THE CHKOMOSOME COMPLEX 01'' CULEX 1'IPIENS. 287

The Chromosome Complex of Culex pipiens-

Part II.—Fertilisation.

By

Monic» Taylor, S.N.D., D.S«:.

W i t h P l a t e 20 and 1 Text-figure.

C O N T E N T S .

P A G E

I N T R O D U C T I O N . . . . . 287

M A T E R I A L A N D M E T H O D S . . . . . 289

T H E R E P R O D U C T I V E O R G A N S . . . . 295

T H E E G G . . . . . . 2 9 5G E N E R A L . . . . • . . 298

S U M M A R Y . . . . . . 299

NOTE . . . . . . . 299

INTRODUCTION.

IN the summary of a paper, entitled " The ChromosomeComplex of Culex p i p i e n s " (4), it was stated that :

(1) The somatic number of chromosomes is three, both inthe male and female.

(2) The number of chromosomes in the spermatogonia, aswell as iu the primary and secondary spermatocytes andsperinatids, is three.

Two alternative suggestions as to the cause of this apparentanomaly were offered:

(1) The non-participation of one of the gametic nuclei inthe formation of the "zygote" in " fertilisation."

288 MONICA TAYLOR.

(2) The fusion of three pairs of homologous chromosomesat some early stage in the life-history, this fusion remainingpermanent throughout later divisions.. In the discussionpreceding the summary it was stated that the question couldnot toe settled until the fertilisation process had beenexamined.

This work on the cytology of Culex, as stated in the intro-duction of the above paper, had been undertaken because ofthe importance of the conclusions given by Miss Stevens inher paper, entitled " The Chi'omosomes in the Germ-cells ofCulex" (3).

The second of the alternative suggestions offered above isa modification to meet the particular needs of the case ofCulex p i p i e u s of Miss Stevens' statement that : " Para-synapsis (parasyndesis) occurs in Cu lex in each cellgeneration of the germ-cells, the homologous maternal andpaternal chromosomes being paired in telophase, and remainingso until the metaphase of the next mitosis."

Difficulties in obtaining the necessary material, and in thetechnique connected with the food supply of the imagines;delayed, for the time being, the examination of the fertilisa-tion processes, and prevented the demonstration of the wholehistory of oogenesis.

Dr. Woodcock's paper " O n ' C r i t h i d i a ' f a s c i c u l a t ain hibernating mosquitoes (Culex p ip ieus) and on thequestion of the connection of this pai'asite with a Trypauo-some" (5) has, however, incidentally filled up some of thegaps left in the history of oogenesis, and, in this paper, thetechnique of artificial rearing of Culex p ip iens in allstages of its life-history has been described.

I should like to take this opportunity of thankingDr. Woodcock not only for sending me a Reprint of thispaper, but also for giving me full details in writing of hisexperiments, by which I have been enabled to repeat his workwith similar success, and also, as will be shown later, to con-firm his statement that " C u l e x p i p i e n s is essentially the-British mosquito which-likes Avian blood."

THE CHROMOSOME COMPLEX OF COLEX PIPIENS. 289

In the 'Proceedings of the Royal Society/ 1915> a full de-scription of the chromosome cycle of Coccidia and Gregarinesappeared (Dobell and Jameson) (1), a work which elucidatesa cytological condition of affairs superficially somewhat similarto that which obtains in-Culex p ip iens . The authors ofthat paper demonstrate the presence of the haploid numberof Chromosomes at every nuclear division in the life-historyof A g g r e g a t a and Dip locys t i s except in the zygote.

In 'Chromosome Studies of Diptera' (2), July, 1914,Charles W. Metz calls attention to the neglect of Diptera bystudents of cytology—a neglect all the more pronounced whencontrasted with the great amount of energy expended uponother insect-groups—notably Hemiptera, Orthoptera, Coleop-tera. He offers, as a probable explanation of this neglect,the unsuitability of Dipterous material for cytological studyand the great difficulties connected with such study. Theresults embodied in Metz's paper, as will be shown later, havebeen helpful in interpreting the phenomena observed inCulex and in showing that there is no essential differencebetween it and other Diptera.

Dr. Woodcock discovered that, after the summer female ofCulex p ip iens has fed once on the blood of a living bird,the eggs attain their normal size and are ready for fertilisa-tion. The raft is laid almost immediately after the secondfeed—fertilisation taking place in the interval between thetwo meals. This probably accounts for the fact that femalesreared in captivity and fed on a fruit diet appear never to befertilised.

. MATERIAL AND METHODS.

In the summer of 1915 cages similar to those described byDr. Woodcock were set up in the College Laboratory of NotreDame, Glasgow, young pigeons being employed as food. Apigeon was also caged in the near neighbourhood of awooden tub stocked with larvae, and placed in a small gardenat Notre Dame. Control experiments, which will be described

290 MONICA TAYLOR.

later, were also started at Ladywood, Milngavie, the sourceof the material for all these experiments being mainly arectangular iron t rough near the farm-yard of GarscaddenMains, Bearsden. Two rafts were found dur ing the courseof the summer 1915 in the wooden tub , although all a t temptsin previous years to induce the imagines to lay there, or tolay in any of the aquai-ia on the premises, had failed. Thisexperiment would seem to show tha t wild birds are not soeasily bit ten by the gnats as domestic birds, since manysparrows, thrushes, and blackbirds visit the small garden inwhich the tub is situated.

The season's experience of artificial reariDg showed tha tthe number of egg-rafts produced under artificial conditionswas not likely to be sufficient for a thorough investigation ofthe fertilisation processes. Moreover, unfertile egg-raftswere frequently obtained by Dr. Woodcook from the arti-ficially confined imagines, and I too obtained rafts whichproduced no larvss; while, on the other hand, egg-rafts laid inthe open invariably produced larvas. For these reasons theconfinement of the imagines in net ted chambers was aban-doned, and the stocking of a pond at a convenient place inthe country was resolved upon.

I n view of the above experiments, and of the fact that allthe sources of material already used had been situated in thevicinity of farm-yards, it was decided to establish such apond at Ladywood, near a stock of poultry—these latterbirds being evidently easier of access than wild birds.Earl ier in the summer of 1915 several small t roughs, 12 in.in diameter, and about 6 in. in depth, had been stocked withlarvfe to serve as control experiments to those being carriedon at Notre Dame. The results were disappointing. Noegg-rafts appeared on these small ponds (during the summerof 1915). The number of female gnats seeking for hiber-nat ing quarters in the autumn of tha t year around Ladywoodshowed tha t the imagines, developed from the contents ofthe small t roughs mentioned above, must have found a pondmore suitable to the needs of their offspring than those pre-

THE CHEOMOSOMB COMPLEX OF CULEX PIPIENS. 2 9 1

pared for them in the garden of Ladywood. Mr. MacDougall'spermission having been obtained, a hunt over his nurserygardens revealed the secret of the non-appearance of raftson the Ladywood ponds. A large disused iron bath, ellipticalin form (36 in. X 26 in. X by 11 £ in.), and containing stagnantwater, was swarming with larvae and pupte of every agewhich were evidently supplying hibernating imagines. Thisdiscovery was made too late in the year to be utilised for thefurther production of rafts—but it showed that the situationwas a good one—the former experiments having possiblyfailed because of the smallness of the troughs, and becausethey were more or less concealed by the surrounding grass.

Early in 1916, thanks to the kindness of Mr. MacDougall,four ponds, A, B, C, and D respectively, were prepared :One (A), a large circular iron trough, diameter 30in., greatestdepth 24 in.; another (B), a wooden tub, 25 in. in diameter,depth 16 in.; the third (C) was the bath already selected by thegnats in 1915; the fourth (D), a rectangular porcelain sink,20 in. X 14 in. X 10 in.

The first two, A and B, were situated side by side, beingprotected on the north by shrubs, and on the east by a wall.The iron trough (A) was exposed to all the sunshine ofmorning, afternoon, and evening; the wooden one (B), beingnearer the wall, was more shaded. Both, however, werequite unhidden by vegetation. The tinned iron bath (C) wassurrounded on three sides by glass-houses, and exposed onthe southern side; the fourth (D) was placed in the uncutgrass of an open space.

On May 21st, 1916, fifteen egg-rafts were discovered in A.—there was thus no necessity to stock the ponds. No raftsappeared on the water in the wooden pond until June 15th.The preference shown by the gnats for the iron trough, andfor the tin bath, may be due to the higher temperature ofthe water of these, or to the fact that they were in a moreexposed situation, and consequently more easily found. Theporcelain trough has never been popular, comparatively fewrafts being forthcoming.

292 . MONICA TAYLOH..

The rafts found in May and June were evidently those laidby the hibernating imagines seen in the previous autumn, since,after that date, eggs were not abundantly produced until themiddle of July, when a spell of exceptionally warm, moist,weather conduced to an abnormally abundant supply. Thissupply continued to be good until the middle of August,when the spell of hot weather ceased. Odd rafts wereoccasionally found until the end of August.

In one of his letters Dr. Woodcock expressed his opinionthat the rafts were deposited at about 5 o'clock in themorning, and text-books also state that the early hours ofsummer mornings are chosen by the gnats for the purposeof egg-laying. On July 12th, 1915, an egg-raft, cream-whitein colour, was found at the Bearsden pond at 5 a.m. Portionsof this raft were fixed at intervals of a quarter of an hour,and sections showed that it was quite young. After a care-ful study of ponds A, B, G, and D it became clear that, atMilngavie, most rafts are laid between the hours 9.30 p.m.and 12 p.m., very few between 12 p.m. and 4 a.m., and somebetween 4 a.m. and 6 a.m. As the season advances fewer arelaid in the morning hours. Damp heat conduces greatly tothe deposition of rafts, many more being forthcoming after aday of calm, moist, close weather. Numerous imagines werealways hovering over the ponds in the evenings at a height ofabout six feet. These disappeared during the day time.

No difficulty in rearing the larval and pupal stages of thegnat occurred until the summer of 1915—the critical periodin the life-history of the Cu lex being apparently the imagoperiod. Even in exceptionally favourable circumstances thenumber of egg-rafts deposited at any one period bears a verysmall proportion to the number of imagines that escape fromthe pupal cases, and this has been very marked in a longstudy of the naturally occurring ponds. However, in theuminer of 1916 D a p h n i a were introduced into the gnat

cultures at Notre Dame, and since these, like the Culex larvse,also flourish well on a diet of C h l a m y d o m o n a s , the tubquickly became swarming with. Daphnia, while the gnat

THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 293

larvas disappeared almost entirely. Perhaps this accountsfor the more frequent occurrence of Culex in the fairlyclean water of rain-barrels and drinking-troughs, in spite ofthe fact that, as shown by long experience, the larvaa growmore quickly in a good culture of Cl i lamydomonas , andthat, on this account, one would expect the gnats to prefer amore stagnant habitat.

The egg-rafts, for the cytological studies in question, afterbeing laid, which process takes about ten to fifteen minutes,were isolated, along with sufficient water from the pond, inPeti-i dishes, in which they underwent their subsequentdevelopment. Thus it was possible to determine the age ofthe eggs. Rafts just laid, and of ages ranging from halfhour after the deposition of the last egg to two and three-quarter hours, were preserved. Imagines just before, during,and after oviposition were also fixed. Many rafts of unknownage were necessarily found during the season, their colourindicating to si certain extent their age.

My bust thanks are offered to Sr. Carmela, S.N.D., who,during my absence in August, went on with the work oftending the ponds and fixing the rafts at timed intervals.She gives twenty minutes as the period required for thelaying of one raft.

Carnoy proved an excellent fixative for both rafts andimngines. Bouin and Gilson-Petrunkewitsch were used ascontrols. The rafts did not sink in the former fixative, hencethe results were not so good, but the Bouin material wasmore easily sectioned. Portions of whole rafts were em-bedded in paraffin, and sagittal sections of from 5/i to8jumade of the whole mass. Thus many eggs could be examinedat once. The funnel (see Text-fig. ] , / ) was removed beforeembedding:

Mr. P. Jamieson's long experience in microtoming wasplaced entirely at my disposal in the matter of sectioning theproverbially difficult dipterous egg, and I should like to expressmy gratitude to him for this help, and also for actuallycutting many of the sections used in this investigation.

294 MONICA TAYLOR.

TEXT-FIG. 1.

- -osSections through mature female gnat parallel to the sagittal

plane (b under higher magnification), ch. Chorion. e./. Youngegg follicle. / . Funnel, m. Mieropyle. m. e. Mature egg.o. t. Ovarian tube. v. in. Yitelline membrane.

THE CHROMOSOME COMPLUX OF OULEX PIPIENS. 295

The E e p r o d u c t i v e O r g a n s in the younger stages havealready been described. In the mature ovary (Test-fig. 1)the egg is elongated, and is provided with a chorion, verybeautifully sculptured, and a second membrane—the vitellinemembrane—both of which are perforated before fertilisationby the micropyle. Separating one egg from its neighbour ofthe same size, or from a young egg follicle, is a chitinousstructure (see fig. 30, ' Natural History of Aquatic Insects,Miall.), like a funnel in shape, which remains attached tothe laid egg at its broad anterior end. This funnel is per-forated above the micropyle, and apparently serves to guidethe spermatozoa to the micropyle. A somewhat similarstructure is shown in Sedgwick's ' Student's Text-book ofZoology/ vol. 3 (fig. 400), for the egg of Drosophi lace l l a r i s . The future head end of the embrjro lies in theanterior position in the ovarian tube, as is usual in insects—thehind end of the egg being the first to emerge from the imago.

Dr. Woodcock (5) expressed his opinion that an imago wasprobably capable of depositing more than one raft in theseason. Sections of gnats, which were fixed immediatelyafter depositing their rafts, showed that the contents of thespermathecEe were by no means exhnusted. Moreover, thegonad showed some large eggs, as well as very many smallegg follicles, which evidence seems to support his opinion.The gnats, flying away after laying eggs, were perfectlyvigorous, and showed no tendency to die.

The three spermathecae communicate with the hind end ofthe common oviduct by three minute tubes, so that thespermatozoa make their way into the funnel as the eggpasses out.

In view of the foregoing description and the fact thateggs laid in captivity often produce no larvse, the first hypo-thesis as to the origin of the haploid number of chromosomesin the somatic tissues of Culex can be rejected, and thisis justified by actual observation of the two pronuclei inthe egg.

The Egg .—I have not been able to observe the formation

296 . MONICA TAYLOR.

of any polar bodies. The egg nucleus lies in the middle ofthe egg, and is a typical resting nucleus. It is surroundedby the large yolk globules which constitute the greater bulkof the egg. The cytoplasm, which is sparse, is denselystaining and provided with numerous deeply-stained particles,as was the case in the tissues of the larva and pupa, andthere are large numbers of small bodies surrounding theyolk globules which resemble chromatin in their stainingreactions. These chromatin-like bodies, " yolk nuclei ", seemto be intimately concerned with the digestion of the globularyolk-masses, and with their conversion into protoplasm, andsometimes resemble very minute chromosomes (PI. 20, fig. 1).When surrounded by the digestion products, which haveresulted from their activity, they do not stain so clearly, andeventually they appear in the fully-formed protoplasm as theparticles already described. This quality of the protoplasmgreatly detracts from the beauty of the histology.

The sperm nucleus (male pro-nucleus) assumes the restingcondition very quickly. In one egg of a raft, fixed a fewminutes after deposition, the two pro-nuclei are already inclose contact. In one egg of a raft half an hour old, thespermatozoan has reached the egg nucleus, but is still sperm-like. In one-hour rafts there are several segmentationnuclei. (It must be remembered that there is a difference inage between the first laid egg of the raft aud the last laidegg, of from ten to twenty minutes, hence these times are onlyapproximate.)

The segmentation nuclei, which lie in little islands ofprotoplasm (PI. 20, fig. 2) pass into a decided resting-stageafter dividing, and several blocks have to be sectioned tomake sure of obtaining mitotic nuclei.

The prophases (PI. 20, figs. 4-9) of the dividing segmenta-tion nuclei of Cu lex show six chromosomes. In earlyprophase a tendency to emerge in pairs from the restingnucleus is evident (PI. 20, fig. 9). These six chromosomes(PI. 20, fig. 10) arrange themselves on the equator of aperfectly typical spindle, split longitudinally, and in anaphase

THE CHROMOSOME COMPLEX OP OULEX PIPIENS. 297

and telophase six thin chromosomes can be seen going toeach daughter nucleus (PI. 20, figs. 11-13). In contrast toformer experience of larval, pupal, and imaginal material fcliedividing nucleus is most frequently found in the metapliaseand anaphase stage in the segmenting egg of Culex.What takes place in late telophase and in the passage of thechromosomes into the resting nucleus recticulum is difficultto follow.

There is thus no difficulty in demonstrating six chromosomesin the segmenting nuclei of Culex pi pi ens—fertilisationand the early stages of development are perfectly typical.No reduction in the " Zygote," as found by Dobell andJameson in Coccidia and Gregarines, takes place. Thesecond hypothesis set forth in the beginning, viz. " thefusion of three pairs of homologous chromosomes at someearly stage in the life-history, this fusion remaining perma-nent throughout later divisions/' most probably explains thecase. Early prophases show decided tendency of the chromo-somes to come out of the reticulum in pairs, full prophasesshow six chromosomes, among which pairing can sometimesbe detected (PI. 20, figs. 4, 7, 8, 9). As has been shown byStevens and Metz a side-to-side pairing of homologous chromo-somes is a characteristic of Dipterous cytology. Metz (2)states that Miss Stevens records it in nine species of Muscidte,and four species of Mosquitoes, and that he has verified it infive of these species of Muscidce, and extended it to eightothers, ID addition to species of Drosophi la . An increas-ingly closer proximity of these homologous chromosomes oneto the other, would produce, eventually, an actual fusion ofthe maternal and paternal constituents, with the result, in thecase of Gulex p ip i ens , that in full prophase three, and notsix, would be the apparent chromosome number. The threechromosomes that appear so persistently in the somatic tissueof larvEe, pupee, and imagines of Culex p ip iens are, therefore bivalent in character; they are really three groups ofchromosomes, with two in each group. I have not beenable to trace out more fully the development of this tendency

298 MONIOA TAYLOR.

of homologous chromosomes to fuse into an apparentlysingle chromosome. This would entail a study of raftsranging from a quarter hour to three and a half to four daysold. (The development of the larva in Scotla.nd occupiesfrom three and a half to four days.) Moreover, the eggcapsule becomes so hard and brittle as it changes from creamwhite to dark brown, that a special technique would have tobe devised for sectioning tliese eggs without destroying thenuclear detail. All that can be stated with certainty up tothe present is, that the homologous chromosomes have notfused in segmenting nuclei—while this fused condition hasbecome a characteristic of Culex p i p i e n s in the early larvalstage.

It would seem, then, that parasyndesis has reached its limitin the somatic tissues of Culex p i p i e n s , resulting in thenctual fusion of homologous chromosomes, and that extremepurasyndesis is responsible for the apparent anomaly de-scribed in the'Chromosomes Complex of Culex p i p i e n s ' , I.

GENERAL.

There is general agreement that we are justified inassuming (1) that hereditary qualities are represented bymaterial substance, and (2) that this substance is eitherchromatin or is inextricably involved in chromatin. Grantingthese two assumptions, we seem logically bound, by thegenerally occurring accurate longitudinal splitting of thechi-omosomes in mitosis, to admit that the patches of materialrepresenting definite hereditary qualities are arranged inlinear fashion along the course of the chromosome or threadof chromatin. But this involves necessarily a teudency ofthe hereditary substance representing one particular quality,or group of qualities, to segregate together. In other words,there must be, in the case of hereditary substance, anattraction of like for like. If this be so, there will be atendency for chromosomes composed of corresponding patchesof hereditary material arranged in like order, to come

THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 2 9 9

togdther side by side with homologous poles together, theopposite of what happens in the case of magnetic attraction.

Such a hypothesis will account for the freqaent occurrenceof parasyndesis, and for the further accentuation of thisinto complete fusion as is supposed to take place in Culexpipiens .

SUMMARY.

(1) The egg-rafts of Culex pipiens are laid mostcopiously between hours 10.30 p.m. and 12 p.m. They arealso laid between 4 a.m. and 6 a.m.

(2) Fertilisation in Oulex pipiens is normal. Segmenta-tion begins in less than an hour after the deposition of thelast egg.

(3) The chromosome number in the segmenting nucleiis six.

(4) A tendency to parasyndesis is exhibited by the seg-menting nuclei.

(5) Parasyndesis probably effects the condition of thechromosomes in the nuclei uf larva, pupa, and imago, i.e.is responsible for the presence of the apparently " haploid "character of the nuclei in the somatic cells.

NOTE.—After the completion of the foregoing paper,Metz's "Chromosome Studies on the Diptera I I .The Paired Association of Chromosomes in the D p-te ra , and its Significance' , 'Jouru. Exper. Zool.J, xxi.,came into my hands.

With regard to certain criticisms passed on my work,I should like to state :—

(1) The fixatives employed by me were precisely thoseemployed by Metz (see p. 379, ' Quart. Journ. Micros. Sci.',vol. lx).

(2) Although I have not specified this, all the precautionsrecommended by Metz to secure good fixation and penetra-tion were employed by me. . I cannot, therefore, put myresults down to bad fixation.

300 MONICA TAYLOE.

(3) I have failed absolutely in my former work on Culex ,although I devoted much time and study to this point, to findany figures resembling Metz's figs. 166 and 167, or Stevens'sfigs. 8,9, and 10. This failure may be attributed to differencesin the material studied. It could not be due to bad fixation.

(4) Setting aside the Diptera, my,figs. 68 and 69 (4),would be interpreted, by the vast majority of cytologists,as a split spireme, as chromosomes precociously split formetaphase.

(5) My conclusions in the present paper, made after astudy of fertilisation, do not differ in principle from those ofMiss Stevens. I state, for my variety of Culex p ip i ens ,that parasyndesis has resulted in actual fusion. Only theassumption that some such fusion has taken place can accountfor the striking recurrence of three chromosomes in theprophase of somatic tissues in larval, pupal, and imaginalmaterial.

(6) In a preliminary investigation of Chironomus sp.,an account of which I hope to publish later, I have found aconfirmation of the results obtained in Culex.

LITERATURE LIST.'

1. DobelJ and Pringle Jameson (1915).—" The Chromosome Cycle inCoccidia and Gregarines," 'Proo. Roy. Soc.,' vol. lxxxix.

2. Metz, C. W. (1914).—" Chromosome Studies in the Dipteva. I. Apreliminary survey of five different types of chromosome groupsin the genus Drosophi la ," ' Journ. Exp. Zool.,' xvii.

3. Stevens, N. M. (1910).—" The Chromosomes in the Germ Cells ofCulex," 'Journ. Exp. Zool.,' viii.

4. Taylor, Monica (1914).—"The Chromosome Complex of Culexpipiens," 'Quart. Journ. Micr. Sci.' (N.S.), vol. 60.

5. Woodcock, H. M. (1914).—"On ' C r i t h i d i a ' f asc icu la ta inHibernating Mosquitoes (Culex pipiens) and the Question ofthe Relation of this Parasite with a Trypanosome," ' Zool. Anz.,'Bd. xviii, 8.

THE CHROMOSOME COMPLEX OF CULEX P1PIENS. 301

EXPLANATION OF PLATE 20,

Illustrating Miss Monica Taylor's paper on "The ChromosomeComplex of Culex pipiens.—II: Fertilisation."

All figures were drawn with the Abbe camera to the given scale*(Leitz T!J oil itnm., Zeiss compensating oculars).

Fig. 1.—Section through egg fixed Gil. Pet. Stained in thionin;differentiated to show " yolk nuclei." " Yolk nuclei " remain deeplystained; chromosomes lose stain when sections are left for some timein absolute alcohol.

Fig. 2.—Section through egg of a raft laid at 4.15 a.m. ch. Chorion.c. in. Closed micropyle. / . Funnel. s. p. n. Segmentation restingnucleus surrounded by mass of protoplasm, v. in. Vitelline membrane.

Fig. 3.—Resting nuclei; daughter nuclei of fertilisation nucleus ;" yolk nuclei" around yolk globules.

Fig. 4.—Prophase of segmentation nucleus ; egg one hour old.Fig. 5.—Prophase.Fig. 6.—Early prophase.Fig. 7.—Prophase.Fig. 8.—Prophase.Fig. 9.—Early prophase.Fig. 10.—Metaphase.Figs. 11 and 12.—Anaphase.Fig. 13.—Telophase.

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