EVect of freezing on the length of the penis in Nucella lapillus (L.)† Annagh Minchin* and Ian M. Davies FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, UK AB11 9DB. E-mail: minchina@marlab.ac.uk; daviesim@marlab.ac.uk; Fax: +44 1224 295511; Tel:+44 1224 876544 Received 16th December 1998, Accepted 1st February 1999 When quantifying imposex in Nucella lapillus, two indices are used: the Vas Deferens Sequence Index (VDSI) and the Relative Penis Size Index (RPSI).Freezing and thawing increase the length of the penis in both male and female Nucella lapillus. In the population studied, this had no significant eVect on the RPSI, but was potentially an important source of additional variance in the estimation of the mean penis length. The sex and VDS of each snail were recorded.The penis Introduction length of each snail was measured immediately after dissection The dogwhelk, Nucella lapillus, has been widely used as a bio- to the nearest 0.01 mm by microscope eyepiece graticule. The indicator of the eVects of tri-butyl-tin (TBT) on marine variance of these measurements was estimated by repeated ecosystems.1–4 Exposure to TBT results in the superimposition observations of a series of snails and, for example, penis length of male characteristics on females, known as imposex.5 The ranged from 2.4 mm to 2.6 mm for a single snail with four quantification of imposex involves two indices: the Vas measurements out of six of 2.5 mm.The snails were then Deferens Sequence Index (VDSI) and the Relative Penis Size placed into numbered segmented trays and stored for 19 h at Index (RPSI).VDSI gives an indication of the reproductive -20 °C. The trays were wrapped in polythene bags while in competency (mean of the females in the population) of the the freezer to prevent the snails from drying out. The snails population, and the RPSI expresses the relative development were then allowed to thaw for 2.5 h and two measurements of of the penis in males and females: penis length were repeated, firstly soon after thawing and secondly a further 4 h later.RPSI= (Mean female penis length)3 (Mean male penis length)3 ×100% Results Imposex has been adopted by the Oslo and Paris The VDSI values for adults and juveniles at Skatey Shore Commission (OSPAR) as a component of their international were 3.95 and 3.88 respectively. The RPSI value in adults was Joint Assessment and Monitoring Programme (JAMP), rec- 9.4%.The mean shell height of toothed adults was 24.96 mm ommending that snails should be examined alive (without (s=1.45) and of juveniles 24.53 mm (s=1.79). narcotisation). The use of Nucella lapillus has been adopted The VDS of individual Nucella ranged from 3 to 4.The for Laboratory Performance Studies under the international VDS of each individual was identified before and after freezing. QUASIMEME quality assurance programme using the revised Freezing therefore had no eVect on VDSI determination. JAMP Guidelines.6 Freezing caused an increase in penis length in both males and It is sometimes impractical, for example during extended females (Fig. 1) and an increase in the variance of the penis periods of fieldwork, to maintain Nucella alive after collection. length measurements. There were only small changes in the Preservation of samples is then necessary. Chemical preserv- RPSI values (Table 1). atives, such as formalin, commonly change the texture of the Linear regressions were fitted to the relationships (Figs. 2 tissues of the snail making them diYcult to handle. Freezing and 3) between the initial penis length (PL0) and the penis is a convenient form of preservation which has less eVect on length immediately after thawing (PLF) and, for adults, after the texture of the tissues. However, it is not known what eVect a further 4 h (PLF4): freezing may have on imposex measurements. This paper examines the eVects of freezing on the length of the penis and, juveniles: PLF=1.133PL0+0.165 (R2=0.851, n=97) consequently, on the RPSI.adults: PLF=1.28PL0+0.171 (R2=0.842, n=196) PLF4=1.32PL0+0.154 (R2=0.835, n=196) Methods The two regression lines for adults were not significantly diVerent (Student t-test). Nucella lapillus were collected from below mid-shore level at Skatey Shore, Stonehaven, east Scotland on 22 January (97 juveniles) and 4 June (200 toothed adults) 1998.Snails were Discussion maintained at ambient temperature in an aquarium at the Freezing Nucella resulted in an increase in penis length in both laboratory for no more than 4 d prior to analysis. males and females, but had no eVect on VDSI and only a The shell height of each snail was measured to the nearest small eVect on RPSI (Table 1).The penis length in adult males 0.1 mm, using electronic callipers. The snails were then crushed and females increased by factors which were approximately with the aid of a vice and the soft tissues carefully removed. log normally distributed. The median value of the factor for males was 1.36 (interquartile range, 1.23–1.50) and for females was 1.36 (interquartile range, 1.10–1.57).No significant †© Crown copyright. J. Environ. Monit., 1999, 1, 203–205 203Table 1 Summary table of the eVects of freezing on Nucella lapillus Mean penis length/mm s VDSI RPSI (%) Adult Live male 2.72 0.50 Live female 1.24 0.37 3.95 9.4 Thawed male 3.73 0.69 Thawed female 1.67 0.42 3.95 8.9 Thawed for 4 h male 3.82 0.75 Thawed for 4 h female 1.69 0.41 3.95 8.7 Juvenile Live male 1.50 0.54 Live female 0.69 0.19 3.88 9.7 Thawed male 1.87 0.71 Thawed female 0.95 0.26 3.88 13.0 Fig. 1 Ratio of mean penis lengths after thawing (PLF) and 4 h later (PLF4) to initial mean penis length (PL0) in adult Nucella lapillus. Fig. 4 Predicted ratios of RPSI values calculated after thawing (PLF and PLF4) to the initial RPSI value (RPSI0) for adult and juvenile Nucella lapillus.Also shown is the predicted eVect of 6 h delay in measuring live dogwhelks (PL6) from Minchin and Davies.7. changes in penis length occurred during a further 4 h of storage after thawing. The marked changes in penis length with time observed in live dogwhelks by Minchin and Davies7 did not occur in frozen specimens (Fig. 4).Application of the regression equations above to a range of initial mean penis lengths can be used to estimate the potential eVect of freezing on RPSI measurements on populations showing a range of initial RPSI values. This eVect is relatively minor at initial RPSI values greater than 1%. The increases in penis length after freezing and thawing show considerable diVerences between individual dogwhelks.Fig. 2 Relationship between penis lengths after thawing (PLF) and In the population studied, the consequential increase in the initial penis length (PL0) for adult Nucella lapillus. standard deviation of the mean penis length in the adults was from 15% (females) to 40% (males). Snails of equal initial penis length showed wide ranges of penis lengths after thawing.For example, after thawing, the penis length in males of initial penis length 2.7 mm ranged from 2.97 to 5.13 mm. To investigate the importance of freezing as a source of additional variance of penis length, subgroups of males (or females) of equal initial mean penis length, but diVerent variance, were selected from the data set. The relationships between the standard deviation of the penis lengths of these subgroups before and after freezing (Fig. 5) show that freezing increases the variance of penis length in both males and females. The eVect is most marked in subgroups of low initial variance, and relatively small in subgroups of females where the initial standard deviation is >0.25 mm (RSD >~20%). While narcotisation leads to an underestimation of the RPSI,8 and a delay in the measurement of untreated snails Fig. 3 Relationship between penis lengths after thawing (PLF4) and initial penis length (PL0) for adult Nucella lapillus. leads to an overestimation,7 freezing introduces little bias to 204 J. Environ. Monit., 1999, 1, 203–205Acknowledgements We would like to thank Patricia Barrett for her help with the preparation of the samples.References 1 P. E. Gibbs, G. W. Bryan, P. L. Pascoe and G. R. Burt, J. Mar. Biol. Ass. UK, 1987, 67, 507. 2 G. W. Bryan, P. E. Gibbs, L. G. Hummerstone and G. R. Burt, J. Mar. Biol. Ass. UK, 1988, 66, 611. 3 G. W. Bryan and P. E. Gibbs, in Metal Ecotoxicology: Concepts and Applications, ed. M. C. Newman and A. W. McIntosh, Lewis, Ann Arbor, 1991, pp. 323–361. 4 E.Stroben, J. Oehlman, U. Schulte-Oehlmann and P. Fioroni, Malacol. Rev., 1996, Suppl. 6 (Molluscan Reproduction), 173. 5 B. S. Smith, Proc. Malacol. Soc. London, 1971, 39, 377. Fig. 5 The eVect of freezing on the standard deviation of the penis 6 I. M. Davies, A. Minchin and M. Harding, Marine Laboratory, length in adult Nucella lapillus. Aberdeen Report No 9/97, FRS Marine Laboratory, Aberdeen, 1997. 7 A.Minchin and I. M. Davies, Marine Laboratory, Aberdeen Report the RPSI measurement, but is potentially a source of consider- No 4/98, FRS Marine Laboratory, Aberdeen, 1998. able increased uncertainty in the estimation. 8 M. Huet, P. Fioroni, J. Oehlmann and E. Stroben, Hydrobiologia, 1995, 309, 29. Conclusion Paper 8/09806I Freezing and thawing increase the length of the penis in both male and female Nucella lapillus. In the population studied, this had no significant eVect on the RPSI, but was potentially an important source of additional variance in the estimation of the mean penis length, particularly in populations showing relatively narrow ranges of initial penis length. J. Environ. Monit., 1999, 1, 203–205 205