Leptocephalus energetics : an examination of a unique larval developmental strategy

Leptocephalus energetics : an examination of a unique larval developmental strategy

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Leptocephalus energetics : an examination of a unique larval developmental strategy
Bishop, Ren'ee E.
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Tampa, Florida
University of South Florida
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xi, 124 leaves : ill. ; 29 cm.


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Leptocephalous larvae -- Metabolism ( lcsh )
Dissertations, Academic -- Marine Science -- Doctoral -- USF ( FTS )


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Includes vita. Thesis (Ph. D.)--University of South Florida, 1997. Includes bibliographical references (leaves 105-118).

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University of South Florida
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University of South Florida
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024600993 ( ALEPH )
39135458 ( OCLC )
F51-00199 ( USFLDC DOI )
f51.199 ( USFLDC Handle )

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LEPTOCEPHALUS ENERGETICS: AN EXAMINATION OF A UNIQUE LARVAL DEVELOPMENTAL STRATEGY by RENEE E. BISHOP A diss ertat ion submitted in partial fulfillment of the re quirements for th e degree of Doctor of Philo sophy Department of M arine Sc ience University of South Florida December 1997 Maj or Professor : Joseph J Torres, Ph D


Graduate School University of South Florida Tampa Florida CERTIFICATE OF APPROVAL Ph D Dissertation This i s to certify that the Ph.D D i ssertat i on of RENEE E. BISHOP with a major in Marine S cie nce has been approved by the E x amining Comm i t t ee on October 31, 1997 as satisfactory for th e diss e rtation requirement for the Doctor of Phi losophy degree Examining Committee : Ma jor Professor : Joseph J Torres Ph D M e mb e r : Roy E. Crabtr ee, Ph. D Member : Kent A. Fanning, Ph. D Member : Mark M. Leiby, M S Member : Edward Pf e il e r Ph .D. M emb er : Gabr iel Vargo Ph. D


ACKNOWLEDGEMENTS I wish to express my appreciation to Dr. Joseph J. Torres for his guidance and to all my committee members for their adv ice and support I would like to thank the Gulf Coast Research Laboratory, Florida Institute of Oceanography, Dr Hopkins, and the Florida Marine Research Institute for shiptime Special thanks are due to the capta ins and crews of the RN Tommy Munro RN Hernan Cortez RN Bellows, and RN Suncoaster for assistance that was above and beyond their normal duties This work would n eve r have been compl ete d without the help of Joseph Donnelly and Steven Gei ger, not only due to their valuable assistance in the laboratory, but for their advice and moral supp ort I also would like to thank Chad Edmisten for his as sis tance with graphi cs Finally, I could ne ver have completed this dissertation without five very important people: Lewis and Sharon Bishop, Kat hryn and Gilbert Pierce, and most of all my husban d Daryl Pierce Thank you.


Copyright by Renee E. Bishop 1997 All Rights reserved


TABLE OF CONTENTS LIST OF TABLES Ill LIST OF FIGURES v ABSTRACT viii CHAPTER 1. INTRODUCTION 1 CHAPTER 2 LEPTOCEPHALUS METABOLISM 9 Introduction 9 Materials and Methods 11 Results 16 Discussion 28 CHAPTER 3 ENERGY LOST TO EXCRETION 33 Introduction 33 Materials and Method s 35 Results 38 Discu ssion 40 CHAPTER4. GROWTH INDICES IN LEPTOCEPHALI 44 Int roduction 44 Materials and Meth ods 46 Res ults 50 Discussion 58 CHAPTER 5 PRO X IMATE AND NUCLEIC ACID COMPO SITION 64 Introduction 64 Materials and Methods 65




LIST OF TABLES Table 1 Leptocephalus species used in respiration analyses shown by years collected and gears used 12 Table 2 Mass specif i c respiration and enz yme activities (CS citrate synthase LOH -lactate deh yd rogenase) with respect to wet mass (WM) and dry mass ( OM) 19 Table 3. Oxygen consumption rates (uL m g oM-1 hr -1 ) for various tele os t larvae 30 Table 4. Leptocephal us species used in e xcreti on analyses shown by ye ars co llected and gear used. 36 Table 5. Individual NH3 excre t ion ra tes (Y, umo l NH3 i ndiv i dual -1 hr-1 ) against we t mass (X, g WM) 38 Table 6. Wet mass(WM ) and dry mass (OM) specific NH3 excretio n rates (umol NH3 gM-1 h r -1). 40 Table 7. Lepto cep hal us spec ies used in growt h analyses shown by ye a r s c ollected and gear use d 46 Table 8. Linear regr ess ions of total larval lengt h (Y mm TL) on preserve d leng th (X, mm PL) 50 Table 9. Mean i n crement widths (um) (SE) and r ela tionships of incr ement numbe r on otolith radius a nd tota l larval length 53 Table 10. Larv al gr owth equations obtained by regressing otolith age (X days) agai nst growth par amete rs (Y, total length ( TL) wet mass ( WM) dry mas s (OM), a nd ash-f ree dry mass (AFOM)). 56 iii


Table 11. Mean protein growth, Gp1 (% day-1 ) and RNA: DNA ratios for each species of larvae. 58 Table 12. The relationships of protein growth (Gp1 ) (% day-1 ) against total length (mm TL), wet mass (g WM) and ash-free dry mass (g AFDM) for each species 59 Table 13. Growth rates and duration of the larval phase for elopmorph larvae 61 Table 14. Relationships of proximate and nucleic acid composition with total length (TL), wet mass (WM), dry mass (OM) and ash-free dry mass (AFDM). 69-71 Table 15. Ranges of the concentrations of pro x imate composition by wet mass (%WM) and ash-free dry mass (%AFDM) for the three species 73 Table 16 Regressions of the total energy in Joules required per day on wet mass (g WM) for each species. 92 Table 17. Ranges of th e joules (J indiv -1 day-1 ) and percent of total energy(%) allocated to met abolism growth, and excretion for the four spec ies. 94 IV


LIST OF FIGURES Figure 1. Phase I leptocephalus illustrating principal anatomical features (Smith 1989) 2 Figure 2. Location of sampling area off the Florida west coast. 12 Figure 3. Water-jacketed respirometer designed to accommodate the leptocephalus body form. 14 Figure 4. Paraconger caudilimbatus respiration incubation with oxygen solubility (ml L-1 ) as a function of temperature and salinity on theY-axis and time in minutes on the X-axis. 17 Figure 5. Plots of individual respiration (Y, ul02 indiv. -1 hr1 ) with wet mass (X, g WM) 18 Figure 6. Regressions of wet mass-specific respiration (Y, ul02 g wM-1hr1 ) against wet mass (X g WM) 21 Figure 7. Regressions of dry mass-specific respiration (Y, ul 02 mg DM-1 hr-1 ) against dry mass (X mg OM). 22 Figure 8. Plots of indi vid ual citrate synthase ac tivity (Y umol substrate conve rted min -1 ) with wet mass (X, g WM). 23 Figure 9. Plots of individual lactate dehydro gen ase activity (LDH) (Y umol substrate converted min -1 ) with wet mass (X, g WM ) 24 Figure 10. Regression s of mass-specific citrate synthase (CS) activities (Y umol of substrate converted min-1 g wM-1 ) on mass (X g WM). 25 v


Figure 11. Regress i ons of mass-specif i c lactate dehydrogenase (LDH) activities (Y, umol of substrate converted min -1 g wM-1 ) against mass (X g WM). 26 Figure 12. Plots of individual Na + -K+ ATPase activities (Y, units) with wet mass (X g WM). 27 Figure 13. Regressions of mass-specific Na+-K+ ATPase activ i ties (Y, units g wM-1 ) aga i nst wet mass (X g WM). 27 Figure 14. Regressions and plot of individual e x cretion rates (Y umol NH3 hr -1 ) against mass (X g WM). 39 Figure 15. Regressions of mass-spec i fic e x cret i on rates (Y, umol NH3 g wM-1hr -1 ) against mass (X g WM). 41 Figure 16. Otolith from a 17 day A. balearicu m ( 30 mm) 52 Figure 17 Sagittal otolith of 20 day P caudili m batus (43 mm) showing the primordium. 52 Figure 18. Regressions of larval total length (Y, mm TL) on otolith age (X days). 54 Figure 19 Regressions of w et mass (Y g WM) o n otolith age (X days) 55 Figure 20. Regressions o f protein growth ( Y % day -1 ) against wet mass (X g WM) 57 Figure 21. Regressions of w et mass (Y g WM) aga i nst total length (X, mm TL) 68 Figure 22. Regress ions o f protein content (Y mg protein) against mass ( X g W M). 74 Figure 23. Regressi on an d plots of protein conce ntration (Y, % WM) with mas s (X g WM). 75 Figure 24. Regress ions of l i pid content (Y m g li p id ) against mass ( X g W M). 77 vi


Figure 25. Regressions of carbohydrate content (Y mg carbohydrate) against mass (X g WM). 78 Figure 26. Regressions of RNA content (Y, ug RNA) against wet mass (X, g WM). 80 Figure 27. Regressions of RNA concentration (Y, ug RNA g AFDM-1 ) against ash-free dry mass (X g AFDM) 81 Figure 28. Regressions of the DNA content (Y, ug DNA) against wet mass (X, g WM) 82 Figure 29. Regressions of DNA concentration (Y, ug DNA g AFDM-1 ) against ash free dry mass (X g AFDM) 83 Figure 30. Regressions of the total joules required by each species per day (Y, J day -1 ) against larval wet mass (X g WM) 91 Figure 31. Regressions of the mass-specific joules expended per day (Y J g WM "1 day -1 ) against wet mass (X, g WM) 93 Figure 32. Regressions and plot of the percent of the total energy per day that is allocated to metabol ism (Y % day-1 ) against wet mass (X g WM) 95 Figure 33. Regressions of the percent of the t o tal energy required by each speci e s per day that is allo c ated to growth (Y, % day -1 ) aga i nst wet mass (X g WM) 96 Figure 34. Regressions of the predicted percent of the total energy required by each species per day that is allocated to excretion (Y % day-1 ) against wet mass (X g WM) 98 Figure 35. Geographi c d istribution of Paracon ger caudilimbatus and P. guianen sis ( from Bohlke et al. 1989) 120 Figure 36. Geographi c distribution of the hig h and low myomere count forms of A. ba learicum (from Boh lke et al. 1989) 122 Figure 37. Geograp hic distribution of the G saxi cola-nigromarginatusace/latus comp le x (from Bohlke e t al. 1989) 123 vii


LEPTOCEPHALUS ENERGETICS: AN EXAMINATION OF A UNIQUE LARVAL DEVELOPMENTAL STRATEGY by RENEE E. BISHOP An Abstract Of a diss e rtation submitted in part ial fulfillment of th e requirements for the degree of Doctor of Philoso phy Department of Marine Science Univ ersity of South Florida December 1997 M ajor Pro fessor : Joseph J. Tor res Ph .D. VIII


Leptocephali, the larvae of the superorder Elopomorpha, possess a developmental strategy that is unique among fishes. They grow rapidly by accumulating acellular mass in the form of glycosaminoglycans, instead of by large increases in cell number Glycosaminogl yca ns serve as an energy depot for metamorphosis and as a gelatinous skeleton in the l arvae Besides their unique mode of growth leptocephali are highly unusual in that for more t han 230 years the trophic niche occupied by the long-li ved larvae was unknown Recent histological evidence has revealed that leptocephali consume larvacean houses and particulate organic m atte r The present study examined the developmental strategy of leptocephalus larvae and their ener getic requ ir ements by determin i ng the energy budgets for four species of leptocep hali common to the Gulf of Mexico : Paraconger caudilimbatus, Ariosoma balearicum Gymnothora x sa xi cola, and Ophichthus gomesii Metabolism was d eterm ined by analysi s of oxygen consumption rate and the activities of three enz y mes : citrate syntha se lactate dehydrogenase and Na + K + -ATPase. Mas s s p ec ific o xyge n consumption rates { Y V02 g wM-1 ) (250 1200 ul02 g wM-1hr-1 ) de cline d precipitousl y with increasing wet mass {X, g WM) according to th e r e l a tions hip Y = a WM b with b value s ranging from -0 3 to 0 8 Mass spec if ic ox yge n co nsumption rat es expressed versus wet mass were I X


substantially lower than rates for other larval fish. However, dry mass-specific oxygen consumption rates were similar to those of other teleost larvae Mass specific enzyme activities (Y activity WM-1 ) decreased with increasing mass (X g WM) follow i ng the equation Y =a WMb. Slopes ranged from -1. 04 to -1.23 for CS -0 805 to -1. 739 for LDH and were -1. 23 for Na+K+ ATPase. The greatest changes in the slopes of mass-specific respiration and enzyme activities occurred in larvae less than 0 5 g WM or during Phase 1 a of leptocephalus development. As the leptocephali i ncreased in mass they did not increase substantially in metabolizing tissue Absolute ammonia excretion rates ranged from 0 .21 to 0 65 umol NH3 indiv:1 hr-1 The mass specific excretion rates (Y, umol NH3 g wM-1 ) scaled significantly with mass (X gWM) again follo wing the relationship Y =a WMb, with the maximum change in slope occurring during Phase 1 a Rate of growth was ex amined using otoli th age estimates, RNA: DNA, and protein growth. Larvae, ranging from 7.8 to 240.0 mm total length (TL) were 4-111 days old. Growth rat es fit multiplicative re lation ships (TL = a Ageb) with slopes ranging from 0 37 to 0 96. Mean p r o tein growt h (% day -1 ) ranged from 16.1% to 37. 3%. RNA:D NA values were low with means for all four species ranging from 0 62 to 1 1 Energy i nvested i n somatic growth was exam ined by proximate and nucleic acid compositi on a nalys i s Protein car bo hydrate, and lipid increased with increasing total l ength and mass in a s i m ilar ma nner to other larval f i sh X


Concentrations of the proximate components were different from convent i onal larvae Carbohydrate concentration ranged from 2% to 25% of the ash-free dry mass The transition from Phase 1 a to Phase 1 b was evident in the nucleic aci d concentrations as a decrease in both RNA and DNA with increasing mass Comparison of the assembled energy budgets suggests that leptocephali require 50% less energy than conventional larvae of the same dry mass Leptocephalus larvae grow rapidly with minimal energy expenditure develop a substantial energy depot and avoid the i ncreased energy demands assoc i ated w i th increased mass by accumulating non-metabolizing tissues Abstract Approved: Professor\--d'oseph J Torres Ph .D. Professor Department of Marine Science Date approved : xi


CHAPTER 1 INTRODUCTION Two fundamentally different developmental strategies characterize the larval periods of marine teleosts (Pfeiler 1986). In Type 1 larvae, representative of most larval fishes, increases in somatic mass are achieved primarily by accumulating protein in the form of muscle (Balbontin et al. 1973, Ehrlich 197 4a, b, 1975, Cetta and Capuzzo 1982) There is little or no energy storage; all available energy is devoted to growth (Brightman 1993) and growth rate in some instances, controls the duration of the larval phase (Werner and Gilliam 1984) The length of the larval phase for Type 1 larvae r anges from days to several months (Werner and Gilliam 1984) The Type 1 developmental strategy minimizes the period of vulnerability through rapid growth and a short larval phase, regardless of the potential for starvat i on The Type 2 deve l opmental strategy approaches survival from a different perspective. Growth is rapid but concomitan t w i th substantial energy storage Residence times in the plankton range from several months to more than a year (Schmidt 1922, 1925 Castonaguay 1987, Crab t ree et al. 1992) This developmental strategy is closely linked to the l a rval form's distinctive morphology It is decid e dly laterally compressed almost leaf-like in 1


appearance with a perfectly clear body and a slender head that gives it its name : the leptocephalus (Figure 1 ) i i ,..,..l ...... ...... v ..... ( L V & V I Figure 1. Phase I leptocephalus illustrating prin c ipal anatomical features (Smith 1989) Leptocephali were d iscovered in 1763 b y W illiam Morris More than one hundred years passed be fo re it was realized t hat these transparent organisms were larval forms of eels a nd related fishes Rese archers were then faced with the daunting task of pairi ng the larval form with the adult. To date, there are eels for which there is no de s c ription of the l eptoce ph a lus and vice versa. For many species, the f ertilized eggs and newly h atche d larvae remain undescribed Fishes possessing th e leptoc ephalus larval f orm c u r rently belong to the superorder Elopomor pha a nd are conside re d one of the most ancestral taxa of teleosts (Greenwood et al. 196 6 Robins 19 89). The l e ptocephalus larval form unites five orders of bony fis h e s : the Albulifo rmes ( th e b onefish), the Elopiformes 2


(the tarpon and ladyfish}, the Notacanthiformes (the spiny eels), the Saccopharyngiformes (the gulper eels), and the Anguilliformes (the true eels) Leptocephalus larval development proceeds in two phases Phase I, or premetamorphic period, has been further divided into two stages, engyodontic and euryodontic based upon morphological features (Le i by 1979). In the engyodontic stage the leptocephalus has only a few needle-like teeth. In some species, the teeth are so pronounced that the larvae are incapable of closing their mouth The euryodontic stage begins with the shedding of the needle-like teeth which are replaced with much shorter broad-based teeth Phase I is characterized by very rapid growth ranging from 0 5 mm total length day1 in Anguilla anguilla (Castonaguay 198 7) to 2.5 mm total length day1 in Ariosoma balearicum (this study). Unlike Ty pe I larvae leptocephali accumulate a substantial e nergy depot concu r r e nt with rapid growth. This is accomplished through the i nc orporation of gly cosa minoglycans, large mucopolysaccharides, an d lipids into the body matrix (Rasquin 1955, Pfeiler and Luna 1984 Pfeiler et al. 1991 ). As the larva e accu mulate the glycosaminoglycans, the bo dy walls separate to acc ommodate storage, creating a "mucinous pouch" (Smith 1989d). Becaus e th e gelatinous material is a non metabolizing tissue it allows a substant i al gro wt h in mass with little increase in energy demands. The length of Phase I in leptocephali is pro tracted and highly variable. Leptocephali may r emain in the plankton for severa l months (Crabtree et al. 3


1992) for Megalops at/anticus to 2 5 to 3 years for Anguilla anguilla (Schmidt 1922 Schmidt 1925). Leptocephali are truly pelagic larvae ; they are found predominantly in offshore waters during premetamorphic development (Hulet and Robins 1989 Crabtree et al. 1992) The integument is very thin and fragile, consisting of only 2 to 3 cell layers (Hulet 1989) In early premetamorphic development the larvae are virtually isotonic with seawater and it has been proposed that they do not possess the ability to osmoregulate (Hulet et al. 1972, Hulet 1978) As metamorphosis approaches and as the larvae get closer to juvenile habitat, they develop osmoregulatory cap ability surviving in broad euryhaline conditions (4 .2 to 52 ppt) (Pfe i ler 1 98 1 and 1984a) Larvae are either carried passively by currents or actively sw i m (Miller and McCleave 1994) to suitable juvenile habitat w he re they enter Phase II (Ts e ng 1990) Metamorphosis, or Ph ase II of le ptoc ephalus d evelopment is accompanied by profound physiological chan ges in the larvae. After attaining a species-specific maximum siz e some more th an a meter (Smith 1989d) the larvae begin to use their su b s tantial energy r eserv es to fuel their rather amazing transformation into elvers. The combustion of these re se rves results i n a greater than 60% reduction in tota l length and a mor e tha n 80 % decrease in mass (Pfeiler 1984b Padro n et al. 1996, Bishop a n d Torre s i n prep) This complex transition to the juvenil e form is unpa r alleled in any oth e r larval fish Leptocephali a r e ubi quitous and epis odica lly important in the blue water micronekton/macro zooplankton co m munity i n trop ic al a nd temperate regions of 4


the world's oceans Leptocephali comprise 3 to 25% of wet biomass in the upper 200m of the Gulf of Mexico in spring and summer months (Hopkins and Lancraft 1990) Abundance estimates for Ariosoma balearicum, one of the most common eels in the western Atlantic, are greater than 225 larvae in 1 05 m3 of water in the Subtropical Convergence Zone (Smith 1989d Miller and McCleave 1994) In a 2 5-month survey conducted in the waters west of Exuma Sound Thorrold et al. (1993) coll e cted 2753 leptoceph a li, mainly metamorphic A. balearicum in channel nets positioned to sam ple d uring the incoming tide The number of leptocephali m o ving into the shallow w ater habitat far exceeded the abundance of any other t yp e of fish larva Despite their abun d ance, the leptoceph alus l arval form remains surrounded by mysteries F rom the discov ery of leptoce phali in 1763 until 1993 a period of 230 years le ptoce ph a lus ing es t ion a nd d i e t had remained undescribed and thousan ds o f leptoceph a li with no ap p a rent gut contents had been examined (Hulet 19 78 K r acht and Tesch 198 1 ) This posed the question where are the leptocepha li ac quiring the en e r gy to accumulate an extensive reserve i n an oligotrophi c env ironment? Two potent i a l sources of nutrition have been proposed for l eptocep h a lus larvae : di ssolv e d organic material (DOM) and particulate organic m atter (P OM) in the form of zoop l a n k ton fecal pellets and larvcean houses Uptake of D OM acro ss t h e integum ent is no t un iq ue to larval fish This mode of supplem en t al nutr i t io n has been o bs erve d in e ggs and yolk -sac larvae 5


of herring and rockfish, respectively (Siebers and Rosenthal 1977, Korsgaard 1991, Yoklavich and Boehlert 1994). Hulet (1978) examined the epidermis of A. balearicum and found the surface to be covered with microvilli-like structures similar to those found in fish digestive tracts Though not yet tested, the uptake of DOM appears to be a possible source of leptocephalus nutrition. Based primarily upon the works of Otake et al. (1993) and Mochioka and lwamizu (1996) it has become apparent that leptocephali feed on particulate organic material. Examination of the gut contents and ultrastructure of the midgut mucosal cells in Conger myriaster reve a led that zooplankton fecal pellets were present in more than 78% of the larvae. Low levels of gut pigments in C myriaster indicated that the larvae were not feeding on phytoplankton Stable isotope ratios (o15N), indicative of an organism's trophic position in food webs, in C. myriaster w ere equivalent to lev els for POM Mochioka and lwamizu (1996) examined the gut contents of l eptocep hali from 8 eel species spanning 5 families The g ut contents primaril y co nsisted of the gelatinous feeding webs of appendicularians and attached z ooplankton fecal pellets. These results indi ca te that leptoceph ali may be occupy i ng an unusual niche for a larval mari ne vertebrate Marine snow, or POM, along with DOM constitute an enormou s reserv oir of carbon in t he ocean Without question, both POM and DOM are cent ral to bacterial produ ction, and therefore global carbon cycling The relation ship of the microbial and m e tazoan food web has been addressed (Azam et al. 1993 Azam and Ammerm an 1984) and a current model 6


suggests that the two food webs are connected by the consumption of microheterotrophs by zooplankton. Whether leptocephal i util i ze mainly POM or DOM or a combination of the two they essentially short circu i t this model by occupying the niche normally filled by bacteria. The daily energy required by leptocephali may be estimated using the well establ i shed techniques of en e rgetics Comparing the daily energy estimates to the ene r getic value of the proposed food sources provides the invest i gator w i th insight in t o their feasibility Th e p r i ncipal of energetics requires that each component of the energetics equation be e x amined in detail : I=M+E+G where I is the amount of ing est e d energy, M i s the metabolic expenditure of the organism E is the en ergy l os t to exc r e ti o n a n d G i s th e e n e rgy i nvolved in growth (Brett and Grove s 1979) By using th e e ner g e ti c s equation, it is possible to determ i ne the a m ou n t of e n e r g y leptoc ephali must in ge st per day to meet energetic e x p e nses The present study ex a m ine d eac h comp o ne nt of the energy budget in four species of leptocephali. The s p e c ies of larv a e wer e selec t e d bas e d on the i r larval abundance in th e G u lf o f M ex ico Th e f our spec i e s i nclude two congr i ds Paraconger cau dilim b a tu s and A rio som a balea r ic um, a mura e nid Gymnothora x saxi co l a and a n ophi c hthid Ophi chthus g omesii The diss e rt at i o n co ntai n s six cha p ters In Chapte r 2, m e t a b o lic requirements wer e addr es s ed through the d i r ect meas ur e me nt of r e spiration and 7


intermediary metabolic enzyme activities The energy lost to excretion (Chapter 3) was measured by the amount of ammonia excreted per unit of time. Growth was examined in two parts Chapter 4 looks at individual growth rates per day Several growth indices are presented and compared with conventional Type 1 larval growth. In the second portion of the growth analysis (Chapter 5), proximate composition of the larvae was determined and changes in the chemical composition were examined with respect to ontogeny Finally, in Chapter 6, the energetics equation was assembled with energy requirements assigned to each of the components of the equation The total daily energetic requirements for the leptocephali were summed and compared to the availability and energetic values of the proposed food sources. 8


Introduction CHAPTER2 LEPTOCEPHALUS METABOLISM The larval period in fishes is a time of great vulnerability and high mortality; a period of constant struggle between maintaining metabolic rates and maximizing growth Metabolism in conventional larval fishes scales isometrically with increasing mass (Giguere et al.1988 Manahan 1990, Torres et al. 1996 ). Therefore, as the larva grows, the absolute o xygen consumption correspond ingly increases, requiring more combustible energy. The larger larva is in an improved ecological position ; it has increased the prey spectrum by improving its locomotory capabilities and by enlarging its gape and it has reduced the predator spectrum by enhancing its escape pot ent ial and exceeding its predators' gape Howev er now the larva must inge st more to meet the higher metabolic energy deman ds that come with incr ease d size The first priority in the allocation of energy is to meet metabolic requirements (Brett and G roves 1979) In larval fish, little energy is stored (Brightman et al. 1997 ) w ith most of the ingest ed energy that is not allocated to 9


metabolism being devoted to increasing tissue mass Thus, a larger larva is nearly as vulnerable to starvation as a smaller one Leptocephali, the larval form uniting the members of the superorder Elopomorpha, possess a unique developmental strategy (Pfeiler 1984a, Donnelly et al. 1995) Rapid growth in mass is achieved by the deposition of a metabolically inert storage compound: glycosaminoglycan By using this unique mechanism for growth, the larvae can hypothetically direct less energy to metabolism. Leptocephal i are less vulnerable to starvation than conventional teleost larvae, since increases in mass are in the form of nonmetabolizing tissue The metabolic rate of an organism is frequently determined by closed vessel respirometry where the oxygen consumption rate is an indirect calorimetric measure of the rate at which food is converted to energy. The activities of enzymes in m eta bolic pathways that ge nerate energy are also representative of metaboli c rate Citrate synth ase in the Krebs cycle and lactate dehydrogenase in anaero bi c glycolysis are indicators of an organism's capacity for ATP production Citra te synthase (CS), loc ate d within the mitochondrion, is positioned at the begin ning of the citric acid cy cle and catalyzes the formation of citrate from acetyi-CoA and oxaloacetate (Lehninger 1975) CS activity correlates directly with o xygen consumption rate in fishes (Torres and Somera 1988). Lactate dehyd rogenase (LDH) is the terminal enzyme in anaerobic glycolysis in vertebra tes an d is an indicator o f anae robic potential (ct. 10


Hochachka and Somero 1984) Na-K -adenosine triphosphatase utilizes the ATP produced to regulate monovalent ion concentrations in marine teleosts (Epstein et al. 1980) and is directly related to osmoregulatory costs. The objectives of this study were to determine the metabolic rates of four species of leptocephali, Paraconger caudilimbatus, Ariosoma balearicum, Gymnothorax saxicola, and Ophichthus gomesii, by direct measurement of oxygen consumption rate. Oxygen consumption rates were corroborated by determinations of citrate synthase and lactate d e hydrogenase activities The third objective was to examine the activities of Na -K -adenosine triphosphatase in P caudilimbatus and A. balearicum Methods and Materials Leptocephalus larv a e were collected at th e edge of the continental shelf in the eastern Gulf of Me xic o on three cruises f rom 1995 to 1996 (Table 1 ) Sampling was conducted f rom approximately 26 N to 28 N between 84 W and 86W (Figure 2). The coll e ction gear consist ed of a 2 m plankton net with 505 urn mesh, and a 9m2 mou t h area Tucker trawl co n s tructed of 6.8 mm mesh with a quick-release blind cod e nd Nets were towed a t 1 to 2 knots in a double oblique pattern from th e s urface to a depth of 1 00 m Tow times varied from 10 to 60 minutes dependi n g up on plankton den s it y All sampling was conducted at night to maximize coll e c t io ns 11


Table 1. Leptocephalus species used in respiration analyses shown by years collected and gear used Species Year Collected Gear Used P caudi l i mbatus 1996 2m plankton net 3 05m x 3 05m Tucker trawl A. balearicum 1995 1996 2m p lank ton net 3 05m x 3 05m Tucker trawl G sa x icola 199 5 2m pl a n k ton n et 0 gomesii 1995, 1996 3 05 m x 3 05m Tucker trawl Figure 2. L o c a t i o n of sam p l i n g area o n t h e F lorida west co ast. 1 2


Leptocephali were sorted immediately after each tow. Active, undamaged larvae were removed from the catch and placed in filtered (0.45 urn) seawater for respiration analysis. The remaining larvae were measured to the nearest 0 1 mm total length, rinsed with deionized water, blotted, and frozen in liquid nitrogen. Larvae were maintained at -80C until enzyme analysis. Active, undamaged larvae were placed in respirometers consisting of water-jacketed lucite chambers specially designed to accommodate the long, thin, leptocephalus body form (Figure 3) Chamber volumes varied with the size of the larvae The chambers were filled with filtered (0.45 urn) seawater and maintained at the experimental temperature of 25 C ( 0 2C) through the use of a circulating, refrigerated waterbath The larva w as placed in the respirometer, which was then sealed and the oxygen electrod e was inserted in the chimney (Figure 3). Oxygen partial pressures, P02 wer e c ontinuously monitored using Clark microcathode polarographic oxygen electrodes (Clark 1956) as individuals reduced the oxygen levels to low (0 to 40 mm H g) partial pressures The system was covered with black pl as tic to eliminate vi s u a l stress to the larvae The electrodes were calibrat ed before and after ea ch r e spirat i on run using air-and nitrogen-saturated sea wate r at the experiment al te mperature. After removal from the chambers larv ae we re frozen followin g t he above procedures To determine the effect of micro bial oxygen cons ump tion, larvae were removed after selected incubations, th e i r volume replaced wi t h fre sh seawater, and oxygen consumption again m easure d for 2 to 1 0 h 13


RESPIROMETER Vent to relea se water displaced by electrode Stainle ss steel bolt s Figure 3. Water-jacketed respirometer designe d to accommodate the leptocephalus body form. Data were recorded c ontinuously for th e durat ion of the respiration analysis using a data logging system and Keith ley multichannel analyzer that sampled each of ten chan nels for one second during every minute of the run Respiratory measurements were of the routin e type, where activity was monitored hourly but was not controlled. Ma ximum and minimum oxygen consumption rates wer e taken from the maximum 30minute rate and the minimum 30-minute rate respec tively, after th e first 3 0 minutes the larvae were in the chambers. For enzyme anal yses an d dry mass dete rmina tions tissue was introduced frozen into the homog enizing medium, ice-col d dist illed water, at a dilution of 14


24: 1 and homogenized at 0 to 4C using a sonifier and by hand, using conical glass homogenizers having ground-glass contact surfaces (Kontes Glass Co "Duall" models). Duplicate 1 ml aliquots were dispensed into preweighed crucibles dried for 72 hours at 60C, allowed to cool to room temperature and reweighed Samples were combusted for 3 hours at 600 C to obtain ash weights Homogenates were centr i fuged at 2500g for 10 minutes and the supernatants saved for citrate synthase and lac ta te dehydrogenase analyses Citrate synthase (CS EC 4 1 3 7 ; Citrate:O x aloacetate-Lyase (CoA Acetylating)) activity was assayed using the meth o ds of Torres and Somera (1988). L-Lactate dehydro ge nase (LDH EC 1 .1.1.2 7 ; Lactate : NAD+ Oxidoreductase) activity was assayed in the pyr uva te reductase direction using methods described in Torr es a nd Somera (1988) P caudilimbatus an d A. balearicum w ere se lect e d for the determination of Na+ -K+adenosine triphos pha tase enzyme a ctivitie s In procedures following a modification of Gibbs and So m era (1990) h omogena t e was prepared in a buffer of 50mM imidazole 250 m M s u c rose 1 mM E DTA a nd 5 mM 2 mercaptoethanol pH 7 5 at 20 C Homogen ates w e re centrifu ged at 2500g for 15 min and the supernatants saved and spu n for 90 min at 1 9 ,000 x g The resulting pellets were resuspended in 1 ml hom ogenization b u ffer pe r gr a m of original tissue ATPase activities of t hre e rep l ic ates of each samp l e w e re measured at 20C using the coupled p y r uv a te k in a se/lactate d ehydroge n as e assay system described in Gibbs a n d Somera (1989) 15


Enzyme activities were assayed on a Cary spectrophotometer at 20C ( 0.1 C) and are reported in umol of substrate converted to product per minute All enzymes were assayed in triplicate and the mean was used for analysis To compare the CS enzyme activities to oxygen consumption, a suite of determinations were made at both 20C and 25C, and the remaining enzyme activities were corrected using the resulting 05 value (05 at 20C to 25C = 1.17) Oxygen consumption rates (002 ) were reported in absolute oxygen consumption (Y, ul 02 ind iv idual -1 h-1 ) on mass (X, g M), with mass expressed in wet mass (g WM) and ash-free dry mass (g AFDM), and as mass-specific rates (Y ul 02 g M-1 hr1 ) on mass (X, g M) in WM and AFDM Regressions were generated using the least-squares method with sig nificance at p less than 0.05 Analyses of covariance (Z ar 1996) were perfo rmed on mass specific respiration and enzyme activities to d isce rn differenc es between species. Results Respiratory determ inat ions were made on a total of 58 premetamorphic leptocephali. Respiration inc ubations rang ed in du r at ion from 2 to 18 hours. Oxygen consumption rat es showed little vari abilit y during the course of the incubation (Figure 4); m axi mum and minimu m o x yge n co nsumption rates were not significantly diffe rent fro m the mean (Stud ent s t-test, p >0.05). No obvious critical oxygen partial press ures (Pc) were ob serve d in leptocephalus' respiration A Pc is norm a lly obse rved as a decline in oxygen consumption 16


below a critical P02 and indicates an inability by the gas exchange system to extract oxygen below that P02 (Prosser 1973) usually indicated by a rapid decline in oxygen consumption rate Individual respiration rates ranged from 15.8 to 1655.5 ul02 hr1 (Figure 5) .,.... E .......... c 0 :.;::: <0 c Q.) (.) c 0 (.) c Q.) Ol >. X 0 5.0 4 0 3.0 2.0 1.0 0 0 ,...__ T 200 [ r--.. \ \ 400 600 800 1000 Tim e (min) Figure 4 P. caudilimbatus res piration incub ation wit h oxygen solubility (m l L "1 ) as a function of temperatur e an d sa linity on theY-axis a nd time in minutes on the X-axis The lack of a signifi cant correlation b etween oxygen consumption and mass indicated that meta b o lism did not sca le with inc r eas ing individual wet mass (Figure 5) or with tot al l en g th. However, ma sss p e ci fic oxy gen consumption (002 ) decreased p recipit o usly with increasin g w e t a nd dry mass in all four species (Table 2). Slo p es, orb values, of the equat ions relating mass-specific respiration and mass ran ge d fro m -0.52 in G s ax i c ola to -1. 00 in 0. gomesii (Table 2). The gre atest cha nge in 002 with incr eas i ng mas s occurred at less 17


P. ca udilimbatus A. ba /ear icum 400 400 300 300 200 200 100 ... 100 ......... ..... .!.. ... ..c ..... 0 0 I 0 0 2 0.4 0.6 0.8 1 0 1 2 3 4 5 6 "0 c -N 0 ...J :::1 ......... c: 0 -..::; G. s a x i c o / a 0 go m e s i i ro .... a. en 2 4 0 200 Ql a: 200 160 160 120 120 80 80 40 4 0 0 0 0 0.3 0 6 0.9 1 2 1 5 1 8 0 0.1 0.2 0.3 0.4 0.5 W e t M a s s ( g ) Figure 5. P lot s of indi vidual resp i rat io n ( Y ul02 ind i v :1 hr-1 ) with wet mass (X g WM ) 18


Table 2. Mass specific respiration and enzyme activities (CS-citrate synthase, LDHlactate dehydrogenase) with respect to wet mass (WM) and dry mass (OM) A and bare least -squares scal i ng parameters in the equatio Y = AMb, where Y= mass-specific respiration or mass specific enzyme activities ; n = number of larvae assayed and r = coefficient of determination. Sp ecies Respiration Intermediary Metabolic Enzymes A b n A b n r2 A P. caudilimbatus W M s p e cifi c 1 232 -0 8 7 6 36 0 855 0 2 2 6 -1. 043 36 0 663 13 851 -0.843 46 0.462 OM s pecific 43.341 -0 986 35 0.689 A. b a l e ari c um WM s p e cific 146 .51 -0 .573 6 0.845 0 167 -1. 151 7 0 855 6 323 0.805 5 0.457 OM s p e cifi c 446 93 -0 .562 6 0 .872 G. saxico/a WM specific 1.744 -0.529 11 0.225 0.197 -1.236 37 0.897 4.6 29 1 739 11 0 554 OM specific 85.85 0.909 10 0. 2 79 0 go m es ii WM s pecific 68. 358 1 050 5 0.426 0 .20 5 1 039 5 0 752 * NS** OM specific 62. 78 -1. 039 5 0 504


than 0 .20 g WM (Figures 6 and 7) for P. caudilimbatus, G saxicola, and 0. gomesii and less than 0 50 g WM for the much larger A. balearicum Mass -s pecific oxygen consumption leveled-off in larvae greater than 0 50 g WM As a result of the leveling-off in larvae greater than 0 50 g WM, P caudilimbatus larvae between 0.40 to 0 60 g WM did not respire significantly more per unit of mass than larvae 0 .61 to 1 00 g WM (Student's t-test, p > 0.05) Mass-specific oxygen consumption was homogeneous between the four species (analysis of covariance ; p > 0 05) CS and LDH individual activities also did not increase with increasing body mass (Figure 8 for CS, Figure 9 for LDH) indicating that maximum aerobic and anaerobic metabol ic pot e ntials were not dependent upon whole individual mass Mass-specific enzyme activities decrea sed precipitously i n strongly significant power relationsh ips with increasing WM (Figure 10 for CS, Figure 11 for LDH) and OM. The only exce ption oc curred in 0 gomesii LDH mass specific enzyme activities, which showed no significant relation with increasing mass The greatest decre ase in enzyme activiti es occur red in larvae less than 0 2 g WM for P caudili mb atus, G. saxicola and 0. gomesii and less than 0 .5 g WM for A. balearicum ; after whic h all activities subseq u en tly leveled-off. Analysis of covarian ce (p > 0 05) indicat ed that the mass-specific enzyme activities for both CS and LDH were homogeneou s between species. There was no signi fica nt relationship between abso lute Na+K+-ATPase activities and mass (Figu r e 12). Mass -specific NA +K+ -ATPase activities 20


...... I 0 (j) E N 0 ...J ::J .......... c 0 ro '-Q. (/) (!) 0::: P. c audilimbatus 1 8 15 1 2 9 6 3 a a 2 a 4a 6a 8a G. s a x ico/a 8 6 4 2 0 2 0 4a 6a 8a A ba/e ar icum 1 a 8 a a 1 oa 20a 3aO 4aO 0. gom esii 24 2a 16 12 8 4 a 1 0 2 0 3 a 40 5 0 Dry Mass (mg) Figure 7 Regress i o ns of dry mas s-sp eci fi c r espiration ( Y ul 02 mg oM -1 hr-1 ) agai n st dry m ass (X mg OM). E q u at i ons and r2 values a re reported in Tabl e 2. 22


P. caudi/imbatus A. ba/earicum 0.8 2 0 .5 0.72 0.4 0 6 2 0. 3 0.52 0.4 2 0.2 0 .32 0. 1 0.22 .. 0 -0 0 .2 0.4 0.6 0 8 1.0 0 1 2 3 4 5 6 (/) :!:: c :J .._.. >. ..... ::;: :.;:; () ro 0 gomesii (/) G. saxicola () 0.4 3 0. 34 0.38 0.30 0. 2 E 0 .33 0.28 0.22 0.1 8 0.23 0.18 0.1 4 0. 13 0.1 0 0.3 0 .6 0.9 1.2 1.5 1.8 0 0. 1 0. 2 0. 3 0.4 0. 5 W e t M ass ( g ) F i gure 8. Plots of i ndiv idual citrate synthase activity ( Y umol substrate converted min -1 ) with wet mass ( X g WM) 23


P. caudilimbatus A. b a /earicum 60 4 0 5 0 3 0 40 3 0 20 2 0 ;,. ... 1 0 1 0 0 -0 (/) 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 6 c :J ......... >. .... ::.; u ro I 0. go m esii 0 G. saxico/a .....J 40 1 80 150 3 0 1 2 0 2 0 90 6 0 1 0 3 0 0 0 0 0 .3 0.6 0.9 1 2 1 5 1.8 0 0 1 0.2 0. 3 0 .4 0. 5 W e t M ass ( g ) Figure 9. P l ots of ind ividu a l l act ate d e hydrog e n as e ac t i v i ty ( LDH) ( Y umol substrate co n verted m i n -1 ) w i t h w et mass ( X g WM). 2 4


P. caudilimbatus A. ba/earicum 8 4 6 3 4 2 2 1 0 0 0 0.4 0.8 1.2 0 1 2 3 4 5 6 G. saxicola 0. gomesii 4 3 3 2 5 2 2 1.5 1 1 0.5 0 0 0 0 3 0.9 1 5 0 0.1 0.3 0 5 W e t Mass (g) Figure 10. Regress ions o f cit rate synthase (CS) a c t iv ities (Y, umol of substrate converted min -1 g1 ) on wet mass (X g WM). Equ at io ns and r2 values are reported in Table 2 25


P. c audiliTnbatus A. b a /ear icuTn 0.2 4 0.05 0.2 0.04 0.16 :s: 0.0 3 = .!!3 0.1 2 c: 2- 0.0 2 0.08 <( 0.01 0.04 .... I .... 0 0 0 0.3 0 6 0.9 1.2 1 5 0 1 2 3 4 5 M ass ( g ) Figure 12. Plots of ind iv idua l Na+/K + ATPase ac t ivit i es { Y units ) with wet mass {X g WM). P. c audiliTnbatus 0.8 0 6 0.4 0.2 0 0 0.3 0 6 0.9 1.2 1 5 M ass (g) A ba/ear icuTTI 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 Figure 13. Regressions of mass specif i c Na + -K+ ATPase acti v it i es {Y, un i ts g wM-1 ) agai nst we t m ass { X g WM ) 27


decreased precipitously with increasing mass for larvae less than 0 3 g WM (Figure 13) in the same manner as CS and LDH activities Discussion In the relationship of oxygen consumption to mass, V02= aMb, the slope value b provides a quantitative expression of the increase in respiration with increasing individual size The slope can be used to examine the influence of body size on metabolism to body size ; a slope value of 0 67 indicates that respiration is scaling with surface area and a slope of 1 indicates that respiration is scaling directly with mass (Schmidt-Nielsen 1990) In leptocephali, there is no significant relationship between increasing whole larval mass and oxygen consumption rates This is also substantiated by the lack of a significant relationship between intermediary metabolic enzymes activities and increasing larval mass. Mass-specific oxygen consumption conformed strongly to the equation V0.jMb = aMb b For a wide variety of organisms b ranges between 0 .33, indicating that respiration scales with surface area and 0, indicating that respiration scales with mass (Schmidt-Nielsen 1990). For most larval fish this value is close to 0 indicating that mass has the greatest impact upon respiration rates (d. Giguere et al. 1988, Torres et al. 1996). The slope values obtained for leptocephali (b = -0. 5 to 1 0) indicated a precipitous decline in mass-specific respiration with increasing mass; much greater than any previously described for a larval fish The significance of this result is that it demonstrates that a lower 28


proportion of the mass of the leptocephalus is invested in metabolizing tissue than in other larval fish This is a very unusual situation for a larval fish, or for any organism. The wet mass-specific metabolic rates obtained (250-1200ul 02 g wM-1 hr-1 ) were low and were not comparable to any reported wet mass-specific metabolic rates obtained for larval fishes at similar temperatures A comparison of the dry mass-specific leptocephalus respiration rates (extreme values ranged from 0.52-20.39 ul02 mg DM-1 hr-1 ) to the values for sea bream (Houde and Schekter 1983) (3 .14-9 .39 ul02mg DM-1 hr-1 for 25 .5-66 ug individuals) and red drum larvae (Torres et al. 1996) (2.38-4 12 ul02 mg DM-1 hr-1 for 21 68ug larvae) reveals that leptocephalus respiration was comparable to and even higher in small leptocephali than in other teleost larvae at similar experimental temperatures when examined as a function of dry mass. The only published data on respiration in premetamorphic leptocephali for comparing the rates obtained in the present study are from electron transport system analysis (ETS) (Schalk 1988, Pfeiler and Govoni 1993) Mass-specific respiration rates obtained in the present study were substantially higher than those collected by Pfeiler and Govoni (1993) (WM specific median-83ul 02 g wM-1 hr1 ) respiration rates reported in Table 3) Even after adjusting their estimates for a 10% loss of activity due to storage (Yamashita and Bailey 1989), the ETS values remained substantially lower than direct respiration values. The activities obtained by Pfeiler and Govoni (1983) could be anomalously low if larvae were damaged 29


dead or dying when collected Only active, undamaged larvae were used for this respiration analysis. ETS activity is assumed to represent the maximum potential oxygen consumption (Owens and King 1975) This assumption is only valid when the ratios of respiratory rate to ETS activity (R:ETS) are similar in both groups R: ETS were not compared for non-metamorphic leptocephali. The inference of oxygen consumption to premetamorphic leptocephali from metamorphic larvae appears to drastically underestimate metabolic processes, as a result of the reduced metabolism during metamorphosis (Bishop and Torres, unpublished data) Table 3. Oxygen consumpt i on rates (uL mgDM-1 hr-1 ) for various teleost larvae. (SD) =standard deviation, *indicates respiration values obtained through electron transport system analysis using the conversion 0 083 ug-at 0 uL02 1 T = experimental temperature Species ul mg DM"1hr1 (SD) T Source Achirus lineatus larvae 2 .00-19.70 28 Houde and Schekter (1983) Anchoa mitchel/i larvae 4 .0-8.20 26 Houde and Schekter (1983) Archosargus rhomboida/is larvae 5 6 (0 3) 26 Houde and Schekter (1983) Ophichthus sp. *0.70 -1.57 17 Pfeiler and Govoni (1993) Pseudopleuronectes 1 .80-8.00 5 Cetta and Capuzzo (1982) americanus Sciaenops ace/latus 10 d 5.47 (0 83) 25 Torres et al. (1996) 17 d 4.72 (2 63) 30


Citrate synthase activity serves as a biochemical proxy for aerobic respiration It is the rate limiting step in the citric acid cycle and it therefore represents maximum aerobic potential. As observed in the mass-specific respiration rates, there was a rapid decrease in both CS and LDH activity with increasing mass. The mass-specific CS activities decreased at a greater rate (b = -1. 02 to -1.23) than was observed in larval menhaden (b = -0 8) (Power and Walsh 1992). Citrate synthase activity correlated directly with oxygen consumption rate in the leptocephali allowing the use of CS activity as a predictive tool for future analyses to determine respiration rates from frozen specimens. For both mass-specific oxygen consumption and intermediary metabolic enzyme activities, the greatest change in slope occurred in larvae less than 0.05 g WM for all four species. Donnelly et al. (1995) reported that based upon proximate and nucleic acid composition A. balearicum Phase I development could be divided into two subphases (Ia and lb). Phase Ia was characterized by high cellular proliferation, with growth occurring in length rather than mass In Phase lb there was a leveling off of RNA and DNA at an asymptotic concentration; indicating that a greater proportion of the larvae was comprised of acellular tissue: glycosaminoglycans. The size at which the transition from Phase Ia to Phase lb occurred was approximately 0.05 g WM. The changes observed in the proximate composition of the larvae were also apparent in the oxygen consumption and enzyme activities 31


The most significant result of the present study on metabolism, enzyme activities and mass is the scaling coefficient relating mass-specific metabolism to mass, both in direct determinations of metabolism and in intermediary metabolic enzyme activities. The mechanism responsible for this unusual relationship of mass to metabolism is the formation of an energy depot in the form of glycosaminoglycans (Pfeiler 1984b, Donnelly et al. 1995) The larvae increase rapidly in mass but accumulate little metabolizing tissue, thereby maintaining low overall metabolic costs in very large larvae The rapid growth of a larva decreases the number of its potential predators giving the leptocephalus the ecological refuge of increased size In larvae exhibiting Type 1 developmental strategies where energy storage is virtually nonexistent (Brightman 1993), the absolute costs of being larger result in a higher susceptibility to death by starvation (cf Miller et al. 1988, Torres et al. 1996 ) Because energy storage in the leptocephalus is closely linked to growth in the form of glycosaminoglycans, being larger is beneficial; for leptocephali, bigger is better. 32


CHAPTER3 ENERGY LOST TO EXCRETION Introduction The energy available for larval fish growth is influenced profoundly by two factors: the amount of energy required for metabolism (Chapter 2), and the energy lost in excretion (Brett and Groves 1979) The energy lost in excretion can be further divided into two subcomponents : feces, the portion of ingested energy that is indigestible, and nonfecal nitrogen, in fishes, most commonly evident in the form of ammonia or urea (Brett and Groves 1979) The amount of energy lost in each of the subcomponents of excretion varies with the ontogeny of the fish (Buckley and Dillman 1982). In older fishes, feces can comprise up to 20% of the ingested total. However, in larval fishes, feces are rarely measured due to the small size and difficulty in collection The fraction of ingested energy lost to fecal excretion in larval fishes is therefore usually calculated by the difference between the sum of the energy devoted to growth and metabolism and the total energy ingested (Houde and Schekter 1983, Yamashita and Bailey 1989). 33


Nonfecal excretion in adult fish ranges from 3 to 10% of the total energy ingested (Brett and Groves 1979) Larval fish excretion anges from 23 to 40% of the gross ingested calories (Houde 1989) Jobling (1981) found that rates of ammonia excretion scaled with body weight to the power tot 0 67, suggesting a surface area dependence Teleosts are mainly ammonotel i c excreting the majority of their nitrogen in the form of ammonia (Brett and Groves 1979, Wood 1993) In most fish species 60 to 80% of the ammonia is lost through the gills via the gill epithelium (Brett and Groves 1979 Wood 1993) N i trogen excreted i n the urine constitutes a minor component of the total nitrogen excreted in most teleosts (Ashley 1975 Pandian and Vivekanandan 1985) Urea i s primarily excreted in the uri ne however in adult teleosts urea us u ally i s a m i nor portion of the total n i trogen excretion (Brett and Groves 1979) Data on larval f i sh estimates urea values at 15 to 33% of the total ingested energy lost in excretion (Jobl i ng 1981 Torres et al. 1996) The amount of urea excreted is dependent upon the condit i on of the larvae (Torres et al. 1996) ; elevated urea excretion was i ndicative of starvat i on in very young red drum larvae resulting from the catabo l ism of the fast-growing larvae's tissues Excretion estimates for pelagic larvae are virtually nonexistent. This is a result of t he difficulty in mainta i ning the larvae as well as in the log i stics of the ammonia analysis The objectives of the excretion study were to quantify the ammonia excretion rates of four species of leptocephalus larvae Paraconger 34


caudilimbatus Ariosoma balearicum, Ophichthus gomesii and Gymnothorax saxicola The data on excretion were used to calculate the total energetic requirements for each of the leptocephalus larvae (Chapter 6) Materials and Methods Leptocephali were collected in the eastern Gulf of Mexico on three cruises from 1995 to 1996 (Table 4 ) Sampling was conducted from approx imately 26N to 28N between 84 W and 86W (Figure 2) The collection gear consisted of a 2 m plankton net with 505 urn mesh and a 9 m2 mouth area Tucker trawl constructed of 6 8 mm mesh and a quick-release blind cod end. Nets were towed at 1 to 2 knots in a double oblique pattern from the surface to a depth of 100 m Tow times varied from 10 to 60 minutes depending upon plankton density All sampling was conducted at night to maximize collections Leptocephali were sorted and identified after each tow Active undamaged larvae were immediately removed from the catch for excretion analysis Ammonia excretion rates were determined simultaneously with oxygen consumption rates. Active, undamaged larvae were placed in water-jacketed lucite chambers (Figure 3) containing filtered (0.45 urn) seawater maintained at the experimental temperature of 25C ( 0 2C) through the use of a circulating refrigerated waterbath An initial 20ml water sample was taken after the addition of the leptocephalus The chambers were sealed and larvae were incubated for 2 to 18 hours 35


Table 4. Leptocephalus species used in excretion analyses shown by years collected and gear used Species Year Collected Gear Used P. caudilimbatus 1995, 1996 2m plankton net 3.05m x 3 05m Tucker trawl A. balearicum 1995, 1996 2m plankton net 3.05m x 3.05m Tucker trawl G saxicola 1995 2m plankton net 0. gomesii 1995 2m plankton net Chambers were covered with black plastic to reduce visual stress to the leptocephali. Larvae were monitored hourly throughout the duration of the incubation period; moribund leptocephali were removed immediately At the completion of the incubation, a second 20 ml water sample was dispensed in triplicate. Samples were pipetted into acid-washed, oven-dried polyethylene containers. Phenol-alcohol reagent was added to each sample at analytical concentrations to stabilize the ammonia concentrations during storage Larvae were removed from the chambers, measured to the nearest 0 1 mm total length (TL), rinsed in deionized water, blotted, and frozen in liquid nitrogen Larvae were stored at -80C until further analysis. Between incubations, the chambers were rinsed with filtered seawater. Background ammonia was determined by adding leptocephali to chambers and 36


removing the specimen prior to sealing Background incubation periods lasted from 2 to 1 0 hours. In the laboratory, larvae were weighed to the nearest 0 1 mg and introduced frozen into the homogenizing medium ice-cold distilled water at a dilution of 24:1 and homogenized at 0 to 4C using a sonifier and by hand, using conical glass homogenizers having ground glass contact surfaces (Kontes Glass Co "Duall" models) Duplicate 1 ml aliquots were dispensed into preweighed crucibles, dried for 72 hours allowed to cool and reweighed for dry mass determinations. Ammonia content of the samples was analyzed within one week of collection using the colorimetric techniques of Solorzano (1969). Sample contamination was minimized by acid-washing and dedicating all glassware exclusively for ammonia determination Absorption was measured at 640 nm in a Cary spectrophotometer; results were in umol NH3 Ammonia excretion rates were expressed in absolute (umoiNH3 indiv -1 hr1 ) and mass-specific values (umol NH3 g M-1 hr1 ) both in wet mass (WM) and dry mass (DM). Regressions were generated using the least-squares method with significa n ce at p less than 0 05 Analysis of covariance (Zar 1996) was performed on mass specific excretion rates to determine differences between species 37


Results Background ammonia resulting from the presence of microorganisms was minimal and required no correction of the data All incubations resulted in ammonia concentrations within the detectable limits of the assay Excretion rates were determined for 51 larvae Absolute ammonia excretion rates (Y, u mol NH3 indiv -1 day-1 ) were highly variable and increased linearly with increasing larval mass (X, g WM) in three of the four species (Figure 14). The excretion rates for the four 0 gomesii specimens did not correlate significantly with increasing mass Table 5 shows the linear equations with means and ranges for each species. Table 5. Individual NH3 excretion rates (Y, umol NH3 individual-1 hr1 ) against wet mass (X, gWM). X = mean values (SE) for each species, n = number of incubations and ,-2 = coefficient of determination NS no significant relationship Species Equation ,-2 X n P. caudilimbatus y = 0 1459 + 0.5207 X 0.27 0 302 28 (0 0965) A. balearicum y = 0.3162 + 0 1629 X 0 36 0 654 5 (0.4013) G saxicola y = -0 0596 + 0 6461 X 0 85 0.221 15 (0.1357) 0. gomesii NS 0 2700 4 (0.4152) 38


("t) I z 0 E ::I 1.5 1 2 0.9 0.6 0. P. caudil i mb a tus 0 0.2 0.4 0.6 0.8 1 0 1 2 A. ba l ea r i cum 1.5 1 2 0 0 1 2 3 4 Figure 14. Regressions and plot of individual excretion rates (Y umol NH3 hr1 ) against wet mass (X g WM ) Equations and r2 values are reported in Table 5 39


Mass-specific ammonia excretion (Y, umol NH3 g M-1hr1 ) declined with increasing mass, in both wet mass (X, g WM) (Figure 15) and dry mass (X g OM) (Table 6), according to the power function : E/M =a Mb-1 where E/M is the excretion rate (umol NH3 g M-1 hr1), M is mass in grams and b is the species-specific slope. The maximum decline in mass-specific excretion with increasing mass occurred in larvae less than 0 5 g WM. Mass-specific excretion in larvae more than 0 5 g WM remained approximately constant. Mass-specific excretion rates ranged from a high in small P. caudilimbatus of 3.5 umol NH3 g wM-1 hr1 to 0.5 umol NH3 g WM-1 hr-1 for 0. gomesii (Figure 15). An analysis of covariance (Zar 1996) indicted that mass-specific excretion rates in all four species were not significantly different from each other (p>0 05) Table 6. Wet mass(WM) and dry mass (OM) specific NH3 excretion rates (umol NH3 g M -1 hr\ n =number, r-2 =coefficient of determination WM specific OMs pec ific S pec i e s Equat i on r n Equat ion r P caudilimbatus y = 0.4 2 62 WM .().5882 0 .34 2 8 y = 1 5965 DM .o.s= 0 29 A. balearicum y = 0 2175 WM ..... 0 .7 1 4 y = 0.7185 DM .o.aa7 4 0 .90 G saxi cola y = 0 3466 WM.o -0 .06 15 y = 2.9949 0 .12 0 gomesil y = 0.0909 WM 1 .31 16 0 55 4 y =0 0507 DM 124:ze 0. 7 0 Discussion n 28 4 14 4 Little data are available on excretion in larval fishes largely as a result of the difficulty involved in maintaining the larvae and in the storage of ammon i a 40


..... I Ol 0 E ::J -c 0 0 >< w P. caudilimbatus 4 3 2 1 0 0 .2 0.4 0.6 0.8 1 1.2 G. saxicola 1.5 1.2 0 9 0 .3 -0 0 0. 3 0 . 2.4 2.0 1 .6 0 8 0.4 A.ba/earicum 0 1 2 3 4 0. gomesii 4 3 2 1 0 0 0. 1 0. 2 0 3 0 .4 0 .5 W e t M a ss ( g ) Figure 15. Regressions of wet mass-specific excret i on rates (Y, umol NH3 g wM-1h-1 ) against wet mass ( X g WM). Equations and r2 vlaues are reported in Table 6 41


samples. Storage of ammonia samples is a serious problem in excretion studies because concentrations can change rapidly and shipboard analysis is often impossible. Unfortunately, the information on storage procedures is conflicting Marvin and Proctor (1965) observed no significant change in ammonia concentrations (15-30 ug-atoms liter1 ) in filtered samples of estuarine water when frozen and stored in glass up to 4 months Newell (1967) found that freezing did not prevent changes in ammonia concentrations (0 3 to 0 7 ugatoms liter1 ) in samples of English Channel water stored in polyethylene bottles An increased variability in results has been attributed to mechanical rupture of plant and animal cells during freezing and thawing. Other authors (Thayer 1970, Degobbis 1973) have reported that quick-freezing samples resulted in a slight decrease in ammonia concentration for the first day and stabilized the ammonia concentrations (1.25 ug-atoms liter1 ) for up to 204 hours Degobbis (1973) examined the effects of freezing, freezing rate, filtration, use of preservatives, and storage containers upon ammonia samples and determined that preservation with phenol at the same concentration used in the analytical method stabilized the unfrozen samples for up to two weeks. The data reported here are the first reported on ammonia excretion for a leptocephalus larva and contribute significantly to the limited data describing excretion in larval fishes. Individual excretion rates in the leptocephali were substantially higher than values reported for 9 to 40 d Pleuronectes platessa (0 0018 0. 0338 umol N ind-1 hr-1 ) and 1 0 to 21 d B/ennius parvo (0 0186 42


0.0488 umol n ind-1 hr1 ) both reared at 6 to 9C (Klumpp and von Westernhagen 1986) and higher than those found by Torres et al. ( 1996) for 10 to14 d red drum larvae (0 002 umol NH3 ind -1 hr1 at 25 C). Dry mass-specific excretion rates in the three smaller leptocephali species were similar to the values for red drum larvae (30.58 umol NH3 g DM-1 hr1 ) at a mass of 0 .10 mg. Excretion values obtained in this study may slightly underestimate total nitrogen excretion Jobling (1981) found that 15 to 25% of nitrogenous waste in plaice larvae occurred in the form of urea Torres et al. (1996) found that urea was (18 to 30%) of the nitrogenous waste in red drum larvae. As a result, total nitrogen excretion values for leptocephali may be underestimated by 15 to 30% Leptocephalus excretion when compared to other teleost larvae in absolute values is substant i ally higher than in Type 1 teleost larvae Expressed in dry mass-specific rates, leptocephalus excretion is comparable and higher than other larvae of the same dry mass The pronounced decline in mass specific excretion rates in larvae less than 0.5g WM was indicative of the shift in mass accumulation from high cellular proliferation in the Phase Ia larvae to an accumulation of acellular tissue in Phase lb as described by Donnelly et al. 1995. As the concentration of metabolizable tissue per gram of WM decreases in Phase 1b, the mass-specific metabolic wastes also decrease 43


CHAPTER4 GROWTH INDICES IN LEPTOCEPHALI Introduction Greater size increases a larval fish's prey spectrum by improving locomotory capabilities and by enlarging its gape at the same time greater size provides an ecological refugium from predation because the larvae exceed the gape limitations of predators and their escape potential is enhanced (Ware 1975b, Hunter 1981, Peterson and Wroblewski 1984, Blaxter 1986, Kiorboe et al. 1987, Post and Prankevicius 1987). However metabolism increases with increasing mass in most larval fish, therefore, the increased size comes at the expense of higher metabolic energy demands (Torres et al 1996 Schmidt Nielsen 1972) Only after the daily metabolic requirements are met can the remainder of ingested energy be devoted to growth Two distinctly different developmental strategies exist in larval fish to meet increasing metabolic expenses while continuing to grow rapidly The Type I developmental strategy representative of most larval fishes, is to minimize the period of vulnerability through rapid growth and a short larval phase regardless of the potential for starvation Mass increases in larval fish are manifested primarily through the accumulation of protein in the form of 44


muscle (Balbontin et al.1973 Ehrlich 197 4a, b, 1975, Cetta and Capuzzo 1982). Growth rate has been shown to control the duration of the larval phase (Werner and Gilliam 1984) and there is little or no energy storage All available energy is devoted to maximum growth (Brightman 1993) Type 2 larvae, characterized by the leptocephalus, a unique larval form uniting the members of the superorder Elopomorpha, have a different early life history strategy. Growth, as substantial daily increases in both mass and total length is concurrent with energy storage. Residence times in the plankton are extended, ranging from 30 days to more than a year (Schmidt 1922, 1925, Castonaguay 1987, Crabtree et al. 1992) The onset of metamorphosis to the juvenile phase may potentially be delayed until appropr i ate physical conditions are present (Tseng 1990) Growth in mass is achieved by incorporating glycosaminoglycans into the body matrix (Pfeiler and Luna 1984, Pfeiler et al. 1991 ) Thus the majority of the leptocephalus' growth in mass is in the form of non-metabolizing tissue, minimizing the increased metabolic costs traditionally associated with increased size The objectives of the present study were to address growth in leptocephalus larvae through examination of daily growth using otolith microstructure analysis as well as two b i ochemical indicators of growth : RNA: DNA ratios and percent increase in larval protein 45


Materials and Methods Leptocephali were collected in the eastern Gulf of Mexico on six cru i ses from 1990 to 1996 (Table 7) Sampling was conducted from approximately 26N to 28N between 84W and 86W (Figure 2) The collect i on gear consisted of a 2 m plankton net with 505 um mesh and 9 m2 mouth area Tucker trawl constructed of 6.8mm mesh and equipped with a quick-release blind cod end Nets were towed at 1 to 2 knots in a double oblique pattern from the surface to a depth of 100m. Tow times varied from 10 to 60 minutes depending upon plankton density All sampling was conducted at night to max i mize collections Table 7. Leptocephalus species used in growth analyses shown by years collected and gear used Species Year Collected Gear Used P caudilimbatus 1995 1996 2m plankton net 9m2 Tucker trawl A. balearicum 1990, 1991 2m plankton net 1992 9m2 Tucker trawl G saxicola 1992 2m plankton net 0 gomesii 1992 2m p l ankton net Leptocephali were sorted, identified to species and measured to the nearest 0 1 mm total length (TL) Larvae were rinsed with deionized water, blotted, and frozen in liquid n i trogen for mass protein, and nucleic acid 46


determinations Larvae from each of the four species were also measured fresh and used to determine shrinkage resulting from preservation. Specimens that weren't frozen were preserved in 95% ETOH. Only premetamorphic, or Phase I, larvae were used for analysis Myomere counts were made on those specimens requiring counts for specific identification (See Appendix A). The relationship between fresh total length (TL) and preserved length (PL) was determined by an exponential equation for each species. All preserved lengths were subsequently converted to fresh total lengths For age determinations, the heads of larvae from the four species, including six A. balearicum embryos, were removed, dehydrated and infiltrated with increasing concentrations of low-viscosity embedding media (Spurr 1969). Only otoliths from the largest A. balearicum larvae were removed using a dissecting microscope under polarized light. The dehydration and infiltration times varied with the size of the larvae and ranged from several days to several weeks at each step The heads were cured in blocks of resin and the otoliths were prepared as described by Haake et al. (1982) Sagittal otoliths were used for age analysis due to their consistently larger size and greater increment width. Otoliths were polished until the primordia were exposed at the surface of the sagittal plane To prepare the otoliths for SEM analysis, an etching agent, 0 12 N HCI, was applied to erode the inorganic portions of each increment. The erosion of 47


the otolith was observed under 200x magnification and halted by immersion in distilled water when increment contrast was apparent. Etching times varied with the size of the otolith and ranged from seconds to a minute. Samples were sputter-coated with gold-palladium and examined using either a Cambridge or Hitachi SEM at 15 to 25 Kev, with a working distance of 100 mm and a standard large spot size. First derivative signal processing was used to enhance the increments Each image was photographed or stored digitally using Optimas Image Analysis Software (Optimus 5 0). A minimum of three micrographs were taken of each otolith : the primordium the entire otolith and the best counting plane. For the very large otoliths a series of micrographs were taken using landmarks on the otolith to overlap each micrograph Maximum and minimum primordia diameters, designated as the maximum and minimum distance from the first discontinuous zone across the center of the otolith (Secor et al. 1991) were measured for each otolith. Any otoliths possessing a core diameter greater than the mean two standard deviat i ons were rejected from the age analyses on the basis that all of the increments were not properly exposed in the polishing process. Otolith radius measurements were calculated from the mean of the maximum and minimum distances from the center of the primordium to the otolith margin Daily increment counts began with the first consistent increment dis t al to the primordium Otolith increments were enumerated during three different sessions with two readers In each instance the readings were blind, with the 48


reader unaware of the larval size or any previous age estimates Any otolith varying by more than 1 0% of the mean in any of the s i x separate reads was rejected No significant differences between the mean counts of each reader (pa i red t-test, p <0 05 df = 79) justified pooling the increment counts for further analysis. The mean of the six increment counts represented the larval age in days post-hatch For the analysis of the biochem i cal ind i cators of growth larvae were weighed to the nearest 0 1 mg wet mass (WM) and homogenized in a 24: 1 dilution of ice-cold distilled water using a son i fier and then manually, using a hand-held tissue grinder. Dupl i cates of 1 ml aliquots were dispensed in preweighed cruc i bles and dried for 72 hours at 60C Samples were cooled to r oom temperature, reweighed for dry mass, and subsequently combusted for 3 hours at 600C for ash weight determinat i on Triplicates of homogenate aliquots were dispensed for the determination of RNA DNA and protein Nucleic aci d contents were determined using the techniques of Bentle et al. (1981) with bakers yeast RNA (Sigma Type XI, R-6750) and calf thymus DNA (Sigma D3664) used as standards Prote i n was assayed us i ng a modification of the techniques of Lowry et al. (1951) with human albumin and globulin as the standard Protein growth rates (Gpi) the percent change in protein content pe r day were calculated using the natural logarithm of protein content (ug larvae -1 ) 49


and age in days : GP1 =In protein (ug larvae1 ) 100 where tis the time in days post-hatch or larval age (Buckley 1984) Daily growth was e x pressed in increases in total length, wet mass, and ash-free dry mass All data were examined for normal distribution and homogene i ty of variances Age and growth data were transformed to natural logarithms (Sakal and Rolf 1981 ) Regressions were generated using the leastsquares method with significance at p less than 0 05 Results The relationship of larval fresh total length (Y, mm TL) and preserved length (X mm PL) was linear (Table 8) Table 8. Linear regressions of total larval length (Y, mm TL) on preserved length (X mm PL) n =number of larvae measured r = coefficient of determination Spec i es Equation n r P caudilimbatus TL = 4 008 + 0 978 (PL) 42 0 98 A. balearicum TL = 17 764 + 1 258 (PL) 27 0 92 0 gomesii TL = -1.752 + 1 183 (PL) 21 0 96 G sa x icola TL = 7 .371 + 1 013 (PL) 30 0 89 Only sagittae and lap i lli were present in the ot i c capsules Asterisci were not present in any larvae examined appearing only at the onset of metamorphosis Except in yolk-sac larvae sagittae were consistently largest of the 50


two pairs of otoliths Both sagittae and lapilli exhibited characteristics of daily increment microstructure as described by Dean et al. (1983) (Figure 16). Sagittae of each species possessed a distinct primordium followed by a wide, diffuse band (Figure 17). Mean core d i ameters were determined for each species and ranged from 12 72 (.31) urn for 0 gomesii to 18 8 7 {. 76) urn for P. caudilimbatus with A. balearicum and G saxicola possessing inte r mediate mean core diameters of 13.94 ( 25) urn and 15 17 ( 19) urn, respectively Examination of the otol i ths from six A. balearicum embryos revealed that no increments were formed prior to hatch There were no microstructural otolith features dist i nguishing yolk-sac from post yolk-sac larvae Absorption of the yolk-sac occurred at 4 to 8 mm TL in A. balearicum and P caudilimbatus but due to a lack of small specimens, s i ze at yolk-sac absorption was not observed in G saxicola and 0 gomesii Mean i ncrement widths ranged from 0 .68 ( 0 04) urn for A. balearicum to 1 .58 ( 12) urn for G. saxicola (Table 9). Otol i th radius was linearly related to i ncrement number in three of the four species, suggesting that otolith growth was consistent throughout the larval period Otoliths examined for G saxicola spanned a very narrow size range (19 to 43 urn radius) resulting in an inability to detect a sign i ficant relationship (p>0 05) between increment number and otolith radius 51


Figure 16. Otolith from a 17 day A. balearicum (30mm) Figure 17. Sagittal otolith of 20 day P caudilimbatus (43 mm) showing the primordium 52


Table 9. Mean increment widths (urn) (SE) and relationships of increment number on otolith radius and total larval length. A= otolith radius, 8 = total length X = increment number, n= number of larvae, r2 = coefficient of determination an NS = not significant. Species W i dth Otolith radius r Total length r n P caudilimbatus 0 813 A= 0 2439 + 0 7629X 0 4160 B =16.0697 +0 4915X 0 3826 (0.0413) A. balearicum o .sn A= .{) .8861 +1. 7436X 0 5506 B =-3 6440+4 3284X 0 5148 (0 0365) O gomesi 0 .697 A = 3 8324+0 .8711 X 0 5197 B = 30. 4328+1 .5n8X 0 3471 (0 1092) G saxicola 1 .581 NS NS 39 65 79 Otolith ages ranged from 4 to 111 days for larvae 7 8 mm to 240.0 mm The regressions of age (Y, days) against TL and WM are shown in Figures 18 and 19 Periods of extremely rapid growth in total length occurred in all larvae prior to 20d. The In-transformed data (Table 10) conformed to a linear relationship Growth in total length ranged from 0.6357 (0 0749) mm d "1 in 0 gomesii to 1.4224 (0 .11 06) mm d"1 in P caudilimbatus. At approximately 20 d the rate of growth in total length decreased while growth in mass increased indicating a shift in growth from total length increases to increases in mass RNA/DNA ratios in leptocephali were low and never exceeded 1 .5. Mean ratios ranged from 0 613 ( 0.0315) for G sa xi cola to 1 1100 ( 0448) for A balearicum (Table 11 ) RNA : DNA ratios exhibited no significant trends with TL, WM, AFDM or larval age 53


........... E E ......... Figure 18 Regressions of larval total length (Y mmTL) on otolith age (X days) Raw data i s plotted with Intransformed equations and r2 values reported in Table 10 54


P caudilimbatus A. ba/earicum 0.8 0 6 8 0.4 6 4 0 2 2 ---0 Ol 0 2 0 40 60 80 100 20 40 60 80 100120 -rtJ rtJ cu E ..... Q) s: G. saxicofa 0. gomesii 0.8 1 0 6 0.8 0.6 0.4 0.4 0.2 0.2 0 0 0 2 0 40 60 80 0 20 40 60 80 Age (days) Figure 19. Regressions of wet mass ( Y g WM) on otol it h age (X, days) Raw data is plotted w it h In-t r ansformed equations and r2 values reported in Table 10. 55


Table 10. Larval growth equat i ons obtained by regress ing otolith age (X, days) against growth parameters (Y total length (TL), wet mass (WM), dry mass (DM) and ash free dry mass (AFDM ) ) n =number of larvae and r2 i s the coefficient of determinat i on. Spe c ies Gro wth Equation n r-2 P caudilimbatus TL y = 5.9495 )(15424 49 0 84 WM y = 0 .0007 xum 49 0 84 DM y = 5 .4x1 o x 1.3747 49 0 85 AFDM Y = 5 8 x 10-5x 1 2 427 49 0 86 A. ba/ earic um TL y = 2.5834 )(19688 83 0 .69 WM y = 0 0001 83 0 .69 DM y = 83 0 .69 AFDM y = 1 1 x1 o.o 8 3 0 .73 0 gome sii TL y = 6 0281 )(17452 81 0 .47 WM y = 0 0005 x 1 8665 81 0 48 DM y = 1 1 x 1 o4037 81 0.50 AFDM y = 1 x 1 o7659 81 0 .50 G saxi co l a TL y = 16 1889 )(13752 36 0.42 WM y = 0 .0069 x 1 051 36 0.40 DM y = 5 .4x1 0 5 X 1 '7098 36 0 .42 AFDM y = 2 7x10-5x 1 7407 36 0 42 Mean protein growth rat e s ranged from 16 12 (.0 0 2 4) % (P. caudiJ i mbatus) to 37 33 (.. 018% (G sa x icola) (Table 11 ) Protein growth (Gp1 ) was strongly correlated with increasing TL (Table 12) WM (Figure 20, Table 12) and AFDM (Table 12) with th e greatest changes occurring in larvae smaller than 0 5 g WM, indicating that the rate of protein accumulation p e r unit mass decreased with inc r easing s ize. 56


......... ..-I >. P. caudi/imbatus 40 1 0 A. balearicum 4 5 40 35 30 25 2 0 cf?. 0 0.3 0.6 0.9 1.2 1.5 0 0.4 0.8 1 2 1.6 2 2.4 ..r:::. 0 L-Ol c Q) ..... 0 L-n... 52 48 44 40 3 6 32 2 8 G. saxico/a 0 0.2 0.4 0.6 0.8 0. gomesii 46 43 40 37 3 4 31 2 8 1 0 0.1 0.2 0.3 0.4 0.5 0.6 W e t m ass (g) Figure 20. Regressions of protein growth (Y, % day-1 ) against wet mass (X, g WM). Equations and r2v alues are reported in Table 12. 57


Discussion As with all growth studies, direct validation of daily increment deposition is ideal (Beamish and McFarlane 1983) The difficulty of maintaining fragile, premetamorphic specimens in excess of 24 hours precludes direct Table 11. Mean protein growth, GPI (% day -1 ) and RNA : DNA ratios for each species of larvae Protein growth was calculated as described in the text Values are reported as (SE), n = number of larvae Species Gpt RNA:DNA n P. caudilimbatus 16.12 0 861 51 (0. 984) (0. 1145) A. balearicum 23.75 1 108 84 (3.779) (0.0435) G saxicola 37.36 0 607 39 (0.521) (0. 0344) 0. gomesii 31.56 0 785 82 (0.568) (0.0386) validation. Increments were assumed to be deposited daily because of two lines of evidence First, Umezawa et al. (1989) confirmed the daily deposition of sagittal increments up to five days using sequential samples of artificially hatched larvae of Anguilla japonica Second, daily otolith increment deposition has been validated for metamorphosing A. ba/earicum (Bishop and Torres unpublished data) The consistent core diameter measurements support the assumption that ages were not underestimated due to a failure to expose all of the sagittal plane of the otolith on the surface The core diameters obtained here for all four species are similar to those of Lecomte-Finiger (1992) (7 5 urn) and Castonguay (1986) for Anguilla sp (16 2 urn). By using an average core diameter criterion for acceptance of otoliths, the possibility of underestimating 58


Table 12. The relationships of protein growth (Gp1 ) (% day-1 ) against total length (mmTL}, wet mass (g WM), and ash-free dry mass (g AFDM) for each species Protein growth was calculated as described in the text All equations are GPI = aXb, where X is TL, WM, or AFDM n= number of larvae, r = coefficient of determinat i on. Species P caudi limbatus TL WM AFDM A. ba/earicum TL WM AFDM G saxico/a TL WM AFDM 0 gomesii TL WM AFDM Equation Y=2262.8039X -1 2 153 Y=8.0017X-0 4925 7 Y=23 34 59 0 .9601 0 9923 0 6329 0 9927 0 8386 0 9126 0 4628 0.6098 0.3318 0.5002 0 .3121 0 .1941 n 46 46 45 6 6 6 38 39 37 27 27 25


the number of increments is reduced If underestimation does occur, the resulting error will be standardized and consistent across all otoliths examined Age estimates on yolk-sac larvae of A. balearicum (4-6 d) were similar to data on hatchery-reared Anguilla japonica, indicating yolk-sac absorption occurred at 5 -7 days post-hatch (Umezawa et al. 1995). Examination of the otoliths from A. balearicum embryos demonstrated that otolith increments did not form prior to hatch, thereby reducing the possibility of overestimating larval age. The intercepts of total length on age, corresponding to the size at hatch, for all four species were slightly less than the sizes at hatch reported for each species (Smith 1989) and for Anguillajaponica hatched at a size of 2 to 2 9 mm, (Yammamoto et al. 1975, Umezawa et al. 1989). Growth rates obtained by both length-frequency analysis and otolith analysis for other members of the Elopomorpha are shown in Table 13. Crabtree et al. (1992) using similar otolith preparation techniques obtained growth rates of 0 92 mm day1 for Megalops at/anticus, comparable to and less than the growth rates for the leptocephali in this study. A change occurs in larval development at 0 2 to 0 5 g WM and an age of 20 to 40d where growth in TL slows and that of WM increases. Growth in length continues throughout the premetamorphic phase but at a slower rate than that observed in the first three weeks of life. A change in growth pattern at 0 2 to 0 5 60


g WM was also observed in the chemical composition of the larvae and resulted in a division of Phase I into two subphases (Donnelly et al. 1995) The daily growth rates, both as increases in wet mass and total length, are the greatest reported to date for leptocephalus larvae. The disparity between leptocephalus growth rates and growth in non-leptocephalus larval fish is a direct consequence of the high proportion of gelatinous body tissues found in the leptocephali. In non leptocephalus larvae, water concentrations decline with increased mass (Balbontin et al. 1973, Laurence 1979) In leptocephali, however, water concentrations remain high throughout the premetamorphic period (Donnelly et al. 1995, Chapter 5). Increases in wet mass are in the form of glycosaminoglycans and water Once the water is removed, it is apparent that leptocephalus larvae accumulate metabolizing tissue at a rate comparable to other larval fishes Growth in dry mass values were similar to those for the Table 13. Growth rates and duration of the larval phase for elopomorph larvae TL= total length Species Growth rate (mm TL d-1) A. anguilla 0 .190 .24 0 38 0 .26-0. 30 A. rostrata A.japonica Megalops 0 92 Duration of larval phase 11 18 months 5 1 year 8-12 months 155 169 151-276 d 1 month 61 Source Boetius and Harding (1985) Castonguay (1987) R. Lecomte-Finiger (1992) Kleckner and McCleave (1985) Tabeta et al. (1987) Tsukamoto (1990) Crabtree et al. (1992)


non-leptocephalus larvae, Archosargus sp. (at 28C) (Houde and Schekter 1981 ). Values for RNA: DNA were lower (less than 1.3) than those of conventional Type 1 larvae. In conventional larval fishes, RNA: DNA values less than two are indicative of slow or no growth, i.e. starvation (Wright and Martin 1985). Type 2 larval development may violate the assumptions of the RNA: DNA as an indicator of condition. In leptocephali, protein content increases with increasing mass (Donnelly et al. 1995, Chapter 5), but the greatest gain in mass is a result of acellular tissue accumulation as the leptocephali grow (Donnelly et al. 1995). The protein growth rates for leptocephali (16 .31 to 33 36% day-1 ) were similar to the rates reported by Torres et al. (1996) for red drum larvae 6 to 14 d (1 0.40 to 50.20%day-1 ) at temperatures equivalent to summer Gulf of Mexico surface water temperatures The protein content of leptocephali is lower than that of conventional larval fish (Donnelly et al. 1995) but the percent increase per day (Gp1 ) is similar In comparison with growth rates of other larval fish leptocephalus growth rates in increases in length and wet mass per day appear inordinately high; however the leptocephalus is using a unique mechanism for rapid growth Leptocephali accumulate wet mass and total length in the form of glycosaminoglycans, thereby rapidly increasing mass with minimal energy expenditure Leptocephalus growth in dry mass and percent protein, however, is 62


comparable to that of other larval fish Since the energy allocated to growth receives the lowest priority, the leptocephalus' unique developmental strategy circumvents the necessary tradeoff between energy required for metabolism and the need to rapidly increase in size to prevent predation. 63


CHAPTERS PROXIMATE COMPOSITION AND NUCLEIC ACID CONTENT Introduction The ultimate goal of each stanza in a fishes l i fe h i story is growth whether it is through increases in somatic tissue or in the form of reproduction (Weatherly and Gill 1992) In a larval fish maxim i zing growth is paramount to survival (Ware 1975b, Hunter 1981, Peterson and Wroblewski 1984, Blaxter 1986 Kiorboe et al. 1987, Post and Prankev i cius 1987) The unique growth strategy utilized by the leptocephalus larva of the elopomorph fishes is reflected in the proximate and nucleic acid composition of the larvae (Donnelly et al. 1995). Early studies on premetamorph i c leptocephali indicated high water and salt content (Callamand 1943, Hulet et al.1972, Hulet and Robins 1989) A thorough examination of the proximate composition and nucleic acid content of premetamorphic Ariosoma balearicum (Donnelly et al. 1995) revealed that growth in Phase I leptocephali occurred i n two subphases (Ia and lb). Phase Ia was characterized by cellular proliferation and preferential synthes i s of protein and carbohydrate relative to lipid. Growth was manifested more as increased 64


length than increased mass (Chapter 4). In Phase lb, nucleic acid content leveled off at an asymptotic maximum, lipid deposition increased and mass increased exponentially. The growth in mass was achieved through the accumulation of glycosaminoglycans stored in the gelatinous matrix of the larvae (Pfeiler and Luna 1984, Pfeiler 1988, Pfeiler et al. 1991 ). These energy stores are combusted by the larvae during the metamorphosis to the elver (Pfeiler and Luna 1984, Pfeiler 1996). The purpose of this study was twofold The main objective was to determine the chemical composition of three species of leptocephali, Paraconger caudilimbatus, Gymnothorax saxicola, and Ophichthus gomesii The second goal was to determine if the distinction between Phase 1 a and Phase 1 b observed by Donnelly et al. (1995) was evident in confamilial larvae and in leptocephali from morphologically different families. Materials and Methods Leptocephalus larvae were collected at the edge of the continental shelf in the eastern Gulf of Mexico on three cruises from 1995 to 1996. Sampling was conducted from approximately 26N to 28N between 84 W and 86W (Figure 2). The collection gear consisted of a 2 m plankton net with 505 urn mesh and a 9m2 mouth area Tucker trawl constructed of 6.8 mm mesh equipped with a quick-release blind cod end Nets were towed at 1 to 2 knots in a double oblique pattern from the surface to a depth of 100 m. Tow times varied from 10 to 60 65


minutes depending upon plankton density All sampling was conducted at night to maximize collections Immediately after each tow, premetamorphic leptocephali were measured to the nearest 0 1 mm total length, rinsed with deionized water blotted, and frozen in liquid nitrogen. Larvae were maintained at -80C until proximate composition, nucleic acid, and enzyme analyses were conducted. For proximate composition and nucleic acid analyses, individual specimens were weighed to the nearest 0 1 mg wet mass (WM) Larvae were introduced frozen into ice-cold distilled water and homogenized at 0 to 4 C using a sonifier, and by hand with conical glass homogenizers hav i ng ground glass contact surfaces (Kontes Glass Co., "Duall" models) Duplicate aliquots were dispensed in to preweighed crucibles and dried for 72 hours at 60C for dry mass and water content determinations Ash content was measured from dried homogenate aliquots combusted at 600C for 3 hours Homogenate aliquots were dispensed in duplicate or triplicate for the analysis of the proximate components, protein, lipid, carbohydrate, and nucleic acids Due to limited amounts of homogenate, particularly in very small larvae, the complete suite of analyses were not performed on every specimen. Protein was determined using the method of Lowry et al. (1951) with human albumin and globulin (Sigma, 540-10) as the standard Lipids were dried under a flow of nitrogen at 35C and total lipid was extracted according to the method of Reisenbichler and Bailey (1991 ); extracts were charred following 66


the method of Marsh and Weinstein (1966), with stearic acid (Kodak, 402) as the standard Carbohydrate was determined by the method of DuBois et al. (1956), with glucuronic acid (Sigma, G-4875) as the standard Nucleic acids were measured fluorometrically following procedures from Bentle et al (1981 ) ; calf thymus DNA (Sigma, D-3664) and bakers' yeast RNA (Sigma, Type XI, R-6750) were used as standards. Proximate composition was expressed in two ways: as content of a particular component measured in an individual (ug ind-1 ) and as a percent or concentration of the total mass, either wet mass (% WM) or ash-free dry mass (% AFDM). Changes in measured components were examined in relation to total length (TL), wet mass (WM), and ash-free dry mass (AFDM) Regressions were generated using the least-squares method with significance at p less than 0.05 Results Total length and wet mass were measured on premetamorphic larvae ranging from 29. 56 to 110 74 mm TL and 0 04 to 1 39 g WM. Wet mass increased with increasing length over the entire size range with the greatest variability in G. saxicola (Figure 21, Table 14) The regressions for AFDM on TL were similar to that for WM and are reported in Table 14. The relationships between water content and TL were similar to that between water and WM, with water (g) increasing multiplicatively with increasing 67


-0) ---en en co ..... Q) 1.5 1.2 0.9 0.6 0.3 P. caudilimbatus 0 29 49 69 G. saxico/a 1 0.8 0.6 0.4 0.2 0 33 43 53 0. gomesii 0.6 0.5 0.4 0.3 0.2 0.1 0 37 47 57 TL (mm) 89 109 63 73 67 77 87 Figure 19. Regressions of wet mass (Y, gWM) against total length (X, mmTL) Equations and r2 values are reported in Table 14 68 129 83 97


Table 14. Relat i onships of proximate and nucleic acid composition with total length (TL) wet mass (WM) dry mass (OM) and ash-free dry mass (AFDM) Spec i es abbreviations are : PC = Paraconger caudilimbatus GS = Gymnothorax saxicola and OG = Ophichthus gomesii n = number of larvae analysed ,-2 = coefficient of determination All equations are reported with a significance at p

Table 14 cont. Protein TL (mm) PC Y=3 327 4X1 8648 0.7757 60 c::ontent (mg) GS Y=2 5825x1 O ... X4 0818 0.4886 40 OG Y=5.232x1 0-3X3 1324 0.7248 27 WM(g) PC Y=18 9815x1 o ... xo.ns1 0 7757 49 GS Y=25 8213x1 ox'3982 0 7778 41 OG Y=o o221 x1.2667 0 8022 27 AFDM (mg) PC Y=153 .63x1 o..sX0 70 19 0 8892 59 GS Y=231 0444x1 05X0 8n17 0 8332 39 OG Y=409 0295x1 o 2x'0391 0 9386 25 Protein (%WM) WM(g) PC Y=2 0285X.o ms 0 1221 40 GS NS OG NS Lipid TL (mm) PC Y = 7 .1218x1 0-5X2 550 0 8759 60 content (mg) GS Y=2 1 o20x1 o..sx2.S539 0 3474 29 OG Y=0 0013X3 2865 0 .6799 17 WM(g) PC Y=9 9358X1 0467 0.912 60 GS Y=8 9558X1 0881 0.5946 30 OG Y= 7 5064X0 9531 0.6671 16 AFDM (g) PC Y=67.481 OX0 7 404 0 .6472 59 GS Y=57 9060X0 7295 0 5783 30 OG Y=38.9937X0 6534 0 6671 16 Carbohydrate TL (mm) PC Y=0 0473X1 9996 0 6868 60 content (mg) GS Y=3.1700x1 o7X3 1890 0 3331 36 OG Y=5.3096X1 o'2X5 53 5 0 8336 19 WM(g) PC Y=0.5078X0 7304 0 7046 60 GS Y=0 5604X1 0899 0 4990 37 OG Y=1. 6946X1 8914 0 8548 19 AFDM (g) PC Y=2 1553x1 0-3X0 5660 0.4802 59 GS Y=1. 9436X0 5934 0 .4133 35 OG Y=90 7846X1 4458 0 8781 17 Table 14 continued on next page. 70


Table 14 cont. Carbohydrate WM(g) PC NS (%WM) GS NS OG Y=0 1694X0 8914 0 5666 19 Carbohydrate AFDM(g) PC 0.502 19 (%AFDM) GS Y=0.944X0 4066 0.2485 35 OG Y=9 0784X2.2059 0.4065 17 DNA content (ug) AFDM (mg) PC Y=173.790X0 7 609 0 6673 32 GS Y= 70 1 054X0 4567 0 7727 24 OG Y=133.797X0 6717 0 9600 18 DNA cone WM(g) PC 0.3292 35 (ug) GS 0 5668 29 OG 0.4965 19 AFDM (mg) PC 0 2803 35 GS 0 8278 24 OG 0.8515 18 RNA TL (mm) PC Y=2 .91 x1 o-sX2 9347 0 7112 32 content (ug) GS Y=0 0003X2 3863 0 4559 25 OG Y=3.48x1 o-sx2 7450 0 9135 19 WM(g) PC Y=16 0828X0 8718 0 8545 32 GS Y=15.9199X0 8759 0 9363 28 OG Y=18.3348X0 9682 0 .9600 19 AFDM (mg) PC Y=285 6536X0 9282 0.8442 32 GS Y=63.44600X0 5463 0.8456 24 OG Y=144.2070X0 7478 0 9117 18 RNA cone (ug) WM (g) PC Y=1 0.097 0 2411 35 GS NS OG NS AFDM (mg) PC 0.2563 35 GS 0 7907 24 OG 0.5401 18 71


Tl and WM (Table 14) The water content of P caudilimbatus was more variable than that of G. saxicola and 0 gomesili. Percent water demonstrated no significant trends with respect to Tl or WM (Table 15) with mean percentages of water(% WM) ranging from 91. 75% ( 184%) i n 0. gomesii to 93.88% (.405%) in G saxicola, with P caudilimbatus at intermediate values of 92 23% ( 249%) Ash content (mg) increased multiplicatively w i th increasing larval length (Table 14). As a function of mass, ash content increased with increasing WM (Table 14), again with increased variab i lity in P caudilimbatus resu l ting in no significant regression Ash concentration (% OM) regressed on total length was not significant. Protein content (mg) increased with increasing larval total length (Table 14), WM (Figure 22) and AFDM (Table 14). Variability increased in G sa x icola and 0. gomesii larvae larger than 0 3 g WM. Protein concentration (% WM) regressed against increasing wet mass resulted in a significant re lationship with one species. Only P. caudilimbatus demonstrated a decrease in percent protein with increasing WM; G saxicola and 0 gomesii showed a general increase but the relationships were not significant (Figure 23). Mean protein concentrations (% WM) ranged from 1.67% ( 178%) in 0 gomesii to 15.61% (.497%) in G. saxicola with P. caudilimbatus having values at 2 84% (.113% )(Table 16) As concentrations of AFDM (% AFDM) mean protein values ranged from 34 95% 72


Table 15. Means of the concentrations of proximate composition by wet mass (%WM) and ash-free dry mass (%AFDM) for the three species SE =standard e r ror n =number of larvae analysed and N/A =not applicable. Spec i es Concentration n %WM +SE %AFDM +SE P. caudilimbatus Water 92.23 0 .249 NIA 50 Prote i n 2 .84 0 113 67.01 5.698 50 Lipid 1 .01 0 036 29 .14 3.602 36 Carbohydrate 0.07 0 003 2 .95 0.476 32 G saxicola Water 93.88 0.405 NIA 40 Protein 15 .62 1.498 42 .61 2 291 37 Lipid 0.873 0.059 23.56 2 .395 31 Carbohydrate 0 059 0.005 1 93 0.238 38 0 gomesi i Water 91.75 0 184 NIA 26 Protein 1 .672 0 179 34.95 1 747 28 Lipid 0 .814 0 037 17.44 0 .878 18 Carbohydrate 0 011 0 0909 0 3626 2.4112 16


en E -c::: a:; ....... 0 L-0... 25 20 15 10 5 P. caudilimbatus 0 0 0.3 0.6 0 9 1 2 1 5 1 8 15 12 9 6 3 0 2 4 20 16 12 8 G. saxico/a 0 0.2 0. gomesii 0.4 0 6 0 8 1 0 0 0.1 0.2 0.3 0.4 0.5 0.6 W e t M ass ( g ) Figure 22. Regression s of protein content ( Y mg protein) aga i nst wet mass (X, g WM) Equa tions and r2 values are reported in Table 14 74


-::2 3: 0 -c.) c 0 (..) c: "(]) _. 0 L. a.. P. caudilimbatus 5 1- 4 3 f- 2f- 1 1-0 f-0 0.2 G. saxicola 4 f- 3 f- 2 1 f- 0 f-0 0.2 0. gomesii 5 f4 13 2 11 f,. f-0 0 0. 1 0.2 ,. 0.4 0.6 ,. 0.4 0.6 - ... .. 0.3 0.4 Wet M ass (g) 0.8 1 0.8 1 0.5 0 .6 Figure 23. Regression and plots of protein concentration (Y, %WM) with wet mass (X, g WM) Equation and r value are reported in Table 14 75


(. 746%) (0. gomesil) to67. 011% (.698%) Paracongercaudilimbatus contained 42.61% (.291%) protein (Table 15) Lipid content (mg) increased multiplicatively with increasing TL (Table 14), WM (Figure 24) and AFDM (Table 14). However, there were no significant relationships between lipid concentration and WM or AFDM. Mean values of lipid concentrations as a function of WM ranged from 0 81% (.037%) in 0. gomesii to 1 00% ( 036%) in P caudilimbatus with G saxicola possessing intermediate values of 0 87% (. 059%) (Table 15) Mean lipid concentrations as a function of AFDM were 29. 14% ( 602%), 23 56% ( 394%) 17.44% (.878%) in P caudilimbatus, G saxicola and 0. gomesii, respectively (Table 15). Carbohydrate content (mg) increased multiplicatively with increasing TL (Table 14) WM (Figure 25), and AFDM (Table 14) with the greatest rate of increase observed in 0 gomesii (Table 14). As with protein the percent of carbohydrate was variable and was not related to WM or AFDM As a function of WM, mean carbohydrate concentrations ranged from 0 05% (.005%) in G saxicola and 0. gomesii to 0.08% (.003%) in P caudilimbatus; as a function of AFDM carbohydrate concentrations were 2.95% (.476%), 1.92% ( 238%) and 1 31% (.114%) for P. caudilimbatus, G. saxicola, and 0. gomesii, respectively (Table 15) RNA content (ug) increased with increasing TL in a multiplicative relationship for P caudilimbatus and 0. gomesii but showed no significant 76


-0) E -"'0 0.. :.::i P. caudilimbatus 15 12 9 6 3 0 8 6 4 2 0 5 4 3 2 1 0 0.3 G. saxico/a 0 0. 2 0. gomesii 0 0 0.1 0.6 0.9 0.4 0 .6 0.2 0.3 0.4 W e t M a ss (g) 1.2 1.5 0.8 1 0.5 0.6 Figure 24. Regressions of lipid content (Y mg lipid) against wet mass (X g WM) Equations and r2 values are reported in Table 14 77


...-... CJ) E .......... ....... c: Q) ....... c: 0 () Q) ....... ro L-'0 >. ..r:: 0 ..0 L-ro () 1 2 1.0 0.8 0 6 0.4 0.2 P. caudilimbatus 0 0 0.3 1.5 1.2 0.9 0.6 0.3 0 1 .0 0.8 0.6 0.4 G saxico/a 0 0.2 0 gomesii 0.6 0.9 1.2 1.5 0.4 0.6 0.8 1 0.2 0 0 0 1 0.2 0 3 0.4 0.5 0.6 Wet Mass (g) Figure 25. Regressions of carbohydrate content (Y, mg carbohydrate) against wet mass (X, g WM). Equations and r2 values are reported in Table 14. 78


relationship in G saxicola (Table 14) With WM and AFDM, however, RNA adhered to a strongly significant multiplicative relationship with increasing mass (Figure 26 Table 14). RNA concentration as a function of WM decreased in a multiplicative function for P caudilimbatus decreased but not significantly for G saxicola and was highly variable for 0. gomesii (Table 14) As a function of AFDM, RNA concentration was significantly correlated with increasing mass in all three species (Figure 27, Table 14). As with RNA, DNA content (ug) increased multiplicatively with increasing TL (Table 14) in two of the three species, with G saxicola showing no significant relationship between DNA and TL. DNA content regressed against WM (Figure 28, Table 14) and AFDM (Table 14) resulted in significant multiplicative relationships for all three species DNA concentration decreased multiplicatively with both WM (Table 14) and AFDM (Figure 29, Table 14) with the greatest decreases occurring in larvae less than 0 3g WM. The greatest decrease in DNA concentration occurred in P caudilimbatus (b = -0.4886). Discussion The unique developmental strategy utilized by leptocephali is reflected in the proximate and nucleic acid composition of the larvae. Protein, carbohydrate and lipid contents increased in the leptocephali with increasing total length and mass similar to other larval fish (Donnelly et al. 1995}, however, the accumulation of glycosaminoglycans stored in the larvae's gelatinous matrix 79



P. caudi/imbatus 1000 800 600 400 200 0 -0 0.02 0.04 ....--0 06 0.08 I 0 u.. <1:: 0> G. saxicola <1:: z 3000 0::: 0> 2500 ::3 2000 ........... c: 1500 0 ... +:o 1000 ro ..... -500 c: <1> (.) 0 c: 0 0 0.01 0.02 0.03 0.04 (.) <1:: z 0::: 0. gomesii 790 690 590 490 390 290 190 0 0.01 0.02 0.03 0 .04 Ash-free Dry Mass (g) Figure 27. Regressions of RNA concentrat i on (Y, ug RNA gAFDM.1 ) against ash-free dry mass (X g AFDM). Equations and r2 values are reported in Table 14 81


-<1: z 0 0> :::J .._ ....... c Q) ....... c 0 (,) <1: z 0 40 30 20 10 P. caudilimbatus 0 0 0.3 0.6 0.9 1 2 1.5 40 30 20 10 0 12 10 8 6 4 2 G. saxicola 0 0.3 0. gomesii 0 .6 0.9 1.2 1 5 1.8 0 0 0.1 0.2 0.3 0.4 0.5 0.6 W e t M ass (g) Figure 28. Regressions of the DNA content (Y, ugDNA) aga inst wet mass (X g WM). Equations and r-2 values are r eported i n Table 14 82


P. caudilimbatus 1600 120 0 800 400 = -0 "("'"" 0 0.02 I 0.04 0.06 0.08 0 LL <( 0) <( G. saxico/a z 0 600 0) :::s 500 -c 400 0 300 ro .._ ..... c 200 Q.) (.) 100 c 0 (.) 0 0 0.01 <( 0.0 2 0.03 0.04 z 0 0. gomesii 1110 910 710 510 310 0 0.01 0 02 0 03 0 04 Ash-free Dry M a ss ( g) Figure 29. Regressions of DNA concentrat i on (Y, ug DNA g AFDM-1 ) against ash-free dry mass (X, g WM) Equations and r2 values are reported in Table 14 83


(Pfeiler et al. 1991, Pfeiler 1996), resulted in concentrations of the proximate components that were very different from larvae utilizing a Type I developmental strategy Comparing the data from this study with the only available information on the proximate and nucleic acid composition of premetamorphic leptocephali, protein carbohydrate and lipid were present in quantities similar to the observations made by Donnelly et al. (1995) for A. balearicum larvae. However concentrations of the components showed slightly different trends Protein concentrations were higher than those reported by Donnelly et al. (1995) (1.4 to 4 1 %WM and 29 to 58% AFDM) but lower than Hulet (1979) (6 3% WM) obtained for three unidentified larvae Hulet's (1979) prote i n values were overestimated because they were calculated from total N values without taking into account the relatively high levels of non-protein N in leptocephali. Donnelly et al. (1995) observed that the protein concentrations as a function of WM decreased with increasing mass up to 0.4 g WM This relationship was only observed in P caudilimbatus which showed a slight decrease up to 0.2 g WM but leveled-off for larvae greater than 0 3 g WM As with protein concentrations, the relationships observed by Donnelly et al. (1995) regarding lipid concentrations and WM and AFDM were not observed in this study Lipid and carbohydrate concentrations remained variable with increasing mass. Donnelly et al. (1995) observed an asymptote with relation to RNA and DNA 84

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content and increasing mass An asymptotic relationship was not observed in the three species examined in this study The division of Phase I into two subphases proposed by Donnelly et al. (1995) was not apparent in the protein, carbohydrate and lipid concentrations of the three species examined in this study The subphases, however, were evident in the nucleic acid concentrations Donnelly et al. (1995) based the division of the subphases in A. balearicum based on the shift from an increase in length via cellular proliferation to an increase in acellular mass occurring at approximately 0 5 g WM (90 mmTL) A. balearicum is one of the largest leptocephali found in the Gulf of Mexico, reaching a maximum size of 260 mm (more than 5 g WM) before metamorphosis As a percentage of the larva's maximum size, the transition between Phase Ia and lb occurred at 9.6% of the total premetamorphic wet mass The three species of larvae examined here reach a substantially smaller maximum size; P. caudilimbatus metamorphoses at 120 mm, G. saxicola at 90 mm and 0 gomesii at 110 mm ( cf. Bohlke 1989). In P. caudilimbatus the predicted transition from Phase Ia to Phase lb based on percent of the total wet mass would occur at 0 13 g WM, coinciding with the decrease in protein concentration observed in Figure 21. Only three data points are present for G saxico/a larvae less than 0 09 g WM and two for 0 gomesii less than 0 07 g WM; the mass values corresponding to 9 .6% maximum size The trends of decreasing concentrations observed by Donnelly et al. (1995) are 85

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not evident here most likely as a result of a paucity of small larvae with respect to the species specific maximum premetamorphic size. Interpretation of the data reveals that the proximate composition and nucleic acid content relationships with mass and total length observed by Donnelly et al. (1995) for A. balearicum are consistent for different species of leptocephali. Although these data cannot confirm the division of Phase I development into two subphases based upon the proximate composition analysis the abrupt change in enzyme activities supports the existence of two subphases. 86

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CHAPTERS INGESTION: ASSEMBLY OF THE ENERGETICS EQUATION Introduction The principles of energetics are potentially useful tools for describing the ecological physiology of an organism The approach has been used frequently for juvenile and adult fishes to examine how populations grow and reproduce in relation to available food supplies ( eg Kith ell et al. 1977, Hunter and Leong 1981 Mills and Forney 1981, Peters and Schaaf 1981 ) However at present there are few energet i cs measurements in larval fish The unusual developmental strategy of the leptocephalus larvae of the elopomorph fishes maximizes growth in mass by accumulat i ng non-metabolizing t i ssue in the form of proteoglycans. As demonstrated in earlier chapters, this developmental strategy is unique and results in a very low wet mass-specific metabolism while supporting rapid growth in wet mass However the same parameters measured in relation to dry mass are very similar to those in other larval fishes. It is highly likely that the percent allocation of the components of the energetics equation will also reflect the d i fferent developmental strategy implemented by the leptocephali. 87

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The energy budget expressed in total energy ingested (Brett and Groves 1979) is the summation of the energy required for metabolism, excretion, and growth Examination of energetics provides insight into the allocation of energy in the developing leptocephalus and quantifies the nutritional requirements of the larvae The proposed sources of nutrition in leptocephali, dissolved organic carbon, or particulate organic carbon in the form of discarded Appendicularian feeding webs or "houses" and attached zooplankton fecal pellets, designates leptocephali as unique trophic specialists (Azam and Ammerman 1984). Whether leptocephali use mainly DOM or POM for nutrition, they occupy a niche infrequently utilized by teleosts Leptocephalus energetics will provide insight into the feasibility of each of the proposed food sources as well as an estimate of the total energy consumed per larva The objective of this portion of the study was to provide a picture of the total nutritional and energetic requirements of leptocephali as well as how energy was allocated in the leptocephalus' developmental strategy. Materials and Methods Because a complete suite of energetics measurements were not available for every larva, regressions of wet mass-specific metabolism, excretion and daily growth rates were applied to the concatenated data set to obtain predicted values for each component for each species. The regressions for wet mass specific metabolic rates from Chapter 2 were used to determine daily metabolic 88

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requirements. The oxygen uptake measurements were considered to be routine rates; activity was monitored but not controlled (Brett and Groves 1979). Predicted mass-specific oxygen consumption (ul02 g wM-1 hr1 ) was converted to calories using an oxycalorific equivalent of 0 00463 calories ul 02 1 (Brett and Groves 1979). Nonfecal excretion values (NH3 ) (Chapter 3) in the form of wet mass specific excretion rates (umol NH3 g WM-1 hr-1 ) were converted to calories using a value of 0.00489 cal ug NH3 1 (Elliot and Davidson 1975) Fecal energy was not reported due to the extremely small amounts for larval fish Larval mass was converted to calories using the proximate composition (ug g DM-1 ) of P. caudilimbatus, G saxicola, and 0. gomesii (Chapter 5) and A. balearicum (Donnelly et al.1995) and the caloric equivalents in Brett and Groves (1979) for protein (0 0048 cal ug-1), lipid (0.0095 cal ug-1), and carbohydrate (0 0041 cal ug-1 ) Mass-specific caloric values (cal g wM-1 ) and daily larval growth rates (g WM day-1), determined from the multiplicative growth models (Chapter 4) for each of the species were combined to yield daily growth in calories (cal g wM-1 day-1 ) All caloric values were subsequently converted to joules (J) by multiplying by 4 1868 J cal-1 (Pennycuick 1988). The energy values allocated to each of the components of the energetics equation were reported as ranges and did not include standard error estimates since the values are predicted from equations obtained from each segment of this dissertation 89

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Results The total individual energy required per day (J ind1 day-1 ) increased in a multiplicative relationship with increasing wet mass (g WM) (Figure 30 Table 16) The total energetic requirement per day for the four larvae ranged from 15.70 ( 892) J ind1 day 1 for G saxicola, to 198.49 (. 557) J ind1 day1 for A. balearicum, the largest larva of the four species examined. As with mass specific respiration and excretion the total energy required per gram of mass (J g WM-1 day -1 ) decreased precipitous l y w i th increasing mass (Figure 31, Table 17) with the smallest larvae less than 0 2 g WM, requir in g the most energy, followed by a leveling off for larvae larger than 0 3 g WM All four species allocated the greatest amount of energy to metabolism The energy needed for metabolism ranged from 11.11 J ind1 day 1 in G saxicola to 125 57 J ind1 day1 in A. balearicum (Table 17) The percent of energy necessary to maintain the routine metabolic rate ranged from a minimum of 60. 21% observed in G saxicola to a maximum of 91.49% for P caudilimbatus and decreased significantly with increasing mass in all but P caudilimbatus (Table 17 Figure 32) Growth was the second largest energy expend i ture; 1 56 J ind1 day 1 for 0 gomesii to 73 97 J ind1 day 1 for A. balearicum (Table 17) The energy allotted to growth increased multiplicatively with the greatest rate of increase occurring in larvae smaller than 0 2 g WM (Figure 33). Percentages of the total 90

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--, C/) Q) ::J 0 --, C'O 52 48 44 80 60 P. caudilimba t us 0.3 0.6 0.9 1 2 1 5 G. saxico/a 0 0.2 0.4 0.6 0.8 1 A. ba/earicum 40 0 1 2 3 4 5 0. gomesii 53 5 0 4 7 44 41 ... .. 0 0.1 0.2 0.3 0.4 0.5 0. 6 W e t M ass ( g ) Figure 30. Regress i o n s of the to t al j oules req ui red by eac h species per day ( Y J day-1 ) agai n s t larval wet m ass ( X g WM). Eq u at i o n s and r val ues are repo rt ed i n Table 1 6 91

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Table 16. Regress i ons of the total energy in Joules required per day (Y J day -1 ) on wet mass (g WM) for each species General formula: Joules day 1 = a(g WM)b Standard errors and coefficients of determ i nation are not reported since the equat i ons result from pred i cted values Sp ec i es M eta bol i sm Growth Excreti o n Total P caudilimbat us Y= 40.4452WM0 1720 Y= 6 1138WM0 3022 Y= 0 .3678WM1 092 8 Y=46 2726WM0 1685 A. ba/earicum Y= 67 0936WM0 268 Y= 2 3 7129WM0 68n Y = 0 1244WM"1 0309 Y = 91.9752WM0.a99 G saxicola Y= 48.6888WM0 4 7 05 Y= 26.9955WM0 9532 Y= 0.1325WM1 1 3 19 Y = 72.6410WM0 .5-499 0 gome sii Y= 31. 8023WM0 0503 Y = 21. 3955WM0 8929 Y= 0 .2579WM1 752 Y= 45 3568WM0 0556

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P. caudi/imbatus A. balearicum 800 600 400 2 00 0 0 0.3 0.6 0 9 1.2 1 5 0 1 2 3 4 5 G. saxicola :::::J 0. gomesii 4 00 8 0 0 3 00 6 00 2 0 0 4 00 1 00 200 0 0 0 0 .2 0 .4 0 6 0 8 1 0 0. 1 0. 2 0 .3 0 .4 0 .5 0 6 W e t M a ss ( g ) Figure 31. Regressions of the mass-specific joules e xpended per day (Y, J g wM-1 day-1 ) against wet mass ( X g WM) Equations are reported in Table 16. 93

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Table 17. Ranges of the joules (J indiv-1 day-1 ) and percent of total energy(%) allocated to metabolism growth and excret i on for the four species. Species Total (J) Percent(%) Min Max Min Max P. caudilimbatus Metabolism 23 2663 42 8914 80.8969 91.4993 Growth 2 2960 7 6397 7.1934 16.5773 Excretion 0 0880 3 1981 0 1756 11. 1198 Total 28 7604 50. 1507 NIA A. balearicum Metabolism 30.7224 125. 5718 62 7170 82 7254 Growth 7.1694 73 9759 17 0048 37 2689 Excretion 0 0274 0 8 2 05 1.40770 21.1955 Total 38 7123 198.49 22 NIA G. sa xic ola Metabolism 11. 1069 91. 2124 60 2073 91. 2124 Growth 1.4255 21. 8088 8 3490 39 1864 Excretion 0 1522 3 17066 0 2630 20. 1914 Total 15 7031 62 6377 NIA 0. gomesii Metabolism 32 8646 36 7746 65 3774 90 5921 Growth 1 5580 17 2241 3 8544 34 0849 Excretion 0 2440 2.4493 0 5376 6 0023 Total 38 9914 50. 5327 NIA

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P. caudilimbatus A. ba/earicum 9 2 ., 86 90 8 2 88 ... 7 8 86 r' 74 84 7 0 8 2 66 80 6 2 0 0.3 0.6 0 9 1 2 1 5 0 1 2 3 4 5 G. saxico/a 0. gomesii 9 5 90 90 8 5 y 80 80 7 0 75 .... 7 0 60 6 5 0 0.2 0.4 0 6 0 8 1 0 0.1 0.2 0.3 0.4 0.5 0.6 W e t M ass ( g ) Figure 32. Regressions and plot of the percent of the total energy per day that is allocated to metabo li sm ( Y % day 1 ) against wet mass (X gW M ) 95

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P. c audilim ba tus A ba /ear i cum 17 4 1 1 5 37 13 .. 33 .... 2 9 1 1 25 9 21 7 17 ....-. 0 0.3 0.6 0.9 1 2 1 5 0 1 2 3 4 5 ..--I >. ro "'0 0 ..._, ..c 0 I... G s axicola <.9 0. go m e sii 40 40 .... 3 0 3 0 2 0 2 0 1 0 1 0 0 0 0 0.2 0.4 0.6 0.8 1 0 0.1 0.2 0.3 0.4 0. 5 0.6 W e t M ass ( g ) Figure 33. Regressions of th e p e rce n t of t he total energy r equ i red per day that is a l lo c ated to growth { Y % day-1 ) aga in s t w et m ass ( X g WM) 9 6

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energy per day devoted to growth during the premetamorphic period ranged from 3.85% (0. gomesit) to 39 18% (G sa x icola) (Table 17) The smallest f r action of the energy budget at all sizes in all larvae was the energy lost to excretion ranging from 0 03 J ind -1 day1 (A balearicum) to 3.19 J ind -1 day 1 (P. caudilimbatus) (Table 17) The percent of the total energy lost to exc r etion ranged from a low of 0 55 % in 0 gomesii to 21. 19% in A balearicum (Table 17) with pronounced decreases occurring in larvae l ess than 0 3 g WM for P caudilimbatus G sa x icola, and 0 gomesii and in larvae less than 0 5 g WM for A balearicum (F i gure 34). Discussion Leptocephali use a unique growth strategy that allows them to i ncrease rapidly in size while devoting the majority of their energy not to growth as in most larval fish (Houde and Schekter 1983 Yamashita and Bailey 1989 Keckeis and Schiemer 1992) but to metabolism. The larvae have evolved a means of inexpensively increasing their mass while devoting a l arge portion of their energy to metabolism The incorporation of proteoglycans provides a mechanism for rapid growth in wet mass and serves as a gelatinous skeleton (Hulet 1978 Hascall and Hascall 1981 ). This material prov i des st r ucture for the muscles to work aga i nst in place of an ossified skeleton conserving energy that would potentially be expended in oss i fication It allows for a well developed angu i llifrom locomotory ability and a very large l arval size with no bony skeleton 97

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c 0 12 10 8 6 2 P. caudilimbatus 24 20 16 12 8 4 0 0.3 0.6 0.9 1.2 1 5 G. saxico/a A. balearicum 2.4 0.8 0.4 0 8 6 4 2 0 1 2 3 4 5 0. gomesii 0 0 0 0.2 0.4 0 6 0.8 1 0 0 1 0.2 0 3 0.4 0.5 0.6 Wet Mass (g) Figure 34. Regressions of the predicted percent of the total energy required per day that is allocated to excretion (Y, % day-1 ) against wet mass (X, g WM). 98

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A common thread through all of the components of the energetics analysis is the transition from Phase Ia to Phase lb occurring at approximately 1 0% of the maximum premetamorphic mass. In three of the species, the transition occurred at 0 2 g WM. The transition occurred at O.Sg WM in A. balearicum. Mass-specific energy for metabolism and excretion decreased precipitously in Phase Ia Energy allocated to growth increased at elevated rates until Phase lb and subsequently increased at reduced rates The transition from Phase Ia to Phase lb was the point at which the larvae shifted developmental strategies from growth by cellular proliferation to increases in mass achieved through proteoglycan accumulation (Donnelly et al. 1995). The percentage of the total energy allocated to growth in the four species of leptocephali ( 4 to 40%) agreed favorably with reported values (Brett and Groves 1979, Houde and Schekter 1983, Brightman 1993) for other larval fish In three larval teleosts, Anchoa mitchelli, Archirus lineatus and Archosargus rhomboidalis, percentages devoted to growth ranged from 14 to 41 % at comparable temperatures (Houde and Schekter 1983) The amount of energy reportedly lost to excretion varies among studies It is generally believed to range from 7 to 30% of the total ingested energy (Brett and Groves 1979). The loss to excretion decreases with ontogeny as assimilation efficiencies increase (Klumpp and von Westernhagen 1986) Houde and Schekter (1983) report a large portion of the ingested energy was excreted by larval fish, 32 to 83%. However, Torres et al. (1996) found that the 99

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energy excreted by red drum larvae ranged from 0 5 to 21%, similar to the allocation for leptocephali. In leptocephali, metabolism received 60 to 92% of the total ingested energy, higher than reported values for other larval fish at similar temperatures (Houde and Schekter 1983, Houde 1989, Torres et al. 1996). The high percentages allocated to metabolism may be explained by their mode of nutrition Transport of OOM across the integument, either as the main food source or supplementally, is energetically expensive (Withers 1992) The amount of POM required to power the leptocephalus whether in the form of POM or more specifically as larvacean houses would require substantial energy expenditure for collection and consumption Locomotion is energetically expensive and can account for up to 30% of a fish's total energy expenditure (Ware 1975a). Either mode of nutrition would theoretically result in increased metabolic rates Excluding metabolism, the ranges the percentages of energy allocated to each of the components of energetics in leptocephali are comparable to other larval fish, but total energetic requirements of the four leptocephalus species examined are very different from those of larval fish using the Type 1 developmental strategy Brightman (1993) determined the energetic requirements of growing red drum larvae ranged from 0.0557 to 0 0589 calories per day for day 14 larvae (0 1 09 mg OM). Standardizing the energy required per day for red drum larvae and P caudilimbatus to dry masses of 0 05 g OM, the 100

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red drum would require 96.03 J ( 94 cal) per day compared to 42.48 J ( 0 15 cal) per day, more than double the energy required by the leptocephalus Westerberg (1990) proposed that due to the transparency large size, and peculiar dental arrangement of leptocephali, they must consume equally transparent, soft food items In particular, the discarded houses of the Appendicularia, or larvaceans, were an excellent candidate for leptocephalus prey Moshioka and lwamizu (1996) have largely substantiated Westerberg's deductions They examined the gut contents of leptocephali from eight eel species representing five families. The gut contents primarily consisted of larvacean houses and attached zooplankton fecal pellets Appendicularians are important in the macrozooplankton of the oligotrophic central parts of the subtropical convergence, locations of suspected eel spawning. These pelagic tunicates filter particles through a "house", a unique feeding structure secreted around the animal by glandular epithelium on the body. When the external filters become clogged with phytoplankton or particulate matter, the house is discarded but remains in the water column as an organic aggregate. Larvaceans have been observed to construct and discard up to six houses per day, one every four to six hours (Lohmann 1909 in Alldredge 1976b). Houses, macroscopic mucus aggregates ranging from a few millimeters to 2m in diameter, are h i ghly concentrated energy sources for grazers (Alldredge 1976a). The houses constructed by Oikop/eura rufescens a larvacean species 101

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that produces a house slightly larger than those observed by Mochioka and lwamizu (1996}, combined with attached zooplankton fecal pellets, contain 5.4.3 ug C per discarded house (Alldredge 1976) A 0 5 g WM (80 mm TL) P. caudilimbatus, requiring 41.17 J day-1 (9.833 cal day-1), would need to consume 181.32 larvacean houses and attached particulate matter, sweeping clear more than 1m3 per day (Alldredge 1976) Densities of houses from Oikopleura in the Florida current reach 1,130 m3 (Alldredge 1976a) and could theoretically support the leptocephali. Otake eta/. (1993) examined the midgut mucosal cells of the leptocephali of Conger myriaster and discovered detrital aggregates less than 20 um in diameter and zooplankton fecal pellets. Suspended particulate organic material in the upper layers of the sea, consists mainly of detritus and phytoplankton. The concentration of particulate organic matter in the euphotic zone is usually considerably higher than in the underlying water. Particulate organic carbon occurs in concentrations varying between 0 02x 1 0 -3 to 0 2 x 10-3 g C liter-1 and is an extremely important part of the marine food chain, providing food for organisms at several trophic levels (Parsons 1975) Stable isotope ratios have proven useful in examining trophic position in marine food webs In particular, the heavy isotope 15N is enriched by 3 to 4% per trophic level (DeNiro and Epstein 1981, Wada eta/. 1987). Otake eta/. (1993) examined the stable nitrogen isotopic composition of C myriaster leptocephali, and found it to be at the lowest level, equal to that of POC, 102

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suggesting that particulate organic matter is a possible food source for eel larvae Using Parsons (1975) carbon values for POM, a 0 5 g WM P. caudilimbatus would need to consume all the particulate organic material in 10 92 I seawater per day assuming an unlikely assimilation efficiency of 100% Marine dissolved organic material represents one of the largest act i ve reservoirs of organic carbon in the biosphere (Hedges 1992) DOM contains substances representative of the main biochemical classes : amino acids, carbohydrates, lipids and vitamins DOM in marine ecosystems represents a very large pool of potential energy for organisms adapted to the uptake of these compounds from dilute solution (Manahan and Crisp 1982), leading to the theory that certain aquatic animals acquire nutrients through direct absorption of DOC via the integument. The amount of DOM usually exceeds the particulate organic fraction by a factor of 1 0 to 20. Dissolved organic material concentrations in the western Gulf of Mexico at a bottom depth of 1550 m and sampling depth 2 m is approximately 83 uM (Guo et al. 1994). Using the carbon values of Guo et al. (1994) and the same 0.5 g WM P caudilimbatus would need to absorb all of the DOMin 1 .02 I of water to obtain the energy it requires each day The Type 2 developmental strategy used by leptocephali allows the larvae to avoid the risks involved in the Type 1 strategy The accumulation of glycosaminoglycans serves a threefold purpose in leptocephali. The larvae grow rapidly with little energy expenditure. The large mucopolysaccharides are resistant to compressive forces thereby providing structural support to the larvae 103

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in the absence of ossification. At the same time, the leptocephali avoid the increased energy demands associated with increased mass by increasing in non-metabolizing tissues The final benefit of the Type 2 developmental strategy is the establishment of an energy depot that fuels the larvae through the complex metamorphic period The developmental strategy of leptocephalus larvae takes advantage of the benefits of increases in mass while circumventing the potential drawbacks associated with greater size. 104

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LITERATURE CITED Alberts, B D Bray, J. Lewis, M. Raff, K. Roberts and J. Watson 1994 Molecular biology of the cell. Garland Publishing Inc New York : 864 pp Alldredge A 1976a Discarded appendicularian houses as sources of food, surface habitats and particulate organic matter in planktonic environments Limn and Ocean 21(1) : 14 23. Alldredge, A 1976b Field behavior and adaptive strategies of Appendicularians (Chordata : Tunicata). Mar. Bioi. 38:29-39 Alldredge A 1972 Abandoned larvacean houses: A unique food source in the pelagic env i ronment. Science 177 : 885-887 Ashley L.M 1975. Nutritional pathology In : Fish Nutrition J .E. Halver (ed ) New York Academic Press pp 439-537. Azam, F and J W Ammerman 1984 Cycling of organic matter by bacterioplankton i n pelagic marine ecosystems : Microenvironmental considerations In: Flows of energy and material in marine ecosystems Fasham (ed ) pp 345-358 Azam F T Fenchel J .G. Field J S Gray, L.AMyer-Reil and F. Thingstad 1983 The ecological role of water-column m i crobes in the sea Mar. Ecol. Prog Ser. 1 0 : 257-263 Balbontin, F .S., S DeSilva and K.F Ehrlich. 1973. A comparative study of anatomical and chemical characteristics of reared and wild herring. Aquaculture 2 : 217-240 Bamstedt U 1980 ETS activity as an estimator of respiratory rate of zooplankton populations The significance of variations in environmental factors. J. Exp Mar. B ioi. Ecol. 42 : 267-283 1 05

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Beamish R.J and McFarlane. 1983. The forgotten requirement for age validation in fisheries biology. Trans. Am Fish. Soc 112 : 735-7 43. Bentle L.A. S Dutta, and J Metcoff 1981 The sequential enzymatic determination of DNA and RNA Anal. Biochem 116 : 5-16 Blaxter, J H.S 1986 Development of sense organs and behaviour of teleost larvae with special reference to feeding and predator avoidance Trans Am. Fish. Soc 115:98-114 Boetius, J and E.J Harding 1985. A reexamination of Johannes Schmidt's Atlantic eel investigations. Dana 4 : 129-162 Bohlke E. 1989. Fishes of the Western North Atlantic: Orders Anguilliformes and Saccopharyngiformes Part 9 Vol.1 Sears Foundation of Marine Research, New Haven : 654 pp. Bohlke, E.B J .E. McCosker and J .E. Bohlke 1989 Family Muraenidae. pp 104-206 In: E.B Bohlke (ed ) Fishes of the Western North Atlantic Part 9 Vol.1, Sears Foundation of Marine Research New Haven. Brett, J.R. and T.D.D. Groves. 1979. Physiological energetics. In: W.S Hoar and D.J Randall (eds.) Fish Physiology Academic Press, London-New York : 279-352 Brightman R.I. J.J. Torres J Donnelly, and M .E. Clarke 1997 Energetics of larval red drum Sciaenops ocellatus. Part II: growth and biochemical indicators Fish. Bull. 95:431-444. Brightman R. I. Energetics and RNA/DNA of red drum larvae Sciaenops ocellatus Ph.D. diss Univ South Florida : 178pp Buckley, L. 1984 RNA-DNA ration: an index of larval fish growth in the sea. Mar. Bio. 80: 291-298 Buckley L.J. 1979 Relationships between RNA-DNA ratio prey density, and growth rate in Atlantic cod (Gadus morhua) larvae J Fish Res Board can. 36 : 1497-1502 Buckley, L.J and D W. Dillman 1982 Nitrogen utilization by larval summer flounder Paralichthys dentatus (Linneaus) J exp mar. Bioi. Ecol. 59 : 243-256 106

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Otake, T., K. Nogami, K. Maruyama 1993. Dissolved and particulate organic matter as possible food sources for eel leptocephali. Mar Ecol. Prog. Ser 92:27-34 Otake, T., J. Hirakawa, H Fujimoto, and K. lmaizumi. 1995. Fine structure and function of the gut epithelium of pike eel larvae J. Fish. Bioi. 47: 126-142 Padron, D V A Lindley and E. Pfeiler. 1996 Changes in lipid comosition during metamorphosis of bonefish (Aibu/a sp ) leptocephali. Lipids 31 (5):513519 Pandian, T. J. and E. Vivekanandan. 1985 Energetics of feeding and digestion pp 99-124 In P. Tytler and P. Calow (eds ) Fish energetics : New perspectives. The Johns Hopkins University Press, Baltimore, Maryland. Parsons, T R. 1975. Particulate organic carbon in the sea pp 2 : 365-383/n J P Riley and G. Skirrow (eds ) Chemical oceanography .. Academic Press, London, New York Pennycuik, C J 1988. Conversion factors : Sl units and many others Univ Chicago Press, Chicago and London : pp.36 Perry, S F and P .J. Walsh 1989 Metabolism of isolated fish gill cells contribution of epithelial chloride cells J. Exp Bioi. 144:507 520 Peterson I. and J.S Wroblewski, 1984. Mortality of fishes in the pelagic ecosystem. Can J Fish Aquat. Sci. 41: 1117-1120 Peters, D.S. and W.E. Schaaf 1981 Food requirements and sources for juvenile Atlantic menhaden. Trans Am Fish. Soc. 110:317-324 Pfeiler, E 1981. Salinity tolerance of leptocephalous larvae and juveniles of the bonefish (Aibulidae: Albula) from the Gulf of California. J. Exp. Mar. Bioi. Ecol. 52 : 37-45 Pfeiler, E. 1984 Glycosaminoglycan breakdown during metamorphosis of larval bonefish A/bu/a Mar Bioi. Let. 5:241-249. Pfeiler, E. 1984b Changes in water and salt content during metamorphosis of larval bonefish (Aibu/a) leptocephali. J. Exp. Mar. Bioi. Ecol. 82 : 183-190 Pfeiler E. 1984c. Effect of salinity on water and salt balance in metamorphosing bonefish A/bula. Mar. Bioi. Lett. 5 : 241-249. 113

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Pfeiler E. 1986. Towards an explanation of the developmental strategy in leptocephalous larvae of marine teleost fishes Env. Bio of Fishes. 15 : 3-13 Pfeiler, E., J. Donnelly J.J Torres and R. Crabtree 1991. Glycosaminoglycan composition of tarpon (Megalops at/anticus) and lady fish (Eiops saurus) leptocephali. J. Fish Bio 39:613-615. Pfeiler, E. 1996 Energet i cs of metamorphosis in bonefish (Aibula sp. ) leptocephali: Role of keratan sulfate glycosaminoglycan Fish Physiol. and Biochem. 15( 4 ):359-362. Pfeiler E. and A. Luna 1984 Changes in biochemical composition and energy utilization during metamorphosis of leptocephaluous larvae of the bonefish (Aibula). Env Bioi. Fish 10 : 243 -251. Pfeiler E. and J.J Govoni. 1993. Metabolic rates in ea r ly life history stages of Elopomorph fishes Bioi. Bull. 185 : 277 283 Philpot, C W. 1980 Tubular system membranes of teleost chloride cells : osmotic response and transport sites. Am. J. Physiol. 238: R 171-R 184. Post J R. and A. B. Prankevicius 1987 Size selective mortality i n young-of-the year yellow perch (Perea flavescens) : evidense from otolith microstructure Can J Fish Aquat. Sci. 44 : 1840 1847 Power J. H and P J. Walsh 1992 Metabolic scal i ng, buoyancy, and growth in larval menhaden Brevoortia tyrannus. Mar Bioi. 112 : 17-22 Preston, R.L. and B .R. Stephens. 1982 Kinetic and thermodynamic aspects of sodium-coupled amino acid transport by marine invertebrates Am Zool. 22: 709-721 Prosser, C L. 1973. Comparative Animal Physiology. W. B Saunders Co. Philadelph i a : pp 672 POtter, A. 1909 Die Erna h rung der Wassertiere und der Stoffhaushalt der Gewasser. Jena : Gustav Fisher Rasquin P 1955 Observations on the metamorphosis of the bonefish Albu/a vulpes (Linnaeus). J Morphol. 97 : 77-117 Reisenbichler, K.R. and TG Bailey 1991 Microextraction of total lipid from mesopelagic animals. Deep-Sea Res 38 : 1331-1339. 114

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Robins, C .R. 1989 The phylogenetic relationships of the anguilliform fishes. pp 9-29 In Bohlke, E. B (ed.) Fishes of the Western North Atlantic. Vo 9(1 ) Allen Press, Lawrence, Schalk, P H 1988 Respiratory electron transport system (ETS) activities in zooplankton and micronekton of the Indo-Pacific reg i on Mar Ecol. Prog Ser. 44 : 25-35 Schmidt J 1925 The breeding place of the eel. Annu Rep Smithson lnst. 1924 : 279-316 Schmidt, J 1922. The breeding places of the eel. Phi los Trans. R. Soc. London Ser. B 211:178-208 Schmidt-Nielson, K. 1990 Animal Physiology : Adaptation and environment. Cambridge University Press Cambridge: pp 543 Secor, D.H. J M Dean and E.H Laban 1992 Otolith removal and preparation for microstructural examination. pp 19-57. In: D K.Stevenson and S.E Campana (eds.), Otolith microstructure examination and analysis Can Spec Publ. Fish Aquat. Sci. 117. Sherr, E. B. and B. F. Sherr. 1988. Role of microbes in pelagic food webs : a revised concept. Limnol. Oceanogr 33: 1225-1227 Siebers, D & H Rosenthal. 1977. Ami no-acid absorption by developing herring eggs. Helgol. Wiss. Meeresunters 29: 464-472. Smith D G 1989a. Family Congridae pp 460-567. In: E B Bohlke (ed ) Fishes of the Western North Atlantic part 9 Vol. 1 Sears Foundation for Marine Research, New Haven Smith, D G 1989b Family Congridae : leptocephali. pp 723-763 In : E. B. Bohlke (ed ) Fishes of the Western North Atlantic part 9, Vol. 2, Sears Foundation for Marine Resea rch, New Haven Smith, D .G. 1989c Family Muraenidae : leptocephali pp. 900-916 In: E. B Bohlke (ed.) Fishes of the Western North Atlantic, part 9 Vol. 2, Sears Foundation for Marine Research New Haven Smith, G D 1989d. Introduction to leptocephali. pp 657-668 In: E B Bohlke (ed ) Fishes of the Western North Atlantic, part 9 val. 2 Sears Foundation for Marine Research New Haven. 115

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Smith, G.D 1989e Order Elopiformes pp 961-972 In: E.B.Bohlke (ed.) Fishes of the Western North Atlantic, part 9, Vol. 2 Sears Foundation for Marine Research, New Haven. Sakal, R.R. and F. J. Rolf. 1981 Biometry : The principles and practise of statistics in biological research. W H. Freeman and Co San Francisco : pp 859 Solorzano, L. 1969. Determination of ammonia in natural waters by the phenol hypochlorite method Lim no I. Oceanogr 14: 799-801 Spurr, A.R. 1969 A lowviscosity epoxy resin embedding medium for electron microscopy. J Ultrastructure Res 26:31-43 Stephens G 1988 Epidermal amino acid transport in marine invertebrates Biochimica et Biophysica Acta 947 : 113-138 Stephens, G 1968 Dissolved organic matter as a potential source of nutrition for marine organisms Am. Zool. 8 : Stewart, M 1979 Absorption of dissolved organic nutrients by marine invertebrates Oceanogr Mar Bioi. Ann. Rev 17 : 163-192 Tabeta 0., K. Tanaka, J. Yamada, and W. Tzeng 1987 Aspects of the early life history of the Japanese eel Anguilla japonica determined from otolith microstructure Nippon Suisan Gakkaishi. 53(1) : 1727-1734 Tanaka, K., 0 Tabeta, N Mochioka J Yamada, and S Kakuda 1987. Otolith microstructure and ecology of the Conger eel (Conger myriaster) Larvae collected in the Seta Inland Sea, Japan Nippon Suisan Gakkaishi. 53(4):543549. Thayer, G W 1970 Comparison of two storage methods for the analysis of nitrogen and phosphorus fractions in estuarine water Chesapeake Sci.11: 155-158 Thorrold S. R., J M Shenker, E.D Maddox R. Mojica and E. Wishinski. 1994 Larval supply of shorefishes to nursery habitats around Lee Stocking Island, bahamas II. Lunar and oceanographic influences Mar Bioi. 118:567-578 Torres, J.J and G .N. Somera 1988 Metabolism, enzymatic activities and cold adaptation in Antarctic mesopelagic fishes. Mar. Bioi. 98: 169-180 116

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Torres J J., R.I. Brightman J Donnelly and J Harvey 1996 Energetics of larval red drum Sciaenops ocellatus Part 1 : Oxygen consumption, specific dynamic action, and nitrogen excretion. Fish Bull. 94 : 756-765 Tsukamoto, K. A. Umezawa, and T. Ozawa. 1992. Age and growth of Anguilla japonica leptocephali collected in western North Pacific in July 1990 Nippon Sui san Gakkaishi. 58(3):457 -459 Tseng, W 1990 Relationship between growth rate and age at recruitment of Anguilla japonica elvers in a Taiwan estuary as inferred from otolith increments. Mar. Bio 107:75-81 Utrecht. W L., van and M .A. Holleboom 1985 Notes on eel larvae (Anguilla anguilla Linnaeus, 1758) from the central and eastern North Atlantic and on glass eels from the European continental shelf Bijdragen tot de Dierkunde 55 : 249-262 . Umezawa, A., K. Tsukamoto 0 Tabeta, and H Yamakawa. 1989. Daily growth increments in the larval otolith of the Japanese eel, Anguilla japonica Jap J lchthyol. 35: 440-444 Wada, E., M. Terazadi, Y Kabata and T. Nemoto. 1987. 15N and 13C abundances in the Antarctic Ocean with emphasis on the biogeochemical structure of the food web. Deep Sea Res 34 : 829-841 Ware, D M 1975a. Growth, metabolism, and optimal swimming speed of a pelagic fish. J Fish Res Bd Can 32 : 33-41 Ware, D .M. 1975b Relation between egg size g r owth, and natural mortality of larval fish J Fish. Res Bd Can 32:2503-2512. Westerberg, H 1990 A proposal regarding the source of nutrition of leptocephalus larvae Int. Revue ges Hydrob i ol. 75(6) : 863-864 Westerman, M. and G J Holt. 1994 RHA:DNA ratio during the critical period and early larval growth of the red drum, Sciaenops ocellatus Mar. Bio. 121:1-9 Withers, P.C. 1992 Comparative Animal Physiology Saunders College Publishing, Fort Worth pp 949 Wri ght D A. and F.D Martin 1985 The effect of starvation on RNA:DNA ratios and growth of larval striped bass, Marone sa x atalis. J Fish Bioi. 27 : 479-485 117

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Yamamoto, K., K. Yamauchi, and T Morioka 1975 Pre-leptocephal i c larvae of the Japanese eel. Bull. Jap Soc Sci. Fish 41 : 29-34. Yamashita, Y and K.M Bailey 1989 A laboratory study of the b i oenergetics of larval walleye pollock, Theragra chalcogramma Fish. Bull 87 : 525 536 Yoklavich M. and G Boeh l ert. 1991. Uptake and utilization of 14C-g l ycine by embryos of Sebastes melanops Env Bioi. Fish 30 : 147-153 Zar J.H 1996. Biostatistical Analysis Prentice-Hall Inc New Jersey 662pp 118

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APPENDIX A Species Identification Leptocephali were first descr i bed in 1763 by W illi am Morris. For the next century, they were treated as a distinct group of fishes It wasn't until the mid 19th century that the concept of leptocephali be i ng l arval forms was first introduced. Since that time many of the leptocephali have been paired with their adult forms. Despite the advances made in leptocephali/eel identification there still remain questions regarding the taxonomy of some larvae The larvae used in this study have been i dentified to genus ; however some questions remain with respect to their specific taxonomy Paraconger caudi/imbatus Specific identification of this genus is complicated. The vertebral counts of the two recognized western Atlantic species overlap : P caudilim batus has 121 127 (n=9) vertebra and P. guianensis l:;l, e P araconger cau dlllmbatus P. g ul an ensis, .. 15 30" 95 eo 35 20 5 Figure 35. Geographic distribution of Paraconger caudilimbatus and P. guianensis (from Bohlke et al. 1989) 120

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APPENDIX A cont. has 126-131 (n=6) vertebra Smith (1989a) based the specific identification upon geographic location and considered specimens collected south and east of Trinidad P. guianensis and the northern populations to be P caudilimbatus (Figure 35). Disparate spawning seasons indicates that P caulilimbatus may consist of two sibling species. One group appears to be confined to the Gulf of Mexico and the other occurs in both the Gulf and the Caribbean In the western Gulf of Mexico, two groups have been distinguished by myomere counts; a high count group with 123-129 myomeres and a low count group with 119 -126 myomeres High-count Paraconger were present in the Gulf of Mexico in July and August and the low-count specimens were present during November and February Information on adults indicates that the low-count forms spawn during the summer and the leptocephali predominate in the Gulf of Mexico in the fall and winter (Smith 1989b) The high-count larvae are spawned in the spring Specimens collected in October can only be distinguished by myomere counts The mode of the myomere counts (mode=124, n= 1 00) falls between the two myomere modes for P caudilimbatus Currently, the two proposed groups are not separated. Representative larvae, as well as the bodies of the larvae used in age and growth analyses, were archived for future changes in species designations 121

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APPENDIX A cont. Ariosoma ba/earicum Three populations of A. balearicum exist in the western North Atlantic based upon number of myomeres in the leptocephali and vertebral counts in the e northern high-count low-count T southern high count adults: a northern high-Figure 36. Geographic distribution of the high and low myomere count forms of A. balearicum count form, a southern (from Bohlke et al. 1989). high-count form and a geographically intermediate low-count form (Figure 36) (Schmidt 1912) Smith (1989b) combines the northern and southern groups into one high-count and one low-count population In the Gulf of Mexico, only lowcount leptocephali have been identified but in the Caribbean the Gulf Stream, and the Sargasso Sea both high-count and low-count forms occur The bimodal distribution of myomere counts typical of A. balearicum have modes at 126 and 132. Mean myomere counts of the larvae collected in this study were not significantly different from the low-count population (n=1 00, 122

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APPENDIX A cont. Gymnothorax saxicola The leptocephalus used in this study was a member of the Gymnothorax saxicola-nigromarginatusocellatus complex. Vertebral counts of adult G nigromarginatus and G saxicola are virtually identical, therefore myomere counts cannot 45 30 15 o 15 2o 5 Figure 37. Geographic distribution of the G. saxicola nigromarginatus-ocellatus complex (from Bohlke et al. 1989). be used to identify species The three species are separated geographically and exist sympatrically at the boundaries where Bohlke et al. (1989) described non conforming individuals. The specific i dentification of these larvae is based upon the distribution of the adults Gymnothorax ocel/atus is not found i n the Gulf of Mexico. Adults of G saxicola are abundant from North Carolina to Florida, in the eastern Gulf of Mexico off Florida, and north to Mobile Bay Alabama. West of Mobile Bay, G. saxicola is replaced by G. nigromarginatus (Figure 37) Based upon geographic location, G saxico/a is the best identification possible for the leptocephali collected in this study 123

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APPENDIX A cont. Ophichthus gomesii Leiby (1979) thoroughly identified larvae of 0 gomesii based on the number of total and nephric myomeres, gill arch osteology, number of branchiostegal rays, and condition of the leptocephalus lateral system. There are currently no ambiguities regarding the species identification of this leptocephalus. 124

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Renee E Bishop received her bachelor of science degree from Bowling Green State University, Bowling Green Ohio in 1988 She worked as aquarium director and marine life instructor at the Maria Mitchell Science Center, Nantuc ket Massachusetts until 1989 when she moved to Florida to work at the Florida Marine Research Institute. In 1992, Renee was accepted in the doctoral program at the University of South Florida.


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