Symsagittifera smaragdina, Achatz, Johannes G., Gschwentner, Robert & Rieger, Reinhard, 2005

Achatz, Johannes G., Gschwentner, Robert & Rieger, Reinhard, 2005, Symsagittifera smaragdina sp. nov.: A new acoel (Acoela: Acoelomorpha) from the Mediterranean Sea, Zootaxa 1085, pp. 33-45 : 35-41

Symsagittifera smaragdina sp. nov. ( Figs. 1 , 2 , 3 )

Type Material. Holotype: NHMW­EV­MP­4621; Mollenhauer­embedded specimen, transversal 1­µm­thick sections, stained with HE. Paratype 1: NHMW­EV­MP­4622, Paratype 2: NHMW­EV­MP­4623; Mollenhauer­embedded specimens, sagittal 1­µmthick sections, stained with HE.

Type Repository. Natural History Museum, Vienna, Austria

Type Locality. Giglio, Italy, at the beach of Campese (42°22´N; 10°50´E), 5 September 2003. Animals occurred from 1–2 m water depth, with highest abundance at 1.5 m in coarse sand.

Other Material Observed. Serial HE­stained sagittal sections of four Mollenhauer embedded sexually mature specimens and one immature specimen. TEM sections of three sexually mature specimens and one immature specimen.

Etymology. The specific epithet smaragdina is derived from the Latin smaragdus or the Greek smáragdos emerald, a green mineral, and refers to the deep green colour of the animals, caused by numerous symbionts, which are not just scattered irregularly throughout the animals, but are highly ordered in bands, due to the highly ordered distribution of muscles.

The genus name Symsagittifera refers to the possession of symbiotic algae and sagittocysts. In the original description it is written Simsagittifera because of an error of the printer (personal communication of Y. Mamkaev with Seth Tyler). Mamkaev and Kostenko (1991) mention this lapsus. Consequently, referring to chapter 7, article 33.2 of the 4th Edition of the International Code of Zoological Nomenclature (1999), Symsagittifera is a “justified emendation” and takes the authors and date of the original spelling (for contrary view see Faubel et al. 2004).

Description. Specimens of Symsagittifera smaragdina nov. sp. in culture adhere to the Petri dishes with the posterior­lateral portions of their bodies and extend their anterior ends into the water column, or rest it on the substrate. Swimming worms move in a spiral pattern through the water column, having their lateral parts enrolled ventrally, or glide slowly over the substrate. When a Petri dish of worms is disturbed, the animals cluster together.

Abbrevations: dsp donor spermatozoa, o oocyte, sp spermatozoa, sv seminal vesicle,

Mature animals are 2.5–3 mm long, 1 mm wide, and have enrolled lateral sides. Viewed dorsally, the body is most narrow in the middle ( Fig. 1 A). Specimens appear green due to the presence of symbiotic algae that are distributed evenly both dorsally and ventrally. Prominent red rhabdoid glands are scattered over the dorsal side of the body, most numerously anterior to the mouth.

Setting the total body length to 100 units (100 U), the distance of prominent structures from the anterior tip of the body are as follows: statocyst 4 U, mouth 45 U, female genital pore 75 U, male genital pore 94 U. Testicular and ovarian follicles are positioned laterally in paired strings and mature towards the genital pores ( Fig. 1 A). The epidermis is cellular and multiciliated. The cell nuclei of the epidermal cells are insunken and are located beneath the muscle layers ( Fig. 2 B, C). On the surface, S. smaragdina has an epimyum (sensu Crezée 1975) measuring 3 µm in thickness. The cilia on the dorsal side are longer (10 µm) than the cilia on the ventral side (7 µm). The sensory cilia measure 15 µm in length.

The circular muscles are positioned beneath the epimyum, the longitudinal muscles more distally. Dorso­ventral muscles are scattered through the whole body, with the highest abundance in the anterior portion of the body and around the male copulatory organ. All muscles measure approximately 3 µm in diameter ( Figs. 2 B, C).

The nervous system consists of an insunken brain that lies around the statocyst, six longitudinal nerve cords, and a subepidermal nerve net. Four of the longitudinal nerve cords are easy to observe due to their displacement of symbiotic algae ( Fig. 1 A). The most laterally­positioned nerve cords can be clearly seen in sectioned material (data not shown).

Several different types of gland cells are present. The cell bodies of gland cells lie in the parenchyma. The most obvious glands, rhabdoid glands, are present on the dorsal side. Each cell contains up to 70 orange­red rods that are 8 µm in length and 1 µm in diameter. Hatchlings also possess additional white coloured rhabdoids.

A second type of gland cell composes the frontal organ. The frontal glands open to the outside through the anterio­dorsally positioned frontal pore, and contain a large number of vesicles that stain basophilic. Usually situated anterio­frontally to the brain, the frontal glands occasionally pass through the brain.

A third type of gland cell was observed at the lateral margins of S. smaragdina . Merocrine gland cells, with vesicles attached to membranes, were observed by TEM. The cells were innervated by the most laterally positioned longitudinal nerve cords. In Symsagittifera corsicae Gschwentner et al., 2002 these same glands are also present in the same distribution pattern (unpublished data). The behavior of S. smaragdina , along with our observations of the appearance and distribution of the merocrine glands, leads us to hypothesize that the cells have a function related to adhering and gliding; similar to that of terrestrial snails.

Other gland cells associated with the copulatory organs are discussed below ( Figs. 2 B, C).

The sagittocytes occur ventrally within the parenchyma. They are found in the area from the female opening to the posterior end of the animal. They contain up to three sagittocysts, each sagittocyst lying in a separate vesicle. The distal part of the sagittocyte containing a sagittocyst can be wrapped by a muscle cell. The sagittocysts are delimited to the ventral side from the female opening on posterior ( Figs. 2 A, B, C).

Abbrevations: as algal symbiont, b bursa seminalis, bc bursal canal, bn bursal nozzle, bnc bursal nozzle cell, cc covering cell, cg cyanophilic gland cell, cm circular muscle, cs central syncytium, ds donor spermatozoa, ff female follicle, lm longitudinal muscle, lsg large sagittocyst, m mouth, ma male antrum, mf male follicles, o oocyte, ofo opening of frontal organ, pg penial gland, pm parenchymal muscle, rg rhabdoid gland, sa sorting apparatus, sge sagittocyte, sp spermatozoa, ssg small sagittocyst, st statocyst, sv seminal vesicle, v vagina

There are two size classes of sagittocysts. Large ones measure 38 µm in length and 4 µm in diameter and small ones measure 8 µm in length and 1 µm in diameter ( Fig. 2 B). The small sagittocysts lie around the male opening up to the vagina and are also wrapped by a muscle mantle.

Sagittocysts consist of a concentrically lamellated cortex about 3.5–4 µm in diameter surrounding a central filament. The central filament is 1µm thick and has a rhomboid shape. Between the muscle mantle and the sagittocyst cytoplasmatic organelles of the sagittocyte are visible ( Fig. 3 A).

The symbiotic algae measure 8–12 µm in diameter ( Figs. 2 B, C). They are distributed all over the body on the dorsal and ventral side. Typically they contain one or two pyrenoids, a chloroplast and a nucleus. There is no rigid plasma membrane enclosing them.

The mouth is situated slightly in front of the midbody and opens directly into the digestive syncytium. The mouth opening is a small invagination with an epimyum up to 15 µm in thickness and lacking any special musculature.

During feeding, individuals of S. smaragdina swim forward and transform their frontal region into a basket shape that is used to capture prey. Once contained within the basket, the prey is forced into the digestive syncytium by muscular contractions of the whole body. In the laboratory, specimens of M. lignano fed to S. smaragdina could be seen moving in the digestive syncytium for several minutes after ingestion. One day after feeding, a brownish vacuole appears in the syncytium before being extruded through the mouth.

Immature specimens of S. smaragdina fed on one specimen of M. lignano each day for five days became fully mature after 10 days. Male sexual organs were the first to develop. The male follicles, 50–70 µm in diameter, arise more laterally and ventrally than the female follicles, which consist of one developing oocyte and three abortive eggs ( Fig. 2 A). Early spermatogonia have a relatively small nucleolus (stained intensively violet) and few organelles. Mitotic spindles appear during spermatogenesis. The highest number of observed mitotic spindles in one follicle was 16, suggesting that a male follicle consists of a minimum of 32 spermatozoa. Spermatozoa measure 270 µm in average. The spermatozoa have a small nose (20 µm), a middle part containing granules (100 µm) and a tail (150 µm) (data not shown). During development the two cilia are incorporated. In early stages of developing spermatozoa vestigial grooves are visible. In all cases the axonemes lack central microtubules. The axial microtubules are visible in cross and longitudinal sections ( Fig. 3 B, C).

At the level of the mouth the male follicles transition into strings of sperm passing posterior and terminating at the male copulatory organ. The male copulatory organ consists of an unciliated seminal vesicle, which contains spermatozoa as well as the distal parts of several prostate gland cells (up to 150 µm long). Approximately 12 cyanophilic gland cells are positioned around the genital pore in a region of thick epimyum (10 µm) ( Fig. 2 C). The male antrum is short and ciliated.

Abbrevations: em external matrix, m muscle mantle, n nucleus, r refractile body, s sagittocyte.

The female follicles originate more centrally than the male follicles ( Fig. 2 A). The female follicles have a maximum of four cells per follicle, the nuclei of which are larger than those of the male follicles. Early stages consist of four identical cells that develop into one oocyte and three abortive eggs. Some abortive eggs appear to incorporate symbionts. During development the abortive eggs are assimilated, and the oocyte grows to a size of 200 µm, with a nucleus of 50 µm and a nucleolus 15 µm in diameter. The remainder of the oocyte is filled with yolk. At about the second mitotic division, the dark remains of the nuclei of the abortive eggs can be observed in the eggshell (data not shown).

The vagina is short, ciliated, and surrounded by a few cyanophilic gland cells identical to the penial gland cells surrounding the male pore. The vagina leads to a seminal bursa with an attached bursal nozzle measuring 125 µm in length, and having a slight ventroanterior curve. Three types of cells could be distinguished in the bursal nozzle: ringshaped bursal nozzle cells, gap cells, positioned between the bursal nozzle cells; and covering cells, which, by TEM, can be seen to coat the bursal nozzle ( Fig. 2 B). The sperm within the bursa seminalis was highly ordered in all specimens we examined; with the tails directed toward the proximal opening of the bursal nozzle. At the proximal end of the bursal nozzle we observed approximately 15 cells comprising the sorting apparatus (sensu Brüggemann 1985). These cells are club­shaped, with slender portions that extend into the bursal canal.

Spermatozoans were found in the digestive syncytium of a sectioned species, suggesting S. smaragdina to have sucking behaviour like M. lignano ( Schärer 2004) .

Copulation and egg laying were not observed, but we did find egg masses containing an average of 28– 34 eggs and held together with an unidentified secretion. The egg­balls, measuring 1 mm in diameter, were typically glued to a few sand grains. The embryos we observed were in advanced developmental stages and consisted of hundreds of cells. Under culturing conditions the juveniles hatch approximately four days after egg laying. The juveniles are approximately 150 µm long, transparent, lacking endosymbionts, and possessing about 10 orange rhabdoids, a few white rhabdoids, and a well­established frontal organ. Three types of vacuoles are distinguishable in the hatchlings: one, containing bacteria (suggestive of heterotrophy), and two other types, containing differentsized barbell shaped rods (granules). The first contained ovoid shaped short rods 8–10 µm in length, while the second one contained rods 30 µm in length. We hypothesize that the short vacuoles may be a degraded form of the long vacuoles.

Figures

FIGURE 1. Photomicrographs of Symsagittifera smaragdina sp. nov. A. Slightly squeezed adult animal. Scale bar: 250 µm, arrow points to bursa with bursal nozzle and bursa seminalis filled with donor spermatozoa, arrowhead points to statocyst, double arrowhead points to glandular penis. B. Male copulatory organ. Scale bar: 20 µm, arrow points to penial gland cells, arrowhead points to sagittocyst in sagittocyte, double arrowhead points to sagittocyst wrapped by muscle mantle. C. Female copulatory organ. Scale bar: 20 µm, arrow points to bursal nozzle, arrowhead points to sorting apparatus.

FIGURE 2. Reconstructions of Symsagittifera smaragdina sp. nov. A. Sagittal reconstruction of whole organism. Note that follicles are projected on this plane. Scale bar: 250 µm. B. Reconstruction of female copulatory organ. Scale bar: 20 µm. C. Reconstruction of male copulatory organ. Scale bar: 20 µm.

FIGURE 3. Photomicrographs of Symsagittifera smaragdina sp. nov. A. Cross section of sagittocyst and surrounding muscle mantle. Scale bar: 2.5 µm, white arrow points to mitochondrium, white arrowhead points to the central filament. B. Longitudinal section of a spermatid. Scale bar: 0.5 µm, arrows point to axial microtubules, arrowheads point to axonemes. C. Cross section of a spermatid. Scale bar: 0.5 µm, arrows point to axial microtubules, arrowhead points to axoneme.