Acantharia
Methods

The extreme solubility of the celestite skeletons of acantharians requires special collection and preservation methods. Sampling is restricted to the water-column, as they are not preserved in ocean bottom sediments. Solubility also prevents their retention in sea water samples preserved by conventional methods: addition of strontium to preserved samples is necessary. With these provisos, various collecting techniques applicable to other delicate microplankton are also appropriate for acantharians.

Net samples can be collected by either simple, non-closing plankton nets (Massera Bottazzi and Andreoli 1974, 1982a, 1982b; Tan and Su, 1983), or more elaborate devices allowing multiple, discreet, vertically stratified tows (Schreiber, 1960; Spindler and Beyer, 1990). Nets have been employed in horizontal surface tows, as well as in oblique or vertical hauls from varying depths. Mesh sizes also vary, ranging from 25 to >160 µm, although sizes in the range of 160-350 µm result in gross abundance underestimates. Estimates from nets are in the range of 1-500 ind. mö-3 (Massera Bottazzi et al., 1971; Massera Bottazzi and Andreoli, 1974, 1982a, 1982b) compared to 1,000-50,000 mö-3 based on pump or filtration methods (see below). This suggests that net sampling, especially with coarse meshes, is generally inadequate for these organisms as their small and fragile bodies get extruded through the gauze or destroyed beyond recognition in the samples (Michaels, 1988a, 1988b).

Niskin bottles of 12-30 l capacity at various intervals through the upper 160-200 meters of the water column have been employed successfully by Beers et al. (1975, 1982), and Michaels et al. (1995). To minimize destruction of fragile acantharian cells during collection, Graham et al. (1976) used a 1 liter polyethylene bag attached to a spring-hinged sampling device controlled by a standard heavy-duty fishing rod and reel. The technique was successful at depths from 1 to 300 m, and the number of acantharians collected was two orders of magnitude higher than those previously published.

Pump sampling has been employed by several workers. Bishop et al. (1977, 1978, 1980) used large volume in situ filtration systems to collect particulate matter from 5-30 mö3 of water from the upper 400 m. Water was pumped at a rate of ~6 mö3 hö-1 through 53 µm mesh screens, and then through acid-leached pre-combusted glass filters to provide sample size fractions of 1 µm, 1-53 µm, and >53 µm. Michaels et al. (1995) used a deck-mounted air-driven pump connected to a 200 m long hose to collect water samples from various depths. Canisters mounted close to the hose and near the pump contained 300 and 20 µm mesh Nitex socks to separate sample material into two size fractions.

Acantharian skeletons and cysts may be collected in sediment traps (Bernstein et al., 1987). Short deployment times (on the order of 24 hours), relatively shallow trapping depths, lack of preservatives that might enhance dissolution, and sample processing immediately after trap retrieval were instrumental in yielding well-preserved organisms, although abundances are still considered low (Bernstein and Betzer, 1991). Michaels (1991) used free-floating cylindrical traps over periods of 54 days. Traps were charged with a salt gradient (NaCl, 50 g lö-1) to retain 10 ml lö-1 of formalin preservative and 72 mg lö-1 of Sr to maintain acantharian integrity.

Michaels (1988) compared the efficiency of various acantharian collection and processing techniques to show that (1) acantharian density estimates obtained from Niskin bottles are similar to those from diver-collected samples; (2) plankton net samples (both 25 and 160 µm mesh, and both horizontal and oblique tows) appear to capture far fewer specimens than do Niskin bottles; and (3) Niskin bottle specimens obtained from the spout were more highly clumped than those retrieved through the bottom valve. Possible explanations for acantharian underestimation by plankton nets include overestimation of the amount of water filtered, damage to acantharian cells by the net, leading to distortion and passage through the net, and net avoidance by a buoyancy mechanism triggered by disturbance.

To concentrate acantharian cells, whole samples are often sieved through a series of screens separating the material into discrete size classes (Beers et al. 1975, 1982; Bishop et al., 1977, 1978, 1980; Bernstein and Betzer, 1991; Michaels et al., 1995), or allowed to settle for 1-3 days and the supernatant fluid then gently withdrawn (Michaels, 1991).

Although Acantharia are often the most abundant shelled sarcodines in the upper ocean (Caron and Swanberg, 1990; Michaels, 1988a, 1988b, 1991; Michaels et al., 1995), their skeletons dissolve in traditional preservatives such as buffered formalin (Beers and Stewart, 1970), and thus tend to be missed in assessments of microplankton abundance. Addition of SrCl2 6H2O to fixed samples such that the final Sr concentration equals 80 mg lö-1 has been widely adopted (Beers et al., 1975, 1982; Michaels, 1988b, 1991). Higher Sr concentrations (160 to 240 mg lö-1) in formaldehyde-preserved samples, especially when buffered with sodium borate, tend to produce a white precipitate and are therefore not recommended (Michaels, 1988b). Michaels (1988b) also concluded that in samples with large numbers of acantharians, skeletal dissolution will raise the dissolved Sr concentrations and preclude further dissolution.

Preservative formulations used in modern studies include Bouin solution (Tan and Su, 1983), 2-3% formalin buffered with borax or hexamethylenetetramine (Massera Bottazzi et al., 1971), 5% formalin buffered with hexamine and containing 72 mg lö-1 Sr (Michaels, 1991), 2.5% gluteraldehyde with a sodium cacodylate buffer and strontium chloride (80-160 mg lö-1) (Michaels, 1988b), 4% formaldehyde buffered with sodium borate, and strontium chloride (80-160 mg lö-1) (Michaels, 1988b).

Filtration of samples onto Nuclepore or Millipore filters and subsequent drying is another common technique used to preserve skeletal and cystic material and to facilitate examination using an electron microscope (Bishop et al., 1977, 1978, 1980; Michaels, 1988b; Bernstein and Betzer, 1991), although it preserves the skeleton only.

Acantharians can be examined in wet samples under a regular microscope or an inverted microscope. Larger specimens (>100 µm) can also be counted under a dissecting microscope. Best results are obtained with freshly collected, living specimens. Staining with boric carmine (Schreiber, 1960), or bengal rose (Beers et al., 1975, 1982) may help visualize the soft parts. Scanning electron microscopy (Bernstein et al., 1987, Bernstein and Betzer, 1991) is especially appropriate for high resolution of skeletal geometry.