The Carbon Dioxide-Induced Bioluminescence Increase In

Home , Arachnocampa, Photocyte

bioRxiv preprint doi: https://doi.org/10.1101/2020.03.12.989616; this version posted March 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 The Carbon Dioxide-induced Bioluminescence Increase in

2 Arachnocampa Larvae

4 Hamish Richard Charlton and David John Merritt1

5 School of Biological Sciences, The University of Queensland, Brisbane, Qld 4072 Australia

6 1Author for Correspondence

7 [email protected]

9 WORD COUNT: 7718

11 Short title: Bioluminescence regulation in glow-worms

13 Key-words: glow-worm, anaesthesia, fungus gnat, light organ, photocyte

15 Abbreviations:

16 Carbon dioxide (CO2)

17 Nitrogen (N2)

18 Light:dark (LD)

19 Single lens reflex (SLR)

20 Terminal abdominal ganglion (TAG)

21 Summary Statement

22 CO2 was thought to act as an anaesthetic producing elevated bioluminescence in

23 Arachnocampa. Here we show it acts directly on the light organ and does not act as an 24 anaesthetic. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.12.989616; this version posted March 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

25 Abstract

26 Arachnocampa larvae utilise bioluminescence to lure small arthropod prey into their web-

27 like silk snares. The luciferin-luciferase light-producing reaction occurs in a specialised light 28 organ composed of Malpighian tubule cells in association with a tracheal mass. The

29 accepted model for bioluminescence regulation is that light is actively repressed during the 30 non-glowing period and released when glowing through the night. The model is based upon

31 foregoing observations that carbon dioxide (CO2) – a commonly-used insect anaesthetic – 32 produces elevated light output in whole, live larvae as well as isolated light organs.

33 Alternative anaesthetics were reported to have a similar light-releasing effect. We set out to 34 test this model in Arachnocampa flava larvae by exposing them to a range of anaesthetics

35 and gas mixtures. The anaesthetics isoflurane, ethyl acetate, and diethyl ether did not

36 produce high bioluminescence responses in the same way as CO2. Ligation and dissection

37 experiments localised the CO2 response to the light organ rather than it being a response to

38 general anaesthesia. Exposure to hypoxia through the introduction of nitrogen gas

39 combined with CO2 exposures highlighted that continuity between the longitudinal tracheal

40 trunks and the light organ tracheal mass is necessary for recovery of the CO2-induced light

41 response. The physiological basis of the CO2-induced bioluminescence increase remains

42 unresolved but is most likely related to access of oxygen to the photocytes. The results 43 suggest that the repression model for bioluminescence control can be rejected. An

44 alternative is proposed based on neural upregulation modulating bioluminescence intensity.

45 Count: 242

46 Introduction

47 Bioluminescence, the emission of visible light by a living organism as a result of a chemical

48 reaction, occurs in a remarkable diversity of organisms spanning terrestrial and marine 49 environments (Wilson and Hastings, 1998). Among arthropods, bioluminescence has been

50 observed in crustaceans, insects, and myriapods with functions including sexual 51 communication, aposematic signalling, and prey attraction. In all bioluminescent

52 arthropods, light is produced as the result of the luciferin-luciferase chemical reaction 53 (Viviani, 2002). In this reaction, luciferase enzymes catalyse the oxygenation of luciferins to

54 produce electrically excited compounds and photons of visible light (Kahlke and Umbers, 55 2016).

56 The best-characterised and most conspicuous terrestrial bioluminescent insects are the

57 fireflies (Order Coleoptera: Family Lampyridae) and the members of the genus 58 Arachnocampa (Order Diptera: Family Keroplatidae) (Branham and Wenzel, 2001; Meyer-

59 Rochow, 2007). Among these insects, significant differences in bioluminescence production, 60 utilisation, and regulation have been observed (Lloyd, 1966; Meyer-Rochow and Waldvogel,

61 1979; Meyer-Rochow, 2007). Adult lampyrid beetles emit light in controlled, periodic, 62 patterned flashes to detect and communicate with potential mates (Copeland and Lloyd,

63 1983; Lloyd, 1966). Lampyrid larvae release a steady glow, believed to be used 64 aposematically, corelating with distastefulness (Matthysen, 1999). Arachnocampa larvae are

65 predators that produce light continuously throughout the night to lure arthropods into web-

66 like silk snares (Broadley and Stringer, 2001; Mills et al., 2016). The light-producing organs in 67 Arachnocampa and fireflies are evolutionarily independent and morphologically distinct so

68 bioluminescence production and regulation are expected to differ (Viviani et al., 2002).

69 The genus Arachnocampa is comprised of 9 species endemic to Australia and New Zealand

70 (Baker, 2010; Baker et al., 2008; Meyer-Rochow, 2007). The larvae inhabit cool, dark places 71 including rainforest embankments and the inside of wet caves (Berry et al., 2017; Merritt et

72 al., 2012; Meyer-Rochow, 2007). The lifespan of an adult Arachnocampa is very short with 73 adult males living for a maximum of 6 days and adult females living for 2-5 days. The larval

74 state has the longest duration, lasting for many months, during which it utilises its

75 bioluminescence to attract prey (Baker and Merritt, 2003; Merritt and Baker, 2001; Willis et 76 al., 2011). The larvae are relatively immobile and construct snares consisting of mucous-

77 dotted silk lines that hang downward from mucous tubes anchored to a rocky or earthen

78 substrate (Baker and Merritt, 2003; Broadley and Stringer, 2001; Merritt and Baker, 2001; 79 Mills et al., 2016; Willis et al., 2011). The species used in this study, Arachnocampa flava, is

80 endemic to south-east Queensland with large epigean populations at Springbrook National 81 Park (Wilson et al., 2004).

82 Morphology of the light organ

83 Light is produced by a posteriorly-located light organ (LO), composed of the modified, large- 84 diameter distal cells of the Malpighian tubules in association with a tracheal mass (Green,

85 1979; Wheeler and Williams, 1915). The photocytes have a dense cytoplasm with synaptic

86 contacts on the cells of the light organ containing dense-core vesicles that are indicative of 87 neurosecretory regulation (Green, 1979). A single nerve runs from the terminal abdominal

88 ganglion (TAG), separating into neural processes that innervate the LO (Gatenby, 1959; 89 Rigby and Merritt, 2011). The lateral and ventral surfaces of the LO are covered by a mass of

90 tracheoles with interspersed nuclei, taking on a silvery appearance visible through the 91 cuticle (Green, 1979; Rigby and Merritt, 2011). The tracheal layer is closely associated with

92 the photocytes (Green, 1979), suggesting that access to oxygen is a critical factor in 93 bioluminescence output just as it is in fireflies (Ghiradella and Schmidt, 2004); however, the

94 firefly LO is evolutionary derived from a different tissue, believed to be fat body (Amaral et

95 al., 2017).

96 Neural control of bioluminescence and effect of anaesthetics

97 While the regulatory mechanism of firefly bioluminescence is well-known (Ghiradella and

98 Schmidt, 2004; Lloyd, 1966; Timmins et al., 2001; Trimmer et al., 2001), the regulation and 99 production of light by Arachnocampa larvae is less well-known. Prior to this study, the

100 prevailing model for bioluminescence regulation in Arachnocampa was that

101 bioluminescence is actively repressed when larvae are not glowing such as under daylight or 102 when disturbed, and that the repression is released under darkness (Gatenby, 1959; Rigby

103 and Merritt, 2011). The repression is re-initiated if larvae are exposed to light (Mills et al., 104 2016). The fact that larvae have a capacity to increase their bioluminescence when

105 stimulated by vibration or the presence of prey in their webs led Mills et al (2015) to 106 propose a two-part system where bioluminescence can also be actively promoted. Evidence

107 for the repression-based model came from ligation and gas exposure experiments. Ligating

108 larvae behind the terminal abdominal ganglion anterior to the LO caused the LO to emit

109 light (Gatenby, 1959) and isolated LOs with neural connections removed emitted low levels 110 of light (Rigby and Merritt, 2011), interpreted as being due to the release of inhibition. The

111 model was reinforced by the response to anaesthetics. In A. richardsae, the anaesthetics

112 CO2, ether and chloroform caused light release in whole larvae while methanol and ethyl

113 acetate were ineffective (Lee, 1976). Isolated light organs of A. flava released low-intensity

114 light when removed from the body and then emitted very bright light when CO2 was

115 introduced (Rigby and Merritt, 2011).

116 From the foregoing studies, one possible explanation for the apparent anaesthetic effect of

117 CO2 is that it causes loss of muscle control and that muscle activity could be a requirement

118 for the repression of bioluminescence through modulation of oxygen supply to the LO 119 (Gatenby, 1959; Rigby and Merritt, 2011). It is known that bioluminescence requires oxygen

120 because placing glowing larvae under a vacuum caused them to dim over several minutes

121 (Lee, 1976). In insects, CO2 is generally thought to block synaptic transmission at

122 glutamatergic synapses including those at neuromuscular junctions (Badre et al., 2005b); 123 therefore, muscular control could be the key to restricting or activating bioluminescence

124 output. Rigby and Merritt (2011) saw no obvious muscular sphincters associated with the LO 125 or its tracheal supply but acknowledged that this avenue is worthy of further investigation.

126 Current understanding is that in fireflies neurally-regulated oxygen-gating is involved in the

127 regulation of flash bioluminescence (Timmins et al., 2001). Oxygen transfer to light- 128 producing peroxisomes is interrupted by the mitochondria consuming oxygen between

129 episodes of bioluminescence (Timmins et al., 2001; Trimmer et al., 2001). Flashes occur 130 when uptake of oxygen by mitochondria is repressed by nitric oxide arising from

131 octopamine release. In contrast to adult fireflies, Arachnocampa larvae slowly modulate 132 light emission suggesting that the systems will differ (Mills et al., 2016). Further, the two

133 systems have evolved independently from different organ systems. Nevertheless, a common 134 feature is that both fireflies and Arachnocampa possess a rich and effective tracheal supply

135 to the LO and both require ATP and oxygen for light release (Kahlke and Umbers, 2016; Mills 136 et al., 2016; Timmins et al., 2001). It appears the access to oxygen is a vital factor for light

137 production in both the coleopteran and dipteran bioluminescence systems.

138 Vibration modulation of bioluminescence

139 Arachnocampa larvae brighten substantially when exposed to a vibration stimulus (Mills et

140 al., 2016); vibration of whole larvae in containment produced a 7-10 fold increase in 141 bioluminescence. To incorporate this neurally-based brightening, Mills et al. (2016)

142 proposed a two-part regulatory system; (1) a bioluminescence-inhibiting system that 143 prevents bioluminescence when larvae are exposed to daylight or natural light and (2) an

144 acute vibration response, whereby vibration causes an increase in light output followed by a

145 return to pre-stimulus levels. A key aspect of the inhibition hypothesis was the effect of CO2

146 anaesthesia in releasing the proposed inhibitory control, resulting in uncontrolled light

147 output.

148 Here we explore the physiological and morphological mechanisms of bioluminescence

149 regulation by exposing A. flava larvae to a range of anaesthetic, atmospheric, and vibration 150 stimuli and examining the consequent responses. It complements other studies of the

151 Arachnocampa luciferin-luciferase system, which are revealing similarities with coleopteran 152 systems – the luciferases belong to the same family of enzymes (Sharpe et al., 2015; Silva et

153 al., 2015) – as well as differences – the Arachnocampa luciferin is novel, being different to 154 any described to date (Watkins et al., 2018). Given that bioluminescence is emitted in long

155 bouts and comes under slow neural control, the bioluminescence regulatory mechanisms of

156 Arachnocampa are of significant interest when compared to the well-known adult firefly 157 system.

158 Materials and methods

159 Experimental animals: collection and maintenance

160 A. flava larvae were collected from Springbrook National Park, Queensland, Australia in

161 accordance with a Department of Environment and Science permit (PTU18-001356). Larvae 162 (~ 2 – 2.5 cm length), probably corresponding to the fourth or fifth instar stages, were

163 collected. Larvae were individually housed in halved, inverted plastic containers (7 cm 164 height x 7 cm diameter) with clay pressed into the upturned base, and fronted with

165 transparent plastic. The containers were kept in glass aquaria filled with ~1 cm of water and

166 sealed with a glass lid and plastic wrap to ensure high humidity. The aquaria were placed 167 inside a temperature-controlled (24 ± 1°C) room under 12:12 light:dark (L:D) conditions with

168 artificial light produced by a 12V DC white LED lamp controlled by a timer. Larvae were

169 acclimatised to the conditions for a minimum of one week before being utilised in 170 experiments. Larvae that pupated during or immediately following the experiment were not

171 used in analysis. Once per week during the photophase of a non-treatment day (at ~ 13:00

172 hours), larvae were fed three CO2-anaesthetised Drosophila melanogaster adults by

173 securing the Drosophila to the silk lines of A. flava, taking care to minimise damage to the 174 lines.

175 Measuring bioluminescence

176 Larval bioluminescence was imaged using a SLR camera (Canon EOS 1000D), using identical

177 exposure time, lens and distance between the lens and larva for all experiments. Camera 178 settings were 25 seconds exposure, F5.6, ISO 640. A Pclix XT intervalometer camera

179 controller was used to define intervals between exposures. The application, ImageJ (Version 180 1.52a), was used to analyse the light output from the individual larvae using the method

181 described by (Mills et al., 2016). Briefly, the greyscale images were imported into ImageJ, 182 then a threshold value (Max: 250, Min: 30) was applied to distinguish the image of the

183 glowing LO region from the background and the intensity of the pixels comprising the glow 184 was summed. As the intensity of isolated pixels is a non-SI unit, the bioluminescence output

185 is referred to in terms of relative light units.

186 Light output of live larvae

187 To investigate the effect of CO2, 4 live larvae were pinned to a wax dish, taking care not to 188 damage any internal structures. They were then placed under the camera in a transparent,

189 airtight plastic container (400 ml) with gas ingress and egress connectors and imaging 190 initiated. The pre-treatment bioluminescence output was recorded once per minute for 15

191 minutes, then larvae were exposed to the treatment gas for 1 minute at a rate of 10

192 L/minute. The inflowing CO2 was humidified by bubbling through water. Imaging continued 193 for 15 minutes after treatment.

194 Light output in ligated larvae

195 To investigate light emission when there was no direct connection between the brain and 196 LO, larvae were ligated with a silk thread at two different locations; (1) behind the head or

197 (2) just anterior to the LO, and the LO-containing region separated by cutting anterior to the

198 ligation. The LO section was then placed on a wax dish (head ligation) or in an excavated

199 glass block under saline (2.6 mmol l-1 KCl, 1.8 mmol l-1 CaCl2, 150 mmol l-1 NaCl and 11 mmol

-1 200 l sucrose) (Lozano et al., 2001) and exposed to CO2 using the chamber described above

201 with a 15-minute pre-treatment recording period followed by a 1-minute CO2 exposure, 202 followed by 15 minutes of recording.

203 Light output of isolated light organs

204 To isolate the LO, a larva was pinned to a wax dish and a cut was made along the dorsal 205 midline to expose the internal organs. The LO, visible as a small cell mass associated with

206 the silvery-white tracheal reflector, was removed from the body and placed in saline. For

207 each application (n = 9), a baseline bioluminescence output was established over 15

208 minutes. The saline was then replaced with CO2-saturated saline and imaged for another 15

209 minutes. CO2 saturation was achieved by bubbling the gas through the saline beforehand via 210 an aquarium aerator stone for 60 seconds. As a control, the isolated LOs of 5 larvae were

211 treated with acidified saline at the same pH as the CO2-saturated saline (pH 5.26). As an

212 alternative means of introducing CO2, the gas was introduced for 1 minute into the air-space

213 above an excavated block holding isolated LOs (n = 8) under saline.

214 Light output in semi-intact dissected larvae

215 An apparatus was designed to allow introduction of gas to exclusively the head region or the 216 LO-containing region of a partly-dissected larva. Larvae (n = 5 for each treatment) were

217 pinned and dissected along the dorsal midline on a wax dish. A plastic cylinder (5 mm 218 diameter) was then placed over either the LO or the head region containing the brain and

219 an air-tight barrier was formed using petroleum jelly to isolate the cylinder from the rest of 220 the larva. A glass cover-slip was then placed over the top of the cylinder. A gas inlet and

221 outlet port were drilled into the side of the cylinder. This approach allowed sequential

222 directed introduction of CO2 into the airspace (a) above the light organ or (b) above the 223 brain.

224 After dissection and mounting of the cylinder, a larva was imaged for 15 minutes (1 frame

225 per minute) and CO2 was introduced into the cylinder air-space for 1 minute (10 L/min). The

226 larva was then imaged for a further 25 minutes and the CO2 cylinder was moved to the

227 alternative target area (either LO or brain) not exposed in the initial phase. CO2 was

228 introduced at the second location and imaged for 15 minutes.

229 To apply CO2 in solution, larvae (n = 5 for each treatment) were pinned and dissected on a

230 wax dish to reveal the LO and internal organs, as described above. Before any saline was 231 applied, a petroleum jelly barrier was constructed across the mid-body so that a solution

232 applied to either segment would not contact or flow through to the other, then both the 233 anterior and posterior regions were immersed in a drop of saline. The larvae were imaged

234 for 15 minutes then CO2-suffused saline was placed on either (a) the LO segment or (b) the 235 brain segment and imaged for a further 15 minutes. Standard saline was used to flush away

236 the CO2-suffused saline before imaging for a further 25 minutes. CO2-suffused saline was

237 then placed on whichever target site was not exposed in the initial phase of the experiment 238 and imaged for a further 15 minutes.

239 Effect of hypoxia on light output

240 To investigate the effect of hypoxia, the isolated light organs of 3 larvae in saline were

241 exposed to humidified N2 in the air-space above the LOs for 1 minute. After 15 minutes, CO2

242 was introduced for 1 minute at a rate of 10 L/minute. In a modified approach, isolated LOs

243 (n = 3) in saline were exposed to N2 for 1 minute followed immediately by CO2 for 1 minute,

244 both at a rate of 10 L/minute. After 15 minutes the CO2 exposure was repeated. This

245 treatment was also applied to ligated, head-removed sections of larvae (n = 3). Hypoxia 246 recovery was further explored by exposing either isolated LOs (n = 9) or ligated, head-

247 removed sections of larvae (n = 9) to N2 for 1 min at a rate of 10 L/minute, followed by a

248 recovery time of either 5, 10, or 15 minutes before being exposed to CO2 at the same rate

249 and duration. The imaging set-up and pre-and post-exposure periods were the same as for

250 CO2 exposures described above.

251 Alternative anaesthetics

252 To investigate bioluminescence output when the LO was exposed to alternative

253 anaesthetics, isolated LOs in saline were exposed to ethyl acetate or diethyl ether (n = 4, for 254 each treatment). Approximately 50 ml of either ethyl acetate or diethyl ether was placed in

255 a 500 ml glass conical flask and vapour allowed to accumulate for 15 minutes. To expose the 256 LO, air was pumped through this bottle into the LO-chamber for 1 minute at a rate of 10

257 L/minute with a pre- and post-exposure imaging period of 15 min. An isoflurane vaporiser

258 (Model V300PS) was used to expose the larvae (n = 4) to 4% isoflurane at the same rate and 259 duration as the other anaesthetics.

260 Vibration response

261 Larvae were placed in a humidified aquarium before the onset of darkness. Two 10 mm 262 diameter vibrating AD1201 haptic motors (180-200 Hz) were attached to the sides of the

263 aquarium with duct tape and operated using a programmed Raspberry Pi 3 (Model B V1.2). 264 Light output of the larvae was recorded at 1-minute intervals through the night using a SLR

265 camera (Canon EOS 1000D) under time-lapse control, viewing the larvae from below the

266 aquarium. The vibrating haptic motors were activated in 15 sec pulses at a range of inter- 267 pulse intervals throughout the scotophase (Table 1). Treatment nights were interspersed

268 between control nights with no vibration stimuli, using the same larvae. Light intensity of 269 individual larvae was determined using ImageJ as described above. In this series of

270 experiments the light units are not comparable to those from all other experiments due to 271 the different location of the camera. The light output was displayed as the mean intensity of

272 the larvae in the aquarium. The same larvae were used in multiple treatments; however, 273 data from larvae that pupated during or within three days of an experiment were removed.

274 Statistical analysis

275 Data from physiological experiments were analysed using a paired t-test, comparing the

276 total mean light output for 15 minutes pre- and post-exposure to stimuli. Further, a paired t- 277 test was performed to compare light output in paired trials on the same larvae, as this test

278 compares two different methods of treatments, where the treatment is applied to the same 279 subject. The test determined if the two methods of treatment produced results with

280 significant difference.

281 Results

282 CO2 response

283 The bioluminescence output of live larvae (n = 4) increased when exposed to CO2 in the air-

284 space (Fig. 1a). Very low levels of light were emitted by the larvae before treatment, but this

285 output did not register on the scale that shows the subsequent response to CO2. On

286 exposure, the larval light intensity increased over the next 10 minutes and started to decline

287 after reaching a mean peak light intensity of 6.1 x 106 units.

288 Ligated, head-removed larvae and ligated, LO-isolated larvae (n = 10, 11, respectively)

289 displayed increases in bioluminescence intensity when exposed to CO2 in the air-space (Fig. 290 1b). Both treatments showed an initial increase in bioluminescence followed by a brief

291 decrease and then a steady increase in bioluminescence, culminating at a mean peak light 292 intensity of approximately 4.9 x 106 units (ligated, head-removed) and 2.5 x 106 units

293 (ligated, LO-isolated). In both, the bioluminescence level remained elevated after the 15- 294 minute recording period.

295 When light organs were removed by dissection, they were isolated from the larval body and

296 all contact with nerves and main tracheal trunks removed. After this treatment, the light 297 organs released light at relatively low levels through the pre-exposure period (mean of 3.9 x

5 298 10 units). Exposure to CO2-saturated saline (n = 9) or CO2 in the air-space (n = 8) produced

299 an immediate increase in bioluminescence intensity within 1–2 minutes (Fig. 2). The CO2-

300 saturated saline treatment produced a peak bioluminescence level of 7.1 x 106 units, that 301 exponentially declined over the next 15 minutes but remained above the pre-stimulus levels

6 302 of bioluminescence. The CO2-air-space treatment showed a lower peak of 5.5 x 10 units 303 and returned to pre-stimulus bioluminescence levels after approximately 7 minutes.

304 Localisation of the CO2 effect

305 The high light emission response to CO2 was localised by sequentially exposing the LO and

306 the brain to CO2 using two different approaches (1) applying CO2-saturated saline to either

307 region or (2) exposing the air-space above either region. When CO2-saturated saline was

308 applied to the LO first (n = 5) (Fig. 3a) bioluminescence production increased greatly and no 309 obvious increase occurred when the treatment was applied to the brain. Similarly, when the

310 CO2-saturated saline was applied to the brain first and then the LO (n = 5), bioluminescence 311 intensity increased when the treatment was directed to the LO. The sequential application

312 of CO2 to the air-space above the brain and the LO (n = 5 for each treatment) (Fig. 3b)

313 produced similar results: exposure to the brain region produced no bioluminescence 314 response while exposure to the LO produced a large response. Overall, the bioluminescence

315 responses recorded through exposure to CO2-saturated saline (Fig. 3a) produced less light

316 than those exposed to CO2 in the air-space (Fig. 3b). Air-space exposure produced a shorter-

317 lasting light output than saline exposure.

318 To determine if the application of CO2 was indirectly triggering acute bioluminescence

319 response by altering either the internal pH of A. flava or the pH of the saline, an acidified

320 saline of pH 5.26, identical to that of CO2-saturated saline, was applied to isolated LOs (n =

321 4). No bioluminescence responses were recorded after exposure (data not shown).

322 Combining hypoxia and CO2 exposure

323 To determine the effect of hypoxia on bioluminescence production, isolated LOs (n = 3)

324 were exposed to N2 for 1 minute, immediately followed by the introduction of CO2 for the

325 same period. No bioluminescence responses were detected (data not shown). When a 15-

326 minute recovery period was allowed, and a second CO2 treatment was applied, the isolated

327 LOs (n = 3) produced no bioluminescence responses (data not shown). However, when this 328 same treatment series was applied to ligated, head-removed larvae (n = 3) (Fig. 4), a low

329 response was elicited after the first CO2 exposure and a much higher bioluminescence level

330 was recorded following the second CO2 exposure. As an additional control, introduction of

331 N2 into the air-space above isolated LOs produced no bioluminescence (n = 3, data not 332 shown).

333 To investigate the ability of ligated, head-removed larvae to recover from hypoxia, the

334 segments were exposed to N2 for 1 minute, followed by a recovery period of 5, 10 or 15

335 minutes (n=3 for each treatment) before exposure to CO2 for 1 minute. In all cases, light was

336 released on exposure to the CO2 pulse with the maximum mean light output increasing with 337 recovery time (Figure 5). When the identical treatments were applied to isolated LOs, no

338 light was emitted after the recovery periods.

339 Alternative anaesthetics

340 To determine if the CO2 response was due to an anaesthetic effect, the alternative 341 anaesthetics isoflurane, ethyl-acetate, and diethyl-ether were applied to isolated LOs via air-

342 space treatment (n = 4 for each treatment) (Fig. 6). None of the treatments produced

343 bioluminescence responses at levels comparable to that induced by CO2. A small increase

344 was noted in the ethyl acetate and diethyl ether treatments; however, it did not precisely 345 coincide with exposure.

346 Vibration

347 Undisturbed larvae began bioluminescence shortly after the onset of darkness and reached

348 peak light intensity approximately 1 hour into the 12-hour scotophase (Fig. 7). Following the 349 peak, the bioluminescence intensity steadily declined over the next 11 hours and did not

350 completely cease until lights-on.

351 Exposure of larvae to vibration produced significant increases in bioluminescence output

352 compared with the controls recorded during the preceding scotophase without vibration 353 (Paired t-test, P<0.001) (Fig. 8). Larvae exposed to one minute of vibration at frequencies of

354 1 pulse/hour, 1 pulse/30 minutes, and 1 pulse/15 minutes showed acutely elevated light

355 responses to vibration stimulus (Fig. 8a–c). The bioluminescence outputs peaked following 356 vibration and then exponentially returned to approach the pre-stimulus levels. It is apparent

357 that the average amplitude of the acute vibration response decreased as the interval 358 between pulses shortened. Acute responses followed by declines were not seen when

359 treatments occurred less than 15 minutes apart (Fig. 8d–f). Rather, larvae displayed 360 persistent, elevated levels of bioluminescence without the response-decline seen when

361 treatments were more widely spaced. Larvae vibrated at 1 pulse/minute recorded the 362 greatest bioluminescence increase.

363 When larvae (n = 9) were exposed to vibration pulses at 1 pulse/5 minutes for 3 hr, the

364 bioluminescence level was elevated and responses to the individual pulses were not 365 apparent (Fig. 9). When vibration ceased between 3:00 and 6:55 hours after lights-off,

366 bioluminescence returned to a lower baseline level and approached that recorded in the 367 control treatment. When the pulse series was re-initiated, the larval light output

368 immediately elevated and remained higher than in the rest period.

369 Discussion

370 Elevated Bioluminescence in Response to CO2

371 At the initiation of this work, it was believed that CO2 causes a major release of light in 372 Arachnocampa through its anaesthetic effect (Lee, 1976; Rigby and Merritt, 2011). In

373 insects, CO2 is a widely used anaesthetic that is believed to block synaptic transmission at

374 the neuromuscular junction by influencing glutamate sensitivity (Badre et al., 2005). Diethyl 375 ether is a common anaesthetic used for many years on both vertebrates and invertebrates

376 (Whalen et al., 2005). The halogenated ether, isoflurane, is also widely used, including on

377 insects, where it acts upon voltage-gated K+ channels (Barber et al., 2012; McMillan et al., 378 2017). Ethyl acetate is another insect anaesthetic (Loru et al., 2010); however, its mode of

379 action is unknown. Here, we tested the effect of these anaesthetics on different

380 preparations of the light organ and found low to zero response to any other than CO2,

381 casting doubt on the concept of anaesthesia releasing neural repression of light output. 382 Isoflurane, ethyl acetate, and diethyl ether produced no bioluminescence responses in

383 isolated light organs that approach the CO2-induced response magnitude. A small increase 384 was noted in the ethyl acetate and diethyl ether treatments; however, it did not precisely

385 coincide with exposure. Lee’s (1976) experiments were performed on an unstated and

386 possibly small sample size and responses to the different anaesthetics were not quantified, 387 so it is possible that light levels were not strictly compared among treatments. It has been

388 noted since that the light released upon separation of the LO from the body was very dim

389 compared to that released under exposure of the LO to CO2 (Rigby and Merritt, 2011). The

390 present study confirms that observation, that CO2 produces intense light production.

391 To explore the location of the CO2 effect, either the brain or the LO of whole, semi-dissected

392 larvae (the body wall was opened up and pinned out under saline to reveal the internal

393 organs) was exposed to either CO2-saturated saline or CO2 in the airspace. Using either

394 method, bioluminescence responses were seen only when CO2 was directed to the LO,

395 indicating that its effect is initiated at the LO and that exposure of the brain has no direct

396 effect on bioluminescence. The outcome also supports the above: that the CO2-induced

397 elevation of light is not due to a general anaesthetic effect. The findings cast doubt on the 398 validity of the current models where neural signals from the brain repress bioluminescence

399 and where CO2 acts as a general anaesthetic.

400 How then does CO2 have such a strong effect on bioluminescence? A supply of air to the

401 larva is essential for light emission as shown by dimming of larvae under vacuum, and a 402 return when air was readmitted (Lee, 1976). Previous studies have highlighted the intimate

403 relationship between the LO and the tracheal mass (Green, 1979), indicating that the 404 availability of oxygen is necessary for the oxidative bioluminescence reaction, just as it is in

405 firefly larvae (Carlson, 1965; Hastings and Buck, 1956) and adults (Buck, 1948; Timmins et

406 al., 2001). Here, we confirmed that hypoxia limits the CO2-induced brightening. When CO2

407 was introduced immediately following N2 exposure or after recovery periods of 5 – 15

408 minutes, the isolated light organs showed no bioluminescence. When CO2 was applied to

409 ligated, head-removed larvae immediately after N2, a low light output was recorded and a

410 second CO2 exposure 15 minutes later produced a significantly higher light output, 411 indicating that the ligated section is capable of recovery from hypoxia. Using another

412 approach, ligated head-removed larvae exposed to N2 followed by CO2 after a spaced delay

413 (5 – 15 minutes) showed a recovery the intense CO2 response, with signs of a graded

414 increase in magnitude with increased recovery time.

415 We consider it most likely that recovery is due to the LO tracheal mass maintaining a

416 connection with the main longitudinal tracheal trunks in ligated larvae, whereas that

417 connection had been removed in isolated light organs. Arachnocampa larvae are apneustic 418 (Ganguly, 1959), i.e. they have no spiracles, although the longitudinal tracheal trunks are air-

419 filled. This respiratory strategy is common in aquatic or endoparasitic insect larvae. Gas 420 exchange is cutaneous—across the cuticle—which is probably aided in Arachnocampa by

421 the fact that larvae have thin cuticle and dwell inside a mucous tube. In apneustic insects, 422 air-filling is attributed to the cells lining the trachea having the ability to withdraw fluid from

423 the tracheal lumen and replace it with air (Buck and Keister, 1955). In Arachnocampa larvae, 424 the longitudinal tracheal trunks run directly into the tracheal mass associated with the

425 photocytes (Green, 1979). We propose that the epithelial cells of the main paired

426 longitudinal tracheal trunks modulate the oxygen content of the lumen through active 427 transport. The recovery seen in ligated larvae is attributed to reoxygenation of the tracheal

428 lumen during the recovery period and suggests that available oxygen is consumed during

429 the CO2-induced bioluminescence.

430 There have been no reports of similar acute bioluminescence outputs in larval or adult

431 fireflies upon CO2 exposure (Buck, 1948) so Arachnocampa’s response appears to be unique

432 to its regulatory system. CO2 is known to be a product of light production in the ATP- 433 dependent light production system of fireflies (Plant et al., 1968), which is likely to also

434 apply to Arachnocampa because of the related luciferase system (Lee, 1976). It could be

435 that light production in the photocytes is modulated by both CO2 and O2 levels and that the

436 light regulation mechanism is reliant on a balance between both gases. It is possible that

437 high PCO2 triggers a runaway reaction due to the supra-metabolic levels of CO2 producing

438 maximum light production in the LO cells. Such high and persistent light levels were also

439 seen when larvae were fed prey items dosed with either phentolamine or octopamine, 440 involving biogenic amines in bioluminescence regulation (Rigby and Merritt, 2011) but how

441 neurotransmitters interact with gas regulation remains unknown.

442 Vibration response

443 Larvae are capable of upregulating the level of bioluminescence is situations where prey are

444 detected in their web, during aggressive interactions with other larvae (Mills et al., 2016) 445 and at the onset of rainfall (Merritt and Patterson, 2018). These responses are believed to

446 be mediated through detection of vibration and this stimulus-response system is readily

447 manipulated in the laboratory (Mills et al., 2016). When larvae were glowing at an 448 undisturbed level during the night, a vibration stimulus produced an acute increase in light

449 output to about x times the base level that slowly returned to pre-stimulus levels (Mills et 450 al., 2016). The up-regulation is likely mediated through the nervous system; however, it is

451 not known whether the elevated light emission is attributable to an increased oxygen supply 452 to the photocytes or to the availability of the light-producing reaction involving luciferin,

453 luciferase or ATP. Here we examined whether persistent stimulation would produce 454 habituation or effector fatigue. First, we tested whether light emission decreased with

455 repeated vibration exposures. When larvae were exposed to 1 pulse/15 minutes or less

456 frequently, an acute response was recorded. When pulses were more closely spaced, light 457 output remained persistently elevated above the control level. This appears to be due to a

458 general elevated excitation level because a 3-h gap in stimulus exposures resulted in a 459 gradual decline in light output, approaching the control level, followed by a return to a

460 higher level when stimuli were reinstated. We conclude that habituation does not occur 461 through a 12 h scotophase because the level of response to spaced pulses was consistent.

462 Second, evidence of effector fatigue was seen: the more widely spaced pulses produced 463 consistently higher peak responses. This could be due to reduced availability of light-

464 producing metabolites as the stimuli become more closely spaced. While the experimental

465 observations presented here do not shed light on the mechanism by which vibration 466 elevates light output, the vibration response will be a useful readout for quantifying the

467 effect of gas mixtures on the bioluminescence system.

468 Bioluminescence regulatory mechanism

469 Our findings suggest that the model of light production being suppressed under neural

470 control when it is in the off state should be rejected due to (1) the lack of a bioluminescence

471 response when anaesthetics other than CO2 were applied to the LO, and (2) the fact that

472 CO2 directly affects the light organ. The vibration responses mentioned above are indicative 473 of an active elevation of bioluminescence in response to a neural stimulus mediated by the

474 sensory system, so a simpler control model is that excitatory neural signals initiate 475 bioluminescence, maintain it at a steady state, and mediate the vibration-induced startle

476 reflex. Octopamine is the prime candidate as the neurotransmitter (Rigby and Merritt). The

477 time-course of light elevation and reduction suggests that the neuronal control acts through 478 a second mechanism acting over a longer time-course with the most likely being the

479 modulation of oxygen access to the photocytes. One possibility is a sphincter-based control 480 system that modulates airflow between the longitudinal trachea and the tracheal mass, as

481 mentioned by others (Gatenby and Ganguly, 1956; Rigby and Merritt, 2011) but not yet 482 thoroughly investigated. Another is modulation of oxygen transfer across the junction

483 between the tracheal mass and the photocytes, perhaps akin to the indirect way oxygen 484 transfer between tracheal end cells and photocytes is modulated in adult fireflies (Timmins

485 et al., 2001; Trimmer et al., 2001). At the ultrastructural level, the tracheolar units are very

486 closely opposed to the basal lamina of the photocyte cells with many deep infoldings of the 487 membrane (Green, 1979), all consistent with a functional association between the

488 photocytes and the tracheal supply. There is no barrier of mitochondria between the 489 photocyte cytoplasm and the air supply as seen in firefly LOs—the large mitochondria in

490 Arachnocampa photocytes are distributed throughout the cytoplasm (Green, 1979)— 491 however the firefly arrangement appears to be specifically adapted to flash control. Further

492 experiments exposing larvae to combinations of gas mixtures and excitatory/inhibitory 493 stimuli should reveal more details of light regulation in Arachnocampa.

494

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604 Acknowledgements

605 We would like to thank Dr. Simone Blomberg for statistical advice, Professor Bruno Van Swinderen and 606 Michael Troup for use of isoflurane vaporiser equipment, and members of the Asgari Lab for help with pH 607 measurement. We would like to thank Rory Charlton for his invaluable assistance in designing and 608 constructing the vibration apparatus.

609 Competing Interests

610 The authors have no competing interests.

611 Funding

612 The authors acknowledge financial assistance from the School of Biological Sciences, the University of 613 Queensland.

614 Figure Legends

615 Figure 1: The bioluminescence output of (a) live A. flava larvae (n = 4, mean ± s.e.m.), and (b) ligated, head- 616 removed larvae (n = 10, blue) and ligated LO-isolated larvae (n = 11, red) (means ± s.e.m.) following exposure to

617 CO2 in the air-space. Yellow bar indicates period of gas exposure.

618 Figure 2: The bioluminescence outputs of isolated A. flava LOs following exposure to CO2-saturated saline (n =

619 9, red) and the introduction of CO2 into the air-space (n = 8, blue) (means ± s.e.m.). Yellow bar indicates CO2-

620 diffused saline application or introduction of CO2 into the airspace.

621 Figure 3: The bioluminescence outputs of semi-intact A. flava larvae when exposed to (a) CO2-saturated saline 622 (yellow bars) in the order (1) the LO followed by the brain (n = 5, blue) or (2) the brain followed by the LO (n =

623 5, red) (means ± s.e.m.). (b) Bioluminescence outputs following air-space CO2 exposure (yellow bars) depicted 624 as the same colour codes as (a) (n = 5, for each treatment order) (means ± s.e.m.).

625 Figure 4: The bioluminescence output of ligated, head-removed A. flava larvae (n = 3) in response to N2

626 exposure (orange bar) immediately followed by CO2-exposure (yellow bar). After a 15-minute recovery period,

627 the LOs were again exposed to CO2 (yellow bar).

628 Figure 5: The bioluminescence output of ligated, head-removed A. flava larvae (n = 3) in response to N2

629 exposure (orange bar) followed by CO2-exposure after 5 (n = 3, blue), 10 (n = 3, red), or 15 (n = 3, green) 630 minutes (yellow bars).

631 Figure 6: The bioluminescence response of isolated A. flava LOs (n = 4 for each treatment) (means ± s.e.m.)

632 following exposure to ethyl-acetate (red), diethyl-ether (green), Isoflurane (blue), and to CO2 into the airspace 633 (n = 8) (purple).

634 Figure 7: The generalised bioluminescence output (mean ± s.e.m.) derived from 108 A. flava larvae recorded in 635 laboratory conditions under an artificial light cycle (lights-off at 0:00 and on at 12:00).

636 Figure 8: Bioluminescence output (mean ± s.e.m.) of live A. flava larvae in response to a 15-second (180-200 637 Hz) vibration at a rate of: (a) 1 pulse/hour (n=17), (b) 1 pulse/30 min (n=17), (c) 1 pulse/15 min (n=17), (d) 1 638 pulse/12 min (n=7), (e) 1 pulse/10 min (n=8), and (f) 1 pulse/min (n=9). The bioluminescence output of the 639 same larvae recorded the during prior night’s scotophase is shown in red. The insets show the total 640 bioluminescence output of larvae through the treatment night (blue) in comparison to the total 641 bioluminescence released in the previous control during night (red) (* P < 0.001).

642 Figure 9: Bioluminescence output (mean ± s.e.m.) of 9 live A. flava larvae in response to a 15-second 180-200 643 Hz vibration at a rate of 1 pulse/5 minutes between 0:00-3:00 and 6:55-12:00 (yellow). Vibration responses 644 were compared to the bioluminescence output of an untreated control (red) recorded the previous night.

10 (a) 8

) 6 6

(x10 4

Light Intensity 2

0 5 10 15 20 25 30 Time (min) 645 10 (b) 8

) 6 6

(x10 4

Light Intensity 2

0 5 10 15 20 25 30 Time (min) 646

647

) 6 6

(x10 4

Light Intensity 2

0 5 10 15 20 25 30 Time (min) 648

649

4 (a)

3 ) 7 2 (x10

Light Intensity 1

0 0 10 20 30 40 50 60 Time (min) 650 4 (b)

3 ) 7 2 (x10

Light Intensity 1

0 0 10 20 30 40 50 60 Time (min) 651 652 653

4 ) 7 (x10 2 Light Intensity

0 0 10 20 30 40 50 Time (min) 654

) 15 7

(x10 10

Light Intensity 5

0 0 10 20 30 40 50 Time (min) 655

) 6 6

(x10 4

Light Intensity 2

0 0 5 10 15 20 25 30 Time (min) 656

657

658

10 ) 5 (x10 5 Light Intensity

0 0:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00 8:00 9:00 10:00 11:00 12:00 Hours After Lights-Off (24 h) 659 660

661

662

663

20 20 10 10 (a) 15 (b) 15 ) ) 8 8 10 10 (x10 (x10 8 * 8

Light Intensity 5 Light Intensity 5 * 664 0 0 ) ) 6 6 6 6 (x10 665 (x10 4 4 Light Intensity Light Intensity 2 2

666 0 0 0:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00 8:00 9:00 10:00 11:00 12:00 0:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00 8:00 9:00 10:00 11:00 12:00 Hours After Lights-Off (24 h) 667 Hours After Lights-Off (24 h)

20 20 10 10 668 (c) 15 (d) 15 ) ) 8 8 10 10 (x10 8 (x10 8 * Light Intensity Light Intensity 5 5 * 0 0 ) 669 ) 6 6 6 6 (x10 (x10 4 4 670 Light Intensity 2 Light Intensity 2

671 0 0 0:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00 8:00 9:00 10:00 11:00 12:00 0:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00 8:00 9:00 10:00 11:00 12:00 Hours After Lights-Off (24 h) Hours After Lights-Off (24 h) 672

20 20 10 10 * 673 (e) 15 (f) 15 ) ) 8 8 10 10 (x10 (x10 8 * 8 Light Intensity Light Intensity 5 5

0 0 ) 674 ) 6 6 6 6 (x10 (x10 4 4 675 Light Intensity Light Intensity 2 2

676 0 0 0:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00 8:00 9:00 10:00 11:00 12:00 0:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00 8:00 9:00 10:00 11:00 12:00 Hours After Lights-Off (24 h) Hours After Lights-Off (24 h) 677

) 6 6

(x10 4

Light Intensity 2

0 0:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00 8:00 9:00 10:00 11:00 12:00 Hours After Lights-Off (24 h) 678