The reproductive biology and movement
patterns of the draughtboard shark,
(Cephaloscyllium laticeps ): implications
for bycatch management
by
Cynthia Andrea Awruch
Submitted in fulfilment of requirements for the Degree of
Doctor of Philosophy
January 2007
Tasmanian Aquaculture and Fisheries Institute
School of Aquaculture
University of Tasmania, Australia
Draughtboard shark, Cephaloscyllium laticeps
ii DECLARATIONS
I hereby declare that this thesis is my own work except where due acknowledgement is given, and that the material presented here has not been submitted at another university for the award of any other degree diploma.
This thesis my be made available for loan and limited copying in accordance with the Copyright Act 1968
Cynthia Andrea Awruch
January 2007
iii ABSTRACT
The draughtboard shark ( Cephaloscyllium laticeps ) is the most common shark on temperate reefs in southeastern Australia. In order to implement adequate management plans its reproductive biology and movement patterns were studied.
Females developed a single external type ovary with a maximum follicle diameter of 35 mm. Vitellogenesis commenced at 10 mm follicle diameter. The male reproductive tract consisted of paired testis with spermatocysts undergoing diametric development.
The hormones testosterone, 17 β estradiol, progesterone and 11 ketotestosterone
(males only) were examined to determine their role in reproduction. Testosterone and estradiol showed major changes during follicle development. Estradiol increased as the follicle developed before declining as the follicle reached maturity. Testosterone remained low during the first stages of follicular development and increased as the follicle reached maturity. Progesterone showed a peak just prior to ovulation.
Testosterone was the only hormone that varied with maturity in males and no levels of
11 ketotestosteorne were detected.
Females were able to store sperm for at least 15 months and eggs were laid in pairs at monthly intervals. Juveniles hatched after 12 months.
The size at maturity and seasonality of reproduction were estimated using reproductive parameters obtained from dissected animals and from steroid hormones. The sizes at onset of sexual maturity by both methods were similar. Females laid eggs throughout the year with a peak in deposition between January and June. Elevated values of testosterone and progesterone coincide with this period of egg deposition. Males showed no seasonal pattern in reproduction although both testosterone and the amount of sperm in the seminal vesicle were marginally higher in the first semester of the year.
Movement studies were undertaken using conventional and acoustic tagging. The area of study included a marine reserve and the adjacent bays of southeast Tasmania. Both
iv methods demonstrate that the majority of sharks remained in the same region in which they were tagged, although a few sharks moved large distances. Sharks were active throughout the day and night with peak activity during dawn and dusk. This species could remain stationary on the bottom for periods up to five days. No correlation was found between activity and lunar patterns and both sexes showed similar activity patterns
This study has provided the first information on reproduction and movement of draughtboard shark and demonstrated the potential for hormones to provide reproductive information necessary for management without the need to sacrifice the shark.
v ACKNOWLEDGMENTS
I would like to start these acknowledgments by saying THANK YOU to my supervisors: Ned Pankhurst, John Stevens and Stewart Frusher. I will be forever grateful to them. I could not have done this study without their constant support; it has been a great pleasure and honour to work side by side with you all. I would specially like to say thanks to Ned for giving me the opportunity to come to Australia to complete this degree, for his patience and time in the laboratory, for discussing ideas and opening my mind. To John, for his support and advice since the very beginning and for showing me how to be a good scientist, but also, and more importantly, how to be a good person in science. And finally to Stewart, for absolutely everything, for spending endless hours with me helping with everything, discussing every detail, making me think, and for being always patience and open to see me. I would like to extent my thanks to Chris Carter, for his understanding and help. Thanks to Stewart’s family for accepting me in their house and for showing me such kindness. To the Tasmanian Aquaculture and Fisheries Institute, Marine Research Laboratories, and the School of Aquaculture for their funding support. I could not have done this research without the help of many commercial and recreational fishermen that collected samples and gave me tag returns. I would particularly like to thank Neville Perryman, for collecting so many sharks and for being so helpful. Bryan Hughes offered me great help by sharing his insight and knowledge when studying the gestation period of the sharks. To all the rock lobster section at Marine Research Laboratories, especially, Shane Fava, Craig McKinnon, Pip Cohen and Robbie Kilpatrick for their help on land and aboard the “Challenger”. A big thanks to Caleb Gardner for his kindness and constant support. I am very thankful to all the students at Marine Research Laboratories and members of the endocrine laboratory at the School of Aquaculture, Peter Lee, Hannah Woolcott and Quinn Fitzgibbon. Thanks to all of you for being always so helpful and for creating a fun research environment. Special thanks to Tobias Probst, for his constant help and for making me laugh so much! Matias Braccini and Javier Trovar Avila, thanks for always being there and for our time together, it has been great have your friendship and your scientific advice. To Sarah Irvine for her constant support, for the fun at the conference, and for always cheering me up; to Michelle Treloar, for her constant encouragement, and to Cass Hunter for being such amazing friend through all these years. I feel so lucky for the times that we have shared. Jayson Semmens, Colin Simperdorfer and Michelle Heupel gave me helpful advice in the analysis of the tracking data. Thanks also to Colin and Michelle for their hospitality when I stayed in U.S.A. Julian Harrington offered me invaluable assistance in the use of the GIS software. Malcolm Haddon, Phil Ziegler, Al Hirst, Barry Bruce and Hugh Pederson all assisted with data analysis, for this I thank you.
vi I could not have come to Australia without the invaluable help of my very good friends Cecilia Arighi and Sergio Schuchner. Without them, this PhD would be nothing but a dream. My stay in Australia has been a wonderful experience, I have come to know amazing people and I had the chance to make very good friends for life, thanks to all of you. How can I express my gratitude to my friends Louise Ward and Justin Ho? I will be forever grateful for their constant support, for always being there for me, and for making my life so much easier and happier in a new country. To my Argentinean friends, Lali, Marce and Ferchu, for, as usual, being such a good and important friends and for keeping our friendship without seeing me for all these years. To my uncle Juan, my aunty Raquel, my cousins, and specially my brother Alejandro, without your support and love I would not have done it, thank you.
vii TABLE OF CONTENTS
GENERAL INTRODUCTION 9
CHAPTER ONE : 15
CHAPTER TWO : 18
2.1 INTRODUCTION 19 2.2 MATERIAL AND METHODS 25 2.2.1 Source of samples and data collection 25 2.2.2 Steroid hormone measurement 28 2.2.3 Classification of reproductive stage of the sharks 29 2.2.4 Data Analysis 34 2.3 RESULTS 35 2.3.1 Reproductive development 35 2.3.2 Embryo development 44 2.3.3 Endocrine correlates 46 2.3.4 Seasonality of reproduction 51 2.4 DISCUSSION 57 Females 57 Males 62 Seasonality of reproduction and egg laying behaviour 65 Incubation period 69 CHAPTER THREE : 74
3.1 INTRODUCTION 75 3.2 MATERIALS AND METHODS 78 3.2.1 Source of samples and data collection 78 3.3 RESULTS 83 3.3.1 Size at maturity of all sharks dissected 83 3.3.2 Sharks of known maturity stage (blood taken before dissected) 85 3.3.3 Sharks of unknown maturity 92 3.4 DISCUSSION 98 CHAPTER FOUR : 102
4.1 INTRODUCTION 103 4.2 MATERIAL AND METHODS 106 4.2.1 Acoustic tagging 106 4.2.2 Conventional tagging 115 4.3 RESULTS 118 4.3.1 Acoustic tagging 118 4.3.2 Conventional tagging 140 4.4 DISCUSSION 146 GENERAL CONCLUSIONS 153
REFERENCES 159
General introduction
9 General introduction
The practice of harvesting sharks has a long history. Although sharks were considered edible prior the twentieth century, records associated with shark captures were more likely to be related with rituals rather than eating habits (Budker, 1971;
Taylor et al. , 2005). In the early part of the 1900s, sharks supported small regional artisanal fisheries that targeted individual species for specific products, while towards the 1920s the advance in fishing technology resulted in an increase in harvesting of chondrichthyans globally (Budker, 1971; Taylor et al. , 2005). The commercial exploitation of sharks dates from the period between the two world wars (1930s to
1940s) when attention was drawn to the demand for shark liver oil stimulating a rapid growth in shark fisheries (Budker, 1971; Taylor et al. , 2005). Since the mid 1980s the demand for shark products had greatly increased and by the late 1980s shark fisheries were widespread. A further escalation in exploitation of sharks occurred in the mid
1990s with the high price and increased demand for shark fins in Asian markets (Castro and Brudek., 1999).
Currently, chondrichthyan populations around the world are harvested by commercial, artisanal and recreational fisheries, and are mostly caught as bycatch
(discarded after capture) in the world’s fisheries which target teleost species (Walker,
1998; Stevens et al. , 2000). As such, understanding incidental catch and mortality of bycatch species is becoming an increasing requirement of future ecosystem management plans (Hall et al. , 2000; Pope et al. , 2000). Furthermore, because most sharks exist at or near the top of the food chain (Cortés, 1999; Heithaus, 2004), the removal of upper trophic level predators from their ecosystem can have food web consequences. In addition to their primary prey items, trophic cascades can have significant impacts on non prey species (Pauly et al. , 1998; Stevens et al. , 2000; Schindler et al. , 2002; Scheffer et al. , 2005). With the fear of rapid depletion of world fish stocks because of over exploitation, together with the reduction in sharks as top predators, the effects on marine ecosystems of overfishing could result in an important loss of marine
10 General introduction biodiversity (Coleman and Williams, 2000; Mullon et al. , 2005). Therefore, understanding the role of the top predator sharks in the ecosystem are primary requirements for management and conservation of the shark species but also to accurately address an ecosystem based fisheries management framework.
It is increasingly being recognized that the life history characteristics of chondrichthyans (long lived, slow growth and producing few offspring) make this group a fragile marine resource that is vulnerable to exploitation (Walker, 1998; Dulvy et al. ,
2003). Several species of sharks, rays and skates either targeted or caught as bycatch or byproduct (retained after capture) have demonstrated substantial population declines over the last 20 years (Pauly et al. , 1998; Stevens et al. , 2000; Graham et al. , 2001; Baum et al. , 2003). By 2006, of the 547 chondrichthyan species listed in the International Union of the Conservation of Nature’s (IUCN) Red List, 20% are threatened with extinction; confirming that this taxonomic group is extremely vulnerable to overfishing and is disappearing at an unprecedented rate across the world (IUCN, 2006). In recognition of the expanding global catch of chondrichthyans and the potential negative impacts on chondrichthyan populations, an International Plan of Action for the Conservation and
Management of Sharks (IPOA Sharks) was adopted by the 23 rd session of the United
Nations Food and Agriculture Organisation’s (UN FAO) Committee on Fisheries in
1999 (FAO, 1999; FAO, 2005).
Australia has an extremely rich chondrichthyan fauna with the most recent taxonomic review estimating that of the 1025 species of chondrichthyans worldwide, at least 297 species inhabit Australian waters. Of these species more than half (48% of sharks, 73% of rays) are endemic to Australia (Last and Stevens 1994). In 2001 a Shark
Assessment Report, commissioned by the Department of Agriculture, Forestry and
Fisheries (DAFF), listed 5 species as protected, 6 species as endangered, 6 species as vulnerable, 21 species as near threatened and 3 species as conservation dependent
11 General introduction
(DAFF, 2001). As a member of the UN FAO, Australia committed to producing its own National Plan of Action for the Management and Conservation of Sharks (referred to as Shark plan). The Shark plan was endorsed by the Natural Resource Management
Ministerial Council on 16 April 2004 (DAFF, 2004). The Shark plan recognises that while Australia is not a major shark fishing nation, it is acknowledged that sharks are an important part of the total quantity of Australia’s wild fish production and that
Australian vessels regularly take sharks as target and non target catch (DAFF, 2004).
This thesis has focused on an endemic Australian shark species, the draughtboard shark Cephaloscyllium laticeps (Duméril, 1853); that within the order Carcharhiniformes, belongs to the Scyliorhinidae family. This family, the largest of the shark families, includes up to17 genera and about 100 species. Particularly, 8 genera and 32 species are found in Australia (Springer, 1979; Last and Stevens, 1994). The entire family lives in marine habitats, feeding mainly on small fish and invertebrates. Most of scyliorhinids are near bottom dwellers in shallow waters, although a few genera include species that occur along the continental slopes to depths exceeding 2000 m (Springer, 1979;
Compagno, 1984; Last and Stevens, 1994).
The draughtboard shark is the most common catshark in the coastal areas of southern Australia, where it is a higher trophic level predator of temperate reefs. It is particularly found inshore on the continental shelf of southern Australia from the
Recherche Archipielago (Western Australia) to Jervis Bay (New South Wales) down to at least 60m (Last and Stevens, 1994). Common names for this species are: Australian swell shark, sleepy joe, and nutcracker shark, and local synonymies are: Cephalloscylium isabella laticeps and Cephalloscylium isabella nascione (Last and Stevens, 1994).
Draughtboard sharks form a significant component of the southeastern Australian shark fishery where they are caught as accidental bycatch from rock lobster traps, demersal trawls, long lines and gillnets (Frusher and Gibson, 1999; Walker et al. , 2005).
12 General introduction
This species is usually returned to the water and fishing mortality is low due to its resilience (Brickhill, 2001). In Bass Strait (southern Australia), there was a reduction of approximately 54% in C. laticeps between 1973 1976 to 1998 2001 period. However, this reduction has been attributed to commercial fishers avoiding fishing grounds where these animals are abundant (Walker et al. , 2005). There is currently no targeted commercial fishery for draughtboard shark, although it has recently been marketed in some areas of Tasmania, where there has been a trend for draughtboard sharks that have been caught in commercial gillnets to be retained for local consumption as “flake”
(J. Lyle, TAFI Marine Research Laboratories, Hobart. pers. comm). Although caught as a bycatch, draughtboard sharks are potentially vulnerable to population reduction through fishing due to their high catchability in either pots or gillnets. Despite being a common bycatch species, the lack of commercial value has resulted in this species not being the subject of scientific study. Furthermore, assessing the potential impact of fishing mortality of this top level predator in a temperate reef ecosystem is currently hindered by the poor knowledge of its biology.
The aims of this thesis were to address the biology, ecology and ecosystem role of draughtboard sharks by studying their reproductive biology, movement patterns and habitat utilisation. The results of this thesis will both, increasing the knowledge of draughtboard sharks, but will also be essential to accurately addressing ecosystem based fisheries management programs in Australian temperate reefs, where draughtboard sharks share the habitat with other marine species of very high commercial value, such as the rock lobster Jasus edwardsii (S. Frusher, TAFI Marine Research Laboratories,
Hobart. pers. comm). In addition, as this shark is, in general, not retained as a byproduct there is a need for a non destructive sampling methodology to study reproduction on this species. In consequence, steroid hormones were used as a tool to assess draughtboard sharks reproductive biology. These results will not only help to
13 General introduction understand reproduction of this species, but will also increase the knowledge of the reproductive endocrinology of chondrichthyan species; an area within this marine group that still remains highly unknown. Furthermore, because assessing and protecting threatened species or species living in marine protected areas has become an important part of global conservation activities (Powles et al. , 2000; Blyth Skyrme et al. , 2006), there is a need to find methodology of addressing the reproductive stage of chondrichthyans species without killing the animals.
The thesis consists of the general introduction, four descriptive chapters and the general conclusions. The next chapter, chapter one, describes the area of study. Chapter two initially describes the reproductive characteristics and development of C. laticeps obtained thorough anatomical and histological examination of the reproductive organs.
The second part of chapter two correlates reproductive hormones with basic reproductive parameters to explore the role of steroid hormones on oviparous sharks.
Finally, in the last part of chapter two the seasonal reproductive cycle and embryo development are described. Chapter three evaluates the use of reproductive hormones as a non destructive method to describe reproduction in sharks, and the hormones are explored in the context of providing information required for fisheries management and conservation. Chapter four explores the use of acoustic tagging techniques to assess habitat utilisation and movement patterns and to compare this data with a conventional tagging project that was also undertaken at the same time. Finally, in the general conclusions, the information from the previous chapters was used to understand the linkages between reproduction, movements and habitat selection, essential for understanding the role of draughtboard sharks in the reef habitat and therefore address future ecosystem management programs.
14
CHAPTER ONE : Area of study
15 Chapter one – Area of study
Tasmania is an island located at 39 44°S and 144 149°E (Fig. 1.1 and 1.2a). Three main bodies of water influence the island (Fig. 1.2b). The East Australian Current
(EAC) flows down the eastern seaboard to the southern tip of Tasmania where it converges with colder subantarctic waters in a subtropical convergence zone (STC). The
Zeehan Current is an extension of the Leeuwin and flows down Tasmania’s west coast and around southern Tasmania (Cresswell, 2000). Both the EAC and Zeehan Current are nutrient poor water originating in sub tropical regions. In contrast, the sub antarctic water mass is nutrient rich (Harris et al ., 1987). Water temperatures in Tasmania range from 10.7 to 18.6 (°C) between summers to winters (Cresswell, 2000).
The main marine habitat surrounding Tasmania is rocky reef formed of sandstone and granite. While the reef supports a diverse and abundant fauna, seaweed and seagrass is the predominant living flora (Edgar, 2001).
10 °5'
AUSTRALIA
BASS STRAIT TASMANIA 43 °60'
112 °5' 154 ° Fig. 1.1: Map of Australia. Tasmania is located in the south east of Australia.
16 Chapter one – Area of study
a
40°00'
Derwent Estuary SOUTHERN OCEAN CRAYFISH POINT RESERVE 43°00'
TASMAN SEA Bruny Island 145°00' 147°00' 148°00'
b
BASS STRAIT EAC
TASMANIA
STC ZC
SAW
Fig. 1.2: Map of Tasmania showing (a) the location of the main study site and (b) a satalite image demonstrating the main currents influencing Tasmania during autumn. The EAC and ZC extend further south during summer and retreat further north during winter. EAC : East Australian Current, ZC : Zeehan Current, STC : Subtropical Convergence, SAW : Subantarctic Water. NOAA 14 NLSSTC MOSAIC 11 MAR 1998 05 11Z 0653Z COPYRIGHT 1998 CSIRO. 17
CHAPTER TWO : Reproduction
18 Chapter two - Reproduction
2.1 INTRODUCTION
Reproduction involves one of the most important events in the life of any living organism. The primary requirement for successful propagation of any species and their individuals is the availability to reproduce. Understanding of the overall process of reproduction requires knowledge of the morphology and physiology of the reproductive tract, and reproductive strategies and cycles.
In life history theory, reproductive strategy is defined as a complex mixture of adapted characteristics designed by natural selection to solve ecological problems
(Stearns, 1976). A series of reproductive strategies has been developed by chondrichthyan during their long evolutionary history. The general trend in chondrichthyans reproductive evolution is a progression from oviparity to viviparity, but within this there is still a great diversity of morphological and physiological adaptations (Wourms, 1977; Carrier et al. , 2004). These reproductive strategies are expressed through reproductive cycles, which are regulated by a combination of physical and biological variables to ensure that young fish are produced in the best environment for their survival (Bromage et al. , 2001; Pankhurst and Porter, 2003). Four categories of reproductive cycles can be defined for chondrichthyan females: 1) species that are reproductively active throughout the year, 2) species that are reproductively active throughout the year, but exhibit seasonal periods where a greater proportion of reproductive activity occurs, 3) species with a well defined seasonal cycle, where animals are reproductively active for only a portion of the annual cycle, and 4) species that are pregnant for approximately a full year, after which they spend a year or two non pregnant (Wourms, 1977; Hamlett and Koob, 1999; Koob and Callard, 1999). In chondrichthyan males, sperm production can be either seasonal or occur throughout the year and can be coupled or not with the mating period (Parsons and Grier, 1992).
19 Chapter two - Reproduction
Among oviparous elasmobranchs, both seasonal and non seasonal reproductive activity has been observed. For example, while a clear seasonal reproductive period for both females and males has been reported in Hemyscyllium ocellatum (Heupel et al. , 1999), in Amblyraja radiata both sexes are reproductively active all year round (Sulikowski et al. ,
2005a). Furthermore, male and females of the same species may differ fundamentally in their reproductive tactics. In species such as Leucoraja ocellata , females are capable of continuously laying eggs but show a seasonal peak in activity, while males are able to continuously reproduce throughout the year (Sulikowski et al. , 2004).
In the family Scyliorhinidae, species show single or multiple oviparity, as well as aplacental yolk sac viviparity. Single oviparity, where only one egg case develops in each uterus, has been recorded in the most primitive genera: Cephaloscyllium , Apristurus ,
Scyliorhinus and some species of Galeus . Multiple oviparity, where several egg cases develop in the uterus and the egg capsule is laid when the embryo reaches a certain length, has been found in the genus Halaelurus and some species of Galeus (Nakaya,
1975; Springer, 1979; Compagno, 1984). Aplacental yolk sac viviparity, where embryos are retained in the uterus during the entire period of development and depend solely on yolk sac reserves, has been reported in some species of Galeus and in Cephalurus cephalus
(Nakaya, 1975; Springer, 1979).
Studies on reproduction of the scyliorhinids are limited to only a few species. In the oviparous scyliorhinids, both sexes of Apristurus brunneus and Parmaturus xaniurus are reproductively active throughout the year (Cross, 1988) whereas females of S. canicula
(Craik, 1978; Sumpter and Dodd, 1979) and Galeus melastomus (Costa et al. , 2005) although capable of producing eggs throughout the year, demonstrate seasonal periods of reproductive activity. Embryo development in the scyliorhinid shark, Scyliorhinus retifer
(Castro et al. , 1988) showed an incubation time of 256 days in captivity, while embryo development in S. canicula (Capapé, 1977; Mellinger et al. , 1986; Lechenault et al. , 1993)
20 Chapter two - Reproduction varied (in captivity) from 185 days in warm temperatures to 285 days in colder temperatures.
Histological examinations of chondrichthyans show that follicular organization in females is very similar to that of other vertebrate species (Fasano et al. , 1989). However, in chondrichthyan males, the testicular organization has been classified into three different categories (radial, diametric and compound) (Pratt, 1988) depending on the origin and propagation of the spermatocysts (the unit of structure and function of the testis (Callard, 1991b)). The lamnid alopiid testis type is radial and the testis is comprised of lobes. The germinal zone is localized in the centre of each lobe and development of spermatocysts proceeds radially from the germinal zone towards the end of the lobes. The carcharhinid sphyrnid testis is diametric; the development of the spermatocysts proceeds from the germinal zone across the diameter of the testis. The rajid testis is compound, combining both the radial and the diametric organization of the testis (Pratt, 1988; Girard et al. , 2000).
All scyliorhinid sharks share an external type of ovary (follicles are ovulated into the body cavity where they reach the ostium (Pratt, 1988)). Ovarian follicles are embedded under a single layer of generative tissue, and follicles are ovulated into the body cavity
(Pratt, 1988). Some species contain only one functional ovary, while in others both ovaries are developed (Springer, 1979; Compagno, 1984; Cross, 1988). While the anatomy of the scyliorhinid males have been less studied than that of females, it is known that the entire family shares a pair of testes and accessory ducts, including epididymis, efferent and deferent ducts and seminal vesicles (Springer, 1979; Dodd,
1983; Compagno, 1984). In the only two histological studies of the gonads of scyliorhinids, Scyliorhinus retifer and S. canicula females had follicular organization similar to other vertebrates, and males had a diametric type of testis (Dodd, 1983; Pratt, 1988).
Knowledge of vertebrate endocrinology is an essential component of understanding reproductive processes, as reproductive hormones are involved as either triggers or
21 Chapter two - Reproduction regulators of all aspects of reproduction. The brain pituitary gonadal axis is a cascade system that regulates the entire reproductive process, promoting gametogenesis and subsequent gamete maturation (Sherwood and Lovejoy, 1993; Gelsleichter, 2004;
Pankhurst, 2006). The release of gonadotropin releasing hormone (GnRH) by the brain stimulates the production of the gonadotropins (GTH) from the pituitary gland. These gonadotropins are released into the circulatory system, reaching the target cell where they bind with membrane bound receptors. This gonadotropin receptor complex triggers adenyl cyclase, to form cAMP (cyclic adenosine monophosphate), which in turn activates protein kinases A, leading to the activation or de novo synthesis of steroid synthesizing enzymes resulting in production of steroid hormones (Eckert, 1988). In females, the ovary is the primary producer of steroid hormones, producing three main
gonadal steroids: testosterone (T), 17β estradiol (E 2) and progesterone (P 4) (Koob and
Callard, 1991; Gelsleichter, 2004). Follicular estrogens are necessary for both hepatic vitellogenin synthesis and reproductive tract development. Progesterone has antagonistic actions with estrogens and is considered necessary for ovulation, the continued maintenance of pregnancy, egg retention, and the simultaneous inhibition of vitellogenin synthesis (Callard et al. , 1991; Tricas et al. , 2000; Gelsleichter, 2004). The role that androgens play in female elasmobranchs is less clear. As in teleost fishes, T is
the precursor for biosynthesis of E 2 (Tsang and Callard, 1982; Selcer and Leavitt, 1991;
Pankhurst et al. , 1999; Tricas et al. , 2000) . While some authors have associated T only with the follicular cycle of oviparous elasmobranchs (Sumpter and Dodd, 1979; Koob et al. , 1986), others have found high T concentrations during the egg retention and egg laying process (Rasmussen et al. , 1999; Sulikowski et al. , 2004).
In vertebrate males, the testis is the principal source of reproductive hormones
(Fasano et al. , 1989; Pankhurst, 2006) and although the presence of several gonadal steroids has been reported in male elasmobranchs (Simpson et al. , 1964; Callard, 1991a;
Manire et al. , 1999), the function of most of these hormones remain uncertain.
22 Chapter two - Reproduction
Testosterone seems to be the primary androgen in elasmobranch males, and may play a role in development and maturation of spermatocysts, and stimulation of the development of secondary sex characteristics (Callard et al. , 1985; Cuevas and Callard,
1992; Sourdaine and Garnier, 1993; Tricas et al. , 2000; Gelsleichter, 2004). However, other androgens such as dihydroxytestosterone (DHT), 11 ketotestoterone (11 KT), and 11 ketoandrostenedione (11 KA) have also been reported to play a role in spermatogenesis (Callard et al. , 1989; Garnier et al. , 1999; Manire et al. , 1999). Although
male elasmobranchs do produce estrogens and P 4 (Cuevas and Callard, 1992; Manire and Rasmussen, 1997; Tricas et al. , 2000) and both E 2 and P 4 receptors have been identified in elasmobranch testes (Callard et al. , 1985; Cuevas and Callard, 1992), their roles in male reproduction remain unclear. Several authors (Callard, 1991a; Tricas et al. ,
2000; Sulikowski et al. , 2004) have reported elevated E 2 levels with the middle stages of spermatogenesis, while others (Manire and Rasmussen, 1997; Garnier et al. , 1999) have
found no clear variation in E 2 concentrations within the male reproductive cycle.
Male elasmobranch production of P 4 has been associated with spermiogenesis and spermiation (Gelsleichter, 2004), which suggests that P 4 could act as a precursor for androgen synthesis (Manire and Rasmussen, 1997; Gelsleichter, 2004). In contrast, P 4 was found to peak independently of T in other shark species (Snelson et al. , 1997;
Garnier et al. , 1999; Gelsleichter, 2004). Studies on Squalus acanthias (Simpson et al. , 1963;
Simpson et al. , 1964; Callard, 1991a) have found that P 4 may primarily be the substrate for the production of T and other androgens.
The endocrinology of oviparous elasmobranchs has been reported in only a few species such as Scyliorhinus canicula (Sumpter and Dodd, 1979), Leucoraja erinacea (Koob et al. , 1986), Raja eglanteria (Rasmussen et al. , 1999), Hemiscyllium ocellatum (Heupel et al. ,
1999) and Leucoraja ocellata (Sulikowski et al. , 2004). Of these species, only S. canicula belongs to the family Scyliorhinidae.
23 Chapter two - Reproduction
The aim of the present study was to investigate the reproductive biology of the draughtboard shark, Cephalloscylium laticeps , a common species in southeast Australia. The draughtboard shark belongs to the family Scyliorhinidae (Springer, 1979) and the only information available states that the species is oviparous and that males reach maturity at approximately 820 mm total length (Last and Stevens, 1994).
This study explores the anatomy and histology of the gonads, correlations of changes in gonad condition with plasma levels of gonadal steroids, and assessment of the seasonality of the reproductive cycle. The majority of the data were collected from sharks immediately after capture from the wild, however some sharks were also kept in captivity to address questions associated with embryo development and periodicity of egg laying.
24 Chapter two - Reproduction
2.2 MATERIAL AND METHODS
2.2.1 SOURCE OF SAMPLES AND DATA COLLECTION
Draughtboard sharks were obtained from two different sources:
1) Commercial and research surveys
A total of 636 females and 468 males were collected throughout Tasmanian
coastal waters as bycatch from rock lobster trap, gillnet and hook fisheries between
June 2002 and April 2004 (Fig. 1.1 and 1.2, Table 2.1).
Table 2.1: Number of female and male draughtboard sharks sampled, sorted by date and location of capture in Tasmania. Locations Date Derwent Estuary East Coast South West Coast North West Coast F M F M F M F M Jun 2002 - - 3 2 - - - - Jul 2002 5 25 ------Aug 2002 ------Sept 2002 1 4 ------Oct 2002 1 - 16 18 - - - - Nov 2002 - - - - 55 39 - - Dec 2002 7 29 - - 34 17 - - Jan 2003 - - - - 16 20 - - Feb 2003 - - - - 39 26 - - March 2003 - 2 39 46 25 24 - - April 2003 - - - - 71 24 - - May 2003 5 7 ------Jun 2003 - 1 - - 22 21 - - Jul 2003 - 2 - - 56 22 - - Aug 2003 - - - - 27 13 - - Sep 2003 ------Oct 2003 - - 5 - 2 - - - Nov 2003 ------Dec 2003 ------Jan 2004 4 4 ------Feb 2004 6 40 4 - - - - - March 2004 9 - 10 - 12 - - - April 2004 1 - 33 27 19 7 106 16
25 Chapter two - Reproduction
After capture sharks were euthanased by immersion in a benzocaine bath consisting of
0.5 l of benzocaine solution (40 g benzocaine. l ethanol 1) in 8 l of seawater, and the following measurements were taken from both males and females: total length (mm
TL), total body weight (g TBW), liver weight (g), and stomach weight (g).
In addition, the following data were also recorded for males: calcification, rotation, and length of the clasper (mm) (from the distal end of the metapterigyum to the tip), testes weight (g) and weights (g) of the seminal vesicles: prior to and after expression of any sperm. Ovary weight (g), oviducal gland width (mm) (at the widest part) and weight
(g), presence, condition and length of egg cases were recorded from females. For the first 20 female sharks, the diameter (mm) of all follicles was measured, after which only the largest 20 follicles were measured.
The following indices were calculated:
Gonadosomatic Index (GS I)= (Gonadal (testes or ovary) weight/Total weight)*100
Hepatosomatic Index (HS I)= (Liver weight/Total weight)*100
Where total weight= Total body weight – (Gonadal weight + Liver weight + Stomach weight)
Proportion of sperm within seminal vesicle (PS) = [(weight of seminal vesicle – weight of seminal vesicle after expression of any sperm)/ weight of seminal vesicle]*100
HISTOLOGY
For histological analysis, ovaries, testes and seminal vesicles were fixed in Bouin’s solution, embedded in paraffin, sectioned at 7 m and stained with Haematoxylin eosin.
Stained sections were examined and photographed under a light microscope (Leica
DMLB2 microscope with a Leica DFC 320 camera). Spermatogenic stages in the testis
26 Chapter two - Reproduction were classified according to the descriptions of (Callard, 1991b) and (Parsons and Grier,
1992 ).
BLOOD SAMPLING
To correlate hormone levels with reproductive condition, blood samples (~3 ml) from 118 females and 113 males, were collected by caudal venipuncture using pre heparinized syringes fitted with 22G needles. After extraction, blood samples were placed on ice for 3 6 h and then centrifuged for 5 minutes at 8000 rpm. The plasma was collected and stored at 15°C until thawed for analysis.
2) Captive sharks
Four female and three male sharks caught off Bruny Island (Southern Tasmania) during January 2003 were held in captivity until December 2004 at Woodbridge Marine
Discovery Centre (southern Tasmania). A female shark caught at Bicheno (east coast of
Tasmania) in April 2004 was held in captivity until July 2005 at the Bicheno Aquarium on the east coast of Tasmania. At Woodbridge, females and males were placed in a tank of 7 m length, 3 m width and 1.2 m depth, supplied with water at ambient temperature
(10 11ºC winter and 16 17ºC summer) pumped from the sea. At Bicheno, the female was held in a tank of 2.2 m length, 2.2 m width and 0.8 m depth, under static holding conditions with a complete change of water once a month. The water temperature ranged from 11 12ºC in winter to 21 22ºC in summer. Captive sharks were monitored for the presence of egg laying and development of the embryos.
27 Chapter two - Reproduction
2.2.2 STEROID HORMONE MEASUREMENT
Levels of 17β estradiol (E 2), Progesterone (P 4), Testosterone (T) for both females and males, and 11 Ketotestosterone (11 KT) in males were measured by radioimmunoassay (RIA). Plasma samples (200 l) were extracted twice with ethyl acetate (1 ml) and 100 l aliquots were transferred to assay tubes for evaporation prior
to addition of an assay buffer. Assay reagents for E 2, T and 11 KT were used as described by Pankhurst and Carragher (1992). Progesterone was measured using
[1,2,6,7 3H] Progesterone supplied by Amerhsam Biosciences UK Ltd. The antibody is a polyclonal full serum antibody raised in sheep and was donated by Dr Ken McNatty,
Wallaceville Animal Research Station, Upper Hutt, New Zealand. The assay protocol used was as described by Pankhurst and Carragher (1992). Steroid assays were validated by assessment of the slope of serial dilutions of extracted plasma against assay standards. All samples diluted parallel to standard curves. Extraction efficiency was determined from recovery of 3H– labelled steroid added to pooled aliquots of plasma.
Extraction efficiencies were 86, 74, 86 and 88% for T, E 2, P 4 and 11 KT respectively.
Each sample was analysed in duplicate and the assay values were corrected accordingly to account for the extraction efficiency. The detection limit for all assays was 0.15 ng.ml
1 plasma. Interassay variability was determined by repeat measurement of a pooled
internal standard and was 13 (9), 11 (9), and 9 (7) (%CV (n)) for T, E 2 and P4 respectively. 11 Ketotestosterone data were measured in a single assay.
28 Chapter two - Reproduction
2.2.3 CLASSIFICATION OF REPRODUCTIVE STAGE OF THE SHARKS
Females
In this study the term “follicle” refers to the oocyte and surrounding theca and granulosa layers prior to ovulation, and the term “ovum” refers to the oocyte after ovulation.
The ovarian follicles were classified into four different stages based on follicle size, colour, and histological characteristics as follows: previtellogenic (PV), early vitellogenic
(EV), vitellogenic (V), and mature (M) (Table 2.2, Fig. 2.1).
Based on follicle classification, oviducal gland condition and the presence of egg cases in the uterus, females were then classified into five different reproductive stages as shown in Table 2.3 and Fig. 2.2.
Males Sperm was present in the seminal vesicles of individuals with uncalcified claspers
(Figure 3.4). As calcified claspers are required for copulation (Clark and Von Schmidt,
1965), the presence of sperm could not be used as a reliable indicator of functional maturity. Therefore, clasper condition (determined by assessing the rigidity of the clasper by hand) was used to decide the sexual stage of males. Males were classified as juvenile, sub adult and adult according to Table 2.4, and Fig. 2.5.
29
Table 2.2: Classification of ovarian follicles based on size, colour and histological characteristics.
Follicle type Maximum Follicle Histology Colour (See Fig. 2.1) Diameter (MFD) Follicular epithelium Zone pellucida width Yolk platelets
Previtellogenic (PV) MFD < 7 mm Very white Single row of columnar cells 14 -16 µm No yolk platelets evident within the follicle. Early vitellogenic (EV) 7 mm ≤ MFD ≤10 mm Slightly yellow Pseudostratified 8-10 µm Individual yolk platelets evident within the follicle.
Vitellogenic (V) 10 mm < MFD < 30 mm Yellow Single layer of tall columnar 4-5 µm High density of yolk cells, slightly losing the platelets within the follicle. pseudostratified appearance
Mature (M) * MFD ≥ 30 mm Yellow No histology due to size of follicle
* 30 mm follicular diameter was the smallest follicle found within the initial egg encapsulation stage in pregnant animals.
30 Chapter two - Reproduction
µ (a) 100 µm 100 µm 100 m YP
YP Follicle
Detail of YP
F 50 µm F (b) 50 µm 50 µm BM YP TI F ZP FE FE ZP TH ZP
FE BM TI BM YP TE TE
PV EV V
Figure 2.1: Examples of follicle development (cross sections stained with haematoxylin eosin). a) Presence of yolk inside the follicle. No yolk is present in PV follicle. Yolk started to be distinguished in EV follicle, and follicle is filled with yolk in V follicle. b) Detail of the follicle walls. PV: Previtellogenic, EV: early vitellogenic, V: vitellogenic. F: Follicle, ZP: zona pellucida, BM: basement membrane, FE: follicular epithelium, YP: nucleus of yolk platelets, TH: theca, TI: Theca interna, TE: theca externa. No histology of mature follicle was possible due to size of the follicle.
Table 2.3: Classification of female sexual stages based on maximum follicular diameter (MFD), oviducal gland characteristics and presence of egg cases. PV: Previtellognic, EV: Early vitellogenic, V: Vitellogenic, M: Mature. Type of MFD Oviducal gland Female stages (Refer Table 2.2) Colour Width (mm) ± SE Juvenile (J) PV Translucent 9 ± 0.5 Sub adult (Sa) EV Pink 25 ± 0.6 Adult Stage 1 (As1) V Light red 33 ± 0.9
Adult Stage 2 (As2) V or M Dark red > 35
Adult pregnant (Ap) ** M Dark red > 35 ** Pregnant animals were defined by the presence of either partially formed egg cases in the oviducal gland or fully developed egg cases in the uterus.
31 Chapter two - Reproduction
50mm 50mm 50mm 50mm 50mm
Follicle
Follicle Follicle Follicle Follicle
Juvenile Sub-adult Adult stage 1 Adult stage 2 Adult pregnant
Figure 2.2: Examples of ovary conditions for the different sexual stages in female draughtboard sharks.
For pregnant females, four stages of egg case development were identified (Fig. 2.3)
Anterior
Oviducal gland
Egg case
Tendrils
Stage 1 Stage 2 Stage 3 Stage 4 Posterior
Figure 2.3: Classification of the different egg case developmental stages in draughtboard shark. Stage 1: Posterior coiled tendrils of the egg case are developed and protrude from the posterior part of the oviducal gland. Stage 2 : Posterior half of the egg capsule is developed and protrudes from the oviducal gland. Stage 3 : The entire egg capsule is complete, but only the anterior coiled tendrils are still contained within the oviducal gland. An ovum can be distinguished inside the egg case. Stage 4 : The egg case is free of the oviducal gland. An ovum can be distinguished inside the egg case.
32 Chapter two - Reproduction
Table 2.4: Classification of male sexual stages based on clasper calcification.
Male stages Clasper condition
Juvenile (J) Non-calcified
Sub adult (Sa) Partially calcified
Adult (A) Fully calcified
0.5 mm
Epithelium
Spermatozoa
50 µm
Figure 2.4: Seminal vesicle of juvenile draughtboard shark (cross section stained with haematoxylin eosin).
50mm 50mm 50mm
Juvenile Sub-adult Adult
Figure 2.5: Macroscopic view of the development of the testes in juvenile, sub adult and adult draughtboard sharks. Criteria for classification as given in Table 2.4
33 Chapter two - Reproduction
2.2.4 DATA ANALYSIS
Plasma hormone level comparisons were analysed by one way ANOVA and subsequent Tukey’s multiple comparison tests (Quinn and Keough, 2002). For this and all subsequent ANOVAS, residual plots were undertaken to assess the equality of variances, and data was transformed (square root or logarithmic) where necessary. To determine the relationship between adult and pregnant females a regression analysis was made using SPSS. Assessment of reproductive seasonality of oviducal gland weight,
MFD, sperm accumulated in seminal vesicle and GS I in males, was made using one way
ANOVA and Tukey’s multiple comparison tests. Unless otherwise noted, all data were analysed using SPSS (SPSS® Base 10.0).
For the analyses of monthly variations in GS I and oviducal gland for females, the sample size in the period March and April was considerably larger (n >120) than in the other months. To ensure that sample sizes were not affecting the statistical results, the
ANOVA was repeated 15 times, randomly discarding a number of 50 animals each time.
The significance level was set at P=0.05 for all analysis.
34 Chapter two - Reproduction
2.3 RESULTS
2.3.1 REPRODUCTIVE DEVELOPMENT
Description of reproductive system
FEMALES
The reproductive system of Cephaloscyllium laticeps consisted of a single external ovary, a single ostium, a pair of oviducts, oviducal glands and uteri (Fig. 2.6). The ovary, embedded in the epigonal organ, was attached beneath the vertebral column to the anterior dorsal body by thin connective tissue. The smallest ovaries contained follicles of less than 7 mm in diameter with non discernible yolk. As the follicles grew by acquisition of yolk, a group of 4 6 yellow follicles of similar size began to differentiate.
Follicles began vitellogenesis at about 10 mm diameter and reached the size for ovulation at around 30 mm diameter. Follicles were ovulated in pairs and each ovum enters a separate oviduct.
Macroscopic analysis was unable to separate atretic follicles from corpora lutea although either or both were present in the ovary.
While the MFD of previtellogenic, early vitellogenic and vitellogenic follicles generally correlated with juvenile, sub adult, and adult stage 1 respectively; a small proportion of vitellogenic follicles were also present in adult stage 2. Mature follicles were present in both adult stage 2 and adult pregnant stages (Table 2.3 and Fig 2.7).
The relationship between maximum follicular diameter (MFD) and oviducal gland width was sigmoideal (Fig. 2.8). The oviducal gland initially increased to about 30 mm with small changes in MFD. Between 30 45 mm oviducal gland width, there was a substantial change in MFD. The MFD remained high for the latter parts of the oviducal gland growth (Fig. 2.8).
35 Chapter two - Reproduction
Oviducal gland 1 mm
Ovary
Egg case Follicle
a ostium
Oviduct
Oviducal gland
Epigonal organ Ovary
Uterus c
Epigonal organ
Cloaca b Figure 2.6: Reproductive system of female draughtboard shark. General view of reproductive organs. a) In situ . b) Dissected to show complete reproductive system. c) Cross section of ovary from a juvenile shark (stained with haematoxylin eosin).
36 Chapter two - Reproduction
600 Juvenile Adult pregnant-egg case stage 1 500 400 300 200 100 0 600 500 Sub-adult Adult pregnant-egg case stage 2 400 300 200 20 15 10 5 0 600 500 Adult stage 1 Adult pregnant-egg case stage 3 Number follicles Number of 400 300 200 20 15 10 5 0 600 500 Adult stage 2 Adult pregnant-egg case stage 4 400 300 200 20 15 10 5 0 0-2 3-6 7-10 11-13 14-17 18-21 22-25 26-29 30-40 0-2 3-6 7-10 11-13 14-17 18-21 22-25 26-29 30-40
Follicle diameter
Figure 2.7: Size distribution of follicles in the different maturation stages of the draughtboard shark.
37 Chapter two - Reproduction
40 n=555 35
30
25
20
15
10
5 Maximum follicular diameter (mm) follicular Maximum
0 0 10 20 30 40 50 60 70 Oviducal gland width (mm) Figure 2.8: Relationship between maximum follicular diameter and oviducal gland width in the draughtboard shark.
MALES
A pair of equally developed testes and genital ducts (epididymis, efferent ducts, and seminal vesicles) constituted the macroscopic structure of the male reproductive system. Testes, cylindrical in shape, were enveloped in a thin layer of epigonal organ
(Fig. 2.9). The proportion of epigonal organ attached to the testes decreased from juvenile to adult (Fig. 2.10). Histological sections revealed a diametric spread of spermatocyst development from the germinal zone to the efferent duct zone (Fig. 2.10).
Seven stages of spermatogenesis were distinguished (Fig. 2.11). Each of the three male categories (juvenile, sub adult, and adult) contained at least some spermatocysts with
38 Chapter two - Reproduction spermatozoa although the proportion of spermatocysts containing spermatozoa substantially increased from juveniles to adults.
Histological examination showed an inverse relationship between the different spermatogenesis stages and male sexual categories. The proportion of early stages of spermatogenesis (stage 3 and 4) decreased and the proportion of late stages (stages 6 and 7) increased with the transition from juvenile to adult categories (Table 2.5).
Epididymis
Testis
Deferent ducts
Zone of seminal vesicles Epigonal organ Figure 2.9: Reproductive system of male draughtboard shark.
39 Chapter two - Reproduction
1mm Testis Testis
Epigonal organ Epigonal organ b 1mm a 20 µm Germinal zone Early stages of spermatocyst 1mm development d
Efferent duct
c Spermatozoa
Testis Epigonal Efferent duct 100 µm organ zone e Figure 2.10: Histological sections of testes of draughtboard shark stained with haematoxylin eosin. a: Juvenile , b: Sub adult , c: Adult, d: detail of germinal zone, e: detail of efferent duct zone.
Table 2.5: Percentage of spermatogenesis stages in male draughtboard shark. Proportion of spermatogenesis stages Male sexual stages Stage 1+2+3 Stage 4 Stage 5 Stage 6 Stage 7 Juvenile 0.76 0.12 0.06 0.03 0.03 Sub -adult 0.53 0.23 0.07 0.09 0.08 Adult 0.44 0.25 0.07 0.13 0.11
40 Chapter two - Reproduction
1 A SG SG
SPM µ SPC 100 µm 100 m
2 B 50 µm
SG SG 50 µm
3 C
50 µm SC 1
SC 1
50 µm
4 6 100 µm
100 µm SC 2 SP
5 7 µ 100 µm 100 m
SP
ST
Figure 2.11: Male testes microscopic view. Cross section of testis stained with haematoxylin eosin. 1: Stage 1, spermatocysts containing spermatoblasts with spermatogonia. Sertoli cells can be seen in the interior of spermatocysts. 2: Stage 2, Sertoli cells (nuclei) are seen migrating from the interior to the periphery of spermatocysts. 3: Stage 3, spermatocysts contain primary spermatocytes. Sertoli cells have completed the migration to the basement membrane. 4: Stage 4, spermatocysts contain secondary spermatocytes. 5: Stage 5, spermatocysts contain spermatids with elliptical nucleus. 6: Stage 6, spermatozoa are developed. 7: Stage 7, head of spermatozoa tightly packed forming a spiral shape. A: detail of 1, B: detail of 2, C: detail of 3. SG: Spermatogonia, SPC: spermatocysts, blue arrows: show the basement membrane, red arrows: show the sertoli cell nuclei, SPM: spermatoblasts, SC 1: primary spermatocytes, SC 2: secondary spermatocytes, ST: spermatids, SP: spermatozoa. 41 Chapter two - Reproduction
Female and male tissue indices
In females, GS I remained the same for juveniles and sub adults, but increased significantly in adult females. In contrast, HS I reached its peak in sub adult animals (Fig.
2.12). The increase in GS I was most marked between adult stage 1 (As1) and adult stage
2 (As2), where there was a four fold increase. The HS I steadily increased from juvenile to As1, after which it significantly declined (ANOVA, P< 0.001). In males, GS I showed a linear and significant increase from juveniles to adults (ANOVA, P< 0.001), and HS I was not significantly different at any of the male sexual stages (Fig. 2.12).
The female ovulatory cycle
A general pattern was observed in follicle development in the ovary of female draughtboard sharks. When the follicle size increased beyond 7 mm diameter, a group of 4 6 follicles started to differentiate. These developed until they reached approximately 30 mm diameter when they were ovulated. As these follicles developed, a second, third and fourth group of 4 6 follicles also started to differentiate. Each of these size class groups shared the same diameter, and the differences in diameter between subsequent groups were about 4 5 mm. All pregnant females showed follicles of all size classes indicating follicular development continued throughout the ovulatory, egg retention and oviposition cycle (Fig. 2.7).
Observations from captive sharks demonstrated that eggs were released in pairs with
12 24 h intervals between each egg of the same pair. One female, maintained in a tank by itself for 15 months, laid eggs at routine intervals of approximately 28 days . All the eggs laid by this shark showed embryonic development, confirming that female draughtboard sharks stored sperm for at least 15 months.
42 Chapter two - Reproduction
20 Females c GS I b HS I 15
a a a 10 Index(%)
c c 5
b a a 0 Juvenile Sub-adult Adult stage 1 Adult stage 2 Adult pregnant
20 Males
GS I
HS I 15
10 Index Index (%)
5 c b a 0 Juvenile Sub-adult Adult Sexual stages
Figure 2.12: GS I and HS I for female and male stages in draughtboard shark. Values are mean + SE. For each index, different letters show significant differences between sexual stages. Juvenile (n= 317, 156) Sub adult (n= 69, 36) for females and males respectively. Adult males (n=249), Adult stage 1 (n= 41), Adult stage 2 (n=103), Adult pregnant (n=92). GS I: Gonadosomatic Index, HS I: Hepatosomatic index.
43 Chapter two - Reproduction
2.3.2 EMBRYO DEVELOPMENT
Observations from a single captive shark at Bicheno, showed that the female swam in circles just before and during oviposition to enmesh the tendrils around any object protruding from the substratum. Egg cases found by divers in the field (south and east coast of Tasmania) have always been found attached to objects protruding from the substratum. Observations from 20 eggs laid at Bicheno, showed that at early stages of development, the embryo connected through the yolk stalk to the external yolk sac of 30 mm diameter did not show any movement. When the embryo was about two months old, and had grown to approximately 20 mm in TL, external gills were visible. At this stage the embryo swam vigorously. The external gills reached their greatest development at about four months, when the embryo was approximately 50 60 mm TL. At about five months old, the embryo skin developed pigmentation, the external gills were reabsorbed, and the internal gills became functional. At this stage mouth movements started. At about six months of age, there was a substantial increase in total length (to about 120 mm), gill movements increased and the adult shape was evident. At this stage, the external yolk sac became markedly reduced in size. At about nine ten months of age, the yolk stalk disappeared and the external vitelline sac was about 3 5 mm diameter. Hatching in captivity occurred when the embryo was about 11
12 months old and had reached 160 180 mm in TL (Fig. 2.13). One of the females kept in captivity at Woodbridge, laid two eggs that were successfully hatched 13 months later.
The size of these two juveniles was 160 mm and 177 mm TL respectively.
44 Chapter two - Reproduction
150mm
200mm
SVE A B
150mm 40mm
SVE
SVE
C D
SVE
SVE
E 50mm F 150mm i
60mm 120mm
SVE
G H
Figure 2.13: Embryo development of draughtboard shark. A: Recently laid egg. B, C, D, E, F and G: 1, 2, 4, 5, 6 and 10 months after oviposition. H: Recently hatched embryo. SVE: External vitelline sac. Photos D, E and G by Bryan Hughes. 45 Chapter two - Reproduction
2.3.3 ENDOCRINE CORRELATES
Females
Hormone levels varied among fish with different follicle types. Testosterone began to increase in female sharks with vitellogenic follicles, prior to reaching its highest levels in fish with mature follicles. Estradiol levels increased steadily with follicle
development reaching a peak in fish with vitellogenic follicles. The lowest levels of P 4 were found in early vitellogenic females and the highest levels in sharks with mature
follicles. Similar plasma levels of P 4 were found in fish with previtellogenic and vitellogenic follicles (Fig. 2.14a). Assessment of steroid levels in adult animals showed
that both T and P 4 levels were significantly higher in As2 and Ap (ANOVA, P< 0.001), whereas E 2 did not show a significant difference between any of the adult stages (Fig.
2.14b). To assess the possible role of steroids in pregnancy, plasma steroid concentrations of pregnant females were plotted against the different egg case stages.
Non pregnant animals that contained MFD ≥ 30 mm (type M) were classified as stage 0 and were included in the plot to compare the hormone levels prior to egg case development. Due to the low number of pregnant females encountered during the
study, stages 1 and 2, and stages 3 and 4 were combined. Only P 4 displayed a significant change, reaching a peak of 8 ng.ml 1 during the early stages of egg case development
(ANOVA, P< 0.001) (Fig. 2.14c).
Plasma T levels were low in fish with oviducal glands of width < 50 mm and a significant increase occurred as the oviducal gland reached its maximum size (ANOVA,
P< 0.001). Estradiol increased along with oviducal gland growth. Progesterone showed a small increase in levels in the latter stages of oviducal gland growth before substantially increasing in pregnant animals (Fig. 2.15).
46 Chapter two - Reproduction
6 T (a) E2 c 5 P4 C
4
3 B B
2 b c
1 a,b b A a a a 0 PV EV V M 6 Follicle type b
5 (b)
b 4 A A A )
-1 3
2 b b a 1 a 0 As1 As2 Ap Sexual stages 10 T Plasma steroid (ng.ml steroid Plasma b E2 (c) 8 P4
6
a
4 a A
A 2 B a a a 0 0 1+2 3+4
Egg case stages Egg case stages
Figure 2.14: Plasma levels of T, E 2 an d P 4 in draughtboard shark for follicle types (a) , for adult sexual stages (b) , and for egg case development (c). Values are mean + SE for (a) and (b), and ± SE for (c). For each hormone, different letters show significant differences between follicles, sexual stages and egg cases respectively. PV: Follicle previtellogenic (n= 61), EV: Follicle early vitellogenic (n=10), V: Follicle vitellogenic (n 16), M: Follicle mature (n=27). As1: Adult stage 1 (n=12), As2: Adult stage 2 (n=11), Ap: Adult pregnant (n=20). Stage 0: non pregnant animals with MFD type M (n=12). Stage 1 and 2 represent the development of the first half of the egg case (n=6) and stage 3 and 4 the completion of egg case development (n=14). T: testosterone, E2: 17β estradiol, P4: progesterone. 47 Chapter two - Reproduction
6 T c
E2
5 P4 ) -1 4 c c b,c 3 b
b 2 b a,b b Plasma (ng.ml Plasma steroid 1 a a a a a a 0 <10 ≥10 to ≤ 25 >25 to <50 ≥50 Pregnant
Oviducal gland width (mm)
Figure 2.15: Plasma steroid levels for oviducal gla nd width in draughtboard sharks. Values are means + SE. For each hormone, different letters show significant differences between oviducal gland widths. T: testosterone, E2: 17β estradiol, P4: progesterone.
A diagrammatic summary of the ovulatory and hormonal cycle of the draughtboard shark is presented in Fig. 2.16. Follicular development occurred in parallel with ovulation, egg retention and oviposition. Testosterone remained low during the early stages of follicle development and started to increase in sharks with late vitellogenic follicles, reaching a maximum in sharks with mature follicles. Estradiol steadily increased with follicle development up to the late vitellogenic stage. During the last stages of
follicle maturation E 2 declined slightly, after which it remained relatively high.
Progesterone remained low until just prior to ovulation, when it rapidly increased. After
ovulation, P 4 declined and then remained at a medium level.
48
Follicle development
30mm
PV EV V M P4 Ovulation and encapsulation
Egg retention and Oviposition E2
T Oviposition
next cycle
Follicular phase
20 -28 DAYS IN CAPTIVITY CONTINUOUS
Figure 2.16: A summary of o vulatory and endocrine cycles in female draughtboard sharks. PV: Follicle previtellogenic , EV: Follicle early vitellogenic, V: Follicle vitellogenic, M: Follicle mature. T: testosterone, E2: 17β estradiol, P4: progesterone.
49 Chapter two - Reproduction
Males
Plasma levels of 11 KT were undetectable (< 0.15 ng.ml 1) in all sharks.
Testosterone showed a significant increase from juveniles to adults (ANOVA, P<
0.001). Estradiol and P 4 were detectable but displayed only minor and non significant differences between sexual stages (Fig. 2.17).
7 T c 6 E2 P4 -1) 5 b
4
3
2 Plasma steroidPlasma (ng.ml 1 a
0 Juvenile Sub-adult Adult Sexual stages
Figure 2.17: Relationship between plasma steroid levels and male sexual categories. Values are means + SE. Different letters show significant differences between sexual stages. Juvenile (n=54), Sub adult (n=6), Adult (n=53). T: testosterone, E2: 17β estradiol, P4: progesterone.
50 Chapter two - Reproduction
2.3.4 SEASONALITY OF REPRODUCTION
Females
Females of adult stage 1 formed a greater proportion of the catch in the later part of the year, while pregnant females were more common in the first half of the year.
There was no seasonal trend in the intermediate stage (As2) (Fig. 2.18). A weak inverse relationship was found between adult stage 1 and pregnant females (r 2=0.54).
Concurrent with the apparent decline in pregnant females, and the increase in adult stage 1 females throughout the year, there was a decline in both the MFD and oviducal gland width (Fig. 2.19). Due to the small number of adult females present during most surveys, samples were combined into bimonthly groups.
The low number of blood samples collected each month resulted in the data being
aggregated into periods of two or three months. In general, T and P 4 showed a similar trend to oviducal gland width and MFD with highest levels from the beginning to the
middle of the year and the lowest levels at the end of the year. E 2 was highest at the start of the year prior to falling to the lowest level in the March April period (Fig. 2.20).
51 Chapter two - Reproduction
100 As1 80
60
40
20
0 100
80 As2
60
40
20 Percentage ofadultPercentage females 0
100 Ap 80 11 15 34 1173 9 17 5 7 18 7
60
40
20
0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Figure 2.18: Percentage of adult female sexual stages per month in the draughtboard shark. No adult animals were captured in September. N values are the total numbers of sharks of all stages captured during that month. Ap: Adult pregnant, As2: Adult stage 2, As1: Adult stage 1.
52 Chapter two - Reproduction
b MFD 60 b 90 b Oviducal gland weight 55 80 a,b 26 140 (gr)gland weight Oviducal 50 11 70 a 45 60 22 4 40 50 20 35 40 c MFD (mm) MFD b,c c 30 a,b 30 12 121 a,b 6 25 a 20
22 6 20 24 10
15 0 Jan-Feb Mar-Apr May-Jun Jul-Aug Sep-Oct Nov-Dec
Months Figure 2.19: Monthly variations of maximum follicular diameter (MFD) and oviducal gland weigh t in adult draughtboard shark females. Values are means ± SE. For each variable, different letters show significant difference. Numbers are sample sizes. Because of the low sample size for oviducal gland weight for Sep Oct period, this period was excluded from the statistical analysis.
53 Chapter two - Reproduction
7 a P4 6 5 a 4 a 3 2 b 1 0
7 ) E2 a -1 6
5 a,c 4 b,c
3 b 2 1
Hormone level Hormone (ng.ml 0
2.0 T a,b a 1.5 a,b
1.0 b
0.5
13 11 8 10 0.0 Jan-Feb Mar-Apr May-Jun-Jul Aug-Sep Oct-Nov-Dec Months
Figure 2.20: Mean grouped monthly plasma steroid levels for adult female draughtboard sharks. Values are mean ± SE. Different letters show significant differences between sample times. Numbers are sample sizes. T: testosterone, E2: 17β estradiol, P4: progesterone.
54 Chapter two - Reproduction
Males
No significant monthly difference was found in the proportion of sperm in the
seminal vesicle (PS) and in the GS I throughout the year (ANOVA, P< 0.001). Due to
the small sample sizes, May and June, August to October, and November and
December samples were grouped (Fig. 2.21).
Average T values in males tended to decline throughout the year although the change
was not significant. Due to small monthly sample sizes, the samples were grouped into
periods of four months (Fig. 2.22).
60 10 Proportion of sperm GS I 50 8
40 6 GS I (%) 30
PS (%) 4 20
2 10
17 48 20 37 14 12 11 8 25 0 0 Jan Feb Mar Apr May-Jun Jul Aug-Sep Oct-Nov Dec
Months Figure 2.21: Monthly variations of gonadosomatic index and proportion of sperm in the seminal vesicle of adult draughtboard shark males. Values are means ± SE. There were no significant differences (P> 0.05) between months. Numbers are sample sizes. GS I: Gonadosomatic index, PS: proportion of sperm in seminal vesicle.
55 Chapter two - Reproduction
8 )
-1 7
6
29
5 16
4
Testosterone levels(ng.ml Testosterone 21
3 Jan-Apr May-Aug Sep-Dec Months
Figure 2.22: Monthly plasma testosterone (T) levels for adult male draughtboard sharks. Values are mean ± SE. Numbers are sample sizes. There were no significant differences (P> 0.05) between sampling times.
56 Chapter two - Reproduction
2.4 DISCUSSION
FEMALES
The entire family Scyliorhinidae displays an external type of ovary (Pratt, 1988) although the position of the ovary in the coelomic cavity varies among species.
Cephaloscyllium laticeps showed a single ovary located in the middle of the body cavity. In contrast, several species ( Apristurus brunneus , Halaelurus canescens and Scyliorhinus canicula ) have two ovaries present with only one being functional, and others (e.g. Cephalurus cephalus ) have two ovaries that are equally developed (Springer, 1979; Dodd, 1983; Cross,
1988; Balart et al. , 2000). Although, the genus Cephaloscyllium is considered to occupy the most primitive position within the family and Cephalurus is the most advanced group
(Nakaya, 1975; Springer, 1979; Dulvy and Reynolds, 1997), no correlations appear to exist between reproductive mode and ovarian symmetry (Hamlett and Koob, 1999).
Vitellogenesis commenced in C. laticeps when follicles reach about 10 mm in diameter. Similar to other elasmobranch species (Dodd, 1983; Storrie, 2004), the follicular epithelium in C. laticeps began as a simple structure but as the follicle grew and becomes vitellogenic, the epithelium became slightly pseudostratified. The differentiation of the theca into internal and external layers in C. laticeps was also reported in other elasmobranch species (Dodd, 1983; Prisco et al. , 2002; Storrie, 2004).
However, in some other species such as Urolophus jamaicensis (Hamlett and Koob, 1999) the theca remains undifferentiated.
Female draughtboard sharks were reproductively active all year round, showing a constant overlap between follicular recruitment and development, and egg laying. As a result, the ovary of adult sharks exhibited the full range of developing follicles in addition to atretic follicles and corpora lutea from previously ovulated follicles. Hormone levels were therefore a composite of different stages of follicle development.
Testosterone, E 2 and P 4 were found in all the different sexual stages although their
57 Chapter two - Reproduction
concentrations varied between these stages. Testosterone, along with E 2, were found to be the main steroids present during follicular development and both declined, although
not to baseline levels, during encapsulation and oviposition. In contrast, P 4 was only found in its highest concentrations during the ovulatory period.
Plasma E 2 levels increased during follicle development of C. laticeps , reaching highest concentrations (mean 4 ng.ml 1) in sharks with vitellogenic follicles, prior to declining
(mean 2.5 ng.ml 1) as follicles reached their maximum sizes. These results were also
correlated with the decrease in the HS I in the later stages of maturity. The substantial increase in GS I between stages As1 and As2 and the corresponding decline in HS I suggested that liver resources were being diverted to gonad development at this time.
The increase in E 2 concentrations with the progression from previtellogenic to vitellogenic follicles was related to the increase of steroidogenic activity, and stimulated the liver to synthesize and release vitellogenin for its uptake by the developing follicle
(Dodd and Sumpter, 1984; Ho, 1987). Similar increases in E 2 have been reported for both oviparous and viviparous sharks (Manire et al. , 1995; Heupel et al. , 1999; Tricas et al. , 2002; Sulikowski et al. , 2004). In vitro studies on oviparous sharks also found the
major production of E 2 to be correlated with intermediate sized vitellogenic follicles
(Tsang and Callard, 1982; Koob and Callard, 1991; Callard et al. , 1993).
Estradiol levels were found to decline in C. laticeps as follicles reached their maximum
size prior to ovulation, after which E 2 levels rose to approximately equivalent levels of
1 mature follicles prior to egg case development (3 3.5 ng.ml ). Although a decrease in E 2 levels before ovulation was also reported in Leucoraja erinacea , the plasma steroid concentrations in that species decreased to baseline levels during oviposition (Koob et
al. , 1986). The present results also contrasted with seasonal oviparous sharks where E 2 was at its highest concentration prior to ovulation (Sumpter and Dodd, 1979; Heupel et
al. , 1999; Rasmussen et al. , 1999). High levels of E 2 before ovulation were also observed in viviparous sharks (Manire et al. , 1995; Snelson et al. , 1997; Koob and Callard, 1999;
58 Chapter two - Reproduction
Tricas et al. , 2000). The decline in E 2 as follicles reach maximum size in C. laticeps may be associated with changes in plasma levels of T and P 4. Plasma T levels increased (mean
1.5 ng.ml 1) during the latter stages of maturation of the follicles of C. laticeps , suggesting
a down regulation of P 450 aromatase activity (regulating conversion of T to E 2), leading to accumulation of T rather than its onward conversion to E 2. Similar observations in T levels have been found in several other species of shark (Dodd et al. , 1983; Callard et al. ,
1993; Manire et al. , 1995). Moreover, in teleost fishes, most authors have found maximum T concentrations just prior to ovulation or during final oocyte maturation
(reviewed in Pankhurst, 2006). Current results found that T levels remained high (mean
1.5 ng.ml 1) after ovulation in C. laticeps . Similar findings were reported for Leucoraja ocellata and Raja eglanteria (Rasmussen et al. , 1999; Sulikowski et al. , 2004), but contrast with the results of (Sumpter and Dodd, 1979; Koob et al. , 1986) where T was only elevated during follicle growth in Leucoraja erinacea and Scyliorhinus canicula . Both the
increase in E 2 and the maintenance of high T concentrations after ovulation observed in the present study, may have resulted from the continual recruitment of follicles into vitellogenesis that occured during the reproductive process. This is similar to teleost fishes with asynchronous gamete development where there is often no fall in plasma steroids after ovulation, as there are further clutches of follicles undergoing vitellogenesis (Pankhurst et al. , 1999).
Several studies have also suggested additional roles that estradiol and androgens may perform during the reproductive cycle of elasmobranchs. Estradiol has been suggested to play a role in development and function of the reproductive tract (particularly the oviducal gland). Gilmore (1983) and Koob and Callard (1991) suggested that endocrine
factors may induce egg capsule secretion, and Reese and Callard (1991) identified an E 2 receptor in the oviduct of Leucoraja erinacea . As C. laticeps reproduced throughout the
year, the role E 2 plays in the growth of the oviducal gland was difficult to determine with E 2 levels also being associated with recruitment of the next batch of maturing
59 Chapter two - Reproduction
follicles. A possible role of E 2 in the storage of spermatozoa by the oviducal gland was suggested by (Gelsleichter, 2004). This last author reported that females of Sphyrna tiburo from populations exhibiting high rates of infertility, showed a reduced peak in
preovulatory E 2 related with an apparent decline in the viability of stored spermatozoa by the oviducal gland (Gelsleichter, 2004). As C. laticeps was able to store viable
spermatozoa, the high levels of E2 found in draughtboard sharks after ovulation may indicate that E 2 has also a role in the maintenance of spermatozoa. Estradiol has also been associated with the expression of relaxin, the hormone required to enlarge the cervix to allow egg passage during oviposition. Although relaxin has been identified in the ovaries of several sharks and its effects are considered to be estrogen dependent
(Tsang and Callard, 1983; Callard et al. , 1988; Koob and Callard, 1991), this hormone
and its interactions with E 2 were not investigated in this study. There were, however, no signs of a change in E 2 in females in the present study with fully encapsulated ova that would support this hypothesis.
Androgens may also be associated in the regulation of sperm storage by the oviducal gland. Studies on Sphyrna tiburo females showed that T levels were highest for the 4 5 months between the mating and the ovulatory period suggesting that T is involved in
the regulation of sperm storage by the oviducal gland (Manire et al. , 1995). As E 2 was correlated with oviducal gland function, it is possible that the oviducal gland’s ability to
store sperm is regulated by a combination of both E 2 and T. Androgens might play a role in encapsulation and oviposition, as levels of T increased by the onset of breeding activity and remained high during egg laying in Raja eglanteria (Rasmussen et al. , 1999), and elevated T levels were found during egg case formation and oviposition in Leucoraja ocellata (Sulikowski et al. , 2004). This hypothesis is also supported with the finding in the present study, where T levels remained high in C. laticeps . Further assessment of the
possible roles of both E 2 and T in C. laticeps will require manipulative experiments.
60 Chapter two - Reproduction
1 Plasma levels of P 4 remained low (mean < 1.5 ng.ml ) during the follicular phase in C. laticeps , but showed a marked peak (mean 8 ng.ml 1) in animals carrying partially formed egg cases without an ovum, indicating that ovulation had not yet taken place (Fitz and
Daiber, 1963). A similar peak in P 4 levels just before ovulation was observed in other oviparous species (Koob et al. , 1986; Heupel et al. , 1999; Rasmussen et al. , 1999). After
ovulation, P 4 levels in C. laticeps fell, but still remained elevated during egg encapsulation and oviposition.
Although studies on chondrichthyan endocrinology have advanced in the last few years, there is still insufficient information to present a unified pattern of endocrine control that encompasses all the oviparous species. Several studies have shown that the same steroids hormones appear to behave differently in different oviparous species. For example, while T concentrations were elevated during egg capsule formation and oviposition in Raja eglanteria (Rasmussen et al. , 1999) and Leucoraja ocellata (Sulikowski et al. , 2004) , in Leucoraja erinacea (Koob et al. , 1986) T production was reported to be very low during the egg case production and oviposition. Although there is no explanation for the differences in endocrine patterns among different species, it is important to note that while some studies have been undertaken in the wild (Sulikowski et al. , 2004;
Sulikowski et al. , 2005a), others were done in captivity (Koob et al. , 1986). Furthermore, several authors have reported differences in the reproductive cycle between sharks held in captivity in aquariums and sharks captured from the wild (Carrier et al. , 1994; Heupel et al. , 1999). Heupel (1999) found Hemyscillum ocellatum to adjust its reproductive cycles from a seasonal cycle in the wild to an annual cycle in captivity. Different factors such as stress of capture or husbandry could produce differences in hormone levels or patterns of secretion (Wardle, 1981; Cliff and Thurman, 1984). A decrease in steroid hormone levels in response to confinement or acute stress were reported for the teleost fish
Onchorynchus nerka and Acanthopagrus butcheri (Haddy and Pankhurst, 1999; Kubokawa et al. , 1999). Sampling strategies, particularly the time of sampling in relation to the
61 Chapter two - Reproduction reproductive cycle, could also account for differences in the reported roles of these hormones. However, to date there is insufficient information to understand if the differences in steroid hormone behaviour for oviparous species are a result of the sampling methodology or are related to differences between species. Caution should be applied when comparisons or generalization between oviparous species are made.
MALES
Male C. laticeps had two equally developed testes and reproductive ducts. The origin and propagation of spermatocysts within the testes was characterised by a diametric development, and was consistent with other species of carcharhiniformes that have been studied (Pratt, 1988). In C. laticeps , the proportion of mature spermatocysts and plasma T concentrations increased with sexual maturation. Similar results have been found for both oviparous and viviparous elasmobranchs (Rasmussen and Gruber, 1993;
Heupel et al. , 1999; Manire et al. , 1999; Tricas et al. , 2000; Gelsleichter et al. , 2002;
Sulikowski et al. , 2004). The results of the present study support the view of (Callard et al. , 1985; Sourdaine et al. , 1990; Sourdaine and Garnier, 1993) that T plays a major role in the regulation of testis development. In contrast, detectable levels of 11 KT were not found in C. laticeps . Although, 11 KT is the main androgen reported for teleost fishes
(Pankhurst, 2006), and despite 11 KT being reported in both Sphyrna tiburo (< 2.21 ng.ml 1) (Manire et al. , 1999) and Scyliorhinus canicula (< 0.27 ng.ml 1) (Garnier et al. , 1999), this hormone appears to play no role in C. laticeps .
In C. laticeps , clasper length and testis weight, along with the proportion of spermatocysts containing spermatozoa, increased with increasing T concentrations.
Similar results have been reported for other elasmobranch species (Callard, 1991a;
Rasmussen and Murru, 1992; Sulikowski et al. , 2005b). No androgen receptors have so far been identified in male reproductive tracts (Callard, 1991b; Conrath and Musick,
62 Chapter two - Reproduction
2002), however, it is unlikely that androgens would not be involved in the development of male reproductive organs. As androgens have such widespread actions as anabolic agents in a wide range of vertebrate tissues (Eckert, 1988), it is likely that they would exert similar effects on chondrichthyans. It also cannot be excluded that other unmeasured androgens might also play a role in male reproductive development. As both clasper length and testis weight increased simultaneously in C. laticeps with the increase in T levels, it is suggested that T regulated both events.
Various authors (Heupel et al. , 1999; Tricas et al. , 2000) have associated T concentrations with different stages of spermatogenesis suggesting the possible role of
T in regulating the final stages of sperm maturation. Plasma T concentrations increased during the middle to late stages of spermatogenesis in some species such as Hemiscyllium ocellatum (Heupel et al. , 1999), Leucoraja ocellata (Sulikowski et al. , 2004) and Dasyatis sabina
(Tricas et al. , 2000). Cuevas and Callard (1992) reported that androgen receptors were primarily localized in the early stages of spermatogenesis in the testis of Squalus acanthias , suggesting that androgens may regulate the development of spermatogonia. Although the proportion of spermatocysts containing spermatozoa increased from juvenile to adult animals in parallel with the increase in T concentrations in C. laticeps , more experimental studies need to be done to understand the role of T and possibly other androgens, in regulating spermatogenesis.
Estradiol was present at very low plasma concentrations in male C. laticeps (< 0.20 ng.ml 1), similar to levels in S. canicula where plasma concentrations did not exceed 0.05 ng.ml 1 (Garnier et al. , 1999). In males of the elasmobranch species Dasyatis sabina and
Sphyrna tiburo , relative changes in E 2 levels have been reported throughout the year, but
1 the absolute value of E 2 concentrations (< 0.27 and < 0.072 ng.ml respectively) were similar to, or lower than for C. laticeps (Manire and Rasmussen, 1997; Tricas et al. , 2000).
Several studies have shown elevated E 2 levels to be associated with the early to middle stages of spermatogenesis (Manire and Rasmussen, 1997; Snelson et al. , 1997; Tricas et
63 Chapter two - Reproduction al. , 2000). Furthermore, on the basis that estrogen and androgen receptors were found to be higher in the regions of premeiotic stages of spermatogenesis, (Callard, 1991a) suggested that intratesticular estrogens and androgens may cooperate in regulating the early stages of spermatogenesis. Estradiol is also implicated in spermatogonial proliferation in teleosts (Pankhurst, 2006). Gelsleichter (2004) proposed that circulating
levels of E 2 might not reflect its rate of production or function in testis, if the effects of this hormone were mainly paracrine. Furthermore, studies in S. canicula showed that
1 plasma E 2 reaches maximum values of 0.05 ng.ml , while the maximum value in
1 testicular E 2 was 0.50 ng.ml (Garnier et al. , 1999). It is not known whether E 2 plays a role in modulating spermatogenesis in C. laticeps ; however, any such function is not
reflected in changing plasma levels of E 2.
-1 Detectable levels of P 4 (~ 1 ng.ml ) were found in male C. laticeps at all sexual stages; however, there were no marked changes with changing sexual stage. This contrasts with Sphyrna tiburo and Negaprion brevirostris where P 4 increased with testicular development (Rasmussen and Gruber, 1993; Manire and Rasmussen,
1997). Because P 4 receptors were found to be higher in the post meiotic stage of spermatogenesis, (Cuevas and Callard, 1992) suggested that P 4 is primarily associated with spermiogenesis and spermiation. Several authors (Manire and
Rasmussen, 1997; Gelsleichter, 2004) have identified P 4 as a possible precursor of androgen synthesis, while others reported that P 4 peaks independently of T (Snelson et al. , 1997; Garnier et al. , 1999; Gelsleichter, 2004). The role that P 4 plays in male
C. laticeps remains unclear. As there was no increase in P 4 with sexual development, it is unlikely that plasma P 4 was involved in spermatogenesis, with levels of P 4 instead reflecting the rate at which it was being converted to downstream steroid metabolites such as T.
64 Chapter two - Reproduction
SEASONALITY OF REPRODUCTION AND EGG LAYING BEHAVIOUR
The positive relationship between oviducal gland width and MFD suggests that the draughtboard shark displayed a continuous breeding cycle, although within this cycle there were peaks in both MFD and the oviducal gland weight between January and June indicating that this was a preferred period for egg deposition. Elevated values of T and P4 also coincided with this period. A greater proportion of adult stage 1 females were found towards the end of the year and these females contained ovarian follicles in the advanced stages of vitellogenesis with corresponding elevated levels of
E2. This suggests that C. laticeps was reproductively active throughout the year with an increase in mature egg production in the first half of the year (austral summer and autumn).
This type of reproductive strategy, where animals are reproductively active throughout the year but tend to exhibit one or two peaks in activity (Wourms, 1977), has also been reported in other oviparous elasmobranchs including some scyliorhinid sharks (Sumpter and Dodd, 1979; Cross, 1988; Richardson et al. , 2000; Sulikowski et al. ,
2004). In contrast, other oviparous species have well defined annual reproductive cycles with reproduction restricted to a shorter period of the year. For example, in Hemiscyllium ocellatum , females were found to lay eggs between August and January (Heupel et al. ,
1999), and in Raja eglanteria between January and August (Rasmussen et al. , 1999).
On the basis of the limited information available, there appear to be two basic reproductive strategies in oviparous chondrichthyans. One group has a short incubation period (less than 6 months), a limited time of sperm storage (at least 8 months), and a shorter time between ovulation of each successive pair of eggs (less than about 1 week).
This group displays a seasonal reproductive cycle and tends to be found at lower latitudes. In contrast, higher latitude species tend to be able to reproduce all year round, have longer incubation periods (longer than 6 months), are able to store sperm for
65 Chapter two - Reproduction longer period (at least two years) and oviposition of successive pairs of eggs occurs at longer intervals (longer than one week) (Table 2.6). Similar results were found by
(McLaughlin and O'Gower, 1971) in their study on the genus Heterodontus, suggesting that the breeding season is longer among cool water species. However, in chondrichthyan males, there is no clear trend between the reproductive cycles and fish from low or high latitudes. For C. laticeps males, the lack of marked annual changes in
GS I, proportion of spermatozoa in the seminal vesicles, or T levels would suggest that males are able to produce spermatozoa all year round. Other high latitude oviparous species such as Leucoraja ocellata and Amblyraja radiata , also continuously produce mature spermatocysts throughout the year (Sulikowski et al. , 2004; Sulikowski et al. , 2005a). In contrast, for morphological and hormone data some species from high and low latitudes such as Hemiscyllium ocellatum and Scyliorhinus canicula show clear seasonality in their reproductive cycles (Garnier et al. , 1999; Heupel et al. , 1999). Males of H. ocellatum were reported to have red and swollen claspers (indicating the mating season) from July to
November, with a peak in androgen concentrations from July to October (Heupel et al. ,
1999). In S. canicula males, both gonadal activity and T concentration were found to peak in winter (Garnier et al. , 1999; Henderson and Casey, 2001).
Among teleosts, fish at higher latitudes tend to have a markedly seasonal and synchronised reproductive cycle within the population, while species at lower latitudes are likely to display multiple spawning and less population synchrony (review in
(Pankhurst, 2006)). Teleost fishes tend to produce large numbers of eggs and larvae that are dependent on environmentally driven phytoplankton and zooplankton production cycles to survive. These cycles are more seasonal in higher latitudes. In contrast, oviparous chondrichthyan young hatch as small juveniles and do not undertake a larval phase that is dependent on seasonal planktonic production cycles. The fact that high latitude elasmobranchs do not show seasonality suggests that the large size at hatching uncouples juveniles from dependence on seasonal production cycles.
66 Chapter two - Reproduction
There are still significant gaps in understanding the process by which the environmental signals are transmitted into the endocrine process that control reproduction. However, in teleost fishes, there is evidence that the main factor driving high latitude species is photoperiod, followed by temperature and social interactions, while in low latitudes species this hierarchy may be inverted (Pankhurst and Porter,
2003).
In chondrichthyans, one possible explanation for seasonality among low latitude species relates to sperm storage. In oviparous species found in warm waters the period of sperm storage is usually coincident with the winter months (Rasmussen et al. , 1999)
(Table 2.6). Furthermore, studies on androgen concentration and sperm production by males from warm water species, showed that there is an inverse relationship between androgen concentrations and temperature (Garnier et al. , 1999; Heupel et al. , 1999) , and that sperm production and its accumulation by the seminal vesicle is higher during the winter months (Heupel et al. , 1999). It could be hypothesised that the short periods of sperm storage reported for elasmobranch females in lower latitudes may reflect the inability of sperm to survive for long periods at elevated temperatures. An effect of this type would truncate the period of reproduction among low latitudes species.
It is important to note that other selective forces such as juvenile mortality, growth rate, size and age at maturity, offspring size, fecundity and longevity will influence the differences in life history strategies of chondrichthyan species between high and low latitudes (Stevens, 1999; Frisk et al. , 2001; Cortés, 2004). However, the discussion of these parameters is beyond the scope of the present study.
The relationship between female and male reproductive cycles can vary. While both sexes of several species have a synchronized reproductive cycle (Heupel et al. , 1999;
Kyne and Bennett, 2002; Sulikowski et al. , 2004), there is an un coupling of reproductive activity in others (Ellis and Shackley, 1997; Henderson and Casey, 2001). In Scyliorhinus
canicula , the cycle of females and males was not synchronised with GS I peaking during
67 Chapter two - Reproduction
May in females, and November and December in males (Henderson and Casey, 2001).
In Hemyscillum ocellatum , females lay eggs from August to January and males have the highest volume of sperm in the epididymis during August to November, indicating a synchronous cycle (Heupel et al. , 1999). For females that are able to store sperm (Pratt,
1993), there is no necessity to have a synchronous reproductive cycle between both sexes. In the case of C. laticeps , females and males presented unsynchronised cycles.
Although females were able to reproduce all year round, a peak in egg deposition was found between January to June. However, males did not show a peak in sperm production at any time of the year.
Observation of C. laticeps held in captivity for 15 months, showed that females were able to store sperm for this extended period. Although, migratory behaviour, peaks in sexual aggregations and the benefit of different copulation and fertilization times have all been hypothesized to explain sperm storage in vertebrates (Dodd et al. , 1983;
Birkhead and Moller, 1993; Pratt, 1993; Conrath and Musick, 2002), there is limited biological information available for C. laticeps to support any of these theories. However, the presence of mature adult females and males throughout the year would suggest that sperm storage was not associated with disaggregation of the sexes.
Studies on the mating behaviour of scyliorhinid sharks show that the male initially bites the tail and then the pectoral axilla of the female (Castro et al. , 1988). No mating scars have been distinguished in any of the female C. laticeps sampled. However, the skin of the draughtboard shark is very thick and this could prevent the damage that gives rise to mating scars in other species. On the base of high levels of androgen during the mating season, T has been associated with copulatory activity in elasmobranch species
(Rasmussen and Gruber, 1993; Heupel et al. , 1999; Tricas et al. , 2000). However, Crews
(1984) and Parsons and Grier (1992) suggest that a peak in testicular development or circulating levels of gonadal hormones may not necessarily coincide with the peak in the mating season. In species such us Mustelus griseus and Mustelus manazo there is a 6 month
68 Chapter two - Reproduction
delay between the peak in GS I and the mating season (Parsons and Grier, 1992).
Currently the mating season (if there is one) in C. laticeps is not known.
INCUBATION PERIOD
The different stages of embryonic development in C. laticeps were similar to those reported for other scyliorhinids (Mellinger et al. , 1986; Castro et al. , 1988). In C. laticeps , the incubation period in captivity was similar to the incubation time suggested by Castro
(1998) for Scyliorhinus retifer in the wild. In species from temperate waters such as
Leucoraja erinacea (Richards et al. , 1963) and Raja clavata (Ellis and Shackely, 1995), embryo development takes between six months to one year; while in species from warm waters such as Raja eglanteria (Luer and Gilbert, 1985) and Hemiscyllium ocellatum (West and Carter, 1990) the incubation period takes around four five months (Table 2.6).
However, incubation time in captive Scyliorhinus canicula varied according to the temperature of the aquarium, being shorter (180 days) for egg deposited in warm water compared to those deposited in cold waters (285 days) (Capapé, 1977). For this species, the increased water temperature was considered to increase the metabolic rate of development of the embryo. For sharks with incubation times of less than 12 months, the amount of time the egg is exposed to warmer (summer) or cooler (winter) temperatures may account for variability in incubation times. For C. laticeps , with a incubation time of approximately 12 months, temperature generated variation in incubation period was unlikely as each egg would appear to experience the same total exposure to winter and summer temperatures, irrespective of the time of oviposition.
This assumes that temperature dependent effects on development do not change with stage of development.
69 Chapter two - Reproduction
In summary, female C. laticeps presented an external type ovary with follicles starting
vitellogenesis at 10 mm diameter and maturing at 30 mm. Testosterone and E 2 played a major role during the follicular phase, while P 4 peaked during the ovulatory phase.
Females were reproductively active all year round with a seasonal period between
January to June where a greater proportion of eggs were laid. Deposition of the eggs occurred once a month, and the incubation period is about 12 months. Male C. laticeps presented diametric type testes. Testosterone played a major role changing according to the sexual stage. Males were able to produce sperm all year round. The mating season for this species (if there is one) remains to be determined.
70
Table 2.6: Reproductive information on oviparous species. Data was not included in the table when it was considered unreliable due to sa mpling strategies or small sample size. *Raja eglanteria lives either in both high and low latitudes. ** No data on water temperature was available; but m ost of the studies by Dodd were in captivity in cold water.
Reproductive Time Time Sperm Species name activity between between Incubation time Latitude Location Authors storage Females laying each egg C. laticeps Year round; peak in At least 15 20-28 days 12-24 hrs 12 months High Tasmania (This study) Jan - June months R. eglanteria * Jan-Aug At least 3 4-5 days Min-hours 3 months Low Captivity (20-22°C) (Luer and Gilbert, 1985) months
Year round; peak in No data No data No data 3 months High Delaware Bay, USA (9°C) (Fitz and Daiber, 1963) spring R. clavata Year round No data No data No data 4.5-5.5 months (12-18°C) High Plymouth, England (Clark, 1922)
No data At least 15 0-2 days No data 19 weeks High Captivity (14.9°C) ( Ellis and Shackely, 1995) weeks
No data No data 48 hrs 24 hrs No data High Captivity (11-16°C) (Holden et al. , 1971)
Jan -Sep No data No data No data No data High Southern North Sea (Holden, 1975)
S. retifer No data At least 843 15 days From minutes 8.5 months High Captivity (11.7-12.8°C) (Castro et al. , 1988) days up to 8 days A. brunneus No data No data No data No data 14 months (10°C) Hi gh British Columbia, Canada (Jones and Geen, 1977) P. xaniurus Year round; peak in No data No data No data No data High California, USA (Cross, 1988) Dec-May H. regani Year round No data No data No data No data High Cape Town (98-515m) (Richardson et al. , 2000)
71
S. canicula Year round No data No data No data 271-285 days (14-19°C) High Tunisia (Capapé, 1977)
177-180 days (19-24°C)
Year round; peak in No data No data No data No data High West coast of Ireland (Henderson and Casey, May 2001)
No data More than 2 No data No data No data High No data but probably cold (Dodd et al. , 1983) years waters**
No data No data 15 days No data No data High Captivity (14°C) (Mell inger, 1983)
Year round; peak in No data No data No data No data High Plymouth, England (Ford, 1921) spring and summer
10 months; peak in No data 15 days to No data 5-6 months (8.5-18.1°C) High British waters (Ellis and Shackley, 1997) June-July one month
No data No data No data No data 170-200 days High Captivity (16°C) (Mellinger et al. , 1986)
L. erinacea Year round; peak in No data 7 days No data 6-8 months High Connecticut and Rhode (Richards et al. , 1963) Nov-Jan and June-Jul (16.3-20.3C) Island
Year round; peak in No data No data No data 6 months High Delaware Bay, USA (Fitz and Daiber, 1963) spring and fall (<15°C) H. ocellatum Aug-Jan No data No data No data No data Low Heron Island, Australia (Heupel et al. , 1999) (21-28°C)
Year round No data No data No data 4 months Low Captivity (25°C) (West and Carter, 1990)
A. radiata Year round; peak in No data No data No data No data High Western Maine, USA (Sulikowski et al. , 2005a) September L. ocellata Year round peak in No data No data No data No data High Western Maine, USA (Sulikowski et al. , 2004)
72
G. melasto mus Year round, peak in No data No data No data No data High South of Portugal (Costa et al. , 2005) summer and winter P. extenta Year round; peak in No data No data No data No data High Puerto Quequén, (Braccini and summer Argentina Chiaramonte, 2002)
Year round No data No data No data No data High Northern coast of Sao (Martins et al. , 2005) Paulo, Brazil R.maculata Year round No data No data No data 5 months (13-18°C) High Plymouth, England (Clark, 1922) R. brachyura Year round 5-6 weeks No data No data 4 months (9-18°C) High Plymouth, England (Clark, 1922) R. microocellata No data No data No data No data 5-7 months High Captivity (14.2-16.3°C) (Koop, 2005) H. portusjacksoni Aug and Sep. No data Approx 13 No data No data Low Central coast of New (McLaughlin and days South Wales, Australia O'Gower, 1971) C. plagiosum No data No data No data No data 4 months Low Captivity (24-26°C) (Tullis and Peterson, 2000)
73
CHAPTER THREE : Non lethal assessment of reproductive parameters: draughtboard shark a case study
74 Chapter three - Non-lethal assessment of reproductive parameters
3.1 INTRODUCTION
The reduction and collapse of global fish stocks due to over exploitation is increasing, with several species nearing extinction (Dulvy et al. , 2003; Cortés, 2004;
Mullon et al. , 2005). These declines have called for conservation strategies to be developed for marine resources such as; implementing fisheries management policies, establishing a global system of marine protected areas (MPAs) where fisheries are restricted, or declaring some species as threatened or endangered where their capture is prohibited. Currently, due to the potential for chondrichthyans to be strongly susceptible to overfishing, the impact on fishing chondrichthyan species around the world is the focus of considerable international concern (Stevens et al. , 2000).
Chondrichthyan populations are harvested by commercial, artisanal, and recreational fisheries (Bonfil, 1994; Walker, 1998) and while some species are the direct target of the fishery others are taken as bycatch. It is commonly accepted that chondrichthyans have slow growth, long life span, late sexual maturity, a low fecundity, long gestation period and low natural mortality compared to teleost fish (Cortés, 2000; Stevens et al. , 2000).
These life history strategies make this group very vulnerable to high levels of fishing pressure and have led to a number of conservation and management strategies in an attempt to protect chondrichthyan populations from decline (Simpendorfer and
Donohue, 1998; Stevens et al. , 2000; Musick, 2004).
In order to manage chondrichthyan species, it is necessary to develop demographic models that address their vulnerability to exploitation. Understanding their life history strategies, particularly their reproductive cycles, is fundamental if species are to be managed so that they reproduce to maintain appropriate population levels. Knowledge of the size at which animals mature, is required to ensure that the species has sufficient time to replace the stock prior to being harvested or impacted upon by fishing, and the spatial and temporal timing of reproduction is therefore essential for sustainable
75 Chapter three - Non-lethal assessment of reproductive parameters fisheries management to ensure that fishery activities are minimised during reproductive periods.
Clasper calcification is the most common external method used to assess sexual maturity in male chondrichthyans (Clark and Von Schmidt, 1965). However, not all species (eg: seven gill sharks) alter the degree of calcification in their claspers as they reach maturity, therefore the sacrifice of these males is necessary. In females, as macroscopic examination of the ovaries from dissected animals is the only method to assess sexual maturity, the sacrifice of females is always required. However, there are many circumstances where killing the animal is inappropriate as in the case of endangered or protected species, or species residing in MPAs. Similarly it may be inappropriate to sacrifice bycatch species that would normally be returned to the water alive. For studies on the reproductive biology and management of these species there is a need to obtain data on reproduction without the requirement to kill the animal.
Furthermore, any investigation of the temporal and spatial timing of reproduction, for both sexes, currently requires the examination of gonadal condition after dissection of the animal.
Gonadal steroids, obtained from blood samples, could be used as endocrine markers to determine the reproductive status of sharks without the need to kill and dissect the shark. Only a few studies have compared the levels of plasma steroid hormones between juvenile and adult chondrichthyans, and all of these suggest that hormones could be used as an indicator of maturation status (Rasmussen and Gruber, 1990;
Rasmussen and Murru, 1992; Rasmussen and Gruber, 1993; Gelsleichter et al. , 2002).
However, despite these results, only one study has linked plasma steroid hormones to histological and morphological studies of the gonads to address size at onset of sexual maturity (Sulikowski et al. , 2005b).
76 Chapter three - Non-lethal assessment of reproductive parameters
This study has demonstrated that changes in plasma levels of reproductive hormones are associated with maturation for both sexes in C. laticeps , and that reproductive hormones reflect the temporal timing of reproduction (see chapter 3). This chapter
examines whether the endocrine markers, testosterone (T), 17β estradiol (E 2) and progesterone (P 4) could be used as an unambiguous indicator of sexual maturity in both males (where gonadal sexual maturity might occur in advance of clasper calcification) and females (where there are no external morphological markers of maturation), and therefore eliminate the need for sacrificing sharks for subsequent macroscopic examination of the gonads. The results from the assessment were then applied to draughtboard sharks sourced from a marine protected area where only non destructive sampling methods are appropriate.
77 Chapter three - Non-lethal assessment of reproductive parameters
3.2 MATERIALS AND METHODS
3.2.1 SOURCE OF SAMPLES AND DATA COLLECTION
Draughtboard sharks were obtained from two different sources:
1) Commercial and research surveys : Animals from these surveys (see chapter 3, section 3.2.1) were used to calculate size at maturity and to validate plasma steroid levels against macroscopic examination of the gonads.
2) Surveys at a marine reserve: Eighty two females and 54 males were caught between
May 2002 and May 2003 using rock lobster traps in the Crayfish Point Reserve in southern Tasmania (Fig. 1.2a). Total length and total weight for each sex and clasper length for males were recorded. Blood samples (as described in Chapter 2, section 2.21) were taken prior to releasing the sharks.
For all sharks, steroid hormones were measured as described in section 2.2.2.
3.2.2 DATA ANALYSIS
To determine the maturity of sharks, from the marine reserve of unknown maturation stage, that were released immediately after taking blood, plasma hormone concentrations were compared with the hormone concentrations from sharks of known maturity stages.
78 Chapter three - Non-lethal assessment of reproductive parameters
Size at maturity of sharks dissected
Reproductive stages of the sharks were described in section 2.2.3. For this chapter adult females (As1, As2 and Ap) were combined into a single adult group. For both sexes, juveniles and sub adults were combined into a single group called juveniles.
To establish size at maturity of all sharks sampled in this study, oviducal gland width
(for females) and clasper length (for males) were compared to total length. Oviducal gland width and clasper length were chosen as they were morphological parameters that progressively grew with maturity, and were independent of the reproductive cycle. In contrast, gonadal weight varied within mature animals depending on the cyclic gametogenesis stage of the ovary or testis.
To determine the size at which 50% of the sharks were mature, animals were grouped as either juvenile or adults. Sharks were grouped into 25 mm length classes ranging from 170 to 1020 mm. For dissected sharks, clasper calcification (males) and macroscopic examination of the gonads (females) were used to distinguish between juveniles and adults. A logistic regression was applied to each sex separately. The proportion of adult animals ( P) at 25 mm length class was obtained using the following equation (Neter et al., 1990).
P=e (a+bx )/ [1+e (a+bx )] Equation 3.1
Where a and b are constants and x is the medium value of the length class. Confidence intervals around the logistic model were obtained by conducting 1000 simulations in a bootstrapping routine where data were randomly sampled with replacement for each of the 25 mm length classes (Turner et al. , 2002). The middle 95% of the bootstrap replicates constituted the confidence intervals. Values of P and the 95% confidence limits were obtained from equation 3.1 using Excel (Microsoft ® Excel 2000).
79 Chapter three - Non-lethal assessment of reproductive parameters
Sharks of known maturation stage
LINEAR DISCRIMINANT PREDICTIVE MODEL (LDPM)
For both sexes, weighted averages of the predictive variables: total length (TL),
testosterone (T), 17β estradiol (E 2), and progesterone (P 4) (for females) and clasper length (CL), T, E 2, and P 4 (for males), were used to obtain discriminant function scores
(D) to distinguish juveniles from adult sharks. Discriminant function scores ( D) were calculated as follows:
D = Bo + B 1X1 + B 2X2 + …+ B iXi Equation 3.2
Where X i is the value of each independent variable (i) and B i is the coefficient estimated from the data. From the discriminant scores it was possible to obtain the probability that a shark was either a juvenile or adult. This probability P(Gi/D) was estimated by:
= P(D /Gi )P(Gi ) P(Gi / D) g Equation 3.3 ∑ P()()D / Gi P Gi i=1
Where P (Gi) is the prior probability and is an estimate of the likehood that a shark belongs to a particular group (juveniles or adults). The prior probability was calculated as the observed proportion of sharks in each group. The conditional probability P
(D/Gi) is the probability of obtaining a particular discriminant function value of ( D) if the shark belongs to a specific group. To calculate this probability, normal probability theory (the D scores are normally distributed for each group) was assumed. Each shark was known to belong to a particular group, and the conditional probability of the observed ( D) score given membership in the group was calculated.
80 Chapter three - Non-lethal assessment of reproductive parameters
The predictive function was built using Excel and SPSS (SPSS ® Base 10.0).
MULTI DIMENSIONAL SCALING (MDS)
For both sexes, a multidimensional scaling (MDS) ordination based on the
variables T and E 2, (for females), and T and CL (for males) was used to separate juveniles and adults using normalized Euclidean distances. Data were transformed when necessary. To test the null hypothesis that there were no assemblage differences between groups (juveniles and adults) in the spatial matrix, a one way analysis of similarities (ANOSIM) and a Pairwise test were performed. The MDS and ANOSIM
were performed using the Primer software package (Clarke and Gorley, 2001). Adults were separated using a 95% cut off line. The line was calculated as the position on the
MDS ordination where 95% of adults were correctly classified.
The significance level was set at P=0.05 for all data analyses.
Size at maturity
To determine the size at which 50% of the sharks were mature, animals were grouped as either juvenile or adult using LDPM and MDS analysis. Sizes at maturity estimates were calculated for each method using equation 3.1.
81 Chapter three - Non-lethal assessment of reproductive parameters
Sharks of unknown maturation stage
To determine the size at maturity, sharks were classified as either juvenile or adult based on their (D) scores using LDPM or on their MDS ordination. Size at 50% maturity was calculated using equation 3.1 for both methods.
Reproductive cycle
For both sexes, differences in the proportion of adult sharks that came from the marine reserve and from the rest of Tasmania were compared using a Chi Square test
(Quinn and Keough, 2002).
Hormone comparisons were analysed by one way ANOVA and Tukey’s multiple comparison tests (Quinn and Keough, 2002). Residual plots were undertaken to assess the equality of variances and data were transformed where necessary.
All data were analysed using SPSS and the significance level was set at P=0.05 for all data analyses.
82 Chapter three - Non-lethal assessment of reproductive parameters
3.3 RESULTS
3.3.1 SIZE AT MATURITY OF ALL SHARKS DISSECTED
In females, oviducal gland width increased exponentially between 750 850 mm TL
(Fig. 3.1a). The largest juvenile female found was 850 mm TL and the smallest adult was
730 mm TL. For males, clasper length showed a steady increase as the animal grew until
715 mm TL (Fig 3.1b). From 700 780 mm TL, clasper length rapidly increased (Fig.
3.1b). The largest juvenile male recorded was 830 mm TL and the smallest adult male was 725 mm TL. Size at 50% maturity of females was estimated at 815 mm TL (95% confidence interval = 812.58 – 842.79, r 2=0.80, n=609), and 761 mm TL (95% confidence interval = 754.98 – 789.60, r 2=0.84, n=462) for males (Fig. 3.1c).
83 Chapter three - Non-lethal assessment of reproductive parameters
70 Juvenile 60 Sub - adult Adult 50 n=609
40
30
20
10
Oviducal width glandOviducal (mm) a 0 90 80 Juvenile Sub - adult 70 Adult 60 n=462 50 40 30 20 Clasper Clasper length (mm) 10 b 0
1.0 0.9 Females (n=609) Males (n=462) 0.8 0.7 0.6 0.5 0.4 0.3
Proportionmature 0.2 0.1 c 0.0 100 200 300 400 500 600 700 800 900 1000
Total length (mm)
Figure 3.1: Changes in (a) oviducal gland width (females) and (b) clas per length (males) with total length. Male claspers were classified as non , partially and fully calcified for juveniles, sub adults and adults respectively. Males mature at smaller sizes than females (c) .
84 Chapter three - Non-lethal assessment of reproductive parameters
3.3.2 SHARKS OF KNOWN MATURITY STAGE (BLOOD TAKEN BEFORE DISSECTED )
Linear discriminant predictive model (LDPM)
FEMALES
Discriminant function analysis using TL, T, E 2 and P 4 showed significant differences between juvenile and adult sharks (Wilk’s Lambda, χ 2= 121.697, P< 0.001).
Both the standardized coefficient and the correlation of each variable with the discriminant function showed that total length was the main variable to contribute to the divergence between juveniles and adults. Testosterone and Estradiol contributed in
similar proportion while P 4 did not explain any additional separation between groups
(Table 3.2). Progesterone was found to only play a major role in draughtboard sharks during the ovulatory cycle (see chapter 2), and because its level only varied within adult animals this hormone was unlikely to contribute to the separation between the two
groups. Therefore the model was rerun excluding P 4. Discriminant function scores ( D) generated using TL, T and E 2 were substituted into equation 3.2 as follows:
D= 6.919 + 0.008*TL + 0.90*T + 0.22*E 2
Conditional probabilities under the discriminant scores (D) were generated for both groups. The prior probability of any shark to be juvenile was 0.63 and to be adult was
0.37. The group to which a case belongs is based on its largest posterior probability.
From 118 females, 92% of cases were correctly classified.
85 Chapter three - Non-lethal assessment of reproductive parameters
Table 3.1: Standardized discriminant function coe fficients and correlations of the discriminant linear function for draughtboard shark females. Total length showed the highest standardized coefficient and the highest relationship with the discriminant function.
Standardized Correlation with Variable coefficient discriminant function Total length 0.61 0.90
Testosterone 0.42 0.80
Estradiol 0.40 0.71
Progesterone 0.16 0.58
MALES
Clasper calcification is traditionally used to determine maturity in male sharks, however, the differences between partially and fully calcified claspers can be very subjective. As the calcification of the clasper was related to clasper length (CL) (Fig.
3.2), clasper length was included to separate maturity stages in male sharks. Discriminant
function analysis combining CL, T, E 2 and P 4 showed significant differences between juveniles and adults (Wilk’s Lambda, χ 2= 41.377, P< 0.001). Clasper length and T were
the main contributors to the separation of juveniles and adults. Both E 2 and P 4 played a minor role in the divergence of the two groups and were excluded from the analysis
(Table 3.2). Discriminant function scores ( D) were generated using the following equation:
D = 4.239 + 0.070*CL + 0.234*T
86 Chapter three - Non-lethal assessment of reproductive parameters
Conditional probabilities under the discriminant scores were generated for juveniles and adults. The prior probability was estimated as 0.55 and 0.45 for juveniles and adults respectively. The group to which a case belongs was based on its largest posterior probability. From 111 males, 99% of cases were correctly classified.
1.0 n=435 0.9
0.8
0.7
0.6
0.5
0.4
calcified claspers calcified 0.3
0.2
Proportion fullyof Proportion withmales 0.1
0.0 0 10 20 30 40 50 60 70 80 90 Clasper length (mm)
Figure 3.2: Relationship between clasper c alcification and clasper length for draughtboard shark males.
87 Chapter three - Non-lethal assessment of reproductive parameters
Table 3.2: Standardized discriminant function coefficients and correlations of the discriminant linear function for draughtboard shark males. Clasper length showed the highest standardized coefficient and the highest relationship with the discriminant function. Standardized Correlation with Variable coefficient discriminant function Clasper length 0.89 0.96
Testosterone 0.59 0.78
Estradiol 0.16 0.12
Progesterone 0.07 0.27
Multi dimensional scaling (MDS)
FEMALES
A combination of T and E 2 successfully separated the reproductive stages of female sharks. Based on the discriminant function result of the contribution of P 4 into the separation of both groups, P 4 was not included in the MDS analysis. The majority of adult animals were on the left side of the ordination and the juveniles on the right side
(Stress=0) (Fig. 3.3). ANOSIM analysis showed that there were significant differences between the reproductive stages (Global R=0.61, P< 0.001). A ‘95% cut off’ line for adults resulted in 90% of the females correctly classified; eight juveniles were classified as adults and five adults as juveniles (Fig. 3.3).
88 Chapter three - Non-lethal assessment of reproductive parameters
Stress: 0
Adults Juveniles
Figure 3.3: Multi dimensional scaling (MDS) of juvenile (white circles) and adult (black circles) draughtboard shark females of known maturity, using testosterone and 17 β estradiol. The vertical dashed line represents the “95% cut off” line whereby 95% of adults were to the left of this line.
MALES
Based on the results from the discriminant function analysis, where CL and T
played a major role in the separation between juveniles and adults, E 2 and P 4 were excluded from the MDS analysis. A combination of CL and T separated adult male sharks from most of the juveniles (Stress=0.01) and ANOSIM analysis demonstrated that there were significant differences between the reproductive stages (Global R=0.70,
P< 0.001) (Fig. 3.4). Based on a ‘95% cut off’ lines of adults, 97% of the 111 males sampled were correctly classified (Fig. 3.4).
89 Chapter three - Non-lethal assessment of reproductive parameters
Stress: 0.01
Adults Juveniles
Figure 3.4: Multi dimensional scaling (MDS) of juvenile (white triangl es) and adult (black triangles) draughtboard shark males of known maturity, using clasper length and testosterone. The vertical dashed line represents the “95% cut off” line whereby 95% of adults were to the left of this line.
Size at maturity
Hormone analysis was undertaken on 229 sharks that were also dissected. Size at
maturity was calculated for these sharks using equation 3.1 based on macroscopic
examination of the gonads (destructive sampling) and after classification of the sharks
into juveniles or adults using either LDPM or MDS analysis (non destructive sampling).
For the MDS method, sharks on the left of the ‘95% cut off’ line were classified as
adults and sharks on the right of the ‘95% cut off’ line were classified as juveniles. All
three analyses resulted in a similar size at 50% maturity for both sexes, with females
within 1.8% and males within 0.4% of the estimated values from macroscopic
examination (Table 3.3).
90
Table 3.3 . Comparison of the size at 50% maturity between destructive (visual examination) and non destructive (LDPM and MDS) methods for female and male draughtboard sharks. Percentage differences in the size at maturity using the non destructive method were compared with the destructive method. LDPM: linear predictive discriminant model, MDS: Multi dimensional scaling.
50 % maturity Percentage 95% Confidence interval r2 a and b values n TL (mm) difference TL (mm)
Females (macroscopic) 814 - 0.80 a = -32.16, b = 0.04 798 – 830 118
Females (LDPM analysis) 823 1.10 0.77 a = -59.55, b = 0.07 812 – 832 118
Females (MDS analysis) 829 1.84 0.75 a = -28.54, b = 0.03 811 – 848. 118
Males (macroscopic) 779 - 0.80 a = -47.70, b = 0.06 762 – 790 111
Males (LDPM analysis) 776 -0.38 0.80 a = -63.08, b = 0.08 768 – 783 111
Males (MDS analysis) 782 0.25 0.82 a = -34.23, b = 0.04 760 – 802 111
91 Chapter three - Non-lethal assessment of reproductive parameters
3.3.3 SHARKS OF UNKNOWN MATURITY
Sharks from the marine reserve were categorized as juveniles or adults based on their posterior probabilities for the LDPM analysis. For the MDS ordination, the unknown sharks were overlaid on the MDS plots for sharks of known maturity (Fig. 3.5 and 3.6). Sharks that fell to the left of the ‘95% cut off’ line for adults were classified as adults and those on the right as juveniles.
Stress: 0
Adults Juveniles
Fig. 3.5: MDS ordination of draughtboard shark females (known and unknown maturity) using testosterone and 17 β-estradiol. J (juveniles): white circles, A (adults): black circles, U (unknown): grey triangles. The vertical dashed line represents the “95% cut off” line whereby 95% of adults were to the left of this line.
92 Chapter three - Non-lethal assessment of reproductive parameters
Stress: 0.01
Adults Juveniles
Figure 3.6: MDS ordination of draughtboard shark males (known and unknown maturity) using clasper length and testosterone. J (j uveniles): white triangle, A (adults): black triangle, U (unknown): grey circles. The vertical dashed line represents the “95% cut off” line whereby 95% of adults were to the left of this line.
Size at maturity
For both sexes, the LDPM and MDS analysis resulted in a similar size at 50% maturity (Table 3.4.). There was no difference between the estimates of size at maturity for female and male draughtboard sharks caught in the marine reserve compared to those caught from the rest of Tasmania. The LDPM estimates of size at maturity were closer to the macroscopic estimates for all values except males in the marine reserve.
Similarly, the confidence limits for the LDPM were narrower than the corresponding
MDS for all analyses except for males in the marine reserve. The techniques were sensitive to sample sizes with the smaller sample sizes from the reserve population resulting in larger increasing the 95% confidence limits (Fig. 3.7).
93 Chapter three - Non-lethal assessment of reproductive parameters
Table 3.4 . Size at 50% maturity for female and male d raughtboard sharks based on the hormone results of the linear discriminant predictive model (LDPM) and multi dimensional scaling (MDS) analysis.
50 % maturity 95% Confidence ogive r2 a and b values interval n TL (mm) TL (mm)
Females (LDPM analysis) 818 0.74 a = -72.09, b = 0.09 803 – 855 82
Females (MDS analysis) 828 0.76 a = -9.44, b = 0.01 775 –870 82
Males (LDPM analysis) 768 0.83 a = -43.84, b = 0.06 745 – 803 54
Males (MDS analysis) 757 084 a = -25.73, b = 0.03 740 – 795 54
880
860
840
820
800
780
760
740
720 Visual LDPM MDSLDPM MDS Visual LDPM MDSLDPM MDS Size at 50% 95%at maturity Size limits confidence with Rest of Tasmania Marine Reserve Rest of Tasmania Marine Reserve
Method Figure 3.7 : Comparison of 50% size at maturity and 95% confidence limits for fem ale (circles) and male (triangles) draughtboard sharks caught in a marine reserve and from the rest of Tasmania using linear discriminant analysis (LDPM) and multi dimensional scaling ordination (MDS).
94 Chapter three - Non-lethal assessment of reproductive parameters
Reproductive Seasonality
For both sexes the proportion of adult animals found in the marine reserve was only slightly less than those found from the rest of Tasmania, although this difference was non significant (Table 3.5).
FIG . 3.5: Proportion of mature draughtboard sharks caught in lobster tr aps from a marine reserve and from the rest of Tasmania. Proportion adult animals Location Females Males
Marine Reserve 0.29 0.48
Rest of Tasmania 0.38 0.54
To compare monthly variations of hormones between sharks obtained from the marine reserve and the rest of Tasmania, sharks were grouped into three periods due to
the small sample sizes. For females, there were no significant differences between E 2, or
P4 in sharks from the marine reserve compared with those from the rest of Tasmania. In contrast, T levels were lower in sharks from the marine reserve than from the rest of
Tasmania in the March May period (ANOVA, P< 0.001) (Fig. 3.8). The levels of T were significantly lower for males in the marine reserve for the March May period (ANOVA,
P< 0.001), although the gradual decline in T levels from January to December was consistent in both the marine reserve and the rest of Tasmania (Fig. 3.9).
95 Chapter three - Non-lethal assessment of reproductive parameters
7 P4 6
5
4
3
2
1
0
7 ) E2 -1 6
5
4
3
Hormone level Hormone (ng.ml level 2
1 2.0 T
11 1.5 13 * 1.0 10
* 0.5 14
6 4 0.0 Jan-Feb Mar-Apr-May Oct-Nov-Dec Period
Figure 3.8: Seasonal variations in testosterone (T), 17β estradiol ( E2) and progesterone ( P4) for adult female draughtboard sharks caught in a marine reserve ( ) and the rest of Tasmania ( ). Values are mean ± SE. Numbers are sample sizes. * Values are significant different. 96 Chapter three - Non-lethal assessment of reproductive parameters
8 16
7 ) 29 -1 6 * 21 5 *
4 11 3 9 12 2 Testosterone (ng.ml Testosterone levels 1
0 Jan-Apr May-Aug Sep-Dec Period
Figure 3.9: Seasonal variations in testosterone for adult male draughtboard sharks caught in a marine reserve ( •) and the rest of Tasmania (▲). Values are mean ± SE. Numbers are sample sizes. * Values are significant different.
97 Chapter three - Non-lethal assessment of reproductive parameters
3.4 DISCUSSION
Size at maturity obtained from blood samples was within 2 % of the size at maturity obtained from macroscopic examinations of gonads. For both sexes in C. laticeps, the combination of external features (e.g. total length in females and clasper length in males) and gonadal steroids can be used to obtain reproductive information for management of sharks without having to sacrifice the animal.
From macroscopic examination of dissected animals it was clear that for draughtboard sharks, maturity is strongly size dependent. Sharks larger than 860 and 870 mm TL (females and males respectively) were all adults and sharks below 750 and 710 mm TL (females and males respectively) were all juveniles.
For C. laticeps , both the linear discriminant predictive function and the multi dimensional scaling analysis provided objective methods to classify sharks as juveniles or adults, and therefore address size at maturity and reproductive seasonality. Furthermore, steroid hormones could determine the stage of maturity in the intermediate length size classes where sharks could be either juveniles or adults. For these sharks neither total length or clasper calcification could be used to determine the reproductive stage of the animals, therefore hormones provided a mechanism for determining the reproductive status of these sharks. The LDPM had narrower confidence limits and was, in general, closer to the macroscopic estimates giving a more precise and accurate method than the
MDS, although no significant differences were found between values. The sample sizes would suggest that for C. laticeps , it is necessary to have approximately 100 sharks with a significant proportion in the critical region between 100% adults and 100% juveniles to obtain an accurate estimate of size at maturity.
When selecting the hormones to use to separate juvenile or adult sharks, understanding the role that each of the gonadal steroids play in shark reproduction is important. Hormone analysis is relatively costly, thus knowledge of which hormones
98 Chapter three - Non-lethal assessment of reproductive parameters contributed to the separation of the reproductive stages should enable costs to be minimized. For draughtboard sharks there was a need to measure only two hormones, T
(for both sexes) and E 2 (for females), to separate juveniles from adults. Testosterone and E 2 were found to be the principal hormones during the follicular phase of females, while elevated plasma P 4 was found primarily in the ovulatory phase (see chapter 2). As
P4 only varied in adult females and was dependent on the female ovulatory phase, it was possible to find adult females with low or high levels of P 4, whereas juvenile females always had low levels of P 4. Therefore, P 4 was not a reliable discriminant factor for separating juvenile and adult females. In males, only T showed a significant increase from juvenile to adult animals (see chapter 2) and thus was the main contributor to the separation. In C. laticeps males, clasper length and T contributed to the separation of juveniles from adults. As the degree of calcification and size of claspers were external features that could be readily assessed, clasper length and calcification will be the most cost effective method of identifying the size at sexual maturity of C. laticeps males.
Steroid hormones were also important in providing data on seasonality of reproduction. While clasper calcification can be used to address size at maturity in several chondrichthyans species, dissection of these males is still required to understand seasonality. Although C. laticeps was not found to have a defined seasonal reproductive pattern (see chapter 2), variations in hormone levels followed similar trends in reproductive activity obtained from macroscopic examination of the gonads. In seasonally reproductive species such as Hemiscyllium ocellatum (Heupel et al. , 1999), Raja eglanteria (Rasmussen et al. , 1999) and Dasyatis sabina (Tricas et al. , 2000), strong correlations in hormones and reproductive seasons have been reported.
A concern could be that the lower steroid plasma levels in seasonally reproductive sharks captured during their non reproductively active period could confound the estimates of size at maturity as they could be classified as juveniles (ie. if the hormone
99 Chapter three - Non-lethal assessment of reproductive parameters values fall to values equivalent of juveniles). To obtain size at maturity estimates it is essential to sample animals during the reproductive period.
The population of C. laticeps from the Crayfish Point Reserve showed that the proportion of adult sharks (for both sexes) was similar to the proportion in the rest of
Tasmania, suggesting that although this reserve would offer protection to adult animals, this protection was not preferential. The similar seasonal trends in reproductive hormones for females and males between the reserve population and the rest of
Tasmania is expected, as tagging studies (see chapter 4) demonstrated that this species can move substantial distances and mix between regions. Although the seasonal sample sizes were small, the similarity in trends between the dominant hormones and macroscopic examination of the gonads in each sex demonstrated the potential of hormones to define spatial and temporal variability in reproduction without the need to sacrifice the sharks.
Different methods, such as ultrasonography and endoscopy, have been used to assess gestation period and reproductive condition in females without killing the animal
(Carrier et al. , 2003) (J. Daly, Melbourne aquarium, Melbourne. pers. comm.). To date no estimates of size at sexual maturity or seasonality of reproduction have been reported using these techniques. Ultrasonography or endoscopy also require substantial handling and manipulation of the sharks, which could affect both the shark and its embryos
(Carrier et al. , 2003). Obtaining a blood sample from the draughtboard shark for estimating hormone concentration, involved a minimal handling time (2 3 minutes) before the shark was returned to the water. The sample could be taken at sea in exposed and rough conditions, making hormones a less invasive, quick technique.
Endocrine markers provide a non destructive way to obtain information on somatic, temporal and spatial reproductive parameters for management of sharks. Non destructive techniques are essential for sampling marine species on the world’s threatened and endangered species lists, of which there are many chondrichthyans
100 Chapter three - Non-lethal assessment of reproductive parameters
(IUCN, 2006). Understanding the impact of fishing operations on bycatch is also required for industry accreditation and meeting ecosystem based fishery management objectives (Hall et al. , 2000). In circumstances where the bycatch is not retained, sacrificing the shark to obtain information on reproductive status would no longer be required.
A general trend or common pattern in the MDS ordination or the (D) score values of the LDPM analyses may also be found to distinguish juvenile and adult sharks for the different reproductive modes (oviparity and viviparity). In this case, it would no longer be necessary to sacrifice sharks that are not a target or by product of fishing operations.
If the relationship between steroid hormones and reproduction is reproductive mode specific or generic to all chondrichthyans, then validation for different species would not be required and future reproductive needs (size at maturity, seasonal reproductive activity) for management could be addressed non destructively though blood sampling.
As hormones can also provide information on the seasonality of reproduction, they have the potential to provide necessary information required for the conservation and management of shark populations without the need to sacrifice the animal.
101
CHAPTER FOUR : Movements, activity patterns and habitat utilisation
102 Chapter four - Movement
4.1 INTRODUCTION
Efforts to study the movement of fish at either the population or individual levels have been in progress for over a century (Casey and Taniuchi, 1990; Kohler and Turner,
2001). Tagging is the most used method for studying fish movements, and provides important information on life history and population dynamics (Hilborn, 1990; Eiler,
2000). Recently, the importance of incorporating fish behaviour and habitat utilisation as components of fish movement studies has been recognized for marine management and conservation programs (Shumway, 1999; Koehn, 2000).
Historically, migrations, movement patterns and habitat preferences of fishes were determined by fishery dependent mark and recapture or visual ( in situ ) observations
(Gunn, 2000; Stevens, 2000; Lowe et al. , 2003). Initially, mark recapture studies used conventional tags which are defined as those that can be identified visually without the use of detection equipment (Kohler and Turner, 2001). Conventional tagging experiments of cartilaginous fishes were first reported in the 1930s (McFarlane et al. ,
1990; Kohler and Turner, 2001; Latour, 2004), and have subsequently continued to be an important source of information for understanding chondrichthyan populations
(McFarlane et al. , 1990; Hurst et al. , 1999; Stevens, 2000).
Conventional mark recapture studies rely on recaptures of the tag by a variety of sampling gears used by either researchers or fishers. Bias associated with conventional mark recapture studies can occur when selectivity of sampling gears varies with habitat, or habitat specific movements alter catch rates, or fleet dynamics alter the probability of recapture (Kohler and Turner, 2001; Simpendorfer and Heupel, 2004; Bolle et al. , 2005).
The development of acoustic tracking technology in the 1960’s enabled detection of the animal independent of the need to be recaptured. By either the use of hand held detectors (active tracking) or moored ‘listening receivers’ (passive tracking) detailed information on animal behaviour and movement can be obtained. Passive acoustic
103 Chapter four - Movement monitoring technology allows movement patterns of multiple individuals tagged with acoustic transmitters to be determined (Heupel and Hueter, 2001; Voegeli et al. , 2001;
Heupel and Hueter, 2002). Hydrophone (listening) stations (receivers) record the date, time and identity of an aquatic animal fitted with an acoustic transmitter swimming within the detection range of the receiver (Voegeli et al. , 2001). One of the most promising current applications of acoustic tags to fishery management is elucidation of home range area (Kramer and Chapman, 1999), and habitat utilisation (Sibert and
Nielsen, 2000). While there are many studies applying acoustic technology in sharks and rays (Holland et al. , 1999; Heupel and Hueter, 2001; Klimley et al. , 2002; Nakano et al. ,
2003; Garla et al. , 2006)), only a few have addressed home range or habitat utilisation
(Morrissey and Gruber, 1993a; Morrissey and Gruber, 1993b; Heithaus et al. , 2002;
Heupel et al., 2004; Duncan and Holland, 2006; Heupel et al. , 2006b).
Although Cephaloscyllium laticeps is primarily caught as bycatch in rock lobster traps and other inshore hook and gill net fisheries, there is concern that the small amount of byproduct that is currently caught has the potential to expand (J. Lyle, TAFI Marine research Laboratories, Hobart. pers. comm.). As a precautionary measure, Tasmania has implemented a possession limit of two draughtboard sharks per person, or five sharks per boat per day to constrain future catches.
Walker (2005) reported a 54% decline in draughtboard sharks caught in Bass Strait, southern Australia between 1973 76 and 1999 2001. Although the cause for this decline is uncertain, Walker suggested that it might be due to a change in fishing patterns in an attempt to minimise bycatch of this species rather than a true decline in abundance due to fishing.
Prior to considering any increased utilisation of this species it was important to understand the mixing of populations between regions. Small scale movement patterns and habitat utilisation was also considered important to establish if sharks were more vulnerable to capture at certain times of the day and on certain substrates. Knowledge
104 Chapter four - Movement of the behaviour of draughtboard sharks can therefore be used to both increase exploitation through targeted fishing or to minimise bycatch by avoidance.
Previous studies in other scyliorhinids showed that sharks are characterised as slow swimmers (Springer, 1979; Compagno, 1984) and are often found resting in caves either alone or in aggregations (Nelson and Johnson, 1970; Sims et al. , 2005). Nelson and
Johnson (1970) reported nocturnal activity patterns for the scyliorhinids Heterodontus francisci and Cephalloscylium ventriousus and Sims (2001) found differences in the day night activity between males and females of Scyliorhinus canicula .
This study investigated the movement behaviour of the draughtboard shark using conventional and acoustic tagging. Conventional tags were used to identify longer term movement (> 6 months) over larger geographic regions, while acoustic tagging evaluated short term movements (< 6 months) and habitat utilisation.
105 Chapter four - Movement
4.2 MATERIAL AND METHODS
4.2.1 ACOUSTIC TAGGING
Study site, acoustic receivers and transmitters
An array of 82 VR2 automated acoustic receivers (Vemco Ltd., Nova Scotia) were deployed in October 2002 and retrieved in July 2003 in southeast Tasmania, Australia
(Fig. 4.1a). The sea floor in these areas consisted of sand, silt, seagrass and low profile reef (Barrett et al. , 2001; Jordan et al. , 2001). Each receiver was secured to a vertical steel post on a concrete mooring, approximately 1 m above the sea floor.
An extensive array of receivers was established as a series of acoustic ‘curtains’ separating the main bays and channels in southeast Tasmania (Fig. 4.1b). The depth of receiver placement varied from 2 to 55 m. The distance between receivers was chosen to ensure that detection distances had substantial overlap and varied from 720 to 930 m depending on the habitat type. Receivers were positioned at the entrances of bays and channels to ensure that no shark could move into or out of these areas without being detected.
Within the extensive array, an intensive array was established at the Crayfish Point
Reserve (total area= 800 m 2) and the adjacent areas of Alum Cliff and Taroona High
(Fig. 4.1 c). In the Crayfish Point Reserve, the sea floor includes a complex mix of sand, silt, low and high profile reef (Barrett et al. , 2001; Jordan et al. , 2001). The complexity of this habitat resulted in a reduction of the detection range for the acoustic receivers to a minimum of 60 m (Semmens, unpublished data). Thus, the receivers were placed approximately 100 m apart to provide sufficient overlap for determining position from detection at multiple receivers. The receivers were placed in depths from 2 to 11 m. The receivers that formed a small ‘curtain’ perpendicular to the shore at Alum Cliff and
Taroona High were 400 450 m apart.
106 Chapter four - Movement
The transmitters (V8SC 2H: Vemco, Nova Scotia), were cylindrical in shape, 30 mm
in length, 9 mm in diameter and weigh 3.1 g in water. The transmitters emit a 69 kHz
frequency “ping” code repeated after a random delay of 20 to 60 s. The battery life was
set at 180 days.
Tasmania
41°
43°
148° 147° a
Taroona High
Crayfish Point I J Crayfish Point Reserve E H K Reserve D F C B G
Alum Cliff
b c Figure 4.1: Acoustic receiver positions. a: Map of Tasmania showing out the southwest area. b: Receiver positions in the southwest area. Extensive curtains are labelled as: B: Lower mid channel, C: Upper mid channel, D: Upper channel, E: Upper Derwent, F: Lower Derwent, G: Storm Bay, H: Frederick Henry Bay, I: Norfolk Bay, J: Dunally, K: Eaglehawk Neck. c: Receivers position in Crayfish Point Reserve and adjacent areas. c: Receiver positions at the intensive array area established by the Crayfish Point Reserve, Alum Cliff and Taroona High.
107 Chapter four - Movement
Sampling methodology
Between January and March 2003, 25 (15 females, 9 males, 1 no sex recorded) draughtboard sharks were caught in rock lobster traps. Fifteen sharks were sourced from the Crayfish Point Reserve and 10 sharks from the east coast of Tasmania (42
43°S, 147 148°E) (Fig. 2.1). All sharks were released in the Crayfish Point Reserve. Prior to release, total length, total weight, and clasper length (for males) was recorded. Sharks were fitted with the acoustic transmitters and injected with 25mg/kg of the antibiotic tetracycline dissolved to saturation in seawater.
Initially, two sharks were internally tagged by inserting the tag into the body cavity.
After capture, these sharks were injected with a localised anaesthetic (Xylocaine 0.5%,
25 mg in 5 ml), and a 3 4 cm incision was made in the ventro lateral region (the ventral region was considered unsuitable as the sharks rest on the sea floor) towards the rear of the stomach cavity. The transmitters were coated in 100% paraffin to prevent transmitter rejection and to cover any sharp protrusion on the transmitter surface that might irritate the shark (Heupel and Hueter, 2001). The transmitter was inserted and the cavity closed using surgical glue (Indermil® Loctite Corporation, Dublin) and a disposable skin stapler (Royal 35W, United State Surgical Corporation, Ltd). The sharks were then held in captivity for one week prior to being released in the Crayfish Point
Reserve. However, the tagging wound was observed to re open in several sharks, probably due to the frenetic movements following release. Because of the uncertainty associated with a partially open wound, the remaining 23 sharks were tagged externally.
For the external tagging, two 1.10 mm x 38 mm surgical needles were joined to the distal end of the transmitters. The transmitter was attached to the base of the first dorsal fin by the needles piercing through the fin. The needles passed through buttons on the opposite side of the fin and were then crimped and the excess needle length removed
(Fig. 4.2).
108 Chapter four - Movement
a b c d
Figure 4.2: External acoustic tag attachm ent on draughtboard sharks. a: Acoustic transmitter has been modified with the insertions of needles across both ends. b: The transmitter is inserted in the base of the first dorsal fin. c: The transmitter is attached to the dorsal fin and the residual needles are crimped. d: The attachment is complete.
Analysis of the data
Raw data collected by the receivers, including transmitter number, and time and date of detection was downloaded (in March April and July 2003) using the VR2 data processing software (Vemco Ltd). Data from both arrays was analysed using ArcView
3.2 (ESRI 1999) with the Animal Movement Analyst Extension (AMAE) tool (Hooge and Eichenlaub, 2000) and Microsoft Excel.
PERFORMANCE OF THE RECEIVERS WITHIN THE CRAYFISH POINT RESERVE
Receiver performance could be affected by the interference of acoustic noise (eg: signals from motoring small vessels) in addition to obstruction and ‘bounce’ of acoustic signals in association with different substrate types (eg: sand, high profile reef) and vegetation (eg: density of seaweeds such as kelp). To evaluate the performance of the receivers, data from sharks detected in the inner arrays of the Crayfish Point Reserve and arrays outside the reserve (i.e. Alum Cliff or Taroona High) were compared. The data were examined to determine if sharks detected inside, and subsequently outside the reserve (or vice versa) were also detected by the outer ring of the Crayfish Point Reserve receivers (Fig. 4.3). The performances of the receivers were then assessed by counting
109 Chapter four - Movement the times that the outer receivers detected the movement of 3 (1 internally and 2 externally tagged) sharks that moved, on 232 occasions, in and out of the inner area of the Crayfish Point Reserve. Detection on the outer ring receivers indicated progress of sharks from inner to outer regions and suggested good receiver performance.
Taroona High Receivers
3,4 Crayfish Point Reserve
4 3 4,1 Inner 2,3 1 2
Alum Cliff 1,2 Receivers
Figure 4.3: The Crayfish Point Reserve was subdivided into four outside areas (1 4) and 1 inside area (inner). Sharks moving from the inner region of the reserve to either Alum Cliff or Taroona high (or vice versa) had to be detected by the outer ring receivers (areas 1, 2, 3 and 4) of the reserve.
DEFINITION OF MOVEMENT
For the intensive array, it was possible to obtain large datasets if the sharks remained for periods of time within the reserve and adjacent areas. Due to the complexity of the habitat, sharks could move by swimming between adjacent high profile reefs (e.g. in narrow channels within the reef structure), over higher profile reefs
(e.g. elevated position in the water column) and over low profile reefs and other
110 Chapter four - Movement habitats. Thus, the detection distance of the receivers changed as the habitat increases in complexity (Fig. 4.4). Rocks and differing densities of macroalgae decreased the detection distance and small movements of a shark, particularly vertically within the water column, could result in detections by different receivers suggesting different locations. Thus, movement in this study was defined as either movement from one area to another non adjacent area of the reserve (eg. from area 1 to 3 and from 2 to 4, or non adjacent joint areas from 1,2 to 3,4 and 4,1 to 2,3) or a consistent pattern of a new set of receivers detecting a transmitter (see Fig. 4.3). For the extensive array, Alum Cliff and Taroona High, movement was defined as when more than two non adjacent receivers of the curtain detected a shark.
Reef habitat
VR2 Rock Rock
Figure 4.4: Generalised representation o f the reef habitat. The receiver (VR2) reception is permanently blocked when there are rocks between the sharks and the receivers, resulting in no detection (x). Intermittent blocking of the receiver reception (x√) occurs when there are algae between the sharks and the receivers. A receiver will also intermittently detect a shark, when the shark moves around a rock situated between the shark and the receiver (x√). When there is not obstruction between the shark and the receiver, sharks will be continuously detected (√).
111 Chapter four - Movement
DEFINITION OF STATIONARY OR MINOR MOVEMENT PERIODS
When the shark was detected by the same receiver or set of receivers for at least
60 minutes at intervals of 1 minute or less (60 + detections in an hour) the shark was considered to be stationary. Often during this time it was possible for an additional receiver to detect the shark, but it was unknown if the shark had moved slightly or the detection was associated with movement of the habitat (eg. kelp) between the shark and the receiver. In any case, stationary or minor movements indicated that the shark is not actively swimming.
Differences in the number of sharks with stationary periods were tested by
Student t-test (Quinn and Keough, 2002). To determine if there was a difference with size, sharks were classified as juveniles and adults according to 50% length at maturity (see chapter 2). Sharks smaller or larger than 815 mm (for females) and 760 mm TL (for males) were considered as juveniles and adults respectively .
DEFINITION OF UNACCOUNTABLE TIME
There were times where an individual draughtboard shark could not be accounted during tracking period. There were two possible reasons why the VR2 system did not record the shark positions: 1) sharks were moving to areas outside the detection range of the receivers, 2) sharks remained in areas were the VR2 system was obstructed by external noise (eg. motor vessel) or marine substrate.
DEFINITION OF PRESENCE
As there were a large number of periods in the data set when individual sharks could not be accounted for, the presence of a shark in any given area was defined as when one or a group of receivers recorded at least 10 detections. This minimises the
112 Chapter four - Movement number of false detections (single detection of any transmitter code) caused by external noises, producing false readings of the transmitter identification number (tag) by the receivers.
HABITAT UTILISATION
Habitat utilisation was determined as the 95% probability of a draughtboard shark being found within a certain area calculated as the 95% kernel utilisation distribution
(KUD) (Worton, 1987) using the AMAE tool in Arcview. The spatial use of the habitat through time was evaluated by examining the 95% KUD estimates for each shark per month and by combining all months together. A student t test was used to compare the total number of animals that had stationary periods (see definition above) found in each of the 5 areas of the Crayfish Point Reserve and the overlap areas (Fig 4.3) to determine if sharks preferred specific areas of the Reserve.
HABITAT PREFERENCE
To evaluate the preferred habitats that sharks used to either move or have stationary periods it was necessary to use separate approaches for movement and stationary periods:
1. To determine if sharks preferred to move in any specific habitat, the proportion of
movements in each area was calculated, for each shark, as the number of
movements within an area divided by the total number of movements for that
shark. Differences in the proportion of movements in different areas were tested
using a Chi Square test (Quinn and Keough, 2002).
113 Chapter four - Movement
To determine if there was a preference for certain boundary regions of the Crayfish
Point Reserve for access into and away from the reserve, the proportion of
movements in each area was compared. For this part of the analysis, area 1 of the
reserve includes the overlap between areas 1 and 4 (area 4,1), area 2 includes the
overlap between areas 1,2, area 3 includes the overlap between areas 2,3, and area 4
includes the overlaps between areas 3,4.
To determine if sharks preferred to spend stationary periods in any specific area, the
proportion of the hours that sharks were stationary in each area was calculated as
the number of hours that all sharks were stationary in each area, divided by the total
number of hours that sharks were stationary. Differences in these proportions were
tested using Chi square. As this analysis was based on time, the same analysis was
undertaken based solely on the occurrence of a stationary period irrespective of the
length of the stationary period (providing it was greater than one hour). Thus, a
single stationary period of 20 hours provided a count of 1 whereas 3 separate one
hour stationary periods separated by periods of movement provided a count of 3.
2. Habitat preference for both movement and stationary periods was determined as the
50% kernel utilisation distribution (KUD) using the AMAE tool in Arcview. The
50% contour was chosen to indicate the areas of greatest use.
DAY NIGHT ACTIVITY
Day night habitat utilisation and preferences were calculated using both the 95% and 50% KUDs respectively.
114 Chapter four - Movement
SITE FIDELITY FOR THE CRAYFISH POINT RESERVE
To determine if sharks remained in, or dispersed away from the Crayfish Point
Reserve region, the number of days that each shark was detected in the Crayfish Point
Reserve was compared for each month after release and differences tested by a one way
ANOVA and Tukey’s multiple comparison tests (Quinn and Keough, 2002).
4.2.2 CONVENTIONAL TAGGING
Study site and sampling methodology
Between January 2000 and January 2005, sharks were tagged during routine fishery dependent and independent rock lobster catch sampling trips around southwestern and eastern Tasmania and in the Crayfish Point Reserve (Fig. 1.2a). Each shark was tagged with a 35 mm yellow standard Rototag (Daltons, Henly on Thames,
England) externally attached to the second dorsal fin. For each shark, sex, total length and clasper length (males) were recorded.
Analysis of the data
Differences in the proportion of sexes, sizes and time at liberty were tested by
Chi square. The time at liberty was subdivided into 7 periods: 1) 0 to 6 months, 2) 7 to
11 months, 3) 1 2 years, 4) 2 3 years, 5) 3 4 years, 6) 4 5 years and 7) > 5 years. Size frequency distributions were compared between sexes of released and tagged sharks from different areas, using a randomisation procedure with the Kolmogorov Smirnov test statistic ( D) with data pooled across sexes. Size frequency data from the two distributions being compared were pooled and randomly reallocated to each original distribution and the test statistic ( D) recalculated. The procedure was repeated 1000
115 Chapter four - Movement times and the test of significant difference between the two distributions made by comparing the value of the observed test statistic to the distribution of D values obtained by the randomisation procedure. Significant differences were identified when less that 20 of the D values obtained from the randomisation procedure exceeded the value of D from the original distributions (Haddon, 2001).
To calculate short and long term site fidelity for the Crayfish Point Reserve, data was standardized to account for differing effort (number of trap lifts) undertaken in the different surveys, by the following equation:
Ti (Sri ) Sti Pij = Cj Stj
Where Pij is the proportion of sharks recapture in trip i that where tagged in trip j, where j > i. Cj is the catch rate (number of sharks/trap) of sharks tagged during j, and was calculated as St j/Tj, where St j is the number of sharks caught and tagged in trip j, and Tj is the total number of traps set to capture the sharks in the trip j. The value Ti is the total number of traps set to capture the sharks in trip i, Sri is the number of sharks
recaptured in trip i that were tagged in trip j, and Sti is the total number of sharks caught in trip i.
To calculate the expected catchability the following assumptions were made:
1. Catch rate was a function of effort
2. The Crayfish Point Reserve had no finite carrying capacity
3. Tagged sharks were distributed randomly within the population
116 Chapter four - Movement
Differences in the proportion of sharks recaptured either per month or per year in the
Crayfish Point Reserve were tested using Chi square test.
All statistical analyses, for both acoustic and conventional tagging, were carried out using SPSS (SPSS® Base 10.0). The significance level was set at P=0.05 for all data analysis.
117 Chapter four - Movement
4.3 RESULTS
4.3.1 ACOUSTIC TAGGING
Transmitter performances
Of the 25 sharks that were acoustically tagged, six transmitters did not start
working until one month after attachment (they were incorrectly set to start one month
after the battery was connected), one transmitter (#143) was never recorded and one
transmitter (#156), attached to a shark that was caught on the east coast of Tasmania,
was recorded only twice on the day of tagging. Transmitters #143 and #156 were
excluded from the analysis.
Performances of the receivers within the Crayfish Point Reserve
The performances of the receivers was considered to be high as 97% (± 0.43 SE)
and 86% (± 4.44 SE) of sharks that moved between the inner region of the Crayfish
Point Reserve and Alum Cliff or Taroona High respectively were detected (Table 4.1).
Table 4.1: Movements of three draughtboard sharks between the inner areas of the Crayfish Point Reserve (CPR) and Alum Cliff or Taroona High.
Inner CPR Alum Cliff Inner CPR Taroona High
Percentage of Percentage of Number of sharks movement movements Total number Total number detected by the detected by of movements of movements outer CPR the outer CPR receivers receivers 2 sharks (externally tagged) 92 97% (n=89) 67 88% (n=59) 1 shark (internally tagged) 49 96% (n=47) 24 79% (n=19) Total 141 97% (n=136) 91 86% (n=78)
118 Chapter four - Movement
Movement patterns
For the 17 sharks where transmitters were working on release, 10 were recorded at the marine reserve on the same day of released, six within the first week of being released and one more than a week after release (Fig. 4.5). Presence of sharks was detected in the entire intensive array and some areas of the extensive array (the River
Derwent, the Upper Channel and Storm Bay (Fig. 4.6)).
12 n=17
10
8
6
4
2 Number Number recorded of atsharks the CPR 0 0 1 2 3 4 5 6 7 > 7
Days between release and first detection Figure 4.5: Number of draughtboard sharks recorded at the Crayfish Point Reserve (CPR) soon after being released.
119 Chapter four - Movement
42.75°
Intensive array E
43° D F
G
147.88° 147.38°
Figure 4.6 : Presence of draughtboard sharks in the study area (red triangles). D: Upper channel, E: Upper Derwent, F: Lower Derwent, G: Storm Bay. The intensive array is the area formed by Crayfish Point Reserve, Alum Cliff and Taroona High.
Sharks showed two typical patterns of movement. The majority of the sharks (n=20) were never recorded beyond the Derwent River during the survey period (Fig. 4.7a).
However, three females moved away from the intensive array and beyond the Derwent
River (Fig. 4.7b).
120 Chapter four - Movement
CPR Initial recorded 42.75° position of shark #146 CPR
43° 43° Initial recorded Final recorded F position of shark position of Final recorded #121 shark #146 position of shark #121
F G 43.6° D
147.62 ° 147.50 ° a 147.88° 147.38° b Figure 4.7: Examples of the two ty pical movement patterns of the draughtboard sharks. a) Shark 146 was never recorded beyond the Derwent River, moving between the intensive array, the Derwent River (F) and the Upper mid channel (D). b) Shark 121 left the Crayfish Point Reserve (CPR) soon after it was tagged travelling through the Lower Derwent (F) to Storm Bay (G). Blue arrows show the movement of the sharks.
The minimum time that sharks were recorded before leaving the extensive array was
11 days and the maximum overall time was 188 days. The last records for the 23 sharks were evenly split between the intensive array (n=11) and the extensive array (n=12)
(Table 4.2). Although the sharks that were translocated from the east coast of Tasmania tended to leave the reserve earlier than those captured from the Crayfish Point Reserve
(Fig. 4.8), all of the nine sharks sourced from the east coast remained within the
Derwent River region during the study period.
Neither of the two sharks that were internally tagged demonstrated any behaviour that would suggest an impact of tagging different to the fin tagged sharks. One of the sharks moved away from the Crayfish Point Reserve and out of Storm Bay whereas the other was last located, at the end of the study, within the reserve.
121
Table 4.2: Summary data for draughtboard sharks tracked in the southeast region of Tasmania. All sharks were tagged and released at the Crayfish Point Reserve. Ten sharks were caught on the east coast of Tasmania and translocated to the Crayfish Point Reserve (CPR), the other 15 were caught at the CPR. Two sharks were internally tagged, these sharks are indicated with a single asteric (*). One transmitter did not work (shark ID 143). AC: Alum Cliff, B: Lower mid channel, E: Upper Derwent River, EC: East coast of Tasmania, F: Lower Derwent River, G: Storm Bay, TH: Taroona High. (**) For 6 sharks, transmitters started working one month after insertion; the date of tagging was used as the date of the first record. Sharks with less than two hits (#156, #143) were excluded from the analysis. Date of Date of last Number of Zone of last Shark ID Sex Size Source tagging record hits record 164 F 920 EC 18/03/03 29/03/03 313 F 163 ** F 760 EC 26/04/03 4/05/03 7 AC 162 ** M 760 EC 26/04/03 29/04/03 20 TH 161 ** M 880 EC 26/04/03 28/05/03 137 F 160 F 820 CPR 18/02/03 17/03/03 56 G 159 M 715 CPR 13/02/03 9/05/03 2553 AC 158 F 620 CPR 20/02/03 6/05/03 93 B 157 M 530 CPR 20/02/03 22/03/03 1286 F 156 M 820 EC 26/03/03 26/03/03 2 - 155 F 600 CPR 11/02/03 20/03/03 1458 CPR 154 F 820 CPR 18/02/03 01/07/03 2702 AC 153 F 630 CPR 18/02/03 22/03/03 146 AC 152 F 750 EC 25/03/03 4/04/03 88 AC 151 M 770 CPR 18/02/03 8/06/03 3616 AC 149 F 830 CPR 12/02/03 30/03/03 14846 E 148 M 610 CPR 19/02/03 22/03/03 677 TH 147 M 870 CPR 12/02/03 16/05/03 8541 TH 146 ** M 750 EC 26/04/03 7/07/03 415 F 145 ** M 660 EC 26/04/03 21/05/03 1758 F 144 ** F 650 EC 26/04/03 22/05/03 235 F 143 F 880 CPR 13/02/03 MISSING - - 141 F 870 EC 18/03/03 25/04/03 1903 F 140 F 770 CPR 18/02/03 06/07/03 9685 AC 121 * F 830 CPR 16/01/03 21/02/03 20 G 116 * F 770 CPR 16/01/03 22/07/03 1313 CPR
122 Chapter four - Movement
1.0
0.8
0.6
0.4
0.2
0.0 Proportion at of thesharks CPR 0 1 2 3 4 5 6 Months after being released
Figure 4.8: Monthly proportion of draughtboard sharks at the Crayfish Point Reserve (CPR). 13 sharks were sourced from the CPR (black bars) and 10 sharks were translocated from the east coast of Tasmania (grey bars).
Movement behaviour
Two types of movement behaviour were recorded; sharks that only showed movement and sharks that alternated movement with stationary periods. Nine sharks showed continuous movement and 12 sharks alternated between movement and stationary periods (Fig. 4.9, Table 4.3). Movement behaviour could not be determined for two sharks due to a low number of detections. All sharks were out of the range of detection for several days (Table 4.3). Within the total number of days between the first record and the end of the study period, the percentage of days that sharks were detected averaged 17 % (± 4.11% SE) with 1% and 69% as the minimum and maximum (Table
4.3). The average time spent by sharks having stationary periods was eight hours per day
(± 1 hr SE) with one shark spending a continuous period of five consecutive days stationary. There was no difference in the average time spent in stationary periods for
123 Chapter four - Movement either sex (seven females and five males) or between juveniles (n=7) and adults (n=5).
No correlation existed between the movements of the draughtboard sharks and lunar phases (Fig. 4.9).
Shark# 140 Shark # Shark 147 Shark# 146 2/02 2/02 9/02 2/03 9/03 6/04 4/05 1/06 8/06 6/07 19/01 19/01 26/01 16/02 23/02 16/03 23/03 30/03 13/04 20/04 27/04 11/05 18/05 25/05 15/06 22/06 29/06 13/07 20/07 Day Figure 4.9: Examples of movement behaviour of draughtboard sharks. Nine sharks showed only movement records (eg: shark # 146), while 12 sharks alternated between movements and stationary periods (eg: shark #147 and #140). The figure indicates both movement and stationary behaviour simultaneously as these behaviours could occur on the same day” was added in the figure legend. Dashed lines represent the initial and the last record of the sharks. Black and grey regions represent movement and stationary periods, respectively. Lunar phases are shown at the top of the graph.
124 Chapter four - Movement
Table 4.3: Type of movement of draughtboard sharks. The total number of records were classified as movement, stationary period, or uncer tain. Sharks showed days when they were not recorded. * The percentage of days that had shark records was calculated assuming that all sharks were alive at the end of the study period.
Number of Number of Number of days Percentage of days Number of days between days recorded between last Shark ID Movement Stationary Uncertain within the study period detections first and last between first record and the end that shark was recorded * record and last record of the study period 164 271 18 2 251 12 4 115 3 163 7 7 9 4 79 5 162 16 16 4 2 84 2 161 104 3 0 101 32 3 55 3 160 44 6 0 38 28 4 127 3 159 2219 410 659 1150 86 40 74 25 158 89 6 0 83 76 4 77 3 157 1063 2 814 247 31 10 122 8 155 1215 26 927 262 38 8 124 6 154 2372 26 1622 724 134 55 21 35 153 100 4 0 96 33 2 122 1 152 69 10 0 59 11 8 109 7 151 3233 261 362 2610 111 69 44 45 149 12256 206 9632 2418 47 33 114 21 148 586 48 84 444 32 5 121 3 147 3066 299 2767 0 94 63 67 58 146 346 8 0 338 72 10 15 12 145 1474 468 0 1006 25 6 62 7 144 212 9 129 74 26 4 61 5 141 1610 459 302 849 39 16 88 18 140 8440 40 5702 2698 139 67 16 43 121 17 6 0 11 37 9 151 5 116 1155 120 413 622 182 125 69
125 Chapter four - Movement
Habitat utilisation
To determine habitat utilisation, the analysis was restricted to sharks with ≥ 20 movements. Sharks (n=12) with less than 20 detections, had insufficient information to demonstrate movement behaviour (Fig. 4.10)
14
12
10
8
6
Number Number sharks of 4
2
0 0-20 21-40 41-60 61-80 81-100 > 100
Number of movements
Figure 4.10: The distribution of movements for the 23 acoustically tagged draughtboard sharks.
For the 11 sharks with ≥ 20 movements, movements were recorded in all areas of the intensive array, the Upper and Lower Derwent River and the Upper Channel (Fig.
4.11a and Fig. 4.11b). The majority of the sharks remained within the Derwent River region (Fig. 4.11a). Nine of the 11 draughtboard sharks had ≥ 20 movements in the
Crayfish Point Reserve. These sharks utilised all regions of the reserve, with a minimum
126 Chapter four - Movement
of five sharks being recorded in each region and a maximum of nine sharks being
recorded in region three. No region was visited by all 11 sharks (Fig. 4.11a).
15 Movements Movements (a)
12
9
6
3 Number of sharks of Number detected 0 CPR AC TH Derwent Up CH 1 2 3 4 Inner 1,2 2,3 3,4 4,1 Areas Areas of Crayfish Point Reserve
Shark 116 Shark 164 (b)
CPR CPR
Figure 4.11: a) Number of draughtboard sharks detected in the different study regions estimated by 95 % KUD contours. Sharks (n=11) with at least 20 recorded movements were used to calculate habitat utilisation of movement. CPR: Crayfish Point Reserve. b) Examples of habitat utilisation distribution for draughtboard sharks estimated by 95% KUD contours.
The majority of the sharks (n ≥ 8) were detected as having stationary periods in the
Crayfish Point Reserve and in the Derwent River array, while less sharks (n ≤ 3) were
found to have minimal movements around Alum Cliff and Taroona High (Fig. 4.12a
and Fig. 4.12b). As the Derwent River, Alum Cliff and Taroona High have substrates of
sand with small areas of reef (Table 4.4), it is uncertain if these sharks were in stationary
127 Chapter four - Movement periods on the sand which surrounds these receiver arrays, were in stationary periods on the small reef areas, or are utilising the cement tyre that was used to support the listening stations. In comparison to the broad areas of the Crayfish Point Reserve moved over by the draughtboard sharks, sharks showed a preference for areas within the reserve to have stationary periods. The majority of the sharks were detected in area 1 and its overlap with area 2, followed by a significant decline (t test, P<0.001) in the other areas of the reserve used for stationary periods (Fig. 4.12a).
Table 4.4: Type of marine substrate for the intensive and extensive array. CPR: Crayfish Point Reserve.
Area Substrate type
Area 1 of CPR Mostly high profile reef and some sand and hard sand Mostly silty sand and some patchy reef Area 2 of CPR Mostly different profile sands and some patchy reef Area 3 of CPR Different profile reefs (low, patchy and high) Area 4 of CPR
Inner area of CPR Reefs and some sand and hard sand
Alum Cliff Sand and small areas of reef Sand, silty sand, and small areas of reef Taroona High Silty sand, sand and small patchy reef Derwent
128 Chapter four - Movement
(a)
15 Stationary periods Stationary periods
12
9
6
3 Numberof detected sharks 0 CPR AC TH Derwent Up CH 1 2 3 4 Inner 1,2 2,3 3,4 4,1 Areas Areas of Crayfish Point Reserve
Sh ark 148 Shark 141 (b)
CPR CPR
Figure 4.12: a) Number of draughtboard sharks having stationary periods in the different stu dy regions. Stationary periods were recorded for 12 sharks. b) Examples of habitat utilisation during stationary periods of draughtboard sharks estimated by 95% KUD contours. CPR: Crayfish Point Reserve.
Based on both movement and stationary periods, all monitored sharks showed similar habitat utilisation either by month or when all months were combined (Fig. 4.13 a and b).
129 Chapter four - Movement
Movement March Shark 116 Movement April Shark 116 Movement May Shark 116 (a)
CPR CPR CPR
Movement June Shark 116 Movement July Shark 116 Movement combined months Shark 116
CPR CPR CPR
Stationary March Shark 147 Stationary April Shark 147 Stationary combined months (b) Shark 147
CPR CPR CPR
Figure 4.13: Examples of individual and combined monthly habitat utilisation for (a) movements and (b) stationary periods of draughtboard sharks using 95% KUD contours. CPR: Crayfish Point Reserve.
130 Chapter four - Movement
Habitat preference
Of the total shark movements (n= 2594), 6% occurred when sharks moved between the outside and the inside of Crayfish Point Reserve. When moving between
Alum Cliff and Crayfish Point Reserve, draughtboard sharks used the region of the reserve (area 1) adjacent to Alum Cliff on 80% of occasions. In contrast, sharks that moved between Taroona High and Crayfish Point Reserve used the closest area of the reserve (area 4) equally (43%) to the furthest area of the Crayfish Point Reserve (area 1).
Sharks moving between the Derwent River and Crayfish Point Reserve used the closest area of the reserve (area 3) and the adjacent area (area 1) in equal proportions (38%).
Area 1 was the preferred region for sharks to enter or leave the Crayfish Point Reserve
(Fig. 4.14).
Despite the higher number of receivers in the Crayfish Point Reserve, the greatest number of movements were recorded by the six receivers at the Alum Cliff. These receivers recorded a significantly greater number of detections than all the other sites
(Chi square, P< 0.001) (Fig 4.15a). Although not significantly different, the two receivers at the Taroona High also had a higher number of movements compared to the
Crayfish Point Reserve. The draughtboard sharks tended to move along the shore rather than spend time moving within the reserve. To determine if there was a preference for sharks to move around the Alum Cliff region compared to the Taroona High region, the proportion of detections at Taroona High were compared with the proportion of detections at the two inner (closer to shore) receivers of the six at Alum Cliff. These two receivers are at approximately the same distance from the Crayfish Point Reserve and cover the same detection radius as the Taroona High receivers. The two receivers at the
Alum Cliff detected a significantly greater proportion of shark movements (0.30 ± 0.12
131 Chapter four - Movement
SE) than those at Taroona High (0.10 ± 0.05 SE) (Chi square, P< 0.001), indicating that tagged sharks dispersed out of the Derwent River rather than further up the river.
Within the Crayfish Point Reserve the two sides of the reserve closest to the Alum
Cliff (areas 1 and 2) detected a greater proportion of movements than those facing
Taroona High (Fig. 4.15b). At a finer scale, sharks showed a higher proportion of movements in shallower regions (the overlaps areas 3,4 and 4,1) rather than the deeper region (the overlaps areas 1,2 and 2,3) (Chi square, P< 0.001). Different areas of the
Crayfish Point Reserve overlapped within a 50% KUD for each shark, two sharks were detected only in area 1, two sharks used the composite region of areas 1, 2 and the inner region of the reserve, two shark used only area 1 and 3, and three sharks used the entire reserve (Table 4.5 and Fig. 4.16). All nine sharks monitored were detected in area 1 during this study.
132 Chapter four - Movement
(a) 1.0
0.8
c 0.6
0.4 Proportion of movements 0.2 a a,b b b 0.0 CPR AC TH D UC Areas 0.20 (b)
0.15 a a
0.10 a a a
a,b a 0.05 Proportionof movements
b,c c 0.00 1 23 4i 1,2 2,3 3,4 4,1 Areas of Crayfish Point Reserve
Figure 4.15: Area preferences for draughtboard sharks. a) Intensive and extensive array. b) Areas of the Crayfish Point Reserve (CPR). Values are mean + SE of the proportion of movements of each shark within specific regions. AC: Alum Cliff, TH: Taroona High, D: Upper and Lower Derwent, UC: Upper Channel. Different letter shows significant differences between movements and different areas. 133 Chapter four - Movement
Table 4.5: Areas within the Crayfish Point Reserve (CPR) that overlapped with the estimated 50% KUD of individual draughtboard sharks.
Number of sharks whose 50% KUD overlapped in Areas each area Area 1 and the joint area of 2 and 4 2 Areas 1, 2 and inner area 2 Area 1 and 3 2 The whole CPR 3
Shark 140 Shark 149
CPR CPR
Figure 4. 16: Example of habitat utilisa tion for draughtboard sharks determined by 50% KUD contours. CPR: Crayfish Point Reserve.
In contrast to shark movements within the Crayfish Point Reserve, the areas that
overlapped within the 50% KUD contours for each shark indicated that sharks had
stronger preferences for areas in which to have stationary periods (Table 4.6, Fig. 4.17).
Area 1 and its overlap with area 4 has 66% of the 33 recorded stationary periods and
92% of the 674 hours spent stationary were recorded in these two areas (Fig. 4.18 a and
b). These areas are the only two areas characterised by a high profile reef (Table 4.4),
indicating preferences for this reef type. Although areas 2 and the overlapping area 3,4
had a high proportion of movements they recorded a low number of stationary periods
indicating that they were only transit zones to other areas of the Crayfish Point Reserve.
134 Chapter four - Movement
However, areas 1 and the overlapping area 1,4 were preferred for both visiting and
having stationary periods.
Table 4.6: Areas within the Crayfish Point Reserve (CPR) that overlapped with the estimated 50% KUD of individual draughtboard sharks during their stationary periods.
Number of sharks whose 50% KUD overlapped Areas in each area Area 1 and the joint area of 2 and 4 7 Areas 1, 2 and inner area Area 1 and 3 Inner area 2 The whole CPR
Shark 154 Shark 155
CPR CPR
Figure 4. 17: Example of habitat utilisation for draughtboard shark stationary periods determined by 50% KUD contours. CPR: Crayfish Point Reserve.
135 Chapter four - Movement
0.6 (a) n=674
0.5
0.4
0.3
0.2
0.1
Proportion of Proportion in hoursstationary periods 0.0
20 (b)
18
16
14
12
10
8
6
4 Number ofperiods Number stationary 2
0 1 2 3 4 Inner 1,2 2,3 3,4 4,1
Areas of Crayfish Point Reserve
Figure 4.18: Habitat preferences for draughtboard shark stationary periods. a) Proportion of hours that draughtboard sharks spent in stationary periods in each area. b) Number of stationary periods spent in each area.
136 Chapter four - Movement
Day-night activity, habitat utilisation, and preference
Twelve draughtboard sharks were excluded from the day night activity analysis because they had less than 20 movements (see Fig. 4.10). Of the remaining 12 animals, nine moved mostly at night, while the other three moved predominantly during the day
(Fig. 4.19). Similar patterns were found irrespective of whether the sharks were inside or outside of the Crayfish Point Reserve. No sharks had an even distribution of movements over a 24 hr period.
The majority of the sharks (n=8) showed similar 95% and 50% KUD distributions between day and night suggesting no change in diel activity patterns either for habitat utilisation and preferences (Fig. 4.20 a and c). The remaining four sharks showed distinct diel patterns, although the areas used still overlapped (Fig. 4.20 b and d). Three sharks increased their 50% KUDs during the night moving out of the Crayfish Point
Reserve and one shark increased its 50% KUD during the day.
137 Chapter four - Movement
0.2 Inside the Crayfish Point Reserve Shark 147
0.1
0.0 0.2 Outside the Crayfish Point Reserve Shark 147
0.1
0.0
0.2 Inside the Crayfish Point Reserve Shark 116 Proportion Proportion movement of
0.1
0.0
0.2 Outside the Crayfish Point Reserve Shark 116
0.1
0.0 0 1 2 3 4 5 6 7 8 910 111213141516 1718 19 20 21 22 23
Time (hr) Figure 4.19: Example of daily movement activity of draughtboard sharks. Two distinctive movement patterns were found, 3 sharks moved more during the day (shark 147) and 9 sharks moved more at night (shark 116).
138 Chapter four - Movement
Shark 147 a Shark 149 b
CPR CPR
Shark 147 c Shark 149 d
CPR CPR
Figure 4.20: Example of day and night habitat utilisation (95% KUDs, a and b) and habitat preference (50% KUDs, c and d) for draughtboard sharks. Grey and white spaces represent the day and night movements respectively. CPR: Crayfish Point Reserve.
Site fidelity for the Crayfish Point Reserve
Sharks tended to leave the Crayfish Point Reserve within the first month of being released. The average number of days spent at the reserve significantly decreased from 6 days (± 2 days), just after being released, to 1 day (± 1 day) after 4 to 5 months (t test,
P< 0.001) (Fig. 4.21).
139 Chapter four - Movement
10 n=17
a 8
6
4 a,b a,b
2 b b
Number of daysCrayfish NumberReserve of at Point the 0 1 2 3 4 5 Months at the Crayfish Point Reserve
Figure 4.21: Average number of days spent at the Crayfish Point Reserve for the draughtboard sharks. Values are means (± SE). Different letters show significant differences.
4.3.2 CONVENTIONAL TAGGING
Between January 2000 and January 2005, 1234 draughtboard sharks were tagged in southwest and eastern Tasmania and the Crayfish Point Reserve. The Crayfish Point
Reserve showed the highest recapture rate, 36% of 364 sharks tagged, followed by eastern and southwestern areas where the recapture rate was 9% (sharks tagged n=398) and 3% (sharks tagged n=472) respectively (Table 4.7). However, the fishing effort by researchers in the reserve was higher than for southwest and eastern Tasmania. The maximum time at liberty ranged from one month to up to five years in the Crayfish
Point Reserve, from one month up to four years in both eastern and southern Tasmania
140 Chapter four - Movement
(Table 4.7). There were no significant differences in the proportion of recaptures (for
either sex or size) with time (chi square, P< 0.001).
Table 4.7: Summary of draughtboard sharks conventional tagging. Sharks were tagged in southwestern and eastern Tasmania and in the Crayfish Point Reserve.
Tagged Recaptures Recaptures Maximum time at Area (n) (n) (%) liberty (years) Crayfish Point Reserve 364 132 36.3 5 Southwestern of Tasmania 472 17 3.6 4 Eastern of Tasmania 398 37 9.3 4
Distances travelled
The majority of the draughtboard sharks were recaptured in the vicinity of where
they were released (Table 4.8). The majority of the draughtboard sharks travelled a
maximum distance of up to 10 km over the study period. However, for 4%, 7% and
18% of the sharks tagged in the Crayfish Point Reserve and on the east and southwest
coast, the maximum distances travelled were 75, 250, and 300 km respectively (Fig.
4.22). No relationship between time at liberty and distances travelled was found.
Table 4.8: Recapture areas for conventionally tagged draughtboard sharks.
Recaptures in Recaptures in Area same area (n) different areas (n) Crayfish Point Reserve 118 14 Southwestern Tasmania 15 2 Eastern Tasmania 34 3
141 Chapter four - Movement
Eas t
Crayfish Point Reserve Southwest
Figure 4.22: Examples of maximum reported distances travelled for draughtboard sharks. Tagged in southwestern Tasmania. Tagged in eastern Tasmania. Tagged in Crayfish Point Reserve. The lines represent the shortest possible route between the release and the recapture position.
142 Chapter four - Movement
Length frequency composition of tagged and recaptured sharks
The size composition of female and male tagged draughtboard sharks was similar
in the southwest area and the Crayfish Point Reserve, but was significantly different
from eastern Tasmania which had a larger number of smaller males (Kolmogorov
Smirnov, P< 0.005) (Fig. 4.23). There were insufficient recaptures to compare the size
of the sharks that were recaptured for the southwest and east coast populations. For the
Crayfish Point Reserve, the size of recaptured sharks was not significantly different
from the initial population that was tagged (Fig. 4.23).
0.5 Southwest of Tasmania Females (n=292) Southwest of Tasmania Females (n=7) Tags Males (n=145) Recaptures Males (n=9) 0.4
0.3
0.2
0.1
0.0
0.5 Crayfish Point Reserve Females (n=161) Crayfish Point Reserve Females (n=74) Tags Males (n=167) Recaptures Males (n=51) 0.4
0.3
0.2
0.1 Proportion ofProportion sharks 0.0
0.5 East of Tasmania Females (n=292) East of Tasmania Females (n=23) Tags Males (n=105) Recaptures Males (n=14) 0.4
0.3
0.2
0.1
0.0 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 Total length (mm)
Figure 4.23: Length frequency composition of tagged and recaptured female (black bars) and male (grey bars) draughtboard sharks in different regi ons of Tasmania. 143 Chapter four - Movement
Short and long term site fidelity for the Crayfish Point Reserve
Sharks were found up to five years after release at the Crayfish Point Reserve.
Around 2% (proportion 0.02) of sharks were subsequently recaptured each month during the first 11 months of release (Fig. 4.24). Sharks showed a gradual dispersion away from the Crayfish Point Reserve over the six years since tagging began (Fig. 4.25).
0.30 n=109
0.25
0.20
0.15
0.10
0.05 Proportion Proportion of recaptured sharks
0.00 1 2-3 4-5 6-7 8-9 10-11 Months after being released
Figure 4.24: Monthly proportion of recaptured draughtboard sharks at the Crayfish Point Reserve. Numbers are mean + SE. Numbers above the bars are total number of recaptures. No significant differences were found between months (Chi square test).
144 Chapter four - Movement
0.20
0.15 9
0.10 7
21
0.05 Proportion of recaptured sharks
8 2 0.00 1-2 2-3 3-4 4-5 5-6 Year after being released
Figure 4.25: Yearly proportion of recaptured draughtboard sharks at the Crayfish Point Reserve. Numbers are mean ± SE. Numbers above the bars are total number of recaptures. No significant differences between years were found (Chi square test).
145 Chapter four - Movement
4.4 DISCUSSION
A few studies have been reported using passive acoustic receivers to understand shark movements in complex reef habitats (Chapman et al. , 2005; Garla et al. , 2006).
However, this was the first study using passive acoustic technology to address bottom dwelling sharks moving within a complex environment. In addition to external noise
(produced by shipping traffic, waves, currents and vegetation) that can provide mixed signals to the receivers (Clements et al. , 2005; Heupel et al. , 2006a), the physical nature of the habitat (rocky reef) could also reduce the distance over which signals can be reliably detected. Draughtboard sharks were routinely observed by divers to rest in depressions in the reef and under ledges, which could result in missing or uncertain records.
Consequently, it was difficult to know if sharks were resting in the reef area without being detected or had left the area. In this study, the combination of a complex habitat and the movement dynamics of the bottom dwelling draughtboard shark created uncertainty in positioning an animal. Despite these shortcomings a greater understanding of behaviour of this species was developed, including small scale reef utilisation preferences. By determining the accuracy of the outer ring of receivers in the
Crayfish Point Reserve and finding that the probability of a shark being detected was high (86 to 97%), there was greater confidence in correlating detections to behaviour.
In this study, only two sharks were internally tagged with acoustic transmitters. Initial trials with surgical implantation in aquaria studies were problematic with the sharks opening the insertion wound when they flexed as they were released back into the water.
The attachment of external transmitters required less handling of the shark and did not involve local anaesthesia, surgery and recovery. However, external tags were susceptible to fouling which can lead to a reaction at the attachment site. Skin damage caused by abrasion of a fouled conventional tag was occasionally observed in draughtboard sharks.
Moreover, sharks could rub the external transmitters against the reef or other sharks,
146 Chapter four - Movement and the tags could also increase the probability of entanglement in nets and lines.
McKibben and Nelson (1986) suggested that the behaviour of three grey reef sharks,
Carcharhinus amblyrhynchos , was altered by the continual irritation of the dorsal fin where the transmitter was attached. As no draughtboard sharks have been resighted carrying the external transmitters, the impact of the external acoustic tag on the sharks remains uncertain. The two sharks with internally placed tags did not appear to behave any differently to the externally tagged sharks during the period of the study. Similar conclusions on the behaviour of externally and internally tagged sharks were reported for other species such as: Galeocerdo cuvier (Holland et al., 1999), Negaprion brevirostris
(Gruber et al. , 1988) and Scyliorhinus canicula (Sims et al. , 2001). From this study it could be suggested that although the external tagging procedure was quicker, the uncertainty of possible damage on draughtboard sharks due to the external tags is an important factor that could affect future shark behaviour. Further research is required to determine the benefits of either internally or externally tagging draughtboard sharks.
While it was difficult to determine the impact of in situ tagging on draughtboard sharks, 41% of individuals were not recorded in the Crayfish Point Reserve on the day of release. This suggests that they moved to regions of the reef where they were not detected. As all sharks were released towards the middle of the reserve it was unlikely that they could have swam beyond the detection limits of the outer Crayfish Point
Reserve receivers before transmitting their first signal (ie. Maximum time between
‘pings’ was 60 seconds). It was also unlikely that all these sharks they would have swam through the outer ring of receivers without being detected given the greater than 86% chance of detection. It was possible that tagging affected the initial behaviour of the sharks causing them to rest and recover from capture and tagging in regions of the reef
(eg. caves) where signals could not be detected.
Sharks tagged in this study were either, captured and released in Crayfish Point
Reserve or captured on the east coast of Tasmania and released in the Crayfish Point
147 Chapter four - Movement
Reserve. The majority of the draughtboard sharks that were translocated from the east coast did not stay within the release area for longer than one month, after which they left the intensive array to be finally recorded around the lower section of the Derwent
River. While no significant difference could be detected with the small number of sharks tagged, there were indications that sharks did show some site fidelity as the sharks sourced from the Crayfish Point Reserve were sighted back in the reserve on more occasions and tended to disperse less widely than those sourced from the east coast.
The rapid decrease in acoustically tagged sharks recorded in the Crayfish Point
Reserve was similar to the conventional tag data from the reserve where the majority of sharks were not seen after tagging. The acoustic data suggests that these sharks were still in the general vicinity of the Derwent River but only revisited the Crayfish Point
Reserve at decreasing intervals as time increased. As with the large distances recorded for conventional tag recaptures, two of the acoustically tagged sharks moved out of the
Derwent River and associated Storm Bay. Similarly, (McLaughlin and O'Gower, 1971) found that the demersal shark Heterodontus portusjacksoni undertook both short movements around its reef habitats and occasional long (hundred of kilometres) movements. Only short term movements were reported in the scyliorhinidae Scyliorhinus canicula (Rodriguez Cabello et al. , 1998; Sims et al. , 2001).
Draughtboard sharks of both sexes and all sizes used the complete habitat within the boundaries formed by the intensive array, the Derwent River and the Upper Channel.
Within the Derwent region, the Crayfish Point Reserve appeared to be towards the limit of draughtboard shark habitat as a greater proportion of sharks moved between the
Reserve and the mouth of the Derwent rather than moving in the other direction.
Within the small region occupied by the Crayfish Point Reserve, draughtboard sharks did not use the reserve in a random manner. The great number of movements detected on the Derwent mouth side of the Reserve (area 1) would be expected as a result of the greater movement in this direction (as noted above). However, the high use of area 1 by
148 Chapter four - Movement sharks entering and exiting the reserve from the Taroona High site suggest that sharks were actively using this area as a region to enter and exit the Crayfish Point Reserve.
The main difference between area 1 and the rest of the Crayfish Point Reserve was the increased presence of higher profile reef. As higher profile reef would be expected to interfere with acoustic signal transmission, detections in this region were possibly under represented.
Cooper (1978) and Simpendorfer and Heupel (2004) recommended that the temporal pattern of spatial occupation is crucial for determining whether an animal randomly visits habitat or the habitat is the area usually occupied by it (home range). In species such as neonate blacktip sharks Carcharhinus limbatus (Heupel et al. , 2004), the sixgill shark Hexanchus grisesus (Dunbranck and Zielinski, 2003), and the temperate rocky reef teleost Cheilodactylus fuscus (Lowry and Suthers, 1998) changes over time of the home range or seasonal variations in habitat use were reported. These seasonal movements were related to survival strategies, feeding activity and reproductive behaviour. In contrast, and similar to other species such as juveniles of Carcharhinus perezi (Garla et al. ,
2006), the coral reef fish Plectomorus leopardus (Zeller, 1997) and the snapper Pagrus auratus
(Parsons et al. , 2003), draughtboard sharks showed no temporal patterns of habitat utilisation throughout the study period.
Draughtboard sharks showed a preference for crepuscular and night time activity in comparison to moving during the day. Similarly, theses same activity periods have been reported for other bottom dwelling shark species in their natural environment, such as the angel shark Squatina californica (Standora and Nelson, 1977), the horn shark
Heterodontus francisci (Nelson and Johnson, 1970), the scyliorhinids Scyliorhinus canicula
(Sims et al. , 2001), and Cephaloscyllium ventriosum (Nelson and Johnson, 1970). Movements in draughtboard sharks were probably associated with feeding activity, as the main dietary items are nocturnally active animals such as octopus ( Octopus maorum ), squids, southern rock lobster ( Jasus edwardsii ) and crabs (Awruch, personal observation).
149 Chapter four - Movement
Although night time activity was most common, several sharks also moved during the day. This has also been observed for other bottom dwelling species such as
Heterodontus portujacksoni (McLaughlin and O'Gower, 1971), Heterodontus francisci and
Cephalloscyllium ventriosum (Nelson and Johnson, 1970) which were all found to feed mainly at night with a small number of observations of day time feeding. Although day/night differences in habitat utilisation are common among chondrichthyans
(Gruber et al. , 1988; Holland et al. , 1993; Sims et al. , 2001; West and Stevens, 2001; Sims,
2003) for the majority of the draughtboard sharks there were limited differences between the areas utilised during the day and night. This suggests that the sharks had established feeding areas or recognised certain habitat types (eg: high profile reef) as a more productive region to locate food.
Within the Crayfish Point Reserve, draughtboard sharks preferred high profile reef habitat to have stationary periods. In addition, divers have reported draughtboard sharks resting in rocky crevices by themselves or in groups. Similar activity has been reported for H. portusjacksoni (McLaughlin and O'Gower, 1971) and other species of scyliorhinids such as C. ventriosum , S. canicula , S. stellaris and C. ventriosum (Nelson and Johnson, 1970;
Sims et al. , 2001; Sims et al. , 2005). Although periods of inactivity have been reported in other species, such as H. francisci , C. ventriosum , S. stellaris and S. canicula (Nelson and
Johnson, 1970; Sims et al. , 2001; Sims et al. , 2005), this was the first time that a continuous period of five days in stationary behaviour has been documented for any species. Avoidance of predators, thermoregulation, sexual behaviour and digestion have all been suggested as reasons for periods of inactivity among benthic sharks, especially within the Scyliorhinids (Economakis and Lobel, 1998; Sims et al. , 2001; Sims, 2003;
Sims et al. , 2005). (Sims et al. , 2001) reported different sexual aggregation behaviours in
S. canicula , where the resting periods in males occur on gravel substratum in deep water, while females rest in caves or under rocks in shallow water. In other species such as S. stellaris , no sexual segregation was reported with both sexes found to rest in a rocky
150 Chapter four - Movement habitat (Sims et al. , 2005). In this study, there was no correlation between periods of inactivity and either physical parameters such as lunar phase, diel cycle, months or tides or between biological parameters such as sex, reproductive condition, or size. It is therefore most likely that the reason for extended periods of inactivity was due to digestion of prey. Awruch (unplub. data) found that large prey items (eg: 4 kg octopus) were often present in the stomach of the sharks and these would be expected to take a considerable period to digest. Observations in captivity found that draughtboard sharks swallowed rather than chewed food items, and recreational fishers report that a problem with catching draughtboard sharks is that they swallow the baited hooks. It is postulated that the long stationary periods that were found in this study were associated with sharks digesting large prey items.
The recapture of the majority of the draughtboard sharks in the vicinity of where they were released was most likely a function of the research design as few (16.2%) were returned by non researchers. Research surveys in the Crayfish Point Reserve and the east and southwest coasts revisited the same sites. Thus it is reasonable to expect that the majority of the recaptures would come from these surveys. The higher number of research recaptures in the Crayfish Point Reserve (89%) could be associated with the increased and more frequent sampling undertaken in this region. In contrast, surveys in southwest and eastern Tasmania occurred once a year at similar periods and for the same duration.
The lack of tag reporting by fishers using traps or nets clearly highlights the problems associated with gathering data on by catch species. Although there is greater recognition that fisheries are to be managed under the principles of ecosystem based fisheries management, and that recording and reporting of by catch is important, the reporting of recaptured tagged animals that are returned to the sea ( ie. no commercial value) remains problematic. During this study, the tagging program was published in fishing industry magazines and explained through talks given to both gillnet and trap fishers. Fishers
151 Chapter four - Movement were familiar with reporting tags as many of the Tasmanian target species have been the subject of tagging studies. Despite this lack of reporting, conventional tag returns have indicated a degree of mixing over larger ranges. Large distance movements were recorded between eastern and western Tasmania and between southern and northern
Tasmania. The conventional tag returns have also demonstrated longer term site affinities with several sharks being recaptured in the same location up to five years after tagging. Similarly, long term site fidelity or philopatric behaviour (animals returning to a specific location) has been recorded for other species of sharks. The horn shark H. portujacsoni , the dogfish S. canicula and the hammerhead shark Sphyrna tiburo were reported to return to a specific location after periods of absence that can be measured in months or years (Rodriguez Cabello et al. , 1998; Sims et al. , 2001; Heupel et al. , 2006b).
In summary, although short term, the acoustic tag data has revealed information on specific habitat preferences, movements, activity and stationary patterns about this species that were previously unknown. Together with the conventional tagging data, there was confirmation that while mixing between broad regions does occur, the general pattern of movement was of limited dispersion within Tasmania’s major coastal regions.
152
General conclusions
153 General conclusions
The aim of the present study was to investigate the biology and ecology of the draughtboard shark, Cephaloscyllium laticeps ; a common predator of rocky reef ecosystems in southeastern Australia. This thesis has focused on two important components: reproduction, including endocrine control, and the study of movement, activity patterns and habitat utilisation.
In chapter two, the reproductive biology of Cephaloscyllium laticeps was described in detail. As reproduction is one of the most important events in the life cycle of any living organism, being the primary requirement for successful propagation, understanding the reproductive process was considered the first necessary component for scientific investigation in this species. Sexually mature draughtboard shark females were found throughout the year with a slightly higher proportion of pregnant females in the first six months of the year. Sperm production in males was also higher early in the year with a subsequent slight decline later in the year, evident from both macroscopic examination and steroid hormones levels. Females laid two eggs at monthly intervals and embryo development took approximately one year. Together with the results of other studies, two basic reproductive strategies in oviparous chondrichthyans are apparent. Species from higher latitudes, such as the draughtboard shark, tend to reproduce all year round and have longer periods of egg incubation, sperm storage and time between oviposition of successive pairs of eggs. In contrast, species from lower latitudes tend to have distinctive reproductive seasons and have relatively shorter periods for incubation, sperm storage and oviposition.
In addition to macroscopic examination of gonadal stages, the role of steroid hormones in sexual maturation and egg development was explored, in view of their actions as triggers or regulators of all aspects of reproduction. In the present study, the steroid
hormones testosterone (T) and 17β estradiol (E 2) played a major role during the
154 General conclusions
follicular phase of draughtboard shark females, while progesterone (P 4) was primarily involved with the ovulatory phase. These results follow the general pattern in T and E 2 during follicle maturation reported in other oviparous species. However there appears to be no constant pattern during the latter stages of the reproductive cycle, with a diverse behaviour of these hormones in different oviparous species (Sumpter and Dodd, 1979;
Koob et al. , 1986; Heupel et al. , 1999; Sulikowski et al. , 2004). In contrast, and in accordance with previous studies (Koob et al. , 1986; Heupel et al. , 1999; Koob and
Callard, 1999), the results of this work showed a clear role of P 4 during ovulation and oviposition. In draughtboard males, T was the main steroid hormone produced during sexual development. The results of the present study supported the view that T played a major role in the regulation of testis development and in the final stages of sperm maturation, as was suggested by various authors (Callard et al. , 1985; Sourdaine et al. ,
1990; Sourdaine and Garnier, 1993; Heupel et al. , 1999; Tricas et al. , 2000).
Although studies on chondrichthyan endocrinology have advanced in the last few years
(reviewed in Gelsleichter, 2004), the information is still very limited and insufficient to completely understand the endocrine control of reproduction in all the oviparous species in this group of fish. However, results from the present study provided new information and an improved understanding of endocrine control of reproduction and reproductive strategies in oviparous chondrichthyans.
Based on the positive correlation between sexual maturity and steroid hormone levels described in chapter two, chapter three explored the potential of steroid hormone measurements as a non destructive technique to assess reproduction for applied fisheries research. Hormone measurements were found to produce almost identical results in estimating size at maturity and elucidating the reproductive cycle as macroscopic examination of gonadal stages from dissected sharks. Although only validated for Cephaloscyllium laticeps , it is possible that this technique will be widely
155 General conclusions applicable for describing size at maturity and reproductive seasonality in different chondrichthyan species. Positive correlations found by other authors between macroscopic observations and steroid hormones for different species and different reproductive modes (Rasmussen and Gruber, 1990; Tricas et al. , 2000; Sulikowski et al. ,
2004), suggest that there is the potential to apply these methods to all chondrichthyans.
While validation of the steroid hormone levels for different chondrichthyans will require sacrificing a small number of individuals, the hormone levels may be sufficiently consistent between species or reproductive groups to minimise the need for validating each species.
Whether studying chondrichthyans bycatch for ecosystem based fisheries management or managing vulnerable and endangered species, the need for reproductive data to ensure that populations contribute to future generations is essential (Hall et al. ,
2000; Walker et al. , 2005). Therefore, non lethal sampling for biological assessment will be increasingly important in the management of vulnerable chondrichthyan populations.
These results were the first to demonstrate the potential use of steroid hormones for applied fisheries management and open the way for hormone measurements to become a significant scientific tool for non destructive sampling in chondrichthyans.
In chapter four, a combination of conventional and acoustic tagging studies were used to understand the movements, activity patterns, and habitat used by draughtboard sharks. As a bottom dwelling species it was not surprising to find that sharks alternated between swimming and stationary periods, although the finding that a shark could be stationary for up to five continuous days has never been previously reported. Although the majority of the sharks tended to move at night (probably related to movements of their main prey items), they also make opportunistic movements at other times.
Sharks were found to use all regions of the Crayfish Point Reserve during the six month study, with preferred regions for stationary periods as well as entering and exiting
156 General conclusions the reserve. The preference for sharks having stationary periods in high profile reef areas suggested that the sharks may seek caves as refuge area for predator avoidance.
Although information on draughtboard shark predators has not been reported, this species has been found in the stomach of seven gill sharks, Notorynchus cepedianus (Last,
P., CSIRO Marine and Atmospheric Research, Hobart. pers. comm.). Both the conventional and acoustic tagging data showed a preference for sharks to remain in the general vicinity of tagging. Gradual dispersion rather than established migratory routes appeared to be the general movement pattern, however, recaptures from conventionally tagged sharks did demonstrate that this species is capable of travelling relatively long distances.
The results of this work have increased the understanding of the behaviour of bottom dwelling sharks, particularly within the Scyliorhinidae family. Although, a few studies have described the movement patterns and habitat utilisation of scyliorhinids
(Nelson and Johnson, 1970; Sims et al. , 1993; Sims et al. , 2001; Sims et al. , 2005), no information on longer term movements (>6 months) using acoustic technology have been previously reported. In addition, this was the first work using listening stations to understand movement behaviour in bottom dwelling sharks in complex reef habitats, and it was the first study using passive tracking within the scyliorhinids.
With the move to ecosystem based fisheries management it is important to consider the sustainability of catches of major bycatch species. Fundamental to management of bycatch will be the need to ensure that populations can be sustained through adequate reproduction and available habitat. The results of the present study provided important information on the reproductive cycle, movements, habitat requirements, and activity patterns of draughtboard sharks. This will allow development of management plans that consider the requirement of this temperate, rocky reef predator.
157 General conclusions
Marine protected areas (MPA’s) or fishery closures have been reported as an effective spatial tool for fisheries management (Jamieson and Levings, 2001; Stevens, 2002;
Baelde, 2005; Blyth Skyrme et al. , 2006). Although sharks are usually highly mobile animals which often have an extensive distribution (Stevens, 2002), MPA’s can still play a useful role in their management and conservation; as closed areas effectively reduce fishing mortality protecting parts of the population. However, as draughtboard sharks showed no indication of distinctive reproductive seasons or areas, and no strong site fidelity; the implementation of shark refuge areas is unlikely to be particularly effective in protecting draughtboard sharks. Instead, a minimum legal size above the 50% size at maturity that enables sharks to reproduce for several years before being harvested should be implemented. Limiting catches between January and June, the time of peak egg deposition, should also be considered.
In summary, this study has markedly increased the knowledge of the biology and ecology of the draughtboard shark. With new requirements to address bycatch in integrated ecosystem based management programs, together with the ecosystem consequences of removing upper trophic level predators, it is important to conserve draughtboard sharks to have a healthy southern Australian reef ecosystem. As well as providing important life history information on this species, this work has been some of the first to investigate hormonal control of reproduction in chondrichthyans in the wild.
This study has also pioneered the use of hormone measurements as a non lethal sampling tool for elasmobranch reproductive studies, which has important conservation implications for protected and endangered species.
158
REFERENCES Baelde, P. (2005). Interactions between the implementation of marine protected areas and right based fisheries management in Australia. Fisheries Management and Ecology 12: 9 18.
Balart, E. F., Gonzáles García, J. and Villavicencio Garayzar, C. (2000). Notes on the biology of Cephalurus cephalus and Parmaturus xaniurus (Chondrichthyes: Scyliorhinidae) from the west coast of Baja California Sur, Mexico. Fishery Bulletin 98: 219 221.
Barrett, N., Sanderson, J. C., Lawler, M., Halley, V. and Jordan, A. (2001). Mapping of inshore marine habitats in south eastern Tasmania for marine protected area planning and marine management. Tasmania Aquaculture and Fisheries Institute Technical Report Series 7. Hobart, Australia. 73 pp.
Baum, J. K., Myers, R. A., Kehler, D. G., Worm, B., Harley, S. J. and Doherty, P. A. (2003). Collapse and conservation of shark populations in the Northwest Atlantic. Science 299: 389 392.
Birkhead, T. R. and Moller, A. P. (1993). Sexual selection and the temporal separation of reproductive events: sperm storage data from reptiles, birds and mammals. Biological Journal of the Linnean Society 50: 295 311.
Blyth Skyrme, R. E., Kaiser, M. J., Hiddink, J. G., Edwards Jones, G. and Hart, P. J. B. (2006). Conservation benefits of temperate marine protected areas: Variation among fish species. Conservation Biology 20: 811 820.
Bolle, L. J., Hunter, E., Rijnsdorp, A. D., Pastoors, A., Metcalfe, J. D. and Reynolds, J. D. (2005). Do tagging experiments tell the truth? Using electronic tags to evaluate conventional tagging data. ICES Journal of Marine Science 62: 236 246.
Bonfil, R. (1994). Overview of world elasmobranch fisheries. FAO, Rome. 119 pp.
Braccini, J. M. and Chiaramonte, G. E. (2002). Reproductive biology of Psammobatis extenta . Journal of Fish Biology 61: 272 288.
Brickhill, M. J. (2001). The effect of bycatch species upon the catchability of the southern rock lobster (Jasus edwardsii ) in Tasmanian waters, University of Tasmania, Hobart. 33 pp.
Bromage, N., Porter, M. and Randall, C. (2001). The environmental regulation of maturation in farmed fish with special reference to the role of photoperiod and melatonin. Aquaculture 197: 63 98.
Budker, P. (1971). The life of sharks. Columbia University Press, New York. 222 pp.
Callard, G. V. (1991a). Reproduction in male elasmobranch fishes. In: Oogenesis, spermatogenesis and reproduction. Kinne, R. K. H. (ed.). Basel, Karger. Volume 10: 104 154 pp.
Callard, G. V. (1991b). Spermatogenesis. In: Vertebrate endocrinology: Fundamentals and Biomedical Implications. Pang, P. T. K. and Schreibman, M. P. (ed.). Academic Press, New York. Volume 4 (A): 303 341 pp.
Callard, G. V., Fileti, L. A. and Koob, T. J. (1993). Ovarian steroid synthesis and the hormonal control of the elasmobranch reproductive tract. Enviromental Biology of Fishes 38: 175 185.
Callard, G. V., Mak, P., DuBois, W. and Cuevas, M. E. (1989). Regulation of spermatogenesis: the shark testis model. The Journal of Experimental Zoology 2: 23 34.
Callard, G. V., Pudney, J. A., Mak, P. and Canick, J. A. (1985). Stage dependent changes in steroidogenic enzimes and estrogen receptors during spermatogenesis in the testis of the dogfish, Squalus acanthias. Endocrinology 117: 1328 1335.
159
Callard, I. P., Kay, E., Giannoukus, G., Lamb, T. and Perez, L. (1991). The role of steroids in reproduction in female elasmobranchs and reptiles. Journal of Steroid Biochemistry and Molecular Biology 40: 571 575.
Callard, I. P., Klosterman, L. L. and Callard, G. V. (1988). Reproductive Physiology. In: Physiology of elasmobranch fishes. Shuttleworth, T. J. (ed.). Springer Verlag, London. 277 315 pp.
Capapé, C. (1977). Contribution à la biologie des Scyliorhinidae des côtes tunisiennes. 1. Scyliorhinus canicula (Linné, 1758). Répartition géographique et bathymétrique, sexualité, reproduction, fécondité. Bull. Off. natn. Pêch. Tunisie 1: 83 101.
Carrier, J. C., Murru, F. L., Walsh, M. T. and Pratt, H. L. (2003). Assessing reproductive potential and gestation in nurse sharks ( Ginglymostoma cirratum ) using ultrasonography and endoscopy: An example of bridging the gap between field research and captive studies. Zoo Biology 22: 179 187.
Carrier, J. C., Pratt, H. L. and Martin, L. K. (1994). Group reproductive behaviors in free living nurse sharks, Ginglymostoma cirratum . Copeia 3: 646 656.
Carrier, J. C., Pratt, H. L. J. and Castro, J. I. (2004). Reproductive biology of elasmobranchs. In: Biology of the sharks and their relatives. Carrier, J. C., Musick, J. A. and Heithaus, M. R. (ed.). CRC Press, London. 10: 269 286 pp.
Casey, J. G. and Taniuchi, T. (1990). Recommendations for future shark tagging program. In: Elasmobranchs as living resources: advances in the biology, ecology, systematics, and the status of the fisheries. Pratt, H. L., Gruber, S. H. and Taniuchi, T. (ed). NOOA Technical report, NMFS 90. U.S.A. 511 pp.
Castro, J. I. and Brudek., R. L. (1999). A preliminary evaluation of the status of shark species. FAO Fisheries Technical Paper, Rome. 72 pp.
Castro, J. I., Bubucis, P. M. and Overstrom, N. A. (1988). The reproductive biology of the chain dogfish, Scyliorhinus retifer . Copeia 3: 740 746.
Chapman, D. D., Pikitch, E. K., Babcock, F. and Shivji, M. S. (2005). Marine reserve design and evaluation using automated acoustic telemetry: A case study involving coral reef associated sharks in the Mesoamerican Caribbean. Marine Technology Society Journal 39: 42 55.
Clark, E. and Von Schmidt, K. (1965). Sharks of the central gulf coast of Florida. Bulletin of Marine Science 15: 13 83.
Clark, R. S. (1922). Rays and skates. Journal of the Marine Biological Association of the United Kingdom 12: 577 643.
Clarke, K. R. and Gorley, R. N. (2001). Primer v5: User manual/tutorial, Plymouth. 91 pp.
Clements, S., Jepsen, D., Karnowski, M. and Schreck, C. B. (2005). Optimization of an acoustic telemetry array for detecting transmitter implanted fish. North American Journal of Fisheries Management 25: 429 436.
Cliff, G. and Thurman, G. D. (1984). Pathological and physiological effects of stress during capture and transport in the juvenile dusky shark, Carcharhinus obscurus . Comparative biochemistry and physiology 78 A: 167 173.
Coleman, F. C. and Williams, S. L. (2000). Overexploiting marine ecosystem engineers: potential consequences for biodiversity. Trends in Ecology & Evolution 17: 40 44.
Compagno, L. J. V. (1984). Sharks of the world. FAO, Rome. 251 655 pp.
Conrath, C. L. and Musick, J. A. (2002). Reproductive biology of the smooth dogfish, Mustelus canis , in the northwest Atlantic Ocean. Environmental Biology of Fishes 64: 367 377.
160
Cooper, W. E. J. (1978). Home range criteria based on temporal stability of areal occupation. Journal of Theoretical Biology 73: 687 695.
Cortés, E. (1999). Standardized diet composition and trophic levels of sharks. Journal of Marine Sciences 56: 707 717.
Cortés, E. (2000). Life history patterns and correlations in sharks. Reviews in Fisheries Science 8: 299 344.
Cortés, E. (2004). Life history patterns, demography, and population dynamics. In: Biology of the sharks and their relatives. Carrier, J. C., Musick, J. A. and Heithaus, M. R. (ed.). CRC Press, London. 15: 449 469 pp.
Costa, M. E., Erzini, K. and Borges, T. (2005). Reproductive biology of the blackmouth catshark, Galeus melastomus (Chondrichthyes: Scyliorhinidae) off the south coast of Portugal. Journal of the Marine Biological Association of the United Kingdom 85: 1173 1183.
Craik, J. C. A. (1978). An annual cycle of vitellogenesis in the elasmobranch Scyliorhinus canicula . Journal of the Marine Biological Association of the United Kingdom 58: 719 726.
Cresswell, G. (2000). Currents of the continental shelf and upper slope of Tasmania. In: Tasmania and the southern Ocean. Banks, M.R. and Brown, M.J. (ed). Papers and Proceedings of the Royal Society of Tasmania . 133(3): 21 30.
Crews, D. (1984). Gamete production, sex hormone secretion, and mating behaviour uncoupled. Hormones and Behavior 18: 22 28.
Cross, J. N. (1988). Aspects of the biology of two Scyliorhinid sharks, Apristurus brunneus and Paramaturus xaniurus , from the upper continental slope off the Southern California. Fishery Bulletin 86: 691 702.
Cuevas, M. E. and Callard, G. (1992). Androgen and progesterone receptors in shark (Squalus) testis: characteristics and stage related distribution. Endocrinology 130: 2173 2182.
DAFF (2004). National plan of action for the conservation and management of sharks (shark plan). Commonwealth Department of Agriculture, Fisheries and Forestry. Canberra. 83 pp.
DAFF (2001). Australian Shark assessment report. In: Australian National Plan of Action for the Conservation and Management of Sharks. Australian government, Department of Agriculture, Fisheries and Forestry. Canberra. 211 pp.
Dodd, J. M. (1983). Reproduction in cartilaginous fishes (Chondrichthyes). In: Fish Physiology. Hoar, W. S., Randall, D. J. and Donaldson, E. M. (ed.). Academic Press, New York. Volume IX (A): 31 104 pp.
Dodd, J. M., Dodd, M. H. I. and Duggan, R. T. (1983). Control of reproduction in elasmobranchs fishes. In: Control Processes in Fish Physiology. Rankin, J. C., Pitcher, T. J. and Duggan, R. T. (ed.). Control Helm, London. 221 249 pp.
Dodd, J. M. and Sumpter, J. P. (1984). Fishes. In: Marshall's physiology of reproduction. Lamming, G. E. (ed.). Churchill Livingstone, New York. Volume 1: 2 126 pp.
Dulvy, N. K. and Reynolds, J. D. (1997). Evolutionary transition among egg laying, live bearing and maternal inputs in sharks and rays. Proceedings of the Royal Society of London B 264: 1309 1315.
Dulvy, N. K., Sadovy, Y. and Reynolds, J. D. (2003). Extinction vulnerability in marine populations. Fish and Fisheries 4: 25 64.
Dunbranck, R. and Zielinski, R. (2003). Seasonal and diurnal activity of sixgill sharks ( Hexanchus griseus ) on a shallow water reef in the Strait of Georgia, British Columbia. Canadian journal of zoology 81: 1107 111.
161
Duncan, K. M. and Holland, K. N. (2006). Habitat use, growth rates and dispersal patterns of juvenile scalloped hammerhead sharks Sphyrna lewini in a nursery habitat. Marine Ecology Progress Series 312: 211 221.
Eckert, R. (1988). Chemical messengers and regulators. In: Animal physiology. Eckert, R. (ed.). Freeman and company, USA. 266 328 pp.
Economakis, A. E. and Lobel, P. S. (1998). Aggregation behavior of the grey reef shark, Carcharhinus amblyrhychos , at Johnston Atoll, Central Pacific Ocean. Environmental Biology of Fishes 51: 129 139.
Edgar, G. J. (2001). Australian Marine Habitats in temperate waters. Reed New Holland, Australia. 224 pp.
Eiler, J. H. (2000). Fish movements the missing piece. In: Fish Movement and Migration. Australian Society of Fish Biology Workshop Proceedings. Hancock, D. A., Smith, D. C. and Koehn, J. D. (ed.). Bendigo, Victoria, Australia. 85 92 pp.
Ellis, J. R. and Shackely, S. E. (1995). Observations on egg laying in the thornback ray. Journal of Fish Biology 46: 903 904.
Ellis, J. R. and Shackley, S. E. (1997). The reproductive biology of Scyliorhinus canicula in the Bristol Channel, UK. Journal of Fish Biology 51: 361 372.
FAO. (1999). IPOA International plan of action for the conservation and management of sharks. FAO Fisheries Report M 40. FAO Rome. 11 18 pp.
FAO. (2005). FAO Expert Consultation on the Implementation of the FAO International Plan of Action for the Conservation and Management of Sharks. FAO Fisheries Report 795. FAO Rome. 24 pp.
Fasano, S., Pierantoni, R. and Chieffi, G. (1989). Reproductive biology of elasmobranchs with emphasis on endocrines. The Journal of Experimental Zoology 2: 53 61.
Fitz, E. S. J. and Daiber, F. C. (1963). An introduction to the biology of the Raja eglanteria Bosc 1802 and Raja erinacea Mitchill 1825 as they occur in Delaware Bay. Bulletin of the Bingham Oceanographic Collection 18: 69 97.
Ford, A. (1921). A contribution to our knowledge of the life histories of the dogfishes landed at Plymouth. Journal of the Marine Biological Association of the United Kingdom 12: 468 505.
Frisk, M. G., Miller, T. J. and Fogarty, M. J. (2001). Estimation and analysis of biological parameters in elasmobranch fishes: a comparative life history study. Canadian Journal of Fisheries and Aquatic Sciences 58: 969 981.
Frusher, S. F. and Gibson, I. (1999). Bycatch in the Tasmanian rock lobster fishery. In: Establishing meaningful targets for bycatch reduction in Australian fisheries. Buxton, C. and Eayrs, S. (ed). Hobart, Australia 79 81. pp.
Garla, R. C., Chapman, D. D., Wetherbee, B. M. and Shivji, M. (2006). Movement patterns of young Caribbean reef sharks, Carcharhinus perezi , at Fernando de Noronha Archipelago, Brazil: the potential of marine protected areas for conservation of a nursery ground. Marine Biology 149: 189 199.
Garnier, D. H., Sourdaine, P. and Jégou, B. (1999). Seasonal variations in sex steroids and male sexual characteristics in Scyliorhinus canicula . General and Comparative Endocrinology 116: 281 290.
Gelsleichter, J. (2004). Hormonal regulation of elasmobranch physiology. In: Biology of sharks and their relatives. Carrier, J. C., Musick, J. A. and Heithaus, M. R. (ed.). CRC Press, London. 11: 287 323 pp.
162
Gelsleichter, J., Rasmussen, L. E. L., Manire, C. A., Tyminski, B., Chang, B. and Lombardi Carlson, L. (2002). Serum steroid concentrations and development of reproductive organs during puberty in male bonnethead sharks, Sphyrna tiburo . Fish Physiology and Biochemistry 26: 398 401.
Gilmore, R. G., Dodrill, J. W. and Linley, P. A. (1983). Reproduction and embryonic development of the sand tiger shark, Odontapsis taurus (Rafinesque). Fishery Bulletin 81: 201 225.
Girard, M., Rivalan, P. and Sinquin, G. (2000). Testis and sperm morphology in two deep water squaloid sharks, Centroscymnus and Centrophorus squamosus . Journal of Fish Biology 57: 1575 1589.
Graham, K. J., Andrew, N. L. and Hodgson, K. E. (2001). Changes in relative abundance of sharks and rays on Australian South East Fishery trawl grounds after twenty years of fishing. Marine and Freshwater Research 52: 549 61.
Gruber, S. H., Nelson, D. R. and Morrissey, J. F. (1988). Patterns of activity and space utilization of lemon sharks, Negaprion brevirostris , in a shallow Bahamian lagoon. Bulletin of Marine Science 43: 61 76.
Gunn, J. (2000). From plastics darts to pop up satellite tags. In: Fish Movement and Migration. Australian Society of Fish Biology Workshop Proceedings. Hancock, D. A., Smith, D. C. and Koehn, J. D. (ed.). Bendigo, Victoria, Australia. 55 60 pp.
Haddon, M. 2001. Modelling and quantitative methods in fisheries. Chapman and Hall. 401 pp.
Haddy, J. A. and Pankhurst, N. W. (1999). Stress induced changes in concentrations of plasma sex steroids in black bream. Journal of Fish Biology 55: 1304 1316.
Hall, M. A., Alverson, D. L. and Metuzals, K. I. (2000). By catch: Problems and solutions. Marine Pollution Bulletin 41: 204 219.
Hamlett, W. C. and Koob, T. J. (1999). Female reproductive system. In: Sharks, Skates, and Rays. The biology of elasmobranch fishes. Hamlet, W. (ed.). The Johns Hopkins University press, London. 15: 398 443 pp.
Harris, G., Nilsson, C., Clementson, L. and Thomas, D. (1987). The water masses of the east coast of Tasmania: Seasonal and interannual variability and the influence on phytoplankton biomass and productivity. Australian Journal or Marine and Freshwater Research 38: 569 590.
Heithaus, M. R. (2004). Predator prey Interactions. In: Biology of the sharks and their relatives. Carrier, J. C., Musick, J. A. and Heithaus, M. R. (ed.). CRC Press. 17: 487 521 pp.
Heithaus, M. R., Dill, L. M., Marshall, G. J. and Buhleier, B. (2002). Habitat use and foraging behavior of tiger sharks ( Galeocerdo cuvier ) in a seagrass ecosystem. Marine Biology 140: 237 248.
Henderson, A. C. and Casey, A. (2001). Reproduction and growth in the lesser spotted dogfish Scyliorhinus canicula (Elasmobranchii; Scyliorhinidae) from the west coast of Ireland. Cahiers de biologe marine 42: 397 405.
Heupel, M. R. and Hueter, R. E. (2001). Use of an automated acoustic telemetry system to passively track juvenile blacktip shark movements. In: Electronic Tagging and Tracking in Marine Fisheries. Sibert, J. R. and Nielsen, J. L. (ed.). The Netherlands. 217 236 pp.
Heupel, M. R. and Hueter, R. E. (2002). Importance of prey density in relation to the movement patterns of juvenile blacktip sharks ( Carcharhinus limbatus ) within a coastal nursery area. Marine and Freshwater Research 53: 543 550.
Heupel, M. R., Semmens, J. M. and Hobday, A. J. (2006a). Automated acoustic tracking of aquatic animals: scales, design and deployment of listening station arrays. Marine and Freshwater Research 57: 1 13.
163
Heupel, M. R., Simpendorfer, C. A., Collins, A. B. and Tyminski, J. P. (2006b). Residency and movement patterns of bonnethead sharks, Sphyrna tiburo , in a large Florida estuary. Environmental Biology of Fishes 76: 47 67.
Heupel, M. R., Simpfendorfer, C. A. and Hueter, R. E. (2004). Estimation of shark home ranges using passive monitoring techniques. Environmental Biology of Fishes 71: 135 142.
Heupel, M. R., Whittier, J. M. and Bennett, M. B. (1999). Plasma steroid hormone profiles and reproductive biology of the Epaulette shark, Hemiscyllium ocellatum . Journal of Experimental Zoology 284: 586 594.
Hilborn, R. (1990). Determination of fish movement patterns from tag recoveries using maximum likelihood estimators. Canadian Journal of Fisheries and Aquatic Sciences 47: 635 643.
Ho, S. (1987). Endocrinology of vitellogenesis. In: Hormones and Reproduction in Fishes, Amphibians, and Reptiles. Norris, D. O. and Jones, R. C. (ed.). Plenum Press, New York. 145 168 pp.
Holden, M. J. (1975). The fecundity of Raja clavata in British waters. J. Cons. int. Explor. Mer 36: 110 118.
Holden, M. J., Rout, D. W. and Humphreys, C. N. (1971). The rate of egg laying by three species of ray. J. Cons. int. Explor. Mer 33: 335 339.
Holland, K. N., Wetherbee, B. M., Lowe, C. G. and Meyer, C. G. (1999). Movements of tiger sharks (Galeocerdo cuvier ) in coastal Hawaiian waters. Marine Biology 134: 665 673.
Holland, K. N., Wetherbee, B. M., Peterson, J. D. and Lowe, C. G. (1993). Movements and distribution of hammerhead shark pups on their natal grounds. Copeia: 495 502.
Hooge, P. N. and Eichenlaub, B. (2000). Animal movement extension to Arcview. Alaska Science Center Biological Science Office, U. S. Geological Survey, Anchorage. Alaska. http://www.absc.usgs.gov/glba/gistools/
Hurst, R. J., Bagley, N. W., McGregor, G. A. and Francis, M. P. (1999). Movements of the New Zealand school shark, Galeorhinus galeus , from tag returns. New Zealand Journal of Marine and Freshwater Research 33: 29 48.
IUCN. (2006). The IUCN Red list of Threatened species. Gland, Switzerland. http://www.iucnredlist.org/. Downloaded in December 2006.
Jamieson, G. S. and Levings, C. O. (2001). Marine protected areas in Canada implications for both conservation and fisheries management. Canadian Journal of Fisheries and Aquatic Sciences 58: 138 156.
Jones, B. C. and Geen, G. H. (1977). Observations on the brown cat shark, Apristurus brunneus (Gilbert), in British Columbia coastal waters. Syesis 10: 169 170.
Jordan, A., Lawler, M. and Halley, V. (2001). Estuarine habitat mapping in the Derwent Integrating science and management. Tasmania Aquaculture and Fisheries Institute Final Report. Hobart, Australia. 67 pp.
Klimley, A. P., Beavers, S. C., Curtis, T. H. and Jorgensen, S. J. (2002). Movements and swimming behavior of three species of sharks in La Jolla Canyon, California. Environmental Biology of Fishes 63: 117 135.
Koehn, J. D. (2000). Environmental determinants, barriers and human interference to the movement of fish. In: Fish Movement and Migration. Australian Society of Fish Biology Workshop Proceedings. Hancock, D. A., Smith, D. C. and Koehn, J. D. (ed.). Bendigo, Victoria, Australia. 95 100 pp.
Kohler, N. E. and Turner, P. A. (2001). Shark tagging: a review of conventional methods and studies. Environmental Biology of Fishes 60: 191 223.
164
Koob, T. J. and Callard, I. P. (1991). Reproduction in female elasmobranchs. In: Oogenesis, Spermatogenesis and Reproduction. Kinne, R. K. H. (ed.). Basel, Karger. 10: 155 209 pp.
Koob, T. J. and Callard, I. P. (1999). Reproductive endocrinology of female elasmobranchs: lessons from the little skate ( Raja erinacea ) and spiny dogfish ( Squalus acanthias ). Journal of Experimental Zoology 284: 557 574.
Koob, T. J., Tsang, P. and Callard, I. P. (1986). Plasma estradiol, testosterone, and progesterone levels during the ovulatory cycle of the skate ( Raja erinacea ). Biology of Reproduction 35: 267 275.
Koop, J. H. (2005). Reproduction of captive Raja spp. in the Dolfinarium Harderwijk. Journal of the Marine Biological Association of the United Kingdom 85: 1201 1202.
Kramer, D. L. and Chapman, M. R. (1999). Implications of fish home range size and relocation for marine reserve function. Environmental Biology of Fishes 55: 65 79.
Kubokawa, K., Watanabe, T., Yoshioka, M. and Iwata, M. (1999). Effects of acute stress on plasma cortisol, sex steroid hormone and glucose levels in male and female sockeye salmon during the breeding season. Aquaculture 172: 335 349.
Kyne, P. M. and Bennett, M. B. (2002). Reproductive biology of the eastern shovelnose ray, Aptychotrema rostrata (Shaw & Noder, 1794), from Moreton Bay, Queensland, Australia. Marine and Freshwater Research 53: 583 589.
Last, P. R. and Stevens, J. D. (1994). Sharks and Rays of Australia. CSIRO, Australia. 513 pp.
Latour, R. J. (2004). Tagging methods and associated data analysis. In: Elasmobranch Fisheries Management techniques. Musick, J. A. and Bonfil, R. (ed.). APEC Secretariat, Singapore. 57 78 pp.
Lechenault, H., Wrisez, F. and Mellinger, J. (1993). Yolk utilization in Scyliorhinus canicula , and oviparous dogfish. Environmental Biology of Fishes 38: 241 252.
Lowe, C. G., Topping, D. T., Cartamil, D. P. and Papastamatiou, Y. P. (2003). Movement patterns, home range, and habitat utilization of adult kelp bass Paralabrax clathratus in a temperate no take marine reserve. Marine Ecology Progress Series 256: 205 216.
Lowry, M. B. and Suthers, I. M. (1998). Home range, activity and distribution patterns of a temperate rocky reef fish, Cheilodactylus fuscus . Marine Biology 132: 569 578.
Luer, C. A. and Gilbert, P. W. (1985). Mating, behavior, egg deposition, incubation period, and hatching in the clearnose skate, Raja eglanteria . Environmental Biology of Fishes 13: 161 171.
Manire, C. A. and Rasmussen, L. E. L. (1997). Serum concentrations of steroid hormones in the mature male bonnethead shark, Sphyrna tiburo . General and Comparative Endocrinology 107: 414 420.
Manire, C. A., Rasmussen, L. E. L. and Gross, T. (1999). Serum steroid hormones including 11 ketotestosterone, 11 ketoandrosternedione and dihydroprogesterone in juvenile and adult bonnethead sharks, Sphyrna tiburo . Journal of Experimental Zoology 284: 595 603.
Manire, C. A., Rasmussen, L. E. L., Hess, D. L. and Hueter, R. E. (1995). Serum steroid hormones and the reproductive cycle of the female bonnethead shark, Sphyrna tiburo . General and Comparative Endocrinology 97: 366 376.
Martins, I. A., Martins, C. L. and Leme, A. H. A. (2005). Biological parameters and population structure of Psammobatis extenta in Ubatuba region, north coast of the State of Sao Paulo, Brazil. Journal of the Marine Biological Association of the United Kingdom 85: 1113 1118.
McFarlane, G. A., Wydoski, R. S. and Princ, E. D. (1990). Historical review of the development of external tags and marks. American Fisheries Society Symposium 7: 9 29.
165
McKibben, J. N. and Nelson, D. R. (1986). Patterns of movement and grouping of gray reef sharks, Carcharhinus amblyrhynchos , at Enewetak, Marshall Islands. Bulletin of Marine Science 38: 89 110.
McLaughlin, R. H. and O'Gower, A. K. (1971). Life history and underwater studies of a heterodont shark. Ecological Monographs 41: 271 289.
Mellinger, J. (1983). Egg case diversity among dogfish, Scyliorhinus canicula (L.): a study of egg laying rate and nidamental gland secretory activity. Journal of Fish Biology 22: 83 90.
Mellinger, J., Wrisez, F. and Alluchon Gerard, M. (1986). Developmental biology of an oviparous shark, Scyliorhinus canicula . In: Indo Pacific Fish Biology, Conference Proceedings. Uyeno, T., Arai, R., Taniuchi, T. and Matsuura, K. (ed.). Tokyo. 310 332 pp.
Morrissey, J. F. and Gruber, S. H. (1993a). Habitat Selection by Juvenile Lemon Sharks, Negaprion brevirostris . Environmental Biology of Fishes 38: 311 319.
Morrissey, J. F. and Gruber, S. H. (1993b). Home range of juvenile lemon sharks, Negaprion brevirostris . Copeia: 425 434.
Mullon, C., Fréon, P. and P., C. (2005). The dynamics of collapse in world fisheries. Fish and Fisheries 6: 111 120.
Musick, J. A. (2004). Management of sharks and their relatives (Elasmobranchii). In: Elasmobranch fisheries management techniques. Musick, J. A. and Bonfil, R. (ed.). APEC, Singapore. 1 6 pp.
Nakano, H., Matsunaga, H., Okamoto, H. and Okazaki, M. (2003). Acoustic tracking of bigeye thresher shark Alopias supercilius in the eastern Pacific Ocean. Marine Ecology Progress series 265: 255 261.
Nakaya, K. (1975). Taxonomy, comparative anatomy and phylogeny of japanese catsharks, Scyliorhinidae. Memoirs of the Faculty of Fisheries, Hokkaido University 23: 1 94.
Nelson, D. R. and Johnson, R. H. (1970). Diel activity rhythms in the nocturnal, bottom dwelling sharks, Heterodontus francisci and Cephaloscyllium ventriousus . Copeia 4: 732 739.
Neter, J., Kutner, M. H., Nachtsheim, C. J. and Wasserman, W. (1990). Applied linear statistical models, 4th ed. Irwin, Chicago. 1408 pp.
Pankhurst, N. W. (2006). Gonadal steroids: functions and patterns of change. In: Fish Reproduction: cytology, biology and ecology. Rocha, M. J., Arukwe, A. and Kapoor, B. G. (ed.). Science Publisher Inc., Enfield New Hampshire. In print.
Pankhurst, N. W. and Carragher, J. F. (1992). Oocyte maturation and changes in plasma steroid levels in snapper Pagrus (=Chrysophrys) auratus (Sparidae) following treatment with human chorionic gonadotropin. Aquaculture 101: 337 347.
Pankhurst, N. W., Hilder, P. I. and Pankhurst, P. M. (1999). Reproductive condition and behaviour in relation to plasma levels of gonadal steroids in the Spiny Damselfish Acanthochromis polyacanthus . General and Comparative Endocrinology 115: 53 69.
Pankhurst, N. W. and Porter, M. J. R. (2003). Cold and dark or warm and light: variations on the theme of environmental control of reproduction. Fish Physiology and Biochemistry 28: 385 389.
Parsons, D. M., Babcock, R. C., Hankin, R. K. S., Willis, T. J., Aitken, J. P., O'Dor, R. K. and Jackson, G. D. (2003). Snapper Pagrus auratus (Sparidae) home range dynamics: acoustic tagging studies in a marine reserve. Marine Ecology Progress Series 262: 253 265.
Parsons, G. R. and Grier, H. J. (1992). Seasonal changes in shark testicular structure and spermatogenesis. The Journal of Experimental Zoology 261: 178 184.
Pauly, D., Christensen, V., Dalsgaard, J., Froese, R. and Torres, F. (1998). Fishing down marine food webs. Science 279: 860 863.
166
Pope, J. G., MacDonald, D. S., Daan, N., Reynolds, J. D. and Jennings, S. (2000). Gauging the impact of fishing mortality on non target species. ICES Journal of Marine Science 57: 689 696.
Powles, H., Bradford, M. J., Bradford, R. G., Doubleday, W. G., Innes, S. and Levings, C. D. (2000). Assessing and protecting endangered marine species. Ices Journal of Marine Science 57: 669 676.
Pratt, H. L. J. (1993). The storage of spermatozoa in the oviducal gland of western north Atlantic sharks. Environmental Biology of Fishes 38: 139 149.
Pratt, J. H. L. (1988). Elasmobranch gonad structure: a description and survey. Copeia 3: 719 729.
Prisco, M., Ricchiari, L. and Andreuccetti, P. (2002). Ultrastructural studies on developing follicles of the spotted ray Torpedo marmorata . Molecular reproduction and development 61: 78 86.
Quinn, G. P. and Keough, M. J. (2002). Experimental Design and Data Analysis for Biologists. Cambridge University Press, Cambridge. 538 pp.
Rasmussen, L. E. L. and Gruber, S. H. (1990). Serum levels of circulating steroid hormones in free ranging carcharhinoid sharks. In: Elasmobranchs as a Living Resources: Advances in the Biology, Ecology, Systematics, and the Status of the Fisheries. Pratt, H. L., Gruber, S. H. and Taniuchi, T. (ed.). NOAA Technical Report NMFS 90. 143 155 pp.
Rasmussen, L. E. L. and Gruber, S. H. (1993). Serum concentrations of reproductively related circulating steroid hormones in the free ranging lemon shark, Negaprion brevirostris . Environmental Biology of Fishes 38: 167 174.
Rasmussen, L. E. L., Hess, D. L. and Luer, C. A. (1999). Alterations in serum steroid concentrations in the clearnose skate, Raja eglanteria : correlations with season and reproductive status. Journal of Experimental Zoology 284: 575 585.
Rasmussen, L. E. L. and Murru, F. L. (1992). Long term studies of serum concentrations of reproductively related steroid hormones in individual captive carcharhinids. Australia Journal of Marine and Freshwater Research 43: 273 281.
Reese, J. C. and Callard, I. P. (1991). Characterization of a specific estrogen receptor in the oviduct of the little skate, Raja erinacea . General and Comparative Endocrinology 84: 170 181.
Richards, S. W., Merriman, D. and Calhoun, L. H. (1963). Studies on the marine resources of southern New England. IX. The biology of the little skate, Raja erinacea Mitchell. Bulletin of the Bingham Oceanographic Collection 18: 1 67.
Richardson, A. J., Maharaj, G., Compagno, L. J. V., Leslie, R. W., Ebert, D. A. and Gibbons, M. J. (2000). Abundance, distribution, morphometrics, reproduction and diet of the Izak catshark. Journal of Fish Biology 56: 552 576.
Rodriguez Cabello, C., De la Gándara, F. and Sánchez, F. (1998). Preliminary results on growth and movements of dogfish Scyliorhinus canicula (Linnaeus, 1758) in the Cantabrian Sea. Oceanologica Acta 21: 363 370.
Scheffer, M., Carpenter, S. and de Young, B. (2005). Cascading effects of overfishing marine systems. Trends in Ecology & Evolution 20: 579 581.
Schindler, D. E., Essington, T. E., Kitchell, J. F., Boggs, C. and Hilborn, R. (2002). Sharks and tunas: Fisheries impacts on predators with contrasting life histories. Ecological Applications 12: 735 748.
Selcer, K. W. and Leavitt, W. W. (1991). Estrogens and progestins. In: Vertebrates endocrinology: fundamental and biomedical implications. Pang, P. T. K. and Schreibman, M. P. (ed.). Academic Press, New York. Volume 4 (B): 67 114 pp.
Sherwood, N. M. and Lovejoy, D. A. (1993). Gonadotropin releasing hormone in cartilaginous fishes: structure, location and transport. Environmental Biology of Fishes 38: 197 208.
167
Shumway, C. A. (1999). A neglected science: applying behavior to aquatic conservation. Environmental Biology of Fishes 55: 183 201.
Sibert, J. R. and Nielsen, J. L. (ed.). (2000). Electronic Tagging and Tracking in Marine Fisheries. Kluwer Academic. 468 pp.
Simpendorfer, C. A. and Donohue, K. (1998). Keeping the fish in 'fish and chips': research and management of the Western Australia shark fishery. Marine and Freshwater Research 49: 593 600.
Simpendorfer, C. A. and Heupel, M. R. (2004). Assessing Habitat Use and Movement. In: Biology of the sharks and their relatives. Carrier, J. C., Musick, J. A. and Heithaus, M. R. (ed.). CRC Press. 19: 553 572 pp.
Simpson, T. H., Wright, R. S. and Gottfried, H. (1963). Steroids in the semen of dogfish ( Squalus acanthias ). Journal of Endocrinology 26: 489 498.
Simpson, T. H., Wright, R. S. and Hunt, S. V. (1964). Steroid biosynthesis in the testis of dogfish ( Squalus acanthias ). Journal of Endocrinology 31: 29 38.
Sims, D. W. (2003). Tractable models for testing theories about natural strategies: foraging behaviour and habitat selection of free ranging sharks. Journal of Fish Biology 63: 53 73.
Sims, D. W., Davies, S. J. and Bone, Q. (1993). On the diel rhythms in metabolism and activity of post hatching lesser spotted dogfish, Sclyiorhinus canicula . Journal of Fish Biology 43: 749 754.
Sims, D. W., Nash, J. P. and Morritt, D. (2001). Movements and activity of male and female dogfish in a tidal sea lough: alternative behavioural strategies and apparent sexual segregation. Marine Biology 139: 1165 1175.
Sims, D. W., Southall, E. J., Wearmouth, V. J., Hutchinson, N., Budd, G. C. and Morritt, D. (2005). Refuging behaviour in the nursehound Scyliorhinus stellaris (Chondrichthyes : Elasmobranchii): preliminary evidence from acoustic telemetry. Journal of the Marine Biological Association of the United Kingdom 85: 1137 1140.
Snelson, J. F. F., Johnson, M. R., Rasmussen, L. E. L. and Hess, D. L. (1997). Serum concentrations of steroids hormones during reproduction in the atlantic stingray, Dasyatis sabina . General and Comparative Endocrinology 108: 67 79.
Sourdaine, P. and Garnier, D. H. (1993). Stage dependent modulation of Sertoli cell steroid production in dogfish (Scyliorhinus canicula) . Journal of Reproduction and Fertility 97: 133 142.
Sourdaine, P., Garnier, D. H. and Jégou, B. (1990). The adult dogfish ( Scyliorhinus canicula L.) testis: a model to study stage dependent changes in steroid levels during spermatogenesis. Journal of Endocrinology 127: 451 460.
Springer, S. (1979). A revision of the catsharks, family Scyliorhinidae. NOAA Technical Report. NMFS circular 422. Washington. 152 pp.
Standora, E. A. and Nelson, D. R. (1977). A telemetric study of the behavior of free swimming pacific angel sharks, Squatina californica . Southern California Academy of Sciences 76: 193 201.
Stearns, S. C. (1976). Life history tactics: a review of the ideas. The Quarterly Review of Biology 51: 3 47.
Stevens, J. D. (1999). Variable resilience to fishing pressure in two sharks: the significance of different ecological and life history parameters. American Fisheries Society Symposium 23: 11 15.
Stevens, J. D. (2000). Shark tagging: a brief history of methods. In: Fish Movement and Migration. Australian Society of Fish Biology Workshop Proceedings. Hancock, D. A., Smith, D. C. and Koehn, J. D. (ed.). Bendigo, Victoria, Australia. 65 68 pp.
168
Stevens, J. D. (2002). The role of protected areas in elasmobranch fisheries management and conservation. In: Elasmobranch biodiversity, conservation and management: Proceedings of the International seminar and workshop, Sabah, Malasya, 1997. Fowler, S. L., Reed, T. M. and Dipper, F. A. (ed). Switzerland and Cambridge, UK. 241 242 pp.
Stevens, J. D., Bonfil, R., Dulvy, N. K. and Walker, P. A. (2000). The effects of fishing on sharks, rays, and chimaeras (chondrichthyans), and the implications for marine ecosystem. Journal of Marine Science 57: 476 494.
Storrie, M. T. (2004). Microscopic modifications of the female reproductive tissues of Mustelus antarcticus . PhD, Deakin University, Melbourne, Australia. 153 pp.
Sulikowski, J. A., Kneebone, J., Elzey, S., Jurek, J., Danley, P. D., Huntting Howell, W. and Tsang, P. C. W. (2005a). The reproductive cycle of the thorny skate ( Amblyraja radiata ) in the western Gulf of Maine. Fishery Bulletin 103: 536 543.
Sulikowski, J. A., Tsang, P. C. W. and Huntting Howell, W. (2004). An annual cycle of steroid hormone concentrations and gonad development in the winter skate, Leucoraja ocellata , from the western Gulf of Maine. Marine Biology 144: 845 853.
Sulikowski, J. A., Tsang, P. C. W. and Huntting Howell, W. (2005b). Age and size at sexual maturity for the winter skate, Leucoraja ocellata , in the western Gulf of Maine based on morphological, histological and steroid hormone analysis. Environmental Biology of Fishes 72: 429 441.
Sumpter, J. P. and Dodd, J. M. (1979). The annual reproductive cycle of the female lesser spotted dogfish, Scyliorhinus canicula L., and its endocrine control. Journal of Fish Biology 15: 687 695.
Taylor, L., Walker, T. I. and Sowden, C. (2005). Our relationship with sharks and rays. In: A guide to sharks and rays. Tricas, T. C., Deacon, K., Last, P. R., McCosker, J. E., Walker, T. I. and Taylor, L. (ed.). Fog City Press, USA. Volume 2: 32 63 pp.
Tricas, T. C., Maruska, K. P. and Rasmussen, L. E. L. (2000). Annual cycles of steroid hormones production, gonad development, and reproductive behavior in the atlantic stingray. General and Comparative Endocrinology 118: 209 225.
Tricas, T. C., Scott, W. M. and Sisneros, J. A. (2002). Electrosensory optimization to conspecific phasic signals for mating. Neuroscience Letters: 129 132.
Tsang, P. and Callard, I. P. (1982). Steroid production by isolated skate ovarian follicular cells. The Bulletin of Mount Desert Island Biological Laboratory 22: 96 97.
Tsang, P. and Callard, I. P. (1983). In vitro steroid production by ovarian granulosa cells of Squalus acanthias . The Bulletin of Mount Desert Island Biological Laboratory 23: 78 79.
Tullis, A. and Peterson, G. (2000). Growth and metabolism in the embryonic white spotted bamboo shark, Chiloscyllium plagiosum : Comparison with embryonic birds and reptiles. Physiological and Biochemical Zoology 73: 271 282.
Turner, K., Gardner, C. and Swain, R. (2002). Onset of maturity in male southern rock lobsters Jasus edwardsii in Tasmania, Australia. Invertebrate Reproduction & Development 42: 129 135.
Voegeli, F. A., Smale, M. J., Webber, D. M., Andrade, Y. and O'Dor, R. K. (2001). Ultrasonic telemetry, tracking and automated monitoring technology for sharks. Environmental Biology of Fishes 60: 267 281.
Walker, T. I. (1998). Can shark resources be harvested sustainably? A question revisited with a review of shark fisheries. Marine and Freshwater Research 49: 533 572.
Walker, T. I., Hudson, R. J. and Gason, A. S. (2005). Catch Evaluation of target, by product, and by catch species taken by gillnets and longlines in the shark fishery of south eastern Australia. Journal of Northwest Atlantic Fishery Science 35: 505 530.
169
Wardle, C. S. (1981). Physiological stress in Captive Fish. In: Aquarium System. Hawkins, A. D. (ed.). A subsidiary of Harcourt Brace Jovanovich, London. 15: 403 414 pp.
West, G. J. and Stevens, J. D. (2001). Archival tagging of school shark, Galeorhinus galeus , in Australia: initial results. Environmental Biology of Fishes 60: 283 298.
West, J. G. and Carter, S. (1990). Observations on the development and growth of the epaulette shark Hemyscyllium ocellatum (Bonnaterre) in captivity. Journal of Aquariculture and Aquatic Sciences V: 111 117.
Worton, B. J. (1987) A review of models of home range for animal movements. Ecological Modelling 38:277 298.
Wourms, J. P. (1977). Reproduction and development in chondrichthyan fishes. American Zoology 17: 379 410.
Zeller, D. C. (1997). Home range and activity patterns of the coral trout Plectropomus leopardus (Serranidae). Marine Ecology Progress Series 154: 65 77.
170