- b a = - 99.-u1w*sq._w,

L

2.

l,f/{.figr,/AN/r.4/snr.8?.1

99.-u1w*sq._w,

Definitio

Ehcre ate tDro worda

in cytotaxonou{ir:

., ^.

cYtologXr

=

^etudlr

of

the cell

-

^tarcouoqr

=

^{gcience (laws)}

of

the claseification

cytotaxong ₌ taxono3r (cLaeeificatioa) _{by neans}

of

the

cell

chafectcm Notions of ^cytoJ.ogy

(d

_{etructure of tlre celil}

vegptaL ceLL

=

oba€rtatl@; under nicroscope, of a fragnGnt of thc interaa.l nembraae of an

oionls

sca-le:

x

^uithout co.loration

(in

_{a drop of water)}

x

rithout coleation

(in

_a

@

^{eaccharia eoil.utio)}

x after

colonatio, by the ^tlRouge Neutretf ^(Anneur L).

=

observation of a parameciun, under nicroecqtc:

x

rithout coloratio

x

after coloration by metlrylene blue, by ttre

rlRouge Neutrett, by todo-d-otiscd uater ^(Rnncoc

t).

ia

both caBG6, we distinguish: nenbrare, cytoplaem, nuclcue, vacuolea

(Fig.

I

and tr'ig. 2).

(b)

^gtructure _{of, ttre aucleus}

Ia

a nuclaus, ue d:istiaguieh: mabrane, nucleoplasme, nucleolrrsl chrmati.ne (chroosones). Fig. _5,

(c)

_{etructure of the} chromosme

Oo a chrmoeone, we distinguish:

-

^primary coastrictioa and eecondarlr conEtrictj.on

8rn8 = short arm (S) _{and long} arn

(t), right

arn and lef,t arn.

eatellite

centrcmene

-

^ahrooeor (clrrmosomic filsnente

]

aerie of ^gaua)

8i.6. 4, _{FiB. 5r}

fig.

^6.

(d

_{Notlon of cargotype}

Each epecies hae a speoifi,o nunber of, chromoaomeB rhich

ie

^geaerally patr:

- ²ⁿ =

^{8 tor} eeps$1g}gp-ogg_t-ei.

- b a

^{46 ton} &-eSLrS;1p.

- 2n =

^{5 ton Sjnrrliun dannogun.}

*"

-

^{aninal ceLL}

.!

*

I

)

l{hen the ^perirs of chrmoso@es are aunbered.

ia

a

ce}l, frm

the l.o6eet to the etrontest,

this

constitutee the speciea carlrotype. Fig. 7, Fig. 8, ^tr'Lg. 9.

lfe diEtiagu:ish ordinary chrmoomee (autosoes) and eexual chrcrroEoroa

(heterochrmosooeE). _Fo(' nan, therb are

l*

_{autoEmee + 2} heterochromosmaa

(x _ana

r).

(e)

Bhe chronoa@es, support of heredittrr.

G.l

The ceII diuision ltitose ^{(Rie. 1O)}

It ie

th.is kind of division uhich ensrlrea grotrth _and healing.

It

Leade

to

the formation of ^the .giant chromosoee (pac$tcne

e

pol4yteae chrmosones)

in

the ceLLs of the

ailk

^gr-and6 of {tpterousr drrriag the dlvision, the chronosmic f,ilanents unspiral to na:cXruo

(feaetAenind but donrt eeparate after

splitting

(ttrictreatng)r and

the cytop}am doeentt ctivide

ia

tuo parts.

$leioee

(fig. It)

It ia

thie kind of di.rrleion which ensurets the aemral rcproduc-

tlou

=

^{2 ce]-le with n chrmosmea (ganeteg) unite} (fecondattm)

to ^{g:ive oue} ceIL

rith

2n chromosmee (ornrn).

e.2

^{The seoc} determiration (FiS. ₇₂₎

e.3

Heredity linked to autosmes ^(exanpLee) dminant cturacters ₌

x

fttzzy hairs

x

dark skin

x blood ^{groups AB}

x positive rheeus

recessive ctraracters =

x

albinisn

x

dreponocytos5.e

x ^{bLood group O} x ^{negative rheeus}

s.4

^Soc Linked heredity ^(ocanplee)

clraracters Linked to chrmoaone x ₌ x haeuophilla

x

coLor blindaees

r.

characters linked to chronosome y ₌

x

ichtyoeLs or hyetrix ^gravion

(poncupiae dar).

3.

ftre si.Erliun cartrlottrrpe

(d

$rc E:--eggLo_qg cmglor epecies chromosone urmber

is:

^ps ₌

6.

[trat ig to ^{salr 5} chrmosmes-in

] pirs,

^{atmbered I,r}

^{Alr IIf} r

^{fron the longest} to ^the shorteet.

,

(b) _In

_{the 8j€rt nuc.Leus}

_(rig. U) ^ot

^{the ell,k} El-ands, the chroosmes are

lu

an haplold nunben (n _{= 51}

r

^as

ill

the escual

cells

^(ornrla and, eperaatolOo), and they can be as L5o tines longer ttlan

in

the ondrnaqr cclle ^(graat

chrooemes).

(o)

lftre etraracteristica of each chrmosone ares

4.

tfiorouonphoLogical charactere oa the chrmosoues

(d

Xho chrouoameg have been d:irrided lato IOO segnente, nunbered froo

I

to ^1OO

eegnents 1 to ^It2 correspond to chrmoooue

I (t

to 20 f,or IS and p]. to 4a fon

fr)

-

^{eegnente 43 to} fZ ^comespond to ^chrmoeome

lt (l]

to

5j f,e II{i

^and

55 to 72

tw

II,L)

-

^segnenta 71

lo

10o conrespond to chromoefiie

IIl 0ll

^{to 8O} tor

ttts

aud 84 to IOo fon

IIIL).

Each se8nent

ie

ctivtded

in

ponticrs called A, Br

c,

D, E, F, and Ln cach

portioa, the traneveneaL baads are nunbered.

(b) Ia

ttre.[.-*S-o5gg, (conplex) speciesr g:iant chrmoeomea, we dietinguigh peroaneat micrononphotr ogical characters:

Aa orpanded region (Uc) utrich includes the controere (C) o,

chronosone

I;

the nucleolar organizecr ^(UO) on IS

in

aegnent

l9i

the Bing of BaLbiani (B) _{and ttre doubLe bubbLe (aU) on}

IIS, in

eegueate

5]

^and J2 reepeotively;

the para Balbi^ani (p) _oa

IIt,

irr segoent 5J.

the bLister (u) IILS.

ftre briater and the doubl.e bubbre are Errchrooatic sectione.

theee permanent characterE alrsry the recognigron of ^the chrmosouea aad

their *iffenent Brnsr Fig. J.4.

5.

4-

Ihe inversions

the inversions are 18Oo rerrersala

of

one or Beverel fragmeats of, chr@oames, af,ten breat<ing

at

two poi^ots. Fig.

Il.

ftrey are ideatified by arm and ^nunbened

in

series, takjrtrg Do acaount of ^the

apecies,

Conveutional rmiting:

-

^fixcd inveraLoae = written

ln italicel

IS-1r IIII-I+

-

floati"ng inversions:

rrittea in

vertical characters:

I[,-Il

Ih25

-

iatereBeclflc inversions ₌

yritten

_{on the}

left of

the chrmosmes

-

intraspecific i-avereLoog = dri.tten on the

rigfrt

of, the obrmogonee

t

hmoa6rgote inversionn = Irtu?/? t' IJT--4r6/4.6

-

beterozygote irversions: 1,5-/2t TIJ*3/8

-

^unltlple iaveraione: IIL-4.6.7, lI'fi,-2.6.

CoLlectlng lanne

in

^carnoy

Fon gtatmeu! _{see Annex 2t}

Chrmoeamee stai.ning

for

etatemen{see Annoc 3.

Rea&ing. Identificatioa of ttre speoies

Ue have to recogniee the follotriug specific j.aversions:

5r

7.

B.

S. ^ganctirauH_k chrmosme

I r

chromosome

II

: IS-ArE- JL-Ar It-FQ

:

ILS

- 6b, Ils - ?r IIL - llr IIL - 6r IIIL -

^Ar

Iff. - ^Dr

^LIL

- Xr

^tlJ.r

-

^7.

ohronoqme

III : IIIL -

^CrC2r

IIIL -

^{B (ttre}

^triple

^{Lnversion a€qu€lrce}

4.L?.2 i.s always i.qLied).

s.e:gPgr:g/F.-.fl r$Apeqgy'S...qgEgXHlg/S-..-qLeSLeI'gIlE3

chronosmel ! IS-2r IS-1, f,L-1r'IL-Z' fi-35

chrmoeme

II : IIL - cr ^IIL - 8r ^III, - ,

chrooeone

III : llt - br ^IIIL - ?r ^(ffff' -

²

^is

^{af.ways inplied).}

g* egf gsofl S/S.. Jgle.rF q

chronoeomel : IS-2t IL-J2

chronos@e

II : IIL - 8,

^IIS

-

^b

chrmosme

III 3 ILI

^CS

Cf, Anaor 4 and Anne* 5.

/t

mrvrrlnot

ns

it

y1o;law

c;[";f-rr*e

Yllra ^C.,(

Fi>. L

c" tt*f J

^{o nl} "-.*Qc-

F;x- &

ce dJ,. ^*i6JFJc

i.

I

t I

I

Flc. 9t -- Dtvrlrs NoYAUx au B:Blos. ,

Scnsiblcmcot, m€mc grossisccmcat'ActB;ooyiur pcu'ri{r&ur{i, ^-,,

& larvcs d'Oursin; C, noyou tr& srueutt d'A+Uopodc,(bl&lliunt intcftioal d'Oairtu atcllvt); D, ^{noyeu dc} hrvo &,Saho.o&q-cctluh

du foic; E,racior,d'Allimn ^aap1, (odgioal)- _.,-

F _0' 3

^''**

*#li#"

_..;$

' l l'r,

:- ,-., !l .".

. ,':]Ir,';1.'fl-rii_r :.r:' ^{. !.} *1fl;..' . ^.r:

r'&i\

;i;iI eji;i

i

^{j: r-'}

^$;

^-i

ffi

/o

l"'

aO

),'

Frc. to7 - st-rrr:}r.r, D'h.* cHnouosouE tr ^DE sEs coNsxrTUANI.n.

|lrsprrc ^de .Schratlcr. Cp,.constri&ion prinrairc; C., .*rro er";

Cr, coo5trtition sccoodaire; Clr,_chromon€nw; Ma, mattix; ^pei pllicrlc; f, setcllite.

,4

I

c

F,c, + - Srrucrrrlr scutx -

'' ^{'TregE Du} csf,oro6ortB (Daptts ltcitz]

C- cindtochorc; _C0, chrmomirc;

Co, conStrielion secor&irc; ll,

h6t6rochronatine _;

^&f, rnatricc.

L. bf

"r.a n ₃

l ..,,&j

^4N,

^a'i,

Nfl

ln -T- _,f

Fi6. ⁶

I

'F_iI f-',V"q*z -',i-i!

I-.

l-i

r7-lr

U-3

7_ t2

;8rl-[Fm_

'l . .l!_=*l5

-X-I-[n-]r- -in-0rlf- *l-IhJtr

:

--bit}

r-r

r-t't-

B- tt ¹³

.-I'I,^ lx. -r-r:lil

.. i& ,,

=.$itpryf" "ii;,;, "i"t r"rsplce xurel'ri

, Chcz l'tloriunc af6 cfromoqomcq dom r X a r 'Y. ,;

It- ²⁰ 2t-a

F*, ,l - ^Cenrorrrr NotM,tL oexg I'rsolcr HUltltN&

Chcz b Fcmmc _{: 46} chromosomcs, dont z X.

F;6'9

F,t. +

{fi--[f-flt-trhltr ^,li'

It-u:

*.I

t

t

*'-

U*.. ^<

J*!a

Appi',

^Y*.tn!'

l1" "trri^ rrc-

,

-.r h''*."Lr*a

rto' la

a\Pt'i'Jto

4.o

.

- /^

^,u,

,l'roe-(e^ ^{A-t^r} ) 7I ^. -/q, t ('ieot(en

^{I.^.,f}

)

@-

?{.t.*bL-

,,-..

^h ^-l*f

,Lar6^ rfrrutl

9..r.^

t(p*or

l- \,\, ^f 't^. n,

5f

p -^.1"'-

^

ol.a <. U.r-Q.i

-

fu-

^'

I I

&iqrah'".r.,

prtlirrc d- J,r fl(-,-.r^fr

iln ^t

^no.-

en\^i laa

f;:;*:fi

-h

)r'vll!trr ^J.e.

Znl..f^ .

#fi-l#

Fa ".rqG-

XX

AOt-rnu

xY

Un ac^( Lrp"

.c-

tr*rr-^-eYerl

L

,4-*

Jc-

-

^i,hea

):- ^Fy

Pu

xy

Ga*g..6 .,

!

!

i

-.t

.r.

n ^.lo.Aio n

'' _FoV

P,4. XX

Fi XX ilo

5"F

--

^xY

Got.5ea

Fi

6 .7?, * /to ^A'**,-

^{I va .a F.}

n ^{da Aex}

^oa

I

-"1 "

PTATE

rl

I

firc. rzE

- ^{tloyeu oe}

Br,r.arltxlt

' Glafldcs sativeica dc

Cbinnau plwttt, (d'apes Brbi.oi).

f.6. ⁴³

h

['

I,,

I

b

12r4r678

t 2, 9 t0il t2)

,r-Jr5*r.r**

9 toll

6

suAlEffi,i;58{+-

*f,F _I

I

F,

A. 4+

i .h

;-

1.t! t-.

I

:.

j

ct ."t-t

Pb

ll

{l

li

Itri'

,i. iJ ,,,tTuDE Du cpMpLExE sb4aLIaM.DAMNosaM EN.AFRIQUE DE L'ouEsr - ^I

Billc pb : parahrlbftrni.

ilLo-{

-pb

3

^t

Iih Nilc Sirba Soul>rc I tlaodirrna Frc. 5. - D€termination des 6 cytorypes par comparaison dc l'extr€mit€ du chromosom€ ll.

I

hornoz]gote ou hetCrozygote. D'apres Yertnr (comm.

person-) cett€ inversion chez Yah serait !i6e au sexe, les fcrnelks €tant hornozygotes et les m6tes h€t€rozygotcs

pour IIL* (rappelons que chez << Yah ^>> il y a fusion cctllronEfiqtE). Pour la ^paire Nilc-Sirba on nolc lu ^pr6-

sence $ur le bras long du chromoeome ll ^{d'un complcxe}

d'inversions gue nous avons interpr€tC conune forme de 3 inversions successives llLcr, llLq et IILCT. Chez Nile, consid€r€ cornrne le ptus proche du standard Bille, Vruu"u;

pcnse que cette inrrrsion coraplcxe est egakrn€nt li€e au scxe. Elb est standard pour les fenrclles ct h€tdrozygote pour les mihs. Chez Sirba oir elle n'aurait plus de relation avec le sexe, elle est hotnozygote. Enfin pour Eandama- Soubrd I'inversion lll, est homozygote chez Bandarna et standard chez Soubre. Au cours de notre 6tude de la r6gartition geographique dcs cytotypes qui fera I'objet d'une publication prochaine, nous avons trouv€ de trds nornbreux h€t6rozygotes I I L -r -,/ ^{I I} L.-,i- ? ce qui laisse pcn-

ser qu'en fait Baa&rna et Squbr€ fornrent un seul et n€me cytotyF€, dl} rnoins en C6tc d'lvoirc.

Le chrormsorne Ilf ^(Eg. 6) donac quelqrcs infiorrna- tiors supphmentaires. ll est staadard chez Bilh-Yah; chcz Ban&arn:a-Soubr6 il ^possEde lcs inversions fixcs lllLl, IILI

et lllLr?+2; chez Nile il possede I'inversion fixe IllL3 et ctrez Sirba il ^possedc de plui I'inversion lllh A l'6tst hsnozygote (raremcni Mdrozygorc). Cettc nr0rne invcr- sion se rctrouve ir t'6tat tret6rozygo{c chca Nik.

Ces diverses inversions lixes ou &lerminantes nous permeltent d'etablir rxre cle de deterrninatbr des c)rtotypes.

I

lnvcrsions lixcs

- ^{lsr + lLJ. .}

, Bitlc-Yuh

- ^llLrs hdtdrozygote _i - ^llLrx lromozygtltc. . .

i ^.

Yah ^(mdile) Yah (ll'nrelle)

!!r ⁶

Coh O.R.S.T.O.M., sdr. Ert. md.l. et Porasitol., vol. Xlll, no 2, 1975 : 85-98 93

cYtrotN(olroMY Annerc

I

0bscrvation

of

cel-Is

,;-.,",,:.,'fi.

Ii'l;Silternal r.renb:'anc of an onionrs rxicl scale (oboervation af,ter colo-

'''l;trl j

;nation witir tlrc :iouge llcutre)

.i'-

,r',

?

^{Put thc} r,ie::bra.ne ir.to ⁰ ₁₅

fJt

^{:louge iieutrp}

lor

a feu niJtutes

E

Rinse r,rith cLlstilled water

pui

it

LsL,,rce:r blace ^ancl sIide

i::

e drop of 4dil'saccherine sol-uti. on

, t-

2. Parameci,;.1 : (ob$c:'vatiotr Iodo-ioui:cu '",'rrt..rr = ¹⁰

1 10c

aiter coloration vrith iodo-iodized rtater)

; of potassiun iodide

* of ^iod.ine

crrl of distilletl uater _I

a

I.

ClfpTrl _x,r!6r\^y

A,^

^,.e--tr.

l.pr:tgil

^1b

Collection ancl .t i:cation o:.' .r.,arvae

4. ^On rccel1:t of larvac for c;'toIo.gica1 idcntificettion thc ^Carnoyrs ehould be changcd r-rgaln. ('liris is bocernsc alcoirol antt acctic acid.

quLck3-y rcect to fo::rn cthyl acetatc '.'rh1ch hardens thc chromogomcs and

thus untlL the larvae,-::.c coupletcl.;. fixc<l, it is clesirable to replace the. ftxative. ₎ .

5. ^l?hen thc larvae &rc rcceivecl by thc cytotaxononist, dctails of'

the Locatlon, detez sairplc size, con.litlon (parasitizerlr-sizc; ;;")- should be recorded, attcl cach samplc numbered on the record. sheet and

on the tube, and storcd in thc rcfrigerator until exanination.

once tho larvac _{havc bccn exnminccl} , illc appropricrtc ntrnbor on 6ic sheet can be tlcl:ccl off , ^{rvith any comments}

There is ;, conciccrtrblc .'r;cunt of con-uroversy rcgarciii:g _ti:o colrection and f ixation of .:. da44osun s.1. larvae io:. cirtologicar work. Several authors rccoffiEe-usc of ice to irunobilize the

larvael which are tncn oirene(l, blotterl and plurged into carnoyrs f lxative ^(ounuar lgTz, c. ^r/ajirnc _-:,crs. conm. ).- "oirro, -"o"r,"ro

i:ref er ' whole larvae to be p}-rngecl i-trto Carnoyrs, anci tire fi:cative c:i.:ngccl

af ter 1o seconds, ancl a6ain af ier 10 minutes (n.,r. Irost I i)crs. "o"*. ^{) .}

Ifgw_ever, f have fonnd no ed,vantage in using larvae disseclecl in the

fLeld, in fact they often give inferior resr-rlts due to 1iie si:ecd and dtfflcult cond.itions und.cr which disscction takes p1ace. ^l-,or this reason the following procedures are recommendcd.

1. ^'Before going into ti:c fie1d., tubes of absolute alcohol shoul-d

be ^prepared, ancl a mcrlc ml.tle on thc tube sl:o..,,ing the vortule of gracial acetic acld to _(4-O be pcld.ed.':i rlie tubes sholrrd be put lnto an ice ^box

tuues containerl in a polythonc bil)r-or

" ^{vride raolthecl theruos} flosk. A snaIl, tightly sioppererl botire of graciar acetic acitl shouLd be kept separertc - put ^on ice the acetic acid freezes tiuickly.

A acetlc oeparate acid.container lvitir preparcci labeLs _{should be icept} vrith the

2' In the fie1dl the ftppropn;.rtc volumc of acetic ...cicl is ^:rd.decl to the tube of alcohol im:::eciiately 1,yi6r to plunging. the larv;.e into the fixative. rf necesser;y', piecLs of vegetation containing lo.rvere

can be ptrt _5-nto the tubc.

A 1abe1 ivitlr loca.tion, date, end if possible, site co-ordinates (thcsc can, of coursc, bc srrpplicd i;;";) r-i" prt in ilrc ^{tr-rbc rzhich} is then rcplaced in titc ice-box. _{Tirc tubc's} containing larv*c s}:ou1cl

be kept as ncar to 4cc as possibrc for at lcast ) ^days, or prcferably until thcy are scnt for idcntification.

1. On rcturning to il:c i,abora.tory or field basel thc Carno;,rs should bc replaccd rvith frcsir fi:cati-vc.

Fixation is clirectly relate<l to the ratio of larvae/fixative,

and for this reason not morc than _{about 5o} larvae/tube (25pt. rixaiive) is recommendeC.

* _See ;\ppcndix 1c for details of fixative.

I

'

6. on examination of tlie sanples it is ^advisable to record thc lnverdions used to irlent:fy t::c ^{species, as} vicll ^as the ot::cr heterozygoue ^and floating inversions, ^and thc ^nurobcrs of larTac

parasltlzed ^by ilertithiirls, llicrooporiCia ancl CoelorlJrtes (-etancLard

sfreets havc blcn ^made for il:is ^{purposc which can bc} raodifiecl if

requt rea). the ^{record s5,eets can thcn bc icept} in ring ^binders in order of rl.\rer syotems and rlatcs of ^{samples and can be easiiy} referred ^to.

Statnlng

There are several reethods of staining ^polytenc chromosomes

(see Darllngton ^{and T,a} Cotr-rr 19761 )unbar, ^{1972), howcver} only ² ncthods will bc describcd. ^herr-:.

1. Feulgen staining ^{(see Annc:l} for c-lctails of solutions) ' this is the nethod used ^r.,rlten bul]r staining is requircci. '. ^{niIc1 hyCrolysis} Ln lO/, I{yCrochloric ec:-d pr,-iperes tlie chromosomes ^b1- exposing thc aldehyd.e Sroups of cicsoliy-sli6: r i';ilich ^ti-.cn rcact vritil tttc stlin' Iiydroiy:sis is- the most critic.. I st.;p, a.::i cl'!n bc ^''- sourcc of st;:in failure. For fresh r:;tcrtr.t] in ^gooC cotrditio:--, ? ilinutcs hrrctrolysis

at between 60-65 oC i s acletlltatc . :lcvlever, if tllc uatcrial ^{has been} in fixative sone tine ^{(sevcral montirs)} ti:e hydrolysis ^{tin:e ccn bc} extended, or the tenpereturc raisecl siightly.

*}rocedure

Remove larwae from Carno;'rs ^{and wash} in distilled water, ^pour off the disttlled vrater, blot ttu ltr,\lae ^and put into 1"o/' dJ]- at

GO-6:roC for Z ,i""i""-(,i"p""aing ^on the eondilion of ^the material).

furter hyclrolysis quickly pour off iiCI ^{and add} dlstilled ^I'ratcr,

wash for 1-2 mlnutes, r6tore iarvae ^and blot ^before putting them into a smalL quantlty of ieulgen Etain in.a ^sma11, ttghtly stopperecl bottle.

The statn nust !e ltept in the darkl ^(enpty filn cassettes are vely usefuL for this purploc). fJta"inigg tine ^{dcpends on} ti:e roeterial:

fresh ^and reared-nalerial ^{nay neecl} only It'hoursl ^{whereas Zii-J hours} ts necessary for most other naterial. $tainlng timc ^{can be cxtended} for 4-5 houig, but nith incrcasir:g time in the stainl t5o ^condition of the chromosoroes deteriorates.

Ylhen stalning is judge<i to bc adecluatol the stain is ^pourecl off and the ^la:rrae washc<l 2-j tines :-n tap ^water.

Prenaration of Slidcs

The sallvary ^{glands Lirc disscctcd} out iu ^601" o"otic acirl ^l::d

reraoveil to a ^clear, "i:.a" ^rvith a clrop of 6Ofl zcetic rciC' 3-4 larvac can be put on a slide for disoection, ^and it is often better ^to leave the larvae ^f or a f crv seconcls to sof tcn in tl:e ^acid.

Iffarvachavcalotoff|'tboc]y,orthereisalotsillr attached to the _6;1r-nCs, BO/'acctic acicl can be used'

Acleaneoverslipislowereclontothesalivary_gIandsrcare-

ful1y ^avoldlng alr bublles, ^and the slidc ^and. coverslip gently blottcd'

trror caees when there is fat ^body or siLk gently ^vra:mi'n6 the slide ^over a sBirlt laup beforc ^squashing rryiII heip to ^sprearl the chronosomes'

t

oo ^'spread, the chromosomcs the coverslip can be gently tapped rvith a

penelI or dissecting necdle o.nd flrcn squashed. uy appiyin!-prcssure with the thunb or first and second flnfer. CarL nust- be tiken not to move the eoverslip durlng squashlng, The slide sliould then be

ring.ed' wtth rubber solution to avoid clcssi'cation; (tfre mna nscd. to repalr bleycle tyrcs, }ocaIly ava,.ilabIe ln thc narket). several slldes can then be preparerl Lefore examination unrler the coropowr4

mlcroscope. fhe slides can a.Iso be kept in the frldge for several hours before examinati-on, or for pcrnnancnt preparatlon.

2. Orcein Stainlqg

orcein staining often gives better preparatrons than those stainod wtth feulgcn, but it is very stalned. llowevcr, it is a'uscful slour and. ,;.rtcrr:ative onty ina:-viclual laryae f or snall sa:mp1escan bc

and, also to counterstain fa.int feule;en-stalned larvae.

Procedure

larrrae are taken fron carnoyrs and put into a crop of ^60,lr, Acetic a clean acid _sI1de for with 1-2 rni-nutes. 6Or',.1 .,'.cetic fhe acid glands and gentiy are then dissected warmed. over I spiritout to

Lanp. the orceln ad'ited. acid [h9 slide is gently ts then blotterl off warued and a drop iron trne to of lacto-aceticttnne, after J:9- ls silk _bLotteel removed, rl"ytee the staln rs bloitoa and off fat and 6$, bocry removerr i ^acLd. gTo, aaaea of from orce:.n ana the uirr."nry glands. fhe acid isire-;;;;r";;es otr added. +itlr _,-eo% acetrc ^2_5 _squashed acta minutes added _{as described}the and orceinthe

above,

Penqenept Preparati ons

There are-tvro principal _metl:orls of nalring permanent preparetio:r,s.

lhe fi.rst rnethod involves qulekly rrcezing ih" sridc in riqurd nltrogen or but covers3.tp froeztng 1s d.ry _n6y quickly ico. not bo prized (lr,*--^ii,f6-,: qurclr off rviih a orouBh _c.,.,n to prrvpnt _bo ^yLzor Iroocr: bladc in a r10!rt,E,,tian) ;;ii-;;;i; clecp frceaelcover_, Bhr

srtp and sl-ide prungecl into absolutc arcohol for up to 2 mi-n...rtes.

[he chronosomes are thus frozc:: in placc, and the alcoho] carn rep]aoe

the aqueous Acetic acid solrition. ?hc srlce and coverslly, are removed, sIlde and fron tlre coversli!: rcplaced. tire alcohol, clrained., anci speed a d.rop is essential to of Eupo.rol lut ^{preventon the} desslcation and shrinicage of the chromosomes, ancl it 1s iiportgnt to ensure that the aIcol:ol is rrbsolutc - ^as In the tropics alcohol qulckly becomes hydrated.

the second method is Lrseful vrhcn dry lce or liquid nitrogen ts not avallablc, but gcne-raIl;, 6i.res srides of inferior quality.

?he slldes &re put 1n a petri d,ish and eoyered. with Acetlc - aloohol ^(Carnoyrs ttxitive) *n,1 teft overnight. [he acetic aleohol sLowly repLaces the ^aqueous sorurtion. The iext day the sride is

removed, blotted ancl the sl-ide and. eoversltp ^pasoed _throug5 alcohol ae descrlbed above.

$ehednle of fixativc and stains Ftxatlvbi

T, _1.

_CarhbytE

_fixatlve

_{(r\cetic alcohol).}

ii!'

5 ^Parto'' absolute alcohol 1 part,.$lacial acetic acicl'

Carnoyls

flxativc

should bc

freshly

^{preparc'd prior}

Statns

)- FeuLgbn (Leuco uabic fuchsin)

.

Ingredients

g.

baslc fuchsin sms. f,-S,O-

;,.(

z

^>

10'cc

r0l

scr

to use.

1

2AO cc,,

tli.sttlled

lvater,

r lFroced.ure

IIo*-t ?00 cc d.lstilled water to QOoC, add 19. basic fuchsln.

CoSi:lF9 60oc, adtl 2 gnis. I(aS205.

coq?.lto 5ooo, ^add 1o eic

tof

tict.

Shd'ke'weLl and keep ini a ti6htly stoppered cont:.iner in the

frlfrge for 24 hours, erfter whicli bleaching ^should. be conplete.

.-ir:,_t..

Att'd*.,about 0.5g.

of

activated charcoal (decolourising ca.rbon) t1,',"lfibe etatn and. shalce rvelI.

; ^iLl,.,J i.

6... fdIfr{r.,rapldIy to reno,ve charcoa.l r:nd impuritr-cs. ihe resultnnt s!a{a ^should, be tr colourless 1iqr,.irj, hc''.revcr pocr riua}ity

Fuolietn may rcsult in a ye.liorv ol: orangc solnti:,:. If ti'ris is th'e ease, add no.re cli;r:icoal ^,..nrl. filicjr rS.^i::. ."'rre nust be t;"iien

to ftller or-rt aI.,l. tr.iqes of charcoal fron tlic sr)1Ltt1on.

i

7. ^{Keepl J.n} a welL otol-rpercd bottlc in the rcirigcrator.

I

I

lacto-aeot{c Orceln Stock HoLutlon{:'

I g.

Orbeln.,i

25 ss-.gLactal acettc ^acid 25 cc 85rt

l,actlc

acid

thLE oan be.,|rgpt

tn

a rveli otopporcd.

bottlc for

^long perlOd.s:'ofl t1me.

For countersttlntng Seulgen-treated chromosoues,

it

can be'ueed

dlrec!Ly.

However,

to,

Orcein-stoining )-

part of

the Stock ieolutlon shduldl Le dtluted wtth J parts ^{5a,4 aceitc aci<i}

1Y ,,t ^-

.ii

$

,

5 :{,

ir.t .lri,

#

₊

,*.+l

,t' ''li ,d'.|}'b

t

_'lii

s

-:t ,

_t'

si{',},

? ,ii rlsl1*d

si

.s

|:i

&$ iB*.

:,7

.,

rtli!

#

$

*

CYTOEiri(OlI01ff

Anncx J

Staining of

silli

^glands clrronosomes

1. Pour the carnoy awaY

2o ^Rinse the larvae tirith disti].led trater!

3o ^Dny the larvae ^on hy6iellic papcr, ^then tlo _{Put thc} larvae in ^L0# chlorid acid at

J ^r:rinutes

or. Ll ottin6-irctr,or' 6ooc for

f

rnitrutec ir,

. tubesl ^heating with tirernlostat

5. Rinse l'rith. distiLled trater: J ^tines

6. ^{Dny the larvae} (hygienjc paper, tlen blotli*6-pcrpcr)

?. ^Put the larvae in ^Feuli;en for i hours. In rrell ^drir;tl ,-,,.,ir!-l

The Feur6en stocl'. is ^{alr'lays out} of li6ht (o"rt< bci6) aricl it'rr;' freezexl"

B. ^Rinse tle ^{Larvae vrith tap} water. ^{Leave tircr:} in tire l-i:r;t trrr'

dissection of ^the silli ^glands.

Dissect (extraction of sill;. ^{glancis arrcl} c11'ot.io$ones) j,,i _!,C., . r:'.tic acirl' Set the chromosomes (the _BaLbiuri nuclei) ^bettueen bl;t.."t, lrlri'r;Lirie i:r a

drop of orcein.

11.

Crush under blottin5-Pi:Per.

i.i.i..:oIyse

'u i:tls.

.,'r; in

,' until

9.

Lo.

s

r\

-{

o

-l

x o

z o

,I

lst

b t t

+ r

aB

Hl{

(a-

F

b Hd

i, :

Ix o= o@

o= F

a

Hv, Ho za,

tp

a+o oo PF oo oo

oc)

PP

oo

a+o 4

P.

oF'

ln aats (D5

a

a a

c)o

I5

.Id

tg os+

ox g

P.p oo

a

o2,

(Dx g

P.

(Dp

a a a a a

zo

P.9, op rtP.

|-t P.o B

a

ClToTnxoNoMrE

A ^v^?tsc, 5

C//R lnrvO.94,/y?

Sct(8rc

€/v ^s'€l

4,t1.1 <, c,

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wao/arcxo/rz page 1l FTC. {. DIAGRAII' br CANTOS

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