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Home , Alligator gar, Atractosteus, Cuban gar, Florida gar, Gar, Lepisosteus

Aquaculture Research, 2008, 39, 748^763 doi:10.1111/j.1365-2109.2008.01927.x

Gar biology and culture: status and prospects

Roberto Mendoza Alfaro1, Carlos Aguilera GonzaŁlez1 & Allyse M Ferrara2 1Facultad de Ciencias Biolo¤ gicas, Universidad Auto¤ noma de Nuevo Leo¤ n, Universidad, San NicolaŁ s de los Garza, Nuevo Leo¤ n, Me¤ xico 2Department of Biological Sciences, Nicholls State University,Thibodaux, LA, USA

Correspondence: R M Alfaro, Facultad de Ciencias Biolo¤ gicas, Universidad Auto¤ noma de Nuevo Leo¤ n, Apartado Postal F-96, Cd. Universitaria, San NicolaŁ s de los Garza, C.P.66450, Nuevo Leo¤ n, Me¤ xico. E-mail: [email protected]

Abstract (Fig. 1), bony ¢shes that, together with the bow¢n calva, belong to the order Many lepisosteid populations in have (Nelson 1994) and are sometimes grouped as holos- declined and many are now threatened as a conse- teans. This is an ancient group of ¢sh that is older quence of habitat loss and alteration and commercial than most , dating back to the and sport over¢shing. Over the last two decades, period, approximately 180 million years ago (Wiley morphological, histological and molecular studies 1976). The genus is composed of four allowed distinguishing between di¡erent phases of species (L. osseous, L. platostomus, L. oculatus and development and the nutritional condition of larvae. L. platyrhincus), while the genus con- Ontogeny of the digestive enzymes of larvae indi- tains three extant species (A. spatula, A. tristoechus, cated the possibility to feed them arti¢cial feeds since A. tropicus). early developmental stages. An in vitro digestibility At present, lepisosteids are distributed in North system to test di¡erent feed ingredients has been America, and , including The used. Important characteristics of arti¢cial diets Isle of Youth (Comabella, Mendoza, Aguilera, Carrillo, were identi¢ed through di¡erent feeding experi- Hurtado & Garc|¤a-Galano 2006). The northernmost ments. Endocrinological studies showed the feasibil- limit is reached by L. osseus in southern , while ity of altering larval development and the digestive the southernmost limit is reached by A. tropicus in capacity of larvae. Cloning of gar growth hormone Costa Rica (Mora-Jamett, Cabrera & Galeano1996; Or- opened new avenues to enhance growth in the . lando 2001).This is also the only species that ranges to Plasmatic vitellogenin was isolated and puri¢ed, to Paci¢c slope drainages (from southern to Hon- develop a competitive enzyme-linked immunosor- duras). bent assay, which allowed the straightforward In addition to their close phylogenetic relation- separation of males from females to establish ships, the gars share similar ecological roles as top appropriate proportions for reproduction and also predators. Because of the high trophic position and was used to evaluate hormonal protocols to induce relatively long life span of these ¢sh, many lepisosteid gonad recrudescence and spawning. This review populations have declined due to habitat loss and al- analyzes the biology,ecology and physiology of di¡er- teration (dams, channel straightening, road con- ent gar species as a basis for their domestication, struction, £ow alteration, pollution, etc.) and due to mass production of larvae for repopulation experi- over¢shing. The decline of many gar populations ments and for the culture of commercial-size gar. has resulted in research into the feasibility of using captive culture techniques to reduce ¢sheries pres- Keywords: gar, biology, physiology, , sure and to restore natural populations. Of the seven nutrition, reproduction gar species, those belonging to the genus Atractosteus have recently motivated much interest in their cul- ture. This importance is principally due to their rapid Introduction growth rate and large adult size, as is the case of alli- The family Lepisosteidae is at present represented by gator and Cuban gars (Aguilera, Mendoza, Rodr|¤guez two genera and seven extant species of non- & Marquez 2002). Moreover, culturing gars o¡ers

r 2008 The Authors 748 Journal Compilation r 2008 Blackwell Publishing Ltd Aquaculture Research, 2008, 39, 748^763 Gar biology and culture RMAlfaroet al.

Figure 1 Relative total lengths of the seven extant gar species (based on Suttkus1963; Page & Burr1991; Mora-Jamett et al.1997). several additional advantages such as their ability to Mendoza 2000). This review summarizes recent grow in waters of variable quality due to their capability research on the culture of Atractosteus species. to breathe atmospheric air (Hill, Renfro & Reynolds 1972; Smatresk & Cameron 1982) and toler- ance to high ammonia and nitrite levels (Boudreaux, Reproductive biology Ferrara & Fontenot 2007a, b), their resistance to several diseases (Leo¤ n, Aguiar & HernaŁ ndez 1978; Gars spawn seasonally in the £oodplain of large riv- Kulakkattolickal & Kramer1988) and excellent feed con- ers, shallow lacustrine habitats, and tributaries that version rate (FCR) (Aguilera, Mendoza, Comabella & provide protection for their young from predators. Marquez 2006; MaŁ rquez-Couturier,Alvarez-Gonzale¤ z, Lepisosteids seldom display gregarious behaviours, Garc|¤a-Galano, Contreras-SaŁ nchez, HernaŁ ndez-Fra- with the exception of the spawning season, when nyitti, Mendoza-Alfaro, Aguilera-GonzaŁ lez, Garc|¤a- groups of several individuals (some times 20 or more) Galano, Civera. Cerecedo & Goytortua-Bores 2006). may be observed together. Sex ratios of spawning ag- Within this context, the use of new species for gregations are typically skewed towards males (Dean aquaculture production necessarily implies their do- 1895; Holloway1954; Suttkus1963; Rese¤ ndez & Salva- mestication. This process requires, at least, the study dores1983; Aleman & Contreras1987; Chavez-Lomel|¤, of the capacity of those organisms of interest to live Matthews & Pe¤ rez-Vega 1989; Go¤ mez-Go¤ mez 1989; most of their life cycle under arti¢cial conditions Pe¤ rez-SaŁ nchez 1995; Bejerano, Marquez & PaŁ ramo (FAO/PNUMA 1984). Among the main characteris- 1997). The large spawning aggregations will divide tics desired in a species to be domesticated are the into smaller parties containing a female that is at- ability to reproduce in captivity, the likelihood of tended by several males (from two to eight).The smal- spawning in captivity, the possibility of mass larval ler groups will then enter very shallow waters where rearing, adaptability to the consumption of arti¢cial spawning takes place (Dean 1895). However, sex ra- diets and the capability to grow and be maintained at tios vary among gar species. Di¡ering sex ratios have high densities (AQUACOP & Calvas 1990). However, been reported for from1:1,3:1to 5:1males most currently cultured species do not possess all of per female (Rese¤ ndez & Salvadores 1983; Chavez- these characteristics (Mendoza 2005). In fact, the Lomel|¤ et al. 1989; Pe¤ rez-SaŁ nchez 1995; Bejerano et al. reasons to initiate the domestication of a species have 1997). Sex ratios of 1:1 male per female for L. platyr- been economical (commercial value of the species), hincus and 3:1 males per female for L. osseus have sociocultural (traditional ¢sheries) and ecological been reported (Holloway 1954). Finally, ratios of 2:1 (over-exploited or endangered species) (Mendoza, and 1:1males per female have been reported for alli- Aguilera, Rodr|¤guez & MaŁ rquez 2000; Rojas & gator gar (Morales1987; Rodr|¤guez, Banda, GonzaŁ lez, r 2008 The Authors Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 39, 748^763 749 Gar biology and culture RMAlfaroet al. Aquaculture Research, 2008, 39, 748^763

Herrera & Garc|¤a1998;Ferrara2001).Becausethesex of VTG as a sex-speci¢c biochemical marker (HernaŁ n- of individuals in most populations of lepisosteids dez 2002; Cortes 2003;Vela 2003; SantillaŁ n, Mendoza, cannot be determined externally, or that techniques Revol, Aguilera & Montemayor 2005; SantillaŁ n2006; for external sex identi¢cation may need to be devel- Orlando, Binczik, Denslow & Gillette 2007). oped on a population by population basis, and sex ra- Vitellogenin was selected for sex identi¢cation be- tios vary among species and may vary seasonally for cause it is the precursor of yolk and is thus speci¢c a given area, techniques to accurately identify sex of to reproductive females. Moreover, the blood, muscle are needed to establish proper sex ratios and mucus concentrations of VGT increase concur- forcaptive spawning and to optimize fertilization rates. rently with ovarian development. Until recently,accurate sex identi¢cation of brood- Because of the scarcity of wild gar adults stock was a major obstacle in the culture of gars even in northern and central Mexico, plasmatic VTG and though a few studies have documented sexually di- ovarian lipovitellin (VTL) were isolated and puri¢ed morphic external characteristics. Suttkus (1963) re- from female and estradiol-injected male cultured ported that males of L. platostomus, L. oculatus and . Puri¢cation of both molecules was car- L. platyrinchus have a smaller maximum size and ried out by selective precipitation ethylenediamine reach maturation at a smaller size than females. tetra acetic acid (EDTA^MgCl2), followed by molecu- Female L. oculatus are reported to have lar weight ¢ltration in Sepharose-6B and ion ex- larger bodies (Redmond 1964) and longer snouts change chromatography using DEAE-Sephacel (Hubbs & Lagler1949; Suttkus1963; Love 2001). Dean (Sigma Chemical, Sanint Louis, MO, USA). Puri¢ed (1895) observed that the snouts of male gar (Lepisos- molecules were characterized by their prosthetic teous spp.) had a lighter colour than female gar. Leo¤ n groups and molecular weight was determined by re- et al. (1978) noticed that A. tristoechus adult females gression analysis using the Stokes radius of the mole- showed a swollen abdomen and a prominent and cules according to Mendoza (1992).With the puri¢ed coloured genital papilla before spawning. However, molecules, polyclonal antibodies were raised in rab- these di¡erences are only apparent after several bits following the method of Vaitukaitis (1981). Im- years or only during advanced reproductive stages munoglobulins (IgGs) were obtained by protein ‘A’ (Netch & Witt 1962; Bejerano et al.1997), thus hinder- chromatography.With both puri¢ed antibodies and ing gender selection. The same problem has been ob- antigens, a competitive enzyme-linked immunosor- served for alligator gar (Morales1987), in tropical gar bent assay (ELISA) was developed and submitted to a that reaches sexual maturation more precociously set of quality control tests, including sensitivity, re- (Chavez-Lomel|¤ et al.1989). cuperation, parallelism, reproducibility and speci¢- city. The immunoassay was validated by measuring VTG concentrations in the plasma and mucus of fe- Vitellogenin (VTG) male gar. Furthermore, the ELISA allowed for the Sex of mature gar was typically identi¢ed by gross ex- straightforward separation of males from females to amination of the gonads (Netch & Witt 1962; Rese¤ n- establish the appropriate sex ratios for reproduction dez & Salvadores 1983; Mora-Jamett et al. 1996; and was also used to evaluate hormonal protocols Ferrara & Irwin 2001; Garc|¤adeLeo¤ n, GonzaŁ lez- for the induction of gonad recrudescence and spawn- Garc|¤a, Herrera-Castillo, Winemiller & Banda-Valde¤ s ing.VTG has also been used to follow the fate of key 2001), but this practice requires the sacri¢ce of ani- molecules such as amino acids during gar embryonic mals. In addition to a lack of external sexually di- development (Finn, Marel, Mendoza, Aguilera, Evjen morphic features, other problems impeded the & Fyhn 2002). In conclusion, quanti¢cation of VTG successful reproduction of captive gars. For example, by ELISA is a practical, reliable and quick method to the oviducts of female lepisoteids join in a common identify the gender of alligator gar adults without sa- chamber (the urogenital sinus) before opening to cri¢ce and can be used as a de¢nitive marker for the the external environment (Pfei¡er1933; Netch & Witt onset and progress of maturation in female alligator 1962; Suttkus 1963), thus preventing cannulation, gar.VTG assay in gar females has become a popular which is commonly used in other ¢sh to assess the technique using various methods including qualita- degree of oocyte maturation. Development of an ac- tive determinations by immunoprecipitation and curate, non-invasive and sensitive method for sex PAGE (Cortes 2003; Arias, HernaŁ ndez & Contreras identi¢cation became a priority. Research e¡orts 2007) to quantitative determinations such as single were directed to the puri¢cation and quanti¢cation radial immunodifussion (SRID) (HernaŁ ndez,

r 2008 The Authors 750 Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 39, 748^763 Aquaculture Research, 2008, 39, 748^763 Gar biology and culture RMAlfaroet al.

Contreras, Mart|¤ nez&Arias2005)andELISA(Santil- Mendoza, Aguilera, Corte¤ s & HernaŁ ndez 2003). Since laŁ n et al. 2005; Mendoza, Aguilera, SantillaŁ n et al. 2000, a new broodstock was established using cul- 2006; tured and wild-caught juveniles.Wild individuals were Orlando et al. 2007). In some cases, due to a lack caught in ‘Vicente Guerrero’ reservoir, and ‘Soto la of speci¢city of antibodies, quantitative VTG deter- Marina’ river in Tamaulipas. The new broodstock is mination was essential to prevent misidentifying maintained at the‘El Huasteco’aquaculture facility lo- females and males (HernaŁ ndez et al. 2005). cated in Go¤ mez Farias,Tamaulipas. Each gar has been individually tagged with a microchip to follow their Broodstock development performance. In the United States, two and Wildlife Service Broodstock development is a key phase for the do- National Fish Hatcheries and one Regional Fisheries mestication of a new species particularly for threa- Center have worked with the culture and genetics of al- tened species, in which the availability and ligator gar for re-stocking e¡orts. In 1999, the Private reproductive condition of broodstock will determine John Allen National Fish Hatchery in Tupelo, the future availability of larvae and juveniles for the , began spawning wild-caught adult alliga- maintenance of the species in captivity.Within the tor gar and producing alligator gar larvae and juveniles last two decades, have been established for re-stocking e¡orts in the southeastern US Tisho- for each extant Atractosteus species. mingo National Fish Hatchery,Tishomingo, , has also worked with the culture and production of ju- Alligator gar. In , alligator gars venile alligator gar since 2000 (R. Campbell, US Fish have been spawned in captivity since1982 to supple- and Wildlife Service, pers. comm.). The Conservation ment the low numbers of wild larvae. Nevertheless, Genetics Lab at the Warm Springs Regional Fisheries several obstacles hindered the successful captive re- Center inWarm Springs, , is currently develop- production of alligator gar including the species’ ing microsatellite markers to assist in maintaining ge- short natural reproductive season, which is re- netic diversity for the long-term viability of stricted to a few weeks per year. Additionally, the supplemented alligator gar populations (G. Moyer, US mean age of Mexican broodstock is estimated to be Fish andWildlife Service, per. comm.) 15 years with some individuals as old as 35 years (G. Morales pers. comm.). The number of eggs . The ¢rst attempts to establish a Cuban spawned, fertilization rates and larval production gar broodstock were made in the early 1970s at the have been erratic, probably due to the overall age of Center for Fluvial Repopulation in Loma de Tierra, the broodstock. Owing to irregular annual fertiliza- Habana, Cuba. The observations and experiments tion rates (10^70%) and annual numbers of larvae conducted have produced the majority of the pub- produced (10000^400 000), a protocol that uses the lished information on the biology and culture of this previously describedVTG assay was developed to pro- species (Leo¤ n et al.1978). Currently,a new Cuban gar vide more consistent annual fertilization rates and broodstock has been established at the Center for larval production (Fig. 2). Native Ichthyofauna Reproduction located in the Za- Establishment of a new broodstock from wild and pata Peninsula, Cuba (Comabella et al. 2006). cultured juveniles coupled with the use of VTG assay would allow for gender identi¢cation and for the eva- luation of di¡erent hormones to induce sexual matura- Tropical gar. Within their historical range, tropical tion and spawning outside of the natural season to gar are the most abundant Atractosteus species. The provide year-round availability of larvae and juveniles. tropical gar is the smallest and earliest maturing spe- In the early 1980s, the Aquaculture Center ‘Tancol’, cies of Atractosteus (Fig.1). The abundance of tropical located in Tampico,Tamaulipas, Mexico, collected and gar and the species’relatively small size and early ma- maintained wild adult alligator gar. An unplanned turation as compared with other members of Atrac- spawning without the use of hormones occurred, thus tosteus allowed for the establishment of several prompting the maintenance of these individuals until broodstocks. However, the erratic results of captive the present. This broodstock is composed of 40 indivi- spawning and larval maintenance resulted inthe dis- duals, which have produced larvae annually and have solution of some of these broodstocks. The largest been used to repopulate nearby water bodies (Morales current broodstock assemblage is located at the 1987; Mendoza, Aguilera, Rodr|¤guez et al.2002; Aquaculture facilities of the University JuaŁ rez r 2008 The Authors Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 39, 748^763 751 Gar biology and culture RMAlfaroet al. Aquaculture Research, 2008, 39, 748^763

Figure 2 Protocol for the devel- opment and evaluation of brood- stock reproductive quality in northern Mexico.

Auto¤ noma de Tabasco (UJAT),Villahermosa, Mexico, results were achieved for the three species using syn- which maintains up to 130 individuals (HernaŁ ndez thetic analogues of gonadotropin-releasing hor- 2002). Ongoing e¡orts on wild-caught tropical gar in- mones (GnRH). Gonadotropin-releasing hormones duction to maturation and spawning are being car- are the major regulators of reproduction in verte- ried out at the National University of Costa Rica brates acting as a ¢rst signal from the hypothalamus (M. Protti, pers. comm.). to pituitary gonadotropes. Sherwood, Doroshov and Lance (1991)reported that alligator gar and two other Sexual maturation and spawning induction ancient bony ¢shes, the reed¢sh Calamoichthys cala- baricus and white Acipenser transmontanus, Most lepisosteids spawn naturally during spring and have two forms of GnRH. Furthermore, GnRH ana- summer (Simon & Wallus 1989) when spawning oc- logs are e¡ective in most ¢sh and are more cost e¡ec- curs intermittently, resulting in six to seven e¡ective tive than are other hormones when used in large spawning days during the spawning season (Dean species such as alligator gar and Cuban gar. Implants 1895). An exception is the tropical gar that spawns induced spawning in tropical gar but negative results from March to October (Chavez-Lomel|¤ et al. 1989) were obtained when used in alligator gar.This is pre- with a peak in spawning activity in July and August sumably due to the weight of females in relation to (Pe¤ rez-SaŁ nchez 1995). The prolonged spawning peri- the quantity of hormones released by the implants. od of tropical gar may indicate that the length of the For example, mean weights of female alligator gar spawning season in lepisosteids is related to latitude broodstock range from 6.6 kg (GonzaŁ lez 2007) to and temperature. Captive Atractosteus were typically 20 kg (Cortes 2003), whereas tropical gar females spawned annually during the natural spawning per- are reported to have a mean weight of 2.5 kg iod for each species. In some cases, spawning of cap- (HernaŁ ndez 2002). tive alligator, tropical and Cuban gar occurred without the use of hormones in earthen ponds con- taining vegetation or arti¢cial spawning substrates, Larval culture simulating the £ooded grounds in which they nor- mally spawn. Although spontaneous non-hormone- As in other ¢sh species, the dependence of gar larvae induced spawning of captive broodstock occurred, and juveniles on live food has been one of the main the numbers of larvae produced were not consistent impediments to the culture of these ¢sh.Young gars from year to year. grow rapidly and therefore require constant and ade- The use of di¡erent hormones in the three Atrac- quate supplies of live prey items. Often, the cost and tosteus species was evaluated for the induction of ma- labour associated with the production of live food re- turation and spawning in adults (Table 1). The best sult in losses due to cannibalism and the production

r 2008 The Authors 752 Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 39, 748^763 Aquaculture Research, 2008, 39, 748^763 Gar biology and culture RMAlfaroet al.

Table 1 Homologous and heterologous hormones used to induce maturation and spawning in Atractosteus species

Species

Hormone Atractosteus spatula Atractosteus tristoechus Atractosteus tropicus

Heterologous chorionic gonadotropin Human chorionic Human chorionic gonadotropin gonadotropins (1) But low larval survival gonadotropin ( ) (Bejerano et al. 1997) (Colunga 1996) ( ) ( ) SG100 ( ) (Gonza´lez 2007)Ã Carp hypophisis (Pe´rez-Sa´nchez 1995)z ( ) (Leo´n et al. 1978)w Promoted gonadal maturation but no spermiation was registered Homologous (1) After 35 days (Leo´n ( ) (Pe´rez-Sa´nchez 1995) gonadotropins et al. 1978)w 17b-Estradiol ( ) (Herna´ndez 2002) OvaprimTM (1) Out of the regular spawning season – ( ) from April to August (Pe´rez-Sa´nchez 1995) (Gonza´lez 2007)Ã (1) (Herna´ndez 2002) Des-Gly10-LHRHe (1) (Gonza´lez 2007)Ã (1) (Herna´ndez 2002) 6 D-Ala -LHRHa (1), Out of the regular spawning season LHRHa: (1) (Comabella (1) (Herna´ndez 2002; [43]Herna´ndez & (October) (Gonza´lez 2007)Ã et al. 2006) Contreras 2005) Implants Ovaprim: ( ) (Gonza´lez 2007)Ã GnRH-a: (1) (Herna´ndez et al. 2007)

Plus sign (1) indicates successful induction of maturation and spawning, minus sign ( ) indicates unsuccessful induction of matura- tion and spawning. Ã Inductions reported by GonzaŁ lez (2007) were performed from 2003 to 2006. wInductions reported by Leo¤ n et al. (1978) we re p e r fo r me d f rom 1971 to 1973, zInductions reported by Pe¤ rez-SaŁ nchez (1995) were performed from 1994 to 1995. of larvae and juveniles of di¡erent size. Because et al.1978; Simon & Wallus1989; Aguilera et al.2002) growth rates vary among individuals, maintaining and larvae are characterized by an extended yolk-sac adequate live prey density to prevent cannibalism is period (10^30 days, Aguilera et al. 2002). As a conse- di⁄cult.Therefore, larvae and juveniles have been re- quence, larvae are large (23^30 mm, Pearson et al. leased at young ages to avoid cannibalism when 1979; Ag u i le ra et al. 2002) and well developed when maintained at high densities. Release of smaller, exogenous feeding begins. For alligator gar and tropi- younger larvae rather than larger, older larvae or ju- cal gar, morphometric studies indicated that the veniles may result in reduced survival. In an attempt stage of larval development for both species could be to prevent cannibalism among cultured alligator gar, di¡erentiated by snout length, suggesting that this larval growth and the development of the digestive characteristic may be used to evaluate growth of gar tract were examined in an e¡ort to formulate an ef- larvae (Aguilera et al.2002). Moreover, caudal pedun- fective feeding strategy usingarti¢cial feeds. A multi- cle depth, head width and preanal depth were found disciplinary approach aimed at describing the major to be the morphometric characteristics that best dif- morphophysiological changes that take place during ferentiated between fed and starved gar larvae, and the larval period was used and this helped to develop may be used to evaluate the nutritional condition of a feeding strategy using arti¢cial diets for A. spatula larvae of similar developmental stages. Alligator gar and later forA. tropicus. larvae approached adult snout-length proportion at an earlier size and age than did tropical gar larvae (Aguilera et al. 2002; Mendoza , Aguilera, Rodr|¤guez Morphology and growth of gar larvae et al. 2002). This suggests a faster metamorphosis Morphological characteristics of early-life stages from the larval to juvenile state in alligator gar. have been described for Lepisosteus oss- The fastest larval growth rates (5.6 mm day 1) eous, L. platostomus and spotted gar were observed for 10 days post-hatching (dph) alliga- L. oculatus (Netch & Witt 1962; Pearson, Thomas & tor gar larvae exceeding juvenile growth rates re- Clark 1979; Yeager & Bryant 1983; Simon & Wallus ported for juvenile spotted gar (1.3^1.7mm day 1) 1989; Simon & Tyberghein 1991) and for A. tropicus and longnose gar (2.33^4.5mm day 1) (Netch & Witt and A. spatula (Aguilera et al. 2002). Gar eggs are 1962; Echelle & Riggs 1972; Simon & Wallus 1989). moderately large (egg diameter range 3^5 mm, Leo¤ n Larval growth rates of tropical gar (1.0 mm day 1)

r 2008 The Authors Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 39, 748^763 753 Gar biology and culture RMAlfaroet al. Aquaculture Research, 2008, 39, 748^763 were similar to rates reported for larvae of longnose N˛ue 1995; Gisbert, Rodriguez, Costello¤ -Orvary & gar (0.8 mm day 1) and spotted gar (0.83 mm day 1) Willot 1998). Once gar larvae started feeding, struc- (Pearson et al.1979; Simon & Tyberghein1991). These tural changes were observed, including the develop- data con¢rm that lepisosteids, and particularly alli- ment of intestinal folds and the enlargement of gator gar, are among ¢sh with the fastest larval enterocytes. According to Dabrowski (1986) these growth rates (Netch & Witt 1962). changes are indicative of a rapid maturation process Nutritional stages for alligator gar and tropical gar during this period. In addition, the existence of yolk larvae were distinguished byAguilera et al. (2002) ac- reserves in the bodycavity until 8dph was observed cording to the criteria established by Beccaria, Diaz, through histological analysis, con¢rming the propo- Connes and Chatain (1991). Utilizing these criteria, sal for the di¡erent nutritional phases in gar larvae larvae between 6.8 and 13.5 mm TL (1^4 dph) were (Aguilera et al. 2002). Moreover, the existence of yolk lecithotrophic. At this stage larvae remained at- reserves help to explain the absence of qualitative tached to vegetation and received nutrition only from and quantitative di¡erences (enterocytes height) be- the yolk sac. Larvae between 12.5 and 22.5 mm TL tween starved and fed larvae until 8 dph. Once these (5^8 dph) were lecithoexotrophic, when exogenous reserves are exhausted (9^10dph), conspicuous dif- feeding began, although fed and unfed larvae did ferences in the mid-gut cell height of fed and starved not show di¡erences in growth, indicating that yolk larvae were observed (AŁ lvarez et al. 1999). Thus, mid- reserves are still present. Finally,the exotrophic stage gut cell height served as avaluable tool for evaluating began around 22 mmTL, when morphological di¡er- the suitability of arti¢cial diets in gar larviculture, as ences were observed in unfed larvae, indicating a in other studies. (Oozeki, Ishii & Hirano 1989; Thei- total dependence on exogenous food. lacker & Watanabe 1989; McFadzen, Lowe & Coombs Despite the lack of detailed descriptions of mor- 1994; Theilacker & Porter 1995). At 15dph, gastric phologyand larval development of Cuban gar, the ob- glands of starved larvae were still underdeveloped or servations reported by Leo¤ n et al.(1978)and degenerated when compared with those of fed larvae. Comabella et al. (2006) describe a development Hepatic cells of starved larvae contained condensed pattern similar to alligator gar. cytoplasm with no intracellular spaces (areas of gly- cogen and lipid storage), and muscle ¢bers were atro- phied. From these results, it was possible to assume Organogenesis that the structural degree of development of the di- Embryonic development of lepisosteids shares char- gestive tract of gar larvae would not hamper their acteristics with both telosteans and chondrosteans weaning onto arti¢cial diets. (e.g. egg segmentation) (Dean 1895). The appearance and development of di¡erent structures were histolo- Growth indicators gicallyassessed to determine when the digestive tract of alligator gar was fully developed (Aguilera 1999; The need for sensitive and reliable indicators of AŁ lvarez, Sarmiento & Mendoza 1999; Mendoza & growth and nutritional condition has led to extensive Aguilera 2001; Mendoza, Aguilera, Rodr|¤guez et al. research on molecular indicators, particularly nucleic 2002). Upon hatching, the digestive tract develops acids that play a major role in growth and develop- from an undi¡erentiated epithelium surrounding ment. The relationship between RNA and DNA is an the yolk sac. Di¡erentiation takes place rapidly start- index of the cell’smetabolic activityand has been used ing from the posterior end beginning with the forma- to measure short-term growth in ¢sh (Clemmesen tion of the in 3 dph larvae. At the onset of 1996). The RNA/DNA ratio relies on the theory exogenous feeding (5 dph) the stomach and pancrea- that transcription-dependent protein synthesis is tic tissue can be observed, despite the presence of positively correlated with ribosomal activity and an yolk reserves. These observations suggest that the di- increase in cellular RNA levels, while DNA concentra- gestive tract was completely formed. Digestive tract tion does not increase during transcription-depen- development at the start of exogenous feeding places dent protein synthesis, thus providing a point of gar among a small group of ¢sh, including reference (Mommsen1998).The quanti¢cation of total and some salmonids, which possess a di¡erentiated nucleic acids concentrationallowed for comparison of stomach at this stage of development (Buddington & the parallel increase in weight and size, and DNA Christo¡erson 1985; Buddington & Doroshov 1986; and RNA concentrations in gar larvae that were Dabrowski 1986; Gawlicka, Hung, Hinton & de la fed live prey or arti¢cial diets. The RNA/DNA ratio

r 2008 The Authors 754 Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 39, 748^763 Aquaculture Research, 2008, 39, 748^763 Gar biology and culture RMAlfaroet al. increased until13dph, remaining constant thereafter, detected respectively,and between13 and14 dph the which may imply that growth, as a product of protein remaining four alkaline proteases were distin- accretion, stabilizes at this stage, while larvae con- guished in Cuban gar. Finally,the appearance of pro- tinue growing with the same metabolic intensity. teases in tropical gar was the most delayed.The acidic These data support morphological observations indi- and ¢rst alkaline proteases were detected at 5 and cating the end of metamorphosis, the start of expo- 6 dph respectively, two alkaline proteases were de- nential growth and the stabilization of the cell height tected at 13dph, two additional alkaline proteases of enterocytes. In contrast, in starved larvae, RNA were detected at 19 and at 34 dph the last alkaline and DNA concentrations as well as the RNA/DNA protease was detected (Fig. 3). Acidic activity was ratio remained low throughout larval development. inhibited by pepstatin‘A’for the three species, indicat- These observations allowed for the di¡erentiation ing their similarity to aspartic proteases, particularly between starved and fed larvae, and among larvae pepsin. All of the bands exhibiting alkaline pro- that were fed di¡erent diets. Other indices includ- teolytic activity were inhibited by soybean trypsin ing protein/dry weight, protein/DNA, RNA/protein, inhibitor, known to inhibit serine proteases. Phenyl- DNA/dry weight and RNA/total length were used to methylsulphonyl £uoride, also a serine proteases in- con¢rm the results of RNA/DNA ratio analysis to hibitor, inhibited the activity of three to four bands. identify starved versus fed larvae and to identify lar- Three bands were inhibited for each species by the vae that were fed di¡erent diets (Mendoza, Aguilera, speci¢c trypsin inhibitor N-tosyl-L-lysine-chloro- Carreo¤ n et al. 2002; Mendoza, Aguilera & Carreo¤ n methyl ketone, while only two were inhibited by L-1- 2002; Mendoza, Aguilera, Carreo¤ n, Montemayor & tosylamide-2-phenylethyl chloromethyl ketone, a GonzaŁ lez 2007). speci¢c chymotrypsin inhibitor. Only one or two bands were inhibited for each species by EDTA, which is speci¢c to metaloproteases (aminopeptidase Enzymatic ontogenesis or carboxipeptidase). Based on these results, alkaline As mentioned above, several digestive tract struc- proteolytic activity in lepisosteids is due to the activ- tures were found to be functional at the onset of exo- ity of serine^proteases-like enzymes. genous feeding, thus supporting the ability of gar Despite the close phylogenetic relationships larvae to utilize arti¢cial diets at the onset of exogen- among A. spatula, A. tristoechus and A. tropicus, sev- ous feeding. Using substrate ^ PAGE ^ electrophoresis eral di¡erences in the maturation of the digestive the quantity, type and appearance of digestive pro- tract, important to the feeding strategies of cultured teolytic enzymes were studied for alligator (Aguilera, larvae, were observed.The presence of pepsin-like ac- Mendoza, Garc|¤a & Nolasco 1998; Aguilera 1999; tivity, in all three species, from the onset of feeding Mendoza , Aguilera, Rodr|¤guez et al. 2002), Cuban (5 dph) reveals an unexpectedly rapid development (Comabella et al. 2006) and tropical (Iracheta 2006) of the digestive tract, and is re£ected in the carnivor- gar larvae. Seven di¡erent bands with proteolytic ac- ous food habits of these species. Notwithstanding, tivity have been reported for each species (Table 2). this precocious development is indicative of their Alkaline proteolytic activity corresponded to six ability to be fed arti¢cial diets at the onset of exogen- bands of di¡erent molecular weights. Only one band ous feeding. Diets should be formulated with ingredi- was related to acidic activity in alligator gar and tro- ents susceptible to acidic digestion. The gradual pical gar, whereas ¢ve bands had alkaline proteolytic increase in alkaline digestion implies that the ¢nal activity and two had acidic activity in Cuban gar. On- maturation of the digestive tract takes place in the in- togenetically, alkaline proteases of lower molecular testine and associated tissues (e.g. pancreas), unlike weight appeared before proteases of higher molecu- most marine ¢sh larvae in which the stomach is the lar weight for the three species. The expression of last functional region of the digestive tract to mature. proteolytic activity was the most rapid in alligator The fastest digestive tract maturation rate was ob- gar, where at 5 dph (beginning of exogenous feeding) served in alligator gar, followed by Cuban gar and four alkaline proteases and one acidic protease were tropical gar and was related to growth rate and ¢nal detected, while the two remaining alkaline proteases size (Aguilera et al. 2006). However, in the ¢nal stage were distinguished at 15dph. In Cuban gar, the ap- of digestive tract development, di¡erences in the pearance of proteases was slower. At 5 dph only one number and types of proteolytic enzymes are negligi- acidic protease was detected, at 7 and 8 dph a second ble, suggesting that juveniles of the three species acidic protease and one alkaline protease were share similar digestive abilities. This would allow for

r 2008 The Authors Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 39, 748^763 755 Gar biology and culture RMAlfaroet al. Aquaculture Research, 2008, 39, 748^763

Table 2 Digestive proteases detected by substrate-PAGE (zymograms) in lepisosteids larvae

Gar species

Protease enzyme no. Atractosteus spatula Atractosteus tristoechus Atractosteus tropicus

1 Alkaline: 18.8 kDa Alkaline: 21.8 kDa Alkaline: 25.2 kDa 2 Alkaline: 23.3 kDa Alkaline: 28.4 kDa Alkaline: 28.5 kDa 3 Alkaline: 26.8 kDa Alkaline: 34.9 kDa Alkaline: 37.2 kDa 4 Alkaline: 33.7 kDa Alkaline: 41.4 kDa Alkaline: 44.1 kDa 5 Alkaline: 36.4 kDa Alkaline: 46.4 kDa Alkaline: 53.1 kDa 6 Alkaline: 43.7 kDa Acid: 0.71 Rf Alkaline: 54.8 kDa 7 Acid: 0.85 Rf Acid: 0.88 Rf Acid: 0.95 Rf

The quantity (no.), type (alkaline or acid), molecular weight (kDa) or mobility (Rf) are listed for each digestive protease. the formulation of a single diet that could be used for Medale, Fauconneau, Kaushik & LeBail 1991). Within the culture of all three species. In order to formulate this context, the coding sequence of GH from alligator the most e¡ective diet, ongoing research is devoted to gar was obtained and its expression throughout the puri¢cation of enzymes from alligator gar by larval development was studied (Mendoza, Garza et al. chromatography for use in in vitro digestibility tests 2002; Revol, Garza, HernaŁ ndez, Aguilera, Barrera & to select local ingredients for inclusion in arti¢cial Mendoza 2005).The analysis of the fragment sequence diets. con¢rmed that it corresponded to a GH gene, sharing 98% of nucleotide similarity with the GH previously reported for L. osseus (Rubin, Youson, Marra & Dores Hormones in larval development 1996). A single amino acid change (Val/Ala) in the ¢fth Fish growth hormone (GH) is a pituitary hormone re- exon was observed between the two lepisosteids. The sponsible for somatic growth; however, it can also be few residue changes observed between the GHs of expressed in extrapituitary tissues of adults and in these two ancient ¢sh that diverged 180 million years early embryos. Growth hormone is known to stimulate ago support the theory of very slow evolution of the appetite (Pickford & Atz1957; Higgs, Donaldson, Dye & hormone within the ancient ¢sh, as compared with McBride 1975), food conversion e⁄ciency (Markert, the burst of changes observed in the euteleosts (Gayet, Higgs, Dye & MacQuarrie 1977), lipid mobilization Meunier & Werner 2002).The relative expression of GH (Sheridan 1986), nitrogen retention (Matty 1962), ami- along larval stages indicates substantial expression of no acid incorporation into tissues (Cheema this hormone in unfertilized eggs, declining thereafter & Maty1978; Fauconneau, Mady & LeBail1996) and sti- until 3 dph, then increasing around 5^7 dph larvae, mulation of energy and protein metabolism at the corresponding to organogenesis. In 8 dph larvae the whole body level (Medale, Fauconneau & Kaushick expression of GH decreases again, and is consistent 1988) and at the tissue level (Foster, Houlihan, Gray, with the depletion of yolk reserves and the onset of

Figure 3 Ontogenetic appear- ance of proteases in Atractosteus species.

r 2008 The Authors 756 Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 39, 748^763 Aquaculture Research, 2008, 39, 748^763 Gar biology and culture RMAlfaroet al. exogenous feeding. Lastly, the expression of GH in- the other treatments (Fig. 5). This may have resulted creases in 9^10dph larvae, probably corresponding to from exposure of larvae to exogenousT3 added to the the formation of pituitary gland (Fig.4). naturally high endogenous levels of T3 found in indi- Other key hormones implicated in larval develop- viduals of all groups. Abnormally high levels of T3 ment are thyroid hormones (TH). The nature of the may be related to the high concentrations of cortisol growth promoting e¡ect of TH in larval ¢sh meta- in this group. Both hormones may have contributed morphosis may involve induction of morphological to the lower survival and deformities observed in lar- changes in the digestive tract and accessory organs, vae treated withT3. Higher values of weight gain and stimulation of renewal processes of the intestinal total length were observed in the TU and control (C) epithelium, increasing nutrient absorption capacity, groups. The lower weight attained by larvae exposed and growth enhancement in general (Dabrowski & to T3 could be explained by energy invested in pre- Culver 1991). Thyroid hormones may have a great mature metamorphosis. Snout length was signi¢- practical value in aquaculture because they not only cantly reduced in the TU group, whereas in the T3 act as growth promoters but also trigger and acceler- group snout development was accelerated. These re- ate larval development (Lam1980). In the case of alli- sults re£ect the feasibility of altering snout develop- gatorgar,3,30, 5-triiodo-l-thyronine (T3) levels ment and present novel alternatives for the control during embryonic development are low, as shown by of cannibalism under culture conditions, through the concentrations in recently spawned eggs and eggs the retardation of snout growth by exposure to anti- just before hatching (0.0117 and 0.00995 ng egg 1 thyroid compounds (TU) without a¡ecting growth respectively). However, after hatching these levels in- and survival. crease, reaching 2.22 ng larvae 1 at 10 dph, decreas- ing thereafter to 0.3 ng larvae 1 at 13 dph (Aguilera Weaning 1999).These data, coupled with morphological obser- vations, indicate that the metamorphic climax takes Based on the results obtained through the multidisci- place around10dph. This contention is supported by plinary research described above, it could be as- the occurrence of two main events before and after sumed that gar larvae should readily accept and 10dph, the exhaustion of yolk reserves by 8 dph and ulitize arti¢cial diets. However, there were no reports the beginning of exponential growth at11dph. These con¢rming this presumption for tropical gar or Cu- observations together with the rising levels of GH ex- ban gar. On the contrary, most attempts to culture plain the rapid growth rate of alligator gar at this larvae of these species have failed, due to high mor- stage. Considering the use and importance of exo- tality rates (Leo¤ n et al.1978;Garc|¤a, Marquez & genous TH and glucorticosteroids as potential regu- Paramo 1997; G. Morales 2000, pers. comm.; G. Mar- lators of larval gar development, the e¡ects of the quez 2001, pers. comm.). In addition, poor results exposure of larvae to T3, hydrocortisone (HC) or were obtained when juveniles of these species were thiourea (TU); (Mendoza, Aguilera & Montemayor fed live bait ¢sh. (Maldonado 1991). Therefore, live 2001;Mendoza,Aguilera,Rodr|¤guez et al. 2002) were prey was considered the only alternative for the suc- determined. Concentrations of T3 were three times cessful production of lepisosteid larvae, particularly higher in the T3 treatment, when compared with for tropical gar (HernaŁ ndez, Marquez, PaŁ ramo, Fe¤ lix

Figure 4 Concentration of T3, cortisol and relative GH expression throughout the larval develop- ment in alligator gar. T3, 3, 30,5- triiodo-l-thyronine; GH, growth hormone.

r 2008 The Authors Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 39, 748^763 757 Gar biology and culture RMAlfaroet al. Aquaculture Research, 2008, 39, 748^763

Figure 5 Concentration of T3 and cortisol from alligator gar larvae exposed toT3,hydrocor- tisone (HC) and thiourea (TU). T3, 3, 3 0,5-triiodo-l-thyro- nine.

&HernaŁ ndez 1997; Rodr|¤guez, Marquez & Paramo micro-spheres that have lower £oatability, but whose 1997; He r naŁ ndez1999). Feeding trials to produce alli- particles do not lose their shape and can be individu- gator gar and Cuban gar larvae were of short dura- ally ingested (Mendoza et al. 2007). This determina- tion, and were restricted to early developmental tion of suitable characteristics of arti¢cial diets was stages when food acceptance and cannibalism were paramount for lepisosteids considering their strong not yet problematic (G. Morales 2000, pers. comm.). predatory habits. Between 1997 and 1999, the ¢rst attempts to culture The feeding strategy for gar larvae based on that gar larvae using arti¢cial diets were also unsuccess- established for alligator gar can be summarized as ful. For these trials gar larvae were fed nauplii and follows: Free swimming larvae are transferred to adults of Artemia sp., but the culture ended at ¢bre glass tanks to allow for even distribution of indi- 15dph, when larvae reached a maximum size of viduals at the water surface. Starter feed consists of a 50 mm, before the type of food could a¡ect survival combination of £oating particulate diet (0.5 mm dia- and growth. Although good growth and survival meter) and Artemia nauplii. As soon as yolk reserves rates were not attained, some important characteris- are depleted a complete substitution of arti¢cial feed tics of arti¢cial diets were identi¢ed, and the strategy for Artemia should occur. For alligator gar larvae, for achieving the acceptance of arti¢cial diets was de- Artemia nauplii were eliminated from the diet with- ¢ned (Aguilera1999).The following year, the ¢rst suc- out decreased growth or survival.When the highest cessful results were obtained (Mendoza & Aguilera growth rate is achieved, feed particle diameter 2000; Mendoza, Aguilera, Montemayor et al. 2000). should be increased. At this point it is necessary to Observations of gar feeding behaviour indicated that adjust larval density according to the tank surface larvae feed near the water surface and that chemical area to allow all individuals access to the water sur- recognition of feed, indicated by the static position of face. Culture tanks with a large surface area and low larvae near the feed and the contact with the snout turbulence, as opposed to tanks with a small surface before ingestion, is important. This motivated the ad- area and high turbulence, allow for more e¡ective dition of feed attractants to diets for early weaning distribution of feed and larvae. To reduce cannibal- (Rodr|¤guez, Mendoza, Aguilera & Montemayor ism, feed particle size should be increased when lar- 2000). Feeding must be initiated when larvae still car- val densities are adjusted and individuals should be ry yolk reserves and it is imperative to increase parti- sorted and separated according to size to prevent cle size as snout length increases. Gar larvae can be cannibalism. Unfortunately, cannibalism remains conditioned to the consumption of arti¢cial diets from the ¢rst cause of mortality in alligator gar larvae ran- the ¢rst feeding provided that the feed £oats. Because ging from 50 to 150 mm. However, it is seldom ob- of the need for a £oating feed, extruded diets and mi- served in individuals larger than 200 mm, even cro-spheres were chosen. In fact, these diets resulted under starvation. Survival may be higher than 90% in higher growth rates than did live prey (Artemia sp.). by 15dph. Great care is required during the ¢rst On the other hand, crumbled diets despite their high- month of culture due to continuous handling to er £oatability, tend to agglomerate, in contrast with adjust larval densities. This feeding strategy has been

r 2008 The Authors 758 Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 39, 748^763 Aquaculture Research, 2008, 39, 748^763 Gar biology and culture RMAlfaroet al. successfully used to wean tropical gar larvae onto ar- References ti¢cial feed (Iracheta 2006; MaŁ rquez-Couturier et al. Aguilera C. (1999) Bases ¢siolo¤gicas del desarrollo de larvas de 2006), and will be used to wean Cuban gar larvae cataŁ n (Atractosteus spatula) y perspectivas para su cultivo. onto arti¢cial feed. Thesis doctoral, Facultad de Ciencias Biolo¤ gicas, UANL, Monterrey,NL,186pp. Juvenile production and growth to commercial size Aguilera C., Mendoza R., Garc|¤aF.&NolascoH.(1998)De- sarrollo Ontoge¤nico EnzimaŁ tico del CataŁ n, Atractosteus spa- The adoption of the above-mentioned feeding strat- tula’’ IV Symposium Internacional de Nutricio¤ nAcu|¤cola, egy using commercial trout feeds (45% protein) has November1998 La Paz, Baja California. allowed for the production of alligator gar juveniles Aguilera C., Mendoza R., Rodr|¤guez G. & Marquez G. (2002) of 30 cm and 250 g in 4 months (Mendoza , Aguilera, Morphological description of alligator gar and tropical Rodr|¤guez et al. 2002). Recent results in tropical gar gar larvae, with an emphasis on growth indicators. 131, culture have con¢rmed the utility of this diet and 899^909. feeding strategy (AŁ lvarez, Contreras, Castillo, Santa- Aguilera C., Mendoza R., Marquez G. & Iracheta I. (2005) Al- na & Gallego 2007), achieving weights of 130^155 g ligator gar (Atractosteus spatula) larval development and early conditioning to arti¢cial diets. Aquaculture America in 5 months. This is due in part to the feeding e⁄- 2005.World Aquaculture Society. New Orleans, LA, USA, ciency and rapid growth of alligator gar. Juvenile alli- E nero 19, 2005. gator gar had a speci¢c growth rate (SGR) between 5 Aguilera C., Mendoza R., Comabella Y. & Marquez G. (2006) and 8, a FCR lower than 1 and protein e⁄ciency ra- Comparacio¤n dela actividad proteol|¤tica digestiva en larvas dele- tios (PER) between 2 and 5 (Aguilera, Mendoza, Mar- pisosteidos. IConferencia Latinoamericana sobre el cultivo de quez & Iracheta 2005). In contrast, values of FCR of peces nativos.18^20 Octubre, Morelia MichoacaŁ n, Me¤ xico. 1.6,PERof2andadailygrowthof7%arereported Aleman L. & ContrerasW.(1987) Algunas Consideraciones So- for juvenile tropical gar (MaŁ rquez-Couturier et al. bre el Pejelagarto Lepisosteus tropicus (Gill) y Descripcio¤nde 2006). sus HaŁ bitos Alimenticios. Memorias del IX Congreso Na- cional de Ictiolog|¤a.13^16 de Octubre,Tabasco, Me¤ xico. AŁ lvarez J., Sarmiento M. & Mendoza R. (1999) Estudio histo- lo¤gico del desarrollo del tracto digestivo en larvas de cataŁ n Conclusion Atractosteus spatula en condiciones de inanicio¤n.Resu¤ - At present, the basic technology for the commercial menes del XXIII Congreso Nacional de Histolog|¤a, Mon- terrey,Me¤ xico,35pp. culture of di¡erent species of gar has been devel- AŁ lvarez C., ContrerasW., Castillo K., Santana O. & Gallego R. oped and aquaculture of gar species is expected to (2007) Evaluation of commercial diets on tropical gar, Atrac- increase in Mexicoand Cubabecause gar is a staple tosteus tropicus, growth. World Aquaculture Society, food in some regions. Currently, further analysis of 26 February ^ 2 March, 2007, San Antonio, TX, USA nutritional requirements for lepisosteids is being (abstract 835). carried out (MaŁ rquez-Couturier et al.2006).Poly- Arias G., HernaŁ ndez U. & Contreras W. (2007) Sex identi¢ca- culture trials of alligator gar and cat¢sh in cages tion of tropical gar,Atractosteus tropicus, juveniles by vitello- and earthen ponds are also under way (Mendoza, genin detection in skin mucus. World Aquaculture Society, Aguilera, Montemayor et al.2006). Because of their 26 February ^ 2 March 2007, San Antonio, TX, USA position in the food web, gars are being studied as (abstract 860). indicators of contaminants (Orlando et al. 2007). AQUACOP & Calvas J. (1990) State of the art of IFREMER in A microsatellite library is being developed to study tropical aquaculture. In: Advances inTropical Aquaculture the genetic relationships of alligator gar from di¡er- (ed. by Jaques Calvas, Gerard Cuzon, Jaques Fuchs & ent regions of United States and Mexico (W. Karel Maurice Weppe), pp. 625–642. Aquacop-IFREMER, Actes de Colloque 9, Paris. pers. comm.). Beccaria C., Diaz J., Connes R. & Chatain B. (1991) Organo- genesis of the exocrine pancreas in the sea bass, Dicen- trarchus labrax L., reared extensively and intensively. Acknowledgments Aquaculture 99,339^354. Bejerano G., Marquez G. & PaŁ ramo S. (1997) Propuesta Meto- The authors wish to acknowledge CONACYT-SE- dolo¤gica para la Induccio¤n al Desove del Pejelagarto, Atractos- MARNAT (Ref: SEMARNAT-2002-CO1-0882) for ¢- teus spatula. Memoria de la Semana de Divulgacio¤ ny nancing the Project and Belen Elizondo Morales for Video Cient|¤ ¢co1997. Secretar|¤a de Servicios Acade¤ micos. her editorial assistance. Universidad JuaŁ rez Auto¤ noma deTabasco, pp.80^83. r 2008 The Authors Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 39, 748^763 759 Gar biology and culture RMAlfaroet al. Aquaculture Research, 2008, 39, 748^763

Boudreaux P., Ferrara A. & Fontenot Q. (2007a) Chloride in- Fauconneau B., Mady M. & LeBail P.(1996) E¡ect of growth hibition of nitrite uptake for non-teleost Actinopterygiian hormone on muscle protein synthesis in rainbow trout ¢shes. Comparative Biochemistry and Physiology ^ Part A (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). 147,420^423. and Biochemistry15, 49^56. Boudreaux P., Ferrara A. & Fontenot Q. (2007b) Acute toxi- Ferrara A.M. (2001) Life-history strategy of Lepisosteidae: Im- city of ammoniato spotted gar Lepisosteus oculatus, alliga- plications for the conservation and management of alligator tor gar Atractosteus spatula, and paddle¢sh Polyodon gar. PhD disseration, Auburn University, USA,141pp. spathula. Journal of the World Aquaculture Society 38, Ferrara A.M. & Irwin E.R. (2001) A standardized procedure 322^325. for internal sex identi¢cation in Lepisosteidae. North Buddington R.K. & Christo¡erson P. (1985) Digestible and AmericanJournal of Fisheries Management 21,956^961. feeding characteristics of the chondrosteans. In: North FinnR.N.,MarelM.,MendozaR.,AguileraC.,EvjenN.N.& American Sturgeons: Biology and Aquaculture Potential Fyhn H.J. (2002) Ontogeny of yolk proteins and free amino (ed. by F.P. Binkowski & S.I. Doroshov), pp. 31–41. acids in the alligator gar (Atractosteus spatula). In: DR W. Junk Publishers, Hingham, MA, USA. Proceedings 26th Annual Fish Larval Conference, 22–26 Buddington R.K. & Doroshov S.I. (1986) Development of di- July, Bergen, Norway, p. 52. gestive secretions in white sturgeon juveniles (Acipenser Foster A., Houlihan D., Gray C., Medale F., Fauconneau B., transmontanus). Comparative Biochemistry and Physiology Kaushik S. & LeBail P.(1991)The e¡ects of ovine growth hor- 83A,233^238. mone on protein turnover in rainbow trout. 81, 111 ^ 120. Chavez-Lomel|¤ M.A., Matthews E. & Pe¤ rez-Vega M. (1989) Garc|¤a J., Marquez G. & Paramo S. 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Fish Physiology and Biochemistry 32, sosteiformes. Palaeontology 45,361^376. 147^ 157. Gisbert E., Rodriguez A., Costello¤ -Orvary F. & Willot P. (1998) CortesV.(2003) Puri¢cacio¤nyCaracterizacio¤n de la Lipoviteli- A histological study of the development of the digestive na OvaŁ rica de Ejemplares Adultos deAtractosteus spatula.Te- tract of siberian sturgeon (Acipenser baeri) during early sis de Maestr|¤a, Universidad Auto¤ noma de Nuevo Leo¤ n, ontogeny. Aquaculture167, 195^ 209. 58pp. Go¤ mez-Go¤ mez M. (1989) Reproduccio¤n del pejelagarto en es- Dabrowski K. (1986) Ontogenical aspects of nutritional re- tanquer|¤aru¤stica. Primer seminario sobre Acuacultura PE- querimients in ¢sh. 85A,639^655. MEX-UJAT en el Estado de Tabasco, Marzo,pp.13^14. Dabrowski K. & Culver D. (1991) The physiology of larval Villahermosa,Tabasco, Mexico. ¢sh, digestive tract and formulation of starter diets. Aqua- GonzaŁ lez M. (2007) Control reproductivo y cultivo larvario del culture Magazine17, 49^61. caŁ tan (Atractosteus spatula) Tesis de Doctorado,pp.189. Dean B. (1895) The early development of gar-pike and stur- Facultad de Ciencias Biolo¤ gicas, UANL, Monterrey, NL, geon. Journal of Morphology11,1^53. Mexico. EchelleA. & Riggs C. (1972) Aspects of the earlylife historyof HernaŁ ndez U. (1999) Punto cr|¤tico de no-retorno en larvas gars (Lepisosteus) in lakeTaxoma.Transactions of theAmer- de pejelagarto Atractosteus tropicus (Gill, 1863).Tesis ican Fisheries Society101,106^112. de Licenciatura Ine¤ dita. Divisio¤ n Acade¤ mica de Ciencias FAO/PNUMA (1984) Conservacio¤n de los recursos gene¤ticos de Biolo¤ gicas, Universidad JuaŁ rez Auto¤ noma de Tabasco los peces: Problemas y recomendaciones. Informe de la con- 1997, 46 pp. sulta de expertos sobre los recursos gene¤ ticos de los peces. HernaŁ ndez U. 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