Progress 09/01/20 to 08/31/24
Outputs
Target Audience:This project aimed to, "...enhance the knowledge and technology base necessary for the expansion of the domestic aquaculture industry" by eliminating the constraints to culture of an economically highly?promising marine fish. This technology could not only be used by those farming mahimahi, but could also be applied to virtually any other high-value warm water marine finfish culture. This project aimed to compare growth rates, feed conversion efficiencies, and behavior within fertile all female populations, and sterile all?female populations. The results could thereafter be incorporated into larger offshore fish farm operational and financial models, to evaluate the likely potential returns from such a venture, and - if viable - for incorporation into a business plan, and execution on that plan offshore in U.S. waters. Changes/Problems:There were several notable challenges to completion of this work. These include obtaining mature mahimahi broodstock in Kona; obtaining a Controlled Substances permit; mahimahi shipping of fertilized eggs in viable condition from Miami to Hawai'i; difficulty with mahimahi that arrived successfully being able to survive beyond 40 days post-hatch; and the abnormally high percentage of male juveniles. Obtaining mahimahi broodfish in Kona: All adults caught in Hawai'i by Ocean Era or partners/ fishermen were very large (> 15lbs). These fish are aggressive and energetic, but have very small scales so their skin is easily damaged. Unfortunately, when captured, they thrash violently harming themselves, if not properly handled - an issue which was compounded by long transport times from successful fishing grounds. For example, a fisherman that brought one fish in alive, had allowed the fish to land on the boat deck causing severe abrasions. It died within 24 hours of capture. In the end, more than a dozen brood candidates were captured and transported to Ocean-Era quarantine and recovery tanks, all of which succumbed to their capture and handling stress soon thereafter. After numerous attempts, using several different fisherpeople and Ocean Era's own crews, boats, and transport tanks, the decision was made to forgo further efforts in catching brood fish of this species in Kona. However, our subcontractor partners at RSMAS, University of Miami were able to readily catch young adult mahimahi during certain times of year. The team at RSMAS has a history of successfully bringing mahi broodstock to land, holding them, and spawning them. Ocean-Era therefore turned their focus to shipping fertilized eggs, larvae, followed by fully metamorphosed fingerlings, from Miami to Kona. Using imported eggs, larvae and juveniles. Hawai'i has strict importation rules to protect its environment. Ocean Era therefore had to obtain an Import Permit from HI Dept of Agriculture, Plant Quarantine Branch, which took roughly two months. Every shipment that came in had to be examined by veterinarians in Miami, then fly through Honolulu International Airport, where it was inspected by Plant Quarantine. FedEx is the only company that could get the package to Kona overnight, meaning this was the only carrier option we had. Between June 2021 and February 2022 eleven shipments of either fertilized eggs, post hatch larvae, or fingerlings were sent to Kona from UM. Only eight of the shipments arrived successfully (two were lost in transit, one arrived late and the animals had expired). All larval rearing efforts using fertilized eggs were unsuccessful in producing viable fry, either with or without treatment with MTT. Slightly better larval rearing success was achieved when eggs were hatched before shipping, although overall survival was still less than 1% to juvenile stage. (Generally, a larval survival rate of > 10% to weaning is expected for mahimahi). While no singular cause of poor performance was identified, we expect that temperature swings during transit, and an (at the time, unrealized) issue with water quality at NELHA, in Kona may be at least partially to blame for low survival during the first two weeks after arrival. After the first three cohorts had low survival, temperature loggers were added to shipping containers in Miami. We found that temperatures would swing down to 15 degrees C, and reach as high as 25 degrees C over 24 hours in transit. While this wasn't immediately lethal, it may have contributed to poor animal health, which could have then been compounded by bacterial issues in transit. Additionally, the fish are extremely aggressive and prone to cannibalism, so those that did survive their larval stages would often harm each other. To remedy at least some of these issues, the University of Miami held the larvae and applied the MTT treatments, then shipped the surviving fingerlings to Kona. Fish reared and treated in this manner exhibited higher survival, and ultimately were the animals used for genetic and histological evaluation. The abnormally high percentage of male fingerlings. Two hundred thirty fingerings that had been treated with MTT were sent from Miami to Kona in March and April 2022. Of those, 50 survived to a size and age large enough for histological evaluation of gonad development. The fish were euthanized in August 2022 for final sampling and analysis. A fin clip was taken from each fish and sent to CAT for genetic sex determination, while the fish carcasses were preserved and shipped to University of Maryland Baltimore County (UMBC) for histological evaluation of gonadal development. Only after the results from both laboratories were submitted to OE, did we share the identification information with our partners for result interpretation and reporting. Dr Ten Tsao Wong of UMBC provided summary analysis and a brief report. Of the 50 fish submitted to CAT, 39 were genetically male, 10 were genetically female, with one individual remaining undetermined. Of the10 genetically female fish, three had begun to develop testes rather than ovaries, indicating some measure of discernable success in using the methods discussed Previous data on this species, as well as generally accepted sex ratios from other species with similar life history strategies, strongly suggest a near equal sex distribution under normal circumstances.Thus, one would expect a near even split between genetically male and female fish in our cultured population of mahimahi fingerlings. Since there was such a high percentage of males at the end of the trial, we strongly suspect unaccounted for environmental factors influencing the observed skewed sex ratio in those cultured for this trial.Possible reasons include the naturally aggressive nature of male mahimahi , the onset of which occurs soon after metamorphosis, or the large temperature swings in the shipping could have also had a sex-favoring effect on survival. Males of this species have also been observed to grow more rapidly in early larval stages, which could have led to competition and aggression-related mortality favoring males in the surviving cultured population. Water chemistry concerns at the Natural Energy Laboratory in Kona. In late 2022, a number of other NELHA tenants raised concerns that there was an apparent, undiscernible but widespread problem with the quality of surface seawater from the NELHA reticulation system in various hatchery operations. (The NELHA surface seawater is drawn in from a depth of ~80', and pumped throughout the facility to a variety of aquaculture and other marine science-related businesses). Several fish and shellfish hatcheries had been having significant problems producing larvae for almost two years. For the prior 35 years, NELHA had been known for being a productive place for aquaculture because of the superb water quality. The cause of these unexplained mortality issues across a range of vertebrate and invertebrate species is still under investigation. In the meanwhile, Ocean Era and several other NELHA-based producers have had to install expensive ozone treatment systems to "sanitize" the water before it is provided to larvae. This appears to have resolved most of the concerns, suggesting that some low-level toxicity issue was the causative agent, perhaps related to encrusting biofouling organisms inside the NELHA HDPE seawater distribution system. What opportunities for training and professional development has the project provided?This work provided a significant component of training for staff at Ocean Era, along with students and faculty at the University of Miami, and staff at the Center for Aquaculture Technologies. Five staff members at Ocean Era, some of whom have worked in aquaculture for over 20 years, gained experience working with this species for the first time. The project also allowed two technicians to learn marine finfish larval rearing, and live feeds production. Additionally, Ocean Era hosted an undergraduate intern during the summer of 2022 (Andrew Sugashita, University of North Florida) who gained around two months of experience working directly with the mahimahi fingerlings. This project allowed CAT to develop markers and screening protocols for mahimahi, which to our knowledge has not been previously accomplished. How have the results been disseminated to communities of interest?Results of this project have been presented at Aquaculture Canada 2022 and Aquaculture America 2024. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The initial plan involved using proven technology, that had been demonstrated in other species, such as Atlantic salmon and tilapia, to suppress gonad development using gene knock-down techniques. This had the advantage of not involving genetic manipulation. The project Co-PI, Prof. Yoni Zohar, was the inventor of the technology, but the patent had been exclusively licensed by his institution to a Norwegian aquaculture technology company. Zohar provided contact information, and Ocean Era management reached out repeatedly to the company (and through other intermediaries), but there was no response. Zohar reported that the company was similarly stonewalling several other US company's requests for licenses for application to other species. The revised work plan for the project then had three overarching goals: Produce an all-female population through creating neomale broodstock (i.e. XX genotype with male phenotype, producing sperm rather than ova) or feminizing the population To induce sterility in an all-female stock Develop SNP markers that would reliably detect XX males, and that would differentiate males and females. Produce all female population through creating neomale broodstock or feminizing the population: This goal was partially achieved. Some neomale (XX male phenotype) mahimahi were produced. However, due to constraints in acquiring high quality fertilized eggs, we were not able to produce an entire cohort of mahimahi that were all phenotypically female. Creating an all female population can be done by either: A- feminizing an entire cohort, so that males would become females. B - breeding neomale fish with female fish to produce a stock of all genetically female offspring. Producing neomales There are multiple means to produce neomale fish (genetically female fish that have been "masculinized"- producing viable sperm instead of eggs). These include treating young animals with a masculinizing hormone, such as 17α-methyltestosterone (MTT), that will overpower any naturally produced estrogen. Alternatively, aromatase inhibitors can be given to the fish, which will prevent the female animal from converting its own testosterone into estrogen. In either case, a female animal is "exposed" to high amounts of testosterone, leading to the production of testes instead of ovaries. 17α-methyltestosterone Treating fish with MTT was identified as having the highest likelihood of success, because it has been used successfully in other cultured fish. As such, it was the first treatment attempted. 17α-methyltestosterone is a Schedule III controlled substance (non-narcotic). Consequently, Ocean Era was required to obtain a Controlled Substances permit from the United States Drug Enforcement Agency, as well as a Certificate of Registration for Controlled Substances from the State of Hawai'i. Similarly, our partners at University of Miami also had to obtain the permit. Ocean Era was granted the permit in July of 2021. Treatment protocols were adapted from Weber, et al. (2020)[1]. Adaptations were based on fish age and "degree days", in an effort to expose our mahi larvae to the compound at the same developmental stage as the trout in the successful study. All fish used during this project came from the University of Miami. The first group were shipped as eggs, and survival of fish to 1 gram was very low. Aggression, cannibalism, and potential water chemistry issues contributed to this (see description in Challenges section). The University of Miami also treated mahi larvae with the MTT protocol described. Then those fish were sent to Hawaii as fingerlings for growout. We held these fish until they were large enough to have gonadal development distinguishable by histology. The genetic sex results were corroborated with the histological examination of preserved fish. Of the 50 fish that were sampled, only ten were genetically female. (The reasons for this disproportionate survival of males are unknown; normal mahi development results in a 1:1 male-female ratio). However, three out of the ten females showed male-like gonadal development. The MTT treatment was therefore 30% successful. This would be sufficient for commercial application of the technology, given that fin-clips could discern the neomales from the remaining stock. Letrozole The second option for masculinization of female fish was the use of the aromatase inhibitor, Letrozol. The letrozole administration protocol was based on work by Das et al. (2012)[2], but modified for the mahimahi's faster growth rate. As such, letrozole was added to both the fish's first feeds (rotifers and Artemia), and their juvenile weaning "dry" diet. Additionally, a combined methyl testosterone+letrozole treatment was administered to a third cohort. This was also based on Das et al. (2012). Feminizing a cohort of fish to induce female characteristics in males Increasing the water temperature in fish holding systems can lead to a higher percentage of females. The increased temperature leads to an increase in aromatase production. More aromatase means more estrogen production, leading to female fish. This was proposed in the original application. However, due to a variety of challenges, we did not complete this portion of the study (see Challenges section below). Induce sterility in mahimahi This portion was not accomplished because of the challenges associated with rearing mahimahi larvae. (see below, "Challenges"). Identify SNPs markers that reliably detect XX males, and that differentiate males and females? This was accomplished under this project in collaboration with our subcontractors at the Center for Aquaculture Technologies (CAT). See attached image of a poster presented at Aquaculture Canada 2022. Effective management of this program in a commercial hatchery operation will require the rapid differentiation of normal XY males from XX neo-males. The project therefore partnered with CAT to identify genetic markers to differentiate the sex of larval or juvenile mahimahi. Tissue samples were provided to CAT from fresh, wild-caught mahimahi collected by research partner RSMAS, in Miami (around 30 samples). RSMAS also obtained samples from shipments originating in Panama, imported fresh into Miami Airport (100 tissue samples - 50 males and 50 females). These were all preserved in RNA later, and then shipped to CAT. Phenotypic sex was readily determined by shape of the forehead of mature whole or gilled-and-gutted (G&G) fish. No immature fish were sampled. CAT's bioinformatic analyses of sequence data showed a very large number of candidate markers for sex located in what is likely (and previously reported) a specific region of the mahi genome. The majority of these markers were located on several discrete contigs The markers were either single nucleotide polymorphisms (SNPs) or INDELS (insertions or deletions of DNA sequence) that were highly associated with males or females. CAT selected a few of the INDELS that yielded sequence insertions or deletions between males and females, and designed PCR assays around those regions. CAT developed and tested 4 assays targeting different INDELS - to be visualized on gels after PCR.Three of the four assays tested worked well. One INDEL assay was selected, based on a preliminary screening on a subset of previously sequenced fish, as well as additional samples that were provided from Miami and Panama that were not previously sequenced, but which were of known sex. All males and females matched 100% genotype by phenotype. [1] Weber, G. M. et a. (2020). "Sex reversal of female rainbow trout by immersion in 17α-methyltestosterone". Aquaculture 528. [2] Das, R., M.A. Rather, N. Basavaraja, R. Sharma, and U.K. Udit. 2012. "Effect of Nonsteroidal Aromatase Inhibitor on Sex Reversal of Oreochromis Mossambicus (Peters, 1852)."Israeli Journal of Aquaculture - Bamidgeh64 (January).https:/?/?doi.org/?10.46989/?001c.20646.
Publications
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