After a short salutation and comments on how some scientists view Bonnet's research, Spallanzani sets the stage by describing certain experiments he performed to learn about the anatomy and physiology of the earthworm. Spallanzani provides three drawings of the anatomy of arteries and veins (Figs. 1–3). He then proceeds by outlining six different experiments to study regeneration in earthworm. Drawings outlining these experiments and the results are shown in Supplementary Material.
Transversal section of the earthworm into three parts in a way that the ovary, and the small bags (he means vessels; Fig. 2) remain in the anterior part, or in the part where the head is located. Results: After a few months, the posterior parts, or tails, and the middle parts all died, with the exception of 5 or 6 of them. The anterior part, which contained the ovary and the small bags, after 16 to 20 days started to generate a very small bud; this small bud stopped growing during winter, but continued its development during spring.
Divide the worm transversally into 3 parts in a way that the ovary and the small bags belong to the middle part. Results: All anterior parts or the ends of the head died. The tails (posterior parts) also died with the exception of three of them. Many of the middle parts generated a head at their anterior extremity, and a tail at their posterior extremity.
Spallanzani attempts to provide some explanation of the few survivors that did not contain the reproductive system in experiments 1 and 2. Obviously, the pieces survived for several months and regeneration of the tail was more obvious.
Divide the worm longitudinally into two parts from the tail to two thirds of its length and then remove the anterior part containing the head. Results: The two parts, which were separated, died, and the third part, which remained and contained the head, generated the tail (obviously the part contained part of or the whole reproductive system).
Divide longitudinally the whole worm from the head to tail. Results: All the worms died.
Spallanzani then proceeds to discuss how earthworms grow. The question is: Is growth mediated by the addition of new rings or by the expansion of old ones? This question is related to preformation notions. In order to answer this question, Spallanzani decided to count rings in 11 worms that hatched from eggs and in 11 fully grown ones. He observed 84, 100, 96, 90, 102, 104, 88, 93, 96, 104, 90 in the former and 88, 94, 73, 101, 87, 83, 96, 80, 89, 95, 101 in the latter. Based on this, he concluded that growth is the result of expansion of old rings.
Spallanzani then turns to describe regeneration in another species, the fresh water worm. As for its appearance, he states that the part that contains the head is hidden in the mud, while the tail is pushed at the surface of the water forming a structure that resembles a groove or a boat (Fig. 4). He also describes the anatomy of the circulatory system for this worm. As with earthworms, Spallanzani experimented with regeneration. In one experiment, he cut 50 worms into two more or less equal parts. Almost all the anterior parts regenerated (obviously these parts contained the reproductive system) while most of the posterior parts (36/50) died. In the second experiment, he cut the worm into three parts in a way that the anterior part contained 3–6 rings and no reproductive system, and the middle part contained the reproductive system and the posterior part. At the end of the experiment, all the anterior parts were dead, the middle parts regenerated and many of the posterior parts were alive but without signs of regeneration. Spallanzani also mentions that at the level of the tail, the “boat” structure never failed to form, and he also concludes that these worms regenerate naturally because artificial regeneration (when he cut the worms) was identical with natural regeneration (when he found them in nature).
Having established that worms can regenerate, Spallanzani then attempts to explain the mechanism. Most of his observations are through the circulatory system, because this can be easily observed. First, he identifies a red dot by the edges of the anus. This shape of the anus stretches out. A cone starts to form, but the anus he claims is not at the tip of the cone but more at the rear of the cone (Fig. 5). At the posterior end of the animal (stump), a red line appears in the same direction as the artery of the animal (denoted as O artery in Fig. 6). In the beginning, the line does not show any circulation but later it does, and the blood flows into the O artery. Thus, the red line is the regenerated artery. The cone grows and the edges of the anus acquire more redness. As the cone grows, rings become obvious. Spallanzani also attempted repeated amputations, which were successful. He finally concludes that the shape (boat) is always formed and that the repeated amputations lead to shortening of the animal. Spallanzani does not elaborate here how germs could account for regeneration in earthworms, but he compares this later with head regeneration in snails. At the end of this section, Spallanzani develops some ideas about the respiratory system, but his observations are rather inconclusive. Despite the interesting way that earthworms regenerate and the intriguing necessity of the reproductive system, which implies dependence on some specific factor, not much is known today at the molecular level. Nevertheless, after Spallanzani, many scientists experimented with earthworm regeneration. The necessity of certain segments, which include the reproductive system, has been verified (Morgan,1901). In the earthworm, Eisenia foetida, which has 100 segments, amputation between segments 20–35 results in the failure of regeneration (Supp. Fig. 1). In this worm, regeneration is mediated by the formation of blastema, and ectoderm and endoderm maintain their identities. It also seems that annelids in general rely on dedifferentiation of older tissues, rather than pluripotent reserve cells (Goss,1969). Nevertheless, studies on asexual reproduction in annelids have indicated the regeneration of primordial germ cells from parts of the body devoid of gonads, arising from piwi-expressing germile stem cells (Weisblat,2006).