A COMPILATION OF STUDIES ON THE ORNAMENTAL FISH TRADE IN REGULATORY MANAGEMENT, HEALTH, AND APPLICATIONS FOR EDUCATION By ELISA JEANETTE LIVENGOOD A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2015
Â© 2015 Elisa Jeanette Livengood
To Micheal W illiam Coons and Memaw
4 ACKNOWLEDGMENTS I thank my advisor Dr. Frank Chapman. Without his support this work would not have been possible. I made promises in my masters about pulling back and trying to see the big picture . I will continue to make that promise. Without his ideas, faithful support, and making me a part of his research family, I w ould have never been able to do this. I will continue to strive to be the kind of scientist and educator he would like me to be, and build my own program. Dr. Chapman has set the precedent and taught me by example. I also thank Dr. Fu nicelli for encouragem ent and humor. I would also like to thank Dr. Miles for guidance and great ideas, and Dr. Ruth Francis Floyd for her kindness, caring and support over the years. Dr. Brenner for your guidance and instruction over the years. I could not have done this proje ct without the support of my husband, Micheal Coons . W ithout him I would have never been able to pick myself up after every fall and research fail ure . He make s together. Fermentation forev er! I would like to thank Dr. Ed Noga for much n eeded insight and discussion Quantitative injury project. Dr. Murat Balaban for guidance on picture taking and utilization of the LensEye software. I would like to thank the USFWS for help in obtaining the LEMIS data and Marly Wilson for writing the program to help analyze it. I would like to thank the Analytical Division, of University of Florida Chemistry for their help with HPLC analysis. I would like to thank my parents for never letting me think something was too hard and always expecting excellence when it came to school. So many other people in my family supported me through this overly long journey, including Lynette and John Grout, who let me stay with them when I returned from many trips to
5 wholesalers and farms in Miami. I would like to thank my lab mate Christian Lane for his kindred spirit, help, and always sticking together in the Chapman Lab. I would also like to thank the many scientist s, fishermen, wholesalers, and farmers who helped me complete this dissertation: Enrique Schmalbach, Michael Rambarran, El Llanerito, Elizabeth Aya Baquero, Professor Arias and so many others. I am honored to have learned so much from them and forever grat eful that so many of them opened their homes, businesses, and families to me.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREV IATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION AND STRUCTORAL ORGANIZATION OF THE COMPILATION OF STUDIES EXAMING THE ORNAMENTAL FISH TRADE ....... 13 2 REGULATORY MANAGEMENT OF THE ORNAMENTAL FISH TRADE .............. 16 Publication 1: The Applicability of the Law Enforcement Manageme nt Information System (LEMIS) Database for the Protection and Management of Ornamental Fish Trade ................................ ................................ ........................ 17 Materials and Methods ................................ ................................ ............................ 20 Results and Discussion ................................ ................................ ........................... 21 Conclusions ................................ ................................ ................................ ............ 28 3 ORNAMENTAL FISH HEALTH WITHIN SEGMENTS OF THE SUPPLY CHAIN ... 30 Mortality in Seven Select Species Imported from the Wild ................................ ...... 30 Publication 2: Quantitative Measurement of Epithelial Injury in Ornamental Silver Dollar Fish ( Metynnis orinocensis ) Captured in the Wild, Imported Wild Caugh t, and Aquacultured. ................................ ................................ .................. 33 Materials and Methods ................................ ................................ ..................... 36 Results and Discussion ................................ ................................ .................... 39 Publication 3: Uptake of Metronidazole in Artemia of Different Developmental Stages ................................ ................................ ................................ ................. 44 Materials and Methods ................................ ................................ ..................... 45 Results and Discussion ................................ ................................ .................... 47 4 EDUCATIONAL EXTENSION PUBLICATIONS FOR THE ORNAMENTAL FISH HOBBYIST ................................ ................................ ................................ .............. 50 5 CONCLUSIONS ON THE COMPLIATION OF STUDIES PRESENTED AND OVERALL RECOMMENDATIONS ................................ ................................ ......... 55
7 APPENDIX: EDIS PUBLICATIONS PDF AND URL LINKS ................................ .......... 59 LIST OF REFERENCES ................................ ................................ ............................... 60 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 69
8 LIST OF TABLES Table page 2 1 The composition and number of animal imports into the U.S. in 2010; principally for the pet trade ................................ ................................ ................. 22 2 2 The composition and number of animal imports into the U.S. ............................ 22 2 3 Principal ornamental fishes and their numbers imported into the U.S. in 2010 .. 25 3 1 Mortality as a percent of total shipping lot for selected Amazonian species imported in to wholesaler in South Florida ................................ ........................... 32 3 2 Artemia developmental life stage, supplementation regime and resulting concentration measu red for metronidazole from HPLC ................................ ...... 48
9 LIST OF FIGURES Figure page 1 1 A basic structure of the ornamental fish supply chain. ................................ ........ 13 2 1 Number of freshwater (FWTF) and marine (MATF) ornamental fish imported into the U.S. principally for the pet trade, in years 2008, 2009, and 2010. ......... 24 3 1 Wound pattern as a result of shipping and representative color analysis ........... 41
10 LIST OF ABBREVIATIONS APPA American Pet Products Association EDIS Electronic Data Information Systems DOA Dead On Arrival FAO Food and Agricultural Organization LEMIS Law Enforcement Management Information System
11 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy A COMPILATION OF STUDIES ON THE ORNAMENTAL FISH TRADE IN REGULATORY MANAGEMENT, HEALTH, AND APPLICATIONS FOR EDUCATION By Elisa Jeanette Livengood August 2015 Chair: Frank Chapman Major: Interdisciplinary Ecology This dissertation is principally a collection of articles that were published in refereed journals. They focus on aspects of the regulatory management, health, and education sectors of the orn amental fish trade . R egulatory management of the ornamental fish trade was examined through a study on the importation of ornamental fish into the United States . The purpose of this study was to review an existing database system, the Law Enforcement Management Information System (LEMIS) and its utility for management. While many studies of higher class vertebrates (mammals, birds, reptiles, and amphibians) use the LEMIS dat abase to evaluate trade impacts there was very little p ossibility of this same utility for ornamental fishes. This study did produce useful information on the magnitude of imports of ornamental fishes and corals. For example, the approximate number of individuals labeled as freshwater tropical fish (140 million) or marine aqu arium tropical fish (12 million ) that were imported in 2010. A survey of mortality in wild caught Amaz onian fishes was also conducted . A lso an experiment was conducted monitoring the presence of injury in si l v er dollar fish Metynnis orinocensis that were wild caught, wil d caught i mported, and farmed. The
12 mortality survey indicated mortalities ranging from as low as 1 % to as high as 71 %, with much of the mortality occurring within the 24 hour period post shipment. Injury was also found in Metynnis orinocensis that were imp orted as part of the aquarium fish trade and pattern of injury was identified on their ventral keel. In addition, an article was published proposing Artemia as a live drug delivery system for sick fish . Artemia were found to uptake a therapeutic level of metronidazole when supplemented at the developmental life stage of instar 2, which is their second molt. A series of extension publications were prepared realizing the importance of public education in raising aw areness of the responsibility of having fish as pets.
13 CHAPTER 1 INTRODUCTION AND STRUCTORAL ORGANIZATION OF THE COMPILATION OF STUDIES EXAMING THE ORNAMENTAL FISH TRADE The management of the ornamental fish trade is p roblematic due to concerns for sustainability of the wild resource. Millions of animals and plants are collected annually from the wild owing to the high commercial importance of the species for the aquarium market. There is an extensive supply chain from wild capture to the hobbyists that may compromise fish health (Figure 1 1). Regulatory management of the ornamental fish trade is difficult and overall global oversight almost impossible due to the sheer magnitude of the number of both species and individu als traded and the diversity of countries exporting and importing fish make. Further complicating these issues are concerns for the possibility of some species becoming established in environments outside of their natural habitat (becoming either non nativ e or invasive species). Educating the hobbyist may be a useful tool to effect positive change in this industry. Figure 1 1. A basic structure of the ornamental fish supply chain.
14 The ornamental fish supply chain can vary from country to country and between species groups from freshwater or saltwater origin. A generalized description of the ornamental fish trade begins with the introduction of two stakeholders: the fishermen and the farmer. Both of these stakeholders supply fishes to the markets. The farmer supplies fishes through captive breeding and domestication of certain species and the fisherman collects the fishes from their natural habitat (either salt or freshwater). Next the fish are brought to the exporter where they are packed and shipped v ia international air carriers to the country of import. Importing wholesalers receive the fish and then repackage them in order to sell them to retailers. In some cases, import wholesalers may re export the fishes to other countries. Next fish reach the re tailer which may be small local shops or larger retailers that sell many other pets and pet products. The final A description of the value of this industry shows that it is a relatively valuable market that comprises of m any different countries with several dominant importing and exporting markets. Total import value of this industry was estimated at $350 million USD in 2011 and is just slightly higher than the total export value of $315 million USD (FAO 2014). Europe is responsible for 69% of the total market value of imports and is the largest and most consistent importer of ornamental fish. The United States alone is responsible for 15% of the total market value of imports which makes it the single lar gest importing country (FAO 2014). Currently, Asia is the largest exporting region with 54% of the total market value of exports, followed by Europe at 35% (FAO 2014). This dissertation is principally a collection of published articles in refereed journals that were written after a thorough investigation of the value and trade of the
15 ornamental fish industry. It focuses on three areas that were identified as priority in relation to sustainability practices for the industry: management of the import regulatory system, fish health, and education of the consumer.
16 CHAPTER 2 REGULATORY MANAGEMENT OF THE ORNAMENTAL FISH TRAD E Policies and the management of wildlife trade have been reactionary responding to problems such as the introduction of non native species and public health risk (Smith et al. 2009). Regulations of wildlife trade (of which the ornamental fish trade is an integral part) have been criticized as inadequate (Smith et al. 2009). Without species specific volume and diversity data the possibility of managing the ornamental fish trade industry effectively is highly limited (Rhyne et al. 2015). The USA is the sing le largest importer of ornamental fish and as such exerts the largest influence on fish demand and reflects consumer attitudes. In the U.S., all imported and exported fish and wildlife must be declared to the government by filing a Declaration of Importati on or Exportation of Fish or Wildlife form (USFWS Form 3 177) and the forms are inputted into the Law Enforcement Management Information System or LEMIS database, from the U.S. Fish and Wildlife Service (USFWS). This descriptive study examined the issue of wildlife trade that sought to evaluate the data found in the LEMIS for its utility and application with respect to regulatory management of the ornamental fish trade. An examination of the import trade from a regulatory management perspective, may provide the volume and number of principal species in the trade, and identify those that may warrant protection. This publication is presented from pages 17 29.
17 Publication 1 : The Applicability of the Law Enforcement Management Information System (LEMIS) Databas e for the Protection and Management of Ornamental Fish Trade 1 Ornamental fish have long been sources of wonder, and aquariums are used as small windows into the fascinating underwater world. The practice of keeping fish for beauty and companionship began a s early as the tenth century A. D. with goldfish has diversified to include freshwater and marine fishes, invertebrates such as corals, sea anemones, crustaceans (e.g., crabs, hermit crabs, shrimps), molluscs (e.g., snails, estimate accurately, and using different sets of data, the value of the freshwater and marine aquarium livestock alone could be estimated at between US$400 and US$500 million worldwide. The retail industry worldwide has been estimated to be worth between US$7 billion and US$15 billion when aquariums, air pumps and other aquarium accessories directly associated with the hobby are included (FAO 2014; Andrews 1990). The people of the United States of America (U.S.) have become the caring for some 158 million ornamental fish (Chapman et al . 1997; APPA 2014). To meet consumer demands, hundreds of millions of ornamental fish (including invertebrates) are imported every year into the U.S. which exerts a large influence on the worldwide supply and demand patterns on this natural resource (Chapm an et al. 1997; Cato & Brown 2003; FAO 2014). The imported ornamental fish are from both wild 1 Content published with permission from Aquaculture, Aquarium, Conservation & Legislation International Journal of Bioflux Society. End of publication on page 2 9. Livengood E. J., Funicelli N., Chapman F. A., 2014 The applicability of the U.S. Law Enforcement Management System (LEMIS) database for the protection and management of ornamental fish. AACL Bioflux 7(4):268 275.
18 (Chapman et al. 1997; Chapman 2000; Cato & Brown 2003; Livengood & Chapman 2007; Rhy ne et al. 2012). Propagation of freshwater fish on the farm is the source of approximately 95% of the ornamental fish in the trade (Chapman et al. 1997). However, the vast majority of species are still collected from the wild (Chapman et al. 1997; Cato & B rown 2003). While farms provide significant numbers of ornamental fish into the trade, it does not meet the overall market demand. For example, popular freshwater species still collected from the wild include the clown loach ( Botia macracantha ), glass catf ish ( Kryptopterus bicirrhis ), pictus catfish ( Pimelodus pictus ), arawana ( Osteoglossum bicirrhosum ), cardinal tetra ( Paracheirodon axelrodi ), and the hatchetfishes ( Gasteropelecus spp.) (Chapman et al. 1997; Chapman 2000). While nearly all marine species o f ornamental fish es and invertebrates (e.g., corals, sea anemones, shrimps, Livengood & Chapman 2007). Poorly managed consumptive wildlife use results in their overexploita tion and consequently has an effect on global biodiversity (Wilcove et al. 1998). Likewise concerns have arisen regarding the adverse effects depletion of large numbers of ornamental fish may have on this biodiversity (Andrews 1990; Smith et al. 2008). Stu dies show wild collection of ornamental fish for the trade can negatively impact species. If trade levels exceed the ability of populations to replenish, both the species and income generated from their collection will flounder and could eventually disappe ar (Hemley & Fuller 1994). In the Peruvian Amazon, high fishing pressure reduced
19 abundance and lowered biomass of targeted freshwater ornamental fish species, and caused reduction in the overall total number of species (Gerstner et al. 2006). A threat to wild populations of the freshwater O. bicirrhosum which are sometimes collected by killing the mouth brooding adults to collect the juveniles has been documented (Moreau & Coomes 2006). Also, rapid decrease in local populations densities of targeted marine ornamental reef fishes were reported in Hawaii, and of both sea anemones and anem onefish in the Philippines, respectively (Tissot & Hallacher 2003; Shuman et al. 2005). The high demand and collecting pressure for the ornamental fish trade have contributed to the endangered status of particular stony corals, giant clams, and syngnathid fishes (Bruckner 2001). Determining the levels of removals of catch from the wild are essential for the management of populations in the wild (Pope 1988). The connection between free market economics and demand for wildlife products creates a trade force that directly affects wildlife with respect to population numbers and recruitment (Moulton & Sanderson 1997). Therefore monitoring of production, consumption, and trade data of ornamental species from the wild is critical to their successful management, co nservation, and restoration. However, quantifying trade volumes in wildlife can be difficult to compile with respect to country and species (Broad et al. 2003), especially given the informal nature of rural urban marketing networks. Although many countries have their own systems of reporting that serves to monitor the trade of aquatic species imported or exported, there is no global network of reporting for the ornamental fish trade at this time.
20 Since wildlife trade data exists in many capacities, a databa se of digital data can be an effective tool for the monitoring of the ornamental fish trade. The publication of digital data sets has increased the connectivity of scientists and managers, and helps them make more informed decisions (Huettmann 2005). Altho ugh not per se a science based conservation database but that has been perceived and used as one, is the United States Law Enforcement Management Information System or LEMIS database, from the U.S. Fish and Wildlife Service (USFWS). In the U.S., all import ed and exported fish and wildlife must be declared to the government by filing a Declaration of Importation or Exportation of Fish or Wildlife form (USFWS Form 3 177). Such requirement was imposed in respect to two protection and conservation laws of wildl ife, fish and plants: the Lacey Act of 1900 that prohibits trade in wildlife, fish, and plants that have been illegally taken, poss essed, transported, or sold and the Endangered Species Act of 1973, which purpose is to protect and recover species that are endangered or threatened of extinction. The original copy of Form 3 177 that accompanies every shipment provides the scientific name (genus, and species when known), common name, their quantity (either by number or weight) and monetary value, country of or igin, exporter and importer. The information is then transcribed manually, and compiled into the LEMIS database. By reviewing and analyzing the data of all live animal imports in the LEMIS database, this paper seeks to address the utility of the data repor ted, and usefulness as a tool for ornamental fish management and to make conservation decisions. Materials and Methods Records available in the LEMIS database of all live animals imported into U.S. during year 2010 were requested through freedom of inform ation act provisions. Each
21 record in the LEMIS database refers to an individual shipment, which are separated by import codes in spreadsheets for the entire year of imports. A computer program was written using open source code Ruby which separated the raw data from the records and organized shipments to groups and the taxonomic ranks of Phylum and Class. In essence creating a searchable database that collated shipments into categories of taxa or animal descriptors (like ornamental freshwater or marine fish ), and populated the spreadsheet with its respective information. The program also classified shipments that had complete genus and species identifiers, and shipments without genus or species were classified as missing. Anything the program script could no t specifically identify this point, we could examine how many individuals were imported for each group, Phylum, Class, and subsequent genus and species. Results and Discu ssion The electronic records retrieved from the LEMIS database disclosed 105,010 shipments of exotic live animals were imported into the U.S. during the year 2010 (Table 2 1); a figure which amounted to 219,797,719 exotic live animals imported principally for the pet trade (Table 2 1). Animals were primarily classified to the biological taxonomic rank of Class. Vertebrate animals were placed into the Class groups: mammalia, aves (birds), amphibia (frogs, toads, salamanders, newts), reptilia (snakes, lizard s, turtles, tortoises), fishes (miscellaneous) and cartilaginous chondrichthyan fishes (Table 2 1). The invertebrates were grouped into: cephalopoda (octopuses, squid, chambered nautilus), bivalvia (clams, scallops), gastropoda (snails), hydrozoa (corals a nd sea anemones), scyphozoa (jellyfishes), crustacea (crabs, shrimps, crayfish), arachnida (tarantulas and scorpions), and the insecta (Table 2 1).
22 Freshwater Tropical F ish (FWTF), Marine Tropical Fish (MATF), Crustacean (CRUS), Molluscs (MOLL), Insect (INSECT), and Other (Null, Non CITES listed, other live inverts) (Table 2 2). Table 2 1. The composition and number of animal imports into the U.S. in 2010; principally for the pet trade Taxon or LEMIS Labels Shipments, number of Individuals, number of Species, number of Animals classified to species Reptilia 5,659 854,479 585 93% Mammalia 1,246 483,984 90 98% Aves (Birds) 1,116 171,860 327 91% Amphibia 2,403 3,230,081 136 94% Arachnids 568 157,332 74 62% Anthozoa (Corals & sea anemones) 44,386 33,302,560 362 73% Chondrichthyes 190 8,301 19 58% Gastropoda 358 47,749 36 45% Insecta 3,315 198,196 103 44% Hydrozoa 132 920 3 1% Scyphozoa 4 62 1 100% Bivalvia 507 69,897 17 99% Cephalopoda 876 55,858 8 92% Fish, misc. 4,996 10,146,993 236 51% 39,254 171,069,447 unknown unknown Totals 105,010 219,797,719 1,997 Table 2 2. The composition and number of animal imports into the U.S. Animals were designated only to a generic biological classification scheme Shipments, number of Individuals, number of (Insect) code BUTT (butterflies, non CITES listed) 3,813 815,764 (Crustacean) CRUS 5,392 4,036,160 (Null, Non CITES listed & other live invertebrates) code OTHER 11,866 6,912,604 (Molluscs code) = MOLL 2,288 1,005,726 (Marine Tropical Fish) code = MATF 7,499 12,910,934 (Freshwater Tropical Fish) code = FWTF 7,712 140,702,367 (Phyla comprising less than 1%) code = MISC 684 4,685,892 Totals 39,254 171,069,447
23 Roughly 78% of all import shipments were of aquatic animals, of these more than half contained corals, sea anemones, crabs, shrimps, clams, and scallops. However, fishes constituted the largest volume of the imported live animals, approximately 163,768,595 or 75% of the total number. Corals and sea anemones comprised the second largest group, with 33,302,560, or 15% of the whole group. Crustaceans and other aquatic invertebrates amounted to an additiona l 4.8%. Aside from fishes, very few other vertebrates were imported (N = 4,740,404 or 2.1%), and of these, 68% were amphibians which amounted to 3,230,081 or 1.4% of the total. A total of 1,997 species were tabulated from the import records. Reptiles had t he highest number (N = 585) of species reported, followed by birds (N = 327), marine corals and sea anemones (N = 362), fishes (N = 255), and amphibians (N = 136) (Table 2 1). The remaining species (351) belonging to miscellaneous Phyla and Classes (mollus cs, crustaceans, and insects) (Table 2 1). Most imports of the higher vertebrates (> 92%) as well as octopuses (> 92%) were identified to species level (Table 2 1). A good number (> 73%) of corals and sea anemones were also identified at least to the genus level. However, shipments of most other invertebrates were simply entered as 2). In the same way, the greatest number of imported live animals were freshwater tropical fish (FWTF; N = 140,702,367) and marine tropical fish (MATF; N = 12,910,934) (Table 2 2). In fact, millions of these ornamental fish were imported annually into the U.S. for the pet trade (Figure 2 1). In 2010, approximately 63% of the fish imported for the aquarium trade were labeled uncategorized ornamental fish; that is without any reference to species.
24 Figur e 2 1. Number of freshwater (FWTF) and marine (MATF) ornamental fish imported into the U.S. principally for the pet trade, in years 2008, 2009, and 2010. Source: USFWS Law Enforcement Management Information System (LEMIS). The most numerous ornamental fish, identified to species, imported into the U.S. were goldfish ( Carassius auratus Cyprinus carpio ), and guppy ( Poecilia reticulata ) (Table 2 3); the cardinal tetra ( Paracheirodon sp.) was also prevalent in the records. These species with 20 others (Table 2 3), accounted for some 8,862,464 or 5.4% of the total number of imported fish however, 87.4% of all the fish that were identified, were done so to at least genus. The information necessary for identification of ornamental fish to species level is indeed available on the Form 3 177, and has been used for a detailed analysis of the U.S. trade in ornamental fish (Chapman et al. 1997). For example, a pproximately 201 million fish were imported into the U.S. in 1992. Ornamental freshwater fish accounted for approximately 96% of the total volume while marine aquarium fish the remaining 4% (Chapman et al. 1997). Since that time, there was an apparent redu ction of approximately 25% in ornamental fish imports into the 0 20 40 60 80 100 120 140 160 180 Yr 2008 Yr 2009 Yr 2010 Millions of fish FWTF MATF
25 country, but the percentage of imports of ornamental marine fish almost doubled to 7% by 2010 (this study). Table 2 3. Principal ornamental fishes and their numbers imported into the U.S. in 20 10. Except for two species, all were of freshwater origin. Name Number imported Carassius auratus 4,554,773 Cyprinus carpio 2,427,863 Poecilia reticulata 1,372,598 Xiphophorus spp. 3551 Paracheirodon sp. 287,608 Betta splendens 46,481 Barbus spp. 2,170 Botia spp. 8,391 Ancistrus spp. 1,498 Corydoras spp. 10,229 Otocinclus spp. 7,348 Synodontis spp. 4,420 Symphysodon spp. 47,495 Pterophyllum scalare 15,989 Apistogramma sp. 2,597 Aulonocara spp. 3,504 Astronotus ocellatus 2,578 Cichlasoma spp. 16,762 Tropheus spp. 1,461 Osteoglossum bicirrhosum 6,348 Polypterus spp. 1,561 Pantodon buchholzi 3,350 Hippocampus (marine) 33,889 Aeoliscus strigatus (marine) 8,972 Perhaps as a result of increased hobbyist education and advances in design technology for ease of maintenance in marine aquarium keeping. Also, national production of ornamental fish has been developing considerably and increased steadily, especially for p opular species that were imported entirely from abroad such as barbs and tetras. The number of imported ornamental fish species was 1,539 in 1992 (Chapman et al. 1997), compared to an all total of 255 species, including non ornamental fishes, in 2010 (this study). The names and tally of the imported species in
26 the initial study were obtained directly from copies of the declaration of importation 3 177 forms made available through freedom of information act provisions (Chapman et al. 1997). Such data differe nces clearly illustrate the need for accurate recording of data from the declaration of importation forms to the LEMIS database. In addition, the valuable data that can be obtained from the declaration of importation forms. Interestingly, the top 32 specie s imported in 2007 amounted to approximately 71.9% of the total number of fishes, and similarly in 2010 the top 23 species accounted for 87.4% of the number of recorded fishes. Except for four species, the majority of the 23 species in 2010 were included i n the top species imported into the U.S. in 1992 and 1971 (Ramsey 1985; Chapman et al. 1997). It seems reasonable to conclude that the top 19 fish es presented in Table 2 3 are the ones preferred by hobbyists and most will remain the bulk of the trade in or namental fishes for some time. The LEMIS database is a tool with great potential for monitoring and management of wildlife commerce, beyond simply serving as a clearinghouse of international trade declarations. LEMIS was used to identify the most commonly traded genera and species of amphibians and reptiles, and their overall trade volumes, for the purpose of deriving estimates of the number of wild caught individuals and determine if the harvest of certain species was sustainable (Schlaepfer et al. 2005). The LEMIS database was also used to examine import and export trends of freshwater turtles from the U.S. and derive estimates in harvest (collection) and species composition of exports (Ceballos & Fitzgerald 2004; Mali et al. 2014). In addition, LEMIS was used to develop a risk assessment analysis to address the potential introduction and prevention of zoonotic diseases, carried primarily in mammalian wildlife (Pavlin et al. 2009).
27 Where LEMIS fails to be as useful is with ornamental fish, and is where the emphasis should be placed now, since it is the population group with the greatest number of individuals and species in the trade. Although a great number of fishes are produced in farms, the majority of the species, both freshwater and marine are still cau ght in the wild and not recorded. Due to their high demand, many ornamental fish species may be at risk of over exploitation, as has happened to many populations of fish stocks destined for human consumption. Highlights of the scope and implications of tra de in such a high number of fish were also addressed by Smith et al. (2008). Given the ornamental fish trade is global in nature, the establishment of species in sites where they are not native is also a risk; concerns also addressed by Andrews (1990) and Smith et al. (2008). Having hundreds of species, several of them traded in millions of individuals at a time, but broadly labeled simply as marine aquarium tropical fish (MATF) or freshwater tropical fish (FWTF), greatly limits the opportunity to utilize t he information available to the LEMIS database. It will be of great utility if the data for ornamental fishes be recorded in the LEMIS database as detailed as for other groups of animals. As with many large collections of data, inconsistencies can abound. For example, biodiversity databases often contain incomplete distribution of data because information was collected with many different goals (Hortal et al. 2007). Essentially, a database is only as good as the quality of the compiled data, and with any la rge database, quality control of data is critical to maintaining its utility (Pardo et al. 2013). Detailed information in the declaration of importation forms is necessary for proper evaluation of ornamental fish consumption and needs to be included in LE MIS. Most likely is not included at present simply because of the sheer volume of the
28 shipments and forms that must be handled and transcribed. One simple method that can facilitate greatly managing such large volumes of data is to request the declaration of importation 3 177 forms to be scanned or filed electronically by the exporter. A working example: an exporter in a developing country with a mobile phone can now easily collect and submit data in the field. An open source data kit can be installed in th e mobile phone; the kit enables data collection and submissions to a central server. Therefore, the declaration of importation form 3 177 can be filed electronically, or filled out, scanned, and submitted with the mobile phone. Optical character recognitio n (OCR) software can then be used to recognize/separate the text from the scanned document. Allowing data to be easily accessed, combined, analyzed, and placed into standardized categories. A similar system has been used for healthcare data management in r emote locations (Anokwa et al. 2009). The USFWS does have an electronic declarations system (eDecs), as an alternative means for filing form 3 177, for the purpose of reducing paper and time in processing. It is unclear however, how many importers and expo rters are using this system and how much of that information is directly transferrable to the LEMIS database. The LEMIS database has access to a wealth of information. If criteria were developed that allowed for more complete datasets or better transmissio n of data from the form 3 177 to LEMIS, it can be a very useful tool that can guide conservation policy and action on behalf of ornamental fish. Conc lusions The declaration of import Form 3 177 that accompanies every shipment of live animals into the U.S., provides their common name, complete scientific name (when known), their quantity (either by number or weight), monetary value, country of origin,
29 and names of exporter and importer. Therefore, LEMIS could be a very useful database for the control of exotic species in the U.S. proper, and at the same time give insight into the management and conservation measures of the species in their native range. Especial ly of aquatic animals since these are the predominant species imported, and millions are traded every year. The data reported in LEMIS for species in higher Class vertebrates like mammals, birds, reptiles, and amphibians have been used effectively to evalu ate impacts of the trade on target species and their populations. Unfortunately, the vast majority of aquatic animals are not identified to species in the LEMIS database, most likely due to the sheer volume of entries. Perhaps in a day and age when we are all using computers, something as simple as requesting the import forms to be scanned or digitally filled out by the exporter could greatly facilitate the access to such valuable information.
30 CHAP TER 3 ORNAMENTAL FISH HEALTH WITHIN SEGMENTS OF THE SUPPLY CHAIN This chapter includes three studies related to the health of fishes in the ornamental fish trade. The first study (previously unpublished) examines the mortality of seven different species of wild caught Amazonian fishes imported to a wholesaler. T he second study (publication 2) examines the presence of injury for Silver dollar f ish, Metynnis orinocensis that have been wild caught, i mported wild caught, and farmed . A third study (pu blication 3) proposed use of Artemia as a live drug delivery system for metronidazole. F ish health encompasses more tha n the limited definition of a fully functioning state of equilibrium between organisms and environment, but rather is a state of being characterized by anatomical integrity, the abillity to perform certain ecological roles, the abilitity to tolerate stressors o f physical and biological origin , and freedom from disease and death (Last 2001). Mortality in Seven S elect Species Imported from the Wild One source of concern and criticism of this industry are the losses of ornamental fish es attributed to the trauma of collection, handli ng, and exposure to pathogens , which are inherent stressors of fishes in the trade. Mortality attributed to these events can be significant with estimates of the average mortality a s high as 50% to the complete loss of shipments (Schmidt & Kunzmann 2005; Hemdal 2009). Many studies have focused on mortality resulting from the collection process from the wild with reported values of mortality at an industry accepted standard of 5% for deaths on arrival (DOA) (Lim et al. 2002 ). However, other studies have indicated a much higher direct mortality from the wild and show wide variability depending upon the region and the species collected; with estimates for the cardinal tetra ( Paracheirodo n axelrodi ) industry of Brazil at less
31 than 6% to 80% in some marine tropical fish, such as the Banggai cardinal fish ( Chao et al. 2001; Gil & Martinez 2001; Vagelli & Erdmann 2002; Cato & Brown 2003; Dowd 2003; Lunn & Moreau 2004). One main goal of the in dustry is to reduce the causes of trauma and limit the exposure to disease in much of supply chain for ornamental fish. The exposure of fish to stress ors as part of the normal collection, shipping, and retail process, can enhance the risk of infection and mortality in aquarium fish (Winton 2001); posing a significant animal welfare issue. Many of the concerns on the welfare of animals in the aquarium fish trade stem from direct or indirect post harvest mortality (Schmidt & Kunzmann 2005).To replenish lost s tock, more aquarium fish are imported, placing a drain on the wild resource and resulting in a continuous cycle of loss and replacement. Fish deaths were counted by species for selected shipping lots , for DOA (Dead on Arrival), after 24 hours, 4 8 hours, a nd 72 hours , because m uch of the mortality that occurs as a result of the transport and stress occurs during the first week of the recovery period , post transport (Pramond et al . 2010 ) . Species examined were Amazonian , but from a variety of regions , and includ ed Leporinus faciatus, Cordoras aeneus, Boekkia fredcochui, Carengiella strigata, Paracheriodon axelrodi , Corydoras burgessi, and Hemmigrammus blehri. Total mortality was summed and determined as a percentage for the entire shipping lot (Table 3 1). Findings indicate that mortality varied by species and for many species most mortalities occurred within the 24 hour period post shipping. The greatest mortality event occured for a shipment of Cordoras aeneus , which arrived without water due to a change the exporter made to the plastic shipping bags (personal Communication, exporter).
32 Table 3 1. Mortality as a percent of total shipping lot for selected Amazonian species imported into wholesaler in South Florida. DOA represents dead on arrival, DAA represe nts dead after arrival. Species name DOA DAA (24hr) DAA (48hr) DAA (72hr) Individuals shipping lot Total Mortality % Cordoras aeneus 847 45 14 0 1280 71 Leporinus faciatus 152 44 1 0 960 21 Boeklkea fredcochui 326 56 1 0 4000 10 Carnegiella strigata 165 50 0 0 3000 7 Corydoras burgessi 17 1 0 0 400 5 Hemmigrammus bleheri 59 0 0 0 3000 2 Paracheriodon axlerodi 18 0 0 0 2500 1 Cordoras aeneus are armored catfish from the family Callichthyidae, with sharp spines that punctured the plastic shipping bags. This particular incident resulted in 71 % mortality of the shipping lot. While this might have been an isolated incident, it shows how minor changes can result in unintended consequences through the loss of fishes and consequent economic loss. Other g roups had mortalit ies ranging from 1 20 % (Table 3 1). This study demonstrated that mortality greater than the industry accepted standard of 5% frequently occurs when importing fish from the wild. Greater attention should be paid to conditioning and shippin g practices within exporting countries to improve standards for wild caught imported fish es . END OF STUDY 1. To identify potential problems and sources of trauma from handling an experiment was conducted to quantitatively measure the epithelial injury in o rnamental Silver Dollar Fish, Metynnis orinocensis at several points of the supply chain. This study was published in the Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society and is presented as follows from pages 3 3 43.
33 Publication 2 : Quantitative Measurement of Epithelial Injury in Ornamental Silver Dollar Fish ( Metynnis o rinocensis ) Captured in the Wild, Imported Wild Caught, and Aquacultured. 2 The ornamental fish industry relies on the fish product reaching the mark et al ive, in good health, and of sufficient visual quality to attract both advanced and novice aquarists, after withstanding the stresses and injuries during collection, storage, shipping and handling. At each step in the trade route there is considerable movin g and handling of the fish. Almost any type of net, container, or bowl, and even bare hands, are used to handle the fish. Hand nets, of all different shapes and sizes are one of the most widely used tools for collecting and moving ornamental fish. However, few studies have documented the injuries associated with the collection, handling, and shipping of ornamental fishes. Many of the existing studies have focused on the possible morbidity and mortality that can be incurred during handling and transport (Cha o et al . 2001; Gil & Martinez 2001; Vagelli & Erdmann 2002; Cato & Brown 2003; Lim et al . 2002 ; Lunn & Moreau 2004 ). The journey of collection and storage, through transport and distribution, to their final location in the hobbyist tank, may cause severe s tress to the fish and may also result in their mortality (Lim et al . 2007). The necessity to maintain and transport fishes alive has prompted concern over quality control and increased focus on non destructive fishing practices (Sadovy & Vincent 2002). Stu dies on non ornamental fish show that net handling can cause many physical and physiological problems as well as endocrine imbalances (FSBI 2002; Conte 2004; Content published with permission from Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society. End of Publication on page 43. Livengood E. J., Aya E., Arias J. A., Chapman F. A., 2013 Quantitative measurement of epithelial injury in ornamental silve r dollar fish ( Metynnis orinocensis ) captured in the wild, imported wild caught, and aquacultured. AACL Bioflux 6(5):470 477. 2
34 Brydges et al. 2009). Roughly netting the fish or using an abrasive net will easily remove fish sc ales and disrupt the protective mucus coat (FSBI 2002; Conte 2004). Also, if many fish are handled at the same time they may injure themselves inadvertently as there is the potential to cause a deep cut or puncture to the skin with their fin rays or spines . Fins and eyes can also suffer damage as a result of rough handling (Barthel et al. 2003). Acute cutaneous injuries caused by abrasions and rough handling have been known to significantly increase the ability of pathogens to cause disease in the afflicted fish (Bader et al. 2006). The traditional method used to discern skin injury in fish is by clinical examination aided by a standard body injury assessment chart with a diagram of the external anatomy of the fish. Anatomical diagnostic charts have been des igned to function as a general reference to identify where a readily apparent external skin lesion may be located on the fish and if there is some obvious body part missing (Goede & Barton 1990; Kane et al . 1999; Kane 2005). Such clinical assessments can a lso be expressed in quantitative terms using a health assessment index (HAI) where measured health variables such as damage to skin, body condition factor, and blood parameters like hematocrit are assigned index values or numerical ratings from best to wor st or the like (Adams et al. 1993). However, it still remains difficult to conduct an accurate health or clinical assessment, especially for those injuries that are not yet visible or apparent in the skin that can serve as a portal or point of infection fo r more severe forms of a disease. The error in diagnostic accuracy may also be compounded when handling fish of small size.
35 The purpose of this study was to determine the presence and extent of skin injuries incurred during nor mal handling procedures of th e Silver D ollar fish ( Metynnis orinocensis (Steindachner, 1908) at different stages in the supply chain from capture, to post import at a wholesale facility, to fish cultured on a farm. Silver dollars are amongst the most popular and valuable of ornamental fishes from South America, and are in high demand the world over. Colombia has been a world leader in ornamental fish exports and the largest exporter of wild caught ornamental fishes to the USA (Chapman et al . among the principal species exported from that country (Gil & Martinez 2001). Therefore, Colombia and silver dollars were chosen , as they represented a typical fish in the market, employing standard practices for th eir collection from the wild and subsequent handling. We used the fluorescein test described by Noga & Udomkusonsri (2002) to detect any breaks in the skin, and computer assisted image analysis of digital photographs to determine the spatial extent of the injury (Wallat et al . 2002; Davis & Ottmar 2006). The fluorescein test can be performed on live fish, and can discern low level injuries of the skin (Noga & Udomkusonsri 2002). Understanding how and when injuries to the skin occur are important for maintai ning the health and overall quality of ornamental fish in the trade. Skin injuries will cause a significant reduction in the commercial value of ornamental fishes, which are traded or selected primarily for their overall appear ance. Skin injuries may also compromise the overall health of fish. Even when not visibly apparent or obviously threatening, superficial skin injuries can increase the risk of infection. Importantly, fish handling and skin injury can lead to delayed mortality (Svendsen & BÃ¸gwald 1997; Davis 2005), this being more probable given the
36 relatively small size of most ornamental fish. Delayed mortality rates in ornamental threat that can influence the sustainab ility of natural populations of fish destined for the aquarium trade. Establishing more accurate estimates of skin injuries may point to possible relationships between them and net handling procedures during collection, holding, and transport of ornamental fishes. Results can provide a useful management tool for the conservation of the resource and its sustainability in the market. Materials and Methods M. orinocensis ) were collected in the wild along the as the llanos, near the town of Puerto Gaitan, Colombia. Many other species of ornamental fish are also collected from these flood ed forests during the rainy season. These areas are very remote and require travel for most of the day by truck, then hiking to the collection site. The fish were collected with a seine net made of nylon fiber with a mesh size of 3 mm; a typical method of capture by fishermen. After capture, fish were placed in assorted sizes and types of plastic containers filled with surrounding ambient water. Within an hour of collection, fish were sorted into polyethylene bags filled with water and oxygen (approximately 25 fish per bag), and transported via truck to a holding facility in the city of Villavicencio. However, motorcycles are the most common vehicles used for transporting the fish from collection sites to the holding and culture facilities. The entire proces s including collection and transport to the holding facility took approximately 48 hours. After the initial capture, all sampled fish were manipulated with fine mesh hand nets typically used in brine shrimp culture. The fish holding facility in Villavicenc io served as a fish collection center where artisanal fishermen took their fish
37 for later distribution to wholesalers and retailers. Silver dollars were also farmed for export at the center. They were raised in earthen ponds (5.2 x 4.2 m) at a density of s ome 10,000 fish per pond, and were fed sporadically with commercial pellets used for feeding tilapia. The farmed fish were sampled from the pond with the same seine net used for the collection of fish from the wild. A different stock of silver dollars, als o collected from the wild and exported from Colombia, was sampled at an import wholesale facility in Miami, USA. To examine the fish for skin injuries we updated a computer assisted color image analysis system that we utilize in our laboratory to objective ly measure the skin color in live ornamental fish (Wallat et al . 2002). For this study the fish were photographed under white light (6500K) and UV (254 nm) light conditions. The extent of wounding to the dermis was quantified using fluorescein, which is a compound that perfused across any break in the dermis and can be visualized using the UV light (Noga & Udomkusonri 2002). A portable photography studio (BestLight Studio) illuminated from above with a circular fluorescent light tube was used to control lig ht exposure, and create an environment suitable for taking fluorescent photographic images. When photographed in the dark, the studio light was turned off, and the inside and outside of the studio was covered in black felt. The fish were then illuminated w ith a handheld UV lamp (model UVGL 58; UVP), held at a 45Âº angle so the fluorescein dye will emit fluorescence brightly wherever it was present. A consistent angle was critical to taking good images with maximum fluorescence detection. Photographs were tak en with a Nikon 4500 digital camera with Bower +4 macro (close up) 28 mm lens. To take the UV photographs, no light filter was used, and the camera was set to manual exposure mode, ISO of 200, lens aperture value of F5.7,
38 and shutter speed of 1 sec. Camera distance to the fish had little effect on fluorescence detection only on focus, however, the fish were photographed from a consistent distance. In preparation for the test, a stock solution (200 g mL 1) of water soluble fluorescein sodium salt (F6377; Sig ma Aldrich) was prepared in a plastic 1.4 L tank. Batches of 5 fish were immersed in the fluorescein solution for 6 min, and rinsed with freshwater three times for 2 min apiece in a similar but separate tank; the water was exchanged after each rinsing. To accommodate and pose the fish for a photograph, they were placed sideways on a foam backed bottom of a 100 mm diameter Petri dish. The cover of the dish served as restrainer and to calm the fish. No anesthesia was used; MS 222 is the only anesthetic agent FDA approved for aquaculture but is known to interfere with the fluorescein test (Davis & Ottmar 2006). When the fish stopped moving temporarily, the lid was removed and the picture taken quickly; the procedure taking no more than 1 min. Afterwards the fis h were placed in a tank filled with well aerated water (DO 5 7 g mL 1) for recuperation and observed for 2days. The fish were photographed out of the water, since the water attenuated and scattered the UV light, and interfered with detection of the fluores cence. Digital images were downloaded from the camera to an IBM Thinkpad Lenovo X61 computer and stored in bmp file format. The Microsoft Paint software (Microsoft Corp. Windows XP version) was used to trace the outline of the image of the fish, and the co lor analysis LensEye software (Version 10.1.7) to permit edge detection and quantify the amount of color in the digital color images. Not only did the presence of fluorescence in the digital color images indicated the occurrence of skin injuries but allowe d for edge detection. Edge detection refers to a clear and objective demarcation of abrupt changes in pixel intensity, which
39 characterized the boundaries of the skin lesions in the color images and permitted estimation of the overall extent of the injuries . Both sides of the fish were photographed to estimate approximately the percentage of skin injury covering the body. The quantitative variables of weight, length, size, and percentages of skin injury were presented as mean standard deviation. Quantitative estimates of the presence and extent of skin injury in the groups sampled were analyzed using a Chi square test at a 99% level of confidence. The quantitative analyses were performed in a personal computer using the statistical software JMP 9 (SAS Institu te, Version 9.0.0). Results and Discussion The silver dollar fish collected directly from the wild and sampled on site (n= 20) were 1.6 Â± 0.5 g BW and 42.6 Â± 4.6 mm TL. The aquaculture fish sampled directly on farm (n= 20) were 0.5 Â± 0.4 g BW and 28.7 Â± 6 .2 mm TL. The wild caught silver dollars sampled at the import wholesale facility (n= 20) were 5.9 Â± 2.4 g BW and 52.8 Â± 6.6 mm TL. When fishing for the silver dollars in the wild, net seining caused significant mortalities. In contrast, no immediate morta lities were observed in the silver dollars harvested from the aquaculture pond, nor those imported. All live fish, after capture and handling, had minor skin wounds and the overall extent of injuries covered 3.5 4.6% of their body surface; across study gro ups and regardless of fish size. Fifteen percent (15%) of the silver dollars captured in the wild showed skin injuries immediately after collection. At the wholesale facility, 57% of the imported wild caught silver dollars had skin injuries. The lowest num ber of injured fish (5%) was found in the group sampled at the aquaculture farm which were also the smallest in overall size. This finding is counter intuitive to the idea that organisms of a smaller size would have larger surface area contact with the net or handling and therefore greater potential for injury or trauma.
40 These findings suggest that the source or where the fish were coming from influenced the presence or absence of injury. Although it was expected that some injury would always occur whenever fish are captured and handled, a consistent pattern of abrasion was observed at the import wholesale facility for the fish that were recently received from shipment. Most prominent were traces of fluorescence seen along the ventral surface of the body ind icative of mild skin abrasion, proba bly caused by the net (Figure 3 1 A ). Silver dollars possess a laterally compressed body (flattened side to side), with a pronounced ventral keel. The ventral keel is a hardened ridge extending from the ventral surface of the fish, from just below the gill plate region towards the anal fin. This places their body weight entirely along the keel and positions their body to be held inadvertently along their laterally flat sides when manipulating them with a hand net; the net causing the wound by scraping the mucous or skin. When observed grossly the ventral keel area had missing scales and appeared to have d eeper tissue injuries (Figure 3 1 B ). The high number of fish packed for shipment, approximately 200 fish per bag, can exacerbate this type of injury. To alleviate the possible consequences of this practice, densities should be reduced thereby reducing the number of fish to rub against each other. A much more viable alternative and one that can substantially increase the overall health of the fish is to hold or acclimatized the fish for a few days after capture to allow for rapid healing of superficial injuries. During this time therapeutics can be used to prevent infection of areas exposed to trauma. While in recuperation, they can be fed lightly with addition of a nutritional supplement such as vitamin C, which has been shown to significantly reduce mortality post shipment and improve overa ll health (Lim et al . 2002).
41 A B Figure 3 1. Wound pattern as a result of shipping and representative color analysis. Image A) represents the fluorescein stained individual with the pattern of injury noted in the fish that were shipped from Colombia to USA wholesaler. There is a line of fluorescence seen on the ventral keel, which can also be seen grossly in control image, where the fish appears to be missing scales and having a deep injury in the dermis (B). Images courtesy of Elisa Livengood, Miami Flo rida. Of the 20 fish sampled from the fish group acquired from the aquaculture farm, only one individual showed significant markings that were detected by the fluorescein test. Fish from this group lived confined at higher densities and were captured with same mesh net as those collected in the wild. The lack of injury presence suggests that fish were subject to less handling and had more ability to withstand injury or trauma. It appears that aquaculture fish may be less prone to skin lesions than wild fish . The lack of injury presence further support the suggestion that the culture of ornamental fish provides a viable alternative to the collection of fish from the wild and serves as a useful management tool to maintain the sustainability of the resource, es pecially whenever fish are being caught indiscriminately, which is often the case when fish are in popular demand and found primarily in the wild. The fluorescein test has been used to understand the process of disease and healing in fish, the possible sou rces of skin damage in fish associated with different handling methods, and skin sensitivity after exposure of the skin to assorted chemicals (Noga & Udomkusonsri 2002; Davis &
42 Parker 2004; Fontenot & Neiffer 2004; Davis et al . 2008; Ibrahem & Mesalhy 2010 ; Colotelo & Cooke 2011 ). Most often used by the ophthalmic community as a clinical diagnostic tool for viewing the integrity of the cornea, there is very little research on the exact function of the interaction of ocular cells with the dye (Morgan & Maldo nado Codina 2009). As a relatively new tool for fish diagnostics, further research is necessary to determine how fluorescein adheres to damaged sections of the fish skin and consistently yields fluorescence. Fluorescein detects obvious skin abrasions, ulce rs, and fin fraying in fishes (Noga & Udomkusonsri 2002; Davis & Ottmar 2006; Colotelo 2009). In this study it was observed that fluorescein did not stain all visible injuries like those areas where some of the scales were missing from the body surface. Fu rther experiments using M. orinocensis made with a superficial injury to the dermis from direct scale removal, revealed th at this type of injury has a detection period of less than 48 hours, as the injured areas were reduced in size by half within 24 hours (unpublished data). This could be attributed to a rapid process of re epithelialization that can begin within an hour of injury. During the healing process there is rapid proliferation, organization, and differentiation of cells; the restoration of all ce ll layers typically completed at 4 to 6 d post injury (Fontenot & Neiffer 2004). Therefore, the fluorescein test is a useful tool for detection of superficial acute injury to the dermis and other deeper tissue traumas. Only 3 out of 60 fish died after the fluorescein test. However, these individuals displayed large areas of fluorescence (14% or more of their body surface) and gross morphology of abrasions, attributed to their prior handling during capture or shipment. The fluorescein test, especially in com bination with computer aided color analysis, is a
43 useful tool for rapid assessment of acute injuries in fish that do not require killing of the fish. This test is particularly suited for the ornamental fish industry, especially in time periods of concern: collection, shipping, sorting and the like. END OF PUBLICATION Other studies have shown a high prevalence of ornamental fish collected from Amazonian region with presence of at least one type of external parasite in 9.4% of the population surveyed (Aguinag a et al. 2015). Parasites are major causes of death and disposal of ornamental fish and can represent signifi cant economic losses (Aguinaga et al. 2015). Stressors associated with capture, transport, and handling, which occur as part of the normal practice of the ornamental fish trade can favor parasite infestations (Takemoto et al. 2004). When fish become stressed they are less able to fight off disease and often manifest symptoms of disease (Su llivan 2014). These concepts have been studied in laboratory zebra fish colonies . W hen exposed to handling or a disease causing agent separately there were few mortalities (3%) , but when exposed to stressful handling and the disease agent the mortalities were significantly higher (14%) (Ramsay et al. 2009). The orna mental fish trade often combines both the elements of handling stress and pathogen exposure, with respect to the coll ection and shipping of the fish. This was exemplified in Chapter 3 in the study of injury of shipped M. orinocensis . For this reason, devel oping strategies that will improve the health of ornamental fish es in the supply chain is important for maintaining fish welfare. The potential use of Artemia as a live feed drug delivery system for m etronidazole is present from pages 44 49 .
44 Publi cation 3 : Uptake of Metronidazole in Artemia of Different Developmental S tages 3 The purpose of this study was to examine the capacity of live brine shrimp Artemia spp. to accumulate metronidazole at different developmental life stages. Metronidazole is used in fish as an antiparasitic medication. An effective drug delivery method is to enrich the Artemia with metronidazole and offer them as live feed to the infecte d fish, usually ornamental species, and other small fishes. Artemia cysts were hatched and then soaked in a metronidazole solution (0.05%) at instars 1 3 of larval development. Our findings indicated that Artemia were able to accumulate metronidazole at le vels considered therapeutic to other animals and humans (25 100 mg/kg). However, the levels varied depending on the stage of larval development. Artemia accumulated the highest levels of metronidazole (137 143 mg/kg) when they started filter feeding (insta r 2), whereas newly hatched Artemia (instar 1) contained the lowest level (85 mg/kg). Based on this study and a review of the literature, a new protocol recommended for enriching Artemia with metronidazole consists of soaking the Artemia in a 0.05% metroni dazole solution for 3 h at room temperature. Because metronidazole is relatively insoluble in water, it must first be dissolved in warm water with continuous stirring. Brine shrimp Artemia spp. have been used for decades as a highly palatable and reliable supply of live feed that is indispensable to good nutrition in the larviculture of many fish and shellfish species ( Seale 1933 ; Sorgeloos 1991 ; Sorgeloos et al . 1998, Content published with permission from Journal of Aquatic Animal Health. End of publication on page 49 . Rodriguez L., Livengoo d E. J., Miles R. D., Chapman F. A., 2011. Uptake of Metronidazole in Artemia of different developmental stages. Journal of Aquatic Animal Health. 23(2):100 102.
45 2001). Since Artemia are nonselective feeders, they can be enriched or fortified with nutrient supplements to enhance their nutritional value or with medications to provide health benefits for the animals to which they are fed ( Sorgeloos 1991 ; Lavens & Sorgeloos 1996 ; Touraki et al . 1996 ). Artemia have su ccessfully been the vehicle of delivery for antibiotics such as oxytetracycline, enrofloxacin, trimethoprim, sulfamethoxazole, and flumequine ( Tou raki et al . 1996 ; Roque et al . 1998 ; Majack et al . 2000 ; Yahyazadeh et al . 2007 ). However, in the scientific literature, there are only a few references documenting the enrichment of Artemia with antiparasitic agents. Parasites are among the prime agents affecting health in fish ( Scholz 1999 ). Especially troublesome are intern al protozoans of the genus Hexamita , which live principally in the intestines of fish. The afflicted fish generally will lose weight, exhibit a loss of appetite, and eventually waste away. Ornamental aquarium fishes like cichlids (e.g., oscar Astronotus oc ellatus and discus Symphysodon spp.) are especially prone to Hexamita ( Smith & Roberts 2010 ). An effective treatment for Hexamita in fish is metro nidazole, which is often reported in medical formularies (e.g., Noga 1996 ; Carpenter et al . 2001). The recommended dose is 5 100 mg/L as an in wat er medication (e.g., a bath treatment) or 25 100 mg/kg when mixed with any food the fish will eat, including Artemia . For delivering the drug through live Artemia , general directions call for soaking the Artemia in a 1% metronidazole solution under refrige ration (4Â°C) for 3 h (e.g., Langdon 1992 ). The purpose of the present study was to examine the capacity of live Artemia to accumulate metronidazol e at different developmental life stages. Materials and Methods Artemia cysts purchased from Brine Shrimp Direct (premium grade, Great Salt Lake origin; lot 30332) were stored in a refrigerator at 4Â°C until use. Cysts (50 mg) were
46 hydrated for 60 min and t hen incubated in 50 mL, skirted, conical bottom, screw cap tubes (product number 62.559.001; Sarstedt) that were filled (30 mL) with deionized water mixed with artificial seawater salts at 15 g/L (Natural Sea Salt Mix; Oceanic Systems). Air was supplied to each incubator tube from an aquarium air pump; the air was delivered through aquarium air line tubing fitted at the end with a glass Pasteur pipette. The tubes were placed in a rack under continuous fluorescent illumination in a laboratory room at a tempe rature of 26 28Â°C. The hatched Artemia were separated by shutting off the aeration, waiting 5 10 min, and withdrawing them directly from the water column via suction by using a plastic disposable pipette with the tip cut off. Artemia were selected at speci fic naupliar stages (instars 1 3; Benijts et al . 1975 ), typically after 8, 12, and 56 h post hatch , respectively. Before the Artemia sample was obtained for each treatment, subsamples were taken to confirm the identity of the specific life stage. Two replicates were used for each treatment group and each control group. The selected life stages of Artemia were soaked in a 0.05% metronid azole solution (30 mL) for 3 h at room temperature (26 28Â°C). Metronidazole is relatively insoluble in water; therefore, to dissolve the medication, a stock solution of 0.1% was prepared first by mixing continuously and warming the solution to about 60Â°C. The concentration was then halved to achieve a 0.05% solution. This differed from the standard recommendations found in various fish medical formularies, the hobby literature, and Internet sources, which call for soaking the Artemia in a 1% solution of met ronidazole in a refrigerator at 4Â°C for 3 h (e.g., Langdon 1992 ; Noga 1996 ). In a separate treatment, instar 3 Artemia (adults; older than 1 d) were soaked for 3 h at room temperature in a 0.05% metronidazole solution mixed together with a commercial
47 emulsion that was rich in highly unsatura ted fatty acids (Self Emulsifying Concentrate [SELCO]; INVE Aquaculture). The mixture of Artemia was poured over a glass fiber filter (Millipore AP40, 47 mm; Thermo Fisher Scientific) in a Buchner funnel, and the water was drawn by a vacuum operated with a hand pump. The concentrated Artemia (mean Â± SD = 60.6 Â± 0.9 mg) were carefully scraped off the filter into vials, immediately frozen in liquid nitrogen, and kept in a freezer until analysis. Determination of metronidazole concentration in Artemia followed the high performance liquid chromatography (HPLC) methods described by Cox et al . (2010) . The metronidazole used in the trials and in preparation of standards for the HPLC assay was purchased from the Florida Tropical Fish Farmers Association (chemical purity = 99.89%; lot number 08121601). Methanol used for the assay was HPLC grade (Thermo Fisher Scientific). Estimated means (Â±relative SD; [SD/mea n] Ã— 100) were compared by using analysis of variance and reported with corresponding relative SDs. Results and Discussion Results indicated that 60.6 Â± 0.9 mg of Artemi a immersed in metronidazole for at least 3 h at room temperature (26 28Â°C) will bioaccumulate levels ranging from 85 to 151 mg/kg ( Table 3 2 ). Although the efficiency of metronidazole accumulation by Artemia was very low (<1%), these concentrations are similar to those recommended for other animals and humans (dose of 25 100 mg/kg, delivered in capsules that contain 375 mg of metronidazole in a pill of 500 mg). However, the level of bioaccumulation in Artemia is associated with developmental stage. There were significantly lower detected levels of the medicament in newly hatched (instar 1) Artemia
48 ( Table 3 2 ). After molting into the second naupliar stage (instar 2), the Artemia absorbed or filtered the medication much more efficiently such that metronidazole levels reached 137 143 mg/kg. Artemia that were at least 1 d ay old (instar 3) absorbed metronidazole at a significantly higher level than instar 1 Artemia but at a significa ntly lower level than instar 2 Artemia . Instar 3 Artemia treated with SELCO also accumulated the metronidazole; however, there was variability between the two replicates, indicating a possible interaction between metronidazole and the lipid nature of SELCO ( Table 3 2 ). The recommended protocol of soaking Artemia in a 1% metronidazole solution in a refrigerator was abandoned bec ause metronidazole easily precipitated out of solution and a large proportion of the Artemia died at this cold temperature. We also observed that at room temperature, many Artemia died or considerably slowed down when the metronidazole concentration reache d 1%. Table 3 2 . Artemia developmental life stage, supplementation regime and resulting concentration measured for metronidaz ole from HPLC. Means fo llowed by letters are statistically different (P=0.05). BLOQ: Below Limit of Quantification . Developmental Stage Metronidazole, ppm Relative SD Control Instar 1 BLOQ NA Instar 1 85.16 0.60 Control Instar 2 BLOQ NA Instar 2 137.02b 5.91 Instar 2 143.39b 2.92 Control, Instar 3 BLOQ NA Instar 3 104.19c 0.34 Instar 3 114.01c 0.02 Control Instar 3+ Selco Â® BLOQ NA Instar 3 +Selco Â® 94.74 0.11 Instar 3 + Selco Â® 151.02b 0.09 To improve the effectiveness of the enrichment procedure, further studies must be performed to understand the mechanism by which Artemia accumulate metronidazole in their tissues: (1) simple absorption across the cuticle (exoskeleton), (2) active filtering of particles from the water, or (3) both mechanisms. Newly hatched
49 (instar 1) Artemia are nonfeeding nauplii and are entirely dependent on stored yolk for their nutrition. This might explain the lower concentrations of metronidazole present and suggests that drug accumulation during this stage is by simple absorption across a thin cuticle covering the body. Artemia in instar 2 and later s tages of development have a functional digestive system and can obtain their sustenance and other extraneous substances by filter feeding on particles from the water ( Makridis & Vadstein 1999). The higher levels of metronidazole were found only in older Ar temia , suggesting that filter feeding is a primary mechanism for accumulating the medication, acting in combination with some absorptive mechanism. However, a thicker cuticle may develop with age after each molt (e.g., instar 3) and may reduce absorption o f metronidazole compared with that in younger developmental stages (e.g., instar 2). Even though no fish were tested in this study to determine an effective dose of metronidazole for treatment of fish parasites, the findings from this study support the rec ommendations by many aquarists and by a few documented reports (e.g., Langdon 1992 ; Noga 1996 ; McCoy et al . 2004) that Artemia enrichment with metronidazole is a viable drug delivery system to treat parasites in ornamental fishes and other small fishes. We achieved a reduction in drug usage relative to the levels recommended previously; this is important for safety to the target species and to the environment as lesser amounts of the medication are needed. To increase the amount of medication that accumulates in the Artemia , we recommend begi nning enrichment at least 8 h after hatch, when Artemia molt to their second naupliar stage and start to filter feed (i.e., instar 2). If SELCO is used, first mix the SELCO with the metronidazole and then soak the Artemia in this solution, performing all s teps at room temperature.
50 C HAPTER 4 EDUCATIONAL EXTENSION PUBLICATIONS FOR THE ORNAMENTAL FISH HOBBYIST This chapter introduces a collection of extension publications aiming to educate the ornamental fish hobbyist. Publication 1 (FA124 1 ) presents information on the ornamental fish trade and perspectives for responsible aquarium fish ownership. Publication 2 (FA179) presents information on sharks for aquaria and considerations for their selection. Publication 3 (FA166) presents informatio n on a popular species group in the ornamental fish trade, Symphysodon spp . as a candidate species for aquaculture. Publication 4 (FA 176) presents a lesson plan geared towards students (the future fish hobbyist) on how to hatch a popular live feed, Artemia , often used i n the ornamental fish industry . The publication s introduced can be found as links within the Appendix , that link to their pdfs on the University of Florida extension publication sites . The role of extension from its first establishment was t o give instruction or practical demonstrations in agriculture or home economics, effectively bringing the University to the people (Ra s mussen 1989). The goal of the extension professional is to a subject or in their actions (Cone 2011). Educational extension publications can be a useful tool as a means to disseminate information in order to educate the public. The extension publications were aimed to educate the hobbyist on a variety of topics r elevant to the ornamental fish trade. 1 FA124, FA179, FA166, FA176, Refer to a numbering system for extension publications (EDIS) f or the University of Florida, and are an additional ide ntification for the urls found in the appendix to view publications in their intended extension formats.
51 The hobbyist s are the target audience for these publications because they are the key consumer of the ornamental fish trade . The demands placed on our freshwater and marine resources create an urgent need to link education on the use of the resource and environmental stewardship (Livengood & Chapman 2009 ). First time fish keepers often have a high failure rate due to ignorance, wrong equipment use , or choosing a fish species above their level of expertise (Townsend 2011). T here has also been a change to industry in two ways: the technology to keep fish has gotten more accessible resulting in many novice aquarium keepers entering the hobby, and there is a greater disconnect between certain retailers as many purchases can be made solely online (APPA 2014; Sullivan 2014). An analysis of US based aquarium hobbyist resources and interviews showed a lack of information available on the processes involved in collection, and few hobbyi st oriented media outlets covered topics of the negative consequences of t he trade or human health concerns (M cCollum 2007). Pet fish owners surveyed rarely show ed emotional attachment to their fish pets as compared to other pets and this lack of emotional attachment may mean they are valued les s (Langfield & James 2009; Sullivan 2014). Less attachment could also mean less attention paid making fish es the forgotten pets (Sullivan 2014). Other ways fish differ from other pets is how they are incorrectly perceived by the public as being low mainten ance, they are viewed as a group and not individuals, and in general there seems to be a disconnect between the aquatic and terrestrial environment with poor understanding of fish biology (Sullivan 2014 ). E stablishing the social norm of consumer responsibi lity should be the goal so that the hobbyist does not view aquarium species
52 as disposable commodities and is instead making choices that uphold the welfare of the animal and reflect a consideration for the natural resource base. The first publication linked in the Appendix as ( FA124 ) is titled fish trade: An introduction with perspectives for respo . The purpose of this publication was to convey the background of aquarium industry to the public and to give strategies towards being a responsible con sumer. This publication represented a specific attempt to educate the hobbyist that drives the consumption of aquarium fish. Many hobbyists are not aware of how fish reach their home aquaria and how they can be bet ter owners through prac welfare and the ecological impact of their hobby. The second publication is linked within the Appendix as ( FA179 ) rpose of this publication was to present criteria and considerations for species selection of sharks, and to educate the hobbyist on the popular sharks kept in home versus public aquariums. This publication outlines specific considerations of keeping shark s in aquaria, such as aquarium design, and recommendations of species as well as considerations for their overall welfare. Another purpose of this publication is was to introduce the public to some conservation concerns facing sha rks. The third publication is linked in the Appendix as ( FA 166 ) Florida Aquaculture: Discus Symphysodon spp. , a Profitable, but Challenging Species species profile that included taxonomy, classification, genetics, culture methods, and current conservation issues facing the popular aquarium fish Symphysodon spp . also known as Discus . The fourth publication linked to in the Appendix as ( FA176 ) is part of
53 a grea ter aquaculture curriculum series called Teach Aquaculture. This publication titled through an inquiry based classroom activity the hatching and rearing of brine shrimp, Artemia , a popular live ornamental fish feed. The purpose of this publication was to contribute to future hobbyists and also students and provide an activity that could be used in classrooms to practice the scientific method. It is difficult to measure the adopti on or successfulness in reaching the intended audience for many extension publications; however, the University of Florida maintains statistics of how many tim es publications within the extension system called Electronic Data Information System (EDIS) are downloaded. Between the years 2010 2014, the first publication (FA124) title d was downloaded approximately 2 3 , 00 0 times and has been cited by 39 other sc ientific publications (Google scholar analytics) and was picked up by an E lear n ing site for a scho ol in Thailand and the Texas A& M extension site. The second publication (FA lso quite widely downloaded with approximately 7,000 downloads. The third publication, (FA Species for Florida Aquaculture: Discus Symphysodon spp. , a Profitable, but approxi mately 1 1 , 000 times. This fourth publication (FA176) approximately 3,000 times. Each of these publications represent separate strategies to address specific educational goals for different stakeholders within the ornamental fish trade.
54 The number of downloads and other uses indicate that there is considerable interest in the education al aspects of aquatic resources, i n particular those detailing the ornamental fish trade . This s uggests there was and is a need for information in what actions the consumer of aquarium animals can take to better ensure environmental stewardship and care of their aquatic animals .
55 CHAPTER 5 CONCLUSIONS ON THE COMPLIATION OF STUDIES PRESENTED AND OV ERALL RECOMMENDATIONS This chapter is an overview of the con clusions presented in Chapters 2 4 as they relate to the ornamental fish trade. In addition, this chapter also provides recommendations and suggestion for future areas of study. The purpose of ch apter 2, was to evaluate a potential source of data (LEMIS) for the regulatory management of the ornamental fish trade. While many of the higher class vertebrates (mammals, birds , reptiles, and amphibians) have used the LEMIS database to evaluate trade imp acts (Ceballos & Fitzgerald 2004; Mali et al. 2014) , there was very little possibility of this same utility, as the database currently reports for ornamental fishes. This study did produce useful information on the magnitude of imports of ornamental fishes and corals. For example, the approximate number of individuals labeled as freshwater tropical fish (140 million) and the approximate number of marine aqu arium tropical fish ( 12 million ) that were imported in 2010. The overall application of LEMIS to meet with criteria for regulatory management remains limited as it does not produce complete, meaningful, and useful data for the ornamental fish trade. Lack of meaningful data will always inhibit effective management wh ich in turn limits the sustainability of the industry (Smith et al. 2008; Rhyne et al. 2012; Foster et al. 2014; Rhyne et al. 2015 ). The LEMIS system has the potential to provide useful management data, with slight modifications (such as greater inclusion and transparency of data) and better incorporation of the dec laration of import form 3 177. In order for the LEMIS system to effectively serve as a tool for regulatory management the trade data must be organized in a fashion that is useable for both manage rs and scientists.
56 To effectively communicate information about trade flows in the marine aquarium trade the New England Aquarium and Roger Williams University launched a Marine Aquarium Biodiversity and Trade Flow Database ( https://www.aquariumtradedata.org/ ) using import and export data from trade invoices. This database shows trade flows , exporters to import location and the number of individuals shipped (Rhyne et al. 2015). Expanding these efforts to include freshwater tropical fishes data would allow for a better picture of the volume of imports and exports and the distinction of relevant species in the ornamental fish trade . Chapter 3 includes three studies related to the health of fishes. The first examined mortality of ornamental fishes imported, the second examined the presence of injury in Metynnis orinocensis, and the third examined the use of Artemia as a live drug delivery system. Injury was found in Metynnis orinocensis that were imported as part of the aquarium fish trade . These fish showed a specific pattern of injury on their ventral k eel, likely incurred during shipping. Fluorescein, used to highlight injury, and use of the LensEye software to quantify % area of injury, indicated a relatively non invasive health assessment of recent acute injury in ornamental fishes. This procedure could be used as a rapid assessment of fi sh quality post shipment, and its use should be applied to other species within the trade. A survey of mortality observed in shipping lots was conducted for various species of Amazo nian origin. M ortality ranged from as lows as 1% to as high as 71 % , with mu ch of the mortality occurring within the 24 hour period post shipment . Bot h of the findings of injury and mortality indicate the need for continued industry imp rovements with regards to handling and shipping of ornamental fish es as
57 part of the pet trade. F ish injuries may increase the risk of further infections from pathogens, resulting in mortality ( Ramsay et al. 2009; Loh & Landos 2011 ). Therefore, it is concluded that fishes must be handled carefully throughout the transport process to minimize physical injury (Francis Floyd 1995, Crosby 2006, Livengood et al. 2014). One finding s of the study with M. orinocensis , alluded to the differences in exposure to injury between wild caught and aquacultured fish. Exploring these differences further, and making a c omparison between aquacultured fish quality and the same species collected wild, could potentially highlight the benefits of choosing an aquacultured product. M. orinocensis, when rated for homogeneity, color, and appearance of relative health, had a signi ficantly higher rating for cultured fish versus wild caught (Arias et al. 2008). This comparison should be explored further to explore differences in quality after shipping between fish from aquacultured sources versus those that are shipped from the same exporter and of the same size, but are from wild caught sources. The third publication proposed a new drug delivery method for the treatment of a common parasitic disease in ornamental fish. Artemia were found to uptake a therapeutic level of metronidazole when supplemented at the developmental life stage of instar 2, which is their second molt. The results of this publication indicate the need for a future study to evaluate the delivery system of Me tronidazole dosed Artemia and its effectiveness in treating ornamental fishes infected with the parasite Sprinucleous vortans (Hexamita). Chapte r 4 introduced several extension publications aiming to educate the hobbyist. Publication 1(FA124) presents inf ormation on the ornamental fish trade and
58 perspectives for responsible aquarium fish ownership. Publication 2 (FA179) presents information on sharks for aquaria and considerations for their selection. Publication 3 (FA166) presents information on a popular species group in the ornamental fish trade, Symphysodon spp . as a candidate species for aquaculture. Publication 4 (FA176) presents a lesson plan geared towards students (the future fish hobbyist s ) on how to hatch a popular live feed, Artemia , often used in the ornamental fish trade. Research indicates that most hobbyists want to maintain a level of good health and quality for their kept pet fish, but simply lack the knowledge of practices to do so (Sullivan 2014). These educational extension publications have been downloaded extensively from 2010 2014 (> 40,000), linked to many hobbyist forums, and have been cited in other scientific publications. The number of downloads and external link s demonstrates the public interest in ornamental fish. Howeve r, research shows that not all hobbyists value fish welfare equally to other pets (Etscheidt & Ma n z 1992; Townsend 2011; Sullivan 2014 ). This raises the question of how to educate the ornamental fish hobbyist and produce meaningful change with respect to h ow they care for and view their pet fishes as well as other conservation issues such as the introduction of invasive species . More research is needed to assess the efficacy of publications that inform the hobbyist that result in a positive behavioral chang e. The scope and complexity of the ornamental fish trade requires the development of better regulatory management, i mprovement to health, and greater educational efforts . This dissertation concludes that there is no one overarching solution that will fix the issues both ecological with respect to the sustainable use of a resource to sociological with education of the various stakeholders to effect change.
59 APPENDIX EDIS PUBLICATIONS PDF AND URL LINKS 1. FA124 The ornamental fish trade: An introduction with perspectives for respo nsible aquarium fish ownership http://edis.ifas.ufl.edu/pdffiles/FA/FA12400.pdf http://edis.ifas.ufl.edu/fa124 2. FA179 Sharks for the Aquarium and Considerations for their Selection https://edis.ifas.ufl.edu/pdffiles/FA/FA17900.pdf https://edis.ifas.ufl.edu/fa179 3. FA 166 Candidate Species for Florida Aquaculture: Discus Symphysodon spp. , a Profitable, but Challenging Species for Florida Aquaculture https://edis.ifas.ufl.edu/pdffiles/FA/FA16600.pdf https://edis.ifas.ufl.edu/fa166 4. FA176 Teach Aquaculture Curriculum: Dancing with Brine Shrimp http://edis.ifas.ufl.edu/pdffiles/FA/FA17600.pdf http://edis.ifas.ufl.edu/fa176
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69 BIOGRAPHICAL SKETCH Elisa Livengood was born in Greensbor o, North Carolina and grew up mostly on the outdoors. She spent many hours on that farm; fishing and working with animals. Elisa later attended East Carolina University and received a bachelor biology with a concentration in marine b iology. From there she then went on to pursue a master s degree under Dr. Frank Chapman to examine the adoption and efficacy of aquaculture curriculum and its development for use in the K 12 classroom. In Dr. trade and was shocked at what was learned of this little discussed industry. Elisa has always loved fish, found them fascinating, and has bee n an aquarium hobbyist since childhood. However, the biggest fascination of this industry to her was its magnitude and movement of fish around the world. Post master s degree, she decided to stay for her PhD at the University of Florida focusing on the su stainability of the ornamental fish industry. Since then the associated projects have taken her to aquaculture farms in South Florida, wholesalers in Miami, and collection and wholesale in Colombia, South America. After graduation, Elisa accepted a teachin g faculty position in Aquaculture and Renewable Resources at a state university in New York.