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Les diagrammes suivants illustrent la m6thode. 1 2 3 4 5 6 MIC»OCOfV *iSOlUTK>N TBI CHART 'ANSI and ISO TEST CHART No 2: 1.0 I.I 1^ 128 f^ 13. 1^ 12.2 i:s 110 1.8 i:25 i 1.4 1.6 jS applied IIVMGE In m ■<,,(r.e ■'6) US' ^s "6; ^•iB ■ S'iflJ lo >Mf: ; E»ii-i-'5i«'^8f : : !(»Hii^££i;; t EGG MATURATION, CHROMOSOMES AND SPERMATOGENESIS IN CYCLOPS. BY ROBERT CHAMBERS. M.A.. Ph.D. t ti- I, Tl ^' TABLE OF CONTENTS. PAGE I —Introduction 5 11.— Material and Method 6 III.— The Cyclops "Tetrad" 7 IV.— Maturation of the Ovum i6 v.— Chromosome Number in Cyclops: (a) Chromosomes in the Cierm-tract . 17 (6) Chromosome Counts in Different Species . 18 VI.— Chromosome Size-relations and Arrange- ment IN Cyclops parous ai VII.— Spermatogenesis in Cyclops americanus: I . Literature 33 a. The Keimpolster. 33 3. Multiplication Zone 34 4. Synizcsis and Synapsis Zore 35 5. Early and Late Dialcinesis 36 6. Maturation 36 7. Spermiogenesis 37 VIII.— Summary. 37 Literature Cited 39 Explanation of Plates 34 •*>V5s M ^,^m'j^ r w.^^ EGG MATURATION, CHROMOSOMES AND SPERMATOGENESIS IN CYCLOPS.* I . — I NTRODUCTION. Most species of Cyclops show some degree of periodicity in their breeding habits. Cyclops viridis and its close re- latives, C. parous, brevispinosus, and americanus, with which this paper has most to do, may be found in sexual activity all the year round. They are, however, especially active during the spring months. During copulation, the male, which is about one-third to one-half the size of the female, attaches a pair of sper- matophores to the median ventral aperture of the seminal receptacle which lies in the first abdominal segment of the female (cf. Wolf '05). The peculiar ejaculatory bodies in the spermatophores then swell and drive the spermatozoa into the seminal receptacle. Oogonial mitoses occur periodically so that there is al- ways a number of cells in the same stage being gradually car- ried onward through the ovary. When the oocytes pass into the paired oviducts they grow very rapidly and, by distending the several branches of the oviducts, cause them to occupy the greater part of the interior of the cephalothorax. A gel- atinous material fills the distal ends of the two oviducts, and as the eggs pass out this is pushed out ahead to form a large distensible sac in which the eggs come to lie (cf. Gruber '78). The eggs are fertilized as they roll in rapid succession out of the oviducts. Development is, therefore, compara- tively uniform for all the eggs in both egg sacs. A female may lay si.x to seven batches of eggs, all of which may be fertilized by the spermatozoa derived from one male. These batches, consisting of thirty to fifty eggs, are •Recommended for publication i'l the L'niversity or Toronto Studies by Professor R. Ramsay Wright, Profcosor of Biology, University of Toronto. Paper received May, 1912. '*:^imtm^j§/f^J. 6 Chambers: Chromosomes in Cyclops generally deposited once every two to four days. The eggs are laid usually in the early hours of the morning (cf. Haecker 97)- Ova showing maturation divisions I secured between a and 5 a.m., after having isolated the night before a number of gravid females. These are easily distinguishable because the yolk-laden eggs in their interior makes them appear black m transmitted light. Spermatogonia! mitoses exhibit a certain degree of peri- odicity which is by no means so marked as in the oogonial divisions. Males are usually found in abundance together with the females. Their span of life, however, seems to be shorter than that of the females, and periods occur when few or no males are to be found. II. — Material and Method. An easy method for collecting an abundance of material IS the following: Scour the ditch or pond with a fine cheese- cloth net; invert the net in a jai of water and shake so as to release the catch into the water; pour the water through a coaise sieve so as to get rid of leaves and larger undesired objects. Then pour the water into a funnel plugged with absorbent cotton. Whtr. the water has diained through, pick out the plug of cotton with a pair of forceps, turn it over and dip rapidly into the fixing-fluid. Most if not all the Cyclops will at once liberate themselves from the cotton and soon fall to the bottom of tne fluid. As fixing-fluids I have used sublimate alcohol in various proportions both hot and cold, picro-acetic alcohol (McMur- rich). picro formol (Bouin), Zenkerformol, Gilson's mercuro- nitric mixture, vom Rath's picro-aceto-osmic, Flemming's strong solution, Meves' modification of Flemming, and Car- noy's fluid (improved formula: glacial acetic, I; absolute alcohol, i; chloroform, i; the mixture saturated with corrosive sublimate). For the early stages in the ovary and for spermat(^enesis, i found nothing so good as Flemming's strong solution. For oviduct eggs (showing late diakinesis figures) warm sublimate .g.!.uM-dS.A^v^ft;> III.— The Cyclops "Tetrad". Four principal methods have been described to explain the formation of tetrads preparatory to the maturation divisions. These I have attempted to show in Text-figure I . Text- fig. I, Row I. — The parasynaptic pachytene fila- ment (a) splits to form two parallel filaments (6) presumably the same filaments that went into parasynapsis, except that during synapsis they may have interchanged material. Each filament then splits again longitudinally but in a plane 4,1 Chambers: Chromosomes in Cyclops 7 alcohol (100 C.C. 70% ale, 5 grms. corros. subl., 0.3 gnns. NaCl. ; cf . Braun, '09), picroformol, and Carnoy's improved fluid gave very good figures. For ova in the egg sacs, show- ing maturation and early segmentation stages, I found Carnoy's improved fluid to be the best. MateriaJ in warm (40''C.) sublimate alcohol was left for about 20 to 30 minute, then removed to 70% iodized alcohol. In Camoy the objects were allowed to remain about 3 to 4 minutes when they sank. They were then at once washed in 70% iodized alcohol. Sections were made from 3 to i0/« in thickness. The nucleus of an ovum of C partus in the late diakinesis stage is, on the average, about I 5m in diameter. As staining reagents, Heidenhain's iron haematoxy- lin was found to be the best for the oogonial stages and for spermatogenesis. Iron haematoxylin, Delafield's haematoxy- lin, and Babe's safr^jiin followed by light green were all used to advau.tage for staining late oocytes and the matur- ation and segm<:r.tation stages. Investigation for this paper was begun in the Univer- sity of Toron:o, carried on at Woods Hole and completed in the Biological Laboratory of Columbia University. I wish to acknowledge my indebtedness to Professor F. R. Lillie for his kind courtesy in giving me a table at Woods Hole during the summer of 191 1, and to Professor E. B. Wilson and Professor T. H. Morgan for the privilege of work- ing in the Columbia Biological Laboratory and for their highly esteemed counsel. A ■wrsi 1»V^ W' yt- jl 1 g Jj: ^1 '^^^^M mnm Cha*!beks: Chromosomes ,n Cyclops g at right angles to that of the first sniir r^t tu- take on a variety of sh-,n^ , r ^^- ^^'^ ^'^^''^'J may divergence along „ne or ^h' according to the greater or less cording to the var^"n. th f "• " ''^ ,'^' "'^''^^ ^"^ ^'^^ ^c- rods fLn.ing\i;?.:rst?r^:r'^ ^^----^ -^ ^he four IengthX"to?;"Jl:;rrane'in^'"^' '^'r^'"' ^'^^ ^P"^^ on them.c-Ive. (c) al 1 fh , f''^;""i!« (*)• These fold telosynap.i. filaments St n'' ""^ ""'^ "^ '^' °"g'"^' bondtoproduceTtetrad?J , T-',1 '''"■" ^''•■^^'^ ^^ ^he as in T.'-lJo'^;; ?S"t" ^""'^■^"" '^'^'"^- ^«) «P''ts W. One mS^^ratii/ 3k ■ L ' •"" V' ^"'"^ ^ ^ spiu theotwaio^?;t\.e'c;ri;:rj''""^ '''■ '""^•^"^•-' twice bnStudin:ily''"K' resrh-'^'^*;!;"-^ '''""^"^ ^«^ ^P"ts along the line ol^ynapticun on t^' '''"'"'! ^^^" ^^^^^^ transverse break or rro f" Pr^luce a rfzV^/ra^. The in the matur't, divsio^r'^Tri ^"r^^'^^^f ^'^ "° P-^ Matschek.('io).ancljC^^^^^^^ ^^ "^^^l^^^' pairs, of son^a/ic cuZ^^^.^^^^^^l ""'°"n'" They thus assume that fl .. ,-k ^ gt'rm-tract cells. joined end to end Aft^r r- ■ , ^^ ^"^ "^ chromosf.nies breaksup ii°o ^ods ha f tl ? ^ ^'"^ "u*^""'""'">' "'^' '^^'^'^-^^' TheseMo'ngitudSy tl ^r'cxL^'rr^^^^ a cross-suture which nasserti,, . u u^-' '' '''^' '^^ ^^^^ibit cates their bivalen n.Turc T "'V '^'"' '"'^'"^' '"^"^ '"di- divides these tetrads a",. ,7 '.'%^"'^^ "'f "'^^'ion dixision equatorial, the se^ , pa '^3 t":, '""^'^-'inal split and is reductional. '^ ^'"""^^ ''^^ cross-.uture and is tetradst cIn.hoJinfpT. it ""^ T*^^P-^-'- to the d.fferent interpretation. This^e es \ ^rdinr^o^^.^ ^ "^ 3 I X>M 90 * * a a i a iifi 'ill - ill 1 > S 'is „ I 9 6 — 1 s^" in I nil ■■pnnM Chambers: Chromosomes in Cyclops i, ^ss« twenty-four somatic chromosomes. In the ^erm- (II) i iiN.^1' Tro^ ^hT,S:" f "k '^"«^''^'- telosynaptic union (lU) plh^ • I °l ""^ °"«'"^' During in.erl£,s/he ,wo dya'dTZh^r* '.om.«„ypic. pair, become co„,i8„„„sTd fS a? ,hX •H^;'>' "P""" »ynm,x„ ot both gra„dpa.en„l and pa„dmau™al chro mann dements ,s ,h„, produced wi,hm%he pr^uSs c..iar,y X-shaped, i, eUborafX HafcteTo'rde? S a^! c..n,|rr r Tb^al^rSc-eS^-alclcVer-^ Un ormnately (or both Rilckert and Haecker re^n, i- vesnga.tons have placed the Cyclops tetrad "raXTCn"; 13 Chambers: Chromosomes in Cyclops ""'''' lV* that the peculiarly X-shaped tetrads of C. brevicornis are not produced during interkincsis but exist already in the oviduct egg and are due to the divergence at their ends of the longi- tudinal halves of the tetrads. Compare this with my own observation, PI. i, Fig. 4. The first maturation division separates the two parallel sets of tetrads ; the second separates the diverging longitudinal halves of the tetrads. Matschek ('10) made an extensive Etudy of tetrads in species belonging to six families of the Copepoda. He agrees with Haecker and RUckert in regard to the origin of the tetrads from an incompletely segmented longitudinal spireme. He confirmed Braun's discovery, howevei, that RUckcrt's tetiads are really ditetrads or octads (Text-fig. I, row 4). The oogonial chromosomes being twelve in number, there are six ditetrads in the oviduct egg. The primary longitudinal split is much bioader than the secondary, so that the ditetrad gives the appearance of two tetrads lying parallel to each other. The two maturation divisions divide the ditetrad along the two longitudinal splits. The cross-suture being interpreted as the plate of conjunction of two chromosomes and taking no part in the maturation divisions, Braun and Matschek consider both maturation divisions equational. Lerat ('05) discountenanced the existence of the cross- suture in Cyclops. He worked on a variety of Cyclops strenuus found in ditches, the same spicies studied by Riick- ert. According to Lerat's description leptotene filaments in the young oocyte conjugate in pairs by parasynapsis to form pachytene filaments. These subsequently split along the line of conjugation (cf. the split spireme of Haecker, Riickert and Matschek). As the filaments shorten and thicken, they form paiied chromosomes. These show no signs whatever of a cross-suture. Lerat did not go fieyond the metaphase of the first maturation division. It is interesting to note here that Braun mentions the case of a winter form of C. strenuus, living in ponds which dry up in the summer. In this form the chromosomes are long and U-shaped and the cross-suture is barely noticeable. Other individuals of the same species, living in lakes or in small ditches throughout the summer, possess chromosomes t*»»^mmmnmmmi Chambers. Chromosomes in Cyclops 13 with a very distinct cross-suture. Lerat may have studied only the winter fonn. The apparent contradiction in the statenionts of Lerat and Braun concerning the cross-suture may also be due to the fact that the iwo used different killing and staining fluids. Lerat used C.ilson's mixture and Heidonham's iron haematoxylin. Braun used sublimate alcohol and Delafield's haematoxylin. Miss Krimnitl ('10) is the latest to describe tetrads in Copepoda. She made a study of the generative cells during the late -mbryonic development of Diaptomus coerul- eus. In her preparations she finds cross-sutured chromo- somes both in oogonial and in somatic cells. The chromo- some number in the germ-tract she claims to be thirty-two In the somatic cells, however, she finds the number to vary anywhere from sixteen to thirty-two. Her paper is a pre- liminary report. We may defer criticism therefore, until her complete paper is published. Tetrads whose cross-suture take^ no part in either of the two maturation divisions have been described in few forms outside the Copepoda. TretjakofT (•04a) in the egg maturation of Ascaris megalocephala bivalens described two chromosome groups each consisting of two parallel chromosomes, each of which IS longitudinally split. A transverse suture often appears in the middle of these split chromosomes which later on dis- appears without taking part in the maturation divisions In a later paper (•04b) TretjakofT described the shape of the prophasic spermatocyte chromosomes of Ascaris that may well account also for the transverse suture he saw in the prophasic egg chromosomes. The chromosomes first appear =is ribbon-hke bodies with thickened ends. Later their middle region becomes so narrow as to consist of a mere thread con- necting the two ends. TretjakofI suggested that the middle region of the chromosomes consists of trophochromatin, and the two ends of idiochromatin. During the maturation stages the trophochromatin disappears, thus giving the chro- mosomes the appearance of being broken in the middle. Boveri ('04) and Montgomery ('04), however, and more recently Griggs (06), mention no such suture in the Ascaris HBi^P" mmmm »4 Chambers: Chromosomes in Cyclops ^'' forms studied by them. This and the general appearance of Tretjakoff's figures make one rather sceptical of its normal occurrence in Ascaris. Marcus ('06) in Ascaris canis descril)ed a cross-suture giving the tetrads the appearance of ditetrads. Edwards (II), however, in a closely related species, Ascaris felis, found nothing of the sort. A very recent paper which descrilies a condition similar to that of the Cyclops tetrad as explained by Haecker is one by Blanckertz ('i i). Blanckertz describes eight "chromo- somes m the first maturation prophase of Ascaris megalo- cephala umvaUns. The eight fuse end to end, in pairs, to lorm the four elements of the Ascaris tetrad. Each element of the tetrad is thus bivalent in the sense of Haecker 's bivalent Copepod chromost)mes. In Sponges, Jdrgensen ('09) descril)es in a Sycon eight tetrads which appear in the first spermatocyte equatorial plate During metaphase I the tetrads split into dyads which in the anaphase appear again as tetrads. In meta- phase II these divide again into dyads. The spermatid chromosomes thus appear to be bivalent. Tetrads have U-en described by Buchner ('09) as being found also in the oogonial cells of Gryllus. The artificial production of such structures in somatic cells and in the egg (Haecker, 00; Schiller, '08; Delia Valle 09) through the action of the strychnine and other poisons should render us cautious in accepting statements as to their normal occurrence in cells, at least where chromosomes are known to be diploid in number. My own observations have been limited to Cyclops amertcanus, C. parcus. and C. brevispinosus. Double chromatin filaments in the reduced chromosome number come out of the synizesis stage and persist as such throughout the growth period of the ovum. The two ele- ments of the double filament, which are at first close together separate more and more as they contract to form' short paired rods. During the late prophase of the first maturation division these paired rods are scattered through- out the nucleus (PI. i, Fig. 4.) ^ mm Chambers: Chkomosomes in Cyclom 15 When the nuclea. wall breaks down, the paired rods are drawn into the biserial arrangement (Figs. 5-10). From a careful study of a large number of sectionf of this stage in C. amerkanus, parous and hrevispinosus, I un convinced that the so-called Querkerhe, or cross-suture, ii not an actual hrcak but rather a clear area of the chromo- some due to the faint staining power of that region. The chromosome rod is somewhat larger at its two ends than along its middle. If we assume that this narrower, more faintly staining region is easily broken through when effected by killing reagents, we may account for the presence of a cross-suture in so many recorded instances. Fig. 9 gives the side view of several chromosome pairs from different oviduct eggs in the same individual. All were found on the same slide so as to insure as far as p i he eggs i)a>H out of the oviduct, this secondary nucleus approaches the periphery of the egg. No typical meiaphase tignre is over formed, the chromosome pairs in the l)i>erial arrauKcment passing directly into ana- phase I digs. II, 12, i,i). As Matst In k lias already observed, the first polar Iwdy is lornied within two to three minutes after ihe egg is laid Very liale, if any, cytoplasm is given off with the jiolar Ixxly. As the nucUiis moves to the periphery of the yolk-laden egg, it leaves a , .iih ol cytoplasm Uhind it (l"ig. II). When it reaches the [Hriplu ry it pirotrudes, pushing out in front of it a mend)rane d i^> i.^). During the late anaphase the chromosomes a-^-^iime their original I'-shaix', and from now on no sign wliatever of the clear area ii> their middle can be seen. I'he nuclear membrane remains in- tact until a constriction ai its middle occurs which finally cuts i)lT tht- p'>U\r lK)dy has Iteeii given off and the femaU- prumichus has reicdcd iiitn ihe c^k, surrounded by a very indistinct membrane. The chromosonus are losing their deliiiileness ot outline and will sinm form the reticulum of the female pronucleus. Fig. 22 is a jxilar view in C. partus of the male and female pronuclei lying in the first sej mentation spindle. The three chromosomes of one of the p'.muclei, presumably the female pronucleus, are already detiniiely formed and are fnginning to show signs of splitting. Maecker and his pupils, also KUckert, and ishikawa, have already ilescribed the remarkable autonomy of the mah- and female pronuclei during the earlier segmentation pro- cesses. The autonomy goes so far that one may often observe two almost complete spindles side by side each with its proper chromosomes. v.— Chkomosome NuMFUiR IN Cvriops. (a) — CHROMOSOMF.S IN THK l.KRM-TRACT. There is r > doubt that the chromosomes in the germ- tract cells are unreduced in number. Krimmel ^.'lo) has shown this to occur in Diaptonuis. In Cyclops ameri- canus I have been able to make out ten U-shaped chro- mof,omes in several tissue cells. That the chromosomes occur in the sami- numbvr in the oogonia and spermatogonia of the same form may be seen from Fig. I and Figs. 26, 28, 29. The conclusions of Krimmel and myself are contrary to the statements of vom Rath ('95) who observed thirty- two elements in the mitoses of the alimentary canal cells of Anomalocera patersonii, a marine Copepod, and sixteen I8 Chambers: Chromosomes in Cyclops elements in the mitoses of theoogonia; and Matschek ('lo) who figures an oogonial anaphase in Cyclops fuscus showing seven chromosomes, whereas in the biscrial arrangement seven pairs are to be found. Figs. 2 and 3 show the six oogonial chromosomes in C. parens. The chromosomes are usually U-shaped and apparently very plastic in nature. During the oogonial metaphase they split longitudinally and in the anaphase the slender halves shorten to form thick, semi-curved chromo- somes of about half the length of the mother chromosomes. In none of my preparations is there any figure approaching that of a tetrad such as Krimmel depicts in the Diaptonnis germ- tract cells. (*) — CHROMOSOME COUNTS IN DIFFERENT SPECIES. l\ Braun ('09) and Matschek ('10) have ascertained the chromosome counts for si.xte >f the European species of Cyclo^ i. Braun made a study of the relation between the chromosome number and il.e external specific characters of the species. Taking the condition of the fifth rudimentary foot and the number of antennal segments as criteria, he found in general that those which show least signs of rudi- mentation possess the greatest number of chromosomes. He made the following conclusions: (i) that the highest developed forms (e.g. many marine Cyclopidae) possess the greatest nur-ber, and those which are most higlily specialized possess the smallest number, of chromosomes; and (2) that closely related species possess equal or nearly :;qunl chromo- some counts and, therefore, that the chromosome number may be used in the determination of species relationship. As far as I have been able to make out. the chromosome counts for American species tit well into liraun's phylo.cenetir scheme. On the other hand, his statement that closely re- lated species possess equal or neaih equal chromosome counts is quite untenable, at least for our .American forms. The following table gives the specii s with tiieir • iiploid chromosome counts as I have found them : Chambers: Chromosomes in Cyclops 19 Cyclops fuscus 14 albidus 14 bicuspidatus 18 viridis 12 var. parcus 6 " americanus .... 10 hrevispinosus . . 4 " ntuJestus 8 C. fuscus, albidus, bicuspidatus, and viridis (cf. Cham- bers, '12) arc nicrphologically identical with their European representatives. Their chromosDife numbers are also identi- cal with these founci by Brauii and Matschek. The other species mentioned in the table appear to have no European representatives. In the latest revision of the North Ameri- can species of Cyclops, Marsh ('10) classifies C. americanus, parcus, and brcvispinosus as American varieties of the Euro- pean C. viridis Jurine. C. viridis (typ. sp.) has been described only by me as bf ing found in American waters. In their external features C. atncricanus, parcus, and brcvi- spinosus are barely distinguishable, the only main difference being the number of spines on the terminal segments of the swimming feet (Text-fig. 3). It has been suggested (Byrnes, '10) that parcus and americanus are two phases in the life- history of the same form. I have discussel this matter else- where ('12). Americanus and parcus breed true for generations. Slight variations in the number of spines of the swimming feet among individuals of the same culture occasionally occur, but the chromosome number always remains constant. Cyclops brcvispinosus differs fron. americanus and parcus in frequently becoming sexually mature before the swimming feet attain the number of spines characteristic for that variety. We may therefore have a parcus-like form '"''ext-fig. 3, c) or an americanus-like form (Text-fig. 3, d) except for the pres- ence of a spine on the outer side of the tc/miial segment of the endopodite of the fourth swimming foot and the presence in t'.ie cells of four chromosomes.* • The c.ind.il styles of C. hrevispinosus are slightly thicker and shorter than thr,s<- „f c, pircus and C. americanus. .Ml three are abundant in ditches and p'lols, akhiiugh not associated in the same pool. The three appear equally lafestcd H-ith a unicellular green alga which often covers them completely. tl i 1 1 ■3 s J E 3 - Q [i ill SJ C o lo mt^rrsmem^ Chambers: Chromosomes in Cyclops 31 (' » C. viridis (typ. sp.), averaging 2.2 mm. in length, is the largest form in the viridis group (excluding occasional giant forms of all the varieties). It has twelve chromosomes. C brevispinosus with four chromosomes comes next, averaging 1.6 mm. in length. C. americanus with ten chromosomes, and C. parens with six chromosomes, come last and are barely distinguishable from each other in size. The size of the chromosomes varies greatly in the three varieties, C. brevispinosus possessing by far the largest, and C. americanus the smallest, chromosomes. The proportions for the different forms are such that we could readily assume a relationship between the average sizes and the amount of their chromatin content. An explanation of the discrepancy of chromosome number in closely related forms is offered by Wilson ('09) in the following words: — "It seems to me a natural view that the nucleus consists of many different materials or substances that segregate in a particular pattern; that different chromosomes need net, however, represent a complete separation of different sub- stances but are in many cases perhaps in all, compound bodies; and that the particular form of segregation may readily change -om species to species. Marked or even extreme changes ...ight have taken place in the number and size relations of the chromosomes that would involve little or no change in the essential quality of the nuclear substance, and the apparent anomaly presented by differences in the chromosome groups of nearly related forms would disappear.'' VI. — Chromosome Size-relations and Arrangement in Cyclops parens. In C. parens the six somatic chromosomes occur in three sizes, there being a p'-ir for each size. PI. I, Fig. 2 shows the oogonial chromosomes on the point of being arranged in the equatorial plate. In spite of the fact that they are somewhat bent, one may readily pick out the pairs, two long, two medium-sized, and two short chromo- somes. We may see here as Wilson ('06) has already pointed W' vv?;^ ,«■" 'i*^. w Cbavbers! Chromosomes in Cyi CLOPS out for Anasa and other Hemiptera that the chromosomes of a pair do not necessarily lie together in the nucleus Tn aJ! sumption held by many botanists. .^J^^?''''^^^'}''y^th^hich the chromosomes retain their rdative size during the different stages of maturation may l^ Been from a comparison of Figs. 2, 6. 15, and 22 ^ a pair"7? naral^f^' ^"Tf^KT""' '^^ chromosome, of a pair lie parallel to each other. An exception is the case of one of the pairs in an individual takLn f om a ^^^ °^^■ ^"''"'- '^^'^ •"^'^'^"^' possessed an abnormal number of spines on the terminal segment of the exteS rami of the swimming feet, the numln^r for the four feet Snf 3. 4. 4. 3; respectively, instead of 2. 3, 3. 7, the chararf^r .Stic number for parcus. The chromosomes of the ovTduci eggs were found to be in the biserial arrangement and the smallest pair showed a deviation constant fo^aU the eis some fifty or eighty, in the oviducts. The two chromosfm; rods instead of lying parallel to each er. as was tl Sse for the other pairs in this individual, Jay almost It ri^^ht angles to each other (Fig. 7). It is renLkable tSL thb ab.u,mal arrangement should be so constant for that indi! VII.-Spermatogenesis in Cyclops americanus. I- — LITERATURE. Ishikawa ('93) described the spermatogenesis of a o.T r/tK A if ' if bilobed ap- pearance and the possession of double nuclei. In two in- stances two independent spiremes are shown in one pro- phase nucleus. In the spermatid his figures show double nuc col, very prominently. In two of the spermatocyte nuclei he figures distinct tetrads. Lerat ('05) gave an account of the spermatogenesis in Cyclops strenuus. Although he was unable to count the spermatogonial chromosomes, he assumed them to be un- reduced in number. He claims that reduction takes place through parasynapsis as the chromatin filaments come out of the contraction stage. His studies went no farther than the anaphase of the first maturation division, but in that stage he ngures the daughter chromosomes split lengthwise pre- paratory to the second maturation division. He found no sign whatever of tetrads. In Cyclops americanus the testis is single and median lying immediately under the dorsal wall of the thorax' From Its anterior erd two vasa deferent ia rise, and after wind- ing several times, one on each side of the alimentary canal pass back to open, one on each side of the first abdominal segment. 2. — THE KEIMPOLSTER. In old individuals a cup-shaped depression containing a disorganized mass is to be observed at the blind end of the testis (Hg. 24). This depression indicates tbe location of the Keimpolster, or primitive germ-cell group, from which the cestis IS derived. In immature individuals the Keimpolster is a raoidlv proliferating mass of cells (Fig. 2^). In young .sexually mature individuals it appears as a syncitium contaimng a number of deeply chromatic nuclei rather irregularly disposed but chietlv arranged along the periphery. The nuclei are small, barelv two-thirds the size oi the largest spermatogonial nuclei. Heavy strands of chromatic material cause them to acquire a dense stain 'AiimSJ^^MS^ 1 : V 24 Chambers: Chromosomes in Cycxops The absence of mitotic figures in the Keimpolster of all sexually mature individuals, and the fact that the Keim- polster is separated from the testis proper by a sharply de- fined boundary, renders likely the supposition that, after producing a numbrr of spermatogonia, it becomes inert and soon disorganizes, the growth of the testis henceforth being due entirely to spermatogonial mitoses. This Keimpolster corresponds to that described by Haecker in Cantnocomptus and is, according to him ('95a), to Amma ('10), and to Krinimcl ('10), the direct descendant of the germ-cells differentiated as early as in the first cleavage of the egg. Lerat ('05) was unable to find a typical Keimpolster in C. strenutis, He describes an apical cell from which he as- sumed the spermatogonial cells were derived. It is much more probable that this "apical cell" is merely one of the spermatogonial cells and that he failed to find the true Keimpolster as it may have been already disorganized in the individuals studied by him. 3. — MULTIPLICATION ZONE. The region following the Keimpolster consists of a large number of proliferating spermatogonia forming a msss of closely appressed cells. Lerat figures this region as a syn- citium. My preparations, however, give clear ividence of definite cell boundaries (Fig. 24). The resting nucleus (Fig. 25) possesses an irregularly blotched chromatic reticulum. Division figures are periodically frequent (Fig. 27). Definite spindle fibres are pla: ily visible. The chromosomes in the equatorial plate are diploid in number and are more or less U-shaped (Figs. 28, 29). The size of the cells varies greatly, owing partly to dif- ference in time of growth and partly to the number of sper- matogonial divisions that the cells have passed through, the nuclei and cells near the blind end of the testis (Figs. 25, 28) being considerably larger than those about to pass into the synizesis stage (F:^s. 29, 30). '^^rittSCl; Chambers; Chromosomes in Cyclops 25 4. — SYNIZESIS AND SYNAPSIS ZONE. The term synapsis is generally used indifferently by European writers for the massing of the chromatin filaments in a nucleus and their conjugation. The term synizesis, first proposed by McClung ('05), is much more applicable to the massing of the chromatin, while the term synapsis ought to be restricted to the conjugation of the filaments. In Cyclops there is a decided synizesis stage. The chromosomes o.' the last spermatogonial division do not pass directly into the synizetic filaments, there being an appre- ciable zone of restmg nuclei next to the synizesis region. Figs. 30 to 33 represent a number of contiguous nuclei in which one may see the gradation between the irregular network of the resting nucleus and the entangled mass of fine threads in the synizesis nucleus. A distinct sub-spherical nucleolus is noticeable at thir stage. It is always situated at one side of the synizetic mass. No bouquet-like orientation of loops can be distinguished but the threads are cleariy leptotene filaments. The nucle- olus never attains the great size and irregular shape seen in Lerat's figures. It is somewhat rounded in outline, rather small, and never shows the intimate connection with the chromatin filaments as figured by Lerat. Lerat's figures give one the impression of incomplete extraction of the haematoxylin stain. The nuclei of the cells undergoing synizesis are never larger than the small last spermatogonial nuclei. Gates' interpretation ('09) that synizesis figures may be due to the growth of the nucleus unaccompanied by growth of the chromatin content, cannot apply, therefore, to the case of Cyclops. No positive result was reached as to the likelihood of para- or telo-synapsis taking place during this stage. There- seems to be no doubt, however, that synizetic nuclei con- taining leptotene filaments exist together with synizetic nuclei containing pachytene filaments. The two types of filaments are easily distinguishable, there being no inter- gradations such as Matschek claims to be the case in the Chambers: Chromosomes in Cyclop<« oogenesis of Cyclops. Nuclei with pachytene filaments (Fig. 34) are most numerous in the region farthest from the spermatogonia! zone. K^-'-. i vx::' 5. — EARLY AND LATE DIAKINESIS. As the synizetic coil begins to loosen, the pachytene filaments give the appearance of being lumpy along their lengths (Fig. 34). Numerous short splits longitudinally ar- ranged on the filaments soon appear (Fig. 35). The coil finally resolves itself into five long filaments each consisting of two filaments tightly twisted about each other (Fig. 36). The spirals untwist as these filaments thicken (Figs. 37, 38, 39) until the five paired definite chromosomes of the sperma- tocyte of the first order are formed (Fig. 40). There is a slight growth of the cells during the synizesis and early diakinesit- stages. In the oocyte there appears to be some connection between cell growth, which is enormous, and the simultaneous increase in size of the nucleolus which is very great. In the spermatocyte, on the other hand, where growth is co.nparatively slight, no appreciable increase in size of the nucleolus is to be observed. The two elements of the bivalent chrooiosomes are distinctly elongate dumb-bell-shaped. In one individual I found several spermatocytes of the first order which con- tained four bivalent chromojomes and two single elements lying at some distance from one another (Fig, 41). Un- doubtedly the two single elements are halves of the fifth bivalent chromosome which have accidentally broken apart. 6. — MATURATION. The spindle in Division I is an ordinary one with conical poles and numerous fibres (Fig. 42) and with no resemblance to that in the maturation of the ovum. Insertion of the fibres is either subterminal or median. In metaphase the two halves of the bivalent chromosome usually break away first at one end (Figs. 42, 43). The split in the chromosomes for Division II appears during i*naphase I (Fig. 44). In telo- phase (Fig. 45) the chromosc- es become somewhat massed Chambers: Chromosomes in Cyclops tj together but their distinctness is never lost during interkine- 918. During this time the split in each chromosome becomes very promment (Fig. 46). each half appearing distinctly dumb-bell-shaped. In Division II the chromosome halve* are drawn away from each other much as in Division I (Figs. 47 and 48). The spermatocytes of the second order (t"ig. 49) contain five slender dumb-bell-shaped chromosomes. 7. — SPERMIOUENESIS. The more or less rigid dumb-bell-shaped chromosomes become U-shaped (Fig. 50). They then lose their distinct- ness of outlme through the appearance of irregular projec- tions over their surface (Fig. 51). These projections grow and develop in such a way that a hollow sphere of a reticular chromatm mass is formed (Fig. 52), similar to that described by Montgomery ('12) in the spermiogenesis of the Peripatus The sphere is then drawn out into the form of a spindle (t'lgs. 53, 54). As the spindle lengthens, it becomes com- pressed from side to side. At the same time it increases somewhat in size, and the small amount of cytopla.sm ori- ginally about the sphere appears to be sloughed ofT. The spermatozoon in the vas deferens (Fig. 55) is a slemler, faintly staining, finely reticula mass with a slight spiral curve and long tapering ends. In cross section it appears narrow ovate (Fig. 55a). In the seminal receptacle of the female the spermatozoa are often curled in the form of a corkscrew. VIII.— Summary. EGG MATURATION IN Cyclops americanus. parcus and brevispinosus. J. , '• T^* oogonial and spermatogonial chromosomes are diploid m number. 2. The tendency for the chromosomes, both of the oocyte and 'f the spermatocyte, to assume a characteristic U- shape seems to be subordinated during the prophase of the hrst maturation division to a force which causes them to assume a more or less rigid rod-shape, somewhat swollen .1^- ^VftJ--^ p£(r^mrr^^i^r^^-fn^ wmmx^r^^mm^h-^'^ a8 Chambek^: Chromosomes in Cyclops it; at the ends. In the oocyte this massing of chromatin at the endt leaves a clear area in the middle of the chromosomes. Such a clear area does not appear in the spermatocyte chromosomes. 3. Both egg-maturation spindles are entirely within a nuclear membrane. The spindle fibres, attached subter- minally or nu-dialiy to the chromosomes, appear most dis- tinctly in the second maturation spindle. The spindle poles are very broad so that the fibres appear to run almost parallel to one another. 4. The four American 'varieties" of Cyclops viridis exhibit a constant difference in chromosome number. C. viridis(tyii. .sp.) has twelve, \ ar. americanus has ten, var.^orcwi has six, and var. brevispinosus has four chromosomes. 5- The six chromosomes of C. parens are in three sizes there being a pair for each size. The chromosomes of a pair do not necessarily lie together in the spermatogonia! or oogonial nucleus. l^ Spermatogenesis in Cyclops americanus. t. in the mature Cyclops a Keimpolster distinct from the adult testis may exist. 7. Nuclei in synizesis are smaller, if anything, than the last spermatogonial nuclei. In the testi synizesis is ac- companied with only a very slight growth. 8. The nucleus in synizesis resolves itself into five pachytene filaments, from each of which develop two fila- ments, spirally coiled about one another. The five double filaments uncoil and become the five paired chromosomes of the spermatocyte nucleus. 9. The single elements of the double spermatocyte chromosomes are elongate, dumb-beil-shaped, similar to those of the oocyte. 10. The spermatid chromosomes resolve into a hollow sphere of a reticular chromosome mass. The ripe sper- matozoon consists of the spermatid nucleus drawn out into a slightly spiral spindle-shaped body, with fine tapering ends. r^^^mik^'^^ryz 1^ ~\ *. ..^ ,l*-v ■*'■«' '•4 ■^r ;,,eb. III. The Sexual Dif- ferences of th^ Ciiromosomes in Hemiptera Jour Exp. Z^^ol., 3. Chambers: Chromosomes in Cyclops 33 1909. Differences in the Chromosome- ^ups of Closely- ^if^T""! %"'' ^'^"'''''' *"'' ^'•°'^- Seventh ,r... t!' J- ^°"'- Congress. Aug. 1907. 1911. Siud.es on Chromosomes. VII. A Review of the Chromosomes of Nezara; With Some More Wolf. E. Considerations. Jour. Morph.. 22. chen Copepoden. Zool. Jahrb. Syst.. 22. SMbaCU 34 Chambers: Chromosomes in Cyclops Explanation of Plates. All the figures, except Figs. 23 and 24, were drawn with a Zeiss 1.5 ram. oil-immersion objective and a Zeiss No. la compensation ocular. For Fig. 23, ocular No. 8 was used; and for Fig. 24, ocular No. 6. The drawings were made with a Zeiss camera lucida at the level of the base of the microscope, and the reproductions are of the size of the originals. FlG. 2. Plate i. Cyclops atnericanus. 1. Oogonial equatorial plate, showing 10 chromosomes. (Fixation, Flemming; stain, Heidenhain's iron haematoxylin). Cyclops parens. Oogonial equatorial plate, showing 6 chromosomes. (Fl.iH.H.) Polar view of oogonial anaphase daughter chromo- somes. (FI.:H.H.) Cyclops Sp. Nucleus of oviduct egg, late diakinesis showing 5 pairs of chromosomes. (Camoy; H.H.) Cyclops atnericanus. Biserial arrangement of chromosomes in oviduct egg. A partial side view showing only 4 of the 5 paired chromosomes. Cycloids parens. 6. Biserial arrangement. Polar view showing 3 paired chromosomes. (Sublimate alcohol (Braun); H.H.) 7. The same, showing smallest chromosome pair ab- normally arranged. (Sbl. ale; Delafield's haema- toxylin.) 8. The same. End view of chromosomes. 9. The same. Lateral view of several chromosome pairs to show clear area when present is not always in middle of the chromosome rod. In 9b one rod appears broken in the middle, the other re- maining intact. (Sbl. ale. ; Delaf . H.) 5- Chambers: Chromosomes in Cyclops 35 Cyclops brevispinosus. '°' ^'^*if T^'"?!"'- '^^'^ '^« '^ 'y'"« i" the latter end of the oviduct and is compressed from side to side, giving the cytoplasmic area about the chromosomes a spindle shape. There are two pairs of chronosomes (Carnoy ; Delaf. H ) '(?£,:' SAr''""* ^"- Wn, laid. I a. Anaphase I. 13. Anaphase I. Cyclops parcus. (Sbl. ale; H.H.) (Sbl. ale; H.H.) Plate 2. Cyclops parcus. 14 First polar body given oflF. The chromosomes in the egg are turning on their axes preparatory to the next Qivision. (Sbl. ale; H.H.) / i if 15- Polar view in telophase I. (Picroformol ; H.H.) Cyclops brevispinosus. 16. Polar view in telophase I. (Carnoy; H.H.) Cyclops parcus. 17- Metaphase 11. (Picroformol; H.H.) Cyclops atnericanus. 18. Metaphase I!. Only 4 of the 5 chromosomes are visible m the section of the egg nucleus. (Carnoy ; 19. Chromosomes in metaphase II. 20. Telophase II. (Carnoy; safranin ) 21. Second polar body given oflF. Femak pronucleus retreating into egg. (Carnoy; H.H.) Cyclops parcus. Polar view of contiguous male and female pronuclei preparing for the first segmentation spindle. In one pronucleus the chromosomes are already solit- tmg. (Carnoy; H.H.) 33. 36 Chambers: Chromosomes in Cyclops '3 24 25. 26. 27- 28. 29- 30 31 33. 34 35 36. i7~3li 39 40. 4'- Plate 3. Spermatuoenesis in Cyclops americanus (fixation, sirong Flemming; stain. Heidenhain'i iron haematoxylin). a. Keimpolster. Young Cyclops sp ? Keimpolster lying immediately under dorsal wall of cephalothorax Dismtegrating Keimpolster at tip of adult testis. b. Multiplication Zone. Resting spertnatogonium. Spermatogonial prophase, showing 10 chromosomes bpermatogonial metaphase. Spermatogonial monaster showing 10 chromosomes bpermatogomal monaster taken from the end of the testis farthest from the Keimpolster. c. Synizesis and Synapsis Zone. Resting spermatogonium. Network of spermatogonium forming into filamenu and bemg drawn from nuclear wall. Early synizesis figure. Later synizesis figure. ^TengSr ''^^'' ^'''^'"""^' '"""Py al°n« their Diplotene stage. d. Early Diakinesis. ■ Five double chromatin filaments. The two fila- ments to the right extend out of the plane of the section and are therefore only partially shown 1 he double chromatin filaments untwisting. e. Late Diakinesis. Two double filaments entirely untwisted. One is much contracted and thickened Definitely formed 5 double chromosomes of the sper- matocyte of the first order. The same. Abnormal in that the single elements of one 01 the pairs are separate. Chambers: Chromosomes in Cyclops 37 /. Maturation I. 42. Anaphase I. Lateral view. 43- Chromosomes of metaphase I. 44- Late anaphase I. Polar view. 45. Telophase I. g. Maturation II. 46. Spermatocyte of t' s second order, 47. Metaphase II. 48. Telophase II. h. Spernnogenesis. 49- One spermatid with 5 chromosomes. 50. The same. Later stage. 51-54- Chromosomes being transformed into a hollow ball of chromatm network which becomes drawn out mto a sptndle form. 55- Mature spermatozoon. 55a- Transverse section of a mature spermatozoon. t# ^ * V): ^ • \ K> '^ .* ■ ( • < ^att^'-ja^fa^?»-'-r4W!"g.!W'Md' g!^,^wBgi'^«»i :4"_ I .'i, >^| ■. '(J i^LH t ^, -At .pnif. .n ' i«f». 2, ^ \^ Hv ?l^ #^# -/ i''^^' '■' N . I a*: > I'lH'.'!) ,!,,[. (»s, H,r>Lc 'I,, ^ tS< <3s S> ' ■: ;t'^ V*iTV-w«Vi " -■ s Tsv r, • • ' ■•», ^'f. 'Mi£:xi.<,: ^i'i:) 7' ^7Sr;^W^:i^^^' i.:m\ i-.Ksi rv oi- ioromo stidiks. ISlOl.lH.ll Al. Sl.Hll-S. ^->: O" o- *- CD. ro 00- 00- 00- No. No. j; No. ;: \o. 4 ; No. () T\w ^ametopliyto il liotrychiuin N'iryiniaiuim. v.. C'. Jkkfkkv Thi itomv of the Osmundaceae by J. H. Kai o.p. o.p. On the iJcntil'ication of Meckelian and mvlohyoid ;rooves in the ivvs ol McNOzoic and recent niain- i\\. hv H. .AlMHl R HlNSLKV o.p. The met;aspore-nie K. I?. TllO.MSON nibrane of the Gymnospernis In- The homolot;ies of the s molars of the nklclphyidae, tylar cusps in the upper On pi by B. Arthir Hensi.ky 0.50 ,lystely in roots of Orchidaceae. by J. H. WillTK. o..:;o fa chick, by R. Ramsay Wkk.hi 0.25 .Vn earlv .\nadidymus o Ihe habits and larva 1 state of I'lethrodon Kry- thronotus, by W. H. Pif.ksoi No. If. Spawn ,d larva of .\mbyr \V. H. PlERSlM. No. i<>: Thi CoWllKV iloiir chant;es ot rxVc/)' No The North .Knicric; bv K. M. Wai ki:k l)n toma Jell ■rsonianum. by > vu/x'tiris I.nik., by E v. Hies of the ^cnus Aeshna, •50 ^O. IT, I 2 ; Ascidians Irom HlMSMAN A contribution the co.ists .