Investigations in LED lighting and the Mantella cowani to improve amphibian ex situ conservation efforts

A thesis submitted to the University of Manchester for the degree of Environmental Biology (Masters of Philosophy) in the Faculty of Science and Engineering

2016

Chloe Helsey

School of Earth and Environmental Science

Contents Page

List of Figures 4

List of Tables 7

General Abstract 8

Declaration 9

Copyright Statements 9

Acknowledgements 9

General Introduction 10 − Project Aims 13 − References 14

Chapter 1: Establishing in situ mass, snout-vent length and a body condition 16 index as ex situ guidelines for the endangered frog Mantella cowani − Abstract 16 − 1.1 Introduction 17 − 1.2 Methods 20 − 1.3 Results 24 − 1.4 Discussion 28 − 1.5 References 33

Chapter 2: Quantifying and comparing the spectral and thermal output of 36 LEDs and current lighting in amphibian ex situ conservation − Abstract 36 − 2.1 Introduction 37 − 2.2 Methods 39 − 2.3 Results 43 − 2.4 Discussion 49 − 2.5 References 55

Chapter 3: Assessing the effect of LED lighting on the growth, colouration and 59 behaviour of Mantella betsileo − Abstract 59 − 3.1 Introduction 60

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− 3.2 Methods 62 − 3.3 Results 65 − 3.4 Discussion 70 − 3.5 References 77

General Discussion 82 − Limitations 83 − Further Study 84 − Conclusion 85 − References 86

Final Word Count: 30,336 (including references)

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List of Figures

Chapter 1: Establishing in situ mass, snout-vent length and a body condition index as ex situ guidelines for the endangered frog Mantella cowani − Figure 1.1: Edited Google Earth satellite images of the Fohisokina field 21 of the location of the 9 transects. − Figure 1.2: Edited Google Earth satellite images of the Fohisokina field site 21 of the location of the 9 transects with labels. − Figure 1.3: Bivariate plot of the Snout-Vent Length (cm) vs Mass (g). 23 − Figure 1.4: Bar chart of the mean mass (g) of female and male 26 individuals on the different transects. − Figure 1.5: Bar chart of the mean snout-vent length (g) of female 26 and male individuals on the different transects. − Figure 1.6: Bar chart of the scaled mass index of female and male 27 individuals on the different transects. − Figure 1.7: The Scaled Mass Index (SMI) of 92 individuals over the 25-day 27 sampling period.

Chapter 2: Quantifying and comparing the spectral and thermal output of LEDs and current lighting in amphibian ex situ conservation − Figure 2.1: Absolute irradiance measurements of daylight in Manchester 43 in the visible light spectrum (400-700nm). − Figure 2.2: Relative irradiance measurements of the lighting in 16 tanks in 45 Chester Zoo in the visible light spectrum (400-700nm) 10cm from the light. − Figure 2.3: Relative irradiance measurements of the lighting in 1 tank in 45 London Zoo in the visible light spectrum (400-700nm) 10cm from the light. − Figure 2.4: Relative irradiance of 6 commercially available LED lights in the 45 visible light spectrum (400-700nm) 10cm from the light. − Figure 2.5: Relative irradiance measurements of the lighting in 16 tanks 46 in Chester Zoo in the visible light spectrum (400-700nm) 30cm from the light. − Figure 2.6: Relative irradiance measurements of the lighting in 6 tanks 46 in London Zoo in the visible light spectrum (400-700nm) 30cm from the light. − Figure 2.7: Relative irradiance of 6 commercially available LED lights in 46 the visible light spectrum (400-700nm) 30cm away the light. − Figure 2.8: Thermal imaging picture and corresponding photograph of 47 Tank 1 at Chester Zoo. − Figure 2.9: Thermal imaging picture and corresponding photograph of 47 Tank 3 at Chester Zoo. − Figure 2.10: Thermal imaging picture and corresponding photograph of 47 Tank 4 at Chester Zoo.

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− Figure 2.11: Thermal imaging picture and corresponding photograph of 47 Tank 5 at Chester Zoo. − Figure 2.12: Thermal imaging picture and corresponding photograph of 47 Tank 6 at Chester Zoo. − Figure 2.13: Thermal imaging picture and corresponding photograph of 47 Tank 8 at Chester Zoo. − Figure 2.14: Thermal imaging picture and corresponding photograph of 47 Tank 9 at Chester Zoo. − Figure 2.15: Thermal imaging picture and corresponding photograph of 47 Tank 1 at London Zoo. − Figure 2.16: Thermal imaging picture and corresponding photograph of 47 Tank 2 at London Zoo. − Figure 2.17: Thermal imaging picture and corresponding photograph of 48 Tank 3 at London Zoo. − Figure 2.18: Thermal imaging picture and corresponding photograph of 48 Tank 4 at London Zoo. − Figure 2.19: Thermal imaging picture and corresponding photograph of 48 Tank 5 at London Zoo. − Figure 2.20: Thermal imaging picture and corresponding photograph of 48 Tank 7 at London Zoo. − Figure 2.21: Thermal imaging picture and corresponding photograph of 48 Tank 1 at the University of Manchester. − Figure 2.22: Thermal imaging picture and corresponding photograph of 48 Tank 2 at the University of Manchester. − Figure 2.23: Thermal imaging picture and corresponding photograph of 48 Tank 3 at the University of Manchester. − Figure 2.24: Thermal imaging picture and corresponding photograph of 48 Tank 4 at the University of Manchester.

Chapter 3: Assessing the effect of LED lighting on the growth, colouration and behaviour of Mantella betsileo − Figure 3.1: Effect of light treatment on average change in weight. 66 − Figure 3.2: Effect of light treatment on average change in SVL. 66 − Figure 3.3: Effect of light treatment on average change in SMI. 67 − Figure 3.4: Effect of light treatment on average change in redness. 67 − Figure 3.5: Mean proportion of individuals in the each lighting treatment 68 group for each day recorded as ‘Out’. − Figure 3.6: Absolute irradiance spectrum of lighting in the 6 study 68 enclosures in the visible light spectrum (400-700nm). − Figure 3.7: Thermal imaging picture and corresponding photograph of 69

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Tank 1 with Fluorescent lighting at the University of Manchester. − Figure 3.8: Thermal imaging picture and corresponding photograph of 69 Tank 2 with Fluorescent lighting at the University of Manchester. − Figure 3.9: Thermal imaging picture and corresponding photograph of 69 Tank 3 with Jungle Dawn™ LED lighting at the University of Manchester. − Figure 3.10: Thermal imaging picture and corresponding photograph of 69 Tank 4 with Jungle Dawn™ LED lighting at the University of Manchester. − Figure 3.11: Thermal imaging picture and corresponding photograph of 69 Tank 5 with Solar Stinger™ LED lighting at the University of Manchester. − Figure 3.12: Thermal imaging picture and corresponding photograph of 69 Tank 6 with Solar Stinger™ LED lighting at the University of Manchester.

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List of Tables

Chapter 1: Establishing in situ mass, snout-vent length and a body condition index as ex situ guidelines for the endangered frog Mantella cowani − Table 1.1: Detailed descriptions of transects 1-9. 21 − Table 1.2: Table highlighting the average, minimum and maximum mass 24 for male and female M. cowani in the Fohisokina site. − Table 1.3: Table highlighting the average, minimum and maximum snout-vent 24 length (SVL) for male and female M. cowani in the Fohisokina site. − Table 1.4: Table highlighting the average, minimum and maximum Scaled 24 Mass Index (SMI) for male and female M. cowani in the Fohisokina site.

Chapter 2: Quantifying and comparing the spectral and thermal output of LEDs and current lighting in amphibian ex situ conservation − Table 2.1: Detailed specifications of the 7 enclosures and their lighting at 41 London Zoo. − Table 2.2: Detailed specifications of the 21 enclosures and their lighting at 41 Chester Zoo. − Table 2.3: Detailed specifications of the 6 brands of LED lights. 42 − Table 2.4: Detailed specifications of the 4 enclosures with LED lighting 42 at the University of Manchester.

Chapter 3: Assessing the effect of LED lighting on the growth, colouration and behaviour of Mantella betsileo − Table 3.1: Detailed specifications of the three lighting types used in 63 each treatment group.

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Abstract

Chloe Helsey The University of Manchester Environmental Biology (MPhil) 30th September 2016

Investigations in LED lighting and the Mantella cowani to improve amphibian ex situ conservation efforts

Amphibians are in crisis. Threats to amphibians in the wild are numerous and widespread and thus, numbers declining at an alarming rate. Ex situ conservation efforts are increasingly being utilised to halt this decline and mitigate against external threats. Reintroduction is a crucial aspect of ex situ efforts, yet currently the success of these programmes are generally limited. A significant flaw in many of these ex situ conservation efforts is the insufficient knowledge of lesser-known vulnerable amphibian species that need to be brought into captivity. Increasing our understanding and gaining valuable information, for instance of the threatened Mantella cowani can provide guidelines to help inform ex situ programmes. In this thesis, the typical weight and size of M. cowani is outlined and a body condition index is determined. This will allow the fitness of individuals in captivity to be monitored ultimately maximising reintroduction success. Correct provisioning of environmental conditions within captivity is also crucial to the success of ex situ conservation programmes. Lighting in particular is an important environmental parameter for amphibians. Limited studies have investigated the use of visible lighting in amphibian ex situ conservation. The emergence of Light Emitting Diodes (LEDs), a highly attractive, alternative light source has highlighted the need for greater understanding of the current lighting provided. LEDs are a new form of economically and environmentally sustainable lighting, yet they have the potential to change the make up of captive environments. This study demonstrates the distinct differences between both the spectral and thermal output of LEDs and current lighting typically provided by zoological institutions. Subsequently, the impacts of these differences have been investigated. An increase in the activity of Mantella betsileo individuals under different LED treatments was observed. However, interestingly, the intensity of lighting rather than the spectral output could have a potential impact and play a greater role in the maintenance of healthy populations. Together, these preliminary studies provide an insight into the ways to in which ex situ conservation efforts can be monitored and improved and can help guide future investigations. It demonstrates the need for greater research into the provisioning of visible lighting in amphibian enclosures to enhance the success of ex situ programmes.

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Declaration No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning

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4. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property University IP Policy (see http://documents.manchester.ac.uk/display.aspx?DocID=24420), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.library.manchester.ac.uk/about/regulations/) and in The University’s policy on Presentation of Theses.

Acknowledgements I would like to thank Jade Newton-Youens for her assistance throughout the year, particularly her help and advice regarding amphibian husbandry and during the field trip to Madagascar. I would like to thank Professor Richard Preziosi for his supervision and guidance throughout this degree. I would also like to thank Chester Zoo and London Zoo Herpetology Departments for their invaluable time and help in data collection. I would finally like to thank my sister Charlotte for her endless help and reassurance throughout the year.

9 General Introduction

General Introduction

Amphibians are in the midst of a great extinction event, with numbers declining at an increasingly alarming rate (Zippel et al., 2011). Up to 33% of all amphibian species are currently threatened, with 7% facing imminent extinction and this number is likely to be significantly underestimated (Pavajeau et al., 2008). A combination of threats including climate change, disease, overexploitation and habitat degradation have contributed to this crisis. Problematically, the accelerating nature of these threats is predicted to only further exacerbate their already vulnerable position (Wake & Vredenburg, 2008; Zippel et al., 2011).

A range of conservation efforts are being employed in an attempt to halt this decline and particularly, the value of ex situ conservation for amphibians is gradually being recognised (Zippel et al., 2011; Pavajeau et al., 2008). Captive breeding programmes are now believed to be essential to the survival of many amphibian species (McGregor Reid & Zippel, 2008; Zippel et al., 2011; Moore & Church, 2008). Ex situ conservation programmes are being utilised by many amphibian conservation organisations as a tool for reintroduction (Griffiths & Pavajeau, 2008). This works alongside in situ conservation efforts which aims to mitigate external threats and provide an environment that would support reintroduced populations (McGregor Reid & Zippel, 2008). The implementation of programmes such as the Amphibian Ark, which seeks to maximise the number of amphibians in ex situ conservation, illustrates the shift towards utilising ex situ conservation (Mallinson, 2003; Zippel et al., 2011). Ex situ conservation is an increasingly popular approach in amphibian conservation due to the deteriorating outlook for amphibians and their high suitability for captivity (Tapley et al., 2015). Their small size, high fecundity, limited learned behaviour and short generation time amongst many others traits makes them ideal candidates for ex situ conservation (Tapley et al., 2015; McGregor Reid & Zippel, 2008). Many amphibian captive breeding programmes have effectively maintained populations and seen reasonable success in breeding (Banks et al., 2008; Griffiths & Pavajeau, 2008). Accordingly, this has resulted in successful reintroductions attempts (Banks et al., 2008). However, despite this, the number of vulnerable species of amphibian successfully reintroduced into the wild remains fairly small (Griffiths & Pavajeau, 2008). This thesis will seek to explore and overcome some of the obstacles which hinder successful maintenance of amphibians in captivity and their subsequent reintroduction (Griffiths & Pavajeau, 2008; Tapley et al., 2015).

Although amphibians have many traits that make them ideal for captive breeding programmes, they also have a number of problematic characteristics. This includes their complex life histories, high sensitivity to environmental change and a variety of specialised needs regarding their diet, temperature and provisioning of UVB (Tapley et al., 2015; Ferrie et al., 2014). Alongside these specialised requirements, limited knowledge of their optimal environmental conditions has made providing suitable captive environments far more difficult than anticipated (Densmore & Green, 2007; Ferrie et al., 2014; Tapley et al., 2014). Ex situ programmes seek to

10 General Introduction maintain populations and ultimately breed individuals ready for reintroduction. However, so far both the attempt and success rates of amphibian reintroductions has been limited (Michaels et al., 2014c). In fact, less than 20% of amphibians in the Amphibian Ark ex situ conservation programme have been successfully maintained and bred to even a second generation, and consequently even less have been reintroduced (Michaels et al., 2014c). Michaels et al. (2014c) stipulate that maintaining healthy populations of amphibians is a major issue with ex situ conservation programmes. The quality and suitability of the captive husbandry has an impact on amphibian fitness and survival (Michaels et al., 2014a). Thus, a lack of accurate information detailing their needs results in inadequate provisioning of the necessary environmental requirements and accordingly leads to a decline in the fitness of captive populations (Michaels et al., 2014c).

Challengingly, whilst the number of amphibian species requiring ex situ conservation programmes is increasing, sufficient life history information and environmental requirements is still only available for a limited number of species (Michaels et al., 2014c). Many species already held in captivity have little or no life history data to support and inform their captive husbandry (Michaels et al., 2014c). This is exacerbated as vulnerable species are often brought into captivity rapidly due to their dangerously small population sizes (Michaels et al., 2014c). Therefore, efforts to study threatened species in the wild needs to be maximised and information including their typical size and environmental needs delineated before they are brought into captivity. Alongside this, establishing the fitness of individuals in the wild could be utilised to enable the monitoring of individuals fitness in captivity. Body condition is the most popular method of determining fitness of individuals (Speakman, 2001). Therefore, determining the typical body condition of a wild population, will allow programmes to more accurately monitor the fitness of ex situ populations. Captive husbandry methods can therefore be altered in order to maintain body condition between ex situ and in situ populations ensuring that captive populations remain as fit as they would be in the wild. This will maximise the likelihood of successful reintroduction and ensuring that upon reintroduction existing wild populations do not experience a reduction in their fitness by breeding with reintroduced individuals.

Besides a need for greater information from wild populations to inform amphibian husbandry practice, difficulties also emerge within ex situ conservation when attempting to replicate the natural environmental conditions (Wake & Vredenburg, 2008). Amphibian husbandry is a key component that determines the success of ex situ conservation programmes. The design of captive environments is a crucial aspect of ex situ conservation, yet it is often overlooked in favour of other parameters (Michaels & Preziosi, 2015). Provisioning of factors such as diet and UVB lighting are widely known to be crucial to the maintenance of ex situ populations of amphibians and appropriate steps have therefore been taken in amphibian husbandry protocols in order to provide these appropriately. Yet Michaels and Preziosi (2015) suggest there is a lack of investigation into the direct impacts of an artificial captive environment on amphibian fitness.

11 General Introduction

There is empirical evidence to suggest that environmental complexity in captive environments may influence fitness and behaviour in amphibians (Michaels & Preziosi, 2015; Michaels, 2014a). Surprisingly, whilst much research has been conducted exploring the light requirements for amphibians, specifically in UVB provisioning, there has been very little investigation into the use of artificial lighting in amphibian captive environments (Bancroft et al., 2008). It has been concluded that UVB lighting is essential in captivity to maintain healthy populations of anurans yet may also have significant detrimental effects if inadequately provisioned (Licht & Grant, 2008; Tapley et al., 2014; Michaels et al., 2014b). Similarly despite various studies highlighting the ecological consequences of artificial visible lighting for amphibians, it has not been investigated to the same extent as UVB (Perry et al., 2008; Baker & Richardson, 2006). Light conditions are responsible for regulating many physiological processes within amphibians including growth and reproduction, both essential for survival. Yet no studies have examined the impact of artificial visible lighting on these or any other traits (Buchanan, 1993). Therefore, institutions that house amphibian species typically use ‘best practice’ as the guideline for provisioning visible light, as there are no standardised visible lighting protocols for amphibian husbandry (Pough, 2007). Currently, ‘best practice’ involves the use of fluorescent, halide and incandescent lights, all of which have various drawbacks.

However, Light Emitting Diodes (LEDs) are becoming a popular source of lighting in captive environments (Haitz & Tsao, 2011; Pimputkar et al., 2009). LEDs are leading a revolution in lighting; they provide energy efficient, environmentally friendly, cost effective and longer lasting lighting (Pimputkar et al., 2009). This revolution has extended to artificial lighting for ex situ enclosures as a range of LED lights specifically “designed for amphibians and reptiles” are now available. However, despite several studies demonstrating the potential risks and health problems that can result from exposure to LEDs in humans and other species, their production for use in amphibian enclosures has had no regulation or testing (Falchi et al., 2011; Behar- Cohen et al., 2011; Stone et al., 2012). Currently, these potential risks remain untested in amphibians but due to the sensitive nature of amphibians, in depth research into the effects of LEDs is necessary before they are utilised as the major light source in captive environments (Wake & Vredenburg, 2008). Light is crucial for amphibians and there is a great potential for LEDs to change the make up of captive environments. LEDs could alter the intensity and spectrum of light received in captivity and thus could potentially impact the functioning of important behavioural and physiological processes (Falchi et al., 2011). Consequently the need to establish any impacts of LEDs on amphibian welfare is of paramount importance. In order to assess LEDs effectively, a comparison and evaluation with the current artificial lighting provided (“best practice”) is required. However, there is a distinct lack of information regarding the visible lighting amphibians typically receive in captivity. In order to accurately compare LEDs to lighting currently provided by institutions for amphibian species, the spectral and thermal output of these forms of lighting needs to be quantified. Only then can LEDs be assessed as a suitable replacement for current lighting and investigations into welfare risks be carried out.

12 General Introduction

Project Aims

In this thesis I aim to inform husbandry protocols by increasing the understanding of a wild population of amphibians and investigating visible lighting in amphibian captive environments to improve ex situ conservation efforts. • Chapter 1 seeks to establish the general size and body condition of a wild population of M. cowani to provide guidelines for ex situ conservation. This species is facing an imminent extinction risk and is therefore expected to be brought for ex situ conservation. This information can inform and alter husbandry practices in order to maximise the success of ex situ conservation and reintroduction programmes. • Chapter 2 aims to investigate and quantify the temperature and light spectral output of lighting currently used in amphibian enclosures in zoological institutions. This information allows for a comparison and evaluation of new LED lights to assess their potential for replacing these lights. • Chapter 3 determines any potential implications for the welfare of amphibians of using LED lights in enclosures, specifically the impacts on growth, colouration and behaviour.

These studies aim to supplement existing information for captive ex situ populations of amphibians to increase the success rate of reintroduction. This thesis aims to evaluate whether LEDs can provide a safe and effective alternative to current lights with the overall aim to provide recommendations regarding the use of LEDs in amphibian husbandry.

13 General Introduction

References Baker, B.J. & Richardson, J.M.L. (2006) The effect of artificial light on male breeding-season behaviour in green frogs, Rana clamitans melanota. Canadian Journal of Zoology, 84 (10), p1528–1532. Bancroft, B.A., Baker, N.J. & Blaustein, A.R. (2008) A meta-analysis of the effects of ultraviolet B radiation and its synergistic interactions with pH, contaminants, and disease on amphibian survival. Conservation Biology, 22 (4), p987–996. Banks, C.B., Lau, M.W.N. & Dudgeon, D. (2008) Captive management and breeding of Romer’s tree frog Chirixalus romeri. International Zoo Yearbook, 42 (1), p99–108. Behar-Cohen, F., Martinsons, C., Viénot, F., Zissis, G., Barlier-Salsi, A., Cesarini, J.P., Enouf, O., Garcia, M., Picaud, S. & Attia, D. (2011) Light-emitting diodes (LED) for domestic lighting: any risks for the eye? Progress in Retinal and Eye Research, 30 (4), p239–57. Buchanan, B.W. (1993) Effects of enhanced lighting on the behaviour of nocturnal frogs. Animal Behaviour, 45 (5), p893–899. Densmore, C.L. & Green, D.E. (2007) Diseases of amphibians. ILAR Journal, 48 (3), p235–254. Falchi, F., Cinzano, P., Elvidge, C.D., Keith, D.M. & Haim, A. (2011) Limiting the impact of light pollution on human health, environment and stellar visibility. Journal of Environmental Management, 92 (10), p2714–22. Ferrie, G.M., Alford, V.C., Atkinson, J., Baitchman, E., Barber, D., Blaner, W.S., Crawshaw, G., Daneault, A., Dierenfeld, E., Finke, M., Fleming, G., Gagliardo, R., Hoffman, E.A., Karasov, W., Klasing, K., Koutsos, E., Lankton, J., Lavin, S.R., Lentini, A., Livingston, S., Lock, B., Mason, T., McComb, A., Morris, C., Pessier, A.P., Olea‐Popelka, F., Probst, T., Rodriguez, C., Schad, K., Semmen, K., Sincage, J., Stamper, M.A., Steinmetz, J., Sullivan, K., Terrell, S., Wertan, N., Wheaton, C.J., Wilson, B. & Valdes, E. V. (2014) Nutrition and health in amphibian husbandry. Zoo Biology, 33 (6), p485–501. Griffiths, R.A. & Pavajeau, L. (2008) Captive breeding, reintroduction, and the conservation of amphibians. Conservation Biology, 22 (4), p852–861. Haitz, R. & Tsao, J.Y. (2011) Solid-state lighting: ‘The case’ 10 years after and future prospects. physica status solidi (a), 208 (1), p17–29. Licht, L.E. & Grant, K.P. (1997) The effects of ultraviolet radiation on the biology of amphibians. American Zoologist, 37 (2), p137–145. Mallinson, J.J.C. (2003) A sustainable future for zoos and their role in wildlife conservation. Human Dimensions of Wildlife, 8 (1), p59–63. McGregor Reid, G. & Zippel, K.C. (2008) Can zoos and aquariums ensure the survival of amphibians in the 21st century? International Zoo Yearbook, 42 (1), p1–6. Michaels, C.J., Antwis, R.E. & Preziosi, R.F. (2014a) Impact of plant cover on fitness and behavioural traits of captive red-eyed tree frogs (Agalychnis callidryas). PloS one, 9 (4), pe95207. Michaels, C.J., Antwis, R.E. & Preziosi, R.F. (2014b) Impacts of UVB provision and dietary calcium content on serum vitamin D3 , growth rates, skeletal structure and coloration in

14 General Introduction

captive oriental fire-bellied toads (Bombina orientalis). Journal of Animal Physiology and Animal Nutrition, 99 (2), p391–403. Michaels, C.J., Gini, B.F. & Preziosi, R.F. (2014c) The importance of natural history and species-specific approaches in amphibian ex-situ conservation. Herpetological Journal, 24, p135–145. Michaels, C.J. & Preziosi, R.F. (2015) Fitness effects of shelter provision for captive amphibian tadpoles. Herpetological Journal, 25 (1), p21–26. Moore, R.D. & Church, D.R. (2008) Implementing the Amphibian Conservation Action Plan. International Zoo Yearbook, 42 (1), p15–23. Pavajeau, L., Zippel, K.C., Gibson, R. & Johnson, K. (2008) Amphibian Ark and the 2008 Year of the Frog Campaign. International Zoo Yearbook, 42 (1), p24–29. Perry, G., Buchanan, B. & Fisher, R. (2008) Effects of artificial night lighting on amphibians and reptiles in urban environments. In: J. C. Mitchell, R. E. Jung Brown, & B. Bartholomew eds. Urban Herpetology. Society for the Study of Amphibians & Reptiles, p239–256. Pimputkar, S., Speck, J.S., DenBaars, S.P. & Nakamura, S. (2009) Prospects for LED lighting. Nature Photonics, 3 (4), p180–182. Pough, F.H. (2007) Amphibian biology and husbandry. ILAR Journal, 48 (3), p203–213. Speakman, J.R. (2001) Body Composition Analysis of Animals: a Handbook of Non-Destructive Methods. Cambridge: Cambridge University Press. Stone, E.L., Jones, G. & Harris, S. (2012) Conserving energy at a cost to biodiversity? Impacts of LED lighting on bats. Global Change Biology, 18 (8), p2458–2465. Tapley, B., Bradfield, K.S., Michaels, C. & Bungard, M. (2015) Amphibians and conservation breeding programmes: do all threatened amphibians belong on the ark? Biodiversity and Conservation, 24 (11), p2625–2646. Tapley, B., Rendle, M., Baines, F.M., Goetz, M., Bradfield, K.S., Rood, D., Lopez, J., Garcia, G. & Routh, A. (2014) Meeting ultraviolet B radiation requirements of amphibians in captivity: a case study with mountain chicken frogs (Leptodactylus fallax) and general recommendations for pre-release health screening. Zoo Biology, 34 (1), p46–52. Wake, D.B. & Vredenburg, V.T. (2008) Are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proceedings of the National Academy of Sciences of the United States of America, 105, p11466–11473. Zippel, K., Johnson, K., Gagliardo, R., Gibson, R., McFadden, M., Browne, R., Martinez, C. & Townsend, E. (2011) The Amphibian Ark: a global community for ex situ conservation of amphibians. Herpetological Conservation and Biology, 6 (3), p340–352.

15 Chapter 1

Establishing in situ weight, snout-vent length and a body condition index as ex situ guidelines for the endangered frog Mantella cowani

Abstract Amphibians are in the midst of an extinction crisis with numbers declining rapidly across the globe. Ex situ conservation efforts are now essential for the maintenance of reserve populations and therefore an integral part of amphibian conservation. For reintroduction initiatives to be successful, ex situ conservation programmes must maintain healthy populations. However, healthy and fit populations are often difficult to sustain in captivity over a long period of time. Thus there is generally little success seen in reintroduction attempts. Amphibians are often brought in for ex situ conservation with a limited understanding of their biology and specialised needs. This is due to the insufficient studies on wild populations. Efforts to maximise information regarding vulnerable species is therefore required. This study seeks to determine the typical size of M. cowani in the wild in order to provide a guideline for future ex situ conservation efforts. Captive programmes can subsequently utilise this information to guide husbandry techniques. This study also provides valuable information regarding the population of M. cowani in one of its three remaining extant locations. It determines that body condition is largely stable over time and in all areas of the site. Yet, habitat type may have an impact on the overall size of individuals, though the mechanism or reasoning for this is unknown. This study provides an insight into the fitness of this population of M. cowani and will allow for comparisons between other wild populations to ensure that in situ programmes are directed to the most appropriate sites. This preliminary study will be useful for future ex situ conservation programmes providing in situ information of a wild population of M. cowani, guidelines for their maintenance in captivity and demonstrates methods for surveillance of wild populations.

16 Chapter 1

1.1 Introduction Amphibians are in danger; climate change, habitat destruction and disease, amongst many other factors, threaten their survival (Wake & Vredenburg, 2008; Zippel et al., 2011). Termed the Global Amphibian Crisis, 1/3 of the remaining species of amphibians are currently threatened and up to 7% are on the brink of extinction (Zippel et al., 2011). The Harlequin Mantella, Mantella cowani, is categorised as endangered on the IUCN Red List (IUCN, 2015). With a significantly limited distribution, found only in 3 geographically isolated individual sites on the high plateau of Eastern Madagascar, its population is highly vulnerable (Andreone et al., 2005). M. cowani has previously experienced a population crash as a result of overexploitation in the pet trade, however habitat destruction is now the mounting issue in the majority of their areas of distribution (IUCN, 2015). The 3 remaining sites of M. cowani are small and isolated, totalling an area of only 258km2. This largely due to habitat degradation for agricultural use, specifically slash and burn deforestation (Andreone et al., 2005). Though the population has stabilised since 2003, as a result of the introduction of a moratorium in the pet trade, populations remain small, geographically isolated and their habitats are in poor condition (Andreone et al., 2005). There is little national protection to maintain these crucial habitats and limited attempts to mitigate in situ threats have been made (IUCN, 2015).

Increasingly ex situ conservation is being utilised to attempt to halt the decline of amphibian populations. Most of the threats faced by amphibians, including M. cowani, involve habitat alteration or degradation and disease spread. Problematically, these threats are simply occurring at too fast a rate to mitigate in the wild (Antwis & Browne, 2009). Therefore, in situ programmes alone are not viable strategies to prevent extinctions (McGregor Reid & Zippel, 2008; Zippel et al., 2011). As such efforts to maximise the number of amphibian species in captivity are being made (Moore & Church, 2008; Michaels et al., 2014c). Amphibian species in Madagascar in particular, are now considered to a high priority for amphibian ex situ conservation attempts due to the high endemism and species richness (Edmonds et al., 2012). As a result captive populations of M. cowani are due to be established in the near future (G. Garcia, Chester Zoo 2016, pers comms., 2nd October).

However, problems emerge with ex situ conservation programmes due to insufficient information of wild populations regarding species life history, breeding behaviour and environmental requirements (Michaels et al., 2014c). According to the IUCN, 600 species of amphibians need to be brought into captivity, and there is a distinct lack of information to care adequately for many of these species whilst there is no information on ¼ of all amphibian species (Pough, 2007; Zippel et al., 2011). Equally, most of the amphibian species identified as a priority for ex situ conservation are highly specialised and extremely vulnerable (Michaels et al., 2014c). Therefore whilst there is a critical need to bring M. cowani into captivity, there is a distinct lack of research examining this species. Rather, existing research has mostly focused on the genetics and distribution. Although this information is valuable, life history data are

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required in order to supplement this information (Andreone et al., 2005). As little is known about M. cowani, information obtained from populations in the wild is required before effective ex situ conservation programmes can be established.

Inevitably, captive populations often differ drastically from their wild counterparts (Terranova & Coffman, 1997). An essential aspect of ex situ conservation is maintaining fit and healthy populations for eventual reintroduction (Michaels et al., 2014c). However, many captive amphibian populations suffer a decline in fitness throughout their time in captivity, compromising their potential for successful reintroduction (Tapley et al., 2015; Michaels et al., 2014c). For example, studies investigating the differences between wild and captive lemurs have determined that lemurs in captivity are all significantly overweight compared to individuals in the wild (Terranova & Coffman, 1997; Schwitzer & Kaumanns, 2001). This is a widespread issue observed in a variety of species in captivity such as chimpanzees and rhinos (Clauss & Hatt, 2006; Brent, 1995). Crucially, obesity is also commonplace in amphibian captive populations as a result of excess food and inactivity (Michaels et al., 2014b). Studies determining explicit differences between captive and wild populations such as this have subsequently helped to improve existing husbandry techniques by making recommendations to alter nutritional provisioning (Schwitzer & Kaumanns, 2001). In order to mitigate a gulf in differences between captive and wild populations, information regarding the typical size and weight of individuals in the wild should be obtained. This information can then be used to provide appropriate targets for body size (Terranova & Coffman, 1997).

Obtaining a measure of fitness from a wild population can also be valuable for ex situ conservation programmes seeking to bring amphibian species into captivity. However, assessing the fitness, i.e. the physiological state and reproductive success of individuals can be difficult (Jakob et al., 1996). Fitness cannot be determined directly, and therefore is often estimated via proxies (Jakob et al., 1996). Typically, body condition indices are used as a proxy for fitness (Jakob et al., 1996; Green, 2001). Body condition is used as a measure of nutritional state e.g. lipid content or energy stores, to give an estimate of foraging success and therefore fitness (Jakob et al., 1996; Wilder et al., 2016; Peig & Green, 2009). There are a range of body condition indices that are used in ecology, and specifically amphibians and these are calculated in a variety of ways. Predominantly ecologists use calculations relating body mass and body length (Jakob et al., 1996; Green, 2001; Peig & Green, 2010).

The Scaled Mass Index (SMI) is a body condition index used frequently in amphibian studies (Green, 2001; Peig & Green, 2009). The SMI uses the principle of scaling to account for the allometric relationship between mass and body structure (Peig & Green, 2009). It standardises the body mass at a set linear measurement based on the scaling relationship between the mass and length (Peig & Green, 2010). Standardisation allows for comparisons between populations, adequately controls for the variation in body size and effectively accounts for differences in

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ontogenetic variation and sexual dimorphism (Peig & Green, 2009). It removes the effects of growth to give an accurate assessment of health (Peig & Green, 2010). Several studies have successfully used the SMI to assess body condition in various species of amphibians (MacCracken & Stebbings, 2012; Michaels, et al., 2014a). These studies have determined it as an accurate body condition index for amphibians as it reflects the true energy stores of individuals, and therefore lipid content and foraging success (MacCracken & Stebbings, 2012). SMI has been validated by invasive measures of body composition such as body fat, protein and lean mass (Peig & Green, 2009; MacCracken & Stebbings, 2012). SMI is consequently viewed as a suitable predictor of fitness (MacCracken & Stebbings, 2012).Similarly, unlike most other body condition indices it also does not indicate any systemic bias toward larger individuals and is resistant to statistical assumptions (Peig & Green, 2010). Therefore, SMI is commonly used to determine the body condition of amphibian populations and can also be used for comparisons between wild and captive populations in ex situ conservation.

Calculating and monitoring the SMI of ex situ populations can help avoid the introduction of potentially less fit individuals that will hamper reintroduction attempts and reduce the fitness of the existing wild population. Additionally, as little is known about M. cowani, it would be useful to determine if there is a distinguishable range in the body condition of individuals observed in the wild (IUCN, 2015). This would provide an effective range guideline for captive populations. Determining the stability of body condition will also be informative as it can help to identify if fluctuations in body condition occur regularly and provide a useful guideline of full range of safe body condition values for future captive populations.

Yet, determining the body condition of a wild population may also provide valuable information for in situ conservation efforts. Body condition indices are powerful tools for in situ conservation particularly for amphibians (Janin et al., 2011; Smets et al., 2010). Various studies have successfully utilised body condition indices to determine the fitness and state of wild populations of amphibians (Janin et al., 2011; Smets et al., 2010; Reading, 1995). These assessments of body condition have been used in amphibian conservation efforts to provide indications of habitat quality, food availability and abundance and environmental stress amongst other environmental parameters as high body condition indices correlate with high quality environments (Janin et al., 2011; Smets et al., 2010; Sztatecsny & Schabetsberger, 2005). Therefore obtaining a measure of body condition from a wild population of M. cowani will be useful for future in situ conservation efforts as not only will it provide an indication of the health and quality of the population as a whole, it will also help to identify areas of poor habitat quality and in need of greater conservation efforts (Smets et al., 2010; Sztatecsny & Schabetsberger, 2005). Similarly, investigations between different habitat types within this site of M. cowani will help determine if habitat and microclimates can influence the body condition of individuals in the wild (Stevenson & Woods, 2006).

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Aims In this study I aim to establish ex situ guidelines for the weight, length and body condition from an in situ population of M. cowani. Body condition measurements will also be examined to assess the influence of site on the population fitness and stability to inform in situ conservation efforts.

1.2 Methods Ethics Statement All methods used in this study were non-invasive. The University of Manchester Ethics Committee approved this study prior to its commencement.

Study Species The species investigated in this study is Mantella cowani or the Harlequin Mantella. M. cowani was recently re-classified from critically endangered to endangered on the IUCN Red List in 2014 (IUCN, 2015). M. cowani population size was decimated by the pet trade, and though this had been halted, issues such as habitat destruction remain (Andreone et al., 2005). It has a limited distribution to 3 distinct sites in Madagascar; Anoetra, Itremo and Antakasina (IUCN, 2015). It has a total area of occupancy (AOO) of only 253km2 (IUCN, 2015). There is limited knowledge of this species. As with other species of Mantella, females are generally larger and stocky compared to males. There is no known information on oviposition site choice and eggs and tadpoles have never been reported in the wild (IUCN, 2015).

Field Site Fieldwork was carried out at a known site of M. cowani, the Fohisokina Mountain site of Anoetra in Eastern Madagascar. Though it is not a nationally protected area, since 2010 the community, as part of the FOMISAME programme, has protected it locally (IUCN, 2015). The study site comprised of a range of terrain including savannah grassland, rivers and streams, small sites of larger foliage and mountainous rocks. The site had an elevation ranging from 1558 – 1628m. Sampling took place along 9 transects that had been established in a previous pilot study in Fohisokina as areas frequented by M. cowani (Randrianantoandro et al., 2015). Each transect was 50m long and covered the different substrates such as savannah, the stream banks and rock faces at different elevations. See Table 1.1 for a brief description of the 9 transects and for a satellite image of the transects see Figures 1.1 and 1.2.

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1.1

1.2

Figure 1.1 (above) and 1.2 (below): Google Earth satellite images of the Fohisokina field site (20°42’12.67” S, 47°17’18.17” E, Elevation = 1605km) obtained and edited within Google Earth (Google Inc., 2015). Transects are highlighted by the 50m lines. Stream, savannah and rock transects are indicated by blue, yellow and red lines respectively.

Table 1.1: Detailed description of transects 1-9. Elevation information obtained from Google Earth (Google Inc., 2015).

Transect Type Description Elevation 1 Stream Bank of a small stream with a combination of 1558- savannah grassland, rocks, crevices, caves and larger 1563m foliage. The steam was approximately <1m wide along the length of the transect. 2 Savannah Savannah grassland with shorter grasses and a large 1564- cave situated at the end of the transect. 1579m 3 Stream Bank of a small stream with a combination of rocky 1568- and savannah terrain with large foliage, caves and 1585m crevices. The steam was approximately <1m wide along the length of the transect. 4 Rock Vertical rock face with both larger and small moss and 1577- plant islands distributed across it. Some running water 1601m was observed at the top across the rock and moss. The rock is uneven with many crevices and small caves.

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5 Savannah Savannah grassland with mostly high grasses and 1595- occasional large rocks on a vertical slope. 1628m 6 Rock Mostly horizontal rock face at the highest elevation 1617- with several small streams running across and various 1620m plant and moss islands. The rock was uneven and had several crevices and no caves. 7 Rock Vertical rock face with no running water and limited 1606- plant islands and no moss islands. Rock was uneven 1580m with many crevices and no caves. 8 Savannah Savannah grassland with tall grasses and ferns with no 1578- rocks or caves. 1568m 9 Stream Bank of a small river with very wet and marshland 1568- terrain. Width of the river range from <1m - 2m. 1572m

Sampling and Data Collection The study was undertaken for 4 weeks at the start of the rainy season in Madagascar from the 27th November – 21st December 2015. Sampling was conducted twice a day for two sets of ten day periods between the hours of 05:00-08:00, at dawn, and 16:00-19:00, at dusk. These times had been established in a previous pilot study as the peak times of M. cowani activity (Randrianantoandro et al., 2015). During sampling the 9 transects were surveyed by 5 individuals, at a steady pace. The order and therefore the start and ends of the 9 transects was rotated at each data collection. Each observed individual of M. cowani was temporarily caught. A limited number of unsuccessful captures were not recorded in our data. Snout to vent length (SVL) and weight measurements were taken from each individual successfully caught. SVL was measured with callipers and recorded to 0.1mm. Body weight measurements using portable Smart Weigh™ Scales to 0.01g. Photographs of the ventral side of each individual were taken. The date, time (am or pm) and the transect on which they were caught was recorded. Sex cannot be determined precisely by a specific characteristic but males are typically smaller than females. Larger individuals were therefore classified as females (>28cm) and smaller individuals classed as males (<28cm). 28cm was the midpoint in the lengths of individuals. Individuals were then immediately released where they were captured.

Hotspotter Animal Recognition Software A total of 467 individuals were captured, measured and photographed but sufficient data was only obtained for 391 specific individuals as result of various reasons such as incorrectly functioning equipment, bad weather or repeated captures in the same time period. Individuals may have been captured multiple times over the course of the month. In order to distinguish how many specific individuals were captured, Hotspotter (Crall et al., 2013), an animal individual recognition software was used. The photographs of the ventral side of each individual were uploaded into the software. The orientation of the frog was indicated on each individual photo. The software identified the unique ventral patterning of the individual and gave a probability estimate of the match to every other photograph. The final decision of the individual match was done by eye. Overall 391 sampling individuals were identified to 102 distinct individuals. Of these 9 were identified as juveniles (SVL of <24cm) and removed from the subsequent calculations.

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Body Condition Index To calculate the body condition index, the scaled mass index (SMI) was used. The SMI was calculated for the individual in each photograph, excluding the photographs of the 9 juveniles. The SMI was calculated following the procedure laid out by Peig and Green (2009) and followed by Michaels et al. (2014a), and MacCracken & Stebbings (2012). This was carried out in the statistical program R. Firstly a bivariate plot of Weight vs SVL was produced to highlight any outliers to the data (Figure 1.3). As a result of the field nature of this data collection it is assumed that these outliers were caused by incorrect data collection and as repeated data was obtained for individuals these outliers were subsequently removed from the data set (2).

Figure 1.3: Bivariate plot of the SVL (cm) vs Weight (g). Two outliers are highlighted in the red. Outliers were removed from the data set.

The Scaled Mass Index was then calculated via the equation:

!!"# !! SMI = M! !! where Mi is the Individual Weight (g), Li is the Individual SVL (mm), L0 is the average SVL of the population and bSMA is the scaling component as determined by the standardised major axis regression on the natural log of Mass and SVL of the population (Peig & Green, 2009; Peig & Green, 2010). This was calculated for separately for Males (<28mm) and Females (>28mm). The Scaled Mass Index value was then calculated for each individual measurement. The mean SMI was calculated for each specific individual and used in subsequent analysis.

Statistical Analysis The average mass, SVL and SMI was calculated for each specific individual of which there were 92 (excluding juveniles (9) and outlier data (1)). These values were used in subsequent statistical analysis in R. One-way ANOVAs were conducted to determine the differences in Mass, SVL and SMI among transects in males and females. Post Hoc Tukey tests were conducted to identify specific differences.

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1.3 Results Guidelines for Weight, SVL and SMI

Table 1.2: Table highlighting the average, minimum and maximum mass for male and female Mantella cowani in the Fohisokina site. ±SEM is indicated for the average weight.

Average Weight (g) Minimum Weight (g) Maximum Weight (g) Male 1.50 ±0.020 1.06 2.20 Female 1.99 ±0.016 1.44 2.55

Table 1.3: Table highlighting the average, minimum and maximum snout-vent length (SVL) for male and female Mantella cowani in the Fohisokina site. ±SEM is indicated for the average SVL.

Average SVL (mm) Minimum SVL (mm) Maximum SVL (mm) Male 26.58 ±0.075 24.2 27.9 Female 29.24 ±0.060 28.0 32.2

Table 1.4: Table highlighting the average, minimum and maximum Scaled Mass Index for male and female Mantella cowani in the Fohisokina site. ±SEM is indicated.

Average SMI Minimum SMI Maximum SMI Male 1.50 ±0.016 1.13 2.13 Female 2.04 ±0.030 1.21 3.44

Mass The average weight for male M. cowani is 1.50g and females weigh on average 0.5g more than males, (Table 1.2). Both males and females have a range in weight of just over a 1g.

SVL There is over 2.5mm difference in SVL between the larger females and smaller males (Table 1.3). Both males and females have a range in length of around 4mm.

SMI The average SMI index for both male and female M. cowani is very similar to the average mass (Table 1.4). There is a wide range of 2.23 in the SMI of females, whereas only a difference of 1 is seen in SMI of males. Female body condition differs more and has a wider range than male body condition.

Impact of Transect on Weight, SVL and SMI Weight

Mass differs between males and females across every transect (F1,351 = 237.613, p < 0.001). Overall mass does not differ between males on different transects, nor females on different transects (F5,351 = 1.108, p = 0.356), Figure 1.4. However, mass differed in males between specific transects. The average mass of males located on transect 1 was lower than males

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located on transect 6 (p = 0.002). The average mass of males located on transect 2 was also less than males located on transect 6 (p < 0.001) and transect 4 (p < 0.001). The average mass of males similarly was lower in those located on transect 3 (p < 0.001) and transect 6 and transect 4 (p < 0.001).

Snout-Vent Length

SVL also differs between males and females in every transect (F1,351 = 572.898, p < 0.001). Overall SVL is consistent between males on different transects and females on different transects (F5,351 = 1.638, p = 0.149), Figure 1.5. Between some specific transects SVL did differ in males. Similar to the differences in mass, male individuals located on transect 2 were smaller in length than males on transect 4 (p = 0.001) and transect 6 (p = 0.025). Males were also significantly also smaller in length on transect 3 compared to males on transect 4 (p < 0.001) and transect 6 (p < 0.001).

Scaled Mass Index

The SMI of individuals differs between males and females on each transect (F1,112 = 42.143, p < 0.001). The SMI of individuals does not change between different transects in either males or females (F1,112 = 0.158, p = 0.9771), Figure 1.6.

Stability of SMI Overall the SMI stayed fairly constant in individuals across the duration of the 25 day study (Figure 1.7). Most individuals had similar or minor changes in their SMI when measured at different points in the study. However there were severe changes in SMI in 12 of 92 individuals. 4 individuals saw large increases in their SMI when measured on different days, whereas 8 individuals saw signficant decreases in their SMI over the study period. Overall there were limited instances of these large changes.

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Figure 1.4: Bar chart illustrating the mean mass (g) of female and male individuals on the different transects. Transects 4, 6 and 7 were rock transects, transects 1 and 3 were stream transects and transect 3 was a savannah transect. Error bars indicate the ±SEM. There are no distinct differences in mass between males or females located on different transects, p>0.05.

Figure 1.5: Bar chart illustrating the mean snout-vent length (g) of female and male individuals on the different transects. Transects 4, 6 and 7 were rock transects, transects 1 and 3 were stream transects and transect 3 was a savannah transect. Error bars indicate the ±SEM. There are no distinct differences in SVL between males or females located on different transects, p>0.05.

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Figure 1.6: Bar chart illustrating the scaled mass index of female and male individuals on the different transects. Transects 4, 6 and 7 were rock transects, transects 1 and 3 were stream transects and transect 3 was a savannah transect. Error bars indicate the ±SEM. There are no distinct differences in SMI between males or females located on different transects, p>0.05.

Figure 1.7: The Scaled Mass Index of 92 individuals over the 25-day sampling period. Individuals only caught once represent the single points. Individuals caught multiple times are indicated by the line and the change in SMI over time can be seen.

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1.4. Discussion Mass and SVL Previous studies investigating M. cowani have identified its geographic range, genetic variation, threats to its survival and to an extent the total population size (Chiari et al., 2006). Although important, this information is not especially valuable for ex situ conservation efforts. This study, however, provides an outline of the typical size of M. cowani in terms of its length and weight. No study prior to this has investigated the morphology of M. cowani and thus there is no existing consensus on the typical size of individuals. This study has provided a typical body mass and SVL of a wild population of M. cowani. These results indicate that although weight can vary in both males and females by up to 1g, female M. cowani should typically weigh around 1/3 more than males. Generally, females are also longer than males, yet, this difference is not as pronounced as the difference in weight and therefore females are typically stockier than males. However, this is impacted by the categorisation of males and females to a specific length, where females (>28cm) will always be longer than males (<28cm). Longer males and shorter female data will have been lost and therefore the true difference in length between males and females may not be shown.

Monitoring the size of individuals in captivity can allow for comparisons to individuals in wild populations. Determining the average weight and length of individuals in this wild population can provide a target weight and length for individuals in captivity. These minimum and maximum values also outline the acceptable range of weight and length for individuals in captivity. If the size of individuals differs significantly between wild and captive populations, it can result in changes to amphibian husbandry practices to ensure that populations remain similar. For example, a difference in weight could indicate that a change is required in diet, whereas differences in body condition or SVL may indicate that changes in the captive environment are required (Hayes et al., 1998; Michaels et al., 2014b). Equally enrichment has been hypothesised as a method which may improve the quality and fitness of individuals in ex situ populations (Michaels et al., 2014b). Maintaining healthy and fit populations is an essential aspect of ex situ conservation, as reintroduction attempts rely upon consistent fitness between those in captivity and in the wild (McGregor Reid & Zippel, 2008). Therefore, these results will help inform and enhance the ability of ex situ conservation projects to maintain healthy individuals, increasing the likelihood of success upon reintroduction.

Body Condition This study has also provided the typical body condition index for a wild population of M. cowani in terms of a Scaled Mass Index (Peig & Green, 2009). There is a considerably larger range in the SMI of female M. cowani, indicating a greater range in body conditions in females. This likely reflects the added weight of gravid females relative to their body size. Similarly to body weight, these results can be used as a guideline for ex situ populations of M. cowani, to ensure that captive populations are maintained at the correct weight for their size, as obesity is a

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common issue in captivity particularly for amphibians (McWilliams, 2008; Schwitzer & Kaumanns, 2001).

Effect of Transects As well as providing valuable guidelines for ex situ populations of M. cowani, the SMI of individuals can also be used to examine the in situ population of M. cowani. The impact of other factors including their location on individual fitness and size within this population can be investigated. Mass and SVL differed significantly between transect sites, as individuals on the rock transect sites were generally found to be larger than those on the stream or savannah transects. However, interestingly this was not reflected in the SMI results, where body condition did not differ significantly between individuals on different transects. This indicates that whilst individuals may be bigger, this does not necessarily correlate with their body condition. The use of SMI, which accounts for the variation in size, demonstrates that the individuals in the sites examined are in a similar condition. This suggests that other factors must play a role in the difference in the size of individuals among the transects.

One potential factor responsible for the difference in the general size of the individuals between transects is territoriality. There may be greater competition for space (e.g. caves or females) on the rockier transects rather than at the stream, so the larger individuals displace smaller individuals from these sites. Though territoriality has not been studied extensively in Mantella, and not at all in M. cowani, territoriality and male-male aggression has been observed as a behavioural trait in Mantella laevigata (Heying, 2001). Therefore, territoriality could play a role in the difference in size. However, additional behavioural observations would need to be carried out in order to fully assess this hypothesis. Alternatively, it is possible to hypothesise that a difference in size may be attributable to greater food acquisition during development, which results in larger individuals in adulthood. Greater numbers of individuals were observed on the rock transects, and of those observed most were seen feeding. If individuals are predominantly located in refugia near the rock transects rather than the stream transects as juveniles, they may have a greater access to food, which may result in a greater growth in earlier life. Although this study did not seek to investigate the reason for these differences and thus cannot definitively postulate an explanation, it nonetheless provides valuable details of the wild population of Mantella cowani and provides avenues for further research.

Stability of Individual Body Condition Similarly, this study calculates the SMI for the specific individuals over the course of the study in order to see if SMI fluctuates or remains relatively stable within a wild M. cowani population over time. This study indicates that typically the SMI body condition was stable for most individuals throughout the duration of this study. Whilst there were instances of significant change in the SMI of individuals, the instances of this occurring were largely infrequent. There was no discernable pattern in these fluctuations i.e. no majority in reductions or increases in body

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condition, suggesting that any large changes were random. More likely, it appears that significant changes were the result of taking recordings of individuals just after feeding or following a long period of no feeding. In general the SMI of individuals remained fairly consistent, which indicates that body condition is typically stable and therefore should be maintained as such in captivity. Yet, interestingly it also demonstrates that individuals can withstand large changes in SMI if necessary. This information can similarly help to guide ex situ husbandry methods.

Population Fitness Not only can these results guide husbandry methods for captive populations, measures of body condition could provide information regarding the population at the Anoetra site as a whole. Anoetra is one of the three remaining locations of M. cowani and assessments of body condition are increasingly being used as a tool in conservation to assess the health and quality of population habitats (Stevenson & Woods, 2006; Smets et al., 2010). Habitat degradation is undoubtedly impacting survival and therefore population size in many amphibian species across the globe. However a reduction in habitat quality also has an indirect impact on the fitness of remaining populations (Stevenson & Woods, 2006). This has been highlighted by various studies undertaken investigating the impact of habitat quality, food availability and environmental stress on the body condition and therefore fitness of amphibians (Smets et al., 2010; Reading, 1995; Janin et al., 2011). These studies indicate that high quality vegetation and high food abundance and an abundance of nutritious food sources correlates with high BCI measurements (Smets et al., 2010; Reading, 1995; Janin et al., 2011). Therefore BCI measurements are indicative of habitat quality, and can be used to identify low quality habitats and suffering populations. By identifying these habitats, in situ conservation efforts can be directed to poorer quality habitats (Sztatecsny & Schabetsberger, 2005; Stevenson & Woods, 2006). Poor body condition indices are also caused by reduced habitat availability and habitat fragmentation. Calculating BCIs would therefore be highly informative as it can help identify habitats subject to these pressures and identifying areas for greater in situ conservation efforts (Janin et al., 2011). By measuring the body condition of populations across the various locations of M. cowani, the populations with the poorest BCIs and most at risk, likely to be within poor quality habitats, can be identified and therefore conservation efforts can be effectively directed relative to the proportion of the threat (Stevenson & Woods, 2006).

Despite a few exceptions, the body condition of individuals observed at the Anoetra site remains fairly stable over the period of this study. Further studies could explore the stability of body condition in other sites of M. cowani distribution. Slash and burn was observed in the Itremo site during the study period and the health of populations in these areas may be suffering as a result. If comparisons showed that body condition varied quite significantly between different sites or habitats showed signs of instability in body condition, it could indicate populations at risk potentially due to reduced habitat quality (Sztatecsny & Schabetsberger, 2005). Similarly,

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establishing the best environment for consistent body conditions in the wild will be a useful guideline for ex situ conservation efforts to help replicating this environment in captivity.

Limitations By calculating body condition indices (BCI), rather than simply the typical body mass and length, which as demonstrated in this study does not always reflect the true body condition of individuals, a more informative measure for individual fitness can be determined. However the use of BCIs as an accurate measure of fitness has been a topic of discussion in ecology and several drawbacks have been acknowledged. Some suggest that BCIs are uninformative, poorly defined, open to interpretation and therefore may be inconsistent (Wilder et al., 2016; Green, 2001). Additionally, there are concerns with how they are calculated and their ability to provide a reliable indication of lipid content, which limits their suitability as indicators of fitness (Wilder et al., 2016; Green, 2001). Wilder et al. (2016) suggests that in many cases body condition indices are poorly correlated with lipid content and further still lipid content may not accurately represent the body composition of individuals. He suggests that BCIs and lipid content are unrelated to fitness as lipid content is regulated to avoid extremes in body composition (Wilder et al., 2016). Similarly, the Scaled Mass Index specifically replaces the values for absolute body mass with a scaled mass value. Removing the absolute body mass information may result in misleading body condition values. Ecologists therefore suggest that other measures of body composition, such as lean muscle, bone content and amino acids or hormone assays are more direct and accurate indications of fitness (Wilder et al., 2016). However, problematically, these measurements typically require individual sacrifice, which is not a viable option when investigating endangered populations and undertaking field studies. Non- invasive measurements such as body condition indices are as such considered to be the next best alternative (Speakman, 2001). Therefore, although BCIs do have some limitations, ecologists still consistently use them (Speakman, 2001). They can be a powerful, easy to obtain and non lethal tool for conservation efforts and provide an indication of population fitness and habitat quality (Smets et al., 2010; Janin et al., 2011). Improvements have been made to indexes and investigations have concluded that some body condition indices, such as the Scaled Mass Index can be an accurate measure of fitness though all body condition indices measure of fitness should be taken with caution (Speakman, 2001).

Conclusion The information obtained regarding M. cowani’s size (length and weight) alongside the SMI provides a useful guideline for future ex situ conservation programmes that aim to bring the species in captivity. It can be used to help inform husbandry practices and ensure that populations can maintain the high level of fitness necessary for reintroduction. Mass, SVL and SMI are all non-invasive and easily attainable measurements, which can be obtained for other vulnerable and relatively unknown species before they are brought in for ex situ conservation programmes. Determining the SMI as a measure of body condition in amphibian studies

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provided valuable information for this population, providing an initial indication of fitness which will be useful for effective ex situ conservation programmes. These measurements allow for a species-specific approach to ex situ conservation, maximising our understanding of individual species due to brought in. It can be utilised for all captive populations with aims for reintroduction. In order to maximise the success of reintroduction attempts, species should be evaluated against their wild counterparts (Michaels et al., 2014c). Therefore, this study has demonstrated a useful tool for ex situ conservation efforts. Measurements of body condition can be also used to help in situ conservation efforts by assessing population fitness and stability. This study recommends that to fully utilise this information, body condition indices should be measured at the other sites of M. cowani to help determine which populations are the most threatened and require more urgent protection.

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1.5 References Andreone, F., Cadle, J.E., Cox, N., Glaw, F., Nussbaum, R.A., Raxworthy, C.J., Stuart, S.N., Vallan, D. & Vences, M. (2005) Species review of amphibian extinction risks in Madagascar: conclusions from the Global Amphibian Assessment. Conservation Biology, 19 (6), p1790–1802. Antwis, R.E. & Browne, R.K. (2009) Ultraviolet radiation and Vitamin D3 in amphibian health, behaviour, diet and conservation. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 154 (2), p184–90. Brent, L. (1995) Feeding enrichment and body weight in captive chimpanzees. Journal of Medical Primatology, 24 (1), p12–16. Chiari, Y., Andreone, F., Vences, M. & Meyer, A. (2006) Genetic variation of an endangered Malagasy frog, Mantella cowani, and its phylogeographic relationship to the widespread M. baroni. Conservation Genetics, 6 (6), p1041–1047. Clauss, M. & Hatt, J.-M. (2006) The feeding of rhinoceros in captivity. International Zoo Yearbook, 40 (1), p197–209. Crall, J.P., Stewart, C.V., Berger-Wolf, T.Y., Rubenstein, D.I. & Sundaresan, S.R. (2013) Hotspottter Recognition Software (Version 1.0.0). [Computer Program]. Available at http://cs.rpi.edu/hotspotter/ (Accessed April 2016). Edmonds, D., Rakotoarisoa, J.C., Dolch, R., Pramuk, J., Gagliardo, R., Andreone, F., Rabibisoa, N., Rabemananjara, F., Rabesihanaka, S. & Robsomanitrandrasana, E. (2012) Building capacity to implement conservation breeding programs for frogs in Madagascar: Results from year one of Mitsinjo’s amphibian husbandry research and captive breeding facility. Amphibian and Reptile Conservation, 5 (3), p57–69. Google Inc. (2015) Google Earth Pro (Version 7.1.0). [Computer Program]. Available at https://www.google.co.uk/earth/download/ge/agree.html (Accessed May 2016). Green, A.J. (2001) Mass/length residuals: measures of body condition or generators of spurious results? Ecology, 82 (5), p1473–1483. Hayes, M.P., Jennings, M.R. & Mellen, J.D. (1998) Environmental enrichment for amphibians and reptiles. In: D. J. Shepherdson, J. D. Mellen, & M. Hutchins eds. Second Nature: Environmental Enrichment for Captive Animals. Washington D.C., Smithsonian Institution, p205–235. Heying, H.E. (2001) Social and reproductive behaviour in the Madagascan poison frog, Mantella laevigata, with comparisons to the dendrobatids. Animal Behaviour, 61 (3), p567–577. IUCN (2015) IUCN Red List of Threatened Species. Version 2015-4 [Internet]. Available from: (Accessed 11 April 2016). Jakob, E.M., Marshall, S.D. & Uetz, G.W. (1996) Estimating fitness: a comparison of body condition indices. Oikos, 77 (1), p61–67. Janin, A., Léna, J.-P. & Joly, P. (2011) Beyond occurrence: Body condition and stress hormone as integrative indicators of habitat availability and fragmentation in the common toad. Biological Conservation, 144 (3), p1008–1016.

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MacCracken, J.G. & Stebbings, J.L. (2012) Test of a body condition index with amphibians. Journal of Herpetology, 46 (3), p346–350. McGregor Reid, G. & Zippel, K.C. (2008) Can zoos and aquariums ensure the survival of amphibians in the 21st century? International Zoo Yearbook, 42 (1), p1–6. Michaels, C.J., Antwis, R.E. & Preziosi, R.F. (2014a) Impact of plant cover on fitness and behavioural traits of captive red-eyed tree frogs (Agalychnis callidryas). PloS one, 9 (4), pe95207. Michaels, C.J., Downie, J.R. & Campbell-Palmer, R. (2014b) The importance of enrichment for advancing amphibian welfare and conservation goals: a review of a neglected topic. Amphibian & Reptile Conservation, 8 (1), p7–23. Michaels, C.J., Gini, B.F. & Preziosi, R.F. (2014c) The importance of natural history and species-specific approaches in amphibian ex-situ conservation. Herpetological Journal, 24, p135–145. Moore, R.D. & Church, D.R. (2008) Implementing the Amphibian Conservation Action Plan. International Zoo Yearbook, 42 (1), p15–23. Peig, J. & Green, A.J. (2009) New perspectives for estimating body condition from mass/length data: the scaled mass index as an alternative method. Oikos, 118 (12), p1883–1891. Peig, J. & Green, A.J. (2010) The paradigm of body condition: a critical reappraisal of current methods based on mass and length. Functional Ecology, 24 (6), p1323–1332. Randrianantoandro, C.J., Tsiorisoa, G.A. & Razafimanahaka, J.H. (2015) Measuring activity patterns of the critically endangered harlequin mantella (Mantella cowani) at savannah and forest sites in Madagascar, to inform future population monitoring and conservation efforts. Unpublished. Reading, C.J. & Clarke, R.T. (1995) The effects of density, rainfall and environmental temperature on body condition and fecundity in the common toad, Bufo bufo. Oecologia, 102 (4), p453–459. Schwitzer, C. & Kaumanns, W. (2001) Body weights of ruffed lemurs (Varecia variegata) in European zoos with reference to the problem of obesity. Zoo Biology, 20 (4), p261–269. Smets, J., Cogălniceanu, D., Băncilă, R.I., Hartel, T. & Plăiaşu, R. (2010) Comparing three body condition indices in amphibians: a case study of yellow-bellied toad Bombina variegata. Amphibia-Reptilia, 31 (4), p558–562. Speakman, J.R. (2001) Body Composition Analysis of Animals: a Handbook of Non-Destructive Methods. Cambridge: Cambridge University Press. Stevenson, R.D. & Woods, W.A. (2006) Condition indices for conservation: new uses for evolving tools. Integrative and Comparative Biology, 46 (6), p1169–90. Sztatecsny, M. & Schabetsberger, R. (2005) Into thin air: vertical migration, body condition, and quality of terrestrial habitats of alpine common toads, Bufo bufo. Canadian Journal of Zoology, 83 (6), p788–796. Tapley, B., Bradfield, K.S., Michaels, C. & Bungard, M. (2015) Amphibians and conservation

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breeding programmes: do all threatened amphibians belong on the ark? Biodiversity and Conservation, 24 (11), p2625–2646. Sztatecsny, M. & Schabetsberger, R. (2005) Into thin air: vertical migration, body condition, and quality of terrestrial habitats of alpine common toads, Bufo bufo. Canadian Journal of Zoology, 83 (6), p788–796. Terranova, C.J. & Coffman, B.S. (1997) Body weights of wild and captive lemurs. Zoo Biology, 16 (1), p17–30. Wake, D.B. & Vredenburg, V.T. (2008) Are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proceedings of the National Academy of Sciences of the United States of America, 105, p11466–11473. Wilder, S.M., Raubenheimer, D. & Simpson, S.J. (2016) Moving beyond body condition indices as an estimate of fitness in ecological and evolutionary studies. Functional Ecology, 30 (1), p108–115. Zippel, K., Johnson, K., Gagliardo, R., Gibson, R., McFadden, M., Browne, R., Martinez, C. & Townsend, E. (2011) The Amphibian Ark: a global community for ex situ conservation of amphibians. Herpetological Conservation and Biology, 6 (3), p340–352.

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Quantifying and comparing the spectral and thermal output of LEDs and current lighting in amphibian ex situ conservation

Abstract Effective ex situ conservation programmes are integral to the success and maintenance of healthy captive amphibian species. A crucial aspect of amphibian ex situ conservation is the provisioning of natural environments in captivity. One such provision is lighting. LED lighting is becoming increasingly popular due to their high-energy efficiency, low environmental impact and reduced costs. Yet, speculated harmful impacts of LEDs have resulted in the need to investigate their potential use. However, to investigate the use of LEDs in amphibian captive environments, lighting currently used first needs to be considered. Whilst there has been a significant focus on the importance and provisioning of UVB lighting in amphibian ex situ conservation programmes, limited studies have investigated or outlined the use of visible lighting in captive environments. This study investigates and quantifies the thermal and spectral output of visible lighting typically provided in zoological institutions. These results demonstrate multiple sharp spikes in irradiance at various wavelengths across the spectrum. There is also a significant thermal output of current lighting such as fluorescent and mercury vapour lamps, producing large temperature gradients and thermal microclimates. Alternatively, whilst LEDs produce a spike in spectral output at shorter wavelengths ~440-450nm, they also have a broader output of light across the whole spectrum and a limited heat output. Currently, the potential impact of these differences on amphibian welfare is unknown. This investigation provides an insight into the differences between LED and lighting currently used. It concludes that further investigation into the potential health implications of LED lighting is necessary before their implementation in captive environments.

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2.1 Introduction Ex situ conservation is an essential aspect of amphibian conservation, crucial for the maintenance of ‘reserve’ populations of endangered species (Moore & Church, 2008). Amphibian husbandry is a key component determining the success of ex situ conservation programmes, as the provisioning of specific and appropriate environmental conditions is needed for amphibian survival (Michaels et al., 2014c). Light is a basic environmental parameter and is critical for many physiological functions and processes in amphibians such as growth, reproduction and development (Verschooren et al., 2011; Ferrie et al., 2014). Thus captive environments will need to replicate natural light cycles experienced in the wild to ensure these processes are carried out correctly (Browne et al., 2007). “Light” encompasses a wide range of wavelengths within the electromagnetic spectrum and studies investigating the importance of lighting in amphibian ex situ conservation have typically focused on the provisioning of UV light (100-400nm), rather than visible light (400-700nm) (Antwis & Browne, 2009; Bancroft et al. 2008). There are no studies investigating the provisioning of visible lighting for amphibians despite suggestions that light intensity, wavelength and colour temperature can potentially impact animal wellbeing in captivity (Morgan & Tromborg, 2007). This is also surprising due to the high sensitivity of amphibians and their vulnerability to changes in environmental parameters (Boeuf & Le Bail, 1999). Accordingly, not only is there limited information available regarding the current provisioning of artificial visible lighting in amphibian enclosures but few studies have investigated the potential effects this may have on the fitness of captive populations.

For most establishments, natural lighting is difficult to provide in captivity, yet artificial lighting that most accurately mimics natural lighting should be employed (Morgan & Tromborg, 2007). Natural irradiance from the sun has a broad spectral output, across the range of wavelengths within the visible spectrum (Ellis et al., 2014; Thorington, 1985). There is a high irradiance level at shorter wavelengths in the visible spectrum and declining in the longer wavelengths (Thorington, 1985). Though, irradiance values change depending on the time of day and year, and the location of the irradiance, typically the spectral curves for global natural irradiance are similar across the year (Thorington, 1985). The spectral distribution is unaltered by the time of year, cloud cover and turbidity (Thorington, 1985). Lighting that is most similar to this natural daylight irradiance is most sought after.

However, there are no standardised amphibian husbandry guidelines with respects to lighting, visible or UV that outlines spectral distribution requirements (Pough, 2007). As a result, a range of commercially available artificial lights including fluorescent tubing, incandescent, mercury vapour lamps and metal halide lamps are used to provide visible lighting in amphibian enclosures. Whilst these forms of lighting are widely used, each has various limitations. Fluorescent tube lighting, the most prevalent form of artificial lighting for amphibians enclosures, has a relatively good operating life and higher energy efficacy in comparison to other forms of

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lighting such as incandescent and mercury vapour lamps (El-Zein, 2013). However, in addition to a high heat output, they contain small amounts of dangerous mercury, which requires specialised disposal (El-Zein, 2013; Arik & Setlur, 2010). Generally, fluorescent, incandescent and mercury vapour lights are costly to run, have a considerable heat output, consume large amounts of energy and often have a short operating life (Pough, 2007; Bergh et al., 2001; Tsao et al., 2010b). Therefore, recent developments in lighting technology, including the development of Light Emitting Diodes (LEDs) for amphibian enclosures are providing an attractive alternative.

LEDs are transforming the lighting industry as a high quality, energy efficient, cost effective and environmentally friendly form of lighting (El-Zein, 2013; Pimputkar et al., 2009). They currently have widespread use in domestic and commercial lighting and many predict they will eventually replace all everyday forms of lighting (Haitz & Tsao, 2011; Pimputkar et al., 2009; Tsao et al., 2010a). There has been an upswing in the use of LED lighting in captive enclosures, particularly for amphibians. Prominent brands of amphibian lighting such as ExoTerra™ and Reptisun™ have already developed their own LED lighting ranges designed specifically for amphibian enclosures. LEDs are extremely attractive to consumers as a result of their advantages over fluorescent and mercury vapour lamps, particularly their energy efficiency. Most forms of lighting have a very inefficient conversion of electricity to visible light, fluorescent lighting has 38% energy conversion whilst incandescent lights has only 5% conversion rate, consuming large amounts of energy whilst only generating a small output (Haitz & Tsao, 2011). However, LEDs perform considerably better with a higher energy conversion efficiency of around 60%. It is therefore predicted that a switch to LEDs in general lighting will reduce energy consumption by 50-75% (Haitz & Tsao, 2011; Arik & Setlur, 2010). Forecasts suggest that this efficacy will only continue to improve (Schubert & Kim, 2005). LEDs also provide considerable environmental benefits (Arik & Setlur, 2010). Increasingly, the impact of climate change is being recognised and the environmental costs of using energy consuming, incandescent is exacerbating this impact. As a result there has been a significant push towards energy efficient or ‘greener’ technologies as people and companies look to reduce their carbon footprint. Studies suggest that if all current lighting were replaced with LEDs around 200-270 million tons of CO2 could be saved (El-Zein, 2013; Pimputkar et al., 2009).

Using LED lighting is also attractive due to the greater cost efficiency. Lighting is often the most expensive part of any artificial environment as a result of the upfront costs and the subsequent on-going energy and maintenance costs (Bourget, 2008). This may be particularly true for artificial environments for amphibians due to their extensive light requirements (Bourget, 2008). Although LEDs can have an expensive upfront cost, they have significantly reduced energy consumption, which results in a lower running cost (Bergh et al., 2001; Haitz & Tsao, 2011). Similarly, their longer operating life can reduce costs as replacement lights will not need to be bought as frequently. Bergh et al. (2001) suggested that in 2012, an LED lamp would pay for itself after only 500-1500 hours of use. Thus, this initial cost can be quickly made up by a longer

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operating life and reduced daily operational costs (Bergh et al., 2001). Furthermore whilst the upfront costs of LEDs have been exceedingly high in the past, this cost has come down significantly in recent years and is only expected to decline further (Haitz & Tsao, 2011). Overall, there is a substantial cost incentive for using LEDs; as much as $100 billion dollars could be saved each year in the US with adoption of LED lighting (Bergh et al., 2001).

LEDs have many advantages that make them attractive to both consumers and institutions such as zoos. A significant proportion of the running cost of zoos is taken by lighting (Lacy, 1991). Around 22% of all electricity use globally is in lighting and zoos in particular, are likely to divert more than 1/5th of their electricity resources to lighting, due to the display nature of zoos and specific lighting requirements of animals (Pimputkar et al., 2009). Therefore, the significant environmental and economic savings that LEDs can produce would undoubtedly be attractive for zoological institutions eager to reduce costs and their carbon footprint (Bergh et al., 2001). Already LEDs are being implemented, or at least considered, by a number of zoological institutions (Ben Baker, Chester Zoo 2016, pers comms., 2 January). However, despite the availability of these lights for personal and commercial use, there has been little investigation into their use in captive environments. No studies have been undertaken to determine if they are an adequate or safe replacement for current lighting in amphibian enclosures. This may be due to the lack of information regarding the current provisioning of lighting within amphibian enclosures, including their spectral and temperature outputs. In 2007, there were no established standard husbandry methods for the majority of amphibian species, including the provisioning of artificial visible lighting (Pough, 2007). As such, there are no universal guidelines for daylight lighting and accordingly, lighting used in amphibian enclosures is largely dependent on the institution in question (Pough, 2007). This makes it difficult to identify what is required from alternative light sources such as LEDs. The development of LEDs has increased the need to assess current lighting in order for comparisons to be made between them.

Aims This study will quantify the thermal and light spectral output of the current lighting provided by zoos, to identify the conditions typically provided in amphibian enclosures. This will enable an accurate comparison with new LED lights to determine whether LEDs are an adequate replacement for current lighting.

2.2 Methods Ethics Statement The methods used in this study did not require the use of any study species. As a result neither a UK Home Office Licence nor ethical approval from the University of Manchester was required. Permission was granted for data collection at ZSL London Zoo and Chester Zoo from the ZSL Zoological Department and the Chester Zoo Research Council respectively.

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Spectrophotometer Calibration and Set Up The Relative Irradiance and Absolute Irradiance were measured using an Ocean Optics Spark Vis Spectrophotometer (Ocean Optics, FL, USA). The Spark Vis Spectrophotometer measures the irradiance and spectral output from the visible spectrum, 400nm-700nm, to a resolution of 4.5-9nm. The absolute irradiance spectrophotometer was calibrated for absolute irradiance prior to its use at the Ocean Optics facility in Florida and connected to its appropriate attachments, including the SMA Base, SMA Adapter, 400µm diameter premium VIS NIR fibre and the cosine corrector (a spectral diffuser attached in order to diffuse the light into the spectrophotometer across a 180° view). This set up was not dismantled in order to maintain the absolute irradiance calibrations. Absolute irradiance measurements were taken by an Apple MacBook Pro in the OceanView Spectroscopy Software (v.1.5.2) (Ocean Optics, 2016). Calibration information was obtained from a disk and imported into the software (Ocean Optics, 2016). The spectrophotometer used for relative irradiance measurements was connected to an SMA Adapter and SMA Base, 400µm diameter premium VIS NIR fibre and cosine corrector also. The spectrophotometer for relative irradiance was calibrated with a black body light source (FHSA- 2000) via the relative irradiance calibration wizard in the OceanView software (Ocean Optics, 2016). This calibration for relative irradiance was carried out before measurements were taken on each separate use.

FLIR Thermal Imaging Camera Thermal imaging photographs were taken using the FLIR ONE Personal Thermal Imaging Camera for iOS connected to an iPhone 6S, using FLIR ONE Application for iOS. Enclosure doors were opened to take photographs to ensure accurate temperatures were obtained. The FLIR Tools Application for iOS was used to edit photographs, providing the scale and average temperatures at the top, middle and bottom of the enclosure.

Daylight Measurements Absolute irradiance recordings for daylight were obtained in Manchester (September 2016) on a cloudy and sunny day using the OceanView Software and the spectrophotometer calibrated for absolute irradiance. The spectrophotometer was held at ground level.

Data Collection at London and Chester Zoo Investigations were carried out at the anuran enclosures at Chester Zoo in 21 exhibits both on public display (6) and behind the scenes (15). Investigations were also carried out in the Anuran and Reptile enclosures at ZSL London Zoo in 7 exhibits both on public display (6) and behind the scenes (1). Using the OceanView software, relative irradiance measurements were taken at each enclosure using the spectrophotometer calibrated for relative irradiance. Measurements were taken at a specific distance. In small tanks (<30cm) this was at a distance of 10cm and 30cm in larger tanks (>30cm). FLIR ONE Thermal Imaging photographs were taken of each enclosure. Enclosures information was also recorded and is displayed in Tables 2.1 and 2.2.

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LED Lights Measurement The spectral output of 6 commercially available LED lights was measured at the University of Manchester. The lights and their specifications are highlighted in Table 2.3. The lights were secured at specific distances above the end of the fibre connected to the spectrophotometer. These distances were 10cm and 30cm to cover the broad range of sizes of tanks utilised for exhibits and storage of amphibians in zoos and represents the same distances for recordings taken at Chester and London Zoo. The relative irradiance from each light was measured at each distance. FLIR ONE Thermal imaging photographs were also taken of 4 tanks with LED lighting at the University of Manchester with LED lighting see Table 2.4 for details.

Table 2.1: Detailed specifications of the 7 enclosures and their lighting at London Zoo.

Tank UVB Lighting Daylight Tank Species Lighting Size 1 300W Osram Ultravitalux Mercury N/A >30cm Shinisaurus Vapour Lamp crocodilurus, Metal Halide Rhacophorus feae 2 Arcadia D3 Mercury Vapour Lamp N/A >30cm Acrantophis Metal Halide dumerilii 3 Arcadia T5 12% Fluorescent tube + D3 Prolight >30cm Trioceros jacksonii 12%160W Mercury Vapour Lamp Metal Halide 4 Arcadia D3 160W Mercury Vapour N/A >30cm Plica plica Lamp Metal Halide Lamp 5 Arcadia D3 100W Mercury Vapour Lightwave >30cm Rhynchophis Lamp boulengeri 6 Arcadia D3 160W Mercury Vapour Prolight >30cm Aspidites Lamp melanocephalus 7 Arcadia T5 12% Fluorescent tube N/A <10cm Alytes obstetricans

Table 2.2: Detailed specifications of the 21 enclosures and their lighting at Chester Zoo.

Tank UVB Lighting Daylight Tank Species Lighting Size 1 Arcadia T5 12% Fluorescent N/A >30cm Agalychnis moreletii tube Solar Raptor Halide Bulb 2 Arcadia T5 12% Fluorescent N/A >30cm Dyscophus guineti tube Solar Raptor Halide Bulb 3 Arcadia T5 12% Fluorescent Prolight Grow >30cm Excidobates tube mysteriosus 4 Arcadia T5 12% Fluorescent Prolight Grow >30cm Mantella expectata tube 5 Arcadia T5 12% Fluorescent Prolight Grow >30cm Mantella aurantiaca tube 6 Arcadia T5 12% Fluorescent N/A <30cm Ingerophrynus tube divergens 7 Arcadia T5 12% Fluorescent N/A <30cm Sylirana maosonesis tube 8 Arcadia T5 6% Fluorescent N/A >30cm Staurois parvus tube

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9 Arcadia T5 12% Fluorescent N/A <30cm Dendrobates tube azureus 10 Arcadia T5 12% Fluorescent N/A <30cm Excidobates tube mysteriosus 11 Arcadia T5 12% Fluorescent N/A <30cm Phyllobates terribilis tube 12 Arcadia T5 6% Fluorescent N/A <30cm Mantella aurantiaca tube 13 Arcadia T5 12% Fluorescent N/A <30cm Mantella aurantiaca tube 14 Arcadia T5 12% Fluorescent N/A <30cm Mantella expectata tube 15 Arcadia T5 12% Fluorescent N/A <30cm Mantell aurantiaca tube 16 N/A Osram lumilux <30cm Neacomys pictus skywhite 17 N/A Prolight grow <30cm Sylirana maosonesis 18 Arcadia T5 6% Fluorescent N/A <30cm Staurois gutus tube 19 Arcadia T5 12% Fluorescent N/A <30cm Polypedates tube otilophus 20 Arcadia T5 12% Fluorescent N/A <30cm Polypedates tube otilophus 21 Arcadia T5 12% Fluorescent N/A <30cm Rhacophorus tube eichopygus

Table 2.3: Detailed specifications of the 6 brands of LED lights.

Brand Watts Colour Temperature Cost Solar Stinger™ 6W 6500K £80 Jungle Dawn Arcadia™ 9W 6500K £16 Repsitun™ 1W 6500K £53 White Python™ 3.6W 6000-6500K £25 ExoTerra™ 3W Unknown £30 Lucky Reptile™ Sunspot 5W Unknown £20

Table 2.4: Detailed specifications of the 4 enclosures with LED lighting from which thermal imaging photographs were taken at the University of Manchester.

Tank UVB Lighting Daylight Lighting Tank Species Size 1 Arcadia™ T5 6% Fluorescent Jungle Dawn™ LED <30cm Mantella tube 9W betsileo 2 Arcadia™ T5 6% Fluorescent Jungle Dawn™ LED <30cm Mantella tube 9W betsileo 3 Arcadia™ T5 6% Fluorescent Solar Stinger™ LED <30cm Mantella tube 6W betsileo 4 Arcadia™ T5 6% Fluorescent Solar Stinger™ LED <30cm Mantella tube 6W betsileo

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2.3 Results Natural Irradiance Daylight

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Figure 2.1: Absolute irradiance measurements of daylight in Manchester in the visible light spectrum (400-700nm).

Natural daylight produced a high level of absolute irradiance over all wavelengths in the visible light spectrum (Figure 2.1). There were no spikes in irradiance and it gradually increase to its highest level of irradiance between 500-600nm before declining.

Relative Irradiance in Small Enclosures (10cm) Lighting in 10 of 14 enclosures at Chester Zoo had the highest spike in relative irradiance at 543.6nm at a distance of 10cm (Figure 2.2). Tank 6 and 7 had a similar high peak in relative irradiance at 542.6nm. Tank 10 and Tank 14 had the highest peaks in relative irradiance at wavelength 445.2nm. The tanks followed a similar pattern with smaller sharp spikes in relative irradiance at 430nm and 610nm, with broad smaller peaks between 480-85nm and 575-90nm. The majority of enclosures typically have the highest peak in relative irradiance between wavelengths 540-550nm.

In the one small enclosure at London Zoo, the lighting produced the peak of relative irradiance at wavelength 543.6nm at a distance of 10cm from the spectrophotometer (Figure 2.3), similar to the majority of lighting assessed in Chester Zoo. It also showed small spikes in relative irradiance at approximately 430nm and 610nm, similar to the pattern of lights in Chester Zoo.

The 6 LED lights had two clear spikes in relative irradiance at a distance of 10cm (Figure 2.4). Both Lucky Reptile™ and Jungle Dawn™ lights had peak relative irradiance values at 577.6nm, alongside high relative irradiance values in all other lights at this wavelength. White Python™

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and ExoTerra™ lights had peak relative irradiance values at 449.6nm, whilst Solar Stinger™ and Reptisun™ lights had similar peak irradiance values at 449.2nm and 447.2nm respectively. Lucky Reptile™ and Jungle Dawn™ lights also had high levels of irradiance at these wavelengths.

Relative Irradiance in Large Enclosures (30cm) From Chester Zoo, 4 of the 6 lights measured at a distance of 30cm had spikes in relative irradiance measurements but peaking at 543.6nm (Figure 2.5). Tank 1 had the peak relative irradiance at a similar wavelength of 544nm. Tank 2 had peak relative irradiance at 408.4nm. All tanks followed a similar wavelength pattern, with small sharp spikes in irradiance at approximately 430nm, 575nm and 610nm, but with a highest intensity spike typically in the 540- 550nm range.

In the larger enclosures at London Zoo, the spectrophotometer was measured at a distance of 30cm (Figure 2.6). Three of these enclosures had the highest spike in relative irradiance at 544.3nm, whilst 2 enclosures had the highest spike at 543.3nm. The remaining tank had its peak relative irradiance at 408.4nm. These results mirror results obtained at Chester Zoo. The tanks followed a pattern of irradiance similar to that of enclosures at Chester Zoo with some smaller spikes in irradiance at approximately 430nm and 575nm, but with the peak in relative irradiance in the range of 540-50nm for the majority of tanks. 5 tanks also had a small broader spike in relative irradiance at 610nm.

The 6 LED lights had two peaks in relative irradiance, one sharp high spike around the 445nm and a broader high peak rising from 500nm before declining at around 600nm to low levels of irradiance from 650nm – 700nm (Figure 2.7). Solar Stinger™, ExoTerra™, Reptisun™ and Jungle Dawn™ had a peak relative irradiance at 445.2nm, whilst White Python™’s peak in irradiance was similar at 445.5nm. Lucky Reptile™ peak relative irradiance was at 577.2nm.

Thermal Imaging Photographs The different lighting in the enclosures produced vastly different thermal ranges within the tanks (Figures 2.8-2.20). Temperatures were mostly uniform across the LED tanks differing only marginally by a maximum of 2.1°C (Figures 2.21-2.25). LED tanks exhibited a less severe thermal gradient throughout the tank, whereas enclosures typically in Chester and London Zoo with fluorescent and mercury vapour lighting showed with a greater difference in temperature ranging from 1.6°C-11.2°C. They also exhibited a greater variety of thermal microclimates with areas of cooler and warmer temperatures. Temperature was largely uniform in LED tanks and there was greater thermal heterogeneity in enclosures from Chester Zoo and London Zoo.

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Figure 2.2 (top): Relative irradiance measurements of the lighting in 16 tanks in Chester Zoo in the visible light spectrum (400nm-700nm) 10cm from the light. Figure 2.3 (middle): Relative irradiance measurements of the lighting in 1 tank in London Zoo in the visible light spectrum (400-700nm) 10cm from the light. Figure 2.4 (bottom): Relative irradiance of 6 commercially available LED lights in the visible light spectrum (400-700nm) 10cm from the light. Lines represent different brands of LEDs, Lucky Reptile™ (pink), Jungle Dawn™ (green), Reptisun™ (orange), Solar Stinger™ (blue), ExoTerra™ (red) and White Python™ (purple).

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Figure 2.5 (top): Relative irradiance measurements of the lighting in 16 tanks in Chester Zoo in the visible light spectrum (400nm-700nm) 30cm from the light. Figure 2.6 (middle): Relative irradiance measurements of the lighting in 6 tanks in London Zoo in the visible light spectrum (400-700nm) 30cm from the light. Figure 2.7 (bottom): Relative irradiance of 6 commercially available LED lights in the visible light spectrum (400-700nm) 30cm from the light. Lines represent different brands of LEDs, Lucky Reptile™ (pink), Reptisun™ (orange), Jungle Dawn™ (green), Solar Stinger™ (blue), ExoTerra™ (red) and White Python™ (purple).

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2.8 2.9

2.10 2.11

2.12 2.13 2.14

Figures 2.8-2.14 (left to right): Thermal imaging pictures and corresponding photograph of seven selected reptile and amphibian enclosures at Chester Zoo (Tanks 1, 3, 4,5, 6, 8 and 9 respectively). Scale is in °C. The temperature difference from top to bottom is 2.1°C, 4.6°C, 2.8°C, 1.6°C, 3.4°C, 3.3°C, 3.7°C in figures 2.8, 2.9, 2.10, 2.11, 2.12, 2.13 and 2.14 respectively. Average ambient temperature for date and time of images obtained: 12°C

2.15 2.16

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2.17 2.18

2.19 2.20

Figures 2.15-2.20 (left to right): Thermal imaging pictures and corresponding photograph of six reptile and amphibian enclosures at London Zoo (Tanks 1, 2, 3, 4, 5 and 7). Scale is in °C. Temperature difference between the top, middle and bottom of the enclosures is 5.7°C, 6.5°C, 6.1°C, 9°C, 6°C, 11.2°C in figures 2.15, 2.16, 2.17, 2.18, 2.19, 2.20 respectively. Average temperature for date and time of images obtained: 32°C

2.21 2.22

2.23 2.24

Figures 2.21-2.24 (left to right): Thermal imaging pictures and corresponding photograph of four LED lights in amphibian enclosures at the University of Manchester. Scale is in °C. Temperature difference between the top, middle and bottom of the enclosures is 1.7°C, 1.4°C, 0.8°C, 2.1°C respectively in figures 2.21, 2.22, 2.23, and 2.24. Note the heat spots in the centre of the tanks are the reflection of the lighting on the back glass of the enclosure. Average temperature for date and time of images obtained: 23°C.

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2.4 Discussion Spectral Output The spectral output of LED lighting and lighting typically provided in zoological institutions is invariably different and highly distinctive. Whilst the spectral output of lighting presently used in enclosures differs significantly to that of LEDs, it is very similar between the different zoological institutions. This suggests that although institutions may use different best practice guidelines with regards to visible lighting, they generally use the same types of lighting. Typical lighting used in zoological institutions comprises a variety of different types, including fluorescent tubing, metal halides and mercury vapour lamps. These lights produce a spectral output with multiple high, sharp spikes in irradiance, with the highest peak of irradiance typically at the 540nm wavelength. This contrasts considerably with LEDs which produce only 2 peaks; one sharp spike at a shorter wavelength, around 440-450nm and one broad peak in irradiance over a wider range of wavelengths from 530-610nm. LEDs produce high levels of irradiance of light at shorter wavelengths in comparison to fluorescent, mercury vapour and metal halide lighting which produce high irradiance at longer wavelengths. These results are consistent with other studies which indicate that LEDs produce a vastly different spectrum of light in the visible spectrum compared to these forms of lighting (Ellis et al., 2013; Aubé et al., 2013).

However, this difference in spectral output may have implications for the amphibians’ welfare. Differences in spectral output alters the composition of lighting in amphibian enclosures. Should zoos choose adopt LEDs as their primary method of lighting, amphibians will receive vastly different lighting to that which they have previously experienced and is similar between zoological institutions. Therefore, there may be some potentially negative consequences of such a significant change in the spectrum of lighting received. Morgan and Tromborg (2007) suggest that artificial lighting is a significant stressor for many species in captivity. Therefore changes in provisioning of light could result in the activation of a stress response. Amphibians are particularly vulnerable and highly sensitive, thus any change in the environmental conditions or provisioning of environmental parameters is likely to have an impact (Wake & Vredenburg, 2008; Halliday, 2008). The resulting stress response may have a number of knock on effects for the welfare of the individuals (Pough, 2007).

Shorter wavelengths may have also have a direct effect on the activation of the stress response in some species. This is supported by Miguad et al. (2007) who found that in salmon, there was an observed increase in cortisol indicating an acute stress response in response to exposure to high intensity blue LEDs with shorter wavelengths. However, this stress response was not sustained. This supports Morgan & Tromborg’s (2007) claim that the change in the artificial captive environment, rather than the difference in wavelength, results in the activation of the stress response. This may indicate that once the animals have adapted to the new lighting, there will be no adverse reaction to the use of LEDs. Longer-term investigations into the impact of LEDs are however, required to determine the true impact and assess this hypothesis.

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Alternatively, these short wavelengths of light produced by LEDs are also hypothesised to have further direct negative impacts on the health of various species (Falchi et al., 2011; Migaud et al., 2007). Morgan and Tromborg (2007) suggest that the wavelength of light is an important factor affecting animal welfare as species often have different sensitivities to various wavelengths of light. Provisioning of appropriate wavelengths of light is therefore required for the maintenance of fit populations in captivity (Morgan & Tromborg, 2007). LEDs produce a sharp spike at around 450nm. These short wavelengths and narrow spectral output allow for the increased energy efficiency of LEDs, but are believed to be more harmful for vertebrates (Behar-Cohen et al., 2011; Migaud et al., 2007). Thus, Morgan & Tromborg (2007) suggest that artificial lights such as LEDs and their different wavelengths may have an impact on the welfare of amphibians.

Similarly, alterations in photoperiods, namely a continuous exposure to light, have been found to supress melatonin production in various species of amphibians (Edwards & Pivorun, 1991; Delgado et al., 1987). Studies have determined that LED lighting, specifically narrow wavelengths, can have a similar inhibitory effect on melatonin production as continuous lighting in humans (Falchi et al., 2011; Behar-Cohen et al., 2011; Hong et al., 2015). Spectral differences in lighting therefore could alter melatonin production, which previous studies have shown may have an impact on the growth of various species (Aubé et al., 2013; Delgado et al., 1987). In amphibians, melatonin has been experimentally proven to alter amphibian growth, by regulating the production of a growth hormone (Edwards & Pivorun, 1991; Delgado et al., 1987; Chowdhury et al., 2008). Therefore, the potential effect of LEDs on the production of melatonin in amphibians could have implications for amphibian growth.

Several studies have also highlighted various ecological impacts of replacing existing artificial lighting with LED lighting. Artificial night lighting has many ecological consequences for amphibians, as well as a variety of other species such as bats and moths (Perry et al., 2008; Stone et al., 2015; Longcore & Rich, 2004). This includes changes in behaviour such as reduction in anti-predator behaviour or a reduction in activity (Stone et al., 2012). The use of LED lights however, has been suggested to have similar, or even greater ecological impacts for some species such as bats and moths (Stone et al., 2012; Wakefield et al., 2015; Pawson & Bader, 2014). Pawson and Bader (2014) suggest that the differences in output of LEDs exacerbates the ecological impact of night lighting, attracting almost 50% more insects than high-pressure sodium lighting. The potential greater impact of LEDs has not been investigated directly in amphibians and it is unclear if this greater effect would translate to changes in diurnal artificial lighting. Yet, the potential for LEDs to exacerbate the impacts of artificial lighting requires further study.

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Further to this, the shorter wavelengths produced by LED lighting are closer to the wavelengths of ultraviolet light. Artificial sources of UV have been empirically proven to have a number of deleterious effects on amphibians (Hays et al., 1996). In particular, elevated levels of UVB have been found to have an effect on the behaviour, colouration and growth and development of amphibians (Bancroft et al., 2008; Licht & Grant, 1997). The use of LEDs with similar wavelengths to those found in UV wavelengths accordingly may result in similar effects on amphibians. Furthermore, Wakefield et al. (2015) found that UV and LEDs had similar effects on moth behaviour, as both led to a reduction in predator avoidance responses. This could potentially be a result of their similar spectral composition i.e. the production of short wavelengths. Investigation into LEDs before implementation is therefore required as they may have other similar implications for amphibians as demonstrated in UV studies (Stone et al., 2015; Wakefield et al., 2015).

Alongside these potential welfare implications, many studies have also indicated high sensitivity of anuran retinas. Retinas of amphibians are highly vulnerable to light and the provisioning of lighting in captivity, particularly the implementation of LEDs may have detrimental impacts for amphibian eyes. Retinal damage has been identified in a range of amphibian species in response to high levels of UVB lighting, similar to the alterations observed in the behaviour, growth and colouration of amphibians under UVB lighting (Flamarique et al., 2000). Higher incidences of cataracts, the development of lens opacities, in Rana aurora and Hyla regilla has been documented as a result of exposure to increased levels of UVB (Flamarique et al., 2000; Fite et al., 1998). Similarly, short wavelengths produced by UVB caused morphological damage to the retina as well as changes in photoreceptor density in both Rana cascada and Rana pipens (Fite et al., 1998). These impacts together can lead to a decline in the visual ability of amphibians in captivity and have implications for survival upon reintroduction (Fite et al., 1998). These studies have highlighted the vulnerability of amphibian retinas to substantial damage and therefore LEDs with short wavelengths may have a similar impact. This is further supported by studies in a range of other species, which demonstrate significant retinal damage in response to exposure to LED lighting, particularly the short blue wavelengths (Kuse et al., 2014). In humans, retinal photoreceptor cells were damaged under LED lighting, more severely under blue short wavelengths, though not exclusively, by increasing the production of reactive oxygen species in the eye resulting in extensive cell damage (Kuse et al., 2014). The previously observed high sensitivity of amphibian retinas when exposed to short light wavelengths suggests this may be an area where LEDs could impact the welfare of individuals and as a result requires specific investigation.

However, although it is possible to hypothesise some of the potentially negative implications of LEDs, the lack of studies that examine the use of artificial daylight lighting means that the true impact of LEDs is still largely unknown. Certainly, whilst the spectrum of light produced by LEDs differs significantly from the currently lighting used in amphibian enclosures, this does not

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necessarily mean that LEDs are harmful. Rather, some have suggested that instead of the sharp spikes at various wavelengths, the broader spectrum of light across a wide range of wavelengths produced by LEDs mimics natural daylight more accurately (Ellis et al., 2013; Aubé et al., 2013). Fluorescent lighting produces a narrower range of wavelengths compared to natural sunlight whereas LEDs produce a broader spectral output with a greater likeness to natural irradiance spectra (Castelhano-Carlos & Baumans, 2009; Thorington, 1985). Thus, providing artificial lighting which replicates natural lighting more accurately may promote natural behaviour and normal functioning of the processes regulated by light (Ferrie et al., 2014). Studies investigating the use of LEDs in poultry found that LEDs actually improved the well- being of chickens when compared to those reared under compact fluorescent lights (Huth & Archer, 2015). Therefore, whilst the production of narrow wavelengths is typically recognised as potentially harmful for many species of vertebrates, this needs greater clarification. Narrow wavelengths of UVB though dangerous are also vital for correct bone development in amphibians and thus, must be provisioned in captivity (Michaels et al., 2014b). Nevertheless, the true effect of LEDs is still unknown in amphibians and should be determined in order to safely use them in amphibian captivity.

Thermal Output Alongside the differences in the spectral output of fluorescent, metal halide and mercury vapour lights compared to LEDs, the thermal imaging photographs also illustrated a difference in their heat outputs. Within the individuals enclosures, the temperature difference is greater in enclosures with fluorescent, mercury vapour and halide lighting compared to those with LEDs. As expected with current lighting in enclosures, higher temperatures were observed at the top of the enclosure where the lights were found, due to the high heat output typically produced by these lights (El-Zein, 2013). Greater thermal heterogeneity is observed in enclosures with fluorescent and metal halide lighting. Higher temperatures were observed at the top, whilst the temperature gradually declined within the enclosure, creating specific areas of cooler or warmer temperatures. Although this pattern is also seen in enclosures with LED lighting the gradient is reduced and there is less variation in temperature throughout the enclosures. LEDs are highly energy efficient, producing less heat waste and a lower heat output, resulting in a more uniform temperature across the enclosure (Pimputkar et al., 2009; El-Zein, 2013). Temperature declined only marginally within the enclosure, there were no hotpots and the temperature at the top and bottom of the enclosure is relatively similar. This is consistent with other studies which observe a significantly lower heat output of LEDs (El-Zein, 2013; Yaxiao Qin et al., 2009).

However, the potential implications of a difference in temperature distribution within amphibian enclosures is still unknown. The low heat output of LED lighting may be beneficial as the room temperature of enclosures are typically regulated to the precise requirements of amphibians ensuring the temperature remains fairly constant and successfully adheres to the amphibians specific thermal requirements (Pough, 2007). LEDs may limit interference with the temperature

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that has been predetermined, successfully replicating the microclimate conditions experienced in a natural habitat. LEDs therefore, may provide a more stable and accurate environment in terms of temperature. This may allow for the normal functioning of thermally regulated physiological processes and a more constant environmental condition may be less stressful (Morgan & Tromborg, 2007).

On the other hand, various studies investigating the regulation of temperature in amphibian enclosures suggest that it may be beneficial to provide a thermal gradient throughout the enclosures (Tapley et al., 2014; Pough, 2007). Many amphibian species bask in order to help regulate body temperature. Behavioural thermoregulation can modulate a number of physiological process including growth, development, immune function and reproduction in amphibians (Browne & Edwards, 2003). Gradients are typically observed in the wild as a result of natural sunlight and canopy cover in the rainforest (Lillywhite et al., 1998; Hayes et al., 1998). This suggests that captive environments should provide conditions which allow for behavioural thermoregulation. Therefore, a good captive environment should provide a gradient or mosaic of light, as it allows for natural behaviour where organisms can seek out environmental conditions they prefer (Tapley et al., 2014; McWilliams, 2008; Pough, 2007). Many studies advocate the use of lighting to provide thermal gradient (Ferrie et al., 2014; Pough, 2007; Tapley et al., 2014). Equally, Hayes et al. (1998) highlights the issues of providing heat through non-light sources as he suggests that this can promote unnatural behaviour. Further still, Pough (2007) argues that incandescent bulbs, with high heat output resulting in large photothermal gradients, are the best choice of lighting for long term care of amphibians. Therefore, it appears that the use of LED lighting in captive environments will limit the amphibians’ ability to regulate temperature through behavioural thermoregulation. Despite this, the impact of lighting for heat regulation may be overestimated for non-basking species of amphibians and those with specific thermal microclimate conditions (Michaels & Preziosi, 2013). Nevertheless providing a gradient and variety of temperatures will allow individuals to choose the environment they favour.

Limitations However these results may not be conclusive. It is important to distinguish whether these differences in thermal imaging photographs are as a result of the difference in heat output or instead the greater environmental complexity in captive enclosures in zoological institutions. Foliage, ponds, basking logs are often present in enclosures which may block light from penetrating to the bottom of the enclosure and result in in the production of thermal hotspots or cooler areas. Additionally, enclosures where fluorescent and mercury vapour lighting were investigated i.e. in Chester and ZSL London Zoo were generally larger in comparison to the tanks with the LED lights. Therefore the size of the enclosure may also account for the temperature differences observed. Additionally using infrared thermography to determine enclosure temperature may result in inaccurate temperature recordings. Often infrared temperatures do not correspond with conductive temperatures, and therefore they can be a

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poor indicator of temperature in many circumstances (Bach, et al., 2015). Further, rather than measuring radiated infrared temperature, the thermal imaging camera may instead be recording reflected infrared temperature, particularly from the glass backs of enclosures. Therefore a number of factors may play a role in the difference in temperature, not simply the differences in the heat output produced by lighting. This is consistent with studies investigating the impact of plant cover in enclosure design, which show that the presence of plants results in a wider variety of thermal microclimates within enclosures (Michaels et al., 2014a). Future investigations into lighting should use enclosures with similar set ups in order to achieve an accurate comparison.

Conclusion This study cannot conclude that these differences that result between LEDs and current lighting provided will have any notable impacts. Current literature suggests that the use LEDs may have both negative and positive effects for amphibians. Despite a number of identified benefits of LEDs and their increasing popularity, the positivity surrounding their use may be overshadowing some potential problematic consequences. Whilst LEDs may have a number of environmental benefits, they may also have a number of significant ecological side effects which will only be realised once implemented (Kuvlesky et al., 2007). The observed difference in output between compact fluorescent lighting and LEDs has resulted in a number of investigations assessing the welfare implications of LEDs in chicken and fish rearing industries. If these industries deem it necessary to consider if a switch to LEDs compromises animal welfare, studies should also be conducted on the welfare of amphibians in captivity (Huth & Archer, 2015; Migaud et al., 2007). Thus, further study must be undertaken to determine if the observed changes in wavelength will have an impact on amphibians welfare (Adkins et al., 2003; Ellis et al., 2013; Falchi et al., 2011). Therefore, before their use becomes widespread, further investigation should be undertaken in order to determine any potential underlying effects of LEDS on amphibian welfare. Only then can recommendations regarding the use of LEDs in amphibian husbandry be determined.

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2.5 References Adkins, E., Driggers, T., Ferguson, G., Gehrmann, W., Gyimesi, Z., May, E., Ogle, M. & Owens, T. (2003) Ultraviolet light and reptiles, amphibians. Journal of Herpetological Medicine and Surgery, 13 (4), p27–37. Antwis, R.E. & Browne, R.K. (2009) Ultraviolet radiation and Vitamin D3 in amphibian health, behaviour, diet and conservation. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 154 (2), p184–90. Arik, M. & Setlur, A. (2010) Environmental and economical impact of LED lighting systems and effect of thermal management. International Journal of Energy Research, 34 (13), p1195– 1204. Aubé, M., Roby, J. & Kocifaj, M. (2013) Evaluating potential spectral impacts of various artificial lights on melatonin suppression, photosynthesis, and star visibility. PloS one, 8 (7), pe67798. Bach, A.J.E., Stewart, I.B., Disher, A.E. & Costello, J.T. (2015) A comparison between conductive and infrared devices for measuring mean skin temperature at rest, during exercise in the heat, and recovery. M. Buchowski ed. PloS one, 10 (2), pe0117907. Bancroft, B.A., Baker, N.J. & Blaustein, A.R. (2008) A meta-analysis of the effects of ultraviolet B radiation and its synergistic interactions with pH, contaminants, and disease on amphibian survival. Conservation Biology, 22 (4), p987–996. Behar-Cohen, F., Martinsons, C., Viénot, F., Zissis, G., Barlier-Salsi, A., Cesarini, J.P., Enouf, O., Garcia, M., Picaud, S. & Attia, D. (2011) Light-emitting diodes (LED) for domestic lighting: any risks for the eye? Progress in Retinal and Eye Research, 30 (4), p239–57. Bergh, A., Craford, G., Duggal, A. & Haitz, R. (2001) The promise and challenge of solid-state lighting. Physics Today, 54 (12), p42–47. Boeuf, G. & Le Bail, P.-Y. (1999) Does light have an influence on fish growth? Aquaculture, 177 (1), p129–152. Bourget, C.M. (2008) An introduction to light-emitting diodes. HortScience, 43 (7), p1944–1946. Browne, R.K., Odum, R.A., Herman, T. & Zippel, K. (2007) Facility design and associated services for the study of amphibians. ILAR Journal, 48 (3), p188–202. Castelhano-Carlos, M.J. & Baumans, V. (2009) The impact of light, noise, cage cleaning and in- house transport on welfare and stress of laboratory rats. Laboratory animals, 43 (4), p311– 27. Chowdhury, V.S., Yamamoto, K., Saeki, I., Hasunuma, I., Shimura, T. & Tsutsui, K. (2008) Melatonin stimulates the release of growth hormone and prolactin by a possible induction of the expression of frog growth hormone-releasing reptide and its related reptide-2 in the amphibian hypothalamus. Endocrinology, 149 (3), p962–970. Delgado, M.J., Gutiérrez, P. & Alonso-Bedate, M. (1987) Melatonin and photoperiod alter growth and larval development in Xenopus laevis tadpoles. Comparative Biochemistry and Physiology Part A: Physiology, 86 (3), p417–421. Edwards, M.L.O. & Pivorun, E.B. (1991) The effects of photoperiod and different dosages of

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melatonin on metamorphic rate and weight gain in Xenopus laevis tadpoles. General and Comparative Endocrinology, 81 (1), p28–38. El-Zein, N. (2013) The LED lighting revolution. In: A. Sayigh. Sustainability, Energy and Architecture: Case Studies in Realizing Green Buildings. Massachusetts: Academic Press, p171–194. Ellis, E. V, Gonzalez, E.W., Kratzer, D.A., McEachron, D.L. & Yeutter, G. (2013) Auto-tuning daylight with LEDs: sustainable lighting for health and wellbeing. In: Proceedings of the 2013 ARCC Spring Research Conference. Charlotte: University of North Carolina, ARCC Past Conferences, p465–473. Falchi, F., Cinzano, P., Elvidge, C.D., Keith, D.M. & Haim, A. (2011) Limiting the impact of light pollution on human health, environment and stellar visibility. Journal of Environmental Management, 92 (10), p2714–22. Ferrie, G.M., Alford, V.C., Atkinson, J., Baitchman, E., Barber, D., Blaner, W.S., Crawshaw, G., Daneault, A., Dierenfeld, E., Finke, M., Fleming, G., Gagliardo, R., Hoffman, E.A., Karasov, W., Klasing, K., Koutsos, E., Lankton, J., Lavin, S.R., Lentini, A., Livingston, S., Lock, B., Mason, T., McComb, A., Morris, C., Pessier, A.P., Olea­Popelka, F., Probst, T., Rodriguez, C., Schad, K., Semmen, K., Sincage, J., Stamper, M.A., Steinmetz, J., Sullivan, K., Terrell, S., Wertan, N., Wheaton, C.J., Wilson, B. & Valdes, E. V. (2014) Nutrition and health in amphibian husbandry. Zoo Biology, 33 (6), p485–501. Fite, K. V., Blaustein, A., Bengston, L. & Hewitt, H.E. (1998) Evidence of retinal light damage in Rana cascadae: a declining amphibian species. Copeia, 1998 (4), p906–914. Flamarique, I.N., Ovaska, K. & Davis, T.M. (2000) UV-B Induced Damage to the Skin and Ocular System of Amphibians. The Biological Bulletin, 199 (2), p187. Haitz, R. & Tsao, J.Y. (2011) Solid-state lighting: ‘The case’ 10 years after and future prospects. physica status solidi (a), 208 (1), p17–29. Halliday, T.R. (2008) Why amphibians are important. International Zoo Yearbook, 42 (1), p7–14. Hayes, M.P., Jennings, M.R. & Mellen, J.D. (1998) Environmental enrichment for amphibians and reptiles. In: D. J. Shepherdson, J. D. Mellen, & M. Hutchins eds. Second Nature: Environmental Enrichment for Captive Animals. Washington D.C., Smithsonian Institution, p205–235. Hays, J.B., Blaustein, A.R., Kiesecker, J.M., Hoffman, P.D., Pandelova, L., Coyle, D. & Richardson, T. (1996) Developmental responses of amphibians to solar and artificial UVB sources: a comparative study. Photochemistry and Photobiology, 64 (3), p449–456. Hong, S.-K., Kim, K.-S., Kim, I.-T. & Choi, A.-S. (2015) Melatonin Suppression under LED Lighting Focused on Spectral Power Distribution Differences. Journal of Korean Institute of Illuminating and Electrical Installation Engineers, 29 (8), p7–17. Huth, J.C. & Archer, G.S. (2015) Comparison of two LED light bulbs to a dimmable CFL and their effects on broiler chicken growth, stress, and fear. Poultry Science, 94 (9), p2027– 2036. Kuvlesky, W.P., Brennan, L.A., Morrison, M.L., Boydston, K.K., Ballard, B.M. & Bryant, F.C.

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(2007) Wind energy development and wildlife conservation: challenges and opportunities. Journal of Wildlife Management, 71 (8), p2487–2498. Kuse, Y., Ogawa, K., Tsuruma, K., Shimazawa, M. & Hara, H. (2014) Damage of photoreceptor- derived cells in culture induced by light emitting diode-derived blue light. Scientific reports, 4, p5223. Lacy, R.C. (1991) Zoos and the surplus problem: an alternative solution. Zoo Biology, 10 (4), p293–297. Licht, L.E. & Grant, K.P. (1997) The effects of ultraviolet radiation on the biology of amphibians. American Zoologist, 37 (2), p137–145. Lillywhite, H.B., Mittal, A.K., Garg, T.K. & Das, I. (1998) Basking behavior, sweating and thermal ecology of the Indian tree frog, Polypedates maculatus. Journal of Herpetology, 32 (2), p169. Longcore, T. & Rich, C. (2004) Ecological light pollution. Frontiers in Ecology and the Environment, 2 (4), p191–198. Michaels, C. & Preziosi, R.F. (2013) Basking behaviour and ultraviolet B radiation exposure in a wild population of Pelophylax lessonae in Northern Italy. Herpetological Bulletin, 124 (124), p1–8. Michaels, C.J., Antwis, R.E. & Preziosi, R.F. (2014a) Impact of plant cover on fitness and behavioural traits of captive red-eyed tree frogs (Agalychnis callidryas). PloS one, 9 (4), pe95207. Michaels, C.J., Antwis, R.E. & Preziosi, R.F. (2014b) Impacts of UVB provision and dietary calcium content on serum vitamin D3 , growth rates, skeletal structure and coloration in captive oriental fire-bellied toads (Bombina orientalis). Journal of Animal Physiology and Animal Nutrition, 99 (2), p391–403. Michaels, C.J., Gini, B.F. & Preziosi, R.F. (2014c) The importance of natural history and species-specific approaches in amphibian ex-situ conservation. Herpetological Journal, 24, p135–145. Migaud, H., Cowan, M., Taylor, J. & Ferguson, H.W. (2007) The effect of spectral composition and light intensity on melatonin, stress and retinal damage in post-smolt Atlantic salmon, Salmo salar. Aquaculture, 270 (1), p390–404. Moore, R.D. & Church, D.R. (2008) Implementing the Amphibian Conservation Action Plan. International Zoo Yearbook, 42 (1), p15–23. Morgan, K.N. & Tromborg, C.T. (2007) Sources of stress in captivity. Applied Animal Behaviour Science, 102 (3), p262–302. Ocean Optics (2016) OceanView (Version 1.5.2). [Computer Program]. Avaliable at http://oceanoptics.com/support/software-downloads/ (Accessed February 2016). Pawson, S.M. & Bader, M.K.-F. (2014) LED lighting increases the ecological impact of light pollution irrespective of color temperature. Ecological Applications, 24 (7), p1561–1568. Perry, G., Buchanan, B. & Fisher, R. (2008) Effects of artificial night lighting on amphibians and reptiles in urban environments. In: J. C. Mitchell, R. E. Jung Brown, & B. Bartholomew

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eds. Urban Herpetology. Society for the Study of Amphibians & Reptiles, p239–256. Pimputkar, S., Speck, J.S., DenBaars, S.P. & Nakamura, S. (2009) Prospects for LED lighting. Nature Photonics, 3 (4), p180–182. Pough, F.H. (2007) Amphibian biology and husbandry. ILAR Journal, 48 (3), p203–213. Schubert, E.F. & Kim, J.K. (2005) Solid-state light sources getting smart. Science, 308 (5726), p1274–8. Stone, E.L., Jones, G. & Harris, S. (2012) Conserving energy at a cost to biodiversity? Impacts of LED lighting on bats. Global Change Biology, 18 (8), p2458–2465. Stone, E.L., Wakefield, A., Harris, S. & Jones, G. (2015) The impacts of new street light technologies: experimentally testing the effects on bats of changing from low-pressure sodium to white metal halide. Philosophical Transactions of the Royal Society B: Biological Sciences, 370 (1667), p20140127. Tapley, B., Rendle, M., Baines, F.M., Goetz, M., Bradfield, K.S., Rood, D., Lopez, J., Garcia, G. & Routh, A. (2014) Meeting ultraviolet B radiation requirements of amphibians in captivity: a case study with mountain chicken frogs (Leptodactylus fallax) and general recommendations for pre-release health screening. Zoo Biology, 34 (1), p46–52. Thorington, L. (1985) Spectral, irradiance, and temporal aspects of natural and artificial light. Annals of the New York Academy of Sciences, 453 (1), p28–54. Tsao, J.Y., Coltrin, M.E., Crawford, M.H. & Simmons, J.A. (2010a) Solid-state lighting: an integrated human factors, technology, and economic perspective. Proceedings of the IEEE, 98 (7), p1162–1179. Tsao, J.Y., Saunders, H.D., Creighton, J.R., Coltrin, M.E. & Simmons, J.A. (2010b) Solid-state lighting: an energy-economics perspective. Journal of Physics D: Applied Physics, 43 (35), p354001. Verschooren, E., Brown, R.K., Vercammen, F. & Pereboom, J. (2011) Ultraviolet B radiation (UV-B) and the growth and skeletal development of the Amazonian milk frog (Trachycephalus resinifictrix) from metamorphosis. Journal of Physiology and Pathophysiology, 2 (3), p34–42. Villamizar, N., García-Alcazar, A. & Sánchez-Vázquez, F.J. (2009) Effect of light spectrum and photoperiod on the growth, development and survival of European sea bass (Dicentrarchus labrax) larvae. Aquaculture, 292 (1), p80–86. Wake, D.B. & Vredenburg, V.T. (2008) Are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proceedings of the National Academy of Sciences of the United States of America, 105, p11466–11473. Wakefield, A., Stone, E.L., Jones, G. & Harris, S. (2015) Light-emitting diode street lights reduce last-ditch evasive manoeuvres by moths to bat echolocation calls. Royal Society Open Science, 2 (8), p150291. Yaxiao Qin, Y., Deyan Lin, D. & Hui, S.Y. (2009) A simple method for comparative study on the thermal performance of LEDs and fluorescent lamps. IEEE Transactions on Power Electronics, 24 (7), p1811–1818.

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Assessing the effect of LED lighting on the growth, colouration and activity of Mantella betsileo

Abstract The success of ex situ conservation efforts for amphibians is largely dependent on the ability to maintain healthy populations in captivity by replicating natural environments. The use of Light Emitting Diodes (LEDs), although increasingly attractive for zoos, has the potential to alter these ex situ captive environments for amphibians and may impact the maintenance of healthy populations in captivity. Light produced by LEDs is vastly different to light produced by current lighting in captivity in terms of both spectral and thermal output, yet any potential implications of this are still currently unknown. These changes in spectral output are posited to have an impact on the growth, colouration and behaviour of amphibians. Contrary to various hypothesised impacts, there were no, or limited changes observed in the growth and colouration of Mantella betsileo under LED lighting. Despite this, there were observed differences in the behaviour of individuals, where individuals under some LED lighting displayed greater levels of activity. The impact of this increase in activity on amphibian welfare is yet to be determined, yet provides an avenue for further investigation. Although primarily the change in activity is attributed to the difference in spectral output of LEDs, other factors such as light intensity may also be playing a role. Thus far, the use of LEDs in amphibian captive environments does not appear to demonstrate any detrimental effects on their welfare. However, certainly this study has highlighted a considerable gap in the knowledge of amphibian captive environments, demonstrating the need for investigations into artificial light as a whole, including the role of both spectral output and intensity.

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3.1 Introduction Ex situ conservation has an important role to play in the protection of many amphibian species (Moore & Church, 2008; Pavajeau et al., 2008). Amphibians are increasingly vulnerable in the wild primarily due to extensive habitat degradation and the spread of disease (Wake & Vredenburg, 2008). As a result, increasingly ex situ conservation programmes are being utilised in order to mitigate against population declines and to supplement in situ efforts (McGregor Reid & Zippel, 2008; Zippel et al., 2011). However, the success of ex situ conservation programmes and subsequent reintroductions of amphibians remains limited (Tapley et al., 2015). Provisioning of the correct environmental conditions is a factor which influences the success of these initiatives as it ensures the maintenance of fit and healthy populations of amphibians for potential reintroduction attempts (Michaels et al., 2014c; Michaels & Preziosi, 2015). An important aspect of the captive environment is lighting (Pough, 1991). Light is particularly essential for amphibian survival. It is an important regulator of a range physiological processes for amphibians, including growth and reproduction, and promotes natural behaviour (Lentini, 2013). While other housing conditions have been investigated, there are no studies exploring the effects of artificial visible lighting on amphibians (Michaels et al., 2014a; Michaels et al., 2014b). Accordingly, there are no published guidelines regarding the appropriate use of visible lighting in amphibian husbandry. As such fluorescent, mercury vapour and incandescent lighting are used and provided in a manner that each individual zoological institution has deemed ‘best practice’ (Ferrie et al., 2014; Pough, 2007).

However, the development of LED lighting units for amphibian enclosures has provided an alternative form of artificial lighting. LEDs are at the centre of a lighting revolution and are becoming increasingly popular as a result of their economic, environmental and user benefits (Schubert & Kim, 2005; El-Zein, 2013). The spectral composition of light from LEDs is vastly different from the spectra of light currently provided by lighting and has the potential to change the make up of captive environments (Helsey, 2016 [Chapter 2]). LEDs produce a high peak in spectral irradiance at narrower wavelengths towards the end of the visible spectrum around 440-450nm (Helsey, 2016 [Chapter 2]). This contrasts the spectral output of the lighting currently provided in zoological institutions where there are spikes in irradiance at longer wavelengths around 540nm and 610nm (Helsey, 2016 [Chapter 2]). Additionally, LEDs may provide a more uniform thermal environment (Helsey, 2016 [Chapter 2]). However, the impact of these changes for amphibian welfare must be determined.

A range of studies have investigated the potential impacts of LED use and a growing body of research has outlined some negative effects of LED lighting (Stone et al., 2012). Shorter wavelengths are suspected to have harmful side effects for many species (Falchi et al., 2011). Whilst shorter wavelengths allow for a narrow spectral output which increases energy efficiency, they are typically more harmful for vertebrates and in some species can have consequences for health and survivorship (Behar-Cohen et al., 2011; Migaud et al., 2007). Further to this, the

60 Chapter 3 short wavelengths in several studies have been recognised as disrupting the production of melatonin in a variety of species (Shin et al., 2011; Cajochen et al., 2005). Disruption of melatonin is postulated to have a variety of physiological effects in amphibians, including potentially in growth and colouration (Rich & Longcore, 2005; Vanecek, 1999).

Similarly the effect of artificial night lighting on amphibians is well documented indicating that lighting can have an impact on amphibian physiological processes (Perry et al., 2008; Rich & Longcore, 2005; Buchanan, 1993). This is important as a number of studies comparing LEDs and other forms of artificial night lighting have determined a greater impact of LEDs on a variety of behaviours (Stone et al., 2012; Villamizar et al., 2009). Though several studies exist highlighting the potential risks of LEDs, no study or investigation to assess the potential harmful impacts for amphibians has been conducted.

LEDs typically produce a high peak in spectral output between 440-450nm which is closer to the wavelengths produced by UV than fluorescent lights (Helsey, 2016 [Chapter 2]). Therefore, similar to the effects of UVB, LEDs may potentially impact amphibian welfare in captivity. Narrow wavelengths like those produced by UVB light are thought to be responsible for various negative impacts on health in amphibians (Licht & Grant, 1997). UVB has been empirically proven to impact survival rate, skin quality, behaviour, colouration and even behaviour in amphibians (Licht & Grant, 2008; Bancroft et al., 2008; Michaels et al., 2014b). UV producing lights and LED lights had a similar effect in moths, both resulting in anti-predator behaviour (Wakefield et al., 2015). Thus the effect of LEDs demands greater investigation in amphibians due to the considerable effect of UVB on amphibians (Wakefield et al., 2015). The impacts of UVB on amphibians can thus guide areas of investigation into the effects of LEDs on amphibians.

Despite the indications that LEDs can have potentially negative impacts on vertebrates, and therefore amphibians, there may also be some positive effects of LEDs. LEDs produce a broader spectrum of light ranging from 500nm-650nm, compared to typical lights in amphibian enclosures which have one or two narrow peaks within this range (Helsey, 2016 [Chapter 2]). Natural daylight produces the full spectrum of visible lighting and this broader spectrum of light production by LEDs may more accurately mimic this natural light (Ellis et al., 2013). Artificial environments are most successful when they most accurately replicate conditions from the wild. Thus LEDs do have the potential to be beneficial for captive populations of amphibians. It may allow for greater maintenance of fitness consistent with wild populations which will subsequently facilitate their reintroduction into the wild.

Differences in the thermal output of LEDs may also have an impact. LEDs have a significantly reduced heat output as a result of their greater energy efficiency, and accordingly have a much smaller effect on the temperatures of enclosures. This contrasts with fluorescent and mercury

61 Chapter 3 vapour lighting which produce greater gradients of temperature and greater thermal complexity (Helsey, 2016 [Chapter 2]). The impact of these differences, if any, is still unknown but the use of LED lighting clearly limits the extent to which amphibians can control body temperature through behavioural thermoregulation.

Although many investigations have explored the light requirements of amphibians, specifically in UVB provisioning, there has been very little investigation into the use of artificial lighting within amphibian captive environments. Light is essential for amphibians, however artificial lights, and particularly LEDs, have many potential risks. These need to be investigated and if necessary, mitigated, for successful ex situ conservation programmes. Whilst several studies have demonstrated to an extent the potential risks and health problems occurring as result of exposure to LED lights in a variety of species, specific investigation in amphibians is limited (Falchi et al., 2011; Villamizar et al., 2009; Shin et al., 2011). However, it is important to determine the impact of LEDs, as the correct provisioning of environmental conditions is necessary to ensure the fitness of individuals in captivity (Michaels et al., 2014a). LEDs are increasingly regarded as an environmentally friendly alternative to current lights, and provide both cost and energy savings (Bergh et al., 2001). Nonetheless any impacts of LED use on amphibian welfare needs to be established before they can be utilised.

Aims To asses the impact of LED lighting on anuran welfare, specifically M. betsileo. This study will assess changes in colouration, weight, snout-vent length and activity under LED treatment compared to the fluorescent lighting typically used in anuran enclosures. This will determine the potential welfare implications, if any, of using LED lighting.

2.2 Methods Ethical Statement The methods used in this study were non-invasive and as such did not require a UK Home Office Licence. The University of Manchester Ethical Review Committee approved this study.

Study Species In this study M. betsileo were bought from trade and had imported directly from Madagascar. Mantella betsileo is often held in private collections and is not currently threatened in the wild, categorised by the IUCN as least concern (Andreone et al., 2005; IUCN, 2015). There is limited life history information regarding M. betsileo and its captive requirements are not well known (Andreone et al., 2005; IUCN, 2015).

Animal Husbandry 29 M. betsileo individuals were included in this study imported directly from the Madagascar. The study group contained 3 treatment groups (10, 10 and 9 per group). Individuals were

62 Chapter 3 housed in 6 small ExoTerraTM terranium tanks (30x30x45cm), 5 per group (4 in one group). These were held at the University of Manchester in climate controlled rooms with a diurnal temperature of 24°C and a nocturnal temperature of 22°C. Each group was fed twice weekly approximately 10g of hatchling crickets (Gryllus bimaculatus) lightly dusted with Nutrobal®, the powdered calcium supplement. All tanks were sprayed twice a week to maintain humidity. Each terrarium contained a layer of Hydroleca, a layer of soil and coir (1:1 ratio) and a small pond containing stones. A small bromeliad (Neoregella sp.), moss (Sphagnaceae sp.), a leafy plant (Ficus promelia) and half a coconut shell were also provided for variety and shelter. UV lighting was standardised across the enclosures and provided by a Zoomed Reptisun™ 10.0 UVB bulb placed alongside the visible lighting.

Experimental Treatments Visible lighting was the only parameter altered between the enclosures. Three treatment groups were investigated using different visible lighting sources. The lighting was provided in a 12:12 hour light:dark cycle. See Table 3.1 for detailed specifications of the different lights. • Treatment Group 1 –fluorescent lighting Polylux™ T8 XLR. • Treatment Group 2 – Jungle Dawn Arcadia™ LED lights. These lights are available commercially at a low price. • Treatment Group 3 - Solar Stinger™ LED lights. These lights were used as they have been specifically designed for aquaria and terraria and have been used to some degree in Chester Zoo (Ben Baker, 2016, pers comms., 2 January). They are typically more expensive than most LED lights commercially available for amphibian husbandry.

Table 3.1: The specifications of the three lighting types used in each treatment group.

Treatment Type Brand Colour Watts Cost Group Temperature (W) (K) 1 Fluorescent Polylux™ T8 XLR 4000K 15W £2-6

2 LED Jungle Dawn 6500K 9W £10-14 Arcadia™ 3 LED Solar Stinger™ 6500K 6W £89

Morphometric Data Collection Individuals were randomly separated into groups of 5 per tank and 2 tanks were assigned to each treatment group. One individual was removed before the start of the study, resulting in 4 individuals in treatment group 6. Prior to commencement of the study measurements from each individual were taken. Individuals were photographed against a sheet of graph pad paper (1mm2) and an orange colour standard (X-Rite Colour Checker Classic, Munsel Color, USA), Photographs taken on graph pad paper were analysed in ImageJ (Rasband, 1997) to measure snout-to-vent length (SVL) of the individuals. Photographs with the orange colour standard were analysed with the Digital Colour Meter application on an Apple MacBook Pro (iOS Maverick) to measure the relative changes in colouration of the individuals. A position on the top of the head

63 Chapter 3 between the eyes was used as the colour patch in each photograph. A colour patch was also analysed for the orange colour standard in the same photograph. The Digital Colour Meter provided the mean generic red, blue and green pixel values of the colour patch. The proportion of red pixels within the area were calculated for both the colour patch on the frog and on the ! orange colour standard using the equation � = . The red pixels (R - frog) were then !!!!! calculated as a proportion of the red pixels in the colour standard (R – colour standard) as in Ogilvy et al. (2012) and Pike et al. (2007). Individuals were also weighed using Smart Weigh Digital Pocket Scales (SWS10) to 0.01g. The Scaled Mass Index was also calculated using the weight (g) and SVL (mm) measurements via the equation:

!!"# !! SMI = M! !! where Mi is the Individual Weight (g), Li is the Individual SVL (mm), L0 is the average SVL of the population and bSMA is the scaling component as determined by the standardised major axis regression on the natural log of Mass and SVL of the population (Peig & Green, 2009; Peig & Green, 2010). These measurements were carried out both at the start of the study and 3 months later at the end point of the study.

Behavioural Investigations Individuals were allowed to acclimatise for a period of 6 weeks in their new treatment environments after which scan sampling behavioural observations were carried out (Simpson & Simpson, 1977). Behavioural observations were carried out for approximately 6 weeks, though camera malfunction resulted in several lost days of recordings for various tanks. A Bird Box Camera (wired or wireless) by SpyCamera CCTV was set up inside each tank, in the centre of the tank facing downwards to provide a birds eye view. These cameras were connected either directly or through a wireless receiver to a PC. iSpy Surveillance Software on the PC (iSpy, 2007) was then used take video recordings. Lighting was provided in a 12:12 cycle between 8:00 – 20:00. The cameras have night vision capabilities so recordings could be observed after 20:00. Video recordings were taken between 6:00 – 22:00. Recording was undertaken for 2 hours before and after the lights were turned on as preliminary viewing indicated individuals were active at this time. At 30-minute intervals, the recordings were viewed for one minute to ascertain the location of the individual. The number of individuals in each location was recorded. If individuals moved throughout the minute, the location was recorded as where the individual was first observed. The location of the individuals was recorded as a defined category: - Sheltered – Individuals could not be seen and are assumed to be either in the coconut hut or under the foliage (i.e. hidden) and thus not exposed to the light. Individuals were still classed as hidden if only a small part of the individual was observed or if individuals were observed through the foliage. - Out – individuals were located in the open space of the tank. - Bromeliad – individuals were located within or on the bromeliad leaves. - Pond – individuals were located within the pond.

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- Plant – individuals were located on the plant. - Hut – individuals were located on the coconut hut. - Glass – individuals were located on the glass sides of the tank. The location of individuals is used as a proxy for activity levels of the individuals. This information was collected for each of the six tanks. Spraying and feeding of the frogs was avoided at the 30-minute marks. However room entry may have occurred during these times for other purposes.

Statistical Analysis The categories Out, Bromeliad, Pond, Plant, Hut, Glass were grouped as ‘Out’ for statistical analysis. Analysis was conducted in R and graphs were produced in Microsoft Excel and R. As fewer individuals were in the Solar Stinger™ treatment group, counts of individuals were converted to a proportion in the behavioural data. The average proportion of individuals ‘Out’ was calculated for each day for each treatment group alongside the 95% confidence interval. Before and after measurements of Weight, SNV and Redness for each light treatment group were analysed using a Paired T Test. Changes in Weight, SNV, Redness and Activity were analysed using a one-way ANOVA.

3.3 Results Weight, Snout-Vent Length and Redness Measurements In treatment groups, Fluorescent (t = -2.752, df = 9, p = 0.0224) and Solar Stinger™ LED (t = - 2.925, df = 8, p = 0.0191) weight increased over the course of the 3 months. Weight did not change significantly under Jungle Dawn™ LEDs (t = -1.5501 df=9, p = 0.1555). However, the light treatment did not have a differential effect on change in weight of individuals over the course of the study (F2,26 = 1.712, p = 0.2000), Figure 3.1.

Changes in length were also not affected by the light treatment over the course of the study

(F2,26 = 1.764, p = 0.1910), Figure 3.2. There was no change in length throughout the course of the study for all treatment groups, Fluorescent (t = 1.6806, df = 9, p = 0.1271), Jungle Dawn™ LED (t = 1.6569, df = 9, p = 0.1319) and Solar Stinger™ LED (t = -0.6693, df = 8, p = 0.5221).

The SMI was unaffected by the difference in light treatment (F2,26 = 0.4995, p = 0.6125), Figure 3.3. There was no difference in SMI across the course of the study in the Solar Stinger™ LED treatment group (t = 1.389, df = 8, p = 0.2022). There were significant increases in the SMI of individuals in the Jungle Dawn™ (t = 3.620, df = 9, p = 0.0056) and Fluorescent lighting (t = 2.449, df = 9, p = 0.0368).

Light treatment did not impact the change in redness of individuals in the study (F2,26 = 0.801, p = 0.4600), Figure 3.4. There were no significant changes in colouration before and after the

65 Chapter 3 study for all treatment groups, Fluorescent (t = -1.694, df = 9, p = 0.1245), Jungle Dawn™ LED (t = -1.367, df = 9, p = 0.2047) and Solar Stinger™ (t = 0.0377, df = 8, p = 0.9709).

0.16

0.14

0.12

0.10

0.08

0.06

Change in Weight (g) Changein Weight 0.04

0.02

0.00 Normal Jungle Dawn Solar Stinger

Light Treatment

Figure 3.1: Effect of light treatment on average change in weight (g). The effect was not significant for any light treatment, p > 0.05. Error bars indicate ±SEM.

0.60

0.40

0.20

0.00

-0.20 -0.40

Change in SVL (mm) Changein SVL -0.60

-0.80

-1.00 Normal Jungle Dawn Solar Stinger Light Treatment

Figure 3.2: Effect of light treatment on average change in SVL (mm). The effect was not significant for any light treatment, p > 0.05. Error bars indicate ±SEM.

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0.2 0.18

0.16

0.14 0.12 0.1

0.08 Changein SMI 0.06 0.04

0.02

0 Normal Jungle Dawn Solar Stinger Light Treatment Figure 3.3: Effect of light treatment on average change in Scaled Mass Index calculated from Mass and SVL of individuals. The effect was not significant for any light treatment, p > 0.05. Error bars indicate ±SEM.

0.05

0.04

0.03 0.02

0.01 0

-0.01

-0.02 Changein Redness (RProportion) -0.03 Normal Jungle Dawn Solar Stinger Light Treatment

Figure 3.4: Effect of light treatment on average change in redness calculated from the proportion of red pixels. The effect was not significant for any light treatment, p > 0.05. Error bars indicate ±SEM.

Behaviour Measurements There was an increase in activity in both LED treatment groups across the course of the study. This increase however was not consistent and individuals in Solar Stinger™ lighting saw a greater increase in activity (Figure 3.4). An interaction between time and lighting treatment was observed (F2,211 = 3.1228, p = 0.046). The change in activity over time was different between the lighting treatments. Activity of individuals in Solar Stinger™ light treatment significantly increased over time compared to the Fluorescent lighting (p = 0.0137). The increase in activity in Jungle Dawn™ was not significantly greater than Fluorescent lighting (p = 0.2751).

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Figure 3.5: Mean proportion of individuals in the each lighting treatment group for each day recorded as ‘Out’. Lines of best fit are indicated and the grey border represents the 95% CI (Confidence interval). Proportion of individuals out in Solar Stinger™ is significantly higher than in enclosures with Fluorescent™ and Jungle Dawn™ LED lighting. Both LED treatment groups showed an increase across the study, and Solar Stinger™ lighting increased significantly higher than the other treatment groups.

30

25 /nm) 2

20

15

10

5 AbsoluteIrradiance (µ/W/cm

0 400 450 500 550 600 650 700 Wavelength (nm)

Figure 3.6: Absolute irradiance spectrum of lighting in the 6 study enclosures with Fluorescent and LED lights in the visible light spectrum (400-700nm) measured at a distance of 10cm away from the light. Treatment Groups 1 and 2 with fluorescent lights are highlighted in red, Tank 1 (dark red) and Tank 2 (light red), are shown in red. Treatment groups 3 and 4 with Jungle Dawn™ LED lights are illustrated in green Tank 3 (dark green) and Tank 4 (light green) are shown in green. Treatment groups 5 and 6 with Solar Stinger™ LED lights are shown in blue, Tank 5 (dark blue) and Tank 6 (light blue).

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3.7 3.8

3.9 3.10

3.11 3.12

Figures 3.7-3.12 (left to right): Thermal imaging pictures and corresponding photograph of the 6 amphibian enclosures in the study at the University of Manchester. Scale is in °C.

Tank 1 (Fig, 3.7) and Tank 2 (Fig, 3.8) with fluorescent lights have a temperature difference of 2.2°C and 2.7°C respectively between the top and bottom of the tank.

Tank 3 (Fig, 3.9) and Tank 4 (Fig, 3.10) with Jungle Dawn™ LED lights have a temperature difference of 1.7°C, and 1.4°C respectively between the top and the bottom of the tank.

Tank 5 (Fig, 3.11) and Tank 6 (Fig. 3.12) with Solar Stinger™ LED lights have a temperature difference of 0.8°C and 2.1°C respectively between the top-middle and the middle-bottom

Average temperature for date and time of images obtained: 23°C

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Enclosure Details Spectral Output – Tanks 1 and 2 with fluorescent lighting had a different spectral output compared to Tanks 3-6 with the LEDs (Figure 3.5). Two large sharp spikes in absolute irradiance were observed at 542nm and 509nm, with smaller spikes in irradiance across the spectrum. In contrast to this, Tanks 3-6 displayed similar spectral output with a sharp peak in irradiance around 445nm and then a broader spike in irradiance increasing from 490nm, peaking at around 570-580nm before decreasing. Despite the similarities in the spectral output pattern recorded in tanks 3-6, different levels of absolute irradiance were recorded. At the peak in absolute irradiance at 445nm in tanks 5 and 6, absolute irradiance was 5.06 µ/W/cm2/nm, this is almost half the irradiance level of tanks 3 and 4 at the same wavelength with 9.72 µ/W/cm2/nm. Between wavelengths 530-580nm, the irradiance level is more than double in tanks 3 and 4 (~2.3-2.9µ/W/cm2/nm) compared to 5 and 6 (~5.4-7.3 µ/W/cm2/nm) despite showing a similar pattern. Tanks 1 and 2 produced a similar level of irradiance as tanks 3 and 4 with 9.15 µ/W/cm2/nm at 542nm at their peaks. However, tank 1 and 2 did produce a higher spike in absolute irradiance at 12.8 µ/W/cm2/nm at 609nm, greater than tanks 3 and 4 highest level.

Thermal Imaging Photographs - the different lighting in the enclosures produced marginally different thermal ranges within the tanks. Temperatures were mostly uniform across the LED tanks differing only by 1.7°C and 1.4°C in tanks with Jungle Dawn™ LEDs (Figure 3.8 and 3.9 respectively) and 0.8°C and a maximum of 2.1°C in Solar Stinger™ LEDs (Figure 3.10 and 3.11 respectively). The temperature difference in tanks with Fluorescent lighting was higher with differences of 2.2°C and 2.7°C (Figure 3.6 and 3.7 respectively). Temperature was mostly uniform in LED tanks representing the temperature of the climate controlled room, yet there is greater thermal heterogeneity in enclosures with fluorescent lighting as illustrated by the cooler and warmer areas. Temperature was to some extent more variable across the enclosure with fluorescent lights compared to the enclosures with both LED lights.

3.4 Discussion Survivorship Mostly significantly there was no difference in survivorship between LED and fluorescent lighting treatments. For the duration of the study, all individuals survived and there were no obvious indications of any ill health. There was no evidence to suggest there was a direct impact on the survival of species under LED lighting. This contrasts the impact of UVB, which is able to disrupt DNA, and results in a reduction in survivorship for many vertebrate species including amphibians (Licht, 2003; Bancroft et al., 2008). Bancroft et al. (2008) analysed studies investigating the impact of exposure to high levels of UVB and concluded that 35% of the studies showed a decrease in survivorship (Bancroft et al., 2008). UVB however has the greatest impact on survivorship in early amphibian life stages, which have not been investigated in this study, and perhaps would useful to be studied in future to assess the full impact of LEDs

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(Belden et al., 2000; Hakkinen, 2001).

Growth and Development In comparison to fluorescent lighting, LED lighting had no significant effect on the growth and development of M. betsileo individuals. In both the Solar Stinger™ LEDs and fluorescent light treatments, weight significantly increased across the duration of the study. However an increase in weight was consistent across all treatment groups. Similarly, there was no significant change in snout-vent length in any treatment group across the duration of the study and this was consistent across all treatment groups. Correspondingly, though significant increases were observed in the SMI of individuals in Jungle Dawn™ and fluorescent lighting, the SMI of individuals increased consistently across all treatment groups over the duration of the study. This suggests that LED lighting is not differentially affecting amphibian growth and does not have an impact on their weight or body condition. LEDs appear to have little impact, detrimental or not, on amphibians in this respect.

Such results are inconsistent with previous studies, albeit in other species, which highlight the influential role of LEDs in growth. A number of studies have investigated the impact of LED lighting in a variety of fish species. These studies largely conclude that LEDs have a significant enhancing impact on growth (Shin et al., 2011; Taylor et al., 2005). In Amphirion clarkii short wavelength LED lighting was found to increase growth, which the authors infer is due the increase in the production of a growth hormone modulated by the increase in melatonin (Shin et al., 2011). Shorter wavelengths induce melatonin production which results in greater expression of growth hormones (Shin et al., 2011; Chowdhury et al., 2008). Similarly, in Dicentrarchus labrax and Danio rerio larvae, growth was enhanced when reared under blue LED lighting (Villamizar et al., 2009; Villamizar et al., 2014). Villamizar et al. (2009) attributes this to the shorter wavelengths of LED lighting and a spectral output more consistent to that in the wild.

However, contrary to this, Migaud et al. (2007) determined that melatonin was supressed by blue LED lighting in Salmo salar. This is supported by studies undertaken in humans, which suggest that LED lighting, particularly the narrow wavelengths, cause a significant reduction in melatonin production (Falchi et al., 2011; Cajochen et al., 2005; Hong et al., 2015). Cajochen et al. (2005) specifies that short wavelengths of ~460nm induced melatonin suppression. Studies in fish appear to suggest that this suppression of melatonin would result in a reduction in growth. Yet, several studies have suggested that although melatonin regulates the expression of growth hormones, it may also have an inhibitory effect, particularly in amphibians (Vanecek, 1999; Rich & Longcore, 2005). Suppressing melatonin could potentially reduce its inhibitory effect and thus, may also result in an increase in growth. Though LEDs have been identified as significant repressors of melatonin production in other species, this has yet to be determined in amphibians. Photoperiods in amphibians, however, can modulate the production of melatonin and therefore LEDs may have a similar effect (Delgado et al., 1987; Edwards & Pivorun, 1991).

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However, despite no significant changes in the growth of individuals within this study which indicates that there was a limited impact of LEDs on growth, some observations can be made. Individuals in the Solar Stinger™ LED treatments did see an increase in snout to vent length in comparison to individuals under fluorescent lighting. This coincided with an increase in weight across both LED treatments in comparison to fluorescent lighting. This resulted in a limited change in the SMI index under the Solar Stinger™ treatment, as weight and growth increased simultaneously unlike only an increase in weight under fluorescent and Jungle Dawn™ light treatments. These may be initial indications that LEDs are resulting in minor positive impacts for growth, potentially via a change in melatonin production. Nevertheless, further investigation is required.

Previous studies in a variety of species indicate there should be an impact of LEDs on amphibian growth and initial trends in this study in weight gain and increases in SNV length agree there could be. Whilst inferences and hypothesises can be made regarding the impact of LEDs on melatonin production and its subsequent effects, this mechanism has not be defined in amphibians. The effect of melatonin is not yet fully understood in amphibians and many studies provide evidence of both inhibitory and excitatory role of melatonin on growth hormones, as well as positive and negative effects of LEDs on melatonin production (Filadelfi & Castrucci, 1996; Chowdhury et al., 2008; Edwards & Pivorun, 1991). The differential role of melatonin is dependent on a variety of physiological mechanisms and the species and life stage of individuals (Filadelfi & Castrucci, 1996). The true impact of LED lighting on melatonin production is unclear without quantifying the melatonin levels. Therefore future studies should be undertaken to clarify the role of melatonin and quantify melatonin levels.

Furthermore, to determine the full implications of LEDs on growth and more importantly development, it may be more useful to assess all life stages of amphibians reared under LED lights from tadpoles to juveniles and adults. UVB has greater sub-lethal and lethal effects in amphibians as early life stages compared to adults, due to their greater vulnerability (Bancroft et al., 2008; Licht, 2003). Additionally the impact of LEDs on growth in fish species has only been found in early life stages (Shin et al., 2011). This further highlights the need for assessment of LEDs at earlier life stages as this may allow for more accurate assessments of the impact of LEDs on amphibian development (Belden et al., 2000). Further to this, as indicated by the various studies undertaken on amphibians and UVB, UVB has a differential effect on amphibians depending on the species and their specific needs (Bancroft et al., 2008). Therefore although this indicates there are limited differences in weight and growth in this species, we cannot rule out other impacts on other species of amphibian, which may have more specific requirements or tolerances of short wavelengths. This initial investigation suggests there is a limited effect of LEDs on the growth of individuals, but further investigation is undoubtedly required to further support this conclusion.

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Colouration Studies have indicted that UVB exposure and narrow wavelengths in amphibians can result in skin darkening. However, there was no evidence of a change in the redness of M. betsileo skin in all treatment groups and no difference in the colouration of individuals was observed (Belden & Blaustein, 2002). Contrary to Vanecek (1998) who suggests that a change in melatonin levels would alter amphibian skin colouration, no change in redness was observed (Vanecek, 1998; Rich & Longcore, 2005). This suggests that LEDs had no impact on the colouration of M. betsileo across the duration of this study (Belden & Blaustein, 2002). In fact, the individuals under the normal treatment showed the greatest change in redness.

Activity This study largely indicates that LEDs are not having an impact on the physiological welfare of amphibians. However, differences in the activity of M. betsileo individuals were observed in one of the LED treatment groups. A greater number of individuals in the Solar Stinger™ LED lighting treatment were seen out in the enclosure and not sheltered by either the foliage or cover provided. Individuals in this treatment group therefore demonstrated an increase in activity levels, as they were observed to be moving and out in the open of the enclosure more frequently than those in the other lighting treatments. This difference in visibility and movement is similar to results from studies investigating the impact UVB, where amphibians demonstrated altered locomotion and orientation behaviour patterns as a result of consistent exposure to high levels of UVB (Blaustein et al., 2000). However, this contrasts with studies investigating the impact of LED impact on bat activity where compared to high-pressure sodium artificial night lighting, bat activity was significantly reduced under LED light treatments (Stone et al., 2012). LED lighting had a similar impact on moth behaviour, where activity was reduced under LED treatment (Wakefield et al., 2015; Longcore et al., 2015).

Despite this difference in activity, it is unclear if this will have positive or negative implications for amphibians, yet a number of hypothesises can be made. One hypothesis is that LED lighting is mimicking more closely natural lighting and promoting natural behaviour that would be displayed in the wild (Ellis et al., 2013; Aubé et al., 2013). This may be positive by maintaining populations with more natural behaviour and therefore most likely to be successful in possible reintroduction attempts. There may also be similarly positive implications for the zoological institutions housing the amphibians, as increased activity will result in a greater attraction for visitors. If LEDs promote natural behaviour, are environmentally friendly and cost effective, as well as encouraging greater activity of individuals which is better for displays in zoos, they would undoubtedly be used for amphibian ex situ conservation environments.

However alternatively, it may be an indication of increased unnatural behaviour, as it is contrasts significantly to the behaviour exhibited in the other light treatments. This may demonstrate a similar effect to that observed in alpine newts who displayed erratic behaviour

73 Chapter 3 after high UVB exposure (Nagl & Hofer, 1997). However, this observed change in activity could also be inferred as a reduction in anti predator behaviour. In Bufo boreas and Rana cascada anti-predator behaviour reduced following exposure to excessive UVB radiation, as both species exhibited greater movement and hid less (Kats et al., 2000; Belden et al., 2000). Individuals in the Solar Stinger™ LED treatment displayed a similar change in behaviour as individuals hid less under shelter and displayed a greater level of activity. Undermining their ability to avoid predators could have implications for the fitness of captive populations of amphibians. This could have potentially detrimental impacts for the effectiveness of reintroduction efforts. This is further supported by the observed change in behaviour in moths (Wakefield et al., 2015). Investigations determined that compared to other artificial lights, under LED lighting moths reduced last ditch anti predator manoeuvres (Wakefield et al., 2015). Wakefield (2015) showed that the use of LEDs resulted in similar changes in moth behaviour as UV wavelengths. These similar changes in behaviour between individuals in this study and other species in response to changes in LEDs may be indicative of LEDs having a similar impact to UV in amphibians (Longcore et al., 2015; Wakefield et al., 2015).

The difference observed in the temperature of enclosures with different lighting may also have an effect on behaviour. A small difference in thermal heterogeneity was observed between enclosures under the different light treatments, although this was markedly less than the difference between LEDs and current lighting in zoos (Helsey, 2016 [Chapter 2]). Amphibians are ectotherms and thermoregulation in amphibians is fundamentally behavioural though basking or microclimate selection. A gradient of temperatures, as typically seen in fluorescent lighting, is thought to promote natural thermoregulatory behaviour (Tapley et al., 2014). However, as previously demonstrated, activity was significantly lower in enclosures with fluorescent lighting, suggesting that M. betsileo individuals did not noticeably seek out their preferred temperature. Nevertheless, this is highly dependent on the species and many species of amphibians do not bask to regulate body temperature. Basking however can be an important behavioural response utilised by amphibians (Michaels & Preziosi, 2013; Lillywhite et al., 1998). Therefore the conditions required for basking should be adequately provisioned through appropriate light sources in captivity (Browne & Edwards, 2003).

Limitations Though informative, the results in this study could be improved through a greater standardisation between the lighting treatments that would allow more accurate conclusions to be made. The wattage, illumination levels (which were not recorded), the size of each light and the number of bulbs were different between the three light treatments. Though due to the limited types of lighting currently produced for amphibian enclosures, minimising the differences in these parameters between the lighting types will allow for more accurate direct comparisons and limit potential confounding factors. Similarly, only fluorescent lighting was used as a comparison against LEDs and though commonly used in amphibian captive environments

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(Helsey, 2016 [Chapter 2]), metal halide and mercury vapour lamps are typically used alongside these. It would be valuable to investigate the effects of these other lighting treatments and the effects of these lights together, with both fluorescent and LED lighting. Though this preliminarily study indicates some potential effects of LED lighting compared to fluorescent lighting, limiting potential confounding factors and investigating a greater number of different types of lighting as a control would provide a more valuable and complete study.

Further Investigation Interestingly individuals in the Solar Stinger™ LED treatment group often had opposite responses to individuals in the other treatments. For example, individuals displayed an increase in SVL and a decrease in redness. It also resulted in a greater increase in weight and activity in comparison to the other two treatment groups, which had a remarkably similar effect to each other. These findings may be a result of differences in the intensity of lighting rather the differences in spectral wavelength output. Solar Stinger™ lighting had a considerably lower level of irradiance in comparison both the Jungle Dawn™ LEDs and fluorescent lighting, which had high levels of irradiance and therefore intensity. High intensities of artificial light at night was found to alter amphibians foraging behaviour and reduce their activity (Rand et al., 1997; Rich & Longcore, 2005). A study investigating the reduced activity in bats suggested it may be a result of the high intensity of the LED treatment, rather than the difference in the spectral output (Stone et al., 2015). This suggests that alongside the difference in spectral output, the intensity of lighting could be having on impact on the health of amphibians. Amphibians in the high intensity lighting, Jungle Dawn™ and fluorescent lightings showed significantly lower levels of activity, similar to the reduction of activity in other species in response to high intensity lighting. Similarly, Aube et al. (2013) suggests that LEDs had less effect on melatonin production than high pressure sodium lamps (HPS) due to the reduction in intensity (Aubé et al., 2013). This highlights the need for greater investigation into the impact of the intensity of light. This is an important finding indicating that further investigations into ambient lighting in general, not just in LEDs should be undertaken.

Conclusion Although this study cannot precisely guarantee that LEDs are safe for use in amphibian enclosures, initial signs suggest that there are no immediate threats to using LED lighting. These preliminary investigations suggest that LEDs have little impact on amphibians as there were no significant changes in weight, growth, or redness. Significant increases in activity was observed in amphibians under one LED treatment, yet cannot be identified as either a positive or negative effect and it is likely that the impact will not affect fitness in captivity. Interestingly, it appears that the potentially positive implications of LEDs, i.e. the greater energy efficiency cost and environmental savings, alongside an increase in amphibian activity, appear to outweigh the currently unverified potential negative effects that have been speculated. However, understanding the impact of LED lighting across all life stages and in a range of species with

75 Chapter 3 varying needs is paramount to determining the complete effects of LEDs. This study has also highlighted a substantial gap in information regarding amphibian husbandry, indicating the need for research to investigate the provisioning of visible lighting in amphibian enclosures, particularly the role of intensity of artificial visible light.

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3.5 References Andreone, F., Cadle, J.E., Cox, N., Glaw, F., Nussbaum, R.A., Raxworthy, C.J., Stuart, S.N., Vallan, D. & Vences, M. (2005) Species review of amphibian extinction risks in Madagascar: conclusions from the Global Amphibian Assessment. Conservation Biology, 19 (6), p1790–1802. Aubé, M., Roby, J. & Kocifaj, M. (2013) Evaluating potential spectral impacts of various artificial lights on melatonin suppression, photosynthesis, and star visibility. PloS one, 8 (7), pe67798. Bancroft, B.A., Baker, N.J. & Blaustein, A.R. (2008) A meta-analysis of the effects of ultraviolet B radiation and its synergistic interactions with pH, contaminants, and disease on amphibian survival. Conservation Biology, 22 (4), p987–996. Belden, L.K. & Blaustein, A.R. (2002) UV-B induced skin darkening in larval salamanders does not prevent sublethal effects of exposure on growth. Copeia, 2002 (3), p748–754. Belden, L.K., Wildy, E.L. & Blaustein, A.R. (2000) Growth, survival and behaviour of larval long- toed salamanders (Ambystoma macrodactylum) exposed to ambient levels of UV-B radiation. Journal of Zoology, 251, p473–479. Bergh, A., Craford, G., Duggal, A. & Haitz, R. (2001) The promise and challenge of solid-state lighting. Physics Today, 54 (12), p42–47. Blaustein, A.R., Chivers, D.P., Kats, L.B. & Kiesecker, J.M. (2000) Effects of ultraviolet radiation on locomotion and orientation in roughskin newts (Taricha granulosa). Ethology, 106, p227–234. Browne, R.K. & Edwards, D.L. (2003) The effect of temperature on the growth and development of the endangered green and golden bell frog (Litoria aurea). Journal of Thermal Biology, 28 (4), p295–299. Buchanan, B.W. (1993) Effects of enhanced lighting on the behaviour of nocturnal frogs. Animal Behaviour, 45 (5), p893–899. Cajochen, C., Münch, M., Kobialka, S., Kräuchi, K., Steiner, R., Oelhafen, P., Orgül, S. & Wirz- Justice, A. (2005) High sensitivity of human melatonin, alertness, thermoregulation, and heart rate to short wavelength light. The Journal of Clinical Endocrinology and Metabolism, 90 (3), p1311–6. Chowdhury, V.S., Yamamoto, K., Saeki, I., Hasunuma, I., Shimura, T. & Tsutsui, K. (2008) Melatonin stimulates the release of growth hormone and prolactin by a possible induction of the expression of frog growth hormone-releasing reptide and its related reptide-2 in the amphibian hypothalamus. Endocrinology, 149 (3), p962–970. Delgado, M.J., Gutiérrez, P. & Alonso-Bedate, M. (1987) Melatonin and photoperiod alter growth and larval development in Xenopus laevis tadpoles. Comparative Biochemistry and Physiology Part A: Physiology, 86 (3), p417–421. Edwards, M.L.O. & Pivorun, E.B. (1991) The effects of photoperiod and different dosages of melatonin on metamorphic rate and weight gain in Xenopus laevis tadpoles. General and Comparative Endocrinology, 81 (1), p28–38.

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El-Zein, N. (2013) The LED lighting revolution. In: A. Sayigh. Sustainability, Energy and Architecture: Case Studies in Realizing Green Buildings. Massachusetts: Academic Press, p171–194. Ellis, E. V, Gonzalez, E.W., Kratzer, D.A., McEachron, D.L. & Yeutter, G. (2013) Auto-tuning daylight with LEDs: sustainable lighting for health and wellbeing. In: Proceedings of the 2013 ARCC Spring Research Conference. Charlotte: University of North Carolina, ARCC Past Conferences, p465–473. Falchi, F., Cinzano, P., Elvidge, C.D., Keith, D.M. & Haim, A. (2011) Limiting the impact of light pollution on human health, environment and stellar visibility. Journal of Environmental Management, 92 (10), p2714–22. Ferrie, G.M., Alford, V.C., Atkinson, J., Baitchman, E., Barber, D., Blaner, W.S., Crawshaw, G., Daneault, A., Dierenfeld, E., Finke, M., Fleming, G., Gagliardo, R., Hoffman, E.A., Karasov, W., Klasing, K., Koutsos, E., Lankton, J., Lavin, S.R., Lentini, A., Livingston, S., Lock, B., Mason, T., McComb, A., Morris, C., Pessier, A.P., Olea‐Popelka, F., Probst, T., Rodriguez, C., Schad, K., Semmen, K., Sincage, J., Stamper, M.A., Steinmetz, J., Sullivan, K., Terrell, S., Wertan, N., Wheaton, C.J., Wilson, B. & Valdes, E. V. (2014) Nutrition and health in amphibian husbandry. Zoo Biology, 33 (6), p485–501. Filadelfi, A.M.C. & Castrucci, A.M. de L. (1996) Comparative aspects of the pineal/melatonin system of poikilothermic vertebrates. Journal of Pineal Research, 20 (4), p175–186. Helsey, C. (2016) Investigations in LED lighting and body condition of Mantella cowani to improve amphibian ex situ conservation efforts. MPhil Thesis, The University of Manchester. Hong, S.-K., Kim, K.-S., Kim, I.-T. & Choi, A.-S. (2015) Melatonin Suppression under LED Lighting Focused on Spectral Power Distribution Differences. Journal of Korean Institute of Illuminating and Electrical Installation Engineers, 29 (8), p7–17. iSpy (2007) iSpy Surveillance Software (Version 6.5.3). [Computer Program]. Available at https://www.ispyconnect.com/ (Accessed April 2016). IUCN (2015) IUCN Red List of Threatened Species. Version 2015-4 [Internet]. Available from: (Accessed 11 April 2016). Kats, L.B., Kiesecker, J.M., Chivers, D.P. & Blaustein, A.R. (2000) Effects of UV-B radiation on anti-predator behavior in three species of amphibians. Ethology, 106 (10), p921–931. Lentini, A.M. (2013) Husbandry and Care of Amphibians. In: M. D. Irwin, J. B. Stoner, & A. M. Cobaugh eds. Zookeeping : an introduction to the science and technology. Chicago: The University of Chicago Press, p339. Licht, L.E. (2003) Shedding light on ultraviolet radiation and amphibian embryos. BioScience, 53 (6), p551. Licht, L.E. & Grant, K.P. (1997) The effects of ultraviolet radiation on the biology of amphibians. American Zoologist, 37 (2), p137–145. Lillywhite, H.B., Mittal, A.K., Garg, T.K. & Das, I. (1998) Basking behavior, sweating and thermal ecology of the Indian tree frog, Polypedates maculatus. Journal of Herpetology, 32 (2),

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p169. Longcore, T., Aldern, H.L., Eggers, J.F., Flores, S., Franco, L., Hirshfield-Yamanishi, E., Petrinec, L.N., Yan, W.A. & Barroso, A.M. (2015) Tuning the white light spectrum of light emitting diode lamps to reduce attraction of nocturnal arthropods. Philosophical Transactions of the Royal Society B: Biological Sciences, 370 (1667), p20140125. McGregor Reid, G. & Zippel, K.C. (2008) Can zoos and aquariums ensure the survival of amphibians in the 21st century? International Zoo Yearbook, 42 (1), p1–6. Michaels, C. & Preziosi, Ri.F. (2013) Basking behaviour and ultraviolet B radiation exposure in a wild population of Pelophylax lessonae in Northern Italy. Herpetological Bulletin, 124 (124), p1–8. Michaels, C.J., Antwis, R.E. & Preziosi, R.F. (2014a) Impact of plant cover on fitness and behavioural traits of captive red-eyed tree frogs (Agalychnis callidryas). PloS one, 9 (4), pe95207. Michaels, C.J., Antwis, R.E. & Preziosi, R.F. (2014b) Impacts of UVB provision and dietary calcium content on serum vitamin D3 , growth rates, skeletal structure and coloration in captive oriental fire-bellied toads (Bombina orientalis). Journal of Animal Physiology and Animal Nutrition, 99 (2), p391–403. Michaels, C.J., Gini, B.F. & Preziosi, R.F. (2014c) The importance of natural history and species-specific approaches in amphibian ex-situ conservation. Herpetological Journal, 24, p135–145. Michaels, C.J. & Preziosi, R.F. (2015) Fitness effects of shelter provision for captive amphibian tadpoles. Herpetological Journal, 25 (1), p21–26. Migaud, H., Cowan, M., Taylor, J. & Ferguson, H.W. (2007) The effect of spectral composition and light intensity on melatonin, stress and retinal damage in post-smolt Atlantic salmon, Salmo salar. Aquaculture, 270 (1), p390–404. Moore, R.D. & Church, D.R. (2008) Implementing the Amphibian Conservation Action Plan. International Zoo Yearbook, 42 (1), p15–23. Nagl, A.M. & Hofer, R. (1997) Effects of ultraviolet radiation on early larval stages of the Alpine newt, Triturus alpestris, under natural and laboratory conditions. Oecologia, 110 (4), p514– 519. Ogilvy, V., Preziosi, R.F. & Fidgett, A.L. (2012) A brighter future for frogs? The influence of carotenoids on the health, development and reproductive success of the red-eye tree frog. Animal Conservation, 15 (5), p480–488. Pavajeau, L., Zippel, K.C., Gibson, R. & Johnson, K. (2008) Amphibian Ark and the 2008 Year of the Frog Campaign. International Zoo Yearbook, 42 (1), p24–29. Peig, J. & Green, A.J. (2009) New perspectives for estimating body condition from mass/length data: the scaled mass index as an alternative method. Oikos, 118 (12), p1883–1891. Peig, J. & Green, A.J. (2010) The paradigm of body condition: a critical reappraisal of current methods based on mass and length. Functional Ecology, 24 (6), p1323–1332. Perry, G., Buchanan, B. & Fisher, R. (2008) Effects of artificial night lighting on amphibians and

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reptiles in urban environments. In: J. C. Mitchell, R. E. Jung Brown, & B. Bartholomew eds. Urban Herpetology. Society for the Study of Amphibians & Reptiles, p239–256. Pike, T.W., Blount, J.D., Bjerkeng, B., Lindström, J. & Metcalfe, N.B. (2007) Carotenoids, oxidative stress and female mating preference for longer lived males. Proceedings of the Royal Society of London B: Biological Sciences, 274 (1618), p1591–1596. Pough, F.H. (2007) Amphibian biology and husbandry. ILAR Journal, 48 (3), p203–213. Pough, F.H. (1991) Recommendations for the care of amphibians and reptiles in academic institutions. ILAR Journal, 33 (4), pS1–S21. Rand, A.S., Bridarolli, M.E., Dries, L. & Ryan, M.J. (1997) Light levels influence female choice in Tungara frogs: predation risk assessment. Copeia, 2 (2), p447–450. Rasband, W.S. (1997) ImageJ. (Version 1.50i). [Computer Program]. Available at http://rsb.info.nih.gov/ij (Accessed April 2016). Rich, C. & Longcore, T. (2005) Ecological consequences of artificial night lighting. Washington D.C.: Island Press. Schubert, E.F. & Kim, J.K. (2005) Solid-state light sources getting smart. Science, 308 (5726), p1274–8. Shin, H.S., Lee, J. & Choi, C.Y. (2011) Effects of LED light spectra on oxidative stress and the protective role of melatonin in relation to the daily rhythm of the yellowtail clownfish, Amphiprion clarkii. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 160 (2), p221–8. Simpson, M.J.A. & Simpson, A.E. (1977) One-zero and scan methods for sampling behaviour. Animal Behaviour, 25, p726–731. Stone, E.L., Jones, G. & Harris, S. (2012) Conserving energy at a cost to biodiversity? Impacts of LED lighting on bats. Global Change Biology, 18 (8), p2458–2465. Stone, E.L., Wakefield, A., Harris, S. & Jones, G. (2015) The impacts of new street light technologies: experimentally testing the effects on bats of changing from low-pressure sodium to white metal halide. Philosophical Transactions of the Royal Society B: Biological Sciences, 370 (1667), p20140127. Tapley, B., Bradfield, K.S., Michaels, C. & Bungard, M. (2015) Amphibians and conservation breeding programmes: do all threatened amphibians belong on the ark? Biodiversity and Conservation, 24 (11), p2625–2646. Tapley, B., Rendle, M., Baines, F.M., Goetz, M., Bradfield, K.S., Rood, D., Lopez, J., Garcia, G. & Routh, A. (2014) Meeting ultraviolet B radiation requirements of amphibians in captivity: a case study with mountain chicken frogs (Leptodactylus fallax) and general recommendations for pre-release health screening. Zoo Biology, 34 (1), p46–52. Taylor, J.F., Migaud, H., Porter, M.J.R. & Bromage, N.R. (2005) Photoperiod influences growth rate and plasma insulin-like growth factor-I levels in juvenile rainbow trout, Oncorhynchus mykiss. General and Comparative Endocrinology, 142, p169–185. Vanecek, J. (1998) Cellular mechanisms of melatonin action. Physiological Reviews, 78 (3), p687–721.

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Vanecek, J. (1999) Inhibitory effect of melatonin on GnRH-induced LH release. Reviews of Reproduction, 4, p67–72. Villamizar, N., García-Alcazar, A. & Sánchez-Vázquez, F.J. (2009) Effect of light spectrum and photoperiod on the growth, development and survival of European sea bass (Dicentrarchus labrax) larvae. Aquaculture, 292 (1), p80–86. Villamizar, N., Vera, L.M., Foulkes, N.S. & Sánchez-Vázquez, F.J. (2014) Effect of lighting conditions on zebrafish growth and development. Zebrafish, 11 (2), p173–181. Wake, D.B. & Vredenburg, V.T. (2008) Colloquium paper: are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proceedings of the National Academy of Sciences of the United States of America, 105, p11466–73. Wakefield, A., Stone, E.L., Jones, G. & Harris, S. (2015) Light-emitting diode street lights reduce last-ditch evasive manoeuvres by moths to bat echolocation calls. Royal Society Open Science, 2 (8), p150291. Zippel, K., Johnson, K., Gagliardo, R., Gibson, R., McFadden, M., Browne, R., Martinez, C. & Townsend, E. (2011) The Amphibian Ark: a global community for ex situ conservation of amphibians. Herpetological Conservation and Biology, 6 (3), p340–352.

81 General Discussion

General Discussion

A common shortfall of ex situ conservation programmes for amphibians is the husbandry care they receive, due to their complex and specialised requirements and the difficulties of provisioning these appropriately in captivity. An accurate environment is essential to maintaining fit and healthy populations of amphibians in ex situ conservation programmes which seek to reintroduce populations into the wild (Michaels et al., 2014b). These studies have provided invaluable information that can be used to supplement existing ex situ conservation knowledge and guide future amphibian ex situ conservation research.

Like many endangered amphibian species, almost nothing is known about M. cowani, including its dietary needs, environmental conditions and typical appearance (IUCN, 2015; Andreone et al., 2005). This investigation has outlined the typical size of male and female M. cowani as well as determining the typical body condition and its stability within a wild population. Such information can provide important guidelines for the accurate Mass, SVL and SMI that zoological institutions should seek to maintain in ex situ conservation programmes. With this knowledge of the wild population, the fitness of captive populations can be monitored and regularly evaluated to prevent a reduction in fitness over time. Assessment can bring about changes in husbandry methods. Optimising the quality and fitness of individuals in ex situ conservation programmes is essential to maximise the success of reintroduction (Michaels et al., 2014a). If the body condition index is successfully used to help maintain fit captive populations of M. cowani, this could be replicated in other amphibian ex situ programmes. Calculating the body condition index from in situ data highlights an area where information can easily and non invasively be obtained to help inform ex situ conservation programmes. Equally, this study also determines that body condition is typically stable within this wild population of M. cowani and that the location of individuals within the site has little effect on the fitness of individuals. Therefore assessments of body condition can also be used to compare wild populations of M. cowani in order to direct and inform in situ conservation efforts

Chapter 2 is one of the first recognised investigations into the visible lighting used in captive amphibian environments. Limited understanding of the spectral and thermal output of visible lighting and the potential subsequent effects has resulted in a lack of guidelines on the provisioning of visible lighting in amphibians enclosures. This study therefore, fills a substantial gap in amphibian husbandry information by quantifying the current lighting in captivity and highlights the significant similarities between different zoological institutions. It also demonstrates there are considerable differences in both spectral and thermal output of LED lighting. LEDs produce a broader spectrum of wavelengths, yet peak spectral output is at much shorter wavelengths of around ~450nm, whilst lighting currently provided in zoological institutions produces a spectral output with narrow spikes at various wavelengths, at around ~550nm. The thermal output also differs between fluorescent, mercury vapour and metal halide

82 General Discussion lighting and LED lighting. The reduced thermal output of LEDs results in a more homogenous captive environment, whilst the high heat output of lighting currently used produces an environment with large temperature gradients and microclimates within enclosures. This study highlights the distinct differences between the two lighting types and demonstrates the need for further investigations to determine any potential impacts of using LEDs in captive environments

This thesis provides a preliminary insight into some of the potential impacts of LEDs on amphibian welfare. Initial signs suggest that LEDs may not have many implications for the fitness of amphibians in captivity and therefore their overall welfare. It has found little demonstrable evidence that growth, survivorship and colouration have been affected by the use of LEDs. The potential dangerous effects of LEDs on amphibian welfare are not apparent. However, the studied did observe changes in the activity of amphibians under LED treatment. In one LED treatment, individuals significantly increased their activity, suggesting that there may be some positive impacts of using LEDs. A broader spectral output may promote more natural behaviour resulting in the increase of activity as observed under the LED treatment (Ellis et al., 2013). Conversely, studies in other species suggest that greater activity may be undermine anti predator behaviour, which in the long term will be highly detrimental for reintroduction success (Kats et al., 2000). Further clarification is undoubtedly needed. Although there is evidence to suggest that the use of LED lighting in enclosures may impact amphibian behaviour, the full implications of this are still unclear. Despite this, initial signs suggest there is no reason to reject the use of LED lighting in amphibian ex situ conservation efforts.

Limitations Current literature suggests that LEDs may have both negative and positive effects on amphibian welfare, and to an extent these results support this demonstrating a change in activity. As with the provisioning of UVB, the specific impacts of LEDs are likely to be dependent on a number of factors (Adkins et al., 2003; Ellis et al., 2013; Falchi et al., 2011). The physiology of the species in question, the individuals’ life stage, the mechanisms they posses to respond to narrow wavelengths, the environment they typically occupy and the typical levels of exposure and penetration of narrow wavelengths may affect the impact of LEDs (Blaustein et al., 1998; Licht & Grant, 1997). The results of this study are not definitive, and thus cannot be applied to every species. Therefore, further investigations with a wider scope, examining a range of species, should be undertaken to support and verify these findings (Ferrie et al., 2014).

Similarly, the short duration of this study may not demonstrate the true effects of LED lights over a longer period or lifetime. Whilst preliminary results suggest that there is no significant physiological impact of LEDs, there were noticeable increases in weight and SVL observed in the Solar Stinger™ LED treatment group. This suggests that differences in LEDs treatment groups may have been more apparent if the study was larger or conducted over a greater duration of time. These results therefore, cannot definitively conclude that LED lighting had no

83 General Discussion effect. Further investigation should be considered, namely to determine if the minor changes would be seen to on a greater scale. Similarly, amphibian larvae and juveniles, which were not looked at in this study, are highly vulnerable to changes in environmental parameters and are known to be susceptible to changes in UVB (Bancroft et al., 2008; Belden et al., 2000; Licht & Grant, 1997). Longer studies comprising different stages of development, i.e. from tadpoles to juveniles to adulthood, would supplement this study and provide a greater overview of the impact of LEDs. This combined with a greater sample size may provide a more complete understanding of the effect of LEDs. Recommendations from this study can be made, but a greater sample size would give greater validity to these conclusions.

Further Study These initial results suggest that LEDs seem to have a relatively small impact on the welfare of amphibians in captivity. Yet, undoubtedly greater clarity is required. Further investigations are needed to expand on the results obtained. Firstly, greater refinement in the focus of investigations could provide more informative and conclusive results. In particular, a specific focus on the role of melatonin in amphibians, rather than just an overarching view of the impact of LED lighting would be highly informative. This would allow for observation into the potential change in melatonin production that LEDs confer and the positive or negative impacts of such changes. Whilst these investigations show the initial and potential effects of LEDs, specific investigation to understand the mechanisms underpinning this is required. Equally, further study is required to determine the implications of the change in behaviour observed i.e. if a greater level of activity results in positive or negative effects for amphibians in captivity. Inferences can be suggested, however these remain speculative. Hypotheses which suggest the use of LEDs in amphibian enclosures may have positive or negative implications, lack supporting empirical evidence. However, it is essential to determine the long-term implications of these effects, particularly if the implications are harmful.

Equally, there is a lack of clarity surrounding how LEDs may have an impact. Though spectral output is deemed be the cause of the observed difference in amphibian activity there is evidence to suggest intensity may play a role. Currently, the intensity of lighting used in zoological institutions has not been investigated and lighting provided in zoological institutions typically follow ‘best practice’ husbandry techniques and there is a lack of alternatives. This study suggests that successive studies need to examine all aspects of the provisioning of lighting as it is an integral part of amphibian husbandry (Ferrie et al., 2014). In this investigation, rather than the LEDs showing similar responses, the Solar Stinger™ LEDs resulted in a significant difference in activity in individuals compared to both those under Jungle Dawn™ LEDs and fluorescent lighting treatments. Correspondingly, Solar Stinger™ LEDs had a considerably lower level of irradiance in comparison to both Jungle Dawn™ LEDs and fluorescent lighting. This indicates that the different intensities of light, rather than spectral output, may be the determining factor resulting in the differences in amphibian activity. Previous

84 General Discussion studies support this, suggesting that the intensity of the artificial light correlates with the ecological consequences (Rich & Longcore, 2005). Similarly, in bats, high pressure sodium (HPS) lamps had a similar impact on behaviour to that of LEDs with the same intensity (Stone et al., 2012). Further yet, in humans, LEDs at low intensities had a smaller impact on melatonin production, compared to high intensity HPS lamps (Aubé et al., 2013). Intensity therefore may be the significant factor influencing the impact of lighting rather than the spectral output. Therefore a comprehensive assessment of a range of lighting types, at different intensities and with varying spectral output is required to fully determine the impact of visible artificial lighting in amphibian enclosures. This highlights a significant avenue for further investigation into the provisioning of ex situ captive environments for amphibians.

Conclusion Collecting information regarding wild populations of amphibians is essential to inform ex situ conservation efforts and husbandry practices. Determining the size of unknown species and the body condition of a population in the wild can be used to guide ex situ conservation programmes and ensure the successful maintenance of fit, captive populations. This thesis concludes that maintenance of healthy captive populations is unlikely to be significantly impacted by the introduction of LEDs. The following studies suggest that LEDs have a limited impact on amphibian welfare. Therefore, currently there appears to be no reason to reject their use in captivity. However, these studies are not comprehensive and further research on visible lighting in captivity in general is necessary. A significant gap in the knowledge of amphibian husbandry has been highlighted. The use of artificial visible lighting as a whole, which has the potential to comprise ex situ conservation efforts and reintroduction programmes, requires extensive further research.

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