Larval Rearing of Giant Crabs Pseudocarcinus gigas (Lamarck). Aquaculture,
Abstract
Chemicals were screened for prophylactic treatment of epibiotic fouling and fungal mycosis in larvae of giant crabs Pseudocarcinus gigas. The following treatments were applied as indefinite baths: oxytetracycline, trifluralin, carbendazim, copper oxychloride, malachite green, and formalin. Most effective treatments for improving survival were oxytetracycline (25 mg l-1, despite increased deformity), trifluralin (0.01 mg l-1), carbendazim (0.001 mg l-1),
and copper oxychloride (0.05 mg l-1). Three of these treatments affected size and shape of
the megalopa carapace with relatively smaller megalopas developing in carbendazim and trifluralin, and relatively broader megalopas in copper oxychloride. Toxic effects, measured by increased mortality, deformity, death during ecdysis, or delayed ecdysis, were recorded with oxytetracycline (≥25 mg l-1), trifluralin (≥0.03 mg l-1), malachite green (≥0.1 mg l-1),
and formalin (for all concentrations tested: ≥2.5 mg l-1).
Introduction
Optimising survival during larval rearing of crustaceans is important in aquaculture and is also of benefit in research of crustacean taxonomy, physiology, and fisheries biology. Laboratory cultures of crab larvae often suffer severe mortality from disease, particularly from epibiotic bacteria and larval mycosis (Armstrong et al., 1976; Ebert et al., 1983; Hamasaki and Hatai, 1993). Larval mycosis occurs when fungal hyphae, commonly Lagenidium and Sirolpidium species (Brock and LeaMaster, 1992), invade body tissues,
developing zoospore discharge tubes which protrude through the crustacean’s cuticle in the later stages (Fig. 1; Paynter, 1989). Infected larvae become immobilised and their surface develops a fouled appearance from the external processes of the fungi.
Figure 1. Fouling diseases on giant crab zoeas. A stalked fungal sporangium is present in the center and filamentous bacterial fouling is visible as fine threads.
In research involving culture of giant crab larvae, larval mycosis has consistently caused high mortality which prompted assessment of the various chemical controls available. The pelagic zoeal phase of P. gigas is relatively long, around 60 days, and larval mycosis occurs throughout this period, rather than just in early stages as is more typical in crustaceans (Brock and LeaMaster, 1992; Lightner, 1993). Consequently, prophylactic treatments must be administered for prolonged periods which can produce toxic effects, not evident in short term trials, such as delay in moulting (Caldwell et al., 1978) and deformity at megalopa (Ebert et al., 1983).
Prophylactic treatments are often assessed by separately establishing toxic levels of a given chemical for fungal zoospores and for the larvae to be treated (Armstrong et al., 1976; Lio- Po et al., 1982; Lio-Po and Sanvictores, 1986). While this is an invaluable technique for evaluating treatments rapidly, it can fail to assess the value of a treatment in culture situations. Adverse effects of the treatment may be underestimated because chronic toxicity can become apparent late in development and there may be interaction with bacterial species (Gil-Turnes and Fenical, 1992).
In this chapter results are presented from prolonged clinical trials with giant crab larvae comparing several chemicals reported to immobilise fungal zoospores: malachite green (Armstrong et al., 1976); trifluralin (Armstrong et al., 1976); and formalin (Hamasaki and
Materials and methods
Source of larvae
Ovigerous females were collected from depths in the range of 300 to 380 m off the east coast of Tasmania (41°17'S; 148°40'E) in June 1995. Females ranged in size from 2.2 to 3.5 kg and were held communally in 4 m3 tanks with flow through, unfiltered water supply.
To ensure that only freshly hatched larvae were used, the tanks were thoroughly flushed prior to collecting larvae for the trial. Although attempts were made to collect larvae from several females, most of the larvae used for this trial appeared to be from one female weighing 2.6 kg.
Culture methods and experimental design
Newly hatched larvae were rinsed in 0.2 µm filtered seawater (34‰ salinity) then
transferred to 1.8 l vessels. Fed controls, starved controls, and chemical treatments were randomly allocated to vessels. Fifty larvae were placed in each vessel and were maintained in a temperature control room at 16°C with 700 lux, 8 l :16 d photoperiod. Zoeas were fed Protein Selco™ enriched artemia nauplii but megalopas required larger, 10 day old artemia. Larvae were pipetted into fresh, 0.2 µm filtered seawater every 2 days. The concentrations for each chemical tested to control fouling were: carbendazim (Hoechst and Schering™), malachite green, and trifluralin (DowElanco™) at 0.001, 0.003, 0.01, 0.03, and 0.1 mg kg-1;
copper oxychloride (ChemSpray™) at 0.025, 0.05, 0.1, 0.2, and 0.4 mg kg-1; formalin at 2.5,
5, 10, 20, and 40 mg kg-1; and oxytetracycline (Norbrook™) at 10, 25, 50, 100, and 200 mg
kg-1. Four replicates were used for each concentration. Treatments were run as indefinite
baths for 115 days after which all live animals were censored in survival analyses.
Response data collected
The effect of different concentrations was monitored by recording the number and instar of exuvia and mortalities which allowed calculation of survival and time to each moult. All
mortalities were also classed into possible causes of death: fouled (severe fouling of exoskeleton), moulting (died during ecdysis), deformed, and normal (where no cause was apparent). Larvae were classed as fouled when at least 50% of the external surface was covered. Epibiotic bacterial fouling increased rapidly after death, so the fouling index only gives a general indication of disease. When dead larvae could be ascribed to more than one possible cause of mortality, such as moulting and fouled, each cause was counted and treated as independent. Although wet preparations and microbiology were performed, the cause of fouling was not determined in all cases; animals classed as fouled may have had fungal or bacterial infections. Microbiological cultures were made on TCBS, blood, and Ordal’s medium.
In normal development, giant crab larvae pass through 5 zoeal stages (Gardner and Quintana, 1998; Chapter 3). In this study, many larvae developed to a sixth zoeal stage, which was intermediate between zoea 5 and megalopa (termed 5-a-lopae by Ebert et al., 1983; Fig. 2). Chelae were present and the pleopods bore setae, while the carapace retained the form of the zoea. None of these larvae survived and they were classed as deformed.
The effect of treatments on size of dead megalopas or megalopa exuvia was assessed by measuring carapace length, carapace width, and rostrum width (Fig. 3). The shape of the rostrum varied between individuals so carapace length measurements were made slightly off centre. All measurements of megalopas were made by image analysis using NIH- Image™ 1.6 software.
Figure 2. Giant crab larva intermediate between zoea 5 and megalopa. As with a megalopa, the chelae are differentiated and the pleopods are setose, however zoeal characters such as the dorsal carapace spine and bifurcated telson have been retained. These larvae were classed as deformed.
Figure 3. Morphological measurements taken to assess the effect of treatment on megalopa size. RW = rostrum width, CW = carapace width, CL = carapace length. Note that there was considerable variation in the form of the rostrum so CL was measured slightly to the left of center.
Statistical analysis
Statistical analysis was performed with JMP 3.0™ software (SAS Institute). Where comparisons between treatments and controls are presented, the controls are the fed group. The effect of concentration on moult timing was tested with repeated measures analysis. As survival was poor in some treatment replicates, analysis was restricted to data collected from the start of the trial to the moult from zoea 3 to zoea 4. Data were arranged for multivariate analysis and significance determined for between effects (concentration) with Wilk’s lambda (Mardia et al., 1979). Where a significant effect of concentration was found, comparisons were made between concentrations by analysis of the canonicals (Mardia et al., 1979).
Survival data were analysed by the Kaplan-Meier method with significance between groups determined by Wilcoxon’s test (Miller, 1981). To prevent an increase in type 1 errors, a Bonferroni adjustment was made to alpha for comparisons between concentrations, so that comparisons were only treated as significant where P<0.003 (Sokal and Rohlf, 1995). The effect of treatments on size of megalopas and cause of mortality was assessed by one way ANOVA. Cause of mortality data were arc-sine square-root transformed to produce normality and remove heteroscedasticity. Where a treatment effect was observed, means were compared by Tukey Kramer HSD (Sokal and Rohlf, 1995). Percentage survival to crab 1 data are presented although low survival prevented meaningful statistical analysis.
Results
Larvae classed as fouled appeared to be afflicted with several types of infection. Peritrich ciliates were present in low numbers and were not regarded as pathogenic. Apart from larvae cultured in oxytetracycline, mixed bacterial flora consisting predominantly of Vibrio
Larvae in starved controls remained alive for a mean time of 18.8 days although all were dead by day 20 (survival data from starved and other treatments are presented in Appendix 8). A small number of these successfully moulted to zoea 2 (1.5%). Larvae from both starved and fed controls had low level surface infections of mixed Vibrio spp. The proportion of larvae which died with high levels of fouling were significantly higher (P<0.001) in fed controls than in starved controls: 45.5% compared with 10.5%.