ü t to of R.J.

ASP]'CTS OF THE CYTOTAXONOMT OF THE SIMULTJM DAIVINOSUM @MPL&I(

R.J.

POST

Report

of

^OCP Consultancy Januar;r

to

March L986

t

_{Permanent address :}

Liverpool

school

Tropical

Medecine Pembroke Place

Liverpool ü

5qA

L) 2)

4)

E\

6)

7>

A)

TABLE OF ^COTITENTS

qE/-T T nil

I t ¹ nerary

l-llriaa*irraa

Diagnostic

ioversions for S, sanÇtipauli

and

S,

soubrense

OP-insecticide

resistance

1n

the S.

sanctlpry_l_i €ubcomplex Cytotaxononic

status of

forme konkourd

Àn assessuent

of the potential practlcal

value

of studies

of intraspecif

ic

chrornosonal

varlation

The cytotaxonouy

of

the

fu1ry

subcornplex New cytospecies

distribution

records

Àcknowled6ements References

Appendices 1-7

PAGE

APPEI{DICES

1.&2

5&6 7 3 +

L

)

3 6 e

L2 L7 DC

24 25

ITINERARY

Januarlr 20 1986

January 21

January 28

January 29

February 12

to 1l

I,larch 1

l'larch 6

l'larch

7

¹⁹⁸⁶

Departed Liverpool Amived Ouagadougou

Departed Ouagadougou

Amived Banrako

Prospected Guinea

rivers

by Departed Banako and

arived

Departed Ouagadougou

Arived

Liverpool

helicopter

Ouagadougou

-2-

OBJECTIVES

1)

To assess new

diagnostic inversions for

the

sanctipauli

and

§.

soubrense, and

to traia

OCp

of

such inversions.

identificatioa of

^Sinnrliun

cytotaxononists

ia the

use

2)

To assess which species

of

the

resistance to

OP

insecticides iu

inversions.

S. sanctipauli

subcomplex have evolved

the light of the

new species

- èiefostic

3)

^To.assess

the

cytotaxonomic

status of tioauli

subcomplex.

forne

lonlcouré

within _the s.

^6arrc-

4) ro

make a preliminar5r assessneat,

within the s. sanctipauli of

the

possible

use

of intraspecific

posmorphic

inversioas

as

Iation

markers

for

reinvasion studles.

subcoaplexl

aatural

popu-

5) ro

^a§sess

the

cytotaxonomj.c

status of §.

^{squamosun and}

§.

^{yaheuse, with}

particular

emphasis on

the

Uestern Znîe.

6) ro assist in

mapping rytospecies

distribution in the

l,Iestern Zone.

:.)

pracxosrrc rNvrrisroNs

ron

s SA}ICIIPAULI &

S.

^SOIIBRMISE

I.1

INTRODUCTION

vajime and Drnbar (L9?5) derined

the

use

of inversion 2I-l for the

idea-

tification of s. sanctipauli

and,

s.

soubrense.

Essential§, _larvae

_homozygous

lor 2L-7//

were

usual§ identified

as

!. sanctipauli,

_{and, those}

in

which

â-Z

was absent were

usual§ identified,

_as

S.

soubrense. Howeverl

using the criteria it

was

difficult or

impossible

to identify

heterozygotes, and such heterorygotes are

very

vridelJ

distributed,

haviag been found

in

samples

fron the rivers,

oshun, ogunr ouemé, Mono, Tano, Blach

vo1tal

Conoé, Bandana, sassandra, Boal

Milo,

Balé,

Bafing,

Téné, Konkouré, Mongo and

others

^(see

Quillévérê

]:9?5t Meredith

et

^a:L

1981, Post

in press'

Post new data and Boa§e & tr'iasorgbor

various

consuLtancÿ reports

).

In a recent

cytotaxonomic

revision

Poet

(in

_press

-

^{sêe Appendix}

l)

described a new§ recognised inversj-on

(2I-A)

_{which had}

not

been found hetero-

zygous§,

even when both homorygotes

(Zt-ÿ1,

_and

2I-st/St)

occured together

in the

same sarnple. Re-exa.mination

of

vajime and Dunbarrs holosrpes revealed

that s' sanctipauli

was homozygous

for ?J*A/\

and

§.

soubrense laclced

2r-A

corapret§.

rt

was concluded thatw

hilst 2I*?

vas

clears not

_behaving

like a

species diaSnostic

inversion, _2I-A

was. Hence,

it

has become

pertinant to

assess

the

va-

lue of 2I-[ 1s,

accurate species

identification in the

ocp

area. I,tris

has beea achieved

by the direct

exanination

of

^new

materiar (post

_new

data)r

and

by

the

training of

OCp consultant cytotaxonomists.

I.2

TRAINING

D'

Boalgre and

F.

^Mosha were shom,

â-A

during Noveuber 1984.

since

then

they

have made

routine identifications

according

to ü-7

1y^rime & Drnbar :]g?S)t

but

have

also

noted

the

prescence and abscence

of

2L-A

for nears ar1

specir[êDso

îhe

accuracy

of

Boase,s and. Moshats scoring

of

2Ir.A rras assessed by _{rys_}

tematic reexamination

of their _oId

permanent

slidesl

and,

§r

^checking

their

iden-

tifications of

new

material-

Both

D.

Boa§re and

F.

Mosha were found

to

be

ful\y

competent

in their

assessments

of ZI-A.

Hence

it is possible to

incLude

their

o1d data

for the

asse§sEent

of

2r,-A as

a

species

diagnostic inversion in

the

OCP area-

G' Fiasorglor

was

ful\y traineà in the recognition of 2I-A

during Fe- bruary 1986, and

is

now

fu1§

competento

-4-

RESIILTS

saroples

of larvae

have been examined from

ovcr

80

difrerent sites

acttoaa

I'ligeria,

Togol Ghana,

Ivory

Coastl

Liberia,

Guinea,

Mali

and

Sierra

Leoae. ^No heterorySotes

for

2L-A have been observed

in

arly sanple

(Post in press,

^post

new

data,

Boa}rye & Mosha personal communication),

including

sanples

ia

^which

both homozygotes occured together.

There can be

little

doubt

that S.

sanctipauLi

g

and

S.

soubrease srn

(S.".

_{= §9rrsü novor}

to indicate that identifications

were nade according to 2L-A _and

not

according

to ZI-?)

do

not interbreed

and shorüd be regard,ed as good

B.iological

Species (Mayr

L9?ù.

The

old anbiguity in identification

using

ù-7

can be explained, because when

identification is

nade using ?J-A, both spe-

cies _are

found

to

be posmorphic

for ZL? (post in pressl ^post

_new

_data,

_Boase

& Mosha personal communication).

Sample data

collected

by

post

_{(ney data)} A reassessment

of

a.J.I

oId identification§,

_{as are}

in a future report

by

D. Boase

and

F.

}bsha.

I.4

CoNCLUSIoNS

D. Bofire,

G. Fiasorgbor _and

F.

Mosha are nol,

ful\y

conpetent

in

scoring

inversion

?JnA. Examination

of al.l

^possibLe

material to

date has

not

revealed any

anbiguity in

the use

of

2L-A

for ideatification of s. sanctipauli

_sotr. âDd

s.

soubrense

s.n. 0f

which

S. sanctipauli s.rr. is

always homozygou

s

ZI_A/A, and,

S.

soubrense

s.n.

never has ?J,-A.

are presented

in

Appendix 2.

possible,

_should be collected

S. sanctipauli area,

using An immediate reassessment

of the detailed distribution of

§:n.

^and

s-

soubrense

s.n. is possible

form some

parts of the

ocp new data and

old

records ⁽

if

2I_A was noted).

1"5

RECOI,IYüI'IDATIONS

I) nfter

discussion

with _D.

Boasre,

that in future

OCP cytotaxonomists

pauli

and

§.

^soubrense according to

G. Fiasorgbor _and

F.

Mosha

it is

reconnended should be

instructed to identify S. sancti-

ü-A,

and

not

accordiag

to ZI,-/,

and

that

this is indicated

by use

of the

term ^rrs.nrt,

for

exanple

; s. sanctipauli

s.ao

2)

-

.

^,3oa1rye and

F.

l'losha shoutd be

instructed to

compile

a

comprehensive

list of sltes

and new

distribution

maps

for S. sanctipauli s.n.

and

S.

soubrense

s.n.

ar

^, oon as

possible

using

all possible

data.

3)

^rilren

old identifications

were _made according

to 2L-?,

and

available

data :.s

not

such as

to allow a

reassessment according

to

2L-A, such

identifications

silould no

longer

be regarded as accurate, and l-arvae can

on\r

be regarded as

Seneral§

^belonging

to the S. sanctipauli

subcomplex

(i.e. _{either S.}

_sanctipau-

Ii s.n. o: !.

;oubrense

j,.n.).

4) ror ir,:ffrrl

konkouré, soubrense

rBr

_and

Djodji

form see

sections 3

and 4.

5) tt is ^strong§

recomrnended.

that in future

OCP cytotæcononists _{should. be}

instructed to

make two

or three

representative perrnanent slid.es from

all

san- pl-es examined

cytological§.

This migirt

require

an increase

in technical

as-

sistance, but the

advantages

are

obvious.

In the

present, case

it

would have been

possible to

redraw

the

complete

d.istribution

map according

to

ZI-A by

rapid

examination

of

an extensive

collection of oId

permanent

slides.

2.2

2)

QlLNSEU'l'rurDu lrr^ rsT'AllcE rN THE

s.

sAt'tc'1'rpAulr suBOoMpLEx

.1 D,I'I'i-]ODUCTION

The proposed acceptance

of

2L-A as

a

more

reliable

means

for the identi- fication of S. sanctipauli

and.

S.

soubrense than

the

use

of &-?

atso involves

the rccognition that

most

old identifications _are of

unlorown accuracy, and, some

are

undoubtab§

incorrect. rt is therefore possible that within the S. sancti- pauli

subcompl-ex

on§

one species

is resistant to OP-insecticides, but this

species has been

consistant§ misidentified in

some

populations.

_This

point

was

first raised

by Post

(in

_press

-

^{see Appendix}

1),

because two loowa

resis- tant

populations vrere both

identified

as

§. sanctipauli §:1.,

^although

in

the

past

one

of

then rvould have been

identified,

as

S.

soubrense according

lo

Z1-?.

Therel'ore,

it

was

pertinant to

exarnin as much knoi'ln

resistant naterial

a6 pos-

sible to test the

hypothesis

that within the S. sanctipauli

subcomplex,

on\r S'

sanctipauLi

s.n. is resistant to oP-insecticides

and

not S.

soubre[sê sono

],iA1'IGlf ;\LS AI{D ME1'HODS

:) ^')

Data was compiled from

fqrr

sources :

I

)

Population _sarnples already described by

post

(

in

^press

)

^{and Imovrn}

to

have been

resisti-rnt at the

time

of

sampling

(Kurtak,

_personal communication).

2)

he-exa:nination

of oId

permanent

slides,

made over a number

of

years by G.

Fiasorgbor as representative

of

known

oP-resistant

popuJ-ations

(Kurtak

_personal

cornrnuni cation

).

3) rcentifications

made from new samples and

insecticid.e tests, of

knorm

resis- tant rnaterial

(Kurtak personal communication).

4)

reassessment

of

ol-d data presented, by Mereclith

et af (in

press).

Ii jii _ul,.ts

OP-resistant _Ia-rvae (Kurtak personal communication) were examined.

from 20

sites i.

31 samples, and data

is

^presented

in

Appendix 3.

All resistant material

was found

to

be homozygous

for â-A,

and can

tlieref'ore be

identified

as

§. sanctipauli _s.n.

In the past

much

of this resistant _material

_{had been}

identified.,

using

2L-7t

as

S.

soubrense, and some

of it

could

not

be

identified,

because

it

^was

i.etcrozygous

for

ZL-7

(=

_So/Sa

in old terminolory).

a l,

Itr

It

can be noted

that the

numbers examined from

old

samples,

for the

present stu{r

r

^rr/as

^not

^always

^large

^because

on§ a

few representative permanent

slides

vrerc

available.

However,

the

widespread geographic consistanry

of the results ir; strikj-ng.

CONCLUSIONS

It is clear that S. sanctipauli s.rt. is resistant to

OP-insecticideel

but there is

no evidence

for

resistance

in s.

soubrense

s.n.

Survivors

of

two

insecticide tests

from

the R.

Pra

at

^Eemang on L?.5.82 and november

r98l

lacked

inversion

2L-A

(lteredith et aI in-press), but

since they were

on§

reported

as

^rTrear

the limit for

susceptiblert,

they

are

not

con- sidered

to

be

trues resistant.

These

results strong§

suggest

that within _the §. sanctipauli

subcomplex

only S. sanctipauli-È!. is resistant

and

not S.

soubrense

s.n. This

does not preclude

the possibility that S.

soubrense_§È. might evolve resistance

in

the

future, but at

^present

there is

no evidence

that this is the

cBSêo

RECOI"MD.IDATIONS

it

SE

Now

that

ocP rytotaxonomists

are

fu11y comp

tent in

the

i.s

recomrnended

that future identifications of §. sanctipauli

should be made according

to this inversionr

_arld

_not

_2L-?.

2) Reinterpretation of identifications

mad.e by Meredith et

3) p-

_{Boa§e and}

F.

Mosha should be

instructed to

conpire

scoring

of

ZL-A and

§.

soubren-

a.J.

(in

_press).

a

comprehensive

list

from

all suitable

da- communication, aoê Eiec-

material that

does aot

old identifications

made accorcing

to

2L-? shorüd

not

be regard.ed as

accurater

but it

may be

possible to reinterpret

some

oId insecticide test

da-

ta using three

sources ³

l) fne rei-dentifications

presented.

in

^Append.ix

l.

of

as ma.q1r

reidentifications (using 2t-A)

as

is

^possible

ta that is

already

available

^(noatge and Mosha personal

tion r)r

and such data may

include

some

i.nsecticide test

appear

in

Appendix 3.

-8-

3)

cyroruoNot,trc srATUs

or

^roRJ.tE rouround

')l I]'I'LItODUCTION

Forme konkouré vras

first

observed

by

Qui11évéré

vrithin the S. sanctipauli

subcomplex from

the

Fouta

Djallon in

Guinea. A fu11

description

has

not

been

published, but the

form was

principally

characterised

by

complex

flooting in- versions,

one on each

of

chromosome arms 3L and 2I,

(euil1évéré et

^a.]. t982r

Quillévéré

^personal comnunication).

Post

(i-n

_press

-

^{see Appendix}

1)

has described a nel, rytospecies

with- in the S. sanctipauli

subcomplex from

Sierra

Leone.

This

species, naned

s.

soubrense ^rB

t,

_{can be}

easi§ identified by the fixed diagnostic inversion lL-[.

I'his _inversion

has never been found. heterozygous§

(l-t*l/Sù, _eve[ in rivers

where

S-

soubrense

rB'

_occurs

with S. sanctipauli gg.

and

S.

soubrense

s.n.

However,

S.

soubrense

lBr

al.so possesses complex po§morphic

inversions in

chro-

mosome arms 2T' and

ll,r

^{and these appeared}

superficial§ similar to

those

of

forme konkouré.

In

view

of this

and

the fact that their

^known

distributions

were

virtuall-y

contiguous

i-t

vras considered

possible that S.

soubrense

tBt

_vra.6

the

same as forme konkouré

(post iu

^press).

A

detailed

cytotaxonomic

stuSr of

forme konkouré was undertal<en

to

cIa-

ri-fy its

exact

status within _the S. sanctipauli

subcomplex, and.

specifical§

to cietermin

its relationship with S.

soubrense rBr.

1^' I'iAT]JRIA],S A}ID I,IEIi{ODS

l'lew

material

was coLLected from

the

Fouta DjaJ.lon area

of

Guinea and

subject to full

karyotype

ana§sis for

comparison

with _other

members

of

the

S- sanctipauli

subcomplex collected. from OCP Zone !'lest and elsewhere.

IRESULTS

FuIl

sampling

details

and

inversion

frequency data are presented

in

Appendix 4.

Larvae sampled from

the rivers

Konkourd and Kakrima were found

to

^pos-

high§

complex po§morphic

inversions, in both

chronosome arms 2L and 3tr were

indistinguishable

from those described

by

QuiLlévéré

fron forne

kon-

( Quilré'véré

È al

^1982, euj.llévéré persoual comrnunication).

§amples examined from

the rivers

Konkouré and Kakrima possessed,

neither

rnversion

IL-A nor L-A,

which

are

homozygous and

diagnostic of S.

soubrense ^rBl

ard S. sanctinauli

sorlo

sess whi ch KOUre

-9-

Furthermore, these specimens were

not

found

to

possess anùr

fixed

iuversions which vtould

differentiate

them from

S.

soubrense sollo1 although they were

found

to

have

a distinctive array of

po§morphic

inversions, particularly the

complex i-nversions aLrea$r mentioned

by

Qpillévéré.

The complex rearrangements

in

chromosome arms 2L and

lL

^were

subject to

com-

plete

sequence

ana§sis,

and hence

their constituents accurats

determj.ned.

In

2L

there

was found,

to

be two

alternative

sequences, ZJ^-4.6.?.D(which

is very

common

in S.

soubrense

s.n.)

and 2L-4.X (which

is

unique

to

forme koakouré).

These sequence§ were

not

found

to

be sex

linked

and a-LI three kar;rotypes hrere

observed. _The heterozygotes appear

high§

complex because they

involve

overlap- pj-ng

inversion

loops

(B--6

_{& 7}

u

D &

X). In

3L

there

were

also

found two com- mon

;rLternative

sequences, 3L-4.L7.2 (which

is

extrerne§ cornmon throughout the

S. sanctipauli

subcomplex) and 3L-St (which has

not previouss

been described from

the S. sanctipauli

subcomplex, being

typical of §.

^squanosun

ed

&--trI3- hense). The JL ^sequences !ÿere found

to

be sex linl<ed (see _Appendix

4). I third

secuence,

)L-1,

Irâs rârêe

A

population

sampled from

the river ^tolente

was found,

to

be chromoso_

maf\y

indistinguishable

_{from those .-ampIed. from}

_the rivers

ÿonkouré and

thkri-

ma. I{owever,

S"

soubrense

fBt

_was

also

found

in the

salne sanple

fron the river

Kclenter and no

rl-A/st

heterorygotes were observed.

Populations _sampled from

the river

Bafing and one

of its

major

tribu- taries (river

Tene) were found

to

possess some chromosomal

features

^(incru_

cling

2L-It'X)similar to

those populations _{samplcd from}

the rivers

KonkourJana Kakrima. However,

in certain other

respects

the

Bafing

river

sanples were

similar to

those

of S.

soubrense

s.n. further east in

Guinea. Hence the Bafing

river

popuiations

are

considered

to

be forme konkoure,

but

with

some cytogenetic

introgression

from

S.

soubrensê sorlo

_"4

coi{c],usroNs

In

view

of the location of

^breeding

sites,

and

the fact that the

san-

ples

examined from

the rivers

^(cnkouré and Kakrima were chromosomaLs

indis- trn;uishable

from

euillévérérs description of

forme konkouré

(Quilevaré { al

1982, and

Qrillévéré

^personal comrnunication). There can be

litt1e

doubt

that the

new materia.l- examined was indeed forme konkourJo

Forme konkouré rvas

not

found

to

possess

inversion

2L-A,

ald

hence

it

rc not S. sanctioauli

s.n"

-

^10-

not

in

Forme konkouré was

not

found

to

possess

inversion IL-A,

and hence

it is

tl.re sarne as

S.

soubrense

rBr.

_Indeed,

the

abscence

of

LL-A/St heterorygotes

the river

Kolente sample

is positive

evj.dence

that S.

soubrense

fBr

_and

for-

rne konkouré do

not interbreed

where

they are

sympatrico

No

fixed inversions

were found

that

would have

distinguished

forme kon- l<ouré from

§.

^soubrense

s.n. fherefore

forme konkouré

is

considered

to

be a

Seoi;raphic race

of S.

soubrense sorlo' and should be

referred to as

3

rS.

_sou-

brense

s.n.

forme konkouréil.

The cytota-xonomic

status of §.

^soubrense

s.n.

forrne konkouré

is

very s1nrLar

to that of §.

^soubrense

s.n. Beffa form.

Both

are

geographic races

of

§'

soubrense

LA. r

^and

^neither

^possesses a4)r

fixed diagnostic inversions,

beiag

clifferentiated by characteristic

polymorphims. _Hence

in

both forms

there exist incivrduals

which

are

chromosomal§

identical to §.

^soubrense

s.n.

from other

€rreast and such individuaLs present a problem

for identification. ^practical

cytotaxonomy needs

objective criteria

by rvhich everlr 1arrrae can

be classified.

Tltese uiere provided

for Beffa

form by

picking out the

rnost

characteristic

chro- mosomal

features of

each sex and

defining individuals of Beffa

accord.ing to

their

exact karyosrpes (Merertith

gt aI

1983).

In this

way populations _which contain

Beffa

form

will

always aLso contain

s.

soubrense

g,g. ^(unless

^samples

are very

sma-l-l), although

all

these

flies

come from

the

sarne rand,on-mating po-

pülation.

It ^j's possible to

adopt

a similar

approach

for the identification of

forme konkouré. The most

characteristic inversions are

those complex rearran- gements

initially

noted

by euillévéré et

a1

(1982) ;

^{2L*4.X (which}

is

unique

to

konkourd) ana

3l-st.

^However

_3L-st

might

easis

be confused

with 3t-4

⁽

v;hich

is

videspread

in the S. sanctipauli

subcomplex), because they have super-

ficial§ similar

heterozygotes. Hence 2L-4.X

is the

most

sultable inversion for

use

in identification.

Larvae which

are

heterozÿgous

(zl-4.x/4.6.?.D) or

homo- zy8ous

(2L-4;{/4.X)

_{can be}

identified

as

S.

soubrense

s.n.

forme konkouré,

whilst

those homorygous 2T,-4.6.?.D/4.6.?.D _can be

classified

as

S.

soubrense

s'n' ijoth

these taxa

rvi1l free§ interbreed

and

S.

soubrense

.n. will

pro- bably always occur

vrith §.

soubrense

s.n.

forme konkouré. However

the rela-

tive proportions of

these two

taxa identified will

varJr, and

give

an

inùica-

tj-on

of

^whether the

population

as

a

whole

is

more

or less differentiated _fron

typical S.

soubre[sê sorro

L R I]COI'ii.iU{ DAT ION S

1)

^Forme konkouré should be

treated

as a

!t".Se s.n.,

and should be

general§ referred to

me I<onkourért.

2)

Trrere a"re no

fixed diagnostic inversions,

and hence

it is

rêcofi-

rnended

that for routine identification the following

method ⁽

similar to that

used

for Beffa forn) is

ad.opted. Larvae homozygous

or

heterozÿgous

for

2L-

4-l(

⁽ 2L-4.X/4.X

or

2L-4.X/4.5.?.D) should be

identified

as

s.

soubrense

s.n.

forme konkouré,

vrhilst

those vrithout ?J,-4.X

(l.e ef,-4.6.7.D/4.6.?.D)

should.

be

identified

as

S.

soubrense

§.n.

Hortrever,

it

should be remembered

that

the- se two

taxa

are

part of the

sarne species and

will free§ interbreed

whea rym-

patrlc.

I'levertheless the

proportion of

forme konkouré

identified

from a popu-

Lation rvill give

_an

indication of the

degree

of genetic differentiation that exists

between

the

population as a whole, and.

typical S.

soubrense

s.pr fur- ther

east.

l) ^the

^simple

definition

proposed above

is

based on Qui1lévérérs ^own

criteria for the identification of

forme konkouré, and

will suffice for

rou-

tine

work. However

specific

research

projects

maÿ

occassional§ reguire

a more

accurate

picture

using

detailed karyotypic ana§ses, rather

than

a

slrltrmarlr

consideration

of

2L-4"X

alone.

An example would be arly

stu{r of patterns of

mi-gration usi-ng inversi.ons as

natural

popuration markers.

4) tne distribution of

forme konkourJ should be mapped

in detail.

This

will require

new

collections

from

the

Fouta

Djallon area, with particular

emphasis

in the

east where forme konkourd

is

expected

to

^nerge

with typical S.

soubre[§ê €iolto

geographic race

of S.

sou- as ItS. soubrense

s.D. fof-

4)

-12-

AN ASSESSMINT OF THE POTENTIAL PRACTICAL VALUE OF STUDIES OF INTRASPECIFIC CHROMOSOMAL VARIATION

4.L

INTRODUCTION

Routjne cytotaxonomic

jdentification of larval

samp'les

generally

reveals

very little

information concerning

intra-specific

chromosomal

variability.

However, more complete

karyotipic

analyses

usually reveal a

wealth of

variability

which would otherwise have remained

undetected.

Such

variability

may sometimes provide new

criteria for the identification of

species ^(see

for

example

2L-A, in

Appendix

1),

and

often reveals the

existence

of intraspecific

geographic

varjation.

To date

very little

account has been taken

of the intraspecific

geographic chromosomal

variatjon

which occurs

in

^members

of the S.

^damnosum

complex,

but

nevertheless

it is ^possible to identify at'Ieast

two areas where

such stud i es ^mi

ght

be f

ru'itf

^u I :

(1) Intraspecific

geographic chromosomal

variation,

by

definition, indicates that

populatjons

are to

some

extent genetically d'ifferentiated,

and

it is ^possible that

sometimes such

differentiatjon

might

also

extend

to

^non-

cytologica'l trajts,

such as those which might

affect vectorial

importance.

(2)

The

different arrays of

polymorphic

inversjons at characteristic

frequencies

in different

geographical areas might be used as natural

population _markers

for studies of

reinvasion.

The

simplest

approach

to the descrjption of

geographic chromosomal

variat'ion'is

through

the

designation

of 'marker' inversions.

The

distribution of

these marker inversions can be

easily

mapped,

individually or

ⁱⁿ

conjunct'ion w'ith

other

marker inversions.

-11-

4.2

DESCRiPTION t^JITH MARKER INVERSIONS

hlithjn the S.

damnosum complex

particular

inversions have

in ^the

^past

usually

been chcsen

as'markers'

because

they

belong

to a

classes of

inversions

for

which

there js

considered

to

be

a priori

evidence

of

^taxonomic

importance,

for

example

sex-linked inversions

(Rothfers _1979).

Three named'forms'have been described

in this

way;

Beffa

form (Meredith

et al

1983), forme Konkourd

(Quillev{r{ et al

^19S2), and

Djodji

form (Surtees

in ^press,

and see appendix

6).

^The marker inversions which characterjse these forms

are, 2S-6b,2L-4.X

and 15-"1

respectively,

and hence members

of

^these

forms

are identified

using these

inversjons.

However, 2S-6b and

15-o(

are

largely Y-linked,

and

therefore

most females cannot be

positively identified.

Similarly

2L-4.X

is

polymorphic

in

^forme Konkour((see

section 3.4),

arr,J hence

there will

always be

a proportion of flies

which

lack

2L-4.X and cannot be

positively identified

as

Konkouri. This

should

not

necessarjly present much

of a

^problem

in practice.

For example,

if a

sample

of

reinvading

flies

were

found

to

^be

carrying

?L-4.X

this

would be

positive

evidence

that at least

some

of

those

flies

were coming from

rivers

where forme Konkourdwas breeding, and

the

frequency

of

2L-4.X would g'ive an

ind'ication

as

to the proportion of

the

immi grants.

Besides these marker inversions which have been used

to define

formal cytotaxonomic

'forms' there are other

polymorphic invers'ions which show

geograph'ic

variation

and m'ight be used

to trace

reinvading

flies.

For example

within S. sanctipauli s.n. the inversion

1S-A has been found

at

frequencies up

to

30%

in

Ghana,

fixed

^(100%)

in Ivory

Coast and does

not

exceed 3%

in

S'ierra Leone (Table

2,

Appendix

1). Similarly inversion

2L-D

varies

geographicaliy

in S.

soubrense

s.fl., ^but

^here more complete data

are presently

ava'ilable than

for S. sanctipauli s.n. Inversion

2L-D has

not

been found

in lvory

Coast and

eastern

Liberia, but it

^is

Table

2,

Appendix

1).

In

S.

soubrense

s.n.

^(Post,

although

'it js _also

_very

data) -

see Append'ix 5.

The immediate problem using marker

that their

geographical

distribut'ion

is Appendix 5 ) ^.

fjxed in Sjerra

Leone and western

Ljberia

^(see

Guinea 2L-D seems

to

^be

restricted to the

south in new

data,

Boakye and Mosha personal communication), common

in S.

soubrense

s.n.

forme Konkoure'(Post new

inversions

such as IS-A and 2L-D

is

still

incompletely understood ^(see

4.3

^DISCUSSION

Using marker

inversions there is

^already considerable evidence

for significant geographi variability

and

in the S. sanct'ipauli

subcomplex

th's

has been

partly

formalised by

the description of Beffa

form ^(Meredith

et

al 1984), forme

Konkouri(QuilleîirJet al

^{1982) and}

Djodji

form (Surtees ^jn

press,

_see

also

Appendix

6).

^Besides

these'forms' the inversions

1S-A and

2L-D show

distinct

geographic

patterns,

as described

earlier.

Outside

the

S.

sanctipauli

subcomplex 2L-18 shows geograph'ic

variation in S.

yahense (Vajime and Dunbar 1975, Boakye OCP consu'ltancy

report

Jan-Dec 1985, Post new data) such

that in the east (lvory

Coast and Ghana) 2L-18

is

^near'ly

fixed

on both ^X and Y chromosomes,

whilst further

west

(L'iberia, Sierra

Leone and Guinea)

it is at a

low frequency on

the

Y chromosome such

that

most males are

heterozygous.

At

present these

are

^probably

the best

understood examples of

intraspecific

chromosomal

variation,

and

it is

^possible

that

they might also

reflect other genetic

d'ifferences

jn _vectorial importance.

Furthermore the

variation

might

also

be

useful in tracing the

source

of

reinvading

f1ies,

although

for this

^purpose

it will

be necessary

to get better

geographical

-15-

data. it

should be

possible to

compjle such data

during

routine

'identjficat'ion of larvae, without a signjficant

^jncrease

jn _{the length} of

^time

spent exam'in i ng each spec imen .

There

are

no 'intraspec _i f i

c

marker i nvers ions wh'ich

are

assoc i ated wi th

insecticide

res'istance

in

those

larvae

karyotyped

for the

present study and

listed in

Appendjx 3.

4,4

RECOMMENDATIONS

(i) It is

recommended

that during routine

work ^OCP cytotaxonomists continue

to'identify Beffa

form and forme Konkoure

(using

inversjons 2S-6b and 2L-4.X

respectively) for ^possible future

epidemiological

correlat'ion

and reinvasjon stud i es.

(2) It is

recommended

that

^OCP should now confirm

the status

and

distribution of S. sanct'ipauli s.n. Djodji form.

Simple

cytodiagnostic

should be ^produced

for later

epidemiological

correlation

and

reinvasion studies. This

would ^be

best

ach'ieved by

a

consultant cytotaxonomist (such as

Mr.

D.P. Surtees, who

has most experience

of Djodji form)

over

a period of

about

five

weeks.

It

^is

further

recommended

that until this

has been achieved

Djodji

form should not be

distinguished in routine'identificatjon.

(3) It is

recommended

that

OCP

consultant

cytotaxonomjsts should

routinely

score

the

jnvers.ions _{1S-A and} 2L-D

in S. sanctjpauli s.n.

^and

S.

^soubrense

s.0., to yield

more exact geographic data

for future

ep'idemio'logical

correlat'ion

and reinvasion

studies.

OCP

consultant

cytotaxonomists have

already been given accurate chromosome maps

for

scoring these two inversions,

when requ'ired.

-16-

(4) It ^'is

recommended

that for future

epidemiological

studies wjthjn

S.

yahense an OCP cytotaxonom.ist should be charged

with the

respons'ib j I

ity

of assessing

the relative distributjons of the

eastern and western forms

of

^S.

yahense, by

a

new

analysis of

2L-18 frequency data from

exjsting

OCP records.

This

should

take

two weeks

of ^ful l-t'ime

work.

-17-

5.

^THE CYTOTAXONOMY OF THE SIMULIUM SQUAMOSUM SUBCOMPLEX

5.

i

INTRODUCTION

Vajime and Dunbar (1975)

originally

separated

S.

squamosum from S.

yahense on

the

basis

of inversion 2L-18. This ^jnversion

was described ^as almost absent from

S.

squamosum and

sex-linked in S.

yahense, such

that it

^was

almost

fixed

on

the

X-chromosome and

at

variab'le frequency on

the

Y-

chromosome. Hence, female heterozygotes should always be extremely rare.

These

djfferences

seem

to

hold

well in

most

parts of

^West

Africa,

^but

recent

observations from

Sierra

Leone (Post unpubljshed) and Gu'inea ^(Boakye

OCP consultancy

report

Jan-Dec 1985) have revealed

significant

^numbers of female

heterozygotes.

Many

of

these populat'ions

are'in

genetic

equilibrium

^as judged by Hardy-Weinberg analys'is, which suggests

that they consist of

^a

s'ing1e interbreeding populat'ion,

i.e.

one

species.

^There are

four

^plausible explanations

for

these observations:

(1) _In

Guinea and

Sierra

Leone

there is

mass'ive

hybridisation

squamosum and

S.

yahense (such phenomena

are

somet'imes known as

between S.

swarms; see Mayr 1971), such

that they

can no longer be regarded

a

hybrid as separate spec r es.

(2) These'djfficult'

populatjons

jn

_{Gu'inea and}

_Sierra

_{Leone are}

_S.

_squamosum

whjch shows geographic

variat'ion in

2L-18.

(3

)

^{These popu I at i ons i}

ⁿ

^{Gu'inea and S'ierra}

geographic

variat'ion in

^2L-18.

(4)

These populations

in

^{Gu'inea and} St'erra undescribed, cytospec'ies

different

from both

Leone are

S.

^yahense which shows

Leone

are a

nev'r, as yet

S.

squamosum and

S.

^yahense.

The summary analys'is

of

cytotaxonomic data, which has been accumulated

serendipitously

over

five

^years and

is

^{presented be1ow,}

^strongly

^{suggests that}

the

second

of

these explanations

is true.

A complete

analysis is

^being

prepared

for later publication

(Boakye,

Post,

Mered'ith and

Surtees,

in preparation).

5.2

^RESULTS

Considering

only

samples from

Sierra

Leone and Guinea which were large enough

for statist'ical ^{analysis, three sorts} of

populatjon can be recognised:

(A) This sort of popu'lation'is in

^genetic

equilibrium

and appears

in al1

ways

to

be

cytologically identical to typical S.

^yahense w'ith 2L-18

very

strong'ly

sex-linked

^(Appendix

7, table

1).

(B)

The second

sort of population is also

^'in Hardy-Weinberg 2L-18

is

polymorphic,

but not sex-linked

^(Appendix

7,

table

indeterm'inate centromeric rearrangement

(3C-split),

which

is

squamosum elsewhere,

is often

sex-l'inked ^(Appendix

7,

table

equi 1

ibrjum

^and

1).

^An

known from 2).

S.

(C)

The

third sort of population is not in

Hardy-tJeinberg

equilibrium,

and

2L-18

is

polymorphic

with

weak sex-linkage ^(Appendix

7, table 1). 3C-spfit is also

weakly

sex-linked

^(Appendix

7, table ?).

l,Jjthin these populatjons

'it _is

not just _{?L-lB that} is out of

Hardy-We'inberg,

but

^when complete karyotypes are scored

it'is

^found

that a

^number

of the

^common

inversions are in

^linkage

disequil'ibrium (for

example see Appendix

7 table 3 for inversion

3L-E).

5.3

^DISCUSSION

The samples designated as coming from type A populations

are

undoubtedly

-19-

composed

of a

s'ing1e spec'ies, because

they

occur r'n Hardy-Weinberg

equilibrium.

Furthermore

that

species

js

_most

_{1ikely S.}

_yahense, because the samples

are

chromosomally

identical to typical S.

^yahense as described by

Vajime and Dunbar (1975).

The samples designated as coming from type B populations

are

also undoubtedly composed

of a single

spec'ies;

but they contain

female ?L-LB/st heterozygotes and hence

they

cannot be

jdentified

_according

to ^the

usual

criteria of

^Vajjme and Dunbar (1975).

The samples designated as com'ing from type C populatjons can

best

be

explained as being a mixture

of

species from type

A (i.e. S.

yahense) and type B

populatjons.

Th'is would account

for the

observed

deficiencies

of

heterozygotes and

linkage disequilibrium,

which would be

difficult to

explain

in

any

other way.

Furthermore,

it is unlikely that there is

^any

sign'ificant hybridisation

between

the

two species because

the selective forces

which would be necessary

to

exp'lain

the

observed

disequ'ilibria

would

also

tend to

el'im'inate

the rarer

species (Barton 1979).

The chromosomal evidence

therefore

po'ints

to there

being two separate species one

of ^whjch'is S. yahense. This

conclusion excludes

the first

^and

thjrd

"explanations" postulated ^(see Section

5.1), ind'icating that

the

"diff icult"

populat'ions are

e'ither S.

squamosum

or a

new

species.

There are no

fjxed inversion

d'ifferences between

the "diffjcult"

populations and S.

squamosum, as defined by Vajime and Dunbar

(1975),

_and

cytolog'ically they

are

quite similar.

Hence,

in ^the

^abscence

of

evidence

for

seperate

specific

status

the principle of

^parsimony

dictates that

these

"djfficult"

populations should be consjdered

to

be

a

geographic

variant of S.

^squamosum.

The

electrophoresis

data presented by Mered'ith ^(OCP consu'ltancy report July-August 1985)

very strongly

supports

the

cytotaxonomic conclus'ions above.

In

Guinea

the Loffa river

supports

a type

C populat'ion (Appendix

7, table

1)

wh'ich

is

postulated here

to

be a mjxture

of ^S.

^yahense and

"difficult"

^S.

-20-

squamosum. Mered'ith's

electrophoretjc

^'identif

ications also

revealed both S.

yahense _and

S.

squamosum,

but wjth

no

hybrids at all! In other

words, using electrophores'is every specimen was

clearly ident'ifiab'le

by Meredith as

either S.

squamosum

or S.

^yahense.

5.4

CONCLUSiONS

Both

S.

^yahense and

a cytological variant of S.

squamosum seem

to

^occur

in the

Western Zone, although

in that

area 2L-18

is ^not

always

strict'ly diagnostic.

Therefore cytotaxonom'ic

identifjcatjon'in the

Western Zone is

sometimes

difficult, but jt _{is usually}

_{possible on}

_a

population

basis

by

consjdering

the

karyotype frequency

distributjons of

^2L-18 and

3C-split.

In

S.

^yahense 2L-18

is sex-linked

and

3C-split rare, whilst in S.

squamosum ZL-18

'is

autosomal polymorphic and

3C-split is often sex-linked.

However,

it

^seems

that identification of jndividuals

_{can be more}

reliably

achjeved using isoenzyme

electrophoresjs

than by cytotaxonomy

in the

western Zone.

5.5

RECOMMENDATIONS

(1) It is

recommended

that'in the

t^Jestern Zone populations

of S.

squamosum

and

S.

yahense should be

routinely identified

using

the

cytotaxonomic

criteria

ind'icated

in section 5.4.

Th'is should

allow correct identification of

over 95%

of

larvae.

(2) It is

recommended

that for special studies in the

l,lestern Zone when

reliable identification of individuals is crucial,

isoenzyme electrophoresis should be used.

(3) It is

recommended

that

throughout

the rest of

^OCP area

the

standard

-21

-

cytotaxonomic

criterja

defjned by Vajjme and Dunbar (1975) shou'ld contjnue to be used.

(4)

For

S.

yahense

intraspec'ific variability

see Section 4.5.

6)

^{NEt^l} CYT0SPECTES DiSTRiBUTI0N RtCoRDS

6.1 S.

DAMNOSUM SUBCOMPLEX

New

distribution

records obta'ined

during this

consultancy

will

^be

jncluded as

part of the routjne lists of identificat'ions

issued by the

cytotaxonomy

laboratory in

^Bamako. However, two

special points

may be noted.

Simulium dieguerense larvae were

collected

from

the river

Tene ^(11001'N/

11049'tJ) on

13.2.86. This indicates that the distribution of ^S.

dieguerense extends cons'iderably

further

south than was

previously

thought.

Simulium sirbanum larvae were

collected

from

the river

^Kakrima near Souloudje (10042' N/12o56' t,l),

(10035'N/12o35't,J) and Ganiya

of S.

sirbanum breeding

sites

reported by Garms and Vajime

and

the river

Konkourinear Manangare

(10029'N/12059'hl).

This

conf irms

the

existence

jn the KonkourJriver basin,

as previously (1e75).

6.2 S.

SANCTIPAULI SUBCOMPLEX

The

S. sanctjpaul'i

subcomp'lex has been

the subject of a

recent cytotaxonomic review

(Post in ^press,

see Appendix

1),

and new

criteria

( invres'ion 2L-A) have been proposed

for

species-ident'if

jcation

_(see

_section

₁

of this report). New'identifications are ljsted in

Annex

2,

along

with

data from

a variety of ^other

^sources

all re'interpreted

according

to 2L-A.

Most old

identif icat'ions,

accord'ing

to ^2L-7,

should be regarded as suspect

but a

report

is

be'ing prepared which

will

draw

together all valid

records

with

redrawn

distrjbution

^maps (Boakye and Mosha

in

preparation).

-27--

6.3 ^S.

SQUAM0SUM SUBCoMPLEX

All

^new

distribution

records obtajned

during this

consultancy

will

^be

included as

part of the routine lists of identifications

issued by the cytotaxonomy

laboratory in

^Bamako

^(but

^see

also

sect'ion

5 of this report).

7

)

ACKNOl^lLEDGEMENTS

I

^am

^grateful to

^OCP/VCU personne'l

in

general (and

D. Baldry, B.

Colussa,

P. Guillet

and

B. Philippon in particular) for logistic support,

advjce and

background

informatjon. I

am

also

indebted

to D.

Boakye, G. Fiasorgbor, F.

Mosha and D.P. Surtees

for allowing

me

to

use

their

unpublished

data in

^the

comp i 'lat

i on

of

^th i

s report.

-t6-

B

)

^REFERTNCES

Barton,

N.H.

(1979)

The dynamics

of hybrid zones.

Heredjty

43,

341-359.

Garms,

R.

and Cheke,

R.A.

⁽¹⁹⁸⁵⁾

Infections

w'ith Onchocerca

volvulus

in

different

members

of the

Simulium damnosum complex

in

Togo and Benin. Z.

Angw. Zool. 3L,

479-495.

Garms,

R.

and

vajime, c.G. (L975)

0n

the

ecology and

distribution

of species

of the

sjmulium damnosum complex

jn _different

bioclimat.ic

the zones

of L'iberia

and

Guinea.

Tropenmed.

parasit.26,375-390.

Mayr,

E. ⁽¹⁹⁷¹⁾

Populations specjes and

evolution.

Harvard

University

^press.

Mered'ith,

S.E.0.,

Cheke, R.A. and Garms,

R. (1983) Variation

and

d'istribution

of

^forms

of

S'imulium soubrense and

S. sanctipauli in tlest

Afpica.

Ann.Trop.Med.

Parasit. 77,

^OZ7 -640.

Mered'ith,

s.E.0., Kurtak, D.

and Adiamah,

J.H. (in press)

Following movements

of resistant

populations

of

^Simul'ium soubrense/sanctjpauli by means

of

chromosome

inversions. Proc. xvII Int.congr.Ent.

Hamburg 1994.

Post, R.J. (in press)

The cytotaxonomy

of

^simulium

sanctjpauli

and s.

soubrense.

Genetica.

Quillevere, D.

and Pendriez,

Afrique

de

l'Ouest. II d'Ivoire.

Cah. ^ORSTOM

B. ⁽¹⁹⁷⁵⁾

Etude du complexe Simulium damnosum en

Repartit'ion geographique des cytotypes en Cote

ser. Ent.

med.

Parasi!01. L3,

L6S-172.

Quillevere, D., Guillet, P.

and Sechan,

y. _(i982)

_La

repartition

geographique des especes du complexe simulium damlosum dans

ra

zone du

projet

Senegambie (ICPlMPD/007). Cah. ORST0ivl

ser. Ent.

med.

parasitol. _19,

_303-

309.

Rothfels, K.H. (L979)

cytotaxonomy

of blackflies.

Ann.Rev.Entomol

.

^24,

507-539.

Surtees,

D.P. (in press)

A new cytotype

within

Simllium

sanctipauli

from

-)')

-

Togo.

(Abstract)

Proc.R.Soc.Trop.Med.Hyg.

Vaj'ime, C.G. and Dunbar, R.W.

(i975)

Chromosomal'identifjcation

of

^eight

species

of the

subgenus Edwardsel lum near and 'includ'ing Simul'ium

(Edwardsellum) damnosum

Theobald,

Tropenmed.Parasit.

26,

111-138.

R.J.

^POSI' CONSULTAT{ CT ^it]iJPO}TT

APPEI{DIX 1

Appsndix 1

The cytotaxonomy

of

Simulium

sanctipauli

and SimuLium soubrense

(Diptera:

Simuliidae)

R. J.

^post

Department

of fiedical

Entomology

Liverpool

_School

of Tropical

lyledicine Pembroke Place

Liverpool L3 SQA

Runninq

headline:

cytotaxonomy ofl simulium

sanctipauri

sub-comprex

In

press Genetiea (rq86)

Abstract

^r\r\J\,avvv

It is

noted

that the

chromosomar

inversion

2L-2, uhich has been used

in the past to

separate

s. sanctipauri

flrom

s.

soubrense, occurs as an

intraspecific

polymorphism and hence cannot be considered

diagnostic,

_although

in

some popurations

?r-?

can

stilr

strongry

indicate _the

presence

of tuo species.

Houlever, tulo ner.lry recognised

inversions,

_{1L-A and} 2L-A, can be used

in

combination

to

identiPy

5' sanctipauri, s.

soubrense and

a

neul species

s.

soubrense rBr.

The absence

of the

rerevant heterozygotes

for

these

tuo

neul inversions confirms

the

separate

specific _status of S. sanctipauli

from S.

soubrense rrom

s.

soubrense

r'r

_as

_uerr

_{as providing}

a

reriabre

means

of

larva-r

identiflication.

_{The misus}

_{e o1}

_{2L-? as}

_a

species

diagnostic

_inversion has undoubtedly

led to

past

misidentifications of s' sanctipauri

_and

s.

soubrense, _and

it is

possibre, por _exampre,

that _only s. sanctipauri is resistant to

organophosphate insecticides

in lvory

_{coast and}

_not s. soubrense. _Beffa

_{Form appears}

to

be a

distinctive

geographic race

of s.

soubrense,

but

forme konkourJ remains as

yet unassigned.

A cytotaxonimic key

for the

identiFica_

tion of

members

of the s. sanctipauli

sub-comprex

is

^presented.

TEg:*g

Simulium (tduardsellum)

sanctipauli

and Simulium (Eduardsellum) soubrense uere described by vajime and Dunbar

(tgzs)

_on

_the

_basis ofl

inversion

diFflerences _observed

in the

porytene chromosomes of

the larval silk

glands.

These

tuo sibling

species uere separated _Êrom

al1

other members

ofl

the s.

damnosum species _eomplex by

at least six fixed inter- specific

inversions

distributed

_betueen

arr

three chromosomes

(11-6,

2L-4&6 and

3L-4&flez).

fiore

recentty post

(1984) has shourn

that the

breakpoints

of

one

of

these

inversions

(1L-6) _r.rere

incorrectly

_mapped;

this fixed

rearrangement

is not in fact

a

single inversion but

tr,lo overrapping inversions nou designated

It-o/3.

Despite

this

minor

correction there is

no doubt as to

the status of s. sanctipauri

_and

s.

soubrense as species

distinct

flrom

other

members

oî the S.

damnosum complex.

Vajime and ûunbar (tgZS) _separated

S.

sanctipauLi and S.

soubrense from each

other largely

on

the

basis

of the

chromosomar

inversion

_{2L-z uhich overlaps}

the

doubre

inversion

sequence zL-A.6.

2L-7 uas

at First

considered

to

be polymorphic

in

both species (vajime & Dunbar, 1974),

but

subsequently uas described as absent flrom

s'

soubrense

(the text of

vajime & Dunbar

,

^1975,

is

quite

clear

on

this point,

although 2L-?

is

arso

irrustrated

on

their

idiogram as polymorphic

in s.

soubrense; presumably

in error)

and polymorphic,

usually at a

very

high

flrequency

or fixed, in

S. sanctipauli.

_Vajime and Dunbar (tgZS) _described

only

tuo