Reproduction of Cubaris Murina (crustacea: Isopoda) under Laboratory Conditions and Its Use in Ecotoxicity Tests

Reproduction of the isopod Cubaris murina was studied in the laboratory in order to observe the offspring number per brood, the number and duration of the incubation periods, and the influence of repeated mating on number of young per brood. An experiment was conducted with two different groups: female/male pairs and females that were isolated after mating. No significant difference was found between the two reproductive female groups for the mean number of young per brood (x = 25) and the duration of the incubation periods (x = 17 days). One mating was enough for at least five broods, with no reduction in brood size. There was no significant difference between the number of adult males and females. Low-density cultures had a significantly higher growth rate compared to high-density cultures. Furthermore, the experimental results for optimization isopod culture conditions for terrestrial ecotoxicity testing are reported. eficiência dos cultivos em laboratório para ensaios ecotoxicológicos terrestres são apresentados.


Introduction
Interest in soil ecotoxicity tests has grown considerably in recent years (Van Straalen and Van Rijn, 1998).Aside from earthworms (oligochaetes), terrestrial isopods are one of the most frequently used terrestrial invertebrates for testing the effects of chemicals (Drobne, 1997).These organisms play an important role in the recycling of organic matter, because changes in terrestrial isopod feeding rate affect decomposition processes and subsequent matter and energy flow through ecosystems (Drobne, 1997).
Although there is not yet an international standard protocol to use terrestrial isopods in ecotoxicity tests, many studies with several endpoints have been conducted with these organisms.Examples of such studies include the effects of chemicals on survival and body size

Reproduction experiment and sex ratio
Ovigerous females at different ages were collected from cultures and transferred individually or with a male to plastic boxes (6 x 6 cm, 5 cm height), containing 2 cm of soil, similar to the culture boxes.As a whole 22 boxes with a single ovigerous female (Group A) and 10 boxes with a female/male pair (Group B) were observed daily over four months.For each female, the number and duration of ovigerous periods, the offspring number per brood, and the molt frequency were recorded.Neonates were removed from the box at the moment of counting.
The ovigerous period was considered from the parturial molt, because the passage of the fertilized eggs to marsupium coincides with this molt (Heeley, 1941), until birth of the young.The results of reproduction of solitary females (Group A) was compared to that of females with a male (Group B), using one-way ANOVA (α = 0.05).The successive broods were compared for the mean offspring number using Tukey-Kramer multiple comparison tests (Zar, 1996).
The C. murina adult's sex-ratio was analyzed in 30 culture boxes from neonate organisms kept in the laboratory.Adult males can be identified externally by the modifications of the first 2 pairs of pleopods, which form the external genitalia used in sperm transfer (Sutton, 1980).The number of females versus males was compared using a Student t-test (Zar, 1996).

Density experiment
Five different densities were established: 6, 12, 18, 24 and 30 animals per culture box (260 cm 2 ), employing the previous cultivation procedure.For each treatment, 3 replicates were used with animal ages ranging from 5 to 8 days.Animals were not weighed at the beginning of the experiment to avoid additional stress, but were distributed at random to avoid bias.The weight of a separate randomly chosen sample of 75 young individuals, with age ranging from 5 to 8 days, was averaged as initial weight.Specimens were weighed at the 40 th , 77 th and 110 th day of life and fed ad libitum.Dead organisms were continuously removed and not replaced.The parameters measured were survival, growth and reproductive performance (number of young /female, and the time of the first reproduction).The weight from individuals was compared in the different time periods using one-way ANOVA, after testing the data for ANOVA requirements (Zar, 1996).In the event of significant differences, a Tukey-Kramer multiple comparison test was conducted.The offspring number/female among different densities was compared using nonparametric Dunn's multiple comparison test (Zar, 1996).

Reproduction experiment and sex ratio
The offspring number from 111 broods was recorded and analyzed, together with the time from 90 ovigerous periods.The results of 3 successive broods are shown in Table 1.(Jones and Hopkin, 1998), adult mortality and number of juveniles (Jänsch et al., 2005), biomass (Niemeyer et al., 2006), acetyl-cholinesterase activity (Ribeiro et al., 1999), energy budget (Khalil et al., 1995), bioaccumulation (Odendaal and Reinecke, 2004), exposure routes and toxicokinetics (Vink et al., 1995;Sousa et al., 2000;Loureiro et al., 2002), locomotor behavior (Sorensen et al., 1997) and avoidance behavior (Odendaal and Reinecke, 1999a;1999b;Niemeyer et al., 2006;Loureiro et al., 2005).Most of these tests have been developed using species from temperate regions, and therefore, were not able to provide an appropriate answer to the tropical contamination problem, due to the lack of an ecological perspective.One of the first conditions in establishing a standard protocol to use certain organisms in an ecotoxicity test is their cultivation under laboratory conditions, as Caseiro et al. (2000) did for Porcellio dilatatus Brandt, 1833, species from temperate climate.
Information regarding reproductive aspects such as sex-ratio, that is important for understanding mating relations and reproductive potential of dioecius organisms (Nair, 1984), the offspring number per brood, the number and duration of the ovigerous periods, and whether repeated mating influence the offspring number per brood, is required to assess the sustainability of any organism to be used in ecotoxicity tests.These types of tests require test organisms that reproduce rapidly and generate a sufficient offspring number.Furthermore, these organisms should be readily available in the field and well adapted to laboratory manipulation.
The present study aimed to assess a population of the pantropical terrestrial isopod Cubaris murina Brandt, 1833, under laboratory conditions.Aspects related to C. murina reproduction were determined and conditions necessary to provide continuous offspring for ecotoxicity tests were also evaluated.

Materials and Methods
C. murina is a pantropical species, widely distributed in Brazil, after its introduction and found mainly in synanthropic habitats (Lemos de Castro, 1971).The species is registered also for several other zones throughout the world (Leistikow and Wägele, 1999).
The isopods used in these experiments were collected once on a farm in Simões Filho (BA, Brazil), identified and cultured under laboratory conditions, at 27 ± 2 °C.Organisms were kept in closed plastic boxes (±300 cm²) on a 2 cm layer of natural soil (pH 6.5 and 3.8% of organic matter, sieved in 2-mm mesh), collected in Cruz das Almas (BA, Brazil), and small holes were made on the box cover to ensure ventilation.
In all experiments, isopods were fed with commercial fish food (AlconBasic ® ) and tiny slices of potatoes (Carolina, 2004).Periodic spraying of distilled water maintained an adequate moisture level to avoid either desiccation or fungal growth, and moldy food items were immediately removed.
The mean number of young per brood was 25 (confidence limit 95% = 23.3 to 26.8), with a minimum of 7, and a maximum of 54 young/brood.
No significant difference was found between group A and group B concerning offspring number and duration of gravid periods (p < 0.05).No statistical differences were found among successive broods either (p < 0.05).
One mating was enough to obtain at least five broods.After 4 months, 100% of the females generated at least three broods.Although in group A mating could occur only before the experiment beginning, the females became just as frequently gravid as the females from group B, where mating could occur more frequently.
Most specimens consumed their own exuviae, as observed by Nair (1984) in a population of Porcellio laevis (Latreille, 1804).Moreover, three cases of cannibalism were observed, where the female ate the male during its molting.Concurrently, some isopods had difficulties during molting and died, because they could not remove their own exuviae.
A sex-ratio of 1:1.2, male to female, was found, with no significant differences between the number of males and females.

Density experiment
In the first weighing (40 th day), the weight from organisms at different densities (18, 24 and 30), showed a significant difference (p < 0.05), when compared to the lowest density (6 animals/box) (Figure 1).However, at the end of the experiment (110 th day), there was no significant difference in weight between the different densities.It is possible that the higher level of mortality occurring in the higher density groups led to this result, hence lower densities showed far more favorable conditions to survival (Figure 2).
The first individuals were born at the 67 th day, except for the highest density (30 animals/box), which took a few more days to generate the first neonate.All offspring were counted at the 110 th day.The total number in each replicate was divided by the number of females, and then, the mean offspring number/female in each density was obtained (Figure 3).The females in the lowest densities generated more offspring, when compared to higher densities.However, cause there was a positive relationship between female size and fecundity (Nair, 1984;Hassal and Dangerfield, 1990).Therefore, intraindividual competition at high densities affected the individual body size, which then influenced survival and reproduction success.
Considering that the isopods were fed ad libitum, the negative effects on survival, growth and reproduction were not related to lack of food.Dangerfield (1989) found intraspecific density affected growth, survival and reproduction in a laboratory population of Armadillidium vulgare, when food was not a limiting resource.Thus, these effects were due to competition between individuals rather than resource exploitation, confirming the results generated in this study.Ganter (1984) suggested that the nature of intraindividual competition seemed to be related to pheromonal responses of isopods, which, according to Hassall et al. (2005), should be the subject of further investigation.
The fact that reproduction was not affected by density, as no statistical difference was found between the results, might be due to the great variability observed in the data.This variability might be associated with several conditions not addressed here, such as female size, randomness of the experiment (individuals were distributed without any previous sex determination) and the inherent variability of the species.
Cannibalism is common in laboratory isopod cultures (Sutton, 1980) and was observed throughout the present experiments.Molting distress was also a conspicuous feature during the experiments; however, no clear cause could be determined for this.Araújo et al. (2004), working with Atlantoscia floridana (van Name, 1940), recorded several cases of mortality during molting, especially in the final stage, when the exuvia collapses with the body wall and the animal cannot remove it.
the nonparametric Dunn's multiple comparison tests did not reveal any statistical difference among treatments.

Reproduction
Females of C. murina were able to store sperm and use it for at least 5 broods without a reduction in brood size.Continuous reproduction under laboratory conditions for at least 4 broods was found by Vink and Kurniawati (1996) for Porcellionides pruinosus (Brandt, 1833), and for at least 6 broods by Mocquard et al. (1989) for Armadillidium vulgare (Latreille, 1804).Vink and Kurniawati (1996) found no reduction in the size of successive broods after one mating for a population of P. pruinosus.The results of the present study are in good agreement with these previous findings and confirm the ability of isopods to reproduce without continuous mating.
The absence of a statistical difference between the number of males and females born in the laboratory indicates a potential equal effort on male and female reproductive tactics.Whether under natural conditions males die earlier than females as in Armadillo officinalis Duméril, 1816 (Warburg and Cohen, 1992), the fact that C. murina females were able to reproduce continuously under laboratory conditions without copulation, could indicate compensation to the lower number of males in the population.
C. murina appears to be suitable for use in ecotoxicological studies, because supplying a sufficient number of animals for testing can be obtained in a short period.A comparison between C. murina and other isopods are provided in Table 2.

Density
The density of individuals in the maintenance boxes plays an important role in the survival of the organisms, because the lowest densities (6 and 12 animals per box) provided higher growth rate and reproductive output (but not in a significant way).These results are in good agreement with Caseiro et al. (2000), who observed that density affected survival, growth and reproductive output of P. dilatatus in laboratory cultures.
The decreased growth rate of individuals in high density boxes had a direct effect on reproduction, be- Animals have been easily grown under laboratory conditions, and the use of a standardized commercial fish food, as seen for other crustaceans (Nelson and Brunson, 1995;Maranhão and Marques, 2003), supplemented with a carbohydrate source (slices of potatoes), employed also by other authors (Brody and Lawlor, 1984;Carefoot, 1984;Caseiro et al., 2000), provided the nutritional requirements for their growth and reproduction.

Conclusion
Our results indicate that C. murina could be cultivated under laboratory conditions and be used as a test organism in ecotoxicology.This species provides great offspring numbers in a relatively short time and is also easily manipulated and maintained in laboratory cultures.Furthermore, C. murina is well adapted to tropical conditions and may provide an exceptional tool to assess environmental impact in terrestrial ecosystems in the tropics.Finally, the importance of density in the maintenance boxes was also confirmed and such conditions are to be followed in future cultivation protocols.

Table 1 .
Reproduction statistics for female isopods kept individually after mating (group A) or kept with a male continuously (group B), standard deviation (SD).

Table 2 .
Mean offspring number per brood and duration of ovigerous period of C. murina and other terrestrial isopod species.Mean offspring numbers per female (±SD) of C. murina, in the different density groups, under laboratory conditions, at the 110 th day.No statistically significant differences were found among the densities.