Overview of Larval Identification

 

Monitoring for larval zebra mussels (veligers) can provide substantial advantages to a control program. Since veligers are planktonic, i.e., drifting in the water column for some weeks before they settle and attach to structures, their presence can provide an early warning system for assessing the risk of biofouling to an in-line water system and allow control procedures to maximize benefit at minimal cost. The disadvantage of monitoring for veligers is that their small size requires the use of microscopes and trained personnel to verify identification. The purpose of this section is to present techniques for identifying the larval stages of zebra mussels, quagga mussels, and Asian clams. Actual sampling protocols for collecting larvae are discussed in the Monitoring section.

 

EQUIPMENT NEEDED

Working with veligers requires a dissecting microscope, with a base light source. Cross-polarized light is not required, but highly recommended. The polarized light can either be a factory-installed unit or attached camera filters and polarized film as described in Johnson (1995).

 

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Schematic diagram of microscope retrofitted with cross-polarizing filters.

 

Polarized light is used to quickly detect mineralized material in the sample, including anything with a shell. This type of light requires the use of glass, not plastic dishes to hold samples. For further information on identification using cross-polarized light see Using Cross-Polarized Light.

 

SAMPLE HANDLING

Veliger identification can be done on either preserved or live samples. It is better to preserve samples in 70 percent ethanol than 5 percent formalin, as formalin may dissolve the shell of the veliger. Certain larval characteristics are more easily seen on live individuals. Nonpreserved samples should be held on ice or under refrigeration until processed.

 

IDENTIFICATION

Visual identification is still the main technique used to differentiate between the various species of bivalve larvae. Larval identification through the use of genetic markers or molecular probes is possible for some species including zebra mussels, quagga mussels, false dark mussels, and wedge clams (Baldwin et al 1996, Claxton et al. 1997, Claxton and Boulding 1998, Frischer et al. 1995). These probes are more reliable than visual microscopic identifications, but are not yet readily available for general field use. For further information on the species included in the interactive identification system, see Species Descriptions.

 

The first step in processing samples is to determine if larvae of any type of bivalve are present. The cross-polarized light source is critical to this process, as the shells of bivalve larvae, as well as the shells of some zooplankton, reflect polarized light. Shells are birefringent and will glow white against a dark background under cross-polarized light (see Using Cross-Polarized Light).

 

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Two plankton samples viewed under polarized light (a. and c.) and under cross-polarized light (b. and d.) to illustrate the enhanced visualization of bivalve larval when using this techniques. a. and b.: ‘cluttered’ nearshore plankton sample with four veligers present is shown in a. and b.; scale bar = 500mm. A silty sample from larval collector with three veligers present is shown in c. and d.; scale bar = 500mm.

 

Ostracods, a type of common zooplankton, have shells and appear similar to bivalve larvae under cross-polarized light.

 

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Various plankton samples seen under polarized light (a., c., and e.) and under cross-polarized light (b., d., and f.) (Polarized light was used instead of unpolarized light because the use of nonpolarized light would have required the repetitive removal and installation of the polarizing filters.) Zebra mussel veligers without extraneous material are shown in a. and b. 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 = 200mm. c. and d.: veligers and sand grain (arrowhead) are shown in c. and d. Note that the sand grain does not produce the ‘Maltese cross’ pattern. Sand grains are usually multi-colored, while veligers are always white; scale bar = 200mm. e. and f.: mixed collection of veligers and ostracods (arrowheads) is shown in e. and f. 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 are not visible at this magnification, e.g., shell ornamentation; scale bar = 200mm.

 

Ostracods can be separated from bivalve larvae in preserved samples by examining the edge of the shell under higher power. Ostracod shells have ornamentation (spines) at the anterior and posterior edge. In live samples, separation is easy, since ostracods have legs. Ostracods are typically larger and have a shape similar to a jellybean.

 

image\video2715_shg.gif Ostracods have legs, which makes them easy to distinguish from zebra mussel veligers in live samples.

 

image\video2815_shg.gif In addition to possessing legs, ostracods are also typically larger than the veligers of zebra mussels, and they have a shape similar to a jellybean.

 

A number of physical characteristics are used to identify the type of bivalve larvae found. There are presently three freshwater bivalves in the United States that produce free-living larvae or veligers - two morphs of Asian clams and two species of dreissenids. Portions of the geographic range of three of these bivalves overlap (Corbicula fluminea, Dreissena polymorpha, and Dreissena bugensis), causing problems with larval identification. Larvae of freshwater unionids are also occasionally found in samples, although these larvae, called glochidia, are generally parasitic on fish hosts. Larval identification becomes more complicated along coastal areas, with the addition of veliger-type bivalve larvae of estuarine species such as the false dark mussel (Mytilopsis leucophaeata) and the wedge clam (Rangia cuneata), and the sporadic influx of truly marine species. Unfortunately, the physical appearance of any type of veliger larvae is very similar and some species, particularly the estuarine forms, are difficult to distinguish from either type of Dreissena larvae.

 

Characteristics allowing for the most rapid and accurate separation of the various types of larvae are hinge length, shell length/height proportions (see Shell Orientation and Measurements), shell shape, size, and the presence or absence of the foot and velum. These characteristics can be used to age the larvae or identify the stage as well (D-shell, umbonal, pediveliger, plantigrade, juvenile). They may also determine if older larvae (i.e., those preparing to attach to the substrate) are present. Only three species are fully discussed: the quagga mussel, zebra mussel, and Asian clam. Information regarding identification of larval false dark mussels and wedge clams is limited, but presented where available. Only those physical characteristics readily visible under a light microscope and suitable for rapid identification of specimens collected in routine monitoring programs are used. The development of internal organs and taxonomic features visible under compound microscopes or scanning electron micrographs are not discussed. Descriptions of changes that occur in internal structures and development of Asian clams and zebra mussels can be found in studies such as Meisenheimer (1901), Chanley and Andrews (1971), Waller (1981), Mackie (1984), Kraemer et. al. (1986), Kraemer and Galloway (1986), and McMahon (1991).

 

SOURCE OF LARVAE

The appearance of all the larval stages was determined from laboratory-bred and field-caught individuals for the Asian clam and both dreissenids.

 

Identification of spawning stock used to produce Asian clams, quagga mussels, and zebra mussels:

Morphological characteristics were used to identify the adult clams and mussels from which laboratory-bred larvae were produced. Asian clams were identified as C. fluminea using the characteristics described in McMahon (1991). The features used to identify adult quagga and zebra mussels were those linked by May and Marsden (1992) and Rosenberg and Ludyanskiy (1994) to genotypic differences. Mussels were identified as quaggas if they were laterally compressed (oval in cross-section), had a rounded ventral surface, a round ridge between the dorsal and ventral surface, and a right shell valve larger or more convex than the left. Adults identified as zebra mussels were triangular in cross-section, had a flattened ventral surface with a distinct, angular ridge between the dorsal and ventral regions, and shell valves equal in size and convexity.

 

Adult dreissenid mussels of both species originated from the lower Great Lakes, including Lake St. Clair, Lake Erie, Lake Ontario, the St. Lawrence River, and associated drainage. The profundal and shallow-water forms of quaggas were not differentiated, although there appears to be no genetic difference between the two (see Baldwin et al. 1996).

 

Source of wild-caught larvae:

Wild-caught larvae originated from the lower Great Lakes, including Lake St. Clair, Lake Erie, Lake Ontario, the St. Lawrence River, and associated drainage.

 

Using Cross-Polarized Light

Shell Orientation and Measurement

Species Descriptions

 

Larval Identification Information

Zebra Mussel Identification