RESEARCH NOTES
10. HALL, J.R.C.A., DOLLASE, W.A. & CORBATÓ, C.E. 1974.
Palaeogeogr., Palaeoclimatol., Palaeoecol., 15: 33–61.
11. FORSTER, C.R. 1981. J. Mar. Biol. Ass. U.K., 61: 881 –883.
12. KELLER, N., DEL PIERO, D. & LONGINELLI, A. 2002. Mar.
Biol., 140: 9–15.
13. ZENETOS, A. 1997. J. Mar. Biol. Ass. U.K., 77: 463 –472.
14. SOKAL, R.R. & ROHLF, F.J. 1987. In: Introduction to biostatistics
(N.W. Freeman, ed.), New York.
15. HENDERSON, S.M. & RICHARDSON, C.A. 1994. J. Mar. Biol.
Ass. U.K., 74: 939– 954.
16. RICHARDSON, C.A., SEED, R., BROTOHADIKUSUMO, N.A.
& OWEN, R. 1995. Asian Mar. Biol., 12: 39–52.
17. SEED, R. & RICHARDSON, C.A. 1990. In: The neurobiology of
Mytilus edulis (G.B. Stefano, ed.), 1–37. Manchester University
Press.
18. RICHARDSON, C.A., CRISP, D.J. & RUNHAM, N.W. 1979.
Malacologia, 18: 277–290.
19. VON BERTALANFFY, L. 1938. Hum. Biol., 10: 181– 213.
20. GASPAR, M.B., SANTOS, M.N., VASCONCELOS, P. & MONTEIRO, C. 2002. Hydrobiologia, 477: 73–80.
21. RICHARDSON, C.A., SEED, R. & NAYLOR, E. 1990. Mar. Ecol.
Prog. Ser., 66: 259 –236.
22. RAMÓN, M. & RICHARDSON, C.A. 1992. Mar. Ecol. Prog. Ser.,
89: 15–23.
23. PANELLA, G. & MAC CLINTOCK, C. 1968. J. Paleontol., 42:
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maximum size (around 85 mm) when it is 11 –14 years old, which
are in accordance with the results of the present work.
In conclusion, the morphometric relationships studied here
imply that shell growth reflects mainly ontogenetic adaptations.
The annual pattern of wide and narrow growth bands has been
successfully used to determine the age and to estimate the growth
rate of C. chione. The smooth clam has an extended lifespan and a
slow growth rate. Since recent observations suggest that the
populations are very sensitive to rapid environmental disturbance, such as dredging,6 there is a need for further investigation of
the biology of this species.
This work is a part of an MSc Shellfish Biology, Fisheries and
Culture project supported by the European Social Fund. The
senior author would like to thank Antonis Argyrokastritis, Ageliki
Adamidou and Dr Dimitris Vafidis for their assistance during
sampling and laboratory work. David Roberts and Graham
Forsythe helped with photography. Dr Alexis Tsangridis is
acknowledged for critically reading the manuscript.
REFERENCES
1. TEBBLE, N. 1966. British bivalve seashells: a handbook for identification.
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2. GASPAR, M.B. & DIAS, M.D. 1999. Relat. Cient. Tec. Inst. Invest.
Pescas, 43: 14.
3. GREMARE, A., AMOUROUX, J.M. & CHARLES, F. 1998. J. Sea
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doi: 10.1093/mollus/eyi022
Chirality in snails is determined by highly conserved asymmetry genes
Beerend P. Hierck 1 , Brigitta Witte 1,3 , Robert E. Poelmann 1 , Adriana C. Gittenberger-de
Groot 1 and Edmund Gittenberger 2,3
1
Department of Anatomy and Embryology, Leiden University Medical Center, P.O. Box 9602, NL-2300 RC Leiden, The Netherlands
2
National Museum of Natural History, P.O. Box 9517, NL-2300 RA Leiden, The Netherlands;
3
Institute of Biology, Leiden University, Leiden, The Netherlands
sinistrality may be dominant, as has been found in a few dextral
and a single sinistral species, respectively.1,8 Some authors
emphasize that both dextral and sinistral individuals occur in
Lymnaea species in nature, whereas for example in Physa only
sinistrals are found.7,9,10 That is incorrect. Both sinistral Lymnaea
and dextral Physa have been found in nature.11 Mirror-image
specimens are known as aberrations for a large number of
gastropod species, nearly always as isolated individuals among
normal ones in the field. Starting from such aberrant specimens,
mirror-image populations have been produced in a few cases in
the laboratory.
In the better-known vertebrates many organs develop
asymmetrically. The evident phase of development is during
The heredity of the coiling direction in snails is a classical
example of the maternal effect, in which the genotype of only the
mother determines the phenotype of its progeny.1,3 On the basis
of heredity it is generally accepted that this chirality is
determined by a single gene with two alleles.1,3 The unknown
chirality gene also gained interest since it may be instrumental in
unique, single-gene speciation on the basis of premating isolation
between mirror-image individuals.4,6
Most snail species are dextral, some are sinistral, and only very
rarely is a species comprised of a mixture of dextral and sinistral
individuals. Contrary to Wandelt & Nagy,7 either dextrality or
Correspondence: E. Gittenberger; e-mail: [email protected]
192
RESEARCH NOTES
(Hybond-N þ, Amersham Biosciences, Piscataway, NJ), and
UV cross-linked by standard procedures.
BLAST analysis (www.ncbi.nlm.nih.gov) revealed data on
conserved regions from which chicken and C. elegans primers were
designed. Chicken primers were used for those genes of which no
C. elegans sequence was available. Five micrograms of chicken
(stage 10 embryo) and C. elegans (kind gift of Dr Ronald Plasterk,
NIOB, The Netherlands) total RNA were reverse transcribed
using M-MuLV Reverse Transcriptase (Amersham) and random
hexamers (Promega, Madison, WI) into first strand cDNA. This
was used as a template for the preparation of gene-specific probes
of approximately 600 bp by subsequent PCR in the presence of
DIG-labelled nucleotides (Roche, Basel, Switzerland).
Spotblots were hybridized with DIG-labelled heterologous
DNA probes according to the manufacturers’ directions
(Ambion, Austin, TX) and processed for luminescent detection
of hybridized probes. Spot intensities (Fig. 1G) were measured
with Labworks software (UVP, Upland, CA), mean intensities
were calculated (Fig. 1A –F), and a t-test was used to compare
the means from dextral and sinistral snail populations (Fig. 1).
P values # 0.05 were considered significant.
The results are summarized in Figure 1. Nodal, Bmp4, Fgf 8, Inv,
and Lrd are expressed unequally in sinistral and dextral snails. All
showed a clear season-dependent expression. Altered RNA levels
could only be detected in ovipositing snails (Fig. 1). In resting
individuals neither the complete bodies nor the isolated
reproductive organs (not shown) showed significant differences
in expression. Distinct contrasts in expression levels in the
ovipositing snails were found most prominently in the isolated
reproductive organs containing developing eggs, whereas only
Nodal showed significant differences also in the complete snail (see
Fig. 1). This implies that the unequal gene expression (Fig. 1) can
be assigned to the activity of the reproductive system of the snails.
Bodies without reproductive organs did not show different
expression levels in dextral vs sinistral populations (not shown).
These genes, except Brachyury, are involved in the coiling
direction in snails. They also have a role in asymmetrical
development in vertebrate animals, where they are functionally
linked.15 Recently, Brachyury was shown to be involved in the
determination of the anterior-posterior (AP) axis (not in left–
right asymmetry as in vertebrates) in the marine prosobranch
snail Patella vulgata.21 Apparently the AP and left-right signalling
pathways are strictly separated in snails. The fact that: (1) in
resting animals expression levels are not different between dextral
and sinistral snails, (2) significant differences are most clearly seen
in the genital organs (only Nodal in the entire body) of
reproductively active animals, and (3) Brachyury differs in
expression pattern from the other genes, supports our view that
the differences in expression level are connected to chirality.
Others have shown differential gene expression in sinistral vs
dextral populations of L. stagnalis, but these genes could not be
correlated with genes known to regulate asymmetry.10 The
sinistral snails used by these authors are from our sinistral
population, whereas their dextral specimens have another origin.
As a consequence there will be more differences between the
individuals of both forms used in those studies.9,10
The pathways by which left – right determination is directed in
snails remain largely unknown, but our data suggest a high level of
evolutionary conservation. From these data we conclude that the
single chirality gene responsible for the Mendelian ‘delayed
inheritance’ may be among our candidate genes, but remains
undetermined. The fact that expression levels were increased in
sinistral snails compared with dextral ones indicates that the
mutation should be searched for in terms of a repressor. It is
currently known that the determination of the three body axes
follows a strict hierarchy, partly regulated by gene repression.22
Initially, the antero-posterior axis is determined by the distribution of Bicoid and Oskar in Drosophila and by the position of the
formation of the primitive node, by which the germ layers will
become defined. In the mouse node motile cilia evoke the socalled nodal flow, while non-motile mechanosensory cilia register
the changes in shear stress.12 The direction of flow determines left/
right-handedness.13 Many genes are involved in ciliary activity
and subsequent signalling.14 Among these are Nodal, Left-Right
Dynein and Inversin, whereas Fibroblast Growth Factor 8, Bone
Morphogenetic Protein 4 and many others may have up- and
downstream functions.
Five fully grown, phenotypically sinistral specimens of the
normally dextral pulmonate freshwater snail Lymnaea stagnalis
(L.), received in 1993, were kept alive for breeding, with the hope
that they might carry copies of the mutant allele for sinistrality.
The animals were collected by Gerhard Falkner, together with
normal, i.e. dextral, specimens in a small pond along the Danube
in southern Germany. By artificial selection, separate, homozygous populations of both dextral and sinistral L. stagnalis were
obtained in the laboratory within a few years. Consequently, all
inbred individuals in one population differ from all those in the
other population by at least the chirality allele. This provided the
opportunity to compare gene expression in both groups in a
search for the hypothetical chirality gene.
We hypothesized that the gene responsible for the maternal
effect in Lymnaea, by dictating the initial cleavage pattern of the
fertilized egg,2,15,16 might be highly conserved and that
homologous genes might still be recognizable in other animal
taxa, including the better characterized vertebrates. Since
reversed chirality coincides with a complete inversion of the
body situs4,6 we decided to select six candidate genes (Nodal,
Bmp4, Fgf 8, Lrd, Inversin, and Brachyury) involved in left-right
asymmetry in vertebrates,14,17,18 and analysed our homozygous
populations of dextral and sinistral snails for differential gene
expression. As no Lymnaea sequence information was available we
prepared heterologous probes from the nematode Caenorhabditis
elegans (Nodal, Brachyury, Inv) and from chicken (Fgf 8, Lrd, Bmp4),
based on availability. Special care was taken to include highly
conserved regions into the approximately 600 bp cDNA probes.
These probes were hybridized to isolated RNA from individual
adult dextral and sinistral snails. Positive hybridization signals
indicated a considerable level of correspondance, and detailed
analysis revealed that five out of the six genes showed chiralityspecific levels of RNA expression, the exception being Brachyury.
In invertebrates as well as in mammals, maternal effect genes
control vital functions of the cleaving egg and mutations impair
early embryonic development and implantation.19,20 Because
inheritance of chirality is a maternal effect and coiling direction
can be manipulated by cytoplasm transfer,2 we reasoned that the
gene involved should be expressed during oogenesis, before the
first cleavage of the zygote. It might be hypothesized to be
instrumental in the direction of highly conserved laterality genes
as described in vertebrates, where they are effective in designing
left-right asymmetry in internal organs.12,14 Therefore, we
assumed that differential gene expression might be observed
primarily in the genital organs of reproductively active snails, in
which eggs are formed and kept for some time. This assumption
was tested by comparing complete bodies of dextral and sinistral
snails, both while reproductively active and in rest (in late
autumn), bodies of these groups without genital organs, and the
isolated genital organs of the two categories.
Total RNA was isolated (Rneasy kit, Qiagen, Valencia CA)
from four individual dextral and sinistral snails that were either
ovipositing or non-ovipositing (in late autumn). In addition,
reproductive organs, i.e. the gonads and the tracts that may
contain eggs in various stages of development, from snails in these
categories were isolated and used for RNA preparation (four in
each group). One microgram of each sample was spotted
in duplicate on a positively charged nylon membrane
193
RESEARCH NOTES
Figure 1. Differential gene expression in sinistral and dextral populations of L. stagnalis and season dependency of expression levels. A–F. Relative
expression levels of Nodal (A), Lrd (B), Bmp4 (C), Inv (D), Fgf8 (E), and Brachyury (F) in ovipositing and non-ovipositing dextral and sinistral snail
populations. The ovipositing group is subdivided into complete snail body and isolated reproductive organs. Bars represent the standard errors of the
means. Asterisks identify P values , 0.05 (t-test comparing mean intensities between sinistral and dextral populations). Note that resting, nonovipositing snails do not show differential gene expression between sinistral and dextral populations. Apparently, expression changes in ovipositing snails
can be attributed to the reproductive organs that produce and contain eggs and early staged snail embryos. G. Example of a spotblot on which total RNA
of individual dextral (D) and sinistral (S) snails was hybridized to a chicken probe for Bmp4. The signal intensities in sinistral ovipositing snails are much
higher than those of dextral ovipositing snails. Reproductive resting results in levelling of gene expression.
paternal pronucleus in C. elegans.23 The dorso-ventral axis follows
by bozozok repressing bmp.22 Finally, the left-right asymmetry is
fixed.18 We show for the first time that five chirality-related genes
in snails have their homologous counterparts in vertebrates, where
they have a function in determining left-right asymmetry. We also
show that in snails these genes are already expressed in the
reproductive organs, most likely the eggs, shortly before oviposition. This is reminisent of Levin & Mercola’s24 statement that
there is ‘an interesting set of observations that suggests that even in
mammals, chirality is determined as early as the first few cell
divisions, and certainly before the streak appears’. Unravelling the
genetic background of chirality in our populations of sinistral and
dextral conspecific snails may result in a better understanding of
asymmetrical early development in animals in general.
We thank Mr G. Falkner who donated the valuable snails, Dr
R. Plasterk for providing C. elegans RNA for probe preparation,
and Mr J. Lens for the artwork. BPH is supported by a grant from
the Netherlands Heart Foundation (NHF 2000.016).
194
RESEARCH NOTES
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15. BOORMAN, C.J. & SHIMELD, S.M. 2002. BioEssays, 24:
1004–1011.
16. CONKLIN, E.G. 1903. Anat. Anz., 23: 577–588.
17. HAMADA, H., MENO, C., WATANABE, D. & SIJOH, Y. 2002.
Nat. Rev. Genet., 3: 103 –113.
18. SCHNEIDER, H. & BRUECKNER, M. 2000. Am. J. Med. Genet.,
97: 258 –270.
19. BOWERMAN, B. 1998. Curr. Top. Dev. Biol., 39: 73–117.
20. CHRISTIANS, E.S. 2003. Med. Sci. (Paris), 19: 459–464.
21. LARTILLOT, N., LESPINET, O., VERVOORT, M. &
ADOUTTE, A. 2002. Development, 129: 1411–1421.
22. LEUNG, T.C., BISCHOF, J., SÖLL, I., NIESSING, D., ZHANG,
D., MA, J., JÄCKLE, H. & DRIEVER, W. 2003. Development, 130:
3639–3649.
23. LYCZAK, R., GOMES, J.E. & BOWERMAN, B. 2002. Dev. Cell, 3:
157–166.
24. LEVIN, M. & MERCOLA, M. 1998. Genes Dev., 12: 763 –769.
1. STURTEVANT, A.H. 1923. Science, 58: 269 –270.
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3. FAIRBANKS, D.J. & ANDERSEN, W.R. 1999. Genetics: the
continuity of life. Wadsworth, Belmont, CA.
4. GITTENBERGER, E. 1988. Evolution, 42: 826 –828.
5. BATENBURG, F.H.D. VAN & GITTENBERGER, E. 1996.
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doi: 10.1093/mollus/eyi023
Relocation of the freshwater mussel Diplodon chilensis (Hyriidae) as a strategy
for its conservation and management
S. Peredo 1 , E. Parada 1, , I. Valdebenito 2 and M. Peredo 3
1
Departamento de Ciencias Biolo´gicas y Quı´micas, Facultad de Ciencias, Universidad Cato´lica de Temuco;
Escuela de Acuicultura, Facultad de Ciencias de la Acuicultura y Veterinarias, Universidad Cato´lica de Temuco, Casilla 15-D, Temuco, Chile;
3
Departamento de Ingenierı´a Hidráulica y Medio Ambiente, Universidad Polite´cnica de Valencia, Spain
2
The commonest species of freshwater mussel in Chile is Diplodon
chilensis (Gray, 1828), with a distribution in the country
extending from 348580 S to 468370 S in both lentic and lotic
environments in a number of hydrographic basins.1 It is also
present in Argentina between 328520 S and 458510 S.2 The role of
this species in the ecosystem function has been amply documented. Through their filter-feeding3 and because they are long-lived
organisms,4 they may influence the abundance of phytoplanktonic communities, water quality and the nutrient cycle.5 In
recent decades, a considerable reduction in abundance and/or
disappearance of populations in lotic environments has been
noted (personal observations). This decline may be attributed to
degradation of water quality, destruction of habitat (damming,
canalization, etc.) and probably to the introduction of fish species
for tourism, or installations for industrial fish production, which
have displaced the native species that are the hosts for the
glochidium larvae. To date there have been no proposals in Chile
for the protection of populations of hyriids in the face of
anthropogenic disturbance. The aim of this study was to evaluate
the effectiveness of the relocation of a population of D. chilensis as
a strategy for its conservation and management, through the
long-term evaluation of survival and recruitment.
In the summer of 1983, 400 specimens of Diplodon chilensis with
a valve length of 1.6 – 6.5 cm, originating from a natural
population in Villarrica Lake (VL) in the River Toltén basin,
were transferred to the Gibbs Channel (GC), Temuco (Fig. 1).
The mussels were transported in coolers filled with water taken
from the lake. The area of the channel selected for the relocation
is a site with muddy substrate and profuse vegetation along the
banks. Sampling conducted prior to the relocation, at the
relocation site as well as 3 km upstream and downstream, found
macroinvertebrates and fish, but no specimens of D. chilensis. The
features of the area indicated that this was a suitable site for
relocation of D. chilensis. The specimens were placed at random
along 6 m of the south bank of the channel, at an average density
of 66 individuals/m2. The specimens were not marked, in order to
avoid stress of manipulation. However, lack of marking
prohibited a true quantitative assessment of individual mussel
survival, recovery and growth.
In addition, 100 individuals from the Villarrica Lake
population were taken at random by hand, to measure valve
length (L), valve dry weight (VDW), tissue dry weight (TDW)
and gravid gill dry weight (GGDW); and to estimate the
population parameters sex ratio, density of adults, percentage of
gravid females, size structure, physiological state or condition
index (CI), fertility, recruitment and adult mortality, in
accordance with the methods developed by Parada et al.6 During
the summer of 1986, 100 specimens of the GC relocated
population were recaptured and processed to assess and compare
their biometric and population parameters.
In 2001, 18 years after the relocation, samples were taken from
three sites in GC (site A corresponding to the area in which the
population was relocated in 1983; site B, 3 km downstream; and
site C, 4 km upstream from the relocation site) to evaluate the
Correspondence: S. Peredo; e-mail: e-mail: [email protected]
195