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Journal of Heredity Advance Access originally published online on October 26, 2005
Journal of Heredity 2005 96(6):713-717; doi:10.1093/jhered/esi120
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© The American Genetic Association. 2005. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.

Brief Communication

Maternal Effects on Encystment in Crosses Between Two Geographic Strains of Artemia franciscana

C. Saavedra, and F. Amat

From the Instituto de Acuicultura de Torre la Sal, CSIC, Ribera de Cabanes, 12595 Castellón, Spain

Address correspondence to Carlos Saavedra at the address above, or e-mail: saavedra{at}iats.csic.es.


   Abstract
Top
 Abstract
Materials and Methods
 Results and Discussion
 References
 
Maternal effects can have environmental or genetic causes. A method that can be used to demonstrate the genetic basis of a maternal effect is to look for grandfather effects in a backcross following reciprocal crosses. The absence of a grandfather effect would exclude a chromosomal basis for the maternal effect when the male sex is heterogametic (XX-XY sex determination system). However, in organisms in which the female is heterogametic (ZW-ZZ sex determination system), the absence of a grandfather effect does not rule out a chromosomal basis of the maternal effect, since the genes responsible for that effect can be located in the W chromosome, which is transmitted matrilineally. Conversely, the absence of a grandfather effect would point to a W-chromosome basis for the trait, provided that a maternal effect has been previously demonstrated. Distinguishing between W-located and autosome or Z-located maternal effects is important to understand the evolutionary dynamics of a trait. Here we report on a study of the chromosomal basis of maternal effects on two life-history traits related to encystment in the branchiopod crustacean Artemia franciscana, in which females are heterogametic. We performed crosses of two populations that differ in the number of cysts they produce. The proportion of encysted broods showed a maternal effect and was not affected by the grandfather's genotype, pointing to a W-chromosome basis. The average number of encysted offspring per brood showed a strong paternal effect and also a slight maternal effect. This trait also showed a grandfather effect, which suggests that the geographical variation has an autosomal or Z-chromosomal basis.

Maternal effects occur when the phenotype of a mother or the environment she experiences affects the phenotype of her offspring. Maternal effects often affect traits of adaptive importance [reviewed in Mousseau and Fox (1998)]. The causes of maternal effects can be environmental and genetic. Maternal genetic effects can be determined by cytoplasmic genes (e.g., mitochondrial genome) or by nuclear genes, either residing in the autosomes or in the sexual chromosomes. Specific breeding schemes allow the determination of the different sources of genetic maternal effects. The characterization of a trait as affected or unaffected by maternal effects, and whether the maternal effect is genetic or not are aspects of basic importance to study its evolution (Lynch and Walsh 2000; Roff 1997, 1998).

A simple way to detect genetic maternal effects consists of conducing reciprocal crosses between strains that have been kept in the same environment for several generations (Lynch and Walsh 2000; Roff 1997, 1998). If strains do not have a common environmental history, the genetic basis of the maternal effect can still be demonstrated by searching for "grandfather effects" in the progeny of the F1 females backcrossed to the parental strains (Reznick 1981, 1982; Roff 1997, 1998). This method is valid when the female is homogametic, as it occurs in organisms with an XX-XY chromosomal sex determination mechanism. However, in organisms in which females are heterogametic (ZW-ZZ sex determination mechanism), a maternal effect can occur, but not be associated with a grandpaternal effect if the genes responsible for the maternal effect reside in the W chromosome, since this chromosome is not transmitted through males. Conversely, we can use the presence or absence of grandfather effects as a cue for the location of the genes responsible for a given trait in species in which the female is heterogametic, provided that a maternal effect has been shown previously by other means (such as reciprocal crosses). In this case, the absence of a grandfather effect would point to a W chromosome-based maternal effect. The alternative observation of a significant grandfather effect would imply that the genes responsible for the maternal effect are located in the autosomes or in the Z chromosome, but not in the W chromosome.

Here we apply this reasoning to study the genetic basis of encystment in the brine shrimp Artemia (Branchiopoda, Anostraca), which has a ZZ-ZW sex determination system (Bowen 1965; Stefani 1963). Encystment is an outstanding feature of the complex life cycle of this crustacean adapted to life in marine salterns and inland hypersaline lakes all over the world. Encystment allows Artemia populations to persist in these ephemeral and unpredictable environments, and facilitates dispersal (Green et al. 2005; MacDonald 1980; Proctor and Malone 1965; Proctor et al. 1967). Moreover, knowledge of the genetics of encystment in Artemia is of great interest for those involved in fish and prawn aquaculture (Lavens and Sorgeloos 2000).

Artemia females can produce two types of broods. In the naupliar or ovoviviparous broods, embryos develop normally inside the female ovisac until they reach the nauplius larval stage (instar I), and then they are delivered in the water. In the oviparous or encysted broods, the embryo stops its development at the 16-cell stage, enters into diapause (Anderson et al. 1970; Morris and Afzelius 1967), and then is covered with a membrane or corion to produce the cyst, which is subsequently liberated in the water. Cysts can persist for years in a state of anhydrobiosis. When cysts meet favorable conditions, they can resume their embryonic development to produce nauplii. Females can alternate the production of oviparous and ovoviviparous broods during their lives, but mixed broods are never produced. Whether a female produces cysts or nauplii at a given moment depends on still poorly known environmental cues, of which suboptimal food availability appears to be the most important (Amat et al. 1991; Wurtsbaugh and Gliwicz 2001).

There is considerable variation among wild and cultured populations of Artemia species in the relative numbers of cysts and nauplii produced by females (Amat 1980, 1982). Geographic strains show differences in the proportions of each type of offspring when allowed to reproduce in the laboratory, which suggests that interpopulation differences for this trait may have a genetic basis (Browne 1980; Browne et al. 2002). Similar differences have been observed among clones of parthenogenetic species (Browne and Spencer 1987). Studies have also shown that changes in the rearing conditions of a given strain often lead to changes in the proportions of each kind of progeny, which points to an important genotype by environmental interactions (Browne and Wanigasekera 2000).

During a study of American populations of Artemia we observed that the proportion of cysts in the offspring of crosses among different geographical strains was similar to that observed in pure crosses of the maternal strains. We report here an experiment specifically designed to test the existence of maternal effects on the number of cysts produced by females in Artemia franciscana, its genetic basis, and the chromosomal location of the underlying genes.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results and Discussion
 References
 
Experimental Strains and Rearing Techniques
Laboratory populations of A. franciscana originating from the Laguna Mar Chiquita, province of Cordoba, Argentina (Amat et al. 2004; Papeschi et al. 2000) and from San Francisco Bay, California (USA) were obtained after hatching cysts kept in the cyst collection of the Instituto de Acuicultura de Torre de la Sal. These two populations were chosen because previous experiments showed that they differed markedly in the proportion of encysted broods produced under standard culture conditions.

Cysts were hatched in seawater (salinity 37 g/L), at 28°C, constant illumination, and gentle bubble aeration. Nauplii were grown in cylindrical 5 L plastic containers, in 70–80 g/L of filtered brine (seawater plus crude sea salt) at 24°C, mild aeration, and 12 h light:12 h dark illumination, on a mixed diet of Dunaliella salina and Tetraselmis suecica (105 cells/ml). The medium was monitored and renewed every 3 days.

Adult specimens were allowed to reproduce in the same container, and the new cysts produced were collected and processed according to standardized methods (Vanhaecke et al. 1980). Matings were performed in 50 ml plastic beakers. A female and a male were placed together in the beaker. The medium was monitored and renewed every 2 days. Surviving individuals and female reproduction mode were controlled and successive broods were collected and counted. Salinity and food conditions were the same as described for parental stocks.

Crosses
Cysts were used as described above to produce adults, which were allowed to mate and produce more cysts. These cysts were induced to hatch to produce a second generation of adults, which constituted the parental strains C (Mar Chiquita) and S (San Francisco Bay) used in this experiment. After two generations under laboratory conditions, the environmental parental and grandparental effects due to the native locality should have been removed (Lynch and Walsh 2000; Roff 1997). Males and females from both strains were crossed reciprocally, producing four types of crosses (male x female): S x S, S x C, C x S, and C x C. The offspring of these crosses will be referred to as SS, SC, CS, and CC, respectively. Thirteen to 15 female offspring from each of the four F1 crosses were subsequently mated to males from each of the two parental stocks, producing eight types of crosses which provided the data for our analyses: S x SS, S x SC, S x CS, S x CC, C x SS, C x SC, C x CS, and C x CC.

Data Collection and Analysis
Data collection was restricted to the first five broods. Previous observations indicated that this number of broods was enough to show differences in the variables of interest between the two strains. Broods produced by each female were counted and classified as naupliar or encysted. In addition, individual cysts or nauplii in each brood were counted. Since, in Artemia, the progeny are produced in discrete broods, the total number of cysts produced by a female is the result of combining the number of encysted broods produced (NEB) and the average number of encysted offspring per brood (AEO). Since the number of broods scored varied among females, we used the proportion of encysted broods (PEB) instead of NEB. Raw data on the number of cysts and nauplii produced per brood by each mating pair are not presented here, but can be obtained from the authors upon request. All statistical analyses were carried out with the SPSS software package (version 9.0) (SPSS Inc., Chicago, IL) unless stated otherwise.

The variables were tested for normality and variance homogeneity. The effect of the parental and grandparental genotypes on AEO was studied by standard analysis of variance (ANOVA) by fitting linear models. However, in the case of PEB, we detected a strong heteroskedasticity and lack of normality of the residuals in the majority of the crosses, which could not be normalized by different types of transformations. We therefore performed nonparametric analyses of variance (NPANOVAs) by the method of Scheirer et al. (1976), as described in Sokal and Rohlf (1995). To calculate the H test statistic of NPANOVA, mean squares of ANOVA performed on PEB ranks were imported into an Excel spreadsheet.


   Results and Discussion
Top
 Abstract
 Materials and Methods
 Results and Discussion
References
 
Demonstration of Genetic Maternal Effects in Reciprocal Crosses
A total of 113 matings were established for the eight types of crosses. All produced three or more broods. In total, 549 broods were scored. The average values of PEB and AEO for the eight types of crosses are shown in Figure 1. We first analyzed a subset of crosses which allow the comparison of reciprocal crosses with the parental strains (SS x S, SS x C, CC x S, CC x C). A significant effect of the maternal strain was detected for the two variables (Tables 1 and 2). AEO showed an additional significant effect of the paternal strain. Since the strains used had been maintained in the same environment for at least two generations before the onset of the experiment in order to erase the environmental effects of the presumably different original habitats, the maternal effects observed have to be genetic. Therefore we can conclude that there are genetic differences between the Mar Chiquita and the San Francisco Bay populations of A. franciscana, which accounts for the observed differences between the two strains in the number of encysted offspring produced in pair mating. Maternal effects on several features of diapause have been shown previously in invertebrates, notably insects (Hockam et al. 2001; Ingrisch 1987; Milonas and Savopoulou-Sultani 2000; Mousseau and Dingle 1991; Tachibana and Numata 2004), but also crustaceans (Deng 1996; Pfrender and Deng 1998; Wyngaard 1988). However, the genetic nature of maternal effects has been demonstrated in only a few cases (Mousseau and Dingle 1991; Roff and Bradford 2000; Takeda 1998). Maternal effects are expected especially for traits expressed early in offspring development. In the case of PEB, the maternal effect can be related to the mechanism of encystment, which starts in the ovary before mating (Bowen 1962).



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  		Figure 1.. (A) Average percentage of encysted broods and (B) average number of encysted offspring produced by mating pairs in the eight types of crosses (male x female). Error bars ±1 SE.

 

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  		Table 1.. Summary of the results of the three-way NPANOVA on PEB

 

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  		Table 2.. Summary of the results of the ANOVA on AEO

 
Grandfather Effects and the Chromosomal Basis of Cyst Production
Genetic maternal effects can be due to genes transmitted maternally, such as mitochondrial genes, or to nuclear autosomal genes expressed only in the female (Roff 1998). In Artemia, in which the female is the heterogametic sex, an additional possibility is that maternal effects are due to genes residing in the W chromosome, which is transmitted matrilineally. While autosomal or Z chromosome-based factors would result in grandfather effects, cytoplasmic or sex-linked factors would not. In the present study, the cross design allowed us to have a set of progeny completely pedigreed for two generations, and therefore to trace the effects of the chromosome sets inherited through females only (mitochondrial and W chromosomes) or through males as well (autosomes and Z chromosomes), and to test for the presence of grandfather effects.

The effects of the grandparental genotypes were tested by three-way ANOVA. The effect of the grandpaternal strain on PEB was nonsignificant (Table 1). This suggests that encystment is under the control of genes located on the W chromosome, although a mitochondrial basis cannot be excluded because the two effects are confounded. There are not many studies in which the chromosomal basis of maternal effects on diapause has been established. Takeda (1998) found that the summer and winter diapause polymorphisms exhibited by two geographic ecotypes of the rice stem maggot (Chlorops oryzae) were partially controlled by a major X-linked gene.

The previous results may have implications for the understanding of the evolutionary dynamics of encystment in Artemia. The equilibrium genetic variance for fitness due to sex-linked maternal effect genes is larger when the female is the heterogametic sex, and therefore isolated or allopatric populations can be expected to diverge more rapidly from maternal effects than for other traits by the combined action of random genetic drift and local selection (Wade 1998). Also, a larger variance in fitness for PEB might help Artemia to adapt quickly to local habitats and to colonize new localities, for example, after introduction by migratory birds.

The three-way ANOVA of AEO showed a significant effect of the paternal strain and also of the grandpaternal strain (Table 2). The paternal effect was also observed in the previous analysis of the RC subset and points to an important paternal contribution to fecundity that deserves further study. The grandfather effect on AEO indicates that genes inherited by the mothers from their fathers had an effect on the fecundity component of cyst production. This result excludes the cytoplasmic genomes and the W chromosome as the sole residence of the genes responsible for the variation observed in AEO. They should be located principally in the autosomes or Z chromosomes. However, the present results do not exclude that some genetic factors implied in the trait could be present in the W chromosome or in the mitochondrial genome, but showed weaker effects than the autosomal/Z genes and therefore were masked by them. It also should be stressed that these conclusions refer to the differences between strains. It is possible that other factors involved in the determination of AEO, which are not segregating in the two strains used in our experiments, or which lack variations in gene frequencies or gene effects across populations, reside in mitochondrial or W gene regions. The experimental design used in this work does not allow detection of these factors. It is also possible that the genes involved in the between-population variation in AEO are located in the W chromosome, but in a region homologous to the Z chromosome that can recombine. Bowen (1965) showed that recombination between the homologous regions of Z and W chromosomes can be as high as 20% in experiments carried out with different geographical strains.


   Acknowledgments
 
This research was partly supported by MAR1998-0871-CO2-01 and AGL2001-1968 Spanish government I+D+i projects. C. Saavedra is the recipient of a "Ramón y Cajal" contract of the Spanish Ministry of Education and Science.


   Footnotes
 
Corresponding Editor: Rob DeSalle

Received March 11, 2004
Accepted March 30, 2005


   References
Top
 Abstract
 Materials and Methods
 Results and Discussion
 References
 

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