Field collection and handling of larvae

Plankton was caught several times daily in September and October of 2004 and 2005 from the pier of the Sesoko Laboratory, Tropical Biosphere Research Center, University of the Ryukyus, on the southeast coast of Sesoko Island (26°38'N 127°52'E). We used a hand-thrown net with a 62 μm mesh width, and complete daytime samples were sorted for larvae. Dusk and nighttime samples were prefiltered at 1000 μm to exclude larger organisms and make sorting easier. Samples were inspected and sorted under a dissection microscope with transmitted light. As in other thecostracan Crustacea, facetotectan species can have either feeding (planktotrophic) or non-feeding (lecithotrophic) nauplii, while cyprids never feed [16]. From each plankton catch, we isolated the non-feeding y-nauplii into a 30 mm diameter plastic Petri dish with coarsely filtered (62 μm) seawater; any y-cyprids were isolated separately. The dishes were maintained at ambient room temperature (24–28°C). Those containing nauplius batch cultures were inspected at least three times daily and dead larvae were removed. When noted, stage-five nauplii, identified by the development of the pigmented compound eyes of the y-cyprid, were isolated singly into Petri dishes. After each stage-five nauplius had molted into a y-cyprid, the shed naupliar cuticle (exuvium) was individually mounted on a glass slide in glycerine jelly. This allowed us to document, which morphological type or 'species' it represented based on our extensive, unpublished records and collections of mounted larvae from this area. Occasionally y-cyprids appeared in the batch cultures between examinations, but apart from these and a few caught directly from the plankton, the great majority of all y-cyprids used in our induced metamorphosis trials (around 400) can thus be backtracked to a known naupliar type and be allocated to species, when a formal taxonomy for the Facetotecta is eventually established [3, 4, 6, 25].

Experimental setup

For the induction of metamorphosis, single cyprids were introduced into standard plastic plates with 15 mm wells containing the test solutions and incubated at 24–28°C. A preliminary experiment was set up to test the effect of three different metamorphosis-inducing compounds on the y-cyprids. They were dissolved in Millipore-filtered seawater and used in concentrations tested previously on cyprids of parasitic barnacles (Rhizocephala) [17]: 3-isobutyl-1-methylxanthine (IBMX at 4.5 μM), 20-hydroxyecdysone (20-HE at 208 μM, 20.8 μM, 2.08 μM, 1.04 μM and 208 nM), and juvenile hormone (JH at 37.5 nM, 375 nM and 3.75 μM). This screening showed that only 20-HE was able to induce the y-cyprids to metamorphose, and another preliminary trial established that the most efficient concentration was between 1.04 and 2.08 μM. All subsequent experiments were conducted within this range.

Photo/video documentation

All y-cyprids tested were used within 24 hours after the molt from the nauplius stage five. Well plates were inspected several times daily using an Olympus inverted microscope. Stages of metamorphosis were photographed with an Olympus C5050 digital camera and digital videos obtained with a Sony HAD Power Head. The plastic well plates do not allow optimum conditions for microphotography. To obtain better micrographs and videos, selected specimens were temporarily transferred to glass slides with seawater and observed using a Nikon Microphot with up to 40× objectives. Digital images were optimized for contrast and color using Corel X3 suite. The only other manipulation was to touch up the background, not the specimen, in some scenes to remove extraneous particles.

Transmission electron microscopy

For TEM, selected stages of metamorphosing y-cyprids and free juveniles were fixed and stored in 2.5% glutaraldehyde in Millipore-filtered seawater buffered to pH 7.4 with 0.1 M sodium cacodylate. Following three rinses in the same buffer, specimens were postfixed in 1% OsO4, dehydrated with dimethoxypropane and embedded in TAAB 812 ('Epon') resin. Sections were mounted on slot grids, stained with uranyl-acetate and lead-citrate and inspected using a JEOL JEM-1011 transmission electron microscope fitted with a GATAN digital camera.