Morelia Spilota Imbricata): Is There Evidence for Mesopredator Release in Response to Fox Baiting?

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Biology of the south‐west carpet python (Morelia spilota imbricata): is there evidence for mesopredator release in response to fox baiting?

This thesis is presented for the degree of Doctor of Philosophy

Gillian Lee Bryant

BSc (Hons)

School of Veterinary and Biomedical Sciences

Murdoch University

Perth, Western Australia

2012

"In the end we will conserve only what we love. We will love only what we understand. We will understand only what we are taught." Baba Dioum, 1968

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I declare that this thesis is my own account of my research and contains as its main content of work, which has not previously been submitted for a degree at any tertiary education institution

Gillian Lee Bryant

ABSTRACT

The complexity of ecological networks and ecosystem function has been debated amongst ecologists since the early 1900s. Increasing anthropogenic interference to ecosystems, including habitat loss through land clearing and introduction of exotic species, have provided additional complexity to the functioning of native ecological networks. Greater understanding of such complex and dynamic ecosystems is informed by gaining recognition of the role of top-order (apex) and mesopredator (lower-order, usually smaller-sized) species in the regulation of energy transfer within an ecosystem.

This thesis investigates the mesopredator release hypothesis (MRH) by examining the biology of the Near-Threatened south-west carpet python (Morelia spilota imbricata) as a native mesopredator species to a population control program of an apex exotic species, the red fox (Vulpes vulpes).

Conventional methods, including mark-recapture studies, to quantify population trends of M. s. imbricata are impractical in this cryptic species, which cannot be trapped by enticement. Focal animal studies examining the ecology, physiology, and behaviour of pythons through radiotelemetry monitoring were carried out between 2006 to 2008, in fox baited and unbaited areas of coastal woodland and in fox baited areas of the northern jarrah forest in south-west Western Australia. Pythons were initially captured after they had ingested a radio-collared western ringtail possum (Pseudocheirus occidentalis, n=14) or through opportunistic sightings (n=32). Forty-six pythons were surgically implanted with radiotransmitters into their coelomic cavity under anaesthesia; where body size permitted, temperature data loggers were also attached to the transmitters. Initially pythons were able to expel the implanted radiotransmitters. A novel technique was developed to prevent radiotransmitter expulsion involving anchoring the radiotransmitter to the rib-cage of the python with a non-dissolvable

vi suture. Implementation of this technique led to the successful retention of all the implanted devices, and this surgical implantation technique has been published as an independent study (see Chapter 3).

Haematological and biochemical analyses were undertaken to investigate whether there were any health effects of surgically implanting the radiotransmitters.

Blood samples from 43 wild-caught M. s. imbricata were collected into lithium heparin from the ventral coccygeal vein at various stages over the study. References ranges are reported for 35 individuals. Since pythons had radiotransmitters surgically implanted, repeated sampling of the same individuals could be carried out allowed further investigations of how various factors (study site, season, sex, time in captivity and the presence of haemogregarine parasites) affect haematology and biochemistry measures.

There was no significant effect of sex, time in captivity or the presence of a haemogregarine parasite on blood parameters. Erythrocyte measures were influenced by study site, season, stage of radiotransmitter implantation and time in captivity. In terms of leucocytes, only basophil numbers were influenced by the python’s anaesthetic state and stage of radiotransmitter placement. Albumin, globulin (and the ratio between the two) and calcium and phosphorous were influenced by season, anaesthetic state, and the stage of radiotransmitter placement. No intrinsic or extrinsic factors appeared to affect creatinine kinase, aspartate aminotransferase, uric acid, or total protein. This study demonstrates that various factors including season, time in captivity, location and anaesthetic state will influence blood parameters of M. s. imbricata. Because these animals are increasingly common as pets, veterinarians should be aware of the influence of these factors when diagnosing the health status of individuals. This haematological investigation has been published as an independent study (see Chapter 4).

To measure the physiological and behavioural response of M. s. imbricata to fox control, weekly radiotelemetry monitoring of animals at the study sites was carried out

vii over the three years. Body condition changes, age differences, and mortality events, were assessed to determine differences in the survivorship of pythons. Counterintuitive to the MRH, higher mortality rates and faster body condition declines occurred at the fox baited sites.

Information on reproductive output was gathered to investigate whether pythons had a greater fecundity in the presence of fox baiting. Adult females were regularly palpated and any mating events were recorded. This intensive animal monitoring suggested that there were higher rates of reproductive output at the fox baited coastal woodland area. However, there were also very high rates of female mortality following parturition at this study area, which may negate or slow overall population increases, particularly as pythons are capital breeders and require an accumulation of body reserves to be reproductively successful.

As pythons and foxes share similar prey resources, it was predicted that there would be greater diversity of prey consumed by pythons at the fox baited site. This prediction was investigated through diet analysis of 47 scats collected from 24 individuals at the coastal fox baited and unbaited sites. The ability to collect numerous scats from all individuals was limited given pythons have very low metabolic rates in comparison with endothermic carnivores (e.g. cats), and that they spend long periods over winter months inactive, often sequestered in tree hollows. Although sample size varied between the fox baited and unbaited site, the analysis indicated that the diversity of prey consumed by pythons across treatment areas was similar.

Pythons were assessed for potential behavioural avoidance to the presence of foxes. Pythons at the fox baited sites did not show temporal segregation during hunting to avoid the intraguild killer, as there was no difference in the proportions of diurnally- active prey or nocturnally-active prey in the diets of pythons at either site. However, a higher proportion of pythons at the unbaited site were observed in the most cryptic

viii microhabitat (tree hollows) more often than pythons at the baited sites and also used tree hollows for longer over winter. However, in contrast to predictions for landscape- mediated avoidance (to avoid potential exposure to foxes), pythons at the unbaited site were seen more often in the most exposed body position (stretched) compared to pythons at the baited site.

Focal animal monitoring through radiotelemetry also allowed further investigation into python ecology, physiology and behaviour. Through dietary analysis coupled with thermal biology, a current foraging theory in snake biology was challenged: I tested whether the temporal window for foraging by smaller-sized snakes

(which have a low thermal inertia and therefore cool rapidly at nightfall when air temperatures fall and opportunities for basking are reduced) is restricted to diurnal hunting, cf. larger-sized snakes that may better retain body heat (higher thermal inertia).

There was no evidence that having a small body size thermally-restricted the temporal window for ambush foraging in M. s. imbricata as hourly measures of heating and cooling (rates of change in python body temperature) were not different from those of larger-bodied snakes. Small pythons were able to successfully hunt on crepuscular and nocturnal prey species such as small carnivorous marsupials as indicated through scat analysis. This study has been published as an independent study (Chapter 5).

As habitat selection can influence many physiological facets of animal life, the thermal conditions of microhabitat retreat sites may be very important for ectotherm physiology. The use of tree hollows by pythons over winter was investigated for possible thermal advantages over the alternative microhabitat resources. Temperature data loggers were placed within a range of microhabitats and were coupled with recordings of python body condition and temperature as well as air temperature. When sequestered in tree hollows, pythons had colder daily average and maximum body temperatures (cf. pythons that used other microhabitats), but this did not give them a

ix metabolic advantage (in terms of body condition scores) over winter. It was found that a greater proportion of pythons at the unbaited study site used tree hollows as winter retreats more, and for longer, compared with pythons at the baited coastal woodland site, suggesting that foxes may influence this microhabitat selection behaviour in pythons. Tree hollows provide a critical refuge over winter when python body temperature is low and their responsiveness is limited, rendering individuals extremely vulnerable to predation by terrestrial predators such as the introduced red fox. As a result of land clearing practices, habitat fragmentation, and competition with other hollow-using species it is very likely that tree hollows are a limited resource for pythons. This study indicates the conservation importance of tree hollows for M. s. imbricata. This study has been published as an independent study (Chapter 6).

This PhD research is the first attempt to quantify the impact of removal of an apex predator (the red fox) upon a native snake species. This was achieved by intensive focal animal monitoring and outlines the functional and numerical responses of this ectothermic species to the potential change in prey density as a consequence of removal of the fox. This study also investigates additional biological information essential to predict any future responses of other temperate ectothermic species to wide-spread control of introduced or native apex predators.

ACKNOWLEDGMENTS

My deepest thanks go first and foremost to my primary supervisor, Dr Trish Fleming, for her assiduous support, advice, encouragement and feedback throughout the entire

PhD process. Trish has given me an immense amount of her time, thought and help without ever turning me away. Her mentorship and friendship have taken me through this journey from field work, analysis, writing, conferences and the more difficult and trying circumstances along the way. I will be forever grateful to her.

I offer sincere gratitude to my co-supervisors Dr Kris Warren (Murdoch

University) and Paul de Tores (Department of Environment and Conservation) for their fantastic idea to choose me for this PhD. Having being offered this opportunity certainly has given me so many more experiences I never suspected I would do. Thank you for your comments to drafts, guidance, assistance with surgery techniques, time and financial support you have invested in this PhD.

Thank you to Dr Bill Bateman who has flown from South Africa and the USA to help in the field, with additional experiments that kept us awake into the small hours of the night and has also provided encouragement and support in more ways than one. Dr

Paul Eden (Perth Zoo) was an ever-so-patient teacher showing me techniques on surgery, taking blood samples, and provided additional veterinary care, advice and assistance to pythons whenever they needed it. Your generosity has been terrific.

I want to thank the Department of Environment and Conservation (DEC),

Western Australia for providing funding and support to this project. I have enjoyed meeting many of the research staff over the years, Dr David Pearson and Dr Russel

Palmer, and working with the Mesopredator Research Team: Keith Morris, Dr Nicky

Marlow, Dr Adrian Wayne, Dave Algar and Paul de Tores and Steve Lapidge (Invasive

Animals CRC). I also want to thank all the DEC technical officers that have supported

xi these research scientists, some of which captured pythons for me. I extend my thanks also to the Mesopredator Research external review panel, Dr Dave Choquenot, Dr Peter

Jarman, and Dr Andrew Burbidge for their professional criticism and encouragement to the overall project and individual advice for my PhD.

I have been lucky to have had many volunteers willing to help with fieldwork and surgery and I am indebted to them for donating their time so generously and giving me welcome company in the field. Thank you to Stuart Dawson, my cousin Jade Crow,

Tracey Moore, Judy Dunlop, Lenny Bloomfield, Anna Nowiki, Collin Sheppard Koers,

Sabrina Trocini, Tina Burrows, Dannielle Stokeld, Victoria McLarty, Doug Armstrong,

Yu Ki Wong, Christian Nîkolacson, Freya White, Oli Mallie, Jordan Hampton, Sophie

Arnal, Lynette and Kim Traish, Simon Booth, Meghan Bell, John Clark, Bonita Clark,

Christopher and Greg McCluney, and Carly Palmer. In particular, Shannon Dundas was a champion helper in the final year of field work. I will treasure the hours spent putting iButtons into tree hollows. Thank you Shannon, for your friendship, laughter, and support, and best of luck with your own PhD.

Dwellingup colleagues and friends have provided extra incentives to pursue the drives down south; the weekly McLeod’s night was one of them. My extra thanks extend to Jennifer Jackson who patiently taught me how to radio-track, introduced me to pythons and the unpleasantness of ticks! Wes Manson, I’ll never forget the ‘water tank incident’ at Martin’s Tank, or the tiger snake down the well, and I’m sure you won’t either. You have enriched my research by subjecting me to your critical questioning and debate, I look forward to more of it! I want to thank postdoctoral researchers Dr

Duncan Sutherland and Dr Al Glen for their understanding and advice on the perils of doing a PhD. Thanks to Jess Mann for your welcome hospitality and friendship in

Dwellingup.

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To Judy Clarke, Jen Cruz, Helen McCutcheon, Sabrina Trocini, Carlo Pacioni,

Kobus Wentzel, Tracey Moore, and Kathryn Napier, fellow PhDs, you have made the last five years considerably more enjoyable. Thank you for your support.

I offer thanks to my close friends outside of university; Zoe Campbell, Amberlee

Laws, Chelsea Hikins, Sonja Farrow, Erinn Litchfield, Greta Puddey, Melly Greaves and my cousin Danielle Denniss. I thank James Foley for his mutual understanding and encouragement of deadlines and computer-art help for most diagrams. My life has been enriched thanks to Glen Alexander and the momentous amount of love and support you have shared to get me through the last few hurdles the last couple of years. Celebrating small milestones with all of you over bubbles has been rewarding and I look forward to the inevitable continuation of this practice!

I have a fantastic family, who in particular gave much of their time to help me in the field, with surgery and have listened to me to talk incessantly about all things pythons over the last five years. I will be forever thankful to my sister Meagan for her loving support, understanding, encouragement and friendship. Meg, you have been a pillar of strength and stability for me. I thank my sister Courtney who volunteered many of her school holidays and weekends to help me, not only in the field, but in return for small bribes, entered data into spread sheets when I didn’t want to. James

Connor, my brother-in-law, thank you for being a big part of this journey and I am glad

I was able to introduce you to terms such as ‘predation’ and ‘faeces’. Maybe one day you will look at Leschenault fondly and not sware it’s the worst place on earth!

Finally, but most importantly, I owe enormous thanks to my loving parents,

Jenny and Alan for never wavering in their support of my academic pursuits. Their love, support, enthusiasm, and encouragement over my life are truly appreciated. I cannot thank you enough for everything you have done for me in order to finish my

PhD.

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TABLE OF CONTENTS

1. INTRODUCTION ...... 1 FERAL AND INTRODUCED PREDATORS IN AUSTRALIA ...... 1 MESOPREDATOR RELEASE HYPOTHESIS ...... 5 1.1. Endothermic and ectothermic predators ...... 6 1.2. Functional response ...... 8 1.3. Numerical response ...... 11 1.4. Total response ...... 11 STUDY AIMS ...... 12 THESIS STRUCTURE AND OUTLINE OF CHAPTERS ...... 12

2. GENERAL METHODS ...... 14 FIELD SITES ...... 14 2.1. Leschenault Peninsula Conservation Park ...... 14 2.2. Yalgorup National Park ...... 15 2.3. Jarrah forest ...... 16 STUDY ANIMALS ...... 17 2.4. Animal ethics permits and associated licences ...... 17 2.5. Capture of pythons ...... 18 2.6. Housing of animals in captivity ...... 19 2.7. Marking and identification of individuals ...... 20 FIELD METHODS ...... 21 2.8. Radiotelemetry ...... 21 2.9. Body measurements ...... 22 2.10. Air temperature ...... 23 2.11. Quality of sighting ...... 23 2.12. Microhabitat ...... 24 2.13. Behaviour ...... 24 2.14. Moving: obviously moving from one place to another ...... 25 2.15. Body position ...... 25

3. IMPROVED PROCEDURE FOR IMPLANTING RADIOTRANSMITTERS IN THE COELOMIC CAVITY OF SNAKES ...... 27 AUTHOR CONTRIBUTION: ...... 28 INTRODUCTION ...... 29 ABSTRACT ...... 29 MATERIALS AND METHODS ...... 29 3.1. Study animals ...... 29 3.2. Radiotelemetry device ...... 30 3.3. Surgical procedure ...... 30 3.4. Recovery and monitoring ...... 31 RESULTS ...... 31 3.5. Initial radiotransmitter implantation technique ...... 31 3.6. Anchored radiotransmitter implantation technique ...... 32 3.7. Statistical analysis ...... 32 DISCUSSION ...... 32 ACKNOWLEDGMENTS ...... 33 REFERENCES ...... 33

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4. WHAT FACTORS AFFECT HAEMATOLOGY AND PLASMA BIOCHEMISTRY IN THE SOUTHWEST CARPET PYTHON (MORELIA SPILOTA IMBRICATA)? ...... 34 AUTHOR CONTRIBUTION: ...... 35 ABSTRACT ...... 36 INTRODUCTION ...... 36 MATERIALS AND METHODS ...... 36 4.1. Study animals and sample collection ...... 36 4.2. Hematology analysis ...... 37 4.3. Plasma biochemistry analysis ...... 37 4.4. Statistical analysis ...... 38 RESULTS ...... 38 4.5. Reference ranges ...... 38 4.6. Factors affecting hematologic measures and plasma biochemistry ...... 40 DISCUSSION ...... 41 ACKNOWLEDGMENTS ...... 44 LITERATURE CITED ...... 45

5. DOES BODY SIZE INFLUENCE THERMAL BIOLOGY AND DIET OF A PYTHON (MORELIA SPILOTA IMBRICATA)? ...... 50 AUTHOR CONTRIBUTION ...... 51 ABSTRACT ...... 52 INTRODUCTION ...... 52 METHODS...... 53 5.1. Study sites and ambient conditions ...... 53 5.2. Study animal ...... 53 5.3. Diet analysis ...... 54 RESULTS ...... 55 5.4. Diet analysis ...... 55 5.5. Thermoregulation ...... 56 DISCUSSION ...... 56 ACKNOWLEDGMENTS ...... 59 REFERENCES ...... 59 SUPPORTING INFORMATION ...... 60

6. TREE HOLLOWS ARE OF CONSERVATION IMPORTANCE FOR A NEAR- THREATENED PYTHON SPECIES ...... 62 ABSTRACT ...... 64 INTRODUCTION ...... 64 MATERIALS AND METHODS ...... 65 6.1. Study sites and environmental temperature ...... 65 6.2. Tree hollow use by pythons - Radiotelemetry ...... 65 6.3. Do tree hollows provide a more favourable ambient temperature? ...... 66 6.4. Is there an energy saving by using tree hollows? ...... 67 6.5. Diet ...... 68 RESULTS ...... 68 6.6. Tree hollow use by pythons ...... 68 6.7. Do tree hollows provide a more favourable ambient temperature? ...... 68 xv

6.8. Is there an energy saving by using tree hollows? ...... 69 6.9. Do pythons access more arboreal prey? ...... 70 6.10. Do tree hollows offer protection from predation? ...... 70 DISCUSSION ...... 70 6.11. Do tree hollows provide a more favourable ambient temperature? ...... 70 6.12. Is there an energy saving by using tree hollows? ...... 71 6.13. Do pythons access more arboreal prey? ...... 72 6.14. Do tree hollows offer protection from predation? ...... 72 ACKNOWLEDGEMENTS ...... 73 REFERENCES ...... 73

7. IS THERE EVIDENCE FOR MESOPREDATOR RELEASE IN THE SOUTHWEST CARPET PYTHON? ...... 76 PREY DENSITIES ...... 76 7.1. Rabbits ...... 76 7.2. Western ringtail possums ...... 77 7.3. Other prey ...... 80 PREDATOR DENSITIES ...... 80 7.4. Fox densities ...... 80 7.5. Cat densities ...... 81 7.6. Python densities ...... 81 PREDICTING MESOPREDATOR RELEASE RESPONSE IN PYTHONS ...... 82 7.7. Predictions ...... 82 EVIDENCE FOR MESOPREDATOR RELEASE RESPONSE IN PYTHONS ...... 85 7.8. Survivorship ...... 85 7.9. Diet diversity ...... 102 7.10. Behavioural avoidance ...... 104 CONCLUSIONS TO MESOPREDATOR RELEASE RESPONSE ...... 110

8. SYNTHESIS OF THE THESIS ...... 119

9. REFERENCES ...... 120

10. APPENDICES ...... 136 APPENDIX 1. POST MORTEM REPORTS FOR PYTHONS ...... 137 10.1. Post-mortem report for python Msi9 ‘Kerry’ ...... 137 10.2. Post-mortem report for python Msi14 ‘Pamela’ ...... 140 10.3. Post-mortem report for python Msi23 ‘Peter’ ...... 143 10.4. Post-mortem report for python Msi38 ‘Chris’ ...... 146 APPENDIX 2. CHEMOSENSORY PAPER ...... 151

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LIST OF FIGURES

Figure 1.1. The study sites for radiotelemetry monitoring of Morelia spilota imbricata indicating the fox baited and unbaited control areas of the northern southwest jarrah forest and the two coastal sites; Leschenault Peninsula Conservation Park (baited) and Martin’s Tank (unbaited) campsite area within Yalgorup National Park...... 4

Figure 1.2. When there is a reduction in the number of apex predators in the system, mesopredators are able to functionally and numerically respond given: 1. Reduced exploitative competition as more prey to be available, and 2. Reduced intraguild predation is reduced...... 8

Figure 1.3. Holling’s (1959) Type I, II and III functional response curves of predators to increasing prey densities...... 9

Figure 2.2. The distal edge of three (or four) lateral scales (coloured scales) were cut in line with the ventral scale corresponding to the python’s unique identification number...... 21

Figure 2.3. A). Antenna attached to a pole to maximise the chance of hearing the python’s signal whilst driving slowly around the area. B). The aircraft used with Yagi antenna attached to aeroplane fuselage for radiotracking pythons from the air...... 22

Figure 3.1. Diagram of the position of an implanted radiotransmitter in relation to major organs within the coelomic cavity of a snake...... 30

Figure 3.2. Radiograph of implanted radiotransmitter inside a male python’s coelomic cavity...... 31

Figure 3.3. Diagram of the process anchoring the radiotransmitter to a rib in the coelomic cavity of a snake to prevent it from migrating and potentially being expelled from the body...... 31

Figure 3.4. Radiograph of a radiotransmitter and antenna that had been implanted in the coelomic cavity (without anchoring to the ribcage) of an adult female python (Morelia spilota imbricata), which was re- captured in the field (Leschenault Peninsula Conservation Park, Western Australia) to enable examination of the migrating radiotransmitter after the python had carried it for 2 years...... 32

Figure 3.5. Schematic of the movement of an unanchored radiotransmitter towards the three chambers of the snake’s cloaca. The lower panel shows how the radiotransmitter may be forced into one of the cloacal chambers and then expelled with or without with faecal material...... 32

Figure 4.1. Peripheral blood films of a southwest carpet python (Morelia spilota imbricata) showing: a) Heterophil (black arrow), b) Lymphocytes (black arrows) and thrombocytes (open arrow), c) Basophil (double black arrow), a lymphocyte (single black arrow) and thrombocyte (open arrow) are also visible, d) Azurophil (black arrow) and e) Erythrocyte containing a Hemogregarina sp. (black arrow)...... 38

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Figure 4.2. Of a total of 77 blood samples collected from 43 Morelia spilota imbricata, a greater proportion of blood samples collected from pythons during spring tested positive for intracellular hemoparasites 2 (September-November; (χ 1 = 8.44, p <0.01), whilst a greater proportion of samples collected at the time of radiotransmitter removal 2 tested positive (χ 1 = 8.30, p <0.01) compared to pre-radiotransmitter and post-radiotransmitter placement...... 42

Figure 5.1. Seasonal changes in prey class consumed by Morelia spilota imbricata...... 55

Figure 5.2. Comparison between prey body mass (reported Log10 adult average) and individual carpet python body mass (Log10 individual average body mass)...... 55

Figure 5.3. Differences in the type of prey consumed by small and large Morelia spilota imbricata pythons...... 55

Figure 5.4. Relationship between python body mass and three measures of measures of thermal biology. The measures shown are the (A) thermal differential (difference of python body temperature above air temperature), (B) maximum hourly heating rates and (C) maximum cooling rate. Each column of data points represents monthly values for an individual python (separated by their overall average body mass measures)...... 57

Figure 5.5. The proportion of observations (B, D) and average (± SD) body temperature (A, C) of small (white) and large (black) pythons observed in each of five microhabitat categories (left-hand panel) and in five body positions (right-hand panel)...... 58

Figure 6.1. Average (± 1 SD) daily air temperature at the coastal woodland and jarrah forest study sites were similar for most of the year except during winter/early spring months (June-September), where the jarrah forest has lower daily average ambient temperatures. *Indicates significant difference between the study sites p<0.001...... 66

Figure 6.2. Scatter plot of the body mass (Mb, g) of Morelia spilota imbricata relative to their snout–vent length (SVL, cm)...... 68

Figure 6.3. The use of five microhabitats by Morelia spilota imbricata shown by calendar month. Tree hollow use increased during winter months whilst use of hollow logs decreased. See methods for detailed description of each microhabitat...... 69

Figure 6.4. Box plot illustrating temperature for the five microhabitats used by Morelia spilota imbricata. There was no measureable difference in the winter (June-August) in daily average, minimum, maximum or range of temperatures for five microhabitat types (p>0.06)...... 69

Figure 6.5. Four measures of python body temperature recorded over a seven- month period (April to October) when Morelia spilota imbricata made use of tree hollows...... 70

Figure 6.6. The rate of body condition decline (β-BCS) was not significantly reduced for Morelia spilota imbricata that used tree hollows (TH-C for coastal sites and TH-JF for the jarrah forest) over winter compared with those that did not (NTH)...... 71

Figure 7.1. The number of western ringtail possums (Pseudocheirus occidentalis; WRP) translocated into study areas since 1991...... 78

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Figure 7.3. Observations of declines in python body condition over time (y = - 0.0375x + 1479, R² = 0.09)...... 90

Figure 7.4. Morelia spilota imbricata pythons that died during this study represented the full range of body sizes for animals that were radiotracked, but mortality was marginally more likely for heavier animals...... 95

Figure 7.5. Male Morelia spilota imbricata increased in body length (SVL) at a faster rate than females. Although, there was no significant difference between the baited (LPCP) and unbaited (MT) study sites. Sites are shown separately. Juvenile Msi52 indicated by larger dot at end of line)...... 98

Figure 7.6. A species accumulation curve where the prey species identified in the scats were sorted chronologically...... 103

Figure 7.7. The prey species diversity identified in scats collected from Morelia spilota imbricata at the fox baited Leschenault Peninsula Conservation Park (LPCP) was not significantly different to the unbaited site at Martin’s Tank (MT)...... 103

Figure 7.8. There was no significant difference in the percentage of prey items taken by Morelia spilota imbricata in terms of diurnally active (reptiles: e.g. mainly bobtail lizards and two unidentified skink species; and birds: e.g. parrots) and nocturnally active prey (e.g. mammals including rabbits, brushtail possums, western ringtail possums, house mice, black rats, and dunnarts) at the fox baited site (LPCP) compared with the unbaited site (MT)...... 106

Figure 7.9. Morelia spilota imbricata animals at the unbaited site (MT) were observed proportionately more in tree hollows and less on tree branches and using vegetation cover compared with pythons at the fox baited site (LPCP)...... 108

Figure 7.10. Morelia spilota imbricata animals at the unbaited site (MT) were observed proportionately more in the least cryptic body position (stretched) compared with pythons at the fox baited site (LPCP)...... 110

Figure 7.11. A schematic representation of the most parsimonious density response of sympatric predators (foxes, cats and pythons) and the response of a prey species, the western ringtail possum (Pseudocheirus occidentalis), following implementation of fox baiting over a period of two decades at Leschenault Peninsula Conservation Park (LPCP)...... 115

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LIST OF TABLES

Table 2.1. The number and the type of captures of Morelia spilota imbricata and monitored in the study...... 19

Table 2.2. Five body position categories allocated to each Morelia spilota imbricata field observation...... 26

Table 3.1. Summary of the two surgical techniques used to implant radiotransmitters in the coelomic cavity of southwest carpet pythons and the association with expelling of the device ...... 32

Table 4.1. Hematology and plasma biochemistry values for 35 apparently healthy wild-caught southwest carpet pythons (Morelia spilota imbricata) sampled prior to radiotransmitter implantation...... 39

Table 4.2. Average ± S.D. values for each hematological measure analyzed by multiple regression for all factors...... 40

Table 4.3. Average ± S.D. values for each plasma biochemistry measure analyzed by multiple regression for all factors...... 41

Table 5.1. Results of multiple regression analyses testing for the effect of environmental, individual and behavioural factors on four measures of thermal biology of south-west carpet pythons...... 56

Table 6.1. Multiple regression output for analyses addressing three questions: 1. What factors influence python rate of body condition decline (β-BCS)? 2. What factors influence how long pythons stay sequestered in tree hollows? and, 3. What factors affect the rate of body condition decline (β-BCS) for pythons that use tree hollows over winter? ...... 71

Table 6.2. The spatial ecology of prey species which were identified in scats opportunistically collected from radiotracked Morelia spilota imbricata in coastal woodland and jarrah forest of Western Australia...... 72

Table 6.3. Possible predators of Morelia spilota imbricata pythons at the two study sites in south-west Western Australia and evidence for predation upon M. s. imbricata and M. s. spilota (eastern Australia)...... 72

Table 7.1. Predicted demographic outcomes for a python population under the scenario where fox density is modified through fox baiting. The hypotheses (H) listed are explained further in the text...... 84

Table 7.2. Average body measurement values for all Morelia spilota imbricata pythons over the study period at each of the three study locations...... 87

Table 7.4. Reproduction and observed mating events for female pythons at the baited (LPCP) and unbaited (MT) study sites...... 101

Table 7.5. The measured and tested hypotheses for the Morelia spilota imbricata at the fox baited site, Leschenault Peninsula Conservation Park (LPCP) compared with the unbaited site, Martin’s Tank (MT)...... 114

Table 7.6. Predicted responses of pythons to fox baiting at Leschenault Peninsula Conservation Park where it is likely that feral cats have xx shown a mesopredator release response and increased in density and there has been exploitative competition between the mesopredators...... 117

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1. INTRODUCTION

FERAL AND INTRODUCED PREDATORS IN AUSTRALIA

The introduction of carnivorous endotherms by humans has undoubtedly contributed to exceptionally high levels of native mammalian extinctions and species distribution declines across the Australian landscape (Abbott 2002; Burbidge 2008; Burbidge and

McKenzie 1989; Dickman 1996a; Kinnear et al. 2002). The dingo (Canis lupus dingo) was most likely introduced to Australia by humans travelling to the country in boats

(approximately 3,500 – 4,000 years ago; Corbett 1995; Savolainen et al. 2004) and today is the largest mammalian predator. European colonisation brought the introduction of the red fox (Vulpes vulpes) in 1871 (Dickman 1996a) and feral cats

(Felis catus) between 1824-86 (Abbott 2002). In some areas across the continent (e.g. southwest Western Australia and southeast Australia), persecution of the dingo by landholders has allowed the fox to occupy the apex terrestrial predator position in the system (Johnson et al. 2007). Australia has experienced around 30% of the world’s recent mammal extinctions (Burbidge 2008), many of which can be associated with these predators. This extinction rate has been referred to as “an unprecedented wildlife catastrophe on a continental scale” (Kinnear et al. 2002).

Various management programs have been initiated in Australia in an attempt to control foxes. Control measures include lethal baiting, shooting, trapping, den fumigation, den destruction and exclusion fencing (Saunders et al. 1995). Lethal baiting is currently considered the most effective method to control foxes, however, bait delivery methods vary across the country as fox management strategies are currently governed by the various Commonwealth, State, and Territory laws (Saunders and McLeod 2007). A variety of meat baits containing sodium monofluoroacetate poison (known as “1080”) is

1 the most commonly used poison across Australia (Saunders et al. 1995; Thompson and

Algar 2000). Native fauna species in Western Australia have a degree of resistance to this toxin due to its presence in native plant species (e.g. Gastrolobium spp.). The effectiveness of bait-uptake by foxes using different deployment methods (e.g. aerial drops, ground baiting with tethering and burying the baits), over different landscapes, and in different habitat types, still requires further research (Saunders and McLeod

2007).

In Western Australia, aerial baiting of a dried meat sausage-shaped baits (Pro-Bait®, made from kangaroo mince, pork fat and dog food flavour enhancer) with a set quantity of the 1080 poison are used to control foxes (Saunders and McLeod 2007). In 1996, the

Western Australian Department of Environment and Conservation (DEC; formerly

Conservation and Land Management) introduced the fox baiting program, ‘Western

Shield’. Western Shield uses Pro-Baits® that are dropped from aircraft along transects four times a year to reach a desired bait density of five baits per square kilometre. In total around 780,000 baits are delivered per year over 3.5 million hectares of designated land (Armstrong 2004; Saunders and McLeod 2007). Other delivery methods used by

Western Shield include perimeter ground baiting along pre-existing tracks (to achieve the same density of 5 baits per square kilometre) or burying baits (to depths > 2 cm) in

‘high risk’ areas (i.e. hobby farms, special rural zones, farms and reserves close to town sites and settled areas) to reduce bait uptake by non-target species (Saunders and

McLeod 2007). Landholders also use fox baits in agricultural and pastoral land.

Research into effective bait densities has occurred in some parts of Western Australia, but remain unclear across most Australian landscapes (de Tores et al. 2003; Saunders and McLeod 2007).

Although lethal control of introduced predators has been seen as an important conservation measure, only recently it has been recognised that changing the balance of top order predators is likely to have both direct and indirect impacts on the ecosystem in question. It is therefore important to monitor competitive interactions over prey resources between coexisting predators particularly following lethal control programs of one, or all of introduced species (i.e. dingo, fox and cat). There may be significant consequences to native prey and predator species following lethal control of an apex predator in a system (Elmhagen et al. 2010; Prugh et al. 2009; Ritchie and Johnson

2009).

The present study formed part of a large scale project run by the DEC and the Invasive

Animals Cooperative Research Centre. Since 2006, this project has monitored the responses to 1080 fox baiting by coexisting native predator (including the chuditch,

Dasyurus geoffroii, varanid lizards, Varanus gouldii and V. rosenbergi and the southwest carpet python, Morelia spilota imbricata) and introduced predators (F. catus and V. vulpes). The project has examined baited and unbaited areas of the jarrah forest

(Fig. 1.1). A complimentary study has been undertaken through coastal woodlands at sites where reintroduction of the threatened western ringtail possum (Pseudocheirus occidentalis) has been monitored by DEC researchers since 1991. Some possum translocation sites have been included under Western Shield whilst there are other translocation areas that have been left unbaited. This research examines the biology and some biological responses of the south-west carpet python (M. s. imbricata) to the fox management program in jarrah forest and parts of the Swan Coastal Plain of southwest

Western Australia.

MESOPREDATOR RELEASE HYPOTHESIS

Reduction or elimination of apex (or top order) predators has the potential to destabilise the food-web present in the ecosystem (Prugh et al. 2009; Soulé et al. 1988).

Elimination or reduction in the number of the apex predator in an ecosystem is likely to allow the increase in abundance of smaller, lower-order predators (‘mesopredators’) by releasing the competitive influence of the apex predator on these mesopredators. This response has been described as mesopredator release (MR; Courchamp et al. 1999;

Crooks and Soule 1999; Soulé et al. 1988).

The MR hypothesis predicts an increased density of mesopredators in the system following the elimination or reduction of the apex predator in response to increasing prey numbers. As a result of removal of the apex predator, it is predicted that there will be: 1. A greater number of prey available to mesopredators. This may be due to a reduction of exploitative competition (and possibly interference competition), where resources, such as prey, are less contested as the result of removal of the apex predator

(Glen and Dickman 2005). 2. Reduced intraguild predation (i.e. the killing to consume mesopredators as prey by the apex predator; Fig. 1.2) (Glen and Dickman 2005).

Mesopredator outbreaks have the potential to lead to extinction of prey (Courchamp et al. 1999; Glen and Dickman 2005; Ritchie and Johnson 2009). Cats may experience a

MR from the lethal control of foxes, since, as sympatric predators they exhibit significant overlap in their diet, home range, and habitat use (Catling 1988; Molsher et al. 1999; Risbey et al. 1999; Risbey et al. 2000). We have less evidence for release of

Australian native species in response to fox control. To investigate the potential impact of fox baiting on the south-west carpet python (Morelia spilota imbricata), it is 5 important to understand the potential impacts of fox control on different mesopredator species. This is discussed in the following section.

1.1. Endothermic and ectothermic predators

Predator-prey studies on vertebrates have traditionally focused on a few taxa, particularly marine species (e.g. fish and marine birds) and terrestrial mammals (e.g.

Bodey et al. 2009; Courchamp et al. 1999; Soulé et al. 1988; Strayer 2009; Yodzis

2001). Substantial attention has been directed to the importance of endothermic carnivores in regulating biodiversity, through direct and indirect interactions with other predators, prey and primary producers (Alkama et al. 2005; Banks 1999; Boutin 1995;

Courchamp et al. 2003; Crooks and Soule 1999; Glen et al. 2009; Glen and Dickman

2005; Glen et al. 2007; Johnson and VanDerWal 2009; Letnic et al. 2009; Letnic and

Koch 2009; Lunney et al. 1990; Pech et al. 1995; Pech and Hood 1998; Zavaleta et al.

2001). By contrast, predator-prey dynamics of terrestrial ectothermic predators has received little attention, especially their potential for mesopredator release response

(Brown and Shine 2007; Nowak et al. 2008; Olsson et al. 2005; Russell et al. 2003).

Snakes may exert considerable pressure on prey populations. Australian south-west carpet pythons (M. s. imbricata) can exceed two metres in length and weigh up to six kilograms (Pearson 2002). Pythons are able to swallow relatively large whole prey given many structural adaptations of the skull which allow great freedom of rotation between elements of kinematic linkage chains that permit independent movement of unfused mandibular tips (Kardong 2002). It is important not to ignore the potentially significant effects of ectothermic mesopredators within these ecosystems, and these species should be included in the design of predator-prey monitoring or management control programs. 6

There are, however, fundamental differences between carnivorous endotherms and ectotherms in their energy use and allocation, which may lead to significantly different functional and numerical responses to changes in apex predator and prey population numbers (Nowak et al. 2008). Awareness of these biological responses is required to best predict the role and response of ectothermic mesopredators in driving changes to food webs upon removal of the apex predator. A key to understanding these responses is the knowledge that the metabolic rates and therefore, reproductive rates of endotherms and ectotherms are vastly different. Apex predator control may result in biological changes in mesopredators which can be explained in terms of functional and numerical responses to increases in prey populations (Fig. 1.2). Together, the functional and numerical responses result in the ‘total response’. In this thesis, I discuss the likely responses of an ectothermic mesopredator, the south-west carpet python (M. s. imbricata), and compare this with the likely responses of an endothermic mesopredator, the feral cat (F. catus).

1. Reduced exploitative competition as more prey is available to MP mesopredators (MP) FUNCTIONAL RESPONSE More prey Reduced consumed Faster MP TOTAL number of apex by MP growth rate predators (e.g. RESPONSE red fox) in the system Increased MP reproduction MP NUMERICAL Greater MP RESPONSE survivorship

2. Reduced intraguild predation of mesopredators (MP)

1.2. Functional response

Functional response describes an increase in the number of prey consumed by predators as a consequence of increasing prey population density (Holling 1959). As more prey become available to predators in the system (due to increased population size), predators may functionally respond by consuming more prey until satiation is reached.

Holling (1959) described three functional response curves to explain the predator-prey dynamics based on observations of small mammalian predators and invertebrate prey

(Fig. 1.3).

III

Number of prey consumed per predator

Number of prey

Figure 1.3. Holling’s (1959) Type I, II and III functional response curves of predators to increasing prey densities. Type I response describes predators that maintain a constant, yet random search pattern for prey across increasing prey density until satiation. Type II response describes an initial rapid increase in the number of prey taken per predator, followed by a progressively slower rate due to either the time-consuming nature of prey-handling and digestion, or satiation. Type III response describes a slow increase in the number of prey taken as density increases, due to the time predators may take to learn to recognise and/or capture the prey, or alternate between prey species. This slow increase is followed by a rapid increase, which reaches a plateau.

Ectothermic predators may also respond to an increase in prey availability by consuming more prey, however, there are fundamental biological differences between ectotherms and endotherms that could result in different patterns of functional response.

Pythons are predicted to have slower or reduced functional response to increasing prey abundance compared with endothermic predators, such as feral cats (Table 1.1).

Pythons are ambush (sit-and-wait) predators and have the ability to eat very large prey

9 relative to their body size, however, they are precluded from further foraging (post- feeding) due to the time taken for a large meal to be digested (Shine 1991; Slip and

Shine 1988a). Therefore, prey handling time (restraining, engulfing prey and consequently restricted foraging during digestion) is longer in snakes compared with endothermic predators of similar-size (e.g. feral cats). Additionally, temperate-zone pythons (and other snakes) will cease feeding during colder parts of the year (they often brumate during winter), whereas most endotherms will continue to feed throughout the year. It may therefore be predicted that pythons respond to an increase in prey numbers similar to Holling’s (1959) Type I response (Fig. 1.3) as pythons should maintain a constant, but random search pattern (or ambush site selection) for prey, although they may reach satiation quicker compared with endothermic predators (e.g. cats).

Table 1.1. Comparison of the feeding behaviour of an endotherm (e.g. feral cat, Felis catus) and an ectotherm (e.g. southwest carpet python, Morelia spilota imbricata) to highlight the types of functional response to an increase in prey abundance based on Holling’s (1959) curves.

Endotherm = Type II or III response Ectotherm = Type I response e.g. Feral cat e.g. Southwest carpet python Prey detection Visual Olfactory (chemoreception) Auditory Infrared vision Olfactory - both temperature-dependent Foraging behavior Active foraging Sit-and-wait ambush foraging Prey size Ability to manipulate small and large prey Ability to consume very large prey in (i.e. teeth, claws) relation to body size Prey handling time Short Long (prey is swallowed whole) Prey digestion time Short Considerable (high metabolic rates independent of (low metabolic rate and digestion is temperature) dependent on temperature) Activity period Year-round Restricted (prey consumed throughout the year) (cessation of feeding during winter months)

1.3. Numerical response

Predators may respond numerically to changes in the abundance of their prey, i.e. by increasing their reproductive output (Holling 1959; Weatherhead and Madsen 2009).

Endothermic predators are often income breeders and can respond quickly to increases in prey abundance, either by increasing litter size or being able to reproduce earlier in life due to increased growth rates in response to an abundant food supply (Nowak et al.

2008). Cats for example, are able to reproduce within their first year of life (Deag et al.

2000; Feldman and Nelson 2004) and have several litters per year, with average litter sizes of feral cats reported between 2 to 5 kittens (Stoskopf and Nutter 2004).

Relative to mammalian predators of similar body size, the potential for pythons to numerically respond to prey abundance is likely to be restricted by the temporal pattern of reproduction in these snakes. Morelia spilota imbricata appear to be capital breeders, requiring accumulated body reserves to reproduce (Bonnet et al. 1998;

Pearson 2002; Shine 2003; 2005). Therefore, even in the presence of high prey densities, reproductive output may only be expressed in the following or subsequent years (Nowak et al. 2008; Shine 2003). Additionally, post-parturition anorexia in female snakes is high, potentially compromising their survival, as has been observed in vipers (Bonnet et al. 1998; Shine 2003).

1.4. Total response

The total response of predators to an increase in prey populations is the sum of both their functional and numerical responses (Holling 1959; Nowak et al. 2008).

Ectotherms have an energy-efficient life style compared to similar-sized endothermic mammalian predators; they have lower metabolic rates, shorter activity periods and an

11 ability to consume large prey, but this increases their prey handling time (Nowak et al.

2008). They also require a relatively long time to transform energy gained from consuming prey into reproductive outputs through offspring production (Shine 2003).

STUDY AIMS

I set out to measure the potential mesopredator release response of a cryptic ectothermic species (M. s. imbricata) in coastal woodland of southwest Western Australia, comparing areas that have been baited for the red fox (the apex predator in this system), with unbaited areas. I also studied pythons in fox baited areas of jarrah forest however, no pythons could be located from unbaited jarrah forest for comparison. Given M. s. imbricata is an elusive species and could not be enticed into traps to measure population abundance, I examined some biological and physiological responses of pythons across the study sites to quantify the potential of a mesopredator release response.

THESIS STRUCTURE AND OUTLINE OF CHAPTERS

In order to measure mesopredator release response in M. s. imbricata, animals were captured and monitored through radiotelemetry (focal animal sampling). The following chapter outlines the general methods of the research, describing the general study sites and field methods involved in monitoring pythons through radiotelemetry. Chapter 3 investigates the expulsion of surgically-implanted radiotransmitters experienced during the course of this research, and explains a new methodology that was developed which prevented radiotransmitters from being expelled with 100% success. Additionally, the potential health risks associated with the surgical procedure of radiotransmitter implantation were investigated through haematological and biochemical analysis at three time stages: 1. pre-radiotransmitter implantation, 2. post-radiotransmitter 12 implantation, and 3. removal of radiotransmitter and also considers how other factors such as sex, season, anaesthesia, time spent in captivity and presence of haemoparasites affect blood values (Chapter 4).

Surgical implantation of radiotransmitters was necessary to investigate the potential mesopredator release of southwest carpet pythons (outlined in Chapter 7) but also offered the opportunity to investigate other aspects of python ecology. Chapter 5 investigates differences in the diets of pythons and relates thermal biology measures and thermoregulation behaviour to dietary differences between pythons of two body size groups. Chapter 6 focuses on python tree hollow use over winter months and investigates the physiological importance of their use to pythons by measuring thermal properties of both microhabitats and the pythons themselves. Both Chapter 5 and 6 have further developed biological and physiological understanding of the python across all study sites. Chapter 7 presents all available information on the investigation of whether M. s. imbricata have experienced a mesopredator release response by comparing the biology of individuals in fox baited and unbaited areas. I conclude with a short synthesis of the research in Chapter 8.

2. GENERAL METHODS

This study was carried out between 2006 – 2008 at three sites (two coastal woodland and one inland jarrah forest area; Fig. 1.1).

FIELD SITES

The study sites included two main habitat types; coastal woodland and jarrah forest.

Both habitats experience hot Mediterranean climates with cool, wet winters and warm, dry summers. Two coastal woodland sites were included; Leschenault Peninsula

Conservation Park (LPCP) and Martin’s Tank in Yalgorup National Park (YNP), located on the Swan Coastal Plain, and have an average rainfall between 600 – 1,000 mm per annum. For the most part, the jarrah forest experiences slightly higher average rainfall compared with coastal sites (700 – 1100 mm per annum).

2.1. Leschenault Peninsula Conservation Park

Leschenault Peninsula Conservation Park (33°26’ S, 115° 41’ E) is a 1,071 hectare elongate peninsula of the Swan Coastal Plain between the Indian Ocean and the

Leschenault Estuary, located 22 km north of the town of Bunbury and 150 km south of

Western Australia’s capital city, Perth (Fig. 1.1). The width of the peninsula varies between 0.8 to 1.5 km and is approximately 11 km in length (CALM 1998). The peninsula consists of a series of mobile and vegetated dunes and some woodland plains

(CALM 1998; Trudgen 1984). Vegetation comprises 122 native flowering plant species and the most abundant species include peppermint (Agonis flexuosa) and tuart

(Eucalyptus gomphocephala) trees, several shrub species (Templetonia retusa,

Spyridium globulosum, Diplolaena dampieri, Acacia rostellifera, and A. cochlearis), and liana species (Hardenbergia comptoniana and Clematis microphylla; Trudgen

1984). The peninsula consists of a series of mobile and vegetated dunes and some woodland plains. Vegetation density tends to be greater on the dune systems than on the woodland plains. The peninsula was exempt from prescribed burning over the duration of the study (de Tores Pers. comm)1.

Leschenault Peninsula Conservation Park (LPCP) has been actively managed by DEC to control foxes since 1991. Fox baits have been deployed by hand-baiting and by burying baits in specific locations. The intensity of the regime (i.e. number of baiting rounds per year) has varied over the last two decades (de Tores et al. 2003). Currently monthly ground baiting (tethering baits along regular transects) occurs at LPCP (Fouler

Pers. comm.)2.

2.2. Yalgorup National Park

Further north along the coastline is Yalgorup National Park (Fig. 1.1; 12,888 hectares),

105 km south of Perth. The park includes 10 natural lakes (CALM 1995). The majority of YNP consists of a series of dune ridges and plains with vegetation varying with soil depth, but includes peppermint/tuart woodland and open jarrah (E. marginata), marri

(Corymbia calophylla) and Banksia (Banksia spp.) woodland with understorey shrub assemblages including dense stands of species such as T. retusa, S. globulosum,

Hibbertia spp., and Melaleuca spp. Near the lake edges the vegetation is dominated by

M. rhaphiophylla and sedges (CALM 1995). Pythons were found (Table 2.1) and

1 Paul de Tores 2010. Personal communication. Western Australian Department of Environment and Conservation, Wanneroo. 2 Tiffany Fouler, 2010. Personal communication. Western Australian Department of Environment and Conservation, Collie District. 15 monitored in bushland surrounding the Martin’s Tank campsite (32° 51´ S, 115° 40´ E).

This area (516 ha) was exempt from prescribed burns throughout the study period (de

Tores Pers. comm)1, however, much of the western half of the area was burnt during summer 2007 (accidentally ignited/human error).

Yalgorup National Park is patchily fox baited, principally at translocated sites of the threatened native western ringtail possum, Pseudocheirus occidentalis (WRP; e.g.

Preston Beach Road; de Tores et al. 2005b; de Tores et al. 2003). There has been no fox baiting carried out at Martin’s Tank camp site (MT; despite western ringtail possums being translocated to this area). Western ringtail possums have been the main mammalian species in these areas to be extensively monitored (Clarke 2010; de Tores et al. 2003).

2.3. Jarrah forest

Pythons were opportunistically hand-captured within State Forest surrounding the township of Dwellingup, approximately 100km south-south-east of Perth. Different land use practices of the forest include residential, mining, forestry, and recreation. The study area in which pythons were monitored lies within the northern jarrah forest, where

Archaean granite overlies metamorphic rocks capped by an extensive, but dissected lateritic duricrust (Churchward and Dimmock 1989). The vegetation is comprised mainly of jarrah and marri open forest with a diverse mosaic of understory woodland plant species (Beard 1975; Havel 1975a; b; Havel 2000; Mc Dougall 1996). The jarrah forest experiences regular prescribed burns (DEC), which aim to create a mosaic of forest burnt at different times. Although pythons were sought throughout the general

16 northern part of the southwest jarrah forest area, all individuals that were found were located within fox baited sites.

Northern sections of the jarrah forest are aerially baited (and perimeter baited) 6 times per year under the management by DEC while the southern section of the forest remains unbaited for foxes. At both the baited and unbaited sections, V. vulpes, F. catus, D. geoffroii, V. gouldii, V. rosenbergi and common brushtail possums (Trichosurus vulpecula) have been monitored through trapping programs and radiotelemetry.

Unfortunately for the present study, southwest carpet pythons were only captured (and able to be monitored) in the baited area of the jarrah forest (no animals were captured in the unbaited jarrah forest). For this reason, the majority of comparisons made on python biology between fox baited and unbaited areas are focused on pythons monitored at the two coastal sites. Comparison between coastal and jarrah forest areas is limited due to vast differences in the environment in addition to differences in fox presence.

STUDY ANIMALS

2.4. Animal ethics permits and associated licences

All activities were approved by relevant Animal Ethics Committees. Murdoch

University approved this project under the Animal Ethics Committee Permit No.

W2028/07, “Long term health and survivorship monitoring of the western ringtail possums (Pseudocheirus occidentalis) following translocation”. I was also registered as a researcher through the Animal Competence and Experience Registration process at

Murdoch University according to the Animal Welfare Act (2008).

This research fell formed part of two projects with DEC: DEC AEC 54 / 2006; “The importance of fox, cat and native predator interactions to sustained fauna recovery in the northern jarrah forest – is there a mesopredator release effect?”, and DEC AEC / 55 /

2006; “Translocation outcomes and monitoring of naturally occurring populations of the western ringtail possum, Pseudocheirus occidentalis”.

I was granted a DEC Regulation 17 licence to take (i.e. capture, collect, disturb, study) fauna for scientific purposes under the Wildlife Conservation Act (1950) and a DEC

Regulation 4 Authority (permit to enter DEC lands and/or waters for the purpose of undertaking research).

2.5. Capture of pythons

Pythons were captured by hand. Thirteen pythons were located because they had eaten species that were being monitored through radiotelemetry (12 P. occidentalis and one woylie Bettongia penicillata) and one python had eaten a domestic pet bird. Pythons that had taken telemetered prey were held in captivity until the remains of the prey were defecated or regurgitated with the radiotransmitter collar, either voluntarily or by careful palpation after sufficient time had elapsed for the digestion of the prey. No pythons appeared to be harmed by this process. Thirty-two pythons were opportunistically sighted and captured when crossing roads (during night and day-time driving) or when during radiotracking other pythons at the field sites (Table 2.1). Pythons were physically restrained using conventional methods for handling snakes (pythons were captured with care using the conventional ‘head-grab’ behind the quadrate bone) and transported in calico bags.

Table 2.1. The number and the type of captures of Morelia spilota imbricata and monitored in the study.

Study site Radio-collared prey Opportunistic capture Predation of pet Total Jarrah forest 1 10 1 12 Leschenault Peninsula 9 18 0 27 Conservation Park Yalgorup National Park 3 4 0 7 (Martin’s Tank camp site) Total 13 32 1 46

2.6. Housing of animals in captivity

Pythons were housed in individual custom made cages in a temperature controlled

(25°C) room at the DEC Dwellingup Research Centre (Fig. 2.1). Each cage was built from plywood and had a ventilated roof (fine wire mesh) and a Perspex glass front door.

A heat pad was placed underneath each cage to warm an area (approximately 15% of floor area). Pythons were provided vertical cage furniture (small branches), given several rocks and logs to allow a rough-point for snakes to shed their skin when necessary, and a hide box of appropriate size for each snake (made from a cardboard box with an entrance hole cut into one side). Ad lib water was provided in a container that was large enough for snakes to immerse themselves in if required. The floor of the cage was lined with 2 cm thick recycled newspaper kitty litter (Old News Cat Litter,

Ciber Cycles Pty Ltd, Toowoomba, QLD). To reduce disease risk from contact transmission between different animals using the cages, each cage was de-furnished and cleaned with disinfectant solution of F10SC Veterinary Disinfectant at a dilution 1:125.

Water containers and rocks were similarly cleaned. Branches and hide-boxes were replaced with new items for each animal. Animals that were held for longer than 3 weeks were offered dead (defrosted) mice or rats (depending on the size) until the animal’s hunger appeared satisfied (they no longer took prey). Rats and mice were purchased from the Murdoch University Animal Resource Centre where they are bred specifically for laboratory, research and pet food orders.

Figure 2.1. Photographs of the temperature controlled room at the Department and Environment and Conservation Dwellingup Research Centre, where custom-built cages were installed to temporarily house pythons before and during recovery of surgical implantation of radiotransmitters. A heat-pad was placed under the plywood flooring of each cage, the roof was fully ventilated. Pythons were given a hide-box, cage furniture, ad lib water and the floor was lined with recycled newspaper cat-litter.

2.7. Marking and identification of individuals

Each python was given a unique identification number code (e.g. Msi1 was the first python to be captured in the study, Msi37 was the 37th python captured in this study;

Pearson 2002) which was usually assigned in order of their capture. A microchip

(Trovan®) was subcutaneously inserted on the dorsal surface approximately 10 cm posterior to the left mandible bone. The position of the microchip was remeasured at the time of radiotransmitter removal, and all microchips remained <1 cm from the insertion position. Each python was also given an identifiable mark: the distal half of three (or four for very large pythons) lateral scales from the left lateral scale-line were removed while under anaesthesia (Fig. 2.2; Pearson 2002). The scales were selectively cut according to the python’s unique identification number, to provide additional identification in the event that pythons were able to expel both their implanted radiotransmitter and microchip. For example, Msi4 was marked by cutting the distal sections of three lateral scales adjacent to the fourth ventral scale (counted anterior to 20 the cloaca and always excluding the ventral scale immediately anterior to the cloaca).

These half scales were placed into duplicate vials containing dimethyl sulfoxide

(DMSO4) in a saturated salt solution and were stored for later genetic analysis.

th 4 ventral scale Lateral scale

Tail

8 7 6 5 4 3 2 1

Cloacal opening

Ventral scales Cloacal scale (excluded)

Figure 2.2. The distal edge of three (or four) lateral scales (coloured scales) were cut in line with the ventral scale corresponding to the python’s unique identification number. In this example, the python’s identification is Msi4 and the distal edge of the lateral scales were cut adjacent to the forth ventral scale anterior to the cloaca (excluding the ventral scale immediately anterior to the cloaca; the cloacal scale).

FIELD METHODS

2.8. Radiotelemetry

Following surgical implantation of radiotransmitters (see Chapter 3) pythons were released at their point of initial capture and radiotracked on a weekly (rarely fortnightly) basis between mid-2006 – November 2008. Pythons were radiotracked on foot using a

3-element Yagi aerial (Sirtrack Ltd., New Zealand) and receiver (R-1000 Telemetry

Receiver, Communications Specialists, INC. USA). The position of the pythons was recorded using a differential GPS system (Thales MobileMapper™ software, USA). As previous research has found (Pearson 2002), most pythons did not typically flee when

21 approached by researchers in the field, allowing behaviour, body posture, and aspects of microhabitat to be recorded.

Python radiotelemetry signals that were not easily heard from the last known position were searched for by driving around nearby roads and tracks with the radio antenna attached to a pole and positioned out of the car window (or on the roof top) to increase the antenna height and therefore improve the audibility of the radio signal (Fig. 2.3 A.).

Utilising hills also maximised the chance of hearing a distant radio-signal. All frequencies within 10 MHz of the python’s signal were scanned in case the frequency of the radiotransmitter had drifted out of its usual range. On two occasions a light aircraft

(Fig. 2.3 B.) was used to carry out a wide-ranging search in attempt to relocate the signal of elusive pythons.

A. Antenna pole B. Light aircraft

2.9. Body measurements

During fieldwork observations, wherever possible, a set of standard measurements were recorded on repeated occasions for each python. Digital Vernier callipers were used to measure head width (width of quadrate bones on skull), head depth (depth of base of

22 skull measured from top of quadrate bone to the base of mandible bone on the same side of the head), and head length (length from the base of quadrate bone to the tip of the nasal). A tape measure (sewing style) was used to measure snout vent length (SVL) and tail length. SVL was measured from the tip of the nasal bone to the cloacal opening along the ventral surface of the python. The python was stretched (usually in sections due to their length) when measuring SVL and at least two measurements were taken to obtain an average measurement that did not differ by more than 2 cm. Obtaining accurate SVL was usually accomplished with two people as it was easier to handle each animal. Tail length was taken from the cloaca to the tip of the tail. Body mass (Mb) was calculated using calibrated spring balances (100g, 1000g, 2500g, and 5000g PESOLA®

AG, Rebmattli 19, CH-6340 Baar, Switzerland) when pythons were contained within a calico bag (bag weight was subtracted from the total).

2.10. Air temperature

Air temperature (Ta) was recorded using temperature loggers (HOBO® H8 Pro Series;

H08-032-08, Onset Computer Corporation, Bourne, MA, USA) placed in a central location at each study site. HOBOs were attached to a vertical stake and positioned approximately 1m from the ground in a southwest facing direction and covered with a cardboard shelter to prevent rain damaging the device. The loggers measured and recorded air temperature hourly.

2.11. Quality of sighting

The quality of each sighting was noted by recording whether the python was visualised or not:

N. Not sighted due to microhabitat e.g. hollow log, tree hollow, or burrow

Y. Was sighted by researcher

C. Animal was captured

2.12. Microhabitat

The following categories were recorded for the type of microhabitat:

TH. Tree hollow: pythons were radiotracked to a tree and either observed within a

hollow or if the snake could not be visualised, strength of the radio-signal

indicated its presence within a hollow.

TB. Tree branch: where a python was visible on a tree branch.

IV. In vegetation: where the python would have ≥ 30% of its body under or in piles

of dead branches/sticks or vegetation (e.g. bushes) or was lying on top of logs or

fallen branches.

HL. Hollow log: pythons were radiotracked to and observed within a hollow log on

the ground, or if it could not be seen, the strength of the radio-signal indicated its

presence within the log.

B. Burrow: python in a burrow made from soil, limestone or rockery crevices or in

a pipe laying on ground under the road/surface soil.

GC. Ground cover: where the python would lay coiled or stretched on ground debris

(e.g. leafy matter or low grass <5cm in height).

BG. Bare ground: sand, gravel or muddy soil.

2.13. Behaviour

The following categories were used to record python behaviour:

R. Rest: python undertaking no particular behaviour

B. Basking: python positioned in a way that enabled its body to be exposed to the

sun

2.14. Moving: obviously moving from one place to another

M? Most likely moving: but could have been disturbed by researcher’s presence

Mate. Mating: mating with one or more males/females

A. Ambush: in ambush position (striking pose)

2.15. Body position

Where a python could be observed, its body position was categorised into one of five body position categories from stretched out to tightly coiled (Table 2.2). Where pythons could not be observed in the field, body positions were assumed to be a tight coil for animals in tree hollows, burrows or hollow logs, or as coiled if they were in vegetation or in ground cover.

Table 2.2. Five body position categories allocated to each Morelia spilota imbricata field observation.

Category Description Image Stretched Python’s body is stretched out (S) with none of it’s of body length touching.

Very loose coil Python’s body is slightly (VLC) turned over positioned next to itself (U-bend), with <10% of it’s body length touching.

Loose coil Python’s body has more bends (LC) in it, with approximately 20% of it’s body length touching.

Coil Python’s body is obviously (C) coiled, with most parts touching with >40% of it’s body length touching.

Tight coil Python’s body is coiled with (TC) no gaps exposed; with >90% of it’s body length touching. Often head is hidden underneath body coils.

3. IMPROVED PROCEDURE FOR IMPLANTING

RADIOTRANSMITTERS IN THE COELOMIC CAVITY OF

SNAKES

Understanding the ecology of carpet pythons required that they could be tracked in the field to obtain long term data on individuals. This chapter describes development of a surgical technique to ensure that radiotransmitters would remain in the coelomic cavity of pythons.

This chapter has been published in the Australian Veterinary Journal Volume 88,

Issue 11, pages 443-448, November 2010.

AUTHOR CONTRIBUTION:

G.L. Bryant: Initiated the investigation into recording the loss of implanted radiotransmitters, sought veterinary help to modify the surgery technique, organised personnel to do the surgeries, monitored the study animals, entered data, carried out the statistical analyses, and wrote paper.

P. Eden: Provided surgical training, provided surgical expertise during procedures outlined in the paper, provided draft drawing for Figure 3.5, treated sick and/or injured pythons, and provided editorial comments to draft versions of the manuscript.

P. de Tores: Facilitated the initiation of the PhD project, provided the equipment and facilities to conduct surgical implantations, house pythons pre-and-post surgery and provided editorial comments to draft versions of the manuscript.

K. A. Warren: Provided surgical training, provided surgical expertise during procedures outlined in the paper, and provided editorial comments to versions of the manuscript.

WILDLIFE AND ZOOS

Improved procedure for implanting radiotransmitters in the

coelomic cavity of snakesavj_633 443..448

GL Bryant,a* P Eden,b,d P de Toresc and K Warrenb

oviposition and winter hibernacula site selection, and WILDLIFE AND ZOOS Objective To investigate the expulsion of radiotransmitters in thermoregulation.2-9 However, the limitations of force-feeding snakes and modify the surgical technique for coelomic implanta- radiotransmitters to snakes became apparent during the early 1980s. tion to prevent its occurrence. Firstly, those radiotransmitters did not have antennas attached and Design To enable monitoring of snakes for an ecological study, therefore signal range was limited. Secondly, it was believed the pres- radiotransmitters were implanted in 23 south-west carpet pythons ence of the radiotransmitter in the stomach caused changes in forag- (Morelia spilota imbricata) using the standard surgical technique. In ing behaviour and physiology. Thirdly, long-term studies were a further 23 pythons we used a refinement of the technique, which difficult to plan because of the uncertainty of the snake regurgitating anchored the tracking device, using non-dissolvable sutures, to the the radiotransmitter at any stage. In recognition of this, Reinert and snake’s rib-cage. We also investigated the potential mechanisms for Cundall10 developed a method of implanting radiotransmitters expelling the radiotransmitters in one snake that underwent an equipped with a whip antenna to increase the range of the emitted exploratory coeliotomy. signal. Snakes were anaesthetised with halothane gas and the Results Of the initial group of snakes, 12 (52%) expelled the radiotransmitters were surgically implanted in the coelomic cavity radiotransmitter between 4 days and 3 years post implantation. In just anterior to the gonads, with the whip antenna extending through the later group, which underwent the refined technique of implan- the musculature of the body wall and running subcutaneously in a tation, none of the radiotransmitters was expelled and no adverse cranial direction along the length of the snake. Subsequently, Weath- responses were observed. erhead and Anderka described a subcutaneous implantation tech- Conclusion An appropriately sized radiotransmitter anchored to nique for both the radiotransmitter pack and antenna, but suggested the rib-cage of the snake will prevent expulsion of the device and this method was problematic for species or individuals smaller than appears to be well tolerated. Non-attachment of the tracking black rat snakes (Elaphe obsoleta) (i.e. < 375 g), as the subcutaneous device enables it to migrate along the length of the body, particu- bulge created could hinder the snake’s movement and affect the size of larly during feeding and reproduction. Caudal positioning of the shelter sites that could be selected.11 transmitter’s antenna provides a possible pathogenesis for expulsion into the cloaca. Few radiotelemetry studies of snakes have outlined techniques to improve radiotransmitter implantation, although Anderson and Keywords coelomic cavity; pythons; radiotelemetry; radiotrans- Talcott advocated the need for increased reporting of outcomes of this mitter; surgical implantation type of procedure, in order to identify best-practice modern techniques and associated complications.12 Pearson and Shine13 reported a Abbreviations ET, endotracheal; IPPV, intermittent positive pres- significant problem with surgically implanting radiotransmitters into sure ventilation; NSAID, non-steroidal anti-inflammatory drug; PBT, the coelomic cavity of carpet pythons (Morelia spilota). They preferred body temperature implanted radiotransmitters with antennas in the coelomic cavity of Aust Vet J 2010;88:443–448 doi: 10.1111/j.1751-0813.2010.00633.x 75 pythons (with the antenna directed posteriorly) and 14 (18.7%) subsequently expelled it. The authors suggested that the pythons were he use of telemetry has revolutionised the way in which many able to absorb the radiotransmitter into the gastrointestinal tract and animal species, from fish through to elephants, can be stud- expel it with the faeces. A consequence of the loss of tracking devices ied.1 Radiotelemetry was first used in snakes in the late 1960s.2 is that animals are unable to be located in the field and are lost to the T study, which is costly in terms of loss of information and surgical In that study, 68 individuals of eight species of snake were force-fed a radiotransmitter, which lodged in their stomachs, and to prevent outlay (i.e. time, radiotransmitters and consumables). We investigated regurgitation, a nylon thread was tied around the body of the snake, the phenomenon of radiotransmitter expulsion by snakes and anterior to the stomach, and sutured to the ventral scales.2 This meth- describe a refinement of the technique for surgical implantation to odology enabled telemetric research and led to increased interest in overcome this problem. studies of snake ecology, including spatial and movement analysis, Materials and methods *Corresponding author. a School of Veterinary and Biomedical Science, Murdoch University, Murdoch, Western Study animals Australia, Australia; [email protected] bConservation Medicine Program, School of Veterinary and Biomedical Sciences, Our study group comprised 46 south-west carpet pythons (M. s. Murdoch University, Murdoch, WA, Australia imbricata) captured from several sites in southwest Western cDepartment of Environment and Conservation, Western Australian Wildlife Research Ϯ Centre, Wanneroo, WA, Australia Australia: 25 females (average body mass 1535.5 146.8 g, average dPerth Zoo Veterinary Department, South Perth, WA, Australia snout–vent length 177.3 Ϯ 9.0 cm) and 21 males (average body mass

658.0 Ϯ 53.6 g, average snout–vent length 130.6 Ϯ 3.5 cm). All using a paediatric resuscitation bag (Ambu bag™) to ventilate with pythons were captured between 2004 and 2008 and brought into a room air until spontaneous breathing or voluntary movement was captive holding facility at the Department of Environment and Con- observed. At this point, the python was extubated and returned to a servation, Dwellingup Research Centre, Dwellingup, Western Austra- warm enclosure to complete the recovery process. lia for surgical implantation of radiotransmitters. Pythons were Coeliotomy. Surgical implantation of the radiotransmitters was generally held in captivity for 2 weeks to perform the surgery and achieved via routine ventral coeliotomy.17–19 The surgical site was allow a short recovery before release at the point of capture. prepared with 3% chlorhexidine in 70% alcohol and an incision was

WILDLIFE AND ZOOS made longitudinally at the junction between the first and second row Radiotelemetry device of ventrolateral scales. The incision was no longer than 5 cm and Five different sizes of radiotransmitter with a whip antenna (Holohill® located anterior to the cloaca by approximately 30% of the snake’s models: 5 g SB-2, 11 g S1-2T, 13.5 g S1-2T, 16 g A1-2T, 25 g A1-2T; snout–vent length, ensuring it was caudal to the lung (Figures 1, 2). Holohil Systems Ltd, Canada) were used to ensure the radiotransmit- The incision was continued through the body wall to open the coelo- < ter’s mass constituted 5% of the body mass of each fasted python (i.e. mic cavity to allow implantation of the transmitter. no recent meal determined by palpation). The radiotransmitters ranged in operational life from 50 to 150 weeks, depending on battery It is generally considered ideal to sterilise the transmitters using eth- size.The battery unit was covered in a physiologically inert wax and the ylene oxide, zephiran chloride or gamma irradiation.20 However, for antenna (15 cm long) was coated with a fine, flexible, inert tube. logistical reasons, in this study each radiotransmitter was soaked prior to implantation in a disinfectant solution of F10SC Veterinary Disin- Surgical procedure fectant at a dilution 1:125 for a minimum of 2 h and then rinsed with Each python was anaesthetised using isoflurane (Forthane,Abbott Pty sterile saline solution (Baxter Healthcare Pty Ltd) immediately before Ltd) inhalational anaesthesia and maintained at preferred body tem- insertion in the python’s coelomic cavity. perature (PBT) of approximately 26°C14 by placing it on heat pads and In most cases the radiotransmitter was positioned with the antenna conducting the procedure in a heated room. During induction of directed cranially, but in the first three surgical procedures the anaesthesia, each python was physically restrained and, while con- antenna was directed caudally, as in the previous study.13 After one of scious, intubated with a lubricated uncuffed endotracheal (ET) tube of the pythons expelled the radiotransmitter, the procedure was modi- an appropriate size. (A small drop of local anaesthetic (e.g. lidocaine fied and radiotransmitters were subsequently implanted with the diluted to 1%) is recommended to desensitise the glottis.15,16) antenna positioned cranially, because it seemed likely that the caudal position of the antenna was associated with the device’s expulsion. Once the animal was intubated, the ET tube was carefully secured around the jaw using Micropore® tape and the anaesthetic circuit was Absorbable suture material (Vicryl 3/0©, Ethicon Inc.) was used to connected. Anaesthesia was induced using intermittent positive pres- close the muscle layer in a continuous pattern, and the skin with sure ventilation (IPPV) at approximately 12 breaths/min, with 5% horizontal mattress sutures in an everting pattern (polydioxanone isoflurane gas in oxygen (at 1.5–2 L/min). Surgical anaesthesia was 3/0©, Ethicon). An everting pattern is recommended for suturing maintained with IPPV at approximately 2–4 breaths/min on 2–3% reptilian skin, because of its strong tendency to invert and because the isoflurane gas in oxygen at a rate of 1.5–2 L/min. The python’s heart scales prevent true apposition.21,22 A waterproof transparent dressing rate was monitored during the procedure using a small vascular with an absorbent pad (OpSite post-op®, Smith & Nephew, VIC, Doppler (Hadeco Echo Sounder, Doc Stock Medical Supplies, Sydney, Australia) was used to cover the incision site and secured in place with NSW,Australia) at the position of the heart, in addition to monitoring an adhesive dressing (Elastoplast Sport Elastowrap, Beiersdorf Austra- superficial skin perception (i.e. level of response to gentle tactile lia, North Ryde, NSW,Australia).As standard supportive postoperative stimulation), pain perception and righting reflexes. Pythons were therapy, each python was given a non-steroidal anti-inflammatory recovered from anaesthesia by stopping the isoflurane and maintain- drug (NSAID) via intramuscular injection (meloxicam 0.2 mg/kg: ing IPPV at 2–4 breaths/min with 1.5–2 L/min oxygen for 5 min, then Metacam®, Boehringer Ingelheim, North Ryde, NSW,Australia) in the

Figure 1. Diagram of the position of an implanted radiotransmitter in relation to major organs within the coelomic cavity of a snake.

Figure 3. Diagram of the process of anchoring the radiotransmitter to a rib in the coelomic cavity of a snake to prevent it from migrating and potentially being expelled from the body. (a) Sutures looped twice tightly around the radiotransmitter and secured with square knots, then a further two square knots are made to create a loop between the knots. Note: the suture material is not cut. (b) The radiotransmitter is positioned in the coelomic cavity and the needle is pushed from the ventral surface Figure 2. Radiograph of implanted radiotransmitter inside a male of the ribcage (indicated by hatched lines) to the dorsal surface, over a rib python’s coelomic cavity. Note the antenna has moved within the python and back to the ventral surface. (c) The needle is pushed through the loop and is now more looped than originally positioned during surgery. The created by the square knots attached to the radiotransmitter and secured bulb on the radiotransmitter is additional temperature data loggers that completely with several more knots. The excess suture material is then were wax-embedded onto the radiotransmitter before implantation. cut. paravertebral muscles for anti-inflammatory treatment and pain the ribcage being cut and the radiotransmitter and attached suture relief.16,21 Warmed fluids (normal saline and 5% glucose at a ratio of material being carefully removed from the python’s coelomic cavity. 1:1 at 20 mL/kg) were administered subcutaneously. Both injections The site was sutured closed and the snake recovered from anaesthesia were administered in the cranial third of the snake’s body. as per the standard procedure. Radiotransmitters were placed as described in the coelomic cavity of 23 pythons captured early in the study. A further group of 23 pythons Recovery and monitoring underwent a refinement of the technique. Prior to implanting the After surgery the pythons were maintained in a warm (PBT ª26°C14), radiotransmitter, non-absorbable suture (Prolene 3/0©, Ethicon Inc.) quiet environment for a minimum of 5–7 days while being monitored was tightly wrapped twice around its base near the junction of the for signs of postoperative complications. The dressing remained on antenna (where there are two small ridges on the wax covering of the the wound for up to 5 days and was removed before the python was unit) and tightly knotted with two square knots. Without cutting the released into the field. Pythons were then monitored by radiotelem- suture, another two square knots were made to create a small etry (usually weekly) as part of a broader research project carried out (~0.2 mm) loop between the knots (Figure 3a). The radiotransmitter over 3 years. was then positioned in the coelomic cavity as described and the uncut All procedures were approved by Murdoch University Animal Ethics suture material was carefully looped around a rib by passing the Committee W2028/07 and the Department of Environment and Con- needle around the rib and associated muscle tissue from the caudal servation Animal Ethics Committee DEC AEC/55/2006 and DEC surface of the rib through to the cranial surface (Figure 3b). The AEC 54/2006 permits. needle was then passed back to the coelomic cavity and through the small loop between the square knots and the transmitter secured using several square knots (Figure 3c). The suture material was cut and the Results surgical incision site was closed as before. Initial radiotransmitter implantation technique Radiotransmitter removal. The surgical technique for device Of the 23 pythons with radiotransmitters implanted using the stan- removal followed that for implantation, with the suture surrounding dard technique without ribcage-anchoring, 12 (52%) expelled the

device between 4 days to more than 3 years (mean 259 Ϯ 103 days) was bent close to the base of the radiotransmitter, with its remaining post surgery. Only 3 of the 12 expelled radiotransmitters were found length directed cranially (Figure 4). to be associated with faecal material. The python that expelled the radiotransmitter after 4 days was still being held in captivity for post- Anchored radiotransmitter implantation technique operative recovery. It had been fed two small mice the day following None of the pythons expelled radiotransmitters that had been surgi- surgery and the expelled radiotransmitter was contaminated with cally anchored to a rib (Table 1). When all implanted radiotransmit- faeces. On examination of the surgical site, the incision was almost ters were surgically removed during November 2008 upon completely closed and healing well, demonstrating that the completion of the research project, there was no evidence of inflam- WILDLIFE AND ZOOS radiotransmitter had not migrated through the surgical wound. mation within the coelomic cavity of these pythons and there were no However, there was slight inflammation around the cloaca. adhesions surrounding any of the radiotransmitters. At the time of radiotransmitter removal all pythons appeared to be in good health, The phenomenon of radiotransmitter expulsion was investigated based on physical and clinical examinations. further during an exploratory surgical procedure in a large adult female python, which had carried a radiotransmitter for approxi- Statistical analysis mately 2 years. This animal was re-captured in the field because its Logistic multiple regression analysis confirmed that expelling the radiotransmitter, which had been implanted without anchoring to a radiotransmitters was not associated with a python’s sex, snout–vent rib and with the antenna positioned caudally, had migrated towards length or body mass, but was strongly associated with the type of the cloaca. When the python was captured the radiotransmitter was surgery performed (t = –5.039, P Յ 0.001, i.e. whether or not the contained within a thin membranous sac, which contained urate-like radiotransmitter was anchored to a rib). granules, and was protruding from the cloacal opening. The veterinarian undertaking the procedure noted that the thin membranous sac appeared to be mucosal tissue and identified it as cloacal tissue. It Discussion is likely that the radiotransmitter was about to be expelled through the membranous wall of the cloaca. There was also significant accumula- The anchoring technique used by us when surgically implanting tion of faecal material anterior to the radiotransmitter, which was radiotransmitters in 23 carpet pythons proved to be 100% successful confirmed by radiography (Figure 4). Radiographs also indicated that in preventing expulsion of the tracking device. We recommend that the body of the radiotransmitter had moved caudally and the antenna the suture securing the radiotransmitter to the ribcage should be positioned midway from the spinal column and should not be positioned on the distal aspect of the rib, in order to prevent tearing of the muscle tissue and resulting in the radiotransmitter being effectively unanchored.

Surgical implantation of radiotransmitters is a well-established technique used in studies of fish of the ectothermic taxa, but expulsion of the implanted tags is a widespread problem.23–26 Three ways of expel- Figure 4. Radiograph of a radiotransmitter and antenna that had been ling implanted radiotransmitters have been reported in fish studies. implanted in the coelomic cavity (without anchoring to the ribcage) of an adult female python (Morelia spilota imbricata), which was re-captured in All involve the development of proliferative fibrogranulation tissue the field (Leschenault Peninsula Conservation Park, Western Australia) to containing myofibroblasts in response to the transmitter and the enable examination of the migrating radiotransmitter after the python device is ultimately expelled along the path of least resistance.25,26 One had carried it for 2 years. The radiotransmitter had migrated from the route of expulsion is through necrotic muscle tissue at the incision incision site (approximately 10% of the snout–vent length, anterior to the site, as seen in approximately 7% of implanted African catfish (Het- cloaca) towards the cloacal opening (edge of the image). Note the significant build-up of faecal material anterior to the radiotransmitter, which erobranchus longifilis) and 15% of laterally compressed bluegills may have resulted in the movement of the device towards the cloacal panfish (Lepomis macrochirus) at higher temperature treatment opening. conditions (20°C) and attributed to the incision wound not healing

Table 1. Summary of the two surgical techniques used to implant radiotransmitters in the coelomic cavity of southwest carpet pythons and the association with expelling of the device

No. of pythons (av. body Initial technique Anchored technique mass Ϯ SE) Expelled (av. no. of days Retained (av. no. of days Expelled Retained (av. no. of days Total implanted Ϯ SE) implanted prior to implanted prior to removal Ϯ SE) removal Ϯ SE)

Female (1420.5 Ϯ 34.1 g) 6 (416 Ϯ 186) 8 (688 Ϯ112.3) 0 11 (248.4 Ϯ112.3) 25 Male (693.9 Ϯ 16.4 g) 6 (101 Ϯ 48) 3 (420 Ϯ126.4) 0 12 (308.3 Ϯ21.3) 21 Subtotal 12 11 0 23 46

often large in size, following which the stomachs and intestines of pythons are quickly upregulated30–32 and increase substantially in size. This could result in pressure necrosis of the intestinal lumen wall adjacent to the transmitter, causing it to be encapsulated within the intestines and subsequently excreted with the faeces. The minimum number of days taken for African catfish to expel an implant through the intestine was 8 days23 and a python in the present study expelled the implanted radiotransmitter within 4 days,which seems to rapid for encapsulation through the gastrointestinal wall.13 All of the fish that WILDLIFE AND ZOOS expelled implanted radiotransmitters through the intestine had a degree of intestinal inflammation and there was granulation tissue surrounding the radiotransmitter, but our exploratory coeliotomy of a carpet python that was captured while in the process of expelling a transmitter showed no evidence of inflammation at the implantation site or within the coelomic cavity.

Our investigations support the observation that a feeding event may Figure 5. Schematic of the movement of an unanchored radiotransmitter towards the three chambers of the snake’s cloaca. The lower panel shows allow an unanchored radiotransmitter to be pushed towards the how the radiotransmitter may be forced into one of the cloacal chambers cloaca as the digestion process progresses;13 however, only 3 of 12 and then expelled with or without faecal material. expulsion events in our study were contaminated with faecal material, suggesting that a feeding event is not necessarily the initiating factor effectively.23,27 Because of this, in the present study each radiotrans- forcing the radiotransmitter out of the body. mitter and antenna was implanted intracoelomically, rather than posi- Telemetry studies on fish have shown signs of irritation at the antenna tioning the antennae subcutaneously, to minimise the risk of exit wound and suggest that infection, inflammation or tissue necrosis inflammatory reaction and wound dehiscence as result of the anten- may be problematic for implanted radiotransmitters that have antennae wire being near the incision site. Another route of expulsion is nas.26 Additionally, there was no evidence of an inflammatory through necrotic muscle tissue that develops in an intact part of the response to the implanted transmitter. A previous study25 of channel body wall adjacent to the incision,26 which has been seen in Atlantic catfish found that the prevalence of tissue reaction and radiotransmit- salmon (Salmo salar) but with no adverse effects or mortality of the ters being expelled through the incision was greater with devices study animals.28 The third route is through the intestine (transintes- weighing 2.0% of the fish’s body weight, compared with transmitters tinal expulsion) and has been occasionally observed in channel that were 0.5% of body weight. That study also found that radiotrans- catfish (Ictalurus punctatus),African catfish (Heterobranchus longifilis) mitters being expelled from the incision occurred more frequently in and rainbow trout.23–26 During necropsy procedures, implanted gravid females than in males or spent females (females that have radiotransmitters have been found at various stages of encapsulation, produced eggs).We used an arbitrary rule of transmitter weight being including limited fibrous proliferation surrounding the transmitter, <5% of the python’s body weight, but others have provided detailed full encapsulation by fibrous connective tissue or following encapsu- descriptions of quantifying and monitoring the choice of radiotrans- lation when the transmitter has been located partly or fully inside the mitter or device attachment to animals in order to estimate the costs intestinal lumen.25 associated for the animal.33 When researchers plan to attach a device The cloacal region of snakes is a complex anatomical structure that to animals they must predict how it will affect the animal’s biome- has three chambers (the coprodeum, urodeum and proctodeum) with chanics, locomotion and survival, ranging from altered foraging different functions, separated from each other by papillae and other behaviour, grooming regimens, provisioning of offspring, movement structures.29 Pythons are constrictors and the controlled body wall patterns and exposure to predators, and also provide animal ethics muscle contractions and peristalsis associated with capturing and committees with a ‘discomfort index’ of the device.34 In the present digesting their prey could easily move an unanchored radiotransmit- study we did not find evidence of adverse effects on survival or loco- ter caudally through the coelomic cavity. Movement of the body wall motion associated with the anchored technique of radiotransmitter musculature associated with general mobility may also result in the implantation in any of the study pythons. caudal movement of an unanchored transmitter. If the transmitter is pushed down to the most caudal point in the coelomic cavity, it will be We recommend that in future studies the radiotransmitters should be lodged against the cloacal wall and could potentially perforate through appropriate for the size of the snake. A more elongated shape of and be expelled from the cloaca, with or without faeces (Figure 5). The radiotransmitters would be beneficial, allowing substantially more likelihood of this occurring may be increased if the unanchored room for the snake’s organs to change in size during digestion and radiotransmitter is placed with the antenna directed caudally, which reproduction, particularly in gravid females. could create a sharp point where the antenna folds or bends, initiating There are no studies evaluating the efficacy of NSAIDs or local or exacerbating pressure necrosis of the tissue. anaesthetics in reptiles and doses are anecdotal or extrapolated from Pearson and Shine13 suggested that expulsion of transmitters was a mammalian or avian studies.16 We recognised the need to provide result of feeding events involving the consumption of whole prey, postoperative analgesia with meloxicam, but given the increased

understanding of the need for analgesia in reptiles, recommenda- 13. Pearson D, Shine R. Expulsion of intraperitoneally-implanted radiotransmit- tions for analgesic regimens associated with invasive surgical proce- ters by Australian pythons. Herpetol Rev 2002;33:261–263. 14. Pearson D, Shine R, Williams A. Spatial ecology of a threatened python dures, such as a coeliotomy, should involve the use of both (Morelia spilota imbricata) and the eﬀects of anthropogenic habitat change. Aust intra- and postoperative analgesic agents.15,16,21,33,35 However, the Ecol 2005;30:261–274. eﬀects of pre-emptive analgesia on anaesthetic induction or recovery 15. Bennett RA. Reptile anesthesia. Semin Avian Exotic Pet Med 1998;7:30–40. times of reptiles following general anaesthesia are still not fully 16. Mosley CAE. Anesthesia and analgesia in reptiles. Semin Avian Exotic Pet Med 2005;14:243–262. 16,33,35 understood. 17. Reiss A. Current therapy in reptile medicine. Wildl Aust Healthcare Manag Proc 1999;327:99–100.

WILDLIFE AND ZOOS 18. Schumacher J. Reptiles and amphibians. In: Thurmon JC, Tranquilli WJ, Benson Acknowledgments GJ, editors. Lumb and Jones’ veterinary anaesthesia. 3rd edn. Williams & Wilkins, Baltimore, 1996;670–682. This project was funded by Murdoch University, the Department of 19. Schumacher J, Yelen T. Anesthesia and analgesia. In: Mader D, editor. Reptile Environment and Conservation and the Invasive Animals Coopera- medicine and surgery. Saunders Elsevier, St Louis, 2005;442–451. 20. Mulcahy DM. Surgical implantation of transmitters into fish. ILAR J tive Research Centre. We thank D. Pearson for help with the initial 2003;44:295–306. training and advice for implanting radiotransmitters and the numer- 21. Bennett RA. Reptilian surgery. Part 1: Basic principles. Compend Cont Educ ous volunteers for their assistance in the surgeries, in particular Pract Vet 1989;11:10–20. J. Clarke, M. Connor, J. Bryant,A. Bryant, C. Bryant, J. Crow, S. Dundas 22. Mader DR, Bennett RA, Funk RS, Fitzgerald KT. Surgery. In: Mader DR, editor. Reptile medicine and surgery. 2nd edn. Saunders Elsevier, St Louis, 2006;581–630. and J. Hampton. R. Joy, M. Rouffignac and T. Messer helped with 23. Baras E, Westerloppe L. Transintestinal expulsion of surgically implanted tags invaluable veterinary nursing knowledge. by African catfish Heterobranchus longifilis of variable size and age. Trans Am Fish Soc 1999;128:737–746. 24. Chisholm IM, Hubert WA. Expulsion of dummy transmitters by rainbow trout. References Trans Am Fish Soc 1985;114:766–767. 25. Marty GD, Summerfelt RC. Pathways and mechanisms for expulsion of surgi- 1. Priede IG. Wildlife telemetry: an introduction. In: Priede IG, Swift SM, editors. cally implanted dummy transmitters from channel catfish. Trans Am Fish Soc Wildlife telemetry: remote monitoring and tracking of animals. Ellis Horwood, 1986;115:577–589. London, 1992;3–25. 26. Jepsen N, Koed A, Thorstad EB, Baras E. Surgical implantation of telemetry 2. Fitch HS, Shirer HW. A radiotelemetric study of spatial relationships in some transmitters in fish: how much have we learned? Hydrobiologia 2002;483:239– common snakes. Copeia 1971;1971:118–128. 248. 3. Jacob JS, Painter CW. Overwinter thermal ecology of Crotalus viridis in the 27. Knights BC, Lasee BA. Effects of implanted transmitters on adult bluegills at North-Central plains of New Mexico. Copeia 1980;1980:799–805. two temperatures. Trans Am Fish Soc 1996;125:440–449. 4. Parker WS, Brown WS. Telemetric study of movements and oviposition of two 28. Moore A, Russell IC, Potter ECE. The effects of intraperitoneally implanted female Masticophis t. taeniatus. Copeia 1972;1972:892–895. dummy acoustic transmitters on the behaviour and physiology of juvenile Atlan- 5. Brown WS, Parker WS. Movement ecology of coluber constrictor near commu- tic salmon, Salmo salar. J Fish Biol 1990;37:713–721. nal hibernacula. Copeia 1976;1976:225–242. 29. Funk R. Snakes. In: Mader D, editor. Reptile medicine and surgery. 2nd edn. 6. Landreth HF. Orientation and behavior of the rattlesnake, Crotalus atrox. Copeia Elsevier, Missouri, 1996;42–58. 1973;1973:26–31. 30. Secor SM, Diamond J. Adaptive responses to feeding in Burmese pythons: pay 7. Osgood DW. Thermoregulation in water snakes studied by telemetry. Copeia before pumping. J Exp Biol 1995;198:1313–1325. 1970;1970:568–571. 31. Secor SM, Diamond J. A vertebrate model of extreme physiological regula- 8. Shine R, Bonnet X. Snakes: a new ‘model organism’ in ecological research? tion. Nature 1998;395:659–663. Trends Ecol Evol 2000;15:221–222. 32. Secor SM, Diamond J. Evolution of regulatory responses to feeding in snakes. 9. Lutterschmidt WI, Reinert HK. The effect of ingested transmitters upon the Physiol Biochem Zool 2000;73:123–141. temperature preference of the Northern water snake, Nerodia S. sipedon. Herpe- 33. Bertelsen MF. Squamates (snakes and lizards). In: West G, Heard D, Caulkett N, tologica 1990;46:39–42. editors. Zoo animal and wildlife immobilization and anesthesia. Blackwell, Oxford, 10. Reinert HK, Cundall D. An improved surgical implantation method for radio- 2007;233–244. tracking snakes. Copeia 1982;1982:702–705. 34. Wilson RP, McMahon CR. Measuring devices on wild animals: what constitutes 11. Weatherhead PJ, Anderka FW. An improved radio transmitter and implanta- acceptable practice? Front Ecol Environ 2006;4:147–154. tion technique for snakes. J Herpetol 1984;18:264–269. 35. Machin K, L. Wildlife analgesia. In: West G, Heard D, Caulkett N, editors. Zoo 12. Anderson CD, Talcott M. Clinical practice versus field surgery: a discussion of animal and wildlife immobilization and anesthesia. Blackwell, Oxford, 2007;50–51. the regulations and logistics of implanting radiotransmitters in snakes. Wildl Soc Bull 2006;34:1470–1472. (Accepted for publication 17 February 2010)

4. WHAT FACTORS AFFECT HAEMATOLOGY AND PLASMA

BIOCHEMISTRY IN THE SOUTHWEST CARPET PYTHON

(MORELIA SPILOTA IMBRICATA)?

Haematological and biochemical analyses were undertaken to investigate whether there were any health effects of surgically implanting the radiotransmitters. This study has been accepted for publication in the Journal of Wildlife Diseases. *Please note American spelling for journal requirements.

AUTHOR CONTRIBUTION:

G. L. Bryant: Initiated the investigation into haematology and biochemical analysis of free-ranging pythons, collected all blood samples, organised/undertook transport of blood samples, entered data, carried out the statistical analyses, and wrote paper.

P. A. Fleming: Provided assistance with database management, provided assistance with statistical analysis, and provided editorial comments to versions of the manuscript.

L. Twomey: Analysed haematological samples, together with other clinical pathologists at VetPath, provided specialist interpretation of haematological and biochemical results and provided editorial comments to versions of the manuscript.

K. A. Warren: Initiated haematological investigation to assess python health following surgical implantation of radiotransmitters, and provided editorial comments to versions of the manuscript.

Journal of Wildlife Diseases, 48(2), 2012, pp. 000–000 # Wildlife Disease Association 2012

FACTORS AFFECTING HEMATOLOGY AND PLASMA BIOCHEMISTRY IN THE SOUTHWEST CARPET PYTHON (MORELIA SPILOTA IMBRICATA)

Gillian L. Bryant,1,3 Patricia A. Fleming,1 Leanne Twomey,2 and Kristin A. Warren1 1 School of Veterinary & Biomedical Sciences, Murdoch University, South Street, Murdoch, Western Australia 6150, Australia 2 Vetpath Laboratory Services, 39 Epsom Avenue, Ascot, Western Australia 6104, Australia 3 Corresponding author (email: [email protected])

; ABSTRACT: Despite increased worldwide popularity of keeping reptiles as pets, we know little about hematologic and biochemical parameters of most reptile species, or how these measures may be influenced by intrinsic and extrinsic factors. Blood samples from 43 wild-caught pythons (Morelia spilota imbricata) were collected at various stages of a 3-yr ecological study in Western Australia. Reference intervals are reported for 35 individuals sampled at the commencement of the study. As pythons were radiotracked for varying lengths of time (radiotransmitters were surgically implanted), repeated sampling was undertaken from some individuals. However, because of our ad hoc sampling design we cannot be definitive about temporal factors that were most important or that exclusively influenced blood parameters. There was no significant effect of sex or the presence of a hemogregarine parasite on blood parameters. Erythrocyte measures were highest for pythons captured in the jarrah forest and at the stage of radiotransmitter implantation, which was also linked with shorter time in captivity. Basophil count, the only leukocyte influenced by the factors tested, was highest when the python was anesthetized, as was globulin concentration. Albumin and the albumin:globulin ratio were more concentrated in summer (as was phosphorous) and at the initial stage of radiotransmitter placement (as was calcium). No intrinsic or extrinsic factors influenced creatinine kinase, aspartate aminotransferase, uric acid, or total protein. This study demonstrates that factors including season, location, surgical radiotransmitter placement, and anesthetic state can influence blood parameters of M. s. imbricata. For accurate diagnosis, veterinarians should be aware that the current reference intervals used to identify the health status of individuals for this species are outdated and the interpretation and an understanding of the influence of intrinsic and extrinsic factors are limited. Key words: Anesthetic, captivity, hemoparasite, radiotransmitter, reference intervals, reptile, season, snake.

INTRODUCTION to the same extent as in mammals, although various blood values are known Monitoring hematologic measures and to be influenced by factors such as age, serum or plasma biochemical analytes is sex, and nutritional status (Campbell, important for evaluating the health status 2004). Reference intervals have typically of reptiles kept in captivity for research, as not accounted for these variations in pets, or in zoos (Nordøy and Thoresen, intrinsic and environmental factors, mak- 2002). Evaluating hematologic and bio- ing interpretation difficult (Campbell, chemical responses can facilitate the 2004, 2006). diagnosis of stress and disease states in The International Species Information reptile species (Christopher et al., 1999). System (ISIS) reports hematologic mea- Blood analysis can be used to detect sures and plasma biochemical analytes for conditions such as anemia, inflammatory samples collected from 25 pythons (Mor- disease, parasitemia, hematopoietic disor- elia spilota) from 10 institutions. However, ders, and hemostatic alterations by com- no details are provided on source of the paring individual samples with a clinically animals, nor is any information given to normal sample population (Campbell and indicate sex, season, or length of time in Ellis, 2007). Reptilian blood analysis has captivity before blood sampling (Teare, not been evaluated for clinical application 2002). The near-threatened southwest

Journal of Wildlife Diseases jwdi-48-02-03.3d 19/1/12 04:58:31 1 Cust # 2010-08-206 0 JOURNAL OF WILDLIFE DISEASES, VOL. 48, NO. 2, APRIL 2012 carpet python (Morelia spilota imbricata) of hemipenes or by insertion of a lubricated that inhabits southwest Western Australia blunt probe into the cloaca and then directed toward the tail to determine the presence or measures up to 2.4 m snout-to-vent length absence of hemipenes. Sex was determined by (SVL) and can weigh up to 6 kg (IUCN, depth of the probe insertion, as measured by 1998; Pearson et al., 2002). Since 2003 the number of overlying subcaudal scales. it has been legal to obtain a license and Females probed to between one and five keep reptiles as pets in Western Australia, scales, whereas males probed to depths equivalent to 7–20 scales. Adult male pythons including the southwest carpet python weighed an average 6946(SD)285 g (range (Edwards, 2003). As part of a wider 298–1,500 g) and measured SVL 134617 cm ecology study on wild-caught M. s. im- (range 97–170 cm). Adult female pythons bricata, blood samples were collected to weighed 1,4206554 g (range 103–3,731 g) assess whether the health of pythons was and measured SVL 1796119 (range 90– 200cm). adversely affected by having a radiotrans- Upon initial capture, pythons were brought mitter surgically implanted into their into a holding facility at the DEC Research coelomic cavity, which enabled in situ Center, Dwellingup, Western Australia be- monitoring of individuals. This study tween December 2006 and November 2008 to therefore presented a unique opportunity undergo surgical implantation of radiotransmitters (for surgical details see Bryant et al., to carry out repeated sampling for this 2010). When held at the research center, the population of wild pythons over time, animals were housed in a 25 C temperature- before and after surgery. We present controlled room in purpose-built ventilated hematologic and biochemical data col- enclosures with flooring lined to approximately lected from 43 individuals and analyze 2-cm depth with recycled newspaper kitty litter (Old News Cat Litter, Ciber Cycles Pty the effects of season, sex, study site, Ltd, Toowoomba, Queensland, Australia). The anesthesia, duration of time in captivity, enclosures contained a hide box as a cardboard radiotransmitter placement over time, and box with an entrance hole cut into one side, the presence of parasites (Haemogregari- cage furniture including a branch and rocks, na sp.) within red blood cells. ad libitum water, and an external heat pad positioned under the plywood flooring that heated that portion of the cage floor above the MATERIALS AND METHODS heat pad to approximately 30 C. All cages were Study animals and sample collection kept in a room under natural lighting provided by a large window. If they were held for .3wk Forty-three southwest carpet pythons were during warmer seasons, pythons were fed collected through opportunistic hand capture dead laboratory mice and rats that were stored for an ecology and thermal biology research frozen and thawed before feeding. Pythons project. Individuals were captured in the were not fed during winter. Animals were held southwest of Western Australia from two for varying lengths of time because of logistic habitat types (referred to elsewhere as study reasons, including the necessity of customizing site): 1) coastal woodland: included animals a radiotransmitter for surgical implantation. captured and monitored at Martin’s Tank Length of time each python spent in captivity (32u519S, 115u409E) and Leschenault Penin- before sample collection was recorded and sula Conservation Park (33u269S, 115u419E) scored for statistical analyses as 0–30 days and 2) jarrah forest: areas surrounding Dwell- (n533), 30–60 days (n518), and .60 days ingup Township (32u439S, 116u49E). This (n57). project was approved by the Animal Ethics We recorded repeated samples from py- Committees of Murdoch University (W2028/ thons at three time points; however, the 07) and Department of Environment and number of samples collected from pythons Conservation, Western Australia (DEC AEC/ varied between these points. At the initial 55/2006 and DEC AEC54/2006). stage of presurgical implantation of the radio- Body mass (Mb) was determined with a transmitter 38 blood samples were collected calibrated spring balance (60.2 kg), and SVL in the laboratory (pre-TM, n538 individuals). was measured with a tape measure in a straight Twenty four of these 38 pythons were line along the ventral surface of the python anesthetized via inhalation of 1.5% isoflurane from the tip of the mouth to the cloaca. The gas. The remaining 14 pythons were manually sex of each python was determined by eversion restrained by holding carefully within calico

Journal of Wildlife Diseases jwdi-48-02-03.3d 19/1/12 04:58:42 2 Cust # 2010-08-206 BRYANT ET AL.—HEMATOLOGY AND BIOCHEMISTRY OF SOUTHWEST CARPET PYTHON 0

bags, with the tail exposed for blood collection Hematology analysis from the ventral coccygeal vein. Animals were Blood samples were analyzed using a opportunistically allocated to these two treat- TM ments on the basis of logistics around the CELL-DYN (Cell Dyn 3700, Abbott Diag- transportation of blood samples to the clinical nostics, North Ryde, New South Wales, laboratory for analysis. The reference intervals Australia) analyzer using the reptilian/avian were calculated from the samples collected setting to take nucleated erythrocytes into from these 38 animals. Pythons were released account. The blood variables analyzed includ- approximately 2 wk after surgical implantation ed hemoglobin (Hb), packed cell volume and were radiotracked weekly to monitor (PCV), red blood cell count (RBC), mean feeding behavior and habitat use. When cell hemoglobin (MCH), mean cell volume pythons could be captured by hand from their (MCV), mean cell hemoglobin concentration retreat site, 3–12 mo (average 6.8564.86 mo) (MCHC), and total white blood cell count postimplantation of radiotransmitters (post- (WBC) using the automated system. Manual TM, n522), a second blood sample was calculations for PCV were made to correct collected from manually restrained conscious hemoglobin values (Hb, MCH, MCV, and animals in the field. One python was moved MCHC) and if these measures did not back into temporary captivity for surgical correspond (8/77 samples), they were removed replacement of a radiotransmitter due to from further analysis and only PCV was battery failure, and the posttransmitter (sec- included. ond) blood sample was collected under Blood smears were air-dried and stained anesthesia. Pythons were anesthetized at the with Wright’s–Giemsa stain by an automated time of radiotransmitter removal, allowing a slide stainer (Hematek, Siemens, Osborne third (removal-TM, n520) blood sample. Park, Western Australia). The proportions of Twelve individuals lost their transmitters heterophils (including potential eosinophils, during the period of tracking and 13 died which could not be definitively identified by over the 3-yr study (postmortem examinations morphology alone; Stirk et al., 2007), lympho- were performed when possible and the results cytes, basophils, and combined monocytes/ are presented elsewhere; Bryant, 2012). In azurophils (Fig. 1a–d) were classified through total, we collected 77 blood samples from 43 manual counts of blood smears. Parasites in animals. Sixteen pythons were sampled once, red blood cells that were morphologically 17 were sampled twice, and 10 were sampled consistent with a Hemogregarina species were three times. found in some samples (Fig. 1e). Following A range of needle sizes (depending on the descriptions provided by Mackerras (1961) of size of the python; 25 G for SVL,100-cm the seven Hemogregarina species found in pythons, 25 G, 23 G for intermediate sizes, Australian Boidae (O’Donoghue and Adlard, and 21 G for SVL.150 cm) and a 2-ml syringe 2000), the species in this study appears to be TM (BD PrecisionGlide Needle; BD Slip Tip Hemogregarina moreliea. Samples were cate- Syringe; Becton Dickinson, Singapore) were gorized according to the presence or absence used to collect blood samples from the ventral of the hemoparasites in smears. coccygeal vein. Fresh blood smears without anticoagulate were made immediately after Plasma biochemical analysis collection; the remainder of the sample was carefully transferred immediately into 2-ml A minimum of 150 mlofplasmafrom (13375 ml) lithium heparin vacutainers (BD heparinized blood samples was used for bio- VacutainerH, Becton Dickinson, Plymouth, chemical analysis. Plasma was analyzed for UK) and kept refrigerated at approximately creatinine kinase (CK), aspartate aminotrans- 4 C during transportation to a commercial ferase (AST), uric acid, total protein, albumin, laboratory. Analysis was completed by Vetpath globulin, albumin-to-globulin ratio (A/G), Laboratory Services (Ascot, Western Australia, calcium, phosphorous, and glucose using an Australia) within 48 hr of collection. Samples automated chemical analyzer (Olympus AU collected in the field were kept on ice in an 400, Integrated Science, Tokyo, Japan). Be- insulated container or refrigerated when cause of varying lengths of time before possible before transport to the laboratory. laboratory analysis (up to 48 hr) and incomplete Whenever possible, a complete set of hema- data collection, glucose results are variable and tologic and plasma biochemical data was therefore, although range of data is indicated, collected from each blood sample. Incomplete caution should be taken when using the values blood analysis was common, generally due presented here. For these reasons, multiple to insufficient volumes of plasma for the full regression analyses were not performed on biochemistry panel. glucose.

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Proportional difference~ ðÞAverage M: s: imbricata{average M: spilota average M: spilota

Multiple regression analysis (Statistica 9.0, Statsoft Inc) was carried out separately for each blood measure (dependent variable) against the same independent factors: sex (male or female), study site (coastal woodland or jarrah forest), season (summer: December– February; autumn: March–May; winter: June– August; spring: September–November), anesthesia (anesthetized or conscious), the experimental phase of sample collection (three categories: pre-TM, post-TM, and removal- TM), the time in captivity from capture until sample collection (three categories: 0–30 days, 31–60 days, and .60 days; animals sampled in the field were ascribed as 0 days), and hemoparasite presence (positive or negative). The blood variables examined included: Hb, PCV, RBC, MCH, MCV, MCHC, WBC, FIGURE 1. Peripheral blood films of a southwest heterophil, lymphocyte, basophil, and com- carpet python (Morelia spilota imbricata) showing: a) heterophil (black arrow); b) lymphocytes (black bined monocyte and azurophil counts, CK, arrows) and thrombocytes (open arrow); c) basophil AST, uric acid, total protein, albumin, globu- (double black arrow); a lymphocyte (single black lin, A/G ratio, calcium, and phosphorus. arrow) and thrombocyte (open arrow) are also Chi-square analysis was used to determine visible; d) azurophil (black arrow); and e) erythrocyte variation in hemoparasite prevalence across containing a Hemogregarina moreliea hemoparasite seasons with the expected values calculated (black arrow). Images (a–e) stained with Wright– assuming an equal proportion of the popula- Giemsa stain, 1,0003, bar 5 15 mm. tion was positive each season. A similar analysis was carried out for H. moreliea hemoparasite prevalence at each sampling Statistical analysis period, assuming an equal proportion of Reference intervals for M. s. imbricata were samples collected pre-TM, post-TM, or re- investigated for the first time period sampling moval-TM were positive (i.e., expected values point only (pre-TM implantation) for 38 calculated assuming an equal distribution individuals. Blood samples from three female across these three time points). Chi-square analysis was also used to test for a significant pythons were removed as they were consid- difference in H. moreliea prevalence between ered outliers using Dixon’s range test within study sites. the program Reference Value Advisor V 1.4 (Anonymous, 2010); reference intervals were therefore calculated using blood samples for RESULTS 35 individuals. Standard descriptive statistics include sample size, mean, standard deviation, Reference intervals minimum, median, and the maximum values. Reference intervals are shown for 35 Normality tests were performed and if re- pythons for blood collected before surgical quired, data were transformed with Box-Cox transformation. The lower and upper limit of implantation of radiotransmitters (pre- the reference interval as well as the 2.5, 5, 90, TM) only (Table 1). Raw, untransformed and 97.5% confidence intervals are given data are shown for reference intervals. where possible using the program Reference Most average values found in this study Value Advisor V 1.4 (Anonymous, 2010). were similar (,20% difference) to pub- Data from our study (M. s. imbricata) are expressed as a proportion of reported ISIS lished ISIS values for M. spilota (Teare, average values (M. spilota)followingthe 2002). However, MCV were twice those of formula: published ISIS values, WBC counts were

Journal of Wildlife Diseases jwdi-48-02-03.3d 19/1/12 04:58:42 4 Cust # 2010-08-206 BRYANT ET AL.—HEMATOLOGY AND BIOCHEMISTRY OF SOUTHWEST CARPET PYTHON 0 % 97.5 ot conducted igits, or were screpancies in % 90 ) sampled before % 5 % Morelia spilota imbricata Confidence intervals limit 2.5 Upper limit Lower a Transformation Box Cox robust transformation. 5 Mean Median SD Minimum Maximum n 26 117 111 19.8 96.0 178 UT 67.2 152 51.0 86.1 133 170 26 343 342 17.7 315 374 UT 305 380 297 314 369 389 32 84.8 63.0 63.8 17.0 259 BoxCox 17.8 296 13.9 23.6 187 445 35 14.9 13.4 6.94 4.90 29.3 UT 0 29.4 0 2.70 24.8 32.2 32 4.57 3.94 2.76 1.08 12.3 BoxCox 1.08 12.9 0.768 1.50 9.77 17.0 /l) 26 0.669 0.700 0.174 0.200 0.900 BoxCox 0.027 0.905 12 /l) 35 3.09 2.59 2.19 0.400 10.3 BoxCox 0.367 9.07 0.275 0.603 6.91 11.3 10 9 /l) 31 7.72 7.13 5.17 0.410 24.8 BoxCox 0.620 21.7 0.141 1.79 16.8 27.1 3 9 10 /l) 28 0.232 0.155 0.246 0.00 0.940 BoxCox 0 1.217 0 0 0.778 1.83 9 10 3 3 10 3 Value /l) /l) 9 untransformed robust data; BoxCox 9 1. Hematology and plasma biochemical values for 35 apparently healthy, wild-caught southwest carpet pythons ( 10 10 5 3 3 ( (pg) concentration (g/l) ( (U/l) ABLE UT Mean cell volume(fl)White blood cell count 26 339 327 49.5 279 484 UT 219 430 188 260 386 465 Mean cell hemoglobin Uric acid (mmol/l)Total protein (g/l)Albumin (g/l)Globulin (g/l)Albumin-to-globulin 28 ratioCalcium 32 (mmol/l) 32Phosphorus (mmol/l) 0.186Glucose (mmol/l) 73.0 32 0.407 32 0.184 32 32 20.9 0.420 71.5 52.0 0.061 28 1.14 3.43 0.045 21.0 11.7 0.081 50.5 2.84 1.14 0.280 3.46 56.0 0.333 3.03 2.10 9.25 0.490 0.316 0.30 UT 16.0 105 BoxCox 39.0 0.430 1.94 2.90 27.0 UT 0.296 1.86 78.0 0.047 0.100 4.41 0.485 UT 0.301 UT 8.30 UT 0.251 UT 47.0 0.019 0.343 BoxCox 0.088 14.5 96.0 0.468 31.5 0.469 0.267 2.79 0.189 41.0 27.1 0.499 1.78 0.342 70.4 8.21 53.6 13.0 4.02 0.337 26.3 0.051 2.60 0.643 15.7 88.2 37.0 0.491 1.57 103 3.00 25.3 5.75 64.2 1.94 3.82 28.8 10.3 76.0 4.19 Heterophils ( Mean cell hemoglobin Basophils ( Monocytes & azurophils T Hemoglobin (g/l)Packed cell volumeRed blood count ( 26 30 76.5 0.232 79.0 0.240 17.1 0.051 0.080 28.0 0.30 105 BoxCox BoxCox 0.090 30.4 0.307 104 0a 0 0.155 0.293 48.7 0.319 97.4 109 Creatinine kinase (U/l)Aspartate aminotransferase 32 1880 1651 1115 365 5147 BoxCox 379 4767 293 542 3726 5746 Lymphocytes ( radiotransmitter implantation. Sample sizebecause varies of among small measures sampleerythrocyte because size), counts of due (see differences tointerpreted variation in text as in the for time zero volume further to of for detail). analysis blood negative Values (not that results. all were could samples be calculated were collected using analyzed (some Reference for tests glucose Value n because Advisor of v1.4 transport program delays), and or due given to to di three significant d

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TABLE 2. Average6SD values for each hematologic measure analyzed by multiple regression for all factors. Bold values indicate a significant (* P,0.05, ** P,0.005, *** P,0.001) difference between the categories in the analysis. Values to three significant digits.

Category, n5number of pythons (N5number Hb (g/l), PCV (l/l), RBC (31012/l)a, MCH (pg)b, Factor of samples) N565 N564 N564 N564 Study site Coastal, n528 (54) 69.6616.3 0.20960.050 *0.600±0.155 *118±17.5 Jarrah forest, n513 (23) 74.369.62 0.22760.038 0.672±0.102 113±14.7 Sex Female, n519 (33) 68.7616.7 0.20360.053 0.58160.159 122621.7 Male , n522 (44) 72.7613.0 0.22560.039 0.64960.128 113610.7 Season Summer, n523 (25) 80.2610.0 0.24160.031 0.68160.117 **120±23.1 Autumn, n517 (17) 63.7618.0 0.19060.062 0.53660.169 121±14.1 Winter, n511 (12) 65.0612.2 0.19560.042 0.61860.133 107±12.5 Spring, n520 (23) 69.7613.4 0.21060.046 0.61760.138 115±9.32 State during Anesthetized, n529 (42) 72.0615.7 0.21160.047 0.62360.154 117616.2 sampling Conscious n528 (35) 69.8613.7 0.22060.048 0.61760.136 116617.8 Radiotransmitter Pre, n535 (35) *76.5±17.1 *0.232±0.051 **0.669±0.174 116619.8 Post, n522 (22) 66.6±12.6 0.199±0.042 0.575±0.107 118618.4 Removal, n520 (20) 68.1±11.3 0.199±0.036 0.600±0.119 11569.39 Time in captivity 0–30 d, n531 (48) 71.0614.3 0.21560.045 0.61060.137 *120±19.0 30–60, n518 d (21) 69.1617.5 0.21260.058 0.62460.179 112±11.0 .60 d, n57 (8) 76.769.14 0.22060.025 0.68360.075 111±10.2 Parasite presence Negative, n537 (62) 72.6613.3 0.22160.042 0.64260.131 115615.8 Positive, n511 (15) 65.0618.4 0.19260.057 0.54360.170 121620.2

Hb 5 hemoglobin; PCV 5 packed cell volume; RBC 5 red blood cell count; MCH 5 mean cell hemoglobin; MCV 5 mean cell volume; MCHC 5 mean cell hemoglobin concentration; WBC 5 total white blood cell count. a Nonnormal distribution for Box-Cox transformed data. b Box-Cox transformed data with normal distribution.

40%, and heterophil counts were 172% Season, anesthesia, surgical implanta- greater than ISIS values. All other leuko- tion, and time in captivity significantly cyte values were lower than those report- influenced some hematologic and bio- ed in ISIS for M. spilota. Creatinine kinase chemical analytes; however, due to the (+292%) and AST (+239%) were higher lack of independence of the factors tested, than reference values and uric acid the effects of season and radiotransmitter (257%), albumin (230%), calcium placement cannot be differentiated. Sea- (246%), and phosphorous (259%) con- son significantly influenced values for centrations were lower than average val- MCH (t5653.33, P,0.01), MCV (t565 ues reported by ISIS. 2.55, P,0.01), albumin (t6552.07, P,0.05), A/G ratio (t6552.42, P,0.05), Factors affecting hematologic measures and and phosphorous (t6553.23, P,0.01). For plasma biochemistry MCH, MCV, albumin, and phosphorous, Pythons from the jarrah forest had the lowest average values were recorded greater RBC (t5652.04, P,0.05) and in winter (Tables 2 and 3), whereas the lower concentrations of MCH (t565 A/G ratio peaked in summer (Table 3). 22.41, P,0.05) and MCV (t56522.18, Blood values were apparently influenced P,0.05) compared with pythons from by anesthetic state, with anesthetized coastal woodland. There was no statistical animals having higher average basophil difference in any hematologic variable or counts (t5552.10, P,0.05) and globulin plasma biochemical analytes between concentrations (t6552.01, P,0.05; Ta- males and females (all P.0.05, Tables 2 bles 2 and 3). The A/G ratio was also and 3). lower for anesthetized animals (t655

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TABLE 2. extended.

Heterophils Lymphocytes Basophils Monocytes & MCV (fl)b, MCHC (g/l), WBC (3109/l)b, (3109/l)b, (3109/l)b, (3109/l)a, Auzurophils N564 N565 N577 N577 N577 N563 (3109/l)b, N573 *348±59.4 338623.0 14.966.85 7.056.75 3.1162.31 0.3096.326 4.2862.39 327±46.0 346620.7 14.966.23 6.6264.02 2.9462.51 0.34560.240 4.8762.69 358665.5 342620.9 15.868.08 7.2865.49 3.4562.64 0.23860.186 4.4562.83 331646.4 339623.8 14.265.29 6.6663.68 2.7662.10 0.39360.354 4.4462.21 **351±72.7 343622.0 13.265.86 6.6564.11 2.8562.20 0.23360.282 4.2362.03 355±48.5 340630.3 13. 867.14 6.0763.68 2.9862.33 0.34560.278 4.3863.29 323±54.1 333617.8 15.569.02 6.5465.77 2.5162.49 0.20760.234 4.8163.00 333±38.5 341619.4 18.266.93 8.0664.85 3.6262.51 0.47860.315 4.8562.48 336645.4 345619.1 14.466.15 7.2364.65 3.1562.26 *0.368±0.319 4.1962.39 350667.3 334625.3 15.567.20 6.5664.41 2.9462.50 0.270±0.267 4.7862.58 339649.5 343617.7 14.966.94 6.4063.76 3.0962.19 *0.232±0.246 4.5762.76 356675.2 334629.6 13.867.40 6.7665.01 2.5462.38 0.286±0.286 4.1362.29 332638.9 342619.4 16.165.11 8.0265.18 3.5662.61 0.521±0.314 4.5962.25 *356±61.0 337623.3 14.466.71 7.1264.95 2.9462.39 0.26660.214 4.1262.25 320±39.8 343621.6 16.466.79 5.8463.87 3.3462.50 0.36660.375 5.1162.93 313±30.9 354614.4 13.765.70 8.5762.85 3.0061.92 0.55360.414 4.7262.44 341657.6 338622.8 14.866.81 6.5464.54 2.9662.42 0.31360.305 4.5462.59 348653.5 347620.4 15.266.05 8.5264.22 3.4562.10 0.37160.257 4.1162.01

22.09, P,0.05; Table 3). The time of pythons captured from coastal woodland 2 bloodcollection(categorizedbythree or jarrah forest (x 152.43, P.0.05). A stages of radiotransmitter implantation) greater proportion of blood samples col- influenced several hematologic parame- lected from pythons during spring (Sep- ters (Hb: t5752.16, P,0.04; PCV: tember–November) were positive for 2 t5652.78, P,0.01; RBC: t5653.03, H. moreliea (x 158.44, P,0.01; Fig. 2). P,0.01; and basophil count t55522.44, Significantly more pythons sampled at P,0.05) and plasma biochemical analytes removal-TM were positive for hemogreg- (albumin t6553.54, P,0.001; A/G ratio: arines compared with pre-TM and post- 2 t6554.24, P,0.001; and calcium: t6553.50, TM (x 158.30, P,0.01; Fig. 2). P,0.001). All these measures were highest at the pre-TM implantation sampling DISCUSSION period. The length of time in captivity before blood sample collection (0–30 days, We examined the influence of intrinsic 30–60 days, or .60 days) significantly and extrinsic factors on hematologic pa- influenced only MCH (t56522.09, rameters and plasma biochemical analytes P,0.05) and MCV measures (t56522.78, in a Western Australian python species. P,0.05; Table 2), where values were Health screening provides useful baseline higher for animals that had spent less data for conservation management pro- time in captivity. grams of wild populations of threatened The presence of H. moreliea was only species and for species held in captivity for marginally associated with reduced total zoologic collections and as pets (Espinosa- protein (t6551.88, P.0.06), albumin (t655 Avile´s et al., 2009). We provide reference 1.85, P.0.07), and globulin (t6551.88, intervals for wild-caught southwest carpet P.0.06; Table 3) measures. There was pythons that could contribute to health no significant difference in the occurrence screening of these animals for conservation of the intracellular hemoparasite between management. There was no significant

Journal of Wildlife Diseases jwdi-48-02-03.3d 19/1/12 04:58:58 7 Cust # 2010-08-206 0 JOURNAL OF WILDLIFE DISEASES, VOL. 48, NO. 2, APRIL 2012 0.05, 0.255 0.278 0.490 0.211 0.453 0.383 0.168 0.316 0.295 0.437 0.177 0.384 0.388 0.289 0.310 0.395 0.219 0.279 73 , ± 6 6 6 ± 6 6 6 6 6 6 6 6 6 6 6 ± ± P 5 N * P (mmol/l), 1.17 1.00 1.14 1.32 1.13 1.05 * 0.781 , b 1.14 1.13 0.463 1.06 0.309 0.551 1.16 0.508 1.10 0.359 1.06 0.841 0.788 1.21 0.615 1.14 0.452 1.08 0.526 1.18 0.375 0.330 1.17 0.502 0.519 1.05 0.298 0.667 0.523 73 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 ± ± ± 5 N 3.37 3.37 3.06 3.13 3.04 3.23 3.43 Ca (mmol/l) * 0.050 2.90 0.086 2.93 0.078 0.082 3.23 0.076 3.21 0.054 3.22 0.080 0.077 3.26 0.064 3.21 0.079 3.28 0.086 3.33 0.064 0.084 3.20 0.061 0.073 3.14 0.045 0.080 0.062 6 ± ± 6 6 ± ± 6 6 6 6 ± 6 ± 6 ± ± ± 73 5 N Albumin/ 0.338 0.290 0.370 0.326 0.302 0.407 0.351 0.409 globulin ratio, * * *** 12.2 13.0 0.391 11.4 10.5 0.360 10.7 0.360 9.55 11.4 0.340 12.9 10.9 9.15 11.7 0.387 13.1 0.335 11.1 0.353 12.2 0.363 11.4 11.7 0.348 12.8 10.9 0.349 73 ± 6 6 ± 6 6 6 6 6 6 6 6 6 6 6 6 6 6 5 (g/l), N Globulin 52.6 52.6 60.5 53.4 52.5 51.2 59.2 55.8 53.1 * 2.85 3.03 3.64 54.0 4.27 3.59 4.16 3.70 55.5 3.75 54.0 3.02 4.10 3.25 55.7 3.80 51.2 3.88 54.5 4.18 54.2 4.36 3.86 55.1 3.02 3.60 54.8 73 ± ± 6 ± 6 6 6 6 ± ± 6 6 6 6 ± 6 ± 6 5 (g/l), N Albumin 21.1 20.9 17.1 19.4 17.5 16.5 17.4 * * 16.3 20.7 11.5 13.3 18.9 13.0 18.9 12.1 19.4 11.7 12.8 17.9 14.3 13.3 13.9 13.4 21.0 15.9 16.8 13.0 18.7 14.0 19.2 14.4 13.1 18.6 14.0 12.6 18.6 73 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 5 (g/l), Total N protein , a 0.136 73.7 0.158 74.3 0.246 72.0 0.146 74.5 0.061 73.0 0.238 71.6 0.198 76.7 0.213 72.8 0.293 70.1 0.069 76.6 0.349 68.0 0.223 72.7 0.266 72.9 0.321 67.7 0.197 74.0 0.187 75.8 0.140 73.4 0.048 74.7 68 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 5 N Uric acid (mmol/l) , a 33.7 0.223 72.8 0.301 62.4 0.245 63.8 0.186 34.0 0.295 15.7 0.255 33.3 0.284 65.8 0.372 84.1 0.185 21.7 0.316 50.5 0.282 61.2 0.315 23.4 0.365 67.7 0.250 54.7 0.245 56.7 0.221 74.8 0.153 72.0 0.211 73 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 5 N AST (U/l) 709 53.1 889 61.7 569 38.8 907 61.7 761 40.4 904 62.8 776 45.2 715 53.1 841 73.0 568 101 1,210 88.7 1,030 77.9 1,120 84.8 1,060 77.8 1,200 88.6 1,190 66.6 1,320 76.3 1,260 104 71 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 5 N CK (U/l), 1,525 1,330 1,330 1,940 1,830 1,910 1,870 2,050 29 13 5 5 5 n 28 n n 37 20 23 17 (17) 1,860 aspartate aminotransferase. 11 5 19 (33) 1,740 28 (54) 1,630 11 (12) 1,410 31 (48) 1,680 5 5 20 (23) 1,410 5 7 (8) 1,590 n 5 18 d 22 (44) 1,710 5 5 n 5 n 5 n 5 5 n 5 5 n number 22 (22) 1,850 n n 5 5 n n 35 (35) 1,880 n n n 5 n 5 5 N n of samples) ( n Category, SD values for each plasma biochemical measure analyzed by multiple regression for all factors. Bold values indicate a significant ( (42) (35) (62) (20) (15) (23) (21) (25) 6 60 d, number of pythons Anesthetized, Negative, Removal, Conscious Autumn, Positive, 0–30 d, Post, Jarrah forest, Winter, 30–60, Spring, Male , . 0.001) difference between the categories in the analysis. Values to three significant digits. 3. Average creatinine kinase; AST , Factor P 5 sampling presence captivity Nonnormal distribution for Box-Cox-transformed data. ABLE Box-Cox-transformed data with normal distribution. State during Parasite T Study site Coastal, CK a b *** Radiotransmitter Pre, Time in Sex Female, Season Summer,

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FIGURE 2. Of 77 blood samples collected from 41 southwest carpet pythons Morelia spilota imbricata, a greater proportion of blood samples collected from pythons during spring was positive for intracellular 2 hemoparasites (September–November; x 158.44, P,0.01), whereas a greater proportion of samples collected 2 at the time of radiotransmitter removal was positive (x 158.30, P,0.01) compared with preradiotransmitter and postradiotransmitter placement. The morphology of the hemoparasites seen in blood smears was consistent with Haemogregarina moreliea. The numbers shown in the table indicate sample size for each sample group.

effect of sex or the presence of a hemo- Leukocyte counts for M. s. imbricata gregarine parasite, but season, time in differed from ISIS values in a manner captivity, anesthesia, and the stage of consistent with heterophilia and lympho- radiotransmitter implantation did influence penia, which can occur with a stress blood parameters. response to capture and handling in Because of the physiologic flexibility of wild-caught pythons (Campbell, 2004, reptiles (e.g., poikilothermy, long-term 2006; Campbell and Ellis, 2007). Alterna- fasting, and rapid up-regulation of the tively, there may be differences in leuko- digestive system upon feeding; Bedford cyte identification between individual and Christian, 2001; Secor and Ott, 2007), pathologists. The high heterophil numbers reference intervals are difficult to establish by comparison with low lymphocytes, (Campbell and Ellis, 2007). The overall basophils, and monocytes/azurophils may average leukocyte counts in this study for suggest the latter. However, we also M. s. imbricata varied substantially from recorded higher CK and AST in M. s. ISIS reports for the species (all values imbricata compared with ISIS average presented as M. spilota). This discrepancy values, which may suggest muscle damage may reflect differences between subspe- associated with handling of wild-caught cies of Morelia that include varying diet individuals (assuming that the majority of and potential climatic influences such as samples used for the ISIS values are from rainfall, temperature, and humidity, but captive animals that may be habituated to also reflects that ISIS values do not follow human presence and handling). The the current Clinical and Laboratory Stan- differences between values presented in dards Institute (CLSI) guidelines for this study and reported ISIS values may calculation and presentation (C28-A3) therefore reflect real differences for wild- and should be updated (CLSI, 2008). caught animals.

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Although multiple regression analysis The state of pythons during sampling should simultaneously take multiple fac- (anesthetized or conscious) appeared to tors into consideration in its computation, influence some blood measures. Basophil we nevertheless urge caution in interpre- count and globulin concentration were tation of data where there are clearly elevated for anesthetized M. s. imbricata unequal samples collected for each factor (and the reverse for the A/G ratio). We and where multiple factors could influ- have little understanding of how anesthet- ence the results. Multiple regression ics influence hematologic and biochemistry analysis did not distinguish between sam- values in snakes (McDaniel et al., 1984). ples collected from male and female M. s. Further testing including perioperative, imbricata for either hematologic or plasma intraoperative, and postoperative stages of biochemical analytes tested. Morelia spi- anesthesia and surgery would be beneficial lota imbricata are capital breeders and will in deciphering the interpretation of anes- breed only every second year, at best thetic effects on specific hematologic and (Pearson, 2002). Over the 3-yr study, only plasma biochemical values. seven females were identified as gravid The time individuals were sampled in once over this time; only two of these were relation to radiotransmitter placement sampled while gravid (none was sampled appeared to affect several hematologic postoviposition) and they did not stand out and plasma biochemical values. However, as outliers. Therefore, to our knowledge, given that we opportunistically collected the majority of female M. s. imbricata blood samples, it was not possible to sampled were nonreproductive. Similarly, control for the seasonal spread of samples serum biochemistry does not vary between collected at each radiotransmitter implan- male and female black diamond water tation stage. As pythons are most active snakes (Nerodia rhombifera rhombifera; during spring and summer, they were McDaniel et al., 1984), a viviparous opportunistically captured at higher rates species. By contrast, other studies have during that time (20 of 35 samples were identified sex differences in plasma ana- collected in summer; Fig. 2), and the lytes for various species of snakes, with study concluded in spring of 2008 when elevations of plasma calcium, phosphorus, most radiotransmitters were removed (19 and protein concentrations in females of 20 removal-TM samples were collected during estrus and egg production (Des- in spring; Fig. 2). Hemoglobin, PCV, sauer, 1970; Campbell, 2004). It is there- RBC, calcium, albumin, and the A/G ratio fore likely that blood measures only differ were all highest at the pre-TM stage of between the sexes under specific repro- implantation. The majority of pre-TM ductive states. samples were collected during summer, We found significant seasonal differences and although season did not statistically for MCH, MCV, albumin, A/G ratio, and influence Hb, PCV, RBC, and calcium, phosphorous concentration. Wojtaszek (1992) the seasonal effect was significant for noted a significant decrease in RBC, PCV, albumin and A/G ratio. These parameters and Hb concentration in spring, the mating may reflect hydration or nutritional status. period for the grass snake (Natrix natrix A study by Lentini et al. (2011), specifi- natrix), and attributed the hematologic cally designed to test the inflammatory changes to a hormonal influence produced response of implanting radiotransmitters by decreased erythropoietic activity and in rattlesnakes (Sistrurus catenatus cate- from some RBC breakdown during winter. natus), found that 33% of implanted The reduction in albumin and phosphorous snakes had grade 3 or higher reactions concentrations in winter for M. s. imbricata (on the basis of histopathologic tissue is most likely associated with fasting examination) with extensive and active (Campbell, 2004). inflammation. The reaction to the implant

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was reflected with increases in counts of to the immunosuppressive effects of tes- heterophils and monocytes and a decrease tosterone (e.g., Amo et al., 2005). Increasing in globulin concentrations after 6 mo. numbers of invertebrate intermediate host Hemoglobin was significantly lower com- vectors during spring would potentially pared with snakes without implants (Len- drive an increase in the prevalence of the tini et al., 2011). Increases in these hemoparasites and hence the number of leukocyte counts were not as pronounced affected vertebrate (python) hosts (Huyghe in our study at the three transmitter et al., 2010). Furthermore, M. s. imbricata sampling stages; however, reduced Hb show greater movement and activity pat- concentration at the last two sampling terns during spring (Pearson et al., 2005), stages may indicate an anemic response, which may increase exposure to intermedi- similar to that found by Lentini et al. ate invertebrate hosts at this time. About (2011). 95% of samples were collected from Hemogregarine parasites (Phylum Api- pythons in spring at the time of radio- complexa, Family Haemogregorinidae) transmitter removal and there was a signif- require a vertebrate host (e.g., reptiles) icant difference in the presence of hemo- and an intermediate invertebrate host parasites during this sampling period. (e.g., ticks, mites, mosquitoes, or leeches) Although no conclusions can be drawn to complete their life cycle (Diethelm, regarding the relative influences of diet, 2006). Infection with hemoparasites is breeding activity, or stage of capture/sample often subclinical in reptiles; however, collection, this seasonal pattern in hemo- heavy burdens can result in anemia parasites warrants further investigation. (Diethelm, 2006). Hemogregarines are In conclusion, it is important to con- reasonably common among snake species, sider a variety of factors when interpret- including brown tree snakes (Boiga irre- ing hematologic and biochemical values gularis) and slatey-grey snakes (Stegonotus fordiagnosingdiseasesorevaluating cucullatus) from Queensland (Caudell health status of pythons. We found et al., 2002). In these two species, there compounded seasonal differences in was no significant difference between blood analytes and the time of sampling, infected (less than 10% of RBCs) and which may be related to feeding and noninfected snakes for Hb, PCV, albumin, hydration status. Although our data sug- calcium, phosphorus, protein, uric acid, gest an effect of radiotransmitter place- AST, and CK (Caudell et al., 2002). ment, we recommend that additional Similarly, we found no significant differ- research be undertaken. We hope to ences in any hematologic or biochemical provide the impetus to further improve analytes for M. s. imbricata according to the understanding and interpretation of hemogregarine parasite status. The inci- hematology and biochemical analytes in dence of hemoparasites in M. s. imbricata reptile species. did, however, vary significantly seasonally, with a greater prevalence in blood smears ACKNOWLEDGMENTS collected in spring. Changes in seasonal We thank all staff at Vetpath Laboratory prevalence of hemoparasites have been Services involved in analyzing samples for this observed for some lizards, where there is study. Thank you to all volunteers involved in greater hemoparasite prevalence toward helping in fieldwork and assisting GLB with the end of the mating season in spring sample collection and delivery of samples to (Amo et al., 2005; Huyghe et al., 2010), Vetpath Laboratory Services, in particular M. particularly in females (Amo et al., 2005). Connor, S. Dundas, J. Clarke, J. Bryant, and A. Bryant. This project was financially supported In comparison, males often show consis- by the Department of Environment and tent levels of hemoparasite prevalence Conservation, Murdoch University and the across seasons, which has been attributed Australian Research Council LP0562099.

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Journal of Wildlife Diseases jwdi-48-02-03.3d 19/1/12 04:59:04 12 Cust # 2010-08-206 BRYANT ET AL.—HEMATOLOGY AND BIOCHEMISTRY OF SOUTHWEST CARPET PYTHON 0

Powell (eds.). Eagle Mountain Publishing, Eagle data reference values. ISIS, Eagan, Minnesota, Mountain, Utah, pp. 257–268. on CD-ROM. STIRK, N., I. R. ALLENMAN, AND K. E. HARR. 2007. WOJTASZEK, J. S. 1992. Seasonal changes of circu- Circulating and inflammatory cells. In Infectious lating blood parameters in the grass snake diseases and pathology of reptiles, E. R. Natrix natrix natrix. Comparative Biochemistry Jacobson (ed.). CRC Press: Taylor & Francis and Physiology Part A: Physiology 103: 461– Group, Boca Raton, Florida, pp. 167–189. 471. TEARE, J. A. (ed.). 2002. Reference ranges for physiological values in captive wildlife: Interna- Submitted for publication 4 August 2010. tional Species Information System physiological Accepted 1 October 2011.

Journal of Wildlife Diseases jwdi-48-02-03.3d 19/1/12 04:59:04 13 Cust # 2010-08-206 5. DOES BODY SIZE INFLUENCE THERMAL BIOLOGY AND

DIET OF A PYTHON (MORELIA SPILOTA IMBRICATA)?

Dietary differences between large and small pythons were investigated by looking at differences in their thermal biology. This study has been published as an on-line early edition manuscript, with an anticipated print date in 2012 in Austral Ecology.

AUTHOR CONTRIBUTION

G. L. Bryant: Initiated the research idea, collected the scat samples, prepared the scat samples for analysis, collected the temperature and behavioural data, entered the data, analysed the data, and wrote the paper.

P. de Tores: Provided funding of dietary analysis (scat investigation of mammalian hair contained within the python scats), provided editorial comments to a draft version of the manuscript.

K. A. Warren: Provided editorial comments to a draft version of the manuscript.

P. A. Fleming: Provided guidance for statistical analyses for this research and provided editorial comments to draft versions of the manuscript.

Austral Ecology (2011) ••, ••–••

Does body size inﬂuence thermal biology and diet of a

python (Morelia spilota imbricata)?aec_2318 1..9

GILLIAN L. BRYANT,1*PAULJ.de TORES,2 KRISTIN A. WARREN1 AND PATRICIA A. FLEMING1 1School of Veterinary & Biomedical Sciences, Murdoch University, Murdoch,WA 6150, Australia (Email: [email protected]), and 2Department of Environment and Conservation,Western Australian Wildlife Research Centre,Wanneroo,Western Australia, Australia

Abstract Current theory predicts that larger-bodied snakes not only consume larger prey (compared with smaller individuals), but may also have a different range of prey available to them due to their thermal biology. It has been argued that smaller individuals, with lower thermal inertia (i.e. faster cooling rates at nightfall when air temperature falls and basking opportunities are limited), may be thermally restricted to foraging and hunting during the day on diurnally active prey, and have reduced capacity to hunt crepuscular and nocturnal prey species. This predictive theory was investigated by way of dietary analysis, assessment of thermal biology and thermoregulation behaviour in an ambush forager, the south-west carpet python (Morelia spilota imbricata, Pythonidae). Eighty-seven scats were collected from 34 individual pythons over a 3-year radiotelemetry monitoring study. As predicted by gape size limitation, larger pythons took larger prey; however, 65% of prey items of small pythons were represented by nocturnally active, small mammals, a larger proportion than present in larger snakes. Several measures of thermal biology (absolute body temperature, thermal differential of body temperature to air temperature, maximum hourly heating and cooling rates) were not strongly affected by python body mass. Additionally, body temperature was only inﬂuenced by the behavioural choice of microhabitat selection and was not affected by python body size or position, suggesting that these behavioural choices do not allow smaller pythons to vastly increase their temporal foraging window. By coupling dietary analysis, measures of body temperature and behavioural observations of free-ranging animals, we conclude that, contrary to theoretical predictions, a small body size does not thermally restrict the temporal window for ambush foraging in M. s. imbricata. An ontogenetic or size-determined switch from ambush feeding to actively foraging on slower prey would account for the differences in prey taken by these animals. The concept of altered foraging behaviour warrants further investigation in this species.

Key words: ambush predator, body temperature, dietary analysis, foraging, thermoregulation.

INTRODUCTION and capture success is increased when snakes have higher body temperatures (Ayers & Shine 1997), and Most snake species are unable to dismember their temperature therefore plays a significant role in forag- food, and consequently swallow their prey whole. ing strategies of snakes. Ectotherms behaviourally Snakes are therefore ‘gape-limited predators’, where regulate their body temperature by moving between their maximum ingestible prey size is limited by their sun and shade or hot and cold microenvironments to head size (Forsman & Lindell 1993; Shine & Thomas alter heat flux, by modifying posture to alter surface 2005). It is not surprising, therefore, that many studies areas exposed to heat sources or sinks, and by regulat- reveal an ontogenetic shift in the size of prey consumed ing activity times (Huey 1974). Such behaviour allows as snake body size increases (Mushinsky et al. 1982; snakes to achieve an elevated body temperature which Chiszar et al. 1986; Shine et al. 1998; Rodríguez- will optimize feeding success or physiological func- Robles et al. 1999) which is largely determined by tions such as digestion. physical size restriction. However, there are also indi- Thermal inertia is affected by body size, and there- rect effects of snake body size on prey type selection, fore in a varying environment, body size may influence since body size plays a significant role in a snake’s a snake’s ability to maintain a stable, optimal body ability to capture and ingest prey. temperature. For example, maximum rates of heating Body size influences a complex interplay between and cooling are inversely correlated with body mass thermoregulation and feeding capacity. Prey detection and length in the southern African python (Python natalensis), with small pythons heating and cooling *Corresponding author. twice as quickly as large pythons (Alexander 2007). Accepted for publication October 2011. Theoretically, larger snakes with a higher thermal

© 2011 The Authors doi:10.1111/j.1442-9993.2011.02318.x Journal compilation © 2011 Ecological Society of Australia 2 G.L.BRYANTET AL. inertia (Slip & Shine 1988c; Pearson et al. 2003) can site (one at each of the two coastal areas and two loggers therefore retain more heat in a cooling environment within the jarrah forest positioned approximately 70 km (e.g. at nightfall when air temperatures decline and apart). HOBO loggers were attached to a vertical stake and basking opportunities are absent) and could therefore positioned approximately 1 m from the ground in a have an increased temporal window for feeding, with southwest-facing direction and covered with a cardboard shelter to prevent rain damaging the device or direct solar greater capacity to hunt on crepuscular and nocturnal radiation. Daily air temperature for the jarrah forest averaged prey species. By contrast, it has been argued that 21.5 Ϯ 0.20°C in summer and 9.49 Ϯ 0.12°C in winter, smaller individuals (with lower thermal inertia and and coastal woodland averaged 22.2 Ϯ 1.72°C in summer therefore faster cooling rates at the end of the day) may and 12.4 Ϯ 0.26°C in winter (averaged over the 3 years be restricted to feeding during the day on diurnally of the study; 2006–2008). The jarrah forest experienced active prey (Slip & Shine 1988a,c; Ayers & Shine colder air temperature in winter months (June to September) 1997). compared with coastal woodland, but there was no difference Body size therefore affects snake foraging behaviour in air temperature for the rest of the year (Bryant et al. and thermal biology and should also influence the 2011). type of prey taken in addition to prey size. We investigated the effects of body size upon the foraging ecology of the south-west carpet python (Morelia Study animal spilota imbricata, Pythonidae). A long-term radiotracking study enabled concurrent recording of diet, ther- Forty-six carpet pythons were radiotracked over a 3-year moregulatory behaviour and body temperature for period. Body mass (Mb) of the 46 pythons studied averaged free-ranging individuals of a range of body sizes. We 1030 Ϯ 564 g (range: 136–3730 g) and snout-to-vent length Ϯ tested the theoretical prediction that body size influ- (SVL) averaged 151 28.3 cm (range: 90.0–223 cm). ences not only prey size, but also the type of prey Where possible, individual body mass values were used in statistical analyses. For graphical representation and for the taken. More specifically, we tested the hypothesis that purposes of testing the hypothesis that smaller pythons dem- small individuals are thermally restricted in their onstrated different diet selectivity and thermal biology, two ability to take nocturnal prey. size classes were distinguished: small pythons (<1000 g; average 599 Ϯ 229 g) and large pythons (>1000 g; average 1480 Ϯ 448 g). The thermal biology of these animals was examined in the METHODS following two ways:

Study sites and ambient conditions 1. Temperature-sensitive radiotransmitter data Pythons were opportunistically captured from coastal woodland and jarrah forest in Western Australia. The Temperature-sensitive radiotransmitters (Holohil Systems coastal woodland (Martin’s Tank, Yalgorup National Park Ltd, Canada) were surgically implanted into the coelomic 32°51′S, 115°40′E, and Leschenault Peninsula Conserva- cavity using a surgical technique under general anaesthesia tion Park, 33°26′S, 115°41′E 40 km further south) is on (Bryant et al. 2010). After recovery from surgery, pythons the Swan Coastal Plain where sandy soils are dominated were released back to their point of capture. Pythons were by Banksia spp. and Agonis flexuosa woodlands, with subsequently radiotracked weekly (or fortnightly) on foot emergent tuart (Eucalyptus gomphocephala) trees (Trudgen using a three-element Yagi aerial (Sirtrack Ltd, Havelock 1984; CALM 1995, 1998). Animals sourced from the North, New Zealand) and receiver (R-1000 Telemetry jarrah forest were located within State Forest surrounding Receiver; Communications Specialists, Inc., USA). Most the township of Dwellingup (32°43′S, 116°4′E). This area pythons did not flee when approached, allowing direct lies within the northern jarrah forest, where Archaean behavioural observation and, where possible, capture to granite overlies metamorphic rocks capped by an extensive, measure SVL and Mb. but dissected lateritic duricrust (Churchward & Dimmock Each radiotransmitter was calibrated to a unique tempera- 1989). The vegetation comprises of jarrah (Eucalyptus ture calibration curve prior to surgical implantation (and marginata) and marri (Corymbia calophylla) trees with a confirmed after surgical removal). Python body temperature mosaic of understory woodland plant species. Both study could therefore be calculated and directly compared with air sites experience Mediterranean climates with cool, wet temperature (to the nearest hour, see details for recording winters and warm, dry summers. The jarrah forest has above) and behavioural observations each time the python slightly elevated rainfall (700–1100 mm per annum), com- was located through radiotelemetry. Animals were catego- pared with the coastal woodland (600–1000 mm per rized as located within one of five microhabitats: (i) hollow annum). log – within a hollow log on the ground; (ii) ground cover –

Environmental air temperatures (Ta; °C) were logged where the python would lay on ground debris exposed to hourly using temperature and humidity loggers (HOBO H8 potential sunlight radiation (e.g. leafy matter or low grass Pro Series, H08-032-08; Onset Computer Corporation, <5 cm in height); (iii) vegetation cover – where the python Bourne, MA, USA) placed at a location central in each would have Ն30% of its body under or in piles of dead doi:10.1111/j.1442-9993.2011.02318.x © 2011 The Authors Journal compilation © 2011 Ecological Society of Australia PYTHON SIZE INFLUENCES PREY SIZE 3

branches/sticks or vegetation (e.g. bushes) or was lying on maximum hourly DTb) was extracted and then averaged for top of logs or fallen branches (<2 m in height), with reduced each month. exposure to sunlight radiation compared with ground cover; Maximum cooling rate – daily minimum DTb (i.e. daily (iv) tree branch – resting on a tree branch elevated above the maximum cooling rate) was extracted and then averaged for ground; or (v) tree hollow – radiotracked to a tree and either each month. observed within a hollow or if the snake could not be visu- Multiple regression analyses were performed for these alized, the strength of the radio signal indicated its presence three (dependent) factors. Independent factors for the analy- within a hollow. Python body position was scored as: (i) ses included individual python body mass (Log10 - Mb), stretched; (ii) very loose coil; (iii) loose coil; (iv) coil; or (v) study site, month, sex and the unique python ID code (as a tight coil. If the python was sequestered in a tree hollow fixed factor). or hollow log and therefore not directly observed, it was assumed to be in a tight coil. We used 5 ¥ 2 contingency Pearson’s chi-squared analyses Diet analysis to compare the likelihood of observing small and large pythons in each of the five microhabitats and in each of the Thirty-five scats were collected for dietary analysis from five body position categories. Observations were compared pythons when they were initially brought into captivity with expected values calculated assuming an equal propor- to have radiotransmitters implanted, and 52 scats were tion of pythons observed in each category was small or large collected over the course of the study when radio- individuals. tracking animals in the field – 45 through gentle palpation Multiple regression was used to determine what factors of python’s rectum during field telemetry work, one influenced python body temperature (dependent variable). was found lying adjacent to an animal’s known refuge and Independent factors included python body mass (Log10 - six were collected opportunistically when found in close Mb; average value for each individual over all captures), study association to expelled radiotransmitters (Bryant et al. site (coastal woodland or jarrah forest, recorded as 1 or 0), 2010). In total, these 87 samples enabled the diet assess- month (using a sine function of the calendar month, with ment for 34 individual pythons. The months were pooled maximum values during summer and minimum values over into seasons for graphical presentation and analyses: winter), sex (male or female), the unique python identi- summer (December to February), autumn (March to fication (ID) code (as a fixed factor to account for mul- May), winter (June to August), and spring (September to tiple measures made on each individual), air temperature November). (recorded for the nearest hour to each observation), micro- Each scat was carefully washed with gentle running water habitat (one of five categories) and body position (one of five through a series of stainless steel or brass mesh sieves of three categories). aperture sizes (2.0 mm, 250 mm and 1.0 mm), dried and then sorted (macroscopically) into bird, reptile or mammal samples. Mammal species were identified by microscopic 2. Temperature data loggers analysis of hair samples (Triggs & Brunner 2002), reptiles by the size of undigested scales and limbs, and birds were iden-

For 29 individuals that were large enough (Mb > 409 g and tified by the shape and size of undigested beaks, skulls, feet SVL > 112 cm), one or two additional temperature data and feather colour. loggers (Thermochron iButtons DS1922L-F5; Maxim Inte- As pythons eat their prey whole, prey items found grated Products, Sunnyvale, CA, USA) were implanted within the scats represent a whole animal. Therefore, where with the radiotransmitters. The iButtons were taped to the two or more species were found in a single scat, each radiotransmitter and the unit was dip-coated with three prey item was confidently classified as representing a coats of inert wax (paraffin/elvax coating 130-0004-00; Mini- whole individual animal, eaten sequentially. However, Mitter Respironics, OR, USA). The implanted radiotrans- where successive scats were collected from an individual mitter and iButton package was <5% of the individual’s body (i.e. 1 or 2 weeks between sample times) and analysis iden- mass. The two (or one for smaller animals or individuals tified the same prey species present in the scats, only the captured close to the end of the study) iButtons recorded first record of the prey species was used for analysis, since python body temperature hourly; the second was set with a it could not be assumed that this would represent a new delayed start to commence logging when the first had prey item. reached memory capacity. Individuals were monitored for We used a 4 ¥ 2 contingency Pearson’s chi-squared body temperature using iButtons over 6.38 Ϯ 3.20 months analysis to compare the proportions of scats collected over (range 2 to 23 months). each season and the number of times pythons were cap- Three aspects of thermal biology were calculated from the tured, comparing observed numbers with expected values iButton data for each python: calculated assuming an equal number of scats each season. Thermal differential – hourly records of body tempe- Similarly, the proportions of each prey type consumed each rature (Tb) for each individual were compared with air tem- season were compared by a 4 ¥ 4 contingency chi-squared perature (Ta) recorded simultaneously (as outlined above). analysis with expected values calculated assuming an equal

The hourly thermal differential (Tb - Ta) was averaged for proportion of each prey type. The four prey type classes each month. were: (i) small mammals (range 18 to 300 g); (ii) large Maximum heating rate – hourly changes in body tem- mammals (range 1 to 4.5 kg); (iii) reptiles (principally perature (DTb) were calculated as DTb = Tb(Houri + 1) - bobtail lizards, Tiliqua rugosa); and (iv) birds (for a

Tb(Houri). For each day, the maximum heating rate (i.e. daily full species list, including average adult body mass, see

Appendix S1). Finally, we used a 4 ¥ 2 contingency chi- the size of the prey consumed (y = 1.25x - 1.45, squared analysis to determine if small pythons (<1000 g, R2 = 0.197; Fig. 2). n = 18) consume different types of prey from large pythons Small mammals represented in the pythons’ > ( 1000 g, n = 16); expected values were calculated assum- diet included carnivorous marsupials (Sminthopsis ing that the proportion of each prey category was the same griseoventer, Sminthopsis spp. and Antechinus flavipes) between small and large pythons. as well as introduced eutherian rodents (Mus muscu- Multiple regression analysis was performed to investigate factors that influenced prey body size (dependent lus and Rattus rattus). Small mammal prey species variable; average body mass of adults reported in the litera- were all nocturnally active species but may exhibit ture; Appendix S1). Independent variables included indi- some crepuscular activity. The large mammals con- vidual python body mass (Log10 - Mb), study site, month, sumed were mostly nocturnally active and included python sex and the individual python ID code (as a fixed two possum species (Pseudocheirus occidentalis and factor). Trichosurus vulpecula), a macropod (Bettongia penicil- Data are presented as means Ϯ 1 SD throughout. lata ogilbyi) and introduced eutherian species (Oryc- tolagus cuniculus and Felis catus). Diurnally active prey consumed by large and small pythons included both reptiles (principally Tiliqua rugosa; other skink species RESULTS were taken but were not identifiable to species level from remains) and bird species. The array of prey type categories differed significantly between small 2 Diet analysis and large pythons (c 3 = 17.14, P < 0.001; Fig. 3). Small mammals made up the majority (65%) of Because of the distance (approximately 150 km) and small python diets. Large pythons consumed a wider habitat differences between jarrah forest and the coastal woodland, prey data are indicated for these two areas separately (Appendix S1). A greater number of python scats were obtained for warmer months (summer, spring and autumn) and very few for winter months when the snakes were less frequently captured 2 (c 3 = 23.64, P < 0.001). Despite these differences in sample size, there were no differences in the proportions of small mammal, large mammal, reptile or bird 2 prey consumed across the four seasons (c 9 = 11.05, P = 0.272; Fig. 1). Python body mass was the strongest influence in determining the size of prey (reported average adult mass) consumed by pythons (MR analysis: t72 = 3.58, P < 0.001); study site, month, sex or python ID did not influence the mass of prey consumed (all Fig. 2. Comparison between prey body mass (reported

P > 0.05). As python body mass increased, so did Log10 adult average) and individual carpet python body mass.

Fig. 1. Seasonal changes in the type of prey consumed by Fig. 3. Differences in the type of prey consumed by small Morelia spilota imbricata. and large Morelia spilota imbricata pythons. doi:10.1111/j.1442-9993.2011.02318.x © 2011 The Authors Journal compilation © 2011 Ecological Society of Australia PYTHON SIZE INFLUENCES PREY SIZE 5

Table 1. Results of multiple regression analyses testing for the effect of environmental, individual and behavioural factors on four measures of thermal biology of south-west carpet pythons

Temperature-sensitive radiotransmitters iButton temperature loggers

Thermal differential

Body temperature (Tb) (Tb - Ta) Heating DTb Cooling DTb

Variable t P t P tPtP

Body mass (Log10 - Mb) 0.965 0.336 2.24 0.025 1.40 0.166 -1.33 0.186 Study site (coastal -1.76 0.080 -3.89 <0.001 2.53 0.012 -2.41 0.017 woodland/jarrah forest) Month (sine values of 0.156 0.876 -3.38 <0.001 -6.30 <0.001 2.34 0.020 calendar month) Sex -1.52 0.129 3.38 <0.001 2.48 0.014 -2.23 0.027 Python ID code 1.92 0.057 1.55 0.122 -3.13 0.002 3.51 <0.001

Air temperature (Ta, °C) 6.18 <0.001 – – – – – – Microhabitat category -2.09 0.038 – – – – – – Body position category 1.01 0.315 – – – – – –

Table gives t-values and associated probabilities (P). –, independent variables not included in analysis. range of prey size and type; the greatest proportion of sensitive radiotransmitters) was influenced by their the diets of large pythons was made up of large behavioural choice of microhabitat (t189 =-2.09, P = mammals (41%). 0.038) (and air temperature; t189 = 6.18, P < 0.001), body temperature was not affected by the size of the Thermoregulation python (body mass), body position (Fig. 5C), study site, month, sex or python ID (all P > 0.05; Table 1). Thermal differential – heavier pythons were able to Pythons were warmest if they were located in hollow logs or ground cover and cooler when they were maintain a larger thermal differential (t229 = 2.24, P = 0.025). However, by contrast with other factors, found within tree hollows (Fig. 5A). There was a sig- the effect of body mass was small: the thermal differ- nificant difference in the microhabitat use between 2 < ential was more strongly influenced by study site, small and large pythons (c 4 = 33.14, P 0.001; month and sex (all P < 0.001), but not by individual Fig. 5B).There was no difference between the propor- differences (python ID; Table 1). Python body mass tion of observations of small and large pythons in 2 the different body positions (c 4 = 6.17, P = 0.187; accounted for only 4% of the difference in Tb - Ta (y = 1.75x - 2.57, R2 = 0.044; Fig. 4A). Fig. 5D). Heating rate – the maximum heating rate was influenced by study site, month, python sex and ID (Table 1). When all the factors were taken into DISCUSSION account, python body mass was not a statistically significant influence (t239 = 1.39, P = 0.166). The equa- An ontogenetic shift in prey size for snakes is predicted tion describing heating rate compared with body mass based on their physical restrictions on prey ingestion. (y =-3.26x + 22.79, R2 = 0.032; Fig. 4B) indicates However, a number of studies have also reported an that only 3% of the variation in heating rate was attrib- ontogenetic shift in prey type (e.g. fish consumed by uted to body mass. juvenile colubrids but amphibians as adults; Kjaer- Cooling rate – the maximum cooling rate was influ- gaard 1981; Mushinsky et al. 1982; Chiszar et al. 1986, enced by study site, month, python sex and ID lizards consumed by juvenile vipers and death adders (Table 1). Python body mass was not a statistically but mammals as adults (Shine 1980, but see some significant influence on cooling rates (t239 =-1.33, exceptions, e.g. Vincent et al. 2004). Given they are P = 0.186). Only 4% of the variation in cooling rate capable of consuming larger prey, the range of prey was attributed to body mass (y =-2.10x + 12.61, available to larger individuals is greater than that avail- R2 = 0.042; Fig. 4C). able to smaller individuals (Arnold 1993). Thermoregulatory behaviour – even though python While some degree of prey switching may therefore body temperature (as measured through data collected simply reflect prey availability, it has also been sug- during field observation by implanted temperature- gested that switching prey type may reflect thermal

A. Thermal differential reptiles and birds. The diet of adult pythons (deter- 8 mined through faecal sampling of radiotracked individuals) also included principally mammals (crep- 6 uscular and nocturnal species) and the occasional 4 bird, but no reptiles.The absence of reptiles (diurnally

(°C) active prey) in the diet of adults led the authors to a 2 infer a shift in the time of day juveniles and adults fed.

− T This was expanded in a subsequent thermoregulation b 0

T study, where larger-bodied snakes resting in a coiled

–2 body posture demonstrated increased thermal inertia and therefore slower cooling rates; the authors argued –4 that this would allow larger individuals to ambush forage on crepuscular and nocturnally active prey B. Maximum heating (Slip & Shine 1988c). In contrast, it was explained 25 that small individuals (i.e. juveniles), which could not 20 maintain sufﬁciently elevated body temperatures to allow them to ambush hunt on nocturnal species, are 15 therefore more likely to forage on diurnally active (°C)

b species (e.g. reptiles).

T 10 Our research is generally consistent with Slip and Δ Shine’s (1988a) observations for M. s. spilota, with 5 small M. s. imbricata consuming a large proportion of 0 small mammals (which made up 65% of their diet) 2.6 2.8 3.0 3.2 3.4 and larger individuals consuming a broader range of sizes and type of prey. However, smaller M. s. im- C. Maximum cooling bricata were clearly not restricted in terms of their 0 temporal window from foraging on crepuscular and nocturnal prey, since the majority of prey items taken by small pythons were nocturnally active species. –5 Pearson et al. (2002) also found that both juveniles and adult male pythons mainly consumed noctur- (°C)

b nally active prey including M. musculus and spiny- T tailed geckos (Strophurus spinigerus). Δ –10 The selection for mammalian (nocturnally active) rather than reptilian (diurnally active) prey observed in the present study does not simply reﬂect overall prey –15 availability, since data available for the coastal site Log python body mass 10 indicated that although reptiles made up only 14% of Fig. 4. Relationship between python body mass and three small python diet, an abundance of 21 reptile species measures of thermal biology. The measures shown are the were present (Wentzel 2011). About 91% of these (A) thermal differential (difference of python body tempera- reptiles weighed <20 g (Wentzel 2011) and were there- ture above air temperature), (B) maximum hourly heating fore available as prey to small pythons (although fos- rates and (C) maximum cooling rate. Each column of data sorial species may not have been). Prey consumed points represents monthly values for an individual python (separated by their overall average body mass measures). therefore clearly did not simply reﬂect availability. In addition to the activity patterns of prey species taken, we also examined the thermal biology of M. s. imbricata to test the hypothesis that smaller indi- biology. It was suggested that smaller snakes, with a viduals may be thermally restricted in their temporal lower thermal inertia, would cool quicker in the eve- hunting patterns. Our data indicate little difference in nings and therefore have a shorter temporal window three measures of thermal biology (thermal differen- for foraging on crepuscular and nocturnal prey (Slip & tial, maximum heating and cooling rates) due to

Shine 1988a,c; Ayers & Shine 1997). Slip and Shine body mass (Mb accounted for only 3% or 4% of the (1988a) examined stomach contents (museum speci- variability in these values). Similarly, correlation mens) of juvenile Morelia spilota spilota pythons, a analysis of adult and juvenile animals of the same subspecies to our study species and found that small, species at Garden Island and Dryandra Woodland in nocturnal mammals comprised 70% of their diet; the Western Australia showed no difference between these remaining portion of the diet was diurnally active size classes in terms of the degree to which body doi:10.1111/j.1442-9993.2011.02318.x © 2011 The Authors Journal compilation © 2011 Ecological Society of Australia PYTHON SIZE INFLUENCES PREY SIZE 7

40 A. 40 C. 35 35

30 30

25 25

20 20

15 15 Body temperature (°C)

10 10

35 35 B. D. 30 30

25 25 Small pythons Large pythons 20 20

15 15

10 10 Total observations (%)

5 5

0 0 Hollow log Ground cover Vegetation Tree branch Tree hollow Stretched Very loose Loose coil Coil Tight coil cover coil Microhabitat Body position

Fig. 5. The proportion of observations (B,D) and average (ϮSD) body temperature (A,C) of small (white) and large (black) pythons observed in each of ﬁve microhabitat categories (left-hand panel) and in ﬁve body positions (right-hand panel).

temperatures were related to air temperatures temperature), and may critically extend their noctur- (Pearson et al. 2003). Although there were differences nal foraging window allowing the capture of crepuscu- in the thermoregulatory behaviour of small and large lar or nocturnally active mammals (Christian et al. individuals in the present study, these differences were 2006). Additionally, differences in thermal inertia may unlikely to counter the potentially lower thermal only operate over a matter of minutes, not hours, inertia of small individuals (since smaller animals were following sunset (Christian et al. 2006). Individual apparently selecting microhabitats that resulted in python behaviour coupled with the stochastic nature cooler, not warmer, body temperatures, which is con- of ambush foraging (driven by prey availability, sea- trary to the prediction that smaller pythons may use sonal activity patterns, etc.) may make the detection of behavioural thermoregulation to elevate their body differences between small and large snakes extremely temperature). We therefore found little evidence for a difficult. thermal limitation restricting small individuals from If there is no evidence for a thermal restriction feeding on crepuscular or nocturnal prey. on hunting activities, and prey selection does not There is a considerable range of opportunities for directly reflect prey availability, what alternative ectotherms to regulate their body temperature within explanations may be offered? It is possible that the their environment (e.g. through microhabitat selection lack of reptiles in the diet of adult M. s. spilota (Slip or body position), which may increase or stabilize body & Shine 1988a) reflects that larger snakes prefer temperature, minimizing the effects of thermal inertia larger or more active prey, dropping small or less due to body size alone (Christian et al. 2006).This was active prey from their diet completely (Plummer & clearly observed in the wide range of body tempera- Goy 1984; Shine 1987; Arnold 1993; Shine et al. tures achieved by both small and large pythons located 1998). While active foraging snakes move about in in a range of microhabitats. Behavioural choices search of suitable prey, ambush predators sit and wait may therefore allow small individuals to regulate in an area where prey are likely to pass (e.g. adjacent and maintain their thermal energy (i.e. higher body to an animal passage; Slip & Shine 1988b,c; Ayers &

Shine 1997). Ambush predators are therefore more Australia, Department of Environment and Conserva- likely to feed on fast-moving endothermic prey tion and Murdoch University. such as birds and mammals that have field metabolic rates approximately 10–30 times higher than ectothermic reptiles, since there is a higher likelihood REFERENCES of these prey passing the snake’s ambush position (Shine 1980; Huey & Pianka 1981; Arnold 1993; Alexander G. (2007) Thermal biology of the Southern African Ayers & Shine 1997; Nagy et al. 1999). Small reptiles python (Python natalensis): does temperature limit its distri- may therefore be less likely to be detected and bution? In: Biology of the Boas and Pythons (edsR.W.Hend- captured. Alternatively, the consumption of parti- erson & R. Powell) pp. 50–75. Eagle Mountain Publishing, cular prey types (e.g. Tiliqua rugosa skinks or Pseudo- Utah. cheirus occidentalis possums) may suggest that larger Arnold S. J. (1993) Foraging theory and prey-size-predator- pythons are selecting slower prey (compared to small size relations in snakes. In: Snakes: Ecology and Behaviour (eds R. A. Seigel & J.T. Collins) pp. 87–115. McGraw-Hill, mammal prey taken by smaller pythons) which New York. may reflect size-dependent locomotory patterns. Ayers D. Y. & Shine R. (1997) Thermal influences on foraging The potential for different feeding strategies ability: body size, posture and cooling rate of an ambush between small and large pythons warrants further predator, the python Morelia spilota. Funct. Ecol. 11, 342– investigation. 7. We conclude that while body size directly affects Bryant G. L., Eden P., De Tores P. J. & Warren K. S. (2010) the size of prey that can be taken, the link with selec- Improved procedure for implanting radiotransmitters in the coelomic cavity of snakes. Aust.Vet.J. 88, 443–8. tion of prey type is less obvious. The significant pro- Bryant G. L., Dundas S. J. & Fleming P. A. (2011) Tree hollows portion of nocturnally active mammals found in the are of conservation importance for a Near-Threatened diet of smaller pythons (both the present study and python species. J. Zool. On-line 27 September 2011, doi:10. that of Slip & Shine 1988a) suggests that small 1111/j.1469-7998.2011.00852.x. pythons are certainly able to ambush nocturnally CALM (1995) Yalgorup National Park: Management Plan 1995– active prey. There is also little support for an effect of 2005.Management Plan No.29. Department of Conservation body size on temperature regulation of M. s. imbri- and Land Management for The National Parks and Nature Conservation Authority, Perth. cata, and therefore little chance that these animals CALM (1998) Leschenault Peninsula Management Plan 1998– are differentially influenced in terms of thermal 2008.Management Plan No.38. Department of Conservation biology. Small body size does not thermally restrict and Land Management for The National Parks and Nature the temporal window for ambush foraging in the Conservation Authority, Perth. south-west carpet python. The ontogenetic shift in Chiszar D., Radcliffe C. & Feiler F. (1986) Trailing behavior in prey type consumed by M. s. imbricata therefore does banded rock rattlesnakes (Crotalus lepidus klauberi)and prairie rattlesnakes (C. viridis viridis). J. Comp. Psychol. 100, not appear to reflect their thermal biology or prey 368–71. availability, but possibly a shift in foraging behaviour Christian K. A., Tracy C. R. & Tracy C. R. (2006) Evaluating in this species. Future studies could investigate thermoregulation in reptiles: an appropriate null model. potential physical restrictions of strike rate and prey Am. Nat. 168, 421–30. speed for larger snakes. Churchward H. M. & Dimmock G. M. (1989) The soils and landforms of the northern jarrah forest. In: The Jarrah Forest (eds B. Dell, J. J. Havel & N. Malajczuk) pp. 13–22. Kluwer Academic Publishers, London. Clarke J. (2010) Translocation outcomes for the western ringtail ACKNOWLEDGEMENTS possum (Pseudocheirus occidentalis) in the presence of the common brushtail possum (Trichosurus vulpecula): health, sur- Our thanks to G. Storey (Scats About) for the identi- vivorship and habitat use. PhD thesis, School of Veterinary fication of mammalian hair, J. Angus (Department of and Biomedical Science, Murdoch University. Environment and Conservation) for advice on this Forsman A. & Lindell L. E. (1993) The advantage of a big head: process, M. J. Craig and J. J.Wentzel for assistance with swallowing performance in adders, Vipera berus. Funct. Ecol. 7, 183–9. bird and reptile identification. We are grateful to Huey R. B. (1974) Behavioral thermoregulation in lizards: volunteers who helped with radiotelemetry and data importance of associated costs. Science 184, 1001–3. collection, particularly S. Dundas, M. Connor, J. Huey R. B. & Pianka E. R. (1981) Ecological consequences of Connor, J. Clarke, S. Dawson, A. Nowicki and T. foraging mode. Ecology 62, 991–9. Moore. This project was approved by the Animal Johnstone R. E. & Storr G. M. (1998) Handbook of Western Ethics Committees of Murdoch University (W2028/ Australian Birds, Vol. 1. Non-Passerines. Western Australian Museum, Perth. 07) and Department of Environment and Conserva- Kjaergaard J. (1981) A method for examination of stomach tion, Western Australia (DEC AEC/55/2006 and content in live snakes and some information on feeding DEC AEC54/2006) and was financially supported by habits in common viper (Vipera berus) in Denmark. Natura the Invasive Animal Cooperative Research Council Jutlandica 19, 45–8. doi:10.1111/j.1442-9993.2011.02318.x © 2011 The Authors Journal compilation © 2011 Ecological Society of Australia PYTHON SIZE INFLUENCES PREY SIZE 9

Mushinsky H. R., Hebrard J. J. & Vodopich D. S. (1982) giant tropical snake, Python reticulatus. Funct. Ecol. 12, 248– Ontogeny of water snake foraging ecology. Ecology 63, 58. 1624–9. Slip D. J. & Shine R. (1988a) Feeding habits of the diamond Nagy K. A., Girard I. A. & Brown T. K. (1999) Energetics of python, Morelia s. spilota: ambush predation by a Boid free-ranging mammals, reptiles, and birds. Annu. Rev. Nutr. snake. J. Herpetol. 22, 323–30. 19, 247–77. Slip D. J. & Shine R. (1988b) Thermophilic response to feeding Nowicki A. (2007) Analysis of Capture Data: A Case Study of the diamond python, Morelia s. spilota (Serpentes, Using Program MARK for Analysis of Brushtail Possum Boidae). Comp. Biochem. Physiol., Part A Mol. Integr. Physiol. Trapping Data and Its Relevance to Conservation Manage- 89, 645–50. ment of the Western Ringtail Possum. Bachelor of Science Slip D. J. & Shine R. (1988c) Thermoregulation of free-ranging Honours, School of Biological Science, Murdoch Univer- diamond pythons, Morelia spilota (Serpentes, Boidae). sity, Perth. Copeia 4, 984–95. Pearson D., Shine R. & How R. (2002) Sex-specific niche Triggs B. & Brunner H. (2002) Hair ID. Ecobyte Pty Ltd. partitioning and sexual size dimorphism in Australian CSIRO Publishing, Collingwood. pythons (Morelia spilota imbricata). Biol. J. Linn. Soc. 77, Trudgen M. E. (1984) The Leschenault Peninsula–afloraand 113–25. vegetation survey with an analysis of its conservation value and Pearson D., Shine R. & Williams A. (2003) Thermal biology of appropriate uses. Report prepared for The West Australian large snakes in cool climates: a radio-telemetric study of Department of Conservation and Land Management, carpet pythons (Morelia spilota imbricata) in south-western Perth, Western Australia. Australia. J.Therm. Biol. 28, 117–31. Van Dyck S. & Strahan R., eds (2008) The Mammals of Australia. Plummer M. V. & Goy J. M. (1984) Ontogenetic dietary shift of Reed New Holland, Sydney. water snakes (Nerodia rhombifera) in a fish hatchery. Copeia Vincent S. E., Herrel A. & Irschick D. J. (2004) Ontogeny 1984, 550–2. of intersexual head shape and prey selection in the Rodríguez-Robles J. A., Bell C. J. & Greene H. W. (1999) Gape pitviper Agkistrodon piscivorus. Biol. J. Linn. Soc. 81, 151– size and evolution of diet in snakes: feeding ecology of 9. erycine boas. J. Zool. 248, 49–58. Wentzel J. J. (2011) Is tuart (Eucalyptus gomphocephala) decline Shine R. (1980) Ecology of the Australian death adder Acantho- detrimental for fauna? PhD thesis, Murdoch University, phis antarcticus (Elapidae): evidence for convergence with School of Biological Sciences and Biotechnology. Viperidae. Herpetologica 36, 281–9. Shine R. (1987) Ecological ramifications of prey size: food habits and reproductive biology of Australian copperhead snakes (Austrelaps, Elapidae). J. Herpetol. 21, 21–8. SUPPORTING INFORMATION Shine R. & Thomas J. (2005) Do lizards and snakes really differ in their ability to take large prey? A study of relative Additional Supporting Information may be found in prey mass and feeding tactics in lizards. Oecologia 144, the online version of this article: 492–8. Shine R., Harlow P. S., Keogh J. S. & Boeadi (1998) The influ- Appendix S1. Prey species identified from python ence of sex and body size on food habits of a scats.

Appendix S1. Prey species identified from python scats. Data on prey body sizes and habits are from Van Dyck and Strahan (2008) except where indicated: 1 Clarke (2010), 2 Nowicki (2007), 3 Wentzel (2011), 4 Thomson- Dans and Hunter (2002). Average Python size class Total Class of adult prey small python large python number of Study site prey Family of prey Prey species (common name) Habit of prey mass (g) (n=18) (n=16) scats COASTAL MAMMAL Dasyuridae Antechinus flavipes (Mardo/Yellow footed antechinus) Nocturnal 45 2 2 WOODLAND Sminthopsis spp. (Dunnart) Nocturnal ~18 1 1 55 scats Muridae Mus musculus (domesticus) (House mouse) Nocturnal 25 7 4 11 collected from 24 Rattus rattus (Black rat) Nocturnal 280 1 1 2 python Pseudocheiridae Pseudocheirus occidentalis (Western ringtail possum)1 Nocturnal 1000 2 10 12 individuals (19 Phalangeridae Trichosurus vulpecula (Common brushtail possum) Nocturnal 1665 4 4 scats from small Leporidae Oryctolagus cuniculus (Rabbit) Nocturnal 1580 2 2 pythons and 36 Felidae Felis catus (feral cat) Nocturnal ~4550 1 1 scats from large REPTILE Scincidae Tiliqua rugosa rugosa (Bobtail lizard)2,3 Diurnal 460 4 11 15 pythons) Skinkoidae species (Skink species) Diurnal ? 2 2 BIRD Psittacidae Barnardius zonarius (Ringneck/28 parrot)4 Diurnal 118 1 1 Unknown Bird species (Unknown bird) Diurnal ? 2 2 JARRAH MAMMAL Dasyuridae Antechinus flavipes (Mardo/Yellow footed antechinus) Nocturnal 45 4 4 FOREST Sminthopsis griseoventer (Grey-bellied dunnart) Nocturnal 18 3 3 32 scats Sminthopsis sp. (Dunnart) Nocturnal ~18 3 3 collected from 10 Muridae Mus musculus (House mouse) Nocturnal 25 5 1 6 python Rattus rattus (Black rat) Nocturnal 280 4 4 8 individuals (24 Rodent species Nocturnal ? 1 1 scats from small Phalangeridae Trichosurus vulpecula (Common brushtail possum) Nocturnal 1665 1 1 2 and 8 scats from Potororidae Betongia pencillata ogilbyi (Woylie) Nocturnal 1270 1 1 large pythons) BIRD Psittacidae Purpureicephalus spurious (Red-capped parrot)4 Diurnal 99.7 1 1 Parrot species (Parrot) Diurnal ? 2 2 Unknown Bird species (Unknown bird) Diurnal ? 1 1 Total number of scats 43 44 87

6. TREE HOLLOWS ARE OF CONSERVATION IMPORTANCE

FOR A NEAR-THREATENED PYTHON SPECIES

As habitat selection can influence many physiological facets of animal life, the thermal conditions of microhabitat retreat sites may be very important for ectotherm physiology. The use of tree hollows by pythons over winter was investigated for possible thermal advantages over the alternative microhabitat resources. This study indicates the conservation importance of tree hollows for M. s. imbricata. This study has been published in Journal of Zoology 286 (2012) 81–92.

AUTHOR CONTRIBUTION

G. L. Bryant: Initiated the research idea, implanted the animals with

temperature loggers, placed temperature loggers at field sites,

collected and entered data, analysed the data, and wrote the

paper.

S. J. Dundas: Provided field-assistance and advice to G.L. Bryant for placement

of temperature-loggers, and provided editorial comments to a

draft version of the manuscript.

P. A. Fleming: Mentored G. L. Bryant on statistical analyses for this research,

suggested the use of temperature loggers to record both python

and habitat temperatures, and provided editorial comments to

draft versions of the manuscript.

Journal of Zoology

Journal of Zoology. Print ISSN 0952-8369 Tree hollows are of conservation importance for a Near-Threatened python species G. L. Bryant, S. J. Dundas & P. A. Fleming

School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia

Keywords Abstract microhabitat; microclimate; Morelia spilota imbricata; predation; thermal biology; Understanding microhabitat requirements for species vulnerable to anthropo- resource; snake. genic threats can provide important information to conservation managers. This may be particularly true for ectotherms, where behaviour and physiology (e.g. Correspondence digestion, responsiveness and activity patterns) are strongly influenced by thermal Gillian Bryant, School of Veterinary and conditions of microhabitat retreat sites. Retreat sites selected by south-west carpet Biomedical Sciences, Murdoch University, pythons (Morelia spilota imbricata) were identified through radiotracking 46 Murdoch, 6150, Australia. Tel: +61 8 9360 pythons over 3 years. Tree hollows appear to be a very important resource 6650; Fax: +61 89 310 4144 for pythons: 61% (22 of 36 individuals tracked over winter) used tree hollows as Email: [email protected] retreat sites (56% of all observations in winter), and remained in hollows for an average of 124 Ϯ 49 (range 34 to 210) days. If pythons did not use tree hollows Editor: Nigel Bennett over winter, they found refuge in one of four alternative microhabitats: low vegetation cover (26% of winter observations), ground cover (10%), on tree Received 17 March 2011; revised 12 July branches (6%) or in hollow logs on the ground (2%). We tested whether tree 2011; accepted 12 July 2011 hollows provide a thermally distinct environment compared with alternative microhabitats, but found no difference in minimum, average, maximum or range doi:10.1111/j.1469-7998.2011.00852.x of temperatures recorded between microhabitats. When within tree hollows over winter, pythons had colder daily average and maximum body temperatures (cf. pythons that used other microhabitats), but this did not give them an energy saving (in terms of body condition scores). Pythons ate very little over winter and we predict that animals sequestered within tree hollows do not access prey at this time. Tree hollows provide a critical refuge over winter when python body temperature is low, and their responsiveness is limited, rendering individuals vulnerable to predation by terrestrial predators (e.g. introduced red fox). Destruction of hollows through fire, land clearing, competition with other fauna species and the significant age required for hollows to form in trees all contribute to the decline in availability of this important microhabitat.

Introduction with 50% of hollows formed in this manner (Whitford, 2002). Trees of many species take over 130 years to reach a size where Tree hollows are an important resource for many vertebrate hollows will start to form, and some eucalypt species may need species. In the jarrah forest of Western Australia, 16 mammal, to be at least 400 years (Gibbons & Lindenmayer, 2002; Whit- 21 bird and 5 reptile species use tree hollows for a variety of ford & Williams, 2002). Removal of larger trees from the purposes (Abbott & Whitford, 2001). Tree hollows are used as landscape (e.g. forestry, clearing due to farming or urbaniza- diurnal or nocturnal retreat sites for sleeping, feeding, rearing tion, and bushﬁres) therefore has important consequences for young, thermoregulation, and to facilitate predator-safe dis- the availability and turnover of tree hollows (Bradshaw, 2000; persal and ranging behaviour (Saunders, Smith & Rowley, Abbott & Whitford, 2001; Whitford, 2002; Whitford & 1982; Smith & Lindenmayer, 1988; Inions, Tanton & Davey, Williams, 2002; Archibald et al., 2006). Furthermore, aggres- 1989; Lindenmayer et al., 1991; Gibbons & Lindenmayer, sive competition for tree hollows with introduced species (e.g. 2002; Glen & Dickman, 2006; Ruczynski, 2006; Koch, Munks European honeybee, Apis mellifera), as well as locally abun- & Driscoll, 2008). Sizeable hollows will usually only form in dant native species (e.g. galahs, Eolophus roseicapillus), can dead limbs or trunks of large trees (minimum of 45–50 cm result in exclusion of threatened species (Gibbons & Linden- over-bark tree diameter; Whitford & Williams, 2002). Limb mayer, 2002). Competition and anthropogenic disturbance breakage is an important contributor to hollow formation mean it is critical to understand the importance of tree hollow

Journal of Zoology 286 (2012) 81–92 © 2011 The Authors. Journal of Zoology © 2011 The Zoological Society of London 81 Tree hollows and conservation of pythons G. L. Bryant, S. J. Dundas and P. A. Fleming use for threatened species (Webb & Shine, 1997, 1998; behavioural, temperature and dietary data to investigate Gibbons & Lindenmayer, 2002; Ruczynski, 2006; Ruczynski whether there was support for each of these predictions to & Bogdanowicz, 2008). explain why the majority of M. s. imbricata used tree hollows Tree hollow use is reasonably well studied for birds, bats as retreat sites over winter. and other mammals; however, there is a dearth of information on tree hollow use by reptiles and amphibians (Lillywhite & Henderson, 1993). For example, broad-headed snakes Hoplo- Materials and methods cephalus bungaroides move from rocky outcrops to woodland areas and sequester in tree hollows with the onset of warmer Study sites and environmental temperature weather (Webb & Shine, 1997, 1998), and tree hollows are Pythons were opportunistically captured from coastal wood- therefore an important consideration in the conservation of land and jarrah forest of south-west Australia. The coastal this species. woodland (Martin’s Tank, Yalgorup National Park 32°51′ S, The geographic distribution of the south-west carpet 115°40′ E, and Leschenault Peninsula Conservation Park, python (Morelia spilota imbricata) has contracted substan- 33°26′ S, 115°41′ E 40 km further south) is on the Swan Coastal tially since European settlement, and the species is currently Plain, consisting of sandy soil, dominated by woodlands listed as Near-Threatened (IUCN, 1998; Pearson, Shine & of Banksia spp. and Agonis flexuosa, with emergent tuart Williams, 2005; Henderson & Powell, 2007). Understanding (Eucalyptus gomphocephala) trees (Trudgen, 1984). Animals the biology of this animal is therefore a priority given the sourced from the jarrah forest were located within State Forest significant potential threats of anthropogenic environmental surrounding the township of Dwellingup (32°43′ S, 116°4′ E). change (Webb & Shine, 1997, 1998; Webb, Brook & Shine, This area lies within the northern jarrah forest region of 2002). During a 3-year ecological study of M. s. imbricata,it Western Australia, where Archaean granite overlies meta- was noted that the majority of individuals (28 of 46 individu- morphic rocks capped by an extensive, but dissected latertic als tracked; 61%) used tree hollows, spending a vast amount of duricrust (Chrurchward & Dimmock, 1989). The vegetation time over winter sequestered in the hollows (56% of observa- is dominated by jarrah (Eucalyptus marginata) and marri tions during winter months; June–August). Importantly, (Corymbia calophylla), trees with a mosaic of woodland under- pythons often revisited and used the same tree hollow each story plant species, including grass trees (Xanthorrhoea preis- year, despite the presence of other pythons using the same sii). Both habitat types experience Mediterranean climates with hollow or the same tree. wet, cool winters and warm, dry summers, although the jarrah We investigated four potential reasons that may determine forest has slightly elevated rainfall (700–1100 mm per annum) the use of tree hollows by M. s. imbricata over winter. Because compared with coastal woodland (600–1000 mm per annum). body temperature (T ) of ectotherms affects all biochemical b Environmental air temperatures (T ; °C) were logged rates (e.g. digestion and growth, sensory perception, and a hourly by two temperature and humidity loggers (HOBO® H8 behavioural activity, such as movement, foraging and mate Pro Series; H08-032-08, Onset Computer Corporation, searching), thermal conditions of microhabitat retreats are Bourne, MA, USA) placed at a location central in each site likely to be extremely important for these animals (Huey, (one at each of the two coastal areas and two loggers within 1982; Kingsolver & Raymond, 2008), particularly for species the jarrah forest positioned approximately 70 km apart). that spend long periods sequestered in retreat sites (Huey HOBO loggers were attached to the south-west aspect of a et al., 1989; Huey, 1991; Webb & Shine, 1998; Webb, Pringle vertical stake, approximately 1 m from the ground, and each & Shine, 2004). Therefore, firstly, the microclimate within tree was covered with a cardboard shelter to prevent rain damag- hollows in large trees may buffer temperature extremes, pro- ing the device or direct solar radiation. Data were averaged for viding a warmer or more stable environmental temperature the two loggers for the jarrah forest and similarly for the two (Vonhof & Barclay, 1996). Secondly, pythons forfeit basking at the coastal woodland. For each month, the average of daily opportunities by sequestering inside a tree hollow and should average air temperature (T ) was tested for differences experience body temperatures similar to temperatures within a between coastal woodland and jarrah forest using single- the hollow itself. Daily body temperatures of sequestering factor analysis of variance (ANOVA). Jarrah forest (JF) and pythons may not fluctuate (cf. pythons that do bask and coastal woodland (C) sites provided unique thermal environ- increase daily body temperature), allowing them to reduce ments (Fig. 1); therefore, study site (JF vs. C) was included in their metabolic rate and therefore reduce their loss of body all analyses. mass over winter. Thirdly, potential prey species (e.g. nesting birds, bats, possums, and quolls) use tree hollows for retreat and nest sites (Saunders et al., 1982; Smith & Lindenmayer, Tree hollow use by pythons – radiotelemetry 1988; Inions et al., 1989; Lindenmayer et al., 1991; Glen & Dickman, 2006; Ruczynski, 2006; Koch et al., 2008). Preda- Forty-six carpet pythons were radiotracked over a 3-year tors, such as snakes, could arguably therefore enter a tree period. Body mass (Mb) of the 46 pythons studied averaged hollow to seek prey. Finally, being sequestered in an appro- 1,030 Ϯ 564 g (range: 136–3,730 g), and snout-to-vent length priately sized and shaped hollow (cf. roosting on branches, (SVL) averaged 151 Ϯ 28 cm (range: 90–223 cm). Pythons under vegetation cover or on the bare ground) may be an were surgically implanted with temperature-sensitive radi- advantage in avoiding terrestrial predators. We analysed otransmitters (radiotransmitter selection for each individual

82 Journal of Zoology 286 (2012) 81–92 © 2011 The Authors. Journal of Zoology © 2011 The Zoological Society of London G. L. Bryant, S. J. Dundas and P. A. Fleming Tree hollows and conservation of pythons

1. Tree hollows (TH) – pythons were radiotracked to a tree and either observed within a hollow, or, if the snake could not be visualized, strength of the radio signal indicated its presence within a hollow. 2. Tree branches (TB) – where a python was visible on a tree branch. 3. Hollow logs (HL) – pythons were radiotracked to and observed within a hollow log on the ground, or if it could not be visualized, the strength of the radio signal indicated its presence within the log. 4. Ground cover (GC) – where the python would lay coiled or stretched on ground debris exposed to potential sunlight radiation (e.g. leafy matter or low grass <5 cm in height). 5. Vegetation cover (VC) – where the python would have Ն30% of its body under or in piles of dead branches/sticks or vegetation (e.g. bushes) or was lying on top of logs or fallen Figure 1 Average (Ϯ1 SD) daily air temperature at the coastal woodland branches, with reduced exposure to sunlight radiation com- and jarrah forest study sites were similar for most of the year except pared with GC, above. during winter/early spring months (June–September), where the jarrah Other microhabitats (e.g. burrows, small wetlands and roads) forest has lower daily average ambient temperatures. *Indicates sig- accounted for only a total of 4% of total observations; these nificant difference between the study sites P < 0.001. were not included in analyses for the present study. Thirty-six pythons were observed over at least one winter period (five were observed over two winters; for these individuals, the length of time spent sequestered in tree hollows was based on the individuals body mass). Five model types/ was averaged). The minimum duration of time pythons spent sizes were used: SB-2T: 5 g (10 months battery life), S1-2T: using tree hollows was recorded from the first incident where 11.0 g (18 months battery life), S1-2T: 13.5 g, and A1-2T: the python was radiotracked to a tree hollow (usually around 16.0 g (24 months battery life), and A1-2T: 25.0 g (36 months April) until the python was first recorded back on the ground battery life) (Holohil® Systems Ltd, Ontario, Canada). Each (usually September/October). These data have the caveat that python was anaesthetized using isoflurane (Forthane, Abbott only weekly observations were carried out and pythons could Australasia Pty Ltd, Botany, NSW, Australia) inhalational have moved about between observations. Only five pythons, anaesthesia, and radiotransmitters were aseptically inserted however, were recorded to move position from a tree hollow into the coelomic cavity following routine ventral coeliotomy. over the winter months; one changed trees and hence tree Transmitters were removed under reversal of this surgical pro- hollows, and the remaining four were observed on the ground cedure between October–November 2008 at the conclusion of (nearing the end of winter) but subsequently retreated back the study (see Bryant et al., 2010 for details). Radiotransmit- into the same tree hollow. For some individuals, this ters were implanted for 354 Ϯ 290 (range 4–1,251) days; two descending/ascending behaviour was repeated over several pythons underwent radiotransmitter replacement due to weeks. The minimum amount of time spent sequestered in the battery expiry. tree hollow (days) was used for analyses and could therefore Pythons were released back to their point of capture and be a slight underestimate in such cases. tracked on a weekly (or rarely fortnightly) basis on foot using a three-element Yagi aerial (Sirtrack Ltd, Havelock North, Do tree hollows provide a more favourable New Zealand) and receiver (R-1000 Telemetry Receiver, ambient temperature? Communications Specialists, Inc. Orange, CA, USA). The exact position of each individual was determined using a Microhabitat temperatures differential GPS system (post-processing using Thales MobileMapper software, Thales Navigation, Santa Clara, To test whether tree hollows provide a thermally distinct envi- CA, USA). Most pythons did not flee when approached, ronment to other microhabitats, over winter of the final field allowing direct behavioural observation, and, where possible, season (June–August 2008), data loggers (iButtons Thermo- hand capture for measurement. Snout–vent length (SVL; cm) chron iButtons DS1922L-F5, Maxim Integrated Products, was recorded using a tape measure (sewing style) from the tip Sunnyvale, CA, USA, logging hourly) were placed into the of the nasal bone to the cloacal opening along the ventral five microhabitat types. All iButtons were covered in two surface of the python. Body mass (Mb) was calculated using layers of inert wax (paraffin/elvax coating 130-0004-00, Mini- calibrated spring balances (Pesola® AG, Rebmattli 19, Mitter Respironics, Bend, OR, USA) and were attached to a CH-6340 Baar, Switzerland) when pythons were contained string to facilitate retrieval. Four replicates of groups of five within a calico bag (bag weight was subtracted from the total). iButtons (one per microhabitat, within 10 m of each other)

Values for Mb and SVL were Log10-transformed for analyses. were placed at each of coastal and jarrah forest sites (i.e. 40 Microhabitats used by pythons were classiﬁed as: iButtons in total).

Journal of Zoology 286 (2012) 81–92 © 2011 The Authors. Journal of Zoology © 2011 The Zoological Society of London 83 Tree hollows and conservation of pythons G. L. Bryant, S. J. Dundas and P. A. Fleming

1. Tree hollows (TH) – seven iButtons were placed into tree (they remained in vegetation and used ground cover). Unfor- hollows that animals were using at the time. Due to access tunately, iButton temperature data could not be collected for reasons, two were placed into different hollows within the these individuals (due to uncontrollable logistical issues at same tree, and in another case, a different tree as close as the time of implanting both animals); therefore, comparison possible to a sequestered python was used. The last three tree of body temperature for TH versus NTH pythons over hollows may or may not have contained a sequestered winter months was not possible for the jarrah forest. python (none of the radiotracked individuals were present Four measures of python body temperature (monthly aver- but we could not preclude use by non-marked individuals). ages of: daily average Tb, daily minimum Tb, daily maximum iButtons were placed as far into each hollow as possible Tb and daily range in Tb) were assessed for each month over a (between 20 and 40 cm depth) by accessing the hollow with a 6-month window. Data were analysed by two-way ANOVA tall ladder and additionally using 3–5 m telescopic aluminium (site: JF vs. C and microhabitat: TH vs. NTH as independent poles. factors) for months where more than three pythons were 2. Tree branch (TB) – 10 iButtons were placed on a branch recorded for each category (April, September and October). adjacent to a visible python, or on the nearest branch proxi- Single-factor ANOVA was used for May, June, July and mate to the positions of the tree hollow iButtons. August for C-NTH, C-TH and JF-TH (insufﬁcient data for 3. Hollow logs (HL) – 10 iButtons were placed within JF-NTH category over these months). a hollow log (as close as possible to each monitored tree hollow), approximately midway along its length (using Is there an energy saving by using tree telescopic aluminium poles). 4. Ground cover (GC) – 10 iButtons were placed in exposed hollows? locations on ground debris in the vicinity of each monitored Python body condition score was calculated as the residual tree hollow. scores of Mb relative to body size (SVL), expressed as a per-

5. Vegetation cover (VC) – 10 iButtons were placed within centage of predicted Mb (Madsen & Shine, 2002): vegetation and fallen branches in the vicinity of each moni- − tored tree hollow. ()observedMMbb predicted BCS (%) = ×100 To assess temperature differences between microhabitats, a predicted Mb mixed-model ANOVA was carried out with two fixed factors (site: JF vs. C and microhabitat: TH, TB, HL, GC or VC), and Effectively, body condition score (BCS) indicates how much a one random factor (iButton replicate number) to measure the python deviates (positively or negatively) from a null model temperature variables: daily minimum, daily average, daily where the python’s body mass is proportional to body length. maximum, and daily range (each treated as dependent Where SVL could not be measured (for many individuals, this factors). required two persons to measure accurately) but body mass could, the average SVL for that python was used (<5% of Python body temperature records). There was no statistically significant sex difference in the relationship between Log10-Mb and Log10-SVL (Fslope 1,42 = > For 29 individuals that were large enough (Mb 409 g and 0.19, P = 0.666, StatistiXL). Therefore, predicted Mb was SVL > 112 cm), one or two additional temperature data calculated using a common equation derived from data col- loggers (iButtons) were implanted with the radiotransmitters. lected for 45 individuals (average values for each individual The iButtons were taped to the radiotransmitter, and the unit excluding their first capture to reduce bias, as a number of was dip-coated with three coats of inert wax. The implanted pythons were initially caught because they had consumed a radiotransmitter and iButton package was <5% of the indi- radiocollared prey; the only juvenile python was excluded vidual’s body mass. The two (or one for smaller animals or from analysis) (Fig. 2; R2 = 0.876): individuals captured close to the end of the study) iButtons ()=× ()− recorded python body temperature hourly; the second was set Predicted Log10 -Mb g 290..Log 10 -SVL cm 334 with a delayed start to commence logging when the first had reached memory capacity. Individuals were monitored for The BCS was calculated for every capture of each individual. body temperature using iButtons over 6.38 Ϯ 3.20 months Body condition could not be determined for animals that were (range 2–23 months). sequestered in a tree hollow because they could not be hand- For the purpose of this study, we were interested in captured to measure. Therefore, to calculate the effects of whether pythons wintering in tree hollows (TH) achieved utilizing tree hollows as a winter retreat or not, the change in different body temperatures compared with individuals that body condition over time for each individual was calculated did not use tree hollows (all other microhabitats pooled (StatistiXL): change in body condition was expressed as the as ‘non-tree hollow’, NTH). We therefore classified iButton slope or beta values (b) for the relationship between body temperature recordings according to the weekly microhabitat condition scores against real time (date) (b-BCS). Only indi- observations: pythons were classified as using a tree hollow viduals that had three or more records of BCS and only indi- or not (i.e. TH vs. NTH) each week. Eight of 10 (80%) viduals that were monitored over a winter period were used in jarrah forest pythons sequestered in tree hollows for winter, this analysis. Three multiple regression analyses were per- which meant only two pythons used other microhabitats formed to ascertain:

84 Journal of Zoology 286 (2012) 81–92 © 2011 The Authors. Journal of Zoology © 2011 The Zoological Society of London G. L. Bryant, S. J. Dundas and P. A. Fleming Tree hollows and conservation of pythons

Statistical signiﬁcance was set to a < 0.05. Values are shown as means Ϯ 1 sd.

Results

Tree hollow use by pythons A number of observations indicated that tree hollows are an important resource for M. s. imbricata. Firstly, a large number of pythons used tree hollows. Twenty-two (61%) of the 36 pythons (individuals that were monitored over 3 years and able to be tracked for at least one winter period) used tree hollows over winter months as their Figure 2 Scatter plot of the body mass (Mb,g)ofMorelia spilota retreat site. At the coastal woodland site, 14 pythons used tree imbricata relative to their snout–vent length (SVL, cm). There was no hollows and 12 did not, and in the jarrah forest, 8 pythons statistically significant difference between equations describing males used tree hollows but only 2 did not. Average duration of 2 (X: Mb = 2.43 ¥ SVL - 2.36, R = 0.66, n = 21) and females (D: Mb = 2.40 retreat to a tree hollow was 121 Ϯ 52 days (n = 22), ranging 2 ¥ SVL -2.23, R = 0.65, n = 24; Fslope 1,42 = 0.19, P = 0.666), so the from a minimum of 34 days to a maximum of 210 days (7 common equation was used. The least-squares linear regression of months). Although pythons used tree hollows from April best fit is shown by the line, where Predicted Log10-Mb (g) = 2.90 ¥ through to September, there was a clear increase in the pro- 2 Log10-SVL (cm) - 3.34, R = 0.876. portion of observations of pythons using tree hollows over the winter months (June–August), with an average of 56% (max 65% in June alone) of total winter observations tracking the 1. Which factors influence the change in body condition individual python to a tree hollow (Fig. 3). The remainder of (b-BCS)? b-BCS was used as the dependent variable, with observations over winter (June–August) identified pythons as study site (JF vs. C), sex (F vs. M), body size (average using vegetation cover (26%), ground cover (10%), tree Log10-SVL) and tree hollow use (TH vs. NTH) as independent branches (6%) and hollow logs (2%; Fig. 3). variables. Secondly, tree hollows were used repeatedly by the same 2. Which factors influence how long pythons stay sequestered individual: four of the five pythons tracked over two winters in tree hollows? The length of time (days) was used as the used the same hollows over consecutive years. Additionally, dependent variable, with study site (JF vs. C), sex (F vs. M), multiple individuals were found using the same tree hollow, or body size (average Log10-SVL) and change in body condition different hollows within the same tree. (b-BCS) as independent variables. 3. Which factors affect the change in body condition (b-BCS) for pythons that use tree hollows over winter? Do tree hollows provide a more favourable b-BCS was used as the dependent variable, with study site ambient temperature? (JF vs. C), sex (F vs. M), body size (average Log -SVL) and 10 Air temperature was colder for the jarrah forest compared the time spent sequestered in tree hollows (days) as inde- with coastal woodland (F = 28.2, P < 0.001) during June to pendent variables. 11,12 September but was not different over the remaining months (Fig. 1). It was not, therefore, surprising that average daily Diet temperature recorded for all five microhabitats was cooler for the jarrah forest compared with coastal woodland (site: F A total of 87 scats were opportunistically collected from 34 1,3.42 = 21.3, P < 0.05; Fig. 4). However, within each habitat type individual pythons over the course of this study. Each scat was (jarrah forest and coastal woodland), there was no statistically carefully washed with gentle running water through a series of significant difference in the minimum, average, maximum or stainless steel or brass mesh sieves of three aperture sizes range of daily temperatures recorded for each of the five (2.0 mm, 250 mm, and 1.0 mm), dried, and then sorted macro- microhabitats over winter (June–August; microhabitat: P > scopically into bird, reptile or mammal samples. Mammal 0.05). species were identified by microscopic analysis of hair samples In terms of python body temperature, jarrah forest pythons (Triggs & Brunner, 2002), reptiles by the size of undigested were colder compared with coastal pythons for three measures scales and limbs, and birds were identified by the shape and of body temperature (average T , minimum T , and maximum size of undigested beaks, skulls, feet and feather colour. Prey b b T ) from June through to September (Fig. 5a–c). species were classified according to whether they were arboreal b Comparison between body temperature for animals that or terrestrial based on species descriptions of their behavioural either did or did not use tree hollows was carried out for patterns (Van Dyck and Strahan 2008). animals in coastal woodland (as mentioned previously, Statistical analyses were carried out using Statistica 8.0 iButtons had not been implanted into the two pythons that did (StatSoft Inc., 2007) and StatistiXL (statistiXL 1.8, 2007). not sequester in tree hollows in jarrah forest, which precluded

Journal of Zoology 286 (2012) 81–92 © 2011 The Authors. Journal of Zoology © 2011 The Zoological Society of London 85 Tree hollows and conservation of pythons G. L. Bryant, S. J. Dundas and P. A. Fleming

Figure 3 The use of ﬁve microhabitats by Morelia spilota imbricata shown by calendar month. Tree hollow use increased during winter months, while use of hollow logs decreased. See methods for detailed description of each microhabitat. Data is collated over 3 years of radiotelemetry monitoring of 46 pythons at both coastal woodland and jarrah forest sites. N = total number of pythons observed each month.

the same comparison between pythons sequestering in tree hollows or an alternative microhabitat in this habitat). In coastal woodland, pythons that used tree hollows experienced similar minimum Tb. Pythons were colder on average (average Tb; June to October; Fig. 5a) and experienced colder maximum Tb (over all months examined; Fig. 5c) than animals that did not use tree hollows. Therefore, the range of body temperatures (the monthly average of daily range calculated for each individual) was greater for pythons that did not use tree hollows compared with pythons that sequestered in tree hollows (Fig. 5d).

Is there an energy saving by using tree hollows? We recorded a marked decline in body condition score for animals across this study. Since pythons that used tree hollows were colder than those that used alternative microhabitats, we predicted that a lower metabolic rate would mean that animals using tree hollows would show a slower rate of decline in body condition over winter (when, like most ectotherms, pythons rarely if ever feed). None of the four factors analysed, however (use of hollows, study site, sex, and body size), significantly Figure 4 Box plot illustrating temperature for the five microhabitats affected python b-BCS (Table 1a). In terms of factors that used by Morelia spilota imbricata. There was no measureable differ- influenced the length of time animals stayed in tree hollows, ence in the winter (June–August) in daily average, minimum and Ϯ females spent longer in tree hollows (136 50.3 days) com- maximum or range of temperatures for five microhabitat types (P > Ϯ pared with males (101 49.7 days) (t17 =-2.23, P = 0.040), but 0.06). Jarrah forest daily average temperatures for the five microhabi- the length of time was not affected by study site, body size tats were, however, colder than experienced in coastal woodland (F1,3,42 (SVL) or their b-BCS (Table 1b; Fig. 6). The rate of decline in = 21.32, P < 0.05). The median is identified by a line inside the box. The • body condition differed between the sexes (t17 =-2.18, P = length of the box is the interquartile range (IQR). Indicates outlier 0.043), with males that used tree hollows (b-BCS =-0.668 Ϯ values more than 1.5 the interquartile range (IQR), but less than 3 IQR’s 0.264) showing greater decline in BCS than females that used from the end of the box. *Indicates values more than 3 IQR’s from the tree hollows (b-BCS =-0.449 Ϯ 0.461), but there was no end of the box.

86 Journal of Zoology 286 (2012) 81–92 © 2011 The Authors. Journal of Zoology © 2011 The Zoological Society of London G. L. Bryant, S. J. Dundas and P. A. Fleming Tree hollows and conservation of pythons

signiﬁcant effect of study site, body size or the amount of time sequestered in a tree hollow (Table 1c; Fig. 6).

Do pythons access more arboreal prey? Behavioural observations suggest that pythons did feed while in trees, as pythons were observed in ambush position in trees. However, it should be noted that pythons did not feed regularly over winter (when they were using tree hollows extensively). As part of the broader radiotelemetry study on this species, behavioural observations (where pythons were observered or palpated to check whether they had recently fed) revealed that pythons very rarely fed during winter (7.5% of ﬁeld observations) compared with warmer months (31.5% of summer, 12.6% of autumn and 10.4% of spring observations; Bryant et al. in press). Overall, only 29% of prey species in python scats were identiﬁed as being arboreal or mostly arboreal, whereas over 71% were principally terrestrial (Table 2).

Do tree hollows offer protection from predation? There are at least six potential predator species of pythons in Western Australian jarrah forest and coastal woodland. Most of these predators are terrestrial, including the introduced red fox (Vulpes vulpes) and native varanid lizards (Table 3). Two of the 13 deaths recorded as part of the broader ecological study on M. s. imbricata resulted from predation (or scaveng- ing following death). Red fox DNA was conﬁrmed for one of the carcasses and the bite marks on the implanted radiotransmitter from the second also suggest fox predation.

Discussion South-west carpet pythons display distinct patterns of microhabitat selection, with over half the individuals tracked using tree hollows as winter retreats for an average of 3 months, but for as long as 7 months of the year. Observational data revealed numerous pythons using hollows in the same tree, repeated use of the same tree hollows over consecutive years, and even numerous pythons sharing the same hollow. These observations provide strong indication that tree hollows could be a limiting resource for this python species. Given the high level of use of tree hollows over winter months, we investigated whether tree hollows may provide a thermally distinct Figure 5 Four measures of python body temperature recorded over a 7-month period (April–October) when Morelia spilota imbricata made or physiologically advantageous environment compared use of tree hollows. Average (Ϯ1 SD) monthly body temperature (a), with alternative microhabitats. We also investigated whether maximum body temperature (c) and the range in body temperature pythons utilize a large proportion of arboreal prey and could recordings (d) were lower for pythons sequestered inside tree hollows therefore have greater access to prey if they use tree hollows, compared with those that did not use a tree hollow over this period. or whether they have need of protection from predation. We There was no difference for minimum body temperature (b). The closed discuss these alternative explanations for tree hollow use symbols represent jarrah forest pythons, open symbols represent below. coastal pythons. Circles indicate pythons using tree hollows (TH); squares indicate pythons that did not use tree hollows (NTH) (ground to Do tree hollows provide a more favourable <2 m-level vegetation). Letters link groups that are not signiﬁcantly ambient temperature? different (P > 0.05). Data is shown where there are three or more pythons for each category for each month, as indicated in the table Our data did not support the hypothesis that tree hollows below the ﬁgure. provide a stable or warmer thermal environment, since the

Journal of Zoology 286 (2012) 81–92 © 2011 The Authors. Journal of Zoology © 2011 The Zoological Society of London 87 Tree hollows and conservation of pythons G. L. Bryant, S. J. Dundas and P. A. Fleming

Table 1 Multiple regression output for analyses addressing three questions: (1) Which factors inﬂuence the change in body condition (b-BCS)? (2) Which factors inﬂuence how long pythons stay sequestered in tree hollows? and (3) Which factors affect the change in body condition (b-BCS) for pythons that use tree hollows over winter?

(1) Change in (2) Length of (3) Change in body body condition time in tree hollow condition (b-BCS) for tree (b-BCS; n = 36) (days; n = 22) hollow users (n = 22)

Independent factors: t31 P-value t17 P-value t17 P-value Study site (C or JF) -1.65 0.110 1.60 0.129 -0.624 0.541 Sex (F or M) -1.33 0.194 -2.23 0.040 -2.18 0.043

Body size (Log10 avg. SVL) 1.33 0.194 -0.871 0.396 1.09 0.292 Tree hollow user or not 0.542 0.592 --- - Change in body condition (b-BCS) ---1.64 0.120 -- Time in tree hollow (days) --- - -1.64 0.120

2002; Whitford, 2002; Whitford & Williams, 2002), and this may reduce the thermal insulating properties (Sedgeley, 2001). Possibly even more importantly, having a sufficiently large entrance and internal dimensions to allow a python residence is likely to result in rapid heat exchange with air temperature, and therefore these tree hollows are likely to exhibit temperatures similar to air temperature, with only minimal buffering of temperature fluctuations. In summary, there was no indication that tree hollow temperatures differed from air temperature, and therefore we have no data to suggest that this microhabitat provides a more favourable thermal environment over winter compared with other Figure 6 The rate of body condition decline (b-BCS) was not signifi- microhabitats used by pythons. cantly reduced for Morelia spilota imbricata that used tree hollows over Ϯ winter compared with those that did not (NTH; average 1 SD). For Is there an energy saving by using tree pythons that used tree hollows over winter, males spent less time hollows? (101 Ϯ 49.7 days) in tree hollows compared with females (136 Ϯ 50.3 days), yet showed a faster decline in body condition (lower b-BCS) While sequestered inside a tree hollow, pythons forfeit basking compared with females. opportunities that would enable them to increase their body temperature. The choice to remain sequestered inside a tree hollow and forgo basking over winter was reflected in python body temperature measurements. Both monthly average and thermal properties inside tree hollows were not different maximum values of body temperatures were colder for from those of other microhabitats used by pythons. Simi- pythons sequestering inside tree hollows compared with larly, studies of diurnal roost sites used by microbats found pythons that did not use tree hollows. Pythons experienced a trunk hollows had significantly larger entrances and internal greater range of body temperature when they utilized other volumes than knot-hole cavities, and temperatures within microhabitats compared with pythons that sequestered in tree trunk hollows were much less stable, often fluctuating in the hollows. same way as ambient temperature (Sedgeley, 2001). The It could be argued that an ectotherm attempting to mini- large dimensions necessary to allow a python entry would mize energy losses should select temperatures as low as possi- therefore expose these hollows to ambient conditions. Addi- ble (Huey, 1991). Because pythons that used tree hollows had tionally, heat transfer (convection, radiation, and conduc- lower body temperatures (cf. animals occupying other micro- tion) and thus the microclimate of a tree hollow may be habitats) we predicted there may be an energy saving to using affected by differing thickness of the hollow walls, the size of a tree hollow over winter by decreasing metabolic rate and the hollow entrance and the amount of solar radiation reach- consequently reduce their rate of body condition decline. ing the tree (Bakken & Kunz, 1988). Early-decaying However, our data do not support this prediction. We branches or trunks will have thicker hardwood surrounding observed no difference in the overall decline in body condition the internal hollow (Whitford & Williams, 2002), but the of pythons using tree hollows compared with pythons using hollows are likely to be too small for a python to occupy. alternative winter microhabitats. Our prediction assumed that Larger, older hollows suitable for a python are likely to be none of the pythons feed over winter (Slip & Shine, 1988a); we located in a completely decayed branch or trunk, will have note that because pythons that remained on the ground thinner hardwood surrounds (Gibbons & Lindenmayer, achieved higher maximum body temperatures, they may have

88 Journal of Zoology 286 (2012) 81–92 © 2011 The Authors. Journal of Zoology © 2011 The Zoological Society of London G. L. Bryant, S. J. Dundas and P. A. Fleming Tree hollows and conservation of pythons

Table 2 The spatial ecology of prey species identiﬁed from Morelia spilota imbricata scats opportunistically collected from radiotracked individuals in coastal woodland and jarrah forest of Western Australia

Spatial ecology of prey species Examples of prey species identiﬁed Total no. of scats (% of total) Arboreal Pseudocheirus occidentalis 18 (21%) Parrots Mostly arboreal Trichosurus vulpecula 7 (8%) Ground dwelling to mid-storey vegetation Antechinus ﬂavipes 7 (8%) Felis catus Mostly ground dwelling Mus musculus 31 (35%) Rattus rattus Sminthopsis griseoventer Ground dwelling Betongia pencillata ogilbyi 24 (28%) Oryctolagus cuniculus Tiliqua rugosa rugosa Total 87

Table 3 Possible predators of Morelia spilota imbricata pythons at the two study sites in south-west Western Australia and evidence for predation upon M. s. imbricata and M. s. spilota (eastern Australia)

Jarrah Coastal Predator Species forest woodland Habitat Evidence for predation Reference Lizards Varanus gouldii Present Present Terrestrial Yes V. rosenbergi attempted predation on M. s. Varanus rosenbergi Present Present imbricata (This study, Bryant pers. obs.) V. varius predating on M. s. spilota (Shine & Fitzgerald, 1996) Feral mammals Vulpes vulpes Present Present Terrestrial Yes Fox DNA found on radiotransmitter and body of dead python (This study) For M. s. spilota (Shine & Fitzgerald, 1996) Felis catus Present Present Semi-arboreal No Birds of prey Aquila audax Present Present Arial Limited (very few snake Cherriman, (2008) remains found in Brooker & Raidpath, (1980) scats/pellets) Haliastur sphenurus Uncommon Present Arial Limited (small reptiles) Thomson-Dans & Hunter (2002)

taken more prey over this time than animals sequestered in a ity of prey species consumed by pythons were terrestrial with hollow. Taking this caveat into account, however, we have no only a small percentage of prey species identiﬁed as largely support for a slower rate of body condition decline for arboreal. pythons that used tree hollows.

Do tree hollows offer protection Do pythons access more arboreal prey? from predation? It is possible that pythons use tree hollows because they Tree hollows may provide pythons protection from predators provide a source of accessible prey. It is difficult to be certain over winter, when their minimum body temperatures averaged that pythons, particularly those that spent many months 6.25 Ϯ 2.73°C (jarrah forest) and 9.84 Ϯ 2.36°C (coastal sequestered in hollows, did not have access to prey. However, woodland). At such low temperatures, the perception of a even though birds and arboreal mammals use tree hollows for predator and response to its presence (i.e. flight or fight retreat and nesting sites, it is unlikely that pythons are using response) would undoubtedly be hindered. For example, tree hollows at this time of year in order to gain access to tongue-flicking is necessary for chemoreception in snakes prey, since we suspect these animals were unlikely to be (Peterson, Gibson & Dorcas, 1993), and in numerous species, feeding at this time. Usually at Tb <10°C, digestion is inhib- optimal body temperature is important for effective prey ited, and snakes will regurgitate their food (Lillywhite, 1987). detection and may also be important for predator detection, Pythons that used tree hollows were colder, and, therefore, escape and defensive behaviour (Dorcas & Peterson, 2007). less likely to feed compared with animals that remained on Defensive behaviours (e.g. hissing, striking and biting) are the ground. Furthermore, our radiotelemetry data indicated exhibited more often in three species of colubrid snakes at that pythons fed infrequently over winter, and that the major- high air temperatures (between 20–30°C; Vincent, Herrel &

Journal of Zoology 286 (2012) 81–92 © 2011 The Authors. Journal of Zoology © 2011 The Zoological Society of London 89 Tree hollows and conservation of pythons G. L. Bryant, S. J. Dundas and P. A. Fleming

Irschick, 2004), and increasing body temperature exponen- This project was approved by the Animal Ethics Committees tially increases both the crawling and escape speeds (reaching of Murdoch University (W2028/07) and Department of and hiding its body in a rock crevice) of rubber boas (Charina Environment and Conservation, Western Australia (DEC bottae; Dorcas & Peterson, 2007). Cold pythons would there- AEC/55/2006 and DEC AEC54/2006) and was financially fore be more vulnerable to predation. For many reptile supported by Department of Environment and Conservation species, the risk of predation is significantly increased if indi- and Murdoch University. viduals bask in the open such as on rocks or in open vegetation (Dorcas & Peterson, 1998; Martin & Lopez, 1999). Tree References hollow retreats may therefore provide a safehouse over winter while python body temperatures are at their minimum, and Abbott, I. & Whitford, K.R. (2001). Conservation of verte- hence behavioural and physiological responses are slowest. brate fauna using hollows in forests of south-west Western A number of potential predator species were evident at Australia: strategic risk assessment in relation to ecology, these study sites, including the introduced red fox and varanid policy, planning, and operations management. Pac. lizards (Table 3). Most of the predators identified at these Conserv. Biol. 7, 240–255. sites are terrestrial hunters. It could be argued that hollow Archibald, R., Bowen, B., Hardy, G.E., McCaw, L. & Close, logs on the ground have similar features as tree hollows, and D. (2006). Tuart decline research finding. In: Tuart Bulletin therefore provide similar habitat for pythons. However, while No. 6: a series outlining key research findings associated with hollow logs are used extensively over summer months tuart health in south-west Western Australia. Perth, Western (Fig. 3), only 2% of observations of pythons showed that they Australia: Tuart Health Research Group, School of Bio- used hollow logs in winter (cf. 56% of observations over logical Sciences and Biotechnology, Murdoch University. winter were for tree hollows). While there is no difference in Bakken, G.S. & Kunz, T.H. (1988). Microclimate methods. In the thermal conditions inside hollow logs cf. tree hollows Ecological and behavioral methods for the study of bats: there is likely to be a very different predation risk to pythons. 303–332. Kunz, T.H. (Ed.). Washington, DC: Smithsonian While it would be difficult for a terrestrial predator to remove Institute. a python from a tree hollow, over past centuries, man and now extinct carnivorous marsupials may easily have hunted Bradshaw, F.J. (2000). Recommendations for the regeneration and captured a cold-bodied python sequestered in a hollow and maintenance of the tuart forest in the Yalgorup National log, as can terrestrial predators present today. Although we Park. pp 22. Manjimup, WA: Consultants report to the have no direct evidence to suggest that tree hollows offer Department of Conservation and Land Management. protection from predation, this hypothesis warrants further Brooker, M.G. & Raidpath, M.G. (1980). The diet of the investigation. wedge-tailed eagle Aquila audax in Western Australia. Aust. Our results highlight the importance of understanding this Wildl. Res. 7, 433–452. threatened species’ physiology and ecology for improving Bryant, G.L., de Tores, P.J., Warren, K. & Fleming, P.A. (in conservation management outcomes. Tree hollows appear press). Does body size influence thermal biology and there- to provide pythons with an important resource over winter fore diet of a python (Morelia spilota imbricata)? Austral months. We have no evidence to suggest that tree hollows Ecol. provide a more stable thermal environment, although pythons Bryant, G.L., Eden, P., de Tores, P.J. & Warren, K.S. (2010). sequestered within tree hollows maintain lower and less vari- Improved procedure for implanting radiotransmitters in the able body temperature. Although this should have resulted in coelomic cavity of snakes. Aust. Vet. J. 88, a lowered metabolic rate, we did not find a statistically signifi- 443–448. cant saving in terms of reduced decline in body condition over Cherriman, S.C. (2008). Diet of the wedge-tailed eagle Aquila winter. It is unlikely that pythons use tree hollows as a source audax at some DEC reserves near Narrogin, Western Aus- of prey (since they feed infrequently if at all over winter), but tralia. pp. 10. Unpublished report to the Deptartment of they are likely to provide a safe retreat from predators. The Environment and Conservation, Perth. presence of numerous predators at both study sites, including Churchward, H.M. & Dimmock, G.M. (1989). The soils and mammals such as red foxes and feral cats, suggests there is a landforms of the northern Jarrah forest. In The jarrah risk of predation. Tree hollows are a limited resource due the forest: 13–22. Dell, B., Havel, J.J. & Malajczuk, N. (Eds). long time for hollows to form in trees (Whitford & Williams, 2002), the vast expanses of land clearing for forestry, housing London: Kluwer Academic Publishers. and other infrastructure, along with competition with other Dorcas, M.E. & Peterson, C.R. (1998). Daily body tempera- fauna. Their importance to a wide variety of animal species ture variation in free-ranging rubber boas. Herpetologica needs to be recognized and taken into account as part of 54, 88–103. conservation management. Dorcas, M.E. & Peterson, C.R. (2007). Testing the coadaptation hypothesis: the thermal physiology and thermoregulatory behavior of rubber boas (Charina bottae). In Biology Acknowledgements of the boas and pythons: 245–255. Henderson, R. & Powell, We are grateful to volunteers who helped with radiotelemetry W.R. (Eds). Eagle Mountain, UT: Eagle Mountain and data collection, particularly Meagan and James Connor. Publishing.

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THE SOUTHWEST CARPET PYTHON?

Mesopredator theory predicts that in response to removal or reduction of an apex predator such as the red fox, a greater number of prey will be available to mesopredators

(i.e. release of exploitative competition) and that there should be greater survivorship following reduced predation (i.e. decrease in intraguild predation; Fig. 1.2). To determine whether these responses actually occurred as a result of fox baiting of fox baiting at Leschenault Peninsula Conservation Park (LPCP) would require long-term monitoring of the entire ecosystem, including numbers of each of the prey, mesopredator and the apex predator species. In retrospect, very few data have been gathered to address this issue. Outlined below is a description of what is known about these species’ responses and presence at LPCP based on limited monitoring since the start of fox baiting in 1991.

For this section, Pearson’s chi-square analyses have been carried out with expected values calculated assuming there is no difference between baited and unbaited sites.

The beta (β) coefficients of regression analysis have been calculated using

StatisticXL®. Values are presented as means ± 1 S.D.

PREY DENSITIES

7.1. Rabbits

Rabbit (Oryctolagus cuniculus) densities have not been carefully monitored, but historical anecdotal data suggest that rabbits were once very abundant at LPCP. When 76 fox control was implemented in 1991 at LPCP, cyanide lines were used to determine fox presence (de Tores Pers. comm)1, and as a consequence rabbits were also controlled.

Sightings in 2008 suggested there are some rabbits still present at LPCP (de Tores Pers. comm)1, yet in the absence of controlled rabbit monitoring (i.e. through trapping or transect monitoring), it remains unclear whether rabbit populations have fluctuated at these study sites. In Australia, rabbits constitute a major dietary component of both feral cats and foxes (Catling 1988; often around 50% of their total diet; Coman 1973;

Dickman 1996a; b; Martensz 1971; Pech et al. 1992). Pythons consumed relatively very few rabbits during this study (Chapter 5).

7.2. Western ringtail possums

LPCP and Yalgorup Nation Park (YNP) are sites of an extensive translocation program for western ringtail possums (de Tores et al. 2003). Western ringtail possums are captured from coastal habitats south of LPCP and YNP prior to land clearing (e.g. for housing). After successful rehabilitation of injured animals (by local wildlife carers) they are translocated into protected areas of suitable habitat within the species’ historical distribution (Clarke 2010; de Tores et al. 2003). Between 1991-2008 western ringtail possums have been translocated into LPCP, Martin’s Tank (MT; part of YNP) and other parts of YNP. Some of these individuals were monitored with radiotelemetry and the population has been regularly surveyed through spotlight transect monitoring

(Fig. 7.1; Clarke 2010; de Tores et al. 2005a; de Tores et al. 2005b). Following mortality events of radiotracked individuals has indicated that foxes, cats and pythons took western ringtail possums as prey at LPCP and MT (Fig. 7.2; Chapter 5; Bryant et al. 2011b; Clarke 2010). Raptors within the area also took western ringtail possums to varying degrees (Clarke 2010; Fig. 7.2). There has been a notable change in the type of predator that was taking western ringtail possums as prey over time. Between 1991- 77

1998 foxes were the main predator of possums, however, after the fox baiting program was introduced at LPCP, a combination of all predators were found to be taking possums as prey (13-17 years after commencement of fox baiting). At the unbaited site,

YNP, red foxes remained a significant predator of western ringtail possums.

90 LPCP

80 MT YNP 70

20 Number of WRP translocated

0 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 Year

Figure 7.1. The number of western ringtail possums (Pseudocheirus occidentalis; WRP) translocated into study areas since 1991. Animals have been released into the fox baited areas at Leschenault Peninsula Conservation Park (LPCP) and areas within Yalgorup National Park (YNP), as well as into an unbaited area of YNP, Martin’s Tank (MT). Data were sourced from Clarke (2010) and de Tores et al. (2005b; 2003).

BAITED UNBAITED LPCP 1991-1998 Fox WRP: 122 released, 11 monitored Cat Python Raptor Unknown predator

LPCP 2004-2006 MT 2004-2006 WRP: 106 released, 106 monitored WRP: 66 released, 6 monitored

LPCP 2006-2008 MT 2006-2008 WRP: 31 released, 30 monitored WRP: 23 released, 23 monitored

Figure 7.2. Predation fates of radiocollared (monitored) western ringtail possums (Pseudocheirus occidentalis; WRP) at the fox baited site (Leschenault Peninsula Conservation Park; LPCP) and at the unbaited site (Martin’s Tank; MT) over the years when WRP were monitored through radiotelemetry. Values are proportions of the number of individuals monitored that succumbed to each predator category. Data sourced from Clarke (2010) and de Tores et al (2005b; 2003).

7.3. Other prey

There has been some short-term monitoring of terrestrial mammals and reptiles through trapping programs, and monitoring of birds through visual and auditory surveys in recent years between 2007 and 2009 at both LPCP and MT (Nowicki 2007; Wentzel

2011). These data indicated that there was a large range of potential prey at these sites, many of which have been recorded as dietary items of pythons (Chapter 5; Bryant et al.

2011b), foxes (Glen 2005; Glen et al. 2009), and cats (Catling 1988; Crawford 2010;

Paltridge 2002; Triggs et al. 1984).

PREDATOR DENSITIES

7.4. Fox densities

There are few empirical data indicating that fox baiting at LPCP has been effective in controlling fox numbers, although anecdotal evidence exists. In 1991, eleven foxes were killed at LPCP as a result of culling through cyanide lines implemented by DEC, whereas in 1996 only one fox was killed using the same method, suggesting a decrease in density over this time (de Tores pers. comm)1. Fox activity in LPCP has remained low since the commencement of fox baiting in 1991, as measured through sand pad activity plots, particularly in comparison to the unbaited MT area (de Tores Pers. comm)1. Despite fox baiting being initiated in 1991, 95% of all WRP deaths between

1991-1998 at LPCP were attributed to predation by foxes (Fig. 7.2), suggesting either a delay in bringing fox numbers under control, or that even if fox numbers were low in response to fox control, there were few other predators to take western ringtail possums at this time. More recently (2004-2008), the proportion of telemetered western ringtail possums falling prey to foxes had decreased to less than 10% of all deaths recorded (de

Tores pers. comm)1.

7.5. Cat densities

There is anecdotal accounts that cat numbers at LPCP have increased subsequent to fox control. Firstly, there have been an increased number of reported sightings by conservation managers in the area (de Tores Pers. comm)1. Secondly, there has been an increased number of predation events on monitored (collared) western ringtail possums attributed to cats over the past two decades. Between 1991-1998, no deaths were attributed to cats, however, ≥13 years after the initiation of fox baiting (2004-2008) at least 30% of western ringtail possum predation events at LPCP have been attributed to cats, and 16% of predation events at MT (Fig. 7.2; Clarke 2010; de Tores Pers. comm1).

Finally, in 2008/09, a model to derive cat density estimates from sand pad activity plots suggested that multiple individual cats were present within LPCP and MT, with low densities (<1 cat per 6 km2) evident for MT and higher densities estimated for LPCP

(Clarke 2010).

7.6. Python densities

Indirect evidence suggests that, although southwest carpet pythons were always present in LPCP, python numbers have increased since 1991 (de Tores Pers. comm)1.

Identifying the predator from remains of WRP carcasses requires forensic investigation

(i.e. DNA from saliva left on the radiocollar), in the case of foxes, cats or raptors which leave the collar with the animal remains. By contrast predation by pythons is unambiguous when the prey is in the stomach and providing the python is located by radiotelemetry before the WRP radiocollar stops working. Between 1991-1998, only one of the 11 radiotracked western ringtail possum deaths at LPCP was attributed to a python. After a break in monitoring at LPCP (1998-2004), subsequent radiotelemetry monitoring in 2004-2006 of WRP recorded a staggering 45% of predation events

81 attributed to pythons (Fig. 7.2). During the same period at MT no WRP deaths were attributed to pythons. This indirect evidence is, suggestive that the density of pythons had increased 13-15 years following fox control at LPCP. More recently (2006-2008), the proportion of deaths attributed to pythons has decreased to 21% at LPCP (and 28% at MT).

PREDICTING MESOPREDATOR RELEASE RESPONSE IN PYTHONS

Consistent with the mesopredator release hypothesis, the implementation of fox baiting at LPCP may have released subordinate predators (cats and pythons) from exploitative competition and reduced intraguild predation by foxes. Measuring the outcomes of this disturbance is however, extremely difficult due to the cryptic nature of both cats and pythons. The situation with pythons is further complicated by the fact that these animals cannot be trapped, and population estimates via track counts is ineffective, making direct quantification of numerical response by pythons to fox control impossible. Passive capture methods commonly used to monitor endothermic carnivores such as trapping, sandplotting, spotlighting or DNA collection (e.g. collecting hairs and scats) are largely impractical for secretive species, particularly cryptic ectothermic species, like pythons (or other snakes; Dorcas and Willson 2009).

Instead, successful data collection in pythons requires a focus on individual animals to evaluate population status (Dorcas and Willson 2009).

7.7. Predictions

The most likely scenario as a consequence of fox baiting at LPCP is a decrease (↓) in fox density. Under the scenario of decreased fox density, python survivorship, density, and diet diversity are predicted to increase (↑) due to the predicted increase in prey

82 availability. Pythons are also predicted to reduce (↓), or not change (↔) their predator avoidance behaviour (Table 7.1).

Table 7.1. Predicted demographic outcomes for a python population under the scenario where fox density is modified through fox baiting. The hypotheses (H) listed are explained further in the text.

Predicted response for each measured parameter for a given outcome from fox Parameter measured control measures Fox density ↔ Fox density ↓ e.g. MT (unbaited) e.g. LPCP (baited) Python survivorship Body mass and size

H1 Body mass ↔ ↑

H2 Body length ↔ ↑

Body condition

H3 Body condition score ↔ ↑

H4 Body condition decline ↑ ↔ or ↓

Mortality

H5 Number of deaths ↑ ↔ or ↓

H6 Decline in body condition prior to death ↑ ↔ or ↓

H7 Small animals die of emaciation ↑ ↔ or ↓

Population demographics

H8 Growth rate ↔ ↑

Python density

H9 Fecundity ↔ ↑

H10 Body condition of reproductive females ↔ ↑

Python diet diversity

H11 Greater prey diversity ↔ ↑

Python predator behavioural avoidance Temporal segregation

H12 Nocturnal hunting/foraging behaviour ↑ ↔ Habitat-mediated avoidance

H13 Cryptic microhabitat use ↑ ↔ or ↓

H14 Tree hollow use ↑ ↔ or ↓

H15 Length of time sequestered in tree hollow ↑ ↔ or ↓ Landscape-mediated avoidance

H16 Cryptic body positions ↑ ↔ or ↓ ↑ - increases ↓ - decreases ↔ - remains the same (no change)

EVIDENCE FOR MESOPREDATOR RELEASE RESPONSE IN PYTHONS

Using the set of predictions outlined in Table 7.1, it is possible to examine the available data to test for a mesopredator release of pythons at LPCP in response to fox baiting.

Comparison with the unbaited (MT) site allows some test of these predictions. This section discusses data collected from pythons after radiotransmitters were surgically implanted in their coelomic cavities (for details see Chapter 3) and monitored weekly.

Radiotelemetry allows researchers to monitor animals frequently; pythons are conducive to this type of monitoring as they typically do not flee from approaching observers, allowing direct observations of the animals in their natural environment.

7.8. Survivorship

It is predicted that python survivorship will increase in the presence of fox baiting.

Under fox baiting it is predicted there will be more prey available to pythons and there may be reduced intraguild predation by foxes towards pythons. Therefore, pythons at the baited coastal site (LPCP) are predicted to have: 1. greater body mass and length, 2. better body condition, 3. lower mortality, and will be 4. an aged population compared with pythons at the unbaited site (MT; Table 7.1).

a. Body mass and size

Sexual size dimorphism often exists between male and female snakes; with females often larger than males (Stanford and King 2004). For many snake species, males may differ in asymptotic size but not in the rate at which they approach this size (Stanford and King 2004). Sexual size dimorphism has been found in M. s. imbricata, where females grow to over twice the length and reach more than 10 times the mass of adult

85 males (Pearson et al. 2002a; Pearson et al. 2002b). This sexual size dimorphism needs to be taken into account when comparing the size of pythons between study sites.

A total of 27 pythons (15 females, 12 males) were monitored at LPCP, 7 pythons (5 females, 2 males) were monitored at MT, and 12 pythons (5 females, 7 males) were monitored in the jarrah forest (Table 7.2). Pythons were monitored weekly over varying lengths of time (Table 7.2), depending on date of capture, radiotransmitter expulsion problems (see Chapter 3), death, and other factors. Wherever possible, animals were captured and measured (exceptions include where pythons were sequestered in tree hollows or other impenetrable vegetation, or incubating eggs underground). The hypotheses tested were:

H1: Pythons at LPCP (baited) will be heavier than pythons at MT (unbaited).

H2: Pythons at LPCP (baited) will be longer than pythons at MT (unbaited).

Both the first two hypotheses were confirmed through multiple regression analysis.

Pythons at LPCP were heavier (t442= -8.77, p<0.0001) and longer (t442= -7.10, p<0.0001) than pythons at MT. As this species is known to have sexual size dimorphism where adult females can be several times larger than adult males (Pearson et al. 2002a), the sex of the python was taken into account (included as an independent variable) and females were found to be significantly heavier (t442= 17.4, p<0.0001) and longer (t442= 19.4, p<0.0001) compared with males.

Table 7.2. Average body measurement values for all Morelia spilota imbricata pythons over the study period at each of the three study locations. N= number of measurements obtained for each python, BCS= body condition index, 1 = juvenile, 2 = reproductive female (known ova development), 3 = reproductive female (as a mating, or potential mating event was observed).

Study site Sex Animal ID #Days SVL ± S.D. (cm) Body mass ± BCS ± S.D. (N) monitored S.D.(g) Leschenault Female 1 (5)2 513 200 ± 0.09 2083 ± 291 6.02 ± 15.0 Peninsula 4 (9) 2 502 194 ± 4.33 1380 ± 67.1 -23.6 ± 7.19 Conservation 9 (9) 2 106 188 ± 4.19 1280 ± 54.8 -22.8 ± 6.94 Park 10 (42) 692 170 ± 5.75 1299 ± 153 2.57 ± 17.5 11 (2) 184 181 ± 0.00 2125 ± 0.00 40.3 ± 0.00 14 (21) 2,3 582 177 ± 5.90 1612 ± 370 15.3 ± 38.0 16 (8) 337 170 ± 2.56 1765 ± 139 39.1 ± 14.2 27 (3) 2 698 171 ± 0.00 1318 ± 173 1.18 ± 13.3 33 (12) 628 149 ± 4.57 970 ± 138 8.93 ± 19.8 36 (17) 2 258 192 ± 3.01 1630 ± 173 -7.74 ± 11.7 37 (20) 3 552 192 ± 8.65 2077 ± 294 18.6 ± 24.6 44 (3) 3 94 200 ± 3.21 2410 ± 539 22.1 ± 26.0 47 (1) 64 200 - 3012 - 53.6 - 49 (23) 251 174 ± 3.00 1337 ± 50.4 -1.30 ± 6.05 53 (5) 244 134 ± 4.40 715 ± 17.9 5.94 ± 11.9 Male 17 (3) 21 138 ± 0.00 588 ± 0.00 -20.3 ± 0.00 23 (8) 352 108 ± 2.39 454 ± 43.5 18.5 ± 14.7 25 (28) 646 106 ± 4.53 373 ± 33.5 3.31 ± 12.3 31 (20) 423 154 ± 5.82 1225 ± 192 26.0 ± 26.0 38 (11) 283 108 ± 1.48 451 ± 35.1 18.8 ± 11.1 39 (5) 160 135 ± 0.821 741 ± 89.2 7.67 ± 13.0 40 (17) 406 148 ± 5.45 929 ± 153 6.20 ± 21.5 41 (24) 405 139 ± 4.62 870 ± 106 17.2 ± 19.7 42 (12) 330 113 ± 5.05 409 ± 31.6 -3.19 ± 12.7 43 (19) 332 154 ± 4.56 1061 ± 142 9.97 ± 21.2 46 (24) 311 153 ± 2.92 687 ± 51.9 -28.0 ± 8.90 48 (15) 300 151 ± 3.03 844 ± 72.3 -9.09 ± 9.00 Martin’s Female 13 (2) 2 261 181 ± 15.9 2181 ± 875 54.1 ± 93.2 Tank 19 (30) 720 179 ± 5.26 1405 ± 181 -3.37 ± 13.3 30 (10) 279 168 ± 1.05 1245 ± 168 1.02 ± 12.7 34 (1) 5 139 - 1098 - 47.4 - 52 (10)1 258 96.2 ± 2.60 148 ± 6.38 -46.9 ± 4.89 Male 22 (6) 131 106 ± 4.89 323 ± 16.3 -11.5 ± 9.18 24 (15) 663 118 ± 5.44 415 ± 45.7 -11.6 ± 18.9 Jarrah Forest Female 18 (4) 748 214 ± 14.6 3490 ± 394 49.4 ± 10.4 29 (5) 337 128 ± 2.17 707 ± 31.1 19.1 ± 10.5 32 (10) 616 182 ± 5.28 1872 ± 148 23.1 ± 16.1 54 (6) 266 122 ± 2.25 468 ± 12.7 -11.2 ± 3.10 55 (3) 202 123 ± 0.00 590 ± 4.62 9.71 ± 0.86 Male 20 (37) 485 131 ± 3.89 478 ± 69.8 -24.4 ± 14.6 21 (5) 63 117 ± 4.63 431 ± 33.7 -9.22 ± 7.14 26 (5) 85 130 ± 0.00 490 ± 23.4 -21.5 ± 3.75 35 (7) 316 130 ± 2.66 870 ± 17.7 39.8 ± 7.92 45 (19) 293 141 ± 4.57 1015 ± 130 33.7 ± 30.0 50 (10) 288 135 ± 5.04 612 ± 58.3 -11.1 ± 11.6 51 (11) 249 134 ± 4.94 621 ± 19.0 -7.86 ± 10.9 Total/average 46 (562) 335 ± 206 151 ± 27.9 1035 ± 570 2.29 ± 28.1

b. Body condition score

In snakes, body condition may reflect prey abundance. For example, the body condition of water pythons (Liasis fuscus) fluctuates annually in response dusky rat (Rattus colletti) abundance (their main type of prey), which fluctuate in their population abundance in response to varying rainfall. Annual fluctuations in rat numbers corresponded to varying python body condition; the more rats, the better the python’s body condition score (rates of female python reproduction and survival also corresponded with high rat abundance; Madsen et al. 2006). Similarly, it was expected that the body condition of M. s. imbricata may reflect prey availability, and is predicted to be influenced by fox baiting. The hypotheses tested were:

H3: Body condition score for pythons at LPCP (baited) will be higher than

the score for pythons at MT (unbaited).

H4: There will be a slower decline in body condition score (β-body condition

score) for pythons at LPCP compared with pythons at MT.

Differences in python body condition across treatment sites were quantified as residuals of body mass to SVL (Shine et al. 2001) from weekly (where possible) body measurements (Table 7.2). The expected body mass was derived as a function of SVL and expressed as the body condition score (BCS) as:

(observed M – predicted M ) Body condition score = b b x 100% predicted Mb

The relationship between Mb and SVL was not different between males and females and therefore, the same regression equation to calculate predicted Mb was used for all pythons (see Chapter 6 for details).

The β coefficients of BCS for each individual were calculated as the rate of change in its BCS over time. Three or more BCS over time were required to generate a β coefficient (Table 7.2).

The BCS of pythons in this study were clearly not static (Fig. 7.3) and a decline in BCS over time for the population was recorded, regardless of when individuals were captured. H3 was supported as the BCS for pythons at MT (average = 5.02 ± 35.3, n=7) was significantly lower than those recorded for pythons at LPCP (average = 8.21 ± 19.4, n=27; t442= -3.95, p<0.0001). H4 was, however, not supported, and contrary to the prediction as LPCP pythons (average β-BCS= -0.560 ± 0.333, n= 24) actually showed a steeper rate of decline in BCS than MT pythons (average β-BCS= -0.056 ± 0.543, n= 5; t23=2.77, p<0.05). The decline in body condition recorded was also experienced by jarrah forest pythons (average β-BCS= -0.687 ± 0.222, n= 10). There was no significant difference between sexes for either BCS (t442= 0.985, p>0.05) or β-BCS (t23= -1.23, p>0.05). It is important to note that pythons at all three study sites showed a decline in body condition over time. Additionally, pythons which were first captured later in the study (i.e. late 2007-early 2008) had a lower BCS than pythons which were first captured earlier in the study (ie. late 2006-early 2007; Fig. 7.3).

Over the period of monitoring, pythons at the coastal fox baited site were heavier, longer and had been in better body condition compared with MT pythons. However,

LPCP animals are declining in body condition at a faster rate compared with MT

89 animals. This suggests that prey availability is in decline at LPCP more than at MT, and may indicate the complexity of the mesopredator release response. Other predators, such as feral cats may have also been released from competition with foxes at LPCP and therefore the competition between cats and pythons may now be reducing the prey available to pythons.

Figure 7.3. Observations of declines in python body condition over time (y = -0.0375x + 1479, R² = 0.09). Body condition score was quantified as a residual of snout-vent length to body mass (Shine et al. 2001) for weekly capture records, where three or more data points for an individual were available. The length of the line for each animal reflects the length of time that it was monitored. The circles at the end of trend lines indicate the python was known to have died. The open diamonds at the start of trend lines indicate the python was captured after consuming a collared prey (Pseudocheirus occidentalis, n=12; Bettongia penicillata, n=1) or unknown prey (1 and 2). Number 3 indicates the very low BCS measured from March 2008 –November 2008 which were measurements from the only juvenile included in the study (Msi52).

c. Mortality

There is an interaction between predation risk (i.e. risk associated with spending more time foraging or hunting) and risk of starvation where an individual may increase its vulnerability to predation in order to avoid starvation (McNamara 1987). Additionally, prey availability will affect an individual’s feeding rate and therefore, a python’s body condition and ultimate survival. Three hypotheses were generated to compare mortality rates of pythons between fox baited and unbaited areas:

H5 A smaller number of deaths will occur at LPCP (baited) compared with

MT (unbaited).

H6 There will be a smaller number of deaths due to emaciation in LPCP

(baited) compared with MT (unbaited).

H7 Smaller pythons are more likely to die from emaciation as they have less

body-reserve capacity compared to larger pythons.

A high rate of python mortality was recorded throughout the radiotelemetry monitoring period. Contrary to predictions of the MRH (H5), the highest rate of mortality was recorded at the fox baited site (LPCP). The highest mortality rate was found at LPCP where 11 of the 27 (41%) individuals died (Table 7.3). Of the 7 pythons monitored at

MT, one python died (14%), and of the 12 pythons monitored in the jarrah forest, one python died (8%; Table 7.3). Two pythons at LPCP (baited) were believed to be predated by foxes (one confirmed, one unconfirmed). It is not certain if the pythons died due to direct predation, or were scavenged following death. The carcass of python

Msi16 was found within 1-2 days following death. DNA swabs were taken from bite marks left on the radiotransmitter and on the python’s remains and the

91 predator/scavenger was confirmed to be a red fox (Berry Pers. comm)3. The second carcass (Msi31) was found in a more decayed state (python was known to be alive approximately 6 days prior) and no swabs could be taken, however, the pattern of predation was the same as Msi16 carcass, with the head, tail and a small part of the mid- section consumed. The only juvenile (Msi52) monitored in the study was opportunistically found at MT in the process of being attacked by a Varanus gouldii lizard; when approached the lizard retreated. The juvenile survived the attack and was monitored until the conclusion of the study.

Two animals had no cause of death positively identified (Msi23 was found within an adequate time enough for post mortem investigation, but Msi39 was not). The python that died at MT (Msi13) became trapped and drowned in an illegally placed water tank

(therefore not a ‘natural cause’).

For which a rate of body condition decline (β-BCS) could be calculated (10 pythons), eight, were declining in BCS prior to death (Table 7.3). Additionally, post mortem analysis suggested that five of the dead animals were suspected to be suffering malnutrition and/or dehydration before death. Poor body condition may have also been implicated in the death of the two animals that were predated (or scavenged) since poor condition may have made them more susceptible to predation if mobility was reduced.

Despite these findings a comparison of the slope (β coefficients) for BCS over time calculated for each individual revealed that overall, the animals that died (average β-

BCS =-0.532±0.378) were not declining in body condition at a significantly faster rate compared with animals that were still alive at completion of this study (average β-BCS

3 Oli Berry, 2010. Personal communication. University of Western Australia and Invasive Animals Cooperative Research Centre 92

=-0.631±0.387; t38=0.731, p>0.05). Therefore the entire population was in declining body condition over the course of this study.

Only one juvenile was included in this study, and 45 were adult. Pythons that had died during the study (average body mass=1120 ±604 g) were marginally heavier than pythons known to be alive at the conclusion of the study (average body mass=1025

±564 g; analysis carried out on Log10-transformed values; t35=-1.60, p>0.05 (p=0.059);

Fig. 7.4). Therefore, the prediction that smaller pythons would more likely suffer mortality from emaciation compared to larger pythons (H7) was not supported.

Table 7.3. Thirteen mortality events of carpet pythons were recorded during this study. The majority were recorded in Leschenault Peninsula Conservation Park (LPCP), with only one death at Martin’s Tank (MT) and at the jarrah forest (JF) site. The change in body condition (β-BCS) values represent the slope of body condition scores over time for each individual expressed as the % change in body condition score (% loss per day). Cause of death: post-mortem examinations (PM) carried out (Yes=Y, or No=N) by Murdoch University Pathology Department (see Appendices for individual python post mortem reports); DNA fox indicates the results of molecular analysis of swabs of the python carcass and bite marks on the radiotransmitter. *Drowning due to entrapment within an unlawfully placed water tank. Study Animal site ID Sex Reproductive state Β-BCS Year Season PM Suspected or confirmed cause of death LPCP Msi16 F Non-reproductive -0.72 2007 Spring N Predation/scavenge (DNA fox) LPCP Msi4 F Successful incubation of 14 eggs -0.78 2007 Spring N Post parturition malnutrition LPCP Msi23 M 0.167 2007 Spring Y Unknown LPCP Msi36 F 10 developing ova at death 0.047 2008 Summer N Malnutrition/dehydration LPCP Msi39 M -0.95 2008 Summer N Unknown LPCP Msi14 F Successful incubation of 8 eggs -0.74 2008 Summer Y 1. Cloacal obstruction and pre-obstruction putrefaction of intestinal contents. 2. Membranous glomerulonephritis. LPCP Msi44 F Non-reproductive -0.96 2008 Summer N Malnutrition/dehydration LPCP Msi47 F Non-reproductive - 2008 Summer N Malnutrition/dehydration LPCP Msi31 M -0.91 2008 Autumn N Predation/scavenge (unknown predator) LPCP Msi38 M -0.6 2008 Autumn Y Possible radiotransmitter trauma (unanchored radiotransmitter) LPCP Msi9 F Non-reproductive -0.6 2008 Winter Y Malnutrition MT Msi13 F Assumed successful incubation - 2007 Autumn N Drowning * (eggs not recovered) JF Msi20 M -0.9 2008 Winter Y Neurological issues/malnutrition

5 Alive Dead 4

1 Number of animals Number

0 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 Log10 body mass (g)

In summary, the results of this study contradict all three hypotheses for mortality differences between baited and unbaited areas. The only one (and possibly two) predation events of pythons by foxes occurred at LPCP, the fox baited site, and in the absence of fox baiting (MT), only one python died, and this was due to from an unnatural cause (drowning). At LPCP, malnutrition (and/or dehydration) identified as the cause of death of the majority of individuals, a pattern opposite to what is predicted under a mesopredator release response (where prey population is predicted to increase following apex predator control or removal). Larger pythons showed marginally greater mortality, but the converse was predicted where smaller animals would have had fewer body reserves.

d. Population demographics

The growth trajectory of an animal is determined partly by its genes, and partly by the nutritional environment that it encounters (Madsen and Shine 2000). Determinate

95 growers (most endotherms) attain maximal body sizes early in life and therefore display very different growth trajectories compared to indeterminate growers (most ectotherms), where growth continues throughout life (albeit at a slower rate with increasing age/size;

Madsen and Shine 2000). Prey availability throughout an indeterminate grower’s life can strongly influence individual growth trajectories and, for this reason, it is very difficult to determine the age of many snake species from size parameters (Madsen and

Shine 2000; Taylor and DeNardo 2005).

Morelia spilota imbricata males >88 cm SVL were considered adults by Pearson et al.

(2002b). In this study, the smallest male found copulating with a female measured 106 cm SVL (Mb: 373 g). The smallest female known to be reproductive (gravid) averaged

172 cm SVL and 1,318 g, and was 20 cm smaller than Pearson (2002b) recorded as the minimum reproductive size on Garden Island (195 cm SVL, 4,205 g when gravid and

2,907 g after oviposition).

Wilson et al. (2006) found male tropical pythons (Morelia viridis) reach sexual maturity at 84 cm SVL at 2.4 ± 0.8 years of age, whilst females reach maturity later (3.6 ± 1.0 years) at 99 cm SVL. These pythons reach a maximum SVL of 1,284 cm for males and

1,420 cm for females and the authors predicted that these individuals may reach up to

19 years of age. There is no similar growth rate or age data for M. s. imbricata to draw upon. However, using the M. viridis growth rates as an approximation, it is likely that the M. s. imbricata population under study were well over two years of age given their recorded body measurements (Table 7.2; except for the one juvenile python Msi52 at

MT). In the presence of fox baiting it was predicted that:

H8: There will be a faster growth rate (increase in SVL over time) at LPCP

(baited) compared to MT (unbaited)

The change in SVL for each python (β-SVL) was calculated for the duration that each animal was studied. There was no significant difference in the rate of change in SVL between LPCP and MT pythons (t37 = 0.177, p>0.05), however, male pythons (average

β-SVL = 0.696) increased in SVL at a significantly faster rate compared with females pythons (average β-SVL = 0.522; t37 =2.14, p<0.05; Fig. 7.5).

As there is clear sexual size dimorphism in this species, the range and number of prey available may be a factor in the differing growth rates (β-SVL) between the sexes.

Slower SVL increases in female pythons may be due to reduced numbers of larger prey, whereas male pythons may be able to take smaller prey more frequently. However, research has shown that even in captivity M. s. imbricata male pythons will stop feeding and consequently slow or stop growth once they have reached adult size, suggesting that their smaller size compared with adult female pythons reflects genetic control, rather than local prey availability (Pearson et al. 2002b).

210

Female Male

190 LPCP

170

150 SVL(cm)

130

110

90 07 07 08 08 06 06 07 07 07 08 08 08 07 07 08 08 07 08 07 08 07 08 07 08 07 08 ------Jul Jul Jul Oct Oct Apr Apr Jan Jan Mar Mar Jun Jun Feb Feb Nov Dec Nov Dec Nov Sep Sep Aug Aug May May

e. Density

Pythons are capital breeders, requiring accumulated energy reserves before females can

produce a single clutch of eggs (Bonnet et al. 1998). As for most snake species, male

M. s. imbricata do not care for the offspring, nor do they engage in energetically or

dangerous pre-mating activities, so their main energy expenditure for reproduction is for

the production of gametes and energy spent searching for mates (Aubret et al. 2002).

In contrast to males, female reproduction requires a considerable “start up” investment

(Aubret et al. 2002). There are substantial energetic costs to female snakes during both

ovulation and development of offspring (i.e. incubation if oviparous or gestation if

98 viviparous; Aubret et al. 2002). Female pythons may lose a significant amount of body mass during egg-incubation due to lost feeding opportunities and the metabolic costs associated with keeping eggs at an elevated temperature. The average post-parturition incubation time for M. s. imbricata (average clutch size is between 6-15 eggs; Table

7.4) under natural conditions is 58-78 days (Pearson 2002). High levels of female mortality after parturition have also been reported for vipers. Aubret et al. (2002) found that only 75% of female vipers (Vipera aspis) survive through the year following parturition and all females are extremely emaciated following incubation. Similarly, female M. s. imbricata pythons are known to lose up to 31% of pre-reproductive weight

(based on captive measurements and data from two wild pythons; Pearson 2002) and female M. s. spilota can lose approximately 44% (Slip and Shine 1988).

During the present study, in addition to regular body measurements through regular radiotelemetry monitoring, females were palpated for developing ova at every capture and all mating and possible mating events and incubation behaviour were recorded

(Table 7.4). Given the significant metabolic investment female M. s. imbricata require to successfully hatch a clutch of eggs, the following predictions were made based on the

MRH:

H9 Female pythons at LPCP (baited) will have a higher frequency of

oviposition, larger clutch sizes, and better hatching success compared

with female pythons at MT (unbaited).

H10 LPCP (baited) reproductive females will maintain a better body

condition over time than reproductive female pythons at MT (unbaited).

A limited number of females in the study were observed to have produced a clutch of eggs: 4 of 15 females at LPCP, 1 of 5 females at MT (Table 7.4), and none of the 5 females in JF. Another two pythons at LPCP were known to be gravid, however, one of these died while gravid, the other expelled her radiotransmitter within 20 days following release prior to observing oviposition (Table 7.4). An additional mating event was recorded for a female (Msi44) at LPCP but the animal died and no eggs were produced

(Table 7.4). There was also a possible mating event for one female (Msi37) at LPCP as two males were found within 1-3 meters of her suggesting the female was receptive

(Pearson 2002), but no eggs were produced (Table 7.4). No females were found to produce more than one clutch of eggs over the study duration, and there was very high mortality for females that did produce a clutch of eggs, with only one of five females known to have been gravid or oviposited surviving post-parturition (the fate was unknown for one female). There was no indication that the proportion of reproductive females differed between baited (27% of females) and unbaited sites (20% of females).

Retrieval of the egg-clutches was difficult so a comparison of clutch size and success between baited and unbaited sites was undetermined.

100

Table 7.4. Reproduction and observed mating events for female pythons at the baited (LPCP) and unbaited (MT) study sites.

Study site Female ID Reproductive status Fate Date Observations Body mass ± S.D.(g) β-BCS Leschenault 47 Non-reproductive Dead 3012 - - Peninsula 44 Mating only Dead 12/11/2007 3 males (Msi41, 42 & 43) coiled around female Msi44 in hollow log. No eggs produced. 2410 ± 539 -0.947 Conservation 11 Non-reproductive Unknown (TM) 2125 ± 0.00 - Park 1 Oviposition Unknown (TM) 14/03/2006 Msi1 seen with 3 hatching pythons following incubation of ~8 eggs. 2083 ± 291 -0.634 (BAITED) 37 Mating (possible) only Alive 30/08/2007 2 males (Msi39 & 40) found 1-3m from base of Spiridium sp. bush where Msi37 was basking. 2077 ± 294 0.347 No eggs produced. 16 Non-reproductive Dead 1765 ± 139 -0.166

36 Gravid Dead 12/12/2007 Msi36 found dead with 10 developing ova on post-mortem dissection. 1630 ± 173 -0.374

14 Oviposition Dead 23/11/2006 1 male (Msi25) found coiled together with Msi14 under exposed tree-root. Msi14 incubated a 1612 ± 370 -0.749 clutch of 8 eggs between 4/1/2007-27/2/2007. 14 Oviposition Dead 1/12/2007 1 male (Msi46) found submerged in water directly beneath where Msi14 was basking on a branch. No eggs produced. 14 Oviposition Dead 12/12/2007 1 male python (Msi48) found coiled around Msi14 on the ground. No eggs produced. Found dead 2/2/2008 4 Oviposition Dead 13/02/2007 Python incubated a clutch of 14 eggs between 13/2/2007 – 22/3/2007, but found dead on 1380 ± 67.1 -0.784 9/10/2007. 49 Non-reproductive Alive 1337 ± 50.4 -0.182

27 Gravid Alive 14/12/2006 Msi27 known to be gravid with 6 developing ova. Python expelled radiotransmitter on 1318 ± 173 - 4/1/2007 before oviposition was recorded. Python recaptured alive on 11/11/2008. 10 Non-reproductive Alive 1299 ± 153 -0.696

9 Non-reproductive Dead 1280 ± 54.8 -0.662

33 Non-reproductive Alive 970 ± 138 -0.757

53 Non-reproductive Alive 715 ± 17.9 -0.736

Martin’s tank 13 Oviposition Dead 15/11/2006 3 male pythons found coiled around female on exposed soil, next to a fallen hollow log. Msi13 2181 ± 875 - (UNBAITED) (and mating) incubated clutch of eggs. Eggs were not recovered from inside incubation site (hollow log). 19 Non-reproductive Alive 1406 ± 181 0.064

30 Non-reproductive Unknown (TM) 1245 ± 168 0.835

34 Non-reproductive Unknown (TM) 1098 - -

52 Juvenile Alive 148 ± 6.38 -0.262

101

7.9. Diet diversity

One of the main predictions of mesopredator release hypothesis is that removal of an apex predator (in this example, the red fox) will release the predation pressure on prey species allowing the prey to increase in density; mesopredators will then have greater access to prey. This hypothesis was examined for M. s. imbricata by testing whether:

H11 Pythons will consume a greater diversity of prey at LPCP (baited)

compared to MT (unbaited).

A combined species accumulation curve (the number of additional prey species ranked in chronological order) for scats collected at both baited and unbaited study sites indicated that the number of prey species was reaching a plateau (Fig. 7.6). However, more python scat samples are required to capture the variation of prey species available to pythons, particularly for the unbaited site (MT; Fig. 7.6). Of 39 scat samples taken from LPCP, and of 14 scat samples at MT, a range of species and body mass of prey were identified (see Chapter 5 for more detail on prey body mass) with some species not represented at both sites, i.e. common brushtail possums were only represented at the baited site (LPCP) and dasyurids at the unbaited site (MT; Fig. 7.7). However, analysis of the species diversity of the prey found in python scats as measured by both the

Simpson’s diversity index (1/D) and the Shannon-Wiener Diversity index (H’;

Ecological Methodology, Exeter Software Version 6.1, 47 Route 25A, Suite 2, Setauket,

NY 11733-2870 USA) was very similar and relatively high in diversity of prey consumed across the baited (1/D= 0.834, H’=2.778) and unbaited site (1/D= 0.821,

H’=2.288). Therefore, the hypothesis (H11) that pythons at the baited site would consume a greater diversity of prey was not supported.

102

Number of prey species of prey Number Combined coastal sites 4 LPCP (baited)

MT (unbaited) 2

0 1 6 11 16 21 26 31 36 41 46 51 Number of scats collected

Figure 7.6. A species accumulation curve where the prey species identified in the scats were sorted chronologically.

a. LPCP (fox-baited), n=39 Dasyurids Rodents

Rabbit

Reptiles

Parrot

Feral cat

Commom brushtail possum Western ringtail b. MT (unbaited), n=14 possum

103

7.10. Behavioural avoidance

In addition to directly competing with each other, predator species may conflict through direct intraguild aggression or exploitation (Durant 2000). Even when mortality due to predation is low, predator avoidance is likely to play a strong role in structuring species communities. Foxes can kill pythons: at LPCP red fox DNA was confirmed on a python carcass at LPCP (Table 7.3). Cats may also be able take small or juvenile pythons. In a recent study 4 out of 51 cat stomachs analysed (cats were killed for control purposes across a range of locations in southwest Western Australia) contained snakes, each one a different elapid species (Crawford 2010). Adult pythons may have a better chance at defending themselves against predation however, during winter when ectotherm body temperatures are low, python flight or fight responses will undoubtedly be hindered (Chapter 6). Finally, there was no evidence that pythons take foxes, but a python was found to have consumed a cat at LPCP (Chapter 5).

The mesopredator release hypothesis suggests that competitive interactions between predators may cause behavioural avoidance of each other in two main ways. Firstly, through temporal segregation, by altering activity times to avoid the intraguild killer.

Secondly, through habitat/landscape-mediated avoidance to avoid sites where predators are likely to be (Sergio et al. 2007). For ectothermic species, a third way seems apparent; predators may influence the thermal biology of an ectotherm. For example, ectotherms may alter their thermoregulatory behaviour by altering their use of the landscape by varying their body positions due to the perceived presence of predators.

104

a. Temporal segregation

Foxes are primarily nocturnal hunters (Catling and Coman 2008), cats appear to have flexible hunting patterns (Denny 2008), while pythons are ambush hunters and feed on both diurnal and nocturnal prey species (Chapter 5). In the presence of fox-control, mesopredators may be released from competition from foxes and adjust the times they can hunt for prey.

H12 Pythons at LPCP will feed (and ambush hunt) on more nocturnal prey

compared to pythons at MT that may hunt more on diurnal prey to avoid

nocturnally-active foxes.

This prediction that pythons at LPCP (baited) may exploit more nocturnal prey than at

MT (unbaited; H12) was not supported as there was no difference in the proportion of

2 nocturnally-active prey consumed by pythons at the sites (χ 1= 1.81, p>0.05; Fig. 7.8).

Therefore it does not seem apparent that pythons temporally alter their hunting behaviour to avoid foxes.

105

Fox-baited (LPCP) Unbaited (MT)

7% 21% MAMMAL (Nocturnal) MAMMAL (Nocturnal) 34% REPTILE (Diurnal) REPTILE (Diurnal) 59% BIRD (Diurnal) BIRD (Diurnal) 79%

b. Habitat/landscape-mediated avoidance

The presence of foxes may also influence microhabitat selection by mesopredators.

Different microhabitats provide varying levels of protection or crypsis from foxes (and other predators). For example, tree hollows are likely to provide the greatest protection while bare ground provides very little. It was therefore predicted that:

H13 Pythons at LPCP (baited) will spend less time in cryptic microhabitats

compared to pythons at MT (unbaited).

H14 A smaller proportion of LPCP (baited) pythons will use tree hollows

compared with pythons at MT (unbaited).

H15 Pythons at LPCP (fox baited) will spend a shorter period of time

sequestering in tree hollows over winter compared with pythons at MT

(unbaited).

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Six main microhabitats were ranked from least protected from predation by foxes to the most protected: 1. ground cover, 2. vegetation cover, 3. burrow, 4. hollow log, 5. tree branch, 6. tree hollow (see Chapter 2 for more detail).

There was significant differences between the proportion of pythons found across

2 microhabitats (χ 5= 20.5, p<0.001), where LPCP pythons were found more often in

2 2 vegetation cover (χ 1= 6.01, p<0.05) and on tree branches (χ 1= 4.44, p<0.05) compared with MT pythons. However, MT were seen using tree hollows, the most cryptic

2 microhabitat available to pythons, more often than LPCP pythons (χ 1= 11.2, p<0.001;

Fig. 7.9).

Pythons spent a significant proportion of the year (up to 7 months) sequestered in tree hollows with no or little activity, over the winter period including April – October

(Chapter 6). Over 56% of pythons (including jarrah forest animals) sequestered in tree hollows for some duration over winter. For pythons able to be monitored over a winter period, 45% of pythons at LPCP were tree hollow (TH) users (n=10), whereas at MT,

100% of pythons were TH users (n=4). There was no significant difference for the length of time pythons spent sequestered in tree hollows over winter between sites (t12=

-0.143, p>0.05) or between female and male pythons (t12= -0.143, p>0.05). There is no obvious thermal advantage of using tree hollows as a winter roost site and therefore use of tree hollows is attributed to predator-avoidance behaviour (Chapter 6). Pythons inside tree hollows maintain a lower body temperature, forfeiting thermoregulation though basking, compared with pythons that remained in the open vegetation. Over winter pythons inside tree hollows may gain better protection from predators such as

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100% More cryptic

80%

60%

40% % of observations 20%

Less cryptic 0% Fox-baited (LPCP; n=30) Unbaited (MT; n=6) Ground cover * Vegetation cover Burrows Hollow log * Tree branch *** Tree hollow Figure 7.9. Morelia spilota imbricata animals at the unbaited site (MT) were observed proportionately more in tree hollows and less on tree branches and using vegetation cover compared with pythons at the fox baited site (LPCP). Data were compared by chi-square analysis (* p<0.05, *** p<0.001).

foxes and cats than pythons remaining exposed, in open vegetation, where their flight or fight response would be hindered by low body temperatures (Chapter 6).

c. Altered use of the landscape through varying body positions

Ectotherms adjust their body temperatures by behavioural means. For example, they may move to warmer microhabitats (e.g. rocks: thigmothermy; Huey et al. 1989) or by altering their body position. When pythons alter their body position, they change their surface area exposed to the environment (e.g. lying stretched out with a high surface area to mass ratio, or coiling up reducing their surface area to mass ratio) and can modify the rate of heat exchange (both heating and cooling) with their immediate environment (Seebacher et al. 1999). An ectotherm’s thermoregulation behaviour may also influence its risk of predation. While basking, pythons either stretch out and

108 expose most of their body to the sunlight, but in doing so will also expose themselves to predators, or they lie in loose or very loose coils under filtered sun. To conserve heat gained through basking they will coil to reduce the surface area of their body exposed to the environment, often doing this under cover of vegetation, thereby reducing their potential exposure to predators. The hypothesis generated to compare python temporal segregation behaviour in areas under fox control and without states:

H16 Pythons at LPCP (baited) will spend more time in exposed body

positions compared to pythons at MT (unbaited).

Comparison of the percentage of observed python body positions between study sites suggests that pythons did alter their body positions in response to the presence or

2 absence of foxes (χ 4= 9.68, p<0.05). However, contrary to the prediction, python’s at the unbaited site were observed more in the most exposed body position (stretched) than

2 at the baited site (χ 1= 5.85, p<0.05; Fig. 7.10).

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100% More cryptic

80%

60%

40% % of observations

20%

Less cryptic 0% Fox-baited (LPCP; n=30) Unbaited (MT; n=6)

* Stretched Very loose coil Loose coil Coil Tight coil

CONCLUSIONS TO MESOPREDATOR RELEASE RESPONSE

Most studies analysing the mesopredator release hypothesis have examined endotherms, using sightings or captures to quantify a numerical response of mesopredators to the absence of an apex predator (e.g. Crooks and Soule 1999; Gehrt and Prange 2007). The cryptic nature of Morelia spilota imbricata made it impractical to monitor changes in population density as they are not enticed into traps. Therefore, to quantify and compare python responses to removal of foxes, focal animal study of individual pythons was carried out at sites that were baited for foxes and areas that were not baited for foxes. This approach enabled comparison of python survivorship, body condition, mortality, growth rate, reproduction, diet, and behavioural differences of habitat use, foraging and body positions. However, only six of the fifteen hypotheses generated to test whether pythons experience a mesopredator release response was supported by 110 these data while four hypotheses showed results that are contrary to a mesopredator release response to fox baiting (Table 7.5).

As ectotherms, pythons may be expected to show a slower functional response to an increase in prey density compared with endothermic predators, due to their long prey handling time and reduction of feeding over winter. There was no difference in the diet diversity consumed between baited and unbaited sites, however, prey availability data had not been made available in order to specifically analyse whether the prey consumed by pythons reflected the prey available to the pythons at both sites. LPCP pythons were also on average larger (measures of body mass and body length) as would be expected if pythons were functionally responding by consuming more prey. Contrary to expectations, pythons at the fox baited site showed a greater rate of body condition decline than pythons at the unbaited site. Data gathered on python body condition was unable to be directly compared with prey availability data at these sites. These data do however, suggest that pythons have either reached their carrying capacity, or imply there has been a major reduction in prey density over a short period of time (1-3 years) during which pythons were monitored in this study.

Pythons will also numerically respond slower to an increase in prey density compared with endothermic predators. This study recorded low numbers of clutch production and successful incubation of offspring and additionally many of the reproductive females died within the year following post-parturition. The investment in body reserves required by female pythons to reproduce and the high mortality risk following parturition is greater than income breeders (most endotherms; Nowak et al. 2008). In addition to high mortality rates following post-parturition, pythons at the fox baited site had higher mortality largely identified as due to emaciation (less to predation) than

111 pythons at the unbaited site. It is not possible to quantify a numerical response to increasing prey abundance in pythons, but these survivorship and reproduction data suggest that the python population at LPCP (baited) was declining over the course of the study and suggest pythons have reached or exceeded the carrying capacity at LPCP, or that prey density has actually declined over recent years.

There was minimal support for a behavioural response to avoid the apex predator (fox;

Table 7.5). Pythons did not alter their foraging behaviour to hunt more on diurnally active prey at the unbaited site to avoid nocturnally active foxes. However, pythons at the unbaited site were seen to use the most cryptic microhabitat (tree hollows) more than pythons at the baited site. A proportionately higher number of pythons did however, use tree hollows and for longer over winter at the unbaited site compared to the baited site. These results may not simply reflect direct avoidance behaviour of pythons at the unbaited site as there may be a discrepancy in the availability of tree hollows or seasonal temperature differences between study locations. Tree hollow use may indicate a preference over the colder months of the year for this protected microhabitat, as the flight or fight response by a python to a predator would be hindered as a result of lower body temperatures at this time (Chapter 6). However, in contrast to predictions for landscape-mediated avoidance (to avoid potential exposure to foxes), pythons at the unbaited site were seen more often in the most exposed body position

(stretched) compared to pythons at the baited site, but was the only body position that differed between sites. It should be acknowledged here that this study was only able to make comparisons between the coastal baited and unbaited study sites and therefore no replication over other areas was possible. Any number of differences across sites, such as vegetation, climate or prey abundance and availability could have influenced the findings and therefore all interpretations should be viewed with this understanding.

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Understanding mesopredator release of pythons to a fox control program is further complicated by a number of factors. Firstly, is the presence of an endothermic mesopredator (the cat), which is also believed to have responded to the removal of the red fox (de Tores Pers. comm)1. Secondly, is the complexity of studying a dynamic process at a single point in time. Thirdly, is the existence of other predators are also present at these sites (e.g. varanid lizards). Fox baiting was commenced at LPCP almost 20 years previous to this study and in the presence of fox baiting, fox numbers at

LPCP are believed to have fallen and remained low over this time (Fig. 7.11). The population size of western ringtail possums (a prey species that has been monitored to some degree during translocations into these study sites) and the hypothesised numerical impact of cats and pythons is shown in Fig. 7.11. By contrast with endotherms (e.g. cats), ectotherms such as snakes have lower metabolic rates and shorter activity periods. In response to fox baiting, it is therefore likely that cats and pythons will demonstrate very different rates of numerical and functional responses to prey population increase.

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Actual outcome: Parameter measured Support for MT LPCP hypothesis Fox density ↔ Fox density ↓ 1. Python survivorship 1.1. Body mass and size H1: Pythons at LPCP will be heavier than pythons at MT Yes H2: Pythons at LPCP will be longer than pythons at MT Yes 1.2. Body condition H3: Body condition score for pythons at LPCP will be higher than the score for pythons at MT Yes H4: There will be a slower decline in body condition score (β-body condition score) for pythons at LPCP compared with No ↑ decline at LPCP pythons at MT 1.3. Mortality H5: A smaller number of deaths will occur at LPCP compared with MT No ↑ deaths at LPCP H6: There will be a smaller number of deaths due to emaciation in LPCP compared with MT No ↑ deaths at LPCP H7: Smaller pythons are more likely to die from emaciation as they have less body-reserve capacity compared to larger No ↑ death for larger pythons pythons 1.4. Population demographics H8: There will be a faster growth rate (increase in SVL over time) at LPCP compared to MT No Not different 2. Python density H9: Female pythons at LPCP will have a higher frequency of oviposition, larger clutch sizes, and better hatching No Not different success compared with female pythons at MT H10: LPCP reproductive females will maintain a better body condition over time than reproductive female pythons at MT Undetermined 3. Python diet diversity H11: Pythons will consume a greater diversity of prey at LPCP compared to MT No Not different 4. Python predator behavioural avoidance 4.1. Temporal segregation H12: Pythons at LPCP will feed (and ambush hunt) on more nocturnal prey compared to pythons at MT that may hunt No Not different more on diurnal prey to avoid nocturnally-active foxes 4.2. Habitat-mediated avoidance H13: Pythons at LPCP will spend less time in cryptic microhabitats compared to pythons at MT Yes e.g. ↑ tree hollows use at MT H14: A smaller proportion of LPCP pythons will use tree hollows compared with pythons at MT Yes H15: Pythons at LPCP will spend a shorter period of time sequestering in tree hollows over winter compared with Yes pythons at MT 4.3. Landscape-mediated avoidance H16: Pythons at LPCP will spend more time in exposed body positions compared to pythons at MT No ↑ at MT

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The number of python predation events of radiotracked western ringtail possums (the only prey species monitored extensively over this time) increased dramatically over the first 15 years after fox baiting commenced at LPCP (Fig. 7.2): a functional response to an increase in prey availability at the fox baited site. As a result of consuming more prey (functional response), pythons most likely demonstrated a numerical response through increased reproduction and lower mortality. However, because the reproductive rate of pythons is reasonably slow, it is predicted that this response would take a long time to manifest, and could also be impacted by the increasing number of cats present.

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Cats have may have responded extremely rapidly to fox baiting, both functionally and numerically. Cats are efficient predators with flexible foraging and hunting strategies and therefore they are predicted to have a fast functional response to increased prey availability. Cats are income breeders that reach sexual maturity when individuals reach 80% of their adult body size; before 9 months for males and between 6-9 months for females (Feldman and Nelson 2004). Adult females are also polyoestrous, and often have two litters per year with around 4-6 kittens in each litter (Feldman and Nelson

2004). Cats will also mate with different males (often repeated mating events), increasing the chance of spontaneous ovulation and fertilization (Feldman and Nelson

2004). Therefore, their numerical response to increased prey density will be far more rapid compared with pythons.

It is unlikely cats show direct intraguild predation to adult pythons, and a python had consumed a cat at LPCP (Chapter 5). However, it is likely that pythons are subjected to exploitative competition with cats. An explanation as to why this study did not demonstrate pythons to be currently showing a mesopredator release response, may be due to the interactions between pythons and other mesopredators, such as the feral cat, in this system (Table 7.6).

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Table 7.6. Predicted responses of pythons to fox baiting at Leschenault Peninsula Conservation Park where it is likely that feral cats have shown a mesopredator release response and increased in density and there has been exploitative competition between the mesopredators.

Predicted response for each Parameter measured measured parameter Fox density ↓, Cat density ↑ 1. Python survivorship ↓ 2. Python density ↓ 3. Python diet diversity ↓ 4. Python predator behavioural avoidance: 4.1. Temporal segregation ↔ or ↓ 4.2. Habitat-mediated avoidance ↔ or ↓ 4.3. Landscape-mediated ↔ or ↓ avoidance

The second complication with the system under examination is the length of time over which the responses have taken place. As the responses by mesopredators to the removal of the apex predator are dynamic, the time frame of the study becomes extremely important. Both cats and pythons have most likely demonstrated a mesopredator release response to increased prey density under the apex predator removal at LPCP over the first 10 years following the introduction of fox baiting.

However, it is likely, given the evidence collected from this study (high rates of body condition decline, mortality and low reproductive output), that pythons have reached a carrying capacity at LPCP, perhaps due to the presence of feral cats, and may currently be experiencing a population decline. It is predicted that exploitative competition with feral cats in the system has been detrimental to the python population.

A final complication stems from a recent published study that suggests a global pattern of decline in snake populations (Reading et al. 2010). Populations of snake species which demonstrated relative population stability up until 1998, have subsequently declined. Eleven of 17 species studied experienced a steep decline in numbers over the following four years with no subsequent sign of recovery (Reading et al. 2010). The 117 authors suggest the declines are likely be multi-faceted and may include habitat quality decline, decreased prey availability, but could also imply a common underlying cause such as a response to climate change or disease. Reduction of habitat quality and prey availability is likely to contribute to the decline of M. s. imbricata measured in this study. Prey decline or python access to prey may have been hindered due to feral cats competing for the resource. Tree hollows were found to be an important resource for pythons across all study sites, but again, pythons are likely to be experiencing aggressive competition for these important resources.

Without having a complete understanding of fox, cat and prey numbers since fox baiting began, a retrospective investigation of whether there has been a mesopredator release response of pythons is difficult and has many caveats. Few data support the MRH for the south west carpet python in the system studied, although this may be due to the presence of a competing mesopredator (the feral cat), the dynamic nature of the system under study, or general declines in snake population health over recent years. The impact that large ectothermic predators, such as pythons, may have on prey populations should not be underestimated. Failure to acknowledge, by way of monitoring these predators during management programs (e.g. feral predator control), particularly in the presence of other mammalian predators, may prove to have undesired, long term consequences to prey and ectothermic predator popu lations.

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8. SYNTHESIS OF THE THESIS

This study attempted to quantify the impact of removal of an apex predator (the red fox) upon a terrestrial ectothermic species (southwest carpet python). It used focal animal radiotelemetry monitoring to investigate the mesopredator release hypothesis for

Morelia spilota imbricata. Radiotransmitters were surgically implanted and a new implanting technique successfully retained all devices, without adverse health effects

(analysed through haematological and biochemistry investigation). This study has examined possible behavioural, functional and numerical responses of M. s. imbricata to apex predator control to assess if pythons showed a mesopredator release response.

The biology, physiology and behaviour of the pythons was measured and tested across fox baited and unbaited sites in coastal southwest Western Australia. Over the duration of this study, there was a not a directly attributable mesopredator release response shown by pythons. However, exploitative competition with feral cats and the duration of fox baiting at LPCP prior to the commencement of this study, has most likely confounded the response by pythons. This study demonstrates the importance of understanding the ecology of ectothermic predators within an ecosystem. This understanding is essential to predict the response of ectotherms to changed management regimes, such as apex predator control, as their total response will undoubtedly be very different to endotherms.

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Whitford, K.R., and Williams, M.R. (2002) Hollows in jarrah (Eucalyptus marginata) and marri (Corymbia calophylla) trees: II. Selecting trees to retain for hollow dependent fauna. Forest Ecology and Management 160(1-3), 215- 232.

Wilson, D., Heinsohn, R., and Wood, J. (2006) Life-history traits and ontogenetic colour change in an arboreal tropical python, Morelia viridis. Journal of Zoology 270(3), 399-407.

Wilson, R.P., and McMahon, C.R. (2006) Measuring devices on wild animals: what constitutes acceptable practice? Frontiers in Ecology and the Environment 4(3), 147-154.

Wojtaszek, J.S. (1992) Seasonal changes of circulating blood parameters in the grass snake Natrix natrix natrix. Comparative Biochemistry and Physiology Part A: Physiology 103(3), 461-471.

Yodzis, P. (2001) Must top predators be culled for the sake of fisheries? Trends in Ecology & Evolution 16(2), 78-84.

Zavaleta, E.S., Hobbs, R., J., and Mooney, H.A. (2001) Viewing invasive species removal in a whole-ecosystem context. Trends in ecology and evolution 16(8), 454-459.

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10. APPENDICES

136

APPENDIX 1. POST MORTEM REPORTS FOR PYTHONS

10.1. Post-mortem report for python Msi9 ‘Kerry’

137

School of Veterinary & Biomedical Sciences South St, Murdoch Western Australia 6150 Telephone: (61-8) 9360 2356 Facsimile: (61-8) 9310 4144 Duty Pathologist: 04202 77743 ANATOMIC PATHOLOGY NECROPSY REPORT

Pathology No: 08 916 Date In: 16 Jun 2008 Pathologist: Dr PK Nicholls Owner’s Details: Consulting Veterinarian: Dr Kris Warren 01.26.21.0097.2.XXXXX.01023 Date of Consult: 16/06/2008 C/- Kris Warren Vet Clinical Sciences Murdoch University WA 6150 Ph: Fax To: Patient’s Details: Species: Snake Breed: SW carpet python Clinic Number: 101144 (imbricata) Name: Anatomic Pathology Gender: F D.O.B: Current Age: Adult

History: Python – recapture from earlier study (2005) in Feb 2008. Radio transmitter implanted and sutured to ribcage. Python released back into field and monitored weekly. Python condition not improved – under weight since capture. No evidence of meals. Python found very lethargic, pupils dilated and found dead the next morning. Died 13 June 2008 early morning.

Submission: Cadaver in insulated container Status: Post natural death Post mortem interval: ~ 3 days. Examined 14h00 Monday 16th June 2008. Post mortem decomposition: Minimal. Identifying features: 1.5m female south west carpet python (Morelia spilota imbricata). Examination undertaken by Dr Tim Hyndman.

□ External examination □ Lymphoreticular system No visible lesions (poor body condition) Not examined □ Skin and subcutis □ Urogenital system No visible lesions See below □ Body cavities □ Endocrine system See below No visible lesions □ Respiratory system □ Musculoskeletal system No visible lesions No visible lesions □ Cardiovascular system □ Nervous system See below No visible lesions □ Alimentary system See below

Body Cavity – Mesentery running caudal third of body found to have ~50-100 small (10mm x10 mm) patches of muscle evenly spaced along length of this mesentery. Significance unclear. Alimentary system – Faeces well formed, has eaten something (c.f. history). Urogenital system – No urate deposits seen in pericardium or kidneys. Cardiovascular system – Congestion seen in kidneys and liver. Darker than usual, but not distended or swollen. Other – Transmitter still in place. Sutures still well anchored. No evidence of body reaction to this foreign body.

Gross Summary: Weight loss. Mild hepatic and renal congestion, otherwise no abnormalities detected.

Gross Comment: A cause for the weight loss was not clear, and the faeces suggested the python had eaten. The transmitter appeared not to have caused any problems.

Ancillary Tests: Nil Histopathological findings: H08-0631A. Various organs. A section of kidney shows intracytoplasmic golden brown pigment accumulation in the renal tubular epithelium, otherwise normal. Liver has widespread, multifocal, dark brown/black pigment (melanin accumulation). A section of heart appears normal. A section of lung appears normal. Pancreas appears normal. H08-0632B. various organs. Skin appears normal. Trachea normal. A section of stomach appears normal. Sections of oviduct appear normal. Sections of mesentery show only vascularised adipose tissue.

Final Diagnosis: Inconclusive

Final Comment: No cause for the weight loss and death was found. The transmitter appeared not to be associated with any lesions, and is presumed not to have contributed to the clinical signs.

Yours Sincerely,

Philip K. Nicholls PhD FRCPath

Checklist: (√ = no gross lesions; H = sample for histology; C = culture; P = photographed) √ Eyes √HF Lungs √HF Stomach NE Adrenals √HF Mesentery and mm patches √H Skin n/a Bronch LN √ S. intestine Na Testes (list) F √ Head LN √HF Heart Na Caecum NE Ovaries √ Tongue √HF Liver √ Colon NE Meninges NE Oesophagus √ Gall bladder Na Mesent LN √hf Brain NE Thyroid √HF Spleen √HF Kidneys √HF Bone Marrow (rib) NE Parathyroid √HF Pancreas na Bladder NE Thymus na Forestomachs Frozen samples:□ Liver □ Fat □ Kidney □ Brain □ Other (list)

Disease Process 1 DEG Disease Process 2 System 1 SYS System 2 General Cause 1 UNK General Cause 2 Aetiology 1 Unexplained weight loss Aetiology 2 Common Name 1 Common Name 2

Dr Philip Nicholls

10.2. Post-mortem report for python Msi14 ‘Pamela’

140

School of Veterinary & Biomedical Sciences

South St, Murdoch

Western Australia 6150

Telephone: (61-8) 9360 2356

Facsimile: (61-8) 9310 4144

Duty Pathologist: 04202 77743

ANATOMIC PATHOLOGY NECROPSY REPORT

Pathology No: 08 0142 Date In: 18/02/2008 Pathologist: Dr Sandy McLachlan Owner’s Details: Consulting Veterinarian:Dr Alexander McLachlan

Anatomic Pathology Date of Consult: 18/02/2008

Pathology Use Only - No Charges

Ph: Fax To:

Patient’s Details: Species: Reptile Breed: Carpet Python Snake Clinic Number: 106573 Gender: Female Name: 08/142 Pamela Current Age: D.O.B:

History:

Carpet Snake from Gillian Bryant. (Kris Warren).

Surgery conducted in August for removal of transmitter near cloaca. New transmitter placed in lower third. Brought into captivity 1 week ago, as was looking very malnourished. Given fluid therapy for a week.

Submission: Status: post natural death Post mortem interval: Post mortem decomposition: moderate to advanced with a putrid odour Identifying features:

□ External examination □ Lymphoreticular system See Below No visible lesions □ Skin and subcutis □ Urogenital system No visible lesions See below □ Body cavities □ Endocrine system No visible lesions No visible lesions □ Respiratory system □ Musculoskeletal system No visible lesions No visible lesions □ Cardiovascular system □ Nervous system

No visible lesions No visible lesions □ Alimentary system See below

Post-mortem examination findings: The cloacal orifice was occluded by a plug of concreted faeces. There was loose faeces staining the vent. The caudal intestine was distended by foetid liquid faeces. The cranial intestine and stomach were distended by colourless gelatinous liquid. The carcase was in thin condition.

Ancillary Tests: Microbiology, Immunology, Cytology, Urinalysis, Toxicology, Biochemistry, or other. None performed

Histopathological findings: H08-0134A: Heart, lung, stomach, cloaca: No lesions.

Kidney: There is diffuse expansion of the glomerular mesangium and wire-loop prominence of glomerular capillary loops. There is abundant red-brown pigment resembling bilirubin within the cytoplasm of tubular epithelial cells. Kidney: chronic marked diffuse membranous glomerulonephritis. Kidney: marked diffuse tubular epithelial bilirubinosis.

Liver: There are randomly distributed variably sized black siderogranulomas.

Final Diagnosis: 1. Cloacal obstruction and pre-obstruction putrefaction of intestinal contents. 2. Membranous glomerulonephritis.

Yours Sincerely,

A D McLachlan BVSc, PhD, MACVSc, Diplomate ACVP Senior Lecturer in Veterinary Pathology

Disease Process 1 DEG Disease Process 2 System 1 ALI System 2 General Cause 1 PHY General Cause 2 Aetiology 1 CONSTIPATION Aetiology 2 Common Name 1 Common Name 2

Janice Stigwood

10.3. Post-mortem report for python Msi23 ‘Peter’

143

SchoolofVeterinary&BiomedicalSciences SouthSt,Murdoch WesternAustralia6150 Telephone:(618)93602356 Facsimile:(618)93104144 DutyPathologist:0420277743 ANATOMICPATHOLOGYNECROPSYREPORT PathologyNo:071520 DateIn:12/10/2007 Pathologist:DrGKnowles Owner’sDetails : ConsultingVeterinarian :GillianBryant GillianBryant DateofConsult: 12/10/2007 DepartmentofEnvironmentandConservation [email protected] Ph:FaxTo: Patient’sDetails : Species:SWcarpetpython(Moreliaspilota ClinicNumber: imbricate) Name: Breed: D.O.B: Gender:M CurrentAge: History : Theanimalwaslastseen2weeksagoandappearedclinicallynormal.Whentheanimalwasfounddeadtwo puncture/bitemarkswerefoundoverthehead. Submission: Status:postnaturaldeath Postmorteminterval:>24h(animalkeptchilled) Postmortemdecomposition:moderate Identifyingfeatures:none □ Externalexamination □ Lymphoreticularsystem Novisiblelesions Novisiblelesions □ Skinandsubcutis □ Urogenitalsystem Novisiblelesions Novisiblelesions □ Bodycavities □ Endocrinesystem Novisiblelesions Novisiblelesions □ Respiratorysystem □ Musculoskeletalsystem Novisiblelesions Novisiblelesions □ Cardiovascularsystem □ Nervoussystem Novisiblelesions Novisiblelesions □ Alimentarysystem Novisiblelesions Grossfindings: SignificantExternalFindings Theanimalwasinmoderatebodycondition. SignificantInternalFindings Thereweremoderateautolyticchangesevidentinmostorgans.Thestomachandintestinaltractwereempty.A transmitterwasfoundinthecaudalthirdofthebodycavity. GrossSummary: Pleaserefertocomments GrossComment: Therewerenogrossfindingsevidenttoindicateacauseofdeath.Thepuncturebitesnotedinitiallyinthefield werenodistinct(mostprobablyduetochillingoftheanimalielossofswelling).Afullrangeoftissueswillbe submittedforhistopathology. Grosssummary: SlidesH071170 A:heart,lungintestines,kidney,testis,liver B:stomach,brain,intestine C:skeletalmuscle,boneandbonemarrow Histopathologicalfindings: Liver:Therearewidespreadmicrovesicleswithinthecytoplasmofmosthepatocytes. Theheart,lung,kidney,testisstomach,brain,intestine,skeletalmuscle,boneandbonemarrowappearnormal. FinalDiagnosis: Mildhepaticlipidosis FinalComment: Thereisnodistinctcauseofdeathevidentfromgrossormicroscopicfindings.Thehepaticlipidosisisanonspecific findingandmostlikelyunrelatedtothecauseofdeath.Hepaticlipidosiscanbecausedbysuchfactorsas decreasedmetabolisableenergyintakeandincreasedmetabolicdemand,directtoxicinsulttoliver,increased mobilisationoflipidincirculation.Asnotedabovethesmallpuncturewoundsoverthehead,notedinthefield, werenotevidentaftertheanimalhadbeenchilled.Therewerenomicroscopicfindingswhichareoftenassociated withsnakeenvenomationinmammals,suchasdisseminatedintravascularcoagulation(withfibrinthrombiinthe kidney)ormyodegenerationoracutetubularnephrosis.

GraemeKnowlesBSc(vet)BVScMACVSc RegistrarAnatomicPathology Checklist: ( √ =nogrosslesions; H=sampleforhistology; C=culture; P=photographed) Eyes H Lungs H Stomach Adrenals Otherorgans H Skin BronchLN H S.intestine Testes (list) HeadLN H Heart Caecum Ovaries Tongue H Liver Colon Meninges Oesophagus Gallbladder MesentLN H Brain Thyroid Spleen Kidneys H BoneMarrow Parathyroid Pancreas Bladder Thymus Forestomachs Samplesinglutaraldehyde :liver,kidney,spleen,lung,brain DiseaseProcess1 STO DiseaseProcess2 System1 LIV System2 GeneralCause1 GeneralCause2 Aetiology1 Aetiology2 CommonName1 Hepaticlipidosis CommonName2 GraemeKnowles 10.4. Post-mortem report for python Msi38 ‘Chris’

146

School of Veterinary & Biomedical Sciences

South St, Murdoch

Western Australia 6150

Telephone: (61-8) 9360 2356

Facsimile: (61-8) 9310 4144

Duty Pathologist: 04202 77743

ANATOMIC PATHOLOGY NECROPSY REPORT

Pathology No: 08-636 Date In: 28/4/2008 Pathologist: Dr Annika Oksa/ Dr Sandy McLachlan Owner’s Details: Consulting Veterinarian:

01.26.21.0097.2.XXXXX.01023 Date of Consult:

C/- Kris Warren

Vet Clinical Sciences

Murdoch University WA 6150

Ph: Fax To:

Patient’s Details: Species: Boidae Clinic Number: 101144 Breed: Carpet Python

Name: Anatomic Pathology Gender:

D.O.B: Current Age:

History: Research python, found dead in the field 23-24/4/08

Transmitter implanted Aug 2007. Week of Apr 17th, python found with faecal build up as the transmitter had moved towards the cloaca and possibly caused faecal impaction. I moved the transmitter anteriorly towards head, but blood clots passed out the cloaca.

Submission: Young adult male carpet python Status: Post natural death Post mortem interval: Approximately 4-5 days Post mortem decomposition: Moderate to advanced Identifying features: Submitted in a container with pathology number

□ External examination □ Lymphoreticular system See Below No visible lesions □ Skin and subcutis □ Urogenital system See below No visible lesions □ Body cavities □ Endocrine system See below No visible lesions □ Respiratory system □ Musculoskeletal system See below No visible lesions □ Cardiovascular system □ Nervous system See below Not examined □ Alimentary system See below

Visible lesions: Significant External Findings 1. External Examination: The carcase as a whole was covered in maggots, concentrated heavily around the cranial region. 2. Skin and Subcutis: The snake was in some degree of pre-slough. The skin covering the left maxilla and orbital area was absent, with bone visible (poet-mortem artefact).

Significant Internal Findings 1. Body Cavities: The walls of the coelomic cavity were pale grey in colour in the cranial two thirds of the body (autolysis), and diffusely red in the caudal one third (congestion). The muscle tissue in this area was a solid deep red colour (congestion). The zone of demarcation between these regions was not clearly defined. 2. Respiratory System: The lung tissue was a diffuse pale grey colour (autolysis) 3. Cardiovascular System: The heart was small relative to body size. It was approximately 1.5cm in length (base to apex). On dissection of the heart, it was void of blood or fluid content. Autolysis made identification of any of the vessels difficult. 4. Alimentary System: The alimentary tract was traced from the oesophagus to the stomach with some degree of difficulty due to autolysis. The serosa through to mucosa was pale. Mid body, caudal to the stomach, the intestine was distended but remained diffusely pale. The transmitter was encased within this distended region. It appeared to be surrounded by a membrane, which was independent from the gut lumen, attached to the serosa, but not adherent to the coelomic wall. (Mesenteric? Serosal entrapment?) 4cm of faecal material was localized in the lumen immediately cranial to the transmitter. The faeces did not have traces of urate with it. At the junction where the kidney is found next to the alimentary tract, the gut was less freely movable. In this is region the coelomic wall was red and the tract was untraceable beyond this region due to severe autolysis. In this same area was the antenna from the transmitter, entangled with surrounding autolyzed tissues. The antenna was no longer attached to the transmitter. The liver was a diffuse black colour and friable.

Gross Summary: Inconclusive

Gross Comment: Upon necropsy, the intracoelomic transmitter appeared to have a membranous association with the mid region of the small intestine. The degree of autolysis did not allow further investigation as to how long this encasement had been in place. One suggestion is that it might have become encased before migrating caudally, pulling with it the small intestine. This would create some degree of discomfort for the snake, as well as predispose to faecal impaction. Another suggestion is that after manual manipulation to return the transmitter to its original location, an adhesion of the gut serosa or mesentery formed around the transmitter. This may have also been when the antenna separated from the transmitter. The presence of blood clots post-manipulation suggest there was internal bleeding prior to the transmitter being relocated. A definite cause of death or pathological processes could not be determined. Possible colonic rupture or peritonitis.

Yours Sincerely,

Student Name: Destinee Dummer

Yours Sincerely,

Dr Annika Oksa BSc BVMS (Hons) Resident in Veterinary Pathology

A D McLachlan BVSc, PhD, MACVSc, Diplomate ACVP Senior Lecturer in Veterinary Pathology

Checklist: (√ = no gross lesions; H = sample for histology; C = culture; P = photographed) Eyes Lungs Stomach Adrenals Other organs Skin Bronch LN S. intestine Testes (list) Head LN Heart Caecum Ovaries Tongue Liver Colon Meninges Oesophagus Gall bladder Mesent LN Brain Thyroid Spleen Kidneys Bone Marrow Parathyroid Pancreas Bladder Thymus Forestomachs Frozen samples:□ Liver □ Fat □ Kidney □ Brain □ Other (list)

Disease Process 1 INTRA-ABDOMINAL Disease Process 2 FOREIGN BODY System 1 System 2 General Cause 1 General Cause 2 Aetiology 1 Aetiology 2 Common Name 1 Common Name 2

APPENDIX 2. CHEMOSENSORY PAPER

This paper was an opportunistic behavioural study, able to be performed while pythons were brought into captivity at the end of field observations at the time of radiotransmitter removal.

151

AUTHOR CONTRIBUTION

G.L. Bryant: Collected pythons from field, presented olfactory cues to

pythons, analysed video footage, and wrote and edited versions of

the manuscript.

P.W. Bateman: Initiated the study idea, presented olfactory cues to pythons,

wrote and edited versions of the manuscript.

P.A. Fleming: Organised the timing for olfactory cue presentation, entered data,

produced figures and analysed data. Wrote and edited on

versions of the manuscript.

152

CSIRO PUBLISHING www.publish.csiro.au/journals/ajz Australian Journal of Zoology, 2011, 59,42–48

Tantalising tongues: male carpet pythons use chemoreception to differentiate among females

G. L. Bryant A,C, P. W. Bateman A,B and P. A. Fleming A

ASchool of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia. BDepartment of Zoology and Entomology, University of Pretoria, Pretoria, 0002, South Africa. CCorresponding author. Email: [email protected]

Abstract. For animals sparsely distributed across a landscape, finding and identifying a receptive female during a short breeding period can be a challenge for males. Many snakes appear to rely on the production of sex-specific pheromones to synchronise the timing of reproductive behaviour. The rare Australian south-west carpet python (Morelia spilota imbricata) displays non-aggressive mating aggregations of up to six males around a receptive female, suggesting that males are responding to some chemical signal that enables multiple males simultaneously to identify and locate the female. We investigated chemoreceptive response (tongue-flicking) of 10 male pythons under laboratory conditions to 12 (randomly ordered) treatments each presented for three minutes. Cutaneous chemicals (dissolved in hexane solvent) were collected on cotton buds from the skin of six female pythons and male responses to these were compared with six control treatments. Male pythons produced a greater number of tongue flicks during the first minute of each trial, with fewer in minutes 2 and 3. Male chemoreceptive response in the third minute varied significantly between treatments and was only maintained for trials presenting cutaneous chemicals collected from the three relatively largest female pythons. This experiment suggests that male carpet pythons can use chemoreception to obtain information about their social environment, identifying pheromone cues from large, potentially fecund females. This ability would be adaptive for male mate-selection behaviour and is likely to also reduce costs of searching behaviour.

Additional keywords: mate searching, reproductive behaviour, snakes, tongue-ﬂicking.

Introduction searching, recognition and choice. These snakes emerge from Finding a receptive female during a short breeding period can be a communal hibernacula in spring and breed in large, multiple- challenge for males of species or populations that are distributed mating masses. Males of this species show pheromone trailing thinly across a landscape. In order to synchronise the timing of behaviour (Heller and Halpern 1981; Mason et al. 1990;Le reproduction, many snakes rely on the production and accurate Master and Mason 2001; Le Master et al. 2001; Shine et al. 2005a, interpretation of sex-specific pheromones (Mason 1992). 2005b), and are able to differentiate between male and female Pheromones are semiochemicals released by individuals that may trails, following only the latter (Le Master et al. 2001). In addition, elicit changes in the physiology and behaviour of the receiving males not only differentiate between the sexes but are able to individual (Karlson and Luscher 1959; Ford 1986). Squamates identify both how long the female is and her body condition, have a well developed nasal olfactory system and vomeronasal courting longer and heavier-bodied females more vigorously system (mediated by vomeronasal sensory epithelia within (Shine et al. 2003). The males’ preference for larger females is paired vomeronasal organs) that enables individuals to acquire based on qualities of the pheromonal cues rather than merely chemosensory information (including pheromones) via their higher quantities produced by larger females (Le Master and forked tongue (tongue-flicking behaviour: Schwenk 1995;Le Mason 2002; Shine et al. 2003). This ability to identify the sex Master et al. 2001). Snakes of many taxa rely on chemoreception and quality of individuals through olfactory means would be for multiple roles: for instance, prey location (e.g. Chiszar et al. particularly advantageous in the large mixed-sex masses of 1986; Webb and Shine 1992; Kardong and Smith 2002; Stark breeding garter snakes adjacent to hibernacula, where visual et al. 2002; Greenbaum 2004) or location of suitable ambush differentiation is difficult (Shine et al. 2003). sites (e.g. Downes 1999; Clark 2007), avoidance of predators Unlike the garter snake, however, breeding individuals of (e.g. Burger 1989) and chemoreception even plays a role in kin most snake species do not occur in huge, concentrated masses but selection and social organisation (e.g. Pernetta et al. 2009). are sparsely distributed across their habitat. Males therefore need A North American colubrid, the red-sided garter snake to both find and identify suitable potential mates. Male timber (Thamnophis sirtalis parietalis) has been the primary model rattlesnakes (Crotalus horridus), for example, appear to search species in the study of snake olfaction in relation to mate for females by straight-line movements in an attempt to encounter

CSIRO 2011 10.1071/ZO11029 0004-959X/11/010042 Chemoreception in carpet pythons Australian Journal of Zoology 43 a female’s pheromone trail (Coupe 2005). Once male sidewinder (Bryant et al. 2010). Opportunistic collection sites in south-west rattlesnakes (Crotalus cerastes) have found a trail they can follow Western Australia included Leschenault Peninsula Conservation it for more than 100 m and have a 40% success rate in locating Park (33260S, 115410E), Martin’s Tank campsite, Yalgorup females (Coupe 2005), whereas male copperhead pitvipers National Park (32510S, 115400E) and reserves and State Forest (Agkistrodon contortrix) appear to travel long distances to surrounding Dwellingup township (32430S, 11640E); the receptive females by following airborne odour plumes (Smith maximum distance between capture sites was 170 km. It was et al. 2008). beyond the scope of this study to test for the effects of geographic In Australia, pythons of the genus Morelia show substantial origin upon chemosensory responses, since the pythons were differences in their mating systems. Males of all Morelia species collected opportunistically from several locations that would not are capable of breeding annually. Females, however, are capital allow a balanced experimental design. breeders, requiring accumulated body reserves to produce and Because we had to wait for the opportunity to hand capture incubate a clutch of eggs; females, therefore, do not breed animals when pythons were in an accessible location, they every year (Pearson 2002; Shine 2005), resulting in receptive were held in captivity at the Department of Environment and females being a scarce resource. Five Morelia spilota Conservation Dwellingup Research Centre for varying lengths of subspecies (bredli, cheynei, mcdowelli, metcalfei and variegata) time before our experiment (maximum of six weeks). The demonstrate male combat for access to females, whereas two following chemoreception experiment was carried out while the (imbricata and spilota) subspecies apparently do not (Shine and snakes were in captivity before surgery and their subsequent Fitzgerald 1995; Pearson et al. 2002). Mating systems appear release at point of capture, and was carried out under approval largely to have driven sexual size dimorphism; where males are of the Murdoch University (W2028/07) and Department of larger than females, males compete for access to mates, but where Environment and Conservation (DEC AEC/55/2006 and DEC males are smaller than females, no combat between males is AEC 54 / 2006) Animal Ethics Committees. evident (Pearson et al. 2002). Each male was kept individually in a 40 70 40 cm (112 L) The south-west carpet python (Morelia spilota imbricata)is plastic box with a recycled newspaper pellet substrate (Old distributed patchily across a variety of habitats in south-west News Cat Litter, Cyber Cycles Pty Ltd, Toowoomba, Qld), a tray Western Australia. Female M. s. imbricata grow considerably of water with a bark and cardboard refugium. The room larger than males: over twice the length and more than 10 times the containing these boxes was maintained at 29.6 0.4C and mass of adult males (Pearson et al. 2002). This significant sex pythons were exposed to natural lighting (through three large difference in size at maturity translates into a difference in age at windows) and fluorescent lights under a ~12 : 12 L : D first reproduction, and a consequent skew in the adult sex ratio photocycle. The females were kept in a separate room and housed (4 : 1 male : female; Pearson et al. 2002). Additionally, female in purpose-built ventilated enclosures and presented with similar M. s. imbricata only breed every 2–3 years (Pearson et al. 2002), refugia, water and pellet substrate. The females’ enclosures each further skewing the operational sex ratio. This subspecies forms had an external heat pad positioned underneath the plywood apparently non-combative (i.e. no aggression witnessed or bite flooring (heating the cage floor above the heat pad to ~30C) and marks noted) aggregations of up to six males around a single a large window provided natural light supplemented with female during the austral spring (September to November), each fluorescent light under a 12 : 12 L : D photocycle. Pythons were male waiting for the chance to copulate with the receptive female fed (dead laboratory mice and rats that were stored frozen and (Pearson et al. 2002). thawed before feeding) if they were held for longer than As part of a study on the biology of M. s. imbricata, we set three weeks. out to examine the role of male chemosensory ability in mate During experimental trials, males were presented with choice and identification. First we investigated whether adult olfactory cues that had been collected and stored (<48 h) before male M. s. imbricata can differentiate between female python the commencement of the trials. Odours were collected from skin chemicals and control olfactory stimuli. Second, we individual prey and pythons by rubbing the surface of the investigated whether male M. s. imbricata demonstrate greater animal with two cotton buds dipped in hexane for a total of six interest in the olfactory stimuli collected from larger and better- minutes (3 min for each cotton bud). The tip of each cotton condition females, assuming that larger females will be more bud was then clipped off and both were deposited in a vial fecund (Seigel and Ford 1987; Duvall et al. 1993; Shine and containing 10 mL of hexane. We elected to use this standard Fitzgerald 1995; Rivas and Burghardt 2001; Shine et al. 2003). time collection protocol to attempt to collect the scents in a We predicted that, despite the mating system of this consistent manner. For example Shine et al.(2000, 2003) have python being so different from that of the garter snake, male shown that body size influences the amount of skin lipids that M. s. imbricata would show a similar level of chemical cue can be collected through rubbing the skin. Rubbing the entire recognition, an ability that would be adaptive in the face of ventral surface would therefore result in varying amounts of the operational sex ratio and search costs facing males of this chemicals collected from each individual, according to body sparsely distributed python species. size. To ensure that our results reflected quality rather than quantity of skin lipids, we therefore collected our scents by a set time to prevent body size alone biasing the quantity of lipids Materials and methods collected. A separate 200-mm-long cotton bud was used to Eleven male and six female M. s. imbricata were collected to present the scent to the pythons – this was dipped into the storage remove radio-transmitters that had been previously surgically vial and dried for 3 min (to allow the hexane to evaporate off) implanted into the coelomic cavities at least seven months before immediately before each trial. 44 Australian Journal of Zoology G. L. Bryant et al.

Ten males were tested for their chemosensory response to % ðobserved Mb predicted MbÞ control and olfactory cues over trials running over a total of BCSð Þ¼ 100 predicted Mb two days. During experiments, each of the 10 males were presented with a randomly assigned experimental or control where predicted Mb was calculated using the following equation stimulus on a cotton bud held 3 cm from the tip of their snout by derived from average data collected for 46 individuals (excluding one of two observers while the second observer recorded the their first capture to reduce bias as several pythons were initially snake’s behaviour on a camcorder for a duration of 3 min. The caught because they had consumed a radio-collared prey) from scent presented for each trial was prepared by the third the long-term ecological study that these animals were part of experimenter (presented in random order to each randomly (Bryant et al. in press): assigned male), and therefore both observers were blind to the : • : nature of the stimuli presented on cotton buds. Although we Predicted Log10Mb ðgÞ¼2 90 Log10 SVL ðcmÞ3 34 randomly selected the male to test each stimulus, we ensured that R2 ¼ 0:876 each male had a minimum of 30 min between each successive trial. Tongue flick rate (per minute) was counted during the course To test for possible effects of female python body size of the trial and was confirmed by later watching the hand-held (log SVL and logMb), and body condition (BCS) upon sustained fl camcorder footage for each minute over the 3-min period. During male python tongue ick responses, these measures were replay of each trial, the intensity of each tongue flick was also included as covariates in a mixed-model ANOVA (Statistica fi scored (on a scale of 1–3, where 1 = forked tip of tongue visible, ver. 7.1) with female ID ( xed factor) and male ID (random fl 2 = forked tip and shaft of tongue visible, 3 = forked tip and shaft factor) and the number of tongue icks in minute 3 log(number fl of tongue visible and swung down and up). of tongue icks + 1) as the dependent measure. fl The stimuli presented on a cotton bud (with the exception The proportion of each of the three levels of tongue ick of the first non-odoriferous control) to male snakes were as intensity score were compared between trials where controls or follows. python cutaneous chemicals were presented to male pythons by Chi-square analysis, with expected numbers of tongue flicks Three non-odoriferous controls calculated assuming an equal distribution of each of the tongue- flick intensity categories between control items and python ( – ’ 1) Staring the observer leant over the snake s container as in stimuli. the following stimulus trials but did not present a cotton bud (this was the only stimulus that the observers could not be ‘blind’ to); Results ( – 2) Cotton bud an unscented cotton bud; A significantly greater number of tongue flicks was recorded ( – 3) Solvent a cotton bud dipped in hexane and then dried for during the first minute of trials, with fewer in minutes 2 and 3 3 min. (RM-ANOVA time: F2,18 = 35.01, P < 0.001) (Fig. 1). Post hoc fi Three odoriferous controls analysis revealed a signi cant difference in average number of tongue flicks between minute 1 and minute 3 (P < 0.05). ( 4) Cologne (Jean Paul Gaultier) at a concentration of 1 part to 3 In addition to time effects, there were also significant treatment with hexane; effects upon tongue flick number (RM-ANOVA treatment: (5) Prey scent – collected from a dead laboratory rat by rubbing F11,99 = 3.09, P = 0.001) (Fig. 2a). Male M. s. imbricata rarely the surface of the rat for a total of 6 min and stored in hexane; tongue-flicked during the staring treatment. When males were (6) Male python – cutaneous chemicals collected from the randomly selected eleventh male python (not included in the tongue flick experiment) by rubbing the python’s ventral 5 scales for a total of 6 min and stored in hexane. a Stimuli collected from six female pythons 4 (7) Cutaneous chemicals were collected from the ventral surface of each of the females for a total of 6 min and stored in 3

hexane. S.E.) of tongue flicks ± ab 2 Analysis b The number of tongue flicks recorded each minute for each of 1 the 12 treatments was compared by repeated-measures ANOVA log(number of tongue flicks + 1). Post hoc analyses were 0 Average number ( carried out by LSD test (Statistica ver. 7.1., StatSoft Inc., Tulsa, 123 OK). Minute Body condition scores (BCS) of the females were calculated as Fig. 1. Male south-west carpet pythons showed a greater tongue-flick – the residual scores of body mass (Mb, g) relative to snout vent response over all 12 treatments (pooled for this graph) for the first minute of length (SVL, cm), expressed as a percentage of predicted Mb trials, with decreasing numbers of tongue flicks in minutes 2 and 3. Letters (Madsen and Shine 2002): link data that are not significantly different from each other at P > 0.05. Chemoreception in carpet pythons Australian Journal of Zoology 45

(a) Minute 1 9 Minute 2 Minute 3

S.E.) 7 ±

2 Average number of tongue flicks ( 1

0 Staring Cotton Hexane Cologne Prey Male MSI10 MSI19 MSI49 MSI53 MSI37 MSI18 bud

6 (b) Minute 3

5 S.E.) ± 4

1 Average number of tongue flicks (

0 Staring Cotton Hexane Cologne Prey Male MSI10 MSI19 MSI49 MSI53 MSI37 MSI18 bud

Fig. 2. Although male pythons (n = 10) tongue-flicked towards all 12 treatments tested (a), their activity was sustained towards the female stimuli; this is particularly evident for the data for minute 3 (b). Numbers inside each treatment column indicate tongue-flick activitybetweenminutes.Asterisksrepresent posthoc analysisbetweenminutesforeach treatment:asterisksabovenumbersindicate a significant difference between minutes 1 and 2, or between minutes 1 and 3 (asterisks placed above the signs separating each number); asterisks below the numbers indicate significant difference between minutes 2 and 3. *P < 0.05; **P < 0.001. Letters in (b) link data that are not significantly different from each other at P > 0.05. Females (bracket) are ordered from left to right in increasing body condition score; note that female Msi53 was the smallest individual (Table 1). presented with a cotton bud within 3 cm of the tip of their snout, presentation of the plain cotton bud and solvent (hexane), however, most individuals would tongue-flick to examine the the tongue-flick rate declined markedly over minutes 2 and 3. The item. Although the snakes showed some initial response to odoriferous controls (cologne, prey and male python) stimulated 46 Australian Journal of Zoology G. L. Bryant et al. a response; however, the number of tongue flicks also fell by Table 1. Body mass, snout–vent length and body condition score minutes 2 and 3. By contrast, most male pythons showed (% above or below average population values) of the six female carpet sustained, or even demonstrated increased, response to the female pythons from which skin lipids were collected for presentation to male python stimuli in minutes 2 and 3. pythons Females are ordered by increasing body condition score This difference in response for control and female scents was supported by a significant time treatment interaction (RM- Female ID Body Snout–vent Body condition ANOVA interaction term: F22,198 = 1.69, P = 0.033) and is most mass (g) length (cm) score (%) clearly demonstrated in Fig. 2b, where only the data for minute 3 are shown. During minute 3, male pythons maintained a Msi10 1108 181 –32.22 significantly higher tongue-flick response to female pythons than Msi19 1416 191 –25.89 – any other treatment (Fig. 2b). There was no significant effect of Msi49 1318 179 16.73 – female python body mass (P = 0.278) or SVL (P = 0.939) upon Msi53 695 139 8.58 fl Msi37 2414 192 23.68 the number of tongue icks in minute 3 (mixed-model ANOVA); Msi18 3731 223 24.62 however, there was a significant effect of female body condition (P = 0.012), with the greatest numbers of tongue flicks directed towards the females that were relatively heaviest (Table 1). We note that there was also a significant effect of male python ID Discussion (random factor, P = 0.013), with some males not responsive to Our results suggest that male Morelia spilota imbricata can not any scents. only discriminate between different olfactory stimuli presented Most (81%) tongue flicks directed towards controls (i.e. on cotton buds under laboratory conditions, but can also staring, cotton bud, solvent, cologne and prey) were scored as differentiate differences in female pythons, showing more interest Intensity 1 (i.e. they were simple projections of the tongue beyond in the cutaneous chemicals collected from relatively heavier the labial scales); 18% were scored as Intensity 2 and only 1% as females. These observations, coupled with the field observation Intensity 3 (Fig. 3). Significantly more Intensity 3 tongue flicks of mating aggregations, suggest that male M. s. imbricata are 2 (10%; c 1 = 17.09, P < 0.001) were directed towards python likely to use chemosensory detection to identify a receptive scents (both the male and the six females); only 67% of tongue female. 2 flicks directed towards python scents were Intensity 1 (c 1 = 4.70, Although not all females elicited a sustained tongue-flick 2 P < 0.05) and 23% were intensity 2 (c 1 = 2.49, P > 0.05) (Fig. 3). response from males, the highest body condition females

'Intensity 3': forked tip and shaft of tongue visible and swung down and up 'Intensity 2': forked tip and shaft of tongue visible 'Intensity 1': forked tip of tongue visible 100

Percentage of tongue flicks 20

0 Prey Male Staring MSI19 MSI53 MSI10 MSI18 MSI37 MSI49 Hexane Cologne Cotton bud

Fig. 3. A greater proportion of intensive tongue-flicks (Intensity Score 3) were directed towards python scents (male and the six females) compared with the controls tested (the five categories on the left-hand side of the graph). A greater proportion of tongue-flicks directed towards ‘control’ items were Intensity Score 1; there was no significant difference for the numbers of tongue-flick Intensity 2. Females (bracket) are ordered from left to right in increasing body condition score. Chemoreception in carpet pythons Australian Journal of Zoology 47 certainly did, and tongue-flick response in minute 3 (i.e. a that not all males maintained an interest into the third minute sustained response by males) was significantly correlated with (a significant effect of male IDwas noted for minute 3tongue-flick female body condition score. In many species, female size response), with several individuals showing little sustained reflects fertility and/or fecundity as large female snakes can interest towards any scent presented to them. Although none were potentially either lay more eggs than small females or invest more at the ‘blue-eye’ stage of pre-ecdysis, some of these unresponsive energy into each egg produced (e.g. Shine and Fitzgerald 1995; males were observed to moult within the following weeks of the Rivas and Burghardt 2001). It would, therefore, be adaptive for trials. Their physiological state may have contributed to lower male M. s. imbricata to differentiate between females of different tongue-flick response in these males: behavioural changes during sizes. Shine et al.(2003) found that male garter snakes were able pre-ecdysis (when eyes are at the ‘blue’ stage) have been recorded to differentiate females from males in mating masses and even in common garter snakes (Thamnophis sirtalis), including in these masses, males courted longer, heavier-bodied females reduction in feeding frequency and strike rate (at a moving more vigorously than they did smaller females. Being able to stimulus) and an increase in movement latency following distinguish the scent of a larger female could also presumably lead presentation of a stimulus (King and Turmo 1997). to more efficient mate searching, where better-condition females The potential to detect scents may be different between the would be the target of males’ investment in searching. In the sexes. The copperhead pitviper is similar to M. s. imbricata in that present study, even though female python Msi53 was in relatively females are widely dispersed and males must find them over good body condition, she was the smallest female in terms of large distances (Fitch 1960; Smith et al. 2008) and there is sexual body mass and length (Table 1). Males lost interest in her scent dimorphism in the tongue tine length, with males having and showed minimal tongue flicking during minute 3. Under field significantly longer tines (Smith et al. 2008). This increased conditions, this may translate to males not showing sustained tongue bifurcation is likely to result in increased tropotactic pursuit of such a small female. ability in males, reflecting their mate-searching activities. It is In addition to tongue-flick rate, it appears that tongue-flick possible that there is similar sexual dimorphism in the tongues of intensity (type of tongue-flick) also reflects level of interest to M. s. imbricata. different cues. We recorded, on average, more tongue flicks of Our data support the hypothesis that male snakes, whether Intensity 2 and 3 occurring in response to python cues presented. M. s. imbricata that mate in small aggregations or garter snakes We found little interpretation in the literature of the role of that matein large mixed-sex groups,are able to accurately identify different lingual responses in snakes, and this is an area that potential partners through olfactory stimuli. This ability may be warrants further investigation. important during the mating season if male pythons, as found for Even when mating occurs in smaller groups than those other snakes (e.g. garter snakes: Le Master and Mason 2001), observed in garter snakes, the ability to differentiate between follow scent trails (either airborne or substrate-based) leading females and other males would be adaptive in other snake species. them to a receptive female. Under these circumstances, an ability Rivas and Burghardt (2001) report that large male green that minimises the time required in mate searching would be anacondas (Eunectes murinus) often become the recipients of highly advantageous and is therefore likely to be under strong courting and mating behaviour from other males in mating balls selection. around large females. Mating aggregations last several weeks in this highly sexually size-dimorphic species, and although Acknowledgements pheromones are presumably involved in both trailing and mating We extend our gratitude to Jessica Mann for her tolerant and supportive behaviour, it is likely that males become marked with female hospitality in Dwellingup. We thank staff at Department of Environment lipids containing pheromones during mating sessions and larger and Conservation, Dwellingup Research Centre, for accommodating males become mistaken for females (Rivas and Burghardt this research. We acknowledge Murdoch University and the University of 2001). Although they do not form mating balls, aggregations of Pretoria for funding. male south-west carpet pythons have been reported where males presumably associate with females for long periods, some mating References with the female (Pearson et al. 2002). It would therefore be Bryant, G. L., Eden, P., de Tores, P. J., and Warren, K. S. (2010). Improved advantageous for males to differentiate female olfactory stimuli procedure for implanting radiotransmitters in the coelomic cavity of from those of nearby males. snakes. Australian Veterinary Journal 88, 443–448. doi:10.1111/j.1751- The male M. s. imbricata in our experiment responded to 0813.2010.00633.x stimuli presented on cotton buds through tongue flicking, Bryant, G. L., Dundas, S. J., and Fleming, P. A. Tree hollows are of showing a higher tongue-flick rate in minute 1 than in subsequent conservation importance for a Near-Threatened python species. Journal minutes. Most studies of snake and lizard chemosensory of Zoology, In press. responses generally record for only one minute commencing Burger, J. (1989). Following of conspecific and avoidance of predator only after the first tongue flick. The benefit of recording tongue chemical cues by pine snakes (Pituophis melanoleucus). Journal of – flicks over three minutes after stimulus presentation allows Chemical Ecology 15, 799 806. doi:10.1007/BF01015178 examination of the maintenance of interest in an olfactory cue, Chiszar, D., Radcliffe, C., and Feiler, F. (1986). Trailing behavior in banded rock rattlesnakes (Crotalus lepidus klauberi) and prairie rattlesnakes and also allows data to be collected where no response towards a (C. viridis viridis). Journal of Comparative Psychology 100, 368–371. particular cue (e.g. staring) was shown. Maintenance of tongue- doi:10.1037/0735-7036.100.4.368 flick response into the third minute occurred for cutaneous Clark, R. W. (2007). Public information for solitary foragers: timber chemicals collected from the largest female pythons, suggesting rattlesnakes use conspecific chemical cues to select ambush sites. generally sustained interest in these scents. We note, however, Behavioral Ecology 18, 487–490. doi:10.1093/beheco/arm002 48 Australian Journal of Zoology G. L. 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