Acoels may be kept in small dishes in the laboratory for 

 hours, days, or even months. In plain seawater many species will 

 live for a day or so. However, adding a bit of substratum may 

 provide enough food for them to live much longer, and when 

 food preferences are known they may be maintained through at 

 least part of a life cycle. Eggs are often laid by mature aduhs 

 during the first few days in dishes in the laboratory and young 

 hatched from such a "culture" may survive for a time even 

 without further feeding. Apelt (1969) maintained several species 

 by supplying cultured diatoms to the animals and was thus able 

 to study the complete life cycles. 



For the easiest identification by use of the following key, it is 

 best to begin with living animals. In some cases fixation and 

 perhaps sectioning will be necessary after the living animals have 

 been observed. Where only fixed material is available, whole 

 mounts are of some help but in most cases sectioning is 

 necessary. 



Observation of Hving animals is done in a drop of water under 

 a coverslip with the amount of water adjusted so that the 

 coverslip just slightly flattens the acoel, thus holding the animal 

 quiet and also flattening the body to achieve greater 

 transparency. A narcotic such as MgCU may be added to the 

 drop of water, but the difference between achieving just strong 

 enough a solution of the narcotic to quiet the animal and too 

 strong a solution which kills it and causes it to disintegrate is 

 very narrow. Such treatment is difficult to use with acoels. Flat- 

 tening of the animal may also be achieved by the use of a 

 specially built "rotocompressor" such as Heunert and Uhlig 

 (1966) described but, so far as I am aware, these are not 

 available commercially. The larger structures referred to in the 

 key and many of the finer details of organs such as the seminal 

 bursa may be observed by study of living animals with ordinary 

 low power (ICX) x ) or high dry (430 x ) magnification. The use of 

 phase contrast or interference contrast is best for studying these 

 transparent animals, but is not essential. 



Fixing of materials for further examination may be done with 

 any of the standard histological fixatives. The one most com- 

 monly used, especially in field work, is hot or cold Bouin's since 

 specimens may be left in this for an indefinite period of time. 

 Fixation with this solution, although not considered as good for 

 cytological details, is quite satisfactory for routine identifica- 

 tion. Animals can be picked up in a finely drawn-out medicine 

 dropper with as httle water as possible and dropped directly into 

 the fixative. For animals as small as most species of these two 

 orders, the fixation is almost instantaneous, and there is 

 relatively httle contraction or shrinking of the specimens. 

 Specimens fixed in Formalin with other animals, plant material, 

 or both may be used, but the fixation is poor and often 

 specimens are distorted or broken. 



Whole mounts may be stained in Borax Carmine which is pro- 

 bably best for museum specimens since it does not fade as much 

 as many of the hematoxyUns. However, I have had better dif- 

 ferentiation with a trichrome stain (stock solution: 

 Chromotrope 2R, 0.6 g; Fastgreen FCF, 0.3 g; phosphotungstic 

 acid, 0.7 g; acetic acid, 1.0 ml; distilled water 100.0 ml. See 

 Horen 1957), if it is used in a very dilute solution. For this, 

 specimens which have been fixed in Bouin's (10-20 min) should 

 be washed (several changes for about 1 h) in 70% alcohol. After 

 washing they are left in the dilute stain overnight, then 

 dehydrated and mounted. For the best whole mounts, animals 

 should be fixed by allowing the Bouin's to flow over the animal 

 held slightly flattened under a coverslip. 



For sections, any of the standard histological methods may be 

 used. Imbedding in paraffin and sections 6-10 fi m thick has 

 proved satisfactory. Various stains may be used, but since the 

 structure of acoels depends almost entirely on the structure of 

 individual cells, the most satisfactory stains will be those which 

 bring out as clearly as possible the nuclei and shape of cells and 

 muscle fibers. For this the old standby, a combination of iron 

 hematoxylin with a counterstain such as eosin or orange G, is 

 still the most reliable. Mallory's triple stain is fairly good, but 

 many of the stains for special tissues used in histology are not 

 effective since acoels do not have such specialized tissues. The 

 use of electron microscopy is producing much needed informa- 

 tion about these animals but cannot be used primarily for iden- 

 tification of species. 



GLOSSARY 



Terms are defined here as used in this key; included are some 

 equivalent terms common in the literature. Terminology used 

 for reproductive organs has varied considerably in the past and 

 definitions given here are an attempt to render in English the 

 most recent usageofDOrjes (1968a), Antonius(1968), and other 

 European workers. 



Accessory organs Organs which aid in reproduction as acces- 

 sories to the ovary and testis. 



Adenodactyl A bundle of fine cuticular needles lying with 

 inner ends in the wall of the male antrum and with points 

 projecting into the antrum; the inner ends are associated with 

 gland cells. Functions as a stimulatory ("reizorgan") or 

 adhesive organ; compare "prostatoid organ!' 



Antrum See "female antrum," "male antrum" (in some 

 authors "atrium" is used instead of "antrum." 



Archipharynx A type of pharynx unique to the family Nadi- 

 nidae; consists of a short ciliated outer section and long, 

 funnel-shaped inner part; the wall of the outer section has 

 muscle fibers which are extensions of those of the outer body 

 wall; the wall of the inner section has an added inner layer of 

 very heavy circular muscles; a sphincter muscle separates the 

 two parts. 



Atrium See "common genital atrium!' 



Bursal appendage An appendage on the seminal bursa; see 

 "spermatic duct." 



Bursal nozzle A cuticularized structure associated with the 

 seminal bursa or with groups of stored sperm. 



Bursal sphincter See "spermatic duct." 



Bursa seminatis Same as seminal bursa. 



Central parenchyma See "parenchyma." 



Common genital atrium Passage leading from the exterior to 

 both male and female reproductive organs. 



Common genital pore Opening from the exterior to both 

 female and male reproductive organs; may be associated with 

 a common genital atrium or the male and female parts may 

 open directly from it. 



Common germinal center The site in some species where the 

 early stages of both eggs and sperm are intermingled or lie 

 close together. 



Ductus spermaticus Same as spermatic duct. 



False seminal vesicle See "seminal vesicle." 



Female antrum Passage leading to the female organs which is 

 considered to be an inpocketing of the outer body wall as 



