Enhanced early detection and enumeration of zebra mussel - CiteSeerX

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Hydrobiologia 312 : 139-146, 1995 . © 1995 Kluwer Academic Publishers. Printed in Belgium .

Enhanced early detection and enumeration of zebra mussel (Dreissena spp .) veligers using cross-polarized light microscopy Ladd E . Johnson Maritime Studies Program, Williams College - Mystic Seaport, Mystic, CT 06355, USA Correspondence address : Marine Science Institute, University of California, Santa Barbara, CA 93106, USA Received January 1994 ; in revised form 25 October 1994 ; accepted 19 December 1994

Key words : birefringence, bivalva, calcareous larva, ostracod, methodology, plankton, sampling error

Abstract Zooplankton with calcareous skeletons are birefringent under cross-polarized light, and thus this technique can be quite useful, indeed sometimes almost essential, for the detection and enumeration of these types of organisms in plankton samples . A simple and inexpensive application of this technique is described and illustrated with quantitative examples from research on the veligers of the zebra mussel Dreissena polymorpha (Pallas) . The time required to detect veligers in plankton samples was decreased by an order of magnitude ; the accuracy of counts was substantially improved (15% more than controls), and the time required for counts was greatly reduced (41 % of control times) . This technique is especially useful in situations in which veligers are difficult to find or see (e .g., at low densities, in samples `cluttered' with extraneous organisms or material) or when the investigator is inexperienced with plankton sampling and planktonic organisms . The major limitations are its inability to discriminate among various bivalve species that have planktonic larvae and the similar appearance of ostracods which also have calcareous shells . Expanded use of this technique should (1) increase our ability to use plankton sampling for the early detection of veligers during range expansion and reproductive cycles and (2) permit more accurate estimates of veliger abundance .

Introduction Since its introduction to North American waters in the mid-1980s, the zebra mussel Dreissena polymorpha (Pallas) has spread rapidly through contiguous waters . Because of its potential for severe economic and ecological effects (Nalepa & Schloesser, 1993), there has been an enormous effort by scientists and managers to monitor for the presence and abundance of zebra mussels in the environment . As the range of zebra mussels continues to expand, these efforts will increase . Monitoring efforts for detecting and enumerating zebra mussel populations can be directed at either the planktonic larval stage (the veliger) or the benthic adult stage . Although the specific stage selected for monitoring depends on the particular question being addressed and the resources available, sampling of the larval stage may be preferable for several reasons . As the primary dispersive stage, the larva is likely to be the first

stage present during the spread of this species into contiguous waters . Planktonic stages are also more evenly distributed and thus when present, they can provide better quantitative estimates than sampling of benthic stages . Finally, because biofouling is primarily the result of larval settlement, knowledge of the temporal presence or abundance of veligers permits preventative anti-fouling measures to be implemented in a timely and efficient manner . Moreover, a thorough understanding of the dynamics of benthic populations requires knowledge of abundance and distribution of the larval stages (Roughgarden et al., 1985) . Unfortunately, two major problems exist when attempting to detect and enumerate veligers within plankton samples. First is the difficulty of distinguishing veligers from other planktonic organisms, detritus, and inorganic particles (collectively, the 'seston') that are also collected in samples . The second problem is that veligers are difficult to detect when rare (e .g ., in



1 40 the early stages of colonization, at the beginning of the reproductive season, or in areas with low reproduction) . Although one solution to this latter problem is to increase the volume of the water sampled, this compounds the first problem by increasing the amount of extraneous material in the sample . With these concerns in mind, any technique that might distinguish veligers would aid efforts aimed at either detecting or enumerating veligers in samples . Several techniques, both standard and novel, have been suggested for such purposes (Marsden, 1992) . However, these techniques can involve extra processing steps [e .g ., density segregation (Schaner, 1990)] or have limited discriminatory abilities (e .g ., rose Bengal stains all animal tissue) . An alternative technique using cross-polarized light (CPL) offers both ease of use and a high degree of specificity and is entirely nondestructive ; samples are not treated or manipulated in any way. These attributes may greatly increase abilities for early detection, reduce time for examining samples, and increase the accuracy of estimates of veliger densities . The principles, application, and quantitative and qualitative illustrations of its use are described below . Although CPL has long been used for examining biological materials (Bennett, 1950) and has been used for laboratory studies of the development of larval skeletons (Boolootian, 1959 ; Pennington & Hadfield, 1989 ; Gallager et al., 1989) and the identification of ichthyoplankton (Holland-Bartels et al., 1990), I have found no mention of its use in field studies of calcareous plankton in either marine or freshwater environments . Indeed, the value of this technique is not limited to zebra mussel research ; its use could be valuable in the study of any calcareous plankton in any aquatic system .

Principle and application Light waves generally have no particular orientation . However, a polarizing filter will orient the light (i .e ., polarize it) by blocking any component of the light waves that is not parallel to the direction of the long, oriented molecules that make up the filter . If two polarizing filters are oriented at right angles to each other (i.e ., 'crossed'), light oriented by the first filter will be blocked by the second filter, and thus no light will pass through the combination of filters . However, if, in the space between the two filters, the light is reoriented while passing through an object, it can then pass through the second filter. Objects with such capabilities

Table I . Characters that can distinguish planktonic bivalve larvae

from the ostracod species most likely to be collected in plankton samples or larval collectors . Character

Bivalve larvae

Shell shape

Round or oval ; hinge 'Bean-shaped' ; hinge straight or with umbo curved

Behavior

Slow swimming in Rapid swimspiral or circular path ming, often erratic or pulsed Never present Often present

Eyespot

Ostracods

Shell None ornamentation

Valve surface usually pitted, papillate, or setose

Size

Generally larger (3001000 µ.m) except for earlier instars . Antennae (setated)

Appendages

Generally smaller (60-500 µm ; D. polymorpha 60-300 µm) Velum (ciliated)

are called 'birefringent' and typically consist of long molecules that lie in a preferential orientation (e.g ., crystals, polymers) . If polarizing filters are inserted in the light path of a microscope at points above and below the working section so that they cross-polarize the light (Fig . 1), only birefringent objects are visible . In freshwater, bivalve larvae are one of the few objects found in plankton samples that are strongly birefringent . This property is due to the crystalline structure of the calcite in the larval shell which develops several days after fertilization . Thus the veligers stand out as bright spots against a dark background (Fig . 2a, b) . Moreover, because of the concentric arrangement of the crystals within the shell, the portions of the shell in line with the axes of the filters are not birefringent thus making the shells appear as glowing Maltese crosses (Fig . 2b) . This distinguishing feature sets bivalve veligers apart from all other similarly-shaped birefringent objects commonly seen in plankton samples (e .g ., sand grains ; Fig . 2c, d) with one exception, ostracods (Fig . 2e, f) . These crustaceans have a calcified bivalve carapace and thus are also birefringent . To distinguish them from molluscan bivalve shells, one must rely on other features including behavior, size, shape, shell morphology, or anatomical features (Table 1) . Veligers of different species of bivalves will look similar. Thus this approach will not readily distinguish the veligers of various species of Dreissena, other dreissenids (e .g ., Mytilopsis), or the Asian clam Corbicula . Here again, one must rely on size and morphol-



141

Fixed polarizing filter

I !

Light source

Fig.

1. Schematic diagram of microscope retrofitted with cross-polarizing filters .

ogy differences (Conn et al ., 1993 ; Nichols & Black, 1994) although partially CPL can still assist in visualizing these characteristics (S . J . Nichols, NBS, 1451 Green Rd . Ann Arbor, MI, pers . comm .) .

Methods Equipment specifications

Whereas many compound microscopes have the capability of producing a cross-polarized light field, the stereomicroscopes typically used for examining plankton samples often do not . However, such capabilities can easily be added without permanently altering the microscope . An easy way is to purchase a retrofit kit ($150 to $250), if available, from the microscope manufacturer. A more economical approach is the use of polarizing filters obtained from a camera store . One good setup involves using both a polarizing lens filter and a piece of polarizing film (e.g ., Glare-Stop Polarizing Filter, Visual Pursuits, Inc ., Chicago, IL, USA) . The film can be attached to the glass stage (Fig . 1) using adhesive tape (To avoid scratching the film, it should be attached to the underside of the stage) . The lens filter is then attached to the bottom of the microscope head by an adhesive (e .g . silicon sealant, hot melt glue) or tape so that it covers the objective lens (Fig . 1) . Photographic polarizing lens filters are rotat-

able, a valuable feature for being able to switch quickly from CPL to polarized light (e .g ., to examine anatomical features) which is essentially equivalent to nonpolarized light . This feature also allows one to vary the degree to which the light is crossed-polarized . The use of partially CPL still allows veligers to be easily distinguished but reduces eye strain . Alternatively, reducing background light, i.e ., room lights and unshaded windows, will also make viewing the dark background of a CPL field easier. The container in which the sample is held during examination is of critical importance. Most plastics consist of long polymers which make the plastic birefringent thereby reducing the dark background needed for contrast . Unfortunately, the more common laboratory plastics, acrylic (e .g ., PlexiglasTM ) and polystyrene (e .g ., disposable petri dishes), exhibit this property. The problem varies among different products and even within a given container so some care must be taken in their selection (plastic products that are cast instead of extruded are less likely to be birefringent) . The simplest solution is to use glass which, due to its amorphous structure, is generally not birefringent although some problems occur where the glass has been stressed (e .g . curved surfaces) . Glass petri dishes work well for scanning large portions of plankton samples, whereas modified glass slides (e .g ., SedgewickRafter cells) are useful for smaller samples or high magnification work . For enumerating veligers in larger volumes, a suitable `plankton maze' can be constructed by routing or milling out a series of switchbacks in a small sheet of plastic and then gluing the sheet onto a sheet of glass of the same dimensions (Fig . 3) . The resulting trough can then be filled with the sample and examined along its length without inadvertently missing or repeating areas . Because the light path only goes through the glass and the sample, there is no interfering birefringence . Because the birefringence is dependent on the calcium incorporated into the larval shell, any decalcification of the shells from improper preservation will diminish the usefulness of this technique . Therefore, samples should be properly buffered against acidity . Efficacy of use

Several trials were performed to quantify the efficacy of this technique . The first consisted of counting veligers both with and without CPL . Three 180 1 surface water samples were taken on 25 July 1992

1 42

A

B

C

E

Fig. 2.

F

Various plankton samples seen under polarized light (a, c, & e) and under cross-polarized light (b, d, & f) (Polarized light was used instead of unpolarized light because the use of non-polarized light would have required the repetitive removal and installation of the polarizing filters .) a & b: zebra mussel veligers without extraneous material . Note that the distinctive `Maltese cross' pattern of birefringence of veligers on their sides is not evident when the veliger is viewed edge on (arrowhead) ; scale bar = 200 jum. c & d : veligers and sand grain (arrowhead) . Note that sand grain does not produce the `Maltese cross' pattern . Sand grains are also usually multi-colored while veligers are always white ; scale bar= 200 µm. e & f: mixed collection of veligers and ostracods (arrowheads) . Note that size ranges of veligers and ostracods overlap (e .g . star) and that empty ostracod `shells' (lighter individuals in e) are more birefringent, and thus more similar to veligers, than whole animals due to the lack of interfering body tissue. Certain morphological features that distinguish veligers from ostracods (see Table 1) are not visible at this magnification, e.g., shell ornamentation ; scale bar =200 µm .



143 A . Top view

0 polarized light

1 cm

T

cross-polarized light

r20

15

10

V i

5

0 Time required Fig. 4 . Counts and time required for counts for plankton samples containing zebra mussel veligers using crossed polarized light and polarized light . Error bars represent one SE ; n = 4 . Statistical analysis was a matched pair t-test, one tailed . p=0.05 and 0.01 for counts and time required for counts, respectively .

Schematic diagram of plankton maze suitable for use with cross-polarized light microscopy . A . A track (T) is routed or milled from an appropriate sheet of plastic (P) [e.g ., acrylic, polycarbonate, or polyvinyl chloride (PVC)] . B . Routed plastic sheet is glued to sheet of glass (G) . Fig. 3.

from launching docks at the Ensign Public Access Site (EPAS) located at the Clinton River mouth, Lake St . Clair, MI, and passed through a 63 µm plankton net . Counts with and without CPL were made on four different 15 ml splits using a plankton maze at 20 x

to 30 x magnification under a zoom stereomicroscope . Time to complete the count was also recorded . The second trial assessed the usefulness of CPL for the initial detection of veligers . Plankton samples were examined for larvae with and without CPL until the first veliger was found. This 'time-to- detection' was done for 180 1 water samples collected as above from two sites : the boat launching areas of the EPAS and the Metrobeach Metropark on Black Creek approximately 1 km before it enters Lake St . Clair (sites were c . 5 km apart) . Seven samples at c . 5 m intervals along the shore were taken at each site on 9 October 1992. Because it was late in the reproductive season, veligers were large (i.e., the late stage umbonal forms, c . 200250 N,m in length) and their abundances low . Overall veliger abundances were not determined but were estimated to be between 20 and 100 per sample . Samples were examined by a technician with two months of experience in examining plankton samples for zebra mussel veligers and were coded so that the technician

was unaware of the source of the sample or whether it had been examined before . One to three such measurements were made using both techniques on each sample, and the averages for each technique used for statistical comparisons . Data were analyzed by a two-factor ANOVA (Site and Technique) . As an added example of the power of this technique, ten veligers were added to each of two 180 1 plankton samples collected from waters in which zebra mussels were absent (i.e ., inland lakes), and the time-to-detection determined with and without CPL .

Results In the first trial, counts were significantly lower when CPL was not used and ranged from 75 to 95% (mean= 85%) of the counts obtained when CPL was used (Fig . 4). The higher counts were not due to the inclusion of other calcareous zooplankton (e .g ., ostracods, Corbicula larvae) which were rare in these samples . The effect on the time required for completing counts was even more striking : counts using CPL took only 41 % of the time required for counts without using CPL (Fig . 4) . In the second trial, time-to-detection was generally an order of magnitude longer when CPL was not used (Fig . 5) . However, there was a Technique x Site interaction which may be due to the character of the extraneous material in the samples : filamentous algae were more abundant at Metrobeach, making it harder to locate veligers when CPL was not used . In contrast, there were more sand grains in the samples from

144

cross-polarized light

polarized light

Fig. 5.

Time required for initial detection of Dreissena veligers using cross-polarized light and polarized light in plankton samples collected from two public access sites adjacent to Lake St . Clair (EPAS =Ensign Public Access Site ; Metrobeach = Metrobeach Metropark) . Error bars represent one SE ; n = 7 . Statistical analysis was a two-factor crossed ANOVA (Technique and Site) : p = 0 .0001, 0 .02, and 0 .01 for the effects of Technique, Site, and the Technique x Site interaction, respectively .

EPAS (and thus more birefringing objects in the field of view), making the use of CPL less effective . For the two samples in which veligers were added, time-to-detection was >3 x longer when CPL was not used [167 vs 535 s and 107 vs >600 s (the a priori time limit for the trials) for times with and without CPL, respectively] . These samples were particularly cluttered with other material, especially zooplankton . `A picture is worth a thousand words', and the most persuasive evidence of the efficacy of this technique may be photomicrographs of samples with and without CPL (Fig . 6) . The first series (Fig . 6a, b) are from a nearshore plankton sample in which the extraneous material is mostly filamentous algae, sand grains, and unidentified debris, whereas the latter series (Fig . 6c, d) is from a larval collector (see appendix by A . Martel in Marsden, 1992) in which the extraneous material is the fine silt that tends to collect in these devises, especially in calm waters . In both cases, veligers are distinctly more visible under CPL . Indeed, their high visibility and distinctive pattern of birefringence allows one to work at lower magnification when searching for or counting veligers .

Discussion This technique will generally improve the accuracy of counts (i .e ., fewer individuals will be missed) and decrease the time both for initially detecting veligers in a sample and eventually counting them . Conditions that increase the value of this technique are when

(1) veligers are rare, (2) the sample is `cluttered' by other organisms, detritus, or debris, and (3) veligers are either dead or inactive . Swimming veligers are easily recognizable from other objects due to their distinctive movements (Table 1) ; thus the examination of samples immediately after collection is always the best time to look for veligers . The widespread use of this technique will have several major implications . One is that plankton sampling can become a more viable way in which to detect the early stage of range expansion of the zebra mussel . Previous work using plankton sampling as one of several methods for the early detection of zebra mussel invasions found that it was no better than settling plates or reports by the public (Kraft, 1993) . This situation may change as the use of CPL improves our ability to find veligers at low numbers and thus our ability to detect the early stages of an invasion . In current research on the spread of zebra mussels into the inland lakes of Michigan, I was able to detect veligers at concentrations of less than 0 .01 veligers 1 -1 in lakes in which zebra mussels were previously not known to occur (unpubl . data) . In 10 out of 11 of these cases, veligers were the only stage of zebra mussels found in spite of equivalent efforts to find adults . Another implication of these results is that some previous estimates of veliger densities may be underestimated . The inherent difficulties of counting planktonic organisms, especially when samples are not completely sorted, can lead to underestimation of abundances . The magnitude of these errors will depend on the techniques used and may be relatively small compared to the true variation existing in space and time . Still, comparisons with earlier data collected without using CPL should be made with some caution given this potential sampling error . Given the improvements in both the speed and accuracy of counts seen in this study, I believe that this technique will assist anyone interested in quantifying veliger abundances, especially those with limited experience in plankton sampling . For all types of investigators, the use of this technique will greatly aid in the early detection of veligers within a body of water . Moreover, this technique can be valuable in research involving any organism possessing a calcareous skeleton (e .g ., echinoderm larvae, ostracods) that lives in freshwater, estuarine, or marine environments .





1 45

A

B

C

D

Fig. 6. Two plankton samples viewed under polarized light (a& c) and under cross-polarized light (b & d) to illustrate the enhanced visualization

of bivalve larvae when using this technique . a & b : `cluttered' nearshore plankton sample with four veligers present ; scale bar = 500 µm. c & d : silty sample from larval collector with three veligers present ; scale bar= 500 µm .

Acknowledgments

for help with the literature, to Ellen Marsden for early verification of the technique, to Jerrine Nichols, Renata My appreciation to Richard Emlet for my initial expo- Claudi, Cliff Kraft, and Chuck O'Neil for early enthusure to this technique and along with Tim Pennington siasm and dissemination of the technique, to Jim Carl-

146 ton, Hank Vanderploeg, and the anonymous reviewers for comments on earlier drafts, to the USFWS National Fisheries Center - Great Lakes for the use of photographic equipment, to the NOAA Great Lakes Environmental Research Laboratory for hosting me as a visiting scientist, and to the NOAA National Sea Grant Program (CT Sea Grant R/ER-5 to J . T. Carlton) and the Mellon Foundation (08941139 to S . Gaines & M . Bertness) for funding .

References Bennett, H . S ., 1950 . Methods applicable to the study of both fresh and fixed material : The microscopical investigation of biological materials with polarized light . In R . M . Jones (ed.), McClung's Handbook of Microscopical Technique, 3rd edn . Hoeber, New York: 591-677 . Boolootian, R . A., 1959. A simple technique for studying in vivo skeletal structures of echinoderm larvae . Turtox News 37 : 115 . Conn, D . B ., R. A . Lutz, Y. Hu & V . S . Kennedy, 1993 . Guide to the identification of larval and post-larval stages of the zebra mussel Dreissena spp. and the false dark mussel Mytilopsis leucophaeata . New York Sea Grant Institute, Stony Brook (NY), 22 pp .

Gallager, S ., J. P. Bidwell & A . M . Kuzirian, 1989 . Strontium is required in artificial seawater for embryonic shell formation in two species of bivalve molluscs . In R . E . Crick (ed .), Origin, evolution, and modem aspects of biomineralization in plants and animals. Plenum Press, New York : 349-366 . Holland-Bartels, L . E ., S . K . Littlejohn & M. L . Huston, 1990 . A guide to larval fishes of the upper Mississippi River . USFWS National Fisheries Research Center, La Crosse, WI, 107 pp . Kraft, C ., 1993 . Early detection of the zebra mussel . In T . F. Nalepa & D . W. Schloesser (eds), Zebra mussels : biology, impacts, and control . Lewis Publishers, Ann Arbor (MI) : 705-714 . Marsden, J. E ., 1992. Standard protocols for monitoring and sampling zebra mussels. Illinois Natural History Survey Biological Notes 138, 40 pp . Nalepa, T. F. & D . W. Schloesser (eds), 1993 . Zebra mussels : biology, impacts, and control. Lewis Publishers, Ann Arbor (MI) : 810 pp. Nichols, S . J . & M . G . Black, 1994 . Identification of larvae : zebra mussel (Dreissena polymorpha), quagga mussel (Dreissena bugensis), and the Asian clam (Corbicula fluminea) . Can . J. Zool. 72: 406-417 . Pennington, J . T. & M . G . Hadfield, 1989 . A simple nontoxic method for the decalcification of living invertebrate larvae . J . exp. mar. Biol . Ecol. 130 : 1-7 . Roughgarden, J ., W. Iwasa & C. Baxter, 1985 . Demographic theory for an open marine population with space-limited recruitment . Ecology 66: 54-67 . Schaner, T., 1990 . Detection of zebra mussel veligers in plankton samples using sugar solution. Lake Ontario Fisheries Unit Annual Report, LOA 91 .1, Ontario Ministry of Natural Resources, Picton (ON), 3 pp .