Investigation of the Factors Affecting the Population

Dynamics of Captive Partula Snails.

Ana Rita Peres Cardoso Gouveia

Department of Life Sciences, Division of Biology, Silwood Park Campus,

Ascot, Berkshire, SL5 7PY, U.K.

A thesis submitted for the degree of Doctor of Philosophy

Imperial College London

2011

Abstract

Partula snails, also known as Polynesian tree snails, were first recorded scientifically from

Captain Cook‟s voyage 236 years ago. They are small gastropod molluscs which belong to the order Stylommatophora. Their distribution is focused in the Pacific region where the majority of the species are island endemics. Half the species are found in the Society Islands of French Polynesia where most research has been carried out. Wild populations were almost annihilated following the introduction of a predatory snail, Euglandina rosea, to control the introduced giant African land snail (Lissachatina fulica) which had become an agricultural pest affecting the local economy. In 1987 the Partula Propagation Group (PPG) initiated a worldwide captive breeding programme for Partula snails in an attempt to save them from extinction. In captivity, though, episodes of high mortality have led to the extinction of some species. The causes of these declines are as yet uncertain. This study aimed to investigate the factors that have affected the survival of 15 species (including two subspecies) of Partula snails in captivity. In summary, the results indicate that both environmental factors

(temperature, relative humidity and light) and diet (calcium concentration and dog and cat vitamins in the diet) significantly affect the population dynamics of Partula snails. Both excess light and unstable temperatures and humidity had detrimental impacts on the survival and reproductive success of the species. Additionaly, the low concentration of calcium and the addition of dog and cat vitamins in the diet are also significantly detrimental on the survival and reproductive success of Partula gibba. These studies are highly significant because the biology and requirements of Partula snails‟ are poorly understood, and this can jeopardise a successful captive breeding programme and future reintroductions into their natural environment. It is imperative that the factors that have affected the survival rates of snails in captivity for the last 25 years are fully understood. Partula snails face an uncertain

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future, with the mass mortality episodes observed during the years several species were lost.

It is hoped that these findings stimulate further research in this area and can be applied to other species in similar circumstances.

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Declaration

The work presented in this thesis is entirely my own, and any other material is accordingly referenced.

Ana R. P. C. Gouveia

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Acknowledgements

First and foremost I want to express my gratitude to all the people who helped and supported me through these years. I would like to thank everybody who was important to the successful realization of this thesis, as well as expressing my apology for those I do not mention.

In the first place I would like to record my gratitude to my supervisors Simon Leather and

Donald Quicke for their enthusiastic supervision, understanding, advice and guidance during my PhD. I also want to express my gratitude to Paul Pearce-Kelly who has supported and cared for me since the moment we met. Above anything he has been the best friend I could ask for.

I wish to gratefully acknowledge Professor Elizabeth Cunning, Professor Bryan Clarke,

Professor Tim Coulson, Professor Mick Crawley, Dr. Tilly Collins, Dr. Daniel Reuman, Dr.

Nils Bunnefeld, Dr. Joaquin Hortal, Dr. Arpat Ozgul, Dr. Navinder Singh, Dr. David Hunt,

Dr. Barbara Ritchie, Dr. Jake Bundy, Dr. Mike Wilson and anyone else who contributed directly or indirectly to this thesis with advices, ideas, comments and suggestions. A special thanks to Romain Pizzi, Professor Mark Fox and Dr. Sergei Spiridonov. And also thanks to my panel (Dr. Sally Power and Dr. Rob Ewes) who gave me valuable suggestions and constructive criticism.

I am indebted to Dr. Trevor Coote and Shaheed Macgregor for their wise, comforting words as well as valuable technical advice and the help they provided, with thanks also to the “Bugs

Team” at London Zoo.

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I want to express my thanks to the Silwoodian staff (technicians, cleaners, electricians the security team, the ICT team etc). Without them this could not be accomplished, in particular

Diana Anderson, Christine Short, Paul Nicholas, Paul and Jim, Taru, Catherine, and John all of them, in different ways helped me during these years either with comforting and friendly words and/or with technical advice providing a stimulating and friendly environment to work in.

My Silwoodian and non-Silwoodian friends deserve a place in this pages since they helped me in the up and down emotional stages of the PhD, they prevented me from losing my sanity and also gave me valuable advices, I have no words to express how much they actually helped me, I probably never told them but Ana and Joaquin, Arpat, Navinder, Manju,

Francesca and Eva, Ana, Oriana, Ambika, Marcia, Diego, Jenifer, Lena, Catarina, Paula,

Tania, Carla, and particularly Telmo, Claudia Nunes and Parreira, Angela and Nelson,

Bertinho and Vanessa, thank you so much for being always there when I needed you.

I wish express my gratitude and deep appreciation to Martin MacDowell he dried my tears several times, he gave me encouragement, emotional support and all the love one needs to overcome the rough times, and showed me that there was a light at the end of the tunnel.

Thank you for your love, support and putting up with my grumpiness and also thank you for providing me with a fun relaxing and fantastic environment to live in.

I have no words to express my appreciation to my family whose dedication, love and belief, has given me the confidence and strength to get this far. I was extraordinarily fortunate in having them always by my side supporting my decisions and advising me. My mum and dad always motivated and supported me to pursue an intellectual and academic future. I want to thank them for raising me with buckets of love and laughter for which my two brothers also

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contributed a big deal. I wish to thank my father and mother for all the sacrifices they went through to provide me with the chances and fantastic life I had so far. A special thanks to

Nuno and Gonçalo for being the best friends and siblings I could ask for.

Lastly, and most importantly, to them, my parents and brothers I dedicate this thesis.

Gould, (1993) “this essay is a story of genocide in paradise, a preventable wholesale slaughter, just completed in one human generation. You do not know the tale only because it pitted snail against snail, rather than man against man”

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Contents

Pages

Abstract 2

Declaration 3

Acknowledgements 4-6

Contents 7-9

List of Tables 10

List of Figures 11-13

Chapter 1. General Introduction 14-37

1.1. Partula snails 14, 16

1.2. The species and their natural habitat 17-27

1.2.1. Tahitian species 19-21

1.2.1.1. Partula affinis 19

1.2.1.2. Partula clara 20

1.2.1.3. Partula hyalina 20

1.2.1.4. Partula nodosa 21

1.2.2. Species of the Mariana Islands, Guam and Saipan 21, 22

1.2.2.1. Partula radiolata 22

1.2.2.2. Partula gibba 22

1.2.3. Species of Moorea 22-24

1.2.3.1. Partula tohiveana 23

1.2.3.2. Partula mooreana 23

1.2.3.3. Partula suturalis strigosa and P. s. vexillum 23, 24

1.2.3.4. Partula taeniata nucleola and P. t. simulans 24

1.2.4. Species of Raiatea and Huahine 25, 26

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1.2.4.1. Partula faba 25

1.2.4.2. Partula tristis 25, 26

1.2.4.3. Partula hebe bella 26

1.2.4.4. Partula varia 26

1.2.5. Futuna and Alofi species 26, 27

1.2.5.1 Partula subgonochila 27

1.3. Wild populations 27, 28

1.4. Captive breeding programmes 29, 30

1.5. Population collapses and diseases 30, 31

1.6. Environmental 31, 32

1.6.1. Temperature and Humidity 31

1.6.2. Light 32

1.7. Diet 32, 33

1.8. And the story repeats 34

1.9. Thesis outline 35-37

Chapter 2. Effects of different calcium concentrations added to the diet of

Partula gibba (Férussac) on morphometric growth parameters, weight and reproductive success. 38-51

2.1. Abstract 38

2.2. Introduction 39, 40

2.3. Methods 40-42

2.4. Results 43-48

2.5. Discussion 49, 51

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Chapter 3. Investigation of the effect of vitamin supplement added to the diet of Partula snails. 52-60

3.1. Abstract 52

3.2. Introduction 53-56

3.3. Methods 56, 57

3.4. Results 57, 58

3.5. Discussion 59, 60

Chapter 4. Investigating the effects of environmental stress on the population dynamics of endangered Partula snails. 61-77

4.1. Abstract 61

4.2. Introduction 62, 63

4.3. Methods 64-66

4.4. Results 67-74

4.5. Discussion 75-77

Chapter 5. Investigation into the role played by light intensity on the behaviour, reproduction and mortality of Partula snails. 78-86

5.1. Abstract 78

5.2. Introduction 79-81

5.3. Methods 81-83

5.4. Results 83, 84

5.5. Discussion 85, 86

Chapter 6. General Discussion 86-90

References 91-111

Appendix 112-120

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List of Tables

Tables Pages

Table 2.1. Linear model summary, for Partula gibba morphometric

measurements and reproductive success against the addition of calcium 47

to the diet.

Table 4.1. E ffects of temperature and humidity on adult mortality. Significance

levels (*<0.05, **<0.01 and ***<0.001), the symbols - and + indicate 69, 70 the trend of significant effects.

Table 4.2. S ignificant interactions between temperature and humidity and adult

mortality. 71

Table 4.3. E ffects of temperature and humidity on juvenile mortality. No

significant effects were found for either mean temperature or

maximum humidity 72

Table 4.4. S ignificant interactions between temperature and humidity, and

juvenile mortality. 73

Table 4.5. E ffects of temperature and humidity on fecundity. No significant

effects were found for maximum temperature, temperature range,

minimum humidity and humidity range. 74

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List of Figures

Figures Pages

Figure 1.1. The Pacific Islands from where the Partula species under study are

endemic. http://www.pacificpeoplespartnership.org/ 17

Figure 2.1. Snails‟ morphometric measurements; shell thickness (G),

height/length (A-B), width (C-D), lip height/length (B-F) and lip 42

width (E-D) (Crampton, 1916).

Figure 2.2. Effects on the weight of the snails of different calcium carbonate

concentrations added to the diet, including powdered cuttlebone. 43

Figure 2.3. Effects on shell lip thickness of different calcium carbonate

concentrations added to the diet including powdered cuttlebone. 44

Figure 2.4. Effects on shell height/length of different calcium carbonate

concentrations added to the diet, including powdered cuttlebone. 44

Figure 2.5. Effects on shell width of different calcium carbonate concentrations

added to the diet, including powdered cuttlebone. 45

Figure 2.6. Effects on shell lip height/length of different calcium carbonate

concentrations added to the diet, including powdered cuttlebone. 45

12

Figure 2.7. Effects on shell lip width of different calcium carbonate

concentrations added to the die, including powdered cuttlebone. 46

Figure 2.8. Effects of different calcium carbonate concentrations added to the

diet, including powdered cuttlebone on the reproductive success of

Partula gibba, expressed as number of newborns per month over a 48

one year period.

Figure 3.1. Effects of the treatments on the mean mortality of captive Partula

gibba. 58

Figure 3.2. Effects of two treatments on the mean fecundity of captive Partula

gibba. 58

Figure 4.1. Annual mean temperature and humidity (2008) from Faa‟a (Tahiti),

Opunohu (Moorea) and the controlled temperature room (at Imperial

College). 67

Figure 5.1. Selection box. 82

Figure 5.2. Effects of two light regimes on the fecundity of Partula gibba. 84

Figure 5.3. Effects of two light regimes (dark and light) on the mortality of

Partula gibba. 84

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Figure A.1. Phylogenetic relationships of the Caenorhabditis species nematode

from Tahitian snails and closely related Rhabditidae. 115

Figure A.2. ( Fig.A2.1 aliphatic region; Fig.A2.2 aromatic region). Top: P. varia.

Bottom: P. clara. 117

Figure A.3. Sna ils‟ morphometric measurements 119

14 Chapter 1. General Introduction

Chapter 1. General Introduction

1.1. Partula snails

Land snail species of the genus Partula (Family Partulidae), sometimes referred to as

Polynesian tree snails, are small molluscan gastropods that belong to the subclass Pulmonata of the order Stylommatophora. The Partulidae can be classified into three genera: Eua,

Samoana and Partula (Cowie, 1992). Partulids were first recorded in Captain Cook‟s voyage in 1774 (Pearce-Kelly et al., 2006), then collected by the naturalists of the Freycinet expedition in 1819 and the first (Partula otaheitana) was officially described by Férussac in

1821 (Crampton, 1925). They have been studied for more than a century due to their morphological (and genetic) diversity which provides scientists with excellent material for studying adaptation and speciation (Crampton, 1916, 1932; Murray & Clarke, 1966; Clarke

& Murray, 1969; Murray & Clarke, 1980; Murray et al., 1982; Johnson et al., 1987; Murray et al., 1988; Davison et al., 2009). Wells (1995) stated that “their unique combination of ovoviviparity, low mobility, short generation time and extensive polymorphism, combined with the comparative ease of its culture in the laboratory, make them ideal material for the study of ecological and evolutionary genetics.”

Of the three genera - Partula, Samoana and Eua - that comprise the Partulidae family, Partula constitutes the largest with approximately 120 species. In comparison Eua is the least abundant genera with only four species described and 23 species of Samoana. They have been largely separated on the basis of their genital anatomy. Partula is remarkably different due to the absence of an appendix and possessing a very simple penis when compared with other genera and families. In addition, the size of the kidney in Partula snails equals the size of the pericardium which in general is not the case for the others. The

15 Chapter 1. General Introduction thickness of the shell, the pigmentation of the mantle, the type of mucus and the length of the tentacles are other characteristics used to differentiate the genera (Pilsbry, 1909-1910;

Goodacre &Wade, 2001).

Partula snails are thought to be nocturnal/crepuscular and arboreal. They live in a large diversity of tree and bush species often found in high altitude forests (Murray et al.,

1982).

Like most molluscs, they feed by means of a rasping organ known as the radula. This is a narrow, strap-like rasping tongue provided with several transverse rows of flattened tooth-like plates used to scrape up food (Horst, 1965).

The diet of Partula snails in the wild is largely unknown, though it is generally accepted that they are detritivores and have been observed feeding on dead and decaying

Hibiscus tiliaceous leaf stalks which fall from the trees and become trapped in plants containing Partula. Some species have been recorded as feeding on specific plants while others are generalists (Murray et al., 1982). A survey recorded approximately 76% of the populations of P. hyalina (Broderip) and 40% of the wild Partula clara (Pease) populations on the native plant species red ginger (Etlingera cevuga) (Coote, 2007), though both are known to be plant generalists. Hopper and Smith (1992) reported that Partula gibba

(Férussac) was most abundant on the endemic understory plant Piper guamensis, but was also found on coconut palms and in the ferns Microsorum punctatum. Three other species, P. tohiveana (Crampton), P. mooreana (Hartman) and P. mirabilis (Crampton) were previously recorded on the climbing pandanus plant Freycinetia with percentages of snails found ranging from 84% of the P. mirabilis and more than 90% of the P. tohiveana and P. mooreana (Murray et al., 1993).

Numerous species of molluscs are dioecious. Most snails are however, hermaphrodites (Horst, 1965). Partula snails are hermaphrodites and ovoviviparous, although

16 Chapter 1. General Introduction self fertilization has been known to occur (Murray & Clarke, 1966; Clarke & Murray, 1969).

They can produce a completely shelled offspring every six to eight weeks. Before the introduction of invasive species, Partulids lived in a predator free bliss, where, their reproductive strategy (giving birth to a live fully developed offspring) was successful.

Although fecundity rates are reduced this strategy insures a successful survival of the offspring since predation, competition and dessication are reduced in a fully developed individual. Also in their natural environment the rain is unpredictable, ovoviparity insures that the eggs are not washed away and the fully developed offspring has a higher change of survival. “Ovoviviparity is a form of parental care which allows terrestrial gastropods to persist in habitats otherwise unsuitable for oviparous species” (Baur, 1994). Althoug in captivity this stategy is disadvantageous since genetic variation is reduced and fecundity rates are very low.

The class Gastropoda has a fossil record that begins in the early Cambrian period and has undergone the most extensive adaptive radiation of all the major molluscan groups

(Rupert & Barnes, 1994).

17 Chapter 1. General Introduction

1.2. The species and their natural habitat.

Figure 1.1. The Pacific Islands from where the Partula species under study are endemic. http://www.pacificpeoplespartnership.org/

(For the purpose of this study only the genus Partula will be discussed).

Partula are generally distributed throughout tropical Polynesia in the Society, Marquesas,

Austral and Cook Islands, Samoa and Tonga, where around 60 species have been confirmed

(Kondo, 1958). Roughly 30 species were described from Melanesia (Fiji, Santa Cruz,

Solomon, New Ireland, New Britain and Admiralty, New Guinea and Louisiade) and ten species from Micronesia (Pelau, Caroline and Marianas) (Garrett, 1884; Crampton, 1916;

Kondo, 1958).

The underlying principle of this study arises from one of the most important statements of Crampton (1916) “each group and each inhabited island bears characteristic species not found elsewhere, with only rare and peculiar exceptions.”

18 Chapter 1. General Introduction

In general Partula snails are found in high volcanic or coral islands. These islands usually have high temperature and humidity with two distinct seasonal periods.

Following Crampton (1916) Partula snails can be classified into three different types of habitat: ground-dwelling, semi-arboreal and arboreal. The ground dwelling species are found on the ground crawling under stones, dead trees and leaves (e.g. Partula producta

(now extinct)). These snails have a positively geotactic and negatively phototactic behaviour.

The second group, semi-arboreal, comprise the greater number of species. These are arboreal during daytime remaining sealed upon the undersides of leaves up to three to four metres from the ground, although they resume activity and crawl down to ground level in order to feed on the topsoil. The snails belonging to this group display a positive geotactic behaviour at night and negative during the day, as in the first group they are negatively phototactic. The third group, strictly arboreal, spend their life high up on the leaves of trees, where they possibly survive by feeding on algae and fungi present on twigs, branches and leaves (e.g.

Partula (now Samoana) attenuata). These strictly arboreal snails invariably have a negative geotactic behaviour and as in the other groups they are negatively phototactic (Crampton,

1916).

Based on Crampton‟s volumes on the genus Partula (1916, 1925, 1932) follows a brief description of the Partula species and/or subspecies under study and the areas they inhabited. The descriptions are not complete for all species due to the limited information in the literature although, efforts were made to keep the information as accurate as possible, but sometimes generalised descriptions were unavoidable.

The volumes are divided into species inhabiting Tahiti (1916), species of the Mariana,

Guam and Saipan Islands (1925) and the species inhabiting Moorea (1932). For easier display and flow of information the following descriptions will follow the aforementioned structure.

19 Chapter 1. General Introduction

1.2.1. Tahitian Species

Tahiti (17°39‟S, 149°21‟W) is the largest island of French Polynesia. The southern, south- eastern, eastern windward parts of the island have a higher occurrence of rainfall and hence are more humid. Towards the interior of the island where the deepest valleys penetrate humidity is also at its highest, with the coastal regions having noticeable lower humidity levels, and hence are drier. Few Partula were found along the coast but were equally abundant in both wet east coast valleys and drier west coast valleys. The high mountain peaks, characteristic of these volcanic islands, are generally sheltered from sunlight mainly due to the persistent and high cloud density. The flora of these islands are varied and greatly dependant on the environment (Crampton, 1916).

The four Tahitian species used in this study are Partula affinis (Pease), P. clara (Pease), P. hyalina (Broderip) and P. nodosa (Pfeiffer).

1.2.1.1. Partula affinis are predominately dextrally coiled in nature although sinistral individuals are commonly observed. They were once widely distributed along Tahiti, being one of the most widespread species in this island, although in general they were confined to the northern and eastern sectors of Tahiti. Partula affinis can be found in different colour morphs as well as banded and plain individuals. Crampton classifies this species as having an average to high fecundity rate depending on the colour morph and valleys they occupied

(Crampton, 1916). It is believed that this species is now extinct in the wild (Coote, personal communication, 2011).

20 Chapter 1. General Introduction

1.2.1.2. Partula clara are also dextrally coiled; occasional individuals have a parietal tooth.

They were more abundant in southern and western Tahiti, although the north-western regions were devoid of P. clara possibly due to their drier nature and hence the snails were found on foliage in the uppermost portions of the valleys where the humidity levels were found to be higher. Crampton observed that P. clara were more abundant where P. hyalina were not present. It is very interesting to note that Garrett (1884) found this leaf dwelling species to be quite rare and near extinction, although years latter Crampton‟s (1916) observations were that this species was very abundant and frequent in different parts of the island (Garrett, 1884;

Pilsbry, 1909-1910; Crampton, 1916). However, recent genetic studies suggest that P. clara and P. hyalina (see below) may be colour morphs that could be merged into one species (Lee et al., 2007a).

1.2.1.3. Partula hyalina are recorded from the Society, Cook and Austral groups, the only multi-archipelago distribution of a Partula species, but it is now believed that they are a

Tahitian endemic carried by man to other islands as part of inter-island commerce (Lee et al.,

2007b). They are always dextral and have no parietal tooth. High humidity and absence of light has been observed to be a stimulus for P. hyalina to resume activity on the limestone islands of Cook. Partula hyalina seemed to prefer distinctive areas and conditions that would not be favourable to other species. They avoided humid and shaded areas and favoured open areas exposed to sunlight where large forest trees are scarce, consistent with the larger and drier northern regions. Crampton observed that the snails‟ shells in the drier areas differed from those found in the humid areas, and that P. clara and P. hyalina were the reverse in their general relation to moisture (Crampton, 1916).

21 Chapter 1. General Introduction

1.2.1.4. Partula nodosa are primarily dextral with occurrence of sinistral individuals and they have a parietal tooth. This species was once found only in seven valleys on the western coast of Tahiti in great numbers but is now extinct in the wild. Individuals have been found approximately two kilometres from the coast and common on vegetation alongside rocky and rough borders of rivers and streams. They vary greatly in colour and individuals can be both banded and un-banded. They also have a very low reproductive rate (Garrett, 1884;

Crampton, 1916).

1.2.2. Species of Mariana Islands, Guam and Saipan

The Mariana Islands are the dry peaks of a submarine range with the Northern islands of the group being of volcanic nature and devoid of Partula. The remaining regions are characterized by favourable conditions for Partula with a hot and wet climate and thick forests that provide the perfect environment of shade, moisture and climatic conditions.

Saipan (15°12‟N, 145°44‟E) in general is of volcanic nature but an uplifted reef limestone also prevails with the soil being alluvial or rock limestone. The highest regions are volcanic in origin while the remaining ones are composed of coral. The coast is dry and surrounded by large calcareous rocks (Crampton, 1925).

Guam (13°29‟N, 144°46‟E) is marked by two distinct geological regions and consequently ecological conditions. The northern region is marked by heavy and constant rainfall although because it is a rocky terrain, the formation of rivers and streams is challenging. On the other hand, the southern region is of volcanic nature and various streams and water courses can be found (Crampton, 1925).

Following Crampton, as a general rule, the Mariana species differ constitutionally from the majority, as manifested by their existence in exposed and hence drier conditions.

22 Chapter 1. General Introduction

The two Mariana species that were used in this study are Partula radiolata (Pfeiffer) and P. gibba (Férussac).

1.2.2.1. Partula radiolata coil dextrally, they have no parietal tooth and have six different colour classes and patterns of the shell. This species was once abundant and widespread in

Guam. From Crampton‟s descriptions one can assume that this species did not have a specific preference in terms of habitat, since they were found in both the lower and high levels of the valleys as well as in the two major types of soil found in Guam, although they were more abundant in the southwest and southeast regions (Crampton, 1925).

1.2.2.2. Partula gibba have a parietal tooth and coil dextrally. They were once very abundant and diverse, being the only Partula resident of Saipan. Partula gibba have at least seven known colour classes. Following Crampton, this species generally occurred whenever suitable vegetation existed. They dominated the western half of Guam and south central regions. There was a marked decrease in the snails‟ numbers as one moved towards the southwest where the higher altitudes could be experienced. P. gibba prevailed in lower altitudes and were found virtually at the water‟s edge only a few meters from the shore

(Crampton, 1925).

1.2.3. Species/ subspecies of Moorea

Moorea (17°31‟S, 149°48‟W) is the sister island of Tahiti (approximately 9.5 nautical miles apart). This island is of volcanic origin with a central crater and mountainous terrain created by erosion. These mountains form a series of valleys which provide the ideal condition for partulids, with the highest point being Mount Tohivea at 1207 metres. The island is encircled

23 Chapter 1. General Introduction by a barrier reef. Crampton (1932) also noted that the island topography determines the distribution of the vegetation and hence the existence or absence of Partula.

The described species/subspecies of Moorea are Partula tohiveana (Crampton), P. mooreana (Hartman), P. suturalis (two subspecies P. s. strigosa (Pfeiffer) and P. s. vexillum

(Pease)), Partula taeniata (two subspecies P. t. nucleola (Moerch) and P. t. simulans

(Moerch)).

1.2.3.1. Partula tohiveana coil sinistrally and have a parietal tooth. Their distribution is confined to the Mount Tohivea which indicates that this species had a very secluded territory and was usually found at higher altitudes. When compared with the other species, P. tohiveana is one of the largest species under study, with long and large shells. They have a low reproductive rate and there are three colour morphs described by Crampton (1932).

1.2.3.2. Partula mooreana are sinistral in nature with infrequent dextral snails being observed, all with a parietal tooth and an average reproductive rate. There are at least two colour classes described by Crampton (1932) with the colonies varying from valley to valley.

They were once found in the outer circles of the valleys on bushes and small trees particularly on the southeast coast of Moorea (Garrett, 1884).

1.2.3.3. Partula suturalis strigosa and P. suturalis vexillum have a parietal tooth, they are both dextrally coiled in nature but in P. s. vexillum sinistral types exist. They are both arboreal and can be distinguished by their size, shape and colouration which vary from valley to valley. The areas they once inhabited did not overlap and were completely severed from one another; P. suturalis strigosa was confined to a small area in the valleys‟ outer circle in

24 Chapter 1. General Introduction south-eastern Moorea while P. suturalis vexillum was more abundant and widespread throughout the island (Crampton, 1932).

1.2.3.4. Partula taeniata nucleola and P. taeniata simulans are both dextral in nature although very rare forms of sinistral P. t. simulans were found by Crampton (1932). They both have a parietal tooth but the extent of development varies upon the species. Partula taeniata simulans have thicker, more compact and stouter shells with a rounder aperture and a well developed parietal tooth. P. t. nucleola on the other hand are thinner and longer with a much underdeveloped tooth. They both have seven known colour classes described. The area of the species prevalence within the island was very distinct with a very small degree of overlapping. Partula taeniata nucleola were once confined to the north-western region of

Moorea which is characteristically very dry. Large numbers of this species were once found in valleys very close to the shore and drier regions. Partula taeniata simulans once occupied nearly all of the central area of Moorea from west to east with the exception of the innermost central region. They could also be found on bushes close to the shore. Crampton (1932) observed that these species varied greatly in size depending on the region they were found, with the animals being stouter in the outer western valleys than those of the inner western and eastern regions.

In general P. taeniata were found anywhere on Moorea outnumbering all other species. Their geographical distribution was highly dictated by the ecological conditions, which when optimal, they would thrive. They were found in very high altitudes as well as near the coasts tolerating in this way a diversity of environments and habitats provided that the vegetation was thick to provide their optimal conditions (Crampton, 1932).

25 Chapter 1. General Introduction

1.2.4. Species inhabiting Raiatea and Huahine

Raiatea (16°50‟S, 151°26‟W) and Taha‟a two islands separated by a lagoon but encircled by the same reef and hence will be treated as one. Raiatea is the second largest island of the

Society group. Huahine (16°47‟S, 150°56‟W) is a neighbouring island characterized by a much drier climate than the other islands inhabited by Partula, rendering it a poor habitat and sustaining a smaller number of species (Goodacre, 2002). These islands are of volcanic origin and are located north of Tahiti and Moorea in the Leeward group. The number of species on

Raitea accounted for an approximate one quarter of all the species in the genus Partula

(Crampton, 1956; Lee et al., 2008), though the majority are now extinct and none survive in the wild.

The Partula representatives of these islands used in this study are Partula faba

(Gmelin), Partula tristis (Crampton and Cooke) and Partula hebe bella (Pfeiffer) from

Raiatea and Partula varia (Broderip) from Huahine.

1.2.4.1. Partula faba are dextral and have no parietal tooth. This species was first collected in 1769 by Captain Cook, though he wrongly attributed it to Tahiti. It was once found throughout the island of Raiatea on trunks and foliage of trees and bushes ranging from the lowlands near the sea shore to several meters above the sea level. They varied considerably in shape and colour with thick shells and lip being the largest species under study (Reeve, 1851;

Garrett, 1884; Pilsbry, 1909-1910).

1.2.4.2. Partula tristis thin shells coil dextrally and have no parietal tooth. They have a weak development of the lip. Partula tristis were once found in the western side of Raiatea on trees and shrubs towards the central portions of one valley. They have four colour morphs

26 Chapter 1. General Introduction described by Crampton and Cooke (Pilsbry 1909-1910; Crampton & Cooke, 1953 in

Crampton, 1956).

1.2.4.3. Partula hebe bella have dextrally coiled shells and have a parietal tooth. They are the smallest sized species under study. Their solid and thick shells have a globular shape and are characterized by being white with a pink apex. These arboreal snails were once abundant on the eastern southward regions of Raiatea on the foliage of bushes (Hartman, 1881; Smith,

1902; Pilsbry, 1909-1910).

1.2.4.4. Partula varia have dextrally coiled shells with no parietal tooth. These snails were once found throughout the island, dwelling on the foliage of bushes and trees but were particularly abundant on the west coast of Huahine. They have a high degree of variation in respect to colouration, size and shape. Hardly ever two individuals were exactly similar in colour (Broderip & Cuming, 1832; Garrett, 1884; Smith, 1902; Pilsbry, 1909-1910).

1.2.5. Futuna and Alofi species

Futuna (14°18‟S, 178°7‟W) and Alofi (14°20‟S, 178°2‟W) are part of the Horne archipelago which is part of the Polynesian triangle. They are islands of volcanic origin and considered particularly high with altitudes of 524m and 417m respectively. The tropical climate is characteristically humid and hot with two distinct seasons. The topographic nature of the islands provides a diversity of habitats which provide an ideal territory for partulids found mainly on the coastal forest in a limestone rich soil.

27 Chapter 1. General Introduction

1.2.5.1. Partula subgonochila have dextrally coiled shells and have no parietal tooth. They are strictly arboreal and were recently found on ferns, shrubs, pandanus, coconut and small trees. They are very scarce on high altitudes but can be found in high numbers near the coast on eastern Futuna and northern Alofi. Partula subgonochila seem to favour the coastal forests where the soil is predominately made of cliffs of calcareous limestone which in parts is pure karstic rock (Burrows, 1938; John & Smith, 1971; Clarke & Riching, pers. comm., 2008).

1.3. Wild population

Partula snails are endemic to the islands of the Pacific Rim, particularly the Society Islands; they can be found from the Marianas in the west to the Marquesas Islands in the east (Murray

& Clarke, 1966; Cowie, 1992).

Due to the introduction of the invasive carnivorous mollusc species, Euglandina rosea, wild partulid populations started to decline in the mid-1970s, leading to the extinction of several species. Euglandina rosea was introduced on Tahiti in 1974, on Moorea in 1977, and on other Society Islands in the 1980s and 1990s (Coote, 2007; Lee et al., 2009). Against advice Euglandina rosea (Coote et al., 1999; Shine et al., 2003) had been introduced as a biological control method against the giant African snail Lissachatina fulica (Bowdich) which was imported in 1967 as a food source for the local population, and readily became an agricultural pest, invading and destroying crop plantations and affecting the local economy

(Coote et al., 1999). Coote and Loeve (2003) reported the status of Partula snails in the wild:

“on Huahine, the disappearance of P. varia and P. rosea (Broderip) had an economic and social effect on the local community, many of the women of the villages lost their livelihoods, and the artisans‟ association also folded”.

28 Chapter 1. General Introduction

The introduction of these invasive carnivores had been a misguided attempt at biological control against scientific advice (Murray et al., 1988). This action was and still is criticized by scientists due to the lack of evidence that Euglandina rosea has played any significant role in the decline of Lissachatina populations. Additionally there has never been any scientific evidence that Euglandina rosea would be a suitable control for Lissachatina fulica (Christensen, 1984; Civeyrel & Simberloff, 1996; Cowie, 1998). These species seem to decline naturally as part of population cycles without the presence of any kind of control methods (Mead, 1961; Cowie, 1992) as noted on Huahine where Lissachatina populations had declined before the introduction of Euglandina rosea (Tudge & Pearce-Kelly, 1992).

Some Partula species on islands beyond French Polynesia have become in danger of extinction due to habitat destruction and fragmentation and introductions of other invasive species such as the flatworm Platydemus manokwari (De Beauchamp). This triclad species was previously observed in the wild and in captivity, predating on Partula snails (Hopper &

Smith, 1992). The geographical regions where these two species coexist demonstrate a remarkable decline in the Partula snails‟ population (Hopper & Smith, 1992).

As a result of these circumstances, in 1986 the International Partulid Conservation

Programme was implemented (Pearce-Kelly et al., 2006). For 24 years the programme experienced the two sides of the coin, demonstrating in this way an assortment of remarkable accomplishments as well as frustration. Throughout the years the program successfully managed to breed some species of Partula but it also experienced several crashes in the different populations, the most significant case was the one involving Partula turgida (Pease) when at the beginning of 1996 the last individual became extinct worldwide (Daszak &

Cunningham, 1999).

29 Chapter 1. General Introduction

1.4. Captive breeding programmes

Living specimens had been collected for genetic experiments on expeditions to French

Polynesia and cultured at Nottingham University since 1962. When wild Partula populations started to collapse it soon dawned that such collections were of paramount importance for conservation as an entire family was at risk of extinction. After realizing the devastating effects that both the Partula and the local communities were suffering, the Captive Breeding

Specialist Group (International Union for the Conservation of Nature) organized a meeting at the Zoological Society of London in 1987 to consider the circumstances and possible routes of corrective acts. The outcome was the origin of the Partula Propagation Group – PPG

(Tudge & Pearce-Kelly, 1992). The ultimate purpose was to set up as many populations of

Partula in captivity as possible and if practical, to re-establish them in the wild.

In 1994 the captive breeding programme of partulids made their first successful release of a population of Partula taeniata onto the Polynesian flora section at the Royal

Botanical Garden at Kew in London. The snails were observed and monitored for 15 months with the outcome being deemed a success, since the snails maintained their ability to survive and reproduce (Pearce-Kelly et al., 1995; Mace et al., 1998). This test-study emphasised that the idea for the creation of a „multi-purpose reserve‟ on Moorea (Clarke & Wells, 1986) could be successful. Despite some problems emerged from the experiment, the snails demonstrated their capacity to survive in their natural environment, indicating once more the importance of the conservation breeding programme (Murray et al., 1988; Mace et al., 1998).

Later that year two major events occurred. Firstly, the Partula Propagation Group held a three-day workshop at the Zoological Society in London, from which was produced Partula

„94: Action Plan for the Conservation of the Family Partulidae (Pearce-Kelly et al. 1995).

The second event was the construction of a predator-proof snail reserve in prime forest in

30 Chapter 1. General Introduction

Afareaito Valley on Moorea, and the trial release of three species previously found in that valley.

1.5. Population collapses and diseases

Numerous incidents of significantly increased mortality have been experienced by various captive breeding programmes (Cooper & Knowler, 1992).

Cunningham and Daszak (1998) reported what they considered an emerging disease of Partula snails caused by a new species of microsporidian parasite (Steinhausia sp.), which they associated with an increased mortality rate of the captive population of Partula turgida.

This study reported the first proven case of extinction (P. turgida) due to a disease. Although the results from the histology performed by Cunningham confirmed the presence of a pathogenic microsporidian parasite in at least three species of Partula snails held at the

London Zoo collection, only one species became extinct worldwide. Pearce-Kelly (pers. comm., 1998) stated that the histological results were only revealed two years after the suspected infection and hence the prevention of further contamination of other species became an unattainable challenge, “To have a two years gap between the prepared specimens and the subsequent diagnostic examination is surely unacceptable by anyone‟s standards.”

(Pearce-Kelly, pers. comm., 1998). Pizzi (2004) also notes that “unfortunately as no preliminary screening of the initial healthy population was performed, this is difficult to prove, and is of course not possible to fulfil Koch‟s postulates” and so “it remains uncertain whether this could have been an opportunistic infection, exacerbated by environmental conditions, or a primary pathogen”.

Several hypotheses to explain the mortality of Partula snails have been postulated although no direct scientific evidence about the nature of these causes has been published.

31 Chapter 1. General Introduction

Jersey Zoo reported in the 1990 International Zoo Yearbook, that the population fluctuations were due to density dependent diseases. Mites have also been reported as causing increased mortality in Partula populations at both Detroit Zoo and Virginia University (Tonge &

Bloxam, 1990). Additionally, mites were found in London Zoos‟ Partula culture. These were identified as being Tyrophagus putrescentiae (Schrank), which are fungivorous species commonly associated with stored food products (Baker, pers. comm., 1996), thus supporting the observation that Partula diet can be a host to possible pathogenic agents.

1.6. Environmental factors

1.6.1. Temperature and Humidity

Environmental change has been the rule, rather than the exception, in the evolution of living organisms (Carey, 2005).

Munoz et al. (2005) noted that temperature has both direct and indirect effects upon physiological and ecological processes in ectothermic animals. Partula snails are poikilothermic hence they are highly susceptible to environmental stresses. Simultaneously, temperature and humidity can be considered as one of the most important factors affecting the welfare of Partula snails. The role of humidity is described by numerous authors. For example, Dias et al. (2007) describe the humidity dependence of terrestrial pulmonate molluscs as being reflected in their behaviour, activity period, habitat preference and reproduction. A previous study established that two different species (Partula hebe bella and

P. tohiveana) displayed different sensitivities to environmental factors, with clear correlations between temperature and humidity and the dynamics of the populations (Gouveia, 2006).

32 Chapter 1. General Introduction

1.6.2. Light

In many animals, light is the principal synchronizing factor (Dumortier, 1981in Gomot,

1990). Photoperiod is known to play an important role in reproduction of many vertebrates

(Gomot, 1990).

The influence of light on molluscs has been the subject of research for many years; these studies have given rise to analogous observations among scientists showing clear influences on locomotion (McKillop & Harrison, 1982; Pimentel-Souza et al., 1984), activity

(Pieri & Jurberg, 1981), feeding, reproduction and oviposition (Joy, 1971; Hien, 1981;

Williams & Gilbertson, 1983; Rotenberg, 1989; Gomot, 1990; Ligaszewski et al., 2007).

1.7. Diet

The geographical distribution of Gastropods is highly influenced by their diet, in particular the areas where calcium levels are at their highest (Brodersen & Madsen, 2003).

The significance of calcium in molluscs, particularly in snails, is widely described. It is of general acceptance that this nutrient plays a significant role in their shell development, growth, survival and reproduction (Boycott, 1934; Wagge, 1952; Crowell, 1973; Ireland,

1991; Ireland & Marigomez, 1992; Egonmwan, 2008). The effects of low calcium concentration in their diet have repercussive effects on their fitness, consequently, leaving them exposed to desiccation and predation and destroying or greatly reducing their natural defences (Harrison, 1970; Young, 1975; Madsen, 1987; Zalizniak, 2009; Dalesman &

Lukowiak, 2010).

Concerns regarding diet have been discussed throughout the years. The potential toxic effects of vitamin E, the chelate nature of phytic acid, water quality and purity and age related dietary differences are all listed in the Partula nutrition review (Pearce-Kelly, 2001).

33 Chapter 1. General Introduction

The extinction of the entire genus of Partula snails may disrupt other ecological processes worth studying in future research. Little or no work has so far been performed on the impact that the disappearance of Partula snails may play in their ecosystem. Johannessen and Solhøy (2001) point out the importance of snails as a source of calcium in forest ecosystems. “Snail shells, particularly of the larger species, have been shown to constitute a major calcium source for tits (Parus spp.) during egg laying” (Drent & Woldendorp, 1989;

Graveland et al., 1994; Graveland & van der Wal, 1996). In addition, as detritivores, they may play a role in the recycling of nutrients in the ecosystem.

34 Chapter 1. General Introduction

1.8. And the story repeats itself

This thesis has already mentioned Partulas‟ ongoing saga but it is important however, to emphasise that they are not the only species at risk of extinction. Worldwide, this process is being repeated and many other molluscan species face a similar fate. The threats vary from invasive species to habitat alteration, degradation and destruction, climate change and exploitation.

There are numerous molluscan species facing threats across the globe from the close by Vertigo moulinsiana in United Kingdom and Europe (Killeen, 2003; Tattersfield, 2003;

Tattersfield & Killeen, 2006) to the Achatinellidae in Hawaii (Hadfield, 1993; Cowie, 1998;

Holland & Cowie, 2009; Meyer III & Cowie, 2010) and Mandarina in Japan (Chiba, 2003;

Okochi et al., 2004) the list is extensive and periodically new species are added.

It is, however, interesting to consider the Hawaiian Achatinellidae because of their conspicuous similarities with partulids (Hartman, 1881). Both are arboreal, slow growing and have low fecundity rates. Both are endemic to islands of the Pacific Ocean and are nowadays only found in small fragmented populations. They have both been in severe decline for many years caused by several factors: spread of the invasive predator Euglandina rosea (the responsible for the extinction of Partulids), habitat loss and degradation, other introduced alien species such as rats, flatworms, ants etc and exploitation. The encouraging fact is that both Partulidae and Achatinellidae are under the care of captive breeding programs which aim to recover and save the few remaining representatives.

35 Chapter 1. General Introduction

1.9. Thesis outline

The general aim of this research is to investigate and analyse the factors that influence the welfare of Partula snails in captivity. This is of great importance considering that Partula snails are one of the world's rarest animals. Also, in order to achieve a successful re- introduction of Partula snails into the wild it is imperative to fully understand the factors that affect the species‟ biological processes and to eliminate any pathogenic agents that they may carry, which if unattended and subsequently introduced could lead to severe consequences to the natural ecosystem bearing in mind the enormous damage that the introduction of invasive species has already wrought in the Pacific islands.

The principal objective of the following chapters is to investigate possible factors that influence the snails‟ welfare in captivity. The investigation of environmental factors is imperative. It has been widely documented in the current literature their significant influences in several biological processes. In addition, diet is known to have significant impact on a population being especially, those held in captivity.

Hypothesis: Partula snail mortality in captivity is due to environmental factors and diet in addition to natural causes.

This hypothesis was tested taking into account all the possible factors that could affect their mortality in captivity. A study was carried out of the external factors that directly or indirectly could be responsible for the episodes of mortality recorded. These factors are described in detail in the following chapters. They are: diet, temperature, humidity and light.

Note: the welfare or wellbeing of snails refers to standard reproductive success, shell condition and growth rate.

36 Chapter 1. General Introduction

Chapter 2: This chapter investigates the concentration of calcium influences on the wellbeing of Partula snails in captivity. This study is based on the discovery that some species (in particular Partula gibba) were found with shells in very poor condition, which resulted in mortality or permanent shell dysmorphism. Different calcium concentrations influence the reproductive success and growth rate of Partula snails. The understanding of the dietary/nutritional requirements of Partula snails is imperative for their wellbeing and to improve their success in the breeding programme.

Chapter 3: This chapter investigates whether the supply of dog and cat vitamins and minerals to the Partula diet is detrimental, beneficial or neutral to their welfare. The answer to these questions may provide important information that will help improve their wellbeing and could potentially reduce the amount of resources required to maintain Partula snails in captivity.

Chapter 4: An investigation into whether or not environmental patterns (temperature and humidity) correlate with the mortality and fecundity of the 15 Partula species (including two subspecies) under study was performed. This investigation follows up previous work which had significantly correlated the environmental factors and the dynamics of the population of two species of Partula snails (P. hebe bella and P. tohiveana). It is hoped that this chapter will also improve the general understanding of the species requirements in captivity, and provide valuable information for the successful re-introduction of Partula snails into their natural environment.

Chapter 5: This chapter reports on a study into the effects of light intensity on the behaviour, reproduction and mortality of Partula snails. To date, Edinburgh Zoo has had one of the

37 Chapter 1. General Introduction highest success rates with breeding Partula. Even though the Partula care at Edinburgh, as elsewhere, is strictly controlled by a standard protocol, some small differences can inevitably be found from collection to collection. The most obvious difference between Edinburgh Zoo and other collections is that of lower light intensity which is thought to have beneficial influences on snail survivability. (Pearce-Kelly, pers. comm., 2007).

For ease of reading, each chapter is treated as a unitary essay. Overall conclusions are drawn in Chapter 6.

38 Chapter 2 - Calcium

Chapter 2. Effects of different calcium concentrations added to

the diet of Partula gibba (Férussac) on morphometric growth

parameters, weight and reproductive success.

2.1. Abstract

Partula gibba, commonly known as the Fat Guam Partula, is endemic to Guam and Northern

Mariana. The effects of calcium concentration on growth and reproductive success were studied. The snails were divided into six groups, fed ad libitum on a diet based on the formula devised by Frame and Clarke at the Nottingham University approximately 25 years ago or variants in which cuttlebone was substituted by various concentrations of calcium carbonate.

Shell height/length, thickness and width, aperture/lip height/length and width where recorded every month for one year, and all were found to be significantly positively correlated with the percentage of added calcium carbonate in the diet (range 0 – 40% dry wt:wt). Morphometric growth parameters of snails fed 20% added calcium carbonate did not differ significantly from the diet with added cuttlebone (20%), though reproductive success was lower with the cuttlebone treatment. Reproductive success (expressed by the number of newborns per adult per month) was also significantly positively correlated over the range of added calcium carbonate to the diet.

This Chapter has been published as: Gouveia, A. R. P. C.; Pearce-Kelly, P.; Leather, S. & Quicke, D. (2011) Effects of different calcium concentrations added to the diet of Partula gibba (Férussac) on morphometric growth parameters, weight and reproductive success. Malacologia, 54(1-2) 39 Chapter 2 - Calcium

2.2. Introduction

Partula gibba commonly known as the Fat Guam Partula is endemic to Guam and Northern

Mariana Islands, and it is listed as endangered in the IUCN (International Union for the

Conservation of Nature) Red List of Threatened Species (2007). Specimens of this species were first collected by the naturalists of the Freycinet expedition in 1819 and were described by Férussac in 1821 (Crampton, 1925).

Gastropod mollusc distributions may depend on various factors, but, calcium concentration has been found to be a particularly important and limiting element (Brodersen

& Madsen, 2003). The importance of calcium to terrestrial gastropods is well known and the observations are almost always similar to the early description of Boycott (1934) who showed that dietary calcium is an essential nutrient for growth, reproduction and shell construction (Wagge, 1952; Crowell, 1973; Ireland, 1991; Ireland & Marigomez, 1992;

Egonmwan, 2008). Ireland and Marigomez (1992) reported that the shell is the most important site of calcium deposition where it is present almost entirely as calcium carbonate, and this accounts for approximately 97% of shell weight (Heller & Magaritz, 1983).

Several studies have examined the effects of varying calcium concentration on different snail species (Harrison, 1970; Madsen, 1987; Brodersen & Madsen, 2003), and these have mostly concluded that snails are generally able to live under low calcium concentrations in the laboratory, but that growth is inhibited and thinner shells are usually developed as a result of calcium deprivation (Brodersen & Madsen, 2003). Crowell (1973) demonstrated that plant matter is not a sufficient source of calcium, and observed that the shells of snails fed only on plant matter were weak, thin and fragile. He also showed experimentally that the land snail Helix aspersa ingested soil and that the soil subsequently passed as faeces contained 12-32% less calcium than soil with no previous exposure to snails,

40 Chapter 2 - Calcium and therefore that soil is likely to be an important source of calcium in the wild (Heller &

Magaritz, 1983).

The diet of Partula snails in the wild is not well known. They are detritivores and have been observed feeding on dead and decaying Hibiscus tiliaceous leaf stalks

(Cunningham et al., 1996). Coote (2007) recorded approximately 76% of the populations of

P. hyalina and 40% of the wild Partula clara population on the native plant species, red ginger Etlingera cevuga, though these are generalist feeders and were seen feeding on dead leaves of different species. Hopper and Smith (1992) reported that Partula gibba was most abundant on the endemic understory plant Piper guamensis, but was also found on coconut palms and the fern Microsorium punctatum.

A number of concerns regarding the diet of captive Partula snails have been raised over the years, such as the chelate nature of phytic acid in the porridge. Phytic acid is considered as an anti-nutritional factor due to its ability to bond with other components, such as calcium (Abdel-Hamid et al,. 2007).

Partula snails are ovoviviparous hermaphrodites (Pilsbry, 1909-1910; Clarke &

Murray, 1969) and they may produce a completely shelled offspring, every six to eight weeks. Baur (1994) describes ovoviviparity as a form of parental care which allows terrestrial gastropods to persist in habitats otherwise unsuitable for oviparous species.

In order to test whether or not the calcium source plays an important role in the decline or wellbeing status of the Partula snail populations in the breeding programme a calcium investigation was performed. The effects of calcium concentration on the reproductive success of Partula snails, was also studied.

41 Chapter 2 - Calcium

2.3. Methods

The snails used in the study were the offspring from the parents from London Zoo population. Partula gibba were selected because they were available in sufficient numbers

(n=60) and because several incidents of broken and thin shells had been observed in the captive snails. Individuals were maintained in a controlled temperature room with a temperature of 22ºC at night and 23ºC during the day and humidity kept within a range of 70 to 80%. Light was provided by fluorescent tubes on a twelve hour day and night photoperiodic cycle.

Because of the high mortality experienced during the first post natal week, snails were used after they reached a minimum weight of 0.03 grams and a maximum of 0.05 grams.

They were then randomly placed into individual plastic containers (250ml) with a substrate consisting of layers of damp bleach-free paper tissue. All containers were covered with cling film to prevent the snails from escaping and to maintain humidity.

Throughout this study snails were fed ad libitum on a diet made up as follows: 300g of porridge oats, 150g of powdered trout pellets, 600g powdered grass pellets, 25g of vitamin powder (Vetzyme, Seven Seas Ltd.) and 300g of powdered cuttlebone. Calcium carbonate,

(CaCO3, Mineral Technologies Inc.) replaced the powdered cuttlebone for the experiment except in one group (control). The diet was moistened with filtered water to form a runny paste and then microwaved for approximately two minutes for sterilisation.

The snails were divided into six groups. These groups were fed on the same diet with the proportional addition of: 0, 10, 20, 30 and 40% calcium carbonate and a group with 20% cuttlebone which corresponded to the standard diet. For each group there were 10 replicates.

The diet, substrate and the containers were replaced every month, and the numbers of newborns (reproductive success) were counted. All the materials used to handle, feed, house

42 Chapter 2 - Calcium and clean the snails had been previously sterilised/disinfected in a microwave or sterilized with aqueous sodium hypochlorite solution.

Snails were weighed once per month from the beginning of the experiment until the end (one year). Due to their thin, delicate shells morphometric measurements were only conducted once the snails reached the adults stage (traditionally characterized by the formation of the lip (Hartman, 1881)). Shell height/length, thickness and width, aperture/lip height/length and width of each snail were measured using digital vernier calliper (Figure

2.1). The snails‟ measurements were recorded once per month for a whole year.

Figure 2.1. Snails‟ morphometric measurements; shell thickness (G), height/length (A-B), width (C-D), lip height/length (B-F) and lip width (E-D) (Crampton, 1916).

Growth rate and reproductive success per year were plotted against each breeding condition. T-tests and linear regression analysis were conducted to ascertain significant

43 Chapter 2 - Calcium differences between the groups under study. Statistical analyses of the data were performed using the software package R (Ihaka & Gentleman, 1996).

2.4. Results

Results for changes in morphometric parameter between each monthly measurement are shown in Figures 2.2-2.7.

Figure 2.2. Effects on the weight of the snails of different calcium carbonate concentrations added to the diet, including powdered cuttlebone. The top and bottom of each rectangular box denote the 75th and 25th percentiles, respectively, with the median shown inside the box.

Horizontal bars extending from each box represent the 90th and 10th percentiles, and the outliers are shown as “°”. Significant differences among individual treatments are denoted with letters over each box (statistics are presented in the same format for all of the box plots in this study).

44 Chapter 2 - Calcium

Figure 2.3. Effects on shell lip thickness of different calcium carbonate concentrations added to the diet including powdered cuttlebone.

Figure 2.4. Effects on shell height/length of different calcium carbonate concentrations added to the diet, including powdered cuttlebone.

45 Chapter 2 - Calcium

Figure 2.5. Effects on shell width of different calcium carbonate concentrations added to the diet, including powdered cuttlebone.

Figure 2.6. Effects on shell lip height/length of different calcium carbonate concentrations added to the diet, including powdered cuttlebone.

46 Chapter 2 - Calcium

Figure 2.7. Effects on shell lip width of different calcium carbonate concentrations added to the die, including powdered cuttlebone.

All morphometric measurements as well as snail weight were found to be significantly positively correlated with the percentage of added calcium carbonate in the diet

(Table 2.1).

47 Chapter 2 - Calcium

Table 2.1. Linear model summary, for Partula gibba morphometric measurements and reproductive success against the addition of calcium to the diet.

F-stats p-value r2 Intercept

Snail weight F1,48=131.3 P<0.001 0.73 P<0.001

Shell lip thickness F1,42=215.8 P<0.001 0.84 P<0.001

Shell height/length F1,42=75.72 P<0.001 0.64 P<0.001

Shell width F1,42=108.8 P<0.001 0.72 P<0.001

Shell lip height/length F1,42=120.8 P<0.001 0.74 P<0.01

Shell lip width F1,42=77.45 P<0.001 0.65 P<0.05

Reproductive success F1,48=223.6 P<0.001 0.82 Ns

48 Chapter 2 - Calcium

In addition, reproductive success (expressed by the number of newborns per adult per month) was significantly positively correlated over the range of added calcium carbonate to the diet (Figure 2.8.). Nearly all parameters showed an approximately linear relationship.

Figure 2.8. Effects of different calcium carbonate concentrations added to the diet, including powdered cuttlebone on the reproductive success of Partula gibba, expressed as number of newborns per month over a one year period.

Visual inspection of the graphs and t-tests showed that the morphometric growth parameters of snails fed 20% added calcium carbonate did not differ significantly from the diet with added cuttlebone (20%). However, there was a significant difference in reproductive success (t = 2.9, df = 17.986, p < 0.01) where snails fed with the diet with added 20% calcium carbonate these, had a higher reproduction success than those fed with the diet with cuttlebone (20%).

49 Chapter 2 - Calcium

2.5. Discussion

Nutrition is one of the major factors that affect the performance of livestock, and snails are no exception (Oluokun et al., 2005). This study focused on the importance of calcium in the development and reproduction success of Partula gibba.

The results presented in this study concur with those obtained by Oluokun et al.

(2005) who concluded that the height/length, width and thickness of Archachatina marginata shells increased with an increased calcium concentration in the diet.

Numerous studies have demonstrated the importance of calcium as a determinant of the development and wellbeing of snails. Fournie and Chetail (1982), for example, established that the addition of calcium carbonate to the snails‟ diet prevented them from dwarfing, though not those snails maintained on calcareous soils (Elmslie, 1988; Egonmwan,

2008)

Calcium also plays a vital role in the regeneration process of snail shells. Bierbauer

(1957 in Vaidya, 1980) reported that shell repair in Helix pomatia was enhanced following injection of calcium. Voelker (1959) noted that scarcity of calcium may result in thinner and more brittle shells, rendering them less fit to protect the snail properly against desiccation, physical damage and invertebrate predators (in Johannessen & Solhøy, 2001). Egonmwan

(2008) concluded that calcium deficiency in Limicolaria flammea resulted in a decrease in the growth rate of these snails raised in the laboratory. All these studies demonstrate that calcium is an important nutrient in the snails‟ life.

Due to the fact that Partula snails face an uncertain future, with a high rate of species extinction observed over the years, it is of special importance that all the factors that contribute to their welfare are fully understood. To that end, this study demonstrated that an increased level of calcium carbonate in their diet brought great benefits to their wellbeing. All

50 Chapter 2 - Calcium morphometric growth parameters tested and reproductive success increased significantly

(p<0.001) with the increased calcium concentration added to their diet. In addition, the supplementation of 20% calcium carbonate to the diet did not differ from the diet with cuttlebone, the only difference being the reproductive success which is higher in the snails fed with 20% calcium carbonate added to the diet.

It is critical to consider the chelate nature of the phytic acid present in the porridge oats, this acid is reported to bind with calcium and other minerals rendering it as an anti- nutricional factor, it reduces bioavailability and absorption of minerals and proteins and therefore it may reduce the absortion of calcium by the snails (Graf, 1983; Ali et al., 2010).

Partula snails are nutrient recyclers hence they have an important role in their ecosystem. The total disappearance of Partula snails in the wild may well be detrimental to the ecosystem. The role of invertebrate detritivores in tropical ecosystems is little known, their effect on leaf moisture and production, and the overall above ground production. Further studies on the effects of their extinction upon the fauna, flora and soil composition could be very informative. For example, Gärdenfors et al. (1995) noted that the abundance and diversity of snails is closely positively correlated to pH in the soil (Gärdenfors et al., 1995 in

Johannessen & Solhøy, 2001). Johannessen and Solhøy (2001) concluded that snail populations have a positive effect upon bird populations since some species rely on the snails shell as a source of calcium. These studies indicate the importance of snails as a source of calcium in forest ecosystems.

Several cases have been observed at the Zoological Society of London of Partula snails feeding on the substrate (paper tissue); this is thought to be an indication of a need for a higher calorific diet (Crowell, 1973). It is therefore recommended that an increase of calcium concentration (40% instead of 20%) in the diet of Partula snails would be beneficial and that calcium carbonate powder should be used instead of cuttlebone since the cuttlebone diet did

51 Chapter 2 - Calcium not demonstrate any benefits upon the other diets. Additionally, cuttlebone can be contaminated with heavy metals (Miramand et al., 2006) such as mercury (Hg) (Lacoue-

Labarthe et al., 2009), bioaccumulation of Polychlorinated biphenyls (PCBs) (Danis et al.,

2005) and potentially infectious agents. Calcium carbonate, though, is effortless to use and it is simpler to control possible routes of contamination that may be detrimental to snails‟ welfare.

52 Chapter 3 - Vitamins

Chapter 3. Investigation of the effect of vitamin supplement

added to the diet of Partula snails.

3.1. Abstract

Partula snails are arboreal with nocturnal to crepuscular activity but their diet in the wild is largely unknown. It is known that they can starve to death rather than eat something new.

Several concerns regarding the diet of the captive populations have been raised throughout the years: the potential toxic effects of vitamin E, the chelating nature of phytic acid in porridge, the toxicity of metals present in the water and age related dietary differences. These are among the concerns raised in the Partula nutrition review. The Partula diet currently in use in the Partula holding collections is based on the „Nottingham diet‟ formulated by Frame and Clarke at Nottingham University. This diet was refined over a number of years through a combination of trials and feedback from collections in the breeding programme. The aim of this study was to assess whether the vitamin supplement added to the diet of Partula snails has a detrimental, beneficial or neutral effect on their reproductive success and to pick up on any effects on mortality. The Partula gibba population under study was significantly negatively affected by the supplement of vitamins in the diet (standard diet). Statistical analysis of the experimental data revealed a significant correlation between the presence of

2 vitamins in the diet and an increased numbers of deaths (R = 0.25, F1,30= 11.26, p<0.05).

Conversely, it was found that vitamins did not play a role in the reproductive success of the

2 study population (R = -0.03, F1,30= 9.23e-05, p>0.05).

53 Chapter 3 - Vitamins

3.2. Introduction

Regardless of their food source gastropods have always been found to be both efficient digesters and assimilators (Wieser, 1978). Partula snails are predominantly nocturnal and arboreal (Barker & Efford, 2004), living on a wide variety of tree and bush species. Partula snails‟ diet in the wild is largely unknown, in captivity, they were found to be extremely conservative feeders, they can starve to death rather than eat something new (Tudge &

Pearce-Kelly, 1992). Crampton (1932) noted that the host species of plant were not relevant to the diet of Partula since they would descend to the ground at night to “feed on decaying vegetation. The character of the plants which provides this nourishment is a matter of indifference”. Murray et al. (1982) demonstrated that there are “differences in the distribution of the snails within habitats”. Hence “the number of Partula species in any particular locality depends on the diversity of the flora as an expression of both structural complexity and floristic variety in the habitat”. They demonstrated that Partula eat an assortment of both decaying and fresh plant material (Cowie, 1992).

Various unpublished studies on the diet of Partula have been performed in the past.

West (1996) studied diet feeding preference and orientation of two Partula species. In this study Partula snails showed preference for a given diet, although results on orientation proved inconclusive. Wade (2002) studied the role of gut flora in the nutritional physiology of Partula snails and concluded that there was a lack of cellulose-digesting bacteria.

However, a similar study performed two years earlier by Ridge (2000) found evidence of at least four bacteria species with high levels of cellulose activity suggesting a link between the gut flora and cellulose digestion. Other studies on gut flora were carried out by Stallard (2003 and 2004) where the nutrient digestibility of seven Partula species (2003) and Partula clara

(2004) were investigated. Results from the first experiment did not demonstrate significant

54 Chapter 3 - Vitamins differences in the digestibility in any of the nutritional parameters. In contrast, the second study preformed on a single species (Partula clara) showed significant differences in the digestibility of certain nutrients. However the author noted that results from both experiments were deemed inaccurate and unreliable therefore the results should be treated with caution due to the limited amount of faecal material available for analysis. Such ambiguous results and other unpublished comments regarding Partula diet can be deceptive and further investigation needs to be developed.

Several concerns regarding the diet of captive populations have been raised over the years: for example the potential toxic effects of vitamin E supplied in the diet, and the chelate nature of phytic acid in the porridge which, due to its ability to bond with other components, can be considered has an anti-nutritional factor (Abdel-Hamid et al,. 2007). The toxicity of metals in the water and age related-dietary differences are amongst the concerns raised in the Partula nutrition review (Pearce-Kelly, 2001).

Partula diet currently in use is based on the diet formulated by Frame and Clarke at the Nottingham University approximately 25 years ago with a mixture of other ingredients that evolved from years of experience of keeping snails in artificial conditions, experiments, suggestions and ideas (Dave Clarke, pers. comm., 2011).

The aim of this study was to assess whether vitamin supplement added to the diet has a detrimental, beneficial or neutral role in the reproductive success, or impacts on the mortality of Partula gibba. The vitamin supplement used is prepared for dogs and cats

(Vetzyme, Seven Seas Ltd, U.K.) and is composed of a diversity of ingredients: calcium, phosphorous, sodium in the form of (dicalcium phosphate and sodium dihydrogen phosphate), magnesium sulphate, ferrous gluconate, zinc oxide, manganese sulphate, copper carbonate, potassium iodide, cobalt carbonate, lactose, vegetable proteins, vitamins A (retinyl palmitate) and D3 (cholecalciferol). This extensive list includes a wide range of metals, non

55 Chapter 3 - Vitamins metals and vitamins. These can be considered as trace elements or micronutrients, essential metals, heavy metals and toxic metals.

It is known that at high concentrations all heavy metals elicit toxic effects irrespective of their physiological function (Luckey et al. 1975 in Berger & Dallinger, 1989), but the classification of metals can be complicated. According to Duffus (2002) heavy metal is a very imprecise term and “its inconsistent use reflects flaws in the scientific literature”. In the same study Duffus (2002) considers both the use of the term essential and toxic metals as misleadingly since it should be accompanied by a statement of which organisms show a requirement/toxicity for the element. “Toxicity, like essentiality, should be defined by reference to a dose–response curve for the species under consideration (Duffus, 2002). Hence the important factor is to know the dose response curve for a specific species since species- specific properties in metal budget strategies exist (Martin & Coughtrey 1982 in Rabitsch,

1995; Greville & Morgan, 1990).

The negative effects of heavy metals are well described in a wide range of species but few studies are available for terrestrial pulmonates (Berger & Dallinger, 1993). Metals can cause acute, sublethal and lethal effects in snails by damaging or destroying eggs, reducing embryo survival and causing failure of hatching. Metals can also induce developmental problems which result in malformation of the foot, eyes, digestive gland and shell. Other detrimental effects include thinness of shell and growth rate, consumption or feeding inhibition, loss of chemoreception, locomotion, survival, antagonistic behaviour towards other nutrients and destroying lipids, nucleic acids and proteins (Simkiss & Mason, 1984;

Laskowski & Hopkin, 1996 a, b; Cortet et al., 1999; Swaileh & Ezzughayyar, 2000, 2001;

Notten, 2006; Khangarot & Das, 2010; van Dam et al., 2010; Das & Khangarot, 2011).

Taking this into consideration that the vitamins added to the diet of Partula snails can contain

56 Chapter 3 - Vitamins potential toxic ingredients, the present study was undertaken in order to investigate the effects of added vitamin supplement to the diet on the mortality and fecundity of the Partula gibba.

3.3. Methods

Partula gibba was the species selected for this experiment principally because they comprise the largest captive population of the Imperial College collection. This species has previously displayed thinning of the shell, (see Chapter 3) when fed on a calcium-poor diet which induced nutritional problems.

Individuals were maintained in a controlled temperature room with a temperature of

22ºC at night to 23ºC during the day and a relative humidity range of 70 to 80%. Light was provided by fluorescent tubes on a twelve hour day and night photoperiodic cycle.

Adult snails (n=32) were selected and randomly placed into individual plastic boxes

(11cm x 17cm x 5cm). The substrate consisted of layers of moist bleach-free paper tissue. All containers were covered with cling film to maintain humidity and to prevent snails from escaping.

The two treatments contained the same ingredients but the vitamin supplement was added to one. Snails were fed ad libitum on a diet made up as follows: 300g of porridge oats,

150g of powdered trout pellets, 600g of powdered grass pellets, 300g of calcium carbonate,

(CaCO3, Mineral Technologies Inc.) and 25g of vitamin powder (Vetzyme, Seven Seas Ltd.).

The diet was moistened with filtered water to form a runny paste and then microwaved for approximately two minutes for sterilisation.

The snails were divided into two groups: group A was fed on the diet with added vitamin supplement and group B was fed on the same diet exclusive of the vitamin supplement. For each group there were 16 replicates. The diet, tissue and the containers were

57 Chapter 3 - Vitamins replaced every fortnight, and the numbers of newborns (reproductive success) and mortality were counted. All the materials used to handle, feed, house and clean snails were previously sterilised/disinfected in a microwave or sterilized with aqueous sodium hypochlorite solution.

The study lasted approximately ten months.

Data analysis was performed using the statistical software R (Ihaka & Gentleman,

1996). T-tests and linear regression analysis were conducted to ascertain if there were any significant differences between the two groups under study.

3.4. Results

The results drawn from this experiment are described below.

There was sufficient evidence to accept that the treatments affected the mortality of the population (at 95% confidence interval t = 3.35, df = 20.95, p-value < 0.05) but insufficient evidence to reject or accept the fact that the treatments played any role in the fecundity of Partula gibba (at 95% confidence interval t = -0.0096, df = 26.33, p-value >

0.05).

When compared with the vitamin free diet, the Partula gibba population under study was negatively and significantly affected by the supplement of vitamins in the diet (standard diet). Statistical analysis indicated that there was a significant correlation between the

2 presence of vitamins in the diet and an increased numbers of deaths (R = 0.25, F1,30= 11.26,

2 p<0.05). However, no significant effects on fecundity were observed (R = -0.03, F1,30= 9.23e-

05, p>0.05). The effects of the two treatments on mortality and fecundity rate are illustrated in Figure 3.1 and 3.2.

58 Chapter 3 - Vitamins

Figure 3.1. Effects of the treatments on the mean mortality of captive Partula gibba.

Figure 3.2. Effects of two treatments on the mean fecundity of captive Partula gibba.

59 Chapter 3 - Vitamins

3.5. Discussion

Growth and reproduction are directly influenced by diet (Raut, 1992).This study looked at the effects on the fecundity and mortality of captive-bred Partula snails by adding a vitamin supplement to their diet.

The results indicate that the vitamin supplement has an adverse effect on snail survival by increasing the mortality of the population under study, although no significant correlations were observed between vitamin addition and snail fecundity.

Lydeard et al. (2004) noted that biologists know little, except in very general terms, of the food preferences of the vast majority of land snails. This is true for partulids. There are numerous observations and comments about the diet of Partula snails but there are no concrete studies that can be relied upon. The nutrient requirements for this family of snails are unknown but need to be fully understood in order to provide them with the optimal diet to maintain healthy populations and for maximum survivability. It is fair to say that for the past

20 odd years the snails have been maintained on the same diet and they have fared reasonably well. The reality is however, that several incidents of increased mortality and low overall fecundity have been observed over the years without clear correlations between these events and any potential causes. Prospectively this could lead to disastrous results. These problems clearly demonstrate the continuing need for improvement and to fully understand such factors.

Frantz & Mossmann. (1989) stress that animal preference for a given food item does not necessarily mean that a strict diet of this food will provide the nutrition required for ideal growth and reproduction. It could be that this preference is merely a function of higher availability of the product. In addition, both this study and that in Chapter 3 indicate that the

60 Chapter 3 - Vitamins artificial diet currently in use has not guaranteed acceptable growth and survival of Partula species in captivity.

The negative effects on mortality of adding the vitamin supplement to the diet as revealed by this study are of great concern. The complicated effects of vitamin supplements on the snail biochemistry are unknown and were not covered in this study. Nevertheless, they should be taken into consideration in future research. It is not only the nutritional values of food that are significant for development and reproduction but also a combination of texture and structure, as well as digestibility and absorption of items in the diet and hence it is important to consider these factors as well (Meireles, 2008). Finally, Kobayashi and Hadfield

(1996) state that in order to reproduce the optimal captive conditions for growth, fecundity and survivorship of snails, it is essential to understand their basic biology.

Both the investigation of calcium concentration in the diet of Partula snails (Chapter

3) and this study into the effects of adding a vitamin supplement are in agreement. The results point to problems with the current diet used in the international breeding programme. It seems likely that the snails have nutritional constraints which are shown in their development, reproduction and survival.

61 Chapter 4. Temperature and Humidity

Chapter 4. Investigating the effects of environmental stress on the

population dynamics of endangered Partula snails.

4.1. Abstract

Polynesian tree snails (Partula sp., Partulidae) are medium-sized gastropod molluscs belonging to the order Stylommatophora and endemic to the Society Islands in the South

Pacific region. Wild populations have declined since the introduction of the predatory snail,

Euglandina rosea, to control the invasive giant African land snail (Lissachatina fulica), an agricultural pest which was impacting on the local economy. Periods of notably increased mortality rates have been recorded among Partula populations in captive breeding programmes. The causes of these population declines are uncertain. This study investigated the effect of temperature and humidity on the survival and fecundity of 15 species (including two subspecies) of Partula. The total adult mortality of 14 of them was found to be significantly and negatively or positively related to temperature and/or humidity, and/or on interaction between these variables. Temperature and humidity also significantly influenced the fecundity of five of the species.

62 Chapter 4. Temperature and Humidity

4.2. Introduction

Understanding the influences that environmental constraints have upon Partula snails is likely to be of great value for their successful management in captivity, and for possible re- introductions into their natural environment. Dias et al. (2007) describe the humidity dependence of terrestrial pulmonate molluscs as being reflected on their behaviour, activity period, habitat preference and reproduction. Partula, or Polynesian tree snails, are endemic to the islands of the Pacific Rim, especially the Society Islands where they are under severe threat due to the introduction of a predatory mollusc into these islands. Euglandina rosea was introduced as a biological control agent for the already well-established Lissachatina fulica, in the 1970s. Lissachatina fulica, had previously been introduced as a food source for the local population, and had then begun invading and destroying plantation crops, adversely impacting on the economy of the islands (Coote et al., 1999; Shine et al., 2003). However

Euglandina rosea predated the endemic partulids. This species has been largely responsible for the extinction of all but approximately 29 species in the recent past.

Numerous incidents of greatly increased mortality have, however, been experienced in various captive breeding collections (Cooper & Knowler, 1992). A number of hypotheses regarding the causes of mortality of Partula populations have been proposed, including, density dependent diseases (Tonge & Bloxam, 1990), microsporidian parasites (Cunningham

& Daszak, 1998), mites (Tonge & Bloxam, 1990), etc. In captivity, however, no direct evidence about the nature of these mortality factors has been published, with the exception of

Cunningham and Daszak (1998) who reported an emerging disease of Partula snails, caused by a new species of microsporidian parasite (Steinhausia sp.), which was associated with the increased mortality rate, and the extinction, of the captive population of Partula turgida.

Although the results from the histology performed by Cunningham confirmed the presence of

63 Chapter 4. Temperature and Humidity a pathogenic microsporidian parasite in at least three species of Partula snails held in the

London Zoo collection, only one of these three species has become extinct (Pearce-Kelly, pers. comm., 1998).

The most significant factors affecting snails in captivity are environmental factors and diet. A previous study (Gouveia, 2006) established that two different species (Partula hebe bella and P. tohiveana) differed in their sensitivities to environmental factors, with clear correlations between temperature and humidity and the population dynamics. Partula clara showed a significant positive correlation with maximum and minimum humidity and mortality and its fecundity which was significantly negatively correlated with maximum and minimum humidity. In contrast P. tohiveana mortality was not correlated with either temperature or humidity but its fecundity showed a significant positive correlation with both maximum and minimum humidity (Gouveia, 2006).

Here, the results are presented of a two and an half year long study into the effects of temperature and humidity on a total of 15 species (including two subspecies) of Partula. All the study species are listed in the IUCN (International Union for the Conservation of Nature)

Red List as either extinct in the wild (EW) or critically endangered (CR). Collectively, these taxa originated from several different environments. For example, Partula subgonochila is endemic to the islands of Alofi and Fotuna, where they are found in coastal forests on limestone, and where they are exposed to high temperatures. In contrast P. radiolata from

Guam are found in more open drier habitat (Crampton, 1925) and the Society Island species on forested slopes.

64 Chapter 4. Temperature and Humidity

4.3. Methods

The collection from London Zoo consists of approximately 3000 partulid snails, which represent an estimated 30% of the world‟s captive population (Pearce-Kelly, pers. comm.,

2008). Representatives of all 15 species were transferred to a controlled temperature room at

Imperial College London, Silwood Park Campus, and were maintained and studied from

October 2007 until February 2010.

The husbandry methods applied in this study were adapted from the protocol used at

London Zoo, which are developed from many years of experiments in zoos and universities

(Mace et al., 1998), and are described in the Management Guidelines for the Welfare of Zoo

Animals (Pearce-Kelly et al., 2006). Although the methods were modified to some extent for the study reported here, the changes involved a rigorous regime of disinfection and sterilization of all material and food. Calcium carbonate powder was provided as the calcium source instead of cuttlebone since it is easier to control possible routes of contamination. Due to the small population sizes the snails were housed in smaller containers.

The snails were fed ad libitum on a diet first formulated by Frame and Clarke at

Nottingham University approximately 25 years ago with a mixture of other ingredients that resulted from a combination of experiments and suggestions (Dave Clarke, pers. comm.,

2011), consisting of: 600g grass pellets, 300g porridge oats, 150g trout pellets, 300g calcium carbonate powder and 25g multivitamins. All constituents were reduced to a fine powder and mixed with filtered water to create a runny paste which was then placed in the microwave for sterilization and finally spread onto the side of the containers (either 11cm x 17cm x 5cm or

16cm x 28cm x 9cm depending on the size of the population). All of the materials used to handle, feed, house and clean the snails were previously sterilized/disinfected in the microwave or sterilized with aqueous sodium hypochlorite solution.

65 Chapter 4. Temperature and Humidity

The study group consisted of approximately 800 snails as follows: Partula affinis (57 individuals), P. clara (9), P. faba (25), P. gibba (173), P. hebe bella (44), P. hyalina (27), P. mooreana (133), P. nodosa (15), P. radiolata (6), P. subgonochila (25), P. suturalis strigosa

(6), P. suturalis vexillum (43), P. taeniata nucleola (65), P. taeniata simulans (5), P. tristis

(5), P. tohiveana (118) and P. varia (24).

4.3.1. Environmental study

Snails were maintained in a controlled temperature room with temperatures set to 22ºC at night and 23ºC during the day. Light was provided by fluorescent tubes set to a 12 hour day and night photoperiodic cycle. Relative humidity generally varied between 70% and 80%.

The results presented here result from a range of perturbations of temperature and humidity away from the above settings, largely due to technical faults. These fluctuations enabled statistical analyses of the correlations between temperature and humidity and between snail mortality and fecundity. Temperature and humidity were recorded every 15 minutes using an eTemperature probe (OnSolution Pty Ltd, Australia). The eight explanatory variables considered were, minimum, maximum, mean and range of temperature and humidity of over each four week period.

4.3.2. Data analysis

Data were analysed using the statistical software package R (Ihaka & Gentleman, 1996). It was tested whether or not temperature and humidity played a significant role on the mortality and fecundity (expressed as the number of newborns per species per month). Linear models were created using arcsine transformed proportions of adults and juveniles against all the individual explanatory variables and against different combinations with interactions.

Because of the uncontrollable nature of the data, a test was performed for autocorrelation of

66 Chapter 4. Temperature and Humidity the explanatory variables using a linear model of the variable at time period t and at time period t-1. Of the eight tests (four temperature and four humidity explanatory variables), two were significant (minimum temperature and mean temperature). For these, their regression parameters against date were calculated and two new detrended variables created by subtracting the regression slopes. Retesting showed no significant autocorrelation. For each species was then tested the relationship between mortality and fecundity each month and each of the explanatory variables and against pairs of temperature and humidity variables with interaction terms included in the models.

For an understanding of the different conditions experienced by Partula snails in the wild the mean environmental temperature and humidity levels from 2008 from locations on

Tahiti and Moorea, and the temperature and humidity levels of the controlled temperature room used for the experiment during 2008, are shown in Figure 4.1.

4.3.3. Statistical significance

A number of statistical tests were carried out. As an example, adult mortality of 15 species

(including two subspecies) was investigated independently for a total of eight possible variables giving a total of 136 tests. All significant results are presented with an alpha of 0.05 and statistics indicating the magnitude of all significant effects, rather than applying a

Bonferroni correction for the multiple comparisons, because the sample sizes would in many cases make it impossible to achieve significance at an alpha level of 0.000367. With an alpha of 0.05, one would expect to detect six or seven significant effects by chance alone. In the analyses of adult mortality there were 42 significant effects, most at p<0.01, and it is therefore proposed that the magnitude of detected effects is probably meaningful.

67 Chapter 4. Temperature and Humidity

4.4. Results

Several incidents of technical faults occurred during the study period, ranging from temperature increases up to 29 ºC and drops in humidity to 8% (Figure. 4.1).

Figure 4.1. Annual mean temperature and humidity (2008) from Faa‟a (Tahiti), Opunohu

(Moorea) and the controlled temperature room (at Imperial College).

Of the 15 species (including two subspecies) investigated, adult mortality in 14 (P. affinis, P. clara, P. hebe bella, P. hyalina, P. mooreana, P. nodosa, P. subgonochila, P. suturalis strigosa, P. suturalis vexillum, P. taeniata nucleola, P. taeniata simulans, P.

68 Chapter 4. Temperature and Humidity tohiveana, P. tristis and P. varia) was found to be significantly related either to temperature and/or to humidity (Table 4.1) and in some cases significant interactions (Table 4.2) were found between explanatory variables. The total adult mortality of the population in eight species (P. nodosa, P. subgonochila, P. suturalis strigosa, P. taeniata nucleola, P. taeniata simulans P. tohiveana, and P. tristis) was only affected by temperature, two species (P. clara and P. suturalis vexillum) only by humidity, and three species (P. affinis, P. hebe bella and P. mooreana) by both temperature and humidity (Tables 4.1 and 4.2). Adult mortality of three species (P. faba, P. gibba and P. radiolata) did not show any significant correlation with environmental patterns; however, two of these (P. faba and P. radiolata), were either represented by very low numbers or died very early on in the study period.

There was a relationship between juvenile mortality of 12 species (P. clara, P. faba,

P. gibba, P. hebe bella, P. hyalina, P. nodosa, P. radiolata, P. subgonochila, P. taeniata simulans, P. tohiveana, P. tristis and P. varia) and temperature or humidity or both, and/or with temperature-humidity interactions (Tables 4.3 and 4.4). P. nodosa, juvenile mortality was significantly negatively correlated with minimum humidity and P. tohiveana and P. tristis, juvenile mortality was significantly and positively influenced by temperature. Juvenile mortalities of four species (P. mooreana, P. suturalis strigosa, P. suturalis vexillum and P. t. nucleola) were not correlated with any environmental pattern.

Individual tests were also performed on the fecundity data for each species (Table 4.5) with fecundity as a function of humidity, temperature and interactions as above. In four species (P. clara, P. nodosa, P. suturalis strigosa and P. varia), fecundity was significantly correlated with temperature, while for P. subgonochila it was significantly negatively correlated with humidity.

69

Table 4.1. Effects of temperature and humidity on adult mortality. Significance levels (*<0.05, **<0.01 and ***<0.001), the symbols - and + indicate the trend of significant effects.

Temperature Humidity Species Max Min Range Mean Max Min Range Mean

-* -** s. vexillum N.S N.S N.S N.S R2=0.19 N.S N.S R2=0.23 F1,27=7.46 F1,27=9.18

-* -* clara N.S N.S N.S N.S R2=0.13 N.S N.S R2=0.17 F1,25=4.92 F1,25=6.21

-** +* -* -*** +*** -** mooreana N.S R2=0.27 R2=0.1 N.S R2=0.13 R2=0.36 R2=0.37 R2=0.2 F1,27=11.4 F1,27=4.29 F1,27=5.06 F1,27=16.7 F1,27=17.2 F1,27=8.01

-** +** -* +** affinis N.S R2=0.25 R2=0.22 N.S N.S R2=0.18 R2=0.2 N.S F1,27=10.5 F1,27=9.35 F1,27=7.2 F1,27=8.1

-*** +* -** +* hebe bella N.S R2=0.46 R2=0.14 R2=0.28 N.S N.S R2=0.11 N.S F1,27=24.5 F1,27=5.6 F1,27=11.7 F1,27=4.6

+** +** t. nucleola R2=0.24 N.S R2=0.27 N.S N.S N.S N.S N.S F1,27=9.8 F1,27=11.5

70

+* +* nodosa R2=0.13 N.S R2=0.14 N.S N.S N.S N.S N.S F1,27=5.35 F1,27=5.71

+** +* tohiveana R2=0.24 N.S R2=0.17 N.S N.S N.S N.S N.S F1,27=9.88 F1,27=6.83

-*** -** subgonochila N.S R2=0.83 N.S R2=0.57 N.S N.S N.S N.S F1,12=64.5 F1,12=18.1

-* +* tristis N.S R2=0.18 R2=0.21 N.S N.S N.S N.S N.S F1,17=5.06 F1,17=5.66

-* -* s. strigosa N.S R2=0.18 N.S R2=0.18 N.S N.S N.S N.S F1,27=7.27 F1,27=7.27

+* t. simulans N.S N.S R2=0.58 N.S N.S N.S N.S N.S F1,5=9.38

71

Table 4.2. Significant interactions between temperature and humidity and adult mortality.

Interactions Species Min H: Min H: Max H: Mean H: Min H: Mean H: Min T Mean T Min T Min T Max T Max T

-* -* -** 2 2 2 hyalina R =0.19 N.S R =0.12 R =0.24 N.S N.S F3,25=3.25 F3,25=2.34 F3,25=4.03

+* +** +* subgonochila R2=0.87 R2=0.81 N.S N.S R2=0.40 N.S

F3,10=30.0 F3,10=19.0 F3,10=3.92

+* 2 affinis N.S R =0.30 N.S N.S N.S N.S F3,25=5.03

+* varia N.S N.S R2=0.16 N.S N.S N.S

F3,25=2.79

+* +* 2 2 nodosa N.S N.S N.S N.S R =0.27 R =0.27 F3.25=4.39 F3.25=4.22

+* +** tohiveana N.S N.S N.S N.S R2=0.31 R2=0.38 F =5.21 F =6.80 3.25 3.25

72

Table 4.3. Effects of temperature and humidity on juvenile mortality. No significant effects were found for either mean temperature or maximum humidity

Temperature Humidity

Max Min Range Mean Max Min Range Mean Species

+* +** tohiveana R2=0.16 N.S R2=0.20 N.S N.S N.S N.S N.S F1,27=6.39 F1,27=7.92

+* tristis R2=0.18 N.S N.S N.S N.S N.S N.S N.S F1,17=5.01

-** +** nodosa N.S N.S N.S N.S N.S R2=0.20 R2=0.19 N.S F1,27=8.20 F1,27=7.75

-* +* varia N.S N.S N.S N.S N.S R2=0.13 R2=0.12 N.S F1,27=5.31 F1,27=4.70

-* s. simulans N.S N.S N.S N.S N.S N.S R2=0.71 N.S F1,4=13.0

-** +** -** subgonochila N.S R2=0.39 R2=0.52 N.S N.S R2=0.45 N.S N.S F1,12=9.34 F1,12=15.0 F1,12=10.7

-* +** -* hebe bella N.S R2=0.19 R2=0.23 N.S N.S N.S N.S R2=0.12 F1,27=7.61 F1,27=9.32 F1,27=4.74

73

Table 4.4. Significant interactions between temperature and humidity, and juvenile mortality.

Interactions

Species Min H: Mean H: Max H: Mean H: Max H: Mean T: Mean H: Min T Max T Min T Min T Max T Max H Mean T +** clara R2=0.25 N.S N.S N.S N.S N.S N.S F3,25=3.89

-* varia R2=0.28 N.S N.S N.S N.S N.S N.S F3,25=4.57

+* gibba R2=0.24 N.S N.S N.S N.S N.S N.S F3,25=3.9

+** radiolata N.S N.S N.S N.S R2=0.67 N.S N.S F3,6=7.22

-* -* faba N.S N.S R2=0.15 R2=0.19 N.S N.S N.S F3.25=2.62 F3.25=3.18

-* -* -* hyalina N.S N.S R2=0.15 R2=0.16 N.S R2=0.18 N.S F3.25=2.62 F3.25=2.78 F3.25=3.11

-** -* +* t. simulans N.S R2=0.99 N.S N.S N.S R2=0.91 R2=0.93 F3.2=211 F3.2=18.8 F3.2=23.9

74

Table 4.5. Effects of temperature and humidity on fecundity. No significant effects were found for maximum temperature, temperature range, minimum humidity and humidity range.

Temperature Humidity

Species Max Min Range Mean Max Min Range Mean

* + 2 clara N.S R =0.11 N.S N.S N.S N.S N.S N.S F1,25=4.28

*- nodosa N.S R2=0.14 N.S N.S N.S N.S N.S N.S F1,27=5.64

***- **- varia N.S R2=0.32 N.S R2=0.27 N.S N.S N.S N.S F1,27=14.1 F1,27=11.6

*- *- s. strigosa N.S R2=0.11 N.S R2=0.11 N.S N.S N.S N.S F1,27=4.44 F1,27=4.44

*- *- subgonochila N.S N.S N.S N.S R2=0.31 N.S N.S R2=0.31 F1,12=6.8 F1,12=6.84

75 Chapter 4. Temperature and Humidity

4.5. Discussion

The environmental factors (i.e. temperature and humidity) to which Partula snails are exposed in the wild fluctuate significantly, and vary between the different geographical regions and habitats where the genus is endemic (figure 4.1). This variation in the wild suggests that in captivity different species of Partula may require different conditions for optimal survival and reproduction.

Klocek (1993) noted that seasonality in the Pacific is quite distinct from that experienced in Europe. Analysis of the data from Tahiti and Moorea (Metéo-France, 2010) from 1980 to 2008, indicate that temperature and humidity fluctuations in the wild are rather different from the fluctuations experienced in captivity, and as a result they are most likely to influence sustainability of the Partula population (figure 4.1).

In the majority of the Partula populations monitored here, fecundity and mortality were influenced significantly by both temperature and humidity.

For many species, adult mortality was positively correlated with maximum temperature and/or negatively correlated with minimum temperature, indicating that either too high a temperature in a preceding month or too low a temperature increased monthly.

Most significant effects detected with humidity (maximum, minimum and mean) were negative, indicating that affected snails survived better when humidity was consistently high.

Mortality correlated significantly with environmental variables more often than did fecundity (Table 4.1-4.4 versus 4.5). The fact that more significant effects were found with adult snails probably reflect their longer mean standing population sizes and hence greater statistical power in the results.

It is interesting to note that adult mortality in P. subgonochila (Table 4.1) was negatively correlated with minimum temperature and mean temperature. This species

76 Chapter 4. Temperature and Humidity originated on the islands of Alofi and Fotuna, and all of the individuals were found in coastal forests on limestone where the animals were more exposed to high temperatures which might indicate that a drop in the minimum temperature may negatively influence the survival of the adults of the species. Analysis of P. radiolata from Guam did not show any correlation between environmental factors and adult mortality, Crampton (1925), noted that “the Mariana

[Guam] species differ constitutionally from the majority, as manifested by their existence in drier and more open situations”. This can be an indication that the species from Guam have evolved a higher capacity to withstand environmental stress.

It is, however, important to take into consideration that different species responded differently to the same environmental conditions. This is a good indication that, for a successful breeding programme and consequently future reintroductions into the wild,

Partula snails should be kept in improved closer replication to their natural environment, maybe with species maintained in species specific habitats with appropriate environmental conditions.

It is also important to consider the possible benefits of in-situ conservation within

French Polynesia since the environmental patterns i.e. temperature and humidity will be easier to reproduce and control.

It is possible that other associated factors such as light and reduced natural solar radiation, which is reported in the literature as a factor affecting animals in captivity (Morgan

& Tromborg, 2007), may also have significant effects on the population dynamics in the captive populations, and further research and quantification of their possible influence is needed. Indeed, Crampton (1932) stated that “the areas of habitation in the islands of their occurrence are conditioned by a complex of environmental circumstances, such as moisture, shade, higher temperature and altitude”.

77 Chapter 4. Temperature and Humidity

Finally, it may be beneficial to investigate environmental conditions in other captive

Partula collections. This aspect was challenging during this study because most collections had not stored their data.

It is hoped that these findings may stimulate further investigation in this field.

78 Chapter 5- Light Intensity

Chapter 5. Investigation into the role played by light intensity on

the behaviour, reproduction and mortality of Partula snails.

5.1. Abstract

In animals, light can determine idiosyncratic behavioural patterns which make up their adaptations to the environment. Animals, plants and other organisms depend on light as an exogenous cue which is involved in the resetting of circadian clocks and the modulation of activity patterns. The influence of light levels on molluscs has been subject of numerous research studies. These studies have given rise to analogous observation amongst scientists, showing that light clearly influences several biological processes.

In theory one would expect snails to be negatively phototropic or to be nocturnal or crepuscular since light affects their vulnerability to desiccation and predation. However, their behaviour in relation to light intensity is controversial. In addition, there is limited information on behavioural responses to light levels among terrestrial snail species. This study investigates the effects of light levels on the population dynamics of captive Partula snails and assesses their behaviour when exposed to different light intensities.

The results of this study indicate that the snails, at rest, have a negative phototropic and

2 geotactic behaviour. Analysis also found that mortality (R = 0.80, F1,10= 42.19, p < 0.001) was significantly positively correlated with the presence of light. Light was also found to be

2 positively correlated with the fecundity of the population (R = 0.34, F1,10= 6.67, p < 0.05).

79 Chapter 5- Light Intensity

5.2. Introduction

In many animals, light is the principal synchronizing factor (Dumortier, 1981 in Gomot,

1990). It is a determinant of certain environmentally adaptive behavioural patterns. Animals, plants in particular depend on light as an exogenous cue, for the resetting of circadian clocks and the modulation of activity patterns (Newcomb et al., 2004).

The influence of light on molluscs has been subject of research for many years. These studies have given rise to analogous observation among scientists, where light has been shown to have a clear influence on locomotion (McKillop & Harrison, 1982; Pimentel-Souza et al., 1984), activity (Lewis, 1969; Bailey, 1975; Pieri & Jurberg, 1981), feeding, reproduction and oviposition (Joy, 1971; Sokolove & McCrone, 1978; Hien, 1981; Williams

& Gilbertson, 1983; Rotenberg, 1989; Ligaszewski et al., 2007).

The distribution of Partula snails in the wild depends on several factors. Murray et al.

(1982) demonstrated they are highly associated with specific plant species. Also, the distribution of species within the plant varies greatly.

“Despite considerable overlap there are differences in the distribution of the snails within habitats. Partula suturalis is the most arboreal species. It is found particularly on

Hibiscus tiliaceous, often above three meters from the ground. Partula taeniata is more generally distributed closer to the ground and is found chiefly on woody shrubs and

Angiopteris. Partula tohiveana and P. mooreana are strongly associated with Freycinetia demissa and Partula mirabilis, appears to occupy an intermediate position” (Murray et al.,

1982). One can speculate that the differences in light intensities and even the frequency of the radiation encountered by these species will be different, since sunlight is reflected and diffused once it goes through the different plant levels. Crampton (1916) classifies the species into three different types based on their habitat: they can be ground-dwelling, semi-arboreal

80 Chapter 5- Light Intensity and arboreal. The ground dwelling species were found at the lowest level, generally the forest floor, crawling under stones, dead trees and leaves (e.g. Partula producta, P. labrusca).

These snails had positively geotactic and negatively phototactic behaviour. The second group, semi-arboreal, comprise the greater number of species. These species were, and still are (as some species still survive) arboreal during daytime, remaining sealed on the undersides of leaves around three to four meters from the ground, though they crawl down to the ground at night in order to feed of the topsoil. The snails within this group would therefore display positive geotactic behaviour during the night and negative behaviour during the day. Like the ground-dwellers, they are negatively phototactic. The third group, strictly arboreal, spend their life higher in trees, where they possibly survive by feeding on algae and fungi growing on twigs, branches and leaves (e.g. Samoana attenuata). This group of snails have invariably negative geotactic behaviour and, in common with the other groups, are negatively phototactic (Crampton, 1916).

In their natural environment, animals are exposed to a wide variety of factors that will directly and indirectly affect their behaviour. For example, when exposed to conflicting stressors Lymnaea auricularia prioritized temperature over light (Rossetti & Cabanac, 2006).

In theory one would expect snails to be negatively phototropic or to be nocturnal or crepuscular since light affects their vulnerability to desiccation and predation. However, their behaviour in relation to light intensity is controversial (Rossetti & Cabanac, 2006) since similar studies reach different conclusions. In addition, there is limited information on behavioural responses to light levels among terrestrial snail species. Nevertheless, there is a general consensus that light does influence several biological processes in gastropod molluscs, though there is no clear pattern between light intensity changes and their behaviour

(Perea et al., 2007). Given the limited research in this area a study was devised to further

81 Chapter 5- Light Intensity clarify the effects of light on the population dynamics of Partula snails. As a complement to this study, phototactic and geotactic behaviour was also taken into account.

5.3. Methods

In order to effectively establish the influences of light on the population under study, two different methods were used. The first was aimed at the spatial distribution and phototropism of snails while the second relates to the effects of light intensity on population dynamics

(fecundity and mortality). Partula gibba was the study species in both experiments. The snails were housed under controlled laboratory conditions where temperature varied between

22ºC at night and 23ºC during the day. Humidity was maintained between 70 and 80%.

Light was provided by fluorescent Silvania triphosphor polylux XL cool white lamps, colour temperature 4300K driven by high frequency ballast on a twelve hour day and night photoperiodic cycle.

The substrate consisted of layers of moist bleach-free paper tissue and all the containers were covered with cling film to maintain humidity.

The snails were fed ad libitum on a diet made up of 300g of porridge oats, 150g powdered trout pellets, 600g powdered grass pellets, 300g calcium carbonate, (CaCO3,

Mineral Technologies Inc.) and 25g of vitamin powder (Vetzyme, Seven Seas Ltd.). The diet was moistened with filtered water to form a runny paste and then microwaved for approximately two minutes for sterilisation. All the materials used to handle, feed, house and clean the snails had been previously sterilised/disinfected in a microwave or sterilized with aqueous sodium hypochlorite solution.

82 Chapter 5- Light Intensity

5.3.1. Spatial distribution and phototropism during resting period (day).

For five months phototropic behaviour and spatial preference were recorded every week.

Eleven snails were randomly selected and placed in a box (16cm x 28cm x 9cm) divided into three distinct areas (dark, dim and light, Fig. 5.1) allowing space for the snail to actively select the preferred area. Every week the number of snails in each area, and their spatial distribution within the box (categorized has top or bottom), was randomly recorded. To complement this study a video camera was set up for two weeks in the laboratory and behaviour was recorded every day.

Figure 5.1. Selection box

5.3.2. Effects of two different light intensities on the dynamics of the population.

For this experiment adult snails (n=12) were selected and randomly placed into individual plastic containers (11cm x 17cm x 5cm). They were then divided into two groups. Group A was placed in darkened boxes covered with black opaque plastic film, except for the front side of the box, and group B in transparent boxes exposed to the light. For each group there were six replicates. Food, tissue and the containers were replaced every fortnight, and the

83 Chapter 5- Light Intensity numbers of newborns (measure of fecundity) and mortality were counted. The experiment lasted approximately eight months.

Analyses of the data collected were performed using the statistical software R (Ihaka

& Gentleman, 1996).

5.4. Results

Part A of this study provided sufficient data to indicate that captive Partula snails are negatively phototropic. On average 70% of the snails were found either in the dark (54.6%) or in dim (14.7%) areas of the experimental set up. It was also observed that an average of

71% of the snails had a preference for the top of the tank.

Part B of this experiment looked at the effects of two light intensities (dark and light) on the dynamics of the study population. Both Welch Two Sample t-test and linear models were performed to establish the relation between the variables and the dynamics of the population.

There was sufficient evidence from t-test analysis to accept the hypothesis that light plays a role in the mortality of the population (at 95% confidence interval t = 6.18, df = 5, p- value < 0.01). There was also sufficient evidence to accept the premise that light influences the reproductive success of Partula gibba (at 95% confidence interval t = 2.67, df = 5, p- value < 0.05).

Statistical analysis indicates that mortality was significantly and positively correlated

2 with the presence of light (R = 0.80, F1,10= 42.19, p < 0.001). Light was also found to be

2 positively correlated with the fecundity of the population (R = 0.34, F1,10= 6.67, p < 0.05).

Approximately 92% of the deaths occurred among either newborns or juveniles.

Figures 5.1 and 5.2 show graphically, the effects of light on mean fecundity and mortality.

84 Chapter 5- Light Intensity

Figure 5.2. Effects of two light regimes on the fecundity of Partula gibba.

Figure 5.3. Effects of two light regimes (dark and light) on the mortality of Partula gibba.

85 Chapter 5- Light Intensity

5.4. Discussion

Light intensity was found to have a significant influence on the population dynamics of

Partula snails.

Higher light intensity had a negative effect on snail survival. Partula snails in the wild spend most of their time on the underside of leaves below the forest canopy. The intensity and quality of the sun radiation that reaches the animals is difficult to measure and is very difficult to reproduce in captivity. This is an important factor to be considered and given the appropriate relevance.

The majority of the remaining Partula species are semi-arboreal. Following Crampton

(1916) these species would remain sealed upon the underside of leaves during the day, resuming their activity at dusk. This is consistent with constant negative phototactic behaviour.

During the night these species behave positively towards gravity, descending to the ground to feed on the topsoil, while at daytime a negative behaviour is observed, remaining in the trees and bushes protected from the environmental stresses to which they are exposed.

The findings of this study concur with previous observations and to some extent confirm them statistically.

The test population displayed negative phototactic orientation and when at rest, showing a preference to rest at the top of the experimental containers. Despite food being presented on the side of the containers, when active, the snails would spend most of their time on the bottom or sides.

It is well documented in the scientific literature that light influences molluscs in various ways. If this variable is not adjusted to the particular needs of the target species the fine balance that is required is not achieved, and this impacts negatively on their behaviour

86 Chapter 5- Light Intensity and ability to adapt to their environment. Although this is very important, it is clear from these experimental results that the implication of light on the wellbeing of captive populations is rather more complex. From the data collected it was possible to infer that high light intensity increases both mortality and fecundity of the test population. Specifically, high light intensity is critical for the adult development stages (increase in fecundity) but detrimental on newborn and juvenile survival, where approximately 92% of the recorded deaths fell within the juvenile and newborn development stages.

Results indicate that higher light intensities are requirements for a fecund and healthy adult population but at the same time have an adverse effect on the survivability of juvenile and newborn development stages. This conclusion may be extrapolated from the wild where newborn and juvenile snails are at higher risk of desiccation and predation if directly exposed to sunlight.

Zieger and Rochow (2008) concluded that terrestrial pulmonate gastropods use their eyes primarily for monitoring environmental brightness level and move accordingly towards shadier places. This adaptation is imperative in the wild when developing strategies to avoid predation, overheating and/or dehydration should in some ways be highly regarded and when possible keeping this in mind when setting up captive conditions.

87 Chapter 6. General Discussion

Chapter 6. General Discussion

The main objective of this thesis was to investigate the factors that affect the population dynamics of captive Partula snails. Results of the research and their implications have been comprehensively discussed in their respective chapters. This last chapter aims at a brief overall discussion and consolidation of the findings. Other studies were undertaken, although they are not presented in the main document, either because these produced insufficient results or the lack of funding restricted further work; these are still to be considered and can be found in the appendix.

The significance and impact of this work should be taken into consideration when developing or updating protocols for captive Partula as it is evident that the status of the entire Partulidae family in the International Union for the Conservation of Nature Red List

(IUCN 2007) is extremely fragile. In French Polynesia 45 species out of 77 are classified as extinct (EX) with a further 11 extinct in the wild (EW), nine critically endangered (CR) and two vulnerable (VU). There is insufficient data for five species (Coote, pers. comm., 2011).

Therefore, any further understanding of these species will only bring benefits to the conservation efforts of a highly endangered family which has been declining alarmingly for the past four decades. Without such efforts they will unquestionably face an uncertain and gloomy future. Mass mortality episodes observed in captive populations over the years have resulted in the loss of several species.

Partula snails have been studied for more than a century due to their morphological and genetic diversity which has provided scientists with excellent material to study adaptation and speciation (Crampton, 1916, 1932; Murray & Clarke, 1966; Clarke & Murray, 1969;

Murray & Clarke, 1980; Murray et al., 1982; Johnson et al., 1987; Murray et al., 1988;

Davison et al., 2009). They have been placed on the same platform as Darwin‟s finches

88 Chapter 6. General Discussion

(Randerson, 2003) since their study have contributed greatly to our understanding of evolutionary processes, although there is still much to learn about their biology.

This thesis is separated into two different approaches. The first investigates the influence that environmental variables (temperature and humidity and light intensity) play on the dynamics of the Partula population while the second approach investigates the influences of diet on the dynamics of the Partula snail population.

“Temperature and humidity are two of the most critical environmental factors that govern the geographic distribution, health, and survival of all animal and plant life” (Gordon,

2003). As seen in Chapter 4 both temperature and humidity significantly influenced the dynamics (mortality and fecundity) of the Partula species monitored in this study. The species predominantly favoured a stable environmental temperature and high constant relative humidity. As one would expect, given the distribution of Partula snails in the wild, different species responded differently under the same environmental conditions.

Light intensity also influenced the dynamics of the Partula snail populations under study. Higher light intensities resulted in approximately 92% mortality among the juvenile and newborn development stages. Partula snails in the wild spend most of their time on the underside of the leaves, but the intensity and quality of sun radiation that reaches the animals in the wild is not being reproduced in captivity. This should be considered since Partula snails are directly influenced by light. The majority of the remaining Partula species are semi-arboreal and in the wild they spend the day sheltered under the leaves to avoid desiccation. These species resume their activity at nightfall to feed protected from the environmental stresses and predation. The results from this chapter are largely in agreement with Crampton‟s observations (1916). Partula gibba showed positive geotactic behaviour when active and negative at rest. Additionally they displayed negative phototactic behaviour.

Several studies have found that light influences molluscs in a diversity of ways (Lewis, 1969;

89 Chapter 6. General Discussion

Joy, 1971; Bailey, 1975; Sokolove & McCrone, 1978; Hien, 1981; Pieri & Jurberg, 1981;

McKillop & Harrison, 1982; Gilbertson, 1983; Pimentel-Souza et al., 1984; Williams &

Rotenberg, 1989; Gomot, 1990; Ligaszewski et al., 2007). If this is not balanced it will influence the snails‟ behaviour and their capacity to adapt to their environment.

Both Chapter 2 and 3 focused on the influences that the artificial diet, designed for the breeding programme, play on the dynamics of Partula snails. These studies strongly indicate that diet/nutrition is one of the most important factors affecting the development and health of

Partula species in captivity. For this reason, the investigation into the constitution of the diet currently in use is of great importance. The nutrient requirements of Partula snails are poorly studied and further attention should be given to this subject. It is generally accepted that calcium is particularly important for molluscs. Lack of calcium can be a limiting factor on their distribution, because it influences growth and development as well as reproduction, oviposition and several other biological processes. In Chapter 2 it was shown that an increased level of calcium carbonate in the diet of Partula snails brought benefits to their growth, development and reproduction and as a result improved their wellbeing.

The study in Chapter 3 aimed to evaluate the benefit to snails of the addition of dog and cat vitamins to their diet. The results, however, showed a clear positive correlation between the addition of vitamins in the diet and increased mortality in the population under study. In other words, the diet with added vitamins had an adverse effect on snails‟ survivability. The addition of vitamins in the diet is not questionable although in order to provide the snails with the correct vitamins and minerals as well as dosage it is imperative the further understanding of the nutritional requirements for the target species, there is the possibility that they are being fed either the incorrect vitamins and minerals as well as the incorrect dosage.

90 Chapter 6. General Discussion

Despite the fact that no causative pathogenic agents were associated to specific incidents of increased mortality in this study, the possibility of an as yet unidentified infectious agent remains a promising area for further investigation. Even if a microorganism is isolated, both environmental conditions and diet may well be significant in the epidemiology and pathogenicity of any infectious agents present. There is also the possibility of normal gut flora becoming opportunistic pathogens under different stresses experienced by the snails and as a result contributing in part to population fluctuations.

Recommendations

Taking into consideration the finding and deliberating on the status of Partula snails it is recommended that a full review of the snails‟ diet is to be considered. Both the increase of calcium in the diet and the investigation of the vitamins requirements are of great importance for the improved survivability and welfare of Partula snails in captivity. As metioned in chapter 2 and 3 there are unsolved issues with the diet and these needs to be addressed. It is never enough to highlight the fact that the calcium concentration needs to be increased and the vitamins for dogs and cats removed from the diet currently in use.

It was also found that the light and the temperature and humidity influence the dynamics of the Partula population. As indicated in chapter 5 light does influence the dynamics of Partula species under study, it is therefore recommended the separation of the newborns from adults into a lower light intensity area since light significantly positively correlated with mortality, 92% of the recorded deaths being juvenile and newborn snails.

Adult snails should be kept separately from newborns due to the fact that light is essential for a fecund population.

In the case of temperature and humidity it remains a challenge the adaptation to a more natural reproduction of their environment. It is however imperative to adjust this values

91 Chapter 6. General Discussion in order to attain a balance for the different species. It is now known that the different Partula species behave differently under the same environmental factors and so the best outcome would be the immideate near future introduction of all species into their natural environment.

It is of great importance to keep remembering that captive Partula are no longer the same species and if keep in the same condition without an improved reproduction of their environment and diet they will further loss their identity both at the physiological and genetic levels. Partula snails may have a certain degree of domestication, due to selection pressures of long term captivity. This should be carefully evaluated to ascertain the biological effects of long term captivity and to help in the understanding of the improvement of their captive environment.

It is hoped that these findings stimulate further research in this area and that they can be applied to other species which are being maintained under similar conditions.

92 References

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114 Appendix

Appendix

A.1. Pathogen investigation

The decline of Partula snails in captivity has been under the scrutiny for many years, with numerous reports of sudden and increased mortality in different populations. Consequently, several theories have been put forward over the years to try and account for these incidents.

Cunningham and Daszak (1998) reported the extinction of Partula turgida resulting from a microsporidian parasite (Steinhausia sp.), Jersey Zoo reported that population fluctuations in their Partula collection were due to density dependent diseases, and mites have also been reported to cause increased mortality in Partula populations at both Detroit Zoo, and Virginia

University (Tonge & Bloxam, 1990). However, to date, the work of Cunningham and Daszak

(1998) is the only published scientific evidence about the nature of these. Because of these uncertainties all snails that died during the studies described in this thesis were subjected to post mortem investigation as follows.

The examination consisted of an initial macroscopic external observation of the specimen, followed by morphometric measurements. After removing the body from the shell, the viscera were then checked for any gross abnormalities. Both modified Ziehl-Neelsen and

Trichrome Ryan Blue stains were used to identify possible infectious pathogens with particular attention given to Microsporidia. For Trichrome Ryan Blue the protocol used was that of Ryan (1993) and Garcia (2001) while for the modified Ziehl-Neelsen the protocol from pathology department of London Zoo was used. For comparative purposes both methods were used for each specimen examination. As a control Anopheline mosquitoes were

115 Appendix infected with Vavraia culicis spores from the microsporidian parasite. Only exceptionally rare occurrences of microsporidia were observed.

Post mortem results during the study period were unremarkable and bacteria isolated were identified as normal gut flora or secondary microbial activity also found in other snails examined where no population declines were apparent.

Other microorganisms including nematodes and mites were also observed. The mites were all in the juvenile development stage and further identification was not possible. The nematodes, identified as Caenorhabditis elegans, were sent to Dr. Sergei Spiridonov at the A.N.

Severtsov Institute of Ecology and Evolution Centre of Parasitology in Russia, which used the following protocol in Ivanova and Spiridonov, (2011):

The modified protocol by Floyd et al. (2002) was used for DNA extraction from Dicelis samples fixed in 70% Ethanol. Ethanol fixed nematodes were transferred to 5 μl 0.25M

NaOH in eppendorf tubes, centrifuged and incubated at 99 °C for three minutes. Samples were then cooled to room temperature and 1 μl 1M HCl, 2 μl 0.5M Tris-HCl (pH=8.0) and

1.25 μl of 2% Triton X-100 were added. The tubes were re-centrifuged and again incubated at

99 °C for three minutes. About 0.4-0.8 μl of resulting mixture was used as DNA template for

PCR.

Alternatively, the column extraction from the kit Wizard® SV Genomic DNA Purification

System (A2360) of Promega company was used, the reactions were done using Qiagen®

PCR kits according to the manufacturer‟s instructions.

116 Appendix

The second approach was found to be more effective, 500 µl of DNA solution were concentrated in ethanol precipitation into 50 µl (ten times).

Primer pair „A-left‟ (tMY-cYc-Rtt-gat-Yct-gYc) proposed by Dr. Aleshin (Belozerskii

Institute, Moscow State University) and 26R (GCT-TTC-GTA-AAC-GGA-AGA-ATG) were used to amplify part of 18S rDNA. PCR cycling parameters included primary denaturation at

94 °C for five minutes followed by 34 cycles of 94°C for 60 seconds, 54°C for 90 seconds

(for amplification of 18S rDNA) and 72°C for one minute, followed by post-amplification extension at 72°C for ten minutes. The PCR reaction products were visualized in agarose gels and bands were excised for DNA extraction with Qiagen® gel extraction and product cleaning kits. After ligation with pGEM-T vector, PCR products were used to transform JM

109 Escherichia coli competent cells (according to Promega protocol). Transformed colonies were sub-cultured in 1.5 ml of LB medium and incubated for 16 to 22 hours at 37°C. Plasmid

DNA was obtained from transformed E. coli using „QIAprep Miniprep Kit for purification of plasmid DNA‟ according to the manufacturer‟s instructions and used for sequencing using

BigDye® Termination Kits with 3.2 pmol of vector primers T7 and SP6. Termination reaction products were ethanol-precipitated, air-dried and re-suspended in ABI Prism®

Template Suppression Reagent.

For comparative purposes and phylogeny construction, the several closely related 18S rDNA sequences deposited in GenBank were used. These sequences were discovered through

BLAST search in NCBI GenBank.

Sequence alignments were generated using Clustal X under default values for gap opening and gap extension penalties. All alignments were analysed using PAUP* 4.0b10 (Swofford,

1998 in Ivanova & Spiridonov, 2011) for maximum parsimony (MP). Maximum parsimony analysis was performed with gaps treated as „missed data‟. The heuristic search procedure

117 Appendix was used with 100 replicates of random taxon addition. Bootstrap (BS) analysis with 1000 replicates was conducted to assess the degree of support for each branch on the tree using simple addition sequences with TBR swapping (tree-bisection-reconnection). The bootstrap values are indicated near appropriate nodes of the trees. Trees were displayed with TreeView

1.6.1 (Page, 1996).

Figure A.1. Phylogenetic relationships of the Caenorhabditis species nematode from

Tahitian snails and closely related Rhabditidae.

Criterion - parsimony. Of 539 total characters: 258 characters are constant, 113 variable characters are parsimony-uninformative, number of parsimony-informative characters – 168.

118 Appendix

Bootstrap values are indicated near nodes. The length of branch corresponds to the nucleotide differences (scale – 10 base pair difference) (Spiridonov, pers. comm., 2011).

119 Appendix

A.2. Metabonomics

Attempts were also made to look for possible metabolic differences between Partula species using nuclear magnetic resonance (NMR) with Dr. Jake Bundy at Imperial College of

London. The preliminary results for the test samples of two species (Partula varia and P. clara) demonstrated that there were remarkable differences between them Figure A.2.

Although further investigation was not possible due to lack of funding (NMR is an expensive procedure), further investigation in this field should be considered if funding becomes available.

Partula varia

Fig A2.1

Partula clara

Fig A2.2

Figure A.2. (Fig.A2.1 aliphatic region; Fig.A2.2 aromatic region). Top: P. varia. Bottom: P. clara.

120 Appendix

A.3. Discussion

The occurrence of microsporidia in this investigation was very small and solely observed in one snail; this evidently indicates that it cannot be associated with any incident of mortality, since microsporidial spores would ubiquitously spread to the entire population maintained in the same container and later to the entire collection maintained in the same controlled temperature room. Furthermore, the prevalence of the disease, in this case a microsporidian infection, was reduced to one single individual. Additionally the epidemiology of the microsporidia is unknown as the species was not identified. Accepting the scenario where the

Steinhausia species was present, its routes of transmission, pathogenecity, prevalence, incidence, virulence, lethal and infective dosages as well as further aetiological information regarding the hypothesis of microsporidia as a causal agent of mortality in Partula snails is essential in the diagnosis. It is cautionary to infer that the positive identification of a suspected pathogen does not necessarily mean that the microorganism is the causal agent for the disease. It is, however, accepted that this potential infection does not fulfil modern

“Koch‟s postulates” (Fredericks & Relman, 1996) and hence, the microsporidia should not be scientifically identified as a causative agent in the mortality of Partula snails.

C. elegans are facultative necromenic species and therefore do not appear to have contributed to the mortality of captive Partula snails.

Whilst no causative infectious agents have been associated with the specific incidents of increased mortality in these studies, the possibility of an as yet unidentified infectious agent remains a promising area for further investigation. For example, there is the possibility of normal gut flora acting as opportunistic pathogens under environmentally-stressed conditions, and so in part contributing to population fluctuations.

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A.4. Post Mortem Protocol for the Polynesian Tree Snails (Partula and Samoana)

(Pearce-Kelly et al., 2006)

 set up for necropsy

 weigh the snail

 Take measurements of the shell as illustrated on the figure bellow

Figure A.3. Snails‟ morphometric measurements

 Height/Length (A-B),

 Width (C-D),

 Lip height/Length (B-F) and

 Lip width (E-D).

 Note whether the shell coils to the right (dextral) or to the left (sinistral) and whether or

not the aperture is lipped (evidence of sexual maturity).

 Remove the snail from the shell, either by gentle steady traction or by chipping open

the shell using bone forceps.

122 Appendix

 Identify grossly visible anatomical structures including the apex, foot, mantle, tentacles,

digestive gland and lungs if present

 Examine the snail for granulomas, fungal infections, or other obvious external

abnormalities.

 In snails too small for gross recognition of most internal lesions (e.g. Partula and

Samoana), avoid internal dissection.

 Take tissue samples or swabs for bacterial and fungal culture of any lesions.

 If fresh faeces are present, examine a direct faecal wet mount for parasites.

 Amputate the apex using a sterile scalpel blade.

 Make two or more touch impressions of the apex and apply modified Zielhl-Neelsen

and Gram stains.

 Examine cytology preparations, in particular searching for protozoan parasites.

 Freeze a portion of the foot for DNA bank.

 Save the carcase including the amputated apex in 10% neutral buffered formalin or

70% ethanol.

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