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Journal of Experimental Marine Biology and Ecology
xx (2003) xxx – xxx
www.elsevier.com/locate/jembe
Impact of formalin preservation on
Pleurobrachia bachei (Ctenophora)
Delphine Thibault-Botha *, Terra Bowen
Hawaii Institute of Marine Biology, 46-007 Lilipuna Road, Kaneohe, HI 96744, USA
Received 19 May 2003; received in revised form 17 October 2003; accepted 22 October 2003
Abstract
Here, we report on the impact of formalin preservation on the physical dimensions of the
tentaculate ctenophore Pleurobrachia bachei A. Agassiz collected in the North East Pacific Ocean.
Contrary to previously published papers [Oorthuyzen, W., Sadée, C., 1982. Voorkomen en groei van
Pleurobrachia pileus en Aurelia aurita in de westelijke Wadddenzee en hun mogelijke rol als
predator van platvislarven. Netherlands Institute of Sea Research, Texel; Yip, 1982. A note on the
effect of preserving ctenophores in formaldehyde – seawater. Ir. Nat. J. 20 416 – 419] where a
standard 20% shrinkage was applied to whole populations, we found that shrinkage rates are not
constant and vary widely with the size of the specimen. A size reduction of 5 – 80% was observed in
individuals ranging from 23 to 5 mm in boreal diameter when alive. The relationship between dry
weight (DW) and length is also affected by the preservation process. Previously published biomass
data and physiological rates based on preserved samples should therefore be re-examined when
population was largely dominated by small size individuals.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Ctenophore; Formalin preservation; Pleurobrachia bachei; Size
1. Introduction
Length and weight loss due to preservation in chemical solutions (e.g. formaldehyde,
chloroform and ethanol) is almost an inevitable process for most organisms. Marine
organisms placed in 4% formaldehyde solution experience an osmotic pressure more than
double that of the surrounding seawater (Steedman, 1976). The subsequent diminution in
size results from osmotically driven loss of water. Variation in shrinkage is therefore a
* Corresponding author. Tel.: +1-808-236-7457; fax: +1-808-236-7443.
E-mail address: [email protected] (D. Thibault-Botha).
0022-0981/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2003.10.017
JEMBE-43720; No of Pages 7
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function of the species and species specific morphology (i.e. surface/volume ratio, water
content and body structures). Shrinkage due to preservation with formalin has been already
described in several organisms. In Inland Silverside larvae, the percentage of formalin
used (5% or 10%) had a significant effect on the proportion of shrinkage (5% < 10%)
(Cunningham et al., 2000). In 0+ grayling, formalin led to a decrease in length but to an
increase in width (Sagnes, 1997). In copepods, formalin preservation caused a 20% loss in
weight (Hopkins, 1968; Omori, 1969, 1978). In a mixed zooplankton sample, there was no
statistically difference in shrinkage when 5% or 10% formalin solutions were used,
showing an overall shrinkage of 37 –43% (Giguère et al., 1989). In microzooplankton
(20 – 200 Am), formaldehyde preservation may even lead to the disappearance of cells
(Stoecker et al., 1994).
Therefore, gelatinous zooplankton, which have a very high water content and no strong
structural features or hard covering, are probably prone to larger size losses than
crustacean zooplankton. This loss may be even higher when the surface to volume ratio
is high, such as in small sized gelatinous zooplankton. For one group of gelatinous
zooplankton, the tunicates, Nishikawa and Terazaki (1996) reported a length reduction of
about 93% from the live length for Thalia democratica after 153 days and of 86% for
Dolioletta gegenbauri. Moon jellies, Aurelia aurita, collected in the Black Sea (fresh live
size between 7 and 35 cm bell diameter and 5 and 160 g wet weight [WW]) lost about 26%
of their bell diameter and 70% of their wet weight when preserved in formaldehyde
(Mutlu, 1995). De Lafontaine and Leggett (1989) reported a loss in bell diameter between
67.8% and 84.9% for two planktonic species of hydromedusae (Staurophora mertensi and
Catablema vesicarium) when preserved in 10% v/v formalin – seawater. In terms of wet
weight, those species lost between 30% and 38%, respectively, of their initial weight.
However, they also reported that the relationship between dry weight (DW) and wet
weight for those two species does not appear to be affected by formalin preservation.
Most published results on tentaculate ctenophore are based on the very cosmopolitan
species Pleurobrachia pileus. For this species, the shrinkage value due to formalin
preservation most commonly used in the literature is 20% (Oorthuyzen and Sadée,
1982; Yip, 1982). On the other hand, Mutlu and Bingel (1999) reported only ‘‘some
loss’’ in length and weight for this species due to preservation based on Mutlu (1995),
although in this latter article he reported an average loss of 71% in length and 97% in wet
weight (for specimens whose sizes ranged from 7 to 30 mm in body length and were as
large as 2 g wet weight). Båmstedt (1998) reported an average loss of 31% in length, but
like Mutlu (1995) he did not look at size-specific shrinkage. No direct measurements of
body alteration due to formalin – seawater preservation in Pleurobrachia bachei are
available.
In this article, we report on the impact of formalin preservation on length and weight of
different size classes of P. bachei collected from the North East Pacific Ocean.
2. Materials and methods
Specimens of the tentaculate ctenophore P. bachei (Tiffon, 1993) were collected in the
North East Pacific Ocean (41 – 45jN, 123.5 – 126jW) in August 2002 as part of the Global
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Ocean Ecosystems Dynamics—North East Pacific Ocean (GLOBEC-NEP) effort. A large
net (2.5 m long with a 350 Am mesh size), fitted with a large non-filtering cod end, was
towed obliquely (upper 50 m) at low speed (2 m s 1) for sampling of live individuals. Cod
end contents were then transferred gently into large coolers filled with surface seawater.
Ctenophores were then carefully removed for further measurements (dry weight of fresh
specimen, impact of formalin on length and on the length/weight relationship), using large
ceramic spoons.
About 70 freshly caught live ctenophores (live conditions were determined by
movement of the tentacles and the rows of combs) were immediately measured for live
length (L: boreal diameter) under a stereo microscope. The ctenophores were then rinsed
with distilled water to remove potential deposits of salt. Excess water was then removed by
gently rolling the animal over a dry glass petri dish. They were then placed in preweighted, pre-combusted (500 jC) aluminum boats for measurement of their DW. Each
boat containing a single ctenophore was placed in a drying oven at 60 jC for 2 days. Boats
were examined under a dissecting microscope for potential salt residue that may have
accumulated during drying periods before being weighed. One sample was discarded
showing small amount of salt.
Another set of ctenophores from the same net were measured live and then placed in
individual vials filled with 4% seawater buffered formalin solution. Two, three and thirteen
months later, measurements of the length of these individuals were performed and loss in
boreal diameter was computed.
Several specimens of ctenophore collected at the same time were also pooled and
preserved in seawater –formalin solution for determining dry weight after preservation.
After 3 months, these individuals were measured, subsequently dried at 60 jC for 2 days
and then weighted (weighing boats were carefully checked for any trace of salts) in order
to construct DW/L curve and to estimate loss in DW due to preservation. Changes in the
relationship between boreal diameter and dry weight with preservation were then
estimated.
The individuals used in our study were randomly picked, but unfortunately lack of time
did not allow us to collect larger samples to better describe the size distribution of the
population. No attempt was made to measure WW as sea going conditions were not
optimal.
3. Results and discussion
P. bachei specimens collected in the region were as small as 5 mm and largest one
being over 20 mm in boreal length (Figs. 1 and 2), and weighted (DW) from 2 to f 50
mg, although smaller and larger ones could have been missed by our sampling. The
general distribution pattern for freshly caught specimens fits the following relationship
between DW (in mg) and length (L: boreal diameter in mm): log DW = 2.22log L 1.12
(R2 = 0.89, p < 0.0001). Hirota (1972) obtained for fresh specimens of P. bachei the
following relationship: log DW (mg) = 2.924log D (mm) 1.550 with D = equatorial
diameter. For P. bachei, equatorial diameter is equal to 0.80 of the boreal diameter (data
not shown). The ratio between boreal and equatorial diameter is 0.75 for P. pileus
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Fig. 1. Relationship between dry weight (mg) and length (mm) for fresh (open diamond) and preserved (black
circle) specimens of P. bachei.
(Oorthuyzen and Sadée, 1982), which is slightly more oblong than P. bachei. When the
length used in the Hirota’s study is converted to the corrected boreal diameter, the
following equation is obtained: log DW = 2.34log L 1.24. This is not statistically
different ( p = 0.765, t-test) from the relationship found in our study.
Although measurements were only made on a small portion of the population of
ctenophores in our study area, specimens appeared to be older than 45 days according to
the measurements made by Hirota (1972). In this article, he reported for individuals of P.
bachei 45 days old, grown at a temperature of 15 jC, a body size of 3.75 mm. Under our
in situ conditions (f 11 jC), it is expected that specimens of that length would be even
older.
Twenty-eight randomly selected individuals preserved in a formalin – seawater solution
showed an obvious loss in length (Fig. 2). Two specimens were damaged between months
Fig. 2. Shrinkage in length due to preservation with formalin solution, after 60 days, 90 days and 13 months.
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2 and 3. Majority of loss in body length occurred during the first 2 months with some
shrinkage still occurring in the following 30 days. The loss in length was not constant and
appeared to be a function of the initial size of the specimen, with small ones (< 10 mm)
shrinking on average by 49 % and 56% after 2 and 3 months of preservation, respectively,
with a maximum of 77% after 3 months. The middle-sized specimens (10 – 20 mm)
showed an average loss in length of 18% and 26% after 2 and 3 months of preservation,
respectively. Largest specimens (>20 mm) averaged a loss of 14% after 2 months and 20%
after 3 months of preservation. On average, all these specimens shrunk only an extra 1.8%
between the 3rd and 13th months of preservation and shrinkage in boreal diameter then did
not appear to be size-dependent. Therefore, measurements of specimens should be
conducted after three months of preservation, but, due to the fragility of these gelatinous
zooplankton, we recommend doing measurement as soon as possible after this first 3
months waiting period.
Most of the previously published studies based on P. pileus did not report any sizedependent difference in the rate of shrinkage. For P. pileus, Oorthuyzen and Sadée (1982)
and Yip (1982) assumed a constant shrinkage of 20% independent of the size of the live
animal. Båmstedt (1998) used a single shrinkage factor of 31% of the boreal length for P.
pileus although the size range of the individuals was 10 –16 mm. Yip (1982) reported that
after 12 months of preservation, shrinkage could reach 31%, but averaged around 20%.
The size range used in his study was between 4 and 13 mm, but no obvious increase in
shrinkage was observed for smaller individuals (he actually reported that shrinkage was
less for the smallest individuals). Also only eight specimens were used in this study. Mutlu
(1995) measured live and preserved wet weight and body length on several hundreds of
specimen of P. pileus and reported shrinkage on average of 71% in body length and 97%
in wet weight. In his study, high numbers of individuals appeared to have a body length
shorter than 10 mm (Mutlu, 1995, see Fig. 2); we also found high level of shrinkage within
this size range. Larson (1986) cited in Larson (1987) supposedly reported length vs. dry
weight datasets taking into account shrinkage due to preservation, unfortunately this
reference is not accessible.
Formaldehyde preservation appeared to change the relationship between dry weight
and boreal length of the specimens collected here (Fig. 1). A covariance analysis
( p < 0.01) on the sets of data (before/after preservation) showed that both length and
wet weight were affected by preservation with formaldehyde, but in different proportions
as already observed by Mutlu (1995). Bias due to higher number of small specimens used
in the after preservation curve might have occur; larger size specimens should be added to
confirm this finding.
Another important factor in the preservation of ctenophore and gelatinous zooplankton
is the concentration of formaldehyde used. A solution of 4% formalin to seawater seems to
preserve (i.e. less contraction in body shape, less degradation of body) specimens of P.
bachei much better than a more concentrated one (D. Thibault-Botha, personal observation). Adams et al. (1976) proposed alternative fixation and preservation solutions for
Mnemiopsis leidyi, but as our study was being conducted as an add-on to the main
GLOBEC project, such solutions were not available and have not been tested on other
ctenophores. Their concerns about the storage of ctenophores with crustaceans (causing a
potential disintegration of gelatinous specimen) and the steadiness of the temperature at
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which samples were preserved, were both addressed by placing only one specimen per
container and by keeping containers in regulated temperature areas (always below 25 jC).
4. Conclusion
Differences in the degree of shrinkage, as a function of the size of the specimen
observed on preserved samples, do occur in the ctenophore Pleurobrachia. The constant
length loss of 20%, disregarding the actual size of the specimen that has been use in many
published works is not valid anymore. Therefore, published data on biomass and
physiological rates based on preserved specimens must have been so far underestimated
when the population was largely dominated by small size individuals, where shrinkage
was the most important. Although this work was based on P. bachei, these results can be
apply to other species of Pleurobrachia, as no strong characteristic differences exist
among species (roughly similar size, same number of tentacles).
Acknowledgements
We wish to thank the crew and captain of the R.V. New Horizon. We also acknowledge
the help of GLOBEC colleagues during the Mesoscale Cruise #4. We extend our sincere
appreciation to Mark J. Gibbons, Jane Ball and two anonymous reviewers for their helpful
comments on the manuscript. Financial support and equipment were provided through
NSF grant no. 0002713. This paper is contribution no. 412 of the U.S. GLOBEC. [RW]
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