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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 3 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] 8 9 WORD COUNT: 7718 10 11 Short title: Bioluminescence regulation in glow-worms 12 13 Key-words: glow-worm, anaesthesia, fungus gnat, light organ, photocyte 14 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 2 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. 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- 3 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. 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 4 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. 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).