;;Q~ ~9~ THE CABBA~E

EFFEcrS

OF

^SEVERALINSECT GROWTHREGULATORS

;;Q~ ~9~ THE CABBA~E

^{MAGGOT, '"}

DELIA"RADICUMLiunaeus(DIPT ERA: ANTHOMYIIDAE)

St.John'.

BY

Copyright@Terry-LynnYoung,B.Se.(RoDours)

< < / /

A thesis eubmhred totheSchool01GraduateStudies in partial.rulfilb:.nentof therequirements"tor

~ ~he d.~greeor'M8Sterl{~ieb~e.

...

.

;-"'"

DepartmentorBiology. .MemcrlelUDiYe~ity'orNewjcuadteud

September1088

X. .foundlaud

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..

(

.Per.hetanbaa ~eri granted

to the'National L~brary....of Canad a t.o .ierofil _ this thesis and ec le nd or 8e 1 1 copies of the film.

The -author_{copyright owner}. has re s er ved other publication ri g h t s , and .

neither the thesis nor'

exten81ve~·eJ:_tr.ct8.frolll"i t • lillybe.printed or:othenthe. reproduced without' bh/her writtenper.ai••ton.

.

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L_'autorhaUon:.Ittltaccord's

~u laca~~~~l.~t::qU=l c~~tfii~:l:

^A

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eeeee .th~. e et de pre ter au.

~il:~ndz::eC\ell exe lllplairee"

du

L'auteur (titula'ire

d~Oit' -':

d'aute ur ) .e ~:f!.e.t:Ye."Le e ,

a u tre . dfoited'!i,f,ablltbationJ

~'~rlaait:~~:: -~e~ ~: .. ^cion::

dohen t h r 8\i. p r i m': e.-ou autrell.ent"re p r od u i t a.s a n ll 80n'

.'autoria: t.i,on'~~_lte::.

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1:'~r::5:S0'~6C5

r:' ^{~ ., /. r, '''' '~ ' ; . <'.:.} "J''"t'~;

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: <"' x:; :r'~"::~:~:" J;i' ~ :<'~1"'(?"?~:;< 'i';(;;;;(:;:' :::i";'~i}

~. , . . ~ ..", . ,", ;<J~,~

'

..

^{~'. ''''}

... . :

A proeedureweede;eloped rOl.m.aint.dn h.ga'stott

~Iotl,.

or \be tabbage.: .. . ..

~aggot.^Deliar~di~um(Linnaeus),'in'the-gffe~howe. ^'Apehi,'dfsh',ea.r"ing·

m~th~ produc1~g

^ahighy.ieJd

~r &d~lts

^was-

4evel~ - p'ed ^rOt

^laboratory

~reeDi~g.o~'

^;..:,,"

' ?~

insect .growth·regulaton against this pest.

_.

Th~raY~j'- ch~omatogr

^..

~hy a~~ colori~;etric

aD&ly: t '

~~moilm~h

^..

~~t~~~t~,/

36,

hou~.after tre..tmeDt:.o~,':t-reedin.gIan: ae de, ~.~r~tedthtmethop~,~D;,

,causedII.signiC!~antreduction Int,r~ba.lose,^t:he.pred~miDaDlcarbohydr a!.e.~~he ,~,~

hemolymph. Met~oprene~id~o~ ~~~uptbemoly~ph..amino'acid·orp~tein ..^I,;'

: i J

'ODeerration,~

^{. '}

j /' 1

>1 - ^{: ': ;j}

. ', ••, ' , ..;,, . , :>~..

"~'>;:I ^{," ,} .,~J.:,;'.'':.~,;'.{:., .

^>

;~~:'(~-:..~~:;,,~~." ~~;>-~<;'. :>i;:~ ,,·:.~,:~<~,,:;l~~;j·.~,Ji.':..l;:-i)~i\.;;;~1~ii·~>.~:-:";

. ' .

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Topi~al

ap'plicatioDof,thechitinsynthesis

inhi -~itor. dmdbeDI~roD (Di~i~ ~; ~.> ' :: , _';.;l.~

at

'a ,rat~ 'or 1,~

^/IS

~e; egg\i~ific~~tly

^{reducedhat.ehof24-28}

h~ur old.' eW&~d"~-.. .

^.

,~;~

ca~sed

^100%

~~tt:,:lj~y

^cir

fir8~ iDit~r

^{lar:ae;before}

they" ~uld,reed '

^00'

~utabaga' :-~~~'

" sli~es^..Din~~n_z~toD^hadb~^{erlee'OD'mature}larvae·~t.puPile.or^..I2:·r.&di~u·~,^{';.:' , :..}~:~:

:'Methopr'ene (Alt6sid,CB>SE),~^{'j uvenile'hormone},analogue,'.pplied &t thelam~^{. .' :}~~,

dose,didDotadnrsel;'&tlki'egghalch'

b~l p~ev~~ted th~ '~~i~D' ~' ~d~ltl rr~~

^{" :i'}

,:i~

.pupeethatsu"bsequently

'de~eloped' rro~ tr~atel ~

^..

~~p~.adult

in"ier"Mediatel: ' "

~f:

w.er~:.~bse":etlll .~ t~eai~ 'pu~~~-. Tr~at~e~L ~~ ~~tuie; ~r~din{I~';

^{". .}

/~t

and,reCentl¥..rormed.pupae-with-inet~opre~e'alsoprOduced-noD-~IoJIII'liUP~~

ad'u,l{

roosai~. ~ ': ciose

of

.o:~

^'"g·p.er

larv.~·~~Pleieit:".up~~.

^&dult

~bi~D'.'

^{: ...;/-.}

' ,a

However,dil1ubenzutOnappliedtothird instarlarueor eupae didnatalred / ';·f{

aduli. eclosion. Juvenilehor moneIand

th~

anti-j uvenilecompound,

Plecoce~e

0,

t ' :';i,

. , . ' ' . ," .". ' I' . ,,3

'.-1

^{I,did oot}arr~\IQ."~,w~enappliedtothe'eg~l.l~":aI .ortUPal l~~b,.', ;<~i

.:

~1

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4

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/"$1"

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~--

•I 8ckDowiedge and pVe'8pecialthauuto:

ACKNOWLEDGEMENTS

/

,

^,"

.

This stud; could_~othavebeencompleted withouttheguidance,PjaUenc:eand' eoeo~r.gemen~from. m1 supervisor,Dr.ROger.cOrdon;towhomI.',express~y..

.

~est

appreciat ion. I

wou~~

^also

like .:

to

tb~k

^my.•

uP""e~isO~'.CO+~~1

^Dr.I

Gordon Bennett,Dr.'Rich ard NollLl},"and Mr.Ray Morr is,Corttieir-helpful .suggest ions and, ,criticismof tbe menuserlpt; Specialthanks

are·also ~ueto~

^•

.

~~.a~ceJ ·~o.~ne~t ror_ h~ ~ist8~~e duti~~'

^t,h.e

,~ourse , ~i.~~8t~~!~ : ~\~. ~r

Flcke~.: orphotogr~p~i~.'_{~ist~ce1 ~}Dr.D.C._:~e"J.(~~.icul~ur~,Can_~d~

Ch~r~~.t,tetowD^••~.~.J.~ro~.8bi~meots^of~_r&d,icutl^eggs'~d,^to.Mr.K:G.ⁱ .,,"PrclUdfoo( (AgricultureCauda)Corcriticism

o f

the~i.nl,l8Cript.^{.. i}

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·~L',~-_Ci ^.. ~ ^{" " : ;.;' . ,:} _:i,:,;:;,.;{>_",~; ,~ ^-: ;,,;;;;,,;;.:."}.;" ;;.i,.,0;.-i.,,:;,;.~;:.~ ;;;j~,'

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-Table of C ontents

, -

6 10 I' 16 IS I~- 16 - .9 20_ 23•_ 23 24 -24

25 25 26 27 27 28 30 30

34

.\

34 36 36 .3 BACKG R OUND'

- /

-..INTRODUCTION

2.INSECT GRO WT H REGULATORS,

INFO RMATION 2.1~^.Juvenilebor mone analogues

'i.2..Anti-juv ebile,!lormoneegea te.tbepreeccenee '.2.3. Chitinsynthesis inhibitOrs • --3.MATERIALS AND-MET HOD S

3.,;"

^Greenhouserearing ofstockcol60y

1\.:'

3;Ll~n'"earingcompartment- I

".' 3.1.2.'Re~r.iDgP!.o~~dure

' . '':. . ' .

••_•.J- 3.2-.Developm ent of a petridish .rearing.metbOdlorlabora tor y screenin g

~'01insed.growthreguletc re ::

3.2.1.Lanairc!.rhigunits . . . 3.2.1.1.Deter mination

o r

optimum larv ..1 density

3.2.~..Pu{)ationunits .' . , .i,.

3.3.- ~:~t~(:~~~~t~t~r:i:~~a::~fy ser~Dib'~~;

Beyer..

I'~sectl

growth _s.regula to rs againstDeliaradieum..: " . :\. ",.. ...

. .3.3.]:Erred01insect lI'owthr~latorS.on~l

. .'

3.3.2.,Erreet01insectgrowth legulato rs on

s reedin

^{g larvae .}

3.3.3.Erredoriosect growthregUlatorsonpup . 3.4.Physiological Itudi~: eered'or _metbcpree pn htmolymph

carbohydrates,prote insandaminoaddsinpost--/eeding lar vae.' 3.4.1.Separationend quan t ificationorbemolynlph carbohydrates . ' 3.4.2..Qua ntificatic:'D -tlr hem olymph am1boacidsan~proteins

4. RESULTS ; _ - __ " _ . ' _

4,1.bevelopm l.'Dt'or a petri dishreario'gmd bbd:,determina tionor opti mum larvaldensityI~larval re'a:.ringunits '

;..) . 4.2:Toxicologicalstudies:-labora tory screeningor,severalinsectgrowth

'r eglita.tors againlt'Deliaiadicum . .

4.2.1.ErCeu'orinsectgrowt hr~gqlatoCllon egge..

4.2;2,ErredorInsectgrowthregulator sonpost-.reedinglar vae 4.2.3.~rredorinsectgl'9w't~I:egulatoCll011;'pupae . 4.3.

~~i:~le:aiID ::SU ::~in;!f:;~ae

^{or.,meth\prenc.00}

he~?I~mpb

,

.r.

( ,:

"""" "~~'.i" ',''''; '1 ~'.\~ "'.. ,",~··F~·>l~:";+')'!<~'n' Ilk :'1"' ,\:'1'r 0;;~;1~

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...lo"....)~.^1.1.~~."" N'f..,. )-....• ,'

' - ^') List of Tables

Tabl e 2-1, Summary table'oftheinsecticidalpropertiesofthree major insect growth. regula).or groups with representative

compounds ..

•Table A-l : EUect oflarvaldenSityinrearing-units Onrutabaga 31

•consumption and the~tageand a.ctivity~rlarvae

Tabl e 4-2:•Erfect

a t

topical application ofseveralinsect growth 35 "

regul ator s to-De liar8

jd

icumeggs.

Figu re~l:

Figure,3·2:

,Flg\tr~4-1:

/

<,

Li~t· of .F tgu r es

Stockcolonyor Deliaradlcum in the greenhouse 17 Petri dish methodfor~ale.r~!,riDgorI!!ili!~ 21 Erre.etof larval densityin .rearing unitson percentage 32 yieldofpup aeend adults ".~.

""""FIgure '-2; Topical aj.p licat'ionofinsect growth reguletors topost-' 37 feeding~!.!!!iElln larvae:erreet onemergence of;"!

adults .'

Figur e'-3: Dose-response'or'methoprene'againstpoSt-ree3ingDelia .30. .' / ..radicum larv ae:erred00adultemergence , '

Figur e4-4:•Erree,tofinsect,growth regulatqrs00-adultemergence 41'

•' / ' when appliedtoa heterogeneouspupal populat ion': ,."

Figur e4-61 Separationofhernplympb carbohydrateson8thin·l ayer 44

. 7

~b romAtographyplate . ",_ ' . Fig ure4-11:-'Erredof methoprene oil:hemolymphmet abolit es

of

post- 46 /

feedingDelia. n.dicumlarvae 36hou~Artertrea~ent

,"

.

^/

'/• . J , .

"'

/

a

List of Appendices

Append ixA.Critic al natu reof water contentIn larval rearing units

Appendix BrQullntifieattonof hemolymphtrehalose . Appendix<kQu':nt tn eatloD.o( hemo!ymph.ainlnoacids AppendixD.QuantlneatlQll. of hemolymph proteins

APpe.n~lxE. Effectof1~.Beetgrowth~rgulatorson Della radlcum egg s1,expel'l~eDtteeord

<; .

-'fI!l' Appendix F."EfTeet·of Insect 11'Owth regulatorson pos ....reedln g

luvae:.experimentreeoed •

Append ixG.EtTeet.or,~etbopren:eon·pharateadults

/t" -.:

. .

74

7 . .0 .4 \

••

••

"

INTRODUCTION

The c.!bbage maggot(cabbage'r091ar)Delia.radi~umL~naell:8(Hylemva brlLSSicll.eBouche}(Di~,tera: ~~.~~myii~ae)^isam~j~r^pesto(;,)'oot &n'd'st~~

...crucifersinNorth AmericaandEurope..InNort h~erica,it.iiIapatticu~!l seri~uspeerrOfCrueiteeeeinCanadaami. the'northernre~)ns'of-theUnited States, 'butisseldom

injuri~us '

^{southofthe40 N}Jati t u<le...

(Met~ali ~d'

^Flint,

l{lMJ.

In

^the

At~aDt~c-~rovinces;Jhe

^cabbage

maggo\ is'the' mOst'.se~io~ p~t e r

rutabagas (swede;

t~~Dipsj,

^I

c~bbage,

^.

caulin~wer,.

^broccoli

and : Bru~el

^,sprouts'

.(Read,}972).,Delia;adicumis

o r

par't icular

co~eern

^'iolhe

agri~~itural

^{nid:st ry}

-ln

N'~'wrou~dJI4ld1::au'se it _.~

the,limiiingfactor

i~

^the'

comin~~a1

prqduetion'of

~

cabbages and

rutabag~,

^{two01tbethr ee}

princip~ 'cr~p; gro~n

^in'ibis

r~giOl~

<'furris ,l(iS9a):'It:is estimatedtbat75

pe~

^{cent of}

~be

mark;table·crop

w~uid

^be

rorrejt~d ye~ly

^{iftbis pest:*,88} not ;ontro lled

(~~ra. c~m"l" _. M .

^Stapleton,

~

"'P rovincial Agricultur e, ResearchStatio n,SlJobn'sWest, NeWfOlrlrd land).

,~~^{fd U1ts,}similar in appearance,tot~~.~ommon bous~^{Oy butonly} baUaslong,oviposit eggs onorneartbe soil~urface,or undern eatb tbe,foliageof planteicrucife~^{-.Firat inst}ar larv a e'(L1)heteh Irdm eggs'and!~e,d^{on the.roots.of} hostplan tsbelowtbesoilsurface~ These small whit e D).!-ggots generallyattack the

se~ondai-y

₄ roots and then_'

~ove u~

^{into ;he}

~ain

_{, .} r60t ot their_{' ,} host_.'

~

^-they

Ieedandmatu re, developing into second (L

2)lU!.d third (La) instarlarvae(Read, 1972). Whenmaggots are abun dant;the roots of('ab~8ge.c8uli,now~r^:8nd broccoli becomehon eycomb ed and rot t en andyoun g piautaoften.dtefromthe attack. Delia.radlcumlarvae feedingon young rutabagaand radish~ause-''loot '.distortio n'and lat er

i~

^the

se8S9~'

^{tunnel'intothe'rieshyrootS,'}

;e~de;~Dg 'the~

.tin market able(Morris,19598;Read,1012).Attheend of thefeedingperiod,.~~

··.r.,,:

.f~Il~~ingsprirlg(Morr is,105~a): The eniire lifecyele takesapproximate lysix

... ' -.

reeding

~' larvae

leave Qae.bostinsearch'or

a"~'uitable

'pupation_site in'

't~e

surroundingBOil -or~,more rarely,pupet e.intheirgalleries,part icularly.ifthe

\". , ' .

surrounding soilisverydry(Read,1072).:Inside the puparium,the lar vadevelops into a pupaandeventuallyinto an~ult. At eclosion,the adultbreaksopenth!

puparialcase andpushes itsway.,~thesoi~,~urface.

Depending onthe locality'and soiltype,

It

radicum mayhave several

\ generati~nsp,eryear, the maximum recordedbeingfive in some partsofthe U.S.A.(Nair andMcEwen, 1975). InNewfoundland, most F1adults do not emerge from pupariauntil the'fqJlowingspringhowever-!smallpercentageof adultsdo andcomprisea partialsecondgeneration(Morris, 1050a)..InlateMay orearl~June,adultsemergef~ompupsristhatoverwinte rinfiel~sin which cruciferouscropshave,beengrownthe previousyear.Eggs arelaid 3-6 days arter the flies'emergeand thelarvaebat ch within 3-7 days alter ovlposlnc nandfeed wiihinthtro:ot:J,:ofsU-sceptibleplantsfor3:-4.weeks.Someadultswill emerge{rom pupae 2-3we~ksafter'thelar vaehavelettthe'lll~nts^tocompletea secondcycle. However,'most pupae enterdiapa uaeto overwinterand emergeasadults the

The

~abbag{·m~·g~ :;t.

was

r~st

describedby Bouche"in Germany'in1833 andis thoughtto be'pfEuropeanorigin(Slingerland, 189"). Records showitwas a

.serioua pest.intbe early ·1800's inEll..rope,parti~ulllJ'lyin Germanyand England.

In NorthAmeriCA.it was,firstrecorde~ inthe U.S:in 1833 andfiCtryears laterit wesdescribedasaseriou~pest.of eauliflowerinCanada (Slingerland,IS04). The .

most,errectiv~

^ecntrolme tbode recommended

in

the1800'sweretbeuse of tarred

I/ paper cardsfitte daround thebase of plants (as ovipoeitie gd~terrentsi ~d inorganic,iD~ecticidessuch ascarbolicacid andcarbondlsulpblde against early

lar~al lnita~

(Slingerland, IS9..).These

meth~ds ~ere

'costly and inorganics.·were generally- phytotoxic.and'not effectivein:aiIsoiltypes. During the 19208 and 10.1as,thechloridesofmercurywere favored as seedbed drenches, furrow dusts or asseed coatings (Jud.ge~!l.,1968):

Inthe 10405, the'dJseovery.ot'/(he insecticidal.pr~perties of DDTand ,subseque-ntiy, otherorgan~chloridesofun~reced~nre"d'powe~ ~drange o(acitivlty, ledtotheiruse,against'awide arrayof insectPe9:fs,InPrince Edwardl§land, between ttl52-itlSS, maggots werecontr~1Jed-with.weeklYro'i~gesprays'ofDDTI

timed.to~oincide'~ith the emergence ofnics from overwintering'pupae.

However,the timingor thaJti rstsprayW8!jdifficult to .'PredictaDd"control effectivenesswas poor inwet weather (Read',Cannon,19~/81.ThecYcl~ieDes,a classof orgenceblcnde~nsecticide9,weregenerally morepersistent in soilthan DDT·typ~ compounds an.dprovided.-relativelylong-term control. Sev~ral cyciod ieDJ! compounds, particularly al~in, dieldrin and he~~a.chlor,·gave satisfactory control of cabbage.maggotsin Canada"the U.S.and Britain (Stitt, 1953;King and Forbes, 19S4;Read and Cannon,19S8;Morris, 1959b; MetcairAnd Flint, 1962;Coeker~!i.1~g63).,

The su.ccess of the organochloride insecticides was -s£ort-lived. ReportsofR:.

radicum'populations~h~twere no'longerc.ont~olledwitJ:l. cyclodlene eompounds first appeared in tbe literatu re in the'early1960'sfrom Britain and'theU,S.\

(Howitt end Cole,·!tl62;.Coaker

£.1 .!:!.,~

^{!tl63)..Atth'e}

same 'ti~ '.tests

"cnecll ' insectsfro~^severalregi~ns acr~CMi~'ademonstratedthat/our'species ot'root maggots had developedcyclodieneresistance,includingcabbagemaggotsfrom Newfcundlepd'and

P.E.I.'

(Har~is'£!

!i.,

196~;Morris,'lg63iRe~d;]1)64).'Delia ra dicum and otber soil insec'tpests had8159·developedcross-~~~ance

'?

ot~er cyclodieneinsecticides,not previouslyu~ed,ag~nsttbem(Harris,UJ77),. ·The developmentof resistance in'so'irinsects wasat~ribu\e(Jtoselection pressuresof persistent! organocblorideresidues in the soil (<?caker~!I.,19M;McDo\aldand Swailes,·1975;Harris" 1977)..Read and!Brown(1966)demonstratedthat'dieidrin r~ista~cewas inherited inDo:.radicum~ . ,. \

;' I ."

With the,developm~Dtofinseet~ci1eresistance and concern.o~erpersistent residues in the environment,use of organochlorideinsecticides has beenr~tricted since'the'late19509.Otber,lesspersistent. compounds;eueb-eeorganoph~Pb~tes

. I

f

..•"

andcarbamates havebeendevelopedto.~tAketheirplace. Severa l

o f

these ebemiealshavebeen~ownto be effectiveagaill!t thecabbage maggot,altho~gb theygenerallydonot. give.Mconsisten t.cont rolMdidtheorganoebloridespr~r\0 thedevelopmentof resiswce(Judge!!!l.,UI68;Read,1970;~bletl!l.,1~72).

ResistancetoDoo..Qrgl.oocbloride ioseclicides~I.;Sbeenslower todevelop io iD~ts. Lowlevel orgUlopbosphateandcarbamateresistancebasbeenrepor ted intheeabba!emagp andtheonioll.maggot,lli.li!!!!!!mMeigen(Harris, 1977). ThenOD·specil'icorg8!l0pJiospha~resistaocedeveloped by

.!2:.

antiguain someregions of Canllda\~nd t~e U.S.br resulted in cross-resistance \0 carb ofuran,acarbamatei~~tiddecurr ently inuseforcabbagemaggotcont rol (Harrisand Svec,HI66).

Thes~lerelianceonselectedorganophosphate andcarbamatecompoundsto

%

replace o~leteinsecticj;ies iD~reases.the selectionpressure for.resist ance developmentinin.sect pesu. Root. maggotsareparticularly atr~"i~tbattbey, arecontinuouslyexposedtoinsecticideresiduesin{he soiland mayhav eseveral generationcyclesperg,owing'seasoo(Harris,1077). 10the ongoingsearch"for

alter~ati~einseetieides..empb~is^isbeingpleeed onthedev~.J<i"pme~t:of insect--

specific compoundstbatact.onnovelsystemswithinthe~getpest.One class of compounds beingtestedareiusee! K!0wthregulators.·Thisclass encompassesa widevarietyofrompoundsthat actin vartousways to disruptthenormal developmentof

iDSecu~

^{Deat hby;reat mentis}

n~t 'im~ediate b~t

^resultsfroma

eombinat ica ofsecondu J factors related.to disrupted development. The , compoundsinthis el g erally fall into three 1najor'catego,ries: juvenile hormoneenalcgues("vencids], anti-juvenilecompounds aid chitin synt besis inhibitors. As"a(group,insect~owtbregulatorsareetrectiveagainsta variety of insect pests,i~cludingthose belonging-to-thecyelorrbapbidDi~terB.,arerelati~ely , non-toxic tomammalsandether non-targetorganismsanddonot."persist.forlong

periods!nthe eavlrcnmeer(Retnaka~·an£1!l.,108S). _{I •}

To.date, severalinsecttrowth regulatorsbayebeen showntobe effective

....

:

. .

-;.:

. .

^{,~":." :}

^...

agains tthecabbagemawt. Juvenoida areovicidalandprevent adultemergence in the

l ~ra~t1

^{(vande.veb e'anddeLoot.}

tWa)

andehcw promising eontrcl potentialagaw.

t

I!:.radicumi.nthe field(VaD.de

Ve~e

^aD.dde

Loo ~i, .-'

^4),

Chit~ '"

synthesi! inhibitorswereeffectivein,...teducingcropdamagedu tocabbage maggots in field and greenhousetrials(Howard.1m;Philiptl ;Riteey'!!

!!..•.urn;Turnbull,1'182), Laboratorystudiesahowedone'suchchitinSJnthesis inhibito r,dinubenaufOn,

to

be ovicidaland larvicidalagllinn~ ra.dicu~'when'the' . parentalgeneration of&dullsor the eggs were'truted(va. de Veire&D~Deleour,, 1076;Turnbull,1082). Oinubenluronwaso~low toxicityto~bilineat a Gyllenhal.(qo rdon andOomee t,1986), a major·predator/parasit.oid of.the cabba g/mag~t,^{foundtoparasitizeup.to60% offieldpupae}i~ N~wtouDdia~d (Morri s,1050a).Thisisio contrastto theapp licationofconventionalinsecticides which usuallyresults ill. eonsiders ble mOft~lity'of parasites(Chapmanand Eckenrode,1073 ).

T~isv'st~dy you doneto evaluate the~rreetiYenes5·.of four comp?u.ods.-.'.

represent ingth.(threemajorin~c(gro~thregul atorgroups,~gaiDsteggs.1arY~

and pupae

~f

^the

cabba~e'

ma",ot.in.&

·1~boratOry.; ~iin~. · Th~ stud, ·~

providedanopportu nit,toobtain prelU;Un:arydataon thephysiologicalbu b~f

disr~pted d~velopme~t

^ofaselected

~~pol!nd ag~inst

^the

~get

^insect.

/'

'~'...

. .

. ... . .!

I

. INSECT .GRO W T H REGULATORS:

BACKGROUNDINFORMATIpN ...:-'J,

2.1.Juvenile hormone analogues

" . C

10 1956, Car r0vYilIiamsprepa red thefirstactivejuvenilehormone'extract Cro minsect corpora allata and foundit eeeilypenetrate d insect cuticle"toblock . meta morph osis(Will~ams,19S6).Subse~uent physi~gicalstudi,es,led Williams to .suggest}~,!"enilehormone exereets beueed esinsec~specific'insectic\desto'which pestinsectswould'b~unabletodevelopresistance"(Williams,",H~67)~.However, pure

ju~eIiile

^hormone

~mpoun'd~

^al.e

seld~tsJ

^'in

fiel~ eJ;p'erirne~ts b~ca:se

^{of .}

.theircostofsynthesis and',insta~liity,j~tbe .rd..,Two,~aj~r

F v 'ances

have been..

made

i n

the,practical~p.plicationoqu~eniJebormone'-compounds.The

rust

~&S.

".

th~

discoverY of

iUV,e~i1e'h,~rmoite' acti~iiy

^{'in'balsamfir}

(Sl'~~

^and.:Williams;·'

~UI66).Th~.c~mpound,responsiblefortbe juvenilehormoneactivitywasisolated andnamedjuvabion e;

b~ing structur~l'ly

similartoiJseet,juvenlle,

horm~De;

Ii. repr esentedihe'first of thenaturall y occurr ing juvenilehorm~Deanalogues(JHAs'.

juvcnoids) (Bowers!!!l.,1066),orccmpc uude that functionally resemble juvenile

hOl~oDe

^{and may ormaynotbe}

si~l~ to

it.in stru ctur e

(T~~te

^2.1i

(Retn akaran!l!:!!.,19S5).Thesecond majoradvancein the practical applica t ion of juvenoidswas thelaborator y syntbes'is-efsubst ances thatwereseveralhundred- fold -more active thannatural juvenileh'ormone(Bowers,1960).

" ,

T~,da.te,Iourhomologues of'nat uralinsect juvenilehormone(JHo-J1UII),.ell epoxymethyl 'dodecadienoat~~ithdistincteerbcn'chainle~gths,·havebeen. ident ified (RoU.crand Bjerke,1965;Meyer~!I"1968;Judy£!!!'j1973;Bergot!!

!!.,1990). Efforl.!l tooptimize the chemlstryofj~venoidsforuseas insecticides

T&fe 2-11Summarytahle oft~e'insect icidalprope~tiesofthr ee major·

..lnsed .growth regulator groUPs' with repr esentat ive

com p ounds .

· Sterilizetreatedadultsof t!I hemi- and holome tab.o,loll sinsect s

·Disrupt embryogenesis preventing eggeclceion

';Representative . Comp o unds Juvenil eHormoneI (CecropiaJH)

~CauseIncompletemetemcrpbcels inim meturee producingextr!i'l ar,va,l, nymph alor pupal stages

·

Inhibit,~gg ~a~h'aue-

to disru p ted

~~~I~k;:~)~rwe~: ~PPI~ed

^{to .."}

;Act.

as

selective,ertc t oxlns ofinsect corpor a'allata .' .

"

.

·Oeuse precociousmet am orpho sis of nymph sintominiatur e ster ileedulte Com pound

Class

Ani-Juvenile Ho mon eAgents

°Inhibittb~'rimilstage,of chitinsynthesis byepide r mal cel ls

•.Interferewith thedepo sitionof Chiin Synthesis chitin into the"cuticularmatrix lob bitors

o f

new develop.jogcuti~Je.

0,Causeinterstadialmor ,tality:

larvae unabletoshed ohicuticle

·Ovicid al:fullydevelop edlarv ae unable tobreak 'out or egg case .

·Penetra teinsectcuticletoI?event metaml2r~hicchangeill. the

e

i~orri1is

· Fun ct ion~juvenilehor mone egouiatsand/ orant$go n ists }uenile Hormon e.In,terter e,,:,ithgeneactivation .~an Analogs0, preventi ng imaginaldevelopmen t

have result ed'inthe synthesisotthousands,ofcompounds.'that.collectively

i

are. disruptivetomemben.or all the majorlnsec t orderatStaal, 1976;Ret oakaran~.

!l.,19~1)). ^While insectsmay be unabletodevelop'resist an cetothenatural hormone,theY,candevelop'resistancetojuvenile~orP1;0neanalogues(Staal,1982;

.RetD~kar~_

!ll\

^{!l.,198&). To date,',only0 0}comp ounds,.bothproductS

o r

Zoeco~/Sll.nd02Corporation,_have been regiStered

ror

useinN.ol~hAmerica. In

•the U.S.A.,met~OpteDe-lhasbeenappacved bythe Enviro,omentalProtecti on Agen~ylorthe'cont rolorDood water mosquitoesandOeas end kinop rcnerorthe controlor's~veralhomopjer un pests (Table 2·1) (Staal,1982; Retnakaran.tl~., 1985). In Ceeede,n\L.hop rene~register ed (PesticideDivision,Agriculture Canada)tor useagainst mosquitoes..

I," • ~ • •

-J uvemle hormoneis,a natura:! componentor the neurohormo nalmilieu ofineeets

;w'bos e prhe hee,~uppresses qietamo~hic"change'duri~g'a^molt-.:It is,e~id~~t^{th a t}

·pr~eD'tiligexc:;;'~~D:ous)uvenile^hormoneac~ivityduring periods wheni.tis,n?rmally :low or absent;

i'e, ~ prep~~ti~D f~;'

^the

i~~gin61 ~It

ⁱⁿHemim eta.bola or for the ,-

larv~l~p~pal .,

^or.

pup~I.~d~it _~olt

^'in',Holometabole.'.win'

':- ~~~~eIY ''artec~ ~'

metamorphosis.Thetreatme'nt oflhemimetabolou,nympha withj~~enoidaeeuees the

pr~u~tion

^-of

su~~.r'numerary l&r~ae

^or

~~.

^degreesotny m phal-adu lt

·

fdr~s

)ad.Uitoid.' l", T he

treatm~nt ~

^of

I~:-,~

^or"to ung'pup ae or.

sU!l~eptibl~

Holometabo la results in th e production of larval-pupal or pupal-adu lt intermediat~^(Williams,'t9S6 ; Sehnal.andMeyer,1068 ; Srivastava endGilber~, -1968;Jakoba~d^{SchC?Of,1971 ;'}van de Veireand de

Loor,

'1913; Gar;jsand Adk.in9,'Jr,. 1914;,Mckague,andWoo~, 1-""974~,Staal.108 2;,Sparks,19&4;

Retnakaru!1:

!l't

19 8 5)...Theperiod- of~ea~estsensitivit y forine~amorpbie.

'inhjNti~~by'jtivenoidsi;Itheultimate orpenulthnateinatsr(or young,pupainthe

HoJ,o~etabolal (Sl&~a _a~d

^wifl'iams,1966; Wrig ht,U110;Sta8J, 1975):" .

'~

·metamorphOsis.,Th e

c~rpora.

allat&.,but secrete

~f

^insee,tsju v enile^{,ar e}hor mone

inactiv~

a.gam^d'uring'inthe^thead ul t

UI~ima~

-atage^s;ag:'tobrio

t

' about~exual.malU~ajion,^{Generally,the}adm~btra.tioii^ofjuvenoids h~^lilte

j

',,1

•

Tbe lethal morphogenetic:distaibocellofjuYeDoidslateinIlInldevelopment haveresultedintheir

registrat~D

^toruse-Ig ainstJn.se c:L;of

~oti~ ~por~ee

duringtheadultltages, lueh...publichultbaDd ;ete(inuy'~ta

(stui.

1982;

• Retna karaD

n !l-~·108sl .

^{"T he,.}

aIso_s~o

^{...pr om.lseIc r·COIlt rolotltored}

p~~c:ts

rests(Nickl.e,iO'1~Mian.n~Mulla,1083).

erree~ ~D t f ~ult .~i o~/)Q ~ep~~cti~~ .(~ta;aJ:

^__IGs2;

,RetD~aran'

^{'_ ..}

19S5).Cases'where adult.sLerilityisinducedduetojunu lle.ormoneanalogue trea tment'areosuaiJy •res~lt^oJthe~re~ral^ofthecompo. Du·to-deYelopiD', eggs within'the~atmlal ^{body where}tbeJblock embryoaied·eytk.pm~D~^. Juvenoidsinhibit.embryogenm'when..~pliedtotheeggs"of.allinHd~spiel}'-. exam ined,whichinel ude

represe~tativ~

^fromallmajor

~rdeh

^(reviewedby

~,~~,

"."

1975;SehDal,10&3). Insect egp'are-mostsensitivepriortoorjustaflerbetng

. .

cvt p osned (Su'ma&IldWi1Iiams/l066;Riddifordand Willi&m!l,1067 ).Juvenojds block

e~bryogenesis

^{during·blutokinesis(Riddilordand}

WiUia~.

^{1967;,S t u l,}

1082)or

res~It, ~

^{delayedefrectlon}

~et&morphoeis

^{"fu rtherin}

on~tlar

wthosecbeeie ed-b y.lIeatin,immature.stq;eI (WillisandLawrence,1970; / Ridd itordand Truman,J072 )..

.Tbe

~iocbernk~ ~r;~ts

^or

j~~eDOid5 1I~-c:~m;ln' ~·d "fa:., t~

^one

CC:~pou~d: "

:to

an~ther~··

,not)'

~an r~~cI.iOD

^as

jU"ftDiJ~ hoimoD~· ~ioDist.S ~ ~ta~Dist.s- ·or

. ..-botb{H~prick,ID82;.R~tDakar~^etat,f985).···Jl1veriUehormo~~

il

tbougbUo'act " .

.,at.

ibe"'g~Detic: I~:~l.

^A

wldei)'.~~;ted

^{modelofaction}

propoS'~ , that jllv-e~iie

^/..'

hormone

eD t~~.tbe ~pidermal c,el~

^;w,d

lIr~r c.omplexiDg·w~tJ; recepto~

^{,activ ates'.}

orin~ctivateseertain genespreventingim~ginaldevelopment(Llurerand Borst,'.

[083).

- ,

- - )

·2.2.Ant"'juvenlJeh,orm,?ne.

8gen~: tb~

·p r eeoce n es

""i!esearch withjuveucildll as'insecticidesdemoDstj.~tcdthat a morepractic~1 approachto the controlof cropreedinglary¥wouldbe~isruptthe actionbr endogenous juvenilehormone, Application-of&ri&lIti-juvcnile hormone agcnt wouldiDd~cethose resultS historically produced by_surgical allacte'ctomy,i.e.

__) precocious metamorphosis

of

larvaeintominiature sterileadult!! and sterilization

·of treated adults. Bowers and hiscollea~es,led by the previousdiscovery of insect"hormoneactivity -in.pla~ts,randomly searchedIora~tj.ju~enile-hormone activity in plant extracts[reviewedbyBowers,1982).Alter exposing milkweed

b~g§ IO~copeftus r~il\t~sl

^to

a~proximately 3OOpJants,~~o~ng

^nymphs

sk~pped

·instars'totorrri miniature adulta-end exposed aduhs·were sterilized byto~ica.I

.l,,'im~~t· ; ;

^expcsuretovapors 01 aD.extract

r~omAg';~tom

hoo.JD;aDom,aD

·

ornament al

beddin~.plant

^[Bowers

~f!! ... '1~76).

^[.0

SUPPo~t 6ftbe"~pp~e~t ant~.;

., Juvenile

·.ho~m~ne, acti~D

^{of'the.extract,}

~tb .

errects could be reversed .by coricomi~ntappli·~~tion~rexogenous juvenile.hormone. 'Two distinct antl-

·juveDil~· *ormo~e c.~mpounds, bo~h.

^{dichromenecompcunde('Table2-1), were}

isolatedand named preOOcene(I-Of[Bowers-~

.!l.,

}976).,Thou~h'^{theprecoeeaee} havesignificantanti.juvenil~hormone 'aetivity againstseveral\hemimetabolo~s

~pr~~i&~5-de.;elopmentbu been'inducedinonly twospecies among insects with complete metamorphosis (Bowers 1982,lOSS). A number or other anti- juvenilehormoneage'otshave beenreportedbut the preeceenesremain the most well researched~r~viewedby Staal,1986).

• • ' I ' •

Despite'theebeenceof a developmental eUedinholometaholous insects, the precocenesdisrupt

repr~uetioD

^{..In both,hemi- aD'd}holometabolous'species,

·including Dipterans', Steriliution results Crom the inhibitiOn'of ovarian development

(~wers'rl!!"

^1976),

~yt~growth

(Landers and Happ,

1~)

ind tromthe disruption orvitel10gencsis(~une~ ~

.!!.,

ID70iWilson£1

!I.,

1983), Treatingypu~geggs of sensitiyehemlmetebclcuespeeleswith precocene inhibits hatchduetoa disruption'late in embryogenesis or r;riltsin delayed precocious

, ' . .- ^- / '.

metamorph03is)~we" ~

!I"

1976iDorn,1982jAboulafi&.-Baginsky£1.~.,1084).

.:,.:

...

,

/

..~

.:.~ /

.

^;

" "''''j ,

^{~ ~}

\" .. " "

^{I ~\1:tn,..,·1"}<\$"IIiJ':>~~'!l~"")Ir-;":" V"'i"~.l,':::".'~~~.'-:"~\':'::;''I;:';''

, / . ' ~F;::~

Thepredominantphysiologicalerred01preeoeee eatbought'tobeareduction... .':~ .<4 ill

bemoIYID~h- ·jQ~~nile.

^{bormonetite;since}aUol)t.

ad~D'

^uri

~ re~e~

by .

~

applic&lioD

At

^e.xOieQou,

ju~enile

hormone\(Bo";ers

n

!l.,lQ76;

~f~@I" !!

!l-t.

107~; Bowel'B~

^{1082;Bowers,1983;}

Retnak~

^!:l.tlOSS).

~ideDee

^,tronglf

lugge51.!1that'

_,

thisit&dirl"et

~t

^a'brain-m edWed

_.

response(Bowehl

_. ana

Marl me:z-Pard o,1077;Muller!!!!.,lQ1V;BowenandAldrich,1980) aDdillYolns

.. \ ' r , /

the inhibit ion,of the IeCretoryactivity

or

thecorpo ra

..n. t.

^{(PrattandBowera,}

1077;Masner

\ !! ^{!I. _ I}

^{1070;Muller!! !l" 1070jUnnithaD}and Nair, 1070;

Feyer~isen!!:

!i.,

10SIjWi1;son!!.!!'yl983). Thebll5L, ofitscytotoxicenedill believed'tobei~itshioadiv.tioDbyglandcellsoflnseet co;pora &lld a. ~

I { '.

\

. .

. .

.

Preeoeenetreat mentel."Sf¥!aseleetive.~egen~rl.tionof.corpora I.lll.tl.s,eere1<?l')''. . 0011,

(UD~th

^..\!!

^{!.t .,}

^1977,

~;"hIY

^and

S~d.lak,

^{'.197. ' S'hOOD,;,l d,.1079,·}

Unnitha~tl!I..,"1980)and.it.has.b~e~propose~th~tp~eeoc}ne^ismeta~lic~I.I~ ~ ..etivated.by....in~ecrpcr...allatato,pr~uce:~he ~xic~~Cec~..(Brook~.!!!J.t '.:

1970). .Evidence strongly.suggests thai the preeceeee.mcleeuleundergoes

epoJ:i~

^..tionby

+O~XJg~Da.se:' e~zrm~

^to

~ ~i~bI1 ·re~tive:ud. u~ta.ble

^3,4-

epoxideintermediate"_{• I} tbata!kylates cellul&l'.miacromolecules~(Ohta_{,. , - .}!!!l.t1m; Jennings aJid OUr.idee,llngjPraU!!..!!.,tggojSoderlund!!!!.,Ul80~ The monooxygenase

e~~,meJ

ioyolyed lo thefinal epoxidaiio oIUpDC jUYenile horm<!D-e

biosy~tb

^{. have}

~n

)mpli,cated.as

th~ rflllpo~sible for th~

bioactivation. pr:ecocene(1 &tt!!!!...lG8o;

Ha~~tt

^and

Pr.~l,·'

^{]gsa )..The}

orgusped fi,1tyo~thepreccren~towards the corporaa,Data.m.~resuJt.imply. from .ve highlevelofe ldaseinthisgland ofsenlitlve insects(pr.tt!!!!', 1080).Alth th'ediffereotia1 sensitiyityofbem i-andholometabolouslarvaeto' .the

preeoc~n'7 rem~ins

unexplained,

th~ co~po~~

.lI. t. o.fhOlometabolouslosccta

have_i

~n

^shown

~

_i

b e

sensitive_{_} ill_. vitro·(Bowers aDj!·

Feldl~ufe-'t

_{__} ^{1982). The}_.

,observation-tbatprecceenee arerapidly sequesteredby_hemolymph protelosin a1everal

lDsen~itive

^{insects[Soderlund!!!l....1981)}suggests tbaia toxictiter of. preeceeneniay

ne~er

^reac.hth{corpora.allata duetoperipheral

de~xification.

Unfortuuatelyt&Jloactivationpathway'for preeceeaehubeen discoveredinr~t

\

/

bridge(Table2-1).

/

! .

I'

2.3.ChitinsynthesislDhi1?i~ra

I

Tothe courseof developing analogues and derivatives Qfthe:herbicide dichlobenil, scientis ts atPh ili,p9-DuPhar'B.V.

in

^The'NetberlJ dssynthes ized'

a_~

seriesof2,&-diChloro b enzoylp he:6yl ureas. These cOUJPounds!b&.d noherbicidal lLCtivity'butwerehighlytoxicto'insects: ,larvae

8urvived' ing~tion

^butd'ied

d uring

^the

n ext

~olt-^{(Wellinga,'rt}~.;'^1973).Th~^moltdistupti~g'^actioDoCthe- .

b;nzo~",yl ~re~< ~~d ~he

^{distoverythatthey}inhibitedchitin,sy nthesis in

't;eated.inse~tsI~dthem'~be^'knownasthe chitio'syn the!lisinh ibitors.:ri.em~t"­

investigatedo(·t hese compound sis'di'nulieozuroD(Dimillo

(9,

Piitrto,TH 6040) ,.' {Table 2-1}. N~merous^{othe r}c,ompani~^ha:vesyo,tb~ized tbeir:~wn&n&l~gues;

~hichgenerallyconsis tof two subst ituted ringstruct uresconnected byaurea

.The chitin synthes is inhi bito rs generally induce insed death during .irnmedietlyfollowing the mol ting

p~ocess~

^the

ti~e

^when

~b~iiD synth~

^is

particulady crucial to ,urvival,(Re tnakaran ~.!I.,~98S). Characteristic abnor~a1itiesobserve~intreat edlarvaeinclude splitti ngof the,new cutiCle, complete failureto shedtheold cuticle oronly partialcompletion of themolt with

p:ts

^{of the}exuvium st ill attached(Gr auettand Dunbar,1975;Laceyand.Mulla, 1978&jMlKague~.!!.,1978 ;Sharm a !!!I.,107tl;Retnakaran!!!I"HISS).

.Tr~at~en.t^{during thelast}larv'al laatarcan resultinth efall~reofla.rvae to molt successfullytothepupal.stage(Laceyand Mul1a,1977;McKague.~!l.t^1978)or preventionof adult emergence(McKague!!!l.,HI7SjOtteosand Todd,1979).In

\general,yOUDg lltval!are,moresusceptibt;tothe benzo y lpheny l ureesthanolder ones (Laceyand Mulla,Itl71,1918b;Sharma~!l.,1919 ).

...liver whereinjection

o f"; eco",,,

^causes",t••eive llver d"m"g .(111)1"!!.!!!.•I~i Halpin!!'!l.,HI84).

. r

Thefutu reap~licatioDor preeoeene-type agentsinin~eetcontr olwill ove rcoming twomainproble ms of thenaturalpreeocen es:verte brate and inadiv ity~holometabolous·la~a~. . .

\

~

. .

^"

~

..

p

13

Chitinsyntbesu. inhibitors~..ve lit tleor no effeeLon aduU~t.s(TurDbu~, 1082;Retnakaran!!!I."1085)~inee ib~.cuti cle'ofthePhrrgou,"itnearly"form ed bythe adult stag,"(Andernn,IV70)..The moet proncueeedefl'edODadulyisthe redueticeintheDumbu of.vi&ble.ortspring,the.,s ameresul t .eeomplisbed'by treati ng the

eu

't a geof suseepLible-s~ies. ~eat1neDtofegss.pameul ar!)' young ODes(Miura.tl!!.,1976:Laceyand MuDa,1977; ottens~dTod d,'ttn:O) 0;

parenW~~lts(Moore udTaf~1075;Mi~ra!!!l.,1978;':"rightandSpa~

1076;Ivie and Wright,1078;Chug,1970;Jo; dan!!.!l"1979;Oueee&ad Tod d, 1070;Spates and

Wright.<,J,9~; Tur~bull,

19S(Rup and Cho pra;lQ8S)

~auBes

^..

reduct ioninegg hatc;hgenerallyfollowed bya"reduc edBurv ival rateofbatched .

I~tv~e-.

^{Treat ed}

eg~

^contain

ruUy 'develop~d Jarv~

^that

~e·

^{unabletoescepethe}

eggsbell,probl!oblY,duetothe

possessio~

^{'of a}

w~akeDed ~~bryODic'cut.iele {Moore

andTaft,197&;Miura!!!l.,1076;Laceyand Mulla,1977;Miura and Takahash i, I'

Ig70i,r':lrnb~II,.I082)^.Apointinem~l)'ogeneflia^a"t''l!tich mortalityoftr;~t.ed· egpi of thebl~ckny^{Simuliumviltatim.}Zet~r:!t.edt'd~liDed'^sharply'wu' eonelde ed

to.'~Hep~otati,:,e ~f

^bothan"adveaeed

de~itioD'oi embrYoDi~-.an'd..,

serosal'cuticle with acoocomita.ntmc:reas(intheim pe rmea bilityor the chorio n

(l.ac:~y^..~d

'MuU i,

I077t .

StudiesonDip.tera,·particularl~^thor:-~rmed,~c:al^and~eterinary.im~!"&nee.

showthemtobequiteauseeptib~'thebenroylp heoylureu{1uob,1073;. Wright.,1m..;Mulia,aDdDarwueh,1075;Dame!!.!l.t'1g'16;

Miu~

^•.tl!l.,1076;

V~.

£!'!l.,1076i

L8.~ey

aDd'Mull&,

U rn,

1978a;McKague£!

!!.,

Uri8;Chang, Ig70;Jordan !!M.,107g;MaurI.andT~lhashi,1070 ;Sh~a!!

!l.,

(U7Q;Ali a~d^Lord,Io;so;Spates andWrigb~, ^{IOBOjTurnbull,} IGs2;~Rtip^and'Chop ra, ,!g8S). A'review ofprevioua'st udies on eyelorr haphid Diptera,'suchustablerues,

e

^{use flies,rruit.ruesandtsetse rues-revealtbat tbe}bentoylpb enyt'urees are'"

Vic~dal/ ,

^{larVicidalwhen eggs}

~re ~~sed

^eitherby

toPie,~' ·ap.Plicat ,lo~

^or

vi.

thi', materualbody'(Wright.19'14;Vea!!

!I. "

1976;WrightandSpate8,1976;hie and

' . / . "

Wrigh t ,,1978i Cbaog. 1079i....Jorda.n!!!l.,Itp9 iCban gand Borkov ee,1980;

Spatesand Wright,1080;Rupand'Chopra, 1085).

' /

r-.

,',

.,;.... .

.'

Chitins"ntbesi(mbibitonnrcergoodfieldcontrolpotential foraqueOuslarvae of Illes tbatarepesteintheadult stage(MullaandO.,wa zeh,1075;Dame!!!!..;

1076;McKa~etl!I.,19 78;Sharma~!I.,I07Q;

Ali

andLo~d,~980).IntheU.S.,. Dimilin isrpgisleredror useagainst thegpsymoth,eoucepestspd aeftr "

biting

ny

species.InCa.nada,Dirnilinisrep teredtor useagainst theliYpsymoth andmosquitoes. Theirpo~Dtial'valueagainstdefol iatiDginsed sand agricu ltural pestsdeservesd~rattention.

r-«

.. ~ .

^.

Chiti n-S)'nthes~.inhibitondisl~themoltin gprocessofinsectsbyinterlering withtlredeposition01chitinintotheendocuticle orthenewden lopingcuticle alter'apolysls. A;bo;ta georchitindisnptsthestructuralmake-upor theDev.:

formingcuticleduring mahing andISafe!Ult

o r

1bewea.bnedcuticle,la rvaeare tithe:unabletothedtheoldcuticlean d/ ortheDewcu t icle rupturesdu etotbe increased/muscularacti vit)'requireddurin!:ecdysis(FogaJ .1017). Moltedi1l5~t!I

•oftendie event ually Crom'otby s-"nda ry

erree~

^{causedfromthe}

~ion

^01a

Don·functlonalintesum ent~t!!!!.I191. ,Kee,1077;Vincent, 1978;Andersen, '1070;van Eek,1070;Beeman,1082;Retnu aran!!!I"^{lQSSj. Then:ac l'site of•} . bebloytpbenyl urea.c~ivity^atthebiocb~mital'le vel fba tpreve~ts^theCor mation ..-.!'fchit inmier oCibr!bis not .'u n d erstood butthereare'severaltheori~. Tw~

w'iaeJy:.aecept e;(one:s·are:'theydiieetlyinhibit the nulstagesofchitiney nt besis by

epi·dermal.~ells

^tobaittbepolymer izationofN-acetylglucosam'ineun its

by

.chitintyntbetase

o~

^tbatthey

in~ibit

^{aproteasetb8.t activates}the inactive

chit·in~

syntbet asezymogen (Che nandMayer,19S5;Ret~ akuan£!!l.,19 S5).

)

l ..

/

t..^I' ·

-,:'

..

..::",.

15

/

,

MATERIALS AND'METHOPS

3.1.Greenhouserearingofstock colony

Two basicmethodshavebeen developedro~rearin g!!.~ in the ,laboratory. A method "develo pedbyHarris and Svec (lg66): 'and.·p'i'eselltlY'~"i~'

employedat the A,gricultureCanadaReeeareb Station"inLondon,Ontario, ,.

requires environmental

~~ambers~to

roarlarvae and maintain

J1. ~adicu~ adults.

A seecndmethod,developed,and laferimproved'by Read,(lQ65a )(Agrj~tiUurl:!

Oanede ResearchBranch,BxperlmeetelFarm, Charlottetown, P.E.q,

~s~ ~

heatedgreenhousecompa r t mentequi~pedwith artificial lighting~ s~pportyear round rearing. AftervisitiDg.~bins~itutionslIdetermined Read 's methodto be moreadaptabletoavaiJa b ieIacilitiea.The[oliowjng~tocedurewasdeveloped

...(rom Read'smeth~d,withminorchanges andsimplifications.

3.1.1.Re ar ingcomp~tlDeDt

Thegreenhouse compar t m ent.wasIOtat~dat the AgricultureCanada Research Station,.Brookfield~d.,St. John's. Theoomp~tment,in Additiontonatural daylight,received a minimumof1400luxfromfluorescentlightin g for16hours per day. Thecompartmentwas heated-andthe ambientte mpera t ure allowedto....

fluctuatedbetween15-27C throughouttheyear. Ace ili ng fanprovidedthe adultswith a cont inuous flow ofair.

,1

\

-

:;.,'

».

3.1.2.Re~rlDI proeed~re

Rearing of larvaewas donein5-literplasticbuckets,1/4Iilled with moist organicsoil.Astore-boughtrut aba g:a(~!!.!Illi!naoobrassicab approx.1 kg) waspeeledon

.its

lower surface to expose thesucculentr~\tissueand then pressed, cut surfacedow9.into the loose sopto.2/3itslengt h. Three-hundred and flttyembryonated eggs(heldat 20C for 10 hours; original egg! obtainedfrom

•~ Read's greenhousecolony'of an insecticideeueeepuble P.E.!.strain) were distr ibut edaroundtheex portion of the plantedrutabaga, covered with moist soil, andlightly watered, Thebucketswerewatered twice more, once

duri~g

^{thefirst week}erter plan't!ngad once in

t~d

^{week;furtberwetting}

greatly increasedtheriskofrot which let hal to the,.entiremaggot population.

Therutabagas~ereremoved frQr* ebuckets3 weekS-Arterset-up, a timewhen mostlarvaehad emergedfromth rutabagatopupat e in the surroundingsoil.

Adults,emergingfromlarval buck.ets,wereContained by cone-s haped emergence

~ages (Figure3-lA) madefrom 3O-mesh plastic screen, machine-sewnto.shape (66_ x~2em: basecircumferencex height)andtapered to a 12em (circumference)hole at ihe top which'was closed'wit h an elasticband. Within 48 hoursafter

~~l11erging.^{nies were}transferredtocylindricalovipositioncages (Figure~1B)^{(66 x}

II'_ 32 cm.:·circumferencex height),wheretheymated and subsequentlylaideggs.

The

ni es

were

rem~v-ed

^{from the}

~erge~c~ages

^by

in~~rting

^aglasstube

.(o~POlliteendclosed) into,thehole in the·topof thecage. Thenegatively geotactic"9Jr moved~pintothetube and....ere counted,sexed (Smith,1(21)and transferred to'cvlpceulcn cages,Inserting the openendof thetube Into thehole' in theside of an ovipositioncag~(Figure~IC)and blowing.intotbe opposite.end deliveredtheadults totheoviposition.cages,

.Adultswereplacedin ovipcsitlon cages at a densityor 1()().200 percage,with.at

IElMt1/ 3males.TheIlieefedfromfilterpapersmeared with a mixtureofboney, 's'lga~'.Brewer's yeast, whole,wheatrio~r.andwate~.•(approx.'8:8:{3:1)(Read, .~965b)and placed inaninverted position,on theout ersurfaceat..thelop of the

.ci.

Figure3-1:

•

Stockcolonyof Deliaradicum in thegreenhouse. (A) Emergencecage (B) Ovipositioncage(e)Hclein theside of ovipositioncage (D)Filterpaperon the outside ofcage smeared with ad ult food mixture (E)~Jas~icwater tube (F)Ta~kfilled withwater

I "I

18

cage (Figure 3-1D). Water was presented'tothe adul&in

~

pl-"'tic test tube supplied with acotton wickandlaidon topof' the cage(Figure3-IE). Cages

./

.

were )ightly watered each daywitha fine spray nczale.hockedupto'a'garden hose. Allovipositioncages wereplaced in agalvanized aluminiumtank (Figure 3-1F)(120x 88 x20 em)witha12em waterlevel toprovidehigh humidityfor theadults.

Eggs werecollected from ovipositioncages star ting 6-9days after the adults emerged. Tocalledeggsan'eggcup'(6 em petridishbasewith a pieceof rutabaga pressedinto moist,loosebirdgravel) wasplacedinside-each cageviathe hole in thecage side. Females oviposited theireggsintoholes impressed inthe sand surroundingthe rutabaga cube. The,agespa.n ofeggs'col1ect~d was controlled bymanipulatingthe timeIntervelinwhicheggeupswere presented to theadults. To retrievethe eggs,the eggeupwasremoved~mthe cage andits contents emptied into a beakercontaining distilledwat er. Thebeaker was swirled tonoatthe eggs which weresuctionfilteredontofineblack cloth ..The cloth Cilter wasremovedfrom. the'funnel andplacedon topoftwomoistfilter papers in aninverted

19

ern.petri dish: Egp'we~n~ed.under a stereomlcrceccpeand sorted witha bent insectpin; wett ing.the'absorbent layen facilitat ed manipulation. Eggs'int ended for recyclingback into the stock'colony.

werelefttoembryonate.Larvalr: ringbucketswere inn?tulatedhy'rinsing eggs (350 per bucket) ontothesoil surrou ndingtheexposed portion,of a whole, planted ruta baga, witha streamof wat er "fromawashbott le. .The:gp werelightly coveredwithsoil. Theentit~life cycle,(rom the time larvae batched to ovipositionofadults,tookapproximately 6 weeks.

3.2.Developmentor a petri dish rearing method tor laboratory 'screenlngotinsect growth reguletora

..Lahoratory scre'eoing<,>finsect growthregulators against!1radicumrequireda small-scale rearing'procedure, The crit eriatobe met~II!to providereplicate reariDgun.its,t~atwere easily monitored andcapable~(supporting theliCe stages

tliroul bto adultemergeeee.:Intesting~~ f~rinsecticider~istal:lce.~ead (196Sb) placed embryoDatedegg!instatldard9 cm petri disbes supplied witb• thin sliceo~rutaba~e-treatedwith insee.ticide)to sustainlarvae for48bou rs, unt ilmorta~iJcOunts weretaken.1expanded his methodtoprovideforrear ing tbe cabb agemaggotthroughonegeneration cyele.To conserve incubatorspace, larvaland adult unitsweremadefrom&empetridishes(Figure3-2).The entire .set-upwasho~sedin • beech-topincubato ra"'2p'Cunder a16-ho~rdaylengtb.

Rear ingQ.,~in.t he dark inducedpupaJ diapau!e.

3.2.1.Lanai rea ringun l18 '--~-

Theunit base(8 em plasticpetri dish base)wassuppliedwith anabsor~ent layer, consist ingof~wofilterpaperswith.!pieceof fineblacklining cloth placed ontop(figure3-2A). The absorbe ntlayer wasmoiateuedwithapproximat ely1

, ...;

, \ '

ml ofdistilled wat er;excesswaterwasdrawn orr with apasteur pipet pressed

·againsttheabsorbentlay~r. Theabsorbent layerwasremoistened on.the third and filth daya.rterlet-up aDdonce

'in

theeeeced iuld thirdweek of develOpment.

Excessive moist ure'ointbe larval

rearin~

^units

pro~o~

bacterial growth·tbat was highly lethalto

larv~e (Ap~nd~

A)..

~ ~ eua

were pleeedtooneside of theunit andafreshpleee of rutabagl. (approx.3.0x 2.0x 2.0·cm;15.g) ..., Positioned.·alongside the

eggs.

In selectingrutabaga dimenslone, emphasiswee placed en slice

thic~Dess

^{to permitCo}mplete larval

developm~nt

^{and prbent}

overcrowdingof thenegat ivel,g~tadiclarvae(Vereeckeand Herweldt,1971).

The extraspaceprovidedbyreplacing thestandard petri dishcov,erwitba petri dishbaseaccom~atedthe !hickruti.baga piece.Theunitcove~

wu

crackedto permitairexchange andsecuredto the·unit basewith tape.Onceegg hatch wee recorded, all unha tch ed eggs were removedfrom the unita.

\

,-,I

•

Figure3-2: Petri dish methodfor small-scale rearing01f!.rl!!.~.

(A) Larval rearing uiP!:white,elJg8against.blackcloth01 absorbentlayer. Rutabaga positiotiednext,to_eggs(B) Pupation unit: rutilbaga cont aining third instsr.lervae pressed intOmoist 'sapd(C) Adult emergenceunit containing pupae.(D)Kdultemergence unit, sideviewshowingspace"

betweenunitbase andcover . .

...

:: . ..

:'

. r

:22

D \

· t'E~-=-.- ~It'

"

.-::'

"s.s.r.r.Determlnatl~DoroptImum IlU'Yaldenalt1

\ .'.

'.

^{'. ~}

.

, .The.optimum,Dumberof'

P-

!!!!k.!!.mlarvaeluppor~per lanaJ unitwas:'

determinedby •,density~periment.·"Embryonated.etis~ere"'distributedin"

groups of10,20,or 30 egp!l1nittosixunits per density level.AJJ.additionaJset

~fsixunits wereSUp?liedwith'

100.

eggseach,with.the intention.~f ~epl~iDg^the rutabaga at intervalS to_support theIarv.alpopulation,_Units wereexamin edon dayS~^reecrdeggha~hand again duritlgtbelui'week of lart'aJdeYe~pmeDt ~

~valuaierutabagacoDs~mptioDand the stage and activityof·I~ae..Cabbage maggotswererea redthroug!).tothe adult stage. Pupal~dadultyields wir e recorded ateach densitylevel,upto58daysaftersetting up tbe experiment.

Pupat ionunits(Figure3-2B)consisted oftwo 9 cm petri dish bases,oneinverted over the other.toserve'as JheunitCOYer. Theunit base wasfilledWith bird'

,...grevel,mcjstened and waurdrawn'off.with apipet. The'rutabag a piece

(eontairiing ~) w~

^{pr.essedintothe}

s~nd

and'the un.it

c~v.er c~ac'ked

^to-permitair

exchange'andsecuredwith-tapearound.itsent~eedgetoprevent-larvaefrom escaPID)

3.2.2 .PUp~t10Dunits·

.In

nature.'m~ture

^{larvaele; ve}

th~'host pl~nt

^{and burrow}

into:

the lurro.uriding soilinsearchofas~itablepupetionsite.Mostlarvaeemergingfromtherutabaga

r :

^{larvoalunits did}

DO~

^{pupatebutdiedUpOD}

bec~ming:"e~t;~pped"

ⁱⁿ

:at~~

.dr~piet.S co~d;~sed'-inside^{theunit eoveror}~b~.·dessicat~g^"attb~^unitbase.To. ove;eome

th~'aiffieult11

the,rutabaga

pi~e (con'taini~; 1.3)

i.D

eacb- l~aJ '~Dit ~as

~rans!erred

^-to

~ ';1Jpati?~ '~oit

^just.prior

^to ~m~litioD or. l~aJ feed~&' iG

dars .

altersel.--up... /

3.2,3.Adult emerge,neeunits

In order tor~cordthe timing of pupation and the percentage yield of pupae in toxicologicalexperiments,tbe pupae were retrievedIrcmthe sand of pupation units at 24 hour intervah.

Th~L~utabaga

^{piece was}

re~oved

and the unit bas:) (diedwith distilledWifir.

T~e

noatlDgpupae were decant edonto'filterpaper and transferredwithforcepstoadultemergence units{Figure3-2'C}. The rutabagaw~throug~~checked forany pupae that might have(armedinsideit.

Tbe sand wasreturn,ed.t~the::.unit,excess moisture.drawnorr and tberutabaga andanypost- reeding larvae returnedto it.

Adult'emergenceunitaeonsietedotinverted6em petridishes,lined with,ta foldedmoistenedKimwlpe~(Kimberl~Clark^Ltd.,:r~ron~~Canada.). TThe~Dit.s werem: istened,witboutremo~lngthe cover,by/delivering~fewdro~so~isti'"

. wate.rto.the..·.spac~ ~w.eenthe,u,nitbot~~.and....it;sloose fittingcove~,.(F igu..re

'<,3:-2D).Thecove~crackedto permitairexchange..The.unitswe~e'examtned

nearlyeverydaytorflyemergence and'wereviewed under a eterecmiercscope to .

mo~i~r

^the

progr~

^at

p.upal-adul~.

development

:~ithi~ibe

^-

pupari~

^..Pupaewere

;in.sed'and

iransfe~~ed

to new.emergenceunits upon

tb~ ' Cir~t'

^{sign offungal}

contamin ation ,t~epresenc~ofw~icbwestetbelto the developingimagos'~eCt

unattended, ' . I . '

3.3.Toxicologicaletudlea laboratoryscreeningor severalIneeee growth

regu18~ors agai~!It .

^Dena

,r~dicum

^.

Fpurinsectgrowth regulators were.testedat a ra..te of 1.0 IIg toQ:.radicum eggs, post-feedinglarvae and pupae.

Dinu~erizuron

^"[Dimilic

0,

technicalpov/der

.>

Q7%pure),metboprene(AltOsid85E;65~^active,),'juve'niJeh~rmone^I(s~rith"etic mixture ofIscmere] and preeoeeneIl.(cryst alline; SigmaGh~mical"Co~pany.

St.Louie,Missouri)weretopic'all;appii,ed"'.itba'glBS$ micropipet,eellbreeed"to"

-deliver.,1,0 pi ot a 1.0 /J-g/pl

~Iution

^{of,the Compound'}

.d~ived

ⁱⁿ

dimethylsulfoxide.Eachinsect was treated individuelly whilebeingviewedunder

. ' /. .

"'~ ,~

.3.3 .1.EtTect of Insect growth regulators on eggs

'3: 3.2. Errett or Insect growth

r~gti.lator8

on pOllt,;reedlng'larvae

~

Delia radicum eggs(24-28braold) weredistributed at20eggs/unit to the absorbentlayeroClarval units. Under a dissectingmicroscope,1~gorcompound wastopically applied,one eggat a time,to;the entire eggsurface. Six rearing units wereset-up pertreatm ent and control"groups. Theabsorbentlayers

~9ntaillingthetreetedeggs wereplacedinsidethe rearing unitsand24 bours later arutabagapiece waspOSitionednext,to the eggs.'Egg hatchweereecededcathe Iourtbandsixthdayandpupae

·'."~re

^{counted'21and 28}

d~Y8

^aCter

~reatTent~

Adultemergencecommencedat31days andwasrecord ednea rly every day upto. 79days~rtertreatment .

astereomieroecope. A solve,ni-treate;

~nd

an untreated

cont~oi grqup

^were

~ted',"';

aga.inst egg" larvaeand pupae.After treatment,insefttswe~erearedtoa4~lts

b,'

thepetri.dishmetho d. AUtesultingadults were sexed. Tberesults were subjectedtoa SingleClassification AnalysisoCVarianc;(Sakaland Roblf,lU6U).

/

.Two'experiments were cond uctedon pcst-Ieedlng larva;: tbelarval screening' _ test.and~.'oieco~dexperimenttodetermin~th~minimum doseDC methopr'ene requir edto suppressadultemergence whenappliedtolatethir.dinstar larvae. Forbothexperime~ts,larvaewere collected as theyleft therutabaga Cromstock and/(j~labo~atry.cultures,rinsed and'blotted dry on filterp'aper:'Only larvae that weremobile and had not yet

und~go~e

beadinversionweretr'¥ted. 'Each larvawasheldant eriorly, dor sal side upwith forceps and the compounds,were appliedover the entire body surface'ju~t^{post eriorto}~heia rval mouth hooks:

Beforelarv aeweleplacedintothegravel or pupationunits.they wer eheld for 5-10seeafter treatment'toaid in'the penetr ation oftheeompounde.

Forthe larval screeningjest,ten

larva~

'were testedpertrea tmentand contr ol

J

groups.

Th~

experiment was repiicated ,three'times..'T he timingof/pupationwas '...- monitoredd~ringt~ fi~'~treplicat e,byrecording/pupalformation~24'hour'

.Resultsof·pupal.and~ult yield~^{werer;cordedup to} 52 days arter treatmen"i.~dul~startedemerging 1,6daysafter treatment.

F.or thesecondexperiment· with'methopreneltwenty larvae we'retieat edper -doselevel or 1.0,0:5,0.1,0.05,

~.OOl,

^0.0001and

^e.oooo;

^{Pg perlarva}delivered in 1 piofdirnJ:thYlSuu.oXide. Allconcentra tions werepreparedfrolj1a1pg/ plstock' solutionorhe compound.Results or pupal Bond adult yieldswererecordedupto 33 days aft'r treatment. . ,

3:3,3,Erred or Insedgrowth regulators on pupae

~~experiments were~onductedon!1.radicumpupae.Aseresult ofthe pupal screening test,asecondexperi~ent^{to determineif} methoprene,the only compoundtoadverselyaffect pupal-aduhdevelopment ,was errecti~e_against

ins~ts'

^inimaginaldevelopment. Forthe,screening test.'ttl,Dped

~up~ri~

^inpupaJ='

adult developmentwer,recoveredfromJbestockcolony approximately 26'days, .~f~ereg~^ba,tch,rinse~ⁱⁿdistilled'water~^{placedinadult}emerg~nce^{uni,ts. <I>}

Excess water wasdrawnCromtheabsorbentlayersoit wouldnot interlere with topical'application. Each

~~~pound

^was

~pplied d~rsaliy' wi~ r~pect

^{to t.he}

developinginsect,.Asingle replicateoCt,entypuparia.were tested per tre.atmen.t.

andcontrolgroups.The unitswereremoistened aftertreatmeni andmonitored near(y every dayforthenexttwo weeks.

Forthe secondexperiment,pupaefromthe'samegeneration were recovered .almost a weeklat er{32daysfollowingegghat~.h'.^{To selecta}definitive.stageto treat, ooly insects that hadreachedimaginaldevelopmentbut' notyet developed cuticular hairs, or melanized,,;wingsand legs, were treated. Threerepli~ates01

~entypupaeweretreat edpermethoprene and untreatedcoD.!rol.group.Units were cheekedperodically for adultemergencF, whichwasrecorded upto50 days alter treatment.

" ,

3.4.,P bysiologi.cal studies: effect or~~thopre~eonh~~~l)'.mpb. carbohydrates, proteinsand amino.a~idsⁱⁿ~t--feediD.g larvae

Post-reeding larvae were individually treated with 1 ,.g methopreue and placed intopurM't'fon units. Thirty~six"hours aftertreatment,larvae aeleeted tor hemolymphcoUeeti~nwerethose that bad not yetun~rgonebead inversion.

Larvaethathad pupated during tile thirty-sixhourlneubation were not selected.

Ill.preparationforhemolymph collection,each. larva was blotted dry on filter paperand held ;with forcepsunder a stereomicroscope. Pulsatingp~was applied posteriorlyto the larvaby. rhythmically squeezing the forceps.'Tile larva w~pu.nctured on its ventral side [ustbehiad the mout h hooks with a

_,

- -sharp-

. POin}- .

Theclear.hemolymphthat exudedwas collectedby capill!!'ryaction in 1 ,.

micropipete. Eacb.Ia~vayieldedapproximately1-2 pi,hemoly~p~. Collected hemolymphwasimmedia.telyblowninto5ml trichloroacetic acid ror amino acid and proteinquantilic~tion^orontoan activatedthin~li.y~~hroinatograpby^piate Iorsubsequentcar bohydratesepar~tion,,

3.4.1.Separation and quanUncatlon or hemolymph carbohydrates Prior to colorimetr ic quantificationof hemolymphcarbohydrates,thespeciri,c carbohydratecomponentswere separatedby tbin-Iayer~h~matography. A methodofseparatingsug~rsonsilica gel,by FriedandSherma (1082) was adopted.

withslight modificati ons.

/ ( - '

~i1ica

^gel

preco~ted

plates [Sllice,Gel Gj0.25mm, silica. gelwit hgyPSUmr;Ox 20em, Meeherey-Negel,Duren,Germany) were impregnatedwith 0.1M sodium bisulfiteby dipping. The plateswere air dtied~Ddcolumns 2

e m

wide'were delineatedinthe stationaryphase'~ys.crapin~with a sharp point. The plates were activatei:t(IC?OCj 30 min) andspott ed witli 5 piof hemolymph-orstandard

•s~mples2 em fromthe.bottomof the pla.te.Once theapots had drfed,lhepla.tes wereplacedin achromatography tank pre-saturatedwith 100 ml of mobile phase

J . .. ,

.

•