El sentido de lugar teológico en la teología de la liberación

100 worms were observed daily for 14 days after amputation in order to establish a general time course and assess variability in the timing of opercular regeneration. For the first three days, as morphogenesis proceeds rapidly during this time, the intervals between observations were kept as close to exactly 24 hours as possible. Thereafter, animals were observed daily within a few hours of the original time of amputation. For each animal, the following were recorded upon detubing and amputation:

 Sex

 Size (approximate thorax length)

 Whether the operculum was autotomised or amputated by hand  Injuries incurred during detubing

16 Sex was recorded as male or female if the animal spawned upon detubing, and unknown if no gametes were observed. Size (a proxy for age) was estimated as the approximate length of the thorax from the anterior edge of the collar (folded down) to the last thoracic uncinus, measured on the left side of the animal. These landmarks were chosen because they are relatively easy to observe in imperfect photographs and are less affected by the movement of the animal than other body parts. Photographs were taken with a Nikon Coolpix 4500 digital camera mounted on a dissecting microscope and included graph paper to calibrate length measurements. Since experimental amputations were performed at the natural autotomy plane, autotomy and manual amputation were generally regarded as equivalent. However, autotomies were recorded here to test the correctness of that assumption. Injuries recorded were tentacle amputations, abdominal amputations, and miscellaneous abdominal injuries. Preliminary observations suggest that opercular regeneration is generally unaffected by injury to these body regions (S. Miles and D. Ferrier, unpublished; personal observation), but damage was recorded nonetheless to test this assumption. Animals that sustained damage to the thorax were discarded, as thoracic injuries are likely to cause significant illness or death. Following amputation and initial observations, each animal was maintained in an individual well and scored daily for the following developmental landmarks:

A. Morphogenesis

 Regeneration initiated. Amputation wound healed. Stump elongating. Prongs have begun to develop from the corners of the amputation surface. Slight swelling may be present around the middle of the stump, but a swelling was not a necessary criterion for this stage.

 Significant swelling.

 Rim development. There is a clear transition between the distal end of the swelling and the base of the presumptive opercular spine.

 Cup/plate development. The swelling is cup-shaped and the opercular plate is developing between the spine and the rim.

 Calcification. Under a dissecting microscope, initial calcification is visible as small rounded beads/tiles and/or white areas around the base of the spine.  Groove formation. There is a sharp line between the peduncle and the base

of the cup. (This line later deepens into a well-developed groove [Fig. 1.1; Fig3.1G].)

 Easy break point formation. A line marking the easy break point is visible on the dorsal side of the peduncle. This landmark was only scored as present if it was observed two days in a row due to the difficulty of observing it on live regenerates.

 Wing bud formation. The outgrowth of the lateral wings has begun at the distal end of the peduncle.

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B. Pigmentation

After amputation, some pigmentation is often carried over from the proximal peduncle. Residual pigmentation manifests as white patches in the presumptive spine and plate regions, and/or a uniform dark hue in more proximal portions of the regenerate (Section 3.4.1.2, 3.4.2, Fig. 3.4). Only newly formed pigmentation was recorded for staging. The complex pattern of pigmentation that characterises a typical opercular filament (Fig. 1.1) was divided into the following components:

 Scattered dots on the cup wall. Generally red/brown in colour.

 Contiguous dark bands or patches on cup wall. Usually a red/brown colour at first appearance.

 Proximal pigment band of the peduncle. The dark pigment band situated mid-peduncle, immediately distal to the EBP.

 Distal pigment band of the peduncle. Dark band halfway between the middle of the peduncle and the base of the cup.

 White pigmentation on peduncle or cup wall. This excludes residual white pigmentation, which does not extend below the opercular rim.

In addition, common developmental abnormalities of the opercular filament were recorded. These are illustrated in Fig. 3.2. Abnormalities were recorded as present, absent or transient (if present only temporarily).

 Sub-cuticular “bubbles” on the opercular rim or under the plate.  Sideways “kinks” in the peduncle.

 Short peduncles, defined as peduncles short enough to be hidden by the collar in left lateral view.

Animals were observed for 14 days or until they had reached all landmarks, whichever occurred soonest. Four animals were excluded from subsequent analyses: one died on day 4, one kept its regenerate mostly hidden among its tentacles, making accurate observations impossible without removing the tentacles; one had a highly stunted regenerate that failed to develop any pigmentation by 14 days post-operation (dpo), and one still had an open wound on its abdomen at the end of the observation period. Thus, the final time course data are derived from 96 animals. Seven of these aborted and restarted regeneration before 14 dpo; for these worms, only data from the first regenerate were used.