An Overview of Utilizing Flow Cytometry to Investigate Reproductive Capabilities of Goldfish

Previous studies have shown that F1 hybrid females between koi carp (Cyprinus carpio) and goldfish (Carassius auratus) produce diploid eggs due to a transformation of meiosis. Crossing F1 hybrid females with koi results in backcross triploid hybrids with two haploid chromosome sets of koi, and have one set of goldfish (FBK), while crossing these females with goldfish produces triploids with two sets of goldfish and one set of koi (FBGF). The goal of this study was to evaluate the reproductive ability of both variants of triploids. Some triploid hybrid females of both variants produced eggs after injection of CPE. Eggs were fertilized with sperm from diploid males of parental species (koi and goldfish). Progenies obtained from FBGF females did not survive past one month, and their ploidy has not been analyzed. Progenies obtained from FBK females also had low survival; nonetheless about 1,000 5-month-old juveniles were obtained. Approximately 350 juveniles were analyzed for ploidy by analysis of DNA content in erythrocytes. Analyzed juveniles had an average ploidy of approximately 2.5n, with the majority of values being within the range of 2.3n-3.0n. Since aneuploid fish have in their genomes one haploid set from parental males, this data indicate that FBK females produce aneuploid eggs with a ploidy range of 1.3n-2.0n, and a modal ploidy of approximately 1.5n.

Aquatic vegetation is necessary for a healthy ecosystem by providing several services such as: habitat for organisms, breeding territory for fish, a source of food, improved water quality, withholding sediments, and several more. An excess of aquatic vegetation can however be disadvantageous by making it more challenging to harvest fish, and by having a negative impact on biodiversity, water quality, and on recreation via decreased maneuverability for boats. Invasive aquatic weeds are a significant problem globally with large financial implications; the United States alone is estimated to spend over 100,000 million dollars annually on aquatic weed control. Herbicides, manual removal, and biological control are all options for the removal of aquatic weeds. Manual removal of aquatic weeds is not ideal due to being labor intensive, especially in large bodies of water, and is not a long-term solution. Herbicides are the most commonly used option for removal of aquatic weeds, and can be a fast and effective method contingent on the proper identification of aquatic weed species. It is important to consider that the use of herbicides can negatively impact water quality, and is also not an option when dealing with fish for human consumption. These disadvantages have created the desire for non-chemical treatments of aquatic weeds, which has led to the use of biological control of aquatic weeds.

Biological control is not an immediate fix for aquatic vegetation, but is a more environmentally friendly method in terms of the amount of waste produced. There have been several species of fish utilized for biological control of aquatic vegetation via direct consumption, or by bioturbation effects of foraging behavior. One concern of using biological control is the impact that the introduced species will have on the ecosystem such as outcompeting native species for resources, or unintended hybridization. For this reason, it is desirable that organisms being used for biological control are sterile. It is possible to produce polyploid fish that have additional haploid sets of chromosomes via heat or pressure shock; this allows one to obtain triploid fish that have three haploid chromosome sets and are sterile because of this. Triploid grass carp (Ctenopharyngodon idella) are a popular option for biological control of aquatic vegetation. Another method of producing sterile organisms is hybridization; distant hybridization is when two different species are crossed to generate a new species. Crossing two species with incompatible sets of chromosomes impacts meiosis, and can result in sterility of their offspring.

The occurrence of fertile F1 hybrid females warrants further research on the reproductive capabilities of the triploid progenies originated from them; if it is confirmed that they are sterile then koi x goldfish hybrids can potentially be valuable as biological control. The purpose of this study is to investigate the reproductive capabilities of triploid koi x goldfish hybrid females and results of their crosses with parental species. Crossing these hybrids with their parental species will emulate what could happen in the environment. It is necessary to determine the ploidy of the progenies produced in order to understand the reproductive abilities of the hybrids that produced them. The ploidy of fish can be determined in several ways including: Feulgen microdensitometry, manually counting chromosomes, measuring erythrocytes with coulter counters, gel electrophoresis to observe proteins, and by measuring the DNA content of individual cells with flow cytometry.

The most commonly used method to determine the ploidy of fish is flow cytometry due to its convenience. Flow cytometry has been used since the 1980’s and was initially used for detecting cancerous cells by varying amounts of DNA. Flow cytometry has been used on plants, shellfish, finfish, insects, reptiles, amphibians, phytoplankton, and bacteria. Flow cytometry works by individually shooting particles through a light, and a computer records the disturbance of the light for each particle passing through; the data is then displayed as histograms where different sized particles will show up in different locations. Staining the nuclei with fluorescent dyes to determine DNA content is one of the most common applications of flow cytometry; this technology however is broad and is applied in several other fields when there is need to distinguish subpopulations of a specific type of cell or protein. The methodology for this study is based on quantifying the amount of nuclear DNA in erythrocytes obtained from the fish.

This study was performed at the Aquaculture Research Center (ARC) at Kentucky State University in Frankfort, KY. The fertility of female triploid backcross hybrids was assessed, and those deemed fertile were injected with carp pituitary extract and saline, while all of the male triploid backcross hybrids were injected to induce gamete maturation and spawning. A concentration of 3 mg/kg of the body weight was used. Females received 10% of the total dose 24 hours before spawning, and the remaining 90% 12 hours before spawning. Males recieved the full dosage 18 hours before spawning. Eggs were collected in containers, weighed, and then evenly distributed into bowls. Milt was collected in 30 ml containers. Eggs and milt were then mixed in a bowl with a turkey feather. Two minutes after the fertilization, the eggs were placed in a 1:8 cow milk: water solution in order to reduce adhesiveness of the eggs. After being in the cow milk: water solution for one hour, the fertilized eggs were transferred into McDonald jars to incubate until they hatch.

Hatched larvae were collected in hapas suspended in raceway systems, with a flow of dechlorinated municipal water at approximately 20 °C. Swim-up larvae from the hapas were then relocated into 20 m3 outdoor tanks, supplied from a water reservoir. Progenies remained in outdoor tanks approximately 4-5 months, and were then brought into the Aquaculture Production Technologies building at the ARC. The progeny were placed in indoor recirculating aquaculture systems at approximately 25 °C, and fed a commercial diet. Ploidy was determined by quantifying the amount of nuclear DNA for each fish through the use of flow cytometry at approximately three months after stocking as was done in Delomas et al. (2017), and Warner et al. (2018). Blood was used for this as fish erythrocytes contain nuclei. Fish larger than 25 g had their blood collected from the hemal arch with a 21-gauge needle, and samples were collected in 3.0 ml vacutainer tubes that contained lithium heparin to serve as an anticoagulant. Fish smaller than this had their tails cut and were bled into 1.5 ml microcentrifuge tubes containing a 0.85% NaCl saline solution.

Samples were mixed with a Propidium Iodide Staining Solution (PI), as well as Largemouth Bass (Micropterus salmoides) blood which served as an internal control. Diploid koi blood was used as external controls. 100 µl of PI was first added to each 1.5 ml microcentrifuge tubes. Samples from fish larger than 25 grams had 0.5 µl of bass blood added, and then 0.5 µl of sample blood added. The samples from fish smaller than 25 grams had 4 µl of bass blood added, followed by 4 µl of sample blood. Saline solution was then added to obtain a final concentration of 0.2 µl bass blood/ 100 µl saline. Samples were incubated in the dark at room temperature for 10 minutes, then run on an Accuri C6 (BD Biosciences, CA, USA) flow cytometer for 40,000 events. The ratio of hybrid DNA to bass DNA allows us to calculate the ploidy of the hybrids. Flow cytometry detects the intensity of the emitted fluorescence from the PI stained nuclei as they pass through the machine. Histograms are then generated and contain peaks for both the internal control (bass), and the sample (hybrid). Peaks are isolated from one another using gates, and the mean intensity is then measured. Where FIb is the fluorescence intensity of the bass peak, FIs is the fluorescence intensity of the sample, and Rs is the internal sample ratio. Ratios are obtained which are compared to the external control (diploid koi), in order to determine ploidy levels. The ratio for the control koi is 1.7. The haploid level can be determined by dividing the external control ratio by 2, which results in a haploid ratio of 0.85. The sample ratio is then divided by the haploid ratio, which results in ploidy level of the sample.

Some triploid hybrid females of both variants produced eggs after injection of CPE and yielded progenies with a drastic range of phenotypes and abnormalities when crossed to diploid males of parental species. A total of 344 juveniles obtained from FBK females were analyzed for ploidy. Progenies from FBK females crossed with goldfish males had an average weight of 12.64 g and an average length of 9.53 cm, while those crossed with koi had an average weight of 36.96 g, and an average length of 11.86 cm. All progenies obtained from FBK females had very similar ploidy values so results were pooled. Progeny from these crosses had a ploidy range of 2.3n-4.0n, with the majority being in the range of 2.3n-3.0n, and an average of approximately 2.5n. All progeny obtained from FBGF females did not survive long enough for data on their ploidy to be collected. Goldfish x koi hybrids can potentially be a viable alternative to grass carp for aquatic weed control if they are confirmed to be sterile. Previous studies conducted at the ARC have found that some F1 females were fertile, and produced triploid progeny when crossed to diploid males of the parental species without any treatment to the eggs; quantitatively we are able to confirm that the eggs produced were diploid since the sperm from the diploid males was haploid. The obtained backcross triploids were raised in order to monitor their reproductive capabilities. It was found that a small proportion of the females were fertile and produced aneuploid progenies when crossed with males of parental species.

Progenies obtained from FBK females were majority aneuploid with a mean ploidy of approximately 2.5n. These results were consistent with FBK females crossed with both goldfish and koi males, and show that the eggs produced are approximately 1.5n on average. FBGF females produced eggs, and viable progenies were initially obtained when crossed with both goldfish and koi males. When it was time to collect blood to analyze ploidy of these latter progenies, it was found that no fish had survived. We hypothesize that this mortality is due to the presumed aneuploid nature of these progenies, as well as unusually high temperatures this season. Future work will consist of continuing to monitor the progenies obtained from FBK females in order to observe gamete development, as well as attempting to obtain and analyze progenies obtained from FBGF females. Once the ploidy is analyzed from the latter mentioned crosses, it will be possible to make a better assessment on the reproductive capabilities of goldfish x koi hybrids, and whether or not they will be a feasible option for aquatic weed control.