Source: UNIVERSITY OF FLORIDA submitted to
DEVELOPMENT OF AQUACULTURE SPECIES FOR THE FLORIDA ORNAMENTAL INDUSTRY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1005374
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Nov 21, 2014
Project End Date
Nov 20, 2019
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Forest Resources and Conservation
Non Technical Summary
The ornamental fish trade is a valuable sector of global aquaculture production with global retail sales valued at US$3 billion. Freshwater fishes comprise the majority of the volume in the ornamental trade with approximately 90% of the species and varieties captively produced through aquaculture. Conversely, it is estimated that up to 24 million marine fish are sold annually; with the preponderance of specimens resulting from wild capture. Domestic aquaculture production of ornamental species is dominated by Florida growers, however, pressures from fluctuating market demand, competition with foreign importation, and increasing regulatory compliance threatens the continued growth and resiliency of this valuable segment of agriculture. While a vast quantity of ornamental fishes are currently cultured in captivity, empirical evidence to support management decisions and production goals is limited. Fundamental information regarding reproduction, larval culture, and production techniques is critical when evaluating a species for commercial propagation and these bottlenecks will ultimately dictate the success of domestication and cultivation efforts. The research objectives outlined in this proposal were designed to address critical gaps in knowledge when evaluating new aquaculture species. Information generated on captive reproduction, larval culture, and production techniques will allow producers to establish commercial protocols for economically valuable species.
Animal Health Component
50%
Research Effort Categories
Basic
10%
Applied
50%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30137161060100%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3716 - Ornamental finfish;

Field Of Science
1060 - Biology (whole systems);
Goals / Objectives
The overarching objective of the following proposal is to elucidate ornamental species with potential for commercialization within the state of Florida and address critical gaps in knowledge requisite for production. Two specific research foci that will be addressed include:Development of spawning, larval culture, and production techniques for freshwater ornamental fish speciesDevelopment of spawning, larval culture, and production techniques for marine ornamental fish species
Project Methods
1) Development of spawning, larval culture, and production techniques for freshwater ornamental fish species.Freshwater ornamental species of interest will be evaluated for critical deficiencies in knowledge essential for captive spawning, larval rearing, and commercial production. Controlled reproduction experiments will investigate factors such as photothermal regimes, water chemistry, sex ratios, substrate preference, and broodstock nutrition. Fish which do not volitionally spawn following these manipulations will be assessed for hormone induced spawning. Experiments which examine hormone type (hCG, GnRHa), dose, and administration route will provide insight into effective use of these spawning aids. Hatching methodologies will be evaluated to identify methods of disinfection and incubation which result in the greatest percentage of viable larvae for production. Larval culture experiments will focus on variables which promote growth and survival throughout this developmental phase. Experiments will explore various feeding regimes using live feeds such as Artemia sp., Moina sp., Colurella adriatica, and prepared commercial feeds. Additionally, factors such as stocking density, feeding frequency, temperature, aeration, and time for transition from intensive production (recirculating system) to extensive production (ponds) will also be investigated. All experiments will be appropriately designed and replicated to allow for statistical analyses.2) Development of spawning, larval culture, and production techniques for marine ornamental fish species.Captive spawning and larval culture of marine fishes are two significant bottlenecks in marine ornamental aquaculture. Experiments in this objective will be designed to elucidate effective spawning and larval rearing protocols which will have immediate application in the existing industry. Reproductive dysfunction, in particular failure of final oocyte maturation, is relatively common in captive marine fishes. Reproduction experiments will evaluate photothermal regimes, water chemistry, and sex ratios to induce volitional release of gametes. Hormone induction studies (hCG, GnRHa) will be performed for fish which fail to spawn volitionally. Egg disinfection studies will be carried out to establish appropriate chemical concentrations and exposure times which result in reductions in bacterial colony forming units and increased survival of embryos and larvae. Larval studies will examine culture aspects such as stocking density, temperature, aeration, system design, prey density and feeding frequency, light intensity and photoperiod, and probiotics. Additionally, larval feeding trials will assess the use of novel live feed organisms such as copepods (Oithona colcarva, Parvocalanus crassirostris, Pseudodiaptomus pelagicus), Euplotes sp., and rotifers (Branchionus plicatilis) in conjunction with varying concentrations of live microalgae (Isochrysis sp.). Experiments will be appropriately designed and replicated to allow for statistical analyses.

Progress 11/21/14 to 11/20/19

Outputs
Target Audience:The target audience of the proposed research is ornamental fish producers within the state of Florida. Secondary audiences will include public zoos and aquariums, aquaculture researchers, extension specialists and educators, and state agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research project has provided opportunities for education and training of stake holders through interactions during farm visits, laboratory tours, and professional presentations at conferences and workshops. Graduate student participation in ongoing research projects also provides further opportunities for mentoring and professional development through interaction with faculty and staff. How have the results been disseminated to communities of interest?Preliminary findings and culture protocols have been disseminated through presentations at conferences, annual stakeholder meetings of the Rising Tide Conservation initiative, seminars with local aquarists, and online through the use of podcasts, blogs, (http://risingtideconservation.blogspot.com/), websites (http://risingtideconservation.org/), and social media (https://www.facebook.com/risingtideconservation/). Participation in the production of educational videos and interviews with local and national media have also aided in disseminating information to the general public. Site visits, workshops, tours, phone calls, and emails are also frequently employed to deliver research results to industry stakeholders where appropriate. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1.Development of spawning, larval culture, and production techniques for freshwater ornamental fish speciesUse of exogenous hormones may help overcome reproductive dysfunctionin fishes. Dosages of 50, 100, 200 µg/kg of the hormone cGnRH IIa were compared to the ornamental industry standard Ovaprim® (10 µg/kg sGnRHa) using standard metrics for spawning performance. Four commercially valuable ornamental species Epalzeorhynchos bicolor, Epalzeorhynchos frenatum, Synodontis nigriventris and Synodontis eupterus were used to assess spawning aid efficacy. All species exhibited similar ovulation performance across hormone treatments except for E. frenatum injected with Ovaprim (17% ovulation). In general, fertilization and hatch success were comparable for both analogs among species. Embryo and larval morphometric measurements were also similar among treatments across all four species. Taken together, these results suggest cGnRH IIa to be a safe and reliable option for induction spawning of ornamental species. Prophylactic use of disinfectants on developing embryos of teleosts is a common practice in aquaculture. Fertilized embryos from each of the four previously mentioned species were used to assess the safety of 15 minute static exposure to 100 mg/L iodine, 1,000 mg/L hydrogen peroxide and 1,500 mg/L formalin. Results indicated that iodine was detrimental to hatch of all species tested while formalin and/or hydrogen peroxide did not detrimentally affect hatch rate. Data generated from induced spawning and embryo disinfection studies will help to refine commercial production protocols for ornamental fish species. Masculinization research took an applied approach with the aim of developing masculinization techniques in Rosy Barbs and Dwarf Gouramis that would benefit Florida's ornamental aquaculture industry. The results offer a variety of physiological insights into the larval environmental stress response and sexual differentiation in these species, as well as defining basic larval culture parameters. Results from the temperature experiments yielded the first evidence for temperature influence of Dwarf Gourami sex differentiation. Outcomes of the MT based research suggest that it may require relatively high dosages and prolonged durations of larval administration to affect sexual differentiation. Finally, this study produced novel results on the use of MT to influence male secondary sex characteristics in mature female fishes. This strategy may be broadly applicable to the ornamental industry which is comprised of a diverse array of species. Combined these studies offer evidence that the use of MT may be the most effective way for domestic ornamental producers to reliably raise crops of phenotypically male fish. Larval dietetics trials successfully identified microdiets MD's that could be successfully used in place of Artemia at first feeding in six freshwater ornamental fish species: redtailed sharkminnow, bronze corydoras Corydoras aeneus, upside down catfish, featherfin squeaker, freshwater angelfish Pterophyllum scalare, and dwarf gourami Trichogaster lalius. Partial budget analysis indicated for all species except E. bicolor, at least one MD and the associated feeding protocol provided a net economic benefit compared to Artemia nauplii. Characterization of ontogenetic development, and a subsequent weaning trial for Paracheirodon innesi and Gymnocorymbus ternetzi, identified time periods where successful weaning was possible prior to the complete development of the stomach. C. aeneus gastrointestinal characterization revealed a highly precocious pattern of development in this species with gastric glands and PAS positive neutral mucopolysaccharides appearing at 1 dph. Experiments evaluating spawning substrate preference, incubation techniques, embryo disinfectants, first feed dietetics and larval development were carried out to varying degrees with gulf coast pygmy sunfish Elassoma gilberti, blackbanded sunfish Enneacanthus chaetodon, flagfin shiner Pteronotropis signipinnis, and metallic shiner Pteronotropis metallicus. Gulf coast pygmy sunfish and metallic shiners appear most conducive to commercial propagation at this juncture although further research is needed to refine protocols and increase production efficiency. 2. Development of spawning, larval culture, and production techniques for marine ornamental fish speciesThe melanurus wrasse Halichoeres melanurus, yellow wrasse H. chrysus and Pacific blue tang Paracanthurs hepatus, were chosen for the development of culture methodologies due to their popularity in the aquarium trade and lack of empirical information to guide culture efforts. Over the past five years, critical culture aspects were assessed for these species including broodstock spawning characteristics, egg incubation temperature, early larval culture considerations, and first feeding parameters. Broodstock husbandry, spawning, and larviculture methods were described for these species and two generations of melanurus wrasse were produced, effectively closing the life cycle of this species. Additionally, during this project period the Pacific blue tang was successfully cultured in captivity for the first time in the world at the UF-TAL. Larval development and culture requirements of the melanurus wrasse were described across a series of investigations. Initiation of exogenous feeding, swim bladder inflation, and flexion were first observed at 3, 10, and 15 days post hatch (DPH), respectively, and completion of metamorphosis was first observed at 37 DPH. To identify effective culture methods for the yellow wrasse and to further refine culture methods for the melanurus wrasse, a range of studies were conducted to evaluate parameters affecting embryo incubation, larval survival and growth prior to first feeding, and larval feeding success at initiation of exogenous feeding. For both wrasse species, an inverse relationship was found between water temperature and embryo incubation period. An examination of melanurus and yellow wrasse larvae prior to first feeding established that cultures containing algal densities of ≥50,000 cells mL-1 increased larval survival. Results from subsequent experiments evaluating light intensity suggested that the previously noted increase in survival was likely due to the decrease in light intensity and not from the algae itself. Melanurus wrasse first feeding culture trials revealed that algal densities of 300,000 and 500,000 cells mL-1 resulted in elevated larval feeding success and ingestion of Parvocalanus crassirostris nauplii (<75 µm) was significantly increased at prey densities ≥ 5.0 items mL-1. Similar feeding trials conducted with the yellow wrasse resulted in poor feeding performance and larval survival and these investigations did not clearly delineate preferential culture conditions. Results of algal species manipulations suggest inclusion of the microalgae Tisochrysis lutea during early larviculture of the Melanurus Wrasse aids in prey capture whereas the addition of the diatom Chaetoceros muelleri negatively impacts larval survival (P < 0.001). Trials conducted with Pacific Blue Tang show an increased feeding response with inclusion of either T. lutea or Tetraselmis chuii (P = 0.003). The Yellow Wrasse showed no survival or feeding advantage following microalgae inclusion. T. lutea density of 300,000 cells/ml is optimal for both survival (P < 0.0001) and feeding incidence (P = 0.02) for Melanurus Wrasse and for survival of the Pacific Blue Tang (P < 0.0001). Prey density experiments resulted in no difference in feeding incidence across treatments for both species. Inclusion of feed attractants decreased survival (P < 0.0001) and had no effect on feeding incidence for Melanurus Wrasse. Information gathered from these trials will help to advance commercial aquaculture protocols for all three species of marine ornamental fishes.

Publications


    Progress 10/01/18 to 09/30/19

    Outputs
    Target Audience:The target audience of the proposed research is ornamental fish producers within the state of Florida. Secondary audiences will include public zoos and aquariums, aquaculture researchers, extension specialists and educators, and state agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research project has provided opportunities for education and training of stake holders through interactions during farm visits, laboratory tours, and professional presentations at conferences and workshops. Graduate student participation in ongoing research projects also provides further opportunities for mentoring and professional development through interaction with faculty and staff. How have the results been disseminated to communities of interest?Preliminary findings and culture protocols have been disseminated through presentations at conferences, annual stakeholder meetings of the Rising Tide Conservation initiative, seminars with local aquarists, and online through the use of podcasts, blogs, (http://risingtideconservation.blogspot.com/), websites (http://risingtideconservation.org/), and social media (https://www.facebook.com/risingtideconservation/). Participation in the production of educational videos and interviews with local and national media have also aided in disseminating information to the general public. Site visits, workshops, tours, phone calls, and emails are also frequently employed to deliver research results to industry stakeholders where appropriate. What do you plan to do during the next reporting period to accomplish the goals?No further experimentation is planned as this is last reporting period under this project.

    Impacts
    What was accomplished under these goals? 1.Development of spawning, larval culture, and production techniques for freshwater ornamental fish species Research to develop commercially applicable masculinization techniques for two ornamental freshwater species, the Rosy Barb,Pethia conchonius, and Dwarf GouramiTrichogaster lalius has concluded. In 2019, we completed experiments assessing the masculinization effects of 17α-methyltestosterone (MT) on these two species. In Rosy Barbs, none of the 30-day exposures at experimental concentrations resulted in significant masculinization. MT feeding for 60 days did result in male percentages above 85% but these were not significantly different from the control (70%) in that experiment. In Dwarf Gouramis, paradoxical feminization was observed after treatment in high immersion concentrations and following 50 days of MT feed. An additional study investigated the effects of dietary MT administration on adult female Rosy Barbs and Dwarf Gouramis. Male secondary sex coloration was analyzed by a panel of judges on a weekly basis during and after prolonged MT feeding. Growth parameters, survival, and gonadosomatic index (GSI) were also assessed at the end of the feeding regime. All concentrations of MT diets did not affect survival and resulted in coloration changes in both species. However, MT diets caused more pronounced color changes in Dwarf Gouramis and also reduced their GSIs at all diet concentrations. The bright coloration associated with the male phenotype continued for at least 6 weeks after the cessation of MT feeding in this species. In Rosy Barbs, MT diets did induce the expression of male coloration patterns, but these patterns generally did not reach the color intensity of naturally occurring males which is desired by the ornamental market. There were also no differences in resulting GSI values among the treatments in this species. Overall, this study suggests that post differentiation androgenic treatment may be a viable option to induce secondary male sex coloration after sexual differentiation in valuable ornamental species. Partial budget analysis was conducted on previously completed larval dietetics trials, which compared the performance of commercially available microparticulate diets (MDs) to live Artemia for first feeding in six freshwater ornamental fish species: redtailed sharkminnow Epalzeorhynchos bicolor, bronze corydoras Corydoras aeneus, upside down catfish Synodontis nigriventris, featherfin squeaker Synodontis eupterus, freshwater angelfish Pterophyllum scalare, and dwarf gourami. For all species except E. bicolor, at least one MD and the associated feeding protocol provided a net economic benefit compared to Artemia nauplii and the associated feeding protocol. For E. bicolor, the highest performing MD resulted in a net economic cost compared to Artemia. Methods that were previously applied to the development of early weaning protocols in G. ternetzi, including dietetics, characterization of ontogenetic development, and a subsequent weaning trial, were applied to neon tetra Paracheirodon innesi. P. innesi performed particularly poorly when fed MDs from first feeding, exhibiting significantly reduced survival relative to larvae fed Artemia nauplii. Histological, histochemical and biochemical characterization of gastrointestinal development revealed that stomach differentiation, gastric gland formation, and neutral mucopolysaccharide production did not occur until 20 dph. Pepsin activity was first detected at 13 dph. Weaning beginning with cofeeding at 13 dph resulted in similar growth and survival relative to feeding exclusively Artemia from first feeding. Tiger barb Puntigrus tetrazona larval dietetics were evaluated and intestinal development was characterized using histological, histochemical and biochemical techniques. Additionally, potential feed attractants (betaine, taurine, tryptophan and a mix of all three) were incorporated into MDs and resulting survival, growth, feeding incidence, trypsin activity and total protein content were evaluated from first feeding to 18 dph. Characterization of intestinal development revealed a pattern of progressive elongation and folding over the course of the sampling period, presence of pharyngeal teeth and alcian blue (AB) and periodic acid-Schiff (PAS) positive mucin production in the esophagus, as well as acute increases in trypsin and lipase activities. P tetrazona survived at a similar rate when fed MD when compared to those fed Artemia nauplii from first feeding. Feed attractants applied to the most successful MD did not elicit significant differences among treatments in any of the parameters measured. C. aeneus gastrointestinal characterization revealed a highly precocious pattern of development in this species. During this reporting period, experiments were also carried out to develop culture techniques for native Florida ornamental fishes including the gulf coast pygmy sunfish Elassoma gilberti, blackbanded sunfish Enneacanthus chaetodon, flagfin shiner Pteronotropis signipinnis, and metallic shiner Pteronotropis metallicus. Experiments evaluating spawning substrate preference, incubation techniques, embryo disinfectants, first feed dietetics and larval development were carried out to varying degrees with all species. Gulf coast pygmy sunfish and metallic shiners appear most conducive to commercial propagation at this juncture although further research is needed to refine protocols and increase production efficiency. 2. Development of spawning, larval culture, and production techniques for marine ornamental fish species Melanurus Wrasse (Halichoeres melanurus) and Pacific Blue Tang (Paracanthurus hepatus) are of great interest to aquaculture due to their popularity in the marine aquarium trade and lack of established commercial rearing methods. In 2019 we experimentally evaluated different larval rearing protocols from 0 to 3 dph. Algal species, density, and prey densities were evaluated to determine a suitable rearing environment that resulted in increased survival and feeding incidence during the transition from endogenous nutrition to exogenous feeding. Potential feed attractants were also evaluated, comparing a control treatment with no feed attractant to the addition of taurine, tryptophan, or betaine at 10-5 mg/L in the rearing environment. Results of algal species manipulations suggest inclusion of the microalgae Tisochrysis lutea during early larviculture of the Melanurus Wrasse aids in prey capture whereas the addition of the diatom Chaetoceros muelleri negatively impacts larval survival (P < 0.001). Trials conducted with Pacific Blue Tang show an increased feeding response with inclusion of either T. lutea or T. chuii (P = 0.003). The Yellow Wrasse (Halichoeres chrysus) showed no survival or feeding advantage following microalgae inclusion. T. lutea density of 300,000 cells/ml is optimal for both survival (P < 0.0001) and feeding incidence (P = 0.02) for Melanurus Wrasse and for survival of the Pacific Blue Tang (P < 0.0001). Prey density experiments resulted in no difference in feeding incidence across treatments for both species. Inclusion of feed attractants decreased survival (P < 0.0001) and had no effect on feeding incidence for Melanurus Wrasse. These results will help to shape commercial production protocols and inform decisions about feasibility of production for these valuable ornamental species.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ramee, S., T. Lipscomb, and M. DiMaggio. 2019. Effects of 17a-methyltestosterone immersion and feeding on masculinization of the dwarf gourami and rosy barb. Aquaculture 2019. New Orleans, Louisiana.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ramee, S., T. Lipscomb, and M. DiMaggio. 2019. The influence of larval environment on masculinization and endogenous cortisol in two ornamental fishes. Aquaculture 2019. New Orleans, Louisiana.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Lipscomb, T., Q. Tuckett, A. Wood, S. Ramee, J. Patterson, C. Watson, and M. DiMaggio. 2019. Development of aquaculture techniques for two Florida native ornamental fishes: Elassoma gilberti and Pteronotropis metallicus. Aquaculture 2019. New Orleans, Louisiana.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Lipscomb, T., A. Wood, S. Ramee, and M. DiMaggio. 2019. Characterization of digestive enzyme ontogeny and gastrointestinal development in Gymnocorymbus ternetzi for weaning optimization. Aquaculture 2019. New Orleans, Louisiana.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Hutchins, S., M.R. Hauville p, and M.A. DiMaggio. 2019. Optimizing Culture Parameters of the Cyclopoid Copepod Oithona colcarva. Marine aquarium conference of North America. Orlando, Florida.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Sowaske G., S. Hutchins, T. Lipscomb, and M. DiMaggio. 2019. Evaluating microalgae species effect on survival and feeding during early larviculture for three pelagic spawning marine ornamental species. Marine aquarium conference of North America. Orlando, Florida.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Lipscomb, T., A. Wood, S. Ramee, M. DiMaggio. 2019. Characterization of Larval Digestive System Ontogeny in Gymnocorymbus ternetzi: Progress Toward Feeding Optimization. Annual meeting of the Florida Chapter of the American Fisheries Society Annual Meeting, Haines City, Florida.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Sowaske G., S. Woolley, T. Lipscomb, and M. DiMaggio. 2019. Evaluating Larviculture Protocols for Pelagic Spawning Marine Ornamental Species. Florida Chapter of the American Fisheries Society Annual Meeting, Haines City, Florida
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Patterson, J.T., M.A. DiMaggio, C.C. Green, and C.A. Watson. 2019. Volitional spawning of captive reared age-4 Alligator Gar Atractosteus spatula. North American Journal of Aquaculture 81:291-295.
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Sipos, M.J., T.N. Lipscomb, A.L. Wood, S.W. Ramee, C.A. Watson, and M.A. DiMaggio. 2019. Evaluation of cGnRH IIa for Induction Spawning of Two Ornamental Synodontis Species. Aquaculture 511:734226.
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Sipos, M.J., T.N. Lipscomb, A.L. Wood, S.W. Ramee, and M.A. DiMaggio. (In Press). Evaluation of Three Embryo Disinfectants in Four Freshwater Ornamental Fish Species. North American Journal of Aquaculture. DOI: 10.1002/naaq.10118.
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Lipscomb, T., Tuckett, Q., Wood, A., Patterson, J., Ramee, S., Watson, C., and DiMaggio, M. (In Press). Culture protocols for the Gulf Coast Pygmy Sunfish Elassoma gilberti. Journal of the World Aquaculture Society 1-11. DOI:10.1111/jwas.12646.
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Sipos, M.J., T.N. Lipscomb, A.L. Wood, S.W. Ramee, C.A. Watson, and M.A. DiMaggio. 2020. Evaluation of cGnRH IIa for Induction Spawning of Two Ornamental Epalzeorhynchos Species. Aquaculture Research 51:232-241. DOI: 10.1111/are.14369.


    Progress 10/01/17 to 09/30/18

    Outputs
    Target Audience:The target audience of the proposed research is ornamental fish producers within the state of Florida. Secondary audiences will include public zoos and aquariums, aquaculture researchers, extension specialists and educators, and state agencies Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research project has provided opportunities for education and training of stake holders through interactions during farm visits, laboratory tours, and professional presentations at conferences and workshops. Graduate student participation in ongoing research projects also provides further opportunities for mentoring and professional development through interaction with faculty and staff. How have the results been disseminated to communities of interest?Preliminary findings and culture protocols have been disseminated through presentations at conferences, annual stakeholder meetings of the Rising Tide Conservation initiative, seminars with local aquarists, and online through the use of podcasts, blogs, (http://risingtideconservation.blogspot.com/), websites (http://risingtideconservation.org/), and social media (https://www.facebook.com/risingtideconservation/). Participation in the production of educational videos and interviews with local and national media have also aided in disseminating information to the general public. Site visits, workshops, tours, phone calls, and emails are also frequently employed to deliver research results to industry stakeholders where appropriate. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period masculinization research will conclude for the Dwarf Gourami and Rosy Barb. Experiments which investigate the ontogeny of digestive enzymes in commercially valuable ornamental species will be pursued with the resulting data serving as a guide for the development of weaning and larval nutrition protocols. Experiments focused on developing culture techniques for native Florida ornamental fishes will continue examining incubation techniques, embryo disinfectants, first feed dietetics and larval development for four species. Experiments to improve upon culture techniques for the Pacific blue tang, melanurus wrasse, and yellow wrasse will continue as well as investigations into optimization of live feed production for marine species.

    Impacts
    What was accomplished under these goals? 1. Development of spawning, larval culture, and production techniques for freshwater ornamental fish species Over the past year, we have continued to work towards developing commercially applicable masculinization techniques for two ornamental freshwater species, the Rosy Barb, Pethia conchonius, and Dwarf Gourami Trichogaster lalius. This research has continued to investigate the interaction between the sex differentiation and stress response in these two species. Previous experiments have been conducted on the effect of environmental manipulation of the larval environment on the resulting sex ratios. This line of investigation also included multiple trials where cortisol feed was used as a proxy for environmental stress. The majority of these larval experiments were completed before 2018, but the samples from these experiments still required processing. Over the past year, we have analyzed all remaining cortisol samples from this suite of experiments via a competitive binding cortisol ELISA. Remaining grow-out and dissections to determine sex ratios were also conducted in early 2018. Along with data analysis and reporting, this nearly wrapped up this phase of the masculinization studies. In 2018, we also conducted several experiments assessing the masculinization effects of 17α-methyltestosterone (MT) on these two species. This suit of experiments investigated the effects of dosage and duration of MT administered both through the feed and through immersion baths on the survival and sex ratios of the resulting populations. The results of these experiments will be used to determine the minimum effective does of MT required to induce masculinization of the population. This data may be used as evidence for the expanded use of MT under the FDA INAD program. Overall, the data from these studies will provide a clear picture of the sexual plasticity of these two species and provide guidelines for potential commercial masculinization procedures. Applied dietetics trials aimed at evaluating microparticulate diets as alternatives to live Artemia nauplii were continued with larval neon tetras Paracheirodon innesi, chinese algae eaters Gyrinocheilus aymonieri, and upsidedown catfish Synodontis nigriventris. Commercial microparticulate diets performed similarly to Artemia with respect to growth and survival for larvale G. aymonieri and S. nigriventris, but P. innesi exhibited significantly lower growth and survival with the microdiets. Further research into the reason for these differences will include evaluating the digestive system ontogeny of P. innesi using histological techniques as well as digestive enzyme assays to ascertain when acid protease activity and the presence of a stomach begins, thus indicating the ability to digest inert particles. This approach was applied in black tetra Gymnocorymbus ternetzi, where histological preparations over the course of the larval period (2-24dph) indicate a prolonged, agastric larval period in this species. Initial evidence for stomach formation and pepsin producing gastric glands appeared only after 18 dph. These results, coupled with digestive enzyme activities from each sampling date (in progress), will help guide future research into optimizing weaning of these commercially important freshwater ornamental fish. In addition to larval nutrition research, experiments were initiated which focused on developing culture techniques for native Florida ornamental fishes. Of the four species being studied, the gulf coast pygmy sunfish Elassom gilberti has shown the most promise. For this species, incubation techniques, embryo disinfectants, first feed dietetics and larval development were all evaluated. Research evaluating cGnRH IIa as a spawning aid for freshwater ornamental species has concluded. Dosages of 50, 100, 200 µg/kg of the hormone cGnRH IIa were compared to the ornamental industry standard Ovaprim® (10 µg/kg sGnRHa) using standard metrics for spawning performance. Four commercially valuable ornamental species Epalzeorhynchos bicolor, Epalzeorhynchos frenatum, Synodontis nigriventris and Synodontis eupterus were used to assess spawning aid efficacy. All species exhibited similar ovulation performance across hormone treatments except for E. frenatum injected with Ovaprim (17% ovulation). Poor ovulation success excluded the E. frenatum Ovaprim treatment from subsequent analyses. In general, fertilization and hatch success were comparable for both analogs among species. Embryo and larval morphometric measurements were also similar among treatments across all four species. Taken together, these results suggest cGnRH IIa to be a safe and reliable option for induction spawning of ornamental species. 2. Development of spawning, larval culture, and production techniques for marine ornamental fish species Melanurus wrasse larvae initiated feeding, swimbladder inflation, flexion, and metamorphosis at 3, 10, 15, and 37 days post hatch (DPH), respectively, with 0.5% survival. First generation wrasses began spawning at ~8 months of age, signifying closure of the life cycle. For both wrasse species, an inverse relationship was found between water temperature and embryo incubation period. Incubation temperatures of 25 and 28 degrees Celsius produced highest survival and smaller larvae at hatch. Examination of melanurus and yellow wrasse larvae prior to first feeding established that algal densities greater than or equal to 50,000 cells mL-1 or artificial shading resulting in ~300 lux, enhanced growth and survival. Melanurus wrasse first feeding trials revealed that algal densities of 300,000 and 500,000 cells mL-1, provision of Parvocalanus crassirostris nauplii (<75 microns), and prey densities greater than or equal to 5.0 items mL-1 produced elevated larval feeding success. Yellow wrasse first feeding culture trials examining similar parameters did not elucidate any advantageous culture protocols, indicating the necessity for further exploration of these parameters and others affecting larval first feeding success. Continued investigation into larviculture techniques influencing growth, survival, and feeding success should establish methodologies leading to commercial protocols for these and other marine ornamental species.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Sipos, M.J., T.N. Lipscomb, A.L. Wood, S.W. Ramee, E.M. Groover, C.A. Watson, and M.A. DiMaggio. 2018. Evaluation of cGnRH IIa for induced spawning of Epalzeorhynchos bicolor and Synodontis nigriventris. Aquaculture America 2018. Las Vegas, NV.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ramee, S., T. Lipscomb, and M. DiMaggio. 2018. The potential use of environmental stressors for masculinization in two ornamental fishes. Aquaculture America 2018. Las Vegas, NV.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Lipscomb, T., A. Wood, S. Ramee, M. Sipos, E. Groover, and M. DiMaggio. 2018. Replacing Artemia in the larval culture of six freshwater ornamental fish. Aquaculture America 2018. Las Vegas, NV.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Groover, E.M., M. Alo, J. Degidio, S. Ramee, T. Lipscomb, M. Sipos, and M.A. DiMaggio. 2018. Greenwater culture and larval development of the Melanurus wrasse, Halichoeres melanurus. Aquaculture America 2018. Las Vegas, NV.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ramee, S.W., T.N. Lipscomb, and M.A. DiMaggio. 2018 The potential influence of environmental stress on sex differentiation in two ornamental fishes. Florida Chapter of American Fisheries Society Annual Meeting. Haines City, FL.


    Progress 10/01/16 to 09/30/17

    Outputs
    Target Audience:The target audience of the proposed research is ornamental fish producers within the state of Florida. Secondary audiences will include public zoos and aquariums, aquaculture researchers, extension specialists and educators, and state agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research project has provided opportunities for education and training of stake holders through interactions during farm visits, laboratory tours, and professional presentations at conferences and workshops. Graduate student participation in ongoing research projects also provides further opportunities for mentoring and professional development through interaction with faculty and staff. How have the results been disseminated to communities of interest?Preliminary findings and culture protocols have been disseminated through, peer reviewed publications presentations at conferences, annual stakeholder meetings of the Rising Tide Conservation initiative, seminars with local aquarists, and online through the use of podcasts, blogs, (http://risingtideconservation.blogspot.com/), websites (http://risingtideconservation.org/), and social media (https://www.facebook.com/risingtideconservation/). Participation in the production of educational videos and interviews with local and national media have also aided in disseminating information to the general public. Site visits, workshops, tours, phone calls, and emails are also frequently employed to deliver research results to industry stakeholders where appropriate. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period masculinization research will continue to focus on further data collection and analysis from ongoing experiments. Whole body cortisol will be evaluated where appropriate using an enzyme-linked immunosorbent assays validated for use in both the rosy barb and dwarf gourami. Final trials will be completed for the cGnRH IIa induced spawning investigations. Experiments to improve upon culture techniques for the Pacific blue tang and melanurus wrasse are planned. Investigations focused on improving egg quality and larval rearing methods for the yellow wrasse will also be executed. Pancreatic digestive enzymes including trypsin and lipase are critical for early larval fish. The gastrointestinal tract of most larval fish is relatively underdeveloped upon hatching and does not differentiate into more complex structures until many days post hatch. The ontogeny of digestive enzymes is critical for the survival and growth of larval fish and different diets may influence how these enzymes develop. As we move forward with this research we aim to employ established protocols to evaluate the activity of trypsin and neutral lipase at regular intervals in developing ornamental fish larvae being offered commercial microparticulate diets and Artemia. The information garnered from these trials will provide critical insight into optimum larval feeding regimes and weaning protocols for commercially valuable ornamental species.

    Impacts
    What was accomplished under these goals? 1. Development of spawning, larval culture, and production techniques for freshwater ornamental fish species During this reporting period research efforts have continued to focus on the development of alternative masculinization strategies, evaluations of new spawning aids for freshwater ornamental species, and we have initiated investigations into suitable replacements for Artemia in larval culture protocols. We have continued to work towards optimizing commercial production of select freshwater ornamental species with several experiments. One phase of our research has continued to focus on masculinization of Rosy Barb, Pethia conchonius, and Dwarf Gourami Trichogaster lalius, populations by exploiting the cortisol mediated sex determination pathway. The Rosy Barb temperature trial, which began last year with fish being raised at 4 different temperatures (15, 20, 25, 30°C) for 30 days post hatch (DPH), resulted in a positive correlation between temperature and growth (p<0.001) and higher survival at 25°C than 15 or 30°C. However, there was no significant difference in resulting sex ratios. In addition to the survival and sex ratio results, this year, we developed and validated an ELISA to determine whole body cortisol concentrations in larval Dwarf Gouramis. The Dwarf Gourami cortisol feed trial that began last year, evaluating three levels of cortisol feed (0, 0.2, 0.6 mg/g feed) fed over the first 30 DPH resulted in no significant differences in survival, sex ratio, or cortisol levels per larvae. Dwarf Gourami larvae were then used in an acute cortisol feeding trial where replicate tanks were fed cortisol feed (2 mg/g) for either the 1st, 2nd, 3rd, or 4th week post hatch. This resulted in no differences in survival or sex ratios. However, there were differences in the cortisol levels at 1 and 3 weeks post hatch between the groups of fish fed cortisol the previous week and their respective control groups of the same age. A Dwarf Gourami salinity trial investigated the effects of rearing replicate tanks of larvae in five different salinities (0, 3, 6, 9, and 12 g/L) until 20 DPH. While the 9 and 12 g/L treatments did not survive past 20 DPH, the 3 g/L did have significantly higher survival than the 0 or 6 g/L treatments (p=0.002). There were significantly higher concentrations of cortisol/larvae in the 0 g/L treatment at the 20 DPH sampling point (p=0.035) but no differences at the 11 DPH sampling point. There was also no difference in sex ratios resulting from this experiment. Trials with both Dwarf Gouramis and Rosy Barbs have been conducted with the aim of describing the 48-hour response of cortisol levels to a single cortisol feeding. Research has also continued on evaluation of cGnRH IIa as a spawning aid for freshwater ornamental species. Additional trials have concluded and data regarding fecundity (eggs per gram body weight), fertilization, hatch and ovulation success as well as egg and larval morphometrics has been collected and will be incorporated into the larger data set and analyzed. In addition to the red-tailed black shark (Epalzeorhynchos bicolor) and the upside down catfish (Synodontis nigriventris) trials, two supplementary species, the rainbow shark (Epalzeorhynchos frenatum) and featherfin squeaker (S. eupterus) were added to increase robustness of the experiment. Initial trials with the supplementary species have been completed although more replicates will be added at the beginning of next spawning season to increase statistical power for our analyses. Preliminary results from the E. bicolor and S. nigriventris trials indicate that cGnRH IIa has comparable efficacy to Ovaprim® as an induced spawning aid. Further testing with cGnRH IIa will help to elucidate species specific responses to this novel spawning aid. The efficacy of commercially available microparticulate diets as potential replacements for the live feed Artemia was evaluated in several species of freshwater ornamental fish. Larval nutrition represents a significant bottleneck in the successful culture of ornamental fish and very few feed items have been evaluated for this critical developmental period. In the past year, we evaluated growth, survival and homogeneity of growth after 14 days of feeding with Artemia or three microdiets (Larval AP-100, Otohime A1, and Golden Pearls) in 8 economically important ornamental species: tiger barb Puntius tetrazona, rosy barb Pethia conchonius, angel fish Pterophyllum scalare, red-tailed black shark Epalzeorhynchos bicolor, black skirt tetra Gymnocorymbus ternetzi, Corydoras catfish Corydoras aeneus, dwarf gourami Trichogaster lalius, and eupterus catfish Synodontis eupterus. For all species evaluated, results indicate that at least one of the microparticulate diets performed as well as, if not better than Artemia in terms of survival, growth and homogeneity of growth. This research will serve as preliminary data for future projects that will evaluate the ontogeny of pancreatic digestive enzymes in larval ornamental fish. Digestive enzyme ontogeny research is currently underway for dwarf gourami. 2. Development of spawning, larval culture, and production techniques for marine ornamental fish species During this reporting period marine ornamental research efforts have continued to focus on development of sexually mature broodstock for the Pacific blue tang Paracanthurus hepatus, melanurus wrasse Halichoeres melanurus, and yellow wrasse H. chrysus and investigations into larval culture parameters that yield increased survival and growth. Two additional broodstock populations of the melanurus wrasse were established at the University of Florida's Tropical Aquaculture Laboratory (UF-TAL), one of which being an F1 generation. Daily spawning continues to be observed in the two most recently formed melanurus wrasse populations with yields from hundreds to several thousand fertile eggs, averaging 0.604 mm in diameter. Fertilization rates of spawns produced by the F1 population have been found to be far superior, averaging 87% compared to the wild broodstock averaging 65% fertilization. Early hatching trials have found that algal density had a significant effect on larval survival from 0-3 days post hatch. Seven successful larval rearing trials in both small (13 L) and large (125 L) culture tanks have yielded 45 total post metamorphic melanurus wrasses with increasing survival rates up to 0.5%. Recently, six F2 generation melanurus wrasses were brought through metamorphosis at 0.5% survival. Settlement and metamorphosis has ranged from 35-40 days post hatch across all successful larval runs completed in 125 L tanks. While melanurus wrasse settlement continues to be somewhat delayed when compared with previous efforts and literature estimates, percent survival continues to improve with successive culture attempts. Additional brood stock populations of yellow wrasses have been procured with one current population spawning consistently and producing several thousand eggs per day. Early hatching trials have found algal density had a significant effect on larval survival from 0-3 days post hatch. Poor egg quality and low fertilization rates continue to be a problem for this species, however, progress has been made concerning larval rearing with one spawn being raised to 11 days post hatch. Early larval rearing trials examining live feeds, light intensity, flow rate, stocking density, and additional variables will continue to be explored to improve larval survival for both melanurus and yellow wrasses. Two populations of F1 Pacific blue tang previously bred at the UF-TAL continue to be grown out and conditioned, regularly fed on a diet of fresh seafood, commercially available pellet, and Nori algae sheets. The older population of F1 blue tangs is thought to be nearing sexual maturity and has been moved to a breeding tank where they are monitored daily for imminent spawning activity.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2017 Citation: DiMaggio, M.A., E.J. Cassiano, K.P. Barden, S.W. Ramee, C.L. Ohs, and C.A. Watson. 2017. First record of captive larval culture and metamorphosis of the Pacific Blue Tang, Paracanthurus hepatus. Journal of the World Aquaculture Society 48:393-401. DOI:10.1111/jwas.12426.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Ramee, S.W. and M.A. DiMaggio. 2017. Evaluation of masculinization techniques in two sexually dichromic ornamental species. Aquaculture America 2017. San Antonio, TX.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Sipos, M.J., T.N. Lipscomb, A.L. Wood, S.W. Ramee, E.M. Groover, C.A. Watson, and M.A. DiMaggio. 2017. Evaluation of cGnRH IIa for induced spawning of Synodontis nigriventris. Aquaculture America 2017. San Antonio, TX.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Groover, E.M., M.A. DiMaggio, M. Alo, J. Degidio, S. Ramee, and T. Lipscomb. 2017. Captive Culture and Larval Development of the Melanurus Wrasse, Halichoeres Melanurus. American Fisheries Society 147th Annual Meeting. Tampa, FL.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Lipscomb, T., A. Wood, S. Ramee, M. Sipos, E. Groover, and M. DiMaggio. 2017. Evaluating live feed alternatives for the culture of four commercially valuable ornamental teleosts. American Fisheries Society 147th Annual Meeting. Tampa, FL.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Ramee, S., T. Lipscomb, and M. DiMaggio. 2017. Assessing the influence of environmental stressors on sex determination in two ornamental fishes. American Fisheries Society 147th Annual Meeting. Tampa, FL.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Sipos, M., T. Lipscomb, A. Wood, S. Ramee, E. Groover, C. Watson, and M. DiMaggio. 2017. Evaluating cGnRH IIa for spawning induction of two freshwater ornamental fish species. American Fisheries Society 147th Annual Meeting. Tampa, FL.


    Progress 10/01/15 to 09/30/16

    Outputs
    Target Audience:The target audience of the proposed research is ornamental fish producers within the state of Florida. Secondary audiences will include public zoos and aquariums, aquaculture researchers, extension specialists and educators, and state agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research project has provided opportunities for education and training of stake holders through interactions during farm visits, laboratory tours, and professional presentations at conferences and workshops. Graduate student participation in ongoing research projects also provides further opportunities for mentoring and professional development through interaction with faculty and staff. How have the results been disseminated to communities of interest?Preliminary findings and culture protocols have been disseminated through presentations at conferences, annual stakeholder meetings of the Rising Tide Conservation initiative, seminars with local aquarists, and online through the use of podcasts, blogs, (http://risingtideconservation.blogspot.com/), websites (http://risingtideconservation.org/), and social media (https://www.facebook.com/risingtideconservation/). Participation in the production of educational videos and interviews with local and national media have also aided in disseminating information to the general public. Site visits, tours, phone calls, and emails are also frequently employed to deliver research results to industry stakeholders where appropriate. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period masculinization research will focus on further data collection and analysis from ongoing experiments. In addition to quantifying sex ratios from experimental treatments, whole body cortisol will also be analyzed where appropriate using an enzyme-linked immunosorbent assay validated for use in both the rosy barb and dwarf gourami. Further trials will also be pursued in our continued evaluation of cGnRH IIa for induced spawning of ornamental species. Efforts to repeat and improve upon the successes with the Pacific blue tang and melanurus wrasse are also planned and will involve investigations into optimal incubation and larval rearing temperatures, stocking densities, light intensities, live feeds densities, and other culture parameters. Investigations focused on improving egg quality and fertility in the yellow wrasse will also be executed. Additional wild brood stock for all marine species will be procured to increase numbers of reproductively competent individuals currently available at the UF-TAL.

    Impacts
    What was accomplished under these goals? 1. Development of spawning, larval culture, and production techniques for freshwater ornamental fish species During this reporting period research efforts have primarily focused on the development of alternative masculinization strategies and evaluations of new spawning aids for freshwater ornamental species. These investigations will generate data which may help to shape current production protocols and allow stakeholders to increase on-farm productivity and efficiency. Over the past year, several preliminary experiments aimed at optimizing commercial production of select freshwater ornamental species have been conducted. Our research has focused on masculinization of rosy barb, Pethia conchonius, and dwarf gourami Trichogaster lalius, populations by exploiting the cortisol mediated sex determination pathway. Environmental exposure of rosy barbs to cortisol (4 mg/L) from 1 to 7 days post hatch (DPH) yielded no apparent influence on the proportion of males (mean= 51.3% male). Four concentrations of cortisol feed (0, 0.2, 0.4, and 0.6 mg/g feed) were fed to rosy barb larvae from 8-28 DPH. This resulted in no significant difference in male proportion (P=0.06). An experiment was also completed which evaluated the effect of Tribulus terrestris (TT) extract exposure (0, 0.2, 0.4, 0.5 mg/L) on rosy barb masculinization. Although there was a significant effect of the TT concentration on survival, there was no significant effect on resulting sex ratios. Results from a stocking density (5, 10, 20, and 40 larvae/L) experiment with rosy barbs suggested a possible inverse relationship between the proportion of males and stocking density, however this relationship was not significant (P=0.19). Additional experiments evaluating the effect of cortisol feed on dwarf gourami sex ratios and the effects of temperature (15, 20, 25, and 30°C) on rosy barb sex ratios are also underway. Masculinization investigations completed during this reporting period have allowed us to refine our experimental methods and also helped to elucidate the role of the stress response axis in sexual differentiation of the rosy barb and dwarf gourami. Efficacy, reliability, and safety are of principal concern for spawning aids used in reproduction protocols for aquaculture species. Ovaprim® (Western Chemical Inc., Ferndale, WA, USA), a salmon gonadotropin releasing hormone analog (sGnRHa, D-Arg6-Pro9-Net, 20 μg/mL) and a dopamine antagonist (Domperidone, 10 mg/mL) is currently the preferred choice for induction spawning of ornamental fishes, however, this preparation may be unreliable or completely ineffective in some cases. cGnRH IIa (D-Arg6, Pro9-NHet) has garnered recent interest as an alternative GnRH subtype which offers increased biological activity, reliability, and may ultimately help to increase on farm productivity and expand the diversity of species able to be cultured. Investigations of novel spawning aid preparations completed during this reporting period have focused on cGnRH IIa. Several experimental runs have recently concluded which examined the efficacy of GnRH IIa for induction spawning of the red-tailed black shark (Epalzeorhynchos bicolor) and the upside down catfish (Synodontis nigriventris). Hormone concentrations of 50, 100, 200 µg/kg GnRH IIa were evaluated alongside Ovaprim® to better describe the effects of this novel hormone on successful ovulation, latency time, fecundity, fertilization and hatching success, as well as egg and larval morphometrics in two commercially produced ornamental species. Data has been collected for both species and statistical analyses are forthcoming. Results from these investigations will help to assess the viability of cGnRH IIa as a spawning aid for the ornamental aquaculture industry. 2. Development of spawning, larval culture, and production techniques for marine ornamental fish species During this reporting period marine ornamental research efforts have primarily focused on establishing reproductively competent broodstock and elucidation of successful culture protocols for the Pacific blue tang Paracanthurus hepatus, melanurus wrasse Halichoeres melanurus, and yellow wrasse Halichoeres chrysus. Three separate broodstock populations of the melanurus wrasse were established at the University of Florida's Tropical Aquaculture Laboratory (UF-TAL). A varied diet consisting of fresh seafood and a commercially available pellet is fed regularly to promote gonadal maturation and volitional reproduction. Daily spawning has been consistently observed in two of the melanurus wrasse populations with yields from one hundred to over five thousand fertile eggs, averaging 0.604 mm in diameter. Preliminary larval rearing trials exploring the effects of varying environmental conditions in both small (13L) and large (125L) culture tanks have yielded one successful larval run resulting in nine post metamorphic wrasses or approximately 1% survival from egg to juvenile. The first larvae from this run settled and completed metamorphosis at 37 days post hatch. While settlement appeared to be somewhat delayed when compared with our previous efforts, this cohort is the largest group of melanurus wrasses raised thus far at the UF-TAL. Two separate brood stock populations of the yellow wrasse were also established and maintained using similar methods employed with the melanurus wrasses. Inconsistent spawning was noted in both populations resulting in eggs of poor quality and low fertilization. The Pacific blue tang, Paracanthurus hepatus, is consistently among the top twenty species imported into the United States by volume, with all specimens sourced from wild stocks. Captive culture of this species through metamorphosis has not been previously documented and fundamental information regarding reproduction, larval culture, and production techniques is scarce. During this reporting period the Pacific blue tang was successfully cultured in captivity for the first time in the world at the UF-TAL. Large mortality events were observed at 7 and 20 DPH corresponding with starvation and flexion, respectively. By 41 DPH, a behavioral change was noted with the majority of the remaining larvae associating with the bottom of the tank. On day 50, the first signs of blue pigmentation marked the beginning of metamorphosis. A total of 27 juvenile blue tangs were cultured during this trial.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: DiMaggio, M.A., K. Barden, E.J. Cassiano, M. Hauville, and S.W. Ramee. 2016. First record of captive spawning and larval rearing of the melanurus wrasse Halichoeres melanurus. Aquaculture America 2016. Las Vegas, NV.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: DiMaggio, M.A., M. Hauville, E.J. Cassiano, K. Barden, M.L. Wittenrich, and S.W. Ramee. 2016. Aquaculture of the french grunt Haemulon flavolineatum: larval growth and development, first feeding, and impact of photoperiod. Aquaculture America 2016. Las Vegas, NV.


    Progress 11/21/14 to 09/30/15

    Outputs
    Target Audience:The target audience of the proposed research is ornamental fish producers within the state of Florida. Secondary audiences will include public zoos and aquariums, aquaculture researchers, extension specialists and educators, and state agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results from the research into freshwater ornamental species are still preliminary in nature at this time and inappropriate for dissemination to stakeholders. Results and updates regarding ongoing research with the Pacific blue tang and melanurus wrasses have been disseminated through presentations at the annual stakeholder meeting of the Rising Tide Conservation initiative, seminars with local aquarists, online through the use of podcasts and a blog (http://risingtideconservation.blogspot.com/), as well as through interaction with local producers by means of site visits, phone calls, and emails. What do you plan to do during the next reporting period to accomplish the goals?Further replicated experiments focusing on exogenous stressors such as temperature and salinity and their role in cortisol production and masculinization of the Rosy Barb are planned. Expansion of brood stock populations for both the Pacific blue tang and the melanurus wrasse will also help to increase the quantity of eggs available for larval culture experiments. Larval trials with both marine ornamental species will continue to focus on early nutrition as well as optimization of environmental parameters to maximize growth and survival.

    Impacts
    What was accomplished under these goals? Development of effective culture protocols is essential for the commercialization of new aquaculture species. Results of the proposed research goals will address critical gaps in knowledge for such production to occur. Impacts to Florida aquaculture producers include the transfer of species specific information and protocols on captive reproduction, larval culture, and production techniques. This information may then be adapted by producers to establish commercial culture protocols for economically valuable species. 1. Development of spawning, larval culture, and production techniques for freshwater ornamental fish species Over the past year, we have conducted several preliminary experiments aimed at optimizing commercial production of freshwater ornamental species. Our primary focus has centered on masculinization of Rosy Barb, Pethia conchonius, populations via the cortisol mediated sex determination pathway. Initially, we evaluated salinity exposure as an exogenous stressor, where larvae were subjected to concentrations of 0, 5, 10, 15 g/L through 18 days post-hatch (dph). Concentrations above 10 g/L were not tolerated and thus sex ratios could not be determined. The 0 and 5 g/L treatments were grown out for an additional 5 months and sex ratios were determined grossly with no apparent differences. Several cortisol toxicity trials were also executed with Rosy Barb larvae to ascertain safe dosages for environmental administration. Initial exposures examined concentrations of 0, 0.5, 1, 2, 3, and 4 mg/L cortisol from 1-5 dph. Survival ranged from 95% at 0 mg/L down to 83% for the remaining cortisol treatments. A follow up experiment assessed a broader range of concentrations (0, 2, 4, 6, 8, and 10 mg/L) for 1-5 dph larvae. High survival was still observed and no differences among cortisol treatments were noted. Environmental and dietary cortisol administration experiments are currently under way. Fish have been sampled for whole body cortisol and gene expression analyses where appropriate and results will be forthcoming. These preliminary investigations have allowed us to develop practical and effective methods to evaluate the role of cortisol and the stress response axis in the successful masculinization of the rosy barb and validate techniques for reliable identification of sex prior to maturity. 2. Development of spawning, larval culture, and production techniques for marine ornamental fish species Research into production methods for marine ornamental species has primarily concentrated on the Pacific blue tang, Paracanthurus hepatus and Melanurus wrasse, Halichoeres melanurus. Three separate captive brood stock populations of Pacific blue tangs have been established over the past year and held under conditions which should help to accelerate sexual maturity and promote spawning behavior. Consistent spawning from one population of blue tang brood stock has allowed us to conduct preliminary investigations into the effects of larval stocking densities, probiotics, feed densities, and culture systems on blue tang survival. Irrespective of experimental treatment, consistent mortality has been observed through 7 dph with no larval survival beyond 10 dph. Experiments are currently underway to assess further environmental and dietary variables which may increase larval survival and development. Inconsistent spawning and poor egg quality has been observed from the single population of melanurus wrasses currently in culture. An initial larval run proved successful in rearing larvae of the melanurus wrasse past metamorphosis, but issues with captive egg production have not allowed us to replicate this success. Efforts to increase the number of captive melanurus brood stock through acquisition of animals from the wild are currently under way.

    Publications