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SNSAAT Se wuUuE! , pd iat tas of WUT yay rree eres. NAR =< 997 te; | 2b am TL eee thy rit * ve o aL | Oe panne” ad Tt th ywWerge eM © = = eo ee .2 o F, nae ST Ma ta Ue CO Paw”. | SRN tA ¥ Fay", @eae Swe es Oe yeeew™ 5 . eer | ete > We y My ee tw Se. Paty y | yy 5 ain j Vy: a pat ere, * yy 2 oN LLP) TE Never tM ra athygugltdil es i en allt v< By / ee wy, ADA jp AALS bad want supe UE ot 7 Wedd: s A ¢ AL ~ 8 ae /s iS a = y Ww Bee Sree ses , AAS yes I WW *@ ie = Wey af? oe. di SOLU esse tt ess delay Ses ; Ma adil” aad Adsl 1h ile rr riih is © wy “teh, w va gly Meisner = ta. hed ] o+ eit it. ae vy ~~ = Uvew te... Wu , 4 Saicce Pet hd tok PoP led ooh tela itech ttl MRA AAR beh Waive ELT TTT ete we ug Ww é.. Lion anes WASHINGTON, D. C. PUBLISHED BY THE CARNEGIE INSTITUTION OF WASHINGTON 1919 ~ DEPARTMENT OF MARINE BIOLOGY AA OF THE CARNEGIE INSTITUTION OF WASHINGTON ALFRED G. MAYOR, Direcror PAPERS, FROM THE DEPARTMENT OF MARINE BIOLOGY OF THE CARNEGIE INSTITUTION OF WASHINGTON VOLUME XIII WASHINGTON, D. C. PUBLISHED BY THE CARNEGIE INSTITUTION OF WASHINGTON 1919 CARNEGIE INSTITUTION OF WASHINGTON PuBLicaTion No. 281 PRESS OF GIBSON BROTHERS, INC. WASHINGTON, D. C. CONTENTS. I. Giand-cells of Internal Secretion in the Spinal Cord of the Skates. By Cart Caskey SPRIBNE, OS plates ANG a NEULCH ooo oc cS «sis o's «s,s Gemeis ooh stn Ben one we Beir PPRRIRER TONER os 2 Sings, ot eee YAR Sale are Soe a> Zo Se oe ee one Mone ees it MITRE te eee Ne oe ican Oi oieisecaete es oe ae ae RN aR MEARNS ps orig te ayes Pte ets STE RECUR © aries BINNS > SE ae ee eS (SSIS RE) ELT EN Ry Lea ee eS 2 at Ra A ns Ac Se CCRT PIV EYE Bea maacicis oitisicpav paidigee nee co oe Rssine a LE aes aoe ee ee Ieee AO CLIN EI MEU ONIN rat ess ih rence kis diese bas ate ease Ses a On ee Phagocyte Hypothesis and Vital Staining Experiments...................... Chemical Nature of the Granules. . 3.505256). oe ns coo ca behav oe te ene Electrical Stimulation of the Spinal Cord.................. 0.0. cc cece cece Pilocarpine Stimulation of the Spinal Cord..............0...cccceeecccceee Atropine Stimulation of the Spinal Cord................. ccc cccceccccccecs Discussion of Results from Spinal-Cord Stimulation........................ Piece of Amputation Ot the: Uae: oo os on. cs we, a1s.: > dain s eked oe ode eR GION 505: Sys dete scare 28s, aah een oss Goth a ReMAS came ute ocean nae Pipes tied Ol ENV ES MeALION::, 65.55... oe acs 1 25k: he seo. We av co coe ee oe oe SLOT? 771s 1g Zep PaO el gto SRR ry i= NE Sri ea TR ry ental WR chet ol arte. lI. Structure and Ejaculation of the Spermatophores of Octopus americana. By CIEMAN Ay Dew, Tampines ores oo ee ese crs coihs Soa ass c as ete PENNE os On Sisieas oh rcaean ni AD a arnia oe Sluice e ot hi oe noe mere eee ae tee ae iD SCE FT 1 (eR le aa A a Ty Era a rl Si Apes Meee OO Tg cs lr as are ln Haney cia che SS NASI Hale Ss SPO ob are alee Bee Ill. Distribution of the Littoral Echinoderms of the West Indies. By Husert PARMEANKACNCAED YS, OME ER 267 Sst ccs) 5 sont « two ee atecaiwia s Ba SanhE Bee OPEN GSI Bic 3), oS) 2 (oa Pa a HPRMOIGeL | eet RMON et Pe i SG NE Soe yal, stolons Hacisbeic eon IJ. Littoral Echinoderm Fauna of West Indian Islands and Adjacent Regions. 12 CC) 9: CS a im ea IA ah C8 a = ra ee et EEO RAS soos 5s dice ete ce ieee SAG On G2 ie aie i ee RA AARMREN Shc oar = Gin SERS SIS Ook ahah APRA eh soe ZOE RUNRer aE ees MRT tS Tis asia Wucnhie eleva oa te ER saree ea eee MSC ITER ey Sigh Sy. die sp. 3 dian stolons OS Re eh ee SESS AE AP ISL TAIL GIONY «55,9 Sis ais.s d a-s/os SY ors beeen oes wake Rees Emp E LUST OTe EAGER oid 5 <.5ic) a's so Retn sae ps6 See Atle BOLI Ree IV Contents. PAGE. IV. Further Studies on the Chemistry of Light Production in Luminous Organisms. By E, WNEWION HARVIY: obo pie peeem cei ircaas o Sac cc oly eerene'e ieaiels oe 75-110 I. Reversibility of the Photogenic Reaction in Cypridina.................. 77-86 II. Chemical Nature of Cypridina Luciferin and Cypridina Luciferase....... 87-103 Preparation.or Matertall 52)... o inal v Wal oh Pst a eS A n y : fe i 5 Lai ; : haa a Pi . 2 ih es a as ; : ; 7 ‘hy ae i van eM a is on a) - sho a om te wx a - ve ovis ae: a es ae , - We be 2 7 Yt eee ape Y Bi ow - paren ‘ vile ee at es as ms oA. ie n i a ike to SPEIDEL a x3 post.h, . PLATE 1 PLATE 2 SPEIDEL SPEIDEL PLATE 3 PLATE 4 SPEIDEL 15 SPEIDEL PEATE 5 SPEIDEC PLATE 6 PLATE 7 SPEIDEL — + (nt. sp.c A SPEIDEL 26 21 29 29 30 a4 PLATE 8 SD Ne | PLATE 9 SPEIDEL ae ook Me THE STRUCTURE AND EJACULATION OF THE SPERMATO- PHORES OF OCTOPUS AMERICANA. By GILMAN A. DREW, Marine Biological Laboratory, Woods Hole. Three Plates 33 o } eaten A ; 7 : _ ma of ) 7 7 a i. ian oo > : n . : 7 a * - \) 7 : 1 BA ' ; 8 2 ; ca i . rt 7 nt) iv: ie ' cn 3 ~ : a’ & oe : reat . os : ea ane , PON GTS a 7 i 7 _— ae. ; ty er - ay os : e- 7 . : ‘te : oa - —o 200". tL > ee a i ol _ nt sy vet. ; vin? = - as 5 — _ - Ai re, - Pine T None LS Ae Le - : ; on ' - ‘yay ty U : ms ie oF Pa i ee ae aL av ' oA ww 7 a ai : ; — - 7 > ; i : : S , e 7 ro ut es j a Det : 7 - : an ’ . , ‘ = ‘ 7 , ' 7 - a4 S 7 _ : Ts a) oa i -_ Cer -2..*e0 ae i : o = a _ i ae ae = ‘Gc Y Rane wi _¢ api, 0A 7 7 = q a - oe ean ~~ : 7 - as by) - my ff wa f ; : i nF) moe - - ; 7 : — n ae . 7 - “- : Dt ; r - i i) a fo af es ae im pr “ar Rieti Fi , ys e: wee é 0 : es ne ce Unit Po ir a Fy —) Ys _ rr ee HN vy oe ne dc ; co rs iss : en a ") —_ - - 7 ae en > ° is ’ - ao ic ve — aly Jen 3 os i : _ Lp oe c oe. J Lie i= _ aint 6 = i » i) he - 7 : 7 i i 7 7 ' a 7 : ¥ i 7 7 7 = - * = oe iy - a a = ~=> _ ry - - —_ - zi T q » 4 we ry, 5 - as , a ;? =~ i : an 7 7 ad a r Ay @ : Pry) 6 ia i> oa viene =, a = nN ites i” i = j 7 ty - , = a eee - an : a i ra i - f yo 7 -1 = - -* 4) rd : : 7 “2 ; . . ve a C noe ny : ' yey » ‘i a 7 a 7 -s a - ; — ; oe 7 ’ - ' a : mi ‘Sraiy | = ave : 7 ia bah < Joe Da on arn) THE STRUCTURE AND EJACULATION OF THE SPERMATO- PHORES OF OCTOPUS AMERICANA. By Girman A. Drew. It was my privilege to spend several weeks in February and March 1912 at Montego Bay, Jamaica, and to enjoy the privileges of the Carnegie Marine Biological Laboratory established there that year. For several years I had been working at problems relating to the spermatophores of the squid and this gave me an opportunity to study the spermatophores of an octopod. I regret the delay in publishing the results, but the preserved material has kept in very good condi- tion. Advantage was taken of a sojourn of several weeks at Tucson to complete the observations and drawings, and I greatly appreciate the courtesies extended to me by the staff of the Biological Department of the University of Arizona during that period. Octopus is quite abundant in the waters near Montego Bay and as the animals are used as food they are collected by the fishermen and taken to the markets, where they are sold under the name of “‘sea-cats.”? Most of the material used was purchased directly from the fishermen and many of the specimens were alive and vigorous when received. Fishermen frequently get these animals in their baited fish-traps, which somewhat resemble the lobster pots used in northern regions, and they sometimes drag them from crevices in the rock with sharp hooked wires. Immediately upon catching a specimen it is the usual practice to “turn the cap” to keep it quiet. This consists in catching hold of the edge of the mantle near the funnel and forcibly turning it wrong side out. In doing this the connections of the mantle are torn and the viscera are disarranged and usually mutilated. While this serves to “keep the animals quiet” it is not a desirable method for one who wants to study them. By increasing the price paid for unmu- tilated material it soon became possible to get all that were needed, so other methods of collecting did not have to be devised. The spermatophores of these animals are very much larger and not so turgid as those of the squid. They are stored in a spindle-shaped spermatophoric sac that lies along one margin of the spermatophoric organ. Part of the spermatophoric sac extends between the sper- mataphoric organ and the almost globular testis. The testis is inclosed in a membranous capsule and this, together with the vas deferens, spermatophoric organ, and spermatophoric sac, is inclosed in another membranous capsule that adheres to the capsule of the testis but evidently has no communication with it. Through this outer capsule the sexual duct or penis protrudes a short distance. 35 36 Papers from the Marine Biological Laboratory. The outer capsule may be cut and easily slips back, so that the spermatophoric sac, the spermatophoric organ, and the vas deferens are exposed, but all are held together (as by a mesentery) by the wall of the capsule that joins the testis capsule. The inside of the wall of the spermatophoric sac is thrown into a large number of deep longitudinal folds, among which the spermato- phores lie, with their aboral ends toward the penis. As many as 79 spermatophores have been taken from a single spermatophoric sac. Spermatophores of cephalopods are mentioned often by zoological writers, but mostly as mere references. Descriptions and figures when given are usually incomplete or unsatisfactory and show plainly that not much careful study has been made by the authors. Emile G. Racovitza (1894c) has given an excellent account of the structure and ejaculation of the spermatophore of Rossia and I have a rather more extended account of the spermatophores of the squid, Loligo, now in press (1919). In describing the spermatophores of Octopus comparison will be made to the spermatophores of Loligo described in the paper to which reference has just been made and the same system of lettering for the figures will be used. Inasmuch as the octopus spermatophore is simpler than that of the squid and certain structures found in the latter are not present in it, the reader of this paper will find certain peculiarities in naming. In the spermatophores of Octopus there is no inner tunic, and if there is an outer membrane it is so thin that it has not been identified. Nevertheless the terms middle tunic and middle membrane have been retained, although they do not occupy medial positions in respect to other layers. Specimens of spermato- phores removed from the spermatophoric sac and placed immediately in about 10 per cent formalin do not discharge and remain trans- parent and easily studied. Placing them directly into full strength formaldehyde does no harm, but the added strength is not needed. The membranes and tunics are hardened somewhat by this reagent and, when they are to be stained and mounted, somewhat better results can be had if they are not left many hours in the formalin. The chief difficulty is with wrinkling when the membranes are hardened, but this can be overcome by slow diffusion methods. The most successful stain for most purposes has been Ehrlich’s triacid. The stain may be diluted either with water or formalin solution, and it is usually better to use several times as much water as stain. The exact strength does not seem important, but with stronger solutions the spermatophores are stained much quicker. When removed from the stain the spermatophores are washed and placed in 10 per cent formalin for a few minutes and then mounted directly in glycerine jelly. If the membranes have hardened so that wrinkling On the Spermatophores of Octopus Americana. 37 is likely to occur, they may be placed in a diffusion apparatus with glycerine over night and mounted in glycerine jelly the next day. I have found a very simple diffusion method with glycerine is to place the spermatophores in a concave watch-glass filled with formalin. Place this in a shallow stender dish of about the diameter of the watch-glass, fill the space between the two dishes with glycerine and flood with formalin, so that the diffusion may take place over the edge of the watch-glass. Specimens mounted in glycerine jelly will be clearer than those in formalin and may be studied at leisure. The stain fades slowly, but is sufficiently permanent to be effective some months. STRUCTURE. The spermatophores of Octopus differ in size with the size of the individuals from which they are taken. Large ones measure as much as 50 mm. in length, small ones may not be more than two-thirds as long. The spermatophores are slender and taper irregularly from the aboral to the oral end. The sperm mass (fig. 1 sm), which is white and opaque, lies in the aboral end of the spermatophore and occupies about one-third of its length. The ejaculatory apparatus occupies between one-third and one-half of the oral end of the spermatophore and is quite transparent. Between these two portions and extending along the sides of the sperm mass and ejaculatory apparatus is a space occupied by liquid in which is a considerable mass of granular material (fig. 1 z). This material mixes readily with water and it may have an important osmotic property. The bulk of it occupies a position between the sperm mass and ejaculatory apparatus that corresponds to the position of the cement body in the squid, but it evidently has no cementing property and is not inclosed in a special capsule. No cement is needed in this spermatophore, as the sperm are not loaded by the ejaculating spermatophore into a sperm reservoir that has to be stuck into posi- tion, but are evidently introduced directly into the oviducts of the female. Whether there is any homology between these materials in the two forms is not clear. Their similar positions are significant but their functions are evidently entirely different. The spermatophore is turgid and elastic but not nearly so much so as the spermatophore of the squid, which is so turgid that when bent it will, upon release, assume its original shape immediately. The squid spermatophore may even be picked up by one end with forceps and will stick straight out without appreciably bending. The octopus spermatophore may be bent into flowing curves on the bottom of a dish and remain as left, provided none of the curves are abrupt. It is nevertheless under considerable tension, for when the outer cover- ing of a fresh spermatophore is cut, the contents are thrown from 38 Papers from the Marine Biological Laboratory. the cut rapidly. The turgidity, as in the case of the squid spermato- phore, is due to the tough elastic and stretched outer tunic. The outer tunic (figs. 1 and 5 or) is thin and nearly transparent, although faintly amber in color. It is not as colorless as that of the squid and is much thinner. Magnified as shown in figure 1, it is so thin that it is represented by a single line. The fact that, although the spermatophore of Octopus is much larger than that of the squid, the outer tunic is actually thinner, accounts for the difference in turgidity and that in ejaculation Octopus spermatophores are much slower than those of the squid. The outer tunic forms the covering for the spermatophore to the oral extremity. The oral end is covered by the cap (figs. 1 and 2c), which is thinner than the outer tunic, more transparent, and is evi- dently more affected by water. Over the cap is thrown a broad cap-thread (cr) that adheres rather strongly to one side of the spermatophore and extends aborally for a distance considerably greater than the length of the ejaculatory apparatus. The other end of the cap thread seems always to be free from the spermatophore, but is folded well over the extremity of the cap, frequently back to the end of the ejaculatory apparatus, as a broad, striated bandage. Pulling the cap thread seems to start ejaculation of the fresh sper- matophore. How it is used to start normal ejaculation is not known, but it seems probable that the thread is pulled in some way. The spermatophore leaves the penis aboral end first and it seems reasonable to suppose it reverses ends when started down the groove of the hec- tocotylized arm. It is not known how it reaches this groove, but the groove has no connection with the penis and it seems probable that the spermatophore must pass through the funnel in being transferred to the groove. The mechanics of the transfer is not known, but it is probable that the thread is pulled during the process. A spermato- phore placed in sea-water will usually ejaculate rather promptly even when the cap thread is not pulled intentionally. This may be due entirely to osmotic changes and dissolving effects of the water or it may be that the thread is always disturbed enough by removal from the spermatophoric sac to weaken the cap end. Inside the aboral portion of the outer tunic, corresponding pretty closely with the length of the sperm mass, is a closely adhering mem- brane, the middle tunic (fig. 1 mr). This is not nearly as thick and conspicuous as the middle tunic of the spermatophore of the squid and does not show a granular structure. It does not line the extreme aboral end of the outer tunic and gradually thins out and disappears just beyond the oral end of the sperm mass. The middle tunic evidently swells rapidly in water, but it does not have as great osmotic properties as the middle tunic of the spermato- On the Spermatophores of Octopus Americana. 39 phore of the squid. The difference in the strength and stretch of the outer tunics and in the osmotic properties of the middle tunics is doubt- less responsible for the difference in rapidity of ejaculation in the two forms. The whole process is usually complete in less than 10 seconds in the squid and may occupy from 1} to 3 minutes in Octopus. The liquid that occupies the space between the sperm mass and the ejaculatory apparatus is continued along the sides of these two portions between them and the outer coverings, so there is no adhesion between them and the outer tunic or middle tunic except where the ejaculatory apparatus is permanently attached to the outer tunic at the oral extremity. The liquid that occupies this space is not so absolutely transparent as it is in the squid spermatophore, but is noticeably granular throughout. It is much more granular in the space between the sperm mass and ejaculatory apparatus, but nowhere is it entirely free from granules. Along the sides of the sperm mass the space occupied by the liquid is always distinct (fig. 1 st). Along the sides of the ejaculatory appa- ratus it is frequently hard to find except in the grooves between the loops of the spiral into which a portion of it is thrown (fig. 3 st). Ejaculation shows, however, that there are no adhesions at any point and that the liquid serves as a lubricant and to transmit pressure during the act. The sperm mass (fig. 1 sm) consists of a thread of sperm, mixed with a somewhat granular viscid secretion, that is wound into a cylindrical spiral. The separate loops, unlike those of the squid sperm mass, are distinct with the outlines rounded. The loops are not compressed against each other enough to flatten their adjacent sides very much and the mass is not inclosed in an inner tunic. That the inner tunic is completely absent in this form is indicated by the fact that when ejaculation begins the sperm thread begins to uncoil and straighten (fig. 16 sm). This would not be possible if the sperm mass were inclosed in a definite tunic, as is the case in the squid. The sperm thread is not of entirely even diameter throughout and the coiling is not entirely regular, but the irregularities are only imperfections and have no functional significance. The sperm mass is usually referred to by authors as the sperm rope. In using this term it should be borne in mind that unlike a rope it con- sists of but a single coiled strand. The ejaculatory apparatus, while easily compared with that of the squid, differs in the absence of the inner tunic and outer membrane and in its end relationship, for this form has no cement body. It is perhaps questionable whether there is an outer membrane, but I have not been able to distinguish one. The middle membrane is thick, evidently very pliable, and (as in the case of the squid) is composed of many thin longitudinal layers that are presumably due to the 40 Papers from the Marine Biological Laboratory. winding of a thin sheet of material around its longitudinal axis. While the process of formation of the octopus spermatophore has not been observed, the structure of the spermatophoric organ indicates that, as in the squid, the forming spermatophore is kept rotating on its longitudinal axis while a thin sheet of secretion is supplied and wound on, like the successive layers of fabric in a rubber hose. During ejaculation the middle membrane is shown to be very pliable, evidently much more so than in the squid. Orally, the middle membrane is firmly united to the outer tunic, where this tunic is joined by the cap (fig. 2mm’). Toward the aboral ex- tremity of the ejaculatory apparatus the middle tunic becomes thinner and almost if not quite disappears before the extremity is reached. Inside the middle membrane is the inner membrane. This is so thin that in all the figures it has been shown as a single line, but it is always distinguishable under a moderately high-power lens. This is also firmly united to the outer tunic at its oral extremity (fig. 2 mm) and extends throughout the length of the ejaculatory apparatus. Inside the inner membrane is a spirally coiled filament that is present but hard to distinguish near the oral end of the ejaculatory apparatus and becomes very much more prominent toward the aboral end of the ejaculatory apparatus (fig. 1 sr). This filament seems to be united to the inner membrane and has the appearance of an ornament on the inner membrane. It is evidently very flexible and does not break up into small fragments during ejaculation, as in the squid spermatophore. While it possesses elasticity and probably aids in keeping the ejaculatory apparatus from collapsing, there is no evidence that it possesses any spring properties. The diameter of the coil differs greatly. Toward the aboral end of the ejaculatory appa- ratus, where the middle membrane thins and probably disappears, the diameter of the coil of the spiral filament is greatly increased. It then narrows and near its aboral end becomes thin and relatively weak. This arrangement is significant in the act of ejaculation. The lumen of the ejaculatory apparatus inside the spiral filament is filled with a viscid material that adheres to the spiral filament and inner membrane and becomes spread over the outside in an irregular manner during ejaculation (figs. 2,12,and 15 uc). It is not so liquid as in the squid spermatophore and does not form a definite rod-like plug like that described by Racovitza (1894 c) for Rossia. There is frequently evidence that the core of this secretion is much more liquid than the outer parts (fig. 2 Hc’). Extending into the cap of the sper- matophore from this region is an indefinite, hazy appearance evidently due to the escape of material from the lumen. This appearance never involves the entire width of the lumen but only the central part. The remainder of the material is evidently responsible for forming the papille-like ornaments over the outside of the evaginating tube that, On the Spermatophores of Octopus Americana. 41 as evagination continues, becomes a more or less definite layer, often with lump-like masses that cover the outside (figs. 2, 7, 12, and 15). About one-third of that portion of the ejaculatory apparatus nearest the oral end of the spermatophore is spirally coiled. There are from 14 to 20 distinct loopsin this coil. The loops are pressed close together, but there is a distinct groove between them that is filled with the granular liquid. There is considerable difference in spermatophores as to how near the oral end the coiling begins. Frequently (as shown in figure 1) there are a number of loose coils near the oral end, but this is not always the case. When ejaculation begins the loops near the oral end are first to straighten out. EJACULATION. When a spermatophore is placed in sea-water and the cap thread is pulled, ejaculation begins immediately. Ejaculation will begin in spermatophores placed in sea-water without pulling the thread, but the process is delayed. This is doubtless due to largely increased tension due to osmosis and may be aided by softening of the cap. Ejaculation will also begin in the air, probably because drying shrinks the outer tunic and thus increases the tension, but this is of course not normal. It is easiest to keep material for study in a solution of calcium chloride and to remove specimens individually into sea-water when needed for study. Specimens will keep in good condition for study in this solution for some hours. Many other solutions that reduce the osmotic tension in the spermatophores have bad effects on the mem- branes so that normal ejaculation is interfered with when they are returned to sea-water. It is probable that normal ejaculation is started by pulling the cap thread. How this is done has not been ascertained. The spermato- phores are stored in the spermatophoric sac with their aboral ends pointing outward—that is, toward the opening of the penis. Octopus has a hectocotylized arm that has a groove passing along one margin from the base to the tip. There is every reason to believe that the contents of the spermatophores, if not the spermatophores themselves, are passed along this groove from the base to the tip. Racovitza (1894 a and b) has described how in Octopus vulgaris the tip of this arm is inserted into the mantle cavity of the female, and he found by dissection afterward that the oviducts of the female were packed with sperm. In 95 minutes during which the act of copulation continued no movements of the animals were observed except slight movements of the hectocotylized arm. Although the actual passage of the sperm could not be seen, as the arms are very opaque, it is evident that they are passed down this groove. 42 Papers from the Marine Biological Laboratory. Racovitza speaks of finding the “spermatophores” in the oviducts, but later he modifies this by stating that the examination of the spermatophores showed their sheaths had disappeared and that only the part evaginated persisted. He states further that they had been placed by the orifices of the oviducts and in exploding introduced the spermatic reservoirs into the canal. The statement concerning the condition of the sperm in the oviducts corresponds with my observations made on a specimen at Montego Bay, in which the oviducts were filled except that there were no reservoirs. The sperm were free. There were no parts of the tunics or ejaculatory apparatus found. I do not know upon what evidence Racovitza concludes that the spermatophores were placed by the orifices of the oviducts and in exploding introduced the spermatic reservoirs into the canal. Very possibly it was surmise based on known conditions in other forms. There is no known provision for sticking the spermatophores to the body of the female and there is no spermatic reservoir formed in ejaculation in Octopus. It seems probable either that the ejaculating spermatophore is held by the tip of the hectocotylized arm in position for it to introduce the sperm mass into an oviduct as it ejaculates, or that the spermatophore never enters the groove in the hectocotylized arm, but ejaculates into it, and the sperm mass only passes on to the tip and thus into the oviduct. In either case the spermatophores evidently have to reverse ends in passing from the penis to the groove in the hectocotylized arm, for the oral ejaculating end must be directed toward the oviduct while it is functioning. What mechanism is used in transferring the sper- matophore from the penis to the groove is not known, but it is probable that during the process the thread is pulled and ejaculation begins. Ejaculation is very deliberate, occupying from 13 to 3 minutes, and the sperm thread is all unwound, so that it leaves the spermatophore as along narrow thread. Neither of these arrangements seems to be adapted to packing the oviducts directly from the ejaculating sper- matophores, but they are nicely adjusted to delivering the sperm thread to the groove in the hectocotylized arm. I therefore doubt somewhat whether the spermatophore ever enters the groove. It seems more probable that it is retained between the arms near the entrance to the groove and that the sperm mass only, in the form of the uncoiled thread, passes down the groove to the tip of the hecto- cotylized arm. The tip of this arm seems to be modified for intro- duction into the oviducts and by it the sperm would be conducted into position. The cap thread, as stated in describing the structure of the sper- matophore, forms a broad bandage, one end of which is quite firmly stuck to one side of the spermatophore, while the other end is free but passes over the end of the cap. The free end of the thread is On the Spermatophores of Octopus Americana. 43 among the other spermatophores in the spermatophoric sac, and as they move aboral end first toward the penis these threads are in position to be pulled when the spermatophores leave the penis. Arrangement near the base of the penis indicates that a single spermatophore is ejected at a time. When the cap thread is pulled the cap immediately begins to swell and elongate (fig. 7), and the evagination of the ejaculatory apparatus begins. The cap soon goes to pieces (fig. 8) and evagination continues. The middle membrane is very pliable and is extended far beyond the point of folding back (figs. 7, 10, 11, and 14 pr). The hyaline core is composed of two parts, a central core (Hc’) that is quite liquid mixes with the water and disappears, and an outer part (Hc) that sticks to the inner membrane forming small projections and lumps (figs. 7, 12, and 15) that change form considerably as ejaculation is continued. At the beginning of evagination the spiral coiling of the ejaculatory apparatus begins to straighten out. The loops nearest the cap are affected first, and little change is noticeable in the positions of other parts in the spermatophore until all these coils have been straightened (figs. 11 and 12). The twist in the ejaculatory apparatus is shown again in evagination by the spiral into which the tube is again thrown at the evaginating extremity (figs. 11, 12, and 13). When a position about equivalent to the point 4, figure 1, is reached the act of evagina- tion is slowed down considerably. This is probably due to resistance offered by the spiral filament which here becomes better developed. The slowing and the torsion developed by evaginating the spiral fila- ment result in swelling the evaginating end of the tube, crowding and spirally coiling the aboral end of the ejaculatory apparatus into the swelled portion, and in uncoiling the sperm thread and pressing it forward into the part of the tube that has evaginated (fig. 16). The resistance to evagination becomes greater in the region where the spiral filament is best developed and the swelling of the evagin- ating end is correspondingly increased. This continues until the sperm thread reaches the portion of the ejaculatory apparatus that is still to evaginate and becomes crowded along its sides. When evagination reaches the point where the middle membrane thins and disappears it becomes very much more rapid, the remainder is turned rapidly, and the sperm thread immediately begins to escape through the open evaginated end of the ejaculatory apparatus. It commonly happens (fig. 17) that the ejaculatory apparatus ruptures near its narrowed extremity and that the escape of the sperm thread takes place through this rupture. The escape of the sperm thread is even, not hurried, and continues for a minute or more. It begins while a considerable portion of the aboral end is still closely coiled. The coils open one after another and 44 Papers from the Marine Biological Laboratory. the thread moves continuously on until it is entirely free from the outer case. The thread as it uncoils must have a somewhat similar twisting and this probably accounts for the alternate swellings and constrictions along the thread. This appearance is very much more marked near the coils than toward the free extremity where the twist would have time to adjust itself somewhat. Focussing on the narrow parts shows the places of twisting marked by wrinkles and striations in the mass. The outer tunic does not shrink nearly as much as it does in the squid spermatophore and the middle tunic does not swell nearly as much. Indeed they hardly seem to give evidence of all of the power that is needed for ejaculation. The whole process is very deliberate. It is very possible that the liquid that occupies the space between the tunics and the ejaculatory apparatus and sperm mass has osmotic properties that are important in the process of ejaculation. In the spermatophore before ejaculation much lumpy, granular material is present in this liquid. This seems to become much more liquid as ejaculation continues. It would seem that most of this would be thrown out ahead of the sperm thread, but this is not the case. The end of the sperm thread is crowded in against the ejacula- tory apparatus in such a way that, when this finally evaginates and leaves a free opening to the outside, the sperm thread fills this opening at once and the liquid remains inclosed between it and the evaginated tube. It is not free to escape in quantities until the sperm mass has unwound and the thread of which it was composed has been entirely discharged. Evidently the spermatophores of Octopus and the squid, while built on similar plans, are adapted for quite different actions. It may be of service to call attention to some of the important differences. The squid spermatophore is adapted for very quick service and for filling a reservoir with the entire mass of sperm and sticking it to the body of the female. The contents slowly escape from the reservoir and are stored in a special receptacle, or fertilize the eggs as they leave the oviduct. The octopus spermatophore is adapted for very deliberate service. The sperm mass is not stored in a special reservoir and there is no provision for sticking it to the body of the female. The sperm are introduced directly into the oviducts of the female, where they are stored until the eggs are ready to be laid. The squid spermatophore is accordingly more complicated in structure and is under greater tension than that of Octopus. It has a cement body, which is entirely absent in Octopus. It has an outer membrane and inner tunic that are used in making a sperm reservoir, and has these structures attached to the cement body,so it may be ruptured at the right time. These are absent in Octopus. The sperm thread is also closely packed and bound into a mass that is inclosed in On the Spermatophores of Octopus Americana. 45 the aboral portion of the inner tunic, and the whole mass is moved together. The sperm thread of which it is composed is never uncoiled. This differs entirely in Octopus, where the sperm thread is not closely packed, is not inclosed in any kind of membrane, and uncoils as it is discharged. These questions of structure and speed are of course related to the functions they perform. Squid are free-swimming, active creatures, and copulation is a very rapid act. Octopus are for the most part bottom dwellers and copulation is much more deliberate. The quick machine is more complicated, but the two show very similar structure. Without knowledge of the past history of these animals it is very difficult to arrive at reasons for the formation of such complicated structures as these spermatophores to perform functions where simpler arrangements would seem to do as well. Doubtless if the past history were before us for review we would see clearly why such structures have been developed, but in the absence of this history which seems to be permanently hidden from us, we can be certain only that the complicated machine has been formed and serves its purpose. LITERATURE CITED. The papers given are the only ones to which it has been necessary to refer in this paper. There are many others. Drew, G. A. 1919. Sexual activities of the squid, Loligo pealii (Le Sueur). II. The Sper- matophore; its structure, ejaculation, andformation. (In press). Jour. Morph., vol. 32. Racovitza, Emiin-G. 1894a. Sur l’accouplement des quelques Cephalopods Sepiola rondeletti (Leach), Rossia macrosoma (d. Ch.), et Octopus vulgaris (Lamarck). Comp. Rend. |’Acad. des sci., 118. 1894b. Notes de Biologie. I. Accouplement et fécondation chez l’Octopus vul- garis Lamarck. Arch. d. Zool. Expér. et Gén. (3), 2. 1894c. Notes de Biologie. III. Mceurs et réproduction de la Rossia macrosoma (d. Ch.). Arch. d. Zool., Expér. et Gén. (3), 2. 46 Papers from the Marine Biological Laboratory. EXPLANATION OF FIGURES. The figures were all drawn with the aid of a camera lucida from specimens preserved in formalin and for the most part stained and then mounted in glycerine jelly. Stages necessary for study and drawing were secured by deluging with full strength formaldehyde at the moment required. Large spermatophores before ejaculation may measure 50 mm. in length. The process of ejaculation increases the length to over 100 mm. and to this should be added the sperm thread, which, when uncoiled and ejected, measures at least 150 mm. more. ABBREVIATIONS. Cc, cap. cT, cap-thread. HC, hyaline core; the outer stiff portion of the core only is referred to. Hc’, hyaline core; the inner, more liquid material that mixes with the water and disappears is referred to. MM, middle membrane mm’, middle membrane; the point of attachment to the outer tunic. mt, middle tunic; this extends orally only a little further than the sperm mass. oT, outer tunic. PT, point of turning of the middle membrane; during ejaculation the pliability of membranes is such that this point lags far behind the outermost tip of evagination. SF, spiral filament. SL, space filled with liquid; this space is inclosed by the outer and middle tunics on the outside and extends between these structures and the sperm mass and ejaculatory apparatus; as ejaculation takes place the space continues down the evaginating ejaculatory apparatus. ZM, sperm mass; this consists of a spirally coiled sperm-thread that uncoils during ejaculation. s, granular material in the space, SL. Puate I. 1. Spermatophore as taken from the spermatophoric sac, X 10 diameters. The specimen has been drawn in sections and the connections indicated by dotted lines. Speci- mens just removed from the spermatophoric sacs frequently show the sperm mass entirely filling the aboral end to the tunic. It is common to have specimens in which the ejaculatory apparatus, which extends from point 2 to point 6, is closely coiled nearer the oral opening than was the case in this specimen. The numbers 2, 3, 4, 5, and 6 placed along the side of this drawing indicate the positions of more highly magnified drawings that bear the same numbers. 2. Oral end of the specimen shown by figure 1, X 63 diameters. The position is indicated by the side of figure 1 by the number 2. 3. A portion of the specimens shown by figure 1, X 63 diameters. The position is indicated by the side of figure 1 by the number 3. 4. A portion of the specimen shown by figure 1, X 63 diameters. The position is indicated by the side of figure 1 by the number 4. 5. A portion of the specimen shown by figure 1, X 63 diameters. The position is indicated by the side of figure 1 by the number 5. 6. A portion of the specimen shown by figure 1, X 63 diameters. The position is indicated by the side of figure 1 by the number 6. 7. The oral extremity of a spermatophore at the beginning of ejaculation, X 63 diameters. The cap-thread has been stripped back so that it does not show in this figure. This frequently happens in handling the specimens before they are permanently mounted. Soon after this stage is reached, the cap bursts and the fragments disappear. The cone-like projections on the portion extending into the cap are formed by the outer portions of the hyaline core which becomes spread over the outer surface of the evaginating tube. They settle down and are not as prominent after ejaculation has proceeded some distance. On the Spermatophores of Octopus Americana. 47 Puate II. 8. Oral end of a spermatophore after the cap has broken and disappeared, X 63 diameters. A ridge, which is probably more properly referred to as the margin of the outer tunic, remains behind and for convenience in marking the position is labeled in this and subsequent figures the same as the cap by the letter c. The liquid material of the hyaline core Hc’ is shown being liberated into the water. 9. Oral end of a spermatophore at a slightly more advanced stage in ejaculation, X 14 diameters. 10. Oral end of the spermatophore shown by figure 9, X 63 diameters. It frequently happens, as shown in this figure, that the ridge to which the cap was attached (c) is reflected so that the free edge points backward. This is evidently due to the force exerted on the outer tunic by the attached middle membrane which draws the tunic in somewhat and by the enlargement of the softer membranes immediately upon leaving the confining outer tunic. The wrinkling that is shown near the point of turning of the middle membrane (PT) is pronounced as soon as the turning is well started. It is evidently due to the fact that the middle membrane is made up of a thin sheet wound around the central hyaline core. This allows some coats of the tunic to wrinkle while others are stretched. In this as in other specimens the reflecting of the individual layers can be traced from the point pr to the free evaginating end. They have not been shown in the figures, as they would be confusing. 11. Oral end of a spermatophore at a slightly more advanced stage of ejaculation than the one shown by figure 10, X 24 diameters. As the ejaculatory apparatus uncoils the torsion caused by the evaginating tube throws its extremity into a spiral that becomes longer and more pronounced the more the original coil is straightened. 12. Oral portion of a spermatophore during ejaculation, < 12 diameters. The stage is more advanced than that shown by figure 11. The original spiral coiling of the ejaculatory apparatus has straightened and the torsion that causes the end of the evaginating tube to be thrown in a spiral is accordingly at its greatest. This specimen shows the lumps of material derived from the hyaline core adhering to the evaginated tube more prominently than is usually the case. The numbers 13, 14, and 15, placed along the side of this drawing, indicate the positions of more highly magnified drawings that bear the same numbers. 13. Extremity of the specimen shown by figure 12, X 50 diameters. The position is indi- cated by the side of figure 12 by the number 13. 14. Region of the point of turning of the specimen shown by figure 12, X 50 diameters. The position is indicated by the side of figure 12 by the number 14. 15. A portion of the evaginating ejaculatory apparatus of the specimen shown by figure 12, < 50 diameters. The position is indicated by the side of figure 12 by the number 15. This shows a large mass of the material derived from the hyaline core adhering to the surface. Puate III. 16. A spermatophore that has evaginated to the point where the ejaculatory apparatus retards the evagination until the pressure swells the end that is evaginating, X 10 diameters. The specimen has been drawn in sections and the connections indi- cated by dotted lines. The remaining portion of the ejaculatory apparatus has been thrown into a spiral coil, probably because of the torsion exerted by evagina- tion together with the pressure from behind. The sperm thread that forms the sperm mass has uncoiled for some distance. At a little later stage a considerable portion of the remaining ejaculatory apparatus is evaginated and the sperm thread is pushed down against it. At this time the swelling at the end is much greater than that shown in this figure. When the evagination of the ejaculatory apparatus is complete the sperm thread is set at liberty and immediately begins to push out through the opening. 17. Extremity of the fully evaginated ejaculatory apparatus with the sperm thread escaping, X 24 diameters. In this specimen, as frequently happens, the side of the ejacu- latory apparatus has ruptured near its extremity and the sperm thread is escaping through the rupture. 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THE DISTRIBUTION OF THE LITTORAL ECHINODERMS OF THE WEST INDIES. By HUBERT LYMAN CLARK, Of the Museum of Comparative Zoology, Cambridge, Massachusetts. Three plates. 49 wie rebl rer’ Veen Say Serre ors Tas ae LAA Bie ps belek & 6 * aes 7 . my _ ; Hi , : \ _ ‘ n . THE DISTRIBUTION OF THE LITTORAL ECHINODERMS OF THE WEST INDIES. By Husert LyMan Cuark. INTRODUCTION. Among the various groups of invertebrate animals which swarm on the reefs and along the shores of the West Indies none is more conspicuous than the Echinodermata and few are more abundant or diversified. Wherever conditions are at all favorable for the forma- tion of coral reefs, and in many places where corals scarcely grow at all, echinoderms are found in greater or less abundance, and either by their size or color or both are sure to attract the attention of even a casual observer. In April 1896 I first came into contact with this interesting fauna while I was enjoying the privileges of the Johns Hopkins University’s Marine Laboratory at Port Henderson, Jamaica. The following summer my acquaintance with it was renewed at Port Antonio, Jamaica, under the same auspices. In April 1899 I spent two weeks in Bermuda, most of my collecting being in the vicinity of Bailey Bay, Coney Island, and Castle Harbor. In the fall of 1902 I again visited Port Henderson, Jamaica, and in the spring of 1909 I was at Port Antonio again for a week. In the spring of 1912 I enjoyed the privileges of the Carnegie Institution’s laboratory at Montego Bay, Jamaica, while in 1916, under the same auspices, I had the opportunity of spending five weeks at Pigeon Point, Tobago. Finally, the month of June 1917 was spent at the Tortugas Laboratory of the Carnegie Institution of Washington, where every facility was provided for the collection and study of echinoderms. As a result of these unusual opportunities, I have accumulated a large part of the data presented in this report, my attention having been almost wholly given to the littoral echinoderms. The word littoral is used in the strictest sense, only those species being included which I have myself collected on the reefs or in very shallow water, or for whose occurence there the records are indubitable. As a matter of fact, I have collected nine-tenths of these species. In compiling the list, I have been very arbitrary and have omitted a considerable number of species which might be expected in it. Further reference will be made to these under the respective groups into which the list is divided. The collections of the Museum of Comparative Zoédlogy contain a large amount of West Indian material and this has been freely used and of invaluable assistance in the preparation of this paper. I have also made use of all available publications in search- 51 52 Papers from the Department of Marine Biology. ing for records for the different islands, but no doubt scattered records have been overlooked, while many in which I have lacked confidence are ignored. The purpose of this investigation and report is to see if the dis- tribution of these very littoral species throws any light on the faunistic relations of the various islands. But it should be understood at the start that we have nowhere nearly enough data on which to base any important conclusions. Thus, while the littoral echinoderms of Florida and the Tortugas are quite completely known and those of Jamaica are almost equally well listed, our knowledge of the Cuban fauna is, in this group, very incomplete and nothing whatever is known of the Isle of Pines or of the Cayman Islands. Of the echinoderms occurring on the shores of Porto Rico and the United States Virgin Islands, we are fairly well cognizant, but passing eastward and southward we enter a region of which our knowledge is most superficial until Barbados and Tobago are reached. This report is therefore merely introductory to the subject, but it is hoped that it may serve as a useful beginning. One word is necessary as to the geographical limits of the region here designated as ‘‘West Indies.’”’ It extends from Bermuda on the north and the Tortugas on the west to Tobago in the southeast. Per- haps, strictly speaking, these three extremes do not belong in the region at all, but as their littoral faunas are exceptionally well known it would be absurd to leave them out of account. In concluding this introduction, I wish to express my thanks to those whose encouragement and aid have made my work possible. I desire particularly to record my great and lasting obligations to the late Professor William Keith Brooks, of the Johns Hopkins University, who first opened to me the fascinating field of marine zodlogy; to the late Dr. Alexander Agassiz and to Mr. Samuel Henshaw, of the Museum of Comparative Zoédlogy; and to Dr. Alfred G. Mayor of the Carnegie Institution. At Tobago I had the privilege of the constant companionship and help of my honored colleague, Dr. Th. Mortensen, of Copenhagen; it is a pleasure to acknowledge here my debt to him. Another colleague, Dr. W. K. Fisher, of Leland Stanford Junior University, has put me under obligation by permitting me the use of certain field notes made by him during the summer of 1918 at Barbados and Antigua, where he was a member of the party sent out by the University of Iowa, under Professor C. C. Nutting. I am glad to thank Professor Nutting and Dr. Fisher for permitting me to use these notes. There are many to whom my thanks are due for help in collecting, but I forbear attempting to name them. I must, however, offer my particular thanks to Mr. John W. Mills, chief engineer of the Tortugas laboratory, whose interest and help have been invaluable. Distribution of Littoral Echinoderms of the West Indies. 53 ]. WEST INDIAN LITTORAL ECHINODERMS. Recent echinoderms fall so readily into their five classes, without any disconcerting annectant forms, that it is easiest and most natural to discuss each class separately. COMATULIDA. FEATHER-STARS. At Tobago we found a comatulid, Tropiometra carinata (Lamarck), common in very shallow water in Buccoo Bay and on Buccoo Reef. It is a species of wide distribution on the coasts of southern Africa and Brazil, and reaches its northern limit in deep water (200 to 300 fathoms) off St. Lucia. As a littoral species, its northern limit seems to be at Tobago. The only other littoral comatulids of the West Indies are species of the genus Nemaster, though Mr. A. H. Clark tells me that the little-known Antedon diibenii Bohlsche, of Brazil and St. Thomas, appears “to be from very shallow water.” The remarkable and unique occurrence of Nemaster iowensis (Springer) at the Tortugas ‘Gn water less than 3 feet deep” in 1893, is one of the most curious facts of distribution which the West Indian region affords. Its mystery is only deepened by the apparent occurrence of the same species at Bermuda. Mr. A. H. Clark very kindly permits me to thus note the fact that he has a specimen from that northern point. It is certain that other species of Nemaster occur in the West Indies, as is indicated by material in the United States National Museum, but as yet our knowledge of the genus is too fragmentary to make it of any use in discussing distribution. So Tropiometra is the only comatulid at present of value for this purpose and its occurrence at Tobago dis- tinguishes that island at once from the other West Indies. ASTEROIDEA. SEA-STARS. The littoral sea-stars of the West Indies are not numerous in species or abundant as individuals, and it is only occasionally or under special conditions that a species can be called common. Under favorable conditions, in particular spots, Astropecten duplicatus is very abundant, but these spots are as a rule in water more than 3 fathoms deep. On the other hand, Asterina folium occurs at or just above low-water mark, on particular reefs, in sufficient numbers to warrant calling it common, and the same is sometimes true of young individuals of Linckia guildingii, and at Bermuda it is true of Coscinasterias ten- uispina. At Port Royal, Jamaica, Echinaster sentus is fairly common and Oreaster reticulatus is by no means rare. Verrill reports the latter species as “very common in the Bahamas.” No other sea-stars can be called common in the shallow waters of the West Indies, but Ophidiaster guildingii is widely distributed and is by no means rare on the Tortugas reef-flats and on Buccoo Reef, Tobago. 54 Papers from the Department of Marine Biology. The following 14 species make up the list of littoral sea-stars of the West Indies. All have been taken at or near low-water mark, certainly in less than 3 feet of water. Astropecten articulatus (Say). Asterina minuta Gray. duplicatus Gray. Stegnaster wesseli (Perrier). Luidia alternata (Say). Ophidiaster guildingii Gray. clathrata (Say). Linckia guildingii Gray. senegalensis (Lamarck). Echinaster sentus (Say). Oreaster reticulatus (L.). | spinulosus Verrill. Asterina folium (Liitken). ' Coscinasterias tenuispina (Lamarck). The West Indian species of Astropecten are exceedingly perplexing, although Verrill has recently (1915, Bull. Univ. Iowa, Lab. Nat. Hist. 7, pp. 152-187) made an important contribution to their eluci- dation. Liitken’s A. antillensits has been often recorded, but Verrill thinks it is possible that this nominal species is identical with dupli- catus. If he is correct, it is obvious that the forms I listed from Porto Rico as antillensis (1901, U.S. Fish Com. Bull., 2, p. 236) are certainly not that species. They are perhaps Verrill’s A. comptus. All the astro- pectens which I have myself collected at the Tortugas, in Jamaican waters, and at Tobago are referable to either articulatus or duplicatus, and I am inclined to consider these the only two strictly littoral species. Conditions in the genus Echinaster are equally bad. A number of species are listed from Florida and from Brazil, and several of these are recorded from Cuba, Jamaica, Haiti, Porto Rico, and St. Thomas. My experience in Jamaica has satisfied me that there is only one littoral species there and, so far as I can see, it is not distinguishable from the common Florida species, sentus. I have previously called it spinosus, but Verrill has pointed out that no species of Echinaster may properly bear that name. The material in the Museum of Comparative Zodlogy shows that sentus is a very variable species and it may be that a more satisfactory knowledge of it will permit the recognition of varieties and possibly of subspecies. So far as I can see, no other species of Echinaster is known from the West Indies proper, but on the west coast of Florida, as far south as the Tortugas, there is a second well-marked species, spinulosus Verrill. There are specimens, undoubtedly spinulosus, in the Museum of Comparative Zodlogy labeled as coming from off Cape Fear, North Carolina, in 7 fathoms, but there is a possibility of a mistaken label, and the record may be ignored until confirmed by the discovery of additional specimens north of Florida. Verrill says he has seen no specimens from the eastern coast of Florida, and of those in the Museum of Comparative Zodlogy, all (save the lot mentioned) are from western Florida or Louisiana. There can be little doubt of the occurrence of two species of Asterina in the West Indies, as I have found both in Jamaica and at Tobago, Distribution of Littoral Echinoderms of the West Indies. 55 and Hartmeyer found both at Barbados. Verrill doubts the dis- tinctness of the two forms and calls both folium. Déderlein (1910, Zool. Jahrb. Supp. 11, pp. 152-155) gives the name minuta to the form with 2 to 4 spinelets on the actinolateral plates, and describes the form with a single such spinelet as a new species, hartmeyeri. As has long been known, both forms were regarded by Gray as varieties of Linné’s Asterias minuta, but in 1859 Lutken gave the name folium to the form with 2 to 4 actinolateral spinelets, leaving the name minuta for the other. Hence Déderlein’s proposed name seems to me quite superfluous. Verrill (1900, Trans. Conn. Acad., 10, p. 584) gives Ophidiaster guildingit as occurring at Bermuda, but this is obviously a slip of the pen, Linckia guildingii being the species he had in mind. The little sea-star taken by me at Port Antonio, Jamaica, in 1897 and listed (1898, J. H. U. Cire., No. 137, p. 5) as Pentagonaster parvus is of very uncertain identity; it will probably prove to be a young Oreaster reticulatus, the growth stages of which are at present almost wholly unknown. Of the 14 sea-stars listed above, one appears to be tropicopolitan and hence of little service in determining faunal areas within the tropics; this is Linckia guildingii. 1 have examined specimens from the Society Islands; Masthead Island, Queensland; Zanzibar; and Lower Guinea, as well as much material from the West Indian region, and I have not discovered any valid specific differences. Of the remaining 13 species, Luzdia alternata, L. clathrata, and Ore- aster reticulatus are widely distributed in the tropical Atlantic from South Carolina to Brazil, and Oreaster occurs even in the eastern Atlantic. A similar but somewhat more southern range is that of Luidia senegalensis, which, although known from the west coast of Africa, does not occur at Bermuda or on the coast of the United States north of Florida. Of the remaining species, the following 5 are distinctly characteristic of the West Indian region: Astropecten duplicatus, Asterina folium, A. minuta, Stegnaster wesselit, and Ophidiaster guildingi; while Astro- pecten articulatus, Echinaster sentus, and E. spinulosus seem to be restricted to the northern part of the region. If, however, it is true, as reported, that A. articulatus really occurs at Dominica and Martin- ique, and EF. sentus actually lives on the coasts of Brazil, which seems improbable, this distinction has no validity, for EH. spinulosus is a very local species, probably confined to the northeastern coasts of the Gulf of Mexico. The fourteenth species, Coscinasterias tenuispina, is distinctly a Mediterranean and eastern Atlantic form. It is common at Bermuda, but it is not impossible that it was introduced there accidentally. 56 Papers from the Department of Marine Biology. Verrill reports it from Cuba and from Brazil and even says ‘West Indies.”’ He also describes a variety (originally a species) atlantica, the type of which is said to be from Cuba. The occurrence of a lit- toral Coscinasterias in Cuba, however, needs to be confirmed, for it is many years since Verrill’s specimens were taken and nothing of the kind has been found in that region since. OPHIUROIDEA. BRITTLE-STARS. The littoral brittle-stars of the West Indies are numerous in species and very abundant as individuals. Both Ophiactis savgnyi and Ophiothrix angulata swarm wherever conditions are suitable, while every reef which supports echinoderm life at all is sure to abound with ophiocomas and ophiodermas. On a reef where conditions are moderately favorable, one may be reasonably sure of finding, anywhere in the West Indies, at least 15 species of brittle-star and, on an excep- tionally good ground, such as Buccoo Reef, Tobago, the number may rise to 25. It is not always easy to decide whether a given species of brittle- star should be included in the present list or not, but where a species is well defined and well known from a few fathoms depth, like Ophio- thrix lineata, I have included it, even though I have not found it in water less than a fathom deep. On the other hand, where the specific limits of a species are ill-defined and hence the reliability of the records are dubious, as with Ophiactis miilleri Liitken, I have omitted it unless I have myself collected it along the shore or on a reef. I have omitted also Ophiopsila hartmeyeri and Ophiolimna littoralis, recorded by Keehler from St. Thomas, and Amphiodia gyraspis, recorded by me from Porto Rico, because they are not littoral in the narrow sense ised in this paper. Several other Porto Rican species whose identi- fication is dubious are likewise ignored. The following 58 species are those to which the title “littoral brittle- stars of the West Indies,” in my judgment, rightfully belongs: Ophiomyxa flaccida (Say). Astrophyton muricatum (Lamarck). Ophiacantha oligacantha H. L. Clark. Ophiomitrella glabra (H. L. Clark). Amphiura kukenthali Keehler. palmeri Lyman. stimpsonii Liitken. vivipara H. L. Clark. Hemipholis elongata (Say). Ophiophragmus filograneus (Lyman). liitkeni (Ljungman). pulcher H. L. Clark. septus (Liitken). wurdemanii(Lyman). Ophionephthys limicola Liitken. Ophionema intricata Liitken. Amphipholis gracillima (Stimpson). pachybactra H. L. Clark. squamata (Delle Chiaje). Ophiostigma isacanthum (Say). Amphiodia planispina (von Martens). pulchella (Lyman). repens (Lyman). rhabdota H. L. Clark. trychna H. L. Clark. tymbara H. L. Clark. Ophioenida scrabriuscula (Liitken). Amphioplus abditus (Verrill). coniortodes H. L. Clark. thrombodes H. L. Clark. Distribution of Inttoral Echinoderms of the West Indies. 57 Ophiactis cyanosticta H. L. Clark. Ophiopsila riisei Liitken. lymani Ljungman. vittata H. L. Clark. savignyl (Miller & Tros- | Ophioderma appressum (Say). chel). brevicaudum Liitken. Ophiothrix angulata (Say). brevispinum (Say). brachyactis H. L. Clark. cinereum Miiller & Tros- lineata Lyman. chel. cerstedii Liitken. guttatum Liitken. suensonii Liitken. januari Liitken. Ophionereis olivacea H. L. Clark. phoeenium H. L. Clark. reticulata (Say). rubicundum Liitken. squamulosa Koehler. squamosissimum Liitken. Ophiocoma echinata (Lamarck). Ophiozona impressa Liitken. pumila Liitken. Ophiolepis elegans Liitken. riisei Liitken. paucispina (Say). While certain of the above species are subject to great diversity, especially in color, it is fortunately true that nearly all, even in such genera as Amphiura and Ophiothrix, are recognizable with compara- tively little difficulty. Specific limits in the genus Ophiactis are not very well defined; in particular, the species miillert and savignyi have been much confused and young individuals are certainly hard to separate. Adult miilleri seems, however, to be a much larger and darker-colored animal than adult savignyi and, so far as I can learn, it inhabits deeper water and does not occur on the reefs accessible at low tide. It is therefore ignored in this list. The genus Ophiothrix contains the most ill-digested assemblage of species of any genus of ophiurans, but the littoral West Indian species are not hard to separate when once their distinctive characters are understood. The extra- ordinary diversity of color and disk spinulation shown by O. angulata is certainly perplexing but each of the other species has distinguishing characters which are quite easy to see at once. In the genus Ophio- derma, the species appressum and brevispinum are so similar that they are often confused. Typical examples are not difficult to distinguish, but each form is variable and some of the varieties are not readily assigned. Consequently, the records of these common and long- known species are not wholly reliable and the exact limits of their relative distributions is still uncertain. Before separating the West Indian brittle-stars into the half-dozen groups into which the sea-stars were divided, we must eliminate the following 14 species, which are known from only a single locality and not infrequently from only a single specimen. Ophiacantha oligacantha. Amphiodia tymbara. Ophiomitrella glabra. Amphioplus coniortodes. Ophiophragmus filograneus. thrombodes. pulcher. Ophiactis cyanosticta. Amphipholis pachybactra. Ophiopsila vittata. Amphiodia rhabdota. Ophioderma phoenium. trychna. squamosissimum. 58 Papers from the Department of Marine Biology. There are several other species which are so little known that they might perhaps well be included in this list, but as they are known from at least two different localities it seems fairer to include them in one of the following groups: The first or tropicopolitan group includes only two species, each of which is small and well-adapted by its habits for transportation on the foul bottoms of vessels. It seems highly probable that their wide distribution is thus quite artificial and has no significance from the zoogeographical point of view. The two species are: Amphipholis squamata and Ophiactis savignyi. The first of these is really cosmo- politan, for it occurs far outside the tropics, both north and south. Of the remaining 42 species, one-half are widely distributed in the tropical Atlantic and under favorable conditions their occurrence may be expected anywhere between South Carolina and Brazil. Only 5 of them are as yet recorded from north of Florida on the mainland coast, but 15 have been reported from the Bahamas and 14 from the Bermudas; 5 are already recorded from the eastern Atlantic, and when the littoral faunas of Ascension and the western coast of Africa are better known, it is probable that others will be found there; 18 are already reported from Brazil. These 22 tropical Atlantic species are the following: Ophiomyxa flaccida. Ophiocoma echinata. Astrophyton muricatum. pumila. Amphiura stimpsonu. riisel. Hemipholis elongata. Ophiopsila riisei. Ophiostigma isacanthum. Ophioderma appressum. Amphiodia planispina. brevicaudum. repens. brevispinum. Ophioenida scabriuscula. cinereum. Ophiozona impressa. Ophiolepis elegans. paucispina. Ophiothrix angulata. suensonil. Ophionereis reticulata. The line between the preceding group and that which I call the strictly West Indian is not a hard-and-fast one, but the following species are not known from south or east of Tobago and Trinidad and only two occur on the mainland coast north of Florida; one of these and a third species occur at Bermuda. Only one species in the group is known from the Bahamas. These 13 West Indian species are: Amphiura vivipara. Ophiactis lymani. Ophiophragmus liitkeni. septus. wurdemanii. Ophionema intricata. Amphipholis gracillima. Amphiodia pulchella. Ophiothrix brachyactis. cerstedil. Ophionereis squamulosa. Ophioderma guttatum. rubicundum. A number of species in the tropical Atlantic and West Indian groups need a word of comment. Of Amphiura stimpsonii, specimens in really Distribution of Littoral Echinoderms of the West Indies. 59 shallow water have not been taken south of St. Thomas. The dis- tribution of Hemipholis elongata is remarkable, for the species seems to be common at Charleston (South Carolina) and has been taken in Florida, but it has not been met with in the West Indies proper, although it is recorded from Trinidad and Brazil. The occurrence of Amphiodia planispina at the Tortugas, Porto Rico, and Brazil seems to warrant placing it in the tropical Atlantic class. The occurrence of Ophioderma brevispinum in Buzzards Bay, Massachusetts, gives the characteristically West Indian genus Ophioderma a remarkable north- ern extension. The little species Amphiura vivipara is known as yet from only the Tortugas and Tobago, but it has just recently been described and is so small and so secretive in its habits that it has doubtless been overlooked elsewhere. The species of Ophiophragmus are still very imperfectly known; liitkeni (originally taken at St. Thomas) is common at Pigeon Point, Tobago, but is not known from elsewhere; septus (also originally from St. Thomas) is known now from off Cape Hatteras, in 52 fathoms, as well as from Tobago; wurdemanit, originally from South Carolina and Florida, is recorded from Trinidad. It seems to me quite probable that this last record is erroneous. The extraordinary Ophionema intricata, originally described from St. Thomas, is not rare at Sandy Point, Buccoo Bay, Tobago, but is not known from elsewhere. The records of Amphiodia pulchella,from Tortugas and St. Lucia, are indicative of a general West Indian range. Of Ophiactis lymani, we can only say that its small size and secretive habits are the probable reason for the scarcity of records, since it occurs at the Tortugas as well as at Bermuda and Tobago. The Tortugas and Tobago are the only known localities for Ophiothrix brachyactis, while Ophionereis squamulosa is common at both those places and is also known from St. Thomas. It is probable that the brittle-stars, recorded by me from Porto Rico (1901, Bull. U. S. Fish. Com., 2, p. 248) as O. dubia, are really squamulosa. The remarkable Ophioderma guttatum, originally described from St.Thomas, occurs along the north coast of Jamaica but is very rare, while it is common and reaches a large size on Buccoo Reef, Tobago; it is not known elsewhere. There remain 7 species, which seem to represent different faunal elements from those as yet listed. Of these, only one issoutherninits relationships. This is Ophioderma januarit, a Brazilian species, which israre at Tobago. There is no species with a Mediterranean affiliation, though the occurrence of such characteristic West Indian genera as Ophi- opsila and Ophioderma in the Mediterranean must not be overlooked. The following 6 species may be grouped as northern in their distri- bution: Amphiura palmeri. Amphioplus abditus. kukenthali. Ophiothrix lineata. Ophionephthys limicola. Ophionereis olivacea. 60 Papers from the Department of Marine Biology. Of these, A. palmeri is recorded from rather deep water from off Barbados, but none of the others is known from east or south of St. Thomas. The mud-loving Amphioplus abditus, ranging as far north as Woods Hole, Massachusetts, has not been found in the West Indies proper but occurs on the Florida coast and at the Tortugas. The only known stations for A. kukenthali are the Tortugas and St. Thomas. I did not find it at Tortugas and it is quite possible that it is not really a littoral species in the strict sense. The same remark is appli- cable to Ophionephthyslimicola, whichI dredged at the Tortugas, while St. Thomas is the type-locality. The well-marked and easily recog- nized Ophiothrix lineata is known only from Florida and the Tortugas, a remarkably restricted range for an Ophiothrix. The very rare Ophionereis olivacea is known from only two specimens, one from Porto Rico and one from Key West. ECHINOIDEA. SEA-URCHINS. The littoral echini of the West Indies are not numerous so far as species are concerned, but in number of individuals they are often excessively abundant. On and about the coral reefs, the dreaded poisonous “black sea-egg”’ (Centrechinus antillarum) is common and on certain areas it is so numerous that a person can scarcely move about without touching one. On many reefs the boring urchin (Kchi- nometra lucunter) occurs actually by the thousands, and it is almost always common. On suitable grassy bottoms the “‘white sea-egg”’ (Tripneustes esculenta) is very common, and in similar localities Lytechinus variegatus may be so abundant that one can not walk on the bottom without crushing them under foot. Although Eucidaris tribuloides is often uncommon and hard to find, occasionally it occurs in great numbers and may be gathered literally by the bushel. These 5 urchins may be expected anywhere in the West Indian region in considerable numbers, if the bottom and water are suitable. None of the other echini in the following list are common except locally, but any one of them may prove abundant if a particular locality is suitable. Thus I have neyer found Clypeaster rosaceus common until 1917, when it proved to be abundant on the reef-flats at Bush and Bird Keys, Tortugas. The following 18 species are the littoral sea-urchins of the West Indian region: Eucidaris tribuloides (Lamarck). Encope emarginata (Leske). Centrechinus antillarum (Philippi). michelini (Agassiz). Arbacia punctulata (Lamarck). Mellita quinquiesperforata (Leske). Lytechinus variegatus (Leske). sexiesperforata (Leske). Tripneustes esculentus (Leske). Echinoneus cyclostomus (Leske). Kchinometra lucunter (L.). Moira atropos (Lamarck). viridis A. Agassiz. Plagiobrissus grandis (Gmelin). Clypeaster rosaceus (L.). Meoma ventricosa (Lamarck). subdepressus (Gray). Brissus brissus Leske. Distribution of Littoral Echinoderms of the West Indies. 61 None of the above species, when adult, is at all difficult to recognize nor is any one of them of doubtful authenticity. The two species of Clypeaster are quite unlike and the same is true of the Encopes and Mellitas. The two Echinometras are more liable to confusion because E. lucunter is so variable in form, length of spines, and color, but no one who has once seen typical viridis will have any difficulty. The cassidulid, Rhynchopygus caribearum (Lamarck), is probably a littoral species, but it is so rare and little known that I have not ventured to include it in the above list. On the other hand, the spatangoid, Schi- zaster orbignyanus A. Agassiz, is probably not a littoral species, but I found a bare test under a rock in shallow water at Montego Bay, Jamaica, in March 1912, and it may possibly be truly littoral. It is, however, a very rare and little-known species. Of the above littoral species, Echinoneus cyclostomus seems to be tropicopolitan; at least it is known from not only the West Indian region but throughout the Indo-Pacific from Mauritius to Hawaii and Easter Island. Of the remaining 17 species, 9 have a general tropical Atlantic distribution from the Carolinas, or Florida at least, to Brazil. Of these, 5 are already known from the eastern Atlantic and at least two of the others will probably be found there. One of the 9 (Brissus) is not yet known from Brazil, but it almost certainly will be found there. These are the 9: Eucidaris tribuloides. Clypeaster subdepressus. Centrechinus antillarum. Mellita quinquiesperforata. Lytechinus variegatus. sexiesperforata. Tripneustes esculentus. Brissus brissus. Echinometra lucunter. It is a remarkable fact that there is not a single echinoid that can be called distinctively West Indian, unless possibly one or two of the following group, which seems to have a northern range, should prove to extend further south than is at present known. This northern group mcludes 7 of the remaining 8 species, as follows: Arbacia punctulata. Moira atropos. Echinometra viridis. Plagiobrissus grandis. Clypeaster rosaceus. Meoma ventricosa. Encope michelini. Of all these, Arbacia has the most peculiar distribution, for its range seems continuous from the Tortugas and Florida northward along the coast to southern Massachusetts. It does not occur at Bermuda nor is it known from the Bahamas, but it is recorded from both Cuba (northwestern coast only) and Hayti (an old record that needs verification). It does not occur at Jamaica, nor is it known from Porto Rico or the Lesser Antilles, but it does occur in Trinidad and Tobago. It occurs on the coast of Yucatan and has been reported 62 Papers from the Department of Marine Biology. from Brazil; the latter record, however, is probably due to confusion with the Brazilian species, Arbacia lixula (L.). This distribution is quite incomprehensible, but comparison of specimens from Tobago, the Tortugas, and Massachusetts shows no reason to doubt that all are punctulata. The range of Echinometra viridis is quite restricted, extending only from the Tortugas to St. Thomas, but Clypeaster rosaceus ranges northward along the coast to South Carolina, southward at least to Guadeloupe, and is common in the Bahamas. The range of Encope michelini is like that of Echinaster tenuispinus, from the Tortugas northward along the west coast of Florida and thence westward along the Gulf Coast to Mexico. The remarkable spatangoid Moira has a peculiar distribution, being common at Beaufort, North Carolina, but known also from Florida, Texas, Jamaica, St. Thomas, and Gaude- loupe. The finest of all spatangoids, Plagiobrissus grandis, is common near Nassau, Bahama Islands, but reliable records from elsewhere are rare; it is said to occur at Tampa, Florida; a fragment is known from the Tortugas, and there is a specimen recorded in the “Revision of the Echini” from Mexico. The range of Meoma is much greater, extending from Central America, Florida, the Bahamas, and Jamaica to Guadeloupe. The only remaining echinoid, Encope emarginata, appears to have a rather southern range, occurring from Uruguay to Venezuela and even to Martinique. It is reported from Charleston, Florida Gulf Stream, Nicaragua, and Yucatan, but these records are old and indefi- nite and are probably erroneous. HOLOTHURIOIDEA. HOLOTHURIANS. The holothurians are, next to the brittle-stars, the most abundant of the littoral echinoderms of the West Indies, but as they are not easily preserved and as preserved material is not attractive or inter- esting in appearance, they are as yet very inadequately known. They can be accurately determined only by an examination of the calcareous particles in the skin, and the study of these often involves the high power of a microscope. Moreover, we know as yet little about the growth-changes in holothurians, particularly as regards these cal- careous particles, and hence identifications made years ago are of doubtful validity, while many of those made to-day are merely tenta- tive. Many common West Indian holothurians are as yet unnamed and very few are adequately described. The following list of 24 species includes all those named forms, which are recognizable with sufficient ease and certainty to make the records of their occurrence reasonably reliable. It may be of interest to mention that there are in the M. C. Z. collection more than 25 additional littoral species, chiefly from the Tortugas, Jamaica, and Tobago, which seem to be undescribed and Distribution of Littoral Echinoderms of the West Indies. 63 are as yet nameless. It is obvious, then, that our knolwedge of the West Indian littoral holothurians is as yet too fragmentary and un- reliable to give value to any deductions with reference to their distribu- tion. From the list here given I have excluded 3 of Selenka’s species supposed to be from Florida, 4 species of other regions recorded from the West Indies, and several of Sluiter’s recently described species concerning the status or littoral distribution of which I am still in doubt. The 24 holothurians here accepted are the following: Euapta lappa (Miiller). Holothuria captiva Ludwig. Synaptula hydriformis (Lesueur). cubana Ludwig. Leptosynapta acanthia (H. L Clark). densipedes H. L. Clark. inherens (O. F. M.). floridana Pourtales. roseola Verrill. glaberrima Selenka. Chiridota rotifera (Pourtales). grisea Selenka. Cucumaria punctata Ludwig. impatiens (Forskaal). Thyone briareus (Lesson). rathbuni Lampert. fusus (O. F. M.)? surinamensis Ludwig. gemmata (Pourtales). Stichopus moebii Semper. suspecta Ludwig. Actinopyga agassizii (Selenka). Psolidium braziliense (Theel). parvula (Selenka). Of the above species, Leptosynapta acanthia and Holothuria densi- pedes are known each from only one locality and hence may be elimi- nated from any discussion of distribution. The species listed as Thyone fusus may also be ignored, for while it was not rare in Buccoo Bay, Tobago, the specimens taken were all very small, and it is quite improbable that they are really identical with the European fusus. Perhaps the same should be said of Psolidiwm braziliense, which occurred in coralline algee at Buccoo Bay, with the Thyone. None of the specimens taken was nearly large enough to make its identity certain. Of the remaining 20 species, Holothuria impatiens and Actinopyga parvula seem to have a tropicopolitan distribution, but a critical study needs to be made to ascertain it precisely There are six species which may be called tropical Atlantic, as their range extends from Bermuda or South Carolina to Brazil. They are: Synaptula hydriformis. Holothuria grisea. Chiridota rotifera. rathbuni. Thyone gemmata. surinamensis. The first two of these are unmistakable and there is no doubt about their range, but the Thyone is by no means unmistakable and it is not certain that the Carolinian and Brazilian records refer to the same species. The status of H. grisea is somewhat uncertain, as it is very possibly only the young of H.floridana; it is recorded from the eastern Atlantic as well as from Brazil. Both of the other holothurias are well-characterized species and certainly occur at Bermuda, while they 64 Papers from the Department of Marine Biology. are recorded from Brazil and five or six intermediate places. None of the 6 species, except H. grisea, is known from the eastern Atlantic. There are no fewer than 7 holothurians which seem to be charac- teristic of the West Indies; these are: Euapta lappa. Holothuria floridana. Cucumaria punctata. glaberrima. Thyone suspecta. Actinopyga agassizii. Holothuria captiva.! Of these, Huapta lappa is particularly notable for its large size and striking appearance, which prevent its being overlooked; it is not known from Bermuda, nor from north of southern Florida, nor from the Gulf of Mexico, northern South America, or any point south or east of Tobago. The Thyone is known only from Jamaica and Barbados. Both the Cucumaria and the Actinopyga, as well as H. captiva, are known from Bermuda, although the Actinopyga is very rare there and is possibly only accidental. Both Holothuria floridanaand H. glaberrima, which have not been found at Bermuda, are known from the Bahamas to Barbados. The remaining 5 holothurians are all forms whose distribution is more or less distinctly northern: Leptosynapta inherens. Holothuria cubana. roseola. Stichopus meebii. Thyone briareus. The two species of Leptosynapta are not actually known from any point south of Bermuda, and the occurrence of inherens there is known only from a single specimen, which may perhaps have been a young acanthia. The well-known Thyone briareus occurs along the American coast from Texas to Massachusetts; specimens of Thyone in the M. C. Z. collection from Porto Seguro, Brazil, have been identified as briareus, but as their preservation is poor it is quite possible they are not that species. Little is known of H. cubana, but Stichopus mebi is a very abundant species in Bermuda, Florida, and Jamaica. It does not occur at Tobago and there are no records for it south of Antigua. 1There is strong evidence in support of the opinion that Holothuria captiva is identical with Actinopyga parvula. Distribution of Littoral Echinoderms of the West Indies. 65 I]. LITTORAL ECHINODERM FAUNA OF WEST INDIAN ISLANDS AND ADJACENT REGIONS. In the table herewith published showing the littoral echinoderme known from each island, 116 species are listed. One has but to glancs at the table to see how few areas there are where the echinoderms are even superficially known. Almost nothing is known of the coast between New Orleans and Vera Cruz or of that much more extensive, varied, and important region between Vera Cruz and Colon. That which is known of the Vera Cruz fauna is merely tantalizing. Although there are many Brazilian records, at no point between Colon and Rio Janeiro has there been any attempt to make a collection of echino- derms. It is only fair to say, therefore, that we know almost nothing of the littoral echinoderm fauna of the eastern coast of tropical and subtropical America. When we turn to the islands themselves, con- ditions are somewhat better, but we know absolutely nothing of the Caymans’ marine fauna, nor of that of the many islands and islets in the western part of the Carribean Sea, except for half a dozen species from Swan Island, In the Bahamas no one place has been very com- pletely explored and many records donot designate the particularisland. It is, therefore, necessary to place all Bahaman records under a single head. Very little is known of the littoral echinoderms of Haiti and San Domingo except for Dr. Weinland’s collection of many years ago; of the Lesser Antilles, not a single one has been thoroughly explored. There are, however, half a dozen islands, besides the mainland coast of Florida, where more than one-third of the 116 species have been taken and to each of these areas a few remarks are due. FLORIDA. From the coast of Florida, 69 species are known, 2 larger number than from any island except the Tortugas. But it must be remembered that 3 species, Echinaster spinulosus, Ophiophragmus filograneus, Encope michelini, represent a distinctly Gulf Coast fauna, and the distribution of 5 other species is so local or so peculiar as to make their occurrence of special note. These are: Amphioplus coniortodes, A. thrombodes, Ophi- othrix lineata, Arbacia punctulata, and Plagiobrissus grandis. Of the 69 species, the following 13 are known from Florida and not from the Tortugas, but in many cases unfortunately we do not know from just what part of the Florida coast they come. Luidia senegalensis. Ophionereis olivacea. Stegnaster wesseli. Mellita quinquiesperforata. Hemipholis elongata. Moira atropos. Ophiophragmus filograneus. Thyone briareus. wurdemanii. Holothuria cubana. Amphioplus coniortodes. surinamensis. thrombodes. 66 Papers from the Department of Marine Biology. The absence of suitable bottoms at the Tortugas undoubtedly accounts for the absence of some of these, as Luidia senegalensis, Moira atropos, and Thyone briareus; but it is probable that most of them will ultimately be found there, when our knowledge is more complete. It is noteworthy that of the 69 Florida species, 32 (or almost one-half) are not known from Tobago, while 20 are as yet unrecorded from Jamaica and 27 are not listed from St. Thomas. While our knowledge is as yet too imperfect to make deductions very safe, the increasing difference in the faunas as the distance from Florida increases is so obvious and so regular that it can not be overlooked. One other feature of the Florida fauna must be mentioned. More than a third (24) of the species occur on the coast north of Florida, but not one of these has peninsula Florida as the southern limit of its range and only one, Amphioplus abditus, reaches its southern limit at the Tortugas. At least one echinoderm, A sterias forbesii (Desor) occurs on the coast of Florida, finding the southern limit of its range there, but as this species does not occur at Tortugas or Bermuda and is a distinctly northern species, it is not included in the tables. THE TORTUGAS. No fewer than 76 littoral echinoderms occur at the Tortugas. Perhaps 4 or 5 of these are not so strictly littoral as my restrictions require, but there are certainly more than 70 species which may be collected at the Tortugas by hand, without trawl or dredge. Thus the seat of the Carnegie uaboratory is apparently the best place in the West Indies for this particular sort of fauna. Four of the species are as yet known only locally and 6 are tropicopolitan, 35 are of the Tropical Atlantic group, 13 are distinctly West Indian, and 18 are northern. There are 56 species that the Tortugas have in common with Florida, 52 in common with Jamaica, 47 with St. Thomas, and 45 with Tobago. Only 19 species, just one-fourth, occur on the mainland coast north of Florida. BERMUDA. The echinoderm fauna of Bermuda has been quite thoroughly collected and studied during the past 30 years and is probably better known to-day than that of any other area in the region under con- sideration. There are 42 species here listed which occur there, and there is at least one unidentified holothurian not included herein. Of the 42 species, 1 is endemic and 4 are tropicopolitan, 4 are northern, 6 are West Indian, 1 is Mediterranean, and all the rest (26) have a wide distribution in the tropical Atlantic. It is rather remarkable that the ophiuran fauna is somewhat scanty, without an endemic species. Both at the Tortugas and Tobago more than half of the echinoderms are brittle-stars, while at Bermuda they comprise less than 43 per cent. Of the 42 echinoderms found at Bermuda, 34 occur at the Tortugas, 34 at Jamaica, 32 at Tobago, 26 at St. Thomas, Distribution of Littoral Echinoderms of the West Indies. 67 and 26 on the coasts of peninsular Florida. It is notable that all of the echini and brittle-stars occurring at Bermuda are found also at Tobago. It would be hard to bring out more clearly how dis- tinctively West Indian the echinoderm fauna of Bermuda is. The only non-West-Indian elements in it are the sea-star, Coscinasterias tenuispina, of the Mediterranean, which was possibly introduced by means of ship-bottoms, and the northern synaptids, whose occurrence is difficult to explain. JAMAICA. The echinoderm fauna of Jamaica is rich and varied, including 62 species of the present list and more than a dozen as yet unidentified holothurians. Indeed, the holothurians form a very conspicuous feature of the fauna on the reefs and in shallow water. Intensive collecting of echinoderms has been carried on at three widely separated points on the Jamaican coast: Montego Bay and Port Antonio near the western and eastern ends respectively of the northern coast, and in the vicinity of Port Royal on the southern coast. The last is much the best region, the so-called “lakes” at Port Royal, the rocky coast across the harbor entrance and the outside cays, particularly Drunken- man Cay, affording a diversity of habitats that is very productive. Both Port Antonio and Montego Bay yielded species not taken else- where, but it is quite probable that they will be found in the Port Royal region when it is fully explored. Of the 62 species, not a single one is endemic. More than half (33) belong to the general tropical Atlantic fauna, while 14 are char- acteristically West Indian. There are 6 tropicopolitan forms. Only a single species is indicative of southern affinities, but 8 are plainly northern. There are 52 species in common with the Tortugas, 47 in common with St. Thomas, and 42 in common with Tobago, but only 34 in common with Bermuda. More sea-stars and more holothurians are known from Jamaica than from any other place, but the number of brittle-stars is small, there being five other areas from which more brittle-stars are known. There is little doubt that the number of mud-inhabiting ophiurans known from Jamaica will be considerably increased by further collecting in suitable areas. PORTO RICO. The echinoderm fauna of Porto Rico is not rich either in number of species or (in most places) in number of individuals. It is remark- ably like that of Jamaica, all of the sea-stars, all the echini, and all but one of the holothurians being common to the two islands, while all of the 6 brittle-stars recorded from Porto Rico but not yet known from Jamaica are mud-loving species which will very probably be found in suitable localities at the British island. With both St. Thomas and the Tortugas, Porto Rico has 43 species in common but with Tobago only 33 and with Bermuda only 28. Of the Bermudan 68 Papers from the Department of Marine Biology. fauna, however, just two-thirds (66 per cent) occurs at Porto Rico, while of the Tobagoan fauna only a little more than half (53 per cent) is found there. Of the 54 species, 2 are as yet known only from Porto Rico; 29 are common throughout the tropical Atlantic and 11 others are distinct- ively West Indian; 4 are tropicopolitan and 7 have a northern range; only 1 can be considered representative of a southern fauna. The number of sea-stars known from Porto Rico is exceptionally large, nearly one-fifth of the echinoderms belonging in that class, whereas only 12 per cent of the entire West Indian list is made up of sea-stars. ST. THOMAS. The United States Virgin Islands, so long known as the Danish West Indies, are classic ground for the student of echinoderms, as a very large proportion of the West Indian species were first recorded from there, thanks to the industry and great abilities of the celebrated Danish zodlogist, Liitken. In the present paper, I have not attempted to keep separate the records from the different islands, but have included them all under ‘‘St. Thomas,’’ since the name Danish West Indies is no longer correct and the recently coined official name for the group is also open to misunderstanding. None of the 56 species here listed from St. Thomas is endemic, but on the other hand 6 are tropicopolitan. There are 28 tropical Atlantic and 13 distinctly West Indian forms. The remaining 8 species all have northern affiliations. There are 44 species which occur in Porto Rico, or 81 per cent of that island’s fauna; 47 which are found in Jamaica, 75 per cent of that fauna; 27 which are known at Bermuda, 64 per cent of that fauna; 48 which occur at the Tortugas, 63 per cent of that fauna; and only 38 which are found at Tobago, just 60 per cent of that fauna. TOBAGO. The echinoderm fauna of Tobago is largely confined to the vicinity of Pigeon Point on the southwestern part of the island, where extensive coral reefs protect the shallow waters of Buccoo Bay. Although we made brief visits to several points on the southeastern and northeastern sides of the island, we found very few echinoderms indeed in those places. But in Buccoo Bay and on Buccoo Reef, there is an exceed- ingly rich fauna, especially of brittle-stars, which constitute nearly 60 per cent of it. The most notable member of this fauna is the comatulid, T'ropio- metra carinata, & conspicuous representative of a southern fauna, common on the coast of Brazil. Two other representatives are note- worthy—the little bright rose-colored holothurian Psolidium braail- iense and the handsome brittle-star Ophioderma januarwi. The latter is apparently rare at Tobago, but a number of specimens of Psolidium were taken, though all are very small. Distribution of Inttoral Echinoderms of the West Indies. 69 Besides these 3 southern species, not found elsewhere in the West Indies, 7 other species are not yet known from any place but Tobago. One of these is the little Thyone, referred to on page 63, but the others are all brittle-stars. Of these, two are ophiodermas and deserve special attention. One, Ophioderma squamosissimum, has long been known from the unique holotype in the Copenhagen Museum, which is from an unknown locality in the West Indies, almost certainly not Tobago and very possibly St. Thomas. This brilliantly colored brittle-star (plate 3, fig. 2) is rare at Tobago, only 5 specimens being found on Buccoo Reef at extreme low-tide. None is as large as the holotype. The other notable Ophioderma at Tobago is O. phenium H. L. Clark (1918, Bull. M. C. Z., 62, p. 333), which seems to be a fairly common, endemic species. The coloration is conspicuous, some- times all green, sometimes all red, but usually a red disk with green arms (plate 3, fig. 1). Another remarkable Ophioderma, O. guttatum, is common on Buccoo Reef and reaches a large size there. It is pos- sible that this is a southern species, for while it was described from a single specimen taken at St. Thomas, and I have taken it twice in Jamaica, these three specimens are all small, only about half as large as the adults of Tobago. Associated with the ophiodermas on Buccoo Reef were great numbers of Ophiomyzxa flaccida, of very diverse hues; olive-green either with or without white markings is a usual color for this species, but olive-yellow, passing into brilliant yellow (plate 1, fig. 2) or brown passing through red-brown into red of various shades (plate 1, fig. 1) are common. Another brilliant brittle-star found on Buccoo Reef was the unique type-specimen of Ophiothrix erstedii var. lutea H. L. Clark (1918, Bull. M. C. Z., 62, p. 314), whose bright orange coloration (plate 2) is very distinctive.! All of the half-dozen tropicopolitan echinoderms of the West Indies are common at Tobago and there are two species which have northern rather than southern relationships. One of these is the sea-urchin, Arbacia punctulata, whose distribution from Tobago to Massachusetts (along the mainland coast?) is so puzzling. Of the remaining 45 species, three-fifths are typically West Indian. No fewer than 32 species are common to Bermuda and Tobago; this is 76 per cent of the Bermudan fauna and is a very remarkable fact. It can be explained only on the ground that Tobago is the home of 45 tropical Atlantic and West Indian species, and it is from this wide-ranging group that the Bermudan littoral echinoderm fauna has been almost wholly derived. Of the Jamaican fauna, 43 species (69 per cent) occur at Tobago, and of the St. Thomas fauna, 38 (68 per cent). There are 45 species common to Tobago and the Tortugas, but this is only 59 per cent of the Tortugas fauna. 1I am indebted to Dr. Mayor for making colored sketches from living specimens of these bril- liant ophiurans. From these sketches and the preserved specimens, Mr. J. Henry Blake has made the beautiful drawings reproduced herewith. 70 Papers from the Department of Marine Biology. III. CONCLUSIONS. As already stated, the data at present available are too fragmentary to warrant any reliable deductions. We can as yet scarcely guess at the origin of the littoral fauna of the West Indian Islands, but certain things are suggested by this study which may be mentioned as requir- ing further consideration: 1. There is no very close relationship with the Mediterranean fauna. Of the 55 genera concerned, only 25 occur in the Mediterranean, and only one of these is as yet unknown on the western coast of America. 2. There is a notable resemblance to the fauna of the western coast of tropical America, four-fifths of the genera (44) being known to occur there. And in many genera specific differences between the West Indian and West Coast forms are very slight. 3. The fauna of the Bermudas is practically all derived from the West Indies, and so recently that no endemic species have as yet arisen. The only endemic species in Bermuda is Leptosynapta acanthia, which is probably derived from one of the northern members of the genus. 4. The fauna of Tobago unquestionably contains a southern element derived from the Brazilian coast. 5. If we assume that the genus Arbacia arose on the western coast of America, the present distribution of the genus and of the species punctulata can be explained as follows: The Caribbean Sea was at one time an eastward extension, a narrow-mouthed gulf of the eastern Pacific ocean, formed after Arbacia was well distributed north and south of the present isthmus of Panama. The present species punc- tulata entered this gulf on both shores and followed them eastward. After the closing of the gulf and the formation of the Lesser Antilles, the species being exclusively littoral spread, not in all directions, but only northward along the Mexican and United States coasts and eastward along the South American coast to Trinidad and Tobago. Later the genus passed southward to Brazil and eastward to Africa and the Mediterranean in the form of lirula, which may well have been derived from punctulata. Along the coast of the United States, the local conditions have not been favorable to developing a new species. From Florida, punctulata has crossed over to Cuba and perhaps extended along the north shore of that island to Haiti. According to this theory, Arbacia does not and never has occurred in the Lesser Antilles nor in Jamaica and probably does not occur on the south side of Cuba or in Porto Rico. It may be found in the Bahamas. It certainly ought to occur in favorable places along the north coast of South America. Until it can be shown to occur there, the hypothesis here proposed with reference to Arbacia lacks adequate foundation. But the distribution of Hemipholis, Echinaster, Moira, Meoma, Thyone, Stichopus, and similar genera, as shown in the table on pages 71-73, gives some support to the theory and warrants its consideration in interpreting the West Indian fauna. 71 Distribution of Littoral Echinoderms of the West Indies. *prooad [NyZqnop & sayvoIput 2 *pi0da1 a[qQuIjeI B sayBOIpur — “paulmexe Weeq sey Uoloeds oUeyANe UB 4ey4 soyvorpuT + Sete lie ere fifo} /s ape _, . are ° one (es ool fe eocloece ate ats eoeeleae + pusyenetetesieXers\erelensieie.elserrey queoest BUsysorud Sh) 23 shoal loge ee als ca alas ee ahs ae BE Patiala hoo 5) Pee Os OR oe Gini eee oe es ade a Suet Falleeraan. saree ties emongnned seafeoelie eae os * Bells ace a Sh a ee Sun Wu23 syoudradary fe eae os ; Ben: aval ne BE ain. cine sieve 4 een eMoLiyuE SuUCTTIO ae a ce > se [beee | Cs APES ates icoot net ovinateeag ejoormy eAtaudauornds, : fa els ee Of) 9) pet Tueulepina - Bara eiratel ate 5 Alc eee see eOtes a: | ius/8) se \| (80)0)) (e0 @ e16 0 6 661060. 6.81e'8 a bike sngdos efees eoclosele eee a ot O00 Cec eeevessons qeyoind oi ea Walp Ae (ca) Fae Ph | SS a oa pa he pate haves pa tae ee 5 etare 2 aiaie . see + eee see cee eee ee ee eeesrees “snoueis0[y snuiseiqdorgdg . ae : = see . ee wee a= + eoefeecrteceeeeec ees econ eeeeeene eyesuolo stoydiuieyy - a 3 at. 5 sete + Gila f{/eceXe ellie va)||> aie \ie'e ¢ ele) e 8) 06,0) 6.0.0.6 6.06/60 656 BIBCIAIA 2 ‘ + 3 been le d = P88) rc) Poor) |croro| \aic OG ets F \;oro oc COC iO aro SO aoe OGG Truosdwi1}8 she foes i % aL ab a elieca! |feire/a} | lore <6 alle 0 eieie) allel» stalyte| elapse erentie lzowyed se oe hae os eet | (©) Brats SHONO. 4!) ore ei liare (el flee. ¢iaie 4 6)exe 6}0 0 (6-e1070°6 ble.ele Teyjueyny siniqduy oe oe Scien ||P lite (90) | tare }twany (en |ipie(o.0\fteleitel| (olesei|{s co leie"| tala, ellintaie}i{piw iol eis o everavetelerwisiare citie tart Biqn|s ByjoapzTMOTYdGO eee eee . eee ° oer fece + eoefecvciicccleve Tresstssessseceesceradgoesyo eqquBoRIqdG Paap aa ie ee Baws eee aoe ae ee eforne +. ae ste ah eee Trrresssesseess ss cumagormut 10zAydo1sy a = 5 4 vavell scons Ifohece = 5 ae = a th piel elifetesens dts ze Condit | eos Jk aitaietelie al ulehucalelle aleiniatsteva Bplooeg ex Aul01ydo as “paproiniydgQ a aac sfeee ee el Jee ecfenefonefesostoee = eee ere ce ceseccene sutdsmuo4 SBIIOJSVUINSOD pales i o'ohamte| [is efeee a = a i see ei see Uh. FOIE OOD pr Te (54 fab Cols a pas ah ee a GN alee tcc 5.5% a8 eave see A aS oe puuasaeneunca Ge = oe . = seelewe ak . ane == Kalele) [fevers] \ sree S| Hero) | eas + 2s aes) lato aE ae . = Ae oa = + OOO CO CaO OREO OMECE YS CLcrc Usurpping aerate 2 alee 2 oo oe = 3 S| eiclisee © i i oo te ae HS BOOS Oo Oto Gono neon oncnnnnnnnnn Nauippms iayserprydG ule = 2 00f eee ate eerelooe = oe sa8 +. toehecaelese a Ahp ee aliale ell iere, eheketielaie)7a/avsleleterp) avacaie eronuny Tyessom 1o4svus049 eee hee = rasa Pata cosh | Sac Se ee fas) anal eit ts ee cee ected cee erauea veafeees 8 lee) 16) 26nlcal a Sa foc oe om meee | oleleeile eal taleealeosel Gal Glia-al.c Melb. +. > asiecna otke dtr ee tea orroy cman ae eee +] -[-> : ei ea | a male al hice i |r (re eer fer Df | PB | a Bhautiotiae aaseuenly oes rs cee eee alien |e titer 2 2S gt jae a Bee aCe talG-ciicderas actos fee oer eared Say + ps . eoele . eee Per on =e +. eee + ber tact] foe + + PES esos alee ie + 211s Ce RAS TU ETO oe io + eleows]e . + eeele eet e oe nes a3 (Reon + + ms + db + ors) Sills esi lieve ele) eelele ole s,s! ss © eveinlssueveleters By BUII}/e eIpinyT aS ete ps AL . a [oe efewedece = efeee 2 Go ae eeeteees aie |fecte ae SHeiie ello! eleheliefelejeliereiesieieieieho 6 snyeordnp + . es i o- + _— se eee S(O zs + + + 7 Pe Oe snye[noyse uoaq0odo1sy seete ee] Lo few te eee fooe aon tte eae + BAERS S| Cetahe Te Mase He LeEs |} ene) eifieL es Tetons isre eal lKeheve)|rerele[evenet syerellle wieitte eal iexauel lie evetlteeleiel)|epeve!l-ere9)|\elo)6i@i [lee ailis ie aillajeia ee else siene efbheyerateleserete eyeBulied eiyauoidory, an Saausll atu lleve al tecersl| Beers aval meee Pealiees «| oval bors aevarAaeal| bl Gael ceecll dl cael ema © | ee ee ae, sagact aoe taht “Dpynouoy Si] oOo) oO! a] A nl|o| m >| n| 2| wm 4 by Bele slenl ISIS PG TIBET IPICIelSlElT ITI AIS Ble lelaclZlZleclele BSlPIP CIS EIEIEISIZ(S/EIElElEIS/S/FISIE/FIEIFIE"|2 (2 [a7 12 |: Bales ees = S/RBIBIBIGIBIE)S/Sisisa late a|]° lt |melelzo = ele] |elel2IF lFisleleleiele (ale leleigiz |? 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B & mes st vein 1 & g al aS, = “uorngrsysip Buinoys 94D], Papers from the Department of Marine Biology. 72 *piooar [NJ}Qnop &B saqvoIpuT { *‘peururexe Useq sey UsUIIOAds O1yUOYINE UE 4Bqy SoyeOIpUL -++ *plooal 9] qeljer 8 SaywoIpur — eae I Le REA Ue ee ae “Daprouryoe ey surdstoned Ce Ce Oe a od suBde[o sidaporydg, COR ee eC See tt a 2 ‘essorduit suozo1ydg Seo) |/a3e/ 0) | (o:sie)| exe) ei] sceia) liens e)|iezeie!|}6ie.0)fleiere;|\s els'[\s viei|lezave | svere|ie, 6.0. |'eiec6||ieaie [fel s.00,|\ 6) 6e)]+e fe: e/| 'oe,sieil\e a e'|lerens Seereesesessscumurtsstsourenbs ed uUINPNBOTAIIG ee ee ed uingsoidde suliepotqydg ee Ce Ce Oe Ce ee er i are BYCqYIA © .0\e {0.6 00.080 ee e650 © b1e 0/6 6,09 0.00 ee TOSttd episdorydQ, Cee meee merece re rene eeeeens Testy 6 10 Ud 0 65810160 \0 \0 09 16. bce 68 6/ele 6 *e[tund ee er ee ar ByBUIyoo sulos01gdg cere ccefesalscelecevecnsecsccscces ssojnurenbs via elfenunerelsiarelicsenstete’ stehajals ByBNoYEI C5016 Oe sel iéceiw be ee 16 06.06 oe 067016) 6 06 6 6\e)\6 BOOBATIO sto1au01TydG eye! aver[letie Lol] lscabe,]ie\ wiezieiere co 16: 6:70i%s lelloile'a:.eite eure). sein ‘By Boul] Phere! |petseruel|fetenni|(atonel|fanaieelenens jeuetanaeclevele ie) « stowAyousq ee eye naue x1iqyorydg sro leVelle'#\ #18)/6 le ersiale [AUSIARS eects TUeUrAT es ByorysouvAD styoetydgO * *sapoquio1yy siege telsreisiers s8p0q10TUO9 es er a) snyipqs sndorgdury S26 Of ee eve Gis, of 8, ©6018, 0168.04.86 B[Nosn1iqvos eprmoorydg Ps Ge BIBQWAY ceofeoeerwcccesesesecccesas euyoAdy viwi6 we€ :01'0;.0)168/16):8).0 10 6i19)\9, 6 0)8 er eke suadai Ae es eee ee eyjeqornd Peo fee BCI CIOS) [ITEC TR Od [ICC i ke ae TI I POC) suldstueyd erporqdury *penuru09—vaprouniydQ ‘ogloeg-opuy oUEPyY T19}sBIT “puvps, UBMG “OoTxayY pus Wii) SO. SEOD Sick Vi (Sig9 28809 CN *PSPrmurLy “*qUaOUIA “99 ‘sopequrg “ONDIUI4AV IY “eBorTuluo(y ‘adnojepeny “VBIIOSPUOTT “roqdoystryD “9S “MoWOTONIWVY “39 “seuloy,L “3S Oo 0F10g Wey “BOTBUIe f "897835 pevay) jo ysvog jny ‘penurjzu0o—uorngr1sip burnoys 2190 J, “Sprtopa JO qyiou “9880D *S “1 “Bpnuliog “oUIB NT 73 Distribution of Littoral Echinoderms of the West Indies. *pioodal [NJAQnop & sazuorpur 3 *piodal a[quITAI B SayvoTpUuL — s *PeuIuIBXe Teaq sey UOUTIOAds ONUOYANE UB 4vq} SsoyBoIpUr + +]: alee. 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Puate 1. Fig. 1. Ophiomyxa flaccida (Say). Red form. Buccoo Reef, Tobago. Natural size. Fic. 2. Ophiomyxa flaccida (Say). Yellow Form. Buccoo Reef, Tobago. Natural size. PLatTE 2. Ophiothrix cerstedii var. lutea H. L. Clark. Buccoo Reef, Tobago. Natural size PiateE 3. Fic. 1. Ophioderma phoenium H. L. Clark. Buccoo Reef, Tobago. Natural size. Fic. 2. Ophioderma squamosissimum Lutken. Buccoo Reef, Tobago. Natural size. 74 HELIOTYPE CO. BOSTON. Ophiomyxa flaccida (Say). Buecoo Reef, Tobago. Natural size. Fig. 1, red form. Fic. 2, yellow form. H, L. CLARK PLATE 2 HELIOTYPE CO. BOSTON. Ophothrix erstedii var, lutea H. L. Clark. Buceoo Reef, Tobago. Natural size. PLATE 3 H. L. CLARK HELIOTYPE CO. BOSTON. Fig. 1. Ophioderma phoenium H. L. Clark. Buccoo Reef, Tobago. Natural size. Fig. 2. Ophioderma squamosissimum Lutken. Buccoo Reef, Tobago. Natural size. IV. FURTHER STUDIES ON THE CHEMISTRY OF LIGHT PRODUCTION IN LUMINOUS ORGANISMS. By E. NEWTON HARVEY, Of Princeton University. 75 Sara Os ws pt Se We eee ¢ Th =e!’ 'p war ; Vu te - a ay, oe ‘ FURTHER STUDIES ON THE CHEMISTRY OF LIGHT PRODUCTION IN LUMINOUS ORGANISMS. By E. Newton Harvey. The results embodied in this paper are the outcome of experiments made upon the dried luminous organs of a small ostracod crustacean, Cypridina hilgendorfii, abundant in the coastal waters of Japan. The structure of the luminous cells has been well described by Dahlgren in the Journal of the Franklin Institute for June 1916, and by Yatsu (Jour. Morph., vol. 29, p. 435, 1917). These cells contain the luminous substances concerned in the production of light, which are projected into the sea-water as a luminous secretion. This paper deals par- ticularly with the chemistry of the luminous reaction. It will be con- sidered in two sections, viz: I, reversibility of the photogenic reaction in Cypridina. II, the chemical nature of Cypridina luciferin and Cypridina luciferase. Much of the work was performed in the Zoological Laboratory, Impe- rial University, Tokyo, Japan, and it gives me pleasure to acknowledge the kindnesses of Professors Ijuma, Yatsu, Watase, and Goto during my stay at the university. I am also deeply indebted to Professor C. Ishikawa, of the Agricultural College, Tokyo, for much assistance in collecting material, and I express my sincere thanks for his interest in my work. I. REVERSIBILITY OF THE PHOTOGENIC REACTION IN CYPRIDINA. In a previous paper on the question of animal luminescence! I have described two photogenic substances in Cypridina hilgendorfit, which I called photogenin and photophelein. Photogenin is destroyed below the boiling-point, is non-dialyzable, and is prepared by making a cold- water extract of the luminous animal and allowing it to stand in the air until no more light appears on shaking. This indicates that one of the photogenic substances, photophelein, has disappeared, leaving the photogenin. Photophelein is not destroyed by short boiling and will dialyze. It is prepared by making a hot-water extract of the luminous animal. The hot water destroys the photogenin, leaving the pho- tophelein. Whenever two such solutions are mixed light appears. On the grounds of method of preparation, relation to temperature, and dialysis, I regarded photogenin as comparable to luciferase and 1Harvey, E. N., Am. J. Physiol., 1917, xlii, 318; also, The Chemistry of Light Production in Luminous Organisms, Carnegie Inst. Wash. Pub. No. 251, pp. 171-234, 1917. Gt 78 Papers from the Department of Marine Biology. photophelein as comparable to luciferin, two photogenic substances described by Dubois! in the beetle Pyrophorus noctilucans and in the mollusk Pholas dactylus. Dubois believes that luciferase is an oxidizing enzyme which oxidizes luciferin, an oxidizable substance, with light production. Neither luciferase nor luciferin alone in solution can pro- duce light, but light appears if solutions of the two are mixed and it continues as long as any luciferin remains unoxidized. Dubois has also been able to produce light by oxidizing luciferin (alone) with a small crystal of KMnQO,, by H.O:2 (with or without blood containing hemoglobin), BaO:, PbO», and other oxidizing agents. Through the kindness of Professor Dubois, I have received some material of Pholas dactylus preserved in sugar and I can confirm his results on the effect of KMnOQ, and other oxidizing agents in producing light with luciferin of Pholas. I have likewise repeated my own experiments with the photophelein of Cypridina, using a whole series of oxidizing agents applied in the same way as with the luciferin of Pholas, and, as previ- ously, have failed to obtain any light with this substance.? The differ- ence in our results is, therefore, not to be referred to a difference in method of experiment but to a difference in the animals themselves. I found also that if one takes a concentrated solution of photogenin, filtered through a porcelain or silicious filter candle to remove all granules and cell fragments, and adds to it a little saponin powder or amyl alcohol or NaCl or other inorganic salt crystals or tissue extracts of certain invertebrate non-luminous animals, that light would appear. Because NaCl could not possibly be oxidized by photogenin (=lucif- erase), or any other substance, and because of my inability to make photophelein (=luciferin) luminesce with oxidizing agents, I regarded the photophelein itself as the source of the light and the oxidizable body. I have compared photogenin to zymase and photophelein to the co- enzyme of zymase, believing that we are dealing with a system similar to that of the enzyme—co-enzyme system of yeast. Hence the name photophelein or body assisting in the production of light. I now believe that under the term photophelein I have previously included two separate substances. One of these is the thermostable dialyzing substance extracted from Cypridina by hot water. Although this substance can not be oxidized with light production by oxidizing 1 Dubois, R., Compt. rend. Soc. Biol., 1885, xxxvii, 559. 2 The following oxidizing agents (added, where possible, in minute crystal or powder form) all gave light with Pholas luciferin, but no light with Cypridina luciferin: KMnO4, K2Cr207, PbO», NavO2, BaOo, MnOo, KsFe(CN)¢, KeS203, NazBsOs, and H2O2. The following oxidizing agents gave no light with either Pholas luciferin or Cypridina luciferin: KeCrO4, CrO3, KClO3, KC104, FeCl;, KNOs, Cl or Br water, I in KI, Na hypochlorite, hypobromite, or hypoiodite, colloidal Ag or Pt, benzoyl peroxide, potato or turnip juice, or blood containing hemoglobin or hemocyanin. If H,O: in addition to the oxidizing agent is added to Cypridina luciferin, no light appears except a faint momentary flash with Na hypochlorite and hypobromite. As this faint flash also appears with thoroughly boiled extracts of Cypridina, lacking luciferin, it can have no significance. If H.O2 in addition to the oxidizing agent is added to Pholas luciferin, the light is in some cases brighter than with HQ, alone. On the Chemistry of Light Production in Luminous Organisms. 79 agents, it does oxidize spontaneously (also without light production) in the air and loses its power of producing light with photogenin. In the absence of air its solutions are stable for months. Once oxidized, it can again be reduced and will again give light if photogenin is added. It is therefore an oxidizable material and, I believe, similar to the luciferin of Pholas. I propose, therefore, to use Dubois’s word luciferin for the thermostable dialyzing substance of Cypridina in place of photophelein, and to use luciferase for the thermolable non-dialyzing substance in place of photogenin. The source of the photogenic sub- stances can be designated by prefixing the name of the animal, as Cypridina luciferin, Pholas luciferin, etc. I suggest also that luciferin, when oxidized, be designated oxyluciferin. Luciferin is found only in luminous animals. In non-luminous ani- mals and probably also in luminous animals there is a second substance, which I have formerly included in the term photophelein (and which may be properly so called), that acts in a manner similar to the action of saponin, NaCl crystals, etc., upon the extract of Cypridina which has stood until the light disappears. When we allow a Cypridina extract containing luciferin and luciferase to stand, the luciferin is not com- pletely oxidized, even though the extract is thoroughly aerated, but some of it is bound (adsorbed or combined?) by other substances in the extract. The saponin, NaCl crystals, and extracts of non-luminous animals act by setting free the bound luciferin, which is then oxidized and light appears. I suggest that the term photophelein be now applied to these substances in tissue extracts. They are not destroyed by boiling. On standing some are stable, while others are unstable. The best way to rid a luciferase solution of the bound luciferin is to shake it thoroughly with chloroform. Such a solution will give no light with extracts of non-luminous animals or saponin, NaCl crystals, ete., but a brilliant light with Cypridina luciferin. An insight into the modus operandi of saponin, NaCl crystals, or photophelein may be gained from the following experiment: Both luciferin and luciferase are adsorbed by many finely divided precipi- tates and colloidal particles, such as boneblack, Fe(OH)s, kaolin, and others. If we take a colloidal Fe(OH)s solution of the proper con- centration (which can only be determined by experiment), add some dilute luciferase to it, and then (after a minute) luciferin, no light will appear. This is because the luciferase has been completely ad- sorbed by the colloidal Fe(OH);, for if we now add some dilute luci- ferase to the above mixture, light will appear, but it will very quickly disappear, because the new luciferase added is again very rapidly adsorbed, but not so rapidly adsorbed that we fail to get light at first. On adding more luciferase we may again get a momentary light, but the additions can not be made indefinitely, because we finally reach a point where the colloidal Fe(OH); has become saturated with luci- 80 Papers from the Department of Marine Biology. ferase and then the mixture glows for a considerable time. It is obvious that for this experiment to succeed there must be more luciferin present than can be completely adsorbed by Fe(OH); and so little luciferase present that it is completely adsorbed by the Fe(OH);. Suppose we have a mixture of Fe(OH)s, luciferase, and luciferin complying with the above conditions. Can we in any way remove the luciferase from its adsorbed condition on the colloidal Fe(OH);? This might theoretically be done in two ways, and we actually find in practice that both methods are possible. Anything which precipitates the colloidal Fe(OH); will decrease the surface available for adsorption of luciferase, and if the surface area is suffi- ciently decreased some luciferase may be forced into solution again, where it is able to oxidize the luciferin. If we add NaCl crystals to the colloidal Fe(OH);—dilute luciferase—more concentrated luciferin mixture, the Fe(OH); is precipitated and light appears. If, in place of NaCl crystals, we add a trace of saponin, the colloidal Fe(OH), is not precipitated, but light also appears. This is an example of the second method of removing luciferase from an adsorbed condi- tion—namely, by using a material (saponin) which is more strongly adsorbed than the luciferase and which is able to replace it as adsorbed body. I believe these to be the explanations of the effect of NaCl crystals, saponin, etc., in giving light with luciferase solutions, except that the luciferase is in excess and a small amount of adsorbed (or combined) luciferin is present which is liberated by NaCl or saponin and gives light with luciferase. Photophelein probably acts in a manner analogous to the saponin. I have considered the thermostable, dialyzing substance as similar to the luciferin of Pholas, despite the fact that Dubois finds Pholas luciferin destroyed at 70° C., whereas Cypridina luciferin is destroyed only by boiling for several minutes in an open beaker. I find that this destruction of Cypridina luciferin on short boiling is due to the in- creased rate of oxidation at the boiling-point and that no destruction of Cypridina luciferin will occur if boiled in an atmosphere of hydrogen. Cypridina luciferin is truly thermostable, but is oxidized to oxyluciferin on boiling in the air. We may say that Pholas luciferin is similar but certainly not identical with Cypridina luciferin. If so, we should expect to obtain light on mixing Pholas luciferin and Cypridina luciferase, yet no light appears. Neither is there light on mixing Cypridina luciferin and Pholas luciferase, although the Pholas luciferase I was able to pre- 1T have endeavored to repeat this experiment with the luciferin of Pholas sent me by Professor Dubois, but without success. Pholas luciferin boiled in a current of hydrogen for 15 minutes would give no light when a crystal of KMnOQ, was added. The hydrogen was produced in a Kipp generator and may have contained a little air. In my experience short (20 to 40 seconds) boiling of Pholas luciferin does not completely destroy its power of producing light when a crystal of KMnQ, is added. On the Chemistry of Light Production in Luminous Organisms. 81 pare from the material which Dubois sent me gave a rather faint light with Pholas luciferin.’ We have, therefore, at least three substances concerned in light pro- duction: luciferin, luciferase, and photophelein. Luciferin is a body oxidizing with light production, dialyzable, and relatively resistant to heat. Luciferase is destroyed by boiling, is non-dialyzable, and accelerates the oxidation of luciferin. While it may be used up in the reaction if mixed with a sufficient quantity of luciferin, luciferase has many of the characteristics of an enzyme and certainly as much right to be called an enzyme as the peroxidases of plants,which are also used up in the oxidation process. The Cypridina luciferase reaction appears to be specific to an extraordinary degree. Of many tried I have found no substances or plant or animal extracts which can take the place of luciferase? nor any substances? or plant or animal extracts* which can be oxidized with light production by luciferase. The light recorded with various extracts of luminous and non-luminous animals in my former paper is to be referred to the presence of photophelein, the third substance concerned in light production, which probably acts by assist- ing the luciferin-luciferase reaction in the manner already suggest d. Let us now turn to the oxidation product or products of luciferin. When luciferin is oxidized it must be converted into some substance or substances, and I believe this change involves no fundamental de- struction of the luciferin molecule, as it isa reversible process. I shall speak of the principal if not the only product formed as oxyluciferin. Most observers believe that a rather fundamental change occurs 1T believe the faint light obtained on mixing Cypridina luciferin and firefly or Noctiluca luciferase and vice versa, recorded in my former paper (Am. J. Physiol., 1917, xlii, 328), where luciferin is called photophelein and luciferase is called photogenin, is not due to the oxidation of luciferin by luciferase of the second species, but is due to the presence of photophelein. I am led to this con- clusion because the light is so faint, but can not be sure until the cases are reinvestigated. The mixing of luciferin and luciferase of different species or genera of luminous ostracods, especially if the color of their luminescence differed, would shed considerable light on this interesting question of specificity. A non-luminous Japanese species of Cypridina does not contain either luciferin or luciferase, but it does contain photophelein. 2T have tried the blood or extracts of many species of animals or plants, including those con- taining strong oxidizing enzymes both with and without H2O, and have always failed to obtain light with Cypridina luciferin. Among others the juice of Indian pipe (Monotropa), potatoes and turnips (containing strong oxidases and peroxidases), the blood of the ox anda worm (Areni- cola) (containing hemoglobin), the blood of the squid (Loligo), Limulus, and Sycotopus (containing hemocyanin), and extracts of Chetopterus (a luminous annelid) and the molluse Unio (rich in manganese) were tried. Dubois reports that he has obtained light on mixing Pholas luciferin with the blood of divers molluscs and marine crustaceans (Ann. Soc. Linn. de Lyons, 1913). Ican confirm this statement for an extract of Unio, but obtained no light with Limulus blood, Sycotopus blood, squid (Loligo) blood, or turnip or potato juice and Pholas luciferin. Evidently Pholas luciferin is much more readily oxidized with light production than Cypridina luciferin. 3 The following oxidizable substances have been tested: ssculin, lophin, bergamot oil, pyro- gallol, gallic acid, anilin, adrenalin, phenol, a-napthol, para-phenylen-diamine, ortol, orcin, hydrochinon, resorcin, pyrocatechin, tannin, benzidin, gum guaiac, amidol, a-napthylamine, and the chromogen of the false indigo plant (Baptisia tinctoria). Luciferase, with or without H2Os, will not accelerate the oxidative color change in any of the above compounds. 4T have regularly obtained a fair light on mixing luciferase well shaken with chloroform to set free any bound luciferin and boiled potato or turnip juice or boiled Limulus blood. The light is especially marked about the coagulum in the boiled Limulus blood. The significance of these results is not apparent. 82 Papers from the Department of Marine Biology. when the photogenic substance is oxidized. Thus, the crystals of xanthin or some related substance in the reflecting layer of the firefly have been regarded as the oxidation products of the luminous material, thought to be nucleoprotein. Dubois! regards luciferin as a protein and states that it forms the same oxidation products as other pro- teins, amino-acids being mentioned as possible substances formed. It should be pointed out in this connection that the formation of amino- acids from proteins involves no oxidation, but an hydrolysis. If we assume that the oxidation of luciferin changes the molecule but slightly, we at once think of comparing the change luciferin <—, oxylu- ciferin with the change reduced hemoglobin <=; oxyhemoglobin. The condition is, however, not so simple as this, for oxyhemoglobin will again give up its oxygen, providing the partial pressure of oxygen is sufficiently low, whereas oxyluciferin will not do this. We can not re- duce oxyluciferin solution by exhausting the oxygen with an air pump. There is another oxidation—reduction system which can also be easily reversed, but not by merely removing the oxygen—that is, the reduction of a dye such as methylene blue to its leuco-base. I believe the change which occurs when luciferin is oxidized is similar to that which occurs when the leuco-base of methylene blue or sodium indigo- sulphonate is oxidized to the blue dye. My attempts to reduce the oxidation product of luciferin started from the observation that if one places a clear solution of luciferase in a tall test-tube, although it may give off no light at first when shaken, after standing a day or so a very bright light would appear on shaking. This was especially true when the luciferase had become turbid and ill-smelling from the growing of bacteria. Thinking that the bacteria produced a substance which could be oxidized by the luciferase, I tried growing bacteria and also yeast on appropriate culture media and after some days of growth mixing the culture media containing the products of bacterial or yeast growth with luciferase, expecting to obtain light; but no light appeared. However, if a little crude luciferase solution was added to the bacterial or yeast cultures and then allowed to stand for some hours, light appeared whenever they were shaken. Indeed, such cultures behaved much as a suspension of luminous bacteria which has used up all the oxygen in the culture fluid and will only luminesce when, by shaking, more oxygen dissolves in the culture medium. Realizing that in bacterial cultures in test-tubes anaerobic conditions soon appear, and also the strong reducing action of bacteria upon many substances (for instance, nitrates or methylene blue) under anaerobic conditions, it struck me that the bacteria might be utilizing the oxygen of the oxidaticn product of luciferin, reducing it to luciferin again. We must remember that since crude luciferase solution is a cold-water extract of a luminous animal allowed to stand until all the luciferin has 1 Dubois, Ann. Soc. Linn. de Lyons, 1914, Ixi, 169. On the Chemistry of Light Production in Luminous Organisms. 83 been oxidized, it must contain oxyluciferin as well as luciferase and will give light if the oxyluciferin is again reduced and oxygen admitted. This appears to be the correct explanation of the above experiments. Not only bacteria but also tissue extracts have a strong reducing action in absence of oxygen. Thus, muscle tissue stained in methylene blue will very quickly decolorize (reduce) the methylene blue if oxygen (air) is kept away, but the blue color immediately returns if air is admitted. Oxyluciferin (i. e., a solution of luciferin which has been completely oxidized by boiling or standing in air until it no longer gives light with luciferase), if mixed with a suspension of ground frog’s muscle and kept in a well-filled and stoppered test-tube for some hours, is re- duced to luciferin and gives a bright light if now poured into luciferase solution. Frog-muscle suspension alone or oxyluciferin alone give no light with luciferase, nor will a mixture of frog-muscle suspension and oxyluciferin, if shaken with air for several hours. Only if this last mixture be kept under anaerobic conditions is the oxyluciferin reduced. The reducing action of tissues is said to be due to a reducing enzyme (reducase or reductase), itself composed of a perhydridase and some easily oxidized body such as an aldehyde.’ In the presence of the perhydridase the oxygen of water oxidizes the aldehyde and the hydro- gen set free reduces any easily reducible substance which may be present. There is a perhydridase in fresh milk, spoken of as Schar- dinger’s enzyme,” which is destroyed by boiling. If some aldehyde is added fresh milk will reduce methylene blue to its leuco-base or nitrates to nitrites, upon standing a short time. If shaken with air the blue color returns. There is no reduction unless an aldehyde is added or unless some boiled extract of a tissue such as liver is added. The boiled-liver extract has no reducing action of its own, but supplies a substance similar to the aldehyde which has been spoken of as a co- enzyme. Milk will reduce methylene blue without aldehyde if bacteria are present in large numbers. Also, there is no reduction if the milk, methylene blue, and aldehyde are agitated with air. The temperature optimum is rather high, 60° to 70° C. I find that milk is a favorable and convenient medium for the reduc- tion of oxyluciferin and that it acts without the addition of an aldehyde or the presence of bacteria. There is probably a substance acting as the aldehyde in the luciferase-oxyluciferin solution. No light appears if milk is added to a luciferase-oxyluciferin solution, but if the mixture is allowed to stand in absence of oxygen light will appear when air is ad- mitted. The air can be conveniently kept out by filling small test-tubes completely with the solution and closing them with rubber stoppers. Oxyluciferin may also be readily reduced by the use of the blood of the horse-shoe crab (Limulus) allowed to stand until bacteria develop.* 1 Bach, A., Biochem. Z., 1911, xxxi, 443; xxxiii, 282; 1912, xxxviii, 154; 1913, li, 412. 2 Schardinger, F., Chem. Zeit., 1904, xxviii, 704. 3 Alsberg, C. L., Journ. Biol. Chem., 1915, xxiii, 495. 84 Papers from the Department of Marine Biology. This experiment is of special interest because the blood contains hemo- eyanin, which is colorless in the reduced condition and blue in the oxy- condition. The color change thus serves as an indicator of the oxygen concentration in the blood. A sample of foul-smelling Limulus blood full of bacteria will become colorless on standing in a test-tube for 10 to 15 minutes, but the blue color quickly returns if shaken with air. Such a blood has the power of reducing oxyluciferin through the activity of the bacteria which it contains. Fresh blood has very little if any reducing action. As almost all animal tissues contain reductases it is not surprising to find that a freshly prepared and filtered extract of Cypridina con- taining oxyluciferin and luciferase, which gives no light on shaking, will, on standing in a stoppered tube for 24 hours at room tempera- ture, give light when air is admitted. While this may be due to the development of bacteria with a reducing action, it does not seem likely, as under the same conditions methylene blue is not reduced in 24 hours and there is no turbidity or smell of decomposition in the tube. In 48 hours bacteria appear and methylene blue is also reduced. If we add chloroform, toluol, or thymol to the tubes of Cypridina extract to prevent the growth of bacteria, and allow them to stand 48 hours, upon admitting air the tube with chloroform gives no light, but the tubes with toluol and thymol do give light, although it is not so bright as if they were absent. I believe that these sub- stances have a destructive action on the reductases, most complete in the case of chloroform. I have not been able to demonstrate that a Cypridina extract will reduce methylene blue or nitrates to nitrites, either with or without the addition of acetaldehyde. This may be due to the fact that oxylucif- erin, which is also present, may be reduced more readily than either nitrates or methylene blue, and so is reduced first. Dubois? has described in Pholas a precursor of luciferin which he ealls proluciferin, which is converted into luciferin by another enzyme, coluciferase. The proluciferin is not destroyed by boiling and the coluciferase will withstand a higher temperature than luciferase and may be freed of luciferase in this manner. He cites an experiment* to prove the existence of proluciferin and coluciferase in Pholas, but I have been unable to repeat this with Cypridina. One might suppose that on allowing an extract of Cypridina (luciferase) to stand in absence of oxygen some proluciferin, assuming this to be present, would be con- verted into luciferin, which would give light if air was admitted. But we can allow a boiled extract of Cypridina (containing no coluciferase) to stand with milk or muscle-tissue suspensions in absence of oxygen and upon admitting air and adding luciferase obtain light. As lucif- 1 This experiment may also be performed with Pholas luciferase with a similar result. 2 Dubois, Compt. rend. Soc. Biol., 1907, 850; 1917, 964. 3 Dubois, Compt. rend. Soc. Biol., 1917, 964. On the Cnemistry of Light Production in Luminous Organisms. 85 erase is found only in luminous animals it does not seem likely that a coluciferase would be widespread, but we do know that a reducing enzyme occurs in milk and tissue extracts—in fact is widespread. It seems more logical to interpret the above experiments as due to the reduction of an oxyluciferin to luciferin rather than the conversion of a proluciferin to luciferin. Indeed, we can reduce oxyluciferin by means which do not involve the use of animal extracts and consequently are free from the objection that ‘“‘eoluciferase’’ may be responsible for the result, but which, neverthe- less, are perfectly well-known reducing methods. Perhaps the best of these is reduction by palladium black and sodium hypophosphite. The latter is oxidized in presence of palladium and nascent hydrogen is set free! The nascent hydrogen reduces any easily reducible substance which may be present, such as methylene blue or oxyluciferin. Oxy- luciferin is not reduced by palladium alone or hypophosphite alone, but methylene blue is reduced by palladium black alone. If hydrogen sulphide is passed through a solution of methylene blue the dye is very quickly reduced and becomes colorless. If the H25 is driven off by boiling the colorless methylene-blue solution, the blue color again returns on cooling. Oxyluciferin can also be reduced to a certain extent by H.S. Sulphur dioxide or oxides of nitrogen (prepared - by the action of HNO; on Cu) had no reducing action on either methy- lene blue or oxyluciferin. Dilute acid favors the reduction of oxyluciferin. If one saturates an oxyluciferin solution with CO, or adds a little dilute acetic acid and allows the solution to stand for 24 hours, a certain amount of reduction will occur. No reduction occurs if the solution is saturated with pure hydrogen and allowed to stand 24 hours. If one adds some Mg powder to oxyluciferin and then dilute acetic acid in successive addi- tions as the acetic acid is used up in formation of Mg acetate, the oxyluciferin will be reduced relatively quickly. Nascent hydrogen is produced in thereaction and is no doubt the active reducing agent, while theacid accelerates the reduction. Soured milk also has quite a marked reducing action. Acid thus favors reduction and hinders oxidation, while alkali favors oxidation and hinders reduction of the oxyluciferin. While I have not studied the properties of oxyluciferin as fully as those of luciferin, so far as I can judge both substances give the same general reactions and possess identical properties. If we make a concentrated hot-water extract of Cypridina, it will contain all the substances of the animal soluble in hot water and not coagulated by heat and may be spoken of as crude luciferin solution. If air is bubbled through this solution for some time, all the luciferin is oxid- ized and it may then be spoken of as crude oxyluciferin solution. Both crude luciferin and crude oxyluciferin solution are yellow in 1 Bach, Chem. Ber., 1909, xlii, 4463. 86 Papers from the Department of Marine Biology. color, but I do not believe that either luciferin or oxyluciferin are yellow in color, because an ether or benzine extract of Cypridina is also yellow, although luciferase, luciferin, and oxyluciferin are all insoluble in ether and benzine. The yellow pigment which can be observed to make up part of the luminous gland of Cypridina is not luciferin or luciferase. It may be a pigment related to urochrome. When tests are applied and precipitating reagents are added to crude luciferin and crude oxyluciferin solution they give identical results in each case. A complete account of the chemistry of luciferin will be found on pages 87 to 110, but a few of its more important properties are emphasized here.' If crude luciferin is saturated with (NH4).50O. or MgSO, a floceulent precipitate forms which may be demonstrated to contain most of the luciferin (see page 93). Oxyluciferin solution also gives flocculent precipitates on saturation with (NH,).SO,4 and MgSO, and these contain most of the oxyluciferin. To demonstrate this the precipitates, after washing, are dissolved in a small amount of water, mixed with fresh milk (or frog-muscle suspension) and allowed to stand in a stoppered tube for 24 hours. If any oxyluciferin is present it will be reduced to luciferin and give light when luciferase is added. One-half saturation with (NH,4).8O4 or MgSO, or saturation with NaCl salts out no material from either crude luciferin or oxyluciferin solution. Picric acid gives no precipitate, but only an opalescence in both cases. In a similar manner it may be shown that most of the oxyluciferin is precipitated by phosphotungstic acid but not by acetic acid or COs, in this respect also agreeing with the behavior of luciferin. Like luciferin, the oxyluciferin will pass porcelain filters, dialyze through parchment or collodion membranes, is soluble in absolute alcohol, but not in ether or benzine, and is undigested by salivary diastase, pepsin, HCl, Merck’s pancreatin in neutral solution, and erepsin. The salivary diastase and the pancreatin (containing amylopsin, trypsin, and lipase) were allowed to digest for 4 days at 38° C. without showing any evi- dence of digestive action. It is partially but not completely precipi- tated by basic lead acetate and by tannic acid. As luciferin is so easily oxidizable a substance, we should expect to find that it will reduce just as glucose will reduce. However, a concen- trated solution of luciferase has no reducing action on Fehling’s (alka- [ine Cu), Barfoed’s (acid Cu), Nylander’s (alkaline Bi), or Knapp’s (alkaline Hg) reagent. Glucose will reduce methylene blue in alka- line (not in neutral) solution, but luciferin will not reduce methylene blue in alkaline or neutral solution. It would seem, then, that luciferin must contain no aldehyde group. If so, we should expect to obtain reduction of some of the above reagents. Just what group is concerned in the oxidation is unknown at the present time, and in the absence of more experimental data speculation regarding it can be of little value. 1 Dubois regards Pholas luciferin as a natural albumin and luciferase as an oxidizing enzyme made up of iron associated with a protein. ‘‘La Vie et la Lumieére,’”’ Paris, 1914. I]. THE CHEMICAL NATURE OF CYPRIDINA LUCIFERIN AND CYPRIDINA LUCIFERASE. PREPARATION OF MATERIAL. The living animals are dried quickly in desiccators over CaCl, and may then be kept indefinitely in well-stoppered bottles containing a few lumps of CaCl, to remove any dampness in the air contained in the bottle. As the Cypridine dry, crystals of the salts of sea-water form upon them. These salts are hygroscopic and take up water, which results in a slow deterioration of the material unless precautions are taken to prevent access of water-vapor. Some of the salt and much of the débris of the animal’s shell may be removed by a purely mechanical method. The whole Cypridine are finely ground in a mortar and the powder shaken with carbon tetrachloride. On standing, a layer of powder which contains most of the photogenic material, fragments of muscle, etc., is found floating at the surface of the CCl,, while a layer of greater specific gravity at the bottom is found to contain very little photogenic material but much of the ground-up pieces of the shell. The CCl, extracts some of the fatty material and the remainder (with exception of lecithin) can be removed from the powdered animals by ether or petroleum ether in a Soxhlet extractor. These fat solvents do not dissolve or injure the photogenic substances in any way, although they are not sufficient for removal of all the lecithin from dried tissues. For this it is necessary to extract with hot alcohol, but as luciferin is soluble in absolute alcohol and more so in water-aleohol mixtures, we ean not employ alcohol for this purpose. The lecithin in tissues which can not be extracted with ether is probably in combination with pro- teins. The treatment described above gives us a powder which lights briliantly if moistened with water, both of the photogenic substances going into solution. It serves as the raw material for the isolation of luciferin and luciferase. oxyluciferin appears to be very similar to the change leuco-methylene blue— methylene blue. Oxyluciferin can be reduced to luciferin, which will again give light with luciferase by the reductases of muscle tissue, On the Chemistry of Light Production in Luminous Organisms. 107 liver, etc., or by bacteria; by Schardinger’s enzyme of milk; by H.S or the nascent hydrogen from the action of acetic acid on magnesium; and by palladium black and sodium hypophosphite, all well-known reducing methods. The reaction luciferin<—- oxyluciferin is a reversible one and reduction of oxyluciferin no doubt occurs in animals which burn luciferin within the cell, thus tending for conservation of material. Reduction of oxyluciferin will occur even in presence of luciferase if oxygen is absent. Dilute alkali favors oxidation and dilute acid favors the reduction. So far as I have been able to determine, luciferin and oxyluciferin have identical chemical properties. Neither is digested by the enzymes malt diastase, ptyalin, yeast invertase, pepsin, trypsin, steapsin, amy- lopsin, rennin, erepsin, urease, or enzymes occurring in the water extracts of dried spleen, kidney, or liver. Of the above enzymes tried, luciferase is destroyed only by pepsin (probably), trypsin, erepsin, and something in spleen and liver extract. Further properties of Cypridina luciferin and Cypridina luciferase may be noted from the accompanying tables. Luciferase is unquestionably a protein and all its properties agree with those of the albumins. Although used up in oxidizing large quan- tities of luciferin, it behavesin many ways like an enzyme and may be so regarded. Luciferin, on the other hand, is not digested by proteolytic enzymes, is dialyzable, almost but not completely precipitated by saturation with (NH,4)2SOQu,, and is soluble in absolute alcohol, acetone, and some other organic solvents, but not in the strictly fat-solvents like ether, chloro- form, and benzol. There are, however, certain CO-NH linkages which are not attacked by proteolytic enzymes and some peptones soluble in absolute alcohol, so that these two characteristics do not bar it from the group of proteins. Luciferin, in fact, has many properties in common with the proteoses and peptones and may be provisionally placed in a new group of natural proteins on the borderland between the proteose and peptones. 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Property. Luciferase. Luciferin. | ee ence nner ere ee NSE See Salting out— By saturation NaCl......-.---++-- Not precipitated......... Not precipitated. By half-saturation IMieSO@ae 2-1 eer 1D See mois c mcroi ke Do. By saturation MgSOg.....------+- Nearly completely pre- Partially precipitated. cipitated. By saturation MgSO, tacetic acid..|.......+..2e2eseeeeeeess Do. By half-saturation (NHa)2SOz....-- Slightly precipitated. .... Not precipitated. By saturation (NH4)2SO4...-.----- Completely precipitated. . Nearly completely pre- cipitated. By saturation (NHy)2SO¢+acetic |...-..- 20. sere e eee eeee: Do. acid. Solubility in— Methyl alcohol........-----+++++> Insolubles..ciaccic-kr-t Soluble. Ethyl alcohol.........---++s--ee:[eeeee: TD Yee ¢ eee ecnchcee co ac Do. 90 per cent.......--|.-+-+-- WOk cies ose ei Do. 70 per cent.........-|.-----Do pine Bis Ss te ee tere Do. 50 per cent.......-- Slightly soluble.......... Do. Propyl] alcohol........--++-++-++5> Imeolublesee cs cise ar si Do. Isobutyl alcohol.........-+--++++[e eee: WOka ae owo wees Fairly soluble. Amyl alcohol.........--:eseseess[oerees 1D Yap Otic Sido, O.0 aro Slightly soluble. Benzyl alcohol.........-----seee¢[eere WOR chow crow eines Soluble. INGEST He bo DCRR Ome UIE IoI OOo Dl loko Gano 1D Loy nee ee iececien ic Sic Fairly soluble. 90 per cent.......-.-----|-+---: WD Gotan. c ces Meee Soluble. 70 per cent.......----+-> Slightly soluble........-- Do. 50 per cent........-..--- Fairly soluble.........-- Do. Ethyl acetate...........--s+eeee Insolublesoccecn ae ee 0. propionate.......-----eeeecfeeeees Wow asietieeeeesies Fairly soluble. butyratenecericier acer = clin “il siicnn IDE amo oacoenooe Do. MALOU AGC oc dene isa 21.010 1s o's lo el | eorousrs 5 WD) OMe na rictelane overeiek Slightly soluble. ATES do Roe Soto oeool |p moor WO sae eteiste serorenes Very slightly soluble. Glycerine... oc0ct- - se one che pe rena Oo eke ores Ge Oxebsich- Soluble. MED ysis Sc wade > aot ane vecenenre rede le [6'e rele ee IDO a eee RCO Do. TOU hatoes - oe oudiG oan cumin ooo |S ocn os 1DINa ets Sau soaked oe Insoluble. Ghloroform:. ..<- 6 .2c. 0220 = 2a elle mee ee DIDS Gmonancegadme Do. Carbon disulphide. ........---+-+-}erree: DOs ae eet tion Do. tetrachloride. ........--+-]-seee> DO ne oto noose Do. leaiillas codons oe Ode onmccom@bm oo. |aicamicr DOs soa nererseseste = Do Rill sent n oicte eile ioe tere sersvelis avons) lereksie) Iie IDs Ga eceeicne oat Do Belg) se reetescs ns ianesanntn ative c Stehe to siiene'ieys)fpersi= Sirs 1D yer, oe Ore Cotorconc Do. Petroleum ether........-.--++--ees[eeeee? iDYme goo wo aopE ode Do JNtItith, © Opa e Ba cae oeod 0 og obo mad| | manor AD Oe eesecte ensues exer Do. Glacial acetic acid.........---2++s|-eree> IDA so daogsecoo oC Fairly soluble. 110 Papers from the Department of Marine Biology. TaBLe 3.—Properties of photogenic substances. Property. Luciferase. Luciferin. Alkaloidal reagents: Phosphotungstic acid............. Completely precipitated..| Very nearly completely precipitated. Phosphotungstic’and’aceticacidswaiioces « < odieels cco. swe ses Do. Phosphottngstie acid and ECW. ....2 |). ..aeteleeiesics cis wla we sous Completely precipitated. PR ATINIG KA CLG: eharshe, sroyemiers corto eke wie Nearly completely pre- | Nearly completely pre- cipitated. cipitated. Manniciandsacetichacids: «, soca siecle Same ieilete als ceaieve wroness, otcus Do. FRANNICIACIMSE ATMEL Os, cet ais) croton ceases All ceerctatcenvane cae, Sere nae Do. PICHIC* ACI Aa raysqtentor oe a sieie Sirieke aie ator Nearly completely pre- | Not precipitated. cipitated. Picnic andliacetiG ACKAS so oc e ck Sie like arstaie ois e.s Sree mleres we Do IRI CTIO ACI ONAN Gy EL © liste toe crc pastareel| obit oan aotttcoilensu atccla eubare se ccyena eee Do Keb e(CIN) ¢ ANGFAaACetrOv ard seoce coseccosssll ss, cro bsteNOrePeeT a oro, siisre caine fevers Do Heavy metal salts: Basic lead acetate..............-. Completely precipitated..| Not completely precipi- tated. Neutral lead acetate.............. Nearly completely pre- cipitated. Do. Neutral lead acetate and acetic SCIGs 2) OS ii La coe nas Sete rane ncrere Not precipitated. Mereurie*chloride: +. 5...0 4.0. a2. 0 Not precipitated......... Not completely precipi- tated. Mercuric:chloride/and acetic acid’... 4|\2ea Seine k oc es. Almost completely pre- cipitated. Uranyl nitratevand: acetic acid)’. 5.4.c|'s «.s.. eee leeiee ov ersacis.cusie.ae ore Not completely precipi- tated. Acids and alkalies: eee ee mee ee eee ee ee we ewe we we ee | ANOU PICULPILALCU.,...22.06- P1OLS OO OS @ C10 we Ce 06 e100 ee 6 6] 0 0 © 0 ome © Ce C6 6 v6 6 6 0 oe ©)016 (o: fe) 0 ele lotieeie1e. .0 0:6 0.10: 6) je: (el je! es. \9 6:1 ke (0: 1e ja or aU 6: jele, © Lee le ©. (6,6! s ie) ie) 16:5 V: THE OVARY OF FELICHTHYS FELIS, THE GAFF-TOPSAIL CATFISH: ITS STRUCTURE AND FUNCTION. By E. W. GUDGER, Of The North Carolina College for Women, Greensboro, North Carolina. Four plates, one text figure. 111 CONTENTS. PAGE PEON RTOHINS 6 ose 20g di he's avcrat'e ws asad dw Readers ate acdeachcseubite tonciteue, son aks masta tee eee 113 The Breeding Female Gaff-topsail Cathay. .ici..55 56:4. saje are «6 a-ans 208 Siond «jane, pseremearetee 113 PRe' Ovary vieweu xtermany i) 2 ooo... « 5 esi ssa dog Sid OO Mace Ee eioln ee 114 Tnéernal Structure Gr the! Ovary : ace cso. « saree wkgis aioays tare.» olajeons «ects a een ee 115 Relative Sizes of Immature, Spent, Half-ripe, and Ripe Ovaries...................- 117 immature Ovaries. 059) 5.02 Moe sere he ae hoe eee Se nsk s Ee ee Pour © ene 117 pent OVATOS 55 nc 8.8 3s es leila a Oise enw Be Riel n dia ereincle eek Meth eae Ona a 118 Hali-ripeior-nearly ripe: Ovaries as .)..2 i balys eee ta cee owes CO eG ee Sa eee 121 Rane OVETIGs . «sss 285i 4 Re SE < Es Se eee 121 Size:ot ges. and: Number found in Ovary osteo cece sidrenics dias phaeaks « Satoh ee 123 Historical ACCOUNER c).< is dee 3b ash nica desde Sraciet BERENS SAR REE « cee ee 124 Figures 1 and 2 of this paper were drawn by Mr. E. A. Morrison under a grant from the Carnegie Institution of Washington. The photographs were made by the author. 112 THE OVARY OF FELICHTHYS FELIS, THE GAFF-TOPSAIL CATFISH. By E. W. GupcEr. INTRODUCTION. In previous papers (Gudger, 1916, 1918) I have described in some detail the natural history of this interesting fish, and especially the remarkable habit of the male of carrying in his mouth the eggs until they are hatched and the larve until they are capable of fending for themselves. In the present paper attention will be called to the female and her distinctive organ, the ovary, which is at all seasons an interesting structure and at the time of egg-extrusion a most remark- able one. Furthermore, the structure of the ovary of the gaff-topsail is all the more worth study since practically no investigation has been made on this organ in any siluroid, as will be seen when the historical portion of this paper is reached. Moreover, there is but one figure known of the ovary of a catfish. This will be reproduced and described later. A large amount of material and data is at hand for a description of the ovary of the gaff-topsail. All this was incidentally accumulated in the Beaufort (North Carolina) laboratory of the United States Bureau of Fisheries, while collecting material and making notes on the habits and embryology of this fish; and now that work is being done on the ovary, several questions arise to which (unseen before) neither material nor notes give answer. THE BREEDING FEMALE GAFF-TOPSAIL. The ‘‘ripe” female gaff-topsail catfish, 7. e., with eggs ready for extrusion or approaching that condition, may be readily recognized, for her belly becomes greatly distended. Figure 2, plate 2, shows a non-breeding female gaff-topsail and is fairly slender in general outline, but unfortunately I have no contrasting figure of a “ripe” breeding female, showing the balloon-like expansion of the abdomen. The second structure, enabling one easily to recognize the fish ready to extrude eggs, is the extremely vascularized, swollen, and protuber- ant oviducal orifice. This is an infallible sign of ripeness. Males may show a swollen abdomen, due to feeding on fish and crabs, although even at the breeding season they do not have such exaggerated genital orifices, but in the female this is so much enlarged as almost to obscure both the anal and urinary orifices between which it is placed. 113 114 Papers from the Department of Marine Biology. The following measurements will help the reader to realize the relative sizes of adult breeding females: one was 24.5 inches long over all, girth 13 inches; another 23.5 inches long, 13.5 girth; two fish were 23.5 and 24 inches in length, 13 around; one 22 long by 14 around; and greatest of all, 25 inches over all by 19 in circumference. This last fish, huge as she was, was not quite ripe, since the eggs would not come away when pressure was applied to the abdomen. In such a specimen the great distension of the body-walls, due to the swelling ovary, tends to thin these down markedly. In figure 3, plate 2, we have a poor photograph of the 24.5 individual with a girth of 13 inches, as noted above, dissected to show how much of the body-cavity of the fish is occupied by the ovary. This organ was 7.5 inches long, 9.75 inches at its greatest girth, and, although it occupied almost all of the abdominal cavity, was not yet ripe. It weighed 435 grams. In front is seen the stomach crowded close up to the cardiac region, while posteriorly the much reduced intestine emerges from the space between the two lobes of the ovary in which it lies. THE OVARY VIEWED EXTERNALLY. In figure 1, plate 1, an ovary drawn natural size and viewed from the ventral aspect, this organ is seen to consist of two lobes confluent behind and leading into a single oviduct opening between the anus and the urinary orifice. It is of the normal teleostean type, abnormal in size, and in the size of its giant eggs, which may be easily seen through the (at this stage) relatively thin walls. This ovary is7.5 inches in greatest length and 3.5 inches wide, and has one lobe longer than the other, as is generally the case. It is from the 24.5-inch female with a girth of 13 inches, shown with open abdomen in figure 3, plate 2. When drawn, the accidental cut in the ovisac seen in the photograph was not represented. The ovary is slung to the dorsal wall of the body-cavity by the mesovarium, or double layer of peritoneal epithelium. On their dorsal surfaces, the 2 lobes of the ovary are, except at their anterior ends, closely applied, forming a comparatively flattened surface as may be seen in £, figure 8, plate 4. In case the anterior horns of the ovary diverge, the peritoneal covering forms a transparent sheet stretched dorsally across the space separating them. On the ventral surface, however, the lobes curve sharply inwardly and dorsally, leav- ing between them a region inverted V-shaped in transverse section. Dorsally the ovary is closely applied to the kidneys, and ventrally the now much reduced intestine is closely applied to the base of the inverted V, between the two lobes of the ovary, tightly held in the seam where the peritoneal coats of each ovisac are closely applied. As the breeding season approaches, the eggs and their containing ovisacs grow larger, more and more filling the cavity of the abdomen The Ovary of Felichthys Felis, the Gaff-topsail Catfish. 115 and crowding the stomach forward (unfortunately my notes make no mention of the liver, which presumably shrinks in size somewhat), and the intestine becomes greatly reduced, stringy, and filled with mucus. Apparently the female does not feed as the breeding season approaches its height, or if she does feed it must be on very small objects, since there is no room in the stomach for the common, but bulky, diet of crabs. The ovary is richly supplied with blood-vessels which descend from the hinder region of the kidneys and reach the ovary at about the point where the ovisacs unite to form the short oviduct. Here the vessel forks, one branch going to the oviducal part and the other to the ovisacs. INTERNAL STRUCTURE OF THE OVARY. The general internal arrangements of the ovary are, in the main, correctly foretold by the external appearance—two hollow pouches closed in front and confluent behind into a short, common oviduct whose length and diameter are approximately equal. However, each ovisac from its point of junction to its forward extremity is divided roughly into three nearly equal parts, of which only the foremost bears eggs. The posterior third is raised up into closely placed longi- tudinal folds or plaits, which at the time for oviposition allow for the great distention due to the passage of the 18 to 20 mm. eggs. These eggs are developed in the anterior third of the ovisac, each egg in a follicle swung by a long pedicel. Thus we see that only the anterior third of each ovisac produces eggs, while the hinder third is an oviduct pure and simple, which unites with its fellow to form the short, unpaired tube leading to the genital aperture. However, between the plicated hinder section of the gonad and the anterior ovigerous region is a debatable land which belongs hardly to either and yet to both. As the plice extend forward they decrease in height and, losing their distinctive form as mere folds, become covered on both sides with small eggs. Further forward they become mere ridges, but distinct enough for the eggs to show up in rows. Still further forward the ridges disappear, and this region of small eggs finally disappears into that wherein the functional eggs are formed. ‘These structures may be fairly well seen in figures 4 and 5 of plate 3, in figure 6 of plate 4, and in c of figure 8, plate 4. In the forward region the arrangement of the eggs in longitudinal rows is completely lost—there is no longer any definite arrangement whatever’ This division of the ovisacs into anterior egg-bearing and posterior plicated oviducal parts with an intermediate region is not true of immature and spent ovaries only. As the time for oviposition approaches, the eggs in the forward ends of the sacs become greatly enlarged, while the debatable region develops great numbers of small eggs which come away readily and which seem never to become func- 116 Papers from the Department of Marine Biology. tional. The regions of large functional eggs and of small functionless eggs are marked off by an irregular but well-defined line. Forward of this line are found scattered eggs, large and small, destined to become mature and oviposited; behind it are closely aggregated large masses of small eggs, of whose function I am entirely ignorant. These regions and their eggs are very clearly seen in figure 1 of plate 1, an external view, and figure 4 of plate 3, an internal view of the same ovary. However, as the functional eggs ripen, the pedicels of those lying near the line of demarcation greatly lengthen and the eggs pushed backward by the growing ones in front become crowded in among the smaller eggs in the intermediate section, as may be clearly seen in figure 4 of plate 3 and figure 6 of plate 4. Thus it is that in spent or immature ovaries the ovigerous and oviducal portions of the ovary are about equal in length, while in the pregnant organ the developing eggs and the distention brought about thereby cause each ovisac to become apparently divided into two sections of about equal length—the ovigerous section being in front and the oviducal portion behind. These phenomena will be made clearer in the section dealing with the changes of size. When the ripe eggs break from their follicles they fall into the lumen of the ovisac and thence pass into the rugose oviducal section and so to the exterior. The follicles are left behind and are very prominent in an ovary from which the eggs have recently been extruded. This is shown very clearly in figure 5 of plate 3. Shortly after the breeding season these emptied follicles entirely disappear, are completely resorbed, leaving only the 1 to 5 mm. eggs which will develop into those of next season’s laying. Small eggs in small vesicles standing on short pedicels are found under and around the pedicels of the large eggs; they line the whole of the interior of the ovigerous portion of each sac. The walls of the ovary are fairly thick (about one-eighth inch) and tough in the resting stage, but they are very distensible, and, as the huge eggs develop, the walls become as thin and (especially in the ante- rior section) as transparent as oiled paper. The walls of the ovary are composed of two layers, the inner or germinal layer, from which the eggs and their follicles are developed, and the outer or peritoneal envelope. In ovaries which have been in weak formalin for 8 or 10 years, the two layers can be easily separated. To bring in the food materials required for building up the large number of these giant eggs, a generous blood supply must be provided. The ovarian artery descends from the hinder region of the kidneys about the level of the posterior third of the ovary, but before reaching this organ it divides, one branch going forward to the egg-forming section, the other backward to the oviducal portion. There seems to be some sort of mechanism to send the major portion of the blood to that part The Ovary of Felichthys Felis, the Gaff-topsail Catfish. 117 of the ovary needing it. While the eggs are in the process of making, the greater volume of blood goes to and through the forward branch, but as the breeding season approaches more and more blood is sent to the hinder portion, the plaits of which become blood-red in color and very much enlarged, while the genital papilla increases from a small and very indistinct pore to a genuine papilla. The eggs are very large, ranging from 17 to 22 mm. in diameter when ripe. The investing follicles are everywhere permeated with a network of blood-vessels, and just before the eggs ripen the whole anterior section of each sac is almost blood-red in color. Examined more closely each follicle shows beautifully its mesh of interwoven blood-vessels. This is faintly shown in figures 5 and 6, plates 3 and 4. When the follicles burst to set the eggs free into the lumen of the sac — a good deal of blood is lost, and if at this time the fluid from the ovi- duct is examined microscopically large numbers of white and red cor- puscles will be found mixed with small eggs, which have been torn off and are moving toward the exterior. It is surprising that there is not more bleeding. Whether the elastic fibers of the follicles by their contraction check the bleeding, or whether there is some secretion in the blood which causes contraction of the muscular fibers of the arteries, can not be said, but the bleeding shortly stops and the clots are a marked feature of the blood-vessels in the evacuated follicles. Probably the oviducal portion of the ovary secretes a mucus as a lubricant to aid in the outslipping of the eggs, a thing quite necessary when one considers the size of the eggs in proportion to the normal size of the exit channel. RELATIVE SIZES OF IMMATURE, SPENT, AND RIPE OVARIES. It is desirable to make comparison of the sizes, relative and absolute, of ovaries in the three stages indicated above. The large amount of material at hand is presented in the form of the tables appended, which, however, form a fairly graded whole, though in only a few cases can data be given for the size of the fish and the date of capture. These specimens have all been in formalin from 7 to 10 years, and hence their measurements are somewhat less than when they were alive and fresh. The weights are accurate, the lengths and circumferences approximate to within a few millimeters. IMMATURE OVARIES. In the ovaries noted in table 1, there was no evidence that eggs had ever come to maturity and no empty follicles. In the posterior section of the ovigerous portion of the ovisacs were minute straw- colored eggs, while in the forward part the eggs had the yellow of real yolk. The largest eggs ranged from 2 to 3 mm. in diameter, the average being about 2.5 mm. No difficulty was had, even in these 118 Papers from the Department of Marine Biology. small ovaries, in telling where one region left off and the other began; the line between them, while not a definite and straight one, was very clearly discernible. TABLE 1.—Immature ovaries. Notes on ovaries and eggs. No. |Weight.|Length.| Girth. Posterior section. Anterior section. ==" | grams. | mm. mm. 1 21 45 35 Eggs below 0.5 mm. diam..... Eggsupto3 mm. diam. 25] 4 56 36 Eggs below 0.5 mm. diam..... Eggs up to 2.5 mm. diam. 3! 7.4 57 57 Eggs below 0.6 mm. diam..... Eggs up to 2.6 mm. diam. a 4 Theth 67 67 Eggs microscopic........... Eggsupto2 mm. diam. 5 hid 87 67 Eggs below 0.5 mm. diam..... Eggs up to 2.5 mm. diam. 26! S55 67 75 Eggs microscopic........... Eggs up to 2.5 mm. diam. Ave.| 6.4 63 56 Eggs not above 0.5 mm. diam.} Eggs not above 2.6 mm. diam. In this table are given measurements for 6 ovaries, varying in weight from 2.1 to 9.3 grams, in length from 45 to 87 mm., and in circum- ference from 35 to 75 mm., but it should be noted that maximum weight, length, and circumference are not all found in the same ovary. The average weight is 6.4 grams, the mean length 63 mm., while the average girth is 56 mm. On the whole, this set of ovaries presents a fairly graded series, specimens of which are seen in A, B, and ¢, figure 8, plate 4. SPENT OVARIES No. 1 in table 2 is a stray ovary found packed among immature organs of this kind. It is known to be spent because it has a number of torn and empty follicles. As to ovaries 4 to 8, some brief notes were made when the fish were dissected. They were preserved July 26, 1907, and comparison of the measurements made then with the above shows that the preservative has caused but little appreciable shrinking. Their live measurements were 2.25, 2.5, 2.75 inches over all, the average being 2.5 inches. Because these brief data can be given, these ovaries are grouped together in the table. Unfortunately no record was made of the sizes of the fish possessing these organs, but this very fact indicates that they were normal in size, probably running 17 to 22 inches long, with the majority about 18 or 19 inches. It should be definitely noted here that the line is very clearly marked between the anterior region of functional eggs and the posterior one of small straw-colored eggs. The great masses of 3 to 8 mm. eggs found in this region in ripe ovaries have been swept clean away, leaving this portion of the ovisacs sparsely covered with very small eggs in a single layer set closely upon the germinal epithelium. It will also be noted that some ovaries, and they the largest, have eggs in the forward portion of the ovisacs ranging up to 8 mm. in diameter. The sparse references in my notes indicate that these ovaries were The Ovary of Felichthys Felis, the Gaff-topsail Catfish. 119 collected in July, and probably after the 15th, and hence these eggs may be considered as belonging to next year’s laying. However, the presence of these large eggs, together with other data, leads to the conjecture that there may be a second laying later in the season. TaBLE 2.—Spent ovaries. No. Weight. | Length. | Girth. | Posterior eggs. Anterior eggs. grams mm mm. 1 6.1 52 65 Very small. Upto 3 mm. 2 7.9 55 81 oO By 3 OFT 73 83 Do 4 Me 4 9.8 60 (2 Do Dye 5 iy 58 87 Do 4 de 6 IES 62 70 Do 3 4 th 13.8 67 77 Do 3 uy, 8 22.4 70 95 Do 8 1% 9 10 56 74 Do 5 22 10 AD 60 70 Do 3 7 11 13 57 85 Do 5 , 12 14.2 63 80 Do 3 3 13 14.8 61 95 Do 9 ”—_one 18 mm. 14 18 65 80 Do Y ” 15 18 75 93 Do 8 16 23.4 72 98 Do. @ We il7/ 25.4 83 105 Do: 6 18 26.9 67 120 | About 1 mm. Average..| 14.9 64.2 85 Average maximum size of anterior or functional eggs about 5 mm. A brief analysis of table 2 shows that ovary No. 1 is about equal in size (weight, length, and circumference) to the average of the immature ovaries. Beginning with a weight of 6.1 grams, there is a progressive increase to No. 18, with a weight of 26.9 grams, the average being 14.9 grams. In length, the extremes are 52 to 83 mm., and in circumference from 65 to 120 mm. Here again we do not find all three maxima in the same organ. Contrasted with these extremes the averages are: for weight, 14.9 grams; for length, 64.2 mm.; for circumference, 85 mm. Unfortunately these ovaries were all dis- sected before any thought was had of the question of volume, but measurements were made of the largest eggs of next year’s crop, the extremes being about 2.5 to 9 mm., with an average of about 5 mm. These ovaries are known to be all spent, by reason of the numbers of torn and evacuated follicles in their forward regions. These were too small to be accurately counted in most of these organs. For a figure of such an ovary, see No. 5, plate 3. Table 3 lists a number of ovaries collected at various times, but all ‘‘spent.”? All were measured and some dissected. They seemed to be from larger fish than the preceding. It will be seen that the ovisacs are frequently of unequal length, but no data were collected to show whether the right or left was uniformly the longer. No. 5 is probably the ovary shown in figure 5, plate 3. The length of the 120 Papers from the Department of Marine Biology. ovisac is 98 mm. The opened right one contains 23 empty follicles. Toward the hinder portion of this pouch are seen great numbers of much produced folds of the germinal epithelium of the oviducal section of the egg-bag. Some of these folds were as much as 9 mm. TABLE 3.—Spent ovaries—second lot. Girth. Miscellaneous notes. Left ovisac 10 mm. longer than right one. Girth of neck of oviduct 70 mm. Right ovisac had 13 empty follicles. Right ovisac with 31 empty follicles, left with 16. Right ovisac with 23 empty follicles. See fig. 5, pl. 3. Left ovisac 95 mm. long. Girth neck oviduct 53 mm. Largest eggs up to 7 mm. diameter. Right ovisac 90 mm. long and 95 mm. in girth. 1 2 3 4 5 6 7 8 Ave. 62.8 | 53.3 high. These, however, bear no eggs. Anterior to these and in the middle section of the ovary are the many ruptured follicles of the small straw-colored, non-functional eggs which always break away and pass out with the functional eggs. These latter come always from the anterior region of each pouch. Here are seen their empty follicles and between them eggs 3 to 5 mm. in diameter. These are the beginnings of next year’s crop. The wall of this ovary is very much contracted, thick, stiff, leathery, in marked contrast to the thin parchment-like wall of this section distended in the pregnant organ. Equally marked is the delimitation of the ovigerous part of the ovisac into regions bearing functional and non-functional eggs. In the later the larger eggs have the longer pedicels. The data for No. 4 show the shrinkage possible in a spent ovary. This ovary (which is less than twice as large as the smallest one re- corded in this table and but slightly more than half the size of the largest) has 31 empty follicles in the right sac and 16 in the left; it carried 47 eggs, ranging from 17 to 22 mm. in diameter, and yet in the spent state it weighs but 53.3 grams (less than 2 ounces) and is only 82 mm. (slightly over 3 inches) long and 120 mm. in circumference. Ovary No. 7 of table 4 has had its ovisacs split apart and the left one dissected. This sac has a length of 103 mm. It contains 15 empty follicles, partly resorbed, and about 45 eggs from 7 to 12 mm. in diameter. In the other sac 12 mm. eggs were also found. The finding of empty follicles and such large eggs in the same ovary tends to confirm the hypothesis that there may possibly be two layings in a season. The largest ovary of the first lot, No. 18, weighed 26.9 grams and measured 67 by 120 mm.; the smallest of the second lot weighed 31.1 The Ovary of Felichthys Felis, the Gaff-topsail Catfish. 121 grams and measured 83 by 105 mm.; so here again we find no gap in the series. In the present table the extremes in weight are from 31.1 to 101.6 grams, with an average of 62.8; in volume from 30 to 80 E02, with the average (for 4) 53.3 c.c.; the lengths from 80 to 103, average 92 mm.; while the figures for the circumference run from 105 to 150, averaging (6) 122.7 mm. HALF RIPE OR NEARLY RIPE OVARIES. There is now to be considered a small group of half-ripe ovaries, judged to be in this condition because they contain eggs approaching ripeness and because no empty follicles are found in them. They are 7 in number, and all but 2 were dissected as to one or both ovisacs when taken from the fish; hence not all dimensions and volumes can be given. The average weight is about the same as that of the larger- sized spent ovaries, but the average volume, length, and girth of present lot are much greater, on account of the difference in the contents of the two sets of organs. The volumetric measurements are given for two ovaries only. In the case of No. 3, the large measure- ments are explained by the presence of the 40 eggs of 15 mm. diameter, and for No. 7 by 25 eggs of about 13 mm. The average size of the next year’s eggs is about 4.4 mm., and for the unripe eggs of the present year about 14.7 mm. TaBLE 4.—Half-ripe, or nearly ripe ovaries. No. | Weight. | Volume. | Length. | Girth. Remarks. eS Oe eee 1 SOGOW [Pee eee SO: yt) [sir Small eggs to 2.5 mm., adult eggs to 14 mm. 2 SOS, alcrcetocteises 3 TS bn hell onedu eee Small eggs to 4 mm., adult eggs to 10 mm. 3 53 155 103 Small eggs to 7 mm., 40 adult eggs to 15 mm. 4 BO. Se Sa er as eee Small eggs to 5 mm., adult eggs to 8 mm. 5 (CEC: I PR eee 83) ikeeerack Small eggs to 4 mm., adult eggs to 10 mm. 6 Slee lees Ua | ere Small eggs to 3 mm., adult eggs to 17 mm. 7 109.2 90 107 Ovary crowded with 25 eggs 11 to 13 mm. diam. Pee eg a a a dy Te Ave.| 62.3 22 88.5 168.5] Small eggs about 4.4 mm., adult eggs 11 to 13 mm. diam. It is interesting to note that in these ovaries there are found in the strictly ovigerous section two kinds of eggs: the large dark-yellow eggs nearing ripeness (from 8 to 17 mm. in diameter) and standing on long pedicels, while beneath, on short pedicels sitting close to the germinal epithelium, are the smaller straw-colored eggs of next year’s crop. These average from 2.5 to 4 mm. in diameter. In that portion of each sac approaching the plicated folds of the oviduct the eggs are all small, rarely reaching a diameter greater than 2 mm., even in the most advanced organs. For such an organ, see D, figure 8, plate 4. RIPE OVARIES. Table 5 deals with ovaries containing ripe eggs, within a few days of extrusion, both eggs and ovaries attaining a size out of all expec- 122 Papers from the Department of Marine Biology. tation for a teleostean fish, which in hundreds of specimens rarely exceeded 2 feet in total length between perpendiculars. These ovaries were collected during the last 10 days in May or the first week in June, these dates being the limits of the breeding season of this fish at Beau- fort (North Carolina), where it was studied. Exact data for time and TasBLe 5.—Ripe ovaries. No. Weight. | Volume. | Length. | Girth. Miscellaneous notes. grams c.C. mm mm ik 217.4 197.7 150 184 Right ovisac 16 mm. longer than left. 2 237.6 212 115 235 Right ovisaec slightly longer than left. 3 310 265.5 158 230 Left ovisac slightly longer and larger than right. 4 339 307.8 162 213 Fresh; weight 354 g., length 172 mm., girth, 225 mm. 5 395 388.6 155 256 Width 106 mm. 6 401.8 366.2 177 220 Fresh; weight 435 g., length 190 mm., girth 247 mm. 7 433 379.9 170 230 Left ovisac slightly longer than right. 8 469 438 180 243 Girth oviducts at junction, 180 mm. Ave 350.4 319.5 158.4] 226.4 for size of fish can be given for 4 specimens only, the labels on the others having gone to pieces. However, the time limits are correct and it should be noted that the largest fish I ever dissected was 25 inches long. As in the preceding cases, the order of arrangement in this table is ascending. To one accustomed to the ordinary-sized ovaries found in a teleost not exceeding 2 feet in length, the figures in table 5 are almost un- believable. None of these ovaries, it should be noted, was absolutely ripe, for from none would eggs come away, yet the smallest weighed 217.4 grams and displaced 197.7 c.c. of water. At the other end of the list, No. 8 weighed 469 grams and had a displacement of 438 c.c. The average weight of the 8 is 350.4 grams, the average volume is 319.5 c.c., the mean length 158.4 mm. and the mean circumference 226.4 mm. Specimen No. 6 was collected May 30, 1909, from a fish 24.5 inches long. When fresh it weighed 435 grams, and after being in formalin for nearly nine years its weight was 401.8 grams. Its length when fresh was 190 mm. (the left lobe being slightly the longer); to-day it is 177 mm. When just excised its circumference at the junction of the oviducts was 175 mm. and its greatest girth 247 mm. Its greatest circumference is now 220 mm. This ovary is seen in ventral view in figure 1, plate 1, and in horizontal longitudinal section in figure 4, plate 3. On being dissected, in order to make the photo- graph from this latter figure, the right lobe was found to contain 26 eggs, the left 20. Ovary No. 2 of the above table was dissected and found to have 21 eggs in the right sac and 19 in the left. These eggs in their pediceled The Ovary of Felichthys Felis, the Gaff-topsail Catfish. 123 vesicles were all so closely crowded in the anterior section that the sides were flattened. The anterior third of this ovary was greatly distended, the wall of the ovisac being so thin as readily to permit the passage of light. The thin parchment-like peritoneal layer would easily come away from the germinal epithelium. The elongated pedi- cels of the posterior eggs allowed them to be carried back into the region of non-functional eggs in the intermediate section among which they were found nested. Ovary No. 3 was also dissected, and its left lobe was found to con- tain 2 empty follicles and 27 large eggs, about 20 mm. in diameter. Presuming that the right sac contains an equal number, the total for this organ will be 54. The walls of the intermediate section of this ovary are very thick and leathery, measuring 5 mm. from the outside to the lumen of the oviduct. Some of the plicated oviducal folds have considerable height, measuring as much as 7 mm. Wehave here studied 5 sets of ovaries, making a fairly complete series, from very small to very large. We had first very small, immature ovaries; next small, spent ovaries; next larger, spent ovaries; then half-ripe ovaries, and lastly these organs with eggs nearly ripe enough for extrusion. In Nos. 1, 2, 3 the ovaries are comparatively thick- walled, while in Nos. 4 and 5 the great enlargement brought about by the growing eggs is shown in the distention and thinning down of the walls of the anterior section. SIZE OF EGGS AND NUMBER FOUND IN OVARY. In the paper elsewhere referred to (Gudger, 1918) the question of the size of the ripe eggs has been considered in considerable detail. Also, in the section dealing with ripe ovaries, considerable data have been given touching the matter of the size of the eggs. Hence it will be sufficient here to give the mere facts. From the large number of measurements made, the following may be selected as thoroughly representative. Of live eggs, 138 measured as follows: longest diameter, 17.5 mm. minimum to 21.5 mm. maximum. The largest numbers were 39 eggs of 19 mm. diameter; 38 of 20 mm.; 19 of 21 mm.; 22 of 21.5 mm.; average for 138 live eggs, 18.8 mm. Of preserved eggs, 189 measurements were made; the smallest were 16.5 mm. and the largest 22 mm. in diameter. The largest numbers here were 34 eggs of 19 mm., 64 of 20 mm., and 49 of 21 mm. The average for 189 eggs was 20 mm., and the general average for 327 eggs was 19.5 mm. in diameter. The largest numbers of eggs were found in the 19, 20, and 21 mm. sets, the 20 mm. eggs being the most numerous—102 out of 327 eggs measured. Normal eggs in early stages of development may be seen in figure 7, plate 4. In the section dealing with ripe ovaries, the numbers of eggs found therein and nearly ready for extrusion have been noted. Specimen 124 Papers from the Department of Marine Biology. No. 6 of the table, represented in external view in figure 3 of plate 2 and in longitudinal horizontal section in figure 4 of plate 3, contained 46 eggs. Specimen No. 8 of the table, the largest ovary examined, contained only 12 grown eggs in the right sac and 13 in the left. This ovary, for which, unfortunately, no date of capture can be given, since the label has gone to pieces, was, notwithstanding its great size, plainly not ripe. In the right sac were about 20 and in the left about 25 eggs of 10 to 12 mm. in diameter. This tends to confirm the idea previously expressed that there may be more than one egg-laying in a season. If all these eggs were laid in one season, this ovary would have a productive capacity of about 70 eggs. Ovary No. 2 of the table contained 46 fully grown eggs, while No. 3 gave up 55. However, it is probable that these figures do not repre- sent the maximum. Only one live specimen was studied. A female taken May 27, 1909, was kept in a tank for 4 days. She was then spawned and forced to give up 68 of these huge eggs. This is the largest number of grown eggs I have ever obtained from one fish and is probably the maximum number found in the ovary of any female gaff-topsail catfish. HISTORICAL ACCOUNTS. It is surprising to find how little is known about the structure of the ovary of any catfish. So far as the writer knows, no real investigation of this interesting organ has ever been made, and the data now to be presented have been noted by various writers as merely incidental in the course of other investigations on catfishes. As to the ovary of the gaff-topsail, the only two known references will now be briefly con- sidered, and after that the strictly chronological order will be adhered to. In 18848. C. Clarke wrote: ‘“‘The eggs of this species are golden yellow, and of the size of grapes, which they much resemble, in bunches of ten or twelve.”’ And again in 1892, ‘‘He slashed it [the gaff-top- sail] open with his knife, bringing out a bunch of eggs in form and color like golden grapes.”’ In this connection the attention of the reader is called to figure 6, plate 4. The oldest direct reference to the ovary of the catfish, which has been found so far, is by Bonnaterre in 1788. He speaks of the eggs of Silurus ascite as being ‘‘disposed on each side of the abdomen in two packages [ovisacs?], which extend from the diaphragm clear back to the anus.” If his brief description is referred to figure 3 of plate 2, of this paper, it will be seen how accurately he wrote 130 years ago. Next to Bonnaterre, our oldest and most numerous references (for there are no less than 14) are from the Austrian ichthyologist, Rudolph Kner. Writing of Siluride from Brazil, in 1858, he gives the interesting data cited below, together with the only known figure of the ovary of a catfish. Of Arius luniscutis Cuvier and Valenciennes, he says: The Ovary of Felichthys Felis, the Gaff-topsail Catfish. 125 “Figure 6 [reproduced herein as text-figure 1] shows the ovary of the female in natural size [75 mm. long and 44 mm. wide] with the left side partly cut open lengthwise in order to show the already developed large and also the unripe [small] eggs hanging to its walls. This ovary contains 12 to 14 ripe eggs, the largest of 5 lines in diameter, and these like the others, witha small pedicel like an umbilical cord, sit fast on the wall of the ovary and the [thin] yolk skin or sac allows no trace of any embryo to be seen through it: This remarkable difference in the size of the egg at one and the same time and in the same ovary causes one to conjecture that viviparity is not practiced by this fish.” Of Bagrus mesops and Arius quadriscutis, Kner says that their ovarian structures are identical with those of Arius luniscutis; while of Galeichthys gronovit he notes that the ovaries are like those in Arius, and adds that they are made of 2 thick- walled sacs which are confluent behind into a wide oviduct opening out between the anal and urethral orifices. Cetopsis goboides has the long, closed ovarian sacs united along their whole dorsal surfaces by folds of skin to the middle line of the body. At the approach of the breeding season the large eggs cause the ovary to reach forward fully half the length of the body-cavity, but on the other hand at its breeding season the ovaries of Pimelodus sebe occupy the whole length of the body-cavity. P. bufonius also has large egg-sacs and P. laticaudus at breeding time has a markedly turgid urinogenital papilla. Kner examined the ovaries of two species of Auchenipterus. Of these, A. ceratophysus probably was not taken at qyyemcure 1.—Ovary of Arius lu- breeding season, since the ovary occupied _niscutis, after Kner, 1858. With the about half the body-cavity, but of A. exception of those given herein for Felichthys felis, this is the only nodosus he Says: known figure of the ovary of acatfish. “The ovaries of the females occupy at the laying season the belly cavity clear to the base of the pectoral fins, are nearly cylindrical, are bound to- gether at their fore ends by a membrane and open out behind into a very wide oviduct.” Finally Kner says of Centromochlus aulopygius, Ageniosus militaris, A. brevifilis, and Hypothalmus fimbriatus that they show no remarkable ovarian development, this being probably due to the fact that they were not taken at the breeding season. The next reference is to Peter Bleeker (1858) and is merely incidental. In describing Arius arius, of the Indian Archipelago, he says that 126 Papers from the Department of Marine Biology. the females retain their eggs for a long time and that as these eggs gradually attain the size of pigeon eggs “the abdomen often swells up above the ordinary.’’ Hardly more definite is Wyman (1859), in writing of siluroids of Surinam: “The eggs become quite large before they leave the ovaries, and are ar- ranged in three zones corresponding to three successive broods, and probably to be discharged in three successive years: the mature eggs of a jarra-bakka, 18 inches long, measure three-fourths of an inch in diameter; those of the second, one-fourth; and those of the third are very minute, about one- sixteenth of an inch.” Not so indefinite, however, is William Turner, the distinguished anatomist of Edinburgh, to whom Rev. Bancroft Boake had sent some specimens of Arius boakei, an oral gestator of Ceylon. Among these was one female, of which Turner writes (1867): “From the appearance of the abdomen it was evident that the ovaries were distended; and on opening into the cavity I found a sac-like ovary on each side of the middle line. Each ovary measured 21% inches in length, and extended forward almost as far as the pectoral fin, where it formed a rounded free end, whilst posteriorly it was somewhat constricted, and opened by an orifice common to it and its fellow immediately behind the anus. The ovisac contained a very large number of eggs in various stages of growth. Some were like minute granules, others (and these very numerous) like medium-sized shot, whilst a third set equaled in size grapes, or small cherries, and very materially exceeded therefore the size usually attained by the eggs of osseous fishes. These last, only six in number in each ovary, had evidently almost reached the full period of intra-ovarian growth. Each ovum was attached to the inner wall of the ovisac by an independent pedicel, the atrophy of which would necessarily precede the discharge of the egg.” Equally definite is Francis Day (1873), who gives us an account of the structure of the ovary of certain Indian siluroids. In describing the habits of a number of well-known marine catfishes practicing oral incubation, he says: “Next, the females came under observation. On tracing up the ovisacs it appeared that a very large number of eggs existed in them, but no¢ all of the same size [italics Day’s]. On the part farthest removed from the outlet the eggs were of full size (about half an inch in diameter), and about 50 in number; whilst other batches of much smaller size existed, evidently to take the place in due time of the larger ones when they had been deposited. The full-sized eggs were each attached to the inside of the ovisac by a pedicel of varying length, distinctly supplied with blood-vessels of considerable size.” The next notice of the ovaries of an Indian catfish is an incidental one from Edgar Thurston in 1900. On the Malabar coast of south- west India he dissected an Arius (species undetermined) and found that what he calls the ‘double uterine cavities’ contained respectively 56 and 75 eggs of about 13 mm. in diameter. At the 1907 meeting of the French Association for the Advance- ment of Science, Jacques Pellegrin read a paper on buccal incubation The Ovary of Felichthys Felis, the Gaff-topsail Catfish. 127 in two catfishes of the genus Arius from the fresh waters of Guiana. His paper, published in 1908, contains a good description of the ovary of Arius fissus. His statement is as follows: “ Autopsy showed a female (208 mm. in extreme length) with 2 voluminous ovisacs moderately elongated and about equal. The left ovisac was about 50 mm. long, 18 high, and 8 thick. After having been opened it was found to contain eggs in three clearly marked-off stages of development. Princi- pally, in the posterior region there is found a mass of little rounded ovules, more or less ovoidal, grayish-yellow, extremely numerous, and about 0.25 mm. in diameter. On the ‘face externe et inferiere’ and between the large ovules are found some hundreds of medium-sized ovules, more or less ovoidal, grayish-yellow, having a large diameter of 1.5 mm. and a small diameter of from 1 to 1.5 mm. All the rest of the gland is filled with enormous ovules, rounded at maturity, dark green, tightly compressed against one another and superimposed in three ranks like the seeds of a pomegranate, to the number of a score, and having a diameter of about 6 to 7 mm. The right ovisac being exactly similar, there were on the walls some 40 eggs which were deemed ready for laying.” Last of all, Willey, writing of Boake’s Arius falcarius (boaket) of Ceylon, says (1910): “The ovaries of an adult female contain a very great number of eggs in different stages of growth, but of these only a few become mature at a time and there is a great contrast in size between the mature and the immature ovarian ova. In one case there were only 10 large eggs in the right ovary and 8 m the left. In another there were 21 large eggs in the right and 24 in the left.” For another teleost, but one far removed in time and place from Felichthys, Weber (1908) may be quoted concerning A pogon beaufort, a Cheilodypterid fish from New Guinea: “cc : the forward part of whose ovary bore large eggs in long stalked follicles, while the hinder part encompassed numberless small eggs, between which only here and there a large egg lay. Perhaps it is the rule that only a small number of eggs become ripe while the others undergo resorption.’’ Bearing on some of the points referred to in the preceding notes, Louis Agassiz (1868) may be quoted on the turtles of the Amazon: “|'They] always contain several sets of eggs. Those which will be laid this year are the largest; those of the following year are next in size; those of two years hence still smaller; until we come to eggs so small that it is impossible to perceive any difference between their various phases of devel- opment.” LITERATURE CITED. 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