Pseudomugil luminatus (Red Neon Blue-Eye) Spawning Report Submitted by Patchin Crandall Curtis Background: This is a shocking fishy horror story with a happy ending. In April, 2019 a shipment of the club’s fish from The Wet Spot were acclimated at my home for a week prior to the Spring All-Day Auction. After the auction, the tank used to quarantine the Pseudomugil luminatus was left empty for a couple of months. There were no heater, no water changes, and no food. It gets worse... to keep the filter going, I periodically squirted household ammonia into the tank. When Christine Neumeyer and Jamie Smith of Freshwater Exotics visited, Christine asked what I was keeping in the little 15g cube tank. “Nothing” I replied, remembering that I hadn’t dosed it with ammonia for a while. “Not so,” said she, “something’s swimming around in there.” Bonus babies!!! The quarantined Pseudomugil luminatus had dropped eggs in the tank before their trip to the Auction. The eggs had hatched and the fry survived over a 2-month period despite the ammonia dosing not to mention not ever having been fed. A miracle. Colony: The bonus babies developed into 4 striking adults: two males and two females. Food size has to be small for this diminutive species. The adults top out at barely over 1” in length. They also stay at the surface, so floating or very slowly sinking foods are best. At 6 months of age, they were ready to start spawning. The fish were conditioned with Golden Pearls (300-500 micron), frozen copepods and bloodworms, and live foods including scuds and daily newly hatched BBS. Tank Set-Up: The fish are resident in an unheated, rimless 15g cube tank fitted with a corner mattenfilter, and aquascaped with CaribSea Sunset Gold sand, some stones, and a structure of Brazil Nut shells and Savu pods secured with marine-grade stainless steel screws to a Spider wood frame. Plants include Cryptocoryne mioya , Cryptocoryne beckettii 'Petchii', and Hydrocotyle sibthorpioides . The water is stained very dark with tannins due to the pods. Water temperature ranges from 68F in the winter to 73F in the summer. Spawning: After the fish were observed spawning and depositing eggs in the Cryptocoryne leaves without any surviving fry, it became apparent that collecting and hatching the eggs in a separate tank would be required. Pseudomugil are short-lived fish with lifespans in nature of one year, and in the aquarium 18 months, maybe a little more, so there’s no time to waste in assuring the next generation. After consulting with Frank Cowherd (always the first step in any new project), I attached 3 dark green synthetic spawning mops to Styrofoam floats and suspended them in a corner of the tank up against the mattenfilter and Spider wood. Frank advised having a thick mass of spawning mops to increase the amount of cover provided to the eggs thereby lowering the rate of predation. Pseudomugil deposit eggs all day long, so to maximize recovery it’s helpful to inspect the spawning mops more than once a day. Temperature and Gender Distribution: Controlled experiments have shown that certain species of fish can exhibit temperature-dependent sex distribution. Increasing temperatures can affect the sex ratio of a species in one of three ways: increased numbers of males, increased numbers of females or increased numbers of males at high and low temperatures, with a balanced sex ratio at intermediate temperatures. Researchers show that even small changes of just 1-2°C can significantly alter the sex ratio from 1:1 (males:females) up to 3:1 in both freshwater and marine species. It is well-documented that African Butterfly Barb ( Enteromius hulstaerti ) fry hatched and raised above 71F will be predominately male. There is a large body of scientific papers published on temperature-dependent sex determination in Atheriniformes (to which rainbowfishes and blue-eyes belong). For details and excellent references, see Odontesthes bonariensis (Atherinopsidae, Atheriniformes, Teleostei). Evolutionary History and Whole Genome Sequence of Pejerrey (Odontesthes bonariensis): New Insights into Sex Determination in Fishes by Daniela Campanella. The George Washington University, ProQuest Dissertations Publishing, 2015. Although a major sex-determining gene has been identified recently in several species of Odontesthes, the temperature at which pejerrey eggs or larvae are exposed to during the first weeks of life is a major factor determining phenotypic sex, overriding the effect of the genotype. The cited study reveals potential mechanisms to explain how genetic, environmental, and chemical factors interact in a sex determining network during key developmental stages of pejerrey. After the Spring All-Day Auction, a number of people who won or purchased the Pseudomugil lots reported that the fish were all male. Perhaps the suppliers were only selling male fish. But how many East Asian fish farmers, distributors, and exporters do we suppose are sitting there with a light bulb and magnifying glass sexing each individual juvenile schooling fish by the hundreds of thousands in order to sell only males? I’m skeptical. To me, an interesting hypothesis is that many fish coming from subtropical waters (like Badis species, Odessa Barbs, Enteromius species, and Lake Inlé species) are being raised in fish farms under tropical, warm water conditions resulting in populations heavily skewed toward males. I set out to determine if the gender distribution of Pseudomugil luminatus was dependent on the temperature in the hatching and rearing tank, specifically in a realistic range of temperatures the average North American home aquarist would experience. The experiment consisted of placing 50+ eggs collected over a week each in 4-6 gallon hatching tanks with sponge filters, oak leaves, crepe myrtle bark, and java moss. Each tank was to be maintained at a different temperature and hatch rates and gender distribution evaluated. Tank 1: Temperature started at 75F and drifted down to 72F over three weeks (always on 10W heater). 62 eggs were placed in the tank over the course of a week. 12 fry appeared but as the weather and basement got colder, the temperatures in this tank continued to drift down. As the temperature went below 72F, fry started to die and eventually only 6 were left. 63 eggs were collected between 10/23-29/2019. 12 fry hatched between 11/6-18/2019. Result: Low hatch rate and low survival rate. Of 6 surviving fry, 100% were female. Tank 2: Temperatures 69 – 71F (always on 5W heater). 69 eggs were collected over the course of a week with the intention of maintaining a temperature of about 70F to form the low end of the temperature spectrum. At these low temperatures, only 4 fry appeared and eventually all but one died. The little one’s name is Spunky. She turned out to be a girl is now living happily at warmer temperatures with friends and family. Result: All but 1 larval fish died before developing enough to determine the gender. The surviving fry was moved to warmer temperatures and developed into a female. Tank 3: Temperatures 74 – 75F (always on 15W heater). As the weather got colder, temperatures drifted down to 72F and fry started to die. A second heater was added to stabilize the temperature at 74F, and the population stabilized at 9 fry. Result: low hatch rate and low survival rate with 2 males and 7 females making it to adulthood (22% males, 88% females). Tank 4: Temperature 78F (pre-set Aqueon 50W heater). Even this tank with a 50W heater slowly drifted from 78 – 75F. 91 eggs were collected between 1/17-23/2020 of which 23 hatched between 1/26 - 2/6/2020. The hatch rates still seemed very low, perhaps because eggs were placed in the tank where they sank to the bottom and sat in water with limited current. Alternatively, the cool water temperature in the spawning tank may be preventing eggs from fertilizing properly. The fry that hatched at this temperature were quite vigorous and grew quickly. 23 fry hatched and thrived. Result: low hatch rate and high (100%) survival rate with a gender distribution skewed toward males (5 females and 18 males, or 22% females and 88% males). The pivot temperature at which there is even gender distribution appears to be somewhere between 75 - 77 F. Raising Fry: Pseudomugil fry are surprisingly well-developed upon hatching after an extended incubation period of 10 days or more. They come straight to the surface upon hatching and feed from the surface and near surface. Therefore, it’s important to give them first foods that will stay at the surface as well. The fry immediately started taking vinegar eels and 5-50 micron Golden Pearls. They didn’t hesitate for even a second to eat the dry food. They also received live rotifers and euglena. Their small size requires that they be fed 3 times a day to thrive and put on good growth during the first two weeks or so. Once they can eat BBS at about 2 weeks, they grow very quickly. Conclusions: The experiment confirmed that the gender distribution of blue-eyes is indeed highly temperature dependent under typical aquarium conditions with lower temperatures favoring females and higher temperatures favoring males. The pivot point at which the gender distribution will be 50:50 appears to be somewhere in the range 75 - 77F. While this experiment was interesting and provides strong indications, further experiments are required under more stable temperature conditions to pinpoint the ideal temperature at which there is an even distribution. In the future, I would like to repeat the experiment using 4 identical hatching tanks each outfitted with a 100W heater and external temperature controller. Tank temperatures would be maintained at 74F, 76F, 78F, and 80F with no more than + 1F variation over the course of the experiment. Another interesting experiment would be to evaluate the sensitivity of hatch rates to the amount of water movement over the eggs.