedure

was not significant (p = 0.458). n = 12 experimental procedure; n = 3 control procedure. Standard deviation

(±) bar values from left to right are: 0.905, 1.645, 0.689, 1.292, 0.449, 0.869, 0.744, and 0.585.

75

80

85

90

95So

und

Pres

sure

Lev

els

(dB

SPL)

Ultrasonic Mode Audio Mode

EXP CTR

BF AFT

**

*

42

Figure 12: Bar graph showing the difference in dBSPL between ultrasonic (ult) and audio (aud) mode both

before (BF) and after (AFT) the placement of wax in the scraper pit (experimental procedure) and IVM (Sc

and CuA + M) of the right forewing (control procedure). With exception of the measurements taken from

the experimental procedure, after the wax was placed in the scraper pit (*) p = 0.112, the dBSPL between

each mode is significant, i.e. ultrasonic is significantly more intense than audio. Standard deviation (±) bar

values from left to right are: 0.905, 0.449, 1.645, 0.869, 0.689, 0.744, 1.292, and 0.585.

75

80

85

90

95

Soun

d Pr

essu

re L

evel

s (d

BSP

L)

Experimental Procedure Control Procedure

ult aud BF

AFT

*

43

Figure 13: Dorsal view of wing base in an extended

forewing of S. sphagnorum (A-C). A, position of

wing base sclerites in relation to each other B, wing

base sclerites have been outlined in colour to

delineate them. C, magnified to identify specific

regions of the 2Ax, 3Ax, and 4Ax.

Abbreviations: 1- 4Ax: first, second, third, and

fourth axillaries; m, m’: distal, and proximal

median plates; vAA, ventral anterior arm; dPA, dorsal posterior arm; mb, main body. Greek letters (α, β, γ)

and Roman letters (a, b, c) designated specific regions of the 2Ax and proximal median plate, respectively.

44

Figure 14: Underside of left wing to show

pleural pteralia of a male S. sphagnorum. A,

intact pteralia. B, membrane and Sa removed to

expose 2Ax-δ, 3Ax, and 4Ax. C, 4Ax hook

disengaged from base of 3Ax.

Abbreviations: Sc, subcostal vein; PWP2,

mesothoracic pleural wing process; Ba, basalare;

Sa, subalare; 2Ax, 3Ax, 4Ax, second, third,

fourth axillary; mb, main body; vIL. ventral inner

leg; δ, designating lateral region of 2Ax.

45

Figure 15: Thoracic morphology of D. carolina

showing pteralial and thoracic elements. A, wing base

of extended right wing B, lateral view of left dorsal

mesothoracic pleuron C, dorsal view of mesothoracic

tergum D, sagittal view of internal meso and

metathorax of right side with dorsal longitudinal and

dorsoventral indirect flight muscles E, internal view

of mesothoracic elements that enable flight.

Abbreviations: 1Ax, 2Ax, 3Ax, 4Ax: first, second,

third, and fourth axillaries; m and m’, proximal and

distal median plates; 1A-3A, first, second, and third

anal veins; Sc, subcostal vein; Sa, subalare; Ba, basalare; Ph, phragma; Aw, prealar arm; ANP, anterior

notal process; Sct, principal part of scutum; sct, posterior lateral scutum subdivisions; Scl, scutellum; 81

and 112, first and second pair of dorsal longitudinals; W, base of dorsal forewing; D, flexor; E and M’, first

and second pronator-extensors; M”, supinator-extensor; PlR, pleural ridge. Adapted from Snodgrass (1929).

E

A B

D C

46

Figure 16: Mesolateral view of wing base sclerites, in situ, in the right forewing of a male S. sphagnorum.

A, intact wing base simulating rest position. B, wing base with pedestal and 3Ax-dPa excised to show

underlying sclerites. Red dotted-lines with (*) illustrates where pedestal loads atop the median arch in a

stridulating individual, and its attachment to the 3Ax-dH; red dotted-lines with (**) illustrates where 3Ax-

dPA attaches to the 3Ax-mb.

Abbreviations: 1Ax, 2Ax, 3Ax, 4Ax: first, second, third, and fourth axillaries; Ph, phragma; ANP, anterior

notal process; AxInf, axillary inflection (created by the fusion and invagination of the 1Ax and 4Ax); vAA,

ventral anterior arm; dPA, dorsal posterior arm; mb, main body; dH, dorsal head; m, proximal median plate.

Greek letters (α, β, and y) refer to specific regions of the 2Ax.

47

Figure 17: Wing base sclerites of a male S.

sphagnorum individual. A, posterior view of the

second axillary (2Ax) of the right forewing.

Dotted-red line is where the proximal median

plate, which has been excised, would otherwise

attach. Greek letters (α, β, γ, and δ) designate

specific regions of the 2Ax. B, posterior view of the third axillary (3Ax) of the left forewing.

Abbreviations: vOL, ventral outer leg; vIL, ventral inner leg; vAA, ventral anterior arm; dPA, dorsal

posterior arm; dH, dorsal head; mb, main body.

48

Figure 18: Mesothoracic tergum of male S. sphagnorum with attached first phragma (1Ph) and anterior

notal processes (ANP). The black arrows with corresponding black * demonstrate the region where γ of the

second axillary (2Ax) will slide into when stridulation has been initiated; the fusion and invagination of the

first and fourth axillaries (1Ax and 4Ax), not seen here, also provides space for 2Ax- γ to insert into (see

figure 15). The curved red line and associated red * show the region where β of the 2Ax press and pivot

against the tergum when forewings are stridulating; this region occurs right behind the ANP upon which

the 1Ax and 2Ax-α load. (Greek letters denote specific regions of 2Ax.)

Abbreviations: Aw, prealar arm; Sct, principal part of scutum; sct, posterior lateral scutum subdivisions;

Scl, scutellum.

49

Figure 19: A, mesolateral view of left forewing and

its association with the Sc cradle of the PWP.

Dotted-red lines highlight location where 2Ax-δ

positions itself. B, posterior view of right PlR and

associated structures.

Abbreviations: Sc, subcostal vein; PWP2 mesothoracic pleural wing process; PlR2, mesothoracic pleural

ridge; PlA2, mesothoracic pleural apophysis; CxP2, mesothoracic coxal process. Greek letters (α and δ)

designate specific regions of labelled structures.

50

Figure 20: Posterior view of mesothoracic cavity with soft tissue removed to show internal structural

inflections of cuticle. The position and angle of each axillary inflection (AxInf) is visible. The first pair of

phragmata (1Ph) define the anterior region of the mesothorax. Externally the conical protuberance of the

second axillary (2Ax-δ) is visible and its relationship to the forewings is evident. The interaction of the file

and scraper (pit) is also obvious in this figure.

51

Figure 21: Pleural elements of male S. sphagnorum

individuals. A, internal lateral view of right PlR.

Dotted-white oval highlights the dorsally flatted

region of the PlA, which serves as an attachment site

for AxInf-A of the AxInf. B, internal lateral view of

right pleuron and attached mesothoracic tergum to

show relationship of AxInf-A to both the AxInf and

PlA2-α. C, posterior view of left pleural elements

with the 2Ax and 3Ax in situ.

Abbreviations: PWP2, mesothoracic pleural wing

process; PlR2, mesothoracic pleural ridge; PlA2,

mesothoracic pleural apophysis; SA2, mesothoracic

sternal apophysis; CxP2, mesothoracic coxal

process; AxInf, axillary inflection (created by the

fusion and invagination of the 1Ax and 4Ax); AxInf-A/B, respective muscles which attach to the AxInf;

1Ax, 2Ax, 3Ax, and 4Ax: first, second, third, and fourth axillary sclerites, respectively; mb, main body;

vAA, ventral anterior arm. Greek letters (α, γ, and δ) specify designated regions of respective structures.

A B

C

52

Figure 22: Phragmata of a male S. sphagnorum

individual. A, internal top view of phragmata

extending off of tergum. The first and second

phragmata (1Ph and 2Ph, respectively) are well

developed and have the first pair of dorsal longitudinal muscles (81) running between them; the third

phragmata (3Ph) are vestigial. The absence of the second pair of dorsal longitudinal muscles (112), a result

of being flightless, is noted. See figure 14D to compare the well-defined dorsal longitudinal muscles (81

and 112) in D. carolina. B, posterior view of mesothoracic cavity with soft tissue removed to see phragmata.

A

B

53

Table

Ultrasonic

Audio

Song M

ode

7.9

14.9

Tooth Spacing

(µm)

125

67

Tooth D

ensity, TD

(mm

-1)

78.6±3.8

59.1±2.8

Closing W

ing V

elocity, CW

V

(mm

s -1)

264.0±2.8

256.3±2

Scraper V

elocity, SV

(mm

s -1)

5642.0

3959.7

Tooth Strike R

ate 1 (TD

xCW

V)

33000.0

17172.1

Tooth Strike R

ate 2 (TD

xSV)

34.0±1.2

17.2±0.7

fc (kH

z)

Table 1: Morphological and behavioral attributes that contribute to the sound produced by stridulating m

ale S. sphagnorum

individuals. Adapted from Fernando M

ontelagre-Z et al. J. Exp. Biol. 2006; 209:4923-4937.

© 2006 by The C

ompany of B

iologists Limited.