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105
terms of dry weight, carbon, nitrogen and ash content of eggs, embryos at different developmental stages, juvenile, subadults, and adult individuals of M
. mixta.
2. Materials and methods
2.1. Collecting and rearing of mysids Specimens of M
. mixta were collected in a coastal area of the northern Baltic proper 58
8 499 N, 178 359 E, bottom depth of 25–35 m, in August 1995 and August 1996, with
2
a large plankton net 1 m opening, mesh size 0.5 mm. Immediately after collection, specimens covering the entire representative size range were length measured and
prepared for ash content, carbon and nitrogen analysis. Simultaneously, materials were collected for the development of regression models between various body variables, such
as total body length, carapace length, uropod length, wet and dry weight. The rest of the animals were transported to the laboratory where the animals collected in 1995 and 1996
were maintained for 8 and 5 months, respectively, as part of a study on mysid growth, moulting and stable isotope fractionation. These animals were kept in darkness in natural
brackish water 6–7‰ at 9–10
8C, i.e. conditions similar to those in their natural environment. The mysids were fed with a surplus of a freshly hatched brine shrimp
Artemia spp., San Francisco Bay Brand, which were also regularly sampled for analyses of C and N content. Details on rearing, feeding regime, consumption rates, and
food composition are published elsewhere Gorokhova, 1998, 1999. Initial values of the animals used in the experiments were assumed to be those of mysids from the same size
group
60.5 mm sampled at the start of the experiments. 2.2. Experiment I
During the period from August 1995 until April 1996, groups of 30–50 mysids initial length 7–12 mm were kept in 50-l aerated aquaria. The conditions provided permitted
for growth, gonad development and reproduction. After 5–9 months, in December– April, adult individuals 16.9
60.9 mm from the laboratory culture were sampled for analyses, separating males, non-gravid, gravid, and postspawned females. Mysid length,
dry weight, fecundity, size and stage of development for embryos present in the brood pouch were recorded. Embryos were dissected out and transferred into pre-weighed tin
capsules.
While moulting in ovigerous females is arrested, growth might still occur through stretching of the abdominal joints as has been found for some other malacostracan
crustaceans including mysids Mauchline, 1973a. Consequently, this intersegmental growth leads to changes in body morphometry and particularly in carapace to total body
length ratio, due to expansion of segmented abdomen and little changes in rigid carapace. To estimate the intermoult growth in gravid M
. mixta, the regression lines between body length and carapace length was calculated using females with embryos at
earliest and latest developmental stages.
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. Gorokhova, S. Hansson J. Exp. Mar. Biol. Ecol. 246 2000 103 –123
2.3. Experiment II In August to December 1997, specimens of M
. mixta initial length 8–11 mm were kept individually in 1-l plastic beakers. The food was changed daily and the water was
renewed weekly. Every week, three to five individuals were sacrificed, sized, staged in one of the categories described below, and prepared for the analysis. The experiment
lasted for 18 weeks.
2.4. Developmental stages of mysids and embryos Seven life cycle stages were considered according to Mauchline 1980 and Mees et
al. 1994. 1 Juveniles: no secondary sexual characteristics. 2 Immature males: 4th pleopods are elongated but do not reach the posterior edge of the last abdominal
segment; the lobus masculinus is not fully developed and lacks setation. 3 Immature females: oostegites are visible only from the ventral side. 4 Adult males: 4th pleopods
reach the posterior edge of the last abdominal segment; the lobus masculinus is setose. 5 Adult females: marsupia are visible from the lateral side but empty. 6 Gravid
females: marsupia are filled with eggs embryos. 7 Postspawned females: embryos have been released from marsupia.
Embryo stages were classified using descriptions of Mauchline 1980, Cuzin-Roudy and Tchernigovtzeff 1985, and Wittmann 1981a. 1 Embryonic egg: embryos in the
initial state of marsupial development stages of early embryo and ovoid eggs, still within the egg membrane. 2 Eyeless larvae: newly hatched and developing nauplioid
larvae with eye pigmentation and large yolk mass. Wittmann referred to this stage as ‘naupliar’, and Cuzin-Roudy and Tchernigovtzeff as ‘naulioid phase’. 3 Eyed larvae:
moulted nauplioid larvae with the eyes on stalks and almost absorbed yolk. In the terminology of Wittmann and Cuzin-Roudy and Tchernigovtzeff this was ‘postnaupliar’
and ‘postnauplioid’ stages, respectively.
2.5. Length measurements Measurements of the total mysid body length BL, mm were taken from the tip of the
rostrum to the posterior edge of the last abdominal segment, using calipers 60.02 mm
under a stereomicroscope. Length measurements of the carapace, uropods, and embryos were made using a binocular microscope fitted with an ocular micrometer. Carapace
length CL, mm was measured as the distance from the tip of the rostrum to the mediodorsal margin of the carapace. Uropod length UL, mm was from the posterior
margin of the last abdominal segment to the posterior edge of the outer uropod, excluding the setae. Larval size was measured in seawater as the largest diameter for the
egg stage and the length from the terminal to the frontal tip of the body for the eyeless and eyed stages.
2.6. Drying and weighting The samples of mysids, eggs embryos, and Artemia were dried in 60
8C to constant
E . Gorokhova, S. Hansson J. Exp. Mar. Biol. Ecol. 246 2000 103 –123
107
weight 6–8 days for mysids and 3 days for eggs embryos and Artemia. The dry weights DW, mg were determined using a Sartorius M3P microbalance to the nearest
microgram. When preparing mysid abdominal tissue, abdomens were carefully separated while dried and transferred to the pre-weighed tin capsules. Whole abdomen or its
aliquot, depending on size, 15–20 eggs embryos, or 30–50 Artemia nauplii comprised each sample. All the samples were kept frozen
2208C until analysis. 2.7. CN elemental analyses
Elemental carbon and nitrogen content were measured by combusting the tin capsules in a CHN-analyzer CHN-900, 600-800-300, Leco Corporation. For calibration, EDTA
of analytical quality Sigma-Aldrich was used. The standard deviation among replicate standard samples was within 0.2 for both carbon and nitrogen.
2.8. Ash content After dry weight determinations, five to ten replicate samples from the same set that
had been analyzed for elemental composition, were incinerated at 500 8C for 4 h in a
muffle furnace Hirota and Szyper, 1975. The remaining ash, containing only inorganic substances, were cooled in dessicator and weighed in the same way as DW.
2.9. Energy densities Caloric values of mysid body, muscle tissue and eggs embryos were calculated from
formula given in Salonen et al. 1976, using values of mysid DW, carbon, and ash content. The proportion of inorganic carbon was assumed to be the same as in Mysis
relicta 0.044 of DW, Salonen et al., 1976 and to be independent on mysid size. For calculations of the total energy of ovigerous females, we used the observed fecundity,
eggs embryos energy content and energy content of the female muscle tissue.
2.10. Data analysis When comparing two groups, unpaired t-test was applied followed by F-test to
compare variances. Comparisons of three and more groups were performed with a one-way ANOVA with Bartlett’s test for equal variances followed by Bonferroni’s
multiple comparison test. The length–weight both exponential and log-transformed, length–length and fecundity–length relationships were determined by regression analy-
ses. Significant differences between regression lines within a given category by life stage and sex were analyzed by testing slopes for equality by analysis of variance
ANOVA and then by testing elevations of lines with homogeneous slopes for equality by analysis of covariance ANCOVA. Unless specified otherwise, data are given as
means and standard deviations. Ash and element proportions are given in percentage of the DW.
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. Gorokhova, S. Hansson J. Exp. Mar. Biol. Ecol. 246 2000 103 –123
3. Results
