Effects of stage 2 juvenile removal frequency on final survival rates in artificial incubation of crayfish eggs (Pacifastacus leniusculus Dana. Astacidae).ABSTRACT In artificial incubation of astacid crayfish crayfish or crawfish, freshwater crustacean smaller than but structurally very similar to its marine relative the lobster, and found in ponds and streams in most parts of the world except Africa. Crayfish grow some 3 to 4 in. (7.6–10. eggs, stage 2 juveniles must be removed by hand from incubators and a significant decrease in survival rates has been recorded between hatching (stage 1) and the first moult (stage 2). To reduce these losses, eggs of Pacifastacus leniusculus Pacifastacus leniusculus American crayfish. See Table 23. were incubated at a density of 6.6 cm-2 and different removal frequencies of stage 2 juveniles from the incubators were tested: every 5, 4, 3, and 2 days, daily and only once at the end of incubation. Formaldehyde formaldehyde (fôrmăl`dəhīd'), HCHO, the simplest aldehyde. It melts at −92°C;, boils at −21°C;, and is soluble in water, alcohol, and ether; at STP, it is a flammable, poisonous, colorless gas with a suffocating was used to control growth of fungi. The best final survival rate (88.6%) was obtained when stage 2 juveniles were removed daily from the incubators. There were no significant differences nor between removals every 2 and 3 days (79.6% and 76.5%, respectively) neither between 4 and 5 days (72.6% and 69.8%, respectively). Considering all treatments, final survival rates decreased as juvenile removal was delayed up to only one removal at the end of incubation. KEY WORDS: astacid crayfish, artificial incubation, juvenile removal, Pacifastacus leniusculus INTRODUCTION In artificial incubation of astacid crayfish eggs, it is known that fungi can grow over nonviable nonviable /non·vi·a·ble/ (-vi´ah-b'l) not capable of living. non·vi·a·ble adj. Not capable of living or developing independently. Used especially of an embryo or fetus. eggs and invade healthy ones (Celada et al. 2004). This has been limited by periodic removal of dead eggs (Carral et al. 1992, 2004, Matthews & Reynolds 1995, Perez et al. 1998a, 1998b, 1999, 2003, Celada et al. 2001) or by antifungal antifungal /an·ti·fun·gal/ (-fung´gal) 1. destructive to fungi, or suppressing their reproduction or growth; effective against fungal infections. 2. an agent that so acts. treatments (Mason 1977, Rhodes 1981, Celada et al. 2004, Melendre et al. 2006). In all cases, significant losses (usually between 15% and 35%) have been recorded between hatching (stage 1) and the first moult (stage 2), as much without antifungal treatments as with them. For this reason, Celada et al. (2004) did not consider these losses to be attributable to a possible cumulative toxic effect of the fungicide fungicide (fŭn`jəsīd', fŭng`gə–), any substance used to destroy fungi. Some fungi are extremely damaging to crops (see diseases of plants), and others cause diseases in humans and other animals (see fungal infection). . Stage-2 juveniles produced by artificial incubation must be removed by hand from incubators, and this occurs usually at the end of incubation, when the first moult period concludes. Recently, Melendre et al. (2006) observed that losses between stage 1 and stage 2 had decreased from 30% to 10%, probably because of a higher removal frequency of stage 2 juveniles from incubators. This observation suggests that the losses in this period would be partly caused by a possible negative effect of the coexistence co·ex·ist intr.v. co·ex·ist·ed, co·ex·ist·ing, co·ex·ists 1. To exist together, at the same time, or in the same place. 2. of stage 2 with stage 1 and even eggs still not hatched, because after the first moult juveniles have a great mobility and start feeding. The aim of this study is to determine the influence of the frequency of removal of stage 2 juveniles from the incubators on final survival rates. MATERIAL AND METHODS Eggs were collected from 52 signal crayfish The signal crayfish, Pacifastacus leniusculus, is an American crayfish indigenous to the western United States.[1] Members of this species are up to 15 cm in length. (P. leniusculus) berried ber·ried adj. 1. Having or bearing berries: berried branches; a berried plant. 2. Resembling a berry or berries: "an off-dry, berried flavor" females coming from a crayfish farm, where spawning had taken place between October 15 and 30. On February 22, 5,400 eggs were stripped from maternal pleopods at phases XI (embryo with thoracic thoracic /tho·rac·ic/ (thah-ras´ik) pectoral; pertaining to the thorax (chest). tho·rac·ic adj. Of, relating to, or situated in or near the thorax. appendage appendage /ap·pen·dage/ (ah-pen´dij) a subordinate portion of a structure, or an outgrowth, such as a tail. epiploic appendages see under appendix . rudiments)--XII (embryo with pulsating heart). The embryonic phases were identified by techniques developed by Celada et al. (1985, 1987). After stripping, the eggs were pooled and artificially incubated in devices described by Carral et al. (1992) until stage 2 juvenile production. Artesian well artesian well, deep drilled well through which water is forced upward under pressure. The water in an artesian well flows from an aquifer, which is a layer of very porous rock or sediment, usually sandstone, capable of holding and transmitting large quantities of water was supplied in a flow-through system at the rate of 1 L [min.sup.-1]. The parameters of water quality were: pH 8.1, hardness = 5.2[degrees]d (calcium: 32.3 mg [L.sup.-1]), dissolved oxygen about 9 mg [L.sup.-1], nitrite nitrite Any salt or ester of nitrous acid (HNO2). The salts are inorganic compounds with ionic bonds, containing the nitrite ion (NO2−) and any cation. <0.015 mg [L.sup.-1], ammonium ammonium /am·mo·ni·um/ (ah-mo´ne-um) the hypothetical radical, NH4, forming salts analogous to those of the alkaline metals. ammonium carbonate <0.02 mg [L.sup.-1], total dissolved solids Total dissolved solids (often abbreviated TDS) is an expression for the combined content of all inorganic and organic substances contained in a liquid which are present in a molecular, ionized or micro-granular (colloidal sol) suspended form. = 108.5 mg [L.sup.-1], and total suspended solids Total suspended solids is a water quality measurement usually abbreviated TSS. This parameter was at one time called non-filterable residue (NFR), a term that refers to the identical measurement: the dry-weight of particles trapped by a filter, typically of a <0.5 mg [L.sup.-1]. Temperature was 10 [+ or -] 1[degrees]C until eggs reached the eye stage (phase XIII). Thereafter, it was raised to 15.5 [+ or -] 1[degrees]C up to final stage 2 juvenile production. Photoperiod photoperiod /pho·to·pe·ri·od/ (fo´to-per?e-od) the period of time per day that an organism is exposed to daylight (or to artificial light).photoperiod´ic pho·to·pe·ri·od n. was natural. According to according to prep. 1. As stated or indicated by; on the authority of: according to historians. 2. In keeping with: according to instructions. 3. Melendre et al. (2006), density was 6.6 eggs [cm.sup.-2], dead eggs were not removed and control of growth of fungi was performed with formaldehyde (3,000 ppm) administered by means of peristaltic pump A peristaltic pump is a type of positive displacement pump used for pumping a variety of fluids. The fluid is contained within a flexible tube fitted inside a circular pump casing (though linear peristaltic pumps have been made). for 15 min every second day up to the beginning of hatching. From our knowledge, at present the formaldehyde is approved for use in fish culture in the United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. and the Europe. During the last period of incubation (Med.) the period which elapses between exposure to the causes of an infectious disease and the attack resulting from it; the time during which an infective agent must grow in the body before producing overt symptoms of disease. See also: Incubation , six treatments were tested: removal of stage 2 juveniles from the incubators every 5, 4, 3, and 2 days, daily and only once at the end of the incubation. In all treatments, the number of stage 1 (after hatching) and stage 2 (after first moult)juveniles was quantified. Final survival rate was the percentage of stage 2 juveniles obtained from the initial number of eggs. Each treatment was tested on six replicates of 150 eggs. Results were examined by analysis of variance (ANOVA anova see analysis of variance. ANOVA Analysis of variance, see there ), using the STATISTICA 5.0 computer program, with previous arcsine transformation of data. Mean comparison was tested using the Newman-Keuls' test. The significance level was P < 0.05. RESULTS Artificial incubation lasted 40 days and no fungal growth was observed over dead eggs. Hatching began on day 25 and stage 2 juveniles were obtained from day 32. Survival rates to hatching and to stage 2 are presented in Table 1. ANOVA showed a significant influence (P < 0.0000) of removal frequency on stage 2 juvenile production. The best final survival rate (88.6%) was obtained when stage 2 juveniles were removed daily from the incubators. There were no significant differences nor between removals every 2 and 3 days (79.6% and 76.5%, respectively) neither between 4 and 5 days (72.6% and 69.8%, respectively). In all these treatments, the final survival was significantly better than without periodical removal (51.7%). In fact, final survival rate decreased as juvenile removal was delayed. Up to stage 1, losses were lower than 8% in all treatments, without differences among them. Between stage 1 and 2, the losses were significantly lower with daily removal (7.6%). No differences were found when this practice took place every 2 and 3 days, being around 16%. These treatments were more effective than removals every 4 and 5 days, where losses around 23% were recorded. When only one final removal of stage 2 juveniles was carried out, the losses between hatching and stage 2 (40.4%) were greater than for all the other treatments. DISCUSSION Concerning the concentration of losses in the last period of incubation, Perez et al. (1998a, 1998b) suggested that the causes could be related to the increase in metabolic activities and the enormous physiological effort involved in hatching and the first moult. Results from the present experiment indicate that this could be applied to a minor part of the losses between hatching and stage 2, which could be around the percentage recorded when juvenile removal was performed daily (7.6%), but not up to the 22% to 40% recorded when stage 2 juveniles remained for a long time (4-9 days) in the incubators. Because survival rates to stage 1 were high and similar in all treatments, losses are mainly attributable to juvenile stage 2, and the results of the present experiment show that final survival is reduced the longer the stay of stage 2 juveniles in the incubators. This confirms the suggestion of Melendre et al. (2006) that there is a harmful effect of the coexistence of stage 2 with stage 1, because after the first moult juveniles have great mobility, they are aggressive, and soon start ingestion ingestion /in·ges·tion/ (-chun) the taking of food, drugs, etc., into the body by mouth. in·ges·tion n. 1. The act of taking food and drink into the body by the mouth. 2. of food. So, we could observe stage 2 juveniles grabbing with their claws and eating stage 1 only 24 h after the first moult, and it is possible that the same situation occurs between stage 2 juveniles themselves. Moreover, it must be considered that, if stage 2 juveniles are not removed, their numbers increase in the incubators as the new first moults take place, also increasing the effects of aggressive behavior and cannibalism cannibalism (kăn`ĭbəlĭzəm) [Span. caníbal, referring to the Carib], eating of human flesh by other humans. . Nevertheless, these effects do not seem to be harmful for the eggs not hatched, because survival to hatching was not affected. Mason (1977) tried artificial incubation of eggs during the last period of development (May) as single or pooled broods, obtaining higher survival rates by incubating single broods. This author observed that highest mortality occurred after the stage 1 moult, suggesting that pooling eggs from several broods increased mortality, possibly through increased interaction among individuals in contrasting stages of the moult cycle. In our study, eggs were from pooled broods of many females and differences in embryonic development were identified at stripping time. If all eggs from a single brood brood n. See litter. brood offspring or pertaining to offspring. brood mare a mare dedicated to the production of foals. hatch in 2 days or less (Mason 1970), in the present experiment the hatching period lasted 8 days and, thereby, the duration of the first moult period was similar. Thus, depending on the frequency of removal of stage 2 juveniles, a wide spread in hatching times could involve a wide coexistence of stages 1 and 2, allowing an increase in losses. From our current knowledge, when eggs in different embryonic phases are incubated together, the highest final survival rates can be obtained by removing stage 2 juveniles from incubators daily. However, the possibility of reducing the removal frequency may save on labor, overall costs, and time. Therefore, the most convenient method would be to collect all of the stage 2 production at the same time without losses caused by the coexistence of different stages. This could be reached by incubating together eggs with similar development, to synchronize See synchronization. hatching times. For this, at stripping time, broods for artificial incubation should be distributed in different lots according to the embryonic phase reached. ACKNOWLEDGMENTS Funding of this study was from Plan Nacional de I+D I+D Investigación y Desarrollo (Spanish: Research and Development) +i, Ministerio de Educacion y Ciencia, Spain, Research Project AGL (programming) AGL - (Atelier de Genie Logiciel) French for IPSE. 2005-01127. The authors thank the Quinon S.A. crayfish farm (San Esteban de Gormaz San Esteban de Gormaz is a municipality in the province of Soria in the autonomous community of Castile-Leon, Spain. Its population is approximately 3,500. The village is located in the Route of the Wool and the Way of the Cid, the route of the exile of the Cid. , Soria, Spain) for its collaboration. LITERATURE CITED Carral, J. M., J. D. Celada, J. Gonzalez, V. R. Gaudioso, R. Fernandez & C. Lopez-Baisson. 1992. Artificial incubation of crayfish eggs (Pacifastacus leniusculus Dana) from early stages of embryonic development. Aquaculture aquaculture, the raising and harvesting of fresh- and saltwater plants and animals. The most economically important form of aquaculture is fish farming, an industry that accounts for an ever increasing share of world fisheries production. 105:261-269. Carral, J. M., J. R. Perez, J. D. Celada, M. Saez-Royuela, P. M. Melendre & A. Aguilera. 2004. Effects of dead egg removal frequency on stage 2 juvenile production in artificial incubation of Austropotamobius pallipes Austropotamobius pallipes is an endangered [1] European freshwater crayfish, and the only species of crayfish native to the British Isles [2]. Its common names include white-clawed crayfish and Atlantic stream crayfish. Lereboullet. Bull. Fr. Peche Piscic. 372-373:425-430. Celada, J. D., V. R. Gaudioso, P. Paz & R. Fernandez. 1985. Identification et chronologie des phases de developpement des oeufs de l'erevisse (Pacifastacus leniusculus Dana) par l'observation directe. Piscic. Fr. 82:5-8. Celada, J. D., P. Paz, V. R. Gaudioso & R. Fernandez. 1987. Embryonic development of the freshwater crayfish (Pacifastacus leniusculus Dana): a scanning electron microscopic Adj. 1. electron microscopic - of or relating to or involving an electron microscope study. Anat. Rec. 219:304-310. Celada, J. D., J. M. Carral, J. R. Perez, M. Saez-Royuela & C. Munoz. 2001. Successful storage and transport of eggs of the white-clawed crayfish (Austropotamobius pallipes Lereboullet). Aquacult. Int. 9:269-276. Celada, J. D., J. M. Carral, M. Saez-Royuela, P. M. Melendre & A. Aguilera. 2004. Effects of different antifungal treatments on artificial incubation of the astacid crayfish (Pacifastacus leniusculus Dana) eggs. Aquaculture 239:249-259. Mason, J. C. 1970. Maternal-offspring behaviour of the crayfish Pacifastacus trowbridgi (Stimpson). Am. Midl. Nat. 84:463-473. Mason, J. C. 1977. Artificial incubation of crayfish eggs (Pacifastacus leniusculus Dana). Freshwater Crayfish 3:119-132. Matthews, M. & J. D. Reynolds. 1995. In vitro in vitro /in vi·tro/ (in ve´tro) [L.] within a glass; observable in a test tube; in an artificial environment. in vi·tro adj. In an artificial environment outside a living organism. culture of crayfish eggs using a recirculating airlift incubator. Freshwater Crayfish 8:300-306. Melendre, P. M., J. D. Celada, J. M. Carral, M. Saez-Royuela & A. Aguilera. 2006. Effectiveness of antifungal treatments during artificial incubation of the signal crayfish eggs (Pacifastacus leniusculus Dana. Astacidae). Aquaculture 257:257-265. Perez, J. R., J. M. Carral, J. D. Celada, M. Saez-Royuela & M. P. Romero. 1998a. Effects of different thermal treatments during embryonic development on the artificial incubation efficiency of crayfish (Austropotamobius pallipes Lereboullet) eggs. Control of the embryogenetic duration and implications for commercial production. Invertebr. Reprod. Dev. 34:253-258. Perez, J. R., J. M. Carral, J. D. Celada, M. Saez-Royuela, C. Munoz & J. I. Antolin. 1998b. Effects of stripping time on the success of the artificial incubation of freshwater crayfish, Austropotamobius pallipes (Lereboullet), eggs. Aquac. Res. 29:389-395. Perez, J. R., J. M. Carral, J. D. Celada, M. Saez-Royuela, C. Munoz & J. I. Antolin. 1999. The possibilities of artificial incubation of white-clawed crayfish (Austropotamobius pallipes Lereboullet) eggs: comparison between maternal and artificial incubation. Aquaculture 170:29-35. Perez, J. R., J. D. Celada, J. Gonzalez, J. M. Carral, M. Saez-Royuela & R. Fernandez. 2003. Duration of egg storage at different temperatures in the astacid crayfish Pacifastacus leniusculus: critical embryonic phase. Aquaculture 219:347-354. Rhodes, C. P. 1981. Artificial incubation of the eggs of the crayfish Austropotamobius pallipes Lereboullet. Aquaculture 25:129-140. TABLE 1. Removal frequency of stage 2 juveniles and survival rates to hatching (stage 1) and to stage 2. Removal frequency of stage 2 juveniles Stage 1 (% [+ or -] SEM) Once at the end 92.1 [+ or -] 1.4 Every 5 days 93.4 [+ or -] 2.0 Every 4 days 94.8 [+ or -] 0.7 Every 3 days 93.8 [+ or -] 1.5 Every 2 days 94.6 [+ or -] 0.7 Daily 96.3 [+ or -] 0.6 Removal frequency of stage 2 juveniles Stage 2 (% [+ or -] SEM) Once at the end 51.7 [+ or -] 1.2 (c) Every 5 days 69.8 [+ or -] 1.7 (d) Every 4 days 72.6 [+ or -] 1.4 (cd) Every 3 days 76.5 [+ or -] 1.7 (bc) Every 2 days 79.6 [+ or -] 1.5 (b) Daily 88.6 [+ or -] 1.1 (a) SEM: standard error of mean. Values followed by different letters are significantly different (P < 0.05). P. M. MELENDRE, J. D. CELADA, * J. M. CARRAL, M. SAEZ-ROYUELA AND A. AGUILERA Departamento de Produccion Animal, Universidad de Leon, Campus de Vegazana s/n, 24071 Leon, Spain * Corresponding author. E-mail: jdcelv@unileon.es |
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