10-Induced spawning and early ontogeny of New Zealand freshwater eels Anguilla dieffenbachii and - Metodologia Científica (2024)

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Full Terms & Conditions of access and use can be found athttps://www.tandfonline.com/action/journalInformation?journalCode=tnzm20New Zealand Journal of Marine and Freshwater ResearchISSN: 0028-8330 (Print) 1175-8805 (Online) Journal homepage: www.tandfonline.com/journals/tnzm20Induced spawning and early ontogeny of NewZealand freshwater eels {Anguilla dieffenbachii andA. australis)P. Mark Lokman & Graham YoungTo cite this article: P. Mark Lokman & Graham Young (2000) Induced spawningand early ontogeny of New Zealand freshwater eels {Anguilla dieffenbachii and A.australis), New Zealand Journal of Marine and Freshwater Research, 34:1, 135-145, DOI:10.1080/00288330.2000.9516921To link to this article: https://doi.org/10.1080/00288330.2000.9516921Published online: 29 Mar 2010.Submit your article to this journal Article views: 1129View related articles Citing articles: 4 View citing articles https://www.tandfonline.com/action/journalInformation?journalCode=tnzm20https://www.tandfonline.com/journals/tnzm20?src=pdfhttps://www.tandfonline.com/action/showCitFormats?doi=10.1080/00288330.2000.9516921https://doi.org/10.1080/00288330.2000.9516921https://www.tandfonline.com/action/authorSubmission?journalCode=tnzm20&show=instructions&src=pdfhttps://www.tandfonline.com/action/authorSubmission?journalCode=tnzm20&show=instructions&src=pdfhttps://www.tandfonline.com/doi/mlt/10.1080/00288330.2000.9516921?src=pdfhttps://www.tandfonline.com/doi/mlt/10.1080/00288330.2000.9516921?src=pdfhttps://www.tandfonline.com/doi/citedby/10.1080/00288330.2000.9516921?src=pdfhttps://www.tandfonline.com/doi/citedby/10.1080/00288330.2000.9516921?src=pdfNew Zealand Journal of Marine and Freshwater Research, 2000, Vol. 34: 135-1450028-8330/00/3401-0135 $7.00 © The Royal Society of New Zealand 2000135Induced spawning and early ontogeny of New Zealand freshwatereels {Anguilla dieffenbachii and A. australis)P. MARK LOKMANGRAHAM YOUNGDepartment of ZoologyUniversity of OtagoP. O. Box 56Dunedin, New Zealandemail: mark.lokman@stonebow.otago.ac.nzAbstract Knowledge on the reproductive biologyof New Zealand freshwater eels (Anguilla australisRichardson and A. dieffenbachii Gray) is limited tochanges associated with gametogenesis in the adults;details pertaining to embryonic and larval develop-ment are not available, but are of obvious benefit fora better understanding of the eel life history and forartificial propagation programmes, whether foraquaculture or conservation purposes. Therefore,eels were artificially matured and eggs fertilised withsperm from either species to investigate early ontog-eny. Only eggs from A. australis hatched success-fully after c. 45 h of incubation at room temperature,yielding larvae of c. 2.5 mm in length. Larvae sur-vived for up to 5 days, by which time they had at-tained a length of 5.3 mm. Scanning electronmicroscopy of 1- and 2-day-old larvae revealed thepresence of prominent free neuromasts on the headand along the flanks. Although hatching of NewZealand eels was achieved for the first time, it isstrongly recommended that hormone treatment beinitiated immediately following capture, well beforeovarian atresia starts.Keywords induced spawning; Anguilla spp.; eel;New Zealand; fertilisation; hatchingM99002Received 18 January 1999; accepted 15 September 1999INTRODUCTIONFreshwater eels (Anguilla spp.) have a unique lifehistory, which is characterised by a marine and afreshwater phase. The freshwater phase, comprisingthe longer part of the life cycle, from the juvenile topuberal stage, has been intensively studied (e.g.,Tesch 1977; Degani & Gallagher 1995). In contrast,much less is known about the marine phase. Thus,based on the classical studies by Schmidt at thebeginning of this century, the spawning grounds ofseveral anguillid eel species have been discovered(Schmidt 1922; Tsukamoto 1992) and aspects oflarval biology studied. However, mature adults havenever been caught and eel eggs never collected(McCleave & Kleckner 1985; McCleave et al. 1987),whereas the fragile larvae typically do not survivecapture (K. Tsukamoto, Ocean Research Institute,Tokyo University, Japan pers. comm.) and hence,cannot be studied in the live state.Artificial propagation of eels has long been at-tempted not only to address fundamental questionson the reproductive biology of these fish, but alsofrom a fisheries management and eel aquacultureperspective. Increasing fishing pressures on eelshave led to reduced recruitment of juveniles of sev-eral anquillid species, limiting the industry and po-tentially leading to ecological impacts. Despite theseresearch efforts, however, captive breeding of eelshas not yet been achieved. Sexual development infreshwater eels is halted when these fish are held incaptivity (Dufour et al. 1988), but this arrest can beoverridden by hormone treatment. Accordingly,experimentation has been repeatedly undertaken: ( 1 )to mimic "natural" sexual development in captiveeels, allowing morphological, biochemical, andphysiological changes occurring during the spawn-ing migration to be inferred (e.g., Pankhurst 1984);and (2) to obtain mature gametes for fertilisation andstudy of resulting developmental stages (e.g.,Yamauchi et al. 1976; Yamamoto 1981). The firstsuccessful results using exogenous hormone treat-ments in eels date from the 1930s, when male Euro-pean eels (Anguilla anguilla) were injected with136 New Zealand Journal of Marine and Freshwater Research, 2000, Vol. 34urine from pregnant women (e.g., Fontaine 1936).Similar experiments with female eels, treated repeat-edly with human chorionic gonadotropin (hCG), didnot result in significant ovarian development (Bruunet al. 1949; Sorensen & Pankhurst 1988). Instead,repeated injections of teleost pituitary suspensions,first successfully applied by Fontaine et al. (1964),are now routinely used for induction of vitellogen-esis in female eels. However, despite the availabil-ity of artificially matured male and female eels, thelife cycle of anguillids has still not been completedin captivity. Indeed, researchers have successfullyfertilised and hatched eggs of Japanese (A. japonica;e.g., Yamamoto & Yamauchi 1974; Yamamoto etal. 1975; Tanaka et al. 1995) and European eels(Prokhorchik 1986), but larvae never survived formore than a few weeks, possibly as a result of aber-rant yolking or lack of appropriate food (Yamauchi& Yamamoto 1982; Kurokawa et al. 1995).Experiments aimed at artificially inducingmaturation in New Zealand freshwater eels, thelongfinned A. dieffenbachii and the shortfinned A.australis, were carried out by Todd in the early 1970s(Todd 1974, 1979). The longfinned eel appears tobe an especially suitable candidate for suchexperiments, since males and females of this speciesare relatively sexually advanced at the onset of thespawning migration (Todd 1981 ; Lokman & Young1995). Todd's experiments yielded mature male andfemale gametes from both species, but offspringwere not obtained (Todd 1974). Recent renewedinterest in New Zealand eels, together with improvedtechniques for induction of final maturation andovulation (e.g., Ohta et al. 1996, 1997) and therelatively advanced stage of sexual development inmigratory longfinned eels (Lokman et al. 1998),prompted us to once again apply hormone treatmentsfor the acquisition of mature gametes, fertilised eggs,and offspring to further elucidate aspects of thereproductive biology of these fish.MATERIALS AND METHODSAnimalsMigratory ("silver") eels were obtained from GouldAquafarms (Leeston, New Zealand), a commercialeel processing company, in May 1995. Six males andsix females from both species (for identificationcriteria, see Jellyman & Todd 1982; Lokman et al.1998) were used. The males were chosen randomly,provided they were in good external condition,whereas small females (see next section for bodyweights) were selected forpractical purposes (easierhandling and lower hormone requirements). Eelswere transported to laboratory holding facilities atthe Department of Zoology, University of Otago,Dunedin (May 1995), and housed in 200-litre glassaquaria, containing recirculating fresh water. Fishwere acclimated to natural sea water (salinity c. 30ppt) in September 1995. The water temperature wasmaintained at 12-17°C in winter and increased to18-22°C during experimentation, which was startedin November (early summer) 1995. All animals werestarved throughout the acclimation and experimentalperiods.Induction of gonadal development,final maturation, and spawningFemalesEvery third day from the start of experimentation,body weights were recorded and intramuscular (i.m.)injections of 5 mg kg"1 acetone-dried chinooksalmon pituitary hom*ogenate (SPH) in saline (0.9%NaCl) given to both shortfinned {A. australis; bodyweight 544 ± 47 g; mean ± SE, n = 6) and longfinned{A. dieffenbachii; body weight 1603 ± 58 g; n = 6)female eels to re-initiate vitellogenesis. Ovarian sam-ples were first collected by cannulation(polyethylene tubing, 1.2 mm in diameter) withoutanaesthesia after the body weight had increased byc. 5% over initial values. Once cytoplasmic clearingand germinal vesicle migration were evident underthe dissecting microscope, final oocyte maturationand ovulation were induced using a method similarto that described by Ohta et al. (1996, 1997): anintraperitoneal (i.p.) injection of SPH (10 mg kg"')was given, followed the next day (22-26 h later) byan i.p. injection of 2 mg kg~' 17-hydroxypro-gesterone (dissolved in DMSO; stock solution of 10mg ml"' DMSO), administered at 3-4 sites of theabdomen. After treatment with 17-hydroxypro-gesterone, ovulation was assessed by gently squeez-ing the abdomen of anaesthetised (0.3% benzocainein sea water) females every 3^1 h, until eggs couldbe expelled from the vent. Eggs were collected witha spatula or directly stripped into a plastic bowl.Between 28 November 1995 (Day 0) and 18January 1996 (Day 51), two female longfins and onefemale shortfin matured. These animals weresacrificed within 1 day post-spawning (spawning isdefined here as the time of egg release, whetherspontaneously or by manual stripping). Treatment ofthe remaining eels was interrupted for several weeksbecause of unavoidable circ*mstances, but itresumed on 28 February (Day 91), after one of theLokman & Young—Induced spawning of New Zealand eels 137125 n10075125 n10075.£ 125 nDû"« îooH75-o 125 n•1 10075125 n10075125 n100-75A. australisi I Ti i i ri I TMi i i i ii iHT I I I I I Ii i i i i i 1M0 20 40 60 80 100120140A. dieffenbachiii i ii 1T i i0 20 40 60 80 100120140Time (days) Time (days)Fig. 1 Changes in relative body weight (Day 0 = 100%) of individual shortfinned (Anguilla australis) and longfinned(A. dieffenbachii) females artificially matured with 5 ing kg"1 salmon pituitary hom*ogenate. Treatment was inter-rupted from Day 45 until Day 90. Symbols and abbreviations: • , spawning induction by 17-hydroxyprogesterone orspontaneous egg release; M, batch of fertilised eggs reaching morula stage; E, batch of fertilised eggs undergoingembryogenesis, but failing to hatch; and H, batch of fertilised and hatched eggs.female shortfins spontaneously spawned eggs, condition, were induced to mature repeatedly (2 andExcept for one longfinned female, killed on Day 123, 3 times, respectively). On 11 April 1996 (Day 134),all females responded to renewed SPH injections, the oocytes of the remaining two females, oneTwo female shortfins, both in particularly good longfin and one shortfin, reached the migratory138 New Zealand Journal of Marine and Freshwater Research, 2000, Vol. 34Fig. 2 Scanning electron micrographs of eel eggs showing ovulation (Anguilla australis) at A, high and B, lowmagnification and C, micropyle (A. dieffenbachii). Scale bar: 10 (C) or 100 urn (A, B).nucleus stage. These eels were killed after eggs werestrippedon 13 April 1996 (Day 136).MalesAnaesthetised (0.3% benzocaine in sea water) maleeels received weekly i.m. injections of humanchorionic gonadotropin (hCG; Lot CR127, NationalHormone and Pituitary Program, Bethesda, MD,United States) in saline. Doses corresponded to thoseused by Todd (1974): 250 IU hCG for shortfinned(body weight 140 + 5 g; n = 6) and 500 IU hCG forlongfinned eels (body weight 529 ± 62 g; n = 6),irrespective of body weight. Treatment becameirregular once females started to reach maturity (afterc. 6 weeks), with hCG being administered at the timethat females were induced to undergo final oocytematuration and ovulation. Sperm was collected witha syringe from anaesthetised males by gentlysqueezing the abdomen previously dried with atowel. Sperm was either used immediately, or keptrefrigerated (4°C) in the syringe for up to 2 days.All male shortfins survived treatment until termi-nation on Day 136. In contrast, mortality amongmale longfins reached 100% by Day 105. To allowfertilisation of longfin eggs by conspecifics, we col-lected two more male longfins during the autumnspawning migration of 1996 (between 14 and 24March 1996). These fish were treated with hCGimmediately after capture and, after transport to thelaboratory, maintained as described above.Fertilisation and incubation of the eggsEggs were dry-fertilised with sperm from con-specific and/or allospecific donors and activated withsterilised, concentrated natural sea water (c. 35 ppt).Excess sperm was removed by gently washing theeggs after transfer onto a nylon mesh (250 |imdiameter). Following reimmersion in sea water, onlyfloating eggs were collected and incubated at roomtemperature (18.2-22.7°C). White, sinking eggswere removed throughout the incubation period andsea water was changed as considered appropriate.MicroscopyEel embryonic and larval development was moni-tored using a dissecting microscope connected to anOlympus C-35AD-4 camera. A few larvae werefixed in glutaraldehyde (2.5% in 0.15A/cacodylatebuffer) or Bouin's fixative for imaging by scanningelectron microscopy (SEM). Samples for SEM weredehydrated in a graded alcohol series, cryosubsti-tuted in CO2 and gold-coated before scanning un-der a Cambridge Instruments S360 SEM at 7.5-10kV.RESULTSInduction of vitellogenesis, final maturation,and spawningTreatment with SPH resulted in rapid increases ofbody weight after 4-19 weeks in most females (Fig.1). Subsequently, final maturational stages wereinduced in 11 of 12 females. Oocytes underwentfinal maturation and ovulation (Fig. 2A,B) in all tri-als during the following 1-2 days, resulting in ex-posure of the micropyle (Fig. 2C), which measuredc. 3 |xm in outer diameter. Fourteen batches of eggswere collected (some female shortfins were inducedto mature repeatedly; see Fig. 1). Most batchesyielded transparent eggs with multiple small oildroplets, indicative of good quality (H. Satoh,Lokman & Young—Induced spawning of New Zealand eels 139Fig. 3 Eggs of Anguilla dieffenbachii: A, shortly after ovulation (diameter (D) = 0.95 mm); B, after first cleavagedivision, c. 2 h post-fertilisation (D = 1.0 mm); C, morula, 6.5 h post-fertilisation (D = 1.1 mm); D, blastula, 10 hpost-fertilisation (D = 1.0 mm); E, blastula, 12 h post-fertilisation (D = 1.0 mm); F, G, embryo, c. 30 h post-fertilisa-tion (D = 1.0 mm); H, embryo, 33 h post-fertilisation (D = 1.0 mm). Eggs developed, but failed to hatch.Fisheries Laboratory, University of Tokyo,Shizuoka, Japan pers. comm.). However, in fourbatches, only white (presumed immature or overripe)or atretic eggs were present.Fertilisation and incubation of eggsDry-fertilised eggs hydrated upon exposure to seawater, although water uptake was variable as judgedby differences in width of the perivitelline space(hydrated egg diameter between 1.0 and 1.6 mm;results not shown). Eggsdeveloped as described forthe Japanese (Yamamoto & Yamauchi 1974;Yamamoto et al. 1975; Yamamoto 1981) and Euro-pean eel (Prokhorchik et al. 1987). In brief, lipiddroplets fused after seawater activation, yielding asingle oil drop after c. 2 h (Fig. 3A,B). This drop wasalways oriented towards the water surface (not evi-dent from photographs). In both fertilised (seven of140 New Zealand Journal of Marine and Freshwater Research, 2000, Vol. 34c:c©Fig. 4 Hybrids of female Anguilla australis x male A. dieffenbachii: A, embryo 31.5 h post-fertilisation (diameter(D) =1.55 mm); B, newly hatched larva (0-1 h; total length (TL) = 2.5 mm); C, 2-day old larva (TL = c 41 mm)- DE, 3-day old larva (TL = c. 4.6 mm); F, G, 5-day old larva (TL = c. 5.3 mm).Fig. 5 Scanning electron micro-graphs of eel larvae (Anguillaaustralis x A. dieffenbachii): A, 1 ;and B, 2 days post-hatching. Ab-breviations and symbols: E, eye;Y, yolk sac; F, fin; A , neuromast.Bar = 0.5 mm.14 trials) and unfertilised eggs, a blastodisc developed,which, in fertilised eggs, cleaved meroblastically af-ter c. 1.5-2 h post-fertilisation (Fig. 3B). Consecutivecleavage divisions occurred approximately every 30min, resulting in the formation of a morula (Fig. 3C)after c. 6 h. In four of seven trials, the morula stagecontinued to develop, resulting in the formation of ablastocoel (Fig. 3D,E) from c. 7 h post-fertilisationonwards. After gastrulation, the embryonic body wasformed and somites became discernible (Fig. 3F-H),followed by elevation of the tail from the yolk by31.5 h post-fertilisation (Fig. 4A). Development of thehead and lengthening of the tail were apparent in thesuccessive hours (results not shown).Hatching occurred in two trials of developingembryos after 43-45 h post-fertilisation, yieldingLokman & Young—Induced spawning of New Zealand eels 141Fig. 6 Scanning electron micrographs of: A, head andB, C, flank neuromasts of eel larvae (Anguilla australis xA. dieffenbachii) 1 (A,B) and 2 days post-hatching (C).Abbreviations: S, stereocilium; P, pore. Bar= 10 um.larvae of c. 2.5 mm in length (Fig. 4B). Theremaining two batches of developing embryos,originating from one female of each species, failedto hatch. Instead, these embryos continued to growand develop inside the egg for up to 5 days post-fertilisation.Developing larvae were maintained for 5 dayspost-hatching. During this time, the yolk supplygradually decreased (Fig. 4B-G). Growth was ob-served from 2.5 mm at hatching to 4.1, 4.6, and5.3 mm after 2,3, and 5 days post-hatching, respec-tively. Some pigmentation of the tip of the tail wasseen on Day 5 post-hatching (Fig. 4G). Larval feed-ing was not attempted. No differences were appar-ent between survival and development of larvae fromshortfin x shortfin or shortfin x longfin crosses.Imaging of 1 - and 2-day-old larvae by SEM re-vealed that the head region was poorly developed,judged by the poor definition of the eye and theabsence of a mouth (Fig. 5A,B). In contrast, mech-anoreception was evident from the presence ofneuromasts, containing a kinocilium and numerousstereocilia, on the head (Fig. 6A). Furthermore, freeneuromasts occurred along the flanks of 1-day-oldlarvae (Fig. 6B), increasing in number and distribu-tion by 2 days post-hatching (Fig. 6C). Larvae fixedpost-mortem (Day 6 post-hatching) were infestedwith bacteria and hence, mechanosensory structurescould not be observed (results not shown).DISCUSSIONThe primary objective of this study concerned theacquisition of fertilisable eggs from New Zealandfreshwater eels, since the development of techniquesfor consistently obtaining functional, maturegametes could serve as a baseline for future studiesinto larval rearing. At present, larval productionappears to be routinely realised in the Japanese eelonly (e.g., Satoh et al. 1992;Ohtaetal. 1996, 1997),although a few reports describe the production oflarval A. anguilla (Prokhorchik 1986; Prokhorchiketal. 1987).For induction of vitellogenesis, we repeatedlytreated eels with salmon pituitary hom*ogenate, es-sentially a standard procedure. Subsequently, eelswere induced to undergo final maturation and ovu-lation based on the method recently described byOhta and co-workers (1996), since these authorsspecifically focussed on solving problems related toinduction of these stages in the Japanese eel. How-ever, rather than using 17a,20p-dihydroxy-4-pregnen-3-one as the final inducer (Ohta et al. 1996),we chose to use the much cheaper precursor 17-hydroxyprogesterone.Using these protocols, larvae from New Zealandeels were first obtained from eggs of females treatedwith SPH for c. 6 weeks. Attention was initially142 New Zealand Journal of Marine and Freshwater Research, 2000, Vol. 34focused on optimising the timing for induction offinal oocyte maturation and ovulation, and to stripthe eggs. The importance of timing was previouslyemphasised for artificially matured Japanese eels(Satoh et al. 1992; Ohta et al. 1996). In this species,good quality eggs were obtained after the bodyweight had increased over initial values by 20% asa result of hydration (Satoh et al. 1992; Ohta et al.1996). In the present study, ovarian samples werecollected by cannulation after increases in bodyweight of 5-10% had been attained, which is some-what earlier than in a previous study (c. 15%;Lokman & Young unpubl. obs.). Final oocyte matu-ration and ovulation were induced when the nucleuswas in an eccentric, or sometimes peripheral loca-tion and when clearing of the cytoplasm had started.Strip-ripeness was checked frequently, as findingsby Ohta et al. (1996) indicated that overripening ofthe eggs from the Japanese eel resulted in a rapiddecline of quality. Accordingly, fertilisation andhatching were realised in 16 and 0% of experimentswith eggs from longfinned eels, and in 33 and 22%of experiments with eggs from shortfinned eels, re-spectively. Although we considered these values asquite reasonable, they were well below those re-ported for A. japonica by Ohta et al. (1996). Thismay be attributable to greater experience in fine-tuning of timing by these researchers, rather than theuse of a different final inducer in the present study.In response to SPH treatment, sexualdevelopment proceeded somewhat faster inlongfinned than in shortfinned eels. However, unlikea previous experiment (Lokman 1997) performed ayear earlier in the same season, maturation could notbe induced within a 7-8-week period in most eels.This resulted in serious time constraints, makingcontinuation of the experiment impossible for a 1-month period.Attempts to artificially propagate longfinned eelswere not as successful as those with shortfins. Thefailure to reproduce A. dieffenbachii is possiblyrelated to compromised egg quality, since spermfrom longfinned and shortfinned eels seemed equallycompetent in fertilising eggs of A. australis. Poor eggquality, in turn, may have been the result of the longperiod of captivity and/or starvation beforeexperimentation, especially in view of the highincidence of ovarian atresia that occurs in A.dieffenbachii when held under such conditions(Lokman 1997; also see Lokman & Young 1998).In vitro fertilisation was repeatedly achieved(seven of 14 attempts), but gastrulation and succes-sive stages of development were only observed in afew instances (four of 14 attempts). In two batches,originating from female A. australis and males ofeither species, eggs hatched successfully after 42-45 h, which is similar to incubation times reportedfor A. japonica (38-45 h, Yamamoto & Yamauchi1974; 36-48 h, Satoh et al. 1992; 40-60 h,Kurokawa et al. 1995) and A. anguilla (46^48 h,Prokhorchik et al. 1987). The larvae measuredc. 2.5 mm in total length, which corresponds to val-ues given for A anguilla (2.5-2.7 mm, Prokhorchik1986), but is somewhat smaller than those for A.japonica (2.9 mm, Yamamoto& Yamauchi 1974;Yamamoto et al. 1975). Similarly, A. japonica lar-vae were considerably larger than those from A.australis or A. australis x A. dieffenbachii on con-secutive days post-hatching, which may be partlyexplained by differences in incubation temperature.Scanning electron microscopy revealed the pres-ence of a micropyle on the egg of A. dieffenbachii.The morphology of the micropyle was similar to thatdescribed by Prokhorchik et al. (1989) and Ohta etal. (1983) for eggs fromA anguilla and A. japonica,respectively. In addition, the diameter of the micro-pyle of A. dieffenbachii eggs is the same as that ofeggs from A. japonica and A. anguilla (3 (im, Ohtaet al. 1983; Prokhorchik et al. 1989), despite differ-ences in width of the spermatozoon head: thus, in A.anguilla and A. japonica, the spermatozoon headmeasures just over 1 ̂ .m in width (Billard &Ginsburg 1973; Colak & Yamamoto 1974), whereasthis value is tripled for A. dieffenbachii spermato-zoa (Todd 1974, 1976).In two batches of developing eggs, obtained fromone female A. dieffenbachii and one female A.australis, embryonic development was observed, buthatching did not occur. Embryonic developmentappeared aberrant for at least one of these batches,as indicated by the failure of the tail to elevate fromthe yolk. Some unhatched embryos, still alive afterup to 5 days post-fertilisation as evidenced by heartbeat and muscular contractions, were artificiallyremoved from the egg. Although surviving thistreatment, the larvae remained curled up and hence,showed impaired swimming. We suspect that,possibly as a result of the long period of starvationbefore experimentation, some properties of thechorion may have been responsible for the failure ofthe eggs to hatch, especially in view of the largevariation in egg shell hardness and degree ofhydration among batches of spawned eggs. Indeed,hatching was only observed for batches of eggs thathydrated to c. 1.5 mm upon seawater exposure. Thisis in agreement with the observation that in some eelLokman & Young—Induced spawning of New Zealand eels 143species a wide perivitelline space is common(Blaxter 1988). However, illustrations of developingA. anguilla by Prokhorchik et al. (1987) suggest thatin this species a wide perivitelline space does notseem to be a prerequisite for "normal" embryonicdevelopment.The most prominent sensory system of newlyhatched eel larvae seems to be that of mechanore-ception. Unlike vision, which does not appear todevelop until 8 days post-hatching when pigmenta-tion of the retina begins (see observations on A.japonica by Yamauchi et al. 1976), the mechano-sensory system is probably functional at the time ofhatching. Indeed, larvae responded to movement ofthe beaker in which they were kept by swimming ina spiral fashion (pers. obs; also see Prokhorchik1986). The mechanosensory system has not beenpreviously examined in newly hatched, premeta-morphic leptocephali (Pfeiler 1989), but they havebeen recorded from large leptocephali of the Euro-pean eel (Pfeiler 1989; Appelbaum & Riehl 1993).Our observations indicate that, similar to other fishspecies (Blaxter 1988, and references therein) freeneuromasts, with classical kinocilium and stere-ocilia, were present on the head of 1-day-old eellarva. In contrast, very few, apparently less-devel-oped neuromasts were found along the flanks. By 2days post-hatching, the number of neuromasts alongthe flank increased in number and distribution. Fur-thermore, an opening was visible near severalneuromasts, which may correspond to canal poresor they may be fixation artefacts.Cross-fertilisation of A. australis eggs by spermfrom A. dieffenbachii could be achieved as readilyas intra-specific fertilisation. Although notpreviously documented, inter-specific crosses amonganguillid eels {A. anguilla x A. japonica) have beensuccessfully performed before (K. Hirose, NationalResearch Institute of Fisheries Science, Yokohama,Kanagawa, Japan pers. comm.). For the NewZealand eel species, the production of hybrids haspractical research advantages: migratory ("silver")female A. australis are much easier to collect, keep,and handle than female A. dieffenbachii, because oftheir much smaller size (500-1500 g, and »1500 g,respectively), though this may not outweigh thebenefit of greater fecundity and advanced stage ofoogenesis in newly-caught female A. dieffenbachii(Lokman et al. 1998). Male A australis (body weightc. 120 g), although very accessible, only yield smallvolumes of milt (c. 1 ml) compared to male A.dieffenbachii (c. 500 g; milt volume 3-5 ml).Therefore, access to adequate milt supplies requiresa relatively large number of male A. australiscompared to male A. dieffenbachii, making itparticularly advantageous to using the longfinned eelas sperm donor.In conclusion, this paper describes the first pro-duction of New Zealand eel larvae under artificialconditions. Larvae were maintained for up to 5 days,during which time most of the yolk sac was ab-sorbed. Future experimentation may be much facili-tated if hormonal manipulation is startedimmediately after capture of wild migratory speci-mens, especially longfinned eels, thus preventingadverse affects associated with gonadal atresia.ACKNOWLEDGMENTSWe thank Sue Johnstone, Department of Oral Biology andOral Pathology, University of Otago, for assistance withscanning electron microscopy; and Professor JohnMontgomery, School of Biological Sciences, Universityof Auckland for his help with the interpretation of thescanning electron micrographs. We also thank KarenJudge and Rob Wass for technical assistance and wegratefully acknowledge the gift of human chorionic go-nadotropin by the National Hormone and Pituitary Pro-gram, Bethesda, MD. 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