bioRxiv preprint doi: https://doi.org/10.1101/2021.01.19.427229; this version posted April 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license. Development of larvae of the Australian blowfly, Calliphora augur (Diptera: Calliphoridae), at constant temperatures Donnah M. Day1 Nathan J. Butterworth2*, ORCID: 0000-0002-5679-2700, Twitter: @Butterworthbugs Anirudh Tagat3, ORCID: 0000-0002-7707-453X, Twitter: @inhouseeconomist Gregory Markowsky3, ORCID: 0000-0003-1656-337X 1,4 James F. Wallman , ORCID: 0000-0003-2504-6075 1Centre for Sustainable Ecosystem Solutions, School of Earth, Atmospheric and Life Sciences, University of Wollongong, NSW 2522, Australia 2School of Life Sciences, University of Technology Sydney, Ultimo, NSW 2007, Australia 3School of Mathematics, Monash University, VIC 3800, Australia 4Faculty of Science, University of Technology Sydney, Ultimo, NSW 2007, Australia * Corresponding author E-mail address: [email protected] Keywords: Forensic entomology, post-mortem interval, prediction interval 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.19.427229; this version posted April 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license. 1 Abstract 2 Calliphora augur (Diptera: Calliphoridae) is a common carrion-breeding blowfly of forensic, medical 3 and agricultural importance in eastern Australia. Despite this, detailed information on the 4 developmental biology of C. augur is lacking. Here, we present the first comprehensive study on the 5 development of all three larval instars of C. augur, fed on sheep’s liver, at constant temperatures of 6 15, 20, 25, 30 and 35°C. We provide thermal summation models describing instar duration, as well as 7 95% prediction intervals for larval length at each constant temperature, enabling the age of larvae of 8 C. augur to be estimated from their developmental stage and their average length. These data provide 9 a basis for the application of this species to forensic investigations. 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.19.427229; this version posted April 11, 2021. 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-ND 4.0 International license. 31 Introduction 32 Forensic entomology is the study of insects as applied to legal issues. It may relate to medicolegal 33 events, especially murders, for the purpose of uncovering information useful to an investigation 1. This 34 is because decomposing vertebrate remains provide a temporary micro-habitat and food source that is 35 attractive to a variety of insects and other invertebrates 2. Arthropods constitute a major element of 36 this fauna, with insects, especially flies and their larvae, being the predominant group. Blowflies 37 (Diptera: Calliphoridae) are ubiquitous and are typically the first insects to visit a dead body, often 38 well before it is discovered by humans 3. 39 40 Data on the development of blowflies and other important carrion flies can help provide an estimate 41 of the minimum time since death (or minimum post-mortem interval: mPMI), especially when 42 combined with information on the succession patterns of insects on corpses4. Current approaches to 43 age estimation are based on species-specific rates of development such as length, weight, and 44 developmental duration in relation to temperature. While measures based on length and weight are 45 valuable, developmental duration is the most widely used method of age estimation for two primary 46 reasons; 1) because the duration of each stage of larval development (referred to as ‘instars’) is less 47 variable than larval size (particularly in response to differences in diet)5; and 2) because instar stage is 48 not affected by specimen shrinkage during preservation6. Once these measurements are obtained for 49 the entire period of larval development, a variety of models can be used to estimate mPMI; including 50 curvilinear regression 8, isomegalen and isomorphen diagrams 9, and thermal summation models 10,11. 51 Of these, the most widely used are thermal summation models that incorporate temperature (usually 52 as degree-hours) and time spent in each developmental stage 12. 53 54 Calliphora augur Fabricius is a common blowfly species found in eastern Australia 13, the larvae of 55 which are among those most commonly collected from forensic cases, including from inside buildings 56 14. It is ovoviviparous 15, and also an agent of myiasis in sheep and other animals 16–18. Adult females 57 prefer fresh carcasses 19, but will also larviposit on carcasses in active decomposition (i.e., 10-15 days 58 old) 20. While previous studies have considered the larval development of C. augur 16,20–24 none have 59 made fine-scale size measurements across a range of constant temperatures, or used thermal 60 summation models to estimate mPMI. This lack of accurate developmental data means that at present, 61 C. augur cannot be used reliably in a forensic context. To ameliorate this, we present the first 62 comprehensive analysis of larval growth at constant temperatures of this important species. Our 63 investigations are based on measurement of larval length throughout development, as well as 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.19.427229; this version posted April 11, 2021. 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-ND 4.0 International license. 64 observation of larval instar duration. Most importantly, we provide thermal summation models based 65 on instar duration, and calculate prediction intervals based on larval length – for the first time allowing 66 the application of this species to forensic investigations. 67 68 Materials and methods 69 Fly culture 70 A culture of C. augur was established from adults trapped at Wollongong, New South Wales (34° 25' 71 S, 150°53' E). Experiments commenced using larvae of the F3 generation and were continued for 11 72 generations. This culture was refreshed regularly with wild-type (F0) individuals from the same source 73 population as above and maintained with mixed ages of females and males. The average fecundity of 74 each female was estimated by separating 20 females and transferring them to 50 mL specimen pots 75 containing approximately 10 g of freshly defrosted sheep’s liver (defrosted to room temperature 76 immediately prior to experiments). Larvae were counted the following day. Larvae and adults were 77 kept in a temperature-controlled room at 25±3.5˚C and ambient humidity, with a 12:12 light:dark 78 regime incorporating a 15 minute ‘dusk’ and ‘dawn’ transition period of low light. Adult flies were 79 maintained in square plastic cages measuring 330 mm long, 220 mm wide and 250 mm high (external 80 dimensions). The flies in each cage were provided with water and sugar ad libitum, and chopped 81 sheep’s liver in a small plastic weigh boat for larviposition as required. Larvae were reared in square 82 white 2 L plastic containers, measuring 170 mm wide along each side and 90 mm deep. Most of the 83 centre of the lid of each container was cut away and replaced with fine mesh to permit ventilation. In 84 each container, larvae were sustained on sheep’s liver in the weigh boats placed atop a layer of wheaten 85 chaff approximately 20 mm deep to enable pupariation. After pupariation, pupae were manually sorted 86 from the chaff and transferred to a cage. Sheep’s liver was provided just prior to eclosion to provide 87 females with a constant source of protein for ovary maturation 20. 88 89 Development at constant temperatures 90 To assess rates of development at constant temperatures, freshly laid larvae were randomly collected 91 from the main culture. Preliminary studies on larval density indicated that some endogenous heat 92 generation occurred with as few as 25 larvae growing on 50 g of sheep’s liver. To ensure that the 93 larvae developed at the ambient temperature being examined, each replicate (a plastic weigh boat 94 containing larvae and liver defrosted to room temperature immediately prior to the experiment) was 95 limited to 10 larvae. The growth period (between 0 and 600 hours) of each replicate of 10 larvae at a 96 particular temperature was chosen by lottery. Between three and six replicates were used for each time 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.19.427229; this version posted April 11, 2021. 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-ND 4.0 International license. 97 interval (Supplementary Material 1). These replicates were grown at specific constant temperature 98 regimes (15, 20, 25, 30 and 35˚C ± 1.0 °C) and 60 ± 5% relative humidity, in an Axyos (Brisbane, 99 Queensland, Australia) environmental cabinet. To ensure that the temperature inside the cabinet 100 matched the set value, each replicate also contained a temperature logger (iButton; accuracy ±1.0 °C, 101 resolution 0.5 °C; Maxim Integrated Products, Sunnyvale, CA, USA).