II. REVIEW OF LITERATURE

15

2.1. Antennae and Olfactory Sensilla

VJhen considering antennae of different species,

the V8riation in form is striking. To understar:d

the evolution:; ry development of variations i'h size

1 nd shnpe one must consider all of the different

functions the gntennae may serve (e.g. olfaction,

t3ste, touch, perception of air-movement, heat and

C02 ), which vary considerably between species

(5 chne:l der, 1964) • Antennae with feather like

sl-1apes (e.g., in saturniids, lymantriids) seem most

advanced as 'odor filters'. The surL>ce is greatly

increased by thousands of long vertically oriented

hair se:--1silla on the antennal branches. The large

surface area and characteristic geometry enable the

antennae to sieve out odorous molecules effectively

from the air pa :- :"ing through it ( Kaissling, 1971) •

Olfactory sensilla have been classified on the.

b:isis of size and shape as sensilla trichodea, s.

basiconic3, s. placodea (Schneider and Steinbrecht,

1968). In l_epidotera particularly, in moths ani

butter flies the s ensil1 a t ri chodea, Sen sill a

Ch1etica, ?ensil:::.a b8siconica ::md Sensil. a Coelonica

have been sho•m elect rophysiologicall y re sponci to odor

16

{Schneider ~Jnd steinb;-> ··cht, 1968). In males of

Bombyx II!..Q..:d-_, "the s en.sil::_a tr:i.chodea are known to be

extremely s ensi ti ve to the female sex. pheromone

( S c hn ej_ d e r, 1 9 57 ) •

Sensil~.a _qr~a_tics: are similar to sensilla trichodea

but can be distingushed only by a cuticular collar

gt, the base and have sli;;ht longitudinal ridges at

·the base. They oc ·-.::urmainly on the out erolst eral

:;md later:1l portions of each flae;elJar segment. Most

of them are closed type tips.

§_~n~.i-~l.:_q_s;_~~l_Q_conica_: ar~~ receptors in. vvhich the

sonsory portion lies j_n- a pit or depression of-..the

cuticle. Thece pits are always surrounded by cuti­

cular hairs. They :.~relocated mostly ·on the

dis tal half of s egmerrc s and groups o.f 3 or 4 are

frequently found on diot nl edge of the segment.

S ensil)_a ___ ~~lo coni ca: are relatively large and occur

ventr1lly on the distal porti6n of the flagellar

segments.· Although they may be lacking on certain

segments but are concentrated on the 3pi90l two

thirds of the antenna.

Seni?..i.J_j_(!_ll~r.:h_ci@c~: are named by jefferson et al..,

( 1970) • They o ccour in relat:. iv ely large number on

the antenna of noctuids.

17

Sensi_-1:)-..£._td:_c_.t_o_de~: do not have cuti cu.lar collar at

the base, short an~i long Sensillae are found. The

short i -:3 type 1, 17 pm long and having blunt tip

·with annular ridge along the whole length. \rw'ith

scanning election microscope three t~'pes II,III and

IV long Sensillae have been distinguished. Type II

have spiral ridge which appears somewhat annular

n,:;ar the tip of the hair. Type Ill have also spiral

ridge but less evident than tho~\e of Type II. The

tip of Type III appear to be smooth. Type IV have

annular ridge near the base. Th::: tip of Type IV is

blunt and distgl portion of these hair.s is carrugated.

There is a row of type IV" on the proximal border of

e:Jch flag·-:=1:-:._ar se6m=:n t .but th :y also occur elsewhere on

the segments.

In Lepidopter8 the SensilJa trichodea have boen

identified as the sex pheromone l"'eCeptors {Albert

;t 81., 1974). lVl.ore thFm one type of sensillum tri­

chodc;i..nn is foun::l on the ant enn.a of Trichoplusi8 ni

(Jefferson et al., 1970). Four morphologically distinct

LyJY?S oJ' sensilla trichodea have been identifi ~~d. on the

'·n tenn a of Cho ri stan eura fun:h.,ferana . (Albert and

Seabrook, 1973), althoJ.gi1 only one or.' these. (ty.[)e IV)

h8 s sho ,ffi elr;ctrophysiolo.::;ically to resp·:Jnd to the ·

18

sex phe romon2 ( .1.\l bert et al , 19 74) • Sexual dia morphism

is present in some lepidopteran's antennae (Kais:::;ling,

1971; Steinbrecht, 1973) and many lcpido1Jteran females /

do not posses sensill.a trichodea.

In Bo_~_!_~a_L~ mo ri (schneider· and Kai :=;:line;, 19 57) ,

197"3) Sensil·: ::~ tricbod,~a are present on the anten.'l8e

of both sexes; however it hgs not been established

"Jhether or not, th2 Sensilla trichodea of the ferrnle

responds to its own sex pheromone.

There is a considerable diversity in the number

of sex pheromone sensitive sensilla trichodea present

on the anten'1ae of v::n:·ious species of lepi:h_?ter~.

This is rel8t ed primarily to the form of the antennae.

The larg? plumose, nntenng of BomJ2.yx mor.:_i. posseses

upto 16,000 sex pheromone sensills trichodea.

(Schneider, 1970) where as the small filamen-vous

::JnJ~ ~nr1a of Chari stoneur:J fumi f ~.t:.:~n~l posses es only

2300 sex ph2romon0 sr;;;nsil:..a •rrichod•29 ( ~lbert and

seabro k, 1973) of which only 300 are responied Lo :.:.he

.se~~ pheromone (1\lbert et 81., 1974). The size of the t

antenna ;:md the number of t ri chcd ':8 r(:;fle ct UIJtO the

sen:;::itivity of the moth to its sex pheromone. The

large gntenna h.3s a more efficient catchment areg

19

( K.qi s:3li ng, 1 971 } and trap to odorant molecules of

• much lol'ler concentrations. Hovvever, it does not

r2fl(~ct upon the sensitivity of the individual recep-

tors that appesr to be similar in their threshold

sensitivities. sexpheromone Sensilla trichodea are

i.nnerv8l:.ed by a number of primary sensory neurons.

In Bo_.:_~ mori ( Steinbrecht, 1973) two neurons inner----- ..

vat.· the sex phe~ romone sen sill a t ri chodea. El ectroply-

siolu.gi c:::l Lly 3 neurons respond in Argrot eni3 v el~~i-~~na,

although O' Connell ( 1972) sho~vPd that only 2 of' these

are chemosensory.

In light microscope, five neurons ap:)ear to

innervated the type r'v. Se;1silla trichodewn of

Chodst_~'fl~~ra- funif~:r.~G3. (Albert, 1972), althouc3h not

all of these r(-'spon:l to the sex pheromone (Albert et

al., 1974.). Other types were later described in a

nurnber· o.f' sp!~Ci'38 ( Altner and PriL .. inger, 1980;

Steinbrecht and lVlull.er, 1976}. The first two types,

s. trichodea and s. basiconic.g, are hair-shaped

sensiF.a. The s. coeloconica type, also called a

pit-peg, consists of a peg embedded in a cavity of

the cuticle which opens to the surface. s. placodea,

or poreplGtes: consits of a circular

structure 'J~hi.ch is perforated by pores. Hovoever, n

20

on the basis of exL. emal mo rpholo0y. Internal

morpholo:;,-ic31 properties revesled by studies of

fine strJcture do not alvvays correlate with the

outer shape. For example, S· tri.chodea and .S.

b3siconica usuaLLy differ in their long an·3. short

h~irs resp2ctively, but not al~Jys, ani in this C8Se

th" char.1ct<?r of their fine structure is the prlncip1l

·lifference between the two types (Schneid·-,r nnd

S"c.einb.cecht, 1963). Furthermo.re, intermin<~;led wi-'c.h

S· trichodec1 and S. basiconica in B. mori ar·e found I

sensilJa which .sho''" a fine structure of an int.ermeC:.i'J~e

type (Steinbrecht and l"luller, 1971). 'l'he limited vo1ue

o.:.· ...lsi ng the ext ernsl morphology for cla ssi lying

sensil' a is discus sed by Altner and Pril.l_inger ( 19d0).

One sensil iJ.m type for wh.!. c l1 t !w fine st .cu.ct ur•:: is

well studied is S. trichodea of the silk mo'ch ~· mor:,i_

(StP.inbrecht and ~1uller, 1971; Steinbrecht, 19dO).

This h;;:Ji r-shr:iped sensillum ( Fig.1) can· be ch~r13cteri ?:ed

gsa thin-walled (0.) pm at half len~th) cuticular

protr.uston (10 p.rn long, dianeter ca.2 pm). The

vvsll is pPnertr'ted by numorous pores (ca.250C;) over

the entire suxf'.ice, e:-ce:ept 3t th2 most proxirn'J1 part

of the h3i r. The h:.dr is inL ervated by one or more

sensory cells, vvith dendrites penetrating the hair

·· ······· dendrtte branch

.......• pore tubulus

-------- receptor lymph

------ culicuiar wall

··----outer dendrite segment

-------cuticle

------ tormogene cell

Fig. ! : Scheme of a hair~shaped sensillum, showing one sensory cell, embru.ced by the three auxiliary cells. The dendrite of the sensor'y'~dl is divided into a branched outer segment and an unbranched inner segment (containing'Nl'itochondria). The conduction system for the odor molecules (pore and pore tubules) ma'kcs·c011t~ct with the dendrilir. membr.Euw.

21

lumPn ;:md re:::~ching out to the tip. Each dendrite is

divided into an inner and outer s::.egment, separated by

a speci;d cilia r structure which is. short and 'neck-like'

::Jnd contains the typic8l concentric doublets of tubules

found in other sensory cilia. It is only the outer

dendrh. e s egt:J ent which resides v~i thin the hair lumen.

The tvw sc:>grr.ents also differ with respect to the

contents of cell organ~lies. Whereas the outer

s egwent s m::J i nly contain mi c rot ubul e s and no mit :co chondri a,

the latter are abundant -,vithin the inner segment. The

outer segment is usually divided longitudinall)' into

br·anche•o, rC'~lat.iv;:-:ly fevJ inS. trichodea, but up to

50 in others, e.g. S. basiconica (Fig.2). The inner

segment and the cell bodies are surrounded by thTee

auxiliary cells (the the chogen, to nnogen and trichogen)

wh~- c h form a sheath around the sensory cells. Thus an

olfactor:,; sensillum consists of at least four cells.

The auxiliary cells, ~~hich carry out an import ant

:-ecretory function during the ontogenetic development

of the sr:>nsilJ um,' forming h;::~ir cuticle and the microtubles

(Ernst, 197?), may have another important function

during adult life. Th~y apJ,arently msintain the

electric:-:11 potential (ca. + 50 mV) between thE! haemolymph

"lild the receptor lymph (Kaissling and Thorson, 1930).

r·~-"'l''"-...,.,.~·····-·· k .: •

r­~-

. Fig: 2 : Transmission electron microgrJphs of Bomhyx mori sensWa trichodea (above, x 20 000 and 40 000) and s. basiconica (below, x 20 000). Fixation by frer.:e substitution results in almost round cross-sectional profiles of the dendrites, cor:taining a teguiM array of microtubules. Pore tubules are well fixed in the outer parts of the hair: .. ~ (courtesy of Steinbrecht, l980).

22

. The receptor lymph fills the hair lumen around the

outer segment of the d~ndrites and is separated from

the ha emolymph by tight junctions formed between the

au.xillary cells jupt belo1\ the ciliary structure.

ThP pot entic;l of the receptor lymph, which has

relr:Jtively hi,r;_;h K+ contr•nt, is vrobably maintained

. . + uy an elect~lCJgenl.c K -vum]J locc3Led iu thr; folded

mr-•n,branr"s of the trichogen 8nd tormogen cells. The

membrane fol>lds are only present on the side facinG thE.:

receptor lymph. The sensory and auxiliary cell. s are

loc8ted .dthin the epithelium which is separated from

the haemolymph by the bas;:!l membrane. An axon (0.~ pm)

leading from a sensory cell to the brain is s~rrounded

by gli8l cells, and does not interconnect with other

gxonr, either by fusions or synapses, before re.schin[;

th~· br::1in (Stf~inbrecht' 196?).

The co ndu.ctio n system t~1rough ··~hi ch t hr-: orlo r rr.ol ,c.-

cul es reach the dendrite membrj n e or the sensory .;; ·-~L:

ClC ttl' in t1·JO pri.ncip.1lly difi'erent types (Steinbrr·'·:ht

• l·::>qdins from lon_;itudin3l grooves on the h:-lir 3 urf·,c·.::

to '1n in·-:. er cylinder that contains the dr.:>nd~it es

(Steinbrecht and. £Viulter, 1976). However, the conduction

23

system hAs been rnainl y studied in the ot~.tel~ type

(Steinbrecht and 1v1uller, 1971). Here, the molecules

a 1~e adsorb e::l. on the cuticle of the hair w:Jl:. (FiG· 3)

and diffuse through the pore openings ( fun:1el 100 nm

;vide) vvhich oft en l.ead into 8 1i ne ( S run) anJ

,hort ( 25 nm.) pore charr:1el. This 1videns j_n'.::.o a

cavity ( 50-10,! nm). 'l'h:::~ inside of the cavit.y faces

the r'"'CetJtor lymph of the liquor ch-~nnel (200-40!) nrr1

in leoght) ·which penetrates the remaind<'r of the

inside of the cavity 3-7

fine porR tubules pass through the liquor chan:1el

and the hair lumen to the dendrite membrane.

"©.c:>ct.rJn microscopical st.J.dies have est~blished

th~t contact exists between the_ 0 e tubules and

t.he dendrite membrone (Stej_nbrecht and Muller,

1971). Furthermora, Ernst (1969) h9s shown th8t

tr:;cer subst1nceQ penetrate from the outc-ide

through thF: pores into the pore tu.bulPs, g rout:>

by 1'>lh~- ch odorous rnolPcul e::: c3n evidently re:1ch the

dendrite meulJr.cme. Calculations by Ad~m and

Delbruc;_<:: ( 1968)- sug .est a two-dimetts.i.on:-Jl surt'3ce

di ff'..J.sio.:. of the odorous molecules throu0h the

pore-tubule conduction systEm~

•

cutlcu\ar waH

receptor ceU 'dendrite

lli.a.. ___ 3 :

surrounding air . ______ pore

---------- __ . -- pore kettle

1000 A

·internal fluid

Schematic diagram of culticular wall and dendritic branches of a sensillum basiconi.cum of carr ion beetle, l'lecropho~.

24

'!'he specificity of insect olf::1ctory rec~ptors

.has bnpn studied in details by O'Con_ell ( 1975).

This is ab,.olutely relC:ited on the perception of

sex pheromone:;. ln T-GAGS recording from the antenna of

Bomb_E m..?_:ri_ Schneider ( 19&2) found that there were

no qualitative differences in the respone to the

odor of phe-romone~ gl3nds of various saturnid moths.

The differences were fo0.nd only in the rela"cive

gmplitud·-"s of the response::-;. Schneider et al ...

(1Q64) reported by using extra c~llular r~cording

techniques that 2 to 3 neurons were co nstr1ntly

firing as background activity in s ensil2.a t ri cho·dea

of _A_nthe1:~ea and these celLs responded by an incre:Jsed

r1"c.e of dischar,;e to stimulation by the se:xpheromone.

'1'hey further noted that in the s snsill3 basi coni ca

the longer the hair, the fev-ver the compound.:, th·1t

~>t:."i.:nul3ted it. These finJin~~ led to the concr-f't

of oJor generalist neuruns (cel.i.s) and odor ~pc-cL~Jli:;0

neurons (cells)(Boeckh et al., 1965).

Odor generalists art=; neurons of the S8me sen­

sillum type in one individual, which h~ve different

but partly overlap~Jing reaction patterns j_n aJ 1

25

re1cting cells. Examples of such odor journalis;,:,s.

1rr.; sensil i_a basiconica.

Odor specialists are olfactory neurons belonging

to 8 rc:;cognizable type of sep.silJ.um, giving sterotyped

responses to 8 seri<os of compounds. Example8 of

odor spr:>ci~lists a.rf~ thp, cRc:::ptors for sex attra·ct::mts.

Schneider et al., ( 1Q64) noted in Antherea that of

3 cells in comnon sensill u.m basiconicum, one may

be:: excited one m'1'Y be inhibited. CX.YJ.d one may show

no response to a common olf3 cto ry stimulus. A

sirnila r phPnomenon has been found in the sex pheromo)')e

r~·c epto r s ens ill urn of Tri chop~~sia ni.

Kais~:u_ing and Renner ( 1968) by using single

unit recordings ident,ified tvvo cell types respui1ding to

the pheromone of the honeybee. One cell res:Jonded

to scent from Nasanov gland. Vnreschi ( 1971) further

·separated 7 cell types with no overla}> in B}Jecifi-

city; ho•~ever, there was some vari:lt:.ion of speci:.:'i city

within e3ch of these cP.LI types. SchnE:ider 0t 31.,

( 1()64) notod th:::lt gt=?omc>tric isomer·s of bombykol ~'>~ere

u ltO 1000 times l e:;s pffectiv e at the bombykol re c optcr

th;m thP true phenomon~ ( bomhykol) • The sa?:ne rule

>p;Jlied to Tr~.SJ2QE.l!!.:?J.2 ni '•lhere :)8yue et c.ll. ( 1q73)

26

reported th?.t. the more a parapheramone vnries in

its structure from the structurc-; of the true pherommone, " the greater the amount needed to elicit a significant EAG.

This infonnation indicates a considerable det;ree

of receptor cell specificity to compounds of closely

related chemic.'1l structures and 1 end support to the

concept of odor-specialist nPurons. O'Con:·1ell ( 1975)

by using computer an::llysis of single-unit neuronal

rt:;spon!::Oes in sex pheromone receptor sensil.._a of the

red bonded leaf roller has indicated that the two

cells responsive to the sex pheromone and number of

its anolot;ues do not respond in an identical fashion

to each of these va2ious compounds. He found that

the response freqw:"ncy to the pheromone and to six

other behaviorally active compounds varied be:::.ween

the tv-vo cells and cL3ims that these diL'r:;rences in

rP.sponse were due to intrisic factors within the

neurons. He argues that the encoding of odor quality

i ~'not simply in the pr1'~seric e or ab~ence of activity

in anyone neuro, but is encoded by a pattern that may

vary across an ensemble of receptor neurons to produce

~ unique total response.· 0' Connell ( 1 97 5) states that

·as some neurons can respond to 8 out of 9 b~;haviorally

27

active compounds, there could be as many as 8 fun­

ction:ll :_y dif1 p,·~nt receptor sites. He further

concludes ·chat because one cell responds prefe.Len-

t;ially to cis-11-tetrAdecenyl acctat;e (the pherGmone)

and the second cell respond. s preferentially to

tt3ns isomer (1n inhlbitor), there mu:t be ~t lP1St '

tvvo function~::lLLy distinct rt:;C~~ltor sites. Phercrnone

receptors do reSJ:Jond to the isomers r:md analo~ues

of Jche ap,.'ropriate pheromone. This indicates that

gl·chough the receptor may be more sensitive to

the true pher·omone it does r(~spond in a ~vesker

fashion, to the=e ot~e~ compounds (Scnneider et al.,

1C164; Pgyne, 1971). Kaissling ( 1976) has propo:3ed;

t rnt the 8p 9ro p ri ate molecules for a given a c <' epto r,

r·.::2ult in large, i rregulr-J rl:; timed elementary

gene rAt or pot enti:~l~3 • Er~ch potenti:.:Jl co rr:; S.fJOnds to

an individu<Jl stimv..latory molecule encounter with

the ap rep ri 3t ~' acceptor.

On the othc r- hand, other molecules, wit:1 some

structur8l relationship to the correct molecule,

res:..1lt in smooth receptor potential::; wi:,i<.;h aric.e from

the surnmatio;1 of ·many smaLi.er ele1nentary 'generator

potenti:;ls.

28

Seabrook's ( 1977) study showed th~•t the neurons

of the sex phpromone rr:::ceptor of male Choris~~neur~

£~~!!_i_(c~_r:r!Jl·~ r ·;.Jpond to both its sex pheromone, a

mixture of cis and trans-11-tetradecenal (sanders

and "1-v2atherstone, 1976) and 'co trans-11-tetradecenyl)

8cr~tntr; 8 kno•vn inhib:Ltor of the beh~vioral r~~ponse

to the s:::xphrrornone (Sanders et al., 1972). Seabrook

( ·197'7) demon::,trat ed in his cro'ss adoptation exp,eriments

th3'c both the aldehyde and the acct ate are inter­

acting with the same dendrite acceptor site. Boeckh

et aL (1965) shovved the same interaction in the honeybee.

Th~ pheromonl?- receptor when a:.tapted to the

phr->romone :~1 ~::'0, is 8dapted to caproic acid. Payne

and Dickens { 1976) have shov~n the aggregation

ph-=:romone of TJend~.J;_~o-~~ £rontali~,inacted with

the acceptor for all other cofnpou.nds tested.

Thus it lhGY be a:::;r_;umE-~d that as an acceptor may

respond differenti31 Ly to certgin stimulatory

molecc...les, a specialist rec·?ptor sensu stricto

does not exist.

Here, the minor pheromone components are phero­

mone inhibitors ;=uri synergists. Pheromone inhibitors

are those compounds which reduce the tra ppi_ng

efficiency of a· ph2romone or rt~duce the behavioral

t~. sponse o.t' 8n insect to its sex pheromone \vhen -

compou.nd is rele1sed along ;,vith the pheromone.

Pheromone synergists are those compounds which

enhance the rPsponse of the male insect to the

p hero mo n e so uc c e.

Inhibitors and synergists c~n operate at

Pxtremely lo1v concentrations and are often isomers

of the rna jor component.

Inhibitors have be en described for a number

of insect species (Hathaway et al., 1974). In

some instances, the inhi.b:i."c.or is produced by the·

s~1me insect gs is the pheromone and masks the

effect of the pheromone (Casida et al., 1963;

Shorey and Gaston, 1967).

The inhibitor may serve to chemically isolate

tvvo species with a common pheromone and with over-

lapping geographicGl ranges (sower ~tal., 1971+).

There are 3 potent~al neurological mechanisms

for the perception of inhibitors.

1. .rtec:eption by separate sensory neurons.

(MaclaYiughlin et al., 1974) and thus the information

is car,·i(·;d by di::;crete information channels to the

olfactory lobe or deutocerebrum o.r the brain.

2. rtec epti on \;y ac.eeptor molecules, lo cat -=;d on a

common neuron (in this instrance, the pattern

JO

of action pot ::'ntinl dis charge vmul d code the

signal) and

3. neception by a common acceptor in which C3se

the pattern of the acti!l>n potential sequence

co,ld code the information or a binding between

pher.o:none 3nd inhibitor could occur.

sov.Jec et al., (1g7h) have shown that, the males

of Plodia ~-0~ er~)gn_§_t. e~l~ and Cadr§_ cautella can

be ln bi tu.r; ted to inhi bi tors of the sex pheromone

respc5ns e while subseyuently showing a response to

thE; SE~X pheromon·' plus the inhibit-or. A 3 minutes

pre-exposure to the inhibitor trans-trans-9-12-

tetradecadien-1-01 acetate produced larger increased

rP.sponses in both species on subsequent exposure

to the pheromone (trens-tis) 9, ·12-tetradecadien-1-01

than the response of individuals that m d not been

pre-treated. Here the main interpretation is that

the pheromone and inhibitor were being perceived by

-separate sensors. The male of Trichoplusia ni

eli cited EAGS by expo sure to the sex pheromone

inhi bi tor t ra ns-7-dodec en-1-0 1 vvhich were similar

to. EA.GS resui ti ng from exposure of the antenna to

the pheromone trans-7-dodecer'l-1-01 acetate (iVi'ayer,

1973). Trichoplusi a ni and E;:;eudoplu.§..ia ~~ludens

Jl

upe the same pheromone. M.aclahughlin et al. ,. 1S7ld

found that the inhibitor did not interfer with sex

pheromone behavior L1 Ghat males which showed an

upwind flight behavior and clasper ext ens ion when

exposed to the sex pheromone. If did, however,

p rev .:-:nt the moths from o t~i enting to a lo cu.s of

at-.::.raction when the inhib].tor source W<:;s wh,hin

30 em of the pheromone sou r·c e.

Above conclusion is that the inhibitor is

perceived by a sensory neuron separate from the

pheromone receptor neuron, and that modification of

pheromone-elicited behavior is not. peripheral but a

central phenowenon. ·rhis phenomenon is also sho.m

by Knis~ling (1071).

R:::Jelofs and Com~au, ( 196~ 1971) reported that

compounds structurally related to the sex att.ract:mt

for the red-banded leaf roller moth cis-11-tetradecany-

lacota·._,e can have either a synergistic or inhibitory

ef.Pct on attract::mcy in field studies. Thr=y proposed

~that th8se structurally relat8d molecules are intPr-t

ncting wtth the bindin~: sites on the similrJr rr-,ceptors,

vvitll v::1rying dc-;::;rees of affinity.

In single rect=·ptor recondings from the pheromone

rPc eptor of .1\.rgyrota eni a v el.Jtinana, 0 1 Connell ( 19 72)

32

reported that the same two neurons responded to

the sex pheromone cis-11-tetradecenyl acetate

to ·an inhibitor (trans-11-tetradecenyl acetate)

and synergist (dodecyl acetate). The response of

the sensory neurons to st,imulation by a mixture of

cis-11-tetradecenyl acetate+ trr:Jns-11-tetradeccnyl

acetate was d~creased in the activity of one

neuron when compared to stimulation by an equial ent

8mount of the pure cis-11-tetradecenyl acetate.

The response to cis-11-tetradecenyl acetate

+ the synergist (dodpcyl acetate) 'was an incrn;;~se

in neur':"-Jl activity. In a subsequent study, differential

r ponsPs of the tv~o chemosensory cel~s to the

sex pheromone 3nd 7 other beh8vio rally active com-

t)''tl;!d..J led O'Con.'ell ( 1975) to conclude thst

s•:?parJte acceptor:::. are present for the cis-11-

'JC~et~Jte. Roelofs et al. \1975) shovved th·'lt all

thrr.~'- com. ounds :1 re pre~~ ent in the c~1 1 in<.:.~ fe:n·1l e.

by usi.n;; :S;\G.S, conformed O'Con,le.~.i. 's vir.:>'" t:h:n:. the

two j_somc'7rs are pl?rcei.ved by two fun~L..i.cJn:.~lly different

acceptor sites.

33

A similar situation vJas found in Adoxop_J::!Y_es Q_£_ana,

in ~vhich one of the 2 sensory neurons responded to

ci~3-9-vetradec:enyl acetr1te and 2nd neurone responsed

to cis-11-Letradecenyl acetate, which_comprise the

moth's pheromone in a 9:1 ratio. No neurone w8s

turned to this ratio. Thus the determin3tion of

the opti;:mm blend is D central nervou~:: system pheno-

menon (Den Otter, 1976).

Adoxophyes orana uses tr~ns-9-and ~r3n-11-Ce~radecenyl . -----.,..---- -·--:- ---

a c et 8t e, t. he geomet rl cal is orne r.s of its pher·om.one as

i'cs inhibitors. EtiG responses to mixtures of the

pheromone and its inhibitor showed no aLi.1itJ.Ve

effects vvhen comps red •1i'c h EAG !\-; spons es to the

pheromone aione (IVIinks et nl·, 1974).

of the sex ph.:;rornone trans-11-tetradecanal and the

inhibitor trans-11-tetradecenyl acetate are perceived

by the same neurons and react with the same acceptor

molecules (Seabrook 1977). lt has been suggested that

the n -,u ron ad.apts more to the inhi bi tor than to

the pheromone.

This differP.nti<-1l ad8ptation of the nrouron b:; the

two compoun,ts indicates a clii'fet~ent affinity of each

of t.he·~e mol ecul2,':' for the ccmn:.on acceptor ( ,3eabro·)k,

34

1977) and sUj)i.JOi~ts the view of Roelofs and corneau

( 1971) and KaiS'sling ( 1976) for the responses of a

neuron to structurallysimilar compound.

It is of int.erest to speculate on the potential

beh?.vioral role of the so Cc.llled inhibitor compounds

· tha'c supress the activity of sPnsory n·=.,urons. It may

b~ thg",; th~y do not inh.~bit the beh::~viorol rP::-:-ponse

to Lhe pher'Gmone but that they may act as flight

nr:~est::mt,_,, initiating the short range activity between

the male and fan.sl·e moth. This has been demonstrgted

behaviorally ( C"'rde et al., 1975) by the a1j.dition of

do~lecyl al cohc-:1 to the IJheromone blend for the o ri ential

fr:.:it moth. 'fhe:::e com.i:Jou.nds are often present in small

3mourr'cs in the pheromone blend (Roelofs et gl., 1975)

and therefo ce \1\;'0uld only reach a b ehaviral threshold

at di~:;tances very close to the emitting female. In

Choristoneura fumiferan_? the sensory neurons are able

to perceive c0ncentration dif_,_'erences only during

th-:: first 200 ~(lsce of the exposure to it::; pheromone

(;:;e::~brook, 107·/) and ther~fore are not lik~ly to

be able to percei,(e a concentr!3tion t5radient or to

determine their pro}:irnity to 8 female based on the

cone entration of the mnjor pheromone component •

35

Recently more attention has be en devoted

to the study of minor components of the pheromone

system. Thes (= may occ:our as a part of the insects

pheromone system {Young et al., 1973 ·; Minks et al.,

1974; and Roelofs et al., 1975). In addition,

there rn3y be synthetic structural analogue of

the pheromone (Eoelofs and Comeau, 1968, 1971;

Co;rde <:.t al. ~ 197 5} , t hs t when mixed i'Vi th the

pheromone, increased the behCJvio ral response

of the insect.

R.o r-::lofs and Come om ( 1968) reported that

dodecyl :1cetate w.gs str'Ong;ly synergh>tic in HiBluHe

in traping tests with the Red banded leaf roller

moth and they subsequently li~ted other 11 - and 12 -

carbon acetates as synergistic to RiBLURE (Roelofs

ana. (o.me3u 1971) • Similarities in molecular structure,

they sug,;ested that these componds may be reacting

with the same receptor site as does the pheromone.

Little el~ctro-phy~3iological evidence is avail:Jble

on the perception of the minor pheromone components

and synergists. O'Connell (1972) reported th1t

dodecyl acetate alone had little effect on pheromone

receptor activity in the Red banded leaf roller moths.

2.3: Mo.l::EhQ.~'?..gzL_aQ.d Histology of sexpheromone glands of Female .fuloth:

J6

The c; bdomint.ll tip of female moth containing 7th, Sth and

9th segments, is densely closed with scales and Mirs.' Setae

are al ivays on the posterior rna rgin of the 8th t ergum and on

the valves and minute spines cover both scl erotized ana

unscl erotized a rea. Normally, the Sth and 9th segments are

telescoped into the 7th segment. They are·retracted by longi­

tudinal muscles which hnve their origin in the 7th segment and

are inserted on the valves, the common eviduct, the apophyses

anteriores, and the apophyses posteriores. T·wo sets of

protractor mu8cles are present. One set h~s its origin on

the posterior mar;in of the 7th tergum and is inserted

on th:! anterior end of the apophyses anteriores, and ·the

other set ori6ina~es on the posterior margin of the 9th

.ter;um nnd is secreated on the anterior en~ of the

apophyses posteriores. The gland which produces the female

sex pheromone is Yeversible of glandular epithelium

situated dorsoposterior and ventral region of the inter­

segmental mel.llbrane bet~veen the ·8th and 9th segments. When •

the 8th and 9th segment are extruded the gland ap~ears

as a white sac or vesicle above the valves -which is res-

ponsible to produce sex pheromone. VJhen the abdominal

tip i~ in its ext ended calling position, the longitudinal

muscl'."!s are relr:~x.=>c:l. and cuticUlar oblique ones contracted

the distances between the strB~t\tions of the longitudim 1

37

and cu ti cul ar oblique muscles ext end strongly for

their st3tes of relaxation and contraction respectively •

.Uternating relaxation and contraction of longitudi-

nal and circular oblique mu~3cles are essential to.

the rhythmic protraction and retraction of the

abodominal tip during c·alling.

For the identification of the sexpheromone glands,

the external morphology of the concerned part is shown

under sc8n11ing elect·"'nmicroscope (PLATEI b). The

dorsal vievv of the intersegmental meubrane shows a

well defined a rea containg a highly convoluted

i nt egumenta ry st rlil.cture which seems to be strongly

indicative of its glandular nature ( PL A.TEIG) .

The ventral aspects of the intersegmental mernbeane

between the 8th and 9th abodominal segments is also

convoluted (PLAT13': I d). The lriteral aspects of

the membrane betlfjeen the 8th and 9th segments desc-ribe

as smooth in comparision with the topography of the

dorsal or ventral aspects. Histologically, in the

c~.:iLl.i.nt; fanal e moth, the rhythmical protraction

and retraction of the abdominal se;9U mts reveals the

presence of a modified intersegmental membrane. The

modified area is located dornally between the 8th and

9th abodominal segments (PLA'l'E II a). ' sex pheromooo

J8

See PLATE I a,b,c,d

PLATE I:( \.1. Hollsnder, 1932, J.Insect physiol, 28)

a: Lateral view of extended abdominal tip of female moth. With Sth &. 9th abdominal segments (8 \S .&, 9AS) arrl the interseg­mental membrgne (LVI) in between (scale bar 1 mm) .

b: scanning electronmicrograph of extended 3bdomin31 tip. sho.-Ji ng dorsal and ventral cuticular convolutions (arrows) of sex­pheromone gland. (Scale ba1:.. = 0·4'5 mm).

c: Scanning electron micrograph of cuti­cular convolutions (arrow) of the sex pre romone gland located on the dorsal aspect of the. intercegmental membrane between the 8th and 9th abdominal seg­ments (Scale bar= 0•5 mrn).

d: Scanning electron micrograph of cuti­cular convolutions (arro·w) of the sex pheromone gland located on the ventral

. aspect of the int ersegrnental membrane (Scale bar= 0•5 mm} •

•

PLI\TS I I : (J. _; . Percy , 197 1 , Can. f.nl.. . 103}

a: cro ~s section of 8th a bdominal s e sznent of f Amale moth showi ng dors Rl cresc ent shn p ed . S ex ph e romone gland ( g ) x 35.

b: Cross section t h rou.gh s.ex pheromone gland to s how the U sh~ped arrangement of the modified eqiderma~ cells. e, epicuticle; n, endocuti cle; c. cuticle of Sth abdo­mina l s egme.t x ~ 50.

c : Br.3nching of endocuticle in neck of epi d ermal c el ls of setr pheromone gland X 1000.

d: Goblet shape d glandul ·· r c el l . The arro w i ndica te an indi s tinct cell me.mbrc.: ne x 1300 •

•

39

See PLATE II a, b, c,d

' ..

I . · ~

. . , • f

PL . T3 II

40

glarrls th:,t have been studied in the female lepidop­

tera are located in the intersegmental membrane

between the 8th and 9th abdominal segments. Generally

in the moth, the glandular area is cr~scent shaped

( PL :\ TE II a) •

Stnined sActi :)ns of abdominal tips reveal tmt ·

the epicuticle ave:r•laying the gland is smooth and

devoid of scales, and the enQ.ocuticle is considerably

thicker th:3n tmt a overlaying · normal membrane

{PL.AT1l: II b). As in PLATE II c &. d, the modified

epidermis of the glandular intersegmental membrane

ccnsists of goblet shaped cells, but columnar and

cuboidal cells are also present. The nuclei of the

cells' are i-rregular in outline and usually contain

one nucleolus. The cytoplasm contains many tiny

vacuoles (PL~TE II c). The number of cells increases

by a U shaped arcangement and the endocuticle pro­

truders into the folds created b:;r.this arranf:;ement.

The endocuticle also penetrates the necks of the

cells wrere it gives branches further. These small

branches appear to be' closely associated with the

cell membranes of the necks {PLATE II c)".

41

The communi cation system in .the moth as

shovm schematically in Fig.4 illustrates the extent

of the p ~·es~nt und er·s'ca ndi ng of the different fun­

cL.ions involved. No atL empt will be made here to

prPsent a complete picture, but rather to emphasize

sCJme functions and to present some examples.

Some time after emergence f.rom the pupa, the vir­

gin fe.nal e moth ~""pands hey sexual attractant glands.

The perform::mc e of thi-s "calling" behavior 1 Fi·~-5 .)

depends upon a nwaber of factors. .As a rule, cglling

is only o bs~rved before copul3 tio n. In the female

of the domesticat·.?d silkmoth Bomb)%, calling is more

or le~s continuous, .vhile in mo:-t other moths s~::..uclied

C'Jl:J.ing depends upon a diel rhythm 111hich ap,•ears to

be correlated l.Vith the activity rhythm oi' the m0le

(1:"lrtell, 1963; Traynier, 1970; Shorey, 1973;

Fatzinger, 1973). In the 'ivell studied cabba ~e

lo .. per moth Trichoplusi3 ni , it was fot:.nd that the

ciuratic-n of calling and th~~ amOtlnt of attract~nt

ewi tt ed depe nd8d upon the ,>Ji nd speed ( Ka a e and

Shorey, 1972). This ph,:,.nomeno ::1 can be understoud

in relation to the distribution of tre luring odor.

Fig, 4:

Ph.R. C II Mol .

Functional scheme of the pheromone corrununication systera between a female and a male moth. Arrow indicates the di.:rection of the information flux. Small symbol s (circles, triangles, squares, pentagons) symbolize odor rnoh;cules (filled) or respective precursors (open). Open niches in the receptor cells represent the acceptors (receptor sites). Ci,iS = Centra 1 nervous sys tern; ~"~1ot(!f) = expansion rn0chanism for the lure gland; Mot (d') ::=motor system (wings and legs) for approaches to the female; Mot. PhoGl = Motor system

. to expand the · male hair pencil gland j Ph.Gl = Pheromone gland ( cfY and !;? ) ; Ph.R,C. I and II ~ 0') receptor cells for two female sex attractants; Ph.R,C. (fl.) :::: rec~Rtor Cl'~ lls for two fern ale sex attractants; P l.R .c. ( <;f. ) -= Plant odor receptor ce ll.

Fc·Jll:t l ,. i'.YI':;y Ill<> ill (./.<ftll.rJJI.t·r.r d.i::J>.tr)

\vil.il l't:p:tndc·d lurl' l',io'lll<l. i"llgt.ll or In c:1JI Ln1'. Jll>::iti()ll l'l1e [orCI-'ing ·- !0 P:n.

L~2

In some cgses (Shorey, 1973), tha f'Amale moths I

fly to the l-3J~val food plant before they start

c·l.Ling. A particul3rly striking food-pL:mt odor

depc~ndency was reported in hnther~ea ES?l:Jphanus

( Ri dai ford, 19 67; Ridcd. fo r\.i and williams., 1967) •

The f~male of t·his moth only called in the presence

of the odor of the preferred food plant; oak. The

compound 1.., t-2-hexenal, v~hich is found in most

greRn leves. Pure hexenal brings about the S3me

e.i:'.':'ect .as oak leaves, while other leaves- il). spite

of the fact that they contain hexenal - clo not

induce c2lling. 'l'he odor signal is perc~i'f' 0 d vvith

"Jntc:mr1Jl r,·.,c ~pto es. With regard to the failure

01 leaves such as birch to elicit calling, one is led

to 8S sume th-, t le·w es of plr.Jnt s other than oak con-

tain, in addition to the omnipresent h 8..X enal,

some other compounci s .YVhich prevent calling. The ex-

periments oi' Riddiford and Williams ( 1971) h;'ve

enabled us to advance a t"ontative func1:ional scheme

011 thE> basis of e:xp(~rimr-;nts vvith AntheraerJ an<1 other

ac~.-ivate neurosecretory br.::..in cells, the Tr.:ons ol"

which run to the corpora cardia ca, wb ere t h0ir

odor rt>ceptors on an\(L"nna

brain neurosecrel. cells

~- cardiaca

C. alia\ a

ganglion motoneur..ons

lure gland rtttracled

and el'lponded

Functional scheme of the olfactory, nourohormonal, hormonal, andneural control of the calling behavior of the female giant silk moth, ~-~-~~ uolyphemt22..

43

neuros~·cretion subsequently activates. local hormone

cel.ls. The latter prodi.J.ce a '•calling hormone" ~"hich

circulc;tes in the' hemolymph and is the prerequistite

for expansion oi' the lure gland• This gland is

shifted outw:::Jrd by hemolymph pre8sure as the result

of n1nsrular contractio sin the:; 8bdo111Prl, and can

be r"-'trnct,ed by a retractor mus~le. Since hormonal

effects are ~lov~ and lasting, quick expansion and

rstr8ction of the glands arP presumably control] ed

·through neural pathvvays.

'I'he glt3nds which produce the female attrsctant

pheromones aY'e composed of spPcialized intersegmental

2pidermis cells vJith deeply fluted distal cell

menbr3nes, underlying a cuticle with some pores

( S':..einbrecht 1 1964; Jefferson and Rubin, 1973). In

'I'he gypsy moth Lymant ria dispa r for instance the

attnctant is ;:m epoxide (disparlure) which app;,rently

synthesized from its olefinic }!recursor (bierl et al.,

1972; Garde et al., 1973; K~sang (.,tal., 1971~). ln

tbi:.~ ~:pr,ci c-s, as well as in !3om()yx, whrere the 1-1n~curso r

of the ~~lcoholi c pheromone mit;ht be a l'att,y acid,

the ';vei:;ht ratio of prec:n.·::;or; ~Jheromone j s tD the

order of. 1 () : 1 • 'l'ransforYr1E.ticn of the p1.·ecur::~or

44

to the final produc"c mit;ht t'-3ke place in the cell

or even in the cuticle. The pheromone '-lu3nt.ity per

gbnd (Li1e so C!:lllecl female equivalent) is of the

order· of 1 yg ( apiJroximately 101 5 molecules) in

~.orne spec:.i.es (Steinbr~cht, 1964b; Shorey and Gaston,

1q6r;;), b·.1t l'::s:c: in L-,he majority of cases. The-number ;

of pheromone mol~cul es emanating fr.~)m the glan:i sur-

fc-1c e ~as e ::;tim:::t eel in J3om_Q.y_JS_ to be of the order of

10 1 1 molecules/sec, corresponding to a pheromone ,g .·

molecule density of 10 /cm3 in a moderate airstream

paG8ing over·the female gland (Kaissling and Friesner,

1970; Pr:i.8sner, 1973).

The trGnsfer m ... dium is air, \vhich carci.es the

phr.~l~omone to the recc·ptors of th~; male moth anten·ta.

Repo1~t~~ on e:i'l'ective attr;:Jction dist.'·mc es vary from

:;;everal Kilometers to a fe~v llleter (Pri esneri' 1973),

dependtng on the pheromone output rate, the physicgl

trans fer· condition, and eventually on the male moth's

behavior threshold, .. .,.hich in .Bombvx was found to be

rP::JChed at 103 bombykol molecules/cm3 of air

(Kaissling and Friesner, 1970).

On the r('C ei vi ng side, the signal mol ec 11les are

fi.rst adsorbed on the cuticle of the r~ceptor hair

and then migr::rte by surface diffusion through pores

45

::~nd tubules to the membr:>ne of the recentor cell

d.~ncirite, ,Jh,~re they interact with postulated acceptors

(.receptor.s). While the surface· diffusio::-1 and penet-

- tati0n of the cuticle by the pheromone is well documen.:.

.ted (K3s8ns, 1971, 19T3; ~asang and Kaissling, 1972;

.Steinbre.cht and K'lsang, 1972) •; Pheromone molecules

h::1ve for technical reasons not yet been seen in

fl.3 .. ~ranti on th2 dendritic membrane or in the tubules

le3ding to the membrane. The result··of this so •

far postul~3t ed signal-acceptor interaction can be

seen as a depolarization of the dendritic membra~1e '

(the receptor potential of a single cell, or, ih

8n ovP.rall r~::cording, the electtroantennogr.3rn, EAG),

l eadin,; to the elicita-cion of nerve impulses. The

amplitude of the r'?Ceptor potential and consequently I

th2 frequency of the impuls3S are an e;{ponentiu.l

function of the stimulus strc~ngth. In Bomby~ (Kais::;ling

and Priesner, 1970) and presumably in other moths

al·::;o, sir11~l e pheromone molecules elicit ~Jingle impulses

but, the .behAvior threshold of the mGle insect is not

r"'1Chej b"'.'fore seversl hundred cells are ;:;ctivated.

The· nerve :fibers (axons) run ~vithout branching throuGh

106?) where they synaptically cont~~ct seccnd817

46

olf-:1ctory cPlls. As io. the vertebr3tes, the to1~r1l

number or s·-3condary olfactory celLs -in insects is

less than the number of receptor cells(Pareto,

1Q72; Bosckh, 197l,).

The number of olf8ctory r(~cepto.r cells in each

~mt enna of the ~9.m~yx _male is B bout 50,000 of 1·1hich

mor~ th8n 30,000 are specialized for the female

pheromone (Schneider and KRissling, 1957; Schneider

and Steinbrecht, 1965; K,:Jissling. and Friesner, 1970;

3teinbrecht, 1970, 1973); j_n some ~vild silkmoths,

these figures are more '.:.han tvm fold higher (3oeckh

·~t al., 1960). While the specificity of the pheromone

receptor eel" s itt the males of B~b)?.C_ and ~anJ~.J.:L~

is quite advanced, and a very little of the specificity

o1 the oth2r odor r·~C<j~Jtor cells. The=e cel.1s which

do not respond to th2 phe1~omone have b..:;en found in

3n earli:""r study ·i:,o be present in rnther 1:-.Jr::;e

numbers of physiological cell types with dif.r. erent,

but ov,~rluppin6, l~espon oe spectr8 for general odors

(,:)chneider .c.:t al., 1964). HerP. it r1-:main3 to be seen

wheth-~r the~3e cells respond to as yet unkno•·-ln odorc.mts

l"ihi ch are biologically sL;;nifi cant.

Odor molc~cules must for a number of r<::a3ons

inter3ct dir8ctly with the proposed acc,~ptors. The

47

ceL:_'s (or· cell type.'s) specificity is defined by the

v<holc set of comrounds to which the cell responds,

3ni neces~~rily 3lso.by the respective stimulus

r"-'2 1Jonse char::Jcteris.tics (Kais<::ling, 1971). Since

sorEe odor r~=~ceptor C\?-lls seem to have more tlnn one

tyu~:: of '~cc-·~:_.;~or in th::ir mn11br·.ne, th2 r(~spective

sp::~dl'icity of on!'; acceptor is only part of the cell's

specificity (K.<:Jfka et al._, 1973). Interaction probably

i:3 by wc·ak physiC8l, (polar and dispersion) forces

(Kafka, 1970).

The specificity of the o<;ior receptors gener3ll y

i'S rr~ther high bec.suse the effectiveness of the "wrong''

geometrical isomor is in some cases only 1/10()

1/1000 of that of the pheromone, and even chir3lly

different molecules elicit different cell responses

in the receptor cell and can be distinguished behavio­

r.L'._y (l\gfka et.al., 1973; Riley et al., 1974). iVletabc

lie processes which chr:~n.::..e the odor molecule in

JntP.nn;:.Jl tis2.ue are probably not ,fast enough to be

either directly rel nt ed to the transduction or to

irru,lecliate inactiv.Jtion of the~ odor moL;c~le (;<.as<'~ng,

1971, 1973; F'erkovich et al., 1973).

48

The even::.u::1l outcome of g)_l these processes in

the pht>romone receptor cells, which send t·hPir messages

Lo th~ br~in vi8 their axons in the nerves of both

8nten''JSe, is '3 motor reaction of the male insect.

The domesticgted and flightless nl'ale silkmoth Bombyx,

when stimul·,ted with the .female sexual attractant,

.ficst lifts its anten iae and then start::, marchj_ng

upwir1d, vvhile vibrating its anten:-"ae and fluttering

it:::; .vi~c';f, (Schvvincl<, 1954., 1955). Wild moths only

re::Jct if they are stimulated at cert3in hours of the

d::-Jy; they then lift their antenuae like~ and

:,oono.r o c l3t er fly upwind in the a erial pheromone

tr:::il or odor plume. If the¥ lose it, they may start

' . .mdirected circling, searching flights, as do other

insect::: (Steiner, 1953). Systematic studies of all

factors involved in the final appros ch of the male to

the female in the field h8ve not bec:n conducted.

Since odor alons does not t;ive the anim:::~l a directional

clue, it presumably uses its optomotor system to

~teer upwind (Kennedy ::md I·JlJr·sh, 1974). Bui.", wingless

moths ar·-' able to follow a terrestri-al ph~~romone

trial like ants and termites ( Shor!~Y :md Farka3, 1973). 1t.

Theoretic3l consid•3ra~vions of the composition of

f3Uch an odor· plume l e8d to unexpected prf::dictions,

4-9

.1t 1P8st in an ideali?;Pd case (Wright. 195t'i; Boss~rt

1nd l.Jil.:o n, 1963; ~\Tilson and Bossert, 1963). The

gr·;di ent of uecrr-;asing odor concentration is very

steep near the odor source when there is a reasonable·

air flovv speed. Next to the source the air may contain

up to 1 Od ph:c< romone molecules/ cm3, while at a few to

30 meters distance (as a rr-:sult of air turbulence) the

cone ent, r"''ti.') n rangr-; is 1 o3 -104 molecules/ cm3. The

odor plume then extends with little reduction of

concentr3tio11. (dependj_ng upon the rate of air flow

:::riJ. d.i..LJ.tic)n, sower et al., 1973) to more than 1QO

meters .. Effective luring 8t longer distances is

only possible if '~i ther the source is more effective

than is knov,rn at present, or the pheromone catching

rr-1t e of the m.~Jl (?. ant en<~a i :' better than in Bombyx.

The s'2Ylsitivity of the attractant receptor cell

reaches its theor(~tic3l physical limitis· and the

:.112u l~al noi s espontaneous firing of the odor receptor

cel.Ls - (Ka~ss.ling and Priesner, 1970), 0.1 impulses/

nee., is r3ther low for such a sensitive system.

However, theoretically, a reduction of this spontaneous

activity by a factor of ten vwuld allow th2 r(~c.::p-:.:.or

system to r2spond to a greater dilution range o.f

the pheromone ::Jn:J al]_od the nBle to api;roach from

50

In th(! lic:;ht of this theoretical background, it

i interesting to nots> th~?t Trich<?_P.lus~~ ni females

rel3te their pheromone-rdr;ssing behavior to the

wind velocity (Kaae and Shorey, 1972).. A"'v lovv wind

speed, ·111hen the odor i~3 persistent and dilution is

low, ~:.he f enwl es call for longer p(~ riod s. In adui tion

most fem8les vibrate their wings (fan"'ning) 'v'!hen

c::LI ing 8t low wind spe::ds and do not at high wind

spe ''OS.

ri' his com pl et es the story with respect to c h emi c gl

info rmstio n transfer from. the female to the male which

eventu~lly mates with her. The message is biologically

hi;;;hly meaningful; the male moth underst':lnds the

11 1 a ngu 3 g e 11 o f hi :3 rna t e •

While thi.s flmc'donal cycle seems in some moths

tc be completed with the male's approach and inst:YQ ___ 't

m:tin:; :::~fter he r:3.sches the fem3le, some groups of

moths do have male pheromones, ·.tJhich are apparently

desi,gned for short distance effects. In fact, male

scent scales anJ scent brushes or hair pencils

(androconia and coremata) have been described in

m3ny lepidoptera. Many male Noctuids, Sphingids and

Arctiids h:~ve lar:~e hai r_pencils which they can

Pxp:~ nd for the dis serninatio n of an odor.

51

2 ·5· Beh<wio cal Rr;sponse

The functions of the olfactory organs can be

:,~L ;~ '·~d j nr.lir~~ctJ.y through the observ::.1tions of

b<2ha vio ral responses of many moths species.

In sim~les~ case, an application of a

:ccAnt-sLirnulur-5 rr~lA3Ses a rAflex-li.ke response.

For example: l'~lale moth responds to the sexual . '

3ttract8nts or sex pheromones by showing wing vib-

rations and ant ennal elvations and abdominal move-

r.i2nts. Thc-y cannot fly even and start walking to

the source. Such type of response i;.:. called walking

up wind ('tve anemot8xis) . . This response has been

reported in Bo~x ~g_ri ( Schwinck, 19 54, 1955), in

Por~h_Q..t_ri~ dispa.x (Ivlarsh et al.,· 1978) and in

Gra~h_olit~ molesta (Kuenen and Baker, 1982).

When strong stimulation comes from the source,

m:1le moths sho,, a spiral or circling pattern of

the body with movements of the abdomen. This

pattern has been found in ~ombJ!?C mori (Sch,Jinck,

1 q 54-, 19 55) , in T ri c_£!o...£_~ ~~i:3 ni (Hen~ eb e rry and

HovJl:md, 1966) in Diory~triaab:!:_~!-_~ll9_ (Fatzinger

and AshPr, 1970, 1971 ) , 0 stinia nubilalis ( Klun and

no bi n~"i:' n, 1970) and in ~h e~r::h.~ di~J2J!£ (Marsh

!ct al.' 1973).

52

The IIIJings flutt ~ring or vibrations ccm be

m,:;asure 1 mechanically and can be used for determi­

W:lt:Lorl of the m8l es olfactorjr threshold when

stintulatc-;d \IIJith sex pheromones or other chemical

compounds (Schneider et al., 1967; Kaissli ng and

p;·iesnerl1970; priesner, 1979b; and RoelCifs, 1979).

La.boratory experiments usuC:Jlly allovJ a beti~er

yualifi cation of the odor stimulus. Male moths

arc often made to select between the odor free

and odor containing arms of "Y" ma~e or "Y" tube

olfactometer. hey and mills ( 1968); J.Vu1ller ( 1968);

Toh"-J et 3l, ( 1963); Brady ( 1969) and Rejesus ani

R'qnolds ( 1070) succeded to measure the attracti-

.._,. en e? s and repellent response of male moths and

properties of chemical c·:)mpounds in thr:ir olfactometer

studies.

The olfactory organs play two major role during

male moth's orientati.:-m to an odor source. First

is to detect the presence of odor and 2nd is the

quality and intensity of an odor.

Most works h:::1ve been d·esi ._ned to me~.l sure or

yo evaluate the levc:ls of responsivene:·s oi' males.

(Trayni•~r, 1968; Bartell and shorey, 196g; Brady

and Finery, 1969; Fatzinger, 1970; Klun and

53

Robinson,.1970; T~kahashi and Kitamura, 1970; Brady

:1ru TunilinfOn 1971; Fatzinger and Asher~ 1971;

Dater.11an, 1972). Following are the behavioral steps

depend upon the use of labortary techni4ues that

l~>·~~ay r1 p::lJ~tic~~lar type of respont>e.

1 • ~.r:z J?..b.~-r:~~~ix!.c}:.':!:.~~-ci_§_~y uen\~e of behavior

!\C:.:onll.ng to Tr<:lynier ( 196S) observ3tions with

the males of Anagasta kuhniella were released in a

wind tunnel, downwind of the calling females

fehv upvvind. If they lost the sex pheromone scent,

m:jde the cross wind and located the female by

p? rc piving the scent.

Takaha ;:;hi and Kutamure ( 1970) r:::.port ed that

the sex phenomone rele3sed by the female Bomb~

~<2_ri, indue ed the male for matine; purpose's.

r~l8le shovv~"d the irrmedir-Jte response tovvards

virgin fem3le and nttr~ct::;d to the source of

pheromone. Olfactometer studies of Rejesus and

f\.Pynolds (1970) showed that the male of Bu~_C'_!-~,'"Jt~ix

~~~_r.::_l~~!::.~fJ..:.:.~~ responded to the C'"lling ferrrJ. e

shovving the typical performance of wint; vibration,

ant ennol movement, but 100% att ra ctio n s vJ er:; not

tound though the virgiu females were in c0lJing state.

fAmales vverr:~ reported by Fatiznger ( 1970) and.

FstiznLel' and Asher ( 1971 .) in the olfactometer

study by pa ~sing air through a chamber of virgin

females into a chamber containing males. Enit'cing

,'herornone:~ ~vere rPspon:::;j_ble to behavior8l activities

of t,he m:.Jles. Attr3ction studies experiments were

concmcteci by Bsrtell and shorey ( 1969) on male and

' female of :92_i_p_hy_as Postvitan~ using straight ,glass

'cube· Olfactometer Li the laboratory. ,Male showed

irrrnediate respon<c2 to the scent emitting from female

and stR:rt ed orientating to the source of phe.romon e.

GOtz ( 1930) studied the choice experi ent 011 Labesia

bo rt1n8 males. In the tests, males wer·e released --·-----freely and Given a choice between 1 and 3 virgin

f·9males. Mo,~t attraction of males w::1s found tow:Jrds

the scr~nt source, 8!lln::.ting fr)m•3 females. Qw1ntit8tively

the pheromone3 from 3 females were more attractive

to male~:; than one. Klun and Robinson (1970) succed.ed

to see the attr:=Jcti 111 respon:.:;e of male moth of

started from the virgin female, the males got excited

and started orienting to the source of pheromone.

Rem<JrkGble studies were carried out by schwnick ( 1953),

55

Brady and Smith ( 1968), Brady and Finery (1969) and

Br~.dy and Tumlinson; '( 1971) on pheromone indue ed

behaviour in male Pl<2_~i:'.! inte q~u:r:!.:t ~l_la. lVlale was

'·lui ,,e e:xcit ed by the scent of virgin .t'em8l e. 70 perc:-;nt

an.:i above attr·;ction:,; wer·r,; observed.

The fir~.t rPcogni7able step in the seyuence c:f

re·wons e is the activation of a resting male such

activity is represented by elevation of the antennae,

grooming of the antennae and wing vibration. \'Then

a resting male perceives pheromone at a level above

its bc;h':i·Vioral thrt~shold it is stimulated to '<valk

or fly. This study is being supported by Baker

et al.~(1976),&Flint et al., (1977).

I). Orientative beh;::;vior - --- ---------------·-Orient8tive behSJvior rPsult:-:; iD the male

locating the pheromone ~-;outce: the conspectific

femole. Uf cour·se, the analy:-::is of insect orien-

tative beh8vior is not rt~stricted to ori.e~·1tation

to pheromones, nor even to other chemical cu-es. A

V3St library of cherni c<~l kno\~l edge is now available.

It should serve as a verstila tool for the st,udy

'"~nd the manipulation of orientative behrwiot~. Two

56

}lrincip3l m•"'Ch-1ni:;ms have:; be m aug .•·sted whereby

f lying insects could orientate to pheromone source

cheiHotaxis (chemical st ree ring) ane;Hbtoxis (·wind

~-~ t ~ r: ri n g ) .

Farkas and shorr-;y ( 1972) h?.ve r2 por·Gecl ti1at

ms l es o f th2 ~_@.c_t)_r.!2.P.l:!~.rr:!. g_os·~2i.§.~_l~ i s stillair

rr-~m3ined ca pable of orienting along ~an o dor plume

t:.h?t the ins ects wer e ca pable of gAini ng chemo­

tact ic .orientatio:1 cues from th e st?tionary plume

along which thtO} moths . progressed in the former

upvvind di r ection . Kennedy and March ( 1974), hovJever,

chgll.enged this vievv in their experiments _with Pllodia

int ec.2_~l'!_c_·~_ella th-'3t the insects maintain ed up~r;1nJ

orienta tidn by mPanG of anemota Yis, r egul8ted by

optomotor respons es to the visugl surr'Ounding and

m0dul.1t "' d by the phe r' ·fllOn e flowing do wnvdnd. They

concluded' th!'lt althouJ;h the me ch:::nism ofcxior r t:gu­

lsted 01j tornotor anernotaxis rerroins lart;ely unanaly -

. ~ed it is still · the mo s t r.>lausibl~ gu.idance rn:c.hr..nism

for thernoths studied so far. Farkas et :::~1 · (197~. )

lo0k0d more closely at cnvironm .:~ntal factors ~Ji'J'P.ctin~

the aeri::1l odor trial follo;,ving of mal-:=s of E_2ct_i_nophora

gg_~~YJ)iolla. 'fhey found that the behavior or" the

57

males was regulated by the concentration (at source)

of the pheromone, t·he spatial disposition and intensity

of an illumina Ling light source and visual cues

from nearby objPcts. Increa-·ing pheromone

concentrati'Jn tended to reduce the forward air

spr~ -:::d of the rr~spond.in,; m~:lle, both 8lon[; th"'? zig,.,ag

flight path and the axis of the trail. Th-:: reduction

in speed occurs in part, as a result of r-~duction

in wingbeat frequensy and represents a nagative

ortho kin r::si s.

(Oef: speed or feyuency of locomotion is

dependent on the intensity of Stimulat:..on visual

cur.;s and the concentrations of the pheromone appe~~r

to 1,vork jointly in stimulating the slowin;_; of flight

and. landing) •

4. !IE_~~~l'!d __ a_n_e:!!!_o_~~~-xis: The ,dnd flow is a suL .. icient

magnitude for a moth to detect i-cs direction, for

locating a chemical source in such wind. such

orienteition i.s called upwind an·111otaxis. This process

h"-Js been studied in vdnd tunnels in several.moth

species ( Trn yni er, 1C! 68; F'a rka s ;-md sho r',Y, 1072;

K8rk::Js et al., 1974; Ke.Jt1edy and l1ilarsh_,1074; Kennedy

et :::Jl·)19f~0; 1nt31, Marsh et Al., 1978 Ca1.~d~ and

.58

Hug"lm:;n, 1 q7q; Sanders et r1l . , 1981 ; Baker and

Ku~nen, 198~-2; Kuenen and Baker, 1982).

In mo::;t of these ex~mples the flight trsck

::3lon,;; the ;::J:Xis of the pheromone plume has been

charceterized as 8 zig:;>;ag, along an upwind tra-

j~ctory. Th"-" expl3nations of kennedy and Marsh

( 107h) for the zigzag behaviour was that. the

' successive tu.ms were initiated after the loss of

the scent, the turn carried the moth back towards

the direction where the scent was lost sensed. (the

direction to\·Jards the source, however, would be

detected by the optomotor response). Complimentary

evidence that such turns are initiated by the loss

of the plume come from Traynier' s ( 19 68) wirrl '

tun:1el observations with the moth Ana gust§_ KUh't'li ella

in homogeneous pheromone clouds; moths were reputed

to fly upwind in a straight path. Tests designed

to compare the flight track of males in narrow

discrete plumes V.S. homogeneous clouds of phero­

mones in Adox~2he£ orana (Kennedy et al., 1980,

1981)' unlike Tray'nier' s ( 1968) observations W:.'ls

shovm that zigzag path occured under both conditions

in contradiction to the mechanism proposed·by \

I

59

~ennedy and M<Jrsh ( 1974). Lateral revers~1s, tlH.m,

seem to occur in the presence of windbor:Q.e pheromone

and thus represent on internally generated pattern

0 f fl~ght.

The wide lateral excursion characteristic of the

zigzag subject an air borne moth to increase aggular

deflections of path rdative to the upwind direction

over the deflectiun of track that would be. incun~ed

when the heading is aimed continuaLLy upwind.

A zigzag flight track offers an alternating

series of comparisons of deflection and elevated

th~"' navigational inform ?t tion available to the

insect. The upwind could be set by adju:::ting the

heading betiNeen reversals to msi ntain a flo vJ of the

ground pattern that ::Jltern:=:ttes on the right and

left l~?gs of the zigza.s at the s:1me drift angle

between the body aixs (course hegdin!;) anci track.

In the gypsy moth, the position of the moth .1s

it fliPs along the zi~zag is such that tbe moth's

body axif, is ::Jli,~nr~d with the coursr~ he::)J.ing

(body axis). The ad·vantat;e of this str:1t·~t-~ern, .<:Js

coutrasted with mr-Jintaining <.m upwind body axis

heading along the zigzag course, be rela"Ged to an

improvement in flight efficiency.

/

6o

The visu;l effects generAted in .both situatipns

are similar im>:::rms of the amount of angular deflec­

tion, althou:):1 th_e path of the visual field across

the eyes >llould be quite dil'l'(::rent. If the insectbody

is aligned vvith the course direction, although

the psth of the visual field across the eyes 1vould be

quite different. If·th' Irlsect,body i:. ali.....;n---d ,.,:i.th

the COUrse di rP.Ction, the fl01tJ Of the Visual field

beneath the eyes, alternates at an angle relative

to the head-body axis that is ~qual to the drift·

angle (Marsh et al., 1978).

Another explanation for the zigzag 'path J the

moth WAs allowed to fly up in plume by sequential

sampling of the pheromone plume's spatial structure,

~vben the· vvind drop1Jed below the anemotactic threshold.

Another consideration W8S thRt· a zig7,ag pClth

(noting both the lateral and thP. vertical cotnponents)

increased t be pro ba bili ty rc co uta cti ng chemic a l

stimulus after loss of the scent (Kennedy, 19?8;

David et al., 1982).

Tbe effecls of increase~ in pheromone concc~nt.­

rntion upon the zig zag flight of Grc:pholi ta

ffiolesta were to increase.

61'

1) 'rhe frequ~ncy in turns

2) Decrease the airspeed (and therefore the

distance across the plwp.e's axis between

turns). The an~les between turns, however,

rewain relatively constant ( Kuenen and

B:::tkP.r, 1982). rrhP resultant effect is to

provide a. narrower zigzag path and slow the

speed of progress up the plume as the moth

approaches the pheromone source.

The; pr::>cision and success of Grapholi ta mol esta

o ri ent&tion to pheromone was quite dependent on

a narrow range of optimal. pheromone co nc ent ration

{C8rde et al., 10?5; Baker and Garde 1979; Baker

3nd Roelofs, 1981) and this fact of their behavior

differed from most other moths Lymantria dispar:_ which

oriented to a ·rather bro::1d range of stimulus

concentr8tions.

As pheromcne concent ratir_,n increased the

di stance t r.'3vell ed ':m the directly upvvi nd cornpon ent

o1' the flit_:;ht track on each zigzag decr2ased as did

the ground spe t:~d. In contrast of Gr~1 El~o_J}_t_a_ u;_o_l_~_~t-~

j_n the :!:oY 1Jsy n1oth, t..he ir1terle~ angles narro~~ sligh~.-ly

at increased phecomon~~ levels· and the distance across

the plume bet"1een the turns rf'.rn-.ined relatively constant.

When the plumes removed sud,lenly, the gypsy moths

were allowed to cast into a wide homegAneous cloud of

pheromone, the zigza.;::;ing upwind flight resumed, but

with some alternations: the width of the zigza.::;; p3th

v~ 8 s ap:Jreciably increased as the velocity along each

leg, compared to a na rr'Ow pheromone plum e. These trends

in fli;ht behsvior in plume Vs. Cloud remained simi.lar

over rou -:.:~hly 100-i'old pheromonf~ ranges and so seem

at-::.ribJ.t3ble to the spatial structu ,··~ of the chemical

sticomlus rather than pheromone flux.

These observations compliment those with

Adoxop}~es oran~- ( Ken_:v=:dy et al., 1981) in that zigzagging

was an internal J.y ,gener:~ted beh8viour (pre-programned),

but in ad_~_ ition, they shovved that the zig7.8g path

of Lyma~~r:.~'! di spar was influenced by the external.

information about the spatial structure of the

pheromone stimulus.

Tr::qnier ( 1968) reported that the pyralid moth

Anag8~t8_j<·uh~:b_el~'! in a cloud of pheromone, flied

directly up~vind in straight (or at least not zigzag;)

path over A m,_,ter distance.

2.6. S8x Pheromone s Production and Release A.g.~=q):_the_[_~ma-le motiiaruf its Effect­

on the I.Vlale moth ---·--·------·-

l'~lany insect groups produce sex pheromones and

emit than during their entire life span begining at

the time of emergence. I~1any others do riot attain

sexu.ql maturity until they reach at a certain age

or sometimes natural death occurs due to a cease

of pheromone production. Girnila rly t~e· responding

sex may or may not be sexually mature of emergence.

r'emal t~ nun moths Porthetria monacha were most . -------

attr.:~ctive vJhen 1-2 days old ( Jascentkovaski 1932).

Hanno ( 1939) and tun bros ( 19 40) claimed that newly

emerged f emales are only weakly attractive, the

att.raction becomes stron~er in female 1-2 days old;

8t0raction almost disappears af~er mating and com-

pl Pt ely gone by oviposition. The female continues

to attrsct males for about 12 days ( Hanno 1939).

Richards and Tl1ornson ( 1932) sho~~ed that females

or' Pl0_'l.ia interp1!_n.~~ella_ are ready for f!lating ~

very shor c ti!lle after em c~ rt!;enc e. L ehmensi ck and

Liebers ( 1938) showed that females are not -attractive

until they are at least 3 hour.s' old, and male do

not respond until they are at least 50 minutes old.

64

One day old males placed in a dish in which a 1 day

old female was kept, imrni ediately beg:-1n a court ship

dane e and attempted to copulate. ..U though 5 days

old feltlaJes wen! ~ot attracttve, a we·,k male re..-;-

ponse was evoked by 1 5 days old females. According

to Brady and Smithwick (196:1) newly emerged femr?les

contained a concentration of attraction, equal to

5 days old adults. Virgin female released attractant

almost continuosuly during their adult life.

Vohringer ( 1934) reported that Cfall~ria m2llon.::;lla

begins to -relea~3e attractant 12 hours old after

emergence, copul:1tion was observed ~etween 1i·-2

hou1~s old males anl 12 hours old females. Roller

et al.' ( 19 63) showed th8b the males develop a

ch·1 r<1ct erstic odor when the<J are 2-24 hours old;

this odor researches a maximum during the evenlng

hours.

Labor:::tory experirner1ts showed thul:J a 2 d:1y

old female par.slobesi:3 viteana imnediat.ely exceited, -- ----·-- ----when ::3 rne1l e w~s brou[:';ht to her ( GOtz 193q). One

d'1Y old females of Lobesi8 botrgna ami Paralob,o;!"'ia ---·-- ---- . .........,...---- ---!_i_t_~~E.~ are mdlre atl,ractive to males th:::m are

2 day old females although virgin females rPifl3in

attractive for their normal life span (9-13} days

o 1 d ( GOt z , 1 939 ) •

65

M8kino et gl (1956) and Jones&. Jacabson (1964) reported

that male and female g,omb~ mori do not fly but. eager to mate

immedi3tely after emergence. Female ·remains attractive in

the last days of its pupal stage. Perez & Long ( 1964) reported

that females of Diatraea ~~s_haralis begin to emit their

attr:3 ctant soon after emergence and are most attractive

during the 1st 3 days o~ life then attractiveness decrease·

with agee Most Zeadiatrea grandiosell.a. females began to

attract males on the night of emergence. Attractancy drop-

ped sharply after the4th day and was completely absent in

8 d8y old females (Davis & Henderson, 1967). George &

Ho'nerd (1968) found out that virgin female of Grapholitha . molesta increased in attraactiveness with the age atleast

until their 5th day of 'life and Grapholitha funebrana

female shov~ed the great est degree when 1-3 days old

(Saringer et al., 1968). The sensitivity of male almond

moth Cadra £3_UtelJ.a to the female sex pheromone increases

gradu:=illy with age upto the 7th dRy after emergence.

Maximum s ensi ti vtty occurs at 3 days· of age. The phero­

mone is already detectable in female which have begun

eclosion: it increases in amount upto a maximum of 3 days

after emergence and maintains this 1 evel until death if it

does not mate (Kuwaharct et al., 1968).

2:1. Structur~~--'?__~t_iv_~t_Y. __ r!~_l_<?~G-~Q_~sh~J2..s in SE?~ --- -- -?tt::_r:_c:.~_c_~_::J_'Q_t _ _R_h_erorr~~nes

66

i3ec su.s e of the relat i v.e sirnplici ty of thE! chemical

::;~ructt.lT·~; of fP-male sex-att~r~-:1ctant pheromones; a

consider8b1e number of pheromone analogues have br:~en

synthesiz"'d and their biologic activity evalua~ed

1vith var·L.JUS species. Generally any slight changes·

in the chemical structure of pheromones ro2sult in

.1 pronounc~· .. l r•::>du.c:t"i.on in activity. The ap,•rent

biologic activity of pheromone analogues differs

'1ccocJ.in~:; to which assay method is used. The follovJing

.tn•''tho; s :·.in:;ly or in combin~tion de·Germin~ biolo;:;ic

ac'.::.lvity: Field tests for msle attrsction.

Laboratory .tests for sexual ·excitement or

short-range orientation behaviour and EAG responses.

Out of thes"~ 3 methods, the highest specificity in

structure activity relationships is usually obs2rved

by field tests and lovvest by EAG response as sho\vn

in Tri~h~lu~~~ ni. (Gaston et al., 1971) and the

a no. the gypsy moth (Adler et al·, 1972) •

The low specj_ficity of the EAG m<S.thoq for

detennining struct-.J.re-activity relationships is shown

with doubly unsat.ur.:.::ted sex pheromones and their

::malogi.les. For Ex: In the case o~ the sex

phPromone of the almond moth Cadra cauJella Cis-9 1

t.r8ns 12-Getr<-)decadienyl acetate, there were strong

E\G responses with the monounsaturated tetradecenyl

.'3cP.tAtes having the cis-9 or the trans-12 double

bond (Roelofs and Comeau, 1971). The lovv specificity

in the EAG mothod has been successfully utilized for

the structur:"3l elucidation of the sex pheromone of

the codJ_ing moth, trans-8 trans-10 dodecadien-1-0J I

( Ho el o f s et a 1 . , 19 71 ) •

Laboratory assays on a considerable number of

pheromone analogues revealed that some of these

synthetic compounds elicit s exuF.Jl behaviour from

m3le moths such ~s the cabbage looper moth

(,Tacobson et al., 1968; Toba et al., 1968 and

Gaston et al., 1971) , the southern army wo nn moth

(Toba et al., 1968), the codling moth (Butt et al.,

1968); the gypsy moth (Sarmiento et al., 1972) and

thc-' almond moth ( Takaha s:hi et al . , 1971) • lVlo st of

the compounds found to be active in laboratory

bioassays, hovJever, were in active in the field for

at t, ra ctio n of males.

Fi-"'ld tes~s v.Jith various pheromone ,analogues

show that a minor structural change of a pheromone

resi;lts in a · considr.>rable d;>crerlse in attractive

pot en.(; y • /

6g

The first electophysiological recordings from

olfactory receptor cells in the male of Bombyx mori

were performed by schn.eider .( 19 57) who obtained a

slovJ pot entinl!:-'J, g;rsd.pd. potentir:1ls anc1 !"'.Urnu:atc~u

receptor potentials or electroantenogram (EAG).

In 1962 , Bo eckh reported the 1st quantitative

recordin;;s from single olL'lctory CE.:lls (Sensilla

basiconica) in the corrion beetle Necrophorus

vespilloides .Tungsten micro-electrode with a tip

diameter of about 1-10 JU (different electrode) v~AS

inserted in the cuticle in the vacinity of one of

the olfactory sensilJa. .A 2nd broad~.r,. electrode

(Indifferen:t electrode) w~s inserted into the antenrtal

h-;emolymph. 1/Jith excitatory stimuli, the different

electrode usually b"'came-vely charged by the receptor

potenti:Jls. The nerve impulses, hov.Jever, usually

Rt<'earerl. 8!:'. thP. deflections. This tP.chniciue turned

out Lo be well-suited for recordings from sent~illa

of short hairs of coeloconic and placoc..aic type in

several species. Mor'" suitable technique for record-

ins from single cells ( Sensilla t ri chodea) of long

hair was cut off and. a glass capillary micro-electrode

was brought into contact with the sensory cell by

covering the cut end of the hair with the electrode

tip. In that manner, it was possible to record impul-

ses and the slovv receptor potential of the in!lervating

cells. Recording from a single, sensillum, impulses

from one or more cells may be obtained. In the later

cases, the spikes of varioUs cells are distrint;:;uished

by difference in amplitudes. The stim_ulation techniq_ues

chosen for studies of insect olfaction have to be

considered \~ith respect to the small quantities of

phsromone compounds that are sometimes available.

One technique employed i:!'"l stu?ies of lepidopteran

pheromone, is the use of cartridges (Kaissling and

Pr.iesner, 1970). A specific amount of a compound: is

applied on filter paper inside a glass tube (cartridge)

through which a constant air stream (Puff) is

blown over the antenna. A stream of pure air is

blovm over the antenna between each stimulaticn:n

pP.riod. In other techniques, the odor is introduced

as a puff into an air stream, contineously blo,>ing

over the antenna. (Roelofs and Comeau, 1971).

2 .~.1: Responses of Olfacto :ry Receptor cell to general odors

The receptor cells are either activated, inhibited

or 'unaffected Vl'hen an odor :is blown over the antennae.

I

70

When activated a negative slow deflection is recorded

between the receptor lymph of a sincle s ensillum and

the 11aemolywph (Kaissling and Thorson, 1980). '1'his

. defl~:~ction· is assumed to be the summated receptor

potenti<Jl of the innervated olfactory cells of a

particular sensillum. The sum of deflections from

all. olfactory s ensilla is as swned to constitute the

EAG recol~ded with relatively large diameter glass

C"'Jpillary electrod<>s. When recording from a single

sensili.um with glass capillary micro-electrodes,

dy.ring stimulation \'lith a. low pheromone concentr.3tion,

small elementary potentials (0-5 mV) are observed

10-40 ms befor·e a spike occurs. These potentinls

have been interpreted as .the result of opening of

single ion channels involved in the transduction

process. The spikes of extracellular r2cording

co nf',i st of an initial po si ti ve deflect ion ( 2ms)

follovpd by n'"gative one (Kaissling. and thorson, 1960 ) .

ln some ca~es, responses to odors may also be

recorded 8s an inhibition by receptor celLs, when

they shOV'J a slo·,v hyperj..>olarization and reducr:?d the

firing rate ( Bo eckh, 1967a).

71

Th9 specificity of an olfactory cell reflects the

specificity of membrane. receptors. also called acceptors:

which are located in the dendritic membranes r The

possibility that the specificity might be confined

to the conduction system, e.g.; microtubules w3s

excluded by the observation th:Jt two sensorJ ceLLs

innervating the s~me sensilLum, and sharing the same

conduction system, could be activated by different

'key' subtances.

In activating cell, the odor moleculB is thought

to bind to a membrane receptor protein (Hansen, 1978)

wherc.>by a conformational ch:1nge to an active sta.ge of

the receptor occurs, leading to the opening of ion

channels and depol a ri zatio n of the cell membrane

( Kaisding and Thorson, 19 SO). So far, different

olfactory cells have been shovm to process varying

degrer~s of specialization, some respondirg only to

one of the test compounds while others may respond

to IilCJ!lY difl'~rc-!nt odors. Varying coruposltton of

·ctiffArent types of membrune rr•CPIJtors within eoch I.

cell. h;·1s bf'en postulated as a moclel to explr-3in the

d9gr~e of specialization possessed by different

olf'3ctorj cells (Kafka, 1974).

72

In an early study in the saturniid moth,

\ntherea pern;y:h, 'specialists' cells are those

c elj. s which rEspond to phe ramon e compounds while

'generalist cells are those cells which respond

to 1na,1y dif;'erent host compo...cndc (Schneider et al.,

1961~). 'I'h~ eel LS activated by phr-;romones are

specir.1lized for one compound of a pheromone mi~~ture

(Kais~;ling, 1979; priesner, 1979a; Den Otter, 197'1;

T>'iust8pa l~t- , 1979; Angst, 1981 ; Wadhams et al.,

1982). On the other hand, odors with other

functions such as food attractants usually seem to

activate cells which h8Ve broader response spect,ra.

HovJever, the turm "generalists" can be misleadirJ6 . sine e it may inean either cells which all resporrl

difi'er·.~ntly (individual rr-:sponse spectr;=l) or cells

which br::.lone; to a certain response group: where

th('? r(~:>ponse spectra within a group is identical

( v :~ res chi 1 C? 7 ·1 ) • Further mo re , t h es e g ro ups rna y

ei.ther over lap or be non-overlapping.

73

The female sex pheromones specifically activate

sensilla trichodea re·ceptor cells on the m;::;le r-mten-

n::Je and 2licit 8ttraction andsexual beh8vior in

th9. m."l es (Kromc-:T, 1973). 'rhe ph .. ~romone compotwnt,s

which ar identified by chemical an8lysis nre

str'1it;ht chgin unsatur3ted hycirocarbons vdth acetates,

alcohols ~~nd aldehyde ;:.Js on termine1l group; often in

co mv,ctio n with electro ~)hy siolo gi cal tests and field

observations. Synthetic pheromones' availability

m;;;kes this sti1L~lus rece;_.Jtor system most sditable for ' li

studying the structure-Gct.ivity relat-ionship of the

olf::Jctory system (Kaissling, 1971 1 1979; Priesn'?.r,

1970a, 197C!b; Schneider et al., 197'7; Den Otter,1977)

nncl for test:.in;:; various hypothesis on molecul''lr

receptor (acceptor) interaction (Kafk3 &:- i~eu.virt.h,

1975) gnd transduction processes (Kai3sling, 197h).

Th" m.unbc'r of sensilla trichodea SNlsOr:l C"'l2.::;

v:-:1ries from 1 to 5 and clussify A,B,C,D,E, dep~nding

on the moth species {Priesner, 1979a). In some

spociQs, the cell generated the larg0st spike amplitude

in each sensillum consistently belong to th:> sr!me

..

74'

cell tylJe; i.e. maxim3li.y responds to the same "Key

subst<mce", which i~. the major pheromone component.

This cell type "A" is generally present in all

san<;,ory tricbodea vvhile the others gre Keyed to a

minor componont s and frec1uently found. In Bombyx mori_

::ensilla tri chod.e:~ contain two cells,· one larger

tlnn th:::> oth.-.,r. From the lCJrger cell, l.1rgest

Si.Ji k -·s el c ct ted by bombykol ( Kai ssling and Tho-rson,

1 q :.30) • Similn r rels ti ons hip bet ween spike amplitudes

ana sped fi cities of sensilla t ri chod ea sensory

cells seem to exist in m3ny moths (Priesner, 1979b;

K·,is~,;]_ins;, 197g; Den Otter, 1977; ·O'connell, 1975).

Thus, even ~hou~h most species use multicomponent

pheromones, the r:::ceptor cells det.ecting the~~e

sie;nals ar~" 11 sp,~ciGlists"' for one "Key" .subst'1nce.

Other yuest,ions ot interest include th.e

sensitivity and the SlJecificity of the cells for

th8i r ro:: specti ve "Key" compounds. The pi one· ring

•":1. p c'd.tn •"nt l e.'tdi nt; to the conclusion th:'J t one of

t.-vo moleculr.:-s ol' bomby.kol hittin;::; the rece1Jto~·

cell is e:,olu;h for eliciting a spike, WJ, p~r-

fo nned usin,s el ectrophysiologi cal reco n:iin6s

combin-=·d .. vith m~~surc:nwnt::: of adsorption oi'

L1b"7c.led borubykol on the antennae of Bo~by.x n~ori

75

( Kai ssli n; and Priesner, 1970). In other species,

the rd a:_.i v e s ensit:i vit_r of the cells to t h2i r

·r·-"sp.:>ctive "Keyn compound has .been determined by

measuring the amount of t h~~ unlebc=iU ed compoun.J needed

to (~ive ;1 cert:1in respon~·e at lovJ levels. • In that

m~.; uer it v~··s found in Bombyx_ mori, /\nth~~~ polyphyemus

(l\8isslin.;, 1979) and various noctuids and--tortricid

mot.hs (Pr:;.esner, 1979a, Den Or.t.er, 1977) that

approximately the s8me degree of response of the

respective cells was el:Lcited by e4uivalent amounts

ol' thei.c 11 Key" substances.

In severt'll moths, priesner ( 1979a, 1979b) deter-

t:,lned the specificiti::::s of the various pheromone

cells -~ithin sensi u_ a t.ri chodea by measuring their

t.h~ ''Key'' compoun-::.t. The function of each cell. type

:1ppears to be ''A" is only for main pheromont;: component

"B .. :!GD" are fur interspecific inhibitors. ".E" is

for synergi~t only; and 11 C11 is unknown {Hill ~nd

Ho <.:>lofs, 19[)0; M eDonough et al . , 1980; Fa rine et al.,

1081). All ce.Li. s h8vin.; ap;Jl"'Xim::Jtely the s8rne

threshold for their "Key 11 su b.st8. nc es, also r~sponded

to other compounds but o:1ly ~:lt highr,r corne::1t.r9tions.

76

to 8}.dshyci0s And 8lcohol~~ ::m;i the old,c;lydrc: cell (D) I

len~th from th·1t of the "Key" subst8nce, gen~r1ll.y

csused a decr--a~~e of the stimulatory efl'(:!ct. HovJever,

it cioes not s'?~::J.n to be the total ch:1in le,-:gth, but

rgther the len:;th of particular section of th2 ch::Jin

is most important.

The receptors in the Noctuid and Tortricid moths

ar,_, fo ·n<i dif. er: 'The part of chain bAt\P·:n th<:· polar

alq.1l ,.rou_(; and the double bond. being most criticc:ll ~

for the svirol:J.L~t.ory efL'ect on recr::pt,or celLs in noctuid

moL;!~_, and the asher part of chain is wo~-~t criticJl I

in torticid mcchs. (Priesnsr·, 1979a, 1979b). ll~;ain

thP recc•ptor .rp:_::cificity Of 8 p:o.~rticular CPll t:;pe

gr~ner·1lly r:"mains consistent \.vithin clo~ely r.:.l3ted

sn0Ci•"'S· 'l'hu~~ Pri esner ( 1Cl79a, 1?79b) found

. J , S[)!?C1.•"':3 3S ~~·e .. .L 8S in closely r2la'c ed speci •2s. ·rhis

in contr:.st to the receptor cells of taxonomically

for which th2 specificities diff~red :,s de~3C;'ibed above

77

Th~~s·"' findin,~s of const::Jnt specii'icitL='s of phRromone

C'-::LI __ s belon··:in,; to one 'cype, diLi'er fmm thr~ results of .

sen::.:il .a tl~ichod<?a rec<=;ptor ce.:_! s viere_, each found

to be mo,;l:. sr:::n:_~iti.ve to one o::.' the ph2romone compo-

nen"c.s, (Z) 11-13: r;C 01~ (E) 11-1,+: 1\C. As but indi-

vidu.··1lly the ceLLs differed both in respect to ·absolute

~-HLl r·:'!lJ-L-i.ve sensitivie~::; for the:; pheromones and other

rel3'c :::d c-:>rr>~~ounds. Each type of pheromone cell in

moth species possesses only one membr&ne receptor type,

a ~.peciali:::-t for thr: "Key 11 substance, and thst

ph~romone constituent~ in these species can indeed be

iden~.:.ifi .,d electrophysiolo::::,i cally by scceenin:j; the

cells. Screenin~ tests h~ve been c~rried out in many

species usi~ sin:::;le' cell rGcorJin.;s as WC?LL 1s

reco -,.,di n~~s of 1i'•\G lp..,; econp r 'JO'?o., . 107q·o· K'll. c·~ll' n'~ . "" i \ .l -"- • ' ~ .. , ' :A ' ' ' ''. • . t..d

Prie:.>n0.r (1977) rns r::u-.;~nst.ed th:Jt the reC"p1~0l~s

h:=~ve undergone a stepwL:-;e ch71nge in cert;-:lin sp::ocies.

The ide1 vvcls mainly on electrophysiologic.'3l studies

ol' some moths, shovJin"; a grsdual change in r2c:;ptor

responses of male antennae with the degree of

taxonomic separation (Priesner, 1979a).

78

.Pheromone molecules are filtered out of the ait

str2am by the? 1ntenna and its br:mches anJ sensory

hEucc;. Kai~..:oslin.::; ( 1971) has demonstr.:lted "chat the

cros.c:-sP.ction'il area eyur.;l tu the outline a1·ea of

its :.c1nt en:1a.

'I'he phc~romone molecules removed frcm the

~:~irstream are aJsorbed onto th2 surf·,.'s o:~ th...,

antemw and its sensilla. It hc:~s been su;_;:_.ested

tlYlt only the.c::e moleculPs thAt m;:Jk-:; a di11.=~ct hit

on the pore kattl e are stimulatory, although

Ksissling ( 1C)60) st1tes that the entire sensi:Llum

acts as an ausoj:"bti'!e su1~fr1ce for stimulatory

mol P.cul es. l'hi ~' 8 bso qJtio n phenomenon is non- e.iJ cci fie

(Ka~.~-:lny; and K~~isslin.;;1CJ7:2) and does not contribu.L.e ' ' '

to L-hP 3pt?ci i'i.city of olL~ctory rr·-spon:.3e.

K<1san2.., 3nJ K8is~ling ( 1072) rnPntiorv·d that t.he

S!Y"CiJ'icity o:!.' the' sr~nsory respon~"! must be rel'lted

to the slJeciU.city of the acceptor complex (binuing

site). The:(' c:cc ·1Jtor contplexr::-s (binuins sitss) are

loc;~tPd on ths cc·ll manbrane of t.he denrlritcs o:i' the

s"'nsory n~uronc. (K~1isc:lint;, 1969).

79

The number of accept-or complexes for each r3en-

siL. um is L-.nknovm, althout)l' it probably at least

equals the number of pore tubules (Kaissling, 1()69).

Z3charuk (1971) has sug .. ;r>sted that the specificity

of the s ensil: urn may be a function of the fluid. of

th8 pore tubule.

Se8brook ( 1077) has sppculated that the proteineous

outer coat of the sensillum may contribute to its

specificity and that the bindin"; sites may b~

locatc;d th2re.

In stucii es on the fate of the sex pheromone

molecules of Tl~ichoplusic; ;1i cis -7-dodecen-1-01-acr->t"lte

after reaction with anten~al proteins, FerKovich

·- l (107_,) d·s vo ed· "-'n!:l.L. ev a • . ·' ) l co .Jr L, ·~v both enzym~tic 8nl non

' enzymatic bindin1s occurred. The pheromone wr-~s

degraded to i~s alcohol cis-7-dodecen-1-Ct which

is a potent i nhi bi tor of s ·"'X phenomone behavior in

this species . They sug;es t that T, he inhibitor and

pheromone bind to different pnrts of the proi...ein

or to diJ.'l'erent }Jrotein5·· These J?roteins tllal

bind and degrade the sex pherou~ne were lecated in

the heomolymvh and un the legs as well as on antenna

(i\asan~; :% Kaisslj_ng, 1972; F'erkovich et al., 1r173).

Bo

Schneider (1970) hbs sug~ested that enzymes

C'-ij)8ble of dP-gradin:; pheromones m21y play a d.ouole

role in olf~1ction on the antenna and on the bodies

and legs of insects, to prevent a buildup . of phero-

mu nP. rnol r.:cul ~->f: in these regions.

~ builrlup of these molecules on the body of

the in:.Pct \vould interfer I"Jith the chemical

co .. xuunic~01tion system. Mayer ( 1973) found that the

m::jl" antenna de;:;radeci the pheromone to alcohol

tvvice as fast as did the~ female antenna of Trich<2.2_lusia

ni 3nd that the pheromone w~1s hydrolyzed more rapidly

than six closely related isomers and analogues which

indicates some specificity in the acceptor proteins

for the sex pheromone.

K· sang (1974) calculated that 9C% of the

pheromone molecules of Bombyx mori are absorbed

noncovalently onto micromolecular structures and t~t

large amounts of the pheromone are enzymotically

converced into acids. Little is known of the fate

of the pheromone molecules one e they have reacted with

th<=> ;:mt ,:nnal prot einf. and have been enzywati c ally

dr>grnd""d. l\asaQ.£.!:: (1974), ho,.,over, reports thnt

after ~0 min of incub ~ion with Taitium-labeled

bomykol, H3 molc:c:.J.les h;,_i penetr::Jt~'l to the inner

an upt21k2 of me'cabolic by products by the dendritic

branche3 of the neuron~ •

Kaissling (1972) has proposed a two step proce3s

for the iaactiv~'Jtion of the pheromone molecules:

·1 :st stPp is earl:/ inactiv8tion of the molecule a

pro c e :::s t.hat VJould physical iy or chemic aH y reduce

the concentration. of stimul a tiry mol ec ul e.:, nea r~~r·

the ac eptor and n1ay cont:"ibute to the specificity

o t "C he s y s t em ( K a i s sl j_ ng 19'76) • 2nd step , late

inacti'<'.Jtion is a process that remove the odor

molecules or their reaction product from the sen:3ory

hair:3 ov,c. · a period of minutes. Early activation

accounts for certain characceristies of the :SAG

w;weform anu late activation account-s the rnie;ration

of bombykol molecules 'lii~hin the antenna. Six steps

'3r~ considered itl the tmnsduction of the Olfnctory

stimulus to the receptor potential (Kais::;linr.;, 1974).

l. .\dsor·ptiwn o 1 the antennal survace.

Stimulus {S) ->S '.E,·1s ---/ su rf::~c e

2. Diffusion to the ro:;ceptor moleculr.:s.

3su rf':-•" '" · · --·- + ..... • • ..1\...... \., ""

k----

("<

;:)at r2c epto r

4 ·~ctiv:=Jti·.Jn of receptor molecule

-------;;. A'J -4--·----

S· Ch:.; nge o l' membrane conduct8nc e

!\'S . 6G · lnduces --r membnmce

6 · ~~rly in8ctivation of the odor molecule

Sadsorbed )---~ Satr2coptor( .f---- S inactivated

82

It is dif.ficult to associate the slow enzymatic

d.•~gr'-ldation of bombykol (50% in 4 min) .dth wore

rapid res".Jon:~e time of theEAG whic£1 Boeckh et al.

( 1965) h.:.:Jve shown,may r·each it ~3 maximum voltage

cvithin 0-5 sec. and return to its restin;_; m~mbrane

pot enti3l within 3 sec. of t 0rmim ti on of the

stimulus.

B 1 l .... "l .. o ec,-<: 1 e~, a_.:..., ( 19 65) · ex peri men ts shovv that the

1i ni tial reset ton in the olfaction process· i ~' the

adsorrJti~::-n of thr-; odor;:mt molecule onto the anteltnal

surface. This is follov~ed by dif f·usion of the

molecules to the acceptor complex: where bindi.ng

occurs which r~sults i.l a ch3ng':3 in msrnbn;ne

conductr.JnC'"• The stimulatory molecule aCC'3(Jtor

complex i:3 inact1.v~-1ted by the process of r.arly

i !1Acti vgtion of the odor molecule.

This ol f:::Jctory process reli e.s on the stereo-

chemic~:1l fit of the odor molecule and L:.s ac,:e_titor I

complex, a process sUL:; ested by f<o elofs and

Comeau ( 1971 ) •