324
V. Rossi et al.
des at GPI locus and GPI activity of animals in
the same three sites indicated by Patarnello et al.
(1991) and Montero et al. (1994) for genetic and
enzymatic analysis in spring 1989 and from May
1990 to June 1991. The first sampling station was
located in the Industrial Channel where chemical
pollution is very high due to industrial waste discharged directly for decades. The second one lies
in front of the outflow of the electric power plant
cooling system where temperatures are about
10 D C higher than the rest of the lagoon. The third
station is located in the southern part of the
lagoon (Chioggia) where chemical pollution is
relatively low (Brunetti et a1. 1983; Donazzolo et
aL 1984), The aim of our study is to continue the
biomonitoring of such an area by the analysis of
the genetic structure of B. amphitrite at GPI
locus. Moreover we analyse the colonisation
dynamics and evaluate the enzymatic activity of
GPI in relationship to the genotype and the age
of the animals.
Material and Methods
Colonisation dynamics was studied by counting
barnacles that set 88 experimental fibrocement
panels (24x34 cm) that were fixed to steel frames
and suspended within 1 m of the surface in May
1997 approximately at the beginning of extended
settlement season that ranges from late spring to
early autumn. A mean number of 3 panels per
site per date were removed after 20,40,80, 160.
230 and 310 days and transported to the laboratory in 40x30x22 em and 20 I volume PVC boxes
containing at least 101 of sea water. In laboratory
alive and dead (empty shells) specimens of B.
amphitrite were identified and counted. For each
date and site a sub sample of B. amphitrite was
chosen, soft tissues were excised from the shells
and frozen at -80 a C for later genetic analysis and
determinations of GPI enzyme activity and protein content. Starch gel electrophoresis was carried out to identify alleles at GPI locus (Harris
and Hopkinson 1976). For GPI enzyme activity
assay Sigma kits were used. The analyses were
carried out at 30°C on single individuals scored
for GPI genotype according to Montanini et al.
(1998).
To compare colonisation success, mean number of specimens per panel and mean ratio
between dead and alive organisms per panel in
different stations and different dates, two way
ANOVA tests (site, date), with homogeneity
Levene's test, were performed. Panels colonised
by less than 20 individuals were excluded from
these analyses.
Allele and genotype frequencies were calculated and test for conformance to HardyWeinberg equilibrium with Levene correction
for small sample size and coefficient for heterozygote deficiency or excess, D, were computed
by Biosys-l statistical package (Swofford and
Selander 1981).
To evaluate differences in mean enzyme
activity between genotypes (homozygotes and
heterozygotes at GPI locus) among sites and
sampling dates, one way ANOVA tests were computed. Sheffe F-tests were performed for pair
comparisons.
Results
The mean number of colonising B. amphitrite is
significantly different among sampling sites
(Table 1) (Fig. 1). At the Industrial Channel the
number of individuals per panel was less than 20
in all sampling dates. The highest values were
detected at the power plant outflow where the
species reaches a maximum density of 4718
indlm 2 after 310 days from settlement.
Mortality of B. amphitrite was significantly
related to the sampling date (Table 2) (Fig. 2). At
the Industrial Channel the highest mortality
(33%) was recorded after 80 days from panel
immersion. At the power plant outflow, mortality
increased significantly with the sampling date
(r2=0.809, P=O.OOOl) and presents the absolute
maximum value (78%) after 310 days from panel
immersion.
Considering samples with n>20, the frequency of the most common allele at GPI locus
Table 1. Two way ANOVA table for difference in mean number
of colonising individuals per panel among sampling sites and
sampling dates. Industrial Channel station was excluded from
the analysis as the number of individuals per panel was <20
on all sampling dates
Source
df
Mean square
P
Site
1
52701.880
0.016
Date
5
11869.372
0.207
Sitexdate
4
16229.457
0.111
Error
15
7181.433
V. Rossi et al.
des at GPI locus and GPI activity of animals in
the same three sites indicated by Patarnello et al.
(1991) and Montero et al. (1994) for genetic and
enzymatic analysis in spring 1989 and from May
1990 to June 1991. The first sampling station was
located in the Industrial Channel where chemical
pollution is very high due to industrial waste discharged directly for decades. The second one lies
in front of the outflow of the electric power plant
cooling system where temperatures are about
10 D C higher than the rest of the lagoon. The third
station is located in the southern part of the
lagoon (Chioggia) where chemical pollution is
relatively low (Brunetti et a1. 1983; Donazzolo et
aL 1984), The aim of our study is to continue the
biomonitoring of such an area by the analysis of
the genetic structure of B. amphitrite at GPI
locus. Moreover we analyse the colonisation
dynamics and evaluate the enzymatic activity of
GPI in relationship to the genotype and the age
of the animals.
Material and Methods
Colonisation dynamics was studied by counting
barnacles that set 88 experimental fibrocement
panels (24x34 cm) that were fixed to steel frames
and suspended within 1 m of the surface in May
1997 approximately at the beginning of extended
settlement season that ranges from late spring to
early autumn. A mean number of 3 panels per
site per date were removed after 20,40,80, 160.
230 and 310 days and transported to the laboratory in 40x30x22 em and 20 I volume PVC boxes
containing at least 101 of sea water. In laboratory
alive and dead (empty shells) specimens of B.
amphitrite were identified and counted. For each
date and site a sub sample of B. amphitrite was
chosen, soft tissues were excised from the shells
and frozen at -80 a C for later genetic analysis and
determinations of GPI enzyme activity and protein content. Starch gel electrophoresis was carried out to identify alleles at GPI locus (Harris
and Hopkinson 1976). For GPI enzyme activity
assay Sigma kits were used. The analyses were
carried out at 30°C on single individuals scored
for GPI genotype according to Montanini et al.
(1998).
To compare colonisation success, mean number of specimens per panel and mean ratio
between dead and alive organisms per panel in
different stations and different dates, two way
ANOVA tests (site, date), with homogeneity
Levene's test, were performed. Panels colonised
by less than 20 individuals were excluded from
these analyses.
Allele and genotype frequencies were calculated and test for conformance to HardyWeinberg equilibrium with Levene correction
for small sample size and coefficient for heterozygote deficiency or excess, D, were computed
by Biosys-l statistical package (Swofford and
Selander 1981).
To evaluate differences in mean enzyme
activity between genotypes (homozygotes and
heterozygotes at GPI locus) among sites and
sampling dates, one way ANOVA tests were computed. Sheffe F-tests were performed for pair
comparisons.
Results
The mean number of colonising B. amphitrite is
significantly different among sampling sites
(Table 1) (Fig. 1). At the Industrial Channel the
number of individuals per panel was less than 20
in all sampling dates. The highest values were
detected at the power plant outflow where the
species reaches a maximum density of 4718
indlm 2 after 310 days from settlement.
Mortality of B. amphitrite was significantly
related to the sampling date (Table 2) (Fig. 2). At
the Industrial Channel the highest mortality
(33%) was recorded after 80 days from panel
immersion. At the power plant outflow, mortality
increased significantly with the sampling date
(r2=0.809, P=O.OOOl) and presents the absolute
maximum value (78%) after 310 days from panel
immersion.
Considering samples with n>20, the frequency of the most common allele at GPI locus
Table 1. Two way ANOVA table for difference in mean number
of colonising individuals per panel among sampling sites and
sampling dates. Industrial Channel station was excluded from
the analysis as the number of individuals per panel was <20
on all sampling dates
Source
df
Mean square
P
Site
1
52701.880
0.016
Date
5
11869.372
0.207
Sitexdate
4
16229.457
0.111
Error
15
7181.433