Prophylactic use of antibiotics in larval culture of Argopecten ventricosus (Sowerby, 1835).

ABSTRACT Chloramphenicol, erythromycin and furazolidone were used in larval culture of Argopecten ventricosus at 4 concentrations: 0.5, 1.0, 3.0 and 6.0 mg/L to evaluate larval survival and prophylactic effect of these antibiotics. The use of concentrations higher than 1.0 mg/L induced a significant (P < 0.05) increase in larval survival. The bacteriostatic effect of antibiotics was studied with direct and indirect bacterial count methods. Total bacteria and Vibrio spp. showed a reduced density in culture tanks (55.6% and 98.1%, respectively), when larvae were treated with 6.0 mg/L of chloramphenicol and erythromycin. The results of the study indicate that 6.0 mg/L of chloramphenicol and erythromycin enhance larval condition and survival, reducing potential pathogens in culture tanks.

KEY WORDS: antibiotics, larvae, Argopecten ventricosus, Vibrio spp., survival, scallop 

INTRODUCTION

In the past 15 years in Mexico, efforts have been made to develop aquaculture technology for farming A. ventricosus (= circularis) and other bivalve mollusks of commercial importance. However, there is abundant evidence that bacterial contamination can cause high mortality during larval culture (Riquelme et al. 1995, Lambert et al. 1998, Jorquera et al. 2001, Le Roux et al. 2002). Source of opportunistic pathogens in culture tanks are from seawater, air supply, brood stock and microalgae (Sainz et al. 1999). Vibrio species are the main cause of disease in mollusk larval cultures, such as Vibrio anguillarum (Riquelme et al. 1995), V. natriegens (Zhang et al. 1998), V. pectenicida (Lambert et al. 1998), V. tapetis (Borrego et al. 1996), V. splendidus (Sugumar et al. 1998, Gatesoupe et al. 1999) and V. alginolyticus (Riquelme et al. 1996), followed by Pseudomonas sp. and Aeromonas sp. (Lodeiros et al. 1992, Riquelme et al. 1996). Vibrio density is often present at densities of less than 1 x [10.sup.3] cells/mL in the sea (Utting 1986). However, pathogen bacteria may reach 1 x [10.sup.6] cells/mL in culture tanks (Austin et al. 1988) because of their ability to grow in an artificial environment enriched by nutrients (Sindermann 1990).

To enhance survival, rigorous cleaning and aseptic conditions are required. Careful control of water temperature, salinity, pH, stocking densities and nutrition are also important (Beiras et al. 1994, Inglis 1996). Normally, periodic changes of filtered, sterilized seawater (filters from 10-1 [micro]m and UV radiation) prevent bacterial infections in larval cultures. Antibiotics also routinely accompany these preventive measures. The requirement for antibiotic use in a culture depends mainly on the quality of the water (Toranzo 1990). Antibiotics commonly used in aquaculture are furazolidone, chloramphenicol, estreptomycin, erythromycin, kanamycin, oxytetracycline, neomycin and oxolinic acid (Benbrook 2002). Antibacterial chemotherapy has been applied in aquaculture for over 50 years (MacMillan 2001). However, there is widespread concern that antibacterial agents used in aquaculture have led to the emergence of and selection for resistant bacteria. An improved understanding of how resistance emerges and is selected for among bacteria is essential in evaluating the impact in aquaculture of antibacterial agents, identifying high risk procedures and designing ways to reduce these effects. Bacteria acquire resistance by acquisition of foreign DNA or by modification of the chromosomal DNA (Inglis 1996). Despite the risk of selecting bacterial resistant strains, chloramphenicol is the most frequently used antibiotic in hatcheries (Uriarte et al. 2001). Disalvo et al. (1984) were the first to report the use of antibiotics with cultures of Argopecten purpuratus, using 25 mg/L chloramphenicol when bacterial infection was suspected.

This study focuses on determining the dose of commercial antibiotics for enhancing larval A. ventricosus survival rates and evaluating susceptibility of bacteria, associated with larval culture, to antibiotics.

MATERIALS AND METHODS

Broodstock

Healthy adults (shell length, 58.9 [+ or -] 3.4 mm) were collected from a culture facility in Bahia de La Paz Bay near La Paz, Baja California Sur, Mexico and transported to the Universidad Autononoma de Baja California Sur hatchery. They were conditioned for spawning for at least 20 days in a 1,500-L fiberglass tank with 0.2 [micro]m filtered seawater containing 10 mg/L of EDTA under constant aeration at 24 [+ or -] 1.0[degrees]C and 36 psu. Filtered seawater was maintained at pH 7.8-8.2. Broodstock were fed 1.5 x [10.sup.5] cells/mL of a mixture of Isochrysis galbana, Chaetoceros calcitrans and Chaetoceros gracilis (1:1:2) algae. Tank water was changed 50% daily.

Antibiotics

Furazolidone (Roberts Laboratories), chloramphenicol (Mediatech, Inc.) and erythromycin (Abbott Laboratories) were tested in larval culture tanks at 4 concentrations:0.5, 1.0, 3.0 and 6.0 mg/L.

Larvae

Broodstock were placed individually in 5-L plastic containers with filtered seawater (1-[micro]m filter) and were induced to spawn by thermal shock, changing from 18[degrees]C to 28[degrees]C during 20 to 30 minutes at each temperature. After embryonic development, the resulting larvae were cultured in 5,000-L fiberglass conical tanks (5 larvae/mL) with filtered seawater at 24[degrees]C [+ or -] 1.0[degrees]C and 36 psu. A complete water exchange was made every 3 days. Larvae were fed 3.0 x [10.sup.5] cells/mL of L galbana and C. calcitrans (1:1).

Experimental Protocol

Treatment groups of larvae were cultured in 60-L fiberglass tanks at a density of 15 larvae/mL. Larvae were fed 3.0 x [l0.sup.5] cells/mL of L galbana and C. calcitrans (1:1). Duplicate groups of larvae were treated with 0.5, 1.0, 3.0 or 6.0 mg/L of furazolidone, chloramphenicol or erythromycin for 10 days from the early trochophora larvae to the pediveliger stage. A control group was cultured in filtered seawater free of antibiotics. Temperature and salinity were recorded daily. Culture tank water was changed daily every 48 hours. Concentrations of antibiotics in culture tanks were restored with every complete seawater change; mortality and bacterial counts were recorded at day 1, 3, 5, 7 and 9 of larval culture.

Bacterial Count

Direct and indirect methods were used to count bacteria in culture tanks. The indirect method was by plate count using TCBS and marine agar medium to determine density of Vibrio spp. and total bacteria, respectively, and plates were incubated at 30[degrees]C for 24 h. The direct method used epifluorescence microscopy, with samples taken directly from the treated and untreated tanks, filtered and immediately counted under the microscope and expressed as colony forming units per mL (CFU/mL).

Statistical Analysis

Data were analyzed by ANOVA, using the F test to analyze the differences. Values of P < 0.05 were considered significantly dif ferent. Where significant differences were found, Tukey HSD test was used to identify the nature of these differences (P < 0.05). The analytical software was STATISTICA (StatSoft, Inc.).

RESULTS

Larval Survival

Figure 1 shows larval survival of A. ventricosus in response to 0.5 mg/L of antibiotics. The highest survival was obtained using erythromycin (73.31%) followed by furazolidone (63.79%) and chloramphenicol (50.16%). Larvae treated with erythromycin showed a significantly higher (P < 0.05) survival than controls. A decrease in survival occurred in treatments and controls between day 7 and 9 of culture, but the specimens were collected in good condition before settling (active swimming on the column of water, larvae sinking to the bottom with active movement of cilia and full gut).

[FIGURE 1 OMITTED]

Larvae exposed to antibiotics at 1 mg/L showed a significantly higher (P < 0.05) survival than the controls. Higher survival was found with chloramphenicol (53.2%), followed by furazolidone (42.8%) and erythromycin (42.6%). There were not significant (P > 0.05) differences in survival between treatments. Mortality of 40% to 70% occurred in untreated and treated groups at day 5, except in groups treated with erythromycin (Fig. 2). Larvae treated with chloramphenicol and erythromycin showed better growth, and pediveliger eyespot was observed 48 h earlier than in larvae treated with furazolidone and control.

[FIGURE 2 OMITTED]

At 3 mg/L, significantly higher (P < 0.05) survival occurred in treated than in control groups (Fig. 3). At this concentration, chloramphenicol induced the highest survival (65.7%), with furazolidone (58.3%) and erythromycin (54%) in second positions. Larvae treated with antibiotics at 3 mg/L enhanced larval condition than controls.

[FIGURE 3 OMITTED]

Exposure of A. ventricosus to antibiotics at 6 mg/mL induced a significantly high survival compared with the control group (Fig. 4). Progressive mortality occurred during all bioassays in the untreated (control) groups and until day 5 in treated groups. Groups treated with chloramphenicol achieved 69.83% survival, erythromycin at 68.63% and furazolidone at 59.06%. Larvae exposed to chloramphenicol and erythromycin at 6 mg/mL showed a better condition than larvae treated with furazolidone and control group. Furazolidone induced deformity and low growth in larvae.

[FIGURE 4 OMITTED]

Figure 5 summarizes survival at antibiotic exposures from 0.5-6 mg/L. There was not significant (P > 0.05) difference in survival between the several concentration treatments. Statistically speaking, we can use any antibiotic at any concentration between 0.5 and 6 mg/L, but this graph indicates a tendency toward increased survival when the concentration of antibiotics is increased, reaching the greatest difference at 6 mg/L. Larvae exposed to furazolidone did not show a steady increase in survival with dosage, instead there were particular health conditions, such as, delayed growth, velar deformations and empty gut (data not shown).

[FIGURE 5 OMITTED]

Bacterial growth

Based on larval survival and health conditions, a bioassay of bacterial growth in culture tanks of larval specimens exposed to 6 mg/L chloramphenicol or erythromycin was performed. Larvae exposed to chloramphenicol or erythromycin at 6 mg/L induced a significantly higher (P < 0.05) survival than control groups (Fig. 6). Figure 7 shows in vitro growth of total bacteria (indirect count) treated with 6 mg/L of antibiotics. Total bacteria decreased about 55.6% over control groups (Fig. 7). Similarly, Vibrio spp. showed a significant lower growth, 98.1%, than control groups after exposure to antibiotics, almost 0 CFU/mL in treated tanks (Fig. 8).

[FIGURE 6-8 OMITTED]

Direct bacterial count determined by epifluorescence microscopy (Fig. 9), showed higher numbers than those measured by the indirect method (Fig. 7 and Fig. 8). Bacterial density from treated tanks reached 5.5 x [10.sup.9] bacteria/mL at day 5, higher than control tanks (Fig. 9). At the end of the bioassay, bacteria from treated tanks contained 6 x [10.sup.9] bacteria/mL, lower than the control tanks (Fig. 9).

[FIGURE 9 OMITTED]

Apparently, antibiotics did not reduce bacterial content in tanks and the epifluorescence method did not determine the specific bacteria growing in the tanks. Nonpathogen strains may grow in tanks, but did not grow in vitro. Bacterial content at days 5 and 7 remained constant, perhaps restricted by the particular physicochemical conditions in tanks, such as nutrient availability or temperature. In control tanks, total bacteria content were higher than treated tanks at day 7, probably related to the diversity of strains present. In treated tanks, diversity of bacterial strains is reduced.

DISCUSSION

Despite many years of research, there are still many problems in hatchery production in bivalve species, especially related to higher mortality of larval A. ventricosus from early trochophora to pediveliger stages, attributed to the presence of pathogenic bacteria.

In comparing survival rates obtained from applications of antibiotics to those obtained with filtered water, our results showed better survival rates when larvae were treated with chloramphenicol and erythromycin, without significant differences in larval development with increasing concentrations of the antibiotics. Treatments with furazolidone at 6 mg/L showed lower survival than larvae treated with chloramphenicol and erythromycin, exhibiting abnormal morphology and empty gut at day 9 of culture. Braley (1986) reported higher survival in Tridacna gigas treated with 5-7 mg/L of erythromycin, oxytetracycline, streptomycin and chloramphenicol during the first 2 days of larval development. Fitt et al. (1992) studied the effect of streptomycin, neomycin, penicillin and rifampin in scallop larvae F. tridacnidae, reporting optimal concentration of 10 mg/L. Robert et al. (1995) used chloramphenicol and erythromycin at 8 mg/L to enhance larval survival of Pecten maximus. Uriarte et al. (2001) found that 2 and 8 mg/L chloramphenicol provided effective control of contamination of larval Argopecten purpuratus. Moreover, the health of postlarvae was improved with chloramphenicol at 8 mg/L for eyed larvae to postlarvae. Riquelme et al. (1996) suggested that larvae are initially attacked by exotoxins released by V. alginolyticus and the later proliferation of pathogenic bacteria, such as Aeromonas hydrophila. Because V. alginolyticus is sensitive to chloramphenicol (Riquelme et al. 1996), and our results showed increased survival when the antibiotics were tested, we can infer that Vibrio was likely the prevalent bacteria present in larval cultures of A. ventricosus. Although the use of antibiotics may be beneficial to avoid pathogens in larval cultures, its use must be studied with more care (Araya et al. 1999). An important reason for concern is the evidence of secondary effects from antibiotics, such as bacterial resistance, which could increase pathogenicity (Ervik et al. 1994). Applications of antibiotics are advised only when there are positive results for Vibrio in the culture tanks (Uriarte et al. 2001).

In Europe, United States, Mexico and some other countries, some antibiotics are not permitted in food animals because of the known toxicity of the drug to humans; either an improvement in rearing procedures or substitution products are recommended (Benbrook 2002). However, treatment of mollusk larvae with antibiotics may not be relevant when an effluent treatment system is used to prevent environmental contamination and the 2 mm cultured seed is used to grow shellfish to commercial size at sea (Uriarte et al. 2001). Lower concentrations of chloramphenicol and erythromycin have been considered, particularly, erythromycin seemed to be a satisfactory treatment (Robert et al. 1995). However, further studies need to be done to selected agents for bactericidal rather than bacteriostatic activity, selective toxicity for bacteria, activity and stability in seawater. Alternatively, nontoxic antibiotics approved for use in United States aquaculture, such as oxytetracycline and oxolinic acid, are reported to generate satisfactory results (Fitt et al. 1992, Young 2002).

With the epifluorescence method, bacterial growth appeared greater than with the marine agar method because bioassays in tanks were immediate and all live bacteria were countered. In the second method, not all bacteria survive and grow in the medium. Thus, only bacteria that could grow in marine agar medium could be countered.

The epifluorescence method provides information about bacterial density in culture tanks in 2 or 3 hours, and this is the main advantage of direct counting. No differences in bacterial growth were found between controls and treatments using the epifluorescence method because growth of some bacteria, such as Vibrio, were inhibited and that "space" in the whole population could be used by other bacteria or strains, which are not sensitive to antibiotics, to grow in the tanks; strains are not a source of disease for larvae (Jorquera et al. 2001).

This study showed the importance of decreasing bacterial populations in culture tanks, mainly those related to Vibrio spp., as also reported by Lodeiros et al. (1992), Gatesoupe et al. (1999), Le Roux et al. (2002). It is important to note the beneficial role of bacteria as food for larvae (Amouroux & Amoroux 1986, Douillet & Langdon 1993). The axenic cultures are not the best way to increase larval survival, but they may be better to maintain the optimal relationships between bacteria and larvae. Until now, antibiotics have been an important tool for decreasing the incidence of disease in the aquaculture industry, but alternative prophylactic methods should be studied, such as the use of immunostimulants and probiotics (Avendano & Riquelme 1999).

ACKNOWLEDGMENTS

We thank Claudia Perez-Estrada for technical support and the editor at CIBNOR for improving the English text. This work was supported by Centro de Investigaciones Biologicas del Noroeste (CIBNOR grant AC-1.20) and by Mexico's Consejo Nacional de Ciencia y Technologia (CONACYT graduate fellowship 90778) to A.I.C.C.

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A. I. CAMPA-CORDOVA, (1), * A. LUNA-GONZALEZ, (2) M. ZARAIN-HERZBERG (3) AND C. J. CACERES-MARTINEZ (4)

(1) Centro de Investigaciones Biologicas del Noroeste (CIBNOR). Mar Bermejo 195, Col. Playa Palo de Santa Rita. La Paz, B.C.S. 23090, Mexico; (2) Centro Interdisciplinario de Investigacion para el Desarrollo Integral Regional, Unidad Sinaloa. Km 1 Carretera a Las Glorias, Guasave, Sinaloa, Mexico; (3) Centro de Ciencias de Sinaloa, Apdo. Postal 1889, Culiacan, Sinaloa. Mtxico; (4) Universidad Autonoma de Baja California Sur (UABCS), Unidad Experimental de Maricultura, Carretera a Pichilingue, Km. 14.5. Apdo. Postal 19-B, La Paz, B.C.S. Mexico