392
L. Zane et aI.
inhabit today. The remarkable adaptive success
of these animals is testified by the 200 species
described ~o date, with an overall estimate of
about 1000 existent species (Bousfield 1983).
Special adaptations of terrestrial talitrids
include acquisition of a waxy exoskeleton
(Moore and Francis 1985), reduction in the gill
area (Moore and Taylor 1984), acquisition of a
highly specialized hemocyanin (Marsden 1984;
Taylor and Spicer 1986) and the evolution of a
refined osmoregulative system (Morrit 1988).
Like other amphipods, the life cycle of talitrids is
characterized by internal fecundation and formation of about twenty lecitotrophic eggs; eggs
then undergo direct development in specialized
pockets, and subadult forms are finally released.
These characteristics are noteworthy because
they enable reproduction to occur also in
absence of water.
The whole of these adaptations however is
not enough to qualify talitrids as fully terrestrial
organisms. and they are, differently from
Isopods, confined to high humidity habitats
(Wildish 1988). Talitrids, in fact, are commonly
found in sandy littoral habitats all around the
world (Bousfield 1984). To cope with daily variation of environmental parameters, talitrids
evolved a migratory behaviour, synchronized
with tidal and nictemeral cycles, that enable
them to locate the suitable patches of habitat on
the shore (Pardi and Ercolini 1986),
Sandy littoral habitats are highly fragmented, and are ephemeral, because modification of
coast line is a relatively fast process. To be a stable component of the fauna of these habitats
means to possess a strong ability to recolonize
suitable habitats and to disperse. Typically. at
our latitudes, talitrid populations experience,
during the spring-summer period, an exponential growth characterized by repeated cycles of
reproduction, starting from the small number
of individuals that survived to the previous
winter season (Louis 1977). Their ability to
recolonize a given area is enhanced from the
fact that broods are kept by females in specialized pockets; thus rafting of ovigerous females
provides an effective way to offspring dispersal
(Wildish 1988).
Talitrid species are morphologicallyextremely conserved. Females of Orchestia montagui, O.
gammarella and O. mediterranea, the three
species under investigation, are morphologically
indistinguishable. The study of these species is
therefore difficult when they occur in sympatric
conditions, such as in the Venice lagoon.
In the present study we use DNA and protein
markers to develop a molecular tool for the classification of these species. In addition we aim at
understand whether the lack of morphological
differentiation is parallelled by low divergence at
the genetic level. We also examine survival ability at high temperatures, in relation to peculiar
adaptations at the biochemical level. to obtain a
better characterization of the species on a functional basis. With this regard we investigate GPI
biochemical activity, because this enzyme seems
to be under strong selection in many organisms.
Differential fitness associated with different GPI
genotypes has been reported for pollution resistance in marine gastropods (Lavie and Nevo
1982). for resistance to low oxygen concentration
in isopods (Shihab and Heath 1987). and for
resistance to prolonged flight in Colias butterflies (Watt 1992). The latter study is of particular
interest as superiority of GPI genotypes was
temperature dependent. With regard to
amphipods, in Gammarus insensibilis, following
exposure to high temperature, a higher survival
rate was reported for individuals heterozygous at
the GPI locus, possibly due to a higher specific
activity of the enzyme in heterozygous condition
(Patarnello et al. 1989).
Materials and Methods
Population samples of Orchestia montagui, o.
gammarella and O. mediterranea were collected
in the Venice Lagoon (North-Eastern Italy).
Classification of these species is based on the
morphology of the sexually dimorphic second
gnathopod (gn2), that is a diagnostic character
in males only (Bellan Santini et al.1993).Animals
were kept alive until arrival in the laboratory,
where they were either deep frozen (-40°C) or
used for survival experiments. For RAPD-PCR
analysis, the frozen specimens were stored in
individual vials containing ethanol.
Allozyme analysis of 12 different loci was
performed on about 50 individuals per species
(Table 1). For each specimen, a small piece of tail
muscle was mechanically dissolved in 200 pl of
extraction buffer (Tris-HCI 0.02 M, 0.25%
Bromophenol blue, pH 8); one microlitre of the
solution, clarified by centrifugation at 13,000
RPM, was then subjected to electrophoretic
L. Zane et aI.
inhabit today. The remarkable adaptive success
of these animals is testified by the 200 species
described ~o date, with an overall estimate of
about 1000 existent species (Bousfield 1983).
Special adaptations of terrestrial talitrids
include acquisition of a waxy exoskeleton
(Moore and Francis 1985), reduction in the gill
area (Moore and Taylor 1984), acquisition of a
highly specialized hemocyanin (Marsden 1984;
Taylor and Spicer 1986) and the evolution of a
refined osmoregulative system (Morrit 1988).
Like other amphipods, the life cycle of talitrids is
characterized by internal fecundation and formation of about twenty lecitotrophic eggs; eggs
then undergo direct development in specialized
pockets, and subadult forms are finally released.
These characteristics are noteworthy because
they enable reproduction to occur also in
absence of water.
The whole of these adaptations however is
not enough to qualify talitrids as fully terrestrial
organisms. and they are, differently from
Isopods, confined to high humidity habitats
(Wildish 1988). Talitrids, in fact, are commonly
found in sandy littoral habitats all around the
world (Bousfield 1984). To cope with daily variation of environmental parameters, talitrids
evolved a migratory behaviour, synchronized
with tidal and nictemeral cycles, that enable
them to locate the suitable patches of habitat on
the shore (Pardi and Ercolini 1986),
Sandy littoral habitats are highly fragmented, and are ephemeral, because modification of
coast line is a relatively fast process. To be a stable component of the fauna of these habitats
means to possess a strong ability to recolonize
suitable habitats and to disperse. Typically. at
our latitudes, talitrid populations experience,
during the spring-summer period, an exponential growth characterized by repeated cycles of
reproduction, starting from the small number
of individuals that survived to the previous
winter season (Louis 1977). Their ability to
recolonize a given area is enhanced from the
fact that broods are kept by females in specialized pockets; thus rafting of ovigerous females
provides an effective way to offspring dispersal
(Wildish 1988).
Talitrid species are morphologicallyextremely conserved. Females of Orchestia montagui, O.
gammarella and O. mediterranea, the three
species under investigation, are morphologically
indistinguishable. The study of these species is
therefore difficult when they occur in sympatric
conditions, such as in the Venice lagoon.
In the present study we use DNA and protein
markers to develop a molecular tool for the classification of these species. In addition we aim at
understand whether the lack of morphological
differentiation is parallelled by low divergence at
the genetic level. We also examine survival ability at high temperatures, in relation to peculiar
adaptations at the biochemical level. to obtain a
better characterization of the species on a functional basis. With this regard we investigate GPI
biochemical activity, because this enzyme seems
to be under strong selection in many organisms.
Differential fitness associated with different GPI
genotypes has been reported for pollution resistance in marine gastropods (Lavie and Nevo
1982). for resistance to low oxygen concentration
in isopods (Shihab and Heath 1987). and for
resistance to prolonged flight in Colias butterflies (Watt 1992). The latter study is of particular
interest as superiority of GPI genotypes was
temperature dependent. With regard to
amphipods, in Gammarus insensibilis, following
exposure to high temperature, a higher survival
rate was reported for individuals heterozygous at
the GPI locus, possibly due to a higher specific
activity of the enzyme in heterozygous condition
(Patarnello et al. 1989).
Materials and Methods
Population samples of Orchestia montagui, o.
gammarella and O. mediterranea were collected
in the Venice Lagoon (North-Eastern Italy).
Classification of these species is based on the
morphology of the sexually dimorphic second
gnathopod (gn2), that is a diagnostic character
in males only (Bellan Santini et al.1993).Animals
were kept alive until arrival in the laboratory,
where they were either deep frozen (-40°C) or
used for survival experiments. For RAPD-PCR
analysis, the frozen specimens were stored in
individual vials containing ethanol.
Allozyme analysis of 12 different loci was
performed on about 50 individuals per species
(Table 1). For each specimen, a small piece of tail
muscle was mechanically dissolved in 200 pl of
extraction buffer (Tris-HCI 0.02 M, 0.25%
Bromophenol blue, pH 8); one microlitre of the
solution, clarified by centrifugation at 13,000
RPM, was then subjected to electrophoretic