Effectiveness of antifungal treatments during artificial incubation of the signal crayfish eggs (Pacifastacus leniusculus Dana. Astacidae)

Aquaculture 257 (2006) 257 – 265 www.elsevier.com/locate/aqua-online

Effectiveness of antifungal treatments during artificial incubation of the signal crayfish eggs (Pacifastacus leniusculus Dana. Astacidae) P.M. Melendre, J.D. Celada ⁎, J.M. Carral, M. Sáez-Royuela, A. Aguilera Dpto. Producción Animal II, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain Received 23 November 2005; received in revised form 19 December 2005; accepted 24 February 2006

Abstract In artificial incubation of astacid crayfish eggs, the use of effective antifungal treatments is advisable for controlling the spread of fungi from dead to healthy eggs and increasing final efficiency rates. The aim of this study was to evaluate the effectiveness of formaldehyde, malachite green, hydrogen peroxide, isopropyl alcohol, copper sulfate, potassium permanganate and iodine (as polyvidone iodine) on signal crayfish (Pacifastacus leniusculus Dana) eggs incubated at 6.6 eggs/cm2 for long periods (up to 71 days). The administration of 3000 ppm formaldehyde for 15 min every other day up to the beginning of hatching allowed a stage 2 juvenile survival rate of 74.5%, without significant differences with 15 ppm malachite green (81.5%). These treatments did not show differences with the 150,000 ppm isopropyl alcohol dosage (65.5%). Formaldehyde at 3000 ppm can be administered up to the beginning of moulting to stage 2, without affecting the survival rates. Formaldehyde and malachite green treatments effectively controlled fungi. However, isopropyl alcohol and copper sulfate weakly inhibited mycelial growth only at the highest concentrations (150,000 and 18 ppm, respectively). Hydrogen peroxide below to 1500 ppm was insufficient to control fungi resulting in low survival rates to juvenile stage 2 (b 22%). The potassium permanganate treatments (100 and 150 ppm) controlled fungi, but they were toxic to eggs. Iodine showed a light fungicidal effect being lethal to eggs at 10 and 20 ppm, whereas at 5 ppm survival efficiency to stage 2 juvenile was 54.1%. The losses between hatching and stage 2 in the treatments with the best results were around 30%, when stage 2 removal from incubators was carried out every other day. However, when the removal frequency increased (daily) these losses were reduced to 10%. © 2006 Elsevier B.V. All rights reserved. Keywords: Astacid crayfish; Artificial incubation; Antifungal treatments

1. Introduction Our research team has developed artificial incubation techniques of astacid eggs from early embryonic stages covering most of the embryogenesis (Carral et al., 1988, ⁎ Corresponding author. Tel.: +34 9 87291188; fax: +34 9 87291187. E-mail address: [email protected] (J.D. Celada). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.02.064

1992; Celada et al., 2000, 2001; Pérez et al., 1998a,b, 1999, 2003). During this long period, dead eggs are usually invaded by fungi, being able to spread to the surrounding healthy ones. These fungi are usually Saprolegniales belonging to genus Dictyuchus and Saprolegnia (Vey, 1977, 1979). Fungal proliferation can be controlled with the periodic removal of nonviable eggs and stage 1 juveniles (Carral et al., 2004). Nevertheless,

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this is a delicate operation which requires a great labour, being only feasible at low incubation densities, but when higher occupation loads are involved, the administration of antifungal treatments would be advisable. Malachite green has been the most effective fungicide used for many years in aquaculture. However, in 1991 the use of this chemical agent on food fishes and their eggs was forbidden in the U.S.A. (Marking et al., 1994; Schreier et al., 1996; Kitancharoen et al., 1997) because of its teratological effects (Meyer and Jorgenson, 1983). Furthermore, a residue called leucomalachite with carcinogenic activity can persist in fish tissues for a long time (Culp et al., 1999), and could even remain undetectable in market-sized fish reared from eggs exposed to this fungicide (Meinertz et al., 1995). These findings led to its banning from January 1997 in animal species for human consumption in European Union by means of regulation 2377/90/EEC. Currently, formaldehyde at 1000–2000 ppm is an effective fungicide widely used for treating fungal infections of fish eggs in intensive aquaculture in the U.S.A. (Rach et al., 1997; Arndt et al., 2001). Its effectiveness to control fungi has also been proved on crayfish eggs (Celada et al., 2004). Although formaldehyde is approved for use in fish culture in the U.S.A. and the E.U., there is concern about user safety, because of its suspected carcinogenicity, and its potential adverse effects on the aquatic environment (Arndt et al., 2001). Hydrogen peroxide has been considered as an alternative fungicide because of its effectiveness on salmonid eggs at 1000 ppm (Dawson et al., 1994; Marking et al., 1994; Waterstrat and Marking, 1995; Schreier et al., 1996; Barnes et al., 1998, 2003; Gaikowski et al., 1998; Kitancharoen et al., 1997, 1998, Barnes and Gaikowski, 2004) and because it degrades to water and oxygen in the environment (Marking et al., 1994; Kitancharoen et al., 1997; Barnes et al., 1998; Arndt et al., 2001). Nevertheless, this concentration did not control fungi growth on crayfish eggs (Celada et al., 2004). Some other chemical agents with antifungal effect are potassium permanganate, polyvidone iodine, copper sulfate and isopropyl alcohol. Potassium permanganate has been tested on rainbow trout (Oncorhynchus mykiss) eggs (Marking et al., 1994) and on Chinese sucker Myxocyprinus asiaticus eggs (Liu et al., 1995). Polyvidone iodine was tried on largemouth bass Micropterus salmoides eggs (Wright and Snow, 1975). The dosages of copper sulfate and isopropyl alcohol were chosen taking as reference in vitro studies carried out by Lio-Po et al. (1982). Previously, Celada et al. (2004) reported that 4500 ppm formaldehyde could be excessive and 1500 ppm insuffi-

cient. Moreover, 1000 ppm hydrogen peroxide did not control fungal growth. Thus, the aims of the present study were to determine the adequate formaldehyde dosage, testing intermediate concentrations, to try higher hydrogen peroxide doses and to evaluate for the first time on incubating crayfish eggs four chemical agents: potassium permanganate, polyvidone iodine, isopropyl alcohol and copper sulfate. 2. Materials and methods Eggs were collected from signal crayfish (PacifastaDana) berried females coming from a crayfish farm. Spawning took place between 18th October and 5th November. After stripping, the eggs were pooled and artificially incubated until stage 2 juvenile production. The embryonic phases were identified by techniques developed by Celada et al. (1985, 1987). Experimental artificial incubation devices described by Carral et al. (1992) were used. Artesian well water was supplied in a flow-through system at the rate of 1 l/m. The parameters of water quality were: pH = 8.1, hardness = 5.2 °d (calcium: 32.3 mg/l), dissolved oxygen about 9 mg/l, nitrite b 0.015 mg/l, ammonium b 0.02 mg/l, total dissolved solids content = 108.5 mg/l, and total suspended solids b 0.5 mg/l. Temperature was 10 ± 1 °C until eggs reached the eye stage (phase XIII), when it was raised to 15 ± 1 °C up to final stage 2 juvenile production. Photoperiod was natural. During the incubation, different chemical treatments were periodically administered by peristaltic pump for 15 min to the incoming water flow. Dead eggs and stage 1 juveniles were not removed. Two experiments were carried out: 2.1. Experiment 1 A pool of 7200 eggs coming from 52 females was stripped from maternal pleopods on 4th December at phases VIII–IX (embryo with mandibular rudiments– embryo with naupliar appendages). Eight treatments were tested: – Formaldehyde: 3000 ppm administered 3 times a week until the beginning of moulting to stage 2 juveniles. – Formaldehyde: 3000 ppm administered 3 times a week until the beginning of hatchings. – Formaldehyde: 3500 ppm administered every other day until the beginning of hatchings. – Hydrogen peroxide: 1000 and 1500 ppm administered every other day until the beginning of hatchings.

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– Potassium permanganate: 100 and 150 ppm administered every other day until the beginning of hatchings. – Control: without chemical treatment.

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using the Newman–Keuls test. The significance level was P b 0.05. 3. Results

Removal of stage 2 juveniles from incubators was carried out every other day. 2.2. Experiment 2 A pool of 10800 eggs coming from 97 females was stripped from maternal pleopods on 25th December at phases XI–XII (embryo with thoracic appendage rudiments–embryo with pulsating heart). Chemical agents were administered every other day until the beginning of hatchings. Twelve treatments were tested: – – – – – –

Formaldehyde: 3000 ppm. Malachite green: 15 ppm. Copper sulphate: 6, 12 and 18 ppm. Iodine (as polyvidone iodine): 5, 10 and 20 ppm. Isopropyl alcohol: 50,000, 100,000 and 150,000 ppm. Control: without chemical treatment.

Removal of stage 2 juveniles from incubators was carried out daily. In both experiments, eggs were counted every 15 days until hatching, in order to obtain data on changes in viability, and the number of stage 1 (after hatching) and stage 2 (after first moult) juveniles was quantified. Efficiency rates to hatching and to stage 2 juvenile were calculated as the percentage of stage 1 juveniles and stage 2 juveniles obtained from the initial number of eggs, respectively. Each treatment was tested on six replicates of 150 eggs at a density of 6.6 eggs/cm2. Percentages were examined by analysis of variance (ANOVA), using the STATISTICA 5.0 computer program, previous arc-sine transformation of data. Mean comparison was tested

3.1. Experiment 1 Artificial incubation lasted 71 days. Eye stage was reached on day 39, hatchings began on day 53 and stage 2 juveniles were obtained from day 62. Efficiencies to hatching and stage 2 in each treatment are presented in Table 1. The best efficiency to stage 2 juvenile (64.7%) was obtained with the administration of 3000 ppm of formaldehyde 3 times a week up to the beginning of hatchings, without significant differences with 3500 ppm administered every other day. These treatments did not show differences with the administration of 3000 ppm up to first stage 2 juveniles were observed. Both hydrogen peroxide concentrations allowed low survival rates to stage 2 (b22%), although greater than those obtained without antifungal treatment. None of the eggs treated with potassium permanganate hatched. The evolution of survival rates in formaldehyde treatments (Fig. 1) shows a strong decrease (around 30%) between hatching and stage 2. However, in the control, the losses were continuous through the incubation period. As Fig. 2 shows, the efficiency rates in hydrogen peroxide treatments were high up to day 45, when temperature was raised from 10 to 16 °C. From here, survival values decreased more than 40% until hatching and 76% up to stage 2. Fungal growth was observed after day 10 in control and hydrogen peroxide treatments. In the latter case, fungal growth was initially low and increased with temperature rise. No fungal colonization was observed on eggs treated with formaldehyde and with potassium permanganate.

Table 1 Antifungal treatments and survival rates to stage 1 and to stage 2 juvenile in experiment 1 Chemical agent

Ppm

Frequency

Treatment stopping

Stage 1 (% ± S.E.M.)

Stage 2 (% ± S.E.M.)

Formaldehyde

3000 3000 3500 1000 1500 100 150

3 times a week 3 times a week Every other day Every other day Every other day Every other day Every other day

Stage 2 Hatching Hatching Hatching Hatching Hatching Hatching

84.3 ± 1.1 96.2 ± 0.9 94.5 ± 1.4 44.4 ± 9.0 54.8 ± 6.1 0 ± 0.0 0 ± 0.0 15 ± 2.8

57.8 ± 1.5a 64.7 ± 1.5a 61.2 ± 1.7a 15.4 ± 2.3b 21.1 ± 3.6b 0 ± 0.0d 0 ± 0.0d 8.6 ± 2.0c

Hydrogen peroxide Potassium permanganate Control

S.E.M.: standard error of mean. Values followed by differing letters were significantly different (P b 0.05) from the others in the same column.

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100

99 98.7

100

97.6

90

97.1

96.2 94.5

95.4

91.2

80

89.3

84.3

F

70 SURVIVAL (%)

97.8 97.4

66.6

64.7 61.2 57.8

60 50 40 35

30 20

3000 ppm 3 times a week up to stage 2 3000 ppm 3 times a week up to hatching 3500 ppm every other day up to hatching Control

10

16

15 8.6

0 0

10

15

30

45

53-62 (Stage 1)

71(Stage 2)

DAYS OF ARTIFICIAL INCUBATION Fig. 1. The changes of egg survival (%) in formaldehyde treatments of experiment 1. (F) First observation of fungal growth on eggs.

3.2. Experiment 2 Artificial incubation lasted 50 days. Eye stage was reached on day 16, hatching began on day 30 and stage 2 juveniles were obtained from day 38. Survival efficiencies to hatching and stage 2 in each treatment are presented in Table 2. The 3000 ppm 100

100

F F

98.5

98

97.8

97.5

formaldehyde treatment allowed an efficiency to stage 2 of 74.5%, without significant differences with 15 ppm malachite green administration (81.5%). These treatments did not show significant differences with the 150,000 ppm isopropyl alcohol dosage (65.5%). Copper sulfate treatments allowed efficiencies to stage 2 to be significantly lower than those obtained 95.8 95.4

90 F

80

SURVIVAL (%)

70

66.6

60 54.8

50 44.4

40 35

30 20

Hydrogen peroxide 1000 ppm Hydrogen peroxide 1500 ppm Control

10

21.1

16

15

15.4 8.6

0 0

10

15

30

45

53-62(Stage 1)

71(Stage 2)

DAYS OF ARTIFICIAL INCUBATION Fig. 2. The changes of egg survival (%) in hydrogen peroxide treatments of experiment 1. (F) First observation of fungal growth on eggs.

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Table 2 Antifungal treatments and survival rates to stage 1 and to stage 2 juvenile in experiment 2 Chemical agent

Ppm

Stage 1 (% ± S.E.M.)

Stage 2 (% ± S.E.M.)

Formaldehyde Malachite green Copper sulphate

3000 15 6 12 18 5 10 20 50,000 100,000 150,000

91.2 ± 0.9 89.6 ± 1.5 53.7 ± 7.2 42.3 ± 9.3 55.8 ± 4.8 58.0 ± 7.0 5.0 ± 0.8 0.0 ± 0.0 22.0 ± 14.0 39.0 ± 9.4 76.3 ± 4.9 34.2 ± 16.5

74.5 ± 3.1ab 81.5 ± 3.1a 39.0 ± 6.5cde 29.3 ± 9.0def 45.7 ± 6.3ce 54.1 ± 8.1bcd 1.0 ± 0.4f 0.0 ± 0.0f 18.2 ± 12.1ef 35.1 ± 9.5ce 65.5 ± 4.0ac 29.2 ± 14.6def

Polyvidone iodine

Isopropyl alcohol

Control

S.E.M.: standard error of mean. Values followed by differing letters were significantly different (P b 0.05) from the others in the same column.

with formaldehyde and with malachite green, although an acceptable result (45.7%) was obtained with the highest concentration (18 ppm). In the 10 and 20 ppm iodine treatments, hatchings were close to zero. The 5 ppm administration allowed a final stage 2 juvenile efficiency of 54.1%, without significant differences with the formaldehyde and the control treatment. The survival rates in formaldehyde and malachite green treatments are shown in Fig. 3. Differences between them were not significant throughout the different counts. In all cases, efficiency rates were higher than those

100

100

95.2 94.5

90

obtained in the control. Considering all the treatments, the losses between hatching and stage 2 were 9.9%. Fig. 4 shows the survival rates throughout the different counts in copper sulfate treatments. Survival rates were significantly higher than those obtained in the control only after 15 days of incubation. Considering all the treatments, the losses between hatching and stage 2 were 10.7%. The changes in survival rates obtained with iodine administrations are shown in Fig. 5. After 15 days of incubation survival rates obtained with 5 and 10 ppm were significantly higher (around 93%) than those

F

80

91.2 89.6 81.5

77.2

74.5

SURVIVAL (%)

70 60 50 40

34.2

30

29.2

Formaldehyde 3000 ppm Malachite green 15 ppm

20

Control

10 0 0

10

15

0-42(Stage 1)

50(Stage 2)

DAYS OF ARTIFICIAL INCUBATION Fig. 3. The changes of egg survival (%) in formaldehyde and malachite green treatments of experiment 2. (F) First observation of fungal growth on eggs.

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100

100

F F

90

F

80

F

93.2 93 91.2 77.2

SURVIVAL (%)

70 60

55.8 53.7

50

42.3

40 Copper sulfate 6 ppm

30

34.2

45.7 39 29.3 29.2

Copper sulfate 12 ppm Copper sulfate 18 ppm

20

Control

10 0 10

0

15

30-42(Stage 1)

50(Stage 2)

DAYS OF ARTIFICIAL INCUBATION Fig. 4. The changes of egg survival (%) in copper sulfate treatments of experiment 2. (F) First observation of fungal growth on eggs.

obtained with 20 ppm and in the control. Efficiency to stage 2 juvenile in the 5 ppm treatment (54.1%) was significantly greater, while with 10 and 20 ppm dosages results were lower than without an antifungal agent. Considering all the treatments, the losses between hatching and stage 2 were 13.8%. The change in efficiency rates with isopropyl alcohol treatments is shown in Fig. 6. Compared with the other 100

100

F F

90

93.5 92.5

F

80 F

70 SURVIVAL (%)

concentration tested, the survival rates of eggs treated with 150,000 ppm were significantly higher. Considering all the treatments, the losses between hatching and stage 2 were 5.8%. Fungal growth over dead eggs was observed after 10 days of incubation in all the treatments, except in formaldehyde and malachite green treatments, increasing after 23 days with the rise of temperature. However,

77.2 69.4

60

58 54.1

50 40 34.2

30

29.2

20

Polyvidone iodine 5 ppm Polyvidone iodine 10 ppm Polyvidone iodine 20 ppm Control

10

5 0

0 0

10

15

30-42(Stage 1)

1 0

50(Stage 2)

DAYS OF ARTIFICIAL INCUBATION Fig. 5. The changes of egg survival (%) in polyvidone iodine treatments of experiment 2. (F) First observation of fungal growth on eggs.

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100

100

90

F

93.8

F F F

80

263

81.6 77.2

76.3

76.8

70 SURVIVAL (%)

65.5

60 50 39

40

35.1

30

34.2 Isopropyl alcohol 50000 ppm Isopropyl alcohol 100000 ppm Isopropyl alcohol 150000 ppm Control

20 10

29.2

22 18.2

0 0

10

15

30-42(Hatching)

50(Stage 2)

DAYS OF ARTIFICIAL INCUBATION Fig. 6. The changes of egg survival (%) in isopropyl alcohol treatments of experiment 2. (F) First observation of fungal growth on eggs.

in iodine and 150,000 ppm isopropyl alcohol treatments fungal growth was lower. 4. Discussion In previous studies, Celada et al. (2004) noticed that treatments with 4500 ppm formaldehyde administered 3 times a week for 15 min up to hatching effectively controlled fungal growth and increased hatching rates of astacid eggs. Nevertheless, this concentration was lethal over newly-hatched juveniles (stage 1). The present results show that lower formaldehyde concentrations (3000 ppm) are equally effective, allowing similar results to 15 ppm malachite green. Furthermore, the reduction of formaldehyde concentration allows for the maintenance of the treatment after the beginning of hatchings without affecting negatively to stage 1 survival rates. However, treatment beyond the hatching stage appears unnecessary as improvement of final efficiency rates was not evidenced. On rainbow trout eggs, Dawson et al. (1994), Marking et al. (1994) and Schreier et al. (1996) stated that treatments of 1000 ppm of hydrogen peroxide for 15 min every other day controlled fungi and increase hatching rates, whereas Barnes and Gaikowski (2004) on landlocked fall Chinook salmon (Oncorhynchus tshawytscha) recommended daily treatments at the same dose. However, on signal crayfish eggs, Celada et al. (2004) proved that this dose applied up to 3 times a

week did not inhibit fungal growth. In our experiments, treatments of 1000 and 1500 ppm hydrogen peroxide every other day produced a visible fall in fungal growth rate during the low temperature (10 °C) incubation period. Nevertheless, these doses were not effective enough after the rise to 16 °C. Thus, hydrogen peroxide was an effective fungicide at 1000 ppm, or even less, on rainbow trout eggs incubated at low temperature (12 °C) and over a short time period (around 21 days) but it was not effective, even at a higher concentration (1500 ppm), on crayfish eggs incubated up to 16 °C during a longer artificial incubation period. Marking et al. (1994) reported that potassium permanganate treatments at 50 and 100 ppm on rainbow trout (O. mykiss) eggs did not control fungal infection, delayed hatchings and did not improve the final efficiency. Working with M. asiaticus eggs, Liu et al. (1995) reported that doses of 25 and 75 ppm applied for 60 min every other day up to hatching were toxic, decreasing hatching rates, while lower concentrations were unable to control the fungi. Signal crayfish eggs treated with potassium permanganate (100 and 150 ppm) did not show fungal development. Eggs treated with this agent were so heavily tinted that identification of embryonic stage and dead eggs was not possible. Only when the capsule was removed could we determine that embryonic development stopped in phase XII (embryo with pulsating heart).

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In trials carried out in vitro, Lio-Po et al. (1982) proved that copper sulphate was mycostatic at concentrations of 5–100 ppm. In our experiments, copper sulfate at 18 ppm showed a certain antifungal effect, but lower than formaldehyde. Besides, copper sulphate tends to precipitate in the stock solution supplied to artificial incubation devices and thus the concentration received by the eggs cannot be guaranteed. For these reasons, this chemical agent is rejected as a fungicide with crayfish eggs. Treatments of fish eggs with iodine products have revealed their narrow safety margin (Wright and Snow, 1975) and toxic effects at fungicidal concentrations (Marking et al., 1994). In agreement with results obtained in fish species, iodine (as polyvidone iodine) at 10 and 20 ppm has shown a strong toxicity to crayfish eggs, appreciable from early incubation stages by the eggs disintegrating. However, this fact was not observed in the 5 ppm treatment, revealing its narrow safety margin. Lio-Po et al. (1982) reported that alcohols are mycostatic at very high concentrations. In our experiments, isopropyl alcohol showed weak antifungal effect, even at the highest concentration. Besides, this agent led to an excessive egg movement during its administration. During the artificial incubation of crayfish eggs, several authors have observed a critical period in the final stages of embryogenesis with high mortality rates, either using fungicidal treatments (Mason, 1977; Rhodes, 1981; Celada et al., 2004) or without using them (Carral et al., 1988, 1992; Matthews and Reynolds, 1995; Pérez et al., 1998a,b, 1999). Thus, Celada et al. (2004) did not consider these losses due to a possible cumulative toxic effect of the fungicide. In our experiments, the losses between hatching and final count to stage 2 in the treatments with the best results were reduced from around 30% to 10% when removal frequency of stage 2 juveniles from incubators was increased from every other day to daily. According to these results, in this period the losses would be partly due to a possible negative effect of the coexistence of the stage 2 with stage 1 juveniles and even the eggs still not hatched, since after the first moult the juveniles have a great mobility and early start feeding. To sum up, on crayfish eggs formaldehyde at the dose of 3000 ppm administered for 15 min every other day up to the beginning of hatching is the most effective fungicide to replace the banned malachite green. Other chemical agents like hydrogen peroxide, potassium permanganate, copper sulfate, polyvidone iodine or isopropyl alcohol have low effectiveness or are ineffective. Considering the concerns about formaldehyde safety, the search for effective fungicidal treat-

ments must continue. Furthermore, new research needs to be carried out in order to reduce losses between hatching and stage 2 juvenile. Acknowledgements To the Plan Nacional de I+D+i, Ministerio de Educación y Ciencia, Spain. Research Project AGF 99-0190. We would like to express our thanks to the Quiñón S.A. crayfish farm (San Esteban de Gormaz, Soria, Spain) for its collaboration. References Arndt, R.E., Wagner, E.J., Routledge, M.D., 2001. Reducing or withholding hydrogen peroxide treatment during a critical stage of rainbow trout development: effects on eyed eggs, hatch, deformities, and fungal control. N. Am. J. Aquac. 63, 161–166. Barnes, M.E., Gaikowski, M.P., 2004. Use of hydrogen peroxide during incubation of landlocked fall Chinook salmon eggs in vertical-flow incubators. N. Am. J. Aquac. 66, 29–34. Barnes, M.E., Ewing, D.E., Cordes, R.J., Young, G.L., 1998. Observations on hydrogen peroxide control of Saprolegnia spp. during rainbow trout eggs incubation. Prog. Fish-Cult. 60, 67–70. Barnes, M.E., Stephenson, H., Gabel, M., 2003. Use of hydrogen peroxide and formalin treatments during incubation of landlocked fall Chinook salmon eyed eggs. N. Am. J. Aquac. 65, 151–154. Carral, J.M., Celada, J.D., Gaudioso, V.R., Temiño, C., Fernández, R., 1988. Artificial incubation improvement of crayfish eggs (PaciDana) under low temperatures during embryonic development. Freshw. Crayfish 7, 230–250. Carral, J.M., Celada, J.D., González, J., Gaudioso, V.R., Fernández, R., López-Baissón, C., 1992. Artificial incubation of crayfish eggs (Pacifastacus leniusculus Dana) from early stages of embryonic development. Aquaculture 105, 261–269. Carral, J.M., Pérez, J.R., Celada, J.D., Sáez-Royuela, M., Melendre, P.M., Aguilera, A., 2004. Effects of dead egg removal frequency on stage 2 juvenile production in artificial incubation of Austropotamobius pallipes Lereboullet. Bull. Fr. Pêche Piscic. 372–373, 424–430. Celada, J.D., Gaudioso, V.R., Paz, P., Fernández, R., 1985. Identification et chronologie des phases de développement des oeufs de l'écrevisse (Pacifastacus leniusculus Dana) par l'observation directe. Piscic. Fr. 82, 5–8. Celada, J.D., Paz, P., Gaudioso, V.R., Fernández, R., 1987. Embryonic development of the freshwater crayfish (Pacifastacus leniusculus Dana): a scanning electron microscopic study. Anat. Rec. 219, 304–310. Celada, J.D., González, J., Carral, J.M., Fernández, R., Pérez, J.R., Sáez-Royuela, M., 2000. Storage and transport of embryonated eggs of the signal crayfish Pacifastacus leniusculus. N. Am. J. Aquac. 62, 308–310. Celada, J.D., Carral, J.M., Pérez, J.R., Sáez-Royuela, M., Muñoz, C., 2001. Successful storage and transport of eggs of the white-clawed crayfish (Austropotamobius pallipes Lereboullet). Aquac. Int. 9, 269–276. Celada, J.D., Carral, J.M., Sáez-Royuela, M., Melendre, P.M., Aguilera, A., 2004. Effects of different antifungal treatments on artificial incubation of the astacid crayfish (Pacifastacus leniusculus Dana) eggs. Aquaculture 239, 249–259.

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