Hatching of the eggs of Eulimnadia colombiensis (Branchiopoda) at different temperatures and regime of light

Clam shrimps are small crustaceans typical of temporary ponds. To survive in these highly variable and unpredictable environments, they produce resistant eggs. These settle on the bottom of pools where they remain dormant, forming a cyst bank. We investigated the effects of light and temperature, both of which break the dormancy of Eulimnadia colombiensis eggs. Dormancy was not broken below 25°C, irrespective of the effect of light. Hatching was highest (27.8% of eggs) at 30°C in continuous light. Light also influenced the development of eggs, and some nauplii hatched spontaneously without drought stress. Methods are suggested for the cultivation of this species in tropical areas.


INTRODUCTION
Members of the Class Branchiopoda are common in temporary pools. These environments usually occur in hot, semi-arid, flat regions (Rabet and Thiery, 1996;Demeter, 2005). The temporary nature, abiotic factors, as well as the duration of the pools directly influence the life cycle of these animals .
The genus Eulimnadia Packard, 1874 (Family Liminadiidae) found in these microhabitats includes 48 species (Rabet, 2010) and has androdioic sexual system. This reproductive pattern is characterized by the presence of males and hermaphrodites genetically different, but phenotypically similar, differing only by the presence clasper (Sassaman and Weeks, 1993;Weeks et al., 2009).
Survival of populations these genus in this variable environment, especially in the ephemeris, depends entirely on the production of resistant eggs, which are really cysts. The single-celled egg develops inside the adult and the same instant (Benvenuto et al., 2009) those, whose dormancy is not affected, accumulate in the sediment. The accumulation of these cysts, not hatched, and those of other species in the bottom sediment of water bodies form the socalled "cyst banks". These, in turn, can provide us with information ranging from the richness of species in ecosystems (Arnott et al., 1999;Melo and Froehlich, 2001;Vandekerkhove et al., 2005) to temporal and spatial aspects of community dynamics (Brendonck and De Meester, 2003).
Light and temperature are primary factors in the hatching of the cysts of numerous species of zooplankton (Hairston et al., 1990;Fryer, 1996;Gyllstrom and Hansson, 2004). Large temperature variations are expected in temporary pools and studies have shown that this is a fundamental variable in the process of breaking the dormancy of cysts of many zooplankton species (May, 1987;Schwartz and Hebert, 1987;Vandekerkhove et al., 2005).
Eulimnadia colombiensis was described by Roessler (1989), in temporary ponds around Villavicencio and San Juan de Arama (Meta, Colombia). The species is recorded for Venezuela (Pereira and Garcia, 2001), Brazil (Rabet et al., 2012) and India (Weeks et al., 2006). Unlike other species of the genus, little is known of its biology. Nonetheless, studies were not conducted on the factors that influence the hatching rate of the eggs of Eulimnadia colombiensi. Here we investigated the hatching rates under different conditions of temperature in the presence and absence of light.

MATERIAL AND METHODS
Eulimnadia colombiensis adults were randomly collected from one fish pond at CEPLAC (Comissão Executiva do Plano da Lavoura Cacaueira) near Itabuna, Bahia, Brazil (14º46'51.51''S,39º13'29.11''W). They were taken to the laboratory and cultured in aquaria using low aeration (after Weeks et al., 1997) and exposed to ambient light and temperature. After the adults had died, the water was drained and filtered in a fine mesh sieve (80μm), which retained the deposited cysts. The latter were allowed to dry naturally in Petri dishes and stored in 7mL glass flasks.
After 45 days, simulation of time of interval average as that the tank remain dry, fourty two samples were taken of 30 eggs each, randomly selected, totalling 1,260 eggs of different hermaphrodites. Each sample was placed in Petri dishes and 40mL of purified water was added to each Petri dish, 24 hours afterward further 10mL were added.
Each series with three samples (= 90 eggs) was submitted to different combinations of temperature and light for 48 hours as suggested by Weeks et al. (1997). The thermal amplitude recorded for local water ponds varied between 21° (could months) e 29° C (hot months) (Tejedo et al., 2012). However, the environment studied by these authors differs from our local collection by presenting a lower volume of water and constant shade conditions. Preliminary tests were conducted at 15°C e 20°C and no hatching was observed.
A total of 14 treatments were tested at seven temperatures (25°, 26°, 27°, 28°, 29°, 30° and 35°C) in an incubator in the presence or absence of light. The temperature of water of each sample was monitored and maintained the same during time proposed. After this were period removed from the incubator and, with the aid of a stereomicroscope, hatched nauplii were pipetted and counted.
Finality, "bi-factorial ANOVA" test of, a 95% significance level, was used to analyse the existence of significant differences between the number of hatchings at different temperatures, and in the absence and presence of light, for each replica. The a posteriori Tukey test was used to determine the significance of potential differences.

RESULTS
Light strongly influenced the hatch of the nauplii. The highest hatching rates (17.8% and 27.8%) were obtained at 25°C and 30°C, respectively, in the cultures under constant light. Under this light condition, the difference between hatching rates at intermediate temperatures (26ºC, 27°C, 28°C and 29°C) did not show a clear pattern. However, when eggs underwent these same temperatures in the absence of light, there was a gradual increase in hatching rate from 27°C to 29°C (Fig. 1). None of the eggs hatched at 35ºC disrespected of the light regime.
The results showed that there is interaction between temperature and light on the hatching rates, the presence of light being fundamental in increasing the number of hatched nauplii (Tab. 1).
With respect to the different paired temperatures, the a posterior Tukey test showed significant differences between the samples in the presence of light of the pair 25°C X 27°C and in all the combinations with 30°C. In the absence of light no significant differences between pairs of temperatures were observed.
In addition to the above findings, we also observed that light positively affects the development of individuals after hatching. In the presence of light, the nauplii were shown to be active, free-natant and with no trace of egg connected to the body, contrary to what was observed in the absence of light. Dead nauplii were found among the set great of eggs where were extracted the samples for the study, suggesting the occurrence of hatching without water stress.

DISCUSSION
Both temperature and light regime are critical in the breaking of dormancy of Eulimnadia colombiensis cysts. The rates of hatching varied greatly as these environmental factors changed. The highest hatching rates were at 25°C and 30°C in the presence of constant light. However, at small temperature intervals (±1°C) and in absence of light, the gradual increase in the hatching rate strongly suggests that temperature is an important stimulus in breaking dormancy.
The temperature variation in local temporary ponds is very small (Tejedo et al., 2012), with daily values recorded between 21°C and 25°C (Tejedo, pers. comm.). However, our data show that hatching did not occur below 25°C after 48 hours of incubation in either the presence or absence of light so it may be necessary to extend the incubation period. It is likely that under natural conditions the hatching eggs need a period exceeding 48 hours. The situation is less clear at higher temperatures. None of the eggs hatched at 35°C, which was unexpected in tropical species that living in areas where the temperature of the water exceeds these values into higher irradiation times. This result suggests that this species of the genus Eulimnadia, despite the large phenotypic plasticity (Rabet, 2010), the temperature variation can be an important factor in establishing the limits of their biogeographical distribution.
The light influences not only the number of cysts that will hatch but also the subsequent development of nauplii. Nauplii that hatched in the presence of light proved to be active and with no trace of residual egg. Cysts incubated in the dark remained a pre-nauplius, inactive and the residual of the egg remained connected to the body, suggesting that light plays an important role in the species development.
For populations of E. colombiensis from tropical regions with high rainfall, we suggest that cultures should be made at 30°C, under constant presence of cold light. This value is higher than suggested by previous authors (Weeks et al., , 1999(Weeks et al., , 2001Rogers et al., 2010) for other species of the same genus. However, the consulted works dealt with populations of species from regions with different climatic characteristics. Thus, information about the place of origin of the specimens is key to the guidelines of an efficient cultivation for scientific reasons.
When comparing the egg production between E. colombiensis and E. texana, the difference is apparent. The average initial egg production by E. colombiensis, originating from the same area of study in question is 345 eggs.ind-1.d-1 (Chaves, 2014). Much higher than its congener E. texana, studied by Weeks et al. (1997), in which the initial production was ~ 50 eggs.ind-1.d-1. When we compare the contribution to the bank of eggs between coexisting genres, other observations arise. In one experiment (Chaves -unpublished data), comparing as absolute number of eggs of the coexistents Eulimnadia (Conchostraca) and Dendrocephalus Daday 1908 (Anostraca), in 100g of dry sediment from small puddles (~ 1m²) and large (~ 400m²), it was observed that smaller pools have a greater number of Eulimnadia eggs while larger pools have a higher number of Dendrocephalus. This difference in colonizing potential can be reinforced by the difference in the life cycles of the two genera (Eulimnadia ~ 16 days, personal observation, and Dendrocephalus ~ 15-75 days, see Lopes et al., 2011;Rabet and Thiery, 1996;Pereira and Gonzalez, 1994). Thus, individuals of Eulimnadia reach adulthood and reproduce faster, increasing its power to colonize ephemeral environments. Thus, in case of competition between these groups, E. colombiensis has an advantage in the initial colonization. Further studies are needed to test this hypothesis.
Just as in the natural environment, the variations found between hatching plates subjected to the same conditions, and the existence of percentages of hatching without a clear pattern, may be explained by the socalled bet-hedging mechanism. This strategy can be understood as a moment of hatching asynchrony in individuals originating from the same brood, which prevents local extinctions if the environment is change abruptly, without the bodies have time to adjust to changes (Seger and Brockman, 1987;Cáceres and Tessier, 2003;King and Masel 2007;Benvenuto et al., 2009). Several groups of microcrustaceans (cladocerans, calanoid copepods and anostracans) present in ephemeral environments use this strategy as a way to ensure success in this type of environment (Saiah and Perrin, 1990;Hairston et al., 1995. Simovich andHathaway, 1997;Cáceres and Tessier, 2003;Gyllstrom and Hansson, 2004).
The presence of dry nauplii in the material from which the eggs were extracted for the experiment was never reported for this genus. This record shows that nauplii hatch without necessarily going through a period of desiccation, but possibly some other snapshot unidentified environmental factor capable of modifying the dormant stage or avoid it as observed in other species of Branchiopoda (Fryer, 1996). This record suggests that maintaining environmental conditions favourable for longer favours lineages that have sequential development without the need to water stress.