THE INFLUENCE OF TEMPERATURE ON THE EMBRYONIC DEVELOPMENT OF THE ANNUAL FISH Cynopoecilus melanotaenia ( CYPRINODONTIFORMES , RIVULIDAE )

The present study aims to provide data about the time required for Cynopoecilus melanotaenia kept at different temperatures to complete embryonic development. This information can be valuable for optimizing laboratory culture and facilitating future use of this species as a test organism in toxicity tests. Temperature effects on hatching rate are presented as well as information related to embryonic development stages. Eggs were observed daily, from start to finish of embryonic development. Thirteen developmental stages were described. Eggs were kept at two constant temperatures (20°C and 25°C) and at a variable ambient temperature (16-25°C – mean = 21°C, sd = 1.95), to determine developmental rate (velocity) at each temperature. A shorter incubation period was necessary to complete development at 25° ± 1°C. However, all embryos kept at this temperature hatched with morphological defects, which prevented their survival. No significant difference in developmental time period (p = 0.05) was observed at the 20°C and 16°-25°C (mean = 21°C, sd = 1.95) temperatures.


INTRODUCTION
Annual fishes are defined as a group of Cyprinodontiform fish found in seasonally dried out temporary ponds, ditches, and mudholes occurring in some parts of South America and Africa (Myers, 1942).The complete drying out of this aquatic habitat leads to the death of all adults and juveniles.The population survives as buried eggs for up to 18 months (Wourms, 1972a).When the next rainy season comes, a new reproductive cycle begins.Ponds refill and embryos hatch, allowing fast larvae growth.The fishes become sexually mature and spawn repeatedly over a long period (Meyers, 1942(Meyers, , 1952;;Peters, 1963;Wourms, 1972a, b, c;Arenzon, 1996;Arenzon et al., 1999;ARENZON et al., 2001).
The present study was carried out in order to determine under laboratory conditions the effects of temperature on embryonic development of C. melanotaenia.Some information on embryonic development stages is also presented.This type of information is relevant to culture optimization under laboratory conditions and use of this species as a test organism in toxicity tests.

MATERIAL AND METHODS
Fertilized eggs of C. melanotaenia were obtained from a laboratory population in the Centro de Ecologia at the Federal University of Rio Grande do Sul (Brazil).Fish were kept on a photoperiod of 12 h light and 12 h darkness at 20 ± 1°C.The eggs were deposited in plastic trays filled with peat moss.
Less than 24 hours after spawn, a group of eggs was transferred to petri dishes divided into compartments.Eggs were kept in distilled water adjusted to the conditions presented by Arenzon (1996) (hardness = 20 mg L -1 of CaCO 3 and initial pH of 6.5).The antibiotics Penicillin G (84.3881 mg L -1 -1185UI) and Mycostatin (17.7904 mg L -1 -562UI) (Wourms, 1972a) were added to the water.Dissolved oxygen concentration and pH were monitored and adjusted when necessary.
The eggs in the petri dishes were kept in constant darkness, except for brief exposure to light during observation or manipulation.Surface projections of the chorion having been mechanically removed to facilitate observation, developing eggs were observed daily under a dissecting microscope.This information was used as a reference in evaluating the developmental stage of the eggs kept at different temperatures.As a control to the chorion-removed eggs, a group of intact eggs was kept under the same conditions, observed every fortnight, and compared in its development to that of the dechorionated egg group.
Observation of the total developmental time period was performed based on a group of intact eggs kept at three different temperatures: 20 ± 1°C, 25 ± 1°C, and variable ambient temperature (16°-25°C, mean = 21°C, sd = 1.95).Each group of eggs was kept in closed plastic bags with peat moss, as described by Katz (1993).
Every ten days, the eggs were manually separated from the substrate and observed through a stereoscopic microscope in order to verify the embryonic developmental stage.The observations were repeated four times at each temperature.
After completing development (based on the developmental stages previously observed), the eggs were transferred from the substrate to 40 ml glass beakers with adjusted distilled water as previously described.The eggs kept at the same temperature used in the embryonic development experiment.This was to observe the hatching rate induced by substrate hydration.The hatching process starts a few hours after substrate hydration, as reported by Lacerda (1969).
The results were analyzed statistically using Analysis of Variance (ANOVA), and treatment means were compared with the Tukey complementary test; significance accepted was p < 0.05.

RESULTS AND DISCUSSION
As can be seen in Table 1, embryos of C. melanotaenia spent less time at 25° ± 1°C to complete their embryonic development.However, all embryos incubated at this temperature hatched with gross morphological abnormalities, and remained motionless, thus making their survival impossible.At 20°C and 16°-25°C (mean = 21°C, sd = 1.95), the mean time required to complete embryonic development showed no significant difference (p = 0.05) (Tables 1 and 2).
The hatching rate, within a period of 24 hours after substrate hydration, did not show any significant difference (p = 0.05) at the three test temperatures (Table 3).
To provide criteria for determining developmental rates, the development of C. melanotaenia was divided into thirteen stages, based on the first appearance of some easily recognized marked characters.
Stage 1. Perivitelline space is present between the egg envelope and the vitelline membrane.Central lipid droplet is present in the yolk.
Stage No difference in embryonic development was observed between the dechorionated eggs and the control group.Nevertheless, some embryos did not survive nearly as long after the secondary chorionic envelope had been removed.
According to Carter & Wourms (1991), annual fish development differs markedly from that of typical non-annual teleost fish due to the occurrence of embryonic diapause and the presence of a phase in which embryo-forming cells disperse and subsequently reaggregate.The early embryonic developmental stages, corresponding to the dispersion and reaggregation cell phases, described by Wourms (1972a, b, c) and Avni & Soin (1974), have not been described here.
According to Wourms (1972a), three diapause stages are expected to be observed in the embryology of annual fish.In C. melanotaenia the two first stages are facultative and the last is obligatory.The first diapause can occur at the dispersed cell stage, when the amoeboid cells are randomly distributed.The second diapause stage may occur between the forth and fifth stage.Resumption of heart contractions indicates its end.The third diapause occurs during stage thirteen, the pre-hatching stage.This last diapause stage was the most evident after the bradycardia phase was observed.
Regarding the use of juveniles of C. melanotaenia as a test organism in toxicity tests, stocks at 20° ± 1°C or a variable ambient temperature between 16°-25°C (mean = 21°C, sd = 1.95) are recommended.At these temperatures, we did not observe any significant difference in the mean time spent to complete embryonic development (Table 3).Since the embryos need a longer period to complete their development at these temperatures, they can also be stocked for a longer period.Egg incubation at 25° ± 1°C is not recommended since the juveniles quickly perish.