Relative Contributions of the Egg Layer and Egg Cordon to Pheromonal Attraction and the Induction of Mating and Egg-laying Behavior in Aplysia

14 Pages·1991·5.7 MB·English

by  S D Painter

Reference: Biol. Bull 181: 81-94. (August. 1<WI)
Relative Contributions of the Egg Layer and Egg
Cordon to Pheromonal Attraction and the Induction
of Mating and Egg-laying Behavior in Aplysia
SHERRY D. PAINTER, MARIA G. CHONG, MARY A. WONG, ANN GRAY,
JEFFRY G. CORMIER. AND GREGG T. NAGLE
TheMarineBiomedical Instituteandthe Department ofAnatomyand Neurosciences,
UniversityofTexasMedicalBranch, Galveston, Texas 77550-2781
Abstract. Many species of the opisthobranch mollusk not influence the results. The lack of effect may result
Aplysia form breeding aggregations during the reproduc- from the low-probability nature ofegg-laying activity.
tive season. The aggregations contain both mating and
egg-laying animals, and are associated with egg cordons. Introduction
Although pheromonesplayasignificantrolein developing Pheromones appear to play an important role in co-
and maintaining the aggregations, little is known about ordinating reproductive activities in the marine opistho-
theactive factors. Behavioral studies have shown that egg- branch mollusk Aplysia. Field studies ofboth A. califor-
laying animals are more attractive than nonlaying ani- nica {Kupkrmann and Carew, 1974; Audesirk, 1979)and
mals, have shorter latencies to mating, and induce con- A.,fasciata(Susswein et al.. 1983, 1984) indicatethatthese
specifics to lay eggs. As a first step toward isolating and simultaneoushermaphroditesaresolitaryanimalsduring
chemically characterizing the active factors, we exam- mostoftheyear. Duringthesummerreproductive season,
ined the relative importance ofthe egg layer and eggcor- however, they move into breeding aggregations or
don as sources ofpheromonal activity in Aplysia brasil- "brothels." Theaggregationsaretypically associatedwith
iana. T-maze experiments showed that both animal-de- recently deposited egg cordons, often laid one on top of
rived and cordon-derived factors are attractive, and that another, and contain both matingand egg-layinganimals.
theanimal-derived factorsare specifically associated with Most ofthe egg-laying animals simultaneously mate as
egglayers. Extractsoftheatrialgland anexocrineorgan females, even though mating does not cause reflex ovu-
secreting into the oviduct increased the attractiveness lation(A. brasiliana, Blankenshipetal., 1983),suggesting
of nonlaying animals when placed in the surrounding that egg laying may precede mating in the aggregations
seawater,suggestingthatthe"cordon-derived" aggregation rather than result from it.
pheromones may be products ofthe atrial gland. Mating This sequence ofactivities is important because ofthe
studies showed that both animal-derived and cordon-de- reproductive physiology ofAplysia. Four characteristics
rived factors induce mating, and that the animal-derived of the system are worth noting. First, Aplysia are non-
factorsare associatedwith both egg layersand nonlayers. self-fertilizinghermaphroditesthatstorelargeamountsof
In contrast, neither animal-derived nor cordon-derived exogenous sperm for future use (A. californica, Mac-
factors induced egg laying. Comparable results were ob- Ginitie, 1934). Second, many species ofAplysia lay egg
tained with either one or two animals in the chamber, cordons containing from one to several million fertilized
suggesting that the accessibility of a potential mate did eggs (e.g., A. californica. MacGinitie, 1934; A. depi/ans
and A. fasciata, Thompson and Bebbington, 1969; A.
dactylomelaandA.Juliana, Switzer-Dunlap and Hadfield,
Received 20December 1990:accepted 16 April 1991. 1977). In these species, a single bout ofegg laying signif-
ithAeblbiruemv;iAatSiWo,nsa:rtAifGicEi.alasteraiwalatgelra;nBdCePx,trabcatg;-cAelGl-pLeEp,tidater;iaElLgHl,anedg-gl-ilkaeyeipn-g icantly reduces the amount ofexogenous sperm stored,
hormone. although the stores are not depleted. Third, animals lay
81
82 S. D. PAINTER ET AL.
eggs regardless ofwhether sperm are available to fertilize ogous to ELH and one or more sequences homologous
them (.4. californica. MacGinitie, 1934), suggesting that to BCP. Although the gene sequences are highly con-
there is not a strong internal link between the amount of served (ca. 90% identity), the resulting precursorproteins
exogenous sperm stored and egg deposition. Finally, are processed into homologous but nonidentical sets of
sperm are transferred between animals in an immature peptides with similar pharmacological properties but dif-
state, and cannot immediately fertilize the recipient's oo- ferent physiological functions.
cytes(A. depilans, A.fasciata. andA. punctata, Thompson Several ELH-related genes are expressed in the atrial
and Bebbington, 1969). Mating as a female during, or gland (A. californica, Scheller et ai, 1983; Mahon et ai,
shortlyafter, eggdeposition thusensuresthat matureand 1985), an exocrine organ secreting into the oviduct of
capacitated sperm will be available to fertilize oocytes Aplysia (A. californica. Arch et ai. 1980; Beard et ai,
during future egg-laying episodes. 1982;Painterrfai. 1985). Althoughextractsofthisorgan
Laboratory experiments suggest that the egg layer or induce egg laying when injected into receptive animals
its egg cordon are potential sources ofpheromones that (A. californica. Arch et ai, 1978), the exocrine nature of
attract conspecifics and induce mating activity (A. brasil- itssecretoryactivity indicatesthattheatrialgland products
iana. Aspeyand Blankenship, 1976;Jahan-Parwar, 1976; do not functionashormonestoinduceegglaying. Recent
Painter el a/.. 1989; A. californica, Jahan-Parwar, 1976; behavioral studies suggest that the atrial gland products
Audesirk, 1977);thereisalsoevidencesuggestingthat they may function as sexual pheromones instead (Painter et
induceegglaying(A. californica. Audesirk, 1977)and may ai, 1989; also see Susswein and Benny, 1985). Peptide
be responsible for the masses ofegg cordons associated products of a small family of structurally related genes
with the aggregations. Since mating animals are no more may thus act as nonsynaptic neurotransmitters, neuro-
attractive than nonmating animals (A. dactylomela, Le- hormones, and pheromones to regulate and coordinate
derhendler et a/.. 1977) and mating does not cause reflex both male and female reproductive activities.
ovulation (A. brasilianu, Blankenship et a/.. 1983), the The studiespresented in thispaperexamine the relative
layer-derived or cordon-derived factors are likely to be contributions of the egg layer and its cordon to phero-
responsible for developing and maintaining the breeding monal attraction andtothepheromonal induction ofboth
aggregations. mating and egg-laying activity. They demonstrate that the
Although relatively little is known about pheromonal eggcordon playsan important roleinboth attraction and
regulation ofany component ofAplysia reproductive ac- induction ofmating, andsuggestthatproductsoftheatrial
tivity, a great deal is known about the neuroendocrine gland may contribute to these activities.
regulation ofone component egg laying. The bag cells,
two clusters ofneurosecretory cells located at the rostral
margin oftheabdominal ganglion (A. californica. Frazier Materials, Methods, and Results
et ai. 1967; A. brasiliana. Blankenship and Coggeshall. Animals
1976) are part ofthe final common pathway leading to
eggdeposition. These normallyquiescentcellsperiodically Specimens ofAplysia brasiliana (Rang), weighing from
enter a prolonged phase ofrapid and synchronous elec- 100 to 400 g, were collected from South Padre Island,
trical activity (A. californica. Kupfermann and Kandel, Texas, and were used in experiments between June and
1970;A. brasiliana, Dudek and Blankenship, 1977) which August, the normal reproductive season for this species.
releases several peptides into the connective tissue sheath Aplysiabrasilianawasselectedastheexperimentalanimal
surroundingthebag-cell clustersand abdominalganglion because it lays eggs and mates more frequently than A.
(A. californica. Stuart et at., 1980); this activity is consis- californica (Pinsker and Parsons, 1985), and can be col-
tently followed by egglaying(A. brasiliana, Dudek etai, lected in large numbers from the southTexascoast during
1979). Most ofthe released peptides have been isolated the reproductive season. The animals were housed in in-
andchemicallycharacterized, includingtheovulation-in- dividual cages in one offive large aquaria containing re-
ducing egg-laying hormone (ELH) (A. californica. Chiu circulating artificial seawater (ASW; Instant Ocean,
et ai. 1979; A. brasiliana, Nagle et ai. 1988a) and the Aquarium Systems, Mentor, Ohio) at room temperature
autocrine alpha bag-cell peptide (BCP) (A. californica. (20 2C); the salinity ranged from 30 to 32 ppt. A 14:
Rothman et ai, 1983). 10 light:dark cycle was maintained, with the light period
The bag-cell products that have been characterized to starting at 6 am; animals were fed dried laver in the late
date are all encoded by the ELH gene, one member ofa afternoon (4-6 pm) after experiments were completed.
small familyofstructurally relatedgenesthatareexpressed Specimens ofAplysia californica (Cooper) were pur-
in a tissue-specific manner in Aplysia (A. californica, chasedfrom Alacrity MarineBiologicalServices(Redondo
Scheller et ai, 1983; Mahon et ai, 1985). Each gene en- Beach, California) and were used as a source of atrial
codes a precursor protein containing a sequence homol- glands for extracts. They were maintained as described
SEXUAL PHEROMONES IN APLYSIA 83
above forA. brasiliana, except that the ASW was cooled 101 cm
142C.
to
Atrialglandextracts
Sexually mature individualsofAp/ysiacalifornicawere 1 1
1 1
used as sources of tissue for extracts because the atrial 1 1
gland in this species is large and well-defined; the ho-
mologous organ inA. brasiliana [the atrial gland-like ep-
ithelium (AG-LE)] is a thin band ofsecretory tissue that
extends along the length ofthe oviduct and is difficult to
recognize except in fixed or sectioned material (Painter
elal, 1985). Both organssecreteintotheoviduct(Painter
etat., 1985), andthereisevidencethattheirpeptideprod-
ucts are highly conserved: (1) extracts ofeither organ in-
duce egg laying when injected into receptive animals of
either species (Painter et al., 1985; also see Blankenship
et al., 1983); (2) secretory granules of either organ are
specifically labeled by antisera raised against low molec-
ularweight peptidesisolated from theA. californicaatrial
gland (Painter et al., 1985); and (3) restriction enzyme
maps ofthe ELH-related genes in the two species suggest
a high degree ofsequence identity (Nambu and Scheller,
1986).
In mostcases, theextractswere usedasatool toinduce
egg laying 0.1 ml ofan ASW extract ofthe gland was
injected through the foot into the hemocoel, and egg de-
position began within approximately 30 min. Extracts
were prepared by removing atrial glands (approximately
100 mg wet weight per gland) from 10 animals and ho-
mogenizing them in 10 ml offiltered ASW with a Brink-
mann Polytron homogenizer(setting4 for 1 min at4C).
The homogenate was centrifuged at 1500 X gfor 30 min
at 4C, the supernatant removed, and aliquots frozen at
-20C
until use.
Since previous studies have shown that acidic extracts
ofthe A. californica atrial gland induce A. brasiliana to
mate (Painter et al., 1989), acidic extracts ofthe A. cali-
fornica atrial gland were directly assayed for pheromonal
attractiveness in A. brasiliana. In this case, atrial glands
were removed from 10 animals and homogenized in 15
M mM
ml of 1 acetic acid containing 20 HC1 with a
Brinkmann Polytron homogenizer (setting4 for 1 min at
4C). The homogenate wascentrifuged at 48,000 X gfor
20 min at 4C, the supernatant removed, and the pellet
rehomogenized in 5 ml ofthe acidic medium. After cen-
trifugation, the supernatants were combined, distributed
into 20 aliquots, lyophilized, and stored at -20C until
use. Each lyophilized aliquot was resuspended in 1 ml of
ASW
immediately before addition to the maze, and was
equivalent to approximately 50% ofthe material in one
gland.
Pheromonal attraction
Apparatus. AT-maze(Fig. 1A)wasconstructed ofclear
Plexiglas (0.62 cm thick) and sealed with clear aquarium
84 S. D. PAINTER ET AL.
placed in a perforated plastic cage in one arm. A non-
treated sexually mature "test" animal was placed in the Positive
base ofthe maze 5 min later. Both the stimulus and test No choice
ASW
animals were briefly rinsed in non-conditioned be- Negative
fore beingintroduced intothe maze. A response wascon- 15
sidered to be positive ifthe test animal travelled to the
stimuluswithin 20 min and remained in contact with the Etc
stimulus cage for 5 min, negative if it travelled to the
opposite arm and remained for 5 min, and no choice if (0
itdid neither. Animalswerechoosingbetween astimulus 10
and nostimulusintheseexperiments, ratherthan between
two qualitatively or quantitatively different stimuli. Fif-
teen experiments were performed for each potential at- -EQ
tractant, and the attractant was alternated between arms
in consecutive experiments. Statistical significance was
assessed by X2 analyses.
Animals. A total of 183 Aplysia brasiliana individuals
were used in these studies. All were sexually mature
they laid eggs spontaneously or in response to injections
ofatrialglandextract and all had been observed mating
before they were separated into individual cages. Four No animal Nonlayer Egg layer
criteria were used to select animals for each experiment: No eggs No eggs Eggs
( 1 ) the animal must not have laid eggs duringthe preced-
ing 24 h; (2) the animal must not have participated in a
Stimulus
behavioral experiment during the preceding 24 h; (3) the
animal must not have been a test animal forthe stimulus Figure 2. ThereisnodirectionalbiasintheT-maze,asindicatedby
beingstudied; and (4) the test and stimulusanimals must thedistribution ofresponsestothe no-stimuluscontrol (noanimal, no
have been housed in the same aquarium. Egg layers were enggghst):aarpmpsrooxfitmhaetemlayzee.qu(aNlotneu:mfboerrtshiosfqaunanitmiafliscattriaovne,llmedovteomtehentlefttoatnhde
animals that had been injected with atrial gland extract left was defined as a positive response and movement to the nght a
to induce egg laying and, unless otherwise specified, had negative response.)Aplysm hnixilmna is notattracted to nonlayingan-
completed laying eggs within the preceding 30 min; non- imalswithouteggcordons:theresponsepatterndidnotdiffersignificantly
lcaeydeirnsgwe24reha.nOinmlaylsntohnaltahyaerdsnowterleaiduseegdgsadsutreisntgatnhiemaplrse;- c<forro0d.mo9n5ts]h,.athAo.obwsebrevaersrvi:elditahwneiatlheavrteehleaotfntoar-tastctrtaiecdmtuitloounseagcngod-nltparayotiltneg[rXnc2o(on2fs)pr=eecs0ip.foin1c4ss;es0w.di9it0fhfe<ergePgd
both egg layers and nonlayers were used as stimulus an- significantlyfrom thoseobservedwhenanonlayerwithouteggswasthe
imals. stimulus [X2(2) = 34.12;/><0.005]. Thisbargraph isbased on 15 no-
Results. To assess directional bias and chance levels of stimulus control experiments and 30 single-arm experiments, 15 each
attraction in the maze, 15 experiments were performed for"nonlayer. noeggs"and "egglayer, eggs"; inthesingle-arm experi-
ments, animals were choosing between a stimulus in one arm and no
in which no stimulus was placed in either arm. Four of
stimulus in theother.
theanimals(26.7%) moved tothe leftarm and remained,
three (20%) moved to the right arm and remained, while
eight(53.3%)did neither(Fig. 2), demonstratingthat there without egg cordons [X:(2) = 34.12; P < 0.005], dem-
isnodirectional bias in the maze and establishingchance onstrating that egg-laying animals are attractive.
levels ofattraction at 3-4 animals (20-26.7%). Because the "egg-laying animal" stimulus has two
A similar level ofattraction (3 animals; 20%) and pat- components the egg layer and its egg cordon subse-
tern of responses was observed when a nonlaying con- quentexperiments focusedontheirrelativecontributions
specific without an egg cordon was the stimulus (Fig. 2). to pheromonal attraction. More animals were attracted
The two sets ofresponses were not significantly different toegglayerswithout eggcordonsthan had been attracted
[X2(2) = 0.14;0.90 < P< 0.95], demonstratingthat non- to nonlayers without egg cordons, and fewer made no
layers without egg cordons are not attractive to Aplysia choice (Fig. 3); the difference in response patterns was
brasiliana. A higher level ofattraction (12 animals; 80%) statistically significant [X2(2) = 15.38; P < 0.005], dem-
and lower level ofno-choice responses (1 animal; 6.7%) onstrating that the egg layer is a source of aggregation
was observed when the stimulus was a conspecific that pheromones. Identical resultswereobtained whena non-
wasactively layingeggs(Fig. 2). This pattern ofresponses layer with a recently deposited egg cordon was used as
wassignificantly different from that to nonlayinganimals thestimulus(Fig. 3), indicatingthattheeggcordon isalso
SEXUAL PHEROMONES IN APLYSIA 85
in no-stimulus control experiments [Fig. 4; X2(2) = 1.70;
Positive 0.25 < P<0.50]. Thedifferential responsestoeggcordons
No choice and sham cordons suggests that the attraction is chemi-
Negative cally rather than visually mediated. The results ofthese
15 two series ofexperiments, in conjunction with those re-
0}
a ported above, demonstrate that both egg layers and egg
cordonsare sufficient to attract conspecifics, but that nei-
co ther is uniquely required.
10 As a first step toward identifying tissue sources ofthe
cordon-derived aggregation pheromones, acidic extracts
n ofthe atrial gland (equivalent to 50% ofthe material in
3 one gland) were assayed for the ability to increase the
attractiveness ofa nonlaying animal when placed in the
surroundingASW. A higherlevel ofattraction to nonlay-
ing animals and lower level of no-choice responses was
observed when the extract was present (Fig. 5); the dif-
Nonlayer Egg layer Nonlayer Egg layer
No eggs No eggs Eggs Eggs
Stimulus Positive
No choice
Figure 3. Egg layers are attractive to Aplysia braxiliana: a larger
numberofanimalswasattracted toan egglayerwithouteggsthan was Negative
attracted to a nonlayer without eggs, and the patterns ofresponses to 15
the two stimuli are significantly different [X2(2) = 15.38; P < 0.005]. 0)
EggcordonsarealsoattractivetoA.brasiliana:alargernumberofanimals a
E
was attracted to a nonlayer with eggs than wasattracted to a nonlayer c
withouteggs, andtheresponse patternsaresignificantlydifferent [X:(2)
= 15.38; P< 0.005]. Theeffectsofegg layers and eggcordons are not ffl
additiveattheconcentrationstested:theresponsepatternsforanonlayer 10
with eggsand an egglayerwithout eggsare identical, and do notdiffer
significantly from thepattern obtained foranegglayerwith eggs[X2(2) n4)
= 2.83; 0.25 < P < 0.50]. This bar graph is based on 60 single-arm
experiments, 15perstimulus;ineachexperiment,animalswerechoosing
between astimulusin onearm and nostimulus in theother.
a source ofpheromonal activity. Neither pattern differed
significantly from that obtained for an egg-laying animal
with an eggcordon [X2(2) = 2.83; 0.10 < P< 0.25], dem-
onstratingthat the effects ofthe layer-derived and cordon- No animal No animal No animal
derived factors are not additive at the concentrations No eggs Sham eggs Eggs
tested.
Subsequent experiments examined whether animal- Stimulus
derived factors are required for the attractiveness ofthe Figure4. Animalsarenotrequiredforaneggcordontobeattractive
eggcordonandwhethertheattractionisvisuallymediated. to Aplysia brasiliana: a larger number ofanimals was attracted to an
Two series ofexperiments were performed. In the first, egg cordon without an animal than was attracted to a sham cordon
an eggcordon without anyanimal served asthestimulus. without an animal, and the response pattern wassignificantly different
The level of attraction and pattern of responses were [x:(2) = 15.27; P < 0.005]. The sham cordon was a tangled mass of
identical to those obtained usingan egg layerand itscor- snioltasvtiicsutaulblyinmge,di2atmeld:inthveolpautmtee.rnTohfereastptorancsteisvetnoeassshoafmancoergdgoncowridtohnouits
don as the stimulus (Figs. 3, 4), demonstrating that egg an animal did not differsignificantly from the no-stimuluscontrol (no
cordonsare sufficienttoattractconspecifics. In the second animals, no eggs) (X-(2) = 1.70; 0.25 < P < 0.50]. (Note: for the no-
series ofexperiments, a "sham" cordon (a tangled mass stimuluscontrol,movementtotheleftwasdefinedasapositiveresponse
ofsilastic tubing; vol = 2 ml) served as the stimulus. The andmovementtotherightanegativeresponse.)Thisbargraphisbased
on 15 no-stimuluscontrol experimentsand 30single-armexperiments,
lneivfeilcaontflaytftrraocmtitohnosaendobspaetrtveerdnwoifthretshpeoengsgescodridffoenre[dX2s(i2g)- a1r5meaecxhpefroirm"ennotsa,niamnailm,alsshwaemreegcghso"oasnidng"bneotawneiemnala,setgigmsu"l;usinitnhoenseinaglrem-
= 15.27;P<0.005],butdid notdifferfromthoseobserved and no stimulus in theother.
86 S. D. PAINTER ET AL
behavioral experiment during the preceding 24 h; (3) the
Positive animal must not have been tested with the stimulus; and
No choice (4) all ofthe animals in an experiment must have been
Negative housed in the sameaquarium. Onceselected, theanimals
were randomly assigned to treatments. All experiments
15
were begun between 9 and 10 am, because there is evi-
CO dence ofa circadian rhythm in Aplysia mating behavior
'c (A.J'asciata. Susswein et al.. 1983, 1984).
Relative contributions ofthe egg layerandeggcordon:
10 experimentalprotocol andstatistical analyses. Eight an-
imals were used in each experiment (Fig. 6). One animal
wasinjected with0.1 ml ofatrialglandextractand placed
a
E in a 4-1 plastic beaker containing 3 1 ofaerated non-con-
ditioned ASW; this treatment induced egg laying, usually
z ASW
within 30 min, and the egg layer conditioned the
in the beaker. A second animal was handled and placed
in an identical beaker; this treatment did not induce egg
laying, but the nonlayerconditioned the ASW. When the
injected animal finished layingeggs(70.3 4.1 min after
injection; mean S.E.M.), itwasremoved, rinsed in fresh
Nonlayer Nonlayer Nonlayer non-conditioned ASW and transferred to a third beaker;
No eggs AQE Eggs the handled animal was treated in the same way and
transferred to a fourth beaker. Nontreated animals were
Stimulus thendistributed amongthe fourbeakerssothat eachcon-
Figure 5. Secretory products ofthe Aply.iia californica atrial gland tainedtwoanimals. Theresultingexperimental conditions
ASW
(orA. brasilianaAG-LE)maycontributetotheattractivenessofanegg are: (1) two nonlayers in animal-conditioned with
cordon. The number ofanimals attracted to a nonlayer was increased an egg cordon; (2) two nonlayers in animal-conditioned
when an extract oftheA. californica atrial gland (AGE) was placed in ASW without an egg cordon; (3) one egg layer and one
tfhreomsutrhratouonbdsienrgveAdSfWor.aTnhoenlpaaytitnegrnanoifmarleswpiotnhsoeustdeifgfgesre[dX2s(2i)gn=ifi1c0a.n5t8l:y nonlayerin non-conditioned ASWwithoutaAnSeWggcordon;
F < 0.01], but did not differ significantly from that for a nonlaying and (4) two nonlayers in non-conditioned without
animalwitheggs[V(2)= 0.44;0.90<P<0.95].Thisbargraphisbased an egg cordon.
on45 single-armexperiments. 15 perstimulus: ineachexperiment,an- The reproductive activity of each individual was as-
imalswerechoosingbetween astimulusin onearm and nostimulusin sessed at 10-min intervals for 270 min. Three categories
the other.
ofmating activity were recognized (male, female, and si-
multaneous hermaphrodite); egg-laying activity was also
PoTefr<erne0c.se0po1inn].sreIesnstepdroiendssteninopgtaltytd,iefrtfnheserwlseaivsgenlsiiofgfincaiatfntitcrlaayncttfi[roXno2m(a2n)tdh=opsa1et0t.oe5rb8n-; avreanctioimroadnlesdp,etrhbiauottddciowduerrtnesohtaispmsaiwtganesedonorat.la2Fy8o0er-gcmgasilncduullraaittniegonnctyphuefroporobssteehsra,-t
tw<aaiPsnep<dla0wc.he90de]n,insatuhgregeecsseanttmilneygltdohecapatotsipiortnoed[duFiccgto.sn5so;pfeXct2ih(fe2i)Ac.=ecg0ag.l4ic4f;oorr0nd.io7cn5a abficteetircvieaevniuactsyene.dianAtclomtlnehetaaorsuontlg8hlca5ott%nehdiniscotyfaiopttnophserm,oaaattnchiihenmgaeuflnfbsdeecettmrwaeiessteternidemlsaaitttnrieovseenvlgtyelhryesympdaoeilsxfl---
actornitarligbluatnedt(ootrhAe.abtrtraascitliivaennaesAsGo-fLaEn)emgigghctorsdiognn.ificantly perimental condition tested. Statistical significance was
assessed by a one-way analysis ofvariance, followed by
Duncan's multiple range test for pairwise comparisons.
Induction ofmating activity When time courses of mating activity were compared,
Animals. A pool of205 sexually matureAplysiabrasil- statistical significance was assessed by X2 analysis of in-
iana individuals was used in these studies. Small plastic dividual time points. Egg cordon volume was measured
mm ASW
fish tags (1 1 in diameter; Howitt Plastics, Molalla, at the end ofeach experiment by displacement in
Oregon) were sutured to the caudal region of the right a graduated cylinder and averaged 1.7 ml (1.7 0.2 ml;
parapodium so that individuals could be identified. Four mean S.E.M.). Twentyexperimentswere performed in
criteria were used to select animals for each experiment: this series.
ASW
(1)the animal must not have laid eggsduringthe preced- Results. Animal-conditioning the with a nonlay-
ing 24 h; (2) the animal must not have participated in a ing animal resulted in an increase in the percentage of
SEXUAL PHEROMONES IN APLYSIA 87
Handle animal; Handle animal;
inject atrial gland extract no injection
I i
Conditions ASW; Conditions ASW;
lays eggs does not lay eggs
Transfer egg layer to Transfer nonlayer to
ASW ASW ASW ASW
Conditioned Non-conditioned Conditioned Non-conditioned
with eggs without eggs without eggs without eggs
I I I
Add 2 nonlayers Add 1 nonlayer Add 2 nonlayers Add 1 nonlayer
2 nonlayers in Egg layer, nonlayer in 2 nonlayers in 2 nonlayers in
ASW ASW ASW ASW
conditioned non-conditioned conditioned non-conditioned
with eggs without eggs without eggs without eggs
Figure6. Flowdiagram oftheprotocol used in the first seriesofmatingexperiments. Oneanimal was
injected with an extract oftheatrial gland to induceegg layingand placed in a beaker containingaerated
non-conditionedASW;asecondanimalwashandledandplacedinasecondbeakercontainingaeratednon-
conditioned ASW. When eggdeposition wascomplete, the egg layerwas transferred to a third beakerand
the handled nonlayer to a fourth beaker. Additional nontreated animals were then distributed among the
fourbeakerssothateachcontainedapairofAplysia:theresultingexperimentalconditionsareindicatedat
thebottom ofthediagram. Matingand egg-layingbehaviorswerescored at 10-min intervals for270 min:
animals failing to exhibit a behavior during the observation period were assigned a 280-min latency for
calculations.
animals matingat early time periods relative to non-con- or concentration is relatively low under the conditions
ditioned ASW controls (Fig. 7A); the difference was sta- tested. The ideathatanimal-derived factorsinduce mating
tistically significant atthreetime periods 40, 50, and 60 inAplysiaisconsistentwitharecentreportintheliterature
min [X'(1) > 3.84 foreach; P < 0.05]. The mean latency that the amount oftime that A.fasciata spend mating is
to mating was also reduced (Fig. 7B), but did not differ a function ofthe number ofanimals available as copu-
significantly from that ofthe non-conditioned ASW con- latory partners (Ziv el al, 1989) and thus, presumably, a
=
trols (P 0.29; one-way analysis of variance). It is im- function of the concentration of animal-derived factors
portant to note that these effects, although small, were in the ASW.
consistently observed when the ASW was animal-condi- Similar, but quantitatively larger, effects were observed
tioned. Comparable resultswere obtained in an indepen- whentheASWwasanimal-conditioned byanegglayerand
dent series ofexperiments performed in our laboratory contained an egg cordon (Fig. 7A. B). The percentage of
(A. R. Gustavson, unpubl. data). The studies used a dif- animals matingat early time periods wasincreased relative
ferent pool ofA. brasiliana and animal-conditioned the to non-conditioned ASW controls, and the difference was
ASW
for 60 rather than 70 min, but produced quantita- statisticallysignificant foreveryobservation period from 10
tively similar responses. A higher percentage ofanimals through 1 10 min [X2(l) > 3.84 for each; P < 0.05]. The
matedatearlytimeperiodsrelativetothenon-conditioned meanlatency tomatingwassignificantlyreduced(P= 0.002;
ASW controls [X2(l) > 3.84 at 30, 40, 70, 80. 90, 100, one-way analysis ofvariance). Assuming that animal-con-
and 1 10 min; P < 0.05 for each]; the mean latency to ditioning the ASW with an egg layer is comparable to ani-
mating was reduced, but the change was not statistically mal-conditioning with a nonlayer (see below), these results
significant (P = 0.28; one-way analysis ofvariance). The demonstratethatcordon-derived factorsinduce mating, and
consistency ofthese two sets ofresults suggests that ani- suggest that the effects ofthe animal-derived and cordon-
mal-derived factors induce mating, but that theiractivity derived factors may be additive.
S. D. PAINTER ET AL
100 Animals did not distinguish between recent egg layers
ASW
and nonlayers in non-conditioned without an egg
- cordon. There were no significant differences in the time
80
courses of mating activity (Fig. 8A), in the latencies to
mating (Fig. 8B), or in the sexual role first assumed by
the animals (Table I). These results suggest that there is
not a prolonged change in the motivational state ofthe
E egglayer(i.e.. an increase in receptivitytocourtship)that
c
a 40 persistsintheabsenceofaneggcordon. Moreimportantly,
o Nonlayer, No eggs theysuggestthattheeggcordon, ratherthantheegglayer,
01 Conditioned may be primarily responsible for the relatively short la-
Q5. 20 Nonlayer, No eggs tenciesto matingobserved whenanimalsareactively lay-
Non-conditioned ing eggs. The experiments did not address the question
ofwhetherspecific layer-derived oranimal-derived factors
40 80 120 160 200 240 280 are required for the induction ofmating by egg cordons,
however, and this issue was examined in the next series
Time (mini ofexperiments.
Induction ofmatingbyeggcordons in non-conditioned
ASW':experimentalprotocolandstatisticalanalyses. Five
150 animals, selected as described above, were used in each
experiment (Fig. 9). One was injected with atrial gland
_ 125 extractand placedinabeakertolayeggs. Whendeposition
was complete, the egg cordon was removed, quickly
1
rinsed, and transferredtoasecondbeakercontaining non-
100
CD conditioned ASW; a pair ofanimals was then placed in
this beaker and another pairplaced in a third beakerthat
75 contained only non-conditioned ASW. The resulting ex-
perimental conditions are: (1) two nonlayers in non-con-
50 ditioned ASW with an eggcordon; and (2) two nonlayers
in non-conditioned ASW without an eggcordon. Repro-
25 ductive behavior was assessed foreach animal at 10-min
intervals for 270 min and analyzed as in the preceding
experiments. Egg volume was measured after each ex-
Nonlayer Nonlayer Nonlayer periment and averaged 2.0 ml (2.0 0.3 ml; mean
No eggs No eggs Eggs S.E.M. Fifteen experiments were performed.
Non-cond. Conditioned Conditioned ).
Results. Placing a recently deposited eggcordon in the
Stimulus non-conditioned ASW surrounding two nonlaying ani-
Figure 7. Both animal-derived and cordon-derived factors induce mals significantly increased the percentage of animals
mating activity in Aplysia brasiliana. (A) The percentage ofanimals mating in 9 ofthe first 14 observation periods [Fig. 10A;
btAamhynaSedtaWien6ngi0gwgmimaactltmho-ercaado[rXnonld2nyo(in1ttil)iina>omynteih3in.enpg8ge4Ara;tiSnhoPiWedms<;Aalw0St;.ahW0set5]hwi.epineTctdrrhihcfeeeafanesnptreeaeedrgngcecgbese-ynltwwaaaaeyngirsienemgswaiaslagis-nngciinffomiuinfarcidtlacihnaetatnrintaolditnynilc4hner0ieag,gavhsit5eeh0n,derg lPX<a:t(<0e1.n)0c0>5y.0tf13oo;.r8m4oaenataectih-n2]wg,0a-ryae4nl0aadntmailsivieyngs,tniois7ft0iohcfeamnivctnalo,yrnitaarrnnoeclddeu)g1c.re0od0Tu-hpt1eh4s(eF0eiirgm.rimesn1eu:0alBtnP;s
thanthoseobtained in non-conditionedASWwithoutaneggcordonat demonstrate that cordon-derived factors alone are suffi-
every observation period from 10 through 110 min (X'U) > 3.84 for cient to induce mating.
each;P<0.05].Theexperimental protocolisshowninFigure6(n = 20
experiments).BecauseAplysiatendtomateinboutslastingapproximately
60 min and the bouts are often separated by periodsduring which no Induction ofegg-laying activity
matingoccurs(Leonardand Lukowiak, 1987), it isnotpossibletoread
a mean latency to matingdirectly from this graph. (B) The latency to Egg deposition was also monitored in the experiments
mating(mean S.E.M.)wasreducedbyanimal-conditioningtheASW described above. Neither animal-derived nor cordon-de-
withanonlayinganimal,butthedifferencewasnotstatisticallysignificant rived factors significantly affected the percentage ofani-
(ifnProa=mni0tm.ha2al9t;-cooobnnteda-iiwtniaeoydnaefndoarlAnysSoinWs-cowofinvtdahiritaainnocenege)g.dcTAohrSedoWlna,wtieatnnchdyoudwitaffsaenrfeuedrgtsghiegcrnoirrfedidcoaunncte(ldPy cmaallcsullaatyiionngseoggfsm(eTaanblelaItI)e,ncayndtothdeeplooswitpieorncemnetaangiensglmeasdse.
<0.002; one-wayanalysisofvariance). BecauseAplysiabrasilianalayseggsmorefrequentlywhen
SEXUAL PHEROMONES IN APLYSIA 89
100 cordon (cordon-derived factors only); (3) animal-condi-
tioned ASW without an egg cordon (animal-derived fac-
ASW
tors only); and (4) animal-conditioned with an egg
cordon (both animal-derived and cordon-derived factors).
The conditions in each beaker were established as de-
scribed in the section on mating (see Figs. 6 and 9). The
volumes of the stimulus egg cordons were measured at
the end of every experiment and averaged 3.2 ml (3.2
Egg layer, No eggs 0.6 ml;mean S.E.M.). Infiveexperiments, allanimals
Non-conditioned
Nonlayer, No eggs thatwere not induced to layeggswere injectedwith atrial
Non-conditioned glandextracttoverifythattheywerephysiologicallycom-
petent to do so; all laid egg cordons in response to the
injection, demonstrating that the experimental conditions
were not interfering with the activity.
40 80 120 160 200 240 280 Results. Neither animal-derived nor cordon-derived
factors had a significant effect on egg deposition (Ta-
Time (min)
ble II).
150
B Discussion
125 Pheromonal attraction
c
E ThesestudieshaveshownthatAplysiabrasilianaisnot
o> 100 attracted to nonlaying animals in the absence ofan egg
cordon. The results contrast with those ofLederhendler
75 and colleagues (1977), which showed that Aplysia dac-
tylomela is attracted to nonlaying conspecifics and that
50 the magnitude ofthe attraction increases as the number
ofstimulus animals increases. We do not know whether
25 the difference reflects species differences or whetherit re-
sults from differences in experimental design (e.g., from
differences in concentration produced by using 5-min
Nonlayer Egg layer rather than 60-min conditioning periods), and we have
No eggs No eggs not examined the possibility that agroupofnonlayingA.
Non-cond. Non-cond. brasiliana would be attractive. We have, however, tested
Stimulus A. californica in T-maze experimentsand have found that
Figure8. Aplysiabrasilianadoesnotdistinguishbetweenrecentegg A. californica, like A. brasiliana, is not attracted to non-
layersand nonlayers in non-conditioned ASW without an eggcordon. layingconspecifics underthese conditions (S. D. Painter,
(A) Recent egg layers and nonlayers mated at the same frequency in unpubl. data). These results are consistent with earlier
non-conditionedASWwithoutaneggcordon.Theexperimentalprotocol studies by Audesirk (1977), which showed that A. cali-
isshown in Figure6(n = 20experiments). (B)Thereisnodifferencein fornica is not attracted to nonlaying animals in Y-maze
meanlatencytomatingbetweenrecentegglayersandnonlayersinnon-
conditioned ASW withoutaneggcordon. experiments. Although there is electrophysiological evi-
dence that A. californica detects the odors of nonlaying
conspecifics(Audesirkand Audesirk, 1977;Chase, 1979),
cagedalone ratherthan in pairs(Blankenship eta!., 1983), there is no evidence to date that the electrophysiological
the experiments were repeated with one animal in each response istospecies-specific odors(Chase, 1979) and no
beaker rather than two. behavioral evidence that the odors are attractive.
Single-animalexperiments:protocol. Fourtest animals, Aplysiabrasilianaisattractedtoegg-layinganimalswith
selected as described in the mating studies, were used in eggcordons. Theability todistinguish egg-layinganimals
each experiment. One wasplaced in each offour beakers with egg cordons from nonlaying animals without egg
and egg-laying activity assessed at 10-min intervals for cordonswaspreviouslydescribed in burrowingstudiesin
270 min. The ASW in each beaker contained a different this species (Aspey and Blankenship, 1976). In those
combination ofanimal-derived and cordon-derived fac- studies, when a nonlayinganimal was introduced intoan
tors: (1) non-conditioned ASW without an egg cordon aquarium containing a burrowed conspecific, the intro-
(negative control); (2) non-conditioned ASW with an egg duced animal burrowed; when an egg-laying animal was
90 S. D. PAINTER ET AL
Table I
Theeffectsofrecent eggdeposition onsexualrolein Aplysia brasiliana
Conditions