J. Moll Stud (1997), 63, 469-471
© The Malacological Society of London 1997
RESEARCH NOTES
Inheritance of the calmodulin gene-1 intron 3 in Mytilus edulis juveniles
M.A. Del Rio-Portillat and A.R. Beaumont
School of Ocean Sciences. University of Wales, Bangor, Menai Bridge, Gwynedd, LL595EF, U.K.
The genetics of mussels of the genus Mytilus has been
widely studied at the level of allozymes1 and more
recently via the mitochondrial DNA molecule2-5. In
contrast, genomic DNA has been little studied.
Recently, C6rte-Real et al.4 have designed primers
and amplified two calmodulin gene regions (CaM-1
and CaM-2) in the mussel Mytilus edulis L. Using
primers for the intron 3 region, they detected two size
variant alleles, A and B (approximately 525 and 460
base pairs, bp, respectively), in wild populations
which were then tested for Mendelian inheritance in
embryos from single families5. In some cases, inheritance was Mendelian, but rather high frequencies of
unexpected genotypes were found. For example, in
one cross between a female AA and a male BB some
AA offspring were found and in another cross, a
female AA mated with a male AA, a significant number of AB offspring were detected. Possible explanations offered were (a) contamination between
crosses, (b) external input of wild-type mussel
embryos, (c) analysis of embryos with adhered sperm,
(d) PCR contamination and (e) aneuploidy.
Although it was considered that aneuploidy could
account for some of the unusual genotypes, overall,
the high numbers of odd genotypes and the significant
distortion of Mendelian ratios in some crosses was
not easy to explain.
In the study presented here, as part of a larger
genetic analysis of cultured mussels, three families of
M. edulis were cultivated up to the juvenile stage and
characterised genetically using six allozyme loci6.
These juveniles were also used to test for Mendelian
inheritance of the CaM-1 intron 3 putative locus in M.
edulis. Chromosomal abnormalities are unlikely to
survive beyond the embryo stage and therefore it is
more realistic to test for Mendelian inheritance using
juveniles or adults rather than embryos.
Three female M. edulis (A, B and C) were crossed
with a male (M). After settlement all families were
divided into two groups based on larval growth ((a)
fast growing and (b) slow growing larvae) and reared
until the average shell length over all the cohorts was
greater than 3.0 mm6. Females A and B were homozygous for the same CaM-1 intron 3 allele as the male
(M) and only one group from each of the crosses AM
and BM were scored. Both groups (a) and (b) from
the cross CM were scored (Table 1).
t Praent Addreu: CICESE Acuicultura. Apdo. Postal 2732.
Ensenada, Baja California, 22800. Mexico
Corte-Real et al.Ai reported two alleles, A and B,
with approximate sizes of 525 and 460 bp respectively
and these agree with the sizes of the amplified DNA
found in the present study. We have named the alleles
according to their approximate size (525 and 460 bp)
rather than using letters because we have detected an
additional longer variant (600 bp) in a local wild M.
edulis population6.
All offspring from crosses AM and BM produced
only a single band from the amplified segment. In
both groups from cross CM, heterozygote offspring
exhibited two bands of the expected sizes (Table 1).
However, when first scored, a few of these individuals
exhibited an extra third band but this extra band
never occurred again following repeat PCRs and was
therefore a secondary PCR product rather than a real
product from the mussel genome. Agreements with
Mendelian expectations were found in both groups
(a) and (b), and overall, from family CM (Table 1).
Therefore, Mendelian inheritance of the 460 and 525
alleles at the CaM-1 intron 3 locus is confirmed using
relatively small numbers of juveniles produced from
crosses under laboratory conditions.
Confirmation that there was no contamination
between families and that there were no other unexpected irregularities has been obtained by allozyme
analysis. From the six allozyme loci tested (Table 2),
five were polymorphic for cross AM (ldh-1, Idh-2,
IMP, Csr, and Pgm) and CM {ldh-1, Idh-2, Lap, Dia,
and Pgm), whilst three loci were polymorphic for
cross BM (Lap, Dia, and Pgm, Table 2). No significant deviations from Mendelian inheritance at any
loci were found when allele and genotype frequencies
were tested in offspring from families AM and BM.
Offspring from cross CM which had been selected for
fast larval growth (a) differed significantly (p<0.001)
from Mendelian genotype frequency expectations at
two loci (ldh-1 and Pgm) and these differences were
also evident when the data from this family were
pooled (Table 2). However, offspring from cross CM
(b) were in agreement with Mendelian inheritance at
all five polymorphic loci (Table 2). The restriction of
apparent non-Mendelian inheritance to only two of
the five allozyme loci, and in only one selected group
(the faster growing larvae) from the families, is most
likely due to the artificial selection applied at metamorphosis, and therefore does not imply uneven or
unusual Mendelian segregation.
This study has effectively removed any areas of
uncertainty associated with the Mendelian inheri-
RESEARCH NOTES
470
Table 1. Mean shell length (standard error) and observed genotypes for the CaM-1 intron 3 gene
amplification in offspring from a single male (M) crossed with three females A, B and C. Agreement
with Mendelian inheritance was tested with a x2 goodness of fit test (x^oce.i) = 3.841). Sample size
was 16 except for cross BM where only 12 samples produced an amplified band.
Observed genotypes
460-525
Length (s.e.)
(mm)
Cross
AM
BM
CM (a)
CM(b)
CM pooled
2.463(0.197)
6.308 (0.700)
3.775 (0.613)
4.313 (0.379)
525-525
Observed
Expected
Observed
Expected
X2
0
0
10
7
17
0
0
8
8
16
16
12
6
9
15
16
12
8
8
16
1.000
0.250
0.125
Total DNA was extracted from parental frozen adductor muscle and from the whole body of
juveniles following standard protocols13. The sample was homogenised with CTAB buffer (100 mM
Tris-HCI pH 8.0, 1.4 mM NaCI, 20 mM EDTA and 2% CTAB), digested with 15 mg ml" 1 of proteinase
K, followed by four organic extractions: one with chloroforrrrisoamyl (24:1), two phenolxhloroform:
isoamyl alcohol (25:24:1) and a final chlorofornrisoamyl (24:1). DNA was precipitated with
isopropanol, washed with 70% ethanol, dried and redissolved in 0.1 concentration of TE buffer
(0.01 M Tris-HCI, 0.5 M EDTA pH 8.0)13. DNA amplification was similar to that described by C6rte-Real
et af, except that 'Perfect Match' was not included in the reaction tube.
Table 2. Mytilus edulis. Tests of agreement of allele and genotype frequencies at six allozyme loci {Idh1, Idh2, Lap, Dia, Gsr and Pgm) to Mendelian
ratios. * = significant at p < 0.001, ns = not significant. Sample sizes were
between 52 and 60 for each locus.
Crosses
AM
Locus
Idhl
Allele
Genotype
Idh2
Allele
Genotype
CM
BM
a
b
total
ns
ns
*
*
ns
ns
#
*
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
*
ns
ns
ns
*
Lap
Allele
Genotype
ns
ns
Dia
Allele
Genotype
Gsr
Allele
Genotype
ns
ns
Pgm
Allele
Genotype
ns
ns
12% starch gel electrophoresis was used. Enzymes stained were isocitrate
dehydrogenase (IDH, 1.1.1.42 with two loci), leucine amino peptidase (LAP,
3.4.11.-), NADH-diaphorase (DIA, 1.6.2.2), glutathione reductase (GSR,
1.6.4.2) and phospho-glucomutase (PGM, 2.7.5.1)14-15-18. The Idh, Lap, Dia
and Gsr loci were scored on gels run at pH 7.4 and Pgm on gels run at pH
6.0. Data were Bonferroni adjusted for the number of tests carried out".
RESEARCH NOTES
tance of the CaM-1 locus in M. edulis. The genetic
scoring of mussels at the juvenile stage rather than as
embryos will have removed all those possible aneuploid individuals which are likely to be more common
in early larval stages7. Neither cross-contamination
between cultures nor accidental inclusion of foreign
mussels were detected in this study either by the
CaM-1 intron 3 amplification or by allozyme electrophoresis6. PCR contamination (DNA fragments without template DNA which are amplified) was not
detected, since none of the negative controls produced visible banding. Whilst some PCR by-products
were found, they were not repeatable following
further PCR trials. Further optimization of PCR
conditions would reduce or eliminate this problem.
Further research on neutral markers is needed to
understand more about the different genetic processes that might be acting on different parts of the
genome8. PCR can be applied to either nuclear' or
mitochondrial9 DNA from individual larvae. This
could accelerate the processing of samples in laboratory trials of this type compared with allozyme
electrophoresis, which usually requires animals to be
bigger before analysis is possible. Although some
electrophoretic systems can be used on larvae as
small as 250 jim, only a very few allozyme loci can be
scored10-". The ability to score neutral DNA loci in
small organisms or by non-destructive sampling of
adults using PCR based techniques5 increases the
armory of methods available to study the genetics of
aquatic and terrestrial molluscs and can overcome the
difficulties associated with allozymes that may be
subject to selection12.
471
REFERENCES
1. GOSUNG, E. 1992. In: E. Gosling, (ed.) The
mussel Mytilus: ecology, physiology genetics and
culture. Elsevier Amsterdam. 309-382.
2. SKIBINSKI, D.O.F., GALLAGHER, C. & BEYNON,
CM. 1994. Nature, 368:817-818.
3. ZOUROS, E., BALL, A.O., SAAVEDRA, C. &
FREEMAN, K.R. 1994. Nature, 368:818.
4. C6RTE-REAL,
H.B.S.M.,
DIXON,
D.R.
&
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5. CORTE-REAL, H.B.S.M., HOLLAND, P.W.H. &
DEXON, D.R. 1994. Mar. BioL 120:415-420.
6. DEL RIO-PORTILLA, M.A. 19%. Ph.D. Thesis,
University of Wales, Bangor.
7. DIXON, D.R. AND FLAVELL, N. 1986. / . Mar. BioL
Ass. U.K. 66:219-228.
8. SKIBINSKI, D.O.F. 1992. In: A.R. Beaumont (ed.).
Genetics and evolution of aquatic organisms.
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9. OLSON, R.R., RUNSTADLER, J.A. & KOCHER,
T.D. 1991. Nature, 351: 357-358.
10. Hu, Y.P., Luz, R.A. AND VRUENHOCK, R.C.
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13. SAMBROOK, J., FRITCH, E.F. & MANIATIS, T. 1989.
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Acknowledgement
16. BEAUMONT, A.R., DAY, T.R. & GADE, G. 1980.
MADRP was supported by the Consejo National de
Ciencia y Tecnologia of Mexico.
Mar. BioL Lett, 1:137-148.
17. LESSIOS, H.A. 1992. Mar. BioL, 112:517-523.
/. MolL Stud. (1997), 63,471-473
© The Malacological Society of London 1997
Notes on gamete release and fertilisation in Calliostoma zizyphinum (L.)
(Gastropoda; Trochidae)
S. Holmes
The Northumbrian Water Ecology Centre, Sunderland University, Sunderland, SRI 3SD, UK.
The genus Calliostoma is world-wide in distribution,
and is a common inhabitant of both the littoral and
sublittoral zones of the seashore1. However, despite
the relative abundance of several species, surprisingly
little is known about their ecology. Calliostoma
zizyphinum is ubiquitous to the sublittoral shelf zone
of North Atlantic temperate shores2. Features of the
reproductive ecology, egg production and embryonic
development, of C. zizyphinum werefirstreported by
Roberts3. Further work by Lebour 4J corroborated
there initial findings but, unfortunately, the mechanism of fertilisation, whether internal or external and
the subsequent egg changes to the blastula stage preceding fertilisation were not observed by either
author. Subsequently, much work has been published
on the general ecology of C. zizyphinum''-" but there
have been no further accounts of the reproductive
biology of C. zizyphinum. Studies of other species of
Calliostoma have reported both internal and external
fertilisation'10. In this paper both the mechanism of