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
3.1. Sacculina carcini: experiment No. 1 — no-choice exposure in the laboratory 3.1.1. Methods
One juvenile of each of the five most abundant samples Swedish and Australian C .
maenas, Portunus pelagicus, Ovalipes australiensis and Paragrapsus gaimardii were individually placed into 1-l beakers in a constant temperature 15.98C. culture unit.
Beakers were gently stirred every 4 s using mechanical stirring rods, to prevent cyprids from adhering to the surface film, and they were kept in a 12:12 h light–dark cycle. The
infection trial ran overnight. At approximately 1500 h on day 1, a brood of actively swimming 10-day post-hatch cyprids was apportioned into each of the five beakers,
resulting in several hundred larvae in each. The cyprids were estimated to be . 95 female. The crabs were removed from the beakers and checked for attached cyprids later
R .E. Thresher et al. J. Exp. Mar. Biol. Ecol. 254 2000 37 –51
43
that afternoon 1600 h and again at noon the next day. Afterwards, all five crabs were transferred to the long-term rearing facilities, a second set of juveniles placed into the
beakers which still were conspicuously full of swimming cyprids, and the experiment repeated.
3.1.2. Results Cyprids were found attached to three of the five taxa tested, but attachment rates were
extremely low. Among the ten crabs tested and the thousands of cyprids present, we found only five attached cyprids: all four of the C
. maenas two Swedish, two Australian tested each had one attached cyprid on them and one cyprid was found
attached to one of the P . gaimardii.
3.2. S. carcini: experiment No. 2 — choice exposure in the laboratory 3.2.1. Methods
Individual crabs were placed in 1-cm square-mesh green plastic bags, along with a small granite rock to make the bags sink. Each bag was about 15 cm long 3 9 cm
wide 3 6 cm high, large enough to allow the crabs a small amount of mobility. The captive crabs were placed into a recirculating flume tank, in one or more rows
perpendicular to the current flow. Positions were randomized among samples, and recorded to check for position effects. The flume tank was 300 cm long 3 48 cm wide
and filled to a depth of 22 cm with clean seawater 32 ppt at the beginning of the experiment. Current speed was set at 1 cm s.
In the first trial, five specimens were tested a Swedish and an Australian C . maenas,
plus one each O . australiensis, Portunus pelagicus and Paragrapsus gaimardii and
checked daily for cyprid attachment. At the beginning of the experiment day 1, several thousand, very actively swimming 7-day post-hatch cyprids were added. Two broods
were used, both estimated to be . 95 female. When after 3 days the first attached cyprids were found, two more specimens of each crab taxa were added, along with our
only remaining specimens of N
. integrifrons and Charybdis calliannassa, and two L. depurator. We also added several thousand more cyprids, 10-days post-hatch. All crabs
were checked daily for the next 2 days, by which time the number of active cyprids in the tank was declining sharply and the trial was terminated day 6.
A second trial was started immediately, with fresh, 9-day post-hatch larvae. As the specimens of N
. integrifrons, C. callianassa and L. depurator had no attached cyprids from the first trial, they were placed back into the flume, along with fresh specimens of
the other samples. We also added a small thallassinid ghost shrimp as an extreme out-group sample. The second trial was run for 24 h.
All Carcinus and Australian crabs were transferred to holding facilities at the end of the trials.
3.2.2. Results Pooled over the two trials, S
. carcini cyprids attached to 7 of the 8 species tested, including the thallassinid Fig. 1. The highest rate of settlement 4.7 newly attached
cyprids per specimen per day for species tested over more than one day was on the
44 R
.E. Thresher et al. J. Exp. Mar. Biol. Ecol. 254 2000 37 –51
Fig. 1. Number of cyprids found attached to individual crabs in a single night in the flume tank, compiled over the four nights of the experiment. Values specify the number of individuals at the indicated level of infestation.
Swedish C . maenas, and the lowest no cyprids over 2 days trialed for the Australian N.
integrifrons. However, overall the number of attached cyprids did not differ significantly among crab species Anova F
5 0.52, NS. Reflecting this, maximum attachment
7,35
rates were also similar between C . maenas and other species: up to 12 attached cyprids
attached in a single night to specimens of C . maenas, Portunus pelagicus and L.
depurator, 10 on a single Paragrapsus gaimardii, and 8 on O . australiensis. Eight
cyprids were also found attached to the thallassinid. The distribution of attached cyprids among crabs was extremely patchy. The number
of attached cyprids could not be related to the sex, size carapace width or location of the crab in the flume tank statistics for all individual parameters not significant. The
number of attaching cyprids varied with time age of the cyprids in the first trial, with no settlement among cyprids less than 10 days post-hatch. The host range was identical at
the beginning and end of the settlement period, indicating no reduction in specificity as the cyprids neared the end of their competent period.
3.3. S. carcini: experiment No. 3 — choice exposure in the field 3.3.1. Methods
To test specificity under approximate field conditions, specimens of Paragrapsus gaimardii, O
. australiensis, and Australian and Swedish C. maenas were suspended in cages in the harbour of KMRS. Each taxon was in a separate cylindrical mesh 1-cm
square plastic cage, 40 cm high 3 20 cm diameter, each containing six specimens. Two
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45
weeks into the experiment, the crabs were individually confined in nylon mesh bags 12 3 6 3 4 cm inside each cylinder, to prevent cannibalism. A few specimens lost
before then were replaced. The cages were suspended just above the bottom in water 3 m deep, and separated
from one another by 0.5–1 m. There is little discernible tide in the area, so current flow under the bridge was slight. The experiment was conducted from 17 September to 31
October, during which time the crabs were checked for cyprids at 3–4 day intervals. They were fed blue mussels Mytilus edulis weekly. The natural infection rate by S
. carcini at four localities close to the test site, based on the frequency of scarred and
feminized crabs, ranged from 3.4 to 29.5 with the highest rates in inshore waters.
3.3.2. Results We found one attached cyprid on each of two out of the four Australian C
. maenas and on two out of the four P
. gaimardii tested. This equates to an overall attack rate of 16.7.
3.4. S. carcini: development of interna The sole Charybdis callianassa and all Portunus pelagicus died by February, 1997.
Both are subtropical species and appeared to be unable to tolerate the low seawater temperatures in Sweden, even in the laboratory. As well, two C
. maenas one Swedish and one Australian specimen, two O
. australiensis and the last remaining N. integrif- rons also died between 6 and 12 months after the onset of the trials. The remaining
specimens 27 individuals in three species, all of which fed and molted regularly, were killed in April 1998 19 months after attachment. Specimens were frozen after death
and then transferred to ethanol, or preserved immediately in ethanol. All were examined morphologically for evidence of interna. Genetic tests were subsequently conducted on
all but the C
. callianassa and P. pelagicus. Externae developed on three reared crabs — one Swedish and two Australian C
. maenas — approximately 12 months after the experiments. Morphological examination
of these and the other specimens found S . carcini interna in only these three individuals.
There was no sign of roots in any of the Australian native species, irrespective of attack rates by rhizocephalan cyprids in the laboratory. None of the crabs exposed to S
. carcini in the field developed visible externa or interna.
Genetic screening correctly identified all three specimens in which interna could be discerned macroscopically. However, it also detected rhizocephalan genetic material in
three other specimens. One of these a Swedish C . maenas died early in the rearing
period, and was not examined morphologically for interna. The second and third specimens were O
. australiensis, and had been inspected. Neither showed any morphological sign of infection. The genetic signature for S
. carcini was weak in both specimens, but unambiguous Fig. 2 and confirmed by a repeat analysis. There was no
morphological or genetic indication of parasitism in the two O . australiensis or the N.
integrifrons that died prior to the end of the rearing period.
46 R
.E. Thresher et al. J. Exp. Mar. Biol. Ecol. 254 2000 37 –51
Fig. 2. Electrophoretic banding for experimentally infected crabs. For details, see text. Key to symbols: 1, 100-bp DNA ladder Gibco BRL; 2, Paragrapsus gaimardii; 3, Ovalipes australiensis; 4, Swedish Carcinus
maenas; 5, Experimental Australian C . maenas; 6, uninfected Australian C. maenas control; 7, positive
control Sacculina carcini ; 8, negative control no DNA.
Of the 24 specimens attached to S . carcini in the laboratory, the genetic test indicated
6 developed infections: 2 of 5 Australian C . maenas, 2 of 6 Swedish C. maenas, 2 of 6
O . australiensis, and none of either the 6 Paragrapsus gaimardii or the single N.
integrifrons trialed. In the field, the only specimen infected was a Swedish C . maenas.
Overall the rate of infection of C . maenas 4 out of 15 specimens examined genetically,
both source locations pooled did not differ significantly from that of portunids other than Carcinus 2 out of 9 and the only non-portunid tested, Paragrapsus gaimardii 0
2
out of 10 x 54.21, df52, NS. The infections of Swedish C. maenas might reflect
infestation prior to the experimental work, but even if so, deletion of those specimens from the analysis does not change the conclusions drawn.
Among the laboratory-exposed specimens, the number of attached cyprids was a poor predictor of whether or not the crabs developed interna differences in attachment rate
between infected and un-infected crabs, unpaired t 51.31, NS. Restricting the com- parison to only the two species infected C
. maenas and O. australiensis and further restricting it to only crabs on which we found cyprids did not improve the relationship
t 51.12, NS. There were also no differences between infected and un-infected crabs in
2
terms of mean size carapace width t 50.44, NS, sex ratio x 53.67, df51, NS or
the age of the cyprids when they were attacked t 50.10, NS.
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47
3.5. Heterosaccus lunatus 3.5.1. Methods
Two sets of no-choice experiments were conducted. In the first, whole juvenile crabs 3 Charybdis callianassa and 4 C
. maenas were placed for 1–2 h in individual beakers 200–500 ml. containing several hundred day-2 cyprids. Unlike S
. carcini, female cyprids of H
. lunatus attack their hosts via the gills Walker, 1999. Consequently, after the experimental exposure, the crabs were immediately dissected, their gills removed to
individual cavity blocks containing clean seawater and attached cyprids counted using a dissecting microscope. Cyprids were considered attached if they could not be easily
brushed off the gill. The second experiment was similar to the first, but the cyprids were exposed to only fresh gill filaments five each C
. callianassa and C. maenas suspended in the beakers for approximately 1 h.
3.5.2. Results Two hundred and forty-three cyprids were found attached to the gills of the three C
. callianassa; none were found attached to the gills of C
. maenas. The difference between species is highly significant at P ,0.01 t 54.19, df55. Cyprids were most often found
attached to the more posterior gills. In the trials using excised gills, 29 cyprids were found attached to the five C
. callianassa gills, whereas none were found on the five C. maenas gills.
4. Discussion
