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speculated that haemocyanin from other molluscs may have
similar immunological characteristics to KLH allowing hemocyanin to be procured from an organism already successfully
cultured like Helix pomatia.1 Data presented here would support
this possibility.
7. DEHARO, C., MENDEZ, R. & SANTOYO, J. 1996. FEBS Lett., 10:
1378–1387.
8. SYMONDSON, W. & LIDDELL, J.E. 1993. Biocontrol Sci. Tech., 3:
261–275.
9. GHIRETTI, F. 1966. Molluscan hemocyanins. In: Physiology of
Mollusca (K. M. Wilbur & C.M. Younge, eds), 7: 233–248. Academic
Press, London.
10. STOEVA, S., RACHEV, R., SEVEROV, S., VOELTER, W. & GENOV,
N.A. 1995. Comp. Biochem. Physiol., 110B: 761–765.
11. SWERDLOW, R.D., EBERT, R.F., LEE, P., BONAVENTURA, C. &
MILLER, K.I. 1996. Comp. Biochem. Physiol., 113B: 537–548.
12. LOMMERSE, J.P., THOMAS, O.J., GIELENS, C., PREAUX, G.,
KAMERLING, J.P. & VLIEGENTHART, J.F. 1997. Eur. J. Biochem.,
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J. Moll. Stud. (2003) 69: 159–161
© The Malacological Society of London 2003
Evaluation of methods for assessing brood size in freshwater mussels (Hyriidae)
C. R. Beasley, L. de Quadros Miranda, S. T. M. Alves and C. H. Tagliaro
Laboratório de Moluscos, Campus de Bragança, Universidade Federal do Pará, Bragança 68.600–000, Pará, Brazil
branch, and the total number of glochidia was estimated for
each batch using both the plate and pipette methods. The
counts were repeated 15 times for each batch, and a mean and
median estimate of the number of glochidia was obtained for
each combination of method and batch.
For the plate method, the glochidia were liberated into a
square clear plastic counting plate (100 100 15 mm) containing 20 ml of tap water. The plate has a grid on the bottom
marked with letter and number coordinates (Spectrum Laboratory Products, Inc., USA). The plate containing the glochidia
was swirled gently and placed under a stereomicroscope using
transmitted light. Glochidia were counted in each of 15
randomly chosen grid squares. After counting glochidia in a
single grid square, the plate was gently swirled so as to redistribute the glochidia. This was done because, after several
counts, glochidia tended to aggregate in the centre as the
observer manipulated the plate on the stereomicroscope stand.
The mean number of glochidia per grid square was calculated
and the total number of glochida was estimated by multiplying
the mean by 36, the total number of grid squares on the plate.
The pipette method, modified after Jupiter & Byrne,6 consists
of placing the glochidia into a plastic beaker containing 100 ml
of tap water. The beaker was placed on a magnetic stirrer and
the liquid was stirred at 250 rpm. While the water was being
stirred, a hand-held pipette was used to withdraw a 100-l
aliquot containing glochidia. A sample of 15 aliquots was
obtained, and each one was transferred to a glass slide and
examined under a stereomicroscope. The number of glochidia
in each aliquot was determined and the mean number per
aliquot was calculated. The total number of glochidia in the
beaker was obtained by multiplying the mean by 1000, the total
number of possible aliquots in the beaker.
Finally, a mean brood size estimate was calculated from five
repeat estimates, using both methods, for a single gravid female
Information on reproduction in freshwater mussels from tropical regions is scarce. Due to the relatively stable climatic conditions, tropical freshwater mussels are assumed to have a
constant production of gametes and larvae throughout the
year.1 However, there is evidence to suggest that marked seasonality in the production of both gametes and glochidia does
occur in tropical and subtropical Hyriidae.2–5 There is little
information on brood size of tropical or subtropical freshwater
mussels, although the morphology of glochidia has been
studied in detail.4,6,7 As can be seen in a recent review, much of
the literature deals with glochidia and brood size of temperate
species.8 Many decisions regarding the conservation and
management of freshwater mussels require sound knowledge of
their reproductive output9 and the seasonality of their reproductive cycle.10 Furthermore, methods to assess the production
of gametes and glochidia should be both accurate and precise
enough to allow repeatable estimates of these reproductive
parameters.
The aim of this study was to evaluate two methods (plate and
pipette) of assessing brood size in freshwater mussels. For each
method, the inner demibranchs were dissected, washed gently
with tap water and stored in 70% alcohol. The demibranchs
were opened along the anterior, dorsal and posterior margins,
and the glochidia were liberated into a container. The demibranchs were examined under a stereomicroscope to ensure
that all glochidia had been removed.
To determine which of the methods is the most appropriate
for assessing brood size in freshwater mussels, in terms of
accuracy and precision, counts were carried out in which a fixed
number of glochidia were removed from a demibranch of
Triplodon corrugatus (Lamarck, 1819). Batches of 50, 100, 200,
500, 750 and 1000 glochidia were liberated from the demiCorrespondence: C. R. Beasley; e-mail: [email protected]
159
RESEARCH NOTES
of each of the species T. corrugatus, Paxyodon syrmatophorus
(Meuschen, 1781) and Castalia ambigua ambigua (Lamarck,
1819) that had been collected from the Tocantins river, Brazil.11
Because the batch count data exhibited non-normality
and unequal variances, even after log transformation, the nonparametric Mann-Whitney U-test was carried out to check for
differences between estimates obtained by each method.
The results (Table 1) show no significant differences between
median values of estimates obtained by both methods for
batches of 50, 100, 200 and 500 glochidia. However, the median
plate estimate was significantly different from the median
pipette estimate for the 750 and 1000 glochida batches (Table
1). To check the accuracy of the estimates obtained for each
batch using both methods, the 95% confidence interval (CI) of
the median was calculated. The two-tailed null hypothesis H0:
M M0 was tested, where M is the population median and M0 is
an hypothesized median value12 of 50, 100, 200, 500, 750 or 1000
glochidia, according to the appropriate batch. The null hypothesis is rejected if M0 falls outside the limits of the 95% confidence interval.12 The brood size estimates, however, were
normally distributed, and had similar variances; differences
between plate and pipette methods were then verified using the
t-test.
Figure 1 shows the median number of glochidia (95% CI)
estimated for batches of 50, 100, 200, 500, 750 and 1000
glochidia using both methods. The plate method estimates are
both more accurate (closer to the total number of glochidia)
and more precise (lower variability around the estimate) than
those of the pipette method (Fig. 1). The variability (standard
deviation) around the mean is greater for the pipette method
(Table 1). The latter produced several zero estimates, which
considerably reduces its accuracy in estimating the total number
of glochidia. However, both methods tend to under-estimate
the total number of glochidia in each batch (Fig. 1).
For batches of 50 and 500 glochidia, both methods produced
a median estimate not significantly different from M0 (Fig. 1,
Table 1). For batches of 100 and 1000 glochidia, however, both
methods produced median estimates significantly different
from M0. For the batch of 200 glochidia, the plate median estimate was significantly different from 200, but the pipette
Table 1. Median estimates of numbers of glochidia obtained by the plate and pipette methods using batches of 50, 100, 200, 500, 750 and 1000 glochidia.
N ( M0)
50
100
Pipette
Pipette
500
Pipette
n
15
15
15
15
Mean (x̄ )
44.48
71.11
82.72
71.11
Standard deviation (s)
10.96
73.32
19.41
68.85
13.81
117.83
55.08
354.98
69.92
218.89
98.53
243.26
Median (M)
43.20
66.67
86.40
66.67
172.20
133.32
530.40
533.32
763.20
333.32
1056.00
333.32
Mann–Whitney U-test
U15,15 90, ns
U15,15 69, ns
U15,15 105, ns
U15,15 110, ns
U15,15 16, P0.05
U15,15 0, P 0.05
H0: M M0
Accept
Reject
Reject
Accept
Accept
Reject
15
15
170.01
168.89
Accept
Plate
15
545.27
Pipette
15
595.55
Accept
Plate
1000
Plate
Reject
Plate
750
Method
Accept
Plate
200
15
742.35
Pipette
15
351.10
Reject
Plate
15
1115.31
Pipette
15
386.67
Reject
Abbreviations: N, total number of glochidia in batch; n, number of times the counts were repeated; ns, not significant. The null hypothesis H0:M M0 is rejected
(P 0.05) if M0 falls outside the 95% confidence level of the median (M).
Figure 1. Median number of glochidia (95% CI) estimated by the plate and pipette methods using batches of 50, 100, 200, 500, 750 and 1000 glochidia. The
closed circle indicates the total number of glochidia in each batch ( M0).
160
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The results of the batch counts (low numbers of glochidia)
show that the pipette method tends to under-estimate the actual
number of glochidia, whereas the mean brood size estimates
(high numbers of glochidia) show that the pipette method
tends to over-estimate numbers of glochidia in relation to the
plate method. This difference may be due to the fact that,
despite the action of the magnetic stirrer, there is a tendency for
the glochidia to sink at low densities and thus produce lower
estimates because the pipette cannot reach them. Conversely, at
high densities the glochidia interfere with one another and do
not sink as quickly, thus producing higher estimates. All results
show that there is much larger variation associated with pipette
method estimates.
We suggest the use of the plate method to estimate brood size,
since it appears to be more accurate (at least at lower densities)
and more precise in its estimates of numbers of glochida. The
improvement of standard techniques for evaluation of reproductive output in freshwater mussels is an important step
towards providing biological information necessary for their
conservation and management. However, a final caveat is
appropriate here: the mussel species we are working with are
very abundant throughout the Amazon basin and the populations sampled all show recent reproduction. As the assessment
of brood size often involves the sacrifice of individual mussels
(see ref. 13 for non-destructive sampling), care should be taken
to minimize sample sizes, and to be aware of local, national and
international legislation concerning endangered or rare
species of freshwater mussel.
We are very grateful to Aline Grasielle Costa de Melo,
Universidade Federal do Pará (UFPA) for help in the preparation of material in the laboratory and to the Fundo Estadual de
Ciência e Tecnologia of the State of Pará, and the Programa
PROINT, UFPA, Brazil, for financial support.
estimate was within the confidence interval of the median and
therefore not significantly different from M0. For the batch of
750 glochidia, the plate median estimate was within the confidence interval of the median and therefore not significantly
different from M0, but the pipette estimate was significantly
different from 750. The results suggest that the large differences
in individual estimates obtained using the pipette method may
introduce serious error to estimates of brood size.
Figure 2 shows that the brood size estimates using the pipette
method were greater than those obtained using the plate
method when the methods were applied to real animals. This
difference was significant for T. corrugatus (t 4.66, df 8, P 0.01) and P. syrmatophorus (t 6.65, df 8, P 0.01), but there
was no significant difference in mean brood size using the two
methods in the case of the C. ambigua ambigua individual (t 1.95, df 8, ns). Figure 2 also shows that the standard deviation
values are greater for the pipette method indicating greater variation in individual estimates.
A
B
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Figure 2. Mean brood size estimates ( s) obtained from five repeated
counts, using the plate and pipette methods, for a single gravid female of
each of three species of freshwater mussel (Hyriidae) from the Amazon
basin, Triplodon corrugatus (A), P. syrmatophorus (B) and Castalia ambigua
ambigua (C). Mean brood size estimates were significantly greater using the
pipette method, except in the case of C. ambigua (see text for details).
161
10. BYRNE, M. 1998. Hydrobiologia, 389: 29–43.
11. BEASLEY, C.R. 2001. Stud. Neotrop. Fauna Environ., 36: 159–165
12. ZAR, J.H. 1999. Biostatistical Analysis. Prentice Hall, Upper Saddle
River, NJ.
13. YOUNG, M.R. & WILLIAMS, J. 1984. Arch. Hydrobiol., 99: 405–422.