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CHARACTERIZATION OF A SPAWNING PHEROMONE
OF PACIFIC HERRING
by
Joachim Schnorr von Carolsfeld B.Sc., MSc, University of Victoria
A Dissertation Submitted in Partial Fulfillment of the R equirem ents for the D egree of
DOCTOR OF PHILOSOPHY in the D epartm ent of Biology
W e accep t this thesis a s conforming to the required stan d ard
N.M. Sherwood, Sufiervisor (D epartm ent of Biology)
G O. Mackie, D e p a rtrn ^ ta l Member^(Department of Biology)
0 . Hawryshyn, Departm ent ber{D epartm ent of Biology)
J. Ausio, Outsid artm ent of Biochemistry)
N.E. Stacey,''Exterqgl Examiner
(Departm ent of Zoology, University of Alberta)
© Joachim Schnorr von Carolsfeld, 1997 University of Victoria
All rights reserved. This thesis may not b e reproduced in whole or in part, by m im eograph or other m eans, without th e perm ission of the author.
ABSTRACT
Pacific herring, Clupea harengus pallasi, p o s s e s s e s a pherom one in th e milt and te s te s that triggers spawning behaviour in reproductively m ature individuals of both sex es, an d plays a role in synchronizing the school spaw ning th at is distinctive of this sp ecies. T h e p h erom one w as found to b e effective a s a transient olfactory stim ulus in eliciting a behavioural re s p o n se that varied in the d eg ree of ex p ressio n an d time co u rse. Stimulus strength w as found to influence the time c o u rse of the resp o n se , w h e rea s differences in maturity, evident through exam ination of p lasm a levels of steroids, w ere correlated with a propensity to resp o n d to th e pherom one. Input from factors other than the spaw ning pherom one a p p e a r to be n e e d e d to elicit prolonged spawning; so m e of th e s e factors a lso a c t through olfaction. Immediate effects of s tre s s w ere not found to influence the re s p o n se to the spaw ning pherom one.
P lasm a levels of reproductive steroids of herring during the spaw ning se a s o n w ere m easu red with radioim m unoassays. P e a k levels of 17,20p-dihydroxy-4-pregnen-3-one (17,20p-P) w ere found to coincide with final m aturation in fem ales an d the initiation of milt production in m ales, suggesting th at this steroid is the m aturation-inducing steroid of this sp ecies. O ther featu res found to be distinctive of th e reproductive physiology of the herring included low plasm a levels of th e unconjugated m aturation-inducing steroid, high levels of 17 a-p ro g e stero n e (17-P) and 3a,17a-dihydroxy-5p-pregnan-20-one (3a,17-P-5p), a n d high levels of glucuronated steroids. Structural investigation of th e ph ero m o n e with liquid chrom atography/ m ass spectrom etry show ed that it c o n sists of at le a s t two com ponents which do not elicit a behavioural re s p o n se individually. O ne of th e s e com pounds is sulphated 17,20p-P.
Ill
T he structure of proteinaceous horm ones involved in controlling reproduction of the herring w as also investigated. It w as shown th at this sp e c ie s p o s s e s s e s three forms of gonadotropin-releasing horm one (GnRH) in the brain, o n e with a structure that h a s not b een reported before. T h ese results indicate th at the p resen c e of three GnRH forms is a primitive, rath er than derived, condition in the teleosts. The structure of the (3-subunit of gonadotropin II (GtH ll-P) of herring w as also d educed by isolation of a cDNA for this m olecule. T he structure of the herring GtH ll-p w as found to b e quite different from o th er teleost m olecules of this kind, and a phylogenetic analysis of known GtH ll-P structures su g g ests that the p-subunit of both m am m alian gonadotropins m ay be most closely related to the p-subunit of teleo st GtH-l.
N.M. Sherwood, Stzpervisor (D epartm ent of Biology)
G.O. Mackie, Departmental Member (Departm ent of Biology)
C. Hawryshyn, Departm ental M e r^ p r (Departm ent of Biology)
J. Ausio. CXTtgm e Menfl^ ^ B ^ ^ f t m e n t of Biochemistrvl
N.E. Stacey, ExternaL/fcxaminer
TABLE OF CONTENTS
ABSTRACT... ii
TABLE OF CONTENTS... iv
LIST OF TABLES... viii
LIST OF FIGURES... ix
LIST OF ABBREVIATIONS... xiiii
ACKNOWLEDGEMENTS...xv
CHAPTER 1: OVERVIEW OF REPRODUCTIVE PHEROMONES AND THE ROLE OF HERRING RESEARCH... 1
REFERENCES CITED...13
CHAPTER 2. BEHAVIORAL CHARACTERIZATION OF PHEROMONE-INDUCED SPAWNING OF PACIFIC H ER R IN G 24 ABSTRACT...24
INTRODUCTION...25
MATERIALS AND METHODS... 27
SPECIFIC METHODS AND RESULTS... 29
A) CHARACTERIZATION OF THE PHEROMONE RESPO N SE:... 29
8) FACTORS INFLUENCING THE RESPONSE TO THE SPAWNING PHEROMONE:...44
V
DISCUSSION... 64
REFERENCES CITED... 72
CHAPTER 3. REPRODUCTIVE STEROIDS DURING MATURATION IN THE PACIFIC HERRING (CLUPEA HARENGUS PALLASI)... 76
ABSTRACT...76
INTRODUCTION...77
MATERIALS AND METHODS... 78
RESU LTS... 86
DISCUSSION... 109
REFERENCES CITED... 125
CHAPTER 4. PLASMA STEROIDS DISTINCTIVE TO HERRING RESPONSIVE TO THE SPAWNING PHEROM ONE... 135
ABSTRACT... 135
MATERIALS AND METHODS... 137
RESU LTS... 142
DISCUSSION... 143
REFERENCES CITED... 152
CHAPTER 5. IDENTIFICATION OF A COMPONENT OF THE SPAWNING PHEROMONE OF THE PACIFIC HERRING BY LIQUID CHROMATOGRAPHY / MASS SPECTROMETRY (LC/M S)... 155
ABSTRACT...155
INTRODUCTION... 156
MATERIALS AND METHODS...159
RESU LTS... 184
DISCUSSION... 205
REFERENCES CITED...209
CHAPTER 6. CONCLUSIONS... 216
REFERENCES CITED...221
APPENDIX A. PRIMARY STRUCTURE OF A NOVEL FORM OF GONADOTROPIN RELEASING HORMONE O F THE PACIFIC HERRING CLUPEA HARENGUS PALLASI... 223
ABSTRACT... 223
NTRODUCTION... 224
MATERIALS AND METHODS... 226
RESU LTS...232
DISCUSSION...245
REFERENCES CITED...257
APPENDIX B. ISOLATION AND CHARACTERIZATION OF A CDNA FO R GONADOTROPIN ll-p OF PACIFIC HERRING (CLUPEA HARENGUS PALLASI L ) ...266
v i l
INTRODUCTION...267
MATERIALS AND METHODS... 269
RESULTS AND DISCUSSION... 273
LIST OF TABLES
T able 1. Chemical nature of representative reproductive ph ero m o n es...2 T ab le 2. R ep resen tativ e pherom ones o ffish ... 7 T able 3. Evaluation of influence of test factors on resp o n se and
recovery tim es of the spawning pherom one re s p o n se ... 49 T able 4. Influence of stimulus type and concentration on re sp o n se and
recovery tim es of pheromonal re sp o n se ... 50 T able 5. Influence of test factors on proportion of sp o n ta n eo u s and
pherom one-responsive fish during group a s s a y s ... 56 T able 6. Sexual maturity sta g es of herring...81 T able 7. Morphological characteristics offish sam pled for blood...83 T able 8. Correlation betw een plasm a steroid levels and morphological
indicators of sexual maturity... 106 T able 9. Morphological characteristics of herring with different
resp o n siv en e ss to the spawning pherom one...144 T able 10. Relationship betw een pherom one resp o n siv en ess and
steroid horm one profiles in plasm a of herring... 146 T able 11. Principal ion of interest in electrospray-m ass spectrom etry of
herring te s te s and milt extracts... 175 T able 12. C hrom atographic purification of GnRH from herring brains
I X
LIST OF FIGURES
Fig. 1 : Com ponents of the resp o n se to spawning pherom one In m ature
herring... 31 Fig. 2: Representative resp o n ses of ovulated herring to sequential
pheromonal stim ulation... 37 Fig. 3; Classification of herring by concentration of milt or teste s extract
required to elicit a positive resp o n se...42 Fig. 4: Scatter plots of resp o n se times and factors potentially influencing
the response to the spawning pherom one... 4 6 Fig. 5. Proportion of spontaneous fish encountered In screening a ssa y s
plotted against individual variables considered a s possible Influencing factors...57 Fig. 6. Proportion of responsive fish encountered In screening a ssa y s
plotted against variables considered a s influencing fa c to rs ... 59 Fig. 7. S e a so n al c h a n g e s In sexual maturity of captive herring during
the 1994 reproductive s e a s o n ...87 Fig. 8. Light m icrographs of herring ovaries before and after ovulation 89 Fig. 9. Identification of 17-hydroxyprogesterone, 17a,20p-dihydroxy-
progesterone, an d 3a,17a-dihydroxy-5p-pregnan-20-one
immunoreactivity by HPLC... 91 Fig. 10. Identification of 17,20p-P and 3a,17-P-5p Immunoreactivity (ir)
in the glucuronated steroid fraction of pooled plasm a of ripe
m ale herring by thin layer chrom atography (TLC)...93 Fig. 11. S e a so n al c h a n g e s in plasm a steroids of fem ale h e rrin g ...96 Fig. 12. S e a so n al c h a n g e s in plasm a steroids of m ale h e rrin g ... 98 Fig. 13. S e a so n al c h a n g e s in glucuronation ratios of 3 a ,1 7 -P -5 an d
oth er steroids in m ale herring plasm a... 104 Fig. 15. Correlation betw een plasm a levels of m etabolically related
steroids in maturing fem ale fish and ripe fem ale fish... 108 Fig. 16. Correlation betw een plasm a levels of m etabolically related
steroids in maturing m ale fish and ripe m ale fish... 111
Fig. 17. Frequency histogram of concentrations of cortisol
encountered in th e plasm a of h errin g ... 151 Fig. 18. Outline of purification protocols u sed in th e isolation of the
herring spawning p h e ro m o n e ...162 Fig. 19. Determination of m olecular weight of pherom one in S e p -P a k
purified te s te s extract...170 Fig. 20. Elution pattern of pherom onal bioactivity from a HPLC
separation of a Sep-Pak-purified te s te s e x tra ct...178 Fig. 21. C haracterization of positive ion m/z 516 in Sep-Pak-purified
te s te s extract by enzym e digestion an d collision-induced
dissociation with FIA/ESI-MS/MS... 188 Fig. 22. Negative and positive product ion sp ectra of
collisionally-induced dissociation FIA/ESI-MS/MS of synthetic s ta n d a r d s 190 Fig. 23. A negative ion LC/ESI-MS/MS with MRM of th e pool of
bioactive fractions of te s te s extract...193 Fig. 24. A positive ion LC/ESI-MS experim ent with MRM of a bioactive
LH-20-purified te s te s extract... 195 Fig. 25. Elution of pherom onal bioactivity of m ature te s te s from HPLC
with an isopropanol mobile p h a s e ...198
Fig. 26. M ature te ste s negative ion m icrobore LC/ESI-MS
chrom atography of fraction 40 of a HPLC sep aratio n of the
XX
Fig. 27. Immature te ste s negative ion microbore LC/ESI-MS chrom atography of fraction 40 of an HPLC sep aratio n of an
extract of immature te s te s ...201 Fig. 28. Elution of bioactivity from high pH mini-bore HPLC... 203 Fig. 29. Elution of irGnRH during chrom atographic purification of an
extract of herring b ra in s... 233 Fig. 30. Elution of irGnRH during chrom atographic purification of an
extract of pituitaries of h errin g ...236 Fig. 31. Matrix a ssisted laser desorption ionization (MALDI) m ass
spectrum of h G nR H ... 238
Fig. 32. Phylogenetic relationships of GnRH preprohorm one
m olecules a s d ed u ced from a maximum parsimony analysis
an d a neighbor joining analysis of known se q u e n c e s ... 241 Fig. 33. Phylogenetic relationships of known GnRH forms d ed u ced
with PAUP and NJ a n a ly s e s ... 243 Fig. 34. Com parison of known GnRH structures with herring G nR H ...247 Fig. 35. Proposed evolutionary schem e for GnRH multiplicity in
te le o s ts ...250 Fig. 36. P roposed evolutionary relationships of known GnRH
m olecules... 255 Fig. 37. T he cDNA nucleotide se q u en c e of herring gonadotropin ll-(3
subunit (GtH ll-P)... 271 Fig. 38. Com parison of a a se q u en c e of herring GTH ll-p precu rso r
with th o se of GTH ll-p precursors or m ature m olecules of
other teleo sts... 275 Fig. 39. A bootstrapped strict co n sen su s phylogenetic tre e of
representative fish GtH II, GtH I, LH and FSH-p m ature subunits, b a sed on known amino acid se q u e n c e s ... 282
Fig. 40. Neighbour Joining phyiogram of m ature vertebrate GtH-p
XIII
LIST OF ABBREVIATIONS
Chemical names:
11-DOC: 11-deoxycortisol, or21-hydroxy-4-pregnen-3,20-dione 11 -KT : 11 -k etotestosterone
3 a, 17-P-Sp: 3 a. 17a-dihydroxy-5p-pregnan-20-one
17-P: 17a-hydroxyprogesterone or 17a-hydroxy-4-pregnen-3,20-dlone 17,20a-P : 17a, 20a-dihydroxy-4-pregnen-3-one
17,20p~P: 17a, 20IÎ -dihydroxyprogesterone or 17a, 20S-dihydroxy-4- preg n en -3 -o n e
17a,20p,21-P : 17a,20p,21-trihydroxyprogesterone or 1 7 a,2 0 p ,2 1 -hydroxy-4-pregnen-3-one
A: an d ro sten ed io n e AON: acetonitrile
BSA: bovine serum albumin DOM: dichlorom ethane
EGMEE: ethylene glycol monoethyl ether E: estradiol
FSH: follicle stimulating hormone
GnRH: gonadotropin releasing horm one GtH: gonadotropin
LH: luteinizing horm one
MIS: m aturation inducing steroid RNA: ribonucleic acid
T: testo ste ro n e
TEAF: triethylammonium formate TEAP: triethylammonium phosphate
TFA: triflouroacidic acid
Other terms:
+ve; responsive to pherom one (behaviour); positive Ionization (MS) -ve: not responsive to pherom one (behaviour); negative Ionization (MS) cDNA: com plem entary DMA
CID: colllslonally-lnduced dissociation;
ESI-MS: m ass spectrom etry with an electrospray Ionization source; ESI; electrospray Ionization;
FIA: flow-lnjectlon analysis; g.e.: gram equivalent;
GC: g a s chrom atography; GSI: gonadosom atic Index
HPLC: high p ressu re liquid chrom atography;
LC/ESI-MS: linked liquid chrom atography-m ass spectrom etry with an electrospray Interface;
LC: liquid chrom atography; LH-20: LH-20 S ep h ad ex gel;
MALDI/MS, matrix a ssiste d laser desporptlon m ass spectrom etry MRM: multiple reaction monitoring;
MS/MS: tandem m a ss spectrom etry. MS: m ass spectrom etry;
NJ: neighbour joining
PCR: polym erase chain reaction UTR: untranslated region
XV
ACKNOWLEDGEMENTS
I am particularly grateful to my supervisor, Dr. Nancy Sherw ood, for h er a ssista n c e and Input during this work. I also thank Ann and Tim M agnus (University of Calgary), Carol Warby, Ben Koop, Andrew McArthur, an d Kris von Schalburg (University of Victoria), Monica Tester, Pam Thuringer (Victoria, B.C.), Wolfgang Carolsfeld (Nanaimo, B.C.), Rob Meyers (Vancouver, B.C.), Henrik Krelberg, Ian Whyte, Doug Hay (Pacific Biological Station, Nanaimo, B.C.), Sandy Scott (Ministry of Agriculture, Food, and Fisheries, Lowestoft, England), Mike Ikonomou, Tim He (Institute of O cean Sciences), Stephen Pleasance, Pearl Blay, and Pierre Thiabault (Institute for Marine BloSclences, Halifax, N.S.), for assistan ce and advice for this work; John Boom, Andy Spencer, and other staff of the Bamfleld Marine Station for assistan ce and provision of facilities; Jackm an Productions (Bamfleld, B.C.) for u se of their filming tank and access to the videotape of spawning; Lorena Hammer (Pacific Biological Station, Nanaimo, B.C.) and the crew of the seiner. Pacific Skye, for providing fish. Dr. C. Schreck and G. Feist (University of Oregon) for providing advice on the cortisol a ssa y , and my family (Kit, Anna Carolina, Anna Lucia, Tom as, Penny, Sadie, and Mr. Bill) for their patience and support. Financial a ssista n c e for the work w as provided by the National Science an d Engineering R esearch Council (NSERC) of C anada, the Science Council of British Columbia, the D epartm ent of Fisheries and O cean s (C anada), the Industrial R esearch A ssistan ce Program of NSERC, Archipelago Marine R esearch, Victoria, B.C., the Innovation and Developm ent Corp. of th e University of Victoria, P e a rso n Fishing Co., Russell Robinson, and th e T oquaht Band.
AND THE ROLE OF HERRING RESEARCH
P herom ones are com pounds that a re se cre ted to th e exterior by o n e organism to Induce a specific reaction In a conspeclfic (Karlson a n d Lüscher, 1959). T h ese participate In a wide variety of Interactions a m o n g st Individuals of many species, ranging from pro to zo an s and a lg a e to vertebrates, and have been described a s the “first signals put to service In the evolution of life” (Wilson, 1975),. Pherom onal effects have b e en
reported for reproduction, developm ental synchronisation, social
structuring, alarm signalling, trail marking, territory marking, dispersion, aggregation, aggression, and schooling. T h ese effects Include both th e modification of physiological p ro ce sses and the triggering of behaviours, classified a s “priming” and “releasing” pherom ones. respectively (Wilson, 1963).
Structural Identification of pherom ones Is Important for elucidating the m echanism s of pheromonal communication, but their low natural concentration after secretion h as m ade structural determ ination difficult. N evertheless, starting with the Identification of bombykol In th e silk moth (B utenandt e t al., 1959), pherom ones of a variety of anim als have now b e en Identified, many of them pherom ones with reproductive function (“reproductive pherom ones”; Table 1). R eleasing p h erom ones of Insects have b een particularly am enable to study, largely due to the availability of practical b lo assay s (Blum, 1985; Struble and Arn, 1984, Vogt, 1987). T he picture that h a s em erged for this group of anim als Is that their pherom ones a re generally a blend of com pounds. In which both the
Table 1: Chemical nature of representative reproductive pheromones
Chemical
class Compound(s) Precursor Source Occurrence Sex’ Function’ Ref^ Comments
fatty acids
prostaglandins (P G s) P G horm ones? go n ad ? teleosts m/f induce spawning 14
stiort Chain FA vaginal discharge prim ates f excite m blend
long ctialn HC
alcohols fatty acids epiderm al glands Lepldoptera f attract m 2 blends; evolutionary tn
aldehydes a c e ta te s
ketones & ace ta tes - urine m ouse f Inhibit f maturation 5
methyl ketones skin lipids Integum ent garter sn ak e f elicit m mating 10 blend
m uscalure fatty acid s & a cetate cutlcular h o u se fly f attraction 3
terpenes
mono-terp acetate epiderm al Insects m/f attraction 3
& external s o u rc e s g lands
farn e sc e n e . epidermal m ouse m dom inance 11
g lan d s
sq u alen e skin sn a k e s f m copulation 9 blend
stero id s & conjugates maturation horm one gonad? teleo sts ( Induce final maturation 14
sesqulterpenedloi sirenin . fungus f attract sperm 7 m pherom one similar
pyrazlne - urine m ouse f Inhibit f maturation 5 blend
thiazole - urine m ouse m W hitten effect 11 blend w / fa m e sc en e s
brevlcom - urine m ouse m W hitten effect 11 blend w/ fa m e sc e n e s
5o-androstenone pregnenolone urine & m usk pig m f mating behavior 12 possibly part of blend
& 3a-androstenol aromatic
phenylelhyl alcohol phenylalanine epiderm al gland Insect m Induce f copulation 3
dichlorophenol tyrosine? epiderm al ticks f m attraction 3
vanillic acid nem atode f m attraction 4 single com pound
Chemical
class Compound(s) Precursor Source Occurrence Sex’ Function’ Ref^ Comments’
Proteins
aphrodlsn - vaginal? ham ster f m copulation 13 related to carrier protein
a -m a tin g factor - - yeast - induces mating 6 contains GnRH-like segm ent
Nucleotides
CAMP slime mold attraction 1
O thers
cyclopropanes etc. fatty acid fragment - brown algae f sperm attraction 8 taxonomic tren d s evident
dimethyl sulfate - - ham ster f m attraction 13
alkaloids plant alkaloids epidermal hairs Lepldoptera m f copulation 3
’ m=male, f=female
' R eferences: 1. Bonner, 1963; 2. Blum, 1985; 3. Blum, 1987; 4. Jaffe et al., 1989; 5. Jemlllo et al., 1986; 6. LeRoItti & Roth, 1984;
7. Machlls et al., 1968; 8. Maler & Muller, 1986; 9. M ason et al., 1989; 10. M ason et al., 1990; 11. Novotny et al., 1990; 12. R eed et al., 1974; 13. Singer e t al., 1987; 14. S o ren sen & Stacey, 1990.
Chemical nature and relative concentration of th e various com ponents contributes to the potency and species-specificity.
Evolutionary relationships am ongst the chem ical structures of pherom ones is evident in closely related species, but not am ong th o se of m ore distantly related insects or other organism s (Table 1). S ensory m echanism s and transduction pathw ays of the pherom onal signals, on the o th er hand, a p p e a r similar, though homology h a s not b e e n clearly dem onstrated. As rep resen ted by m oths and butterflies, th e resp o n se m echanism to pherom ones co n sists of 1) finely tuned recep to r cells in sensillary hairs of antennules, an d 2) dedicated “labelled-line” primary
axons leading to specialized macroglomeruli in antennal lobes
(M ustaparta, 1984). Details of the su b seq u e n t neural m echanism s responsible for distinctive behavioural resp o n se s to the pherom onal input a re still poorly understood, but also a p p e a r to have pherom one-specific pathw ays in som e c a s e s (H ansson, 1995).
V ertebrate pherom ones have yielded less readily to structural identification, possibly b e c a u se th e s e pherom ones tend to elicit more subtle influences on physiology or behaviour than th e insect releasing pherom ones, often in concert with other cu es or asso ciativ e learning (Singer et al., 1987; Wilson, 1970). Hence, effective b io a ssa y s a re often m ore difficult to establish. However, reproductive p h ero m o n es of a variety of vertebrates a re now known (Table 1). T h e se include priming pherom ones of mice and rats (se e Novotny et al., 1990), an d releasing pherom ones of sn a k es (Mason et al., 1989) and h am sters (Singer et al., 1987). Chemical signalling for a variety of functions is likely to be important to many other vertebrates a s well (Wilson, 1975), but a lack of information on the chemical identity of the pherom ones h a s slowed research on specific reactions. P resen t evidence su g g e sts that, a s in
insects, pherom ones of vertebrates a re diverse in structure (Table 1), while transduction m echanism s may b e hom ologous to o n e another. T he “vom eronasal” organ is an olfactory structure th at is found in m ost tetrapods, and is believed to be primarily involved in pherom one detection (Bertmar, 1981; Eisthen, 1992; Dorries et al., 1997).
Fish a re representatives of a basal vertebrate lin eag e predating the evolution of vom eronasal organs. However, pherom onal comm unication in th e se vertebrates is also well developed (e.g. Colombo, et al., 1982; S orensen, 1992; Table 2) and morphological specialization for olfactory detection of pherom ones a p p ea rs to be p resen t (Dulka, 1993). The reproductive pherom ones of fish are of particular interest in that m any a p p e a r to be related to reproductive horm ones that a re distinctive of the sta g e of reproduction when the pherom onal com m unication is m ost appropriate (Stacey and Sorensen, 1991 ). Thus, th e s e ph ero m o n es may rep resen t an early stag e in the evolution of pherom onal com m unication a s described by W ynne-Edwards (1962), who su g g e ste d th at "all functional odors h av e evolved from m etabolites originally se c re te d for an o th er function". Studies of "primitive" pherom onal system s su ch a s th o se found in fish reproduction may provide an understanding of pherom onal communication in vertebrates.
Spawning of the Pacific herring occurs in large sch o o ls without noticeable pairing betw een sex es or direct behavioural interaction
betw een individuals (Schaeffer, 1937), which is thought to be
characteristic of a primitive reproductive strategy in fish (K eenleyside, 1979). A spaw ning pherom one involved in synchronization of spawning of herring (Stacey and Hourston, 1982; Sherw ood et al., 1991; Carolsfeld e t al., 1992) is unusual in that it is produced by m ales but rapidly elicits spaw ning in individuals of both sex es. Most o th er reproductive
6
pherom ones offish th at have b e e n studied elicit a le ss c le a r physiological or behavioural re sp o n se an d a c t primarily on only th e o p p o site sex (Table 2). T he herring pherom one m ay thus provide an informative co n trast to p h erom ones of o th er fish, an d help answ er so m e of th e principal qu estio n s now challenging this field of research a s d e scrib e d below.
1 ) Are fish pheromones specialized signals for communication ?
Communication h a s b e en defined by Wilson (1975) an d o th ers a s a bilateral p ro cess in which both the signaller and th e recipient participate in a specialized m anner specific to the act of comm unicating. Controlled re le a s e of reproductive pherom ones of fish specifically for comm unication, however, h as rarely b een dem onstrated. Rather, a physiological or behavioural resp o n se to the smell of horm ones and horm one m etabolites ex creted during particular reproductive sta te s a p p e a rs to h a v e evolved in th e recipient without specific specialization of the signal by th e em itter (S o re n se n and Scott, 1994). This level of interaction h a s b e e n term ed "spying", and may b e an evolutionary precursor of true chem ical communication, in which specialization of both th e re le a s e an d th e reception of a signal exists (S tacey and S orensen, 1991).
W e do not yet know if the spaw ning pherom one of herring consists of com pounds produced specifically for communication. T he m ale African catfish, blenny and black goby attract fem ales with androgenic com pounds produced in specialized structures (Resink, 1988; Laum en e t al.. 1974; Colombo et al., 1980). T h ese may be rare te leo st exam ples of
true communication with reproductive pherom ones. No specialized
Pherom one Source
Type Species Tissue Sex’ Maturity R esponse Sex’ Structure^ R e f Releasing:
Alarm
Ostariophysll m innow s & o thers skin m & f all anti-predator m & f 2 2 ,3 2 Kin recognition
Clchlasoma nigrofasclatum cichlld m & f brooding recognition of young m & f 17 Ictalurids catfish skin m ucous m & f all recognition m & f neuropeptides? 5
Oncorhynchus Msutch coho salm on skin m ucous m & f all homing m & f 7 .1 3
Petromyzon marinus s e a lam prey bile m & f larvae homing m & f bile acids 16
Phoxinus phoxinus m innow skin m ucous m & f all attraction for schooling m & f 6 ,3 3
Plotosus lineatus m arine catfish m & f all attraction for schooling m & f 9 Reproductive
Achelloganthus lanceolatus sle n d er bitterling ovarian fluid f ovulated attraction m 11
Bathygobius soporator frillfin goby ovarian fluid f ovulated courting m 29
Belontllds f ovulated attraction m 15
Belontllds m attraction f 15
Blennlus pavo blenny m attraction f 14
Brachydanlo ratio zebrafish ovary f ovulated attraction & courtship m steroid glucuronldes 30
Brachydanlo ratio zebrafish m attraction f steroid glucuronldes 1 ,3 1
Carassius auratus goldfish ovarian fluid f ovulated attraction m eth er soluble 20
Carasslus auratus Goldfish urine f ripe courting m 15kelo-PG Fiu 24
Clatias gatiepinus African catfish sem inal vesicles m attraction f 3 o ,1 7 -P -5 p ? 31
Clupea harengus pallasi Pacific herring milt m sperm lated spaw ning m & f steroid conjugates 2 1 ,2 6
Goblus jozo black goby mesorchlal m attraction & oviposition f etiocholanolone glue 4
Haplochrotnis burtonl cichlld f gravid courting m 7
Hypotnesus olldus pondsm elt ovarian fluid f courtship m 19
HypottKsus olldus pondsm elt te ste s m ( 19
Hypotnesus olldus pondsm elt ovarian fluid f courtship m 19
MIsgutnus angullllcaudalus loach ovarian fluid f ovulated attraction m 12
Oncothynchus masou y a m a m e salm on ovarian fluid f ovulated attraction m 12
Oncothynchus thodums a m a g o sa lm o n ovarian fluid f ovulated attraction m 12
Plecoglossus altlvells ayu ovarian fluid f ovulated attraction m w ater-ether soluble 10
Poecllla chica guppy (
Poecllla reticulata guppy f attraction & courting m 28
Rhodeus ocellatus ro se bitterling ovarian fluid f ovulated attraction m 11
Saltno galrdnetl Rainbow trout ovarian fluid ( ovulated attraction m w ater-ether soluble 11
Salmo gairdnerl Ralnl)OW trout m attraction f 18
Table 2. Representative pheromones o ffish (Cont'd) Pherom one
Type Species
Source
T issue Sex^ Maturity R esponse Sex Structure Ref® Priming; Reproductive Brachydânio rerio Cerasslus auratus Ptewphyllum scalare Growth various zebrafish Goldfish clchlld ovary/urlne m f m ature m
ovarian growth & ovulatl sperm latlon
ovarian growth & ovulatl
growth
f 2
m 17,20p-P; 1 7 ,2 0 p -P s u 2 5 ,2 7
f 3
m & ( liptd-solut>le S. 23
’ f = fem ale; m = m ale
*1 5 keto-PGFja = 15-keto-prostaglandln F j, ; 3a1 7 -P -5 p = 3 a , 17a-dlhydroxy-5-pregan-20-one; 17,20p-P ■= 1 7 a, 20p-dlhydroxy-4-pregnen-3-one
* R eferences; 1. Bloom an d Perim utter, 1977; 2. C hen & Martlnlch, 1975; 3.Chlen, 1973; ^.C olom bo et al., 1980; 5. Colom bo e t al., 1982 (review); 6. Crapon d e C rapona, 198 7 . Doving et al., 1974; 8. G oz, 1941; 9. HayashI et al., 1994; 10. H onda, 1979; 11. Honda, 1980a; 12. Honda, 1980b; 13. Jo h n so n & Halser, 1980; 14. L aum en et al., 1974 15. Lee & Ingersoll, 1979; 16. Ll et al., 1995; 17. Myrberg, 1975; 18. N ew com be & Hartm ann, 1973; 19. O kada e t al., 1978; 20. Partridge et al., 1976; 21. S herw ood e t al. 22. Sm ith, 1992 (review); 23. Solom on, 1977; 24. S o re n sen et al., 1996 (review); 25. S o re n sen , 1996 (review); 26. S ta c e y & Hourston, 1982; 27. S ta c e y & S o re n se n , 1986 28. S ta c e y et al., 1986; 29. Tavolga, 1956; 30. Van den H urk& Lam bert, 1983; 31. Van don H urkft ResInk, 1992 (review); 32, von Frisch, 1941; 33. W re d e , 1932.
Structural elucidation of the spawning pherom one should help resolve the type of chemical interaction th at is u sed by herring.
2) How are teleost pheromones species specific?
T he majority of reproductive pherom ones identified in fish so far a re steroids. However, many of the steroid horm ones a sso c iated with fish reproduction a re common to a variety of fish sp ecies, m any of which may b e sympatric. This raises the problem of w hether an unspecialized pherom one elicits heterospecific resp o n se s. Most of th e p h ero m o n es investigated a re asso ciated with the relatively short period of final m aturation and/or spawning, so potential heterospecific re s p o n s e s may be limited to fish that are in an appropriate physiological sta te and in clo se proximity. Resink (1988), however, also d escrib es ph ero m o n es of African catfish that are active during the m ore prolonged period of vitellogenesis. So far, a definitive m echanism h a s not b e en discovered that explains why individuals of o ther sp e cie s do not also resp o n d to a pherom one-em itting fish, but several h y p o th eses have b e en developed. Van W eerd and Richter (1991) and S tacey and S o ren sen (1991) su g g e st that the steroids of interest a re m etabolised by sufficiently different pathw ays that a species-specific signal is readily formed from individual steroids, mixtures of steroids, or mixtures of steroids with non-horm onal com ponents. In fact, while m ost pherom ones studied so far contain com pounds that have pherom onal activity on their own, th e natural pherom one probably consists of a mixture of com pounds (Colombo e t al., 1982; Sorensen, 1996; Van W eerd and Richter, 1991). A species-specific pherom one signal of this kind could exist without specialization of the signal for communication, particularly if selective olfactory sensitivity
10
evolves In th e responding fish (S orensen e t a!.. 1990). S o re n se n and S tacey (1990) also su g g e st that pherom one-containing urine may be se c re te d in a species-specific pulsatile fashion. This reg u lated m ode of em ission would provide a m echanism for true com m unication.
Herring milt containing pherom one is p re se n t in substantial quantities in th e w ater of a spawning school. T he chem ical identity of the pherom one com ponents may indicate how the p h ero m o n e is sp e c ie s- specific, if th e structures are unique. In addition, a n an aly sis of the factors regulating resp o n siv en e ss to the pherom one in herring should provide important clu es a s to how the pherom one contributes to “school" spaw ning of th e sp ecies.
3) How does reproductive physiology influence pheromonal communication?
Both "priming" pherom ones that trigger final ste p s of m aturation of the reproductive system and "releasing" ph ero m o n es th at attract the o pposite se x or e n h a n c e spawning behaviour h av e b e e n d escrib ed for teleo sts (Van W eerd an d Richter, 1991). T he "horm onal pherom one" hypothesis for fish (Stacey and S orensen, 1991) su g g e sts that ph ero m o n es signal th e reproductive sta te of the em itter by their content of horm one-related su b sta n c e s. Known priming p h ero m o n es of fem ales are related to th e steroid involved in the final m aturation of oocytes, a s
predicted by th e hypothesis. However, the horm onal control of
reproduction of m ale fish is not a s well understood a s th at of fem ale fish, so th e relationship of th e s e pherom ones to reproductive physiology is less clear. Priming p h erom ones of the male zebrafish a re pro d u ced in the te s te s (Van Den Hurk et al., 1987) and a pherom one of th e m ale Baikal
sculpin is related to testo ste ro n e, a steroid important in m ale m aturation (S tacey an d S o ren sen , 1991).
Spaw ning of fem ales is regulated by prostaglandins in th e goldfish, so p rostaglandins c an b e ex p ected to b e fem ale releasing p h ero m o n e for eliciting spaw ning behaviour of m ale fish. However, re le a s e r p h ero m o n es th at h av e b een found a re m ore diverse: the fem ale goldfish a n d arctic c h a r p h erom ones (S o re n se n e t al., 1988; Svensen, pers. com.) a re prostaglandins, w h e rea s th e fem ale zebrafish pherom one contains estradiol an d testo ste ro n e m etabolites (Van den Hurk and Lam bert, 1983) an d th e pherom one of fem ale guppies contains estradiol an d its m etabolites (Jo h an sen , 1985). Elevated levels of estradiol and te sto ste ro n e a re typical of fish undergoing gonadal growth, rath er than spaw ning, so the u se of th e s e steroids a s pherom ones during final s ta g e s in reproduction is anom alous. T he horm one directly a sso c ia te d with spaw ning in m ale te le o sts is not clear, though a p ro g estero n e probably controls sperm hydration ju st prior to spawning (Fostier et a!., 1987) and te sto ste ro n e may influence m ale reproductive behaviour (Liley et al., 1987). T he pherom one of the m ale African catfish is a p ro g estero n e (Resink, 1988); and th e ph ero m o n es of the black goby (Colombo e t al., 1980) an d Baikal sculpin (S tacey and Sorensen, 1991) a re a n d ro g e n s, so th e few m ale pherom ones identified may fit the proposed m odel.
R esp o n siv en ess to the pherom one also a p p ears to b e reg u lated by th e reproductive state, although olfactory sensitivity in so m e s p e c ie s m ay not be. Most of the known pherom ones induce a resp o n se only in fish of th e appropriate reproductive sta te (Stacey and S o ren sen , 1991; Van W eerd an d Richter, 1991). However, S o ren sen et al. (1988) a n d R esink (1988) have show n th at high olfactory sensitivity for p h ero m o n es p ersists throughout the y ear in goldfish an d African catfish, w h e rea s in so m e o th er
1 2
cyprinids a se a so n a l c h an g e In sensitivity occurs (S o re n se n e t al., 1987). Also, Cardwell e t al. (1995) have dem onstrated th at Implanted a n d ro g e n s can selectively e n h an c e olfactory sensitivity to a putative reproductive pherom one In m ale Puntius schwanenfeldi.
Pacific herring resp o n d to the spawning pherom one only w hen they a re producing milt or a re ovulated an d th e te s te s contain pherom one only w hen fully m ature (S tacey and Hourston, 1982). Thus, pherom one production an d reception Is limited to a particular reproductive sta te In this sp ecies. Elucidation of th e hormonal control regulating reproduction In herring could both provide clues to th e chem ical n atu re of the pherom one and Indicate how th e reproductive state Interacts with pherom onal comm unication In this sp ecies.
T he horm ones Involved In the reproductive physiology of herring a re unknown, though generalized m odels of reproductive endocrinology of fish h av e b e e n proposed (N agaham a, 1994; Fostier et al.. 1987). Information on the structure and function of reproductive horm ones of the herring Is of Interest for predicting w hat horm ones may b e available for pherom onal comm unication and for Investigating factors th at m ay regulate resp o n siv en e ss to the pherom one. In addition, this know ledge ab o u t a primitive teleo st such a s the herring contributes to our understanding of horm one evolution.
In this thesis, I characterize the structure and function of the spawning pherom one of Pacific herring and Investigate th e reproductive physiology underlying the pherom onal communication system . This w as d one by; 1) characterizing the resp o n se to th e pherom one an d factors regulating It, 2) characterizing the pattern s of reproductive stero id s found during reproduction In th e herring, 3) Investigating correlations b etw een levels of steroidal horm ones and resp o n siv en e ss to th e pherom one an d
4) characterizing the structure of th e spaw ning pherom one. This work is p resen ted in C hapters 2-5. In addition, I furthered th e developm ent of the herring model for pherom onal comm unication by helping to elu cid ate the structures of gonadotropin releasing horm one an d gonadotropin-ll S subunit p resen t in this sp e cie s of fish (A ppendices A and B). T h e se proteins have a central role in th e control of reproduction in fish
(Sherwood, et al., 1994; Q uerat, 1992; Sw anson, 1992), but
understanding of their function will b e furthered by knowledge of their actions in a primitive teleost such a s the herring. Structural elucidation is a n important first step in pursuing this line of research .
REFERENCES CITED
Bertmar. G. (1981). Evolution of vom eronasal o rg an s in v erteb rates. Evolution 35: 359-366.
Bloom, H. and Perimutter, A. (1977). A sexual aggregating pherom one system in the zebrafish, Brachydanfo rerio (Hamilton-Buchanan). J. Exp. Zoo/. 199: 215-226.
Blum, M.S. (1985). Exocrine system s. IN: Fundamentals o f insect physiology. (Blum, M.S., ed.) John Wiley a n d Sons, New York, pp. 535-579.
Blum, M.S. (1987). Biosynthesis of arthropod exocrine com pounds. Ann. Rev. Entomol. 32: 381-413.
Bonner, J.T. (1963). How slime molds com m unicate. Soi. Am. 209: 84- 93.
Butenant, A., Beckmann, R., Stamm, D., an d Hecker, E. (1959). Ü b e rd e n Sexuallockstoff d e s S eid en sp in n ers Bombyx mori. R eindarstellun und Konstitution. Z. Naturf. 14b: 283-284.
14
Cardwell, J R., Dulka, J.G . an d Stacey, N.E. (1992). Specles-speclficity of olfactory resp o n siv en ess to potential se x pherom ones. IN: Proœedings o f the Fourth International Symposium on the Reproductive Physiology o f Fish. Norwich, July 7-12, 1992. (Scott, A.P.; Sumpter, J.P.; Kime, D.E. a n d Rolfe, M.S., eds.), FishSym p 91, Sheffield, p. 201.
Cardwell, J R., Stacey, N.E., Tan, E .S.P., McAdam, D.S.O. an d Lang, S.L.C. (1995). Androgen in c re a se s olfactory recep to r re s p o n se to a v erteb rate sex pherom one. J. Comp. Physiol. A 176: 55-61. Carolsfeld, J., Sherwood, N.M., Kyle, A.L., M agnus, T.H., P le asan c e, S.
a n d Kreiberg, H. (1992). C haracterization of a spaw ning p h erom one from Pacific herring. IN: Chemical Signals in Vertebrates VI. (Doty, R.L. an d M üller-Schwarze, □., eds.). Plenum P ress, pp. 343-348.
C hen, L.C. an d Martinich, R.L. (1975). Pherom onal stimulation an d m etabolite inhibition of ovulation in th e zebrafish, Brachydanio rerio. Fish. Bull. 73: 889
Chien, A.K. (1973). Reproductive behaviour of the angelfish,
Rerophyllum scalare (Pisces: Cichlidae): II. Influence of m ale stimuli upon the spaw ning rate of fem ales. Anim. Behav. 21; 457- 463.
Colombo, L., Belvedere, P.C., M arconato, A., an d Bentivegna, F. (1982). P h ero m o n es in teleo st fish. IN: Proceedings o f the Second International Symposium on Reproductive Physiology o f Fish. (Richter, C .J.J. and Goos, H.J.T., ed s.) Pudoc, T he N etherlands, pp. 84-94.
Colombo, L., Marconato, A., B elvedere, P.C., a n d Frisco, C. (1980). Endocrinology of teleo st reproduction: a testicular steroid
p herom one in th e black goby, Gobius jozo L. Boll. Zool. 47: 355- 364.
C rapon d e C rapona, M.-D. (1980). Olfactory com m unication In a clchlld fish, Haplochromis burtoni. Z. Tierpsychol. 52: 113-134.
Dorrles, K.M., A dklns-Regan, E., Halpern, B.P. (1997). Sensitivity and behavioral re s p o n se s to the pherom one an d ro sten o n e a re not m ediated by th e vom eronasal organ In dom estic pigs. Brain, Behavior and Evolution 49: 53-62.
Deving, K.B., Nordeng, H., and Oakley, B. (1974). Single unit discrimination of fish odours rele ased by ch ar {Salmo alpinus L ) populations. Comp. Biochem. Physiol. 47A: 1051-1063.
Dulka, J.G . (1993). S ex pherom one sy stem s In goldfish: com parisons to vom eronasal system s In tetrapods. Brain, Behavior and Evolution 42: 265-280.
Dulka, J.G ., Stacey, N.E., Sorensen, P.W., an d Van D er Kraak, G.J. (1987). A sex steroid pherom one synchronizes m ale-fem ale spaw ning read in ess In goldfish. Nature 325: 251-253.
Eisthen, H.L. (1992). Phylogeny of the vom eronasal system and of recep to r cell types In th e olfactory and vom eronasal epithella of v erteb rates. Microsc. Res. Technique 23: 1-21.
Fostier, A., Le Gac, P., and Loir, M. (1987). Steroids In male reproduction. IN: Idler, D.R., Crim, L.W., W alsh, J.M. (eds.). Proc. 3 ^ Intl. Symp. Reprod. Physiol. Fish. Memorial University P ress, St. Johns, Nfld. pp. 239-240.
Goz, H. (1941). Ü b e rd e n Art- und Indivldualgeruch be! FIschen. Z. vergl. Physiol. 2 9 :1 -4 5 .
H ansson, B.S. (1995). Olfaction In Lepldoptera. Experientia 51: 1003- 1027.
16
Hayashi, N., Nakamura, S., Yoshikawa, H., Abe, T., an d Kobayashi, H. (1994). A role of olfaction In schooling In J a p a n e s e s e a catfish, Plotosus lineatus. Japanese Journal o f Ichthyology 4 f : 7-13. Honda, H. (1979). Fem ale sex pherom one of th e ayu, Plecoglussus
altivelis, Involved In courtship behaviour. Bull. Jap. Soc. Scien. Fish. 46: 1375-1380.
Honda, H. (1980a). Fem ale sex pherom one of rainbow trout, Salmo gairdneri, Involved In courtship behaviour. Bull. Jap. Soc. Scien. Fish. 46:1109-1112.
Honda, H. (1980b). Fem ale sex pherom one of th e loach, Misgurnus anguillicaudatus, Involved In courtship behaviour. Bull. Jap. Soc. Scien. Fish. 46:1223-1225.
Jaffe, H., Huettel, R.N., Demllo, A.B., Hayes, D.K., an d R ebels, R.V. (1989). Isolation and Identification of a com pound from soybean cyst nem atode, Heterodera glycines, with se x pherom one activity. J. Chem. Eco/. 15: 2031-2043.
Jo h a n se n , P. H. (1985). Fem ale pherom one a n d th e behavior of male g u p p ies {Poecilia reticulata) In a tem p eratu re gradient. Can. J. Zool. 63: 1211-1213.
Jo h n so n , P.B. and Hasler, A.D. (1980). T he u se of chem ical c u e s In the
upstream migration of coho salmon, Oncorhyncus kisutch
W albaum . J. Fish Biol. 17: 67-73.
Jemlllo, B., Andreollnl, F., and Novotny, M. (1986). Chemical and Biological Investigations of fem ale m o u se pherom ones. IN: Chemical Signals in Vertebrates IV. Ecology. Evolution and Comparative Biology. (Duvall, D., M üller-Schwarze, D. and Sllverstein, R.M., eds.). Plenum P ress, NY.
Karlson, P. and LQscher, M. (1959). “Pherom ones”: a new term for a cla ss of biologically active su b sta n c es. Nature (London) 183: 55- 56.
Keenleyside, M.H.A. (1979). Diversity and Adaptation in Fish Behaviour. Springer-Verlag, Heidelberg.
Laumen, J.; Pern, U. and Blum, V. (1974). Investigations on th e function and hormonal regualtion of th e anal a p p e n d a g e s in Blennius pavo (Risso). J. Exp. Zool. 190: 47-56.
Lee, C.T. and Ingersoll, D.W. (1979). Social ch em o sig n als in five Belontiidae (P isces) species. J. Comp. Physiol. Psychol. 9 3 :1 1 7 LeRoith, D. and Roth, J. (1984). V ertebrate horm ones an d n eu ro p ep tid es
in m icrobes: evolutionary origin of intercellular com m unication. IN: Frontiers in Neuroendocrinology vol. 8 (Martini, L. an d G anong, W.F., eds.) R aven P ress, New York. pp. 1-25.
Li, W., S orensen, P.W., an d Gallaher, D. (1995). T he olfactory sy stem of migratory adult s e a lamprey {Petromyzon marinus) is specifically and acutely sensitive to unique bile acids re le a s e d by conspecific larvae. J. Gen. Physiol. 105: 569-587.
Liley, N.R., Cardwell, J.R. and Rouger, Y. (1987). C urrent sta tu s of horm ones and sexual behavior in fish. IN: Proc. 3 ^ Intl. Symp. Reprod. Physiol. Fish. (Idler, D R., Crim, L.W., W alsh, J.M., eds.). Memorial University Press, St. Johns, Nfld. pp. 142-149.
Maier, I. and Müller, D.G. (1986). Sexual pherom ones of a lg a e. Biol. Bull. 170: 145-175.
Mason, R.T., Fales, H.M., Jo n es, T.H., Pannell, L.K., Chinn, J.W . and Crews, D. (1989). S ex pherom ones in sn a k es. Science 245: 290- 293.
1 8
M ason, R.T., Jo n e s, T H., Fales, H.M., Panned, LK. a n d Crew s, D. (1990). C haracterization, synthesis, and behavioral re s p o n s e s to se x attractiv en ess p h erom ones of red-sided g a rte r s n a k e s {Thamnophis sirtalis parietalis). J. Chem. Eco/. 16: 2 3 5 3-2369. M atsuo, H., Baba, Y., Nair, R.M.G., Arimura, A., an d Schally, A.V (1971)
Structure of th e porcine LH- an d FSH -releasing horm one. I. T he p ro p o sed amino acid se q u en c e. Biochem. Blophys. Res. Commun. 4 3 :1 3 3 4 - 1339.
Machlis, L , Nutting, W.H., an d Rapoport, H. (1968). T h e stru ctu re of sirenin. J. Am. Chem. Soc. 90: 6434-6438.
M ustaparta, H. (1984). Olfaction. IN: Chemical Ecology o f Insects. (Bell, W .J. a n d C arde, R.T., eds.). C hapm an an d Hall Ltd., pp. 37-70. Myrberg, A.A. (1975). T he role of chemical an d visual stimuli in th e
preferential discrimination of young by the cichlid fish Cichlasoma nigrofasciatum (Gunther). Z. Tierpsychol. 37: 274-279.
N agaham a, Y. (1994). M olecular biology of oocyte m aturation in fish. IN: Perspectives in Comparative Endocrinology. (Davey, K.G., Peter, R.E., an d Tobe, S.S., eds.). pp. 193-198. National R e se a rc h Council of C an ad a, Ottawa.
N ewcom be, C. an d Hartmann, G. (1973). Som e chem ical sig n a ls in th e spaw ning behavior of rainbow trout {Salmo gairdneri). J. Fish Res. Bd. Canada 30: 995-997.
Novotny, M., Jemilio, B., an d Harvey, S. (1990). C hem istry of rodent pherom ones: m olecular insights into chem ical signalling in m am m als. IN: Chemical Signals in Vertebrates V. (M acD onald, D.W., Müller-Schwarze, D. an d Natynczuk, S.E., e d s.). Oxford University P ress, New York., pp. 2-22.
O kada, H., Sakai, O.K., and Suglkawa, K. (1978). C hem ical stim ulus in th e reproductive behavior of the pondsm elt. Sd. Rep. Hokkaido Fish Hatchery. 33: 89.
Partridge, B .L, Liley, N.R., and Stacey, N.E. (1976). T he role of p h ero m o n es in th e sexual behavior of th e goldfish. Anim. Behav. 24: 291-299.
Q uerat, B. (1992). Molecular evolution of th e glycoprotein horm ones in v erteb rates. IN: Perspectives in Comparative Endocrinology. (Davey, K.G., Peter, R.E., and Tobe, S.S., ed s.). pp. 27-35. National R esearch Council of C anada, Ottawa.
R eed, H.C.B., Melrose, D R., and Patterson, R.L.S. (1974). A ndrogen stero id s a s an aid to the detection of o e stru s in pig artificial insem ination Br. Vet. J. 130: 61-67.
Resink, J.W . (1988). Steroid glucuronides as pheromones in the
reproduction o f the African catfish, Clarias gariepinus. PhD thesis. University of Utrecht, The N etherlands.
Schaeffer, M B. (1937). Notes on the spaw ning of th e Pacific Herring, Clupea pallasi. Copeia, 1937: 57.
Sherw ood, N.M., Kyle, A.L., Kreiberg, H. W arby, C M., M agnus, T.H., Carolsfeld, J. an d Price, W.S. (1991). Partial characterization of a spaw ning pherom one in the herring Clupea harengus pallasi. Can. J. Zool. 69: 91-103.
Sherw ood, N.M.. Parker, D.B., McRory, J.E., an d Lescheid, D.W. (1994). M olecular evolution of growth horm one-releasing horm one and gonadotropin-releasing hormone. IN: Fish Physiology Vol. X lll (Sherw ood, N.M. and Hew, C.L., e d s.) A cadem ic P ress, S an Diego, pp. 2-66.
20
Singer, A.G., Agosta, W.C., Clancy, A.N. and M acrldes, F. (1987). The
chem istry of vom eronasally d e te c te d pherom ones:
characterization of an aphrodisiac protein. IN; The Terminal Nerve (Nervus Termlnalis). Structure, Function, and Evolution. (Demski, L.S. and Schw anzel-Fukuda, ed s.). Ann. N.Y. Acad. Soi. 519: 287-298.
Smith, R .J.F. (1992). Alarm signals in fishes. Rev. Fish Biol. Fish. 2: 33- 63.
Solomon, D.J. (1977). A review of chem ical com m unication in freshw ater fish. J. Fish Biol. 11: 363-376.
S o ren sen , P.W . (1992). Hormones, pherom ones, an d chem oreception. IN: Fish Chemoreception, (Hara, T.J., ed.). C hapm an Hall, London, pp. 150-170.
S o ren sen , P.W . (1996). Biological resp o n siv en e ss to pherom ones provides fundam ental and unique insight into olfactory function. Chem. Senses 21: 245-256.
S o ren sen , P.W. and Scott, A.P. (1994). The evolution of horm onal sex pherom ones in teleo st fish: poor correlation betw een th e pattern of steroid re le a se by goldfish and olfactory sensitivity su g g e sts that th e s e c u e s evolved a s a result of chem ical spying rather that signal specialization. Acta Physiol. Scand. 152: 191-205.
S o ren sen , P.W. and Stacey, N.E. (1990). Identified hormonal
pherom ones in the goldfish: the b a sis for a model of sex pherm one function in teleost fish. IN: Chemical Signals in Vertebrates V. (MacDonald, D., M üller-Schwarze, D. and Silverstein, R.M., ed s.) Oxford Press, pp. 302-311.
S o ren sen , P.W ., Hara, T.J., and Stacey, N.E. (1987). Extrem e olfactory sensitivity of m ature and gonadally-regressed goldfish to a potent
steroidal pherom one, 17a,20b-dihydroxy-4-pregnen-3-one. J. Comp. Physiol. A. 166: 373-383.
S o ren sen , P.W., Hara, T.J., Stacey, N.E., and Goetz, F. Wm. (1988). F prostagladins function a s potent olfactory stim ulants which com prise the post-ovulatory fem ale sex pherm one in goldfish. Biol. Reprod. 39: 1039-1050.
S o ren sen , P.W., Hara, T .J., Stacey, N.E., and Dulka, J.G . (1990). Extreme olfactory specificity of m ale goldfish to th e preovulatory steroidal pherom one 17a, 20p-dihydroxy-4-pregnen-3-one. J. Comp. Physiol. A 166: 373-383.
Stacey, N.E. and Hourston, A S. (1982). Spawning and feeding behavior of captive Pacific herring, Clupea harengus pallasi. Can. J. Fish. Aquat. Soi. 39: 489-498.
Stacey, N.E. and S o ren sen , P.W. (1986). 17a,20(3-dihydroxy-4-pregnen- 3-one: a steroidal prim er pherom one which in c re a se s milt volume in goldfish. Can. J. Zool. 64: 2412-2417.
Stacey, N.E. and S orensen, P.W. (1991). Function and evolution o ffish
hormonal pherom ones. IN: The Biochemistry and Molecular
Biology o f Fishes. Vol. 1. (Hochachka, P.W . and Mommsen, T.P., eds.), pp. 109-135. E lsevier P ress, Amsterdam.
Stacey, N.E., Kyle, A.L., an d Liley, N.R. (1986). Fish reproductive pherom ones. IN: Chemical Signals in Vertebrates IV. Ecology, Evolution, and Comparative Biology. (Duvall, D., Müller- Schw arze, D. and Silverstein, R.M., eds.). Plenum P ress, pp. 119- 133.
Struble, D.L. and Arn, H. (1984). Combined g a s chrom atography and electroantennogram recordings of insect olfactory re s p o n se s. IN:
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Techniques in Pheromone Research. (Hummel, H.E. a n d Miller, T A., ed s.). S pringer Verlag, New York. pp. 161-178.
S w anson, P. (1992). Salm on gonadotropins: reconciling old a n d new ideas. IN: Proceedings o f the Fourth International Symposium on the Reproductive Physiology o f Fish. Norwich, Jul 7-12, 1992. (Scott, A.P.; Sum pter, J.P.; Kime, D.E. a n d Rolfe, M.S., ed s.) FishSym p 91, Sheffield, pp. 2-7.
T avolga, W.N. (1956). Visual, chem ical, a n d sound stimuli a s c u e s in the se x discrim inatory behavior of th e Gobiid fish Bathygobius soporator. Zoologica 41: 49-65.
Van d en Hurk, R. a n d Lambert, J.G.D. (1983). O varian steroid glucuronides function a s se x ph ero m o n es for m ale zebrafish, Brachydanio rerio. Can. J. Zool. 61: 2381-2387.
Van d e n Hurk, R. and Resink, J.W . (1992). Male reproductive sy stem a s se x pherom one producer in teleo st fish. J. Exp. Zool. 261: 204- 213.
Van d en Hurk, R.; Schoonen, W .G .E.J., Van Zoelen, G.A. an d Lambert, J.G.D. (1987). T he biosynthesis of steroid glucuronides in the te s te s of the zebrafish, Brachydanio rerio, an d their pherom onal function a s ovulation inducers. Gen. Comp. Endocrinol. 68: 179 -
188.
Van W eerd, J.H. and Richter, C .J.J. (1991). S ex p h e ro m o n e s and ovarian developm ent in teleo st fish. Comp. Biochem. Physiol. A 100: 517-527.
Vogt, R.G. (1987). T he m olecular b a sis of pherom one reception: its influence on behavior. IN: Pheromone Biochemistry. (Prestw ich, G.D. an d Bloomquist, G.J., eds.). A cadem ic P re ss, New York. pp. 251-269.
von Frisch, K. (1941). Ü ber elnen schreckstoff d er F isch h au t und se in e biologische B edeutung. Z. Vergl. Physiol. 29: 46-146.
Wilson, E.O. (1963). P herom ones. Sci. Am. 2 0 8 :1 0 0 -1 1 4 .
Wilson, E.O. (1970). IN: Chemical Ecology (Sonderheim , E. and
Sim eone, J.B., eds.). A cadem ic P ress, New York.
Wilson, E.O. (1975). Sociobiology, the new Synthesis. B elknap P re ss, Harvard University P re ss. 697 pp.
W rede, W.L. (1932). V ersu ch e über d e r Artduft d e r Elritzen. Z. vergl. Physiol. 17: 510-519.
W ynne-Edwards, V.C. (1962). Animal Dispersion in Relation to Social Behaviour. Oliver a n d Boyd, London, 653 pp.
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CHAPTER 2. BEHAVIORAL CHARACTERIZATION OF PHEROMONE- INDUCED SPAWNING OF PACIFIC HERRING’
ABSTRACT
A sp aw n in g p h e ro m o n e in th e milt (se m e n ) a n d te s t e s o f th e Pacific herring, Clupea harengus pallasi, trig g ers sp aw n in g in sex u ally m a tu re fish of b o th s e x e s a n d is th o u g h t to facilitate sch o o l sp aw n in g of this s p e c ie s . I fou nd th e r e s p o n s e to th e p h e ro m o n e to b e a s te re o ty p e d b eh av io ral s e q u e n c e co n sistin g o f a g ra d e d e x te n sio n of th e g o n a d a l papilla, r e le a s e o f g a m e te s , a n d s p a w n d ep o sitio n beh av io r. T h e r e s p o n s e is trig g ered by a n olfactory stim ulus, a s d e m o n s tra te d by th e elim ination o f th e r e s p o n s e by o cclu sio n o f th e n a re s . S tim u lu s c o n c e n tra tio n s o f ap prox im ately a 1:500 dilution o f fre sh milt o r th e e q u iv a le n t o f 0 .0 2 g of fully m atu re te ste s/m l w e re req u ired to elicit a r e s p o n s e in 50 % of ripe herring th a t a re re s p o n s iv e to th e p h e ro m o n e . F e m a le fish a p p e a r e d to b e le s s sen sitiv e to th e p h e ro m o n e in milt th a n m a le s early in th e sp a w n in g s e a s o n , b u t n o t th e re a fte r. T h e a v e ra g e d u ratio n of r e s p o n s e s of m a le fish w a s lo n g er in r e s p o n s e to c o n c e n tra te d milt th a n to te s te s e x tra cts, b u t no c o n s is te n t d ifferen ce in r e s p o n s e tim es b e tw e e n th e tw o s e x e s w a s d e te c te d . F a c to rs o th e r th a n th e sp aw n in g p h e ro m o n e , m aturity of th e fish a n d s tr e s s w e re a lso found to influence th e sp aw n in g re s p o n s e . F o r ex am p le, e x p o s u re to shallow (3 cm ) w a te r in a sm all ta n k in d u ced " s p o n ta n e o u s " papilla e x te n sio n a n d spaw ning ap proxim ately 2 0 min a fte r refilling th e tan k; occluding th e n a r e s p re v e n te d th is re s p o n s e . Also, th e p r e s e n c e of floating kelp
’ Portions of ttiis chapter have been accepted for publication in Hormones and Behavior with co-authors M. Tester (Victoria, B.C.), H. Kreiberg (Pacific Biological Station, Nanaimo, B.C.), and N.M. Sherwood (Biology Dept., University of Victoria).
(Macrocystis) resu lted in p ro lo n g ed sp aw n in g in a la rg e ta n k a fte r a p h e ro m o n a l stim ulus.
INTRODUCTION
Pacific herring, Clupea harengus pallasi, a re primitive te le o sts that spaw n in large schools in a œnnubium œnfusum (B rader an d R osen, 1966) considered ancestral to other teleo st mating sy ste m s (Balon, 1975; Turner, 1986). School spawning such a s this, without pair form ation or physical interaction betw een sexes, is distinctive of this su b s p e c ie s of herring (Schaeffer, 1937; Stacey and Hourston, 1982), although so m e controversy about this conclusion exists in th e literature (R ounsefeld, 1930). O ther su b sp ecies of herring may spaw n with g re a te r interaction betw een se x e s (Aneer et al., 1983; Ewart, 1884; Holliday, 1958). T he school spaw ning of Pacific herring is apparently facilitated by a p herom one that is p resen t in the milt of this sp e c ie s a n d induces spaw ning behaviour of sexually m ature fish of both s e x e s (milt-producing m ales an d ovulated fem ales) (Stacey and Hourston, 1982; Sherw ood et al., 1991; Carolsfeld e t al., 1992). The spaw ning behaviour sta rts with the extension of the gonadal papilla and culm inates in th e deposition of trails of a d h esiv e eg g s or viscous milt (S tacey and H ourston, 1982). T he p herom one is one of the clearest exam ples of a rep ro d u ctiv e-releaser pherom one in fish in that the spaw ning behaviour is initiated within m inutes of exposure. Attempts to induce large-scale spaw ning of captive herring in o cean p e n s with herring milt, however, h av e b e e n largely unsuccessful (D. Gillis, pers. comm.; Kreiberg and Carolsfeld, unpub. obs.). T he sexual maturity of the fish an d th e s tre s s of th e captive situation a re the two m ost likely factors to influence re s p o n siv e n e ss to the
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p herom one. Fish a p p e a r to respond to reproductive p h e ro m o n e s only w hen a t th e appropriate level of maturity (Van W eerd an d R ichter, 1991 ) and, indeed, immature herring do not resp o n d to th e spaw ning p h ero m o n e (S tacey and Hourston, 1982). S tre s s is c o n sid ere d gen erally to b e inhibitory to reproduction in fish (S um pter e t al., 1994). However, s tr e s s a lso may b e stimulatory in th at m ature herring often sp aw n after being cap tu red an d /o r crowded during com m ercial fishing, a re s p o n s e th at is generally attributed to stre ss d u e to handling. T hus, th e re is a n e e d to correlate reproductive maturity an d s tr e s s level with differences in ph ero m o n e resp o n siv en e ss am ong adult fish. However, th e practical difficulty of working with herring h a s m ea n t th at information on reproductive horm ones b ecam e available only recently (C arolsfeld e t al., 1996; C h ap ter 3).
A p ro ced u re for maintaining m ature captive Pacific herring in net p e n s for prolonged periods of time w as d ev elo p ed in th e 198 0 's (Kreiberg e t al., 1982). S tacey and Hourston (1982) tran sferred su ch cap tiv e fish to sm aller tanks for the initial description of the herring spaw ning pherom one. More recently, we developed an a q u ariu m -b a se d a s s a y for sc ree n in g pherom one-containing extracts, in which individual herring in small a q u aria a re ex p o sed to a small bolus of pherom one-containing te st solutions (Sherw ood et al., 1991; Carolsfeld e t al. 1992). O ur resu lts su g g e s te d that th e s e captive herring an d th e ph ero m o n e a s s a y could b e useful tools for investigating the factors th at reg u late th e re s p o n s e to the pherom one, o n ce th e behaviour u n d er th e s e conditions w as a d e q u a te ly ch aracterized . In this chapter, I investigate th e re s p o n se to th e spaw ning ph ero m o n e an d explore som e of the factors th at influence re s p o n siv e n e ss to th e p h erom one in the aquarium a s s a y a n d in larger tanks. In C h a p te r 4 I investigate th e relationship betw een re s p o n siv e n e ss to th e p h ero m o n e
