Aspects of the biology and husbandry of portunid crabs relevant to aquaculture-based enhancement and fisheries management

James R. Tweedley1, Theodore I. Campbell1, Neil R. Loneragan1 & Danielle Johnston2

1Centre for Fish and Fisheries Research, School of Veterinary and Life Sciences, Murdoch University 2Department of Fisheries, Western Australia

June 2017

Cover image. Photographs showing the growout of soft-shell Mud Crabs (Scylla spp.) in the tropics using Mub Crab boxes. Photographs taken from Aquatec (http://www.aquatec.vn/mud-crab-box-32/?hl=en).

2 Summary

This review summarises (i) the biology and behaviour of portunid crabs, focusing on the commercially and recreationally important species within the genera Callinectes, Portunus and Scylla, (ii) identifies previously successful aquaculture/restocking efforts for portunids and (iii) detail any successful aquaculture methods/techniques for these species.

Portunids are highly fecund, fast growing, short-lived species, with high natural mortalities and whose populations are characterised by an abundance of immature individuals and fewer older, mature individuals. The dietary composition of these crabs, which are opportunistic, is influenced significantly by ontogeny, moult stage, habitat and season. Bivalves, gastropods, crustaceans and teleosts are the major prey categories consumed, with larger crabs consuming more mobile prey. Portunid crabs are more active during night than day, when many species bury. They have developed intricate mating and moulting behaviours that are influenced by water temperature and salinity. The females of many species undertake migrations associated with reproduction and the release of hatching eggs.

Portunid fisheries occur globally, with the greatest capture rates occurring in the Indo-West Pacific. Global catches of portunid crabs are still increasing despite the relative stability in the catch rates of world finfish fisheries. High fishing pressure on many established portunid fisheries, exacerbated by environmentally driven fluctuations in recruitment, has led to several localised depletions. The management of portunid fisheries varies greatly between and within countries, but all employ input (i.e. licences, spatial and temporal closures) and/or output controls (size, sex and catch limits). Aquaculture-based enhancement has been suggested as a possible means is increasing stocks in depleted fisheries.

A number of restocking and stock enhancement programs have been implemented around the world, including USA, Japan and South East Asia. These were all initiated in response to declines in stocks and, in each case, successful hatchery techniques were developed to enable small-scale production to occur. Releases were found to be most successful when

3 the hatchery-reared individuals were released as crablets and not megalopae. Noting that far smaller numbers of individuals were produced due to cannibalism. Typically, release programs for brachyuran crabs appear to have been successful, albeit too different extents. For the most part, the evaluations of the effectiveness of these releases have been carried out at the pilot scale with only the culture of P. trituberculatus scaled up (~27-35 million juveniles per year). Species with fast growth rates, limited home ranges and a large economic value are likely to be the most appropriate species to culture and release. Any future release program for a portunid is likely to require extensive research and development, particularly to scale up production, and thus the biggest gains in this field to- date have come from increased knowledge rather than increased stocks.

In terms of aquaculture, broodstock are typically collected from the wild using commercial fishers and to increase the success of the hatch, conditions in the culture environment should be similar to those in the waters the broodstock were collected from. If inseminated but not berried broodstock are collected, crabs require access to sandy substrate to spawn. Maintaining appropriate water quality is paramount and P. pelagicus larvae require relatively warm (25-30 °C) water at near-marine salinities (30-35 ppt) that is well oxygenated and of a slightly basic pH (8). The larvae require different feed throughout the transition from zoea to megalopa. Typically, zoea are fed a range of microalgae strains (which also enrich live feeds and provide a greenwater culture) and rotifers, with Artemia being provided once the larvae reach the later zoeal stages. At the crablet stage, diet in the hatchery comprises commercial shrimp/prawn feeds. Stocking density is important, with faster growth and greater survival occurring at lower densities. Cannibalism in portunids starts at the megalopa stages and lower stocking densities and the provision of greater substrate complexity reduce juvenile cannibalism. Portunid species have been shown to actively select more complex habitats, such as seagrass, macroalgae, pebbles and crushed shells over finer sand substrates and artificial habitats, such a nets, have also been successful in increasing survival.

4 Table of Contents

Summary ...... 3 Table of Contents...... 5 Rationale ...... 7 Section 1. Life history characteristics of three genera of portunid crabs, Callinectes, Portunus and Scylla ...... 9

1.1. Overview of portunids ...... 9 1.2. Biology ...... 11 1.2.1. Life cycle ...... 11 1.2.1.1. Duration of life cycle stages ...... 12 1.2.1.2. Description of life cycle stages ...... 13 1.2.2.1. Seasonal abundance ...... 15 1.2.2.2. Habitat utilisation ...... 16 1.2.3. Reproduction...... 17 1.2.3.1. Fecundity ...... 18 1.2.3.2. Size at maturity...... 19 1.2.3.3. Mate selection, courtship and mating behaviour ...... 22 1.2.4. Growth, mortality and life history ratios ...... 23 1.2.4.1. Growth ...... 24 1.2.4.2. Mortality ...... 28 1.2.4.3. Beverton-Holt Life history ratios ...... 31 1.2.5. Diet ...... 34 1.2.5.1. Dietary composition ...... 34 1.2.5.2. Factors influencing dietary composition ...... 39 1.3. Behaviour ...... 40 1.3.1. Rhythms in behaviour ...... 40 1.3.2. Burying ...... 41 1.3.3. Swimming and migration ...... 43 1.3.3.1. Migrations for reproduction ...... 45 1.3.4. Moulting ...... 46 1.3.4.1. Moulting process ...... 47 1.4. Influence of environmental conditions ...... 49 1.4.1. Influence of water temperature and salinity ...... 49 1.4.2. Influence of environmental conditions on fisheries recruitment and production ...... 50 1.5. Conclusion ...... 52

Section 2. Portunid fisheries and aquaculture-based enhancement ...... 55 2.1. Fisheries ...... 55 2.1.1. Overview of portunid fisheries ...... 55 2.1.2. Influence of fishing pressure crab stocks ...... 57 2.1.3. Management strategies ...... 59 2.2. Aquaculture-based enhancement ...... 62 2.2.1. Rational for release programs ...... 62 2.2.2. Release programs for portunids ...... 63 2.2.2.1. Portunus trituberculatus in Japan ...... 64 2.2.2.2. Portunus pelagicus in Thailand ...... 67 2.2.2.3. Scylla spp. in the Phillippines...... 68 2.2.2.4. Scylla paramamosian in Japan ...... 71 2.2.2.5. Callinectes sapidus in the Chesapeake Bay, USA ...... 71 2.2.2.4. Release programs for other brachyuran crabs ...... 79 2.3. Conclusions ...... 81 2.3.1. Wild capture fisheries ...... 81 2.3.2. Commonalities between release programs for brachyuran crabs and lessons learned ...... 82

5 Section 3. Aquaculture of portunids ...... 86 3.1. Overview of portunid aquaculture ...... 86 3.2. Broodstock collection and conditioning ...... 88 3.3. Egg incubation and hatching ...... 91 3.4. Larval culture ...... 92 3.4.1. Water quality ...... 92 3.4.2. Feed ...... 96 3.4.3. Hatchery environment ...... 97 3.4.4. Cannibalism ...... 100 3.5. Nursery ...... 101 3.6. Grow-out ...... 104 3.7. Conclusions ...... 106

References ...... 108

6 Rationale

Portunid crabs are heavily targeted by commercial, recreational and artisanal fishers around the world and also grown in culture, with the exploitation and aquaculture production of these species increasing seven fold between the 1970s and 2000s (Keenen 2004). The greatest wild capture landings occur in the Indo-West Pacific (FOA 2016a) and these species are the focus of major recreational fisheries in countries such as Australia and the United States of America (Sumner et al. 2000, Miller 2001, Ryan et al. 2015). While, in countries such as Indonesia, Malaysia and Thailand, production has risen rapidly (Keenen 2004), catches in some fisheries in the USA and Australia have declined (Miller et al. 2005,

Johnston et al. 2011b). For example, in Chesapeake Bay (USA), the income generated from the Callinectes sapidus fishery has been reduced by 70% from US$ 900,000 to 300,000 in only 15 years (Zohar et al. 2008). As many of these declines are linked to overfishing, stocks are below their carrying capacity and thus aquaculture-based enhancement or release programs (Taylor et al. 2017) are seen as a potential option to increase stocks (Hines et al. 2008a, Johnston et al. 2011b). In recent years, aquaculture technology has been developed for a number of portunids and these individuals have been used in a number of restocking and stock enhancement programs, some of which release up to 35 million crablets per year (Keenen 2004). However, not all of these programs have been successful and, although some have made it to the experimental stage, they were not always progressed to the commercial scale. Thus, before attempting to develop the technology to culture portunids, such as the Blue Swimmer Crab Portunus armatus, in Western Australia, there is value in critically evaluating the successes and failures of previous aquaculture efforts and/or release programs. In addition, Keenen (2004) recgonised the lack of information on the basic biology of portunids, particularly those of interest to aquaculture.

Thus the aims of this literature review were to:

 Summarise the components of portunid biology and ecology that are relevant to the successful aquaculture of members of this species.

7  Identify previously successful aquaculture/restocking efforts for portunids and similar species in other parts of the world.

 Detail any aquaculture methods and techniques, which may have application in Western Australia.

8 Section 1. Life history characteristics of three genera of portunid crabs, Callinectes, Portunus and Scylla

1.1. Overview of portunids

Over 350 species of crabs, representing 33 genera and seven sub-families are included in the family Portunidae (Ng et al. 2008, WoRMS 2016). The portunid family is distributed throughout the tropics, with maximum diversity in the Indo-West Pacific region, though a number of species occur in temperate waters (Poore 2004). Portunids live in a range of habitats, but are found most commonly in shallow waters (i.e. < 50 m deep) over sand or mud. This substrate preference means many species, such as the Blue Swimmer Crab Portunus armatus and the Blue Crab Callinectes sapidus, are most abundant in sheltered marine embayments and estuaries, where they form the basis of commercial and recreational fisheries (Johnston et al. 2011b).

Portunid crabs are amongst the most powerful swimmers of all crustaceans. They achieve this through rhythmic movements of the fourth pair of pereiopods, on which the final two segments, i.e. the propodus and dactylus, are flattened into a paddle shape. This morphological feature distinguishes portunids from other brachyuran crabs (Poore 2004) and is also responsible for their common names of ‘swimmer’ and ‘swimming’ crabs (Hartnoll 1971).

Larger portunid species are targeted by commercial, recreational and artisanal fishers around the world. Global fishing effort and exploitation of portunid crabs increased seven fold from the 1970s to 2000s (Keenen 2004). Moreover, the combined global landings of the Gazami Crab, Portunus trituberculatus, and the Blue Swimmer Crab, P. pelagicus (probably the P. pelagicus complex, see below), increased from ~475,000 tonnes in 2005 to ~815,000 tonnes in 2014, making up nearly half of the global crab landings in those years, i.e. 1.25 and 1.68 million tonnes in each year, respectively (FOA 2011, 2016a). Other important target species in the Portunidae include the Giant Mud Crab Scylla scylla (Grubert et al. 2014), the Blue Crab Callinectes sapidus (Huang et al. 2015), the Three Spot Crab Portunus

9 sanguinolentus (Dash et al. 2013), and the Blue Swimmer Crabs of the P. pelagicus complex sensu Lai et al. (2010), i.e., P. armatus, P. pelagicus, P. reticulatus and P. segnis (Dinshbabu et al. 2008, Kamrani et al. 2010, Johnston et al. 2014b, Kunsook et al. 2014b). For clarity, the species within the P. pelagicus complex will be referred to henceforth by the names adopted by Lai et al. (2010).

The subfamily Portuninae contains 118 of the 355 portunid species (WoRMS 2016; Table 1.1)). Although the Thalamitinae is the most speciose portunid subfamily, comprising 167 species (Table 1.1), it does not contain as many large, commercially and recreationally important species as found in the Portuninae, such as those in the genera Callinectes, Portunus and Scylla (Table 1.1).

Table 1.1. A taxonomic breakdown of the family Portunidae sensu Ng et al. (2008) and WoRMS (2016), showing the sub-families and number of genera, sub-genera and species in each taxonomic category. Values from WoRMS (2016) have been given when differences were found between sources. Note that Portunus is also a sub-genus within the Portunus genus.

Family Sub-family Genera Sub-genera No. of species Portunidae 30 12 355

Carphyrinae 3 28

Carupinae 7 21

Lupocyclinae 2 16

Podophthalminae 3 5

Portuninae 11 7 118 Including Callinectes 15 Portunus 5 99 Portunus 25

Scylla 4

Thalamitinae 4 5 167

10 1.2. Biology

The majority of the published literature focuses on the commercially valuable portunid species. However, a large proportion of the literature on Portunus trituberculatus, which has been widely studied and cultured in Japan and China (Secor et al. 2002), is not available in English. Similarly, the existing literature on Callinectes spp. in South America (mostly Brazil) is largely in Portuguese. Fortunately, Callinectes species are more widespread than P. trituberculatus, and hence literature on members of this genus is also available in English. This section summarises the life cycle, seasonal abundance, reproduction, growth and mortality rates, life history ratios and diet of portunid crabs, concentrating mainly on C. sapidus and members of the P. pelagicus complex because of the extent of available literature in English.

1.2.1. Life cycle

Portunid Crabs follow the general Brachyuran life cycle described by (Warner 1977), with two planktonic larval stages, i.e. the zoea and megalopa, followed by metamorphosis into the benthic juvenile stage and then more continuous ontogeny until maturity is reached (Fig. 1.1). The larval stages are planktonic and typified by four to eight zoeal stages, depending on the species, and a single megalopal stage (Costlow and Bookhout 1959, Dittel and Epifanio 1984, Jose and Menon 2004, Nurdiani and Zeng 2007). Growth occurs only during moulting (Chang 1995), which in larval stages is from one morphological stage to the next, and in post-larval forms is by incremental increases in size (Fig. 1.1). Once mature, breeding occurs following an intricate mating ritual (Section 1.2.3.3). Females of many species migrate after mating prior to the hatching of their eggs (Hyland et al. 1984, Seitz

2001, Forward et al. 2003, Carr et al. 2004).

11

Mature crabs Mating Ovigerous females

Eggs

Zoea Megalopa Juvenile crabs (4 to 8 stages) (1 stage) Fig. 1.1. Generalised stages in the life cycle of portunid crabs developed from the summary of the typical Brachyuran life cycle by Warner (1977). Images sourced from http://ian.umces.edu/symbols/.

1.2.1.1. Duration of life cycle stages

The duration of the larval stages varies among and within portunid species, but in general, larval development and the intermoult period are related to temperature and are faster at lower than higher latitudes (Warner 1977, Nurdiani and Zeng 2007). Salinity also affects the duration of the larval stages, but its influence varies among species (Bryars and Havenhand 2006, Nurdiani and Zeng 2007). For example, at a constant temperature of 25 oC, the total larval duration of Scylla serrata was ~ 4 d longer at salinities of 30 (36.0 d) than 20 (31.9 d; Table 1.2). The larval duration of Callinectes sapidus was also 4 d longer at salinities higher (31) and lower (20) than 27 (larval duration increased from 46 to 50 days; Table 1.2).

Less is known about the duration of the juvenile and adult crabs as it is difficult to age crustaceans and estimates of growth rates rely mainly on carapace width based methods. However, by comparing the size at maturity and the modelled carapace width at age one, the duration of the juvenile stage is probably less than one year in P. armatus (= P. pelagicus) (de Lestang et al. 2003a, b), P. reticulatus (= P. pelagicus) and P. sanguinolentus (Sukumaran and Neelakantan 1996b, 1997), female P. trituberculatus (Oh 2011) and C. sapidus (Sumer et al. 2013). The maximum age of most portunid species is believed to be ~ 3 years (Helser and Kahn 2001, Dinshbabu et al. 2008, Sugilar et al. 2012,

12 López-Martínez et al. 2014), however, the proportion of individuals that reach this age is likely to be low in areas where they are fished (de Lestang et al. 2003b).

1.2.1.2. Description of life cycle stages

Spawning, in most portunid species, takes place in shallow coastal waters during summer, when offshore winds blow frequently at night and onshore winds during the day (Forward et al. 1997). After hatching, zoea are transported to offshore waters by migrating vertically in the water column to the surface at night (McConaugha et al. 1983). The zoea spend a variable amount of time feeding on phyto- and zooplankton, growing with each moult into the next stage (Zmora et al. 2005). Metamorphosis from the last larval stage into the megalopa stage is associated with an onshore movement, facilitated this time through vertical migration to the surface during the day. Settlement begins once the megalopa are in coastal or estuarine waters. The movement of the megalopa of C. sapidus and other estuarine crab species into settlement habitats in estuaries is facilitated by their use of flood-tide transport (FTT). This is a form of selective tidal-stream transport (STST) in which megalopae suspend themselves in the water column during flood tides and shelter on the substratum throughout the ebb tide to prevent them being flushed downstream. This diel movement enables them to move into the upper reaches of estuaries (Pile et al. 1996, Forward et al. 1997, Tankersley et al. 2002, Pardo et al. 2011). The latter larval stages of penaeid prawns also use STST to recruit from the ocean to nursery habitats in (Rothlisberg et al. 1983, Condie et al. 1999).

The behaviour and morphology of juvenile crabs differ from adults. For example, they (i) select habitat that enhances survival rather than growth (Wilson et al. 1990, Dittel et al. 1995, Hovel and Lipcius 2001, Orth and van Montfrans 2002); (ii) feed on different food items (Stoner and Buchanan 1990, Hsueh et al. 1992, de Lestang et al. 2000, Jose 2011; Section 1.2.5); and (iii) show external morphological differences, e.g. the abdominal flap of females widens and becomes loose after the pubertal moult, while the relative size of male chelipeds (claws) increases (Roberston and Kruger 1994, Sukumaran and Neelakantan 1996b, de Lestang et al. 2003c, Zainal 2016).

13 Table 1.2. Duration (in days) of the different larval stages (zoea, megalopa) in the life cycle of species within three genera (Portunus, Callinectes and Scylla) of portunid crabs and the influence of temperature and salinity on the duration of these stages. The durations given are the mean or the mid-point (MP) of the range in days for each stage, with standard devation given when possible. The range of duration is presented on the line below when available.

Portunus Callinectes Scylla

armatus1* pelagicus2 sapidus3 arcuatus4 serrata5*

Water temp. (oC) 20.0 22.5 25.0 25 – 29 25 25 25 24 ± 1 25 25 31 31

Salinity (ppt) 36 – 38 36 – 38 36 – 38 25 – 31 20 27 31 30 ± 0.5 20 30 20 30

Method Mean Mean Mean MP MP MP MP Mean Mean Mean Mean Mean

5.0 3.4 3.1 2.5 8.5 8.0 10.0 5.9 ± 0.9 4.6 ± 0.1 4.9 ± 0.1 3.3 ± 0.1 4.0 ± 0.2 1 4 – 6 3 – 5 3 – 4 2 – 5 6 – 13 7 – 9 7 – 13 4 – 6 4 – 8 3 – 8 3 – 6 10.1 8.4 6.7 5.0 14.0 14.0 15.0 12.0 ± 1.6 7.8 ± 0.2 7.8 ± 0.1 6.3 ± 0.1 7.2 ± 0.1 2 9 – 11 4 – 10 6 – 8 4 – 6 12 – 16 12 – 16 11 – 19 7 – 10 7 – 10 5 – 8 6 – 9 18.9 12.7 11.3 9.5 22.0 18.5 21.0 17.9 ± 2.7 12.3 ± 0.1 11.8 ± 0.2 9.3 ± 0.2 9.7 ± 0.4 3 15 – 24 11 – 15 9 – 19 8 – 11 17 – 27 14 – 23 15 – 27 11 – 20 10 – 17 8 – 13 8 – 17 26.2 17.9 15.3 14.0 27.0 22.0 24.5 25.2 ± 2.6 16.8 ± 0.3 15.9 ± 0.3 12.5 ± 0.3 12.5 ± 0.3 4 Zoeal 22 – 31 16 – 21 13 – 22 12 – 14 24 – 30 18 – 26 20 – 29 15 – 23 14 – 19 11 – 16 11 – 19 Stages 31.0 28.5 31.5 31.2 ± 2.7 22.6 ± 0.2 20.6 ± 0.2 16.9 ± 0.1 16.7 ± 0.2 5 - - - - 28 – 34 22 – 33 24 – 39 20 – 28 19 – 26 15 – 20 15 – 24 38.0 32.0 36.0 28.1 ± 3.3 6 ------38.0 26 – 38 29 – 43 43.0 38.5 41.0 44.4 ± 3.4 7 ------43.0 32 – 45 35 – 47 50.6 ± 3.8 8 ------

39.5 26.2 21.4 21.5 50.0 46.0 50.0 69.3 ± 16.8 31.9 ± 0.7 36.0 ± 3.9 22.8 ± 0.6 24.2 ± 1.9 Megalopa 39 – 40 24 – 29 19 – 24 19 – 24 50.0 39 – 53 45 – 55 31 – 36 29 – 62 22 – 27 21 – 34

* Other temperature salinity combinations tested. 1Bryars and Havenhand (2006); 2Motoh et al. (1978); 3Costlow and Bookhout (1959); 4Dittel and Epifanio (1984); 5Nurdiani and Zeng (2007).

14 Mating takes place long before the transport of eggs from the ovary through the spermatophore and onto the pleopods, the abdominal appendages, and occurs between hard shell males and soft shell females. After copulation, the hard-shelled male protects the soft-shelled female until her carapace has hardened (Hartnoll 1969, Kangas 2000). Females of some species then migrate offshore out of mangrove systems, e.g. S. serrata (Hyland et al. 1984), or near-shore coastal waters, e.g., P. sanguinolentus (Sumpton et al. 1989), to release their eggs. In contrast, others migrate from estuaries to marine waters, e.g., P. armatus (= P. pelagicus) (Potter and de Lestang 2000) and C. sapidus (Turner et al. 2003,

Aguilar et al. 2005), respectively. Other species appear to mate and release their eggs where they live and do not migrate during reproduction (Safaie et al. 2013b, Zairion et al. 2015a; Section 1.2.3).

1.2.2. Variation in abundance and habitat utilisation

The abundance of portunid crabs varies seasonally in temperate locations due to changing environmental conditions. Estuarine species move to stay within their salinity and/or temperature tolerance (Potter et al. 1983, Kaufman 2014) and the abundance and distribution of mature females changes markedly leading up to the reproductive season (Hill 1994, Potter and de Lestang 2000, Aguilar et al. 2005). In addition to this, daily movement between and within habitats optimises the survival and growth of individuals and is frequently observed (Hines et al. 1987). Patterns in habitat utilisation vary with life history stage, gender and moult stage (Hines et al. 1995, Wrona 2004).

1.2.2.1. Seasonal abundance

Seasonal patterns of abundance are more likely to occur in species inhabiting temperate waters, particularly estuaries, as these water bodies exhibit larger fluctuations in water temperature and salinity (Tweedley et al. 2016b).

15 The abundance of P. armatus (= P. pelagicus) in the microtidal estuaries of south-western

Australia is related to salinity, peaking during summer/autumn when freshwater inflow is minimal and salinity is greatest. In contrast, freshwater flow in winter/spring lowers salinity and thus the crabs move to the most downstream parts of the estuary and into nearshore coastal waters (Potter et al. 1983, Potter and de Lestang 2000, Potter et al. 2001). Because of this movement, coastal populations, particularly those near the mouths of estuaries, show the inverse pattern of abundance to that found in the estuary, with abundance of crabs peaking in late winter. This trend is not observed in C. sapidus in Chesapeake Bay, because they also inhabit low salinity areas in estuaries (Posey et al. 2005, Ralph and Lipcius 2014). In this population, crabs inhabit warm shallow waters in summer, but then move into deeper waters in winter, which cool less quickly (Wrona 2004, Kaufman 2014). Similarly, an increase in the abundance of P. armatus in deep waters of a coastal embayment in south- western Australia (Cockburn Sound) during July to September (winter) is thought to be due to the movement away from the lower temperatures in the shallow waters of the embayment (Potter et al. 2001).

1.2.2.2. Habitat utilisation

Callinectes sapidus utilises different habitats throughout its life cycle to optimise the survival of each life stage, gender and moult stage (Hines et al. 1987). Juveniles select habitats with physical structure (e.g. seagrass beds and salt marshes) that offer protection from the high predation rates they experience (Heck and Thoman 1984, Wilson et al. 1990, Hovel and Lipcius 2001, Posey et al. 2005). Habitats that enhance growth (e.g., those with higher prey abundances) are also favoured (Perkins-Visser et al. 1996). Estuaries in North and South

Georgia (USA) commonly lack such physical structure, so juvenile C. sapidus rely greatly on low salinity areas (mesohaline and oligohaline) for protection from predators, which are largely marine and thus stenohaline (Posey et al. 2005). However, the metabolic cost of the

16 osmoregulation required to live in low salinity waters has been linked to slower growth rates in these environments (Cadman and Weinstein 1988, Guerin and Stickle 1992).

Adult crabs suffer much lower predation rates than the earlier life history stages, and thus select habitats that optimise growth and reproductive success (Hines et al. 1995, Ramach et al. 2009). For example, mature C. sapidus are more abundant in the upper reaches of estuaries where salinity is lower and the abundance of their preferred prey is greater (Aguilar et al. 2005). Interestingly, male and female C. sapidus show sexual segregation, with males more abundant in shallower water and females more abundant in the deep (Wrona

2004, Ramach et al. 2009). This difference is further exacerbated in late summer and autumn when mature and ovigerous females migrate downstream and into marine waters to release their eggs (Hines et al. 1995, Aguilar et al. 2005).

Crabs with soft exoskeletons (i.e., moult stages stage A, early B and E, see Section 1.3.4) are more vulnerable to predation and thus utilise similar habitats to juveniles (Wolcott and Hines 1990, Wrona 2004). This is more apparent in males than females because the presence of mature, intermoult males in deeper waters (i.e., suitable mating partners) can lead to moulting in females as moulting is linked to the mating behaviour (Hines et al. 1987, Shirley et al. 1990; Section 1.2.3.3).

1.2.3. Reproduction

Reproduction occurs throughout the year in tropical populations (de Lestang et al. 2003c), but is less protracted in temperate regions, typically occurring in the warmer months (Potter et al. 1983, Keunecke et al. 2011, Zairion et al. 2015b). Most species reach maturity, i.e., undergo the pubertal moult, during their first year of life (de Lestang et al. 2003c, Kamrani et al. 2010, Keunecke et al. 2011). Intricate mate selection, courtship and mating behaviour has been found in many species and is described below.

17 The release of fertilized eggs occurs approximately six months after mating in most species

(Aguilar et al. 2005). Females begin this six-month period by actively feeding to build up the energy reserves needed for egg production (Warner 1977). Eggs are extruded from ovaries through the spermatophore, where they are fertilized, and fixed onto the pleopods, the appendages of the abdomen, where they are carried until they hatch into zoea (Zairion et al. 2015b). The eggs change in colour from yellow to brown to grey as the yolk is used up by the developing embryo and, when ready for release, are helped into the water column by fanning of the abdomen and pleopods by the female (Warner 1977).

1.2.3.1. Fecundity

Fecundity in portunids is extremely high, with most Portunus, Callinectes and Scylla species producing over 500,000 eggs per batch for females with a 130 mm carapace width (CW; Table 1.3). However, the number of eggs produced can vary markedly among species, ranging from ≈ 190,000 to six million (Table 1.3). Moreover, multiple batches of eggs can be released by a female each year (Hamasaki et al. 2006), even when the reproductive season is short, i.e. < 6 months (de Lestang et al. 2003c). The high fecundity and ability to have multiple egg releases during the breeding season, means that the annual egg production per female commonly exceeds one million eggs (Williams and Primavera 2001, Oniam et al. 2012, Severino-Rodrigues et al. 2013), and in some cases, e.g. S. serrata, may reach 20 million eggs (Prasad and Neelakantan 1989; Table 1.3).

In addition to interspecific variation in fecundity, egg production has also been shown to differ within a species due to environmental conditions and fishing pressure (Jivoff 2003, Johnston et al. 2011b, Harris et al. 2012, Pardo et al. 2015). For example, in Cockburn

Sound, south-western Australia, low temperatures prior to the reproductive season led to a reduction in the number of batches of eggs that large females were able to produce and consequently to the collapse of the P. armatus (= P. pelagicus) fishery (de Lestang et al. 2010, Johnston et al. 2011b). Male-biased harvesting of C. sapidus has been implicated in

18 influencing the reproductive output of females because of decreases in the median size of male crabs and associated decreases in the size of the spermatophore and as a consequence, the number of sperm, passed to females (Jivoff 2003, Pardo et al. 2015). This is especially relevant when small males mate with large females, which happens more frequently when large males are removed, as in male-biased fishery harvest strategies. Lastly, the number of eggs produced by a female is directly related (usually exponentially) to size of the female (Prasad and Neelakantan 1989, Prager et al. 1990, de Lestang et al. 2003c, Jose 2013). For example, a female P. sanguinolentus with a 130 mm CW produces 366 300 eggs, ~ 2.6 times the numbers produced produced by a 110 mm CW female (139 100 eggs). However, this increase is dependent on the specific exponential function between fecundity and CW of each species, with the fecundity of species with greater “a” values e.g., increasing more than those with lower “a” values over the same increase in CW (Table 1.3).

1.2.3.2. Size at maturity

The size (carapace width; CW) at which 50% of the population attain maturity is denoted by

CW50, and is typically used as an estimate of the population size at maturity (often gender specific). The pubertal moult, after which maturity is reached, is characterised in females by the abdominal flap widening and becoming loose and in males by the size of the chela relative to that of the body increasing (Roberston and Kruger 1994, Keunecke et al. 2011). Note that this morphological change in males does not necessarily mean they are mature, as a male with larger chela may not have mature gonads, and a male with mature gonads may not have large chela. However, in many cases the estimates of size at maturity based on morphological observations compare closely with those based on gonad histology for

P. armatus (de Lestang et al. 2003c). Maturity is reached towards the end of the first year of life in most portunid species (Potter et al. 1983, Potter and de Lestang 2000, de Lestang et al. 2003c, Kamrani et al. 2010), however, the size of maturity is decreased by increasing fishing pressure (Berrigan and Charnov 1994, Trippel 1995, Pereira et al. 2009). It can also

19 be influenced negatively, by changes in predator and prey abundance (Hines 1989) and environmental conditions, e.g. temperature and salinity (Sukumaran and Neelakantan 1996b, de Lestang et al. 2003c, Fischer and Wolff 2006, Kamrani et al. 2010).

Estimates of the size at maturity are quite variable within Portunus, Callinectes, and Scylla genera (Table 1.3). On average, maturity is reached at 99.2 mm CW, and variation between species is quite large (SD = 19.8 mm, coefficient of variation [CV] = 20.0%; Table 1.3) with estimates of the size at maturity for each species ranging from 67.4 mm for C. ornatus to

131.4 mm for S. serrata. The CV in CW50 within each species (CV range = 1.7 to 22.9 %) is lower than the CV across all species and families (CV = 20.0 %; Table 1.3). The variation in

CW50 was greater among species in Callinectes (CV = 23.8%) that in Portunus (CV = 14.5%) or Scylla (CV = 19.7%; Table 1.3). The variation between species is, in part, due to the variation in maximum sizes between species and thus the CW50 is often standardised against the modelled asymptotic maximum size (CW∞) in the Beverton-Holt life history ratio CW50/CW∞ for comparisons between species (Hordyk et al. 2015b, Prince et al. 2015b; Section 1.2.4.3).

The variation in CW50 within each species and some of the variation between species is likely due to the previously mentioned factors of fishing pressure, predator and prey abundance and environmental conditions.

20 Table 1.3. Size at maturity (CW50), sex ratio and fecundity for portunid species in the Portunus, Callinectes and Scylla generas. The mean and range for CW50 are shown for each species when two or more estimates are available, and standard deviation (± SD) when three or more are available. The batch fecundity (BF) equation has the form: ln(BF) = a·ln(CW) + c”, where CW is Carapace Width. An overall mean for each genera and the Portuninae on a whole are also given for each parameter. Note, the genera and overall means for batch fecundity parameters exclude estimates marked with ^, * or #.

Taxa CW50 (mm) Sex Ratio Batch Fecundity Equation No. Eggs at 130 mm CW Ref Gender Mean ± SD Range Refs Female : Male Ref a c (x 1000)

Portunus 97.0 ± 14.1 1.01 ± 0.28 : 1 2.30 ± 1.29 1.70 ± 6.20 581.1.4 ± 287.9 P. armatus m 89.7 ± 9.1 72.1 – 115.1 1,2,3 1.49 : 1 4 1.82 3.29 188.9 1 f 92.5 ± 5.0 86.2 – 98.2 1,2,3

P. pelagicus m 96.8 95.5 – 98.0 5,6 0.92 : 1 7 19.1^ -1,517^ 965.9 8 f 106.1 ± 1.8 103.0 – 106.2 5,6,7

P. reticulatus f 96.0 – 9 0.74 9.43 456.8 10

P. sanguinolentus m 85.3 ± 10.8 81.4 – 97.5 11,12,13 0.79 : 1 11 3.69 -5.15 366.3 11 96.7 ± 22.1 81.9 – 135.3 11-15

P. segnis f 113.0 – 16 0.83 : 1 16 5.70^ -18.8^ 719.4 16

P. triterberculatus f 131.2 – 17 1.08 : 1 17 2.95 -0.78 773.7 18

Callinectes 87.7 ± 20.9 1.39 ± 1.13 : 1 4.85 -7.42 2690 C. arcuatus f 94.6 – 19 1.16 : 1 19

C. danae m 79.3 72.5 – 86.0 20,21 3.03 : 1 20 f 70.6 ± 9.5 61.0 – 79.9 20,21,22

C. ornatus f 67.4 – 22 0.83 : 1 22 4.85 -7.42 # 23

C. sapidus m 90.0 – 20 0.52 : 1 29 38.0^ -2,250^ 2,690 28 f 124.4 ± 15.0 102 – 147.0 20,24-28

Scylla 117.6 ± 23.2 0.81 : 1 2.98 1.11 4,291 S. olivacea f 90.8 86.0 – 95.5 30,31 0.83 : 1 30 2.98 1.11 6,019* 32

S. serrata m 131.4 ± 29.8 94.0 – 150.7 33,35,36 0.79 : 1 36 51.5^ -2,404^ 4,291 37 f 130.5 ± 6.8 123.0 – 136.1 33,34,36 Overall Mean 99.2 ± 19.8 1.12 ± 0.68 : 1 2.80 ± 1.60 -0.13 ± 6.74 1,308 ± 1,433

^ BF equation has the form: BF = (a·CW + c) * 1000. * CW is replaced by inner carapace width (ICW, the base of the antero-lateral spines). # The estimate of BF at 130 mm was not included as the maximum observed CW was ~70 mm.

21 1de Lestang et al. (2003c); 2Potter and de Lestang (2000); 3Potter et al. (2001); 4Potter et al. (1983); 5Ishan et al. (2014); 6Zairion et al. (2015b); 7Kunsook et al. (2014b); 8Zairion et al. (2015a); 9Dinshbabu et al. (2008); 10Jose (2013); 11Courtney and Haddy (2007); 12Yang et al. (2014); 13Wimalasiri and Dissanayake (2016); 14Lee and Hsu (2003); 15Sarda (1998); 16Kamrani et al. (2010); 17Oh (2011); 18Hamasaki et al. (2006); 19Fischer and Wolff (2006); 20Pereira et al. (2009); 21Araújo et al. (2011); 22Keunecke et al. (2011); 23Mantelatto and Fransozo (1997); 24Sumer et al. (2013); 25Eggleston (1998); 26Steele and Bert (1994); 27Rugolo et al. (1997); 28Prager et al. (1990); 29Archambault et al. (1990); 30Ikhwanuddin et al. (2011a); 31Jirapunpipat (2008); 32Koolkalya et al. (2006); 33Knuckey (1999); 34Roberston and Kruger (1994); 35Prasad and Neelakantan (1990); 36Ogawa et al. (2011); 37Davis (2004).

1.2.3.3. Mate selection, courtship and mating behaviour

Fighting between males, i.e. female-centred competition, is common during mate selection and involves males searching and fighting for females followed by the guarding of any successful mates for prolonged periods (Christy 1987). This behaviour is intensified if a male or males detect/s water-borne pheromones, which are released by females to express their receptivity to mating (Kangas 2000, Dinakaran and Soundarapandian 2009, Eales 2009). Patterns in colouration, such as the red chela dactylus of mature female C. sapidus, allow male crabs to identify and evaluate potential mating partners from distances too great for pheromone reception. As portunids are aggressive and cannibalistic (Hovel and Lipcius

2001, Dinakaran and Soundarapandian 2009), the ability to differentiate between large females and males from a distance increases male-female interactions and reduces fighting between males, and therefore the risk of injury or death (Baldwin and Johnsen 2009). Furthermore, colouration also stimulates courtship behaviour in the absence of pheromones (Baldwin and Johnsen 2012).

Courtship behaviour begins once a potential mate is identified and is influenced by the moult stage of the female and the sex ratio of the population. Early moult stage females

(stages A, B and C) avoid male interactions, while late moult stage (stage D; see Section 1.3.4 for descriptions of each moult stage) individuals actively seek them. Low female to male ratios increase the investment by males into interactions with females, namely the pre- and post-coital guarding duration (Jivoff and Hines 1998).

22 Mating typically occurs between intermoult (hard-shell) males and newly-moulted (soft- shell) females. However, courtship takes place between intermoult males and pre-moult females as females need to be soft in order to mate (Hartnoll 1969). Once a mate has been identified and courted, the male will grab the female and hold her underneath him in a guarding position for a variable duration of time which can exceed 10 days (Kangas 2000). The female is released temporarily to enable her to moult, after which the male retakes her, either in the guarding position, with the female upright, or in an inverted position depending on the genus (Hartnoll 1969). The sperm package passed to the female is used to fertilize the eggs that will be produced in the following breeding season (Christy 1987). Males in the genera Callinectes and Portunus (other genera may also exhibit this behaviour) continue to carry the female after copulation until her shell has hardened and his protection is no longer required (Hartnoll 1969, Kangas 2000). The duration of this post-coital female guarding by males is related to the male size; larger males guard for longer durations, and sex ratio; males guard for longer periods when the female to male ratio is lower (Jivoff and Hines 1998, Jivoff 2003).

1.2.4. Growth, mortality and life history ratios

Juvenile portunid crabs grow rapidly until maturity is reached (within their first year of life), after which growth slows as energy is diverted to reproductive output (Sukumaran and Neelakantan 1997, Defeo and Cardoso 2002). Longevity is short, between two and four years (Sukumaran and Neelakantan 1997, Knuckey 1999, de Lestang et al. 2003b, López- Martínez et al. 2014), and natural mortality rates (M) are correspondingly high and relatively invariable across species. Fishing mortality (F) is often greater than M, although it, and therefore the total mortality (Z; Z = M + F), fluctuates largely across species and populations. The exploitation rate (E) indicates high fishing pressure and overexploitation and common (Section 1.2.4.2).

23 1.2.4.1. Growth

The growth of portunid crabs, like that of all crustaceans, occurs only after moulting their shell during ecdysis (see Section 1.3.4 for details of the moult cycle). Crustacean growth can be characterised by two variables, the moult increment (MI), the proportionate increase in size after a moult has occurred, and the moult frequency (MF), the frequency at which individuals are moulting (Fitz and Wiegert 1991). In tropical areas, growth occurs throughout the year at a similar rate, i.e. continual growth, (Sukumaran and Neelakantan 1996a, Sukumaran and Neelakantan 1997, Sarda 1998). Whereas in temperate regions, growth slows during winter as water temperatures fall (de Lestang et al. 2003a, b), resulting in either smaller moult increments or lower moult frequencies or both (Lee and Hsu 2003).

Population growth is often modelled using the von-Bertalanffy growth function (VBGF),

-1 which has three parameters: k, the growth coefficient (year ); CW∞, the modelled asymptotical maximum carapace width (CW; mm), and t0, the theoretical CW at age 0 (years). Seasonal growth models have an additional sin graph component to model the oscillation in growth rate, which includes the parameters C, the amplitude (0-1), and ts the timing (in years) at which the oscillation in growth begins (a ts value of t produces a phase shift of t / 2π years in the sin oscillation). Multiple methods can be used to fit a VBGF, and produce variation in the parameter estimates for the same data (von Bertalanffy 1934, Walford 1946, Pauly 1983, Jose and Menon 2007). The ELEFAN I and II computer program is commonly used to fit VBGFs from length frequency data, rather than length at age data due to the difficulty of aging crabs (Dinshbabu et al. 2007). These programs analyse the change in monthly modal frequency of size classes. Comparisons of growth rates between species with similar morphologies are often based on the growth performance index (Φ’)

Φ’ = Log(k) + 2 * Log(CW∞)

due to the intrinsic connection between k and CW∞ (Pauly and Munro 1984). For example, if two species have same real growth (the same actual increase in size over a certain time) but one has a larger CW∞, the larger species will have a lower k as it takes longer to reach CW∞.

24 The overall means for the VBGF parameters for Portunus, Callinectes and Scylla species

-1 showing continual growth were 1.24 ± 0.41 year for k and 185.9 ± 24.6 mm for CW∞. The large standard deviation relative to the mean i.e. the coefficient of variation (CV ≈ 30 %) and range in values for k (0.69-2.39), show that the growth coefficient varies markedly between portunid genera, species and regions (Table 1.4). The CV of CW∞ for the three genera relative to the mean (≈ 14 %) was much smaller than that of k. The overall mean growth performance, Φ’, was 4.57 ± 0.13 (CV = 2.8%). The low standard deviation of Φ’ suggests that k and CW∞ may be inversely proportional to one another. If this is the case, real growth

(the actual increases in size) may occur at a similar rate for all species, and lower k values, where present, are only lower because the CW∞ is larger and hence takes a longer time to reach.

The overall means of k and CW∞ for Portunus, Callinectes and Scylla species that exhibit seasonal growth are 1.01 ± 0.42 year -1 (CV = 41.6%) and 198.1 ± 26.4 mm (CV = 13.3%), respectively (Table 1.5). This overall mean CW∞ is larger than it is for species of the same genera exhibiting continual growth (185.9 ± 24.6 mm), indicating seasonal growth species grow larger than their continual growth counterparts, but do so at a slower rate (continual growth species have a greater overall mean k). While the means differ, the variation in these two parameters is comparable between seasonal and continual growth species. The overall mean Φ’ is 4.56 ± 0.06 for species exhibiting seasonal growth which is almost identical to the Φ’ for species exhibiting continual growth (4.57 ± 0.13), which further supports an inversely proportionate relationship between k and CW∞ in portunid crabs. It also indicates that the real growth rates of seasonal and continual growth species may in fact differ less than suggested by the overall means of k and CW∞.

25 Table 1.4. Average von-Bertalannfy growth parameters for portunid species in the Portunus, Callinectes and Scylla genera that exhibit continual growth. The mean and range (line below) are given for each species when two or more estimates are available, and standard deviation (± SD) when three or more are available. An overall mean of each parameter and means for male and female crabs independently, where possible, are given for species in each genera and the Portuninae on a whole.

Taxa Gender von-Bertalanffy Growth Parameters Growth

k (year-1) CW (mm) t (years) Performance (Φ’) ∞ 0 Portunus

Overall 1.26 ± 0.31 186.2 ± 20.0 -0.05 ± 0.16 4.61 ± 0.09 Male 1.30 ± 0.30 186.6 ± 22.3 -0.05 ± 0.18 4.62 ± 0.08 Female 1.21 ± 0.34 185.7 ± 19.5 -0.05 ± 0.16 4.59 ± 0.11 P. armatus1 Male 1.60 175.0 0.20 4.67

Female 1.61 170.0 0.19 4.67

P. pelagicus2,3,4,5 Male 1.60 ± 0.80 160.1 ± 15.3 -0.36 ± 0.52 4.57 ± 0.18 0.93 – 2.75 142.6 – 174.8 -0.96 – -0.04 4.33 – 4.75 Female 1.54 ± 0.33 174.2 ± 8.4 -0.31 ± 0.45 4.66 ± 0.11 1.13 – 1.93 167.3 – 186.0 -0.84 – -0.04 4.50 – 4.76 P. reticulatus6,7,8 Male 1.24 ± 0.29 200.0 ± 17.0 -0.07 ± 0.03 4.67 ± 0.13 0.72 – 1.71 170.0 – 223.0 -0.09 – -0.03 4.41 – 4.81 Female 1.07 ± 0.26 192.6 ± 11.7 -0.06 ± 0.02 4.59 ± 0.12 0.59 – 1.42 169.0 – 205.0 -0.06 – -0.04 4.29 – 4.71 P. sanguinolentus7,8,9 Male 0.98 ± 0.34 181.1 ± 16.0 -0.04 ± 0.03 4.48 ± 0.19 0.54 – 1.57 158.0 – 195.4 -0.06 – -0.01 4.20 – 4.67 Female 0.85 ± 0.34 173.8 ± 18.7 -0.08 ± 0.02 4.38 ± 0.19 0.57 – 1.49 142.0 – 188.0 -0.10 – -0.05 4.06 – 4.47 P. segnis10,11 Male 1.45 179.5 -0.04 4.66 1.20 – 1.70 168.0 – 191.0 – 4.53 – 4.79 Female 1.35 181.5 -0.04 4.64 1.10 – 1.60 177.9 – 185.0 – 4.54 – 4.74 P. triterberculatus12 Male 0.94 224.0 0.00 4.67

Female 0.84 222.0 0.00 4.62

Callinectes Overall 1.03 ± 0.34 190.2 ± 33.6 -0.17 ± 0.22 4.46 ± 0.13 Male 1.46 162.7 -0.02 4.59 Female 1.44 157.8 -0.02 4.55 C. arcuatus13,14,15,16 Comb. 0.84 ± 0.16 146.0 ± 5.9 -0.13 ± 0.02 4.25 ± 0.08 0.63 – 1.00 140 – 152.0 -0.15 – -0.11 4.17 – 4.35 C. bellicosus13,14,15 Comb. 0.96 ± 0.31 174.0 ± 5.6 -0.13 ± 0.04 4.45 ± 0.13 0.68 – 1.30 169.0 – 180.0 -0.18 – -0.11 4.34 – 4.59 C. sapidus17 Comb. 0.77 ± 0.16 211.9 ± 19.8 – 4.53 ± 0.12 0.62 - 0.94 200.3 – 234.7 – 4.39 – 4.61 18 Male 1.46 162.7 -0.02 4.59 1.42 – 1.50 162.7 -0.02 4.58 – 4.60 18 Female 1.44 157.8 -0.02 4.55 1.42 – 1.46 157.8 -0.02 4.55 – 4.56 C. toxotes13,14 Comb. 0.69 188.5 -0.55 4.36 0.47 – 0.9 168.0 – 209 -0.94 – 0.16 4.31 – 4.40 Scylla Overall 1.77 171.8 -0.01 4.68 S. parmamosian19 Comb. 2.39 150.0 -0.01 4.73

S. serrata20 Comb. 1.14 193.6 0.00 4.63

Portuninae Overall 1.24 ± 0.41 185.9 ± 24.6 -0.08 ± 0.17 4.57 ± 0.13 Male 1.32 ± 0.28 183.2 ± 22.3 -0.05 ± 0.16 4.62 ± 0.08 Female 1.24 ± 0.32 181.7 ± 20.7 -0.05 ± 0.15 4.59 ± 0.10

1Sumpton et al. (1994); 2Abdul and Wardiatno (2015); 3Ishan et al. (2014); 4Kunsook et al. (2014b); 5Sawusdee and Songrak (2009); 6Dinshbabu et al. (2008); 7Sukumaran and Neelakantan (1996a); 8Sukumaran and Neelakantan (1997); 9Sarda (1998); 10Kamrani et al. (2010); 11Safaie et al. (2013a); 12Sugilar et al. (2012); 13López-Martínez et al. (2014); 14Hernandez and Arreola-Lizarraga (2007); 15gil-Lopez and Sarmiento-Nafate (2001); 16Fischer and Wolff (2006); 17Helser and Kahn (2001); 18Ferreira and D'Incao (2008); 19Le Vay et al. (2007); 20Knuckey (1999).

26 Table 1.5. Average von-Bertalannfy growth parameters for portunid species in the Portunus, Callinectes and Scylla genera that exhibited seasonal growth. The mean and range (line below) are given for each species when two or more estimates are available, and standard deviation (± SD) when three or more are available. An overall mean of each parameter and means for male and female crabs independently, where possible, are given for species in each genera and the Portuninae on a whole.

Taxa Gender Seasonal von-Bertalanffy Growth Parameters Growth

-1 Performance k (year ) CW (mm) t (years) C t ∞ 0 s (Φ’)

Portunus

Overall 1.02 ± 0.50 194.9 ± 26.7 -0.01 ± 003 0.72 ± 0.30 0.33 ± 0.39 4.55 ± 0.05

Male 0.80 204.4 0.00 0.64 0.34 4.52

Female 0.82 207.7 0.00 0.67 0.41 4.54

P. armatus1,2* Comb. 1.93 ± 0.61 150.3 ± 20.9 -0.07 ± 0.05 1.00 ± 0.00 0.14 ± 0.06 4.62 ± 0.10 1.50 – 2.82 125.9 – 176.6 0.05 – 0.14 1.00 0.07 – 0.20 4.53 – 4.75

P. sanguinolentus3 Male 0.87 204.8 0.00 0.40 0.00 4.56

Female 0.97 194.3 0.00 0.40 0.00 4.56

P. trituberculatus4 Male 0.72 204.0 0.00 0.87 0.68 4.48

Female 0.66 221.0 0.00 0.92 0.81 4.51

Callinectes Overall 0.96 206.0 -0.51 0.61 -0.09 4.60

Male 0.86 230.1 -0.16 0.29 -0.44 4.66

Female 1.06 181.9 -0.85 0.93 0.26 4.54

C. sapidus5 Male 0.86 230.1 -0.16 0.29 -0.44 4.66

Female 1.06 181.9 -0.85 0.93 0.26 4.54

Portuninae

Overall 1.00 ± 0.42 198.1 ± 26.4 -0.16 ± 0.31 0.69 ± 0.31 0.21 ± 0.43 4.56 ± 0.06

Male 0.82 ± 0.08 213.0 ± 14.8 -0.06 ± 0.10 0.52 ± 0.31 0.08 ± 0.56 4.57 ± 0.09

Female 0.90 ± 0.21 199.1 ± 20.0 -0.28 ± 0.49 0.75 ± 0.31 0.36 ± 0.41 4.54 ± 0.03

* Paramters have been transformed from monthly to yearly rates to correspond with the majority. 1de Lestang et al. (2003b); 2de Lestang et al. (2003a); 3Lee and Hsu (2003); 4Oh (2011); 5Sumer et al. (2013).

Growth differed significantly between the sexes of ten species but not for seven species (Tables 1.4, 1.5). Two species, P. armatus (= P. pelagicus) and C. sapidus, had studies that

demonstrated both significant differences in growth between sexes and no difference in growth. A study on the mole crab Emerita brasiliensis found that females grew slower than males but only after maturity was reached (Defeo and Cardoso 2002). The authors suggested that the greater energy requirements of egg production than sperm production

27 was the reason for this slowing of growth in females. Similarly, the growth rate of male and female juvenile P. reticulatus (= P. pelagicus) and P. sanguinolentus did not differ but once mature, the growth of females was slower than that of males (Sukumaran and Neelakantan 1997; Tables 1.4, 1.5).

The consistency of the Φ’ seen in portunid crabs has also been reported by Munro and Pauly (1983), who found that species within taxonomic families show similar enough growth that the Φ’ of an unstudied species could be estimated relatively well from highly-studied species within the family. A similar pattern, i.e. the growth patterns of species within a family being more similar than species in different families, has been noted for the life history ratios M/k and CW50/CW∞ in 123 species of tropical fish (Hordyk et al. 2015a, Prince et al. 2015a, 2015b; Section 1.2.4.3).

1.2.4.2. Mortality

Mortality is represented by the total (Z), natural (M), and fishing (F) mortality rates with Z equal to the sum of M and F (Z = M + F). Total mortalities are estimated by either tag and recapture studies or from length frequency histograms of a population–the rate at which frequency declines after peak recruitment (the length class of highest frequency). The natural mortality of crabs is most commonly estimated using the FISAT program (either FISAT I or II), which is based on Pauly’s M equation (Pauly 1980), and takes into account biological parameters and mean water temperature. Pauly’s M equation is:

log (M) = -0.0066 – 0.279 log L∞ + 0.6543 log (K) + 0.4634 log (T)

where L∞ (CW∞ in crabs) and K are the VBGF parameters and T is the mean annual water temperature (oC)

28 Fishing mortality can then be calculated from Z = M + F. The annual mortality proportion (A) can be calculated by:

A = 1 - e-x where x is one of the three mortality rates listed above.

The natural mortality (M) of portunid crabs is high, with the highest (2.19 year-1) and lowest (0.86 year-1) mean of estimates and the overall mean of estimates of M (1.55 ± 0.33 year-1) equivalent to A values of 0.888, 0.577 and 0.788 year -1, respectively. The overall mean F is

2.57 ± 1.38, while that of Z is 4.12 ± 1.37 (Table 1.6). The overall coefficient of variation (CV) of M (21.3 %) is notably lower than of F (53.7 %), and therefore Z (33.3 %), likely because of the large variation in fishing pressures exerted on different populations. The rates for Z and F are higher for males than females for almost all species for which estimates of both species are available (Table 1.6). This gender discrepancy in F has been noted previously (Jose and Menon 2007, Sawusdee and Songrak 2009, Safaie et al. 2013a) and may be caused by male-biased harvest strategies designed to protect spawning females (Pardo et al. 2015).

The exploitation rate (E) of a stock can be estimated by F/Z (Hilborn and Walters 1992, Safaie et al. 2013a, López-Martínez et al. 2014, Abdul and Wardiatno 2015). The overall mean exploitation rate for portunid crabs is 0.57 ± 0.15 (CV = 26.3 %; Table 1.6), which indicates that, on average, more adult crabs are captured by fisheries each year than die of natural causes. Portunus species appear to be the most heavily exploited, with male P. trituberculatus having the highest E at 0.82. The coefficient of variation of E for P. pelagicus (= P. pelagicus) and P. reticulatus (26.2 and 21.4 % for males and 12.7 and 30.2 % for females of each species, respectively), the only species it was possible to calculate for, are similar in magnitude to that for the overall mean (26.3 %), i.e. E varies as much between populations of these two species as it does between all the species listed in Table 1.6.

29 Table 1.6. Average natural (M), fishing (F) and total (Z) mortality rates (year-1) and the exploitation rate (E = F / Z) for portunid species in the genera Portunus, Callinectes and Scylla. The mean and range (line below) are given for each species when two or more estimates are available, and standard deviation (± SD) when three or more are available. An overall mean of each parameter and means for male and female crabs independently, where possible, are given for species in each genera and the Portuninae on a whole.

Taxa Gender Mortality Exploitation

Natural (M) Fishing (F) Total (Z) Rate (E)

Portunus Overall 1.58 ± 0.35 3.13 ± 1.20 4.68 ± 1.21 0.62 ± 0.09 Male 1.69 ± 0.41 3.53 ± 1.46 5.20 ± 1.36 0.63 ± 0.11 Female 1.48 ± 0.27 2.72 ± 0.83 4.16 ± 0.87 0.62 ± 0.09

P. pelagicus1,2,3,4 Male 2.03 ± 1.32 3.74 ± 3.00 5.68 ± 3.51 0.61 ± 0.16 1.09 – 3.98 1.09 – 7.62 2.80 – 9.50 0.43 – 0.83 Female 1.45 ± 0.52 4.04 ± 2.52 5.49 ± 2.89 0.71 ± 0.09 0.86 – 2.07 1.95 – 7.24 2.95 – 8.85 0.61 – 0.82 P. reticulatus5,6,7 Male 2.19 ± 0.44 3.06 ± 1.13 5.25 ± 0.86 0.56 ± 0.12 1.70 – 2.76 1.78 – 4.10 4.54 – 6.30 0.39 – 0.65 Female 1.83 ± 0.38 2.46 ± 1.45 4.29 ± 1.58 0.53 ± 0.16 1.41 – 2.20 0.92 – 4.10 3.03 – 6.30 0.30 – 0.65 P. sanguinolentus6 Male 1.63 2.06 3.68 0.55 1.60 – 1.65 1.51 – 2.60 3.16 – 4.20 0.48 – 0.62 Female 1.65 2.13 3.64 0.53 1.50 – 1.8 1.86 – 2.40 3.37 – 3.90 0.55 – 0.62 P. segnis7,8 Male 1.34 2.89 4.23 0.63 1.21 – 1.47 1.27 – 4.50 2.48 – 5.97 0.51 – 0.75 Female 1.28 1.98 3.19 0.59 1.13 – 1.42 1.31 – 2.50 2.44 – 3.94 0.47 – 0.63 P. triterberculatus9 Male 1.26 5.92 7.18 0.82 – 5.83 – 6.01 7.09 – 7.27 0.82 – 0.83 Female 1.17 3.00 4.17 0.70 – 1.82 – 4.17 2.99 – 5.34 0.61 – 0.78 Callinectes Overall 1.57 ± 0.33 0.95 ± 0.41 2.60 ± 0.17 0.37 ± 0.16 C. arcuatus10,11 Comb. 1.41 1.29 2.70 0.48 1.32 – 1.50 1.17 – 1.40 2.49 – 2.90 0.47 – 0.48 C. bellicosus10 Comb. 1.95 0.50 2.70 0.19

C. toxotes10 Comb. 1.35 1.05 2.40 0.44

Scylla Overall 1.21 1.83 3.04 0.60 S. serrata12 Comb. 1.21 1.83 3.04 0.60

Portuninae Overall 1.55 ± 0.33 2.57 ± 1.38 4.12 ± 1.37 0.57 ± 0.15 Male 1.69 ± 0.41 3.53 ± 1.46 5.20 ± 1.36 0.63 ± 0.11 Female 1.48 ± 0.27 2.72 ± 0.83 4.16 ± 0.87 0.62 ± 0.09

1Abdul and Wardiatno (2015); 2Ishan et al. (2014); 3Kunsook et al. (2014b); 4Sawusdee and Songrak (2009); 5Dinshbabu et al. (2008); 6Sukumaran and Neelakantan (1996a); 7Kamrani et al. (2010); 8Safaie et al. (2013a); 9Sugilar et al. (2012); 10López-Martínez et al. (2014); 11Fischer and Wolff (2006); 12Knuckey (1999).

30 1.2.4.3. Beverton-Holt Life history ratios

The life history ratios M/k and CW50/CW∞ were first described by Beverton and Holt (1959) and recently have received greater attention because of the demonstration that they can be used to predict the unfished size distribution of a fish stock and in estimating the spawning potential ratio from length measurements (Hordyk et al. 2015a, Hordyk et al. 2015b, Prince et al. 2015a, Prince et al. 2015b). Jensen (1996) quantitatively determined that values of

M/k = 1.5, CW50/CW∞ = 0.66 optimised the trade-off between reproduction and survival for a number of species. Subsequently, 1.5 and 0.66 have been referred to as the life-history invariants and the ratio of M/k = 1.5 has been used to estimate M, when no other estimates of M are available (Pauly 1980, Prince et al. 2015a). Recently, a meta-analysis of growth and reproductive parameters for 123 species from a wide range of taxa found that the ratios for

M/k and CW50/CW∞ vary widely from the life-history invariants, but that they can be categorised into three major groups (i) M/k > 1.3, i.e., fast growing, early maturing species with populations characterised by an abundance of immature individuals and few older, mature individuals; (ii) M/k ranges from 0.8 to 1.3, i.e., populations with an even distribution of small and large individuals; and (iii) M/k < 0.8, i.e., populations with a greater number of larger than smaller individuals (Hordyk et al. 2015a, Prince et al. 2015a).

The life history ratio M/k showed wide variation among the portunid species (Table 1.7), with an overall mean M/k of 1.38 ± 0.31 (CV = 22.5%). However, the M/k values appear to fall into two groups: values of ~1 for S. serrata and P. segnis (= P. pelagicus) and female P. pelagicus; and values of ~ 1.5 for all other species (Table 1.7), suggesting that portunid crabs utilise two possible life history strategies. The relative variation in M/k ratios in the Portuninae sub-family (1.38 ± 0.38) is comparable to those of the teleost families Lutjanidae (0.41 ± 0.14), Lethrinidae (0.62 ± 0.23), Plectropomidae (0.91 ± 0.21) and Scaridae (0.62 ±

0.34; (Prince et al. 2015b). It was only possible to estimate the standard deviation of M/k for P. pelagicus and P. reticulatus (= P. pelagicus), and both were comparable to the standard deviation of the overall mean of the sub-family (Table 1.7). Pauly (1980) considers these ratios to be greatly influenced by environmental conditions, which may be the reason that

31 the variation within species and for the Portuninae on a whole are comparable, i.e. M/k is as dependent on the environment conditions present as it is on the species in question. If this is the case, determining the M/k of a population relies as much on knowledge of the environmental conditions as it does on knowledge of the species biology.

The variation of CW50/CW∞ is notably less than that for M/k, with an overall mean of 0.62 ± 0.07 (Table 1.7) and the genera and species do not seem to fall into separate groupings.

Conversely, the CW50/CW∞ is similar to M/k in that it is comparable with estimates for lutjanids (0.75 ± 0.17), lethrinids (0.70 ± 0.11), plectropomids (0.59 ± 0.13) and scarines

(0.71 ± 0.09; Prince et al. 2015b).

The comparability in the life history ratios M/k and CW50/CW∞ of portunid crabs offers the potential to assume values of M/k and CW50/CW∞ for portunid species where data are not currently available. Casein these circumstances, it is possible to use the information from other species or locations as a starting point to estimate the spawning potential ratio (SPR; a commonly used parameter in fisheries assessments) from length-frequency data using length-based method developed by Hordyk et al. (2015a), (2015b) – see also Prince et al.

(2015a), (2015b).

32 Table 1.7. The Beverton-Holt life history ratios M/k and CW50/CW∞ for species in the Portunus, Callinectes and Scylla genera and the overall means for genera, sexes and the Portuninae subfamily. Mean ± standard deviation (SD) of each ratio are calculated only from studies that estimated both parameters in the ratio.

Taxa Gender M/k CW50/CW∞

mean ± SD range mean ± SD range

Portunus Overall 1.38 ± 0.33 0.59 ± 0.04 Male 1.40 ± 0.38 0.57 ± 0.06 Female 1.36 ± 0.38 0.61 ± 0.04 P. armatus1,2 Male 0.63* 0.58 – 0.63* Female 0.67* 0.57 – 0.77* P. pelagicus3,4,5,6 Male 1.22 ± 0.16 1.17 – 1.45 0.55 – Female 1.03 ± 0.58 0.45 – 1.83 0.60 0.57 – 0.63 P. reticulatus7 Male 1.61 ± 0.19 1.42 – 1.87 Female 1.57 ± 0.27 1.17 – 1.74 0.57 – P. sanguinolentus8 Male 1.90 – 0.52 – Female 1.86 – 0.61 ± 0.09 0.52 – 0.70 P. segnis9,10 Male 0.94 0.86 – 1.01 Female 0.96 0.89 – 1.03 P. trituberculatus11,12 Male 1.34 – Female 1.39 – 0.59 –

Callinectes Overall 1.50 ± 0.00 0.66 Female - - 0.65 C. arcuatus13,14 Comb. 1.49 1.48 – 1.50 0.66 – C. bellicosus14 Comb. 1.50 – C. sapidus15 Female 0.65 – C. toxotes14 Comb. 1.50 – Scylla Overall 1.06 0.74 Male - 0.77 Female - 0.70 S. serrata16 male – 0.77 – 1.06 Female 0.70 –

Portuninae Overall 1.38 ± 0.31 0.63 ± 0.07 Male 1.40 ± 0.37 0.62 ± 0.11 Female 1.36 ± 0.38 0.63 ± 0.05 * Data has been combined from two concurrent studies based on the same sampled data. 1de Lestang et al. (2003b); 2de Lestang et al. (2003c); 3Abdul and Wardiatno (2015); 4Ishan et al. (2014); 5Kunsook et al. (2014b); 6Sawusdee and Songrak (2009); 7Dinshbabu et al. (2008); 8Sukumaran and Neelakantan (1996a); 9Sukumaran and Neelakantan (1997); 10Kamrani et al. (2010);11Safaie et al. (2013a); 12Sugilar et al. (2012); 13Oh (2011); 14Fischer and Wolff (2006); 15López-Martínez et al. (2014); 16Sumer et al. (2013)17Knuckey (1999).

33 1.2.5. Diet

Portunid crabs are opportunistic and feed predominantly after dusk (Paul 1981, Stoner and Buchanan 1990, Reigada and Negreiros-Fransozo 2001, Branco et al. 2002). The main prey items include sessile and slow moving invertebrates, particularly shelled molluscs (i.e. bivalves and gastropods) and crustaceans, along with teleosts (Edgar 1990, Kunsook et al. 2014a, Hajisamae et al. 2015). Studies of stomach contents have found that portunid diet is influenced by ontogeny (Stoner and Buchanan 1990, Hsueh et al. 1992, Mascaro et al. 2003, Jose 2011), moult stage (Williams 1982, de Lestang et al. 2000), habitat and season

(Laughlin 1982, Rosas et al. 1994). But that few differences have been found between the diet of males and females when both sexes co-occur in the same habitat (Laughlin 1982, Hsueh et al. 1992, de Lestang et al. 2000, Jose 2011, Kunsook et al. 2014a). Differences in the diet of males and females, when present, are likely because of variations in the distribution of each gender (i.e. genders are not sympatric), which is likely to affect the availability and abundance of prey.

1.2.5.1. Dietary composition

Compared with research on growth and reproduction, relatively few studies were found on the feeding of species in the Portuninae, and only six of these contained volumetric analyses. The diets of two Necora (Polybidae) species, previously considered as members of the Portuninae (Laughlin 1982, Choy 1986) were summarised, together with six portunids (Table 1.8). The sampling locations of each study, and therefore species, are broadly grouped as marine or estuarine, with estuaries defined loosely as a body of water that experiences notable variations in salinity from that of marine waters because of freshwater discharge or through becoming hypersaline because of the closure of the connection with the sea (Tweedley et al. 2016b).

On average, shelled molluscs, teleosts and crustaceans combined make up over 60% of volume of the diet of Portunids, with bivalves typically contributing significantly more than

34 gastropods to the overall diet (Table 1.8). Teleosts were the third most voluminous category and are eaten predominantly by larger crabs (Hsueh et al. 1992, de Lestang et al. 2000, Kunsook et al. 2014a). Among the crustacean prey, brachyuran crabs, including conspecifics, represent the majority of the volume of this prey group, with notable contributions made by penaeids, shrimp, hermit crabs and amphipods (Stoner and Buchanan 1990, de Lestang et al. 2000). Large amounts of non-identifiable organic matter (NIOM) are present in most species because of the extensive mastication of food by the maxillipeds and mandibles during ingestion, even though the stomach contents analysed were from the foregut and hence are not subject to grinding by the gastric mill. Rubble and plant matter are found in various quantities, and are likely linked to the habitat in which the individuals are feeding, i.e., seagrass or sandy vegetated substrate.

The average volumetric dietary data for each of the eight species, representing the genera, Callinectes, Portunus and Necora, were collated from the nine published studies was analysed using multivariate methods to investigate variation in the composition of the diets among genera. No dietary information was available for species of Scylla. The data were square-root transformed and used to create a Bray-Curtis resemblance matrix, which was subjected to Analysis of Similarities (ANOSIM; Clarke 2014) to determine whether dietary compositions varied amongst the three genera (Callinectes, Portunus and Necora). Non- metric Multi-Dimensional Scaling (nMDS) ordination and shade plots (Clarke 2014, Clarke et al. 2014) were used to examine visually any differences in dietary composition and determine the causes for those differences.

Diets differed significantly among genera (P = 0.1 %), with the magnitude of these differences being moderate (R = 0.54). The greatest pairwise difference in dietary compositions was between Callinectes and Portunus (R = 0.69; P = 0.1 %), with the points representing Callinectes forming a tight cluster in the middle of the ordination plot and well separated from the Portunus samples at the extremities of the top, right and bottom of the plot (Fig. 1.2). A significant difference was also detected between Callinectes and Necora (R = 0.63, P = 0.1 %) with two distinct clusters for each genus (Fig. 1.2). The diets of the

35 three studies of species in the genus Portunus were much more widely dispersed than those for the other two genera, with those in the Callinectes showing least dispersion (Fig. 1.2). The shade plot illustrates that Callinectes species consume, on average, larger quantities of brachyurans, bivalves and teleosts than species in the Portunus and Necora genera, and consequently lower volumes of other crustaceans, other organics and polychaetes (Fig. 1.3). No significant difference was detected in the diets of Portunus and Necora species (R = 0.11, P = 0.400), with both genera feeding on other organics, polychaetes and teleosts (Fig. 1.3).

The diet of P. armatus1 (armatus 1 in Fig. 1.2; de Lestang et al. 2000) was studied in the

Peel-Harvey Estuary in south-western Australia and is greatly dispersed from the diet of the other both P. armatus2 and P. pelagicus3 (Fig. 1.2), the studies of which were both carried out in marine waters (Edgar 1990, Kunsook et al. 2014a). Differences in the availability of prey between marine and estuarine environments, such as the benthic macroinvertebrate, are likely to affect the diet of portunids and may be the cause of the wide dispersino in the diets of the studies on Portunus species (Paul 1981, Williams 1981, de Lestang et al. 2000, Tweedley et al. 2015) Fig. 1.2).

36 Table 1.8. Percenatge dietary composition by volume across all crab sizes for species in the Callinectes and Portunus (Portunidae), and Necora (Polybidae) generas and the number of individual crabs (n) each study was based on. Shaded plots represent the combined percentage volume of the relevant taxa.

Portunus Callinectes Necora 1 2 3 4 5 6 7 7 8 8 9 armatus * armatus * pelagicus arcuatus ornatus sapidus sapidus similis holsatus puber * puber *

Habitat Estuarine Marine Marine Estuarine Marine Estuarine Estuarine Estuarine Marine Marine Marine Taxa n 464 103 262 533 189 3131 21 66 458 1096 396 Mean

Crustaceans 31.0 4.9 21.2 21.1 42.4 30.4 19.8 9.6 40.4 34.8 14.4 24.5

Brachyura 15.2 20.7 21.8 19.8 9.4 7.5 26.1 11.0

Other Decapods 5.0 5.6 18.9 4.9 22.8 5.2 0.2 Other Crustaceans 26.0 0.3 2.8 3.7 10.1 8.7 4.7

Molluscs 25.0 31.3 18.2 27.9 9.8 40.4 21.4 25.9 11.3 9.3 19.2 21.8

Bivalves 21.3 26.9 8.4 35.6 20.3 24.6 10.8 8.3 16.3 15.7

Gastropods 3.7 1.0 1.4 4.8 1.1 1.3 0.5 1.0 2.9 1.6

Teleosts 5.4 29.6 16.5 22.4 11.9 52.2 48.6 10.2 0.2 2.1 18.1

Polychaetes 22.6 15.0 1.8 4.4 0.5 4.5 1.8 32.8 49.8 12.1

Plant Matter 5.1 10.8 3.2 2.9 4.3 6.0 2.7 0.1 1.0 3.3

Echinoderms 0.5 2.5 2.1 0.5

Other Organics 0.3 37.4 23.6 14.5 5.1 9.0 5.7 8.3 21.8 13.5 11. 33.6 Rubble 10.1 4.2 13.8 9.1 1.7 0.9 4 5.7

Total % 100.0 99.4 100.0 98.5 100.0 99.9 100.0 96.9 100.0 99.0 100.0 99.4

*Indicates that data was manipulated, either re-digitised from figures or combined from multiple tables (e.g. from tables of diet of mature and immature individuals into overall diet). 1de Lestang et al. (2000); 2Edgar (1990); 3Kunsook et al. (2014a); 4Paul (1981); 5Branco et al. (2002), 6Laughlin (1982); 7Hsueh et al. (1992); 8Choy (1986); 9Norman and Jones (1991).

37

Fig. 1.2. A non-metric multi-dimensional scaling ordination plot constructed from a Bray-Curtis resemblance matrix employing the square-root transformed percentage volume of the various dietary items in the stomachs of the eight species (from nine studies) listed in Table 1.8 and grouped by genus (Portunus, Callinectes, Necora). Note that some species have more than one estimate of their diet. Numbers refer to the references listed in Table 1.8

Fig. 1.3. Shade plot of the volumetric contribution of each prey item to the averaged diets of Callinectes, Portunus and Necora species. Grey scale represents the averaged square-root transformed percentage volumetric contribution of each prey item to the diets of each genera, with darker shades indicating a greater intake than lighter shades.

38 1.2.5.2. Factors influencing dietary composition

Ontogenetic shifts in diet commonly occur in the portunids (Stoner and Buchanan 1990, de Lestang et al. 2000, Mascaro et al. 2003, Jose 2011), even though their mouth parts are relatively small, even in large crabs. Size-related changes in diet are therefore due to the ability of larger individuals to consume larger and more mobile prey, e.g.teleosts, bivalves and large crustaceans, which would provide more sustenance than amphipods, gastropods and detritus, which typify the diet in smaller individuals (Stoner and Buchanan 1990, Hsueh et al. 1992, de Lestang et al. 2000, Kunsook et al. 2014a).

Habitat also influences the diet of portunids (Kunsook et al. 2014a), while season appears to influence the diet of some portunid populations (Paul 1981, Laughlin 1982, Rosas et al. 1994), but not all (Hajisamae et al. 2015). However, it is important to note that such variations in diet between habitats and seasons are likely to reflect changes in prey availability, rather than changes in feeding behaviour or prey selection (Paul 1981, Williams 1982, Wear and Haddon 1987, Norman and Jones 1991, de Lestang et al. 2000). The magnitude of the influence of season on diet varies, depending on the extent of seasonal variation in the faunal composition of the habitat studied, which can be enhanced by the seasonal movement of crabs.

Moult and ontogenetic stage are known to influence the habitat selection by crabs (Hines et al. 1987, Shirley et al. 1990, Wolcott and Hines 1990, Wrona 2004), and therefore may indirectly affect the diet of a species because of spatial differences in prey availability. However, studies of the diets of P. armatus (= P. pelagicus) (Williams 1982, de Lestang et al. 2000), P. pelagicus (Hajisamae et al. 2015) and N. puber (Norman and Jones 1991) show that diet is directly influenced by moult stage. For example, recently moulted (partially calcified)

P. armatus in the Peel-Harvey Estuary, south-western Australia, ingest much greater volumes of calcareous material, such as weathered bivalve and gastropod shell fragments, than intermoult specimens in that same system (de Lestang et al. 2000). However, the dominance of calcareous material was less evident in recently moulted N. puber (Norman

39 and Jones 1991). Pre-moult P. armatus had a lower stomach fullness than other moult stages, possibly due to the cessation of feeding part way through this stage (i.e. in D2; Section 2.3.4), and a higher frequency (> 50%) of occurrence of calcareous material than intermoult crabs (de Lestang et al. 2000). In the same study, de Lestang et al. (2000) found prey size to increase with crab size (ontogenetic stage), with more mobile prey also being consumed by larger crabs.

1.3. Behaviour

Studies of the behaviour of portunids have typically focused on large and commercially valuable species. This section describes the circadian and circatidal rhythms seen in portunid behaviour, particularly C. sapidus and the P. pelagicus complex, before summarising their burying, swimming and moulting behaviour.

1.3.1. Rhythms in behaviour

The prominent rhythms of behaviour in portunids (and most decapods) are circadian over a 24 h cycle. However, many estuarine species also exhibit circatidal rhythms in their movement, utilising the tidal regime that suits their direction of travel (Cronin and Forward 1979, Tankersely and Forward 1993, Forward et al. 1997, Darnell et al. 2012).

Portunid crabs are most active at night when actively searching for prey, though there are exceptions to this, such as the Mangrove Swimming Crab Thalamita crenata

(Stoner and Buchanan 1990, Cannicci et al. 1996). During the day, many portunids bury themselves in the substratum (Bellwood 2002). This is thought to reduce predation risk, conserve energy and avoid adverse environmental conditions, e.g.temperature and risk of desiccation (Barshaw and Able 1990, Bellwood 2002, Mcgaw 2005, Kaufman 2014).

40 Whether feeding and burying are night and daytime activities or vice versa, circadian patterns in activity levels are present in many species.

Many estuarine species, like C. sapidus, use selective tidal-stream transport (STST) as megalopa to reach suitable habitat upstream (flood-tide) and, in mature female crabs, to migrate downstream during the breeding season (ebb-tide) (Cronin and Forward 1979, Tankersely and Forward 1993, Forward et al. 2003, Darnell et al. 2012; Section 1.3.3). Both forms of STST, ebb-tide and flood-tide, are circatidal in nature, with crabs moving into the water column when the tide is appropriate for their intended movement is flowing, i.e. ebb- tide for downstream movement and flood-tide for upstream. The cues for this cycle are likely to be changes in water temperature and salinity (Darnell et al. 2012). For example, an individual moving downstream would enter the water column when reductions in temperature and salinity are detected, while one moving upstream would leave the water column and shelter in the benthos during these conditions. It is important to note that circadian influences are present in circatidal rhythms and vice versa. For example, light inhibits swimming in C. sapidus, so only the night time ebb or flood tides are utilised in STST (Forward et al. 2003), also known as nocturnal edd/flood tide transport (Tweedley et al.

2016b).

1.3.2. Burying

Many Brachyuran families, including the Portunidae, show burying, or back-covering, behaviour (Bellwood 2002). Burying does not result in the formation of a true burrow, tunnel or cavity, and thus is faster than burrowing. Typically, crabs remain buried for shorter periods of time than burrowing brachyurans, and sediment is in contact with the majority of the shell (Warner 1977). Portunid crabs bury when not feeding, which is generally during the day. The features of their external morphology that enable burying are similar to those that enable swimming, however, additional respiratory adaptations are required to stay

41 buried for long periods (Warner 1977, McLay and Osborne 1985, Barshaw and Able 1990,

Mcgaw 2005).

Portunid crabs bury for a number of reasons. Firstly, it is thought to reduce predation, especially in juveniles and smaller species (Bellwood 2002, Mcgaw 2005, Nye 2010). (Barshaw and Able 1990) experimentally showed that the success of avoiding predators is related directly to burial depth. Secondly, true burrows are known to protect their inhabitants from adverse environmental conditions, especially in intertidal species at low tide (Bellwood 2002), and burying may do the same as both influence the flow of water over an individual (Rebach 1974, Sumpton and Smith 1990, Hines et al. 1995, Kaufman 2014). Lastly, it has been suggested that burying is a method to reduce metabolic demands during periods of inactivity (Bellwood 2002, Mcgaw 2005).

Four main burying techniques are employed by Brachyurans (Bellwood 2002), all of which incorporate a highly coordinated routine of pereiopod movement that change with varying sediment firmness (Caine 1974, Faulkes and Paul 1997, Mcgaw 2005). Bellwood (2002) describes these techniques as: (i) forward movement of the chela and rear movement of the carapace to create a depression; (ii) movement of the pereiopods to loosen sand and pull the carapace downwards; (iii) scooping and or pushing of sand over the carapace by the chela; and (iv) repeated slamming of the carapace against the substrate to pump water and sand out through gaps in the pereiopods. Two studies have documented changes in behaviour and reduced success in burying by crabs parasitised by rhizocephalan parasites (Innocenti et al. 1998, Mouritsen and Jensen 2006).

A smooth, dorsal-ventrally flattened carapace and flattened pereiopods, common morphologies of portunid crabs, are useful in reducing the friction when in contact with substrate, thus enabling more efficient movement into the substrate (McLay and Osborne 1985). These morphological features align so closely with features that make portunids efficient swimmers, that swimming behaviour is suggested to have evolved from crabs that bury (Stephenson 1962, Warner 1977). Burying also requires adaptations to overcome the

42 problem of respiring when surrounded by substrate. This is achieved by reversing the normal flow of water over the gill surfaces, i.e. water enters pre-branchial vents on the anterior surface of the carapace and leaves from base of the pereiopods during reverse- flow, so that the chela, maxillipeds and antennae are able to filter out sand and particulate matter to allow respiration to continue unimpeded while buried (Taylor 1984, Barshaw and Able 1990, Davidson and Taylor 1995, Bellwood 2002).

1.3.3. Swimming and migration

Many families of brachyuran crabs have the ability to swim, with portunid and polybiid crabs being regarded as the most powerful and efficient swimmers in the brachyurans (Allen 1968, Hartnoll 1971). Pre-settled megalopa stage crabs are less capable swimmers than post-larval crabs, but are still able to influence their position in the water column to facilitate large-scale movements by STST. Post-larval crabs are capable of fast, directional movement when swimming sideways, but can also move vertically in the water column without horizontal movement and hover to maintain their vertical position (Plotnick 1985).

The propulsion force during swimming is generated by highly coordinated and alternating movement of the fourth pair of pereiopods (Spirito 1972, White and Spirito 1973), which are highly adapted for swimming (Hartnoll 1971, Hardy et al. 2010). Swimming facilitates migrations to spawning grounds (Hyland et al. 1984, Sumpton et al. 1989, Potter and de Lestang 2000, Aguilar et al. 2005), small-scale movements between habitats and also plays a role in feeding, when searching for prey and during the capture of mobile prey (Edgar 1990, Wolcott and Hines 1990, Wrona 2004; Sections 2.2.2 and 2.5).

The swimming action of post-larval portunid crabs has been described by Spirito

(1972) and Plotnick (1985) through the analysis of high-speed films. During sideways swimming, the chela and pereiopods one to four on the trailing side are fully extended (Fig. 1.4a; b). The chela on the leading side is tucked neatly into the anterior of the carapace, while pereiopods 1, 2 and 3 beat rhythmically, as if walking. The fourth pair of pereiopods

43 beat synchronously at a rate of ~ 4 s-1, approximately twice that of pereiopods 1 to 3 on the leading side (Fig. 1.4c). More detailed accounts of the sideways swimming action have been developed by Hartnoll (1971) and Spirito (1972), while Plotnick (1985) calculated the hydrodynamic forces acting during this motion. The swimming action of megalopa has not been described.

a) c)

b)

Fig. 1.4. The position of the chela and pereiopods 1 to 4 during sideways swimming from an anterior (a) and posterior (b) view, and the movement of the fourth pereiopod during this type of swimming, (c). Large arrows indicate the direction of travel. A and B sourced from Spirito (1972) and C from Plotnick (1985).

Swimming in portunid crabs is very efficient because of a suite of morphological adaptations, namely: (i) the carapace is wider than long, dorso-ventrally compressed and has a short anterior section of the thorax, compared to the posterior section (White and Spirito 1973, Plotnick 1985). (ii) The fourth pereiopod, especially the two most distal segments, i.e. the dactylus and propodus, is flattened into a paddle shape (pereiopods 1-3 show this to a lesser extent in some species)(Stephenson 1962, Hartnoll 1971, Plotnick 1985). (iii) The angle of the thorax-coxa joint of the fourth pereiopod (between the base of the appendage and the thorax) is re-aligned nearly 90o to facilitate movement in the horizontal plain rather than vertical and the coxa-basi-ischium joint (between the first two

44 segments of the appendage) of this same appendage is re-aligned nearly 90o to facilitate movement near vertical rather plain than horizontal; and (iv) the partitioning of the basiichiopodite levitator muscle (moves the fourth pereiopod) into fast, slow and moderate twitch fibre sections that facilitate rapid swimming, holding the appendage at a set angle for long durations, and slow swimming and walking respectively (Hoyle and Burrows 1973, White and Spirito 1973, Hardy et al. 2010).

1.3.3.1. Migrations for reproduction

The migration of females from estuaries to adjacent coastal waters to release fertilized eggs occurs in populations of C. sapidus (Aguilar et al. 2005) and P. armatus (= P. pelagicus) (Potter and de Lestang 2000). The distances travelled are proportional to the size of the estuary inhabited, with crabs in larger estuaries traveling further to reach marine waters. Similarly, populations of P. sanguinolentus, which live in coastal waters, and S. serrata, which inhabit mangrove systems, migrate to offshore marine waters to reproduce (Hyland et al. 1984, Sumpton et al. 1989, Hill 1994, Loneragan and Bunn 1999, Koolkalya et al. 2006).

This movement of S. serrata is stimulated by rainfall and freshwater flow (Loneragan and Bunn 1999, Koolkalya et al. 2006). It has also been suggested that P. pelagicus in Indonesian coastal waters migrates offshore to reproduce (Zairion et al. 2015b). However, it should be noted that these are not spawning migrations because spawning in crustaceans is the fertilization and extrusion of eggs onto the pleopods, where the eggs are tended to and continue to develop until they are released during reproduction (Warner 1977, Zairion et al. 2015b). Whether from estuaries or from coastal to offshore waters, these migrations follow a similar pattern in that mature females mate, begin their migration, fertilize and extrude their eggs onto their pleopods mid-migration and finally release their hatching larvae once their destination is reached. A detailed account of the spawning migration of C. sapidus is described below, which can take more than one year as distances travelled are large.

45 Mature female C. sapidus migrate from low salinity tributaries in upper Chesapeake Bay to coastal waters to release their eggs. The distances travelled often exceed 100 km and are commonly over 200 km (Turner et al. 2003, Aguilar et al. 2005), with net migratory speeds estimated to be ~ 5 km/day (Carr et al. 2004). The onset of the migration occurs from summer to autumn. Individuals that delay their migration and stay in the productive low salinity regions for longer are able to accrue larger energy reserves that boost egg production. However, if they delay their migration for too long, they may need to overwinter in deep waters mid-migration and reproduce in the following year, which can reduce sperm viability (Hines et al. 1995, Aguilar et al. 2005, Kaufman 2014). The C. sapidus migration is two-phased, even if completed in one season. After mating and successful insemination, mature females migrate downstream towards the mouth of the estuary, by either swimming, walking on the bottom or using ebb-tide transport (Turner et al. 2003, Carr et al. 2004). Once ovigerous, which may not be until the following spawning season, ebb-tide transport facilitates the second phase of migration into coastal waters where the eggs hatch and larvae leave the females (Forward et al. 2003, Darnell et al. 2012). The eggs hatch in summer either 12 or 18 months after the start of the migration, depending on whether the migration commences in summer or autumn, with peak hatching in late July (McConaugha et al. 1983).

1.3.4. Moulting

Portunid crabs, like all arthropods, are encased in a rigid exoskeleton that offers strength, mobility and protection from predators (Chang 1995). However, this needs to be shed regularly to allow for increases in growth and to enable mating to occur (Section 2.2.3.3).

Growth occurs only after ecdysis, i.e., the moulting of the current exoskeleton, and is therefore a stepwise pattern. Ecdysis is only a small part of a complex process that involves numerous physiological and biochemical changes (Greenaway 1985, Lachaise et al. 1993,

46 Chang 1995), which affect many aspects of the biology of crabs, such as feeding, and movement and habitat selection (Sections 2.5 and 2.2, respectively)

The moulting process in brachyurans, and all crustaceans, is controlled by the balance of moult inducing and inhibiting hormones (Chang 1985, 1993, Chang et al. 1993). The Y-organ, located in the thorax, produces the moult inducing hormones, ecdysteroids, while the X- organ produces moult inhibiting hormones (MIH) that are stored in the sinus gland (Lachaise et al. 1993, Chang 1995). Moult inhibiting hormones control the moult cycle by regulating the activity level of the Y-organ, and therefore the production of ecdysteroids (Havens and

McConaugha 1990, Chang et al. 1993, Chang 1995). This has been tested experimentally by removing the eyestalks (i.e. ablation), where the X-organ is located; this quickly leads to the onset of pre-moult (Nakatani and Otsu 1979, Havens and McConaugha 1990). The removal of five or more walking legs also induces pre-moulting, though less rapidly than eyestalk removal. As appendages are regenerated during ecdysis, the onset of pre-moult after the removal of a number of appendages may be a physiological response to being more vulnerable.

In addition to hormones, environmental conditions, such as water temperature (Passano 1960, Hughes et al. 1972, Chittleborough 1975, Havens and McConaugha 1990), light availability (Quackenbush and Herrnkind 1983), prey availability (Chittleborough 1975) and conspecific density (Cheng and Chang 1994) also influence moulting – either the moult frequency or moult increment (the size increase after a moult) or both (Chang 1995).

1.3.4.1. Moulting process

The process of moulting in portunid crabs has not been studied in detail, so knowledge from studies of the moulting process of other brachyurans and crustaceans has been used to infer the process for portunids. Such studies have found five broad stages in the moulting process, (A) newly and (B) recently moulted, (C) intermoult, (D) pre-moult and

47 (E) ecdysis, however, each stage can be further split into sub stages, denoted by number in subscript (Warner 1977). These stages are briefly summarised below, noting the defining features and metabolic processes (Warner 1977, Greenaway 1985, Chang 1995).

After ecdysis, mineralisation of the new exoskeleton occurs in stages A, B and partway through C. Growth occurs through water absorption while the exoskeleton is still membranous in stage A1 (the first sub-stage of A). In A2 the exoskeleton is paper-like, and individuals regain the ability to walk. In stage B1 the exoskeleton has become firm, although it is deformable without cracking (the carapace compresses when pressure is applied), and upon reaching B2 feeding resumes, after pausing prior to ecdysis, as the chelipeds and other mouthparts are now rigid. During these stages, energy and mineral stores in the hepatopancreas are used in mineralisation of the exoskeleton.

Mineralisation of the exoskeleton continues into stage C1 and C2, with feeding providing the resources. By C3 the exoskeleton is complete, although the endocuticle, an inner membranous layer, is still being developed and is not completely formed until C4. This stage is the final intermoult stage and the longest of all stages. Feeding in C4 replenishes the exhausted reserves of the hepatopancreas rather than provisioning mineralisation. This stage continues until death in species that undergo a terminal moult.

In pre-moult D1, the epidermis becomes detached from the endocuticle, which is externally visible on the dactylus of the fourth pereiopod of some species, including P. armatus

(= P. pelagicus; Fig. 1.5). Activity slows and feeding stops in D2, and reabsorption of minerals from the exoskeleton begins. This reabsorption continues into D3, and is focused in certain areas in order to weaken them. The exoskeleton splits, at the pre-weakened areas, once reabsorption is completed in D4, and water up take begins. The old exoskeleton is rapidly shed during ecdysis (E), and large amounts of water are absorbed to stretch the new exoskeleton while it is still membranous.

48

Fig. 1.5. The propodus and dactylus of the fourth pereiopod (the swimmeret) of Portunus armatus in pre-moult condition, as indicated by the epidermis (the inner red line), which has separated from the endocuticle (the outer red line; photo by T. Campbell).

1.4. Influence of environmental conditions

1.4.1. Influence of water temperature and salinity

For example, higher temperatures reduce larval duration and salinity either increases or decreases the rate of development depending on the species (Costlow and Bookhout 1959, Bryars and Havenhand 2006, Nurdiani and Zeng 2007). The distribution and abundance of crabs is influenced by both water temperature and salinity, with species moving to stay within their tolerance levels as conditions change with the season, e.g.moving out of an estuary into marine waters as freshwater flow increases or into deeper waters to avoid cooler temperatures (Hill et al. 1982, Potter and de Lestang 2000, Wrona 2004, Kaufman 2014). Water temperature also affects the fecundity of females indirectly in two ways. Firstly, lower temperatures leading into the reproductive season reduces the number of batches that can be produced during that time (de Lestang et al. 2010, Johnston et al. 2011a), and, secondly, low temperatures can slow growth, leading to a smaller size being reached by the time the reproductive season occurs (Sukumaran and Neelakantan 1996b, Fisher 1999, de Lestang et al. 2003c, Kamrani et al. 2010); Section 2.2.3.2). Growth rates are also affected by low salinities because of the energy requirement for osmoregulation, which in turn can negative affect fecundity (Cadman and Weinstein 1988, Guerin and Stickle 1992).

49 Similarly, natural mortality rates (M) are affected by environmental conditions, with M directly proportional to temperature (Defeo and Cardoso 2002).

The behaviour of portunid crabs is also affected by changes in temperature and salinity. Along with light, temperature and salinity are two of the predominant cues in behavioural patterns, whether circadian or circatidal (Forward et al. 1997, Darnell et al. 2012). The avoidance of adverse environmental conditions is thought to be a reason for portunid burying behaviour (Rebach 1974, Sumpton and Smith 1990, Hines et al. 1995, Kaufman 2014) and swimming behaviour, because it facilitates the aforementioned seasonal changes in distribution and abundance. The moult frequency is thought to slow with dropping temperatures, and is the cause of the slowing of growth (Havens and McConaugha 1990).

1.4.2. Influence of environmental conditions on fisheries recruitment and production

The growth and mortality rates of portunids are influenced by environmental conditions, with increasing water temperatures tending to increase both rates, while the effects of salinity are more complex than temperature (Cadman and Weinstein 1988, Defeo and

Cardoso 2002, Nurdiani and Zeng 2007). Mortality rates during larval stages are highly vulnerable to environmental conditions. For example, a study by Bryars and Havenhand (2006) found the cumulative survival rate of P. armatus (= P. pelagicus) to the first crab stage dropped from ≈ 30 % in 25 oC to 0 % in 18 oC. As populations of crabs are adapted to their local conditions, most of the variations in environmental conditions are detrimental to the survival rate of the population, and therefore the fisheries they support. For example, events such as extreme unseasonal rainfall can lead to large mortalities in populations not adapted to freshwater influx (Harris et al. 2012).

Environmental conditions can also reduce the reproductive potential and of population, and therefore the recruitment to fisheries it supports. This can occur in three ways. Firstly, the size at which maturity is reached is reduced if adverse environmental conditions occur, such

50 as low temperatures or abnormally high or low salinities, because they can slow the growth rate of juveniles (Cadman and Weinstein 1988, Berrigan and Charnov 1994). Slower growth can lead to maturity being reached as smaller sizes, and because fecundity increases exponentially with size in most portunids (Table 1.3), even small changes in the size at which maturity is reached can have large consequences on the egg production. Secondly, increased mortality induced by adverse environmental conditions can lead to a lower female spawning stock and therefore the lower reproductive potential (Lipcius and Stockhausen 2002). Finally, lower than average water temperatures leading into the reproductive season can reduce the number of batches that large females are able to produce (de Lestang et al. 2010, Johnston et al. 2011a).

The P. armatus fishery in Cockburn Sound, Western Australia, a sheltered marine embayment of ~ 100 km2, has been operating since the late 1970’s. Annual landings were ~ 30 – 50 tonnes for the first five years, when they started increasing, and peaked in the late 1990’s at ~ 300 tonnes (de Lestang et al. 2010, Johnston et al. 2011a). Catches began to drop in the early 2000’s and, apart from a small increase from 2001/2 to 2002/3, continued to drop until 2006 when the fishery was closed due to low stock abundance (Johnston et al.

2011b). The collapse of this fishery is thought to largely be due to lower water temperatures prior to the breeding season, reducing the length or breeding season, and the number of batches of eggs that females were able to produce and, as a consequence, greatly reducing the population fecundity (Johnston et al. 2011a, 2011b; see also 2.4 above). The mean water temperature in August and September in this embayment was between ~0.6 and ~ 1.8 o C lower than average for four years (from 2002 to 2005), although within the range of normal fluctuations (de Lestang et al. 2010). The drop in reproductive potential and subsequent recruitment, due to the reduced number of batches each female was able to produce, combined with continued fishing pressure on mated pre-spawning females during winter over those four years, led the population to collapse and to the closure of the fishery (de Lestang et al. 2010, Johnston et al. 2011a).

51 1.5. Conclusion

This section summarises the biology and behaviour of portunid crabs in the sub-family Portuninae, focusing on the commercially and recreationally important species within the genera Callinectes, Portunus and Scylla. They all have two pelagic larval forms followed by benthic juvenile and adult stages. Larval durations are relatively short (1-2 months) compared to that of the juvenile (~ 1 year) and adult (1-2.5 years) stages. Growth is rapid and in general very similar for males and females until maturity is reached, when it slows, particularly in females. Natural mortalities are high, as are fishing mortalities, and overexploitation is common. The life history ratio M/k for species within the three genera combined is 1.38 ± 0.31, with estimated ratios for most species (7 of 9) ~ 1.5 and for three species ~ 1.0 (estimates of male and female P. pelagicus are ~ 1.5 and ~ 1, respectively). The size at maturity varies greatly between species, but the relative size at maturity (CW50/CW∞) is very consistent across species and occurs at about 62% of the asymptotic size, with relatively low variation among species in the three genera (coefficient of variation = 11.3%). Fecundity is high in all three genera, ranging from hundreds of thousands to millions of eggs, and increases exponentially with size. The diets of all three genera are influenced by ontogenetic and moult stages, habitat and season, although the diet of Callinectes differs from that of Portunus and Necora. Bivalves, gastropods, crustaceans and teleosts are the major prey categories consumed across all three genera

Portunids show a range of specialized behaviours including intricate mate selection, courtship and mating rituals, the utilization of different habitats at each life history stage and a circadian pattern of nocturnal activity followed by inactivity during the day, when individuals are generally buried. Species also exhibit changes in behaviour over the course of the moult cycle, with feeding behaviour and habitat selection changing markedly when vulnerable in the moult stages after ecdysis but prior to the remineralisation of the exoskeleton. Swimming facilitates a range of behaviours including feeding, habitat selection and the migration of females prior to the hatching of eggs in some species.

52 The biology and behaviour of all three genera are influenced by water temperature, salinity and fishing pressure. Growth is more rapid and constant in warmer regions, whereas populations in temperate regions exhibit seasonal growth rate, and is positively correlated with temperature. Likewise, reproduction, which occurs year round in tropical regions, is limited to warmer months in temperate locations. The reproductive output of females is reduced by increasing fishing pressure, even in male-biased harvest strategies, because of changes in mate selection, courtship and mating behaviour with low female to male ratios.

Changing environmental conditions can influence the biology and behaviour of each species differently. For example, the larval duration of S. serrata is shortened by high salinities, while that of C. sapidus is extended. Moreover, estuarine populations of P. armatus in south-western Australia migrate towards marine waters during winter when freshwater influx peaks and salinities decline, while C. sapidus populations in Chesapeake Bay are not influenced by salinity.

53

54 Section 2. Portunid fisheries and aquaculture-based enhancement

2.1. Fisheries

2.1.1. Overview of portunid fisheries

Portunids are captured in large numbers in both marine and estuarine waters tropical and temperate regions globally (Poore 2004, Johnston et al. 2014b, López-Martínez et al. 2014, CBSAC 2016), although landings are greatest in the Indo-West Pacific because of the abundance of P. trituberculatus and the P. pelagicus complex (FOA 2016a). In addition to commercial fisheries, recreational landings are significant in some areas and artisanal fishing

(cultural and subsistence fishing) occurs, particularly throughout Asia and Africa (Sumner et al. 2000, Mirera 2011, Mirera et al. 2013). Methods of capture include trawling, trapping, netting and diving, with each component of the fishery utilizing its own method(s) (Coleman et al. 2003, Johnston et al. 2014b, López-Martínez et al. 2014).

Global catches of portunid crabs are still increasing despite the relative stability in the catch rates of world fisheries (Ingles 2004, FOA 2008, 2016a, b). Portunus trituberculatus and members of the P. pelagicus complex are the two dominant groups in global landings and their annual landings rose sharply between 2002 and 2014, from 370,000 to 605,000 tonnes and from 155,000 to 216,000 tonnes, respectively (FOA 2008, 2016a). Note, it is assumed that the capture rate for P. pelagicus given above is for the species complex as a whole, as the reported global catch rate did not decline after 2010 when P. pelagicus was split into four species (Lai et al. 2010). Callinectes sapidus is also a heavily exploited species, with annual landings in the Chesapeake Bay fishery alone averaging 29,860 tonnes, worth $73 million US, between 1990 and 2014, with maximum landings of > 50,000 tonnes in 1993 (Huang et al. 2015, CBSAC 2016).

Recreational fishers do not have to record and submit the weight of their catches, and hence the capture rate and effort of the recreational component is harder to determine, and relies on boat ramp and telephone/online surveys (Sumner et al. 2000, Ryan et al. 2015). Such surveys in Western Australia have shown that recreational landings of

55 P. armatus (= P. pelagicus) make up a substantial proportion of the total catch in areas that are easily accessible to a large number of people, such as the Peel-Harvey Estuary, but in general are lower than commercial catches (Lyle et al. 2003, Johnston et al. 2014a). Artisanal fishing catch rates of mud crabs (Scylla spp.; the main target of artisanal fishing) in northern Australia are lower than recreational rates (Coleman et al. 2003, Lyle et al. 2003). However, in areas of Africa such as Kenya, a new trend of capturing small mud crabs and raising them to market size in addition to capturing wild market size crabs is increasing artisanal fishing pressure in poorly or un-regulated fisheries (Mirera 2011, Mirera et al.

2013).

Two main examples of portunid fisheries are discussed below: P. armatus in south-western Australia and C. sapidus in Chesapeake Bay, USA. These two examples were chosen because of their significance to commercial and recreational fisheries, the documented influence of environment on their biology and the extent of the available literature. Other species (e.g. P. trituberculatus) are also discussed where relevant.

The main fishing method used by commercial fisherman in Western Australia, and elsewhere, is baited traps (Johnston et al. 2011b, Anon 2016), although large numbers of crabs are caught by trawlers, whether targeted, as by-product, e.g. Shark Bay, and by gill nets, e.g.the Swan-Canning Estuary (Weng 1992, Ingles 2004, Watson et al. 2006, López- Martínez et al. 2014). Baited traps (drop- and hoop-nets) are also used by recreational fishers, but typically differ from those used by commercial fisherman (Johnston et al. 2011b, Anon 2016, Broadhurst et al. 2016). Recreational fishers, in Western Australia and elsewhere, also use a range of other methods, including scoop nets, line fishing (baited hooks) and diving (capture by hand) (Lyle et al. 2003, Johnston et al. 2014b). The same fishing methods are used by artisanal fishers but in different rates, with capture by hand being the major method (Coleman et al. 2003, Mirera 2011, Mirera et al. 2013).

Unfortunately, the high fishing pressure on many established portunid fisheries, exacerbated by environmentally driven fluctuations in recruitment, has led to population

56 collapses (Lipcius and Stockhausen 2002, Johnston et al. 2011a). Furthermore, even fisheries that began operating more recently, including many in south-east Asia, are heading in the same direction with catch and exploitation rates increasing yearly (Williams and Primavera 2001). The future sustainability of portunid fisheries requires an in-depth understanding of the biology and behaviour of the species being fished and how it is influenced by changing environmental conditions and fishing pressure.

2.1.2. Influence of fishing pressure crab stocks

Portunid crabs are heavily exploited, with fishing mortality rates (F) over double that of natural levels (M) because of high fishing pressure (Table 1.6). Fishing pressure adds additional stresses to the population and can cause growth to slow, resulting in reductions in the size at which maturity is reached, which, in turn, leads to lowered reproductive potential (Jørgensen 1990, Trippel 1995, de Lestang et al. 2003a). Fishery managers have developed management plans that aim to protect this reproductive potential, including minimum size limits set well above the size at which maturity is reached (Roberston and

Kruger 1994, Johnston et al. 2014a, Zairion et al. 2015b), restrictions on the capture of ovigerous female crabs throughout the reproductive season or entire year (Johnston et al. 2014b, Anon 2016) and male-only harvest strategies (Grubert et al. 2014). Male-biased harvest strategies and, to a lesser extent, restrictions on female capture, can affect the sex ratio of a population and reduce the median size of male crabs, which in turn can lead to reduced reproductive potential as documented for C. sapidus in location Chesapeake Bay and Metacarcinus edwardsii in Chilean waters (Jivoff 2003, Pardo et al. 2015).

Both Jivoff (2003) and Pardo et al. (2015) suggest that reductions in the median size of male crabs and increases in the female to male sex ratio affect the reproductive potential of crab populations by reducing the size of the ejaculate received by the female, relative to her size. This can occur in two ways. The first way is by females mating with smaller crabs, which produce smaller ejaculates than larger crabs. The frequency at which this occurs increases

57 with decreasing median male size, because of a lack of competition from large males, and because females are more receptive to, and less selective against small males, at high female to male ratios (Jivoff and Hines 1998). The second way of reducing the reproductive potential is by females mating with males whom have recently mated. High female to male ratios increase the frequency of mating by males, meaning there is less time between copulations to produce sperm, thus reducing the size of the next ejaculate passed to a female. Reductions in the volume of the ejaculate passed onto females can affect their reproductive output because they store sperm and fertilize a number of batches of eggs from one ejaculate and are highly fecund. This affect is compounded if small males are mating frequently with large females. Additionally, smaller males guard females for a shorter period after mating than large males, which may increase the post-coital mortality of females mating with small males (Jivoff and Hines 1998).

The distribution of fishing pressure can cause a similar effect to that of male-biased harvest strategies if one sex is more likely to be captured than the other. While this has not been studied intensively, differences in seasonal distribution of each sex, such as during a reproductive migration, could lead to such problems if fishing pressure is not evenly spread

(Johnston et al. 2014a).

In addition to this, some fishing methods cause detrimental effects to the environment. Trawling, which is widely used, is the obvious example, as it can modify the benthic habitat (Engel and Kvitek 1998, Watson et al. 2006). However, other methods cause less noticeable effects. Baited traps for example, continue to catch and kill crabs and other animals when lost or left in the water, a consequence that has been termed “ghost fishing” (Guillory 1993).

58 2.1.3. Management strategies

The management of portunid fisheries varies greatly between and within countries, depending on jurisdictional boundaries, with estuarine and coastal fisheries often controlled by state or provincial governments (Criddle 2008, Australian 2016). Most management strategies implement both input and output controls, limiting the fishing effort and the methods used, and the total landings and using size restrictions (Helser and Kahn 2001, Lipcius and Stockhausen 2002, Grubert et al. 2014, Johnston et al. 2014b). Target and limit reference points are also used in the more closely managed fisheries to assess the current condition of the fishery stock and to indicate when further management controls need to be implemented (Huang et al. 2015, CBSAC 2016). In some fisheries, stock recruitment models have been developed that predict recruitment to the fishery in the following year, and these are also used to adjust harvest strategies and management controls (Tang 1985, Bunnel and Miller 2005, de Lestang et al. 2010, Johnston et al. 2011b).

Fishing and boat licences, seasonal closures and protected areas are commonly used input controls (Lipcius et al. 2003, Johnston et al. 2011b), while minimum size limits, total allowable catches (commercial), daily bag limits (recreational) and protection of females in general or ovigerous females are commonly used output controls (Helser and Kahn 2001, Svane and Hooper 2004, Johnston et al. 2014a). However, the controls used vary between species, commercial and recreational fisheries and the regulatory body of the fishery. A clear example of this can be seen by comparing the management of S. serrata and P. armatus (= P. pelagicus) fisheries in Australia (Grubert et al. 2014, Johnston et al. 2014b). In Western Australia and New South Wales, commercial fishers are allowed to use haul and gill nets to capture P. armatus, while fishers in Queensland and South Australia are cannot use these methods, and haul nets are not permitted for the capture of S. serrata in any

Australian state.

When a fished population spans several jurisdictional boundaries, fishers in each zone can be managed in differing ways. Three jurisdictions jointly manage the Chesapeake Bay

59 C. sapidus fishery, the states of Maryland and Virginia and the Potomac River Fisheries

Commission, with an overview committee providing management advice to both states (Bunnell et al. 2010, Huang et al. 2015, CBSAC 2016). In 2001, the three jurisdiction adopted a unified management approach to reduce fishing pressure by 15 % and in doing so, keep the female abundance and exploitation rate above the newly set reference points. However, Maryland and Virginia have since modified their management plans because the desired outcome was not fully achieved (Bunnell et al. 2010). For example, in Maryland, the minimum size limit of 127 mm for male and immature females was increased to 133 mm for crabs caught a July 15 each year, while in Virginia, no change was made to this limit (Huang et al. 2015).

The implementation of additional controls is often based on the values of performance indicators and how they compare with the target and limit reference points. The C. sapidus fishery in Chesapeake Bay uses the abundance of spawning age females and the exploitation rate of females as performance indicators for the fishery (Miller 2001, Bunnell et al. 2010, CBSAC 2016). When target levels are not achieved, i.e. if the exploitation rate of female C. sapidus in Chesapeake Bay goes over 25.5 % (CBSAC 2016), additional controls are implemented. These may include bans on the removal of females or the closure of areas where females are known to congregate. Existing controls may also be made more severe, i.e. the fishing season may be shortened or next year’s total allowable catch reduced.

Spawning potential and recruitment index models are other possible indicator variables and, while data intensive (they rely on fisheries independent data), allow the inclusion of environmental variation and fishery selectivity (size, sex, moult stage etc.) to be included (Bunnel and Miller 2005, de Lestang et al. 2010). It is important to clarify that, while these indices are unbiased by commercial fishing methods because they are based on fisheries independent data (Johnston et al. 2015), they still allow information about the fishery (i.e. its selectivity) to be included. There is generally a pre-determined framework that states how the controls will be changed when the target level is breached, with more severe

60 changes to be implemented when the threshold is breached (Rochet and Trenkel 2003,

Livingston et al. 2005).

Collapses in fisheries, when they occur, require specific management strategies to recover the fishery. The use of indicator variables to identify when to bring in additional controls has already been discussed. However, indicator variables such as egg production and recruitment indices are also used in the reverse way to help identify when to re-open a fishery and decide on the permitted fishing effort or total allowable catch (Johnston et al. 2011a, Johnston et al. 2011b). The release of aquaculture raised juveniles through restocking is another method that has been trialled to reduce the recovery time of collapsed fisheries (Bell et al. 2005, Bell et al. 2006, Aguilar et al. 2008, Bell et al. 2008b, Zohar et al. 2008). Restocking is “the release of cultured juveniles into wild population(s) to restore a severely depleted spawning biomass to a level where it can once again provide regular, substantial yields” (Bell et al. 2008b). While not widely used, restocking has the potential to considerably shorten the recovery time of collapsed fisheries, especially if the fishery is recruitment limited, i.e. the population cannot recover as recruitments are continually low (Bell et al. 2006, Bell et al. 2008b, Zohar et al. 2008).

The only major restocking project to be completed on portunid crabs is that of C. sapidus in Chesapeake Bay and this was not at a commercial scale. This research aimed to assess the feasibility of using hatchery-produced juvenile crabs to enhance breeding stocks and, in turn, their overall abundance in Chesapeake Bay. The research was initiated in response to an ~ 80% decline in the breeding stock between 1992 and 2000 (Zohar et al. 2008). Nearly 300,000 juveniles were released into that estuary from 2002 to 2006, but the real the success of the project was the multi-disciplinary research that included the large scale production of juvenile crabs (Zmora et al. 2005, Zohar et al. 2008), the assessment of the hatchery-raised crabs’ fitness (Davis 2004, Young et al. 2008), the creation of a release strategy that optimised survival by determining when, where and at what size to release, and whether juvenile habitats were at carrying capacity (Hines et al. 2008b, Johnson et al. 2008, Seitz et al. 2008). This research provides a framework in which future projects can

61 operate, and gives insights into the biological, ecological, environmental and socio- economic understanding that is required for a restocking project to be successful (Williams and Primavera 2001, Zohar et al. 2008).

Another possible assessment method to inform managers of portunid fisheries is the length- based spawning potential ratio (SPR) assessment developed Hordyk et al. (2015a), (2015b) – see also Prince et al. (2015a), (2015b) – for data poor fisheries. The life history ratios M/k and CW50/CW∞ presented in Table 1.7 may be of use in estimating values of these parameters for understudied populations. If this is viable, the SPR assessment will give data poor fisheries an index around which a management strategy can be developed.

2.2. Aquaculture-based enhancement

2.2.1. Rational for release programs

Globally, landings from wild capture finfish fisheries have plateaued since the 1990s (Pauly et al. 2002), with many stocks suffering from overexploitation and the adverse effects of habitat degradation (Lorenzen 2010). Although, at the international scale, catches of portunids have not declined (see above), localised depletions have occurred. For example, reclamation of tidal flats and water pollution, together with rapid economic growth following the end of World War II, led to the exhaustion of coastal fisheries in Japan (Kitada 1999, Kitada and Kishino 2006, Hamasaki et al. 2011). These effects, coupled with an expected increase in demand for fish protein as a result of global population growth (FAO 2012), highlights the need for management intervention in order to manage existing fisheries sustainably so that fisheries yield can be stabilized, or more preferably, increased.

Management intervention can take the form of the control of fishing effort through input (e.g. spatial and temporal closures and gear restrictions) and output (e.g. size limits and catch quotas) controls. However, such regulation have the potential to place significant social and economic hardship on businesses and communities that previously had less

62 restricted access to fish stocks (Mascia et al. 2010, Bennett and Dearden 2014). An alternative intervention that is becoming increasingly popular is the use of aquaculture- based enhancement (referred to here also as release programs). This is a method that involves the release of cultured juveniles into an environment to enhance, conserve, or restore fisheries (Bell et al. 2006, Lorenzen 2008, Lorenzen 2010, Taylor et al. 2017). Release programs can be separated into three major categories (Bell et al. 2008a, Lorenzen et al. 2013).

(i) Stock enhancement, the release of hatchery seed to improve self-sustaining

populations. (ii) Restocking, the release of hatchery seed to rebuild severely depleted stocks. (iii) Sea ranching, the release of hatchery seed in-put and take operations.

If successful, aquaculture-based enhancement programs have the potential to produce significant social, economic and ecological benefits for fishery stocks and their users. Moreover, the can increase productivity beyond the level achievable by harvest management alone, create economic opportunities for fishery-livelihoods and provide incentives for active management of fisheries resources (Pinkerton 1994, Lorenzen and Garaway 1998, Lorenzen 2005).

2.2.2. Release programs for portunids

An extensive search of the literature has revealed a number of release programs designed to increase stocks of commercially important species. These include; (i) Portunus trituberculatus in Japan, (ii) Portunus pelagicus in Thailand, (iii) Scylla paramamosain in Japan, (iv) Scylla spp. in the Phillippines and (v) Callinectes spaidus in the USA. The following sections detail the rationale being the release program, the approach adopted and, where possible, with a brief summary of the results of the program and associated research. Note that some relevant aquaculture-based enhancement programs for other species, i.e. Chinese Mitten Crab (Eriocheir sinensis), Alaskan Red and Blue King Crabs (Paralithodes

63 camtschaticus and Paralithodes platypus) and the Mangrove Crab (Ucides cordatus), are included due to their scale and the extent of information available.

2.2.2.1. Portunus trituberculatus in Japan

Aquaculture-based enhancement for a variety of fish and crustacean species was initiated by the Japanese government in 1963 in response to the depletion of coastal fisheries following a period of rapid economic growth after World War Two (Kitada 1999, Hamasaki et al. 2011). Portunus trituberculatus was one of the first selected species as it is fished across the country and catches declined by 75 % from 4,267 tonnes in 1956 to 997 tonnes in 1970 (Fig. 2.1a; Hamasaki et al. 2011). Successful hatchery techniques (see Aileen et al. 2000, Ariyama 2000) were developed in the 1960s and the number of P. trituberculatus juveniles release increase rapidly from 6.3 million in 1977 to over 30 million in 1986 and remaining relatively consistent (Fig. 2.1b; Hamasaki et al. 2011).

Currently, 14 hatcheries across Japan are involved in the production of seed stock using 50,000 – 200,000 L tanks and across 330 production runs in 2007 and 2008 achieved a mean survival rate of 11.2 % (± 12 standard deviation) from larvae to the first crab instar (C1; 5 mm CW; Fig. 2.2; Hamasaki et al. 2011). The large standard deviation reflected the fact that survival was as high as 80%, but that in 20% of all culture runs it was as low as 0%. Mass mortality events are common and occur during the zoeal and megalopa stages and also during the metamorphosis between these stages (Hamasaki et al. 1997). In 62% of 174 mass mortality events studied by Hamasaki et al. (2011), the cause of the mortality was unknown, with bacterial disease (~26%), fungal disease (~8%) and moult death syndrome (~3%) being the other causes. For the first 20 years of the stock enhancement program

P. trituberculatus were cultured to the C1 stage (~40 days post hatch; Fig. 2.3), but survival in the wild was found to be very low (<1%). Thus, by the early 1990s, C1 instars were cultured and then transferred to a secondary rearing facility until they reached C4 (17 mm CW) and were released. Survival from the C1 – C4 stages ranged between 12-61% (Secor et al. 2002) and decreases exponentially with increasing size (Hamasaki et al. 2011).

64

Fig. 2.1. Annual (a) total catch of Portunus trituberculatus in Japan and (b) the number of juveniles released. Taken from Hamasaki et al. (2011).

Fig. 2.2. Frequency distribution of survival rates of Portunus trituberculatus (N – 277), Portunus pelagicus (N = 45) and Scylla paramamosain (N = 17) cultured from hatching to the first crab instar in mass seed production facilities in Japan in 2007 and 2008. Taken from Hamasaki et al. (2011).

Fig. 2.3. Flow chart of Portunus trituberculatus aquaculture in Japan. Taken from Aileen et al. (2000).

65 It is thought that releases of P. trituberculatus should occur at at least the C3 stage, as this is equivalent to the C4 stage at which the juveniles settle on to the tidal flats (Hamasaki et al. 2011). At this stage they are also less venerable to predation by fish as they are able to burry in the sandy substrate (Ariyama 2000).

The relationship between the number of juvenile P. trituberculatus released and the minimum values of catches were investigated using linear regression for the Hamana Bay fishery and in six major regional seas. Significant positive relationships were detected for Hamana Bay, Seto Inland Sea, Central Pacific and Western Sea of Japan (p = < 0.05; R2 =

0.586–0.902; Hamasaki et al. 2011). Economic efficiency values (see Kitada and Kishino 2006 for method) for some of the release programs in the regional seas, indicated that the economic benefits of the enhancement would cover the costs of juvenile production and release, although the authors noted that a more comprehensive analysis was needed (Hamasaki et al. 2011). These authors also evaluated the influence of the P. trituberculatus stock enhancement program at the national level in Japan (Fig. 2.4). The estimates varied widely among the years, typically increasing post 1987, and among the seas, being greatest in the Seton land Sea and East China Sea (31 and 30 %, respectively) and least in the Central

Pacific and Western Sea of Japan (25 and 18 %, respectively). On this basis, Hamasaki et al. (2011) concluded that the release program for P. trituberculatus at the C2 stage or greater had sustained fishery production according to the magnitude of releases and yield from the released individuals.

66

Fig. 2.4. Contribution rate of hatchery releases to annual total catch of P. trituberculatus. Rate estimated by multiplying the number of juveniles released in each year by the yield per release. Taken from Hamasaki et al. (2011).

2.2.2.2. Portunus pelagicus in Thailand

Catches of P. pelagicus in Thailand have declined by 43% between 2001 (36,305 tonnes) and 2011 (20,582); resulting in the initiation of release programs (Nitiratsuwan et al., 2014). An investigation into the effectiveness of restocking this species was undertaken in very small (0.19 km2), shallow, seagrass dominated bay in Trang Province, southern Thailand. A survey of P. pelagicus populations were undertaken prior to (i.e. a baseline) and after the release of thirty thousand crablets (22 days post hatch) in July 2013. Catch rates increased from 1 and 2 individuals per crab trap in September and October 2011, respectively to 19 and 11 individuals per trap in the same month in 2013 (Nitiratsuwan et al. 2014). Although that study did not account for inter-annual differences in abundance or mark the hatchery- reared crabs, the results provide evidence that the release program was successful.

67 An alternative and conservation focused release program, ‘Crab Bank’, has been implemented in Prachuap Khiri Khan Province (Arkronrat et al. 2013). In this initiative berried P. pelagicus caught by commercial fishers are translocated to either sea cages or a hatchery where they spawn. Post spawning, females held in sea cages are released to the wild, whereas those in the hatchery are sold at market, with a 50% proportion of the sale going to the commercial fisher and the rest to the maintenance of the hatchery (Arkronrat et al. 2013). The resultant larvae are then subsequently released into the wild. Surveys of the small-scale crab fishers operating in villages involved with the project indicated that, one year after implementation, 60% of the fishers reported an increase in catches of juvenile and immature crabs.

2.2.2.3. Scylla spp. in the Phillippines

Four species of Mud Crab (Scylla spp.) are found in shallow soft-sediment habitats in the tropics and represent an important source of income for coastal communities (Le Vay 2001). The stocks of these species are not protected and thus the commercial harvest for fishery and aquaculture (broodstock) purposed includes mature and berried females, and thus overfishing has occurred, resulting in decreased catches (Kosuge 2001). Attempts to aquaculture Scylla spp. started in the late 1990s, but a lack of hatchery production technology hindered yields (Le Vay et al. 2008). Recent developments in larval nutrition (Nghia et al. 2007, Quinitio et al. 2007), live food production (Hamasaki et al. 2002, Ruscoe et al. 2004), rearing techniques (Hamasaki 2003, Nghia et al. 2007, Ut et al. 2007) have occurred. These advances have enhanced larval survival (15% survival of zoea to crab instars) and enabled the production of juveniles (Fielder and Allan 2003), albeit not at a sufficiently large scale to conduct a release program to enhance the small-scale fisheries for

Mud Crabs (Le Vay et al. 2008). That being said, recapture rates of wild Scylla olivacea were 56% indicating that individuals do not move far from their release site. Moreover, once released, both Scylla paramamosain and S. olivacea have relatively fast growth rates at 2 and 1 cm carapace width per month, meaning that individuals of the former species would

68 attain a reproductive size on only five months (Ut 2002, Moser et al. 2003, Lebata et al.

2009). Small home ranges and fast growth make Syclla spp., and particularly S. olivacea, a good candidate species for a viable release program (Le Vay et al. 2008).

Mark-recapture studies on hatchery-reared S. olivacea and Scylla serrata demonstrated that survival on these cultures crabs was not as great as for wild S. patamamosain, but that conditioning juveniles (20 mm carapace width) in earthern ponds for 4-6 weeks prior to release enhanced survival (Lebata et al. 2009). For example, the recapture rate for unconditioned S. serrata (12%) was significantly lower than for conditioned individuals of

S. olivacea (29%) and S. serata (37%). The study by Lebata et al. (2009) also considered size- at-release ranging from 20-25 to 75-80 mm carapace width. The percentage survival of crabs was found to increase exponentially from 0% at 20-25 mm to 47% at 50-55 mm and remaining essentially the same despite increase in carapace width. Following 28 releases of S. olivacea and S. serrata comprising, in total, 5,273 individuals, between May 2004 and September 2005 the catch per unit effort of the fishery increased by 51% and 42% in terms of numbers and biomass, respectively (Fig. 2.1; Lebata et al. 2009).

Currently, the technology exists to culture Syclla spp. and analysis of their growth rate, survival and contributions to local fisheries are promising, all of which indicate that these species an excellent candidate for large-scale release programs. However, despite the market demands for Syclla spp., further refinement of the larviculture technology is required to increase efficiency and enable any release program to be economically viable (Le Vay et al. 2008). Given the degradation of mangrove habitats and the small home range of crabs, it has also been suggested that habitat availability limits recruitment success and, as a result the abundance of crabs. Thus, restoration of degraded mangroves could enhance the Syclla spp. and other species, such as mangrove-dependent fishes, and provide additional ecological benefits (Walters 2005, Walton et al. 2006). Therefore, until future developments to refine the culture methodology are available, habitat rehabilitation may provide a simple but effective solution in the interim.

69

Fig. 2.5. Monthly catch per unit effort (CPUE) expressed as (a) abundance (crabs gear-1 day-1) and (b) biomass (g gear-1 day-1) of Sycylla spp. caught from mangroves in Naisud and Bugtong Bata, Ibajay, Aklan in the Philippines between April 2002 and November 2005.  total catches of Scylla spp.;  catches of wild Scylla spp. and  catches of stocked Scylla spp. Taken from Lebata et al. (2009).

70 2.2.2.4. Scylla paramamosian in Japan

The Japanese marine stock enhancement program includes large-scale releases of S. paramamosian and, to a lesser extent S. serrata (Fushimi 1983, Fushimi and Watanabe 2000, Ito 2000, Hamasaki et al. 2011). For example, between 2000 and 2004 between 285,000 and 1,026,000 S. paramamosian at 10-12 mm CW and 6-173,000 S. serrata at 16-29 mm CW were released throughout Japan (Hamasaki and Kitada 2008). Due to problems with discriminating between hatchery-reared and wild crabs at the size-at-release (~10 mm CW) and the use of external tags Obata et al. (2006) developed a genetic tag using the mitochondrial DNA D-loop region. Between 1997 and 2002 72,000-149,000 hatchery-reared S. paramamosian were tagged with a specific haplotype and released into a small (7 km 2) embayment, Urado Bay, Kochi Prefecture, Japan. Recapture rates were low ranging from 0.24 to 1.7%. However, while this recapture rate is less than for penaeids speices in Japan (Hamasaki and Kitada 2008), because S. paramamosian grows to a large size and has a high ratio of price per landed individual compared to the cost per hatchery-reared juvenile (i.e. a high YPR – yield per release) stocking was shown, in some years, to be economically viable (Obata et al. 2006, Hamasaki and Kitada 2008). Moreover, subsequent work by

Hamasaki et al. (2011) has demonstrated a significant relationship between the numbers of S. paramamosian released and catches in both Urado Bay and Hamana Bay. These authors also suggested that, on the basis of work done on Scylla spp. in the Philippines (see above; Lebata et al. 2009), that the release strategy of S. paramamosian should be investigated experimentally to determine whether larger release sizes, preconditioning and the selection of different release sites improved survival. It is also noted that, in order to produce larger juveniles, nursery culture techniques would need to be improved (Hamasaki et al. 2011).

2.2.2.5. Callinectes sapidus in the Chesapeake Bay, USA

Chesapeake Bay in the eastern coast of the USA is one of the largest estuaries in the world at ~11,600 km2 (~90 times larger than the Peel-Harvey Estuary, the largest system in south-

71 western Australia) and historically one of North America’s most productive fishing grounds for bivalves (e.g. Crassostrea virginica), crustaceans (e.g. C. sapidus) and finfish (e.g. Morone saxatilis; Miller 2006). In recent decades, however, commercial landings have declined markedly due to sustained fishing mortality and environmental deterioration (Lipcius and Stockhausen 2002, Aguilar et al. 2008), with, for examples, the total fishery harvest for estuarine and coastal species in Chesapeake Bay decreased by 40% between 1995 and 2010 (Huang et al. 2015). Abundances of C. sapidus in Chesapeake Bay, which represent 50% of the USA’s harvest and have a market values of between US$46-103 million annually (2007 dollars), declined by 70% from ~900 million to ~300 million in 15 years (Miller et al. 2005, Zohar et al. 2008, Bunnell et al. 2010, Miller et al. 2011). An estimated 45-44% of the C. sapidus stocks are harvested each year and thus it is not surprising that commercial catches have declined too. Bunnell et al. (2010) estimated that, on average, the market value of the fishery was reduced by $3.3 million per year. In addition to the marked drop in revenue, was that fact between 1988 and 2004 spawning stock abundance and biomass declined by 81 and 84%, respectively (Lipcius and Stockhausen 2002, Miller et al. 2005, Zohar et al. 2008). Despite the declines in environment in the estuary, the drastic depletion of C. sapidus broodstock and juveniles in the nursery area, the population of C. sapidus was considered to be below the carrying capacity of the available habitat and thus a suitable candidate for restocking (Hines et al. 2008a, Seitz et al. 2008).

In response to the declines, a multidisciplinary scientific team (Blue Crab Advanced Research Consortium; BCARC) comprising aquaculturists, crustacean endocrinologists/pathobiologists and benthic/fishery ecologists was established with industry partners. Firstly, an research expedition was undertaken to Japan, which has an extensive history aquaculturing very large numbers of portunids (see above) to determine the context, extent and success of their crab hatcheries, understand how their release programs were implemented and the impact of the releases on the crab fishery and to use this to assess the feasibility of aquaculture-based enhancement to increase stocks of C. sapidus in Chesapeake Bay. Having decided that the development of aquaculture techniques for C. sapidus would also allow

72 increased knowledge on the biology of the species and provide opportunities to develop better fishery management tool the project was initiated. The BCARC set out to achieve four goals, in brief; (i) enhance understanding of biology of C. sapidus; (ii) develop aquaculture and production technology; (iii) assess feasibility of a release program to enhance stocks and (iv) if successful scale up production (see review in Zohar et al. 2008). The work undertaken in this release program provides a framework in which future projects (particularly for portunids) can operate, and gives insights into the biological, ecological, environmental and socio-economic understanding that is required for an aquaculture-based enhancement project to be successful. As a result, the major research themes are address in relative detail.

Investigations into the biology and complex life cycle of C. sapidus were used to help develop detailed protocols to enable the production of larvae (Zmora et al. 2005). As wild individuals only mate during summer (Jivoff et al. 2007, Hines et al. 2008a) natural broodstock would only be available for part of the year. Thus, Zohar et al. (2008) collected mature females that had already mated (i.e. were inseminated) and exposed them to phase- shifted environmental conditions that mirrored the water temperature, salinity and photoperiod found in the natural environment. For example, to maintain inseminated females, individuals were held in low temperatures (15 °C) and experienced short days (8 h daylight), i.e. winter conditions. Then, to induce ovulation and produce broods on demand, water temperature was raised to 22 °C, day length increased to 16 h and salinity to 30 ppt, i.e. summer conditions. By modifying environmental conditions in the hatchery larvae were able to be produced year-round and > 500,000 crablets (20 mm carapace width) were produced and released between 2002 and 2007 (Zohar et al. 2008).

Although the aquaculture was successful, Zohar et al. (2008) recognised that in order to scale-up production methodologies needed to be produced to control and synchronise ovulation, broodstock production and hatching. Work was undertaken to develop an understanding of the physiology and endocrinology of larval development, growth and reproduction (i.e. Chung et al. 2006, Zmora et al. 2007). While it is unclear how successfully

73 this research has been transferred to the hatchery, the aim was to develop techniques to control and synchronise moulting in the larval stages, which would reduce cannibalism and enhance production (Zohar et al. 2008).

Having successfully produced crablets, studies were undertaken to compare the morphological and behavioural characteristics of the hatchery-reared individuals to wild con-specifics as cultured organism often exhibit lower survival than wild spawned individuals (Olla et al. 1994, Brown and Smith 1998, Ochwada-Doyle et al. 2012). The results of these studies are summarised in Table 2.1. In brief, hatchery-reared crabs were morphologically different from wild stock due to their shorted lateral spines and coloration, however, these traits are plastic and could be altered in the hatchery or wild environments (Davis et al. 2004a, Davis et al. 2005a, 2005b, Young et al. 2008). Growth rates of crabs remained similar and hatchery-reared crabs were able to recognised and consume natural prey upon release (Davis et al. 2004a, Zmora et al. 2005, Young et al. 2008). Hatchery crabs remained buried for ~38% of the time, substantially less than the 63% for wild con-specifics (Davis et al. 2004a), but this difference in behaviour could be reduced by exposing them to sediment in the hatchery (i.e. habitat enrichment). Upon release hatchery-reared crabs found suitable habitat and exhibited similar movement and dispersal patterns as wild individuals (Young et al. 2008). Thus, Young et al. (2008) concluded that although some differences existed between wild and cultured C. sapidus and could be mitigrated through conditioning in the hatchery, they were not considered to have a negligible influence on survival and thus did not warrant the additional cost and effort.

74 Table 2.1. Summary of the difference in morphological and behavioural characteristics of hatchery-reared and wild C. sapidus and the extent to which any differences can be reduced or eliminated. Table expanded from Young et al. (2008).

Factor Difference between hatchery and wild Difference eliminated in hatchery? Difference eliminated in wild? Performance affected? References crabs Morphological Yes; both hatchery and wild crabs held in Yes; exposure for 27 days to visual and Yes; no difference after several weeks No (albeit crabs with larger spines had (Davis et al. Body shape culture for 30 days had shorted lateral chemical cues from fish predator (Striped and moults greater survivorship during tethering 2004a, Young et al. 2008) spines, which are a predator defence Bass) and exposure to adult con-specifics [field] and laboratory experiments) Yes; hatchery crabs were brighter, less Yes; colour faded following exposure to Yes; colour faded following exposure to No; crabs change colour quickly and can (Davis et al. saturated in colour and bluer, which sediments for 1-2 days in the laboratory sediments for 1 day in the field match colour within 1-2 hrs and, in any 2005a) make crabs more conspicuous to case, no difference in survival of Colour predators. No; hatchery and wild crabs conditioned and unconditioned hatchery have similar endocrine control of crabs in wild chromatophores No; growth rates of hatchery and wild N/A N/A No (Davis et al. 2005a, Zmora crabs were similar in laboratory. et al. 2005, Growth Observations from field release indicate Johnson et al. hatchery crabs readily adapt to natural 2008) prey and grow at similar rates.

Behavioural Yes; hatchery crabs remained buried for Yes; after 2-4 days of sediment exposure No tested but presumably similar to No; conditioning quickly induces normal (Davis et al. Predator 25% less time than wild crabs no difference was detected laboratory experiment burying behaviour but no difference in 2004a) avoidance survival between conditioned and unconditioned crabs. Maybe; when presented with a single Not investigated Unknown Unknown (Davis et al. 2005a, Young food item, wild crabs outcompeted et al. 2008) Aggression hatchery crabs, but no displacement in

wild stock seen following hatchery releases No; hatchery crabs quickly adapted to N/A N/A No; abundance of hatchery crabs (Davis et al. live food (infaunal clams) and fed on correlated to bivalve density. No 2004a, Young Feeding et al. 2008) them at rates similar to wild crabs. difference in stomach weight of released hatchery and wild crabs. No N/A N/A No; cultured crabs observed mating (Young et al. Reproduction successfully in the wild 2008) No; field experiments indicate that N/A N/A No (Young et al. Habitat patterns of habitat use in hatchery and 2008) selection wild crabs are similar, preferring structured habitats No; hatchery and wild crabs exhibited N/A N/A No (Young et al. Movement similar dispersal and movement pattern 2008) in laboratory and field experiments

75 In an effort to distinguish between hatchery-reared and wild C. sapidus two tagging methods were trialled, i.e. coded microwire tags and fluorescent elastomer tags (Fig. 2.6). Davis et al. (2004b) found that the former tag, was the preferred tagging method, as it has a higher retention rate and applying these tags was faster and thus more time-efficient (see also work by van Montfrans et al. 1986, Fitz and Wiegert 1991). That being said elastomer tags were also effective. In addition to the physical tagging genetic tags were also develop. This first required the mitochondrial genome of C. sapidus to be determined (Place et al. 2005), followed by the isolation and characterisation of microsatellite markers (Steven et al.

2005). Crabs of hatchery origin were able to be distinguished from wild stock with > 95% accuracy based solely on mitochondrial DNA sequences and the addition of microsatellite marked increased discrimination accuracy to almost 100% (Zohar et al. 2008).

Fig. 2.6. Photograph showing a juvenile C. sapidus tagged with both a coded microwire and fluorescent elastomer tag. Taken from Davis et al. (2004b).

Zohar et al. (2008) recognised, as have many others (see earlier), that the survival of hatchery-reared individuals is directly influence by the release program and in particular (i) size-at-release, (ii) site-of-release and (iii) timing-of-release. Survival of tethered wild juvenile C. sapidus was shown to increase with size from 20 (30%) until 40-50 mm CW (~75%), after which survival remained relatively consistent (~75%; Fig. 2.7; (Johnson et al. 2008). The optimal release size was shown to be influenced by season, being important for

76 releases in summer, but less so for spring and autumn due to lower levels of predation.

Importantly release in spring allowed the juveniles to obtain a size of 50-50 mm CW by summer when heavy predation occurs. On the basis of these results and the fact that the cost of production increase with the size (age) of the crabs, Johnson et al. (2008) suggested that, from an ecological perspective, small individuals (∼15–20 mm CW) should be released in spring and autumn when survival is largely independent of size, but that large individuals (>40–50 mm CW) should be stocked in summer.

Fig. 2.7. Proportion (mean ± SE) of tethered wild juvenile C. sapidus of differing carapace widths (20- 70 mm). Crabs allocated to 5 mm size classes. Total n = 1,354 and 22-141 in each size class. Taken from Johnson et al. (2008). In order to ensure stocking densities did not exceed the carrying capacity of the environment into which they were released (i.e. site-at-release), food availability and juvenile growth rate examined (Seitz et al. 2008) and the stocking density at different sites adjusted to ensure the carrying capacity was not exceeded (Davis et al. 2005b, Seitz et al. 2008). With regard to timing-of-release, water temperature was found to be most conducive to survival between April and October, however, the survival of C. sapidus was low, due to relatively high abundances of predators, i.e. larger crabs and fish (Hines et al. 2008b, Johnson et al. 2008). However, growth rates and the timing at which individuals attained sexual maturity also differed seasonally and were considered by Zohar et al. (2008) to be more influential in determining the success of the releases. This is because juveniles released in April-June, i.e. as soon as the water temperature increased, attained sexual

77 maturity (~100 mm CW) within three months post-release and contributed to breeding stocks that year (i.e. 5-6 months post release). In contrast, juveniles released later in summer did not grow sufficiently to breed before water temperatures cooled, and thus overwintered before growing again the subsequent year and spawning when reaching maturity (Zohar et al. 2008). Thus individuals release in early summer spawned one year before those release just a few months later.

The ability to distinguish between hatchery-reared and wild C. sapidus enabled the potential for stock enhancement to supplement populations of crabs to be investigated in the field.

Davis et al. (2005a) released batches of 6-30 mm CW crabs (58-70 days old) totalling 25,000 individuals. Sampling two months post-release indicated that 22-78% of crabs within the predicated size range were hatchery-reared, resulting in an increase in abundance of 28- 366 %. Individuals released earlier in the summer had a survival rate at maturity of 16-20%, which is greater than the 5-15% for late released crabs, which overwintered before attaining sexual maturity (Johnson et al. 2008) and thus were exposed to predation for a longer period. Note that this experiment was conducted prior to Johnson et al. (2008) study, which determined that individuals with a larger CW exhibited a higher survival. Thus survivorship to maturity would likely increase if larger C. sapidus were released, but, given that lower numbers of hatchery-reared individuals would be expected to be released, it is not clear how the contribution of aquacultured crabs to the total catch of C. sapidus would change. It is worth noting also that the 5-20% survivorship to maturity for small C. sapidus is substantially greater than the 0.0007% for Cod (Gadus morhua), 1-3% for Abalone (Haliotis rufescens), 0.0005-1.4% for Queen Conch (Strombus gigas) all after one year and 2.8-3.5% for the penaeids prawns Penaeus indicus and Peanaeus monodon after six months (Tegner and Butler 1989, Stoner and Glazer 1998, Svåsand 1998, Davenport et al. 1999, Davis et al.

2005b).

Estimates of the C. sapidus population in Chesapeake Bay put the population, as of 2008, at 3-8 million spawning female, with the future target of increasing stocks by 10% per year. This would require the release of 3-8 million juvenile females (or 6-16 million juveniles of

78 both sexes; Zohar et al. 2008). While, based on the Japanese Crab enhancement program

(see below), the authors considered this feasible they noted the need for the economic feasibility to be assessed prior to attempted at large-scale production. This is mainly to account for the intensive cannibalism, which creates a bottleneck and results in the need of lower stocking densities and grading (both of which increase the cost of facilities and labour). If this issue was solved, which the authors considered likely following enhanced understanding of the physiology of C. sapidus, the cost of producing juvenile crabs would be US $0.15-0.30/individual and thus $900,000 to $4.8 million (Zohar et al. 2008). It is worth noting that the value of the commercial fishery is worth $46-103 million per annum and was declining at a rate of $3.3 million per year (see earlier; Bunnell et al. 2010).

2.2.2.4. Release programs for other brachyuran crabs

Eriocheir sinensis is extensively cultured in China for use in stock enhancement programs (see review by Cheng et al 2008). Typically, megalogae are caught during their upstream migration in rivers such as the Yangtze, raised in either indoor intensive larviculture or outdoor extensive larviculture systems before being released into lakes and harvested a year later (Cheng et al. 2008). The total production of this species increased rapidly from 8,000 tonnes in 1991 to 714,400 tonnes (a 90x increase) in 2012 and the industry is worth an estimated US $5 billion annually (Wang et al. 2015). The dramatic increase in production is due to advances in hatchery and growout techniques (see Wang et al. 2006, Sui et al. 2011, He et al. 2014, Wang et al. 2016 for details of the methodology). However, before the development of technology to produce broodstock for the enhancement, wild stocks of megalopae were harvested extensively dramatically lowering the wild stocks in the Yangtze

River (Cheng et al. 2008). The implementation of sustainable production, a closed fishing season and a series of stock enhancement programs have seen the catches of megalopae in the coastal area of Chongming Island (Yangtze river) increase from < 500 kg in 1997 to > 3,000 kg in 2004-2006 (Cheng et al. 2008, He et al. 2014).

79 Alaskan King Crabs, Paralithodes spp., are one of the most valuable crustacean in the world and of economic importance to fisheries in Alaska, USA. Stocks of this species, although variable, crashed in the 1980s and have failed to recover since (Stevens et al. 2001, Daly et al. 2009). Numerous natural and anthropogenic perturbations have been proposed to explain the lack of recovery including recruitment limitation, egg predation, disease, overfishing and climate change (Blau 1986, Kuris et al. 1991, Orensanz et al. 1998, Stevens 2006, Daly et al. 2009).

The development of aquaculture techniques for this species (Nakanishi and Naryu 1981,

Nakanishi 1987, 1988, Epelbaum et al. 2006, Kovatcheva et al. 2006, Stevens 2006, 2006 ), albeit at a small scale, have allowed the feasibility of stock enhancement of P. camtschaticus to be investigated. In 2006 the Alaska King Crab Research Rehabilitation and Biology (AKCRRAB) program was created to assess the feasibility of hatching and rearing wild P. camtschaticus and P. platypus in the hatchery and to develop effective release strategies to reverse the low wild stock abundance. To date, detailed hatchery manuals have been developed (see Alaska King Crab Research Rehabilitation and Biology Program 2016 and the references therein), and the production of P. camtschaticus increased from

1,000 juveniles in 2007 to 108,000 in 2010 (Daly 2010). Currently, hatchery production of juvenile P. camtschaticus is limited by cannibalism and slow growth, which can be mediated to some extent through the provision of artificial substrates (i.e. sand), modification of diet, changing the abiotic culture conditions (i.e. water temperature) and regular size grading (Stoner and Glazer 1998, Stevens and Swiney 2005, Borisov et al. 2007, Daly et al. 2009, Stoner et al. 2010a, Stoner et al. 2010b, Daly et al. 2012, Long et al. 2012, Daly et al. 2013). However, large-scale stock enhancement program not currently economically feasible and will not be until additional rearing technologies are developed. As a result, no releases of hatchery-reared P. camtschaticus and P. platypus into the wild have occurred yet (Daly 2010).

The mangrove crab, Ucides cordatus, constitutes an important fishery in Brazil and the development of aquaculture techniques was initiated in 2001 to mitigate the effects of

80 overfishing and mangrove degradation (Ventura et al. 2010a). Efforts in recent years have gained greater significance following mass mortality events caused lethargic crab disease (Ventura et al. 2010b). Larviculture methodologies have been developed (Silva et al. 2006, Silva et al. 2009, Ventura et al. 2011), but production has been inhibited by high mortality during metamorphosis from zoea to megalopae (Silva et al. 2012) and cannibalism by megalopae or zoea (Ventura et al. 2008). Currently, annual production of U. cordatus megalopae exceeds 1 million per season, with the larvae released into targeted mangrove areas. However, work by (Ventura et al. 2010a) demonstrated that the hatchery-reared megalopae were highly susceptible to predation by fish and were out completed and predated upon by Fiddler Crabs (Uca spp.) present at the release sites. As a result, current research focuses on reducing mortality rates in the hatchery to allow substantial numbers crablets to be produced, thus increasing the survival of released individual in the wild (Ventura et al. 2011).

2.3. Conclusions

2.3.1. Wild capture fisheries

Portunid fisheries occur globally, employing numerous methods of capture include trawling, trapping, netting and diving, with the greatest capture rates occurring in the Indo-West Pacific and targeting Portunus trituberculatus and members of the P. pelagicus complex. Global catches of portunid crabs are still increasing despite the relative stability in the catch rates of world finfish fisheries. Portunid crabs are typically heavily exploited, with fishing mortality rates over double that of natural levels. This fishing pressure adds additional stresses to the population and can cause growth to slow, resulting in reductions in the size at which maturity is reached, which, in turn, leads to lowered reproductive potential. The recruitment of crabs to these fisheries and their overall productivity are also influenced by environmental conditions, namely water temperature and salinity. High fishing pressure on many established portunid fisheries, exacerbated by environmentally driven fluctuations in

81 recruitment, has led to several well-documented population collapses, including in

Cockburn Sound, Western Australia.

The management of portunid fisheries varies greatly between within countries, depending on jurisdictional boundaries, with estuarine and coastal fisheries often controlled by state or provincial governments. Most management strategies implement both input and output controls, limiting the fishing effort and the methods used, and the total landings and using size restrictions. Fishing and boat licences, seasonal closures and protected areas are commonly used input controls, while minimum size limits, total allowable catches

(commercial), daily bag limits (recreational) and protection of females in general or ovigerous females are commonly used output controls. Biological indices are also used to asses the health of portunid fisheries against pre-set reference and target level, some of which can account for the influence of temperature, salinity and fishing pressure, and may play a role in making future management strategies more sustainable.

2.3.2. Commonalities between release programs for brachyuran crabs and lessons learned

Despite differences in the geographical location of the releases and the species selected for enhancement, there are a number of commonalities among the release programs. All were initiated in response to declines in stocks, however, although the reasons as for the reductions in target species were different, i.e. overfishing, disease, habitat degradation and climate change, there was evidence to suggest that the there was sufficient carrying capacity in the environment to support released individuals. In each case, successful hatchery techniques were developed to enable small-scale production, however, only in the case of P. trituberculatus was production increased to a commercial scale (~27-35 million juveniles per year). The breeding season of both P. trituberculatus and C. sapidus was able to be manipulated in the laboratory to produce larvae on demand and throughout the year. For species where less research and development has been undertaken, advances in larviculture are needed, particularly to make aquaculture more cost effective to enable

82 large-scale releases. In most release programs, individuals have been stocked at the crablet stage (≥ 10 mm CW) as releasing megalopae was not effective, as was the case with C1 instar crablets (5 mm CW) for P. trituberculatus. However, there is a trade-off between the economic cost of production and size-at-release and thus survival in the wild. This is poorly understood and likely requires detailed biological studies and bio-economic analysis to answer definitely. Cannibalism is a major reason for the increased cost of producing larger numbers of bigger crabs. Techniques such as habitat enrichment and grading can reduce intra-specific predation and it is believed by developing a better biochemical understanding of portunids, techniques to control and synchronise moulting in the larval stages could be developed which would prevent cannibalism.

Release strategies are critical to the success of any stocking and require quantitative data to determine the best site, sites and time-of-release. This was done very successfully with C. sapidus, which can provide a model platform for designing experiments and the release strategy. Although morphological and behavioural difference between hatchery and wild stock occur, these were not thought to significantly limit the fitness and survival of released individuals. In any case, some of these traits were plastic and could be modified via conditioning in the laboratory or the field post-release. Typically, release programs for brachyuran crabs appear to have been successful, albeit to different extents. For the most part, the evaluations of the effectiveness of these releases have been carried out at the pilot scale with only the culture of P. trituberculatus scaled up. Species with fast growth rates, limited home ranges and a large economic value are likely to be the most appropriate species to culture and release. Any future release program for a portunid is likely to require extensive research and development, particularly to scale up production, and thus the biggest gains in this field have come from increased knowledge rather than increased stocks

(e.g. C. sapidus and Paralithodes spp.).

Zohar et al. (2008) when evaluating the successes and lesson learned from the BCARC project for C. sapidus in Chesapeake Bay, raised three points that should be considered carefully during the planning of future release programs.

83 1. Understand the basic biology and life cycle of the target species within the ecosystem where the release will occur.

2. Release programs cannot be successful without integrating adequate management strategies to protect wild and hatchery-reared individuals until sexual maturity and spawning. This requires all stakeholders involved in the resource to be engaged and involved (e.g.fishers, policymakers/managers and scientists).

3. The program must be guided by solid science. Noting that while this science is costly, it is essential to avoid the kinds of expensive mistakes made in the past and ensure programs provide substantial and long-term ecological and financial benefits.

84

85 Section 3. Aquaculture of portunids

3.1. Overview of portunid aquaculture

Aquaculture-based enhancement has been identified as a possible means to improve stocks of several portunid species, including Portunus armatus in south-western Australia (Zohar et al. 2008, Johnston et al. 2011b). Such a program would, however, requires the technology to produce substantial quantities of megalopa, or ideally crablets for release. Aquaculture methodologies have been produced for a number of portunid species including Scylla sp., Callinectes sapidus, Portunis trituberculatus, Portunus pelagicus and Portunus armatus (see above). The culture process typically involves the following steps;

 Broodstock collection and conditioning  Egg incubation and hatching  Larval culture  Nursery  Grow-out (Fig. 3.1; Shelly and Lovatelli 2011)

This section of the review describes the activities undertaken in each of the above main stages and provides a summary of successful methods that may be relevant to the aquaculture of P. armatus in Western Australia, together with suggested areas for further research and development.

86

Fig. 3.1. Photographic overview of portunid crab aquaculture using Mud crabs (Scylla spp. As an example. (a) berried Scylla serrata, (b) S. serrata spawning eggs into a sand tray submerged in a broodstock tank, (c) S. serrata zoea, (d) S. serrata megalopa, (e) larval rearing tank, (f) C1 crablet, (g) nursery tank, (h) crabs ready for export, (i) cellular crab system and (j) earthern pond used for growout. Photographs taken from Shelly and Lovatelli (2011).

87 3.2. Broodstock collection and conditioning

The vast majority of portunid crab aquaculture projects rely on broodstock collected from the wild (Table 3.1; Smallridge 2002, Zmora et al. 2005, Azra and Ikhwanuddin 2015). In the case of the P. armatus aquaculture effort in South Australia, berried females were provided from commercial fishers and thus only available during the natural breeding season, i.e. September to February (Smallridge 2002). However, aquaculturalists in the USA, were able to collect mature C. sapidus females that had already been inseminated from a tributary of Chesapeake Bay and induce ovulation when required (Zohar et al. 2008). This was achieved by holding individuals in low water temperatures (15 °C) and with short photoperiod (8 h daylight), i.e. winter conditions. Then, to induce ovulation, water temperature was raised to 22 °C, day length increased to 16 h and salinity to 30 ppt, i.e. summer conditions (Sulkin and Norman 1976, Zohar et al. 2008).

Being able to control spawning of wild individuals is highly beneficial, not only for logistical reasons, but also as Anand and Soundarapandian (2011) found that the hatching success of eggs from wild caught P. pelagicus was greater than that for conspecific hatchery-reared females. Thus, providing a plentiful supply of berried individuals are available, this strategy is the preferred option. It has been noted that P. pelagicus is not a ‘robust’ species and so the collection, transportation and maintenance of broodstock is paramount to avoid mortality during the incubation phase (Azra and Ikhwanuddin 2015). Another advantage of being able to manipulate the timing of spawning is, as is the case with P. trituberculatus aquaculture in Japan, that juveniles can be produced at a time of year to enable them to experience a lower growing season in the wild and accelerate their entry into the fishery (Hamasaki et al. 1997, Secor et al. 2002).

The limited success of culture efforts for P. armatus in South Australia demonstrates the importance of matching the physical-chemical environment of the broodstock to that in the hatchery, as spawning was only successful when the broodstock were collected from salinities of 44-48 (Smallridge 2002). This likely reflects the hypersaline nature of Spencer

88 Table 3.1. Summary of broodstock management and hatching of Portunus pelagicus. Taken from (Azra and Ikhwanuddin 2015).

Sources of Crab Broodstock Water quality for Water quality during Water Country Feeding Others Reference broodstock size treatment broodstock hatching exchange Wild trawler Fresh Temperature 28 ± 2 °C, salinity 35 ± 1 ppt and (Josileen and India n/a n/a 50% n/a caught clam pH 8.2 ± 0.1 Menon 2004) Temperature 34 ± Temperature 29 ± 5 Wild caught 200 ppm Oyster 1 °C, salinity 29 ± 5 Photoperiod - (Soundarapandian India n/a °C, salinity 34 ± 1 ppt 50% (n/a) formalin meat ppt, pH 7.725 ± 0.25 12hL:12hD et al. 2007) and DO 5.5 ± 0.5 mg/L and DO 5.5 ± 0.5 mg/L Temperature 27 ± 1.5 1 μl/L filtered Wild caught 100 μl/L Temperature 28 ± 1.5 (Castine et al. Australia n/a n/a °C and salinity 34 ± 1 n/a and UV treated (baited pots) formalin °C and salinity 22 ppt 2008) ppt seawater Prawns, Temperature 28 ± 2 Temperature 26 ± 1 °C Wild caught 50 μl/L (Andrés et al. Australia n/a mussels °C and salinity 32 ± 2 and salinity 34 ± 1.5 10% n/a (baited traps) formalin 2010) and squid ppt ppt Temperature 27.5 ± Wild caught Temperature 29 ± 1 0.5 °C, salinity 30 ppt, Sand substrate (Ikhwanuddin et Malaysia (local n/a n/a n/a °C, and salinity 29 ± 1 50% pH 7.45 ± 0.45 and – 3cm al. 2011b) fisherman) ppt DO 5 mg/L Wild caught Chopped Temperature 29 ± 1 °C, salinity 29.5 ± 0.5 ppt, (Ikhwanuddin et Malaysia n/a n/a 100% n/a (gill nets) fish pH 8.35 ± 3.5 and DO 6 mg/L al. 2012a) Wild caught 120 µl/L Not fed Salinity 31 ± 2 ppt, (Talpur and 124 – Malaysia (local formalin and until pH 7.7 ± 0.3 and DO n/a 50% Sand substrate Ikhwanuddin 138mm fisherman) 2ppm KMNO hatching 7.34 ± 0.55 2012)

89 Table 3.1. Summary of broodstock management and hatching of Portunus pelagicus continued. Taken from Azra and Ikhwanuddin (2015).

Sources of Broodstock Water quality for Water quality during Water Country Crab size Feeding Others Reference broodstock treatment broodstock hatching exchange Wild caught 120 µl/L Not fed Temperature 30 °C, salinity 32.5 ± 2.5 ppt, pH Sand substrate – (Ikhwanuddin Malaysia (local n/a formalin and until 100% 7.75 ± 2.5 and DO 5 mg/L 3cm et al. 2012b) fisherman) 2ppm KMNO hatching Wild caught Chopped Temperature 30 °C, salinity 30 ppt, pH 8.35 ± Moderate (Ikhwanuddin Malaysia n/a n/a 100% (gill nets) fish meat 3.5 and DO 6 mg/L aeration et al. 2012c) Temperature 32.15 ± Domesticated 10.76 ± Minced 2.15 °C, salinity 33 ± 2 (Oniam et al. Thailand broodstock n/a n/a 20-30% n/a 0.97 cm trash fish ppt, pH 8.7 ± 0.5 and 2012) (pond reared) DO 4.93 ± 2.47 mg/L Temperature 27.5 ± Wild caught Fresh 0.5 °C, salinity 30 ppt, Temperature 29 ± 1 °C Sand substrate (Ikhwanuddin Malaysia n/a n/a 50% (n/a) squid pH 7.45 ± 4.5 and DO and salinity 29 ± 1 ppt 3cm deep et al. 2012d) 5ppm Raw clam Temperature 28 ± 0.1 (Ravi and Wild caught 140–160 200 ppm and Photoperiod India °C, salinity 35 ppt and n/a 70% Manisseri (n/a) mm formalin cuttlefish 12hL:12hD pH 8.1 ± 0.1 2012) meat Raw clam Temperature 28 ± 1 Sand substrate (Ravi and Wild caught 140–160 200 ppm and °C, salinity 35 ppt, pH 10 cm deep and India n/a 10% Manisseri (n/a) mm formalin cuttlefish 8.1 ± 0.1 and DO 5 photoperiod 2013) meat mg/L 12hL:12hD *n/a, not available; DO, Dissolved oxygen; KMNO , potassium permanganate; hL, hour light; hD, hour dark

90 Gulf, which is due to a long residence time of water (i.e. 11 years). Salinities used in the successful maintenance of P. pelagicus broodstock were between 30 and 35 ppt and likely matched that in the wild (Table 3.1). To reduce bacterial and contamination loads, berried crabs are typically treated in chemicals such as formalin and potassium permanganate (Table 3.1). This was not, however, done in South Australia for the culture of P. armatus, and the authors noted the presence of parasitic nematodes, ciliates and fungi. Instead, the impacts of these parasites were reduced by maintaining water quality (500% water exchange and daily vacuuming of the tanks (Smallridge 2002).

In almost all culture studies, broodstock crabs were fed with food such as molluscs (clams, cockles, mussels, oysters and squid), prawns and pelletized food (Table 3.1; Smallridge 2002, Zmora et al. 2005).

3.3. Egg incubation and hatching

In South Australia, berried P. armatus were held in 1,500 L hatching tanks, with a 500% daily water exchange and temperature of 25 °C (Smallridge 2002). The tanks were subjected to low light conditions and contained 150 mm PVC pipe off cuts, which provided a refuge for the broodstock and reduced stress. Broodstock conditioning methods for C. sapidus in the USA were slightly different. In brief, females of 160-250 g (150-180 mm CW) were held in 2 m3 tanks (10 individuals / tank), with a single recirculating system, biofilter, protein skimmer and ozone treatment unit (Zmora et al. 2005). A complete water exchange cycle took one hour and 7% of the water volume was replaced daily. As the bottoms of each tank were bare, 8-12 30 cm diameter, 4 cm deep plates of sand were provided to allow females to release their eggs and then attach the egg mass to their abdomen. About five days before spawning, ovigerous females were transferred to 800 L hatching tanks and resultant larvae collected on a 125 um screens.

91 A description of the changes that occur in the eggs during development were provided by

Smallridge (2002) for P. armatus. The egg mass changes from orange (post spawning) to a dark grey colour within two days of hatching, mirror the changes described for this species in Western Australia (Jenkins et al. 2017). The length of time between these two stages was approximately two weeks at a water temperature of 25 °C. This is similar to the 8-10 days egg incubation time for P. pelagicus in Queensland (Fig. 3.1; Mann and Paterson 2004). Fecundity has been calculated for P. pelagicus and ranges between 400,000 and 1,500,000 and increases proportionally with the size of the female (Maheswarudu et al. 2008, Oniam et al. 2012). No fecundity estimates were provided by Zmora for C. sapidus and Smallridge (2002) report that P. armatus, in their trials, produced between 300,000 and 400,000 eggs, which is less than in other populations of this species (Jenkins et al. 2017). Eyestalk ablation was not mentioned in the culture of C. sapidus or P. armatus, but is used for P. pelagicus as it stimulates spawning and gonadal development (Bhat et al. 2011).

3.4. Larval culture

3.4.1. Water quality

Larviculture for P. pelagicus in Queensland lasts about two weeks, with the zoea held in 4-10 t tanks for between 14 and 18 days until they reached megalopa (Mann and Paterson 2004). This is similar to the method used in South Australia for P. armatus, where zoea were held in 3,000 L conical based tanks for between 14 and 19 day (Smallridge 2002). Water quality (water temperature, salinity and dissolved oxygen concentration) is a critical factor influencing the growth and survival of larval crustaceans, as these stages are typically most susceptible (Kinne 1963, 1964, Crisp et al. 2017).

In the case of temperature, it can have a positive or negative effect. For example, while increased temperatures can speed up development and lower survival, lower than optimal temperatures can decrease metabolic rates as well as growth and survival. Development of P. pelagicus in South Australia was shown to be highly temperature dependent, with

92

Fig. 3.2. Flow chart showing the production cycle used by the Bribie Island Aquaculture Research Centre in Queensland for mud crabs (Scylla serrata) and blue swimmer crabs (Portunus pelagicus). Status = the current level of success achieved relative to the required for commercial production in Australia. High, good or moderate water quality are relative terms to indicate the more critical requirements of the production process. Taken from Mann and Paterson (2004). individuals reaching megalopa after 14 days at 27 °C, but this transition took 19 days at a water temperature of 24 °C (Smallridge 2002). A study by Bryars and Havenhand (2006), on the same species, in the same location, found that, in addition to influencing growth rate, it

93 also had a pronounced effect on survival. For example, at a consistent temperature, the survival of larvae to C1 was greatest at 25 °C (~30%), relatively low at ~ 20 °C (< 5%) and 0% at 17 °C, with no larvae metamorphosing beyond Z3 at temperature below 20 °C. These authors suggested that low temperature may represent a physical barrier to successful metamorphosis and that larval survival and settlement would be highest in the Gulf of St Vincent, South Australia during years with abnormally warm summers. In their review of larviculture methods for P. pelagicus Azra and Ikhwanuddin (2015) found that water temperatures of 30 °C had provided the best survival in tropical areas such as Malaysia and

India (Table 3.2), but that survival declined markedly at temperatures above that, with no survival in the 40 and 45 °C treatments (Talpur and Ikhwanuddin 2012).

The developmental patterns and survival of many portunids, including P. pelagicus are influenced by salinity (Azra and Ikhwanuddin 2015). In general, salinities approaching full strength seawater (30-35 ppt) have proved most successful (Table 3.2). A range of extreme salinities (0, 40, 60 and 80 ppt) were trailed by Talpur and Ikhwanuddin (2012), however, no survival was recorded in any treatment other than 40 ppt. In their review, Azra and Ikhwanuddin (2015) recommended that the culture of P. pelagicus occur in a salinity of 30-

35 ppt and water temperature of 30 °C. These values match that successfully used to raise broodstock and are also similar to those found in the marine environment there the broodstock were collected from (Table 3.1). One notable exception to the ~30 ppt guideline was the 44 ppt successfully used to culture P. armatus in South Australia. However, these conditions were similar that in Spencer Gulf, where the broodstock were collected from, which is naturally hypersaline due to the high residency time of water in the embayment (Smallridge 2002).

An adequate supply of dissolved oxygen is crucial to survival of any aquaculture species and field and laboratory experiments have shown that crustaceans can detect and move away from hypoxic conditions and that their abundance in the wild is negatively influenced by low oxygen levels (Wu et al. 2002, Tweedley et al. 2016a). In an hatchery context, Talpur and Ikhwanuddin (2012), exposed zoea to hypoxic (< 0.5 mg/L) and normoxic (> 6 mg/L) oxygen

94 Table 3.2. Influence of various water quality parameters on the growth and survival of Portunis pelagicus larvae. Taken from Azra and Ikhwanuddin (2015).

Water quality parameters Country Crab stages Summary Conclusion Reference and optimum value Higher number of larvae reaching each stage from hatching and low stage of Maximum hatching at lower temperature and (Bryars and Australia Larval stages Temperature (25°C) development period at higher better survival at higher temperature Havenhand 2006) temperature Increased ammonia-N concentration - Over 16.86 mg/L caused significant decreased Ammonia-N (<16.68 mg/L) Larval stage decrease larval vigour of survival and moulting rate China (Liao et al. 2011) (Zoea 1-4) Increased nitrate concentration - decrease Over 53.34 mg/L caused significant decreased Nitrate (<53.34 mg/L) larval survival an moulting larval vigour Higher water temperature - better mean Temperature affected survival and moulting Larval stage Temperature (30°C) survival and juvenile production of larvae (Ikhwanuddin et Malaysia to 1st day compared to the ambient conditions al. 2012b) juvenile crab Lower salinity is highly sensitive to the larval Salinity (30 ppt) Higher salinity better growth and survival rearing Higher temperature - better final survival Significant results on survival and Temperature (30°C) but decrease the stage-wise development development at higher or lower temperature Larval stages (Ravi and India Higher salinity - better final survival rate (Z1-M) Lowest salinity affected both moulting and Manisseri 2012) Salinity (35 ppt) and lower salinity - lower stage survival development No survival at highest temperature (up to Temperature directly influence larval rearing Temperature (30°C) 40 °C) and lowest survival at ambient with higher temperature caused detrimental temperature larval survival Zero survival at the 0 salinity and the Salinity cause primary stress to the crab early (Talpur and Larval stage Salinity (< 40 ppt) Malaysia highest salinity up to 60 ppt larval stages Ikhwanuddin (Z1-2) No survival at lowest oxygen (< 0.5 mg/L) DO not only for respiration, but also for 2012) DO (> 6 mg/L) and better survival at > 6 mg/L) maintaining required chemical and hypienic No survival at pH 4.6. 10 and 8.2 produced Acidic pH and higher alkaline pH have adverse pH (82) higher survival effect on mortalilty at early stage Larval stages No significant in survival when larvae Better survival when crab reared pH at 8.0 (Ravi and India pH (8.0) (Z1-M) treated with pH 7.5, 8 and 8.5 lowest H (5.0) is harmful for larvae Manisseri 2013)

95 concentrations and found that the former conditions resulted in complete mortality of the larvae, thus survival was far greater under normoxic conditions (Table 3.2). These authors also tested the influence of pH on survival, with survival being greatest at a pH of 8.2 and no survival being at acidic (4.6) and alkaline (10) pHs. While these changes in pH were relatively large, no significant differences in larval P. pelagicus survival were detected when individuals were exposed to pHs of 7.5, 8.0 and 8.5 (Ravi and Manisseri 2013).

3.4.2. Feed

Successful culture of portunids requires a range of feeds to be supplied throughout larval development. Details of a feeding regime have been best described for C. sapidus (Table 3.3; (Table3.3; Zmora et al. 2005). In brief, zoea were provided with four species of microalgae, i.e. Isochrysis galbana, Nannochloropsis sp., Tetraselmis tetrathele and Chaetoceros gracilis at a concentration of 2.5 mg dry weight L-1, which resulted in the water turning green. In addition to providing a food source for the crab larvae, the microalgae also supported the enriched rotifers (Brachionus rotundiformis or Brachionus plicatilis; Algamac2000 or

AquaGrow®) and Artemia that were added to the culture. Artemia were only added to the feed when the zoea reached Z3 and where provided until the C. sapidus metamorphosed to C4 (Table 3.3.). A frozen copepod diet was introduced daily from Z4 onwards and having reached megalopa stage individuals were fed predominantly a range of commercial shrimp feeds (Zmora et al. 2005).

This sequential change in diet was mirrored by aquaculturists raising P. armatus in South Australia. Here, Isochrysis tahitian and Nannochloropsis occulata were used as a food source and for the crabs, to enrich live feeds to provide a greenwater culture (Smallridge 2002).

Live feeds comprised the rotifer B. plicatilis and Artemia, for the Z1 and Z2 and > Z3 stages, respectively. Using these methods, a survival of up to 80% was achieved through to the megalopa stage. The results of a range of feeding trails are provided in Table 3.4. These support the provision of a wide range of feeds and the need to alter diet with ontogeny. The

96 concentrations of food were checked daily by Zmora et al. (2005), which is importance as subsequent work has demonstrated the P. pelagicus larvae are not resistant to starvation for 48 hours (Table 3.5; (Table 3.5; Talpur and Ikhwanuddin 2012).

Table 3.3. Feeding regime used for culturing the different stages of Callinectes sapidus. Taken from Zmora et al. (2005).

Stage Z1 Z2 Z3 Z4 Z5 Z6 Z7 Z8 Meg C1 C2 C3 C4 C5 C6 Diet Algae (mg/L) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Rotifers (indivs/L) 50 50 50 50 50 50 50 50 Fresh Artemia nauplii 300 300 300 300 200 200 Enriched Artemia nauplii 500 500 800 800 1000 1000 1000 1000 Cyclop-eeze (g/m-3) 5 5 5 5 5 10 10 10 10 Artemia flakes (g/m-3) 5 5 5 10 10 Zeigler 1 mm shrimp diet (g/m-3) 5 5 5 Cargill crumble diet (g/m-3) 10 10 10 Shredded squid (g/m-3) 5 10 10 15 20 30 Cargill shrimp nursery diet 1 (g/m-3) 10 10 20 20 Zeigler shrimp diet 2 mm (g/m-3) 10 10 10 10 Frozen adult brine shrimp 5 10 10 10 Zeigler shrimp diet 3/32 in (g/m-3) 10 10 20 Zeigler shrimp diet 1/8 in (g/m-3) 10 20

3.4.3. Hatchery environment

Several studies have highlighted the important of determining the optimal stocking density as survival and growth rates can be affected due to limited access to food and the impact of waste products of water quality (Azra and Ikhwanuddin 2015). A quantitative relationship between stocking density and survival was developed by Zmora et al. (2005) for C. sapidus; with survival being greatest at 50 larval/L (55%) and declining in a linear manner to 23% at a density of 138 larvae/L (R2 = 0.402). Rearing of P. pelagicus has been undertaken at a range of densities from 10-400 larvae/L (Soundarapandian et al. 2007, Maheswarudu et al. 2008, Ikhwanuddin et al. 2012c, Ikhwanuddin et al. 2012d, Azra and Ikhwanuddin 2015). Generally, lower stocking densities, ~ 50 larvae/L, have proven most successful, with growth rates in these tanks also being greatest (Table 3.5).

97 Table 3.4. Feeding requirements used to culture Portunus pelagicus larvae to the juvenile stages. Taken from Azra and Ikhwanuddin (2015).

Country Experimental diets Crab stages Summary Conclusion Reference Low survival observed from megalopa to Rotifers, Brachionus plicatilis, Artemia Larvae to Lower survival from last (Soundarapandian India 1st crab instar. Fast growth at earlier nauplii and bivalve meat megalopa zoea to megalopa et al. 2007) zoea stages compared to late zoea stages Higher survival of crab fed with fish meal Micro-bound diet (proteinbased diet: fish micro-bound diet compared to live Soybean meal potentially meal, squid meal, krill mean and soybean Megalopa to (Castine et al. Australia Artemia. Artemia resulted shorter larval provide dietary amino acids meal), live Artemia nauplii and unfed crab stage 2008) development and greater body weight and replace live food treatment and carapace length Better survival and development when Larvae to 1st crab fed with combination diet of rotifers Food type influence crab (Ikhwanuddin et al. Malaysia Mixed diatom, Artemia nauplii and rotifer day juvenile and Artemia compared to addition of growth and survival 2012c) crab mixed diatom Early zoea stages crab fed more rotifers, Presence of Brachionus sp Individual ingestion rates of crab for Early larval Brachionus sp. than Artemia nauplii. Late did not influence the (Ikhwanuddin et al. Malaysia Atemia nauplii and rotifers, Brachionus sp. stages zoea stages crab fed more Artemia consumption of Artemia 2012d) nauplii than Brachionus sp nauplii Best survival, rapid development and Larvae to 1st highest number of juvenile crab when fed Instant frozen encapsulated and artificial Food type influence crab (Ikhwanuddin et al. Malaysia day juvenile with combination diet of frozen food, feed growth and survival 2013) crab rotifer and Artemia nauplii compared to the additional artificial diet

98 Table 3.5. Various types of environment on Portunus pelagicus culture. Taken from Azra and Ikhwanuddin (2015).

Crab Country Environment Experimental design Summary Reference stage Rotifer only-Z1 to M, Artemia only-Z1 to M, rotifer-Z1 with Rotifer-Z1 with Artemia-Z2 to M was the Feeding (Redzuari et al. Malaysia Larvae Artemia-Z2 to M, rotifer-Z1 to M with Artemia-Z3 to M and suitable feeding regime with effected regimes 2012) rotifer-Z1 to M with Artemia Z4 to M survival and development Larvae ingested more Atemia after 24 Live prey Artemia only, rotifer only and Artemia + rotifer with 30, 60 (Ikhwanuddin et al. Malaysia Larvae hours at late zoeal stage as compared to ingestion and 30, 60 individuals/tubes 2011b) the initials zoeal stage (Talpur and Malaysia Larvae Starvation test Feed (rotifer and microalgae) and un-fed No survival at unfed stage Ikhwanuddin 2012) Stocking Survival highest at lowest stocking density (Maheswarudu et India Larvae 50 and 100 larvae/L density at each zoea stage (Z1-4) and vice versa al. 2008) Stocking Mass mortality at highest density and (Ikhwanuddin et al. Malaysia Larvae 50, 200, 300 and 400 larval/L density lowest density resulted better survival 2013) Stocking (Ikhwanuddin et al. Malaysia Larvae 10, 20, 40, 60, 80 and 100 larvae/L Highest survival at stocking at 20 larvae/L density 2012c) Photoperiod at 18L:6D is most suitable Australia Larvae Photoperiod 0L:24D, 6L:18D, 12L:12D, 18L:6D and 24L:0D and significantly affected growth and (Andrés et al. 2010) development Photoperiod at 12L:12D - highest survival (Ravi and Manisseri India Larvae Photoperiod 6L:18D, 12L:12D ad 18L:6D but low development 2013) Tank Black colour tank - better survival and red Malaysia Larvae White, orange, yellow, red and black (Azra et al. 2012) coloration colour tank revealed better development Tank White colour tank - worst result and dark (Ikhwanuddin et al. Malaysia Larvae White, dark grey, blue and brown coloration grey resulted better growth and survival 2012c) Water (Ikhwanuddin et al. Malaysia Larvae 0, 25, 50 and 100% 50% water exchange - better results exchange 2012c) No significant in growth and survival with (Ikhwanuddin et al. Malaysia Larvae Antibiotic Treated and not-treated oxytetracycline addition of antibiotics 2012c)

99 Photoperiod and tank colouration have also been shown to influence the growth, development and survival of larval portunids as it influences swimming and feeding behaviour (Table 3.5). For example, Andrés et al. (2010), found that a photoperiod of 18L:6D was most effective and substantially more so than total darkness (0L:24D). Similarly Smallridge (2002) used 18-20 hours of light and Ravi and Manisseri (2013) found that 12L:12D was most effective. Studies by Azra et al. (2012) and Ikhwanuddin et al. (2012c) tested the colouration of the rearing tanks (black, dark grey, brown, red, orange, yellow, blue and white) and both concluded that P. pelagicus larvae grown in tanks with dark colours, i.e. black and dark grey, had the fastest development and survival. In fact, in the case of individuals in the white tanks reaching the crablet stage (Ikhwanuddin et al. 2012c).

3.4.4. Cannibalism

Typically, the larval durations of portunids are relatively short and cannibalism only really becomes an issue once the larvae reach megalopa (Zmora et al. 2005). Asynchronous development at this stage results in the faster developing larvae becoming megalopa and developing chelipeds that allow them to exhibit raptorial capabilities on the less well- developed zoea (Romano and Zeng 2017). While, the larvae of some non-portunid brachyurans, such as the Red King Crab Paralithodes camstschaticus, are highly cannibalistic and stocking densities of zoea must be kept at low levels, no density dependent stocking effects were seen in new hatched C. sapidus or S. paramamosain (40-100 and 50-200 larvae L-1, respectively; Zmora et al. 2005, Nghia et al. 2007). Upon reaching megalopa, however, populations of P. pelagicus were subjected to density-dependent effects between 20 and 50 larvae L-1 (Maheswarudu et al. 2008, Ikhwanuddin et al. 2012c).

A number of strategies have been developed to reduce the effects of cannibalism in the larval stages. For example, Zmora et al. (2005) used net shelters, which have a high surface area to volume ratio and provide adequate shelter, but leave swimming space in the culture tank. Grading, which is commonly used in fish culture to remove the larger and more

100 aggressive individuals, was not considered by Romano and Zeng (2017) to be an effective for crustacean species, due to their larvae being irregular in shape and highly fragile and thus easily damaged during this process. Instead there is value in understanding the endocrine system of species of aquaculture interest and the role hormones play in the synchronization of moulting (Zohar et al. 2008). Laboratory experiments indicate that higher live food densities and/or more benthic food should be provided to help reduce cannibalism (Romano and Zeng 2017). In the case of the Mangrove Crab Ucide cordatus, predation on zoea by the larger magelopa was reduced when the density of Artemia was increased from

0.3 to 6 Artemia mL-1 (Ventura et al. 2008). This strategy of providing a surplus of food was expanded on by Baylon et al. (2004) who increased the density of Artemia fed to S. serrata for several days after metamorphosis to ensure sufficient planktonic prey were available and provided ‘sinking’ prey, e.g. finely chopped prawns and bivalves, to occupy newly settled crablets and reduce the likelihood of them cannibalising.

3.5. Nursery

Transfer from the hatchery to nursey ponds has tended to occur at the megalopa stage as mortality during this and the C1 stage is too high in the hatchery, due to cannibalism and other factors, including the stress of metamorphosis (Allan and Fielder 2004). In Queensland, nursery facilities for P. pelagicus comprise tanks > 20 t and small ponds > 100 t – 2,000 m2 with hapa nets (Mann and Paterson 2004). Megalopa are held for up to four weeks until they reach 0.1-0.3 g (10-15 mm CW or C4-C5), when they are transferred to full grow-out facilities. At this stage, cannibalism is a primary source of mortality (Luppi et al. 2001, Zmora et al. 2005). As demonstrated by the results of stable isotope analysis, which showed that that larger hatchery-reared P. pelagicus had a more enriched δ15N signature than smaller individuals, indicating that these larger individuals were feeding at a higher trophic level due to them becoming increasingly cannibalistic (Møller et al. 2008).

101 A range of factors that could influence the rate of cannibalism in juvenile P. pelagicus were investigated under controlled conditions using time-lapse video recordings by Marshall et al. (2005). Cannibalistic crabs were found to be significantly heavier than their victims, and were predominantly at the intermoult rather than premoult or postmoult stages. However, no sexual bias among cannibals and victims was found and thus, males and females were equally likely to cannibalise and did not discriminate in their choice of victims (Marshall et al. 2005).

Typically, lower stocking densities and the provision of greater substrate complexity reduce juvenile cannibalism in a range of brachyuran species (Romano and Zeng 2017). Numerous crab species, including C. sapidus, have been shown to actively select more complex habitats, such as seagrass, macroalgae, pebbles and crushed shells over finer sand substrates (Van Montfrans et al. 2003, Romano and Zeng 2017). Artificial habitats have also been successful in increasing survival, with, for example, net shelters, increasing the survival of S. serrata from 44 to 77% (Rodriguez et al. 2007). A number of studies have investigated the effects of structural complexity on the survival of P. pelagicus. The presence of increasing quantities of habitat (i.e. number of large paving stones) increased survival of juveniles proportionally, indicating that the quantity of shelter is important for reducing cannibalism in this species (Fig. 3.3; Marshall et al. 2005). The presence of shelter (habitats) was found to increase the survival of megalopa through to the crablet stage vs no shelter (Oniam et al. 2015). However, differences among habitats was detected, with artificial algae made from plastic shield membrane and polyvinyl rope, being more successful than artificial grass made from plastic. Similarly, Ravi and Manisseri (2012), trailed a range of habitat options and found that sand and the seagrass Cymodocea rotundata was the best (88% survival after 15 days) compared to macroalgae (60%), nylon nets (57%) and no habitat

(25%).

102

Fig. 3.3. Temporal patterns of survival of small (<60 mm) and large (>65 mm) size juvenile Portunus pelagicus in high-, medium- and low-refuge availability. Taken from Marshall et al. (2005).

Work in South Australia on P. armatus has demonstrated the importance of stocking density on survival, with 100% survival being recorded over 90 days for crabs at 7, 10 and 25 individuals m-2, but this declined to 57 an 23 % at densities of 50 and 100 individuals m-2, respectively (Smallridge 2002). Given these relationships, there is value in using cost-benefit analysis to help determine appropriate culture conditions. For example, the survival of Scylla spp. after 120 days in ponds at densities of 0.5, 1.5 or 3.0 m-2 was 98.2, 56.7 and

103 30.5%, respectively and cost–benefit analysis recommended stocking densities between 0.5 and 1.5 m-2 be used in the future (Triño et al. 1999).

In regards to feeding during the crablet stage Zmora et al. (2005), fed C. sapidus crablets a varied diet comprising algae, artificial formulated diets (Zeigler and Cargill shrimp diet), together with enriches Artemia nauplii, Cyclop-eeze®, live and frozen Artemia, shredded squid, shrimp pellets of various sizes and fish food several times a day. Crablets were weaned off live food (algae, and Artemia) from C3/C4. The quantity of food was increased in accordance with growth from 20 to 70 g/m3 per day, and not related to the density of crabs

(Zmora et al. 2005).

3.6. Grow-out

Grow out from the C4 stage is unlikely to be beneficial for an aquaculture-based enhancement program, due to the added mortality and potential for the crabs to become maladapted to hatchery rather than wild conditions, however, it is used in the aquaculture of two portunids (P. pelagicus and C. sapidus). In Queensland, two systems are employed, i.e. earthern ponds and cellular recirculating modules (Fig. 3.2; Mann and Paterson 2004). In the former system P. pelagicus of 10-15 mm CW are placed in earthern ponds up to 1 hectare in size and left for 12 weeks, after which they reach 90-100 mm CW (large juvenile). Cellular systems, where crabs are held individually in containers (or cells; Fig. 3.1) are also used. Separating the crabs eliminates the risk of catabolism (Shelly and Lovatelli 2011) and potentially also increase survival further by not exposing the juvenile crabs to fish and avian predators. This point is emphasised by the study of Oniam et al. (2011) who found that the survival of P. pelagicus following 120 days in an earthern pond at three crabs m-2 was 47%. Survival could, however, be increased to 58% through the provision of shelters (bent plastic plates), but this was still far below the 81% survival achieved by culturing the crabs in individual compartments. Compared to earthern ponds, growth rates in cellular systems are faster, with hatchery-raised P. pelagicus reaching the large juvenile size in 8

104 rather than 12 weeks (Mann and Paterson 2004). Although these systems are capital- intensive to establish, they can be automated, which would reduce labour costs (Mann and Paterson 2004).

Low salinity earthen ponds have been trialled in North Carolina as a means of producing juvenile soft and hard-shell C. sapidus (Turano 2012). The ponds were 1,000 m2 in surface area, ~1.4 m deep and filled with bore water (salinity ~ 0.5 ppt; Fig. 3.4). To reduce cannibalism, artificial seagrass was added to both ponds, one of which also contained barnyard grass (naturally growing before the ponds were flooded). A total of 4,800 hatchery-reared juveniles (11.5 mm CW) were added to each pond (stocking density 4.8 crabs m-2) and feed a commercial prawn feed. Harvesting of the crabs occurred 96 days later, producing a survival rate of ~20%. At this time the crabs were, on average, 106 mm CW and thus grew at between 1 and 1.9 mm CW per day (Turano 2012). While these growth rates are some of the highest recorded for C. sapidus, these faster growth rates came from the pond with lower survival, perhaps indicating that density-dependent growth effects play a role together with cannibalism. It is worth noting that the author felt that the survival could be improved if predators were removed from the ponds (Turano 2012).

Fig. 3.4. A low salinity earthern pond with artificial grass habitat used in the trial to produce Callinectes sapidus in North Carolina (USA). Taken from Turano (2012)

105 3.7. Conclusions

Broodstock are typically collected from the wild using commercial fishers, as hatching success is greater for wild-caught and hatchery-reared broodstock. To increase the success of the hatch, conditions in the culture environment should be similar to those in the waters the broodstock were collected from. Water temperature at the time of spawning ranges from 22-34 °C depending on the location the species was collected from and salinities are always around full strength seawater (30-35 ppt), except in the case of P. armatus in South Australia (44-48 ppt), as this mirrors that in Spencer Gulf. If inseminated but not berried broodstock are collected, crabs require access to sandy substrate to spawn.

Maintaining appropriate water quality is paramount for larviculture and the specific preference will vary depending on the species and the location in which they spawn. However, generally for P. pelagicus larvae require relatively warm (25-30 °C) water at near- marine salinities (30-35 ppt) that is well oxygenated and of a slightly basic pH (8). The larvae require different feed throughout the transition from zoea to megalopa. Typically, zoea are fed a range of microalgae strains (which also enrich live feeds and provide a greenwater culture) and rotifers, with Artemia being provided once the larvae reach the later zoeal stages. Stocking density is important, with faster growth and greater survival occurring at lower densities.

Cannibalism in portunids starts at the megalopa stages and to reduce the impact of such interactions megalopa are transferred from larviculture to nursery conditions. Typically, lower stocking densities and the provision of greater substrate complexity reduce juvenile cannibalism. Portunid species have been shown to actively select more complex habitats, such as seagrass, macroalgae, pebbles and crushed shells over finer sand substrates and artificial habitats, such a nets, have also been successful in increasing survival. At the crablet stage, diet in the hatchery comprises commercial shrimp/prawn feeds.

While an aquaculture-based enhancement program for P. armatus would not require a grow-out phase, the success of pond culture for C. sapidus indicates that perhaps inland

106 saline aquaculture could be used to grow larger P. armatus individuals. However, this would require the salinity tolerances on this species to be determined before any initial trials could be conducted, as salinities in the upper reaches of Chesapeake Bay decline to far lower levels than experienced by the lower reaches of estuaries in south-western Australia where P. armatus are found (cf. Gibson and Najjar 2000, Broadley et al. 2017).

107 References

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