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Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects
1980
Morphological, histological, and physiological aspects of Morphological, histological, and physiological aspects of
assimilation in larval spot, leiostomus xanthurus lacepede assimilation in larval spot, leiostomus xanthurus lacepede
John Jeffrey Govoni College of William and Mary - Virginia Institute of Marine Science
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Recommended Citation Recommended Citation Govoni, John Jeffrey, "Morphological, histological, and physiological aspects of assimilation in larval spot, leiostomus xanthurus lacepede" (1980). Dissertations, Theses, and Masters Projects. Paper 1539616672. https://dx.doi.org/doi:10.25773/v5-r4nj-ds72
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ft L 035 87
GOVON1, JOHN JEFFREY
MORPHOLOGICAL, HISTOLOGICAL, AND PHYSIOLOGICAL ASPECTS OF ASSIMILATION IN LARVAL SPOT, LErOSTOMUS XANTHURU5 LACEPEDE
ITie C o lle g e o f W illia m and Mary in V i r g in ia
PH.D. 1980
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niversityMicrcxiims
itemationaJ
MORPHOLOGICAL, HISTOLOGICAL( AND PHYSIOLOGICAL
ASPECTS OF ASSIMILATION IN LARVAL SPOT,
LEIOSTOMUS XANTHUftUS LACEPEOE
A D isse r ta t ion
Presented to
The Faculty o f the School o f Marine Science
The College o f W illiam and Mary In V irg in ia
In P a r t ia l F u l f i l lm e n t
of the Requirements fo r the Degree of
Doctor o f Philosophy
by
John J e f f re y Govoni
1980
APPROVAL SHEET
Tills d is s e r ta t io n 1s submitted In p a r t ia l f u l f i l lm e n t o f
the requirements f o r the degree o f
DOCTOR Of PHILOSOPHY- ) / _ > {
jjbhn J e f f re y Go von 1
Approved * April 1980C '
ohn V. H errlner
Crafg l . Smith
’ Richard L. Wetzel
-Z.David S. PetersNational Oceanic and At.nosphertc A d m in is tra t ion National Marine F isheries Service Southeast F isher ies Center Beaufort Laboratory
11
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ............................................................................................ . v
PROLOGUE......................................................................................................................v1i
LIST OF TABLES..................... , .................................................. . ....................... v111
LIST OF FIGURES................................................................................................ x
ABSTRACT............................................................................................... * xv
INTRODUCTION................................................................................................... . * 2
THE EARLY LIFE HISTORY OFLEI05T0MJS XANTHURUS: A BRIEF REVIEW................................................... 6
ASPECTS OF THE MORPHOLOGICAL, HISTOLOGICAL, AND FUNCTIONAL DEVELOPMENT OF THE ALIMENTARY CANALAND ASSOCIATED STRUCTURES............................................................................ 11
In t ro d u c t io n ................................ , 12
M a te r ia ls and M ethods............................. . .......................................... 13
Results .......................... . . . . . . . 15
D iscuss ion..................................................... 34
EXPERIMENTAL RADIOTRACER STUDIES OF SHORTTEHM CARBON ASSIIMLATION ............................................................................ 39
In tro d u c t io n . ................................ , , . , .......................... 40
Conceptual Framework. . , , ........................................... 44
M a te r ia ls and Methods .............................. . . . . . ...................... 46
R e s u l t s .............................................................. 64
D iscussion ......................................................... 91
1 !1
EPILOGUE
TABLE OF CONTENTS (concluded)
Page
95
APPENDIX.....................................................................................................................97
LITERATURE CITED.............................................................................. « . , , 109
V ITA ........................................................................................................................... 122
i v
ACKNOWLEDGMENTS
I take pleasure In acknowledging Dr. John V. K e rr in e r who in
la rg e part a ffo rded me the o p p o rtu n ity o f fu r th e r graduate study and
who supported me throughout, 1 thank Drs. J . A, Muslck, D. 5. Pe ters ,
C. R. Smith, and R* L, Wetzel f o r th e i r suggestions, c r i t ic is m s , and
review of the manuscript. A dd it io n a l g rad ltude is due Drs* Peters and
Wetiel who endured many long hours of d iscuss ion concerning animal
energetics and rad io tra ce r methodology and who, along w ith Dr. P. L*
Zubko ff, provided necessary la b o ra to ry space and equipment.
Conversations w ith Drs. J . H* S, B la x te r , G. H. B o eh le r t, E. D. Houde,
R. Lasker, G- C* Laurence, and C. P. 0 1Connel were also most h e lp fu l .
This work would have been impossible w ithou t the support o f the
Southeast F ishery Center, N0AA-NMFS, B eau fo rt, North C a ro lin a . I
thank the e n t i re s ta f f fo r t h e i r co rd ia l he lp and assistance* Special
thanks are due D. S. Peters who sponsored my work and A, B. Powell
whose amiable cooperation was in va luab le .
A m u lt itude o f people co n tr ib u te d much to the m ateria l presented
he re in . Among them 1 acknowledge Dr. C, L. Ruddell fo r h is to lo g ic a l
in s t ru c t io n and assistance; J . C. Hoagman and M. H, Peoples fo r
d r a f t in g ; R, T, Doyle fo r photomicroscopy; Dr. J , L* Oupuy, N, T,
Windsor, and A, T. Leggett fo r C h lo re l la and Brachionus p ! 1 c a t i l i s
v
c u l tu re s i J . E. Warrlner f o r techn ica l ass is tance and hand ling o f
Iso topes; J. Colvocoresses fo r computer programing; and A.D. Estes
and H. Powles fo r f ie ld specimens.
I wish to thank Or. R.M. Ibara fo r h is f r ie n d s h ip and continual
In te re s t 1n my progress as a s tudent. I thank my fe l lo w s tuden ts ,
e s p e c ia l ly J. Colvocoresses, B. K l lc h , D* M ark le , L. Mercer, J.
Olney, B. Raschi, J. Ross* G. Sedberry, J. Smith and C, and B. Uenner
f o r t h e i r sympathetic ears*
F in a l ly , and c e r ta in ly most, I an Indebted to Mary jane Govonl fo r
her lo y a l t y , s a c r i f ic e , and enduring support - both m a te r ia l and
mora 1 e .
PROLOGUE
“ I t may be w ortty mentioning here that the o r ig in a l meaning of the
word "organ" is ''working to o l . " This etymology symbolizes the fa c t
that describ ing an organ and studying i t by the techniques o f a l l
science cannot so fa r provide a complete understanding o f i t . The
organ acquires i t s f u l l s ign if icance only when i t fun c t io n s and when
i t s performance is integrated in the l i f e o f the organism."
from " I l lu s io n s o f Understanding"
In Rene Dubos, The Dreams of Reason:
Science and U top ias. 196K
v i i
LIST OF TABLES
Table
1.
2 .
3.
4.
5.
6.
7.
8.
9.
10.
Page
Tooth courts o f la rv a l Lelostomus xanthurus* ■ * * ■ . 26
Gut contents o f la rv a l sp o t, Lelostomus xanthurus ■ . . 28
B loenergetic components o f la rv a l f ishes onvarious d ie ts ..................... 43
Summary o f s t a t i s t i c s c h a ra c te r iz in g development o f d is c re te age cohorts o f la rv a l Lelostomus xanthurus used In carbon a s s im i la t io n experiments * * ♦ * ■ * • * 47
Major n u t r i t io n a l components o f Brachlonus p U c a t l l 1s expressed as a percentage o f to ta l <fry weight - ■ . - ■ 50
Experimental recovery e f f ic ie n c y o f ^CQg.SLBima^ o f re su lts w ith known q u a n t i t ie s o fNaHl4C03 ............................................................................................. 60
Regression table f o r Brachlonus p U c a t iH s body burden re la te d to time 1 n carbon a s s im i la t io n experiments w ith d isc re te age cohorts o f la rv a l Leiostomus xa n th u ru s ........................ 65
Stepwise m u lt ip le regress ion tab le o f uptake re la ted to time, age* leng th * and w e igh t in carbon a s s im ila t io n experiments w ith d isc re te age cohorts o f la rv a l Lei ostomus x a n th u r u s ............................................... 69
Stepwise m u lt ip le regress ion tab le o f re ta ined re la te d to time, age, le n g th , and w e igh t 1 n carbon a s s im ila t io n experiments w ith d isc re te age cohorts o f la rv a l Lelostomus xanthurus * * .................................... * ■ 70
Stepwise m u lt ip le regress ion tab le o f 14c respired re la te d to time, age, le n g th , and w e igh t 1 n carbon a s s im ila t io n experiments w ith d is c re te age cohorts o f la rv a l Lelostomus xanthurus ..................................... . . . 71
vi 11
LIST OF TABLES (concluded)
Table
11. Stepwise m u lt ip le regression ta b le o f PQ^C re la ted to t im e, age, length* and weight in carbon a s s im ila t io n experiments w ith d is c re te age cohorts of la rv a l Lelostomus xanthurus.
1 2 . Surrmary of values used to assess carbon a s s im ila t io nin d isc re te age cohorts o f la rv a l Lelostomus xanthurus.
13. Stepwise m u lt ip le regression ta b le o f carbon re te n t io n , the index o f the c o e f f ic ie n t o f u t i l i z a t i o n * re la tedto age, leng th , weight, and c o n d it io n fa c to r of Lelostomus xanthurus la rv a l age cohorts ..............................
14. Stepwise m u lt ip le regression ta b le o f carbon absorp tion , the index o f absorption e f f ic ie n c y , re la ted to age, length, weight, and con d it io n fa c to ro f Lelostomus xanthurus la rva l age co h o rts ..........................
15. Sunmary o f d u a l- t ra ce r values used to ca lcu la ted absorption e f f ic ie n c y by the in d ic a to r r a t io method . .
16. Surnnary of values used to assess carbon a s s im ila t io n 1n age cohorts o f la rva l Lelostomus xanthurus fed reduced Brachlonus p i i c a t i l 1s conce n tra t io n s ......................
ix
Page
. 11
. 76
, IS
. 80
. 85
. B7
LIST OF FIGURES
Figure Page
1 . Morphological development o f the alimentary canalo f Lelostomus xanthurus. In c ip ie n t gut o f a yo lk-sacla rva 2.2 mm M L ............................... 30
2 . Morphological development o f the a limentary canal of Lelostomus xanthurus. In c ip ie n t gut o f a yolk-sacla rva 2.3 ran NL , 30
3. Morphological development of the alimentary canal o f Lelostomus xanthurus. In c ip ie n t gut o f a la rva 2.B nmNL 1n o l l -g lo b u le absorption ...................................... . . . . . 30
4. Morphological development of the alimentary canal of Lelostomus xanthurus. Alimentary canal o f a larva3.0 run NL. ..................................................... 30
5. Morphological development o f the alimentary canal o f Lelostomus xanthurus. Alimentary canal o f a transform ing la rva 7,6 mm SL......................................... ............................................. 30
G, Morphological development of the alimentary canal of Leiostomus xanthurus. Alimentary canal o f an early Juven ile 25.0 m SL. . . . . . . . ........................ 30
7, Photomicrograph o f the In c ip ie n t gut o f a Leiostomusxanthurus yo lk-sac larva 2.3 mm NL. Longitudinalsec tion showing yo lk mass* o i l g lobule, in c ip ie n t gut* l i v e r * and p a n c r e a s ..................... .................................................. 3L
B. Photomicrograph of the in c ip ie n t gut o f a Lei ostonusxanthurus yo lk-sac larva 2.3 nm NL. Cross sectionshowing mucosal ep ithe lium and s t r ia te d border . . . . . . 31
9. Photomicrograph o f the alimentary canal o f a Lelostomusxanthurus larva 4,4 nm NL, Longitudinal section showingfo re g u t, midgut, h indgut, l i v e r * and pancreas ................ . 31
10. Photomicrograph o f the alimentary canal o f a Leiostomusxanthurus larva 4.4 nm NL, Longitudinal sectionshowing two types o f mucous c e l ls in f o r e g u t ............................ 31
x
LIST OF FIGURES (continued)
F igure Page
11. Photomicrograph o f the a lim en ta ry canal o f a Lelostomusxanthurus larva 4 ,4 nm NL. Long itud ina l se c t ion showingm id g u t ..................................... , . , . ...................................................... 32
12. Photomicrograph of the a lim en ta ry canal o f a Lelostomusxanthurus larva 4.4 mm NL, Long itud ina l sec t ion showinghindgut ep ithe lium w ith s t r ia te d border and in t r a c e l lu la r I n c lu s io n s ................................................................................................. 32
13. Photomicrograph o f a Lelostomus xanthurus la rva4.4 im NL. Longitudinal sec tion showing l i v e r ac in iw ith l i v e r s inus, hepatocytes, and glycogen vacuoles . . . 32
14. Photomicrograph o f a Leiostomus xanthurus la rva4.4 nm SL. Long itud ina l se c t ion showing pancreatica c in i and zymogen granules ................................. 32
15. Photomicrograph o f a transform ing Leiostomus xanthurus la rva 12.3 nm SL. Longitud inal section showing ju n c t io n o f bucco-pharynx and esophogus w ith esophageal fo ld s and Increased number o f mucous c e l ls ....................... . . . . . . . 33
16. Photomicrographs o f a trans fo rm ing Lelostomus xanthurus la rva 12,3 mm SL, Longitud ina l section showing the ju n c t io n o f the esophagus and b l in d sac w ith developing g a s tr ic g la n d s ........................................................... 33
17. Photomicrograph of an early ju v e n i le Leiostomus xanthurus 18,1 mm SL. Long itud ina l section o fg a s tr ic corpus o f a f is h 18.1 nm SL showing uniformsecretory c e l ls o f the g a s tr ic g lands. 33
18. Photomicrograph o f an early ju v e n i le Lelostomus xanthurus 33,0 nm SL. Long itud ina l section o fthe In te s t in e showing in t r a c e l l u la r Inc lus ions ..................... 33
19. Photomicrograph o f an e a r ly ju v e n i le Leiostomus xanthurus 33,0 irm 5L. Long itud ina l section o f therectum showing granu lar in t r a c e l l u l a r I n c lu s io n s ................. 33
20. Weight and length regressions f o r Leiostomus xanthuruslarvae used in carbon a s s im i la t io n experiments 43
2 1 . Experimental design o f carbon a s s im i la t io n w ith d isc re teage cohorts la rv a l Leiostomus xanthurus. .................................. 51
xi
LIST OF FIGURES (continued)
Figure Page
22. Schematic diagram o f re te n t io n chambers used 1n carbon a s s im ila t io n experiments w ith age cohorts o f la rv a l Leiostomus xanthurus* 54
1423. Schematic diagram o f b u bb le -trap fo r recovery o f CO, in carbon a ss im ila t io n experiments w ith age cohorts o f la rv a l Lelostomus xanthurus............................................................ 56
24, Decay o f chemllurnlnescent a c t i v i t y in la rvae andSrachlpnus samples* ....................... . . . . . 5a
£5. Quench curves fo r larvae and Brachlonus samples................. 59
£6 . Quench curves fo r larvae and Brachlonus samples fromd u a l- lab e l experiments* - * * * ........................................................62
1427. Biochemical f ra c t io n a l o f c in Brachlonusduring la b e l in g ........................................................................................... 66
£8 . Carbon-14 uptake by la rv a l Lelostomus xanthurusIn carbon a ss im ila t io n experiments* * ........................................... 67
29. Carbon-14 re ta ined, re sp ire d , and lo s t as P014C byla rv a l Lelostomus xanthurus in carbon a s s im i la t io n experiments* * * * * * .................................................................... 73
30. Carbon-14 re ta ined , re s p ire d , and lo s t as PO ^c byla rv a l Leiostomus xanthurus In carbon ass lm f1a t lo n experiments"! ISata pooled from a l l experim ents.......................... 74
31. Carbon re te n t io n , the index o f the c o e f f i c ie n t o f u t i l i z a t i o n , re la ted to development and c o n d it io nfa c to r of la rv a l Leiostomus xanthurus age cohorts - * * * 77
32. Carbon absorption, the index o f absorption e f f ic ie n c y , re la te d to development and con d it io n fa c to r o fla rv a l Lelostomus xanthurus age cohorts* . . . . . . . . 79
33. Percent CO2 respired re la te d to development and cond it ion fa c to r o f la r v a l Lelostomus xanthurus age cohorts......................................................................... 32
34. Condition fa c to r re la ted to age and notochord o r standard length o f la rv a l Leiostomus xanthurus age cohorts used In ca rb o n a s s im i la t io n experiments- * - 33
x l l
LIST OF FIGURES [continued)
Figure Page5135. Carbon-14 and Cr re ten tion and loss by la rva l
Lelostomus xanthurus In a d u a l- t ra ce r experiment w ith a 15 day age c o h o r t * * * .................... * * BA
35. Carbon-14 re ten tion and loss by la rv a l Lelostomus xanthurus 1n carbon ass im ila t ion experiments w ith reduced Brachlonus p U c a t f l l s concentrations* * * • 86
37. Carbon Ingested per dry weight o f la rva l Lelostomus xanthurus fed high and low concentrations o f Brachlonus p l1 c a t1 l is - ■ * * ......................................... 89
38. The number o f r o t i f e r s ( Brachlonus p U c a t lH s )Ingested re la ted to development o f la rva l Lelostomus xanthurus age cohorts....................................................................... 90
39, Length and age re la t ion sh ip s o f labora to ry-rearedLelostomus xanthurus larvae" * * * * * * * .......................... 98
40. Weight and age re la t ion sh ip s o f labora to ry-rearedLeiostomus xanthurus la rva e .......................................................... 99
41. Schematic surnnary o f the development oflabora tory-reared Lelostomus xanthurus la rv a e ...........................100
42. Head length and notochord or standard length re la t ion sh ip s o f labora tory-reared and w ild Lelostomus xanthurus la rvae- * ................................. 101
43. Head he ight and notochord or standard length re la tionsh ips o f labora tory-reared and w ild Leiostomus xanthurus la rva e .................... . • * , ...........................102
44, Eye diameter and notochord or standard length re la tionsh ips o f labora to ry-reared and w i ld Lelostomus xanthurus la rvae .......................................................... 103
45, Eye height and notochord o r standard length re la tionsh ips o f labora tory-reared and w i ld Lelostomus xanthurus la rva e ................................................................104
46. Snout to pectoral length and notochord o r standard length re la tionsh ips o f labora to ry-reared and w ild Lelostomus xanthurus la rvae .......................................................... 105
47. Pectoral depth and notochord or standard length re la t ion sh ip s o f labora tory-reared and w ild Lelostomus xanthurus la rvae- * * ■ * ■ ♦ * ■ * * » * ■ • l og
x i i i
LIST OF FIGURES (concluded)
Figure Page
48, Preanal length and notochord or standard length r e la t io n ships o f 1aboratory-reared and w i ld Lelostomus xanthurus l a r v a e .............................................................................................* . * * 107
49. Depth a t anus and notochord o r standard length r e la t io n ships o f 1abora tory-reared and w i ld Lelostomus xanthurus larvae , . * , * . . . . , .................................................................... 108
xf v
ABSTRACT
In view o f questions concerning the d ig e s t iv e and a ss im ila t ive a b i l i t i e s o f la rva l marine f is h e s , the changes 1n these processes with development, and th e i r re la t io n s h ip w ith s ta rva t io n - In du ce d m o rta l i ty ; t h i s study has three o b je c t iv e s ; (1 ) to examine the morphological and h is to lo g ic a l development o f the a lim entary canal and associated s tru c tu re s of la rva l spot, Lelostomus xanthurus (P isces: Sciaenldae);(2 ) t o assess short- te rm carbon asslm lTatlon In d is c re te age cohorts o f la rv a l spot; and (3) to re la te any changes In a s s im i la t io n to development. The a lim entary canal and associated s tru c tu re s o f larval spot from hatching through transfo rm ation (metamorphosis) were examined using l ig h t microscopy* These f in d in g s were re la ted to re su lts of e xp e r im e n ta l-ra d io tra ce r studies o f carbon a s s im i la t io n In d iscre te age cohorts o f la rv a l spot.
C yto log lca l evidence suggested tha t the a lim en ta ry canal and associated s truc tu res were func t io n a l In f i r s t - f e e d in g la rvae a fte r the completion of yolk-sac and o i1 -g lob u le absorp tion , and changed l i t t l e p r io r to trans fo rm ation . There 1s no e la b o ra t io n o f t issues during the la rv a l phase. Major developmental changes associated w ith t ra n s form ation were accompanied by changes in h a b ita t and feeding regime.
The behavior of carbon-14 ind ica ted th a t food was q u ic k ly digested and carbon ass im ila ted fo l lo w in g th e ingestion o f a un ifo rm ly labeled r a t io n . A percentage o f newly ass im ila ted carbon entered ra p id ly Into sho rt- te rm metabolic processes and ex ited the la rva v ia re sp ira t io n beginning as e a r ly as 1 h a f te r in g e s t io n . Gut evacuation was complete w i th in 6 or 7 h,
Uptake, short-term re te n t io n , and loss o f were used to compute two parameters, carbon re te n t io n and carbon a b so rp t io n , tha t were taken as r e la t iv e ind ices of the c o e f f ic ie n t of u t i l i z a t i o n and absorption e f f ic ie n c y . The re la t io n sh ip s between these two in d ice s o f carbon a s s im ila t io n and measures o f development Ind ica ted th a t ass im ila t ive a b i l i t i e s did not Improve w ith la r v a l development. Because there were no major changes in a lim entary canal t is s u e s , t h is might be expected.
The ind ices o f a ss im ila t io n were nega tive ly re la te d to la rva l c o n d it io n fa c to r and th is re la t io n s h ip has im portant Im p lica tions to la rv a l growth and s u rv iv a l . Condition fa c to r is an In d ic a to r o f larval growth and robustness. Larvae th a t have been feeding well enough to s u rv iv e , but less than o p t im a lly f o r maximum growth, apparently a ss im ila te more carbon from a ra t io n than do larvae w ith a b e t te r growth h is to ry *
These re s u l ts are p la u s ib le in e vo lu t iona ry terms. Larvae may m it ig a te the r is k of a patchy food d is t r ib u t io n by f u l l y u t i l i z in g a v a i la b le resources. Such compensatory mechanisms may be of great adaptive s ig n if ica n ce to pelagic marine la rvae .
xv
MORPHOLOGICAL, HISTOLOGICAL, AMD PHYSIOLOGICAL
ASPECTS OF ASSIMILATION IN LARVAL SPOT,
LEIOSTOMUS XANTHURUS LACEPEDE
INTRODUCTION
Research presented here is intended as one pa rt o f the m anifo ld
approach necessary to understand the re la t io n s h ip between stock and
recru itm ent of marine f ishes w ith pe lag ic eggs and la rvae . Because o f
the high annual v a r ia b i l i t y In re c ru itm e n t, due in pa rt to d i f f e r
e n t ia l s ta rva tion- induced m o r ta l i t y , t h is research examines the
a b i l i t y of a la rva to ass im ila te a v a i la b le food resources.
The su rv iva l ra te o f many marine f is h e s through th e i r e a r ly l i f e
h is to ry is extremely low and may be the major determinant o f numerical
yea r-c lass s treng th (Beverton, 1962; Q u lland , 1965; Cushing, 1969),
M o r ta l i t y is o ften near 99,9%, e s p e c ia l ly among h ig h ly fecund f ish e s
w ith pelagic eggs and larvae (see review in Dahlberg, 1979}. To
exp la in surv iva l In terms o f any s in g le fa c to r is Impossible f o r many
s y n e rg is t ic and spec ies -spec if ic fa c to rs in f luence m o r ta l i t y * Yet, a
general p a r t i t io n in g o f a c t ive mechanisms has enhanced an under
standing o f the problem, Egg and la rvae m o r ta l i t y can be a t t r ib u te d
to adverse environmental cond it ions ( e .g . , Bonnet, 1939; L i l l e lu n d ,
1965; B la x te r , 1969; Dickson, e t al . , 1974; Le tt and Koh le r, 1976;
Rogers, 1976; Rosenthal and A ld e r ic e , 1976; F o r re s te r , 1977), genetic
defects (V lad im irov , 1970), and predation (Lebour, 1925; Murphy, 1961;
F raser, 1969; Hempel , 1965; Dekhnik, et a l . , 1970; L i l le lu n d and
Lasker, 1971; The llacker and Lasker, 1974; Westernhagen and Rosenthal,
2
3
1976; Kuhlmann, 1977; Laurence, et a l * , 1979), Fish larvae experience
a d d it io n a l m o r ta l i ty a t t r ib u ta b le to s ta rv a t io n * As the major causes
of la rv a l m o r ta l i ty {Hempel , 1974; Cushing, 1976; Hunter, 1976),
s ta rv a t io n and predation probably In te ra c t w ith s yn e rg is t ic e f fe c ts ;
the la rva tha t feeds poorly grows poorly and th is abates feeding
success and predator avoidance (Cushing, 1976).
S ta rva tion induced m o r ta l i t y was recognized as e a r ly as 1897
(Fabre-Domergue and 01etr1x) and re la ted research has progressed along
several l in e s . The " c r i t i c a l period concept", hypothesiz ing th a t year
class s treng th is determined by the a v a i l a b i l i t y o f p lank ton ic food
immediately a f te r yolk-sac a b so rp t io n , was f i r s t proposed by H jo r t
{1914; 1926). Research designed to te s t t h is hypothesis inc luded the
ana lys is of su rv iva l curves f o r natura l popu la tions (Marr* 1955), and
the c o r re la t io n o f la rva l gut contents and microzooplenkton abundance
in the sea w ith la rv a l cond it ion and abundance (see reviews by May,
1974). Though not a l l agree th a t c r i t i c a l periods e x is t or th a t food
l im i t a t io n is s ig n i f ic a n t in nature (Dekhnik e t a l , , 1970; Dekhnik and
Singukova, 1976), catastroph ic m o r ta l i t ie s apparen tly associated with
inadequate feeding conditions In the sea occur during the la rv a l stage
( e .g . , t f lb o rg , 1976). Inasmuch as s ta rv a t io n m o r ta l i t y has never been
proven c a te g o r ic a l ly , more evidence 1s needed using d iagnostic
h is to lo g ic a l , morphological, and biochemical c r i t e r i a (E h r l ic h , 1974a,
1974b; llmeda and Ochfai, 1975; E h r l ich et a l . , 1976; O 'Connell, 1976;
T h e i la c ke r , 1978), and experimental bioassay techniques (Lasker,
1975}.
4
Experimental research has accelerated w ith the a b i l i t y to
successfu lly rear la rva l marine f ishes in the la b o ra to ry . Experiments
have assessed the s e n s i t iv i t y o f larvae to delayed f i r s t - f e e d in g , the
e f fe c ts o f food density and small scale d is t r ib u t io n on la rva l sur
v iva l and growth, and la rva l searching a b i l i t y and feeding e f f ic ie n c y
{see reviews 1n May, 1974; Ah1strom and Moser, 1976; Vlymen, 1977;
Hqude, 1978)* The resu lts o f these studies suggest th a t the in te
grated concentration of food organisms in the ocean ( e .g . , Beers and
Stewart, 1967, 1971; A r th u r, 1977) may be in s u f f ic ie n t to support
la rv a l f is h su rv iva l; though concentrations in bays and estuaries may
be adequate (see review 1n Houde, 1978)* The contagious sm all-sca le
d is t r ib u t io n of food organisms in the open sea may a lso be c r i t i c a l
{Lasker, 1975; Vlymen, 1977; Houde and Schekter, 1978), and the
In te ra c t io n o f food a v a i la b i l i t y , growth, and predation remain
obscure.
Most recen tly , phys io log ica l energetic studies have enhanced our
understanding o f s ta rva t io n , growth, and su rv iva l o f marine f is h
la rvae . Researchers have attempted to e s ta b lish the amount o f food
necessary to support la rva l f is h metabolism and growth by estim ating
the terms of energy budget equations ( I v le v , 1939; Wlnberg, 1956;
Palohelmo and D ick ie , 1966a, 1966b; Warren and Davis, 1967). T he ir
re s u l ts ind ica te tha t energy reserves upon yo lk-sac absorption are
inadequate to support metabolism and growth without feed ing (Lasker,
1964; B la x te r , 1969) and tha t natural d e n s it ie s of food organisms are
5
s y n o t fc a l ly Inadequate to support s u rv iv a l (see review 1n Laurence,
1977, Houde, 1978). Vlyroen's ( 1977) model, based 1n p a r t on swlrnirfng
e n e rg e t ics , p red ic ts th a t the contagious m 1crod is tr1button o f food
organisms 1s a c tu a l ly necessary fo r la rv a l s u rv iv a l .
Host phys io log ica l ene rge tic s tud ies on f is h larvae have estimated
equation terms as instantaneous rates a t a s in g le developmental
in te rv a l and have not attempted to reso lve the changes in m atter*
energy u t i l i z a t i o n through e n t i r e la r v a l l i f e . Larval f ishes a re ,
however, developing animals; m atter-energy u t i l i z a t i o n involves
dynamic processes influenced p o ss ib ly by age as w e ll as ra t io n s ize
and environmental fa c to rs .
The amount o f matter-energy a v a i la b le f o r metabolism and growth
1s determined, in p a r t , by the a b i l i t y o f the la rva to d iges t and
a ss im ila te n u tr ie n ts ( P h i l l i p s , 1969). Some have speculated th a t
a s s im i la t io n 1s i n i t i a l l y low but Improves w ith la rv a l age as the
r e s u l t o f the morphological development o f the a lim en ta ry canal
(Rosenthal and Herrtpel, 1970; Nishlkawa, 1975). Besides Laurence
(1971,1977) there 1s l i t t l e q u a n t i ta t iv e evidence to support t h is .
This d is s e r ta t io n has three o b je c t iv e s : (1) to examine the
morphological and h is to lo g ic a l development o f the a lim entary canal and
associa ted s tru c tu re s o f la rv a l sp o t, Leiostomus xanthurus; (2) to
assess sho rt- te rm carbon a s s im i la t io n in d is c re te age cohorts o f
la rv a l spo t; and (3) to re la te any changes in a s s im i la t io n to
development. I t is hoped th a t the in fo rm a tio n here in co n tr ib u te s to
the understanding o f the broader question o f p h y s io lg ic a l energetics
and s u rv iv a l o f la rv a l marine f is h e s .
THE EARLV LIFE HISTORY OF LEIOSTOMUS XANTHURUS
A BRIEF REVIEW
Spot, Lelostomus xanthurus Lacepede, is a h ig h ly fecund,
coastal-marine, sclaenid f 1sh ranging from Cape Codt Massachusetts, to
Bay o f Campeche, Mexico (Dawson, 1958), Populations f lu c tu a te
d ram a tica lly , and year-c lass strength is probably determined o ffshore
during the pelagic egg and la rva l phases (Joseph, 1972),
Spot spawn over the continental sh e lf in la te f a l l and w in te r
(see review 1n Johnson, 1978)* Ova counts range from 70,000 to 90,000
per female (Dawson, 1958)* Ovarian eggs o f several sizes w ith in
s ing le females (Hildebrand and Cable, 1930) and repeated spawning in
the labora tory suggest m u lt ip le spawning in nature. The spawning
period may not be p ro trac ted , however, as p re l im ina ry analyses o f
d a l ly o to l i t h growth increments ind ica te a s in g le , ra the r s h o r t ,
spawning season1. Spot begin the offshore spawning m igra tion
[Pacheco, 1962) a f te r th e i r second suraner (Hildebrand and Cable, 1930;
Pearson, 1929). The spherica l, pelagic eggs range from 0,72 to
0.87 mm 1n diameter and have e ith e r a s in g le , spherica l o i l g lobule or
m u lt ip le o i l globules grouped together w ith in the v i t e l l i n e membrane
(Powell and Gordy, 1980). Oil globules coalesce during development
leaving a single globule in the newly hatched yo lk-sac la rva (Powell
1 Peters, D, S ., J . C. DeYane, M. T, Boyd, L. C, Clements* and A, B. Powell. 1978. Pre lim inary observations on feeding, growth, and energy budget o f la rva l spot ( Leiostomus xanthurus) . Ann, Rept. of the Beaufort Laboratory to the U.S. Dept, o f Energy, pp. 377-397.
7
8
and Gordy* 1980), Hatching occurs in ca. Z days a t 20°C (see
Appendix),
Larvae have been described by many authors (see reviews 1n Fruge
and Truesdale* 1978; Johnson, 1978)* but t h e i r ecology 1s not well
documented, Newly-hatched, yo lk -sac larvae range from 1.6 to 2*5 mm
notochord length (NL). Yolk-sac and o i l - g lo b u le absorption are
complete 1n 4 to 7 days at 20°C (see Appendix). The larvae* now
2*0 to 2.4 run NL* begin feeding as members o f the plankton community
(Part I ) , Spot la rvae , l i k e most marine f is h la rva e , are v isua l
feeders w ith feeding genera lly r e s t r ic te d to d a y l ig h t hours (K je lson
e t al .* 1976; K je lson and Johnson* 1976). Development to
trans fo rm ation (metamorphosis) takes place in waters ove r ly in g the
con t in e n ta l sh e lf ( e .g . , Powles and Stender, 1976). P re lim inary
evidence suggests th a t la rv a l growth Is extremely rap id*
Transformation begins by ca. 7.0 mn standard length (SL) and is
complete by 16,0 to 18,0 mm SL (Powell and Gordy* 1980; also see
Appendix), The average growth ra te suggests th a t transform ing larvae
and e a r ly ju v e n i le s are not l im ite d by food a v a i l a b i l i t y . 1 Food
l im i t a t io n may, however* be an Important fa c to r In the su rv iva l o f
f i r s t - f e e d in g la rvae ; a short spawning period would am plify the
c r i t i c a l coincidence of zooplankton abundance and f i r s t feeding.
Spot, along w ith many other sc ia e n id s , u t i l i z e es tuaries as
nursery grounds [see review 1n Chao and Muslck, 1977), Transforming
larvae enter es tuaries during the f i r s t h a l f o f the year when as small
as 10.0 m SL. Larvae complete trans fo rm a tion w ith in es tuaries and
9
coasta l bays during spring and sunnier o f t h e i r f i r s t year.
Transforming larvae feed i n i t i a l l y on ep lfaunal Inverteb ra tes followed
by Infauna 1 invertebra tes and vegetable d e t r i t u s {P a rt I ) . Growth 1s
rap id during the f i r s t summer, and by December when most have moved
o f fsh o re to over-w in te r, they have grown to about 150 mn SL w ith some
as la rg e as 190 mm SL (Chao and Musick, 1977). Some sm alle r juven iles
o v e r-w in te r in the deeper channels o f es tua r ies (Chao and Musick,
1977). Juveniles may penetrate freshwater as well as hypersaline
lagoons and marshes {see review in Johnson, 1978).
The to lerance of eggs, la rvae , and ju v e n i le spot to temperature
and s a l i n i t y v a r ia t io n 1s not well documented. Eggs are not buoyant
and hatching success is reduced in water less than 26 ° /o o . Likewise,
ha tch ing success and su rv iva l through yo lk -sac absorption is reduced
below 15°C (A,B. Powell* personal communication). Eggs and larvae
g e n e ra l ly occur in 18 to 22flC and 30 to 35 ° /oo water in the South
A t la n t ic B ight (Powles and Stender, 1976); though la rvae and Juveniles
are taken in temperatures from 6.3 to 32.5°C and s a l in i t i e s from 0 to
34.2 ° /o o (see review In Johnson, 1978). Temperatures o f 4 to 5°C may
be le th a l to juven iles (H ildebrand and Cable, 1930; Dawson, 1958).
The d is t r ib u t io n o f larvae and Juven iles 1s apparently l im i te d by
temperature ra ther than s a l in i t y [Parker, 1971). Temperature
preferences can be in fe rred from the seasonal movement o f larvae and
Juven iles In to and out of es tuarine nursery areas. Transforming
10
larvae and early juven iles enter estuaries 1n spring as the
temperature rises above fi°C; ju v e n i le s generally depart 1n f a l l as the
temperature drops below 1Q°C (Parker* 1971; Chao and Musick, 1977),
ASPECTS OF THE MORPHOLOGICAL, HISTOLOGICAL, AND
FUNCTIONAL DEVELOPMENT OF THE ALIMENTARY CANAL
AND ASSOCIATED STRUCTURES
INTRODUCTION
Starvation-Induced m o r ta l i ty o f pelagic marine f is h larvae as a
determ inant of f lu c tu a t io n s in numerical year c lass s treng th has been
the sub jec t o f controversy fo r almost a century (see reviews by
B la x te r , 1969i May, 1974). S ta rva tion has received much a t te n t io n ,
In c lud ing extensive f ie ld and labora to ry s tudy, but the d ig es tive and
a s s im i la t iv e a b i l i t i e s of la rv a l f ishes remain to be determined
(H unter, 1976). While some have assumed th a t these a b i l i t i e s are
I n i t i a l l y very poor but improve with la rva l age as the re s u l t of the
development o f alimentary canal t issues [Rosenthal and Hempel, 1970;
Nishlkawa, 1975; Laurence, 1977), l i t t l e supporting evidence e x is ts .
Changes in d ig e s t io n and a ss im ila t io n should be concomitant w ith
changes in the alimentary canal and associated s t ru c tu re s .
This study was undertaken to describe c e r ta in aspects o f the
fu n c t io n a l development of the a limentary canal, l i v e r , pancreas, jaws,
and tee th o f spot, Lelostomus xanthurus■ Spat Is a h ig h ly fecund,
s d a e n ld f i s h w ith pelagic eggs and larvae, fe a r-c la s s size
f lu c tu a te s d r a s t ic a l ly , probably due to d i f f e r e n t ia l la rv a l m o r ta l i ty
(Joseph, 1972); s ta rva tion may be a causative fa c to r . Knowledge of
the extent and t im ing of developmental changes in the a lim entary canal
and associated structures should provide a basis f o r eva lua ting
changes In the d igestive and a ss im ila t ive a b i l i t i e s o f spot during I t s
e a r ly l i f e h is to ry .
12
13
MATERIALS AND METHODS
Specimens fo r d issec tion and h is to lo g ic a l study were obtained
from two sources. Eggs were obtained from f is h Induced to spawn
through temperature and photoperiod manipulation and hormone
In je c t io n . Eggs and larvae were reared at 20 + 2°C In 30-35 D/oo
s a l i n i t y seawater and fed sequen tia l ly w ith the d ln o f la g e l la te *
^ymnodlnium splendens, the r o t i f e r , BracHonus p l lc a tH i s . and w ild
plankton- Specimens were f ixed in c h i l le d neutra l bu ffe red
form alin plus ac ro le in , dehydrated 1n methanol. I n f i l t r a t e d w ith
and embedded 1n glycol methacrylate (2-hydroxy-ethyl m e thacry la te ),
and sectioned at 2 and 4 pm according to m odifica tions o f Ruddell
(1971}, For comparison, f i e ld co lle c t io n s of the V i rg in ia In s t i tu te
o f Marine Science (VIMS), f ixed 1n buffered fo rm a lin 1n seawater,
were dehydrated 1n Isopropanol, i n f i l t r a t e d w ith and embedded in
p a ra f f in , and sectioned a t 5 yin. H is to lo g ica l and hlstochemlcal
s ta ins used fo r glycol methacrylate sections were a d d fuchsln -
t o l d d ine blue 0, a lk a l in e blue 6B - neutra l red, and p e r io d ic acid
S c h i f f 's reagent (PAS) - to lu id ln e blue 0. P a ra ff in embedded
specimens were stained in hematoxylin - astra blue - eos in Y.
h e t t l e r , W.F., A.B, Powell, and L.C. Clements. 1978. Laboratory induced spawning o f spo t, Lelostomus xanthurus (Lacepede). Ann. Rept. o f the Beaufort Laboratory to the U.S. Department of Energy- pp. 351-356.
14
The development o f jaws and d e n t i t io n was studied on specimens
cleared and sta ined w ith a l i z a r in red S - a lc ia n blue by a
m o d if ica t io n o f Dlngeraus and Uhler (1977).
Specimens f o r gut content a n a lys is were obtained from f i e l d
c o l le c t io n s o f the South Caro lina W i ld l i f e and Marine Resources
Department, NOAA-NMFS Sandy Hook Laboratory* and VIMS. Food items
were excised from 59 specimens, mounted 1n CMC-5 (T u r to x )* and
id e n t i f ie d to the lowest poss ib le taxon.
Lengths reported herein are notochord lengths (NL) measured
before the complete form ation o f hypural elements and standard
length (SL) measured th e re a f te r .
15
RESULTS
Though development 1s continuous, the a lim entary canal is
discussed w ith in the framework of three phases th a t gen e ra lly
correspond w ith changes 1n gross morphology: the yo lk -sac la rva
{1*6 - 2.5 mm NL), the la rva (2,5 - 7*0 inn NL), and the transforming
la rva (7 ,0 -18,0 ran 5L). A t a rearing temperature o f 20°C these
phases correspond to 2-7, 7-31, and 31-60 days from ha tch ing .
P e r t in e n t d e ta i ls of the e a r ly ju v e n i le a lim en ta ry canal are also
described . While many te rm ino log ies o f f i s h development In te rva ls
e x is t ( e .g . , Hubbs, 1943; Ah ls trom , 1968; Ba lon, 1975), the present
terms a f fo rd s im p l ic i ty and close alignment w ith a lim entary canal
development*
A lim en ta ry Canal
The yo lk-sac la rva . In the yolk-sac la rva the a lim entary canal
1s u n d if fe re n t ia te d along i t s length and not fu n c t io n a l* The mouth is
not open. The bucco-pharynx is l ined w ith squamous e p ithe lium and
sca tte red mucous c e l ls of un iform appearance underla in by f ib ro u s
connective t is s u e . The in c ip ie n t gut 1s a s t ra ig h t tube ly in g in the
dorsa l v isce ra l ca v ity above the yo lk mass and d e f le c t in g v e n t ra l ly to
a closed anus {Figure 1), The narrow lumen is open p o s te r io r ly
(F igu re 7 ) , but is p a r t ia l l y occluded in the a n te r io r h a l f by pressure
from the o i l g lobu le . I t is l ined w ith c lo s e ly packed, columnar
e p i t h e l ia l c e l l s , 12-15 inti t h ic k . The nucle i o f these c e l ls are
16
located basal 1y and are ca. 3 unn 1 n diameter. The cytoplasm 1s
basoph il ic and granular. Mitochondria are not v is ib le . A p ic a l ly ,
the e p i th e l ia l c e l ls have a prominent* a c id o p h i l ic s t r ia te d border w ith
m ic r o v i l l i 3-4 pm deep (F igure 8). A 5 pm laye r o f a re o la r connective
t issue w ith f ib ro b la s ts underlies the ep ithe lium . This l i n in g ,
lack ing rugae, cons titu tes the early mucosa, but a submucosa and
muscularis are not apparent. A th in serosa is d lsce rnab le .
The a limentary canal becomes func t io n a l during the completion o f
yo lk -sac and o l l -g lo b u le absorption. This t ra n s i t io n is ra p id , tak ing
about 3D h at 2G°C, The yo lk is absorbed before the o i l g lobu le .
During I ts absorption 1 t remains as a cap o f yo lk granules enclosed by
the v i t e l l i n e syncytium ly in g over the a n te r io r pa rt o f the o i l
g lobule (F igure 2 ) . The pos te r io r h a l f o f the a lim entary canal w ith a
lumen abuts the o i l globule whereas the a n te r io r a lim entary canal
remains p a r t ia l l y occluded and l ie s between the o i l g lobule and the
dorsal wall o f the v iscera l ca v ity (F igure 2 ), During f in a l yo lk and
o11-globule absorption, the lumen extends the e n t i re length o f the
a lim entary canal, and the alimentary canal In to r ts to form a loop
midway along I t s length. Two c i r c u la r c o n s t r ic t io n s * the p y lo r ic po rt
and the lleocaeca l po rt , develop fu r th e r d iv id in g the a lim en ta ry canal
in to fou r sections: the bucco-pharynx, the fo regu t, m idgut, and
hlndgut (Figure 3). The mouth and anus are now open, and f i r s t
feeding may occur. The number o f PAS p o s i t iv e mucous c e l ls increases
17
along the s ing le la y e r o f squamous e p i th e l ia l c e l ls l in in g the
bucco-pharynx.
The la r v a . The la rv a l bucco-pharynx, f o r e - , m id-, and h lndgut
are d is t in g u ish e d by h is to lo g ic a l d i f fe re n c e s and remain unchanged
u n t i l t rans fo rm a tion . The a lim en ta ry canal length and su rface area,
however. Increase w ith Increased body s iz e .
The w a lls o f the bucco-pharynx are l in e d w ith s t r a t i f i e d squamous
e p ith e l iu m , 3 to 4 deep. Two la y e rs , an Inner 7 to 8 um la y e r of
a re o la r connective t is s u e , and an o u te r 4 m la y e r o f f ib ro u s
connective t is s u e , l i e beneath the ep ith e l iu m . F ib ro b la s ts are seen
1n these connective t issue la y e rs , and a few un iform PAS p o s i t iv e
mucous c e l ls occur In the ep ith e liu m . The v e n tra l w a lls have more
numerous and la rg e r PAS p o s it iv e mucous c e l l s . Taste buds appear in
the dorsa l and v e n tra l w a l ls ; though they lack gus ta to ry pores and are
presumably not fu n c t io n a l . They are more numerous on fo ld s and ridges
o f the ve n tra l w a l l .
The fo regut connects the bucco-pharynx w ith the midgut (F igures
4,9) and d i f fe re n t ia te s from the a n te r io r closed section o f the
In c ip ie n t gut. I t s mucosa cons is ts o f cuboidal e p i th e l ia l c e l l s , 10
to 20 ym th ic k , w ith basoph ilic cytoplasm. The basa lly loca ted nucle i
are a lso b a so p h il ic . Nucleoli are not v is ib le . The surface o f the
mucosa o fte n has jagged p ro jec t io n s th a t are p o ss ib ly remnants o f
sloughed c e l ls . Two types o f mucous c e l l s , d is t in g u ish e d on the basis
o f c e l lu la r morphology and s ta in in g re a c t io n s , are te n ta t iv e ly
id e n t i f ie d In the fo regu t mucosa (F igu re 10). Most numerous (Type 1)
is
are PAS p o s it iv e mucous c e l ls (8-12 pm 1n diameter) which resemble
to p ica l vertebra te goblet c e l ls and are s im i la r to those o f the
bucco-pharynx. They s ta in m etachromatica lly w ith basic dyes
suggesting tha t they contain acid mucopolysaccharides. The second
type {Type 2) are large (20 pm 1n d la m te r) , b a s o p h i l ic , and PAS
negative c e l l s , often aggregated 1n groups. The fo regu t mucosa has
shallow lo n g itud ina l fo ld s . A 30 pm th ic k lamina propria o f fibrous
connective t issue w ith f ib ro b la s ts l ie s subjacent to the ep ithe lium .
There Is no muscular!s mucosa or submucosa. The muscular!s consists
of 6 to 7 pm o f c i r c u la r smooth muscle f ib e rs , and a very th in serosa
is v is ib le .
The m idgut, l im ite d by the p y lo r ic and lleocaecal p o r ts , is
bulbous w ith a much wider lumen than e i th e r the fo re - or hlndgut
(Figure 9 ) , The mucosa becomes h ig h ly convoluted with deep (40-50 pm)
rugae, thus increasing surface area. I t s ep ithe lium co ns is ts of
basoph il ic columnar c e l ls , 25 to 30 pm h igh, w ith a 2 to 3 urn s tr ia te d
border o f a c id o p h i l ic m ic r o v i l l i underla in by a term inal web. Nuclei
are basoph il ic w ith v is ib le n u c le o l i . Numerous supra- and
in fra n u c le a r mitochondria are seen w ith in the cytoplasm. The most
c h a ra c te r is t ic feature of these c e l ls is the presence o f numerous,
supranuclear, vacuolar, in t r a c e l l u la r inc lus ions (Figure 11). The
mucosa lacks mucous c e l ls . A th in (8-10 pm) lamina p ropr ia o f areolar
connective t issue w ith f ib ro b la s ts underlies the ep ith e l iu m . There Is
no d is t in c t muscularls mucosa or submucosa. The muscularis consists
o f a 5 urn th ic k layer of c i r c u la r smooth muscle f ib e rs . A very th in
serosa is v is ib le .
19
The h lndgu t begins a t the l leacaeca l p o r t , ends a t the deve lop ing
anal s p h in c te r (F igure 4, 9 ) , and d i f f e r s from the mldgut m a in ly In
the cy to logy o f i t s mucosal e p i t h e l ia l c e l l s . The h lndgut mucosa has
sha llower (ca . 25 vm) rugae than those o f the m ldgut. I t s . mucosa
cons is ts o f a laye r o f un ifo rm co lu ttnar e p i t h e l ia l c e l l s , s l i g h t l y
s h o r te r (25 ^m) than those o f the m ldgut, w i th a low (1 o r 2 urn)
a c id o p h i l ic s t r ia te d border. There Is no evidence o f a te rm ina l web
beneath the m i c r o v i l l i . The basal cytoplasm o f the e p i t h e l ia l c e l l s
Is b a s o p h i l ic w h ile the a p ica l cytoplasm 1s a c id o p h i l i c . The nucleus
is loca ted b a s a l ly . As In the mldgut e p i th e l iu m , the re are numerous
supra- and in t ra n u c le a r m itochondria and supranuc lear cytop lasm ic
In c lu s io n s , but these in c lu s io n s are g ranu la r and a c id o p h i l ic
(F igure 12). The hlndgut has a th in (8 - 10 ^m) lamina p rop r ia
subjacent to the e p ith e l iu m , but no m uscularis mucosa or submucosa.
The m uscularis cons is ts o f a 1 jim th ic k la y e r o f c i r c u la r smooth
muscle c e l l s . There 1s a th in serosa. E ry th ro cy te s and an assortm ent
o f leucocytes are v is ib le w i th in the mid- and h lndgu t m uscu la r is .
The trans fo rm ing la rva and e a r ly ju v e n i le . The major changes 1n
the a lim en ta ry canal are associa ted w ith tra n s fo rm a t io n
(metamorphosis). The a l im en ta ry canal completes I t s development w i th
the fo rm ation o f the esophagus, stomach, In te s t in e w i th p y lo r ic
caecae, and rectum (F igure 6).
The bucco-pharynx remains unchanged w ith a few exceptions. A
buccal valve develops midway sepa ra t ing the pharynx and the buccal
20
c a v i t ie s . The ta s te buds* now open v ia gustatory pores, become more
numerous, and presumably fu n c t io n a l .
The esophagus d i f fe re n t ia te s from the fo regut and merges
p o s te r io r ly w ith the developing stomach. The esophageal mucosa
cons is ts o f s t r a t i f i e d cuboida! e p i t h e l ia l c e l ls 10 pm th ic k lack ing
c i l i a . The number o f Type 1 mucous c e l ls in the mucosa g rea t ly
increases (Figure 15), hut Type 2 mucous c e l ls were not seen in the
esophagus. The lamina p ropria and submucosa d i f f e r e n t ia te by ca.
13 mm SL. This d is t in c t io n 1s based on the te x tu re o f connective
t is su e fo r no muscularis mucosa develops In the a lim en ta ry canal. The
th in (6 pm) lamina p ropria underlies the e p ith e liu m and consis ts o f
f ib ro u s connective t is s u e . The submucosa, comprised of 15-20 pm o f
a reo la r connective t issu e w ith a few dispersed lo n g itu d in a l muscle
c e l l s , l ie s subjacent to the lamina p ro p r ia . A 10 pm la ye r of
c i r c u la r , smooth muscle f ib e rs w ith a few s t r ia te d muscle c e l ls appear
in add it ion to the inner layer o f lo n g itu d in a l muscle by ca, 13 mn SL.
These layers decussate a t the esophageal g a s tr ic ju n c t io n .
The stomach d i f fe re n t ia te s from the p o s te r io r section o f the
fo re g u t . The stomach anlage appears as a b l in d sac th a t bulges from
the p o s te r io r end of the fo regu t as i t meets the mldgut (F igure 5 ) .
This out pocketing continues to expand and extends as a bulbous sac
dorsal to the ju n c t io n w ith the m ldgut. Uniform secre to ry c e l ls
appear in the b l ind sac by ca. 10 mm SL [F igure 17). These c e l ls
increase 1n number and even tua lly l in e d is t in c t g a s t r ic glands In the
corpus (sensus B a rr ing ton , 1957 ) by IB itm SL (F igure 17). The g a s t r ic
HI
mucosa consists of simple columnar e p i th e l ia l c e l ls w ithou t a s tr ia te d
border- The apical cytoplasm is s l ig h t ly a c id o p h i l ic , perhaps because
o f the presence of mucigen, and contrasts w i th the b a so p h il ic basal
cytoplasm, Mucous c e l ls , s im i la r to Type 1 c e l ls in form, appear
w h ile the mucosa develops deep rugae. A lam ina propria underlies the
developing g a s tr ic ep ithe lium . The stomach has a 15 ym th ic k
submucosa of a reo lar connective t is s u e , A la y e r o f a few c i r c u la r
muscle f ib e rs is seer w i th in the mucosa. The g a s tr ic muscularis
co ns is ts of a 40 urn th ick inner la ye r of smooth, c i r c u la r muscle and a
3 ym ou ter laye r of smooth, lo n g itu d in a l muscle. The serosa is
sometimes v i s ib le . The ju n c t io n o f the esophagus and the stomach is
marked by an abrupt change In ep ithe lium (F ig u re 16}.
The in te s t in e is separated from the stomach by a developing
p y lo r ic sph inc te r and d i f fe re n t ia te s from the midgut, I t is genera lly
J-shaped (Figure 6} and separated from the s t ra ig h t rectum by the
ileocaeca l va lve, Cytol og ica l ly i t s ep ithe lium Is s im i la r to the
mldgut (Figure 16), A few mucous c e l ls , s im i la r to Type 1 c e l ls ,
appear in the mucosa. A d is t in c t io n between the lamina p ro p r ia and
submucosa is not apparent as 1t 1s in the esophagus and stomach.
Ins tead , a gradual t ra n s it io n from inner f ib ro u s to ou te r a reo la r
connective t issue develops, The rugae of th e midgut deepen in the
in te s t in e * The muscularis th ickens greatly and an ou te r laye r o f
lo n g itu d in a l muscle appears by ca. 13 urn SL- P y lo r ic caeca develop as
b l in d outgrowths of an te rio r In te s t in e ju s t p o s te r io r to the p y lo r ic
sph in c te r and are h is to lo g ic a l ly s im i la r to the In te s t in e (F igure 20).
The f i r s t caecum appears a t ca. 10 mm SL. The ju v e n i le complement of
22
seven to e igh t Is complete by 18 mm SL and Is attendant w ith the
completion o f stomach development.
The rectum d if fe re n t ia te s from the hlndgut but shares a s im i la r
epithe lium {Figure 19). As in the In te s t in e , mucous c e l ls appear 1n
the mucosa and Increase In number near the anal sph inc te r. The mucosa
develops secondary fo lds . The lamina propria and submucosa become
d is t ingu ishab le by ca. 23 m) SL, The lamina propria consists o f
aero lar connective tissue whereas the submucosa consis ts of f ib ro u s
co rrec t ive t issue , a reverse of the esophageal, g a s t r ic , and
in te s t in a l cond it ion . U fth ln the muscularis, the inner layer o f
c i r c u la r muscle expands and a th ic k outer layer o f smooth,
lo n g itud ina l muscle appears by ca. 13 mm SL. The d i f f e r e n t i a l l y
s ta in ing cytoplasmic Inc lus ions, so conspicuous 1n the la rva l mid- and
hindgut, are also obvious 1n transform ing larvae and early ju v e n i le s
(Figures 18, 19).
Associated Structures
The l i v e r . The l i v e r 1s d i f fe re n t ia te d a t hatching and l ie s
dorsal to the yolk mass (Figure 7 ) . Hepatocytes are compact,
g ranu la r, basoph il ic , and lack o f vacuoles In yo lk -sac larvae. In
larvae the l i v e r l ie s beneath and p a r t ia l l y surrounding the a lim entary
canal (Figure 9 ) . I t increases in size and becomes b ilobed. Large
(12 um in diameter) hepatocytes surround d is t in c t c a n a l ic u l i . These
c e l ls contain voluminous (4 urn 1n diameter) PAS p o s it iv e vacuoles
(Figure 13) tha t are presumably the s ite s o f glycogen storage (no
diastase con tro ls were used). The g a l l bladder appears on the r ig h t
l i t e r a l lobe o f the la rv a l l i v e r by ca. 3,0 mm NL. The l i v e r in e a r ly
ju v e n i le s occupies more space in the v isce ra l c a v i ty than any o the r
period in the s p o t 's e a r ly l i f e h is to ry . I ts two lobes extend around
the p o s te r io r loop o f the in te s t in e .
The pancreas. The exocrine pancreas, an in s u la r organ in a d u lt
spo t, is d i f fe re n t ia te d a t hatching w ith well developed b a soph il ic
ac ina r c e l ls . Pancreatic ducts and zymogen granules are not ye t
apparent. The la rv a l pancreas l i e s p r im a r i ly along the r ig h t side o f
the mldgut but sends disseminated branches In to the l i v e r . Pancreatic
ducts appear soon a f t e r yo lk abso rp t ion , and conspicuous a c id o p h i l ic
zymogen granules appear w ith in the pancreatic a c in i (F igure 14). The
pancreas remains unchanged during tra n s fo rm a tio n .
Jaws. At hatch ing anlagen o f the upper and lower jaws are formed
as p ro c a r t i la g e w ith basoph ilic chondrocytes ly in g w ith in a th in
m a tr ix o f chondromucen. At f i r s t feeding (ca , 2.0 mm NL) the upper
jaw is comprised o f pa ired prem axillae w ith the premaxi 11 a completely
excluding the m a x il la from the gapei the lower jaw is comprised o f
pa ired Meckel's c a r t i la g e s . Dentarles and a r t i c u la r s w ith angular
processes are d is ce rra b le as separate lower-jaw elements by ca. 4 .0 mm
NL* C a lc i f ic a t io n o f the jaw elements begins by ca. 0 .5 mm SL in the
a n te r io r shafts and ascending processess o f the p rem ax il lae , the
a n te r io r shafts o f the m a x il lae , and the p o s te r io r flanges o f the
a r t i c u la r s . The a n te r io r t i p o f the den ta rles begins to c a lc i f y by
12 Jim SL. C a lc i f ic a t io n proceeds posteriad along the axes o f the
p rem ax il lae , m a x i l la e , and denta rles and spreads in to the la te ra l
24
flanges o f these elements by IS rim SL. Meckel's c a r t i la g e s p e rs is t in
Juven iles as carti lagenous shafts enclosed w ith in each a r t i c u la r .
Caution must be exercised in the in te rp re ta t io n of s ta in in g
re a c t io n s * Premaxillae* m a x i l la e , d e n ta r le s , and a r t i c u la r s are
dermal bones not preformed as c a r t i la g e , ye t they o ften s ta in w ith
a lc ia n blue* A lc ian blue reacts w ith many a d d mucopolysaccharides
In c lu d in g chondromucin (Pearse, L9G8)* H is to lo g ic a l ly , hya line
c a r t i la g e w ith chondrocytes completely f i l l i n g lacunae embedded w ith in
a th ic k chondromudn m a tr ix , predominates 1n endochondral elements of
larvae and early ju v e n i le s . Perichondral bone appears on the
periphery of some elements by ca. 23 mm SL; though the a l iz a r in
re a c t io n is v is ib le In elements o f much sm a lle r specimens* A l iz a r in
red $ che la tes Ca ions (Pearse, 1968} and thus may in d ic a te the
presences o f c a lc i f ie d c a r t i la g e as w e ll as t ru e o s s i f ie d bone*
The gape, terminal in larvae and trans fo rm ing la rvae , becomes
p rog ress lve ly in fe r io r in p o s it io n during the e a r ly Ju ven ile stage.
D e n t l t io n * Developing teeth are f i r s t seen 1n the a reo la r
connective t issue underly ing the bucco-pharyngeal e p ith e l iu m o f the
la rva e , Retrorse conical tee th erupt on the prem axillae a t ca, 4.0 mu
NL and on the dentaries by 5 ,2 nm NL (Table 1), At ca. 16 rm a second
h o r izon ta l row of recurved v i l l i form tee th erupts 1 inquad on the
p rem axillae and dentaries* This row o f v i l l i f o r m teeth replaces
la rv a l tee th as add itiona l rows of recurved v i l l i f o r m teeth erupt
1 inquad forming tooth rows. At complete t ra n s fo rm a tio n , the
polyphyodont d e n t i t io n (Peyer, 1968) o f spot 1s comprised o f three
25
to o th rows, Premaki 11 a ry and dentary te e th w i th in a to o th fa m i ly
(Peyer, 1968) are sh o rte r toward the U nqua l aspect.
V i l l i f o r m pharyngeal tee th begin to erup t on the paired medial
c e r ta to b ra n c h ia ls of b ranch ia l arch 5 and on the pharyngobranch ia ls o f
arches 2 , 3 , and 4 by 5,5 mm NL (Table 1 ) , The complete complement o f
pharyngeal teeth is reached by complete t ra n s fo rm a t io n w ith v i l l i f o r m
tee th on the a n te r io r aspect of the ce ra to b ra n ch ia ls and
pharyngobranchials 3 grading to con ica l te e th toward th e p o s te r io r
aspect,
Gut Contents
Labora tory reared specimens show no evidence o f feed ing as y o lk -
sac la rvae . Green chyme, comprised presumably o f d in o f la g e l1ates and
a lga l c e l l s , is seen d u r ing o i l - g lo b u le a b s o rp t io n . R o t i fe rs and
copepod nau p lH are taken soon a f te r the absorp tion o f the o i l
g lo b u le . The d ie t of w i ld larvae c o n s is ts p r im a r i ly o f copepod
n a u p l i i and copepotMtes w ith some ju v e n i le pelecypods and c ladocerans.
The s ize o f preferred food organisms Increases w ith the s ize o f the
larvae [K je lso n et a l . , 1975; Table 2 ) , and, as the la rv a grows, a d u lt
calanolds e n te r the d ie t . During t ra n s fo rm a t io n , e p ib e n th ic
h a rpac tico id copepods (Ph.vl 1 opodopsvl lus s p p , ) dominate the d ie t
(Table 2 ) . Juveniles feed on d e t r i tu s and ep ifaunal in v e r te b ra te s ,
mainly anne lids (Parker, 1971; Peters and K je ls o n , 1975; Chao and
Musick, 1977; Sheridan, 1978).
26
Table 1, Tooth counts o f la rva l Lelostomus xanthurus. P re m ax llU ry and dentary teeth were counted on the l e f t s ide and counts m u l t ip l ie d by two; pharyngeal tee th were observed but not counted.
Notochord/Standard Premaxi 1 la ry Denta ry Epibranchial Ceratobr;
Age Length Teeth Teeth Teeth Teel
4 2,4 _ _ _
7 2.4 - - -2.7 - - - -3.0 - - -3.'’ - - _ •
4.0 2 - - -
23 4.5 10 - - -28 4,3 B - -28 5.2 10 2 - -30 5.2 10 - - -35 5.5 14 - + +35 6,4 12 fi - -36 5,1 14 2 - -36 10.0 30 22 +41 5.4 12 - - -41 G.9 20 18 + +42 10.5 30 16 + +43 5.B 12 8 - -50 6.6 18 12 - -54 8.0 IB 16 + +54 8.2 16 14 + +
12.6 32 26 + +12.6 44 38 + +12,9 40 32 + +12.9 34 50 + +13.2 52 58 + +13.9 30 48 + +14.5 90 62 + t17.7 108 76 +18.5 116 78 + +19.1 108 72 + +19,4 116 38 + +20.0 108 86 +20.0 76 72 + +20,1 96 76 + +20.3 106 78 +20.5 68 80 +21.0 106 86 + +
27
Table 1. ( cone 1 tided)
Notochord/'Standard P rem axllla ry Dentary Ep ibranch ia l Ceratobranchlal
Age Length Teeth Teeth Teeth Teeth
21.0 ee 85 + +21.5 102 98 + +22 , 9 70 54 + +23.9 64 62 + +24.2 64 74 + +
Table
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Figure 1 -6 . Morphological development o f the a l im e n ta ry canal o f
Lelostomus xanthurus. 1. In c ip ie n t g u t o f a yo lk-sac
larva 2 . 2 wn NL, 2. In c ip ie n t gut o f a yo lk-sac larva
2.3 ran NL. 3. In c ip ie n t gut o f a la rv a 2,8 inn NL in
o i l -g lo b u le abso rp t ion . A. A lim entary canal o f a larva
3.0 ran NL, 5, A lim entary canal o f a transform ing larva
7.6 ran SL. 6, A lim entary canal o f an e a r ly ju v e n ile
25.0 mm SL. Abbrev ia tions : BP, bucco-pharynx; BS, b l in d
sac; E, esophagus; FG, fo re g u t ; HG, h lndgu t; I ,
In te s t in e ; IG* in c ip ie n t g u t; MG, m ldgut; 0Gt o i l
g lobu le ; PC, p y lo r ic ceaca; R, rectum; S, stomach; YM,
yolkmass.
- N
~-~~ r-
) I I I I I I I
l \ I I I
) I
I
I I \ I I
'· I ' I I ' I
I \ I
I
I I
8 I
I (!) H
C!) H
2 2 >- >-
31
Figures 7-8. Photomicrographs o f the in c ip ie n t gut o f a Leiostomus
xanthurus yo lk -sac la rva 2 .3 mm NL. 7. Long itud ina l
section showing y o lk mass, o i l g lobu le , In c ip ie n t gu t,
l i v e r , and pancreas. 8. Cross sec tion showing mucosal
ep ithe lium and s t r ia te d .
Figures 9-10. Photomicrographs o f the a lim en ta ry canal o f a
Leiostomus xanthurus la rva 4.4 mn NL. 9. Long itud ina l
section showing fo re g u t , m ldgut, h lndgu t, l i v e r , and
pancreas. 10. L o n g itud ina l section showing two types
o f mucous c e l ls 1n fo re g u t . Abbrev ia tions: CT,
connective t is s u e ; FG, fo re g u t ; EP, e p ith e l iu m ; HG,
h lndgut; IG, in c ip ie n t g u t ; L, lumen; L I* l i v e r ; MG*
midgut; MCI, mucous c e l l type 1* MC2, mucous c e l l type
2; OG, o i l g lobu le ; P, pancreas; SB, s t r ia te d border;
YM. yolkmass.
' I , ' _. ...,..
\
•
e e
T
. . .
32
Figures 11-14, Photomicrographs o f the a lim entary canal o f a
Lelostomus xanthurus larva 4 ,4 mm NL. 11.
Longitudinal section showing m idgut, 12,
Longitudinal section showing h lndgut ep ithe lium w ith
s t r ia te d border and In t r a c e l lu la r In c lus ions . 13,
Longitudinal section showing l i v e r ac in i w ith l i v e r
s inus , hepatocytes, and glycogen vacuoles, 14,
Longitudinal section showing pancreatic ac in i and
zymogen granules. Abbreviations: EP, e p ith e l iu m ; GJ,
glycogen vacuoles; H, hepatocytes; IC I, i n t r a c e l lu la r
Inc lus ions ; LS, l i v e r sinus; PA, pancreatic a c in i ; SB,
s t r ia te d border; ZG, zymogen granules.
~· 0 I
'l.. • .. I .
' .;\
..... ... 1'
-~ . ' ..
~ ., .. ...
·' " . .. - . ..... ..
•
- 4JY ...
33
F ig u re s 15-
Figure 17*
Figures 18-
16. Photomicrographs o f a trans fo rm ing Lelostomus
xanthurus la rva 12*3 m SL. 15* Long itud ina l
section showing ju n c t io n o f bueco-pharynx and
esophagus w ith esophageal fo ld s and Increased
number o f mucous c e l ls . 16* L o n g itud ina l section
showing the Junction o f the esophagus and b l in d sac
w ith developing g a s tr ic glands*
Photomicrograph o f an e a r ly ju v e n i le Lelostomus
xanthurus 18.1 mn SL* L o n g itud ina l sec tion o f
gas tr ic corpus o f a f is h 18.1 mn SL showing
uniform secre to ry c e l ls o f the g a s t r ic glands.
. Photomicrographs o f an e a r ly ju v e n i le Lelostomus
xanthurus 33.0 mti 5L. 18- Long itud ina l sec tion
o f the In te s t in e showing I n t r a c e l lu la r inc lus ions*
19. Long itud ina l section o f the rectum showing
granular I n t r a c e l lu la r In c lu s io n s . Abbreviations:
BP, bucco-pharynx; BS, b l in d sac; E, esophagus;
GG, g a s tr ic g lands; IC l t i n t r a c e l l u l a r in c lu s io n s ;
MU, mucosa.
34
DISCUSSION
The a b i l i t y to acquire , d ig e s t , and ass im ila te n u t r ie n ts should
be maximized by adaptation throughout the development o f a f i s h . The
numerous mitochondria and I n t r a c e l lu la r Inc lus ions in th e mid- and
hlndgut ep ith e liu m , as well as the h is to lo g y of the l i v e r and
pancreas, suggest th a t the a lim en ta ry canal becomes fu n c t io n a l du r ing
o i l -g lo b u le absorption when f i r s t feeding may occur. There Is no
e labora tion of c e l l types from f i r s t feed ing u n t i l t ra n s fo rm a t io n ,
though the surface area Increases. The l i v e r expands r a p id ly in the
la rva and is a c t iv e in glycogen storage. E h r l lc h (1974a, b) has found
th a t p ro te in and carbohydrate are t ra n s fe r re d to body t is s u e s
p r e fe r e n t ia l ly over t r lg y lc e r id e s in la rv a l Cluoea harengus and
Pleuronectes p la te ss a . Growth is o f prime importance to the pelagic
marine larva 1n enhancing predator avoidance and feeding success.
P ro te in deposit ion is undoubtedly manifested as la rva l growth, wh ile
high carbohydrate storage as hepatic glycogen poss ib ly has a sparing
e f fe c t on p ro te in catabolism; Cowey e t al . (1975) demonstrated such an
e f fe c t in a d u lt P. p la te ssa . The bulbous midgut perhaps serves a
fu n c t io n s im ila r to a morphological stomach in a llow ing the Ingestion
and storage of many food items, an adaptation which is advantageous to
a pelagic marine larva confronted w ith a patchy food supp ly .
The major changes In the development o f the a lim en ta ry canal and
associated s tru c tu re s are accompanied by changes in h a b i ta t and
35
feed ing regimen. The re tro rs e , c o n ic a l, la rv a l te e th are we’l l suited
fo r seizure o f pelagic copepod naupHi and copepodites while the
mucous ce lls o f the bucco-pharynx and fo regu t aid in Inges tion , The
movement of transform ing larvae in to es tua rine nursery areas (Chao and
Musick, 1977) Is concomitant w ith the d i f f e r e n t ia t io n of the
esophagus, stomach. In te s t in e , and rectum, as well as the appearance
o f the adult complement o f v I lH fo r tn jaw and m o la rlfo rm pharyngeal
te e th . This corresponds w ith a d ie t s h i f t to e p i- and Infaunal
in v e r te b ra te s . The fu n c t io n a l development o f ta s te buds must aid in
the se lec t ion o f these food items while the pharyngeal teeth and the
Increase in mucous c e l ls aid ir> m astication and in g e s t io n . Complete
development o f gas tr ic glands presumably a id w ith p re l im ina ry
p ro te o lys i s .
The sequence of the a lim entary canal development in spot is
s im i la r to tha t described fo r o ther marine f ish e s (Harder, 1960;
Fukusho, 1972; others below) w ith some notable s p e c ia l iz a t io n s . The
I n t o r t ion o f the a lim entary canal to form a loop i s common among
paracarthopteryg lan and acanthopteryglan la rvae (elopomorph and
protacanthopterygfan larvae have a s t ra ig h t g u t ) ; but i t Is not always
simultaneous with yo lk-sac or o i l -g lo b u le absorp tion (e .g . , the case
o f Trachurus symmetric us: The llacke r, 1970), Goblet or mucous ce lls
are present in the bucco-pharynx and foregut In spot as well as most
o th e r larvae (Tanaka 1969a, b; Tanaka 1971; Vu 1976), In co n tra s t,
O'Connell (1976} found them only a t the pharyngeal p o r t in Engraulls
mordax and Vu (1976) found goblet c e l ls in the mid- and hindgut o f
36
la rv a l D1centrarchus la b ra x . The occurrence o f two types o f mucous
c e l ls in the la rv a l fo re g u t concurs w i th R e lfe l and T r a v l l l (1977) who
described s ix d i f f e r e n t esophageal mucous c e l l types 1n a d u lts w i th
pa irs o f types associated w i th f iv e p a r t ic u la r fa m i l ie s o f f reshw a te r
te le o s ts . The apparent absence o f Type 2 mucous c e l ls 1n J u ve n ile
spot suggests th a t these c e l l s are e i t h e r t ra n s ie n t la r v a l s t ru c tu re s
o r s imply Type 1 c e l ls th a t have e xp e lle d t h e i r contents and are be ing
sloughed o f f . Taste buds appear 1n the bucco-pharynx o f la r v a l sp o t,
but are presumed not fu n c t io n a l u n t i l the e a r ly Ju v e n i le phase. In
many te le o s ts w ith demersal eggs, however, tas te buds are found 1n
yo lk -sac la rvae (Tanaka, 1969a). The c o n tro v e rs ia l r o d le t o r
pear-shaped c e l l (see reviews 1n Kapoor e t a l . , 1975; H arr ison and
Odense, 1978), a lso I d e n t i f ie d as the sporozoan Rhadbospora the lo h a n i
( e .g . , B a n n is te r , 1966), has been found In the a lim e n ta ry canal o f many
fishes In c lu d in g the young o f la b o ra to ry cu ltu re d Scophthalmus maximus
(Anderson and Roberts, 1976). Smallwood and Smallwood (1931) re fe r re d
to In te s t in a l secretory c e l l s , p o ss ib ly Id e n t ic a l to these pear-shaped
c e l ls (B a r r in g to n , 1957), in la rv a l Cyprinus c a rp io . A lk a l in e b lue 6B
is p a r t i c u la r l y useful in dem onstrating these p a ra s ite s (R u d de ll ,
personal conm unlcation), y e t they were n o t found 1n la rv a l o r e a r ly
Juven ile spo t.
The mechanism o f l i p i d and p ro te in d ig e s t io n in la r v a l and
ju v e n i le f is h e s may w arrent f u r t h e r research. The e p i t h e l i a l c e l ls o f
spo t 's la r v a l mid- and h in d g u t are s im i la r c y to lo g ic a l ly to those o f
s ix species described by Iwai (1969). He concluded th a t the vacuo la r
37
Inc lus ions of the midgut conta in l i p id s , resynthesized a f te r
hydro lys is and abso rp t ion , and th a t the g ranu la r in c lu s io n s o f the
hindgut contain p ro te in , the re s u l t o f p lnocy tos ls from the lumen.
Kawai and Ikeda (1973) and Tanaka et a l . (1972) reported weak pep tic
a c t i v i t y In larvae before the d i f f e r e n t ia t io n o f g a s tr ic glands while
t r y p t i c a c t i v i t y appeared s h o r t ly a f t e r hatch ing and increased
g radua lly w ith growth. The pancreas o f spot and other larvae (e .g .
Urneda and O ch ia i, 1973; O’C onne ll, 1976; Vu, 1976) contains
conspicuous zymogen granules presumably comprised o f t r y p s in , T ryp t ic
a c t i v i t y alone might not completely hydrolyze p ro te ins to amino a d d s
w ithout p re l im ina ry peptic d ig e s t io n , and Yamamoto (1966) and Stroband
(1977) have made specu la tive c o r re la t io n s between the lack o f peptic
d iges tion 1n stomachless adu lt te le o s ts and the incidence of
p ln o c y c to t ic protelnaceous, in t r a c e l l u la r in c lu s io n s . A cco rd ing ly ,
p inocytos is of p ro te in compensates fo r Incomplete p ro te o ly s is as might
be the case 1n la rv a l f ish e s ( Iw a i , 1969). While the lo ca t io n o f
absorption agrees w ith adu lt f ishes (Fange and Grove, 1979), one might
suspect complete p ro te o lys is a f te r the development o f g a s t r ic g lands.
Yamamoto (1966) has suggested th is based on the lack o f p ln o c y to t ic
invag inations and granular in t r a c e l lu la r Inc lus ions in a d u lt Salmo
1r id e u s . Yet, a c id o p h i l ic in t r a c e l l u la r in c lu s io n s occur in ea r ly
ju v e n i le spot even a f te r complete stomach d i f f e r e n t ia t io n , O'Connell
(1976) also reports f in d in g s th a t co n tras t Iw a i ’ s hypothesis.
The alim entary canal and associated s t ru c tu re s o f spo t, s im i la r
to tha t o f o ther marine te le o s ts , becomes fu n c t io n a l at f i r s t feeding
38
and change l i t t l e during the la rva l phase. Other than the expansion
o f a lim en ta ry canal surface area, there are no morphological or
h is to lo g ic a l changes th a t could e f fe c t major changes in d igestion o r
a s s im i la t io n p r io r to trans fo rm ation . The complete development o f a
stomach w ith functional g a s t r ic glands d u r in g trans fo rm a tion may
in f lu e n ce d igestion and a s s im ila t io n and is accompanied by changes in
h a b ita t and feeding regimen.
EXPERIMENTAL RADIOTRACER STUDIES OF THE
SHORT-TERM CARBON ASSIMILATION
INTRODUCTION
Our understanding of la rv a l f is h s ta rv a t io n and growth and the
re la t io n s h ip of these processes to stock recru itm ent has been re ce n t ly
advanced by the a p p l ic a t io n o f p h ys io log ica l energe tic p r in c ip le s . In
phys io log ica l energetic models developed by Iv le v (1939) and Wlnberg
(1956) and la te r re fined by Paloheimo and D ick ie (1966a,b) and Warren
and Davis (1967), a f i s h 's energy budget can be expressed as:
Qf = Q* + Q' + Q_ (1)
where = Q+ = energy of food consumed (m ges ta )
Q* - energy of waste products in feces and u r in e (egesta and
excre ta )
Q‘ - energy o f growth
Q_ = energy o f metabolism.
Rearranging g ives:
Q+ - Q* = Q' + q_ (2 )
or
bq+ = Q' + q__ (3)
(symbols fo l lo w in g Laurence, 1977). The c o e f f i c ie n t , b , represents
a s s im i la t io n and is gen e ra lly expressed as e i th e r a c o e f f ic ie n t , the
c o e f f ic ie n t o f u t i l i z a t i o n (Wlriberg, 1956); o r as a percentage,
absorption e f f ic ie n c y (Kapoor et a l . , 1975). A s s im i la t io n , defined as
40
41
th a t pa rt of the food In take which 1s u t i l i z e d (B re t t and Groves,
1979) 1s d i f f i c u l t to measure d i r e c t l y in aquatic animals la rgely
because i t is d i f f i c u l t to separate egesta from excreta (Johannes and
5atom1, 1967i Conover, 1978), The c o e f f i c ie n t o f u t i l i z a t i o n
represents the fra c t io n o f m etabolizable energy a v a i la b le a fte r losses
In feces (the resu lt o f Incomplete absorp tion from the alimentary
canal) and in urine or through the g i l l s ( la rg e ly the resu lt o f
deamination of p ro te in s ) . Absorption e f f i c ie n c y , the d iffe rence
between the matter-energy In Ingesta and egesta expressed as a
percentage, has been cus tom arily used synonomously w ith ass im ila t ion
e f f ic ie n c y . Such estimates might be b iased , however, i f some egesta
is confounded w ith exc re ta . The c o e f f ic ie n t o f u t i l i z a t i o n d i f fe rs
from absorption e f f ic ie n c y and is e as ie r to measure as defecatory and
excre to ry losses are combined. Because te le o s ts are amnonotelic
[F o rs te r and Goldstein, 1969), a s s im i la t io n should be the dominant
process encompassed by the c o e f f ic ie n t , e s p e c ia l ly 1f carbon 1s used
as a measure.
As o r ig in a l ly fo rm u la ted , these equations used energy units
( c a lo r ie s ) , but others have subs titu ted t o t a l wet o r dry weight,
weights of carbohydrates, fa ts , and p ro te in s , or weights of to ta l
carbon and nitrogen (see reviews in B re t t and Groves, 1979; Conover,
1978). H a tte r and energy are thus used in te rchangeab ly , though d ire c t
analogies are not always v a l id [ e .g . , W legert, 1968).
Important progress has been made in understanding the b io log ica l
processes represented by th e various terms o f ene rge tic models with
42
la rv a l f ish e s (Table 3 ) , but most o f these stud ies d e a lt w ith
freshwater species w ith precocious la rv a e . Furthermore, few attempts
have estimated energe tic parameters o f marine larvae or assessed
changes in these terms during la rv a l development; though i t 1s well
known th a t r e s p i ra t io n and e xc re t io n ra te s , and convers ion
e f f ic ie n c ie s can change w ith body weight (see review by B re t t and
Groves, 1979}, Laurence (1977), an excep tion , estimated ra te s o f
In g e s t io n , metabolism, and growth In i la r v a l marine f is h and used
these estimates to s imulate changes In a s s im i la t io n w ith development.
Rosenthal and Hempel (1970) and Nlshikawa (1975) suggested th a t
d ig e s t io n Improves w ith la rva l age as the re su lt o f the m orphological
development o f the a lim entary cana l, but the re 1s l i t t l e d i r e c t
evidence to support t h is .
This study was undertaken to estim ate short- te rm carbon
a s s im ila t io n in d is c re te age cohorts o f la rv a l spo t, Leiostomus
xanthurus, to assess changes in a s s im i la t io n , and to r e la te such
changes to development. The c o e f f ic ie n t o f u t i l i z a t i o n and abso rp tion
e f f ic ie n c y were used here in to assess carbon a s s im i la t io n .
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44
CONCEPTUAL FRAMEWORK
Radiotracer methodology, u t i l i z in g carbon-14 was used to compute
two parameters that were taken as r e la t i v e ind ices o f the c o e f f ic ie n t
of u t i l i z a t i o n and absorp tion e f f ic ie n c y . Carbon re te n t io n , the Index
o f the c o e f f ic ie n t o f u t i l i z a t i o n , was computed from the body
burden retained in la rvae and corrected f o r re s p i ra to ry loss a f te r
in g e s t io n and complete a lim entary canal evacuation. Carbon
abso rp t ion , the Index o f absorption e f f i c ie n c y , was computed from l^c
ingested and egested as p a r t ic u la te s . Computations fo llowed Sorokin
(1966):
C = a * r (4)where
C - to ta l carbon tra n s fe r re d (mg C)
a = re c ip ro c a l of the food substra te sp e c if ic
a c t i v i t y
r = r a d io a c t iv i t y in la rvae or p a r t ic u la te egesta
j DPM ,,^mgDW'
Carbon retention and carbon absorption were considered re la t ive
ind ices because use o f Equation (4) to estim ate carbon tra n s fe r has
some methodological problems. Carbon-14 can en te r several
phys io lo g ica l processes w+th d i f fe re n t ra tes w ith in an animal and be
lo s t through re sp ira t io n or excre tion . I f th is occurs during
45
Ingestion o r Alimentary canal evacuation, estimates o f to ta l carbon
ingested o r retained ca lcu la ted w ith Equation (4) w i l l be biased
(Conover and Francis, 1973). To co rrec t t h is b ias, Lampert (1977)
co llec ted respired ^CQg and added these DPM to the body burden o f h is
animals. This co rrec t ion 1s not e n t i r e ly adequate, however, because
the s p e c if ic a c t iv i t y o f carbon lo s t as CQg f lu c tu a te s during o f the
experiment: I t increases w ith time a f te r Ingestion as more
ass im ila ted . Because the sp e c if ic a c t i v i t y o f carbon lo s t 1s unknown,
the absolute amount of t o ta l carbon reta ined w ith in an animal cannot
be ca lcu la ted . Such ca lcu la t io n s requ ire multlcampartmerital analys is
(Robertson, 1956; A tk ins, 1969; Jacquez, 1972); but data necessary to
support these models are d i f f i c u l t to c o l le c t (Conover and Franc is ,
1973), Nonetheless, estimates of carbon ass im ila t io n using equations
s im ila r to Equation (4 ), compare well w ith c a lo r lm e tr lc estim ates,
esp e c ia l ly i f estimates are adjusted fo r re sp ira to ry loss (Lampert,
1977), Moreover such estimates o f carbon a s s im ila t io n are comparable
among age cohorts i f any b ias is constant.
Use o f Equation (4) to estimate and assess changes in these
ind ices is ju s t i f i e d by the fo l low ing assumptions, a l l addressed
herein: ( 1 ) the food substrate Is un iform ly labeled, i . e . , the
biochemical f ra c t io n a t io n o f 1s constant; ( 2 ) the s p e c if ic
a c t iv i t y o f the food substra te is constant during the feeding
In te rv a l ; (3 ) resp ira to ry loss does not occur during the feeding
In te rva l (no corrections f o r such bias could be made); (4) the
estimates o f carbon re ten tion and carbon absorption were taken at
complete alimentary canal evacuation; and (5) any biases are constant
among age cohorts.
46
MATERIALS AND METHODS
Experimental Animals
Spot larvae were obtained from labo ra to ry cu ltu red popu la t ions^ .
A r t i f i c i a l l y spawned and f e r t i l i z e d eggs were cu ltu red in tanks at
20 + 2“C and 30-35 ° /o o s a l i n i t y . This approximated cond it io n s
encountered in nature (see E ar ly L i f e H is to ry s e c t io n } . A f te r
hatching larvae were maintained at concentra tions o f < 1 0 /1 w ith a
photoperiod o f 12 h l i g h t and dark and fed se q u e n tia l ly w ith the
d ln o f la g e l la te , Qymnodlnlum splendens. the r o t i f e r Brachionus
p i i c a t i l 1 s . and w ild plankton a t d e n s it ie s o f 5 to 25 p a r t ic le s /m l .
Larvae were removed from the c u l tu re tanks the evening before an
experiment, placed 1n an experimental feeding ta n k , and fas ted
ove rn ig h t. S ta t is t ic s sumnarlzlng development o f la rva l spot age
cohorts are presented In Table 4. Condition fa c to r , o f te n used as an
in d ic a to r o f la rv a l growth and robustness (Hempel and B la x te r , 1961;
B la x te r , 1971), varied among co h o rts . The growth exponents o f weight
and length regressions o f larvae used 1n experiments (F igure 20) are
somewhat lower than those reported by Laurence (1979). Larvae were
ge n e ra lly hea lthy , however, and showed no h is to lo g ic a l signs
d ia g n o s t ic of s ta rv a t io n (Umeda and O ch la i, 1973; O 'C onne ll, 1976;
T h e ilacke r, 1978),
47
Table 4. Sumnary of s t a t i s t i c s ch a ra c te r iz in g development of d iscre te age cohorts o f Tarval Lelostomus xanthurus used 1n carbon a s s im ila t io n experim ents.
Age (days from hatch lng)
Mean Notochord or Standard Length
[mm)
Mean dry weight
(mg)C ond it i ona
Factor
7 1 .8 0 . 0 2 2 37,7
e 2.5 0.050 32.0
9 2,2 0.034 31,9
14 2 , 8 0.075 34,1
15 3*1 0.048 16.1
16 3,2 0.140 42.7
31 0 .6 1.330 46.3
34 6 . 8 1,505 47.9
43 1 1 .4 4.426 29.9
47 9.2 2,320 29.0
* ^ o n a it .o n fa c to r . ^ £ ^ , 3 x 10*
48
Figure 20* Weight and length regressions fo r Leiostomus xanthurus
larvae used In carbon a s s im ila t io n experiments. Upper
l in e fo r postf lex ion la rva e ; lower l in e fo r p re f le x io n
larvae (Ahlstrom, 1968).
20 00
I 0.00- 000- 6 00 -
2 0 0 -
( 00 -
w t => 0,00021
f = 089
n * 2 5 8
o 0 60- c0 40-
u* O 2 0 -»-a.o
0 I 0-o <:m3 -
0 0 4
w t = 0.00280 Lt 3 061 ri
r - 0,78
4 6 S 10o IOO
N O TO C H O ftD / S TANOAHD l E N O T H ( m r i )
49
Brachlonus p l lc a t l1 1 s was chosen as the experimental food
organism because i t is amenable to mass c u ltu re in the laboratory
(The l!acker and HcMaster* 1971J and 1s an adequate n u t r i t io n a l source
f o r la rva l fishes [May* 1971; Solangl and Ogle, 1977J. Prelim inary
experiments suggested tha t i t could be re a d i ly labeled w ith and
th a t la rva l spot consumed them from f i r s t feeding through
trans fo rm a tion ; though the behavioral mode of ingestion changed with
development* This allowed the use o f the same food substrate among
experiments with d i f f e r in g age cohorts o f la rvae . The major
n u t r i t io n a l components o f Brachlonus. cu ltu re d by the fo l low ing
p ro to c o l, were determined and values compared w ith the l i te ra tu re
(Table 5), Total p ro te in and l i p i d e x tra c t io n s fo llow ed Lowry et a l . ,
(1951), B ligh and Dyer (1959), and Hanson and 01 ley (1963),
Carbohydrates were extrac ted by p re c ip i ta t in g p ro te ins from Brachlonus
homogenates with t r i c h lo ro a c e t ic acid, d is s o lv in g l ip id s 1n ethanol,
and p re c ip i ta t in g carbohydrates w ith NaCl. E x trac t ions were assayed
fo l lo w in g Lowry et al ■* (1951), Barnes and Blackstock (1973), and a
m o d if ica t io n o f the anthrone method (S tr ic k la n d and Parsons* 1972).
Experimental Design
The experimental design Included la b e l in g o f Brachlonus and
m onitoring changes in i t s s p e c if ic a c t i v i t y , es tim ating carbon
ingested by feeding la rv a e , and es tim ating carbon re ta ined and lost
during alimentary canal evacuation [F igu re 21). Experiments were
conducted in 20 + LaC* 30-35 ° /oo s a l in i t y * 0.45 pm f i l t e r e d seawater
“ s t e r i l i z e d '1 by u l t r a v io le t i r r a d ia t io n . Experiments began at the
50
Table 5* Major n u t r i t io n a l components of Brachionus p l i c a t lH s expressed as a percentage o f to ta l dry weight.
ComponentPresent
StudyEhrlIch
(1972)Scott and
Baynes (1978)
Oglno and Watanabe
(1978)
Pro te in 38.9 53,6 b 58
L ip id 1.7 4.6a 1 1 . 2 b 10
Carbohydrate 1.9 8 ,6 6 ,Qb 6
Total carbon 41.7 33.1
Total nitrogen 9,0 0 .0
C/N ra t io 4.6 4.2
Ash 7,0 8 .6
a Determined as to ta l t r ig ly c e r id e s .
b Calculated as grand rtean of th e ir experimental values.
51
Figure 21. Experimental design o f carbon a s s im i la t io n w ith age
cohorts o f la r v a l Leiostomus xanthurus.
Ctifartlfw
Labeling
Bw chiortu t
Labeling
Sieving, Resuspension, and got clearance U 2 hl
Brachionus Samples for liqu id sc in tilla tion counting
Sieving and ResuSpenSion in feeding chamber
Feeding Chamber ( 14 C uptake)
1, 2 & 4 h
Transfer tu retention chambers
R etention Chambers ( 1 4 C retention)
Brachionus Samples tor liq u id sc in tilla tion counting
L a r v a e sam ples fo r l i q u i d sc in lilla tion counting
L a r v a e sam ples fo r l i q u i d2,4,0,7 Si 9 h sc in tilla tion counting
1 4
1 4 CO2 TfaP —
ICO 2 bubble trap
Fi ter
' 4 C0? tra P samples tor liqu id sc in tilla tion counting
1 4 COz bubble traps for liqu id scin tilfa tion counting
P O 1 4 C res idue for l i q u i d Scintillation counting
52
beginning o f the 12 h l ig h t photoperiod* Larvae were fed Brachtonus
at high dens it ies (250*10QQ/ml) to a llow the a lim en ta ry canal to f i l l
and provide p h ys io lo g ica lly equ iva lent ra tion s among age cohorts .
Brachlonus was labeled fo l lo w in g Sorokin (1966). Two 1 cu ltu re s
(ca, 3.0 x 10? c e l ls /m l) o f the u n ce llu la r algae Chlorel la s j). ( s t ra in
Va 52) were innoculated w ith 200 pel o f NaHl*C0 3 and cu ltu re d under
constant I l lu m in a t io n fo r 2 weeks to allow maximum popu la tion growth
and isotope f ix a t io n (ca. 8.0 x 10® c e l ls /m l; 3 .0 x 10*^ CPM/cel 1).
The cu ltu re medium (Dupuy et al . t 1977 ) was adjusted to pH 9 w ith
NaGH. Generally, Brachlonus was fed high concentralons (ca . 8 .0 x 10®
c e l ls /m l) o f Chlorel la fo r 5 days o r through 2 to 3 generations before
use in an experiment. P r io r to an experiment, Brachionus was Iso la ted
by gentle sieving on a 35 pm n ltex screen, washed w ith and resuspended
in seawater, and fasted fo r 12 h to c lear t h e i r guts of labeled algae.
To assess label u n ifo rm ity the d is t r ib u t io n o f in biochemical
frac t ions in Brachlonus was determined, Brachionus was labeled as
described and samples co llec ted a f te r 2, 4, 6 , and 8 days o f la b e l in g .
Total p ro te ins , l i p id s , and carbohydrates were ex trac ted from these
samples and the CPM of these ex trac ts were expressed as ra t io s o f
to ta l extracted CPtf,
The spec if ic a c t i v i t y o f Brachionus was monitored by ho ld ing 1 1
o f labeled Brachlonus 1n a beaker, sampling i n i t i a l l y , and 1, 2 , and
4 h th e re a f te r . T r ip l ic a te a l iq u o ts were f l l t e r d through ta red*
g la s s - f lb e r f i l t e r s , and washed tw ice with 0 .5 N HCl and d i s t i l l e d
53
water. They were then frozen , Tyoph ll ized , and stored w ith in a
des icca to r in a fre e ze r.
Carbon-14 uptake by la rvae was estimated by adding labeled
Brachlonus to a feeding tank and sampling larvae at 1 , 2 , and 4 h.
T r ip l i c a te samples were su ffocated on a n ite x screen, placed on ta re d ,
g la s s - f ib e r f i l t e r s , and trea ted as described fo r Brachionus samples.
Dead or moribund larvae were d iscarded.
Carbon-14 re te n t io n and loss by la rvae was estimated by
t ra n s fe r r in g larvae fed labeled Brachionus fo r 1 , 2 , and 4 h to a
series o f re te n t io n chambers (F igure 22) and sampling at 2, 4 , 6 , 7
and 9 h. Retention chambers were sealed w ith rubber stoppers f i t t e d
w ith ^ C 02 traps suspended in the atmosphere above the sea water.
Traps consisted o f a wel 1 w ith a 2.5 cm fo lded g la s s - f ib e r f i l t e r and
two in je c t io n ports (F igure 22}. Sampling involved In je c t in g 200 pi
o f phenethylamine in to the tra p well and a llow ing any respired ^ C 0 2
in the atmosphere above the sea water to react fo r 5 minutes; the
chamber was then opened and the la rvae sampled as described. A f te r
removing la rvae , the chambers were resealed and 0,3 ml o f concentrated
H2 5 O4 was in jec ted in to the w ater. P re lim ina ry experiments ind ica ted
tha t t h is a c id i f ic a t io n lowered the pH below 4 and thus released
H^C0 3 as gaseous to be co l le c te d in the suspended t ra p .
A c id i f ie d chambers then were reopened. The phenethylamine and f i l t e r
were removed, the trap well washed w ith 200 ul o f 95% methanol, and the
phenethylamine, f i l t e r , and washings placed 1n a s c i n t i l l a t i o n v ia l f o r
coun ting . To c o l le c t any remaining ^COg* the chambers were then
54
Figure 22, Schematic diagram of re te n t io n chambers used 1n carbon
a ss im ila t io n experiments w ith age cohorts o f la rv a l
Leiostomus xanthurus.
□ r . ,
P H E N E THYLAM IN E — I N J E C T I O NI N J E C T I O N
CQ, TRAP
55
f i t t e d to a bubble tra p co n s is t in g o f a series o f two s c in t i l l a t i o n
v ia ls con ta in ing 5 ml o f t ra p s o lu t io n (Woeller 1961) and bubbled w ith
COg-free a i r fo r 20 mln to recover any remaining (F igu re 23).
Following recovery o f the re te n t io n chamber sea water was
assayed fo r egested as p a r t ic u la te organic carbon (PO^C) by
f i l t e r i n g through glass f i b e r f i l t e r s . F i l t e r samples were washed as
described above.
Blanks, designed to assess contamination o f re te n t io n
chambers and la rvae , showed th a t contamination was un im portant.
Blanks consisted o f a se r ies o f re te n t io n chambers to which the
approximated maximum volume of water ca rr ie d over during the t ra n s fe r
o f larvae was added. Blanks were sampled fo r and PQl^C as
described. To assess uptake o f d isso lved and p a r t ic u la te from the
water, blank larvae were exposed f o r 1 h to feeding chamber water from
which labeled Brachionus had been seived at the end o f several
experiments and sampled as described. Analysis of blanks showed th a t
contamination o f samples was va r ia b le but s l ig h t and averaged 9.7%
(^C Q jj) , 8,7% (PO ^C), and 0.3% ( la rv a e ) . Because estim ates were
approximate and probably in f la te d , these e rro rs were ignored.
Sample Processing
Samples o f ly cp h lH ze d Brachlonus and la rvae were removed from
the f re e ze r , brought to room temperature w ith in the d e s icca to r ,
weighed to the nearest vg on an e le c troba lance , placed in a glass
s c in t i l l a t i o n v ia l , wetted w ith 300 y l o f d i s t i l l e d water, and
56
Figure 2 3 1 Schematic diagram o f bubb le -trap fo r recovery o f 14C0£ 1n
carbon a s s im ila t io n experiments w ith age cohorts o f la rv a l
Leiostomus xanthurus.
UJCOCO
tft _l
O 5I- Z 3 o
o 5l/» 3O — Z I-£ 5£L O< to £ *
O 0=
z “w 3H < LU XX O
o:u
o - |
ol/l
L
57
digested in 2 ml o f NCS (Nuclear Chicago t issue s o lu b i l i s e r ) f o r 1 to
Z days at 50°C. P a r t ic u la te organic carbon-14 samples were t re a te d
s im i la r ly , but were not weighed. A f te r d ig e s t io n samples were then
cooled to room temperature before adding s c i n t i l l a t i o n c o c k ta i l ( 6 g
POP. 0.05 g P0P0P/1 to lu e n e }. Brachlonus. la rvae , PQ^C, t ra p ,
and ^COg bubble-trap samples were combined w ith 15 ml o f
s c i n t i l l a t i o n c o c k ta i l , held a t room temperature in the dark f o r 36 h
to decay chemlluminescence (F igure 24}, and counted in a l iq u id
s c in t i l l a t i o n counter fo r the e n t i re pulse height spectrum a t 2%
e r ro r . Counts per minute (CPM) were corrected fo r counting e f f ic ie n c y
and converted to d is in te g ra t io n s per minute (DPM) using quench curves
(Figure 25). Mean counting e f f ic ie n c ie s were used to c o rre c t t ra p
( 8 6 .0%) and bubble tra p (85.0%} samples.
Trap samples (DPM) were summed and corrected fo r recovery
e f f ic ie n c y to give to ta l resp ired by la rva e . Recovery of to ta l
1*C0 2 # determined in p re l im in a ry experiments w ith known q u a n t i t ie s of
Na2 ^ 0 3 , was 854 e f f i c i e n t (Table 6 ) .
Notochord length was taken as the mean o f a subsample in
experiments w ith young age cohorts and as the mean of experimental
samples fo r o lder coho rts .
Considerable v a r i a b i l i t y was observed m the dry weight o f larvae
samples, esp e c ia l ly in samples of sm aller la rvae . In experiments w ith
larvae weighing <0 .1 Mg. the mean dry weight o f the age co h o rt ,
ca lcu la ted by w in so r iza t io n , was used in body-burden c a lc u la t io n s * In
other experiments in d iv id u a l sample weights were used w ith values
58
Figure 24, Decay o f chemil u mine scent a c t i v i t y in larvae and
Brachionus samples.
100
8 0 -
70
d
lo 60
<t -
U.O
I-z 40“kiJU<ELlIa.
LARVAE
BRACHIONUSIO -
20100 30~I 40
T f M E ( h)
59
Figure £5. Quench curves fo r la rvae and Braehi onus samples. Curves
f i t t e d by polynomial regress ion.
COUN
TING
EF
FICI
ENCY
(%
}
100
LARVAE/ £ F F = 91163 - 0 ISBWt - 0 002 Wl9 0
8 0
BRACHIONUS EFF=89 9 3 3 - 0 3 55W t - 0 0 0 2 W1
6 0
5 0 — O IO 3020 4 0 6050 70
DRY W E I G H T ( mg )
60
Table 6. Experimental recovery e f f ic ie n c y o f ^CQg. Summary o f resu lts w ith known q u a n t i t ie s o f Nag^COj.
DPM DPM DPM DPM Total RecoveryNa2 ^CG 3 re ten tion bubble tra p bubble tra p DPM e f f ic ie n c y
added chamber tra p 1 2 recovered {%)
138453,0 46544.7 51294.4 14231,4 112070.5 80.954781,6 50874.9 9414.2 115070.7 83.151466,7 53350.6 14717.5 119534.9 86.3
mean 83.4
69226.5 23068,2 29966.1 8350.5 61384.8 83.922386.9 29369.1 6303.a 5B059.8 S3.913063.8 32035.4 7936.9 53036.1 76,7
mean 83,1
34613.2 14162.9 13599.6 3193.9 30956.4 89.412053.1 11290.0 5899.9 26243,0 75.813620.1 11627,8 2818.7 27866,6 80.5
mean 81.9
6922.6 2154.9 3042.5 1074.9 6272,3 90.62132.1 4265.1 579.9 6977.1 100.02734.2 3186.2 347.2 6267,6 90.5
mean 93.7
Mean of a l l observations 05.1
61
pred ic ted front the weight and length regressions used fo r the few
missing o r suspect weights*
Mean to ta l carbon content o f Brachlonus* (41.7%, Table 4 )* used
to c a lc u la te s p e c i f ic a c t i v i t y (a o f Equation 4 ) , was determined by
gas chromatography.
Data Processing
Carbon-14 uptake, re ta in e d , and lo s t , as well as carbon re te n t io n
and absorp tion were re la te d to development by s te p w ise -m u lt ip le
reg ress ion . Percentage scores (transformed to the arcsine 1n degrees)
were regressed on time [ lo g transformed or polynomial fu n c t io n s ) , age,
leng th , and weight (measures of development}, and co n d it io n fa c to r .
In some cases the number o f observations was low compared w ith the
number o f independent v a r ia b le s , but the m atrix o f simple c o r re la t io n
c o e f f ic ie n ts allowed assessment o f assoc ia t ions .
A n c i l la ry Experiments
D u a l- tra ce r experiments were conducted to compare the behavior o f
an ass im ila ted and metabolized t ra c e r ( ^ C ) against tha t o f an
unassim ila ted and in e r t t ra c e r ( ^ C r ) . This design also allowed an
a lte rn a te c a lc u la t io n o f absorption e f f ic ie n c y , Carbon-14 labeled
Brachionus was suspended in 1 1 o f seawater con ta in ing ca, 200 y d
5^CrCl3 and were allowed to f i x S^Cr f o r 12 h. Experiments were
conducted fo l lo w in g general p ro to c o l. Samples were counted by the
Isotope exc lus ion method (Kobayashi and Maudsley, 1970; Sheppard and
Marlow, 1971), Quench curves fo r and 51[> are shown in F igure 26.
62
Figure 26. Quench curves fo r larvae and Brachlonus samples from dual
label experiments* Curves f i t t e d by eye*
COUN
TING
EF
FIC
IENC
Y (%
)
2-0 n
1.5 -
GO
40 -
LAHVAE30-
Channel
20 ■
Channel I =13.77* Channel 2 = 0.07 7(
4030 3020 0 2010 40
DRY WEIGHT (mg)
63
Results were used to c a lc u la te carbon re te n t io n , carbon abso rp t ion ,
and absorption e f f ic ie n c y by the In d ic a to r - r a t io method (Calow and
F le tche r, 1972; Wlghtman, 1975; Caimen, 1977).
Several expertments were conducted with a reduced, more
environm enta lly r e a l i s t i c food concentra tion , because d ig e s t io n and
a ss im ila t io n might p o ss ib ly be less e f f i c ie n t under maximal feed ing .
Experiments were conducted as described above w ith several age cohorts
using a Brachionus concen tra tion of 1-10 r o t i f e r s / 1 .
A comparison was made between the number o f r o t i f e r s Ingested
(ca lcu la ted using the to ta l carbon per dry weight o f Brachionus.
values o f the weight o f an in d iv id u a l r o t i f e r , and values o f carbon
ingested) and v isual counts o f the number of r o t i f e r s in a lim entary
canals excised from la rv a e . Values used fo r the dry weight o f an
in d iv id u a l r o t i f e r were 0,16 yg (The ilacker and McMaster, 1971) and
0.75 yg per r o t i f e r (S co tt and Baynes, 1978). The number of r o t i f e r s
ingested per larva was estimated as fo l lo w s :
mg C Ingested/la rvae_____mg C/mg DW r o t i fe r s
Number o f r o t i f e r s =(c a lc u la te d by Equation [ 4 ] )
mg D lrf?rotifer
These estimates were compared w ith counts of r o t i f e r s excised from a
subsample o f larvae thus serv ing as a check o f experimental accuracy.
64
RESULTS
Carbon-14 Uptake. Retention, and lo ss
Specific a c t i v i t y estim ates o f Brachionus were c o n s is te n t ly high
among experiments and h ig h ly v a r ia b le both w ith in and among
experiments (Table 6 ) . Regressions o f Brachionus body burden on time
indicated that the slope was never s ig n i f i c a n t ly d i f fe re n t from zero
(Table 7), Thus, the s p e c if ic a c t i v i t y o f Brachionus was considered
constant; the mean of a l l Brachlonus s p e c i f ic a c t iv i t y estim ates
w ith in each experiment was used in c a lc u la t io n s of carbon Ingested,
re ta ined, and egested,
Experiments to assess the biochemical f ra c t io n a t io n o f in
Brachlonus ind ica te d tha t maximum la be l f i x a t io n is complete w ith in
2 days of la b e lin g and tha t the f r a c t io n o f in to ta l ex trac ted
p ro te in , l ip id s , and carbohydrates remains constant th e re a f te r
(Figure 21 ), Each experiment u t i l i z e d Brachionus labeled f o r 4 to
11 days (a t le a s t 3 population d o u b lin g s ) , thus the food su b s tra te was
considered un ifo rm ly labe led .
The magnitude o f uptake by la rva l spot varied among
experiments due to d if fe re n ce s in Brachionus sp e c if ic a c t i v i t y , but
the pattern of uptake showed some trends among age cohorts
(Figure 28), M u l t ip le regression o f uptake (expressed as a
Tabl
e 7.
Reg
ress
ions
of
Brac
hion
us
body
bu
rden
(D
PM/m
g DW
) re
late
d to
time
(min
) in
carb
on
assi
mila
tion
expe
rimen
ts
with
di
scre
te
age
coho
rts
of la
rval
Le
iost
omus
xa
nthu
rus.
65
4 O QQ CM O L fl a r - l in CM- 0 —f vo a v CM r--. O r— O in
* p—■+ r - r -- CO O VO O i VO Oin # a * -A ■A r *■ A B
r—1 CO CM r-«. M" <r» 00 $ fO mm in CM r*-
r-1—• i n
<b■at-3 —■c a 3dn cn O E
Ec a . in a
o t vi <1> -C «TCft O t3<£ <_)■—'
o CM 8O v ** =0
CQ lc V0 CM m tT iso > DO m n *
A A m a A A n Ao €0 o i—1 D oa t n 03 r - . CM ** U 3 rOCM CM U T
O CTv 1—* CM 03
1j0 o o O 3 ;a VO in o o m
Lu 4 A » •* 4 4
a o a o o o
o Q o
VO »“H r-—
■c B «■ A A *1
1—1 r~l p----1 •—P 1-----P
C +J□ c
flj
*J <- CO CM «—4 f—1 P—* 1—P
« (J o m CM O o CM4 * ■ * ■ft A
di n- o o o o o at- Vt-
)- a*o oO V_1
£i'4— I %t— £ I—
1— •r—CO I— 1—
o Oi o in
<M cn to LO o Ol
c r\j *- *3- VO <r *
a 4 o■ CM CM Ln
■ i— ■—| r * 4 rf
-U CM CM *T t-H 1-H A
«3 1 1 i + + i
O"UJ CJ O' O CO i—p
VO CTi CM o (M
c rn cn CM ro
o *■ m » 4i *■ B
CM ro Q O m
iA o CM o> VO 10lfl <M n ■-M
a?t- II ii II ■I II N
O1&> 3 3 3 3Q£ □ O tl a Cl Q
i 1 C "£ w D1
Eat
E
3 : TZ £ £a .a £ a.
O % o.a &
O ')O'I--,
ALO
U0<SI
oo
VC
voo
4CM
CO■
roi
3
esx□
CMi/1<M
LflNNJ
N
ffiinco
Cvir*v.
tjiE
&
cO
66
Figure 27. Biochemical f ra c t io n a t io n o f in Brachlonus during
label1ng,
Ra
tio
POPULATION DOUBLINGS
1,5 35 4.5 9.5
CARBOHYDRATEOOl
7A5 5 aoT I M E ( 4 4 y t }
67
Figure Z8. Carbon-14 uptake by la rv a l Lelostomus xanthurus in carbon
a s s im i la t io n experiments* Values expressed as a
percentage o f body burden (DPM/mg DW) a t 1 b feed ing -
% BO
DY
BURD
EN
JDP
M/m
gDW
)
MC UPTAKE
3 0 0
200
A P -
100 -
i— t f0 1 2 3 4
TIME thrsl
68
percentage o f the 1 h estim ate) on log t im e, length , M ig h t , and age
Ind icated th a t uptake was re la te d to time and length (P<0,10;
Table 8 ) . Among a l l age cohorts uptake was rapid fo r the f i r s t hour
and then slowed (F igu re Z8); the marked in f le c t io n in uptake at 60 mln
o f feeding 1s probably due to the onset o f egestion, Visual
observations o f feeding behavior and defecation agreed w ith th is
p a t te rn . Because estimates o f ing e s t io n based on uptake would be
confounded I f egestlon has occurred, 14C re ten tion and loss was
followed fo r the 1 h feeding se r ie s o n ly .
Carbon-14 re ta ined and lo s t a f te r 1 h of feeding showed a
cons is ten t p a tte rn w ith t im e, bu t no trends with development (F igure
£9). Pooled data fo r a l l experiments are shown In Figure 30.
M u lt ip le regressions o f re ta in e d , resp ired , and lo s t as
p a r t ic u la te s (expressed as a percentage o f the 1 h estim ate) on t im e ,
time squared, le n g th , weight, and age Indicated tha t these processes
were re la ted to time squared and tim e, but not to any measure o f
development (P>.QQ1; Tables 9, 10, and 11). Though l^C loss is a
composite of losses v ia egestlon , re s p ira t io n , and e x c re t io n , the
pa tte rn of re te n t io n and PD^C production ind ica ted th a t egestlon
dominates th is loss during the e a r ly minutes fo llow ing t ra n s fe r o f
larvae to a food fre e environment. Egestlon was complete by 6 or 7 h
a f te r feeding stopped (F igure 3 0 ). A percentage o f new ly-ass im ila ted
entered q u ic k ly in to sh o rt- te rm , metabolic processes and was lo s t
as ^C02* I t appears from these data tha t re sp ira to ry loss o f the
labe l began as e a r ly as 1 h fo l lo w in g inges tion ; thus any bias of
carbon Ingestion c a lc u la t io n s due to re sp ira to ry loss o f labe l during
69
Table 8 . Stepwise m u lt ip le regression tab le of l^ c uptake (1) re la te d to time (ra in), age (days), length (ran), and weight (mg) In carbon a ss im ila t io n experiments with d is c re te age cohorts of la rva l Leiostonus xanthurus.
Regression Equation S1mp1 e r d f P a r t ia l F M u l t ip le F
Arcsine Percent Uptake =
+9.620 + 36.212 Lg Time 0.84 1,41 94.81 (<.001)
+0,074 + 37,126 Lg Time 102,05 « . 0 0 l )
+ 1,812 Length 0.03 2,40 2.93 (<.1Q) 51.09 (<.001)
+0,621 + 37,179 Lg Time 99.66 (<.001)
+ 2,950 Length 0.43 (<*75)
+ 0.252 Age 0.04 3,39 0,07 (>■75)
ChCMtfO (< .001)
-14.481 + 37.259 Lg Time 98.36 (<.001)
+ 14,202 Length 0.56 (<■75}
- 1,469 Age 0.44 (>.7&)
- 14,176 Weight 0.02 4,38 0.37 0 . 7 6 ) 24.66 (<.001)
70
Table 9* Stepwise m u l t ip le regression ta b le o f re ta ined ( * ) re la ted to time (m ln ), age (days), leng th {mm), and weight (mg) In carbon a s s im i la t io n experiments w ith d is c re te age cohorts o f la rv a l Lelostomus xanthurus.
Regression Equation Simple r d f P a r t ia l F M u lt ip le F
Arcsine Percent Retained -
-31.000 + 1.202 Time -0.35 65,60{<.001)
- 0.004 T ime^ -0.30 63,43(<.001)
- 0.000 Time^ -0.33 3,64 59,51(< .001) 27.25{< ,001)
-32.704 + 1.200 Time 65.59(< .001)
- 0.417 Time^ 63,43(< .001)
+ 0.394 Time^ 59,4 8 (< .001)
+ i .746 Weight 0,11 4,63 1.32(< .75) 20.87{<.001)
-25.303 +■ 1,198 Time 65.30(< .001)
- 0.004 Time^ 63.15{<.001)
+ 0.000 T ime^ 59.26(< .001)
+ 7.528 Weight 1.43(< .25)
+ 2,645 Length 0.08 5,62 0,89 (< . 50) 16,8 4 {<.001)
-10.560 + 1,202 Time 65,74{< .001)
+ 0,004 Time^ 63,44(< .001)
+ 0,000 Hme^ 59.84(< ,001)
+21,910 Weight 1,8 7 {< .10)
+15,067 Length 1,34(< .25)
+ 1,446 Age 0.06 6,61 0,95(< .50) 14 .ia (< .001 )
71
Table 10, Stepwise m u lt ip le regress ion tab le o f resp ired ( * )re la ted to time (m1n), age (days), length (ran), and weight (mg) in carbon a s s im i la t io n experiments with d is c re te age cohorts o f la rva l Lelostomus xanthurus.
Regression Equation S im pler d f P a r t ia l F M u lt ip le F
Arcsine Percent Respired *
m ,Z a i-7 Z .Q 3 6 Log Time -0 .5 8 1 ,6 8 35.GA(<.001)
F - Level is in s u f f i c ie n t fo r f u r t h e r c a lc u la t io n .
72
Table 11. Stepwise m u lt ip le regression tab le o f P014C (%) re la te d to time (m1n)t age (days ), length (nm), and weight (mg) in carbon ass im ila t ion experiments w ith d is c re te age cohorts o f la rv a l Lelostomus xanthurus.
Regresson Equation Simple r df P a r t ia l F M u lt ip le F
Arcsine Percent P014C -
216,ASS - 74,176 Lg Time -0.71 1,40 39,63(< .001)
211,882 - 74,176 Lg Time 39 ,42(< .00U
+ 0,190 Age 0,10 2,39 0 .7 8 (< .50) 20,09(<,001)
212,284 - 74.176 Lg Time 38.71(< .001)
- 0.612 Age Q.59(< .75)
- 1.962 Length 0.08 3,38 0.30(> ,75) 13 .26 (< .001)
247.214 - 74.176 Lg Time 39 ,20 (< ,001)
3.286 Age l . 9 8 « . 2 5 )
- 27.628 Length 1,67(<,25)
32.436 Height 0.07 4,37 1 ,4 6 (<,25) 10.44(<.001)
73
Figure £9. Carbon-14 re ta in e d , re sp ire d , and lo s t as PG^C by la rva l
Lelostomus xanthurus 1n carbon a s s im i la t io n experiments.
Values expressed as percentage o f body burden (DPM/mg DW)
at 1 h feed ing.
Si.Q
irn>-aoA
4UuItJa
^ 7" C R E TA IM E O
vfc? -<i
C RESPIRED
5 0
100 —I
5 0
0
—
o ID
/
/
--■■ JE?
100
50
0 ✓ * + ? f i j * i^r -t?o s >o
T IM E 1 hr -s 1
- - - J ^
74
Figure 30. Carbori'14 re ta in e d , resp ired , and lo s t as PO^C by la rv a l
Lelostomus xanthurus In carbon a s s im ila t io n experiments.
Data pooled from a l l experiments; brackets in d ica te 95%
confidence in te rv a ls . Values expressed as a percentage of
body burden (DPM/rng DW) a t 1 h feed ing .
100
90
«0
TO
RETAINED
' 50 a
■a.a
xUJo
2.0IE301
10-
t-xUJ
aUJ
£0H
(0
- "T — T t i 1-----1-----1-----r
RE SPIRED
/ 1 !■ 1T 1-----1-----T-----1-----1----- 1---- 1-----1-----
P O C
10 -
t p 1---------------1----------------r-----------t --------------1---------------1-£ 3 4 5 6 7 9 90 I
T IM E I ft J
75
feeding is probably m in im al. The ra te of p roduction , i n i t i a l l y
very rap id* slows a f te r several hours (Figure 30). C a lcu la t io n s o f
carbon re ta ined and egested were made by pooling data from the 6 and
7 h re te n t io n in te rv a ls .
Carbon A s s im i la t io n and i t s R e la tion to Development
Carbon re te n t io n (Table 12)t the r e la t iv e Index o f the
c o e f f ic ie n t o f u t i l i z a t i o n * was not re la ted to development but was
negative ly re la te d to co n d it io n fa c to r (F igure 3L). Cond ition fa c to r
showed the highest c o r re la t io n fo llowed by weight In m u lt ip le
regression o f re te n t io n on leng th , weight, age, and c o n d it io n fa c to r .
Although the p a r t ia l regression c o e f f ic ie n ts were not s ig n i f ic a n t ,
carbon re te n t io n decreases sharply w ith increasing co n d it io n fa c to r
(the simple regression o f carbon re te n t io n on cond it ion fa c to r
suggested th a t the p r o b a b i l i t y o f a zero slope is <0.25) (Table 13).
Carbon absorption (Table 12), the index of absorption e f f ic ie n c y ,
was nega tive ly re la te d to age as well as condition fa c to r (F igure 32).
M u lt ip le regression ind ica ted that the p a r t ia l regressions coe f
f ic ie n ts o f age and con d it io n fa c to r were both s ig n i f ic a n t (P<0*10;
Table 14).
No age-re la ted biases in the Indices of a ss im ila t io n were evident
o r . I f present, they were constant. Carbon re ten tion was corrected
fo r re sp ira to ry loss o f but th is co rre c t io n might I t s e l f be
biased. The percentage of ingested l^C i ost through re s p i ra t io n was
not associated w ith age and on ly weakly associated w ith co n d it ion
Tabl
e 12
. Su
mmar
y of
valu
es
used
to
asse
ss
carb
on
assi
mila
tion
in ag
e co
hort
s of
larv
al
Leio
stom
us
xant
huru
s
76
E +*□ C lja l h °4 i/l LJ -Q <
OC -P - 2 * *
I s/4 /Jt_] 4i O '
v cn o itr t .|— /g 0> 0 / > cr> 5 -----
LlJ CO
fO L * — O T ! V f O t - 1_
5 2L
'S +.>C £
- I - CTi 4Jrc -r- lO+j gi >CO « g i
c t11""—■O T J 4 - £ } Q t - 4.
■8— -CLO Pt (1)a j —
I—N
C u 5' O T5 4t- 5-
3 8 .
rO>U ■ <g gi
t / lSi ^£T /g <t -O
CM,
<71Ol
f * 'i n
ooC5
o* rC 3
CM ■--1P’Lo
00
m
r~-LfJ &
C\J
i s
S 2o o
tO lT)
CO cr>
a i m
i fooa
c n
oo
CMar oa
LT>
a i*
CM00
CM CO» * »
P i O i1X1 m
ao
nc ooo
CO
t o
a iooo
CO
oo
CM»
■r-,4 0
CT)
S 3
CO
o■
o
o*r vo—t oo o
O cn to * n o a o
n -■no-
r o■
CXt
r--
r u
r ooo
IDIDoo
r*^
Mean
42
.9
37,6
77
Figure 31* Carbon re te n t io n , the Index o f the c o e f f ic ie n t of
u t i l i z a t i o n , re la te d to development (A} and c o n d it io n
fa c to r (B) o f la rv a l Lelostomus xanthurus age cohorts*
Regressions ca lcu la te d w ith a rcs ine ( In degrees)
transformed percentages.
H Q 1 1 U 3 H H N Q S IJV3 iN 3 3 U 3 cJ 3 N IS 3U Y
otty
o&-LI1
o
O
D
QIM
M0I1M313U NOflMVD 1N33U34
AGE
(dO
Ki)
C
ON
DIT
ION
FA
CTO
R
76
Table 13. Stepwise m u lt ip le regression ta b le o f carbon re te n t io n (1 ) , the Index of the c o e f f ic ie n t o f u t i l i z a t i o n , re la te d to age (days), length (mm), weight (mg), and co n d it io n fa c to r o f Lelostomus xanthurus la rv a l age coho rts .
Regression Equation Simple r d f P a r t ia l F M u lt ip le F
Arcsine Percent Carbon Retention =■
61.666 - 0.608 Condition Factor -0.54 1*7 2 ,89(< ,25)
60.003 - 0,609 Condition Factor 2 .6 4 (< ,25)
+ 1,555 Weight 0.21 2,6 D,40(<.75) 1.52(< .50)
71.357 - 0.504 Cond it ion Factor 1 ,9 2 (<.25}
+13.560 Weight 2 ,02 (< ,25)
- 5.421 Length 0,07 3.5 1 ,68(< ,50) 1.69(< .60}
83.970 - 0.525 Condition Factor 1,7 4 (<,50)
+24.979 Weight 0 ,9 5 (<.50 J
-15.437 Length 0,54(<.75 J
- 1.186 Age 0,05 4,4 0 ,2 4 0 .7 5 ) 1 .13(<.50)
79
Figure 32. Carbon absorp tion , the Index o f absorp tion e f f ic ie n c y ,
re la te d to development (A) and co n d it io n fa c to r (B) o f
la rv a l Lelostomus xanthurus age co ho rts . Regressions
ca lcu la te d w ith a rcs ine ( in degrees) transformed
percentages.
I DO
NOuauossv NOayu'3 ±N33N3d inisdhv
OO O O O O D O O* * * s is in <7 t f j t j _
o
m
- o
K)
NQliBBOSBV NOBUVO lN33U3d
AGE
tdiiT
*)
CO
ND
ITIO
N
80
Table 14. Stepwise m u lt ip le regression tab le o f carbon absorp tion , the Index o f absorption e f f ic ie n c y , re la ted to age (days), length (urn), weight (mg), and cond it ion fa c to r o f Lelostomus xanthurus la rva l age cohorts .
Regression Equation Simple r d f P a r t ia l F M u l t ip le F
Arcsine Carbon Absorption -
87,542 - 0-648 Age -0,79 1,4 6,57{< .10}
110,530 - 0,596 Age 16,41(<,05)
- 0-693 Condition Factor -0,62 2,3 8,96(<.1C) 14.31{< ,05}
108,894 - 0,212 Age 0 ,8 6 {< .75)
- 0-762 Condition Factor 19,34(<0-5)
- 4,467 Weight -0.75 3,2 3.60{<,25) *
79,086 - 2.803 Age 1.64(< ,50)
- 0.737 Condition Factor 4 .21{<-25)
-31 ,795 weight 1,9 0 (< .25)
+22.958 Length -0.78 4,1 1.47{<-50) 3,19(<-25)
* F - leve l in s u f f ic ie n t fo r fu r th e r c a lc u la t io n .
61
fa c to r (F igure 33); thus any bias 1n carbon re te n t io n was considered
constant. An age-re la ted bias might be Inherent in re la t io n s h ip s
between carbon a s s im i la t io n Indices and co n d it io n fa c to r fo r the
cond it ion fa c to r o f la rv a l f ishes genera lly increases w ith length
a f te r yo lk -sa c absorp tion (e .g . , Sameoto, 1972; Sekiguchi, 1975;
E h r l ich e t al . , 1976). Condition fa c to r o f the age cohorts used,
however, showed no assoc ia tion w ith age (F igure 34) and the
c o r re la t io n o f co n d it io n fa c to r w ith length was even weaker.
A n c i l la ry Experiments
The p a tte rn o f 51Cr re ta ined and lo s t was s im i la r to th a t o f
w ith some exceptions (F igu re 35). A residue o f 51Cr remained w ith in
the la rva l a lim entary canal longer than the experiment. Taken as a
whole, however, these re s u l ts support the in fe rence th a t egestlon is
complete w i th in G or 7 h fo l lo w in g feed ing . Carbon abso rp t ion ,
ca lcu la ted by d i f fe re n c e according to general experimental procedures,
was 99% (15 day age c o h o rt , Table 12); a s im i la r value was ca lcu la ted
using the in d ic a to r - r a t io method (Table 15).
The pa tte rn o f re ta ined and lo s t In experiments w ith reduced
Brachionus concentra tions was s im ila r to the main experiments w ith
high concentra tions and maximal feeding (F igu re 36), but estimates of
carbon re te n t io n were h igher (Table 16), Results o f these experiments
were h igh ly va r ia b le and on ly two o f the s ix were successful .
V a r ia b i l i t y was la rg e ly due to va r iab le feeding success. The mean
c o e f f ic ie n t o f v a r ia t io n o f uptake samples was 78,0 compared to a
mean o f 62.8 fo r experiments w ith high Brachionus concen tra t ions .
82
Figure 33. Percent resp ired re la ted to development (A) and
c o n d it io n fa c to r (B) o f la rva l Lei ostonus xanthurus age
cohorts . Regressions ca lcu lated w ith a rcs ine {1n degrees)
transformed percentages.
30
£□L il
O.Vt
zo<D<E
20 -
10-
r ? 0 OS
~i— 10
T ~l—3020
AGE < d o r i )
—|— 40
30
20
100
50
uiyKUJa_
0.46
a so
3 0
I30
■ r ' 40
2 0
100
50CONDITION FACTOR
ARC
SINE
PE
RC
ENT
CARB
ON
- 14
RE
SF
iRE
C(%
|
83
Figure 34. C ond it ion fa c to r re la ted to age (A) and notochord or
standard length (B) of la rv a l Lelostomus xanthurus age
cohorts used 1n carbon a s s im i la t io n experiments. Lines
(B) in d ic a te p red ic ted c o n d it io n fa c to r from weight and
leng th regress ions.
COND
ITIO
N fA
CTO
R
20
10-
20 6040
20
r = 0 .0 610 -
2 ao 4 G 12
NOTOCHORD f STANDARD LENGTH (mm)
84
Figure 35, Carbon-14 and 5lC r re ta ined and lo s t by la rva l Lelostomus
xanthurus 1n a d u a l- t ra c e r experiment w ith 15 day age
co h o rt. Values expressed as a percentage o f body burden
(DPM/mg DW) at 1 h feeding.
100
* ,
roo
f i
*
aL I 3a 3
u£inuft
t “c40 -F -z X
U J U Ju ut t 1CUJ L*la . a .
fi
a**
ii
I
r
<vF s
- rj
oO) o<0
0■+
On oM
<MQ Au / W^ Q I N30U0B A008 1W30M3J
Table
15
. Su
mnar
y of
dual
-tra
cer
valu
es
used
to
calc
ulat
ed
abso
rptio
n ef
ficie
ncy
by th
e in
dic
ato
r-ra
tio
met
hod.
65
ca > >
f ( J+ J c ID 41
in 4 - v t U J
<
COo.
4 0CT»
4 - U1 O A )
q k J-P - 4 — + J 'v .
o■4-
<SI0
* 1 P
H— 10 □ L -
d j O LJ
'P - iQ+ J U- IO
QC
O 4 0 . .
i f fOCVI CVJ
L .o
LD
4-o -— -
c a tb
* o o > a - £
CO t :
■ g *3CO
r v i—- U > 4 0 4 0 VO 4 0 luO U 1 4 0 O O rO rO*—i p t
oT f
4 -o •—
C Q <U
D D )* - E
D -
f stO
r - .CO o iS o r o ^
s ^
y>a *
S QJU . Ll .
86
Figure 36. Carbon-14 re ta ined and lo s t by la rv a l Lelostomus xanthurus
1n carbon a ss im ila t io n experiments w ith reduced Brachionus
p l l c a t H ls concentra tions (5-10/ml )* Values expressed as
a percentage o f body burden {DPM/mg DW) a t 1 h feeding.
BODY
BU
RDEN
ID
PM
/mq
D
W)
,4C RETAINED
7—r- f t r r y
IOO -I
C R E S P I R E D
J &
87
Table 16, Summary o f values used to asses carbon a s s im i la t io n 1n age cohorts o f la rv a l Leiostomus xanthurus fed w ith reduced Brachlonus concentra tions.
Age{days)
Carbon Ingested per dry weight
of la rvae [mg)
Carbon Retained per dry weight
o f larvae (mg)
Carbon Retenti on
<*>
9 0.000903 0.000556 61*5
15 0*002578 0.002222 86.2
Mean 73.8
88
The q u a n t ity o f carbon fngested per dry weight o f larvae 1n
experiments w ith reduced Brachlonus concentrations compared w ith
carbon Ingested a t high concentrations was ge n e ra lly an order o f
magnitude lower (Figure 37).
14The nuirtber o f r o t i f e r s ingested by a la r v a * ca lcu la ted from C
uptake data* Increased e x p o n t ta l ly w ith age and compares well w ith
courts o f the number of r o t i f e r s excised from a lim entary canals
(F igure 38). The count o f r o t i f e r s th a t f a l l s outs ide o f the range
o f ca lcu la ted values was based on a s in g le specimen ra th e r than a
mean o f three counts. The method o f es tim ating Ingestion used
herein Is v a l id -
89
Figure 37, Carbon ingested per dry weight of la rv a l Leiostomus
xanthurus fed high (25Q-lQDG/m1) and low (5 -10 /m l)
concentra tions of Brachlonus p l i c a t i l i s *
CARB
ON
INGE
STED
PE
R W
EIGH
T OF
LA
RVAE
[m
g D
W}
LQ* IO_l - i
IJOm I0"2 -
LOm I0"3
* 250-1000/mf
a M O / m l
LQ* I0-4 T "
10 20“T"30 40
n50
AGE (Dayi)
90
Figure 38. The nunker o f r o t i f e r s ( Brachlonus p l i c a t i l i s ) ingested
re la ted to development o f la rv a l Leiostotnus xan thurus . A.
Regression ca lcu la ted using dry weight value of The ilacker
and McMaster (1971)* B. Regression ca lcu la te d using
maximum dry weight value o f Scott and Baynes (1979).
Stars in d ica te mean number o f r o t i f e r s counted in
a lim entary canals excised from la rva e .
NUM
BER
ot R
OTI
FER
S IN
GE
STE
D
1 0 0 0 - i9 0 0 - 8 00 *
TOO - GOO -
500 -
300
ZOO
Intarcept = 5-6
8 8 :80 - 70 - 60 - 50 -40 -
30 -
2 0 -
r * 0.89
LQ0IO Number of Rotifers Ingested -
LotJto Intercept + 0.048 Log(0 Age
GO4 0 so10 30oAGE ( day* }
91
DISCISSION
C o n f l ic t in g evidence ex is ts concerning the presence o f carbon
pools w ith rapid tu rn o v e r rates 1n a q u a t ic . po1ki1otherm ic animals
[see review in Conover and Franc is , 1973; also S t r e K , 1975; Lampert,
1977; U e tz e l, 1976), In la rva l spot, carbon is a ss im ila te d ra p id ly
a f t e r ingestion and a t least some o f i t enters a s h o r t - te rm , m etabolic
poo l. Sorokin and Panov (1966) reported rapid carbon less through
re s p ira t io n fo l lo w in g Ingestion by la rv a l freshwater bream, Abramjs
brama. Larval spot evidenced the same pa tte rn .
Mean carbon re te n t io n , 42.9%, was somewhat lower than the
expected c o e f f ic ie n t o f u t i l i z a t io n fo r ju ve n ile and a d u lt f is h e s ,
about 70* (Ware, 1975), This d if fe re n c e may be due to a rea l s h i f t 1n
the c o e f f ic ie n t of u t i l i z a t i o n a f te r transfo rm ation o r poss ib le b ias
associated w ith t ra c e r methodology. Carbon re te n t io n , as estimated
he re in , is a r e la t iv e index of the c o e f f ic ie n t of u t i l i z a t i o n because
of the bias associated w ith re s p ira to ry loss of newly ass im ila ted
carbon. A l te r n a t iv e ly , carbon re te n t io n could be reduced at high
feeding le v e ls . The re s u l ts o f experiments with reduced Brachlonus
concentra tions were h ig h ly va r ia b le , but values fo r carbon re te n t io n
[7 3 ,6 ) were close to th a t expected fo r adu lt f ishes* Using a
ra d io tra c e r design and envlronm enta lly r e a l i s t i c food c o n ce n tra t io n s .
92
Sorokin and Panov (1966) found close agreement between carbon
re ten tion and carbon absorp tion .
Mean carbon absorp tion , 87 .6S, approximated a s s im ila t io n
e f f ic ie n c ie s reported fo r ju v e n i le and a d u lt f ishes (see review in
Conover, 1978; B re tt and Groves, 1979) as we ll as those ca lcu la te d f o r
la rv a l f ishes (Table 3). Brachlonus lo r lc a s conta in e i th e r
in d ig e s t ib le c h t t ln (Danner, 1966) o r s c le ro p ro te in (Hyman, 1951) and
comprise a la rge port ion o f the p a r t ic u la te egesta o f la rvae. Carbon
absorption may improve fo l lo w in g transfo rm ation w ith the d ie ta ry
s h i f t th a t Includes more s o f t bodied Inve rteb ra tes (P a rt I ) .
A ss im ila t io n , as assessed here in , does not appear to improve w ith
the la rva l development o f spot p r io r to trans fo rm a tion . This 1s no t
su rp r is ing f o r there are no major changes in the a lim en ta ry canal o r
associated organs tha t could e f fe c t such a change (P a rt I ) * Surface
area o f the a lim entary canal appears to Increase, bu t t h is might
simply keep pace w ith the s ize increase o f the la rvae . Sorokin and
Panov (1966) found no major increase in a s s im i la t io n during the la r v a l
development o f A. brama. Yet Laurence's (1971, 1977) ene rge tic models
fo r H. salmoldes and P. americanus s im ulate Increases 1n a s s im ila t io n
w ith increasing body weight o f f i r s t - f e e d in g la rvae.
Is the negative re la t io n s h ip between carbon absorption and
development rea l o r a r t i f a c t? W e igh t-spec if ic absorption e f f ic ie n c y
might a c tu a l ly decrease w ith la rv a l age i f the percentage o f carbon
absorbed Is constant or increases a t a slower ra te than the Increase
1r la rva l weight. The negative re la t io n s h ip might be an a r t i f a c t i f
93
younger la rvae f i l l t h e i r a lim entary canal and begin egestion sooner
than o ld e r la rvae w ith a grea te r a lim entary canal c a p a c ity .
Experiments w i th la rva l Anchoa lamprotaenia and Clupea harengus
(C h i t t y , 1900; Werner and B la x te r , 1900), larvae w ith a
m orpho log ica lly s t ra ig h t ra the r than looped a lim entary cana l, suggest
tha t egestion may begin w i th in minutes a f t e r in g e s t io n . I f t h is
occurred in experiments w ith young spot la rva e , estimates of Ingestion
and consequently absorption would be In f la te d due to p a r t ia l d igestion
and a s s im i la t io n o f food as i t traversed the a lim entary ca n a l.
D issections o f la rvae , however, Ind ica ted th a t d ig e s t io n o f Brachlonus
takes longer than 1 h, and egested c u t lc u la r lo r lc a s were f i r s t
observed several hours a f te r the t ra n s fe r o f larvae to re te n t io n
chambers. The accuracy o f carbon absorp tion values ca lcu la te d by
d if fe re n ce was also supported by re s u lts o f the d u a l- la b e l experiment.
The in d ic a to r - r a t io method avoids problems associated w ith estimates
of In g e s tion , ye t gave a s im i la r ly high value fo r absorp tion
e f f1 d e n c y .
The negative re la t io n s h ip between a s s im i la t io n and cond it ion
fa c to r has Important im p lica t io n s to la rv a l growth and s u rv iv a l .
Larvae tha t have been feeding well enough to su rv ive , but less than
tha t requ ired f o r maximum growth, apparently ass im ila te more n u tr ie n ts
from a ra t io n than do larvae w ith a b e t te r growth h is to ry . Perhaps a
larva w ith a lower co n d it ion fa c to r Is b iochem ica lly o r c y to lo g fc a l ly
b e tte r primed f o r a ss im ila t io n o f n u t r ie n ts . Koslomarova (1962)
reported th a t la rv a l fsox 1ucius made up fo r growth delays by more
e f f i c ie n t u t i l i z a t i o n of a va ila b le food.
The present r e s u l ts , suggesting that f i r s t - f e e d in g la rvae are
f u l l y capable o f d ig e s t in g and absorbing n u t r ie n ts , and th a t larvae
w ith a poor growth h is to ry are more e f f i c ie n t 1n a s s im ila t in g
a v a i la b le food, are p la u s ib le in evo lu tionary te rm s. Larvae may
m it ig a te the r is k o f a patchy food d is t r ib u t io n by f u l l y u t i l i z i n g
a v a i la b le resources. Such compensatory mechanisms may be o f great
adaptive s ig n if ic a n c e to pe lag ic marine larvae.
EPILOGUE
The alim entary canal o f spot becomes fu n c t io n a l during the
completion o f o i l -g lo b u le absorption when f i r s t feeding may occur, a
period co inc identa l w ith H Jo rt 's " c r i t i c a l pe riod " (see In t ro d u c t io n )*
Host pelagic marine larvae begin to feed during yo lk absorption ( e .g . ,
B laxter and Hempel, 1963; B la x te r and E h r l ic h , 1974; Houde, 1974),
Whether these larvae are capable o f d iges tion and a ss im ila t io n before
yolk-sac absorption is not known. I f they are, they may s to re energy
fo r u t i l i z a t io n should they encounter inadequate food at f i r s t
feeding. There Is also evidence of thermal optima fo r e f f i c i e n t yo lk
u t i l i z a t io n (Ryland and N icho ls , 1967; Laurence, 1973); i f yo lk is
absorbed too ra p id ly larvae die even in the presence o f adequate food.
Perhaps a lack o f coincidence between f i r s t feed ing and func t iona l
alimentary canal development Is the cause. P re lim inary experiments on
the p o fn t-o f-n o -re tu rn (see review 1n May, 1974) in la rva l spot
Ind ica te tha t the d u ra t ion o f the " c r i t i c a l pe r iod " is about two days
fo llow ing yo lk-sac and o i l - g lo b u le abso rp t ion , a f te r which
i r re v e rs ib le s ta rva t io n occurs3 . Spot la rv a e , reared a t 20°C, appear
to be f u l l y equipped to d igest and absorb a v a ila b le food resources
during the " c r i t i c a l p e r iod ."
3 Powell, A, B, 1979, E ffec ts o f delayed feeding on f i r s t feeding spot ( Lelostomus xanthurus) larvae: Surv iva l and morphologicalchanges. Ann. Rept * o f the Beaufort Laboratory to the u ,S. Dept. o f Energy, pp. 411-421,
95
96
Two l in e s o f evidence suggest th a t the a s s im i la t iv e a b i l i t i e s of
la rv a l Leiostomus xanthurus does not Improve s ig n i f i c a n t l y p r io r to
the completion o f t ra n s fo rm a t io n . Taken separa te ly the ahsence of
both major developmental changes in the a lim entary canal and
d is c e rn ib le changes In carbon a s s im i la t io n are equivocal , but taken
together they are Im p l ic a t iv e .
The development o f the a lim entary canal i s q u ite s im i la r among
te le o s ts w ith no major morphological o r h is to lo g ic a l changes occurring
p r io r to trans fo rm a tion or metamorphosis. Among pelagic marine
la rvae , clupeomorph and protacanthopteryg ian la rvae have a s t ra ig h t
a lim en ta ry cana l; paracanthopteryglans and acanthopteryglans have a
looped a lim en ta ry cana l. The a lim en ta ry canal 1s h is to lo g ic a l ly
p a r t i t io n e d in a s im i la r pa tte rn in both groups (see review 1n Part
I ) . While these groups might d i f f e r in the way food traverses the
a lim en ta ry cana l* they probably d o n 't d i f f e r in the mechanism o f
d ig e s t io n and a s s im i la t io n ; though some c o n f l i c t in g evidence remains
concerning these mechanisms. Thus unchanging a s s im i la t iv e a b i l i t i e s
o f la rv a l Leiostomus xanthurus may be a general phenomenon o f
te leos tean development.
Comparative s tud ies w ith o the r species, p r in c ip a l ly those w ith a
s t ra ig h t and looped a lim entary cana ls , and experiments th a t o f fe r
con tro l over co n d it io n fa c to r may be the next step 1n the ana lys is of
s ta rv a t io n induced m o r ta l i ty in the e a r ly l i f e h is to ry o f f is h e s .
97
APPENDIX
Because labo ra to ry reared f-fsh larvae o f te n d i f f e r b iochemical 1yp
b e h a v io ra l ly , and m crphom etr lca lly from w ild la rv a e { e .g . , B a lb o n t in
et a l . , 1973; B la x te r and E h r l lc h , 1974; A r th u r , 1976), one may expect
p hys io lo g ica l d if fe re n ce s as w e l l . Herein are data on the development
o f Leiostomus xanthurus a t 2Q°C in the labo ra to ry w ith comparisons
with f i e l d caught specimens. S ta t is t ic a l comparisons were not made
because o f the p o s s ib i l i t y o f d i f f e r e n t ia l shrinkage from
p rese rva t io n .
98
Figure 39. Length and age re la t io n s h ip of lab o ra to ry reared
Leiostomus xanthurus la rvae: Bars in d ica te ranges.
■AGE COHORTS USED IN CARBON A SSI Ml LAT ION EXPERIMENTS
a OTHER ESTIMATES
i " 1 i i50 GO 70 60 90
{ Days)
99
Figure 40. Weight and age re la t io n s h ip s of la b o ra to ry reared
Lelostoinus xanthurus larvae: Bars In d ic a te ranges.
DRY
WEI
GHT
{m
g)
a 51,4
20 —
15
J O -
5 -
0 - -0
* AGE COHORTS USED IN CAROON ASSIMILATION EXPERIMENTS
a OTHER ESTIM ATES
-J ]| .Ai
~ r~30
T ~4010 20 30 60
AGE ( D o y t )
100
Figure 41. Schematic sumnary o f the development of la b o ra to ry reared
Leiostomus xanthurus la rvae .
EE
2y
£azf fCD□£iuo
>•
o
o
iq
>>Da
uio<
101
Figure 42. Head length and notochord o r standard length re la t io n s h ip s
o f labora to ry-rea red and w ild Leiostomus xanthurus la rva e .
Head length was taken as the d istance from the t i p o f the
snout to the In te rse c t io n o f the c le ith rum and the
notochord.
• v .
to
£D
ro CM
•t •
(uiui) 0V3H
NO
TOC
HO
RD
/STA
ND
AR
D
LENG
TH
(mm
)
102
Figure 43. Head height and notochord or standard length re la t io n s h ip s
o f la b o ra to ry reared and w ild Leiostomus xanthurus la rva e .
Head height was measured fo l lo w in g EhrHch e t a l . (1976).
I.
i CD
<0~T~
liD"T"<■
T ”fO
" r T~to
tUD
* *
m
-O
a>
-u>
m
{u rn ) 1 HGI3 H 0 V 3 H
NOTO
CHOR
D /
STAN
DARD
LE
NGTH
(m
m)
103
Figure 44, Eye diameter and notochord o r standard length
re la t io n s h ip s o f labo ra to ry reared and w ild Leiostomus
xanthurus la rv a e . Eye diameter was measured fo l lo w in g
Richardson and Joseph (1973).
<n
CD
00
- Q
(?>
a>
oaa
tn
□tr01o
u>
tn
- fO
ew — <\l
(uiui) d313W VlO 3A3
104
Figure 45. Eye height and notochord or standard length re la t io n sh ip s
o f labo ra to ry reared and w ild Leiostomus xanthurus la rvae .
Eye height was measured fo l lo w in g E h r ltch e t a l , (1976).
r C
.1
ic
ID
1
fO
CJ
- (T>
- CO
u>
- tfl
u- OJ
(- ■“l-m
n ------- r~tv —
r t —r t tvi — O
(UiOi) 1H0I3H 3A3
NOTO
CHO
RD
/STA
ND
AR
D
LENG
TH
(mm
)
105
Figure 46. Snout to pecto ra l length and notochord or standard length
re la t io n s h ip s o f laboratory reared and w ild Lelostomus
xanthurus la rv a e . Snout to pecto ra l was measured
fo l lo w in g Mayo (1973).
SNOU
T TO
PECT
ORAL
LE
NGTH
(m
m)
7
54
32
07 -
6 - 5 4
32 H I 0
r f 1 '
1--------1--------- 1 ■ ~ i-------- r------- t ---------r ------ I " i t --------1--------- r --------1---------1---------r-------- 1----1— --------------1
a
.t
rf.r
■% i1 T 1---------1--------T- T ------ T------- I 1---------- T------- 1--------1-------- T ------ 1 ---------1---------1---------1---------1----------1
I Z 3 4 5 6 7 0 9 1 0 II 12 13 W B 16 17 18 19
NOTOCHORD /STANDARD HEIGHT {mm)
106
Figure 47. Pectoral depth and notochord or standard length
re la t io n s h ip s o f 1abora tory-reared and w ild Leiostomus
xanthurus la rvae. Pectora l depth was measured fo l lo w in g
Mayo (1973),
<f
K) o
£?
- <£
- r--
£
_ tr
K>
_ (M
* Q
at
- CD
m
u>
* If)
- ir
ro
- OJ
r~m
—r T “rO
T "<\J - O
(ww) Hidaa ivdoioid
NOTO
CHOR
D /
STAN
DARD
LE
NGTH
(m
m)
107
Figure 48. Preanal length and notochord or standard length
re la t ion sh ip s o f labora to ry-rea red and w ild Lelostomus
xanthurus la rv a e . Preanal length was measured fo l lo w in g
Mayo (1973).
9e?6
54
32
i
087
854
3
2
I
0
A
* *
• A■»— i— t— t— f— i— i— I f
Q
t i t i r - r — i— i— r— i— i— i— i— i— i— t— i— t— iI 2 3 4 5 6 7 8 9 10 II 12 1} 14 15 16 17 10 19
NOTOCHORD /STANDARD LENGTH (m m )
108
F igure 49. Depth at anus and notochord or standaivi length
re la t io n s h ip s o f 1aboratory-reared and w i ld Lelostomus
xanthurus la rvae . Depth at anus was measured fo l lo w in g
Mayo (1973).
• 4
oc\l
- to
CM o
EE
acc<.QZ<I—1/1
QCCOXoo
{ w u l ) snNV I V Hld3G
109
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VITA
John J e ffre y Govoni
Born in viareharn, Massachusetts, 2 August 1948, Educated through
secondary school in Bourne Public School System, Bourne,
Massachusettsj graduated from Bourne High School in June, 1966,
Received A,B. in b io lo g y from St. Anselm* s College, Manchester, New
Hampshire in June, 1970. Entered Southeastern Massachusetts
U n iv e rs i ty , North Dartmouth, Massachusetts, 1971; served as a Graduate
Teaching A s s is ta n t ; took M. S. in bto logy May, 1973, Employed as
Research A s s is ta n t , Marine Research, In c . , Woods Hole, Massachusetts,
June 1972 to August 1974, Entered the School of Marine Science of The
College o f W il l ia m and Mary September, 1974; served as a Graduate
Research A s s is ta n t; admitted to candidacy f o r the Ph.D. August, 1977.
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