General materials and methods

40 R .E. Thresher et al. J. Exp. Mar. Biol. Ecol. 254 2000 37 –51

2. General materials and methods

2.1. Sacculina carcini trials Experiments were carried out at the Kristineberg Marine Research Station KMRS, Fiskebackskil, Sweden, from 4 August 1996 to 7 April 1998. The infection trials took place between August and December, 1996; crabs were held in individual aquaria up to April 1998 to allow interna time to develop. During captivity, they were fed mussels, fish and shrimp. The general experimental protocol was 1 expose potential hosts to healthy, appropriately staged cyprids, 2 examine potential hosts microscopically for attached cyprids, 3 transfer infected crabs to individual aquaria for rearing, followed by 4 sacrifice of the animals and microscopic and genetic examination for evidence of development of an interna. The experimental design encompassed two orthogonal hierarchical structures. First, the parasite and hosts were tested in three settings: 1 no choice of host i.e. single species exposure, 2 choice of alternative hosts i.e. simultaneous availability of natural and potential hosts in the laboratory and 3 choice experiments in the field. The second hierarchy determined the host species tested. These were, in order of decreasing likelihood of infection: 1 the natural host C . maenas from Sweden and Australia, 2 other members of the same family Portunidae, including a non-host species from Sweden and four potential hosts from Australia and 3 an out-group Australian species in an unrelated crab family Grapsidae never previously reported as attacked by Sacculina spp. All crab species were used in the choice experiments in the laboratory. Because of limited numbers of a few species, a smaller range was used in the no-choice and field trials. In order to prevent accidental introductions, only male crabs were used in the field trial. All were recovered and killed at the end of the experiment. Visual inspection of the experimental crabs for attached cyprids was done by all of the authors, using dissection microscopes. Specimens were frequently reexamined by a second observer, to confirm counts. Morphological examination of the ethanol-preserved ¨ crabs for internae was done by M.W., J.T.H. and J. Lutzen at the Zoological Institute, University of Copenhagen, Denmark. Genetic tests for the presence of rhizocephalan tissue in the crabs were done at the CSIRO Marine Laboratory, Hobart, Australia, using ethanol-preserved tissue from the alimentary tract and thoracic ganglia of the hosts, where interna development is first evident. DNA was extracted from crab tissue using phenol chloroform and precipitated from aqueous solution with ethanol using a modified CTAB hexadecyltrimethyl- ammonium bromide protocol Grewe et al., 1993. The small subunit 39 region, internal transcribed spacer 1 ITS1, 5.8S, ITS2 and 59 region of the large subunit LSU ribosomal RNA rRNA was amplified by PCR polymerase chain reaction approxi- mately 1900 bp using a forward SB2 and reverse [28z Hillis and Dixon, 1991] primer. DNA was denatured at 958C for 60 s, primers annealed at 608C for 30 s with chains extended at 728C for 90 s for 30 cycles. Amplified products were purified using QIAQuick spin PCR purification kits Qiagen, Chatsworth, UK, sequenced using dye terminator reactions Perkin-Elmer, CA, USA and run on an automated sequencer ABI R .E. Thresher et al. J. Exp. Mar. Biol. Ecol. 254 2000 37 –51 41 Prism 377 DNA Sequencer. Nucleotide sequence data was collected from the 39 region of the SSU, ITS1 and partial 5.8S rRNA using two forward SB2 and SAC1 primers located in the 39 region of the SSU and one reverse SAC3 primer located in the 59 region of the 5.8S rRNA. Amplification of S . carcini was verified by alignment of rRNA nucleotide sequence data from interna, externa and larvae collected in Sweden, which were identical. Genetic screening used a species diagnostic primer designed from the ITS1 rRNA nucleotide sequence of S . carcini, which amplifies a 350-bp product from infected crabs. Details about the crabs tested are given in Table 1. We used juveniles wherever possible, on the basis that they might be more susceptible than adults to attack Høeg ¨ and Lutzen, 1995. Swedish C . maenas and a portunid not used as a host by S. carcini in Swedish waters, Liocarcinus depurator, were obtained using fyke nets in the Ellosfjor- den, Strommarna and adjacent areas. Individuals with no evident externa were held for several days prior to the experiments in 20–40-l open-flow seawater aquaria. Specimens of five species of Australian native crabs plus specimens of the introduced population of C . maenas were collected by hand at sites along the southeast coast of Australia and overnight air-freighted to Sweden. All of the shipped crabs except for Charybdis callianassa were very active and apparently healthy on arrival; all but one of the Charybdis the smallest died within 6 days of arrival. The animals were held in screened slow-drip open aquaria for 5 days prior to the experiments, during which time they were generally active and fed vigorously on fish and live mussels. In one test, we also included as an extreme out-group a native Swedish thallassinid ghost shrimp, collected from the Ellosfjorden. Sacculina carcini cyprids were obtained by rearing larvae hatched from externa- bearing C . maenas. Individual crabs, collected as described above, were placed in 3-l aquaria. The crabs were checked daily for hatching. After hatching, each brood was siphoned into an individual rearing chamber, which consisted of a 1-l glass beaker half submerged in a constant temperature 15.98C waterbath. The design of the culture system is based on Strathmann 1987. Seawater in the beakers was changed when the larvae were about 4 days old i.e. precyprid stage. Water in each beaker was stirred slowly via a small paddle, to minimise the chances of larvae being trapped by surface Table 1 Characteristics of host crabs tested for susceptibility to Sacculina carcini settlement. Note carapace width of Portunus pelagicus includes prominent lateral spines, which constitutes about 30 Species Family Carapace width Sex ratio Source location mm male female Carcinus maenas Portunidae 15.6–26.2 6 5 Ellosfjorden, Sweden Carcinus maenas Portunidae 15.1–25.3 3 7 Tasmania, Australia Ovalipes australiensis Portunidae 19.3–32.3 6 0 Tasmania, Australia Portunus pelagicus Portunidae 28.6–42.0 6 0 South Australia, Australia Charybdis callianassa Portunidae 41.2 1 0 Queensland, Australia Nectocarcinus integrifrons Portunidae 16.1–19.8 2 0 Victoria, Australia Liocarcinus depurator Portunidae 48.1–51.3 2 0 Ellosfjorden, Sweden Paragrapsus gaimardii Grapsidae 14.5–20.6 10 0 Tasmania, Australia 42 R .E. Thresher et al. J. Exp. Mar. Biol. Ecol. 254 2000 37 –51 tension. In most cases, we used larvae from a single brood for each experiment, in order to avoid confounding effects of variable sex ratio which can differ between broods from all male to all female, with only the females parasitic and larval condition Walker, 1985. In all cases, the only broods used were either predominantly or entirely female based on morphological examination of a sample of cyprids and in good health, as indicated by vigorous swimming activity. 2.2. Heterosaccus lunatus trials Experiments were conducted at the University of Queensland, Brisbane, Australia, in July August, 1996. Protocols generally follow those described above. Heterosaccus lunatus cyprids were obtained by rearing larvae from externa-bearing Charybdis callianassa, collected by trawling in Moreton Bay, Queensland. The crabs were held in closed-circulation aquaria, fed once or twice weekly and inspected daily for darkening of the externa, which indicates imminent hatching. Once darkening had occurred, the parasitised crabs were isolated in small aerated aquaria and inspected hourly. Newly hatched nauplii were collected by attracting them to a light and pipetting them out. One hundred larvae from each batch were measured to determine sex ratio. Only broods of . 50 female were used in the trials. The experiments were conducted using juvenile, non-externa-bearing crabs, ranging in carapace width from 2.5 to 3.5 cm Charybdis callianassa and 1.5–2.5 cm C . maenas. Charybdis callianassa were collected by trawling in Moreton Bay; the juvenile C . maenas were collected by hand in Tasmania and air-freighted to Brisbane. 2.3. Statistical analysis Significance in experiments involving comparisons among multiple host species was tested using analysis of variance. Pair-wise comparisons were done using t-tests. Procedures follow Sokal and Rohlf 1981.

3. Results