We cultured embryos and larvae in 0.45-[micro]m MFSW (with UV treatment at HMS), in gallon jars either with mechanical stirring (M.
Upon arrival, we changed the water in the cultures, and, for each cross, we counted out exactly 300 larvae into each of 3 replicate jars with 1 L MFSW (thus, 1 larva per 3.3 inL), for a total of 12 jars (3 replicate jars for each of the 4 crosses).
Eggs were subsequently washed in MFSW
and then inserted into the injection chambers and injected as described.
The control values were measured using a chamber filled with MFSW
The primary fixative contained 50 ml of 0.2 mol [l.sup.-1] sodium cacodylate buffer (adjusted to pH 7.4) with 40 ml of MFSW
containing 3 g of sucrose and 10 ml of 25% glutaraldehyde.
Larval stages of the other species were relaxed in a 1:3 mixture of chlorotone-saturated seawater and MFSW at room temperature (Bonar and Hadfield, 1974).
Abbreviations: AG, apical ganglion; AN, apical nerve; MFSW, Millipore-filtered seawater; SCP, small cardioactive peptide.
Clear mature eggs from the oviduct of a cold-anesthetized dissected female were collected and washed several times with well-aerated MFSW
. Next, spermatophores (20-30) from the spermatophore duct of a dissected, anesthetized male were placed in 15 ml MFSW
in a syracuse dish and the sperm were discharged with slight pressure.
Each well contained about 10 larvae in 2 ml MFSW
or experimental solutions in MFSW
Gastrulating embryos were fixed with 1% glutaraldehyde dissolved in MFSW
at room temperature for 2 h.
To explore the role of the cytoskeleton in this process, in vitro fertilized  or activated embryos were placed in small petri dishes lined with 0.2% agarose (Sigma, Type II) and filled with 20[degrees]C Millipore-filtered seawater (MFSW
Small portions of the male testis were prepared for transmission electron microscopy (TEM) by excising testis from the body mass, mincing it into pieces of about 1 [mm.sup.3], fixing in ice-cold 2.5% glutaraldehyde (brought to pH 8.0 using NaOH), in Millipore-filtered seawater (MFSW
), and allowing the tissue to come to room temperature overnight.